Gold nanoparticles decorated with mannose-6-phosphate analogues

Article abstract of DOI:10.3390/molecules19011120

Herein, the preparation of neoglycoconjugates bearing mannose-6-phosphate analogues is described by: (a) synthesis of a cyclic sulfate precursor to access the carbohydrate head-group by nucleophilic displacement with an appropriate nucleophile; (b) introduction of spacers on the mannose-6-phosphate analogues via Huisgen's cycloaddition, the Julia reaction, or the thiol-ene reaction under ultrasound activation. With the resulting compounds in hand, gold nanoparticles could be functionalized with various carbohydrate derivatives (glycoconjugates) and then tested for angiogenic activity. It was observed that the length and flexibility of the spacer separating the sugar analogue from the nanoparticle have little influence on the biological response. One particular nanoparticle system substantially inhibits blood vessel growth in contrast to activation by the corresponding monomeric glycoconjugate, thereby demonstrating the importance of multivalency in angiogenic activity.

Full text of DOI:10.3390/molecules19011120

Molecules 2014, 19, 1120-1149; doi:10.3390/molecules19011120
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Gold Nanoparticles Decorated with
Mannose-6-phosphate Analogues
Stéphanie Combemale, Jean-Norbert Assam-Evoung, Sabrina Houaidji, Rashda Bibi
and Véronique Barragan-Montero *
Institut des Biomolécules Max Mousseron UMR 5247 UM2-UM1-CNRS-ENSCM 8 rue de l’Ecole
Normale, Montpellier cedex 5 34296, France
* Author to whom correspondence should be addressed; E-Mail: veronique.montero@univ-montp2.fr;
Tel.: +33-467-147-290; Fax: +33-467-144-344.
Received: 25 November 2013; in revised form: 7 January 2014 / Accepted: 10 January 2014 /
Published: 17 January 2014
Abstract: Herein, the preparation of neoglycoconjugates bearing mannose-6-phosphate
analogues is described by: (a) synthesis of a cyclic sulfate precursor to access the carbohydrate
head-group by nucleophilic displacement with an appropriate nucleophile; (b) introduction
of spacers on the mannose-6-phosphate analogues via Huisgen’s cycloaddition, the Julia
reaction, or the thiol-ene reaction under ultrasound activation. With the resulting compounds
in hand, gold nanoparticles could be functionalized with various carbohydrate derivatives
(glycoconjugates) and then tested for angiogenic activity. It was observed that the length
and flexibility of the spacer separating the sugar analogue from the nanoparticle have little
influence on the biological response. One particular nanoparticle system substantially inhibits
blood vessel growth in contrast to activation by the corresponding monomeric glycoconjugate,
thereby demonstrating the importance of multivalency in angiogenic activity.
Keywords: C-C coupling reaction; click chemistry; glycoconjugate; mannose-6-phosphate;
glyconanoparticle; angiogenesis
1. Introduction
The ensuing article was initially motivated by the biomedical importance of mannose 6-phosphate
(M6P) [1]. While two different mannose 6-phosphate receptors (M6PR) recognize the M6P residues
and mediate the endocytosis of extracellular M6P-containing ligands, only the larger of these

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(CI-M6PR, 275 kDa) has been reported to also bind retinoic acid and IGF-II [2]. The biological
importance of this receptor is found in numerous processes and it has been reported that the angiogenic
action of proliferin was mediated by this receptor [3]. We have recently described the synthesis of a
series of mannose-6-phosphate (M6P) analogues, showing for the first time that these monosaccharides
play a role in angiogenesis [4–7]. The replacement of the phosphate head-group by analogues, mostly
bioisosteres, was intended to provide a better understanding of the chemical factors involved in the
modulation of angiogenic activities. It is known, however, that a monovalent carbohydrate ligand
possesses only a weak binding affinity toward its associated receptor protein [8–10]. To impart
biological relevance to such interactions Nature often utilizes multivalency [11,12]. Therefore, interest
in designing multivalent carbohydrate systems has been growing [13]. In particular, glyconanoparticles
(GNPs), that offer useful tools for investigating carbohydrate-mediated interactions, have been
developed [14]. The purpose of the present study was: (a) to synthesize new glycoconjugates bearing
M6P-like groups and (b) to insert these compounds onto the surfaces of gold particles via a spacer for
angiogenic testing. Our objective, therefore, is to investigate the effect of clustered sugar derivatives
on angiogenesis and to determine whether or not the spacer has an influence on the biological
response. The choice of the M6P analogues has been guided by previous results conducted in our
laboratory including the synthesis of carboxylate and azido analogues (with 123% and 125% relative
angiogenic activity, respectively, compared to phosphate buffer saline (PBS) as control in an egg
membrane assay) [4]. Additional considerations include varying the length, hydrophilic or
hydrophobic nature, and flexibility of the spacer between the sugar headgroups and the nanoparticle
core. In this manner we could modify the presentation of the carbohydrates and, consequently, affect
their accessibility during the molecular recognition events. Many of the mannose derivatives with their
different spacers were assembled using the “click chemistry” strategy introduced by Huisgen and
improved by Sharpless and co-workers in 2001 [15,16]. Within a short time-frame, the click chemistry
reaction has proven to be of remarkable utility and broad scope, not only in organic synthesis but in
chemical biology and drug discovery [17,18]. Although 1,3-dipo lar cycloaddition reaction is central to
click chemistry, the resulting creation of a triazole moiety may have an adverse influence on a
biological response. For this reason two other reactions were used for chain elongation or for
conjugation of two synthons: the Julia reaction and the thiol-ene reaction that was run under
unprecedented ultrasound activation.
2. Results and Discussion
The preparation of the neoglycoconjugates we describe herein took place in three major steps:
(a) the synthesis of a cyclic sulfate precursor to access the ligand head-group by nucleophilic
displacement with the appropriate nucleophile; (b) the introduction of the spacers on the M6P via
either Huisgen’s cycloaddition, the Julia reaction, or the thiol-ene reaction under ultrasound activation;
(c) the coupling between the spacer and the sugar moiety. By this means gold nanoparticles as
functionalized by various carbohydrates could be compared for their effect on angiogenic processes.
Although preliminary biological data are presented at the end of the paper, the emphasis here will be
on the synthetic challenges involved in obtaining the necessary neoglycoconjugates.

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2.1. 1,3Dipolar Cycloaddition
Huisgen’s 1,3-dipolar cycloaddition is the primary example of a “click reaction”. It is the reaction
between a 1,3-dipole (an azide) and a dipolarophile (an acetylene) to form a five-membered heterocycle.
The classical reaction proceeds by a concerted mechanism under thermal conditions to afford a mixture
of 1,4- and 1,5-disubstituted [1,2,3]-triazole regioisomers [19], but when the reaction is catalyzed by
Cu(I), only the 1,4-substituted-triazole is obtained [20]. We selected this reaction as one means for
securing our mannose-6-phosphate analogues. Thus to prepare the nanoparticles, the carbohydrate
moiety had to bear either an azide or alkyne function. The linker chain, in turn, would provide the
complementary group. The cyclic sulfate strategy, utilized in our laboratory to prepare M6P analogues,
demanded that the carbohydrate possess the azide group because an alkyne function would become
oxidized during the preparation of the sulfate. Thus, peracetylated mannose has been coupled in very
good yield to 2-bromoethanol, under classical conditions [21], in the presence of boron trifluoride
etherate (Scheme 1). The azide group was then introduced with sodium azide, and the acetate protecting
groups were removed under Zemplen conditions [22] to give the 2-azidoethyl-α-D-mannopyranoside 3.
After selectively introducing isopropylidene protection at the 2 and 3 positions of the mannose, the
cyclic sulfate 5 was prepared according to a modified published procedure [23,24]. Compound 4 was
converted via thionyl chloride into the cyclic sulfite which was then oxidized by ruthenium oxide
(prepared in situ) into the corresponding cyclic sulfate 5.
Scheme 1. Preparation of the cyclic sulfate intermediate 5.
Reagents and conditions: (a) 2-bromoethanol, BF3·OEt2, CH2Cl2, 91%; (b) NaN3, DMF, 96%; (c) MeONa,
MeOH, 65%; (d) 2,2 dimethoxypropane, APTS, acetone; (e) AcOH, H2O, 85%; (f) SOCl2, NEt3, CH2Cl2;
(g) RuCl3, NaIO4, CH2Cl2/CH3CN, 66%.
A “spacer” refers to a chain that can be used to join our sugar derivatives to the gold particles. One
of the spacers, possessing an alkyne unit for reaction with a sugar-azide, was designed on the basis of
its flexibility and aqueous solubility (Scheme 2). Thus, the reaction of 5-bromopentene with a slight
excess of 50% sodium hydroxide and hexaethylene glycol provided the monoether 6 [25]. Photochemical
addition of thioacetic acid to the double bond gave the thioacetate in good yield [26]. The next step
was to introduce the alkyne function on the spacer in the presence of NaH, but the acetate protecting

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groups, being sensitive to hydrides, were first replaced by 4-methoxytrityl. Thus, compound 7 was
deacetylated by concentrated hydrochloric acid in ethanol to avoid the formation of disulfide under
basic conditions. The thiol was then protected by reaction with 4-methoxytrityl chloride. Finally, the
free hydroxyl of 8 was reacted with 3-bromopropyne in the presence of sodium hydride in anhydrous
THF to introduce the alkyne function required for the click reaction.
Scheme 2. Preparation of the alkyne 9 for the click reaction.
Reagents and conditions: (a) NaOH 50%, 99%; (b) AcSH, THF, 71%; (c) Conc. HCl, EtOH; (d) MeOTrCl,
THF, 91%; (e) 2-bromopropyne, NaH, THF, 97%.
The literature describes a variety of ways in which the Huisgen cycloaddition can be performed to
join two entities. Sources of copper (I) catalyst can be produced in situ by reduction of copper (II)
salts [20] or obtained through disproportionation of Cu (0) and Cu (II) salts [27]. Cu (I) can also be
introduced as copper (I) salts such as CuI or obtained from oxidation of Cu (0) salt [28–31]. In search
for the optimal reaction conditions, we initially tested the most commonly employed system, namely
CuSO4·5H2O and sodium ascorbate as source of copper (I) in tert-BuOH/H2O [32]. Interestingly, no
reaction was observed after 24 h. In addition, several parameters were altered without success: increasing
the concentration of reactants, changing the ratio copper/sodium ascorbate, using a co-solvent (acetonitrile),
or substituting tBuOH with pyridine. The click reaction was also attempted using cuprous iodide in
pyridine as catalyst [33]. Despite many modifications to the original protocol the desired product was
never obtained in good yield. Thus, another copper catalyst system consisting of formation of Cu(I) by
oxidation of copper metal was investigated. The oxidative cycloaddition of Cu(0) with ammonium
chloride [34] in a mixture of tert-BuOH/H2O was also unsuccessful. It should be noted that heating to
40–60 °C, and increasing the reagents’concentration, failed to improve the performance, as they often
do in many examples of click chemistry reactions, but led only to degradation. Ultrasound in place of
classical activation was carried out again without success. Only a system using copper powder, rarely
encountered in the literature, gave positive results, giving compound 10 in 60% yield (Scheme 3).
Starting from compound 10 two mannose-6-phosphate analogues were prepared with only slight
modification to the previously reported protocols [4]. First, the azide function was easily introduced on
the cyclic sulfate 10 by reaction with sodium azide to afford compound 11. Although isopropylidene
and trityl are usually deprotected under acid conditions, our assays did not allow simultaneous

Molecules 2014, 19
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cleavage of the two functions. The final ligand 12 was therefore obtained in two separate steps.
The trityl group was first cleaved by ceric ammonium nitrate (a redox reaction) [35,36] prior to
deprotection of the isopropylidene and the sulfate groups via acidic ion exchange resins. To afford the
carboxylic acid analogue of M6P 14, sodium cyanide was first reacted with the cyclic sulfate 10, and
the nitrile function was then hydrolyzed with sodium hydroxide in a 30% solution of hydrogen
peroxide to give the corresponding carboxylic acid. The ligand 14 was obtained using the same
deprotection conditions as described for the azide analogue 12 (Scheme 3).
Scheme 3. Preparation of the functionalized ligands 12 and 14.
Reagents and conditions: (a) Cu(I), NHEt3Cl, tBuOH/H2O, 60%; (b) NaN3, DMF, 62%; (c) CAN, CH3CN/H2O;
(d) Amberlyst H+, MeOH/THF; (e) NaCN, DMF, 65%; (f) H2O2, NaOH, 52%; (g) AcOH, H2O, 78%.
2.2. Julia Reaction
Among the olefination reactions to form a regio- and stereoselective alkene, the Julia olefination
is one of the well-known methods, along with the Wittig reaction [37,38], the Wittig-Horner
reaction [39–41], the Horner-Wadsworth-Emmons [42,43], the Peterson reaction [44–46] and the
Johnson reaction [47]. The classical Julia olefination, also known as the Julia-Lythgoe olefination, was
developed fourty years ago and is based on a reductive elimination process of β-acyloxysulfones [48].
Since its discovery, significant improvements have been made to the methodology of this reaction, and
it has become a crucial step in the synthesis of many natural products. A new variant of the classical

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Julia reaction, the Julia-Kocienski olefination, also called modified or one-pot Julia olefination, has
recently emerged as a powerful tool for olefin synthesis [49–51]. The process involves the replacement
of the aryl sulfone moiety, traditionally used in the classical reaction, with different heteroaryl sulfones,
thus allowing a direct olefination process.
In our Julia olefination, a carbohydrate block was derivatized with an allyl bromide function (to be
later joined with a sulfone-bearing linker). The initial steps in the sugar portion of the molecule
followed the same strategy as described for compound 5 (Scheme 4). The methyl α-D-mannopyranoside
was previously protected with two O-isopropylidene groups on the 2,3 and 4,6 positions using
2,2-dimethoxypropane and para-toluenesulfonic acid. After the selective opening of isopropylidene at
the 4,6 positions with a mixture of AcOH/H2O, the cyclic sulfite was obtained by reaction with thionyl
chloride and triethylamine, and subsequent oxidation afforded the cyclic sulfate 15 in good yield.
In contrast to the chemistry in Scheme 1 and Scheme 3, the azide and carboxylic acid analogues of
M6P were prepared prior to the coupling reaction. Therefore, sodium azide was reacted with the cyclic
sulfate 15 to give compound 16. A solution of acetic acid in water led to the cleavage of the
isopropylidene and the sulfate. The replacement of the anomeric methyl group by an acetyl group led
to compound 17 in 83% yield. The allyl bromide unit required to perform the coupling reaction was
then introduced by glycosylation with cis-1-bromo-but-2-en-4-ol. The same strategy was applied to
form the carboxylic acid analogue of M6P.
Scheme 4. Preparation of the allyl bromides 18 and 22.
Reagents and conditions: (a) 2,2-dimethoxypropane, APTS, acetone; (b) AcOH/H2O, 63%; (c) SOCl2,
NEt3, CH2Cl2; (d) RuCl3, NaIO4, CH2Cl2/CH3CN, 85%; (e) NaN3, DMF, 92%; (f) AcOH/H2O;
(g) Ac2O, DMAP, pyridine; (h) AcOH, H2SO4, Ac2O; (i) NaCN, DMF, 100%; (j) H2O2, NaOH, 100%;
(k) HOCH2CH=CHCH2Br, BF3·OEt2, CH2Cl2, 78%.

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To create the Julia spacer (Scheme 5), geraniol was reacted with phosphorus tribromide to give
geranyl bromide 23 in good yield followed by a reaction with sodium phenylsulfinate to provide the
desired sulfone 24. Next, functionalization of the other side of the linker was carried out in one step via
oxidation of a terminal methyl by selenium dioxide. The strategy described by Sharpless using
tert-butyl hydroperoxide as an oxidant [52] was utilized: 24 was reacted with a catalytic amount of
selenium oxide in the presence of tert-butyl hydroperoxide which enables the recycling of selenium
dioxide. A 50/50 mixture of alcohol and aldehyde was obtained and, after purification, the aldehyde
was reduced with sodium borohydride to give compound 26 in 63% yield. The alcohol 26 was then
brominated with tetrabromomethane and triphenylphosphine after which the thiol group was introduced
by reaction with potassium thioacetate. A “click-type” reaction was then performed between 18 or 22
and 27 in the presence of lithium bis(trimethylsilyl)amide (LiHMDS) in THF from which compounds
28 and 29 were obtained in 15% and 17% yield, respectively. Deprotection of the acetates and removal
of the sulfone group under basic conditions gave the desired final compounds 30 and 31 in almost
quantitative yield (Scheme 6). The linker in this case is polyunsaturated.
Scheme 5. Synthesis of the sulphone 27.
Reagents and conditions: (a) PBr3, CH2Cl2, 93%; (b) NaSO2Ph, DMF, 93%; (c) SeO2, tBuOOH, HOPhCO2H,
CH2Cl2; (d) NaBH4, MeOH, 63% of 26; (e) CBr4, PPh3, CH2Cl2, 100%; (f) KSAc, DMF, 100%.
Scheme 6. Coupling reaction between the allyl bromides and the sulphone.
Reagents and conditions: (a) LiHMDS, THF; (b) MeONa, MeOH.

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2.3. Thiol-ene Reaction
One reaction that is emerging as an attractive click-type process is the century-old addition of thiols
to alkenes [53], which is currently called thiol-ene coupling. In fact, the thiol-ene reaction is simply the
hydrothiolation of a C=C bond, and proceeds by a radical mechanism, induced photochemical or
thermally, to give an anti-Markovnikov-type thioether [54,55]. The reaction discovered in 1905 by
Posner [56] has been widely used in the mid-nineteenth century, especially in polymer chemistry.
However, the thiol-ene reaction has recently attracted researchers in other areas of synthesis due to
recognition of its ‘‘click-type’’ characteristics: highly efficient and orthogonal to a wide range of functional
groups, as well as compatible with water and oxygen. Thus, the thiol-ene reaction enables the
establishment of a rapid ligation between two entities assisted by the stability of the thioether linkage
in a wide range of chemical environments. To perform the reaction, the thiol function was placed on
the sugar moiety while the spacer carried the vinylic group. As before, the M6P derivatives were prepared
using the cyclic sulfate strategy prior to performing the click-style reaction (Scheme 7). The synthesis
began by replacing the anomeric acetate with bromine on compounds 17 and 21 described previously.
This was accomplished with a solution of hydrobromic acid/acetic acid in quantitative yields. The thiol
function was then introduced in two steps, first via thiourea in acetone then removal of the nitrogens
with sodium metabisulfide. Only thiosugars (34 and 35) having the β configuration were obtained.
Scheme 7. Preparation of the thiosugars 34 and 35.
Reagents and conditions: (a) HBr/AcOH, Ac2O; (b) thiourea, Acetone; (c) Na2S2O5, CHCl3/H2O.
Having chosen to synthesize a fully flexible spacer (Scheme 8), the triethylene glycol was coupled
to allyl bromide in the presence of 50% aqueous sodium hydroxide.
Scheme 8. Preparation of the spacer.
Reagents and conditions: (a) NaOH 50%, 85%.
Then, to facilitate the reaction, and to avoid formation of byproducts, the free hydroxyl of the linker
was brominated and thioacetylated only at the end of the synthesis. Actually, coupling can be
accomplished between a protected thiol group and an alkene or between a thiol and an alkene.
However, the final thioether was obtained with better results when the anomeric thiol was not
protected (Scheme 9). The initiation step can be triggered in several ways, by simple heating or by

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ultraviolet irradiation. Another method of initiation has been developed in the laboratory which is to
perform the coupling under ultrasound (Table 1). When THF was replaced by dioxane, yields
increased by 10%.
Scheme 9. Preparation of the thiosugars 39 and 40 by click thiol-ene reaction.
Reagents and conditions: (a) AIBN, dioxane, US activation, 80%; (b) CBr4, PPh3, CH2Cl2; (c) KSAc, DMF;
(d) MeONa, MeOH.
Table 1. Comparative results for the click thiol-ene reaction.
Compound Reflux, THF, 24 h
UV, THF, 5 h
50%
Sonication, THF, 4 h
Sonication, Dioxane, 3 h
39
40
76%
78%
72%
75%
79%
80%
60%
2.4. Gold Nanoparticles
Research in developing new synthesis protocols to generate gold nanoparticles (AuNPs) with
desired properties has received immense attention due to their considerable applications in biomedical
field [57]. One of the primary prerequisites for using AuNPs in biomedical application is that they are
non-toxic and biocompatible to both in vitro and in vivo environments. Secondly, AuNPs should be
coated with a protective layer to prevent aggregation. Thirdly, AuNPs need to be labeled with
biologically relevant biomolecules to impart specificity for their potential application. The two most
interesting and common methods to prepare AuNPs are the Brust method [58] utilizing NaBH4 (which
can’t be used in our case because NaBH4 would reduce the azide function of our derivatives) and the
citrate method. This latter method includes only three starting materials, namely, auric acid, sodium
citrate (the reducer), and water. Following a report by Turkevich et al. in 1951 [59], this synthetic
scheme has been widely studied and often used for the preparation of AuNPs-based materials [60–63].
We have developed a protocol by adjusting the gold-to-citrate ratio to obtain 10 nm AuNPs (Table 2).
Details are given in the Experimental section.

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Table 2. Size of the nanoparticles in nm.
Nanoparticles
TEM [a]
10
DLS [b]
18–20
14–16
12–13
19–20
15–16
Azide AuNPs (Huisgen)
Azide AuNPs (Julia)
10
Azide AuNPs (thiol-ene)
10
Carboxylic acid AuNPs (Huisgen)
Carboxylic acid AuNPs (Julia)
10
10
[a] Transmission electron microscopy [b] Dynamic light scattering.
2.5. Biological Assays
AuNPs functionalized with M6P analogues have been subjected to angiogenic assays using an
experimental model, the avian chorioallantoic membrane assay (CAM) [64–66]. Paper discs were
saturated with a phosphate buffer saline dispersion of coated AuNPs (60 mg/mL) in PBS or a control
(phosphate buffer saline) and then deposited on chorioallantoic membranes of 7-day-old chicken
embryos for 4 days in ovo at 38 °C. Sutent® (sunitinib, a non-proteic inhibitor) and endothelial cell
growth supplement (ECGS) were used at 60 mg/mL as negative and positive stimuli, respectively.
Quantification of the angiogenic response was carried out by measuring the area of neo-vascularization
on each particular membrane (Figure 1) using Image J software. The experiments have been repeated
at least four times, and the results were reproducible (see experimental part for details).
Figure 1. CAM assays using gold nanoparticles functionalized with mannose-6-phosphate
analogues compared to angiogenic inhibitor Sutent®, angiogenic activator Endothelial Cell
Growth Supplement (ECGS) and Phosphate Buffer Saline (PBS) as control.
Nanoparticles: NP1: azide-AuNPs thiol-ene; NP2: azide-AuNPs Julia; NP3: azide-AuNPs Huisgen; NP4:
Carboxylic-AuNPs Huisgen; NP5: Carboxylic-AuNPs Julia; NP6: Carboxylic-AuNPs thiol-ene.

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These experiments demonstrate that all our prepared AuNPs are CAM-inhibitors. Study of the three
azide-AuNPs synthesized according to the coupling methods (NP1: thiol-ene 58%, NP2: Julia 59%,
NP3: Huisgen 65%) revealed that the length and flexibility of spacers have little influence on the
observed biological response. Interestingly, the azide sugar-monomer is a good angiogenic activator
(125%), whereas the functionalized -N3 nanoparticles, representing a multi-valent collection of sugars,
show a strong inhibitory effect (58%–65%). Similar results were obtained for the three carboxylic
acid-AuNPs (NP4: Huisgen 86%, NP5: Julia 87%, NP6: thiol-ene 88%). Comparison of the activating
effect of the carboxylic acid analogue (123% observed in previous work) [4] and the inhibitory effect
of the carboxylic acid-AuNPs (86% to 88% compared to the control) indicates that multi-valency can
do more than qualitatively affect the magnitude of blood vessel formation; it can convert a significant
catalyzed process into an inhibition.
3. Experimental
3.1. General Information
Reactions were monitored by TLC using aluminum-backed plates coated with silica gel 60 F254
(Merck); spots were visualized with UV light (254 nm) and/or (a) by staining with p-anisaldehyde
solution [anisaldehyde (25 mL), H2SO4 (25 mL), EtOH (450 mL), and CH3COOH (1 mL)], followed
by heating or (c) by immersion in a 10% H2SO4/EtOH solution followed by charring. Column
chromatography was performed on Carlo-Erba silica gel 60A (35–70 µm). Melting points were
determined in capillary with a Büchi melting apparatus 530. Optical rotations were measured at the
1
sodium D-line with a Perkin-Elmer-241 polarimeter. H-NMR spectra (400.13 MHz) and 13C-NMR
spectra (100.62 MHz) were recorded on a Bruker DRX 400 instrument. Chemical shifts (δ) are given
in parts per million and referenced using residual solvent signals (7.24 ppm for CHCl3 and 4.79 ppm
for HOD). The following abbreviations were used to explain the signal multiplicities or characteristics:
s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet), td (triplet doublet), q
(quartet), and m (multiplet). Chemical shifts (δ) are given in parts per million relative to TMS as an
external reference. Electron ionization mass spectra were recorded in positive or negative mode on a
Waters MicroMass/ZQ 2615. Anhydrous solvents were obtained prior to use according to standard
methods [67]. For transmission electron microscopy (TEM) examinations, a single drop (10 μL) of an
aqueous solution (ca. 0.1 mg/mL in Milli-Qwater) of the gold glyconanoparticles (AuNPs) was placed
on a coppergrid coated with a carbon film (Electron Microscopy Sciences). The grid was left to dry in
air for several hours at room temperature. TEM analysis was performed with a JEOL 1200 EXII
microscope, operating at 120 kV. Dynamic Light Scattering (DLS) analyses were performed on a
MALVERN HPPS.
2'-Bromoethyl-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (1): To 1,2,3,4,6-penta-O-acetyl-α-D-
mannopyranoside (540 mg, 1.38 mmol) dissolved in CH2Cl2 (5 mL) were added 2-bromoethanol
(0.2 mL, 2.77 mmol) and BF3·Et2O (870 µL, 6.92 mmol). After 20 h stirring at room temperature, the
mixture was diluted with CH2Cl2, washed with water, a saturated solution of NaHCO3 then water
again. The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. Purification
by chromatography on silica gel (EtOAc/petroleum ether 1:1) gave the title compound as a white

Molecules 2014, 19
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powder (91%). Rf = 0.86 (EtOAc/toluene 1:1); mp: 116–118 °C (lit. 115–117 °C); ሾߙሿଶ଴ = +42.1
஽
(c = 0.5 in chloroform); 1H-NMR (CDCl3): δ = 2.00, 2.05, 2.11, 2.16 (4s, 12H, 4CH3); 3.52 (t, J = 6.0 Hz,
2H, CH2Br); 3.93 (m, 2H, CH2CH2Br); 4.13 (m, 2H, H5 and H6a); 4.27 (dd, 1H, J = 5.8 Hz, J = 12.6 Hz,
H6b); 4.88 (d, J = 1.6 Hz, H1); 5.27 (dd, 1H, J = 2.0 Hz, J = 3.2 Hz, H2); 5.29 (t, 1H, J = 1.6 Hz, H4);
5.35 ppm (dd, 1H, J = 3.6 Hz, J = 10.0 Hz, H3);13C-NMR (CDCl3): δ = 20.67, 20.70, 20.75, 20.87
(4CH3); 29.60 (CH2CH2Br); 62.41 (C6); 66.00 (C4); 68.48 (CH2Br); 68.93 (C5); 69.02 (C3); 69.42 (C2);
97.75 (C1); 169.76, 169.86, 170.03, 170.62 ppm (4CO); MS (ESI) m/z: 477.01, 478.95 [M+Na]+.
2'-Azidoethyl-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (2): Sodium azide (1.64 g, 25.05 mmol)
was added to a suspension of compound 1 (5.7 g, 12.53 mmol) in DMF (50 mL). After 4 h at 65 °C,
the mixture was poured into brine and extracted with CH2Cl2. The organic extracts were dried
(Na2SO4) and concentrated in vacuo. The residue was purified by flash chromatography on silica gel
(petroleum ether/EtOAc 4:1) to give the appropriated intermediate as a white solid (96%). Rf = 0.86
(EtOAc/petroleum ether 1:1); mp: 80–82 °C (lit. 81.8–82.1 °C); ሾߙሿଶ଴ = +39.0 (c = 0.6 in chloroform);
஽
1H-NMR (CDCl3): δ = 2.0, 2.05, 2.11, 2.16 (4s, 12H, 4CH3); 3.47 (m, 2H, CH2N3); 3.67 (m, 1H,
CH2CH2N3); 3.87 (m, 1H, CH2CH2N3); 4.05 (ddd, 1H, J = 2.4 Hz, J = 5.2 Hz, J = 9.7 Hz, H5);
4.13 (dd, 1H, J = 2.6 Hz, J = 12.2 Hz, H6a); 4.29 (dd, 1H, J = 5.2 Hz, J = 12.4 Hz, H6b); 4.87 (d, 1H,
J = 1.6 Hz, H1); 5.30 (t, 1H, J = 10.0 Hz, H4); 5.28 (dd, 1H, J = 2.0 Hz, J = 3.2 Hz, H2); 5.36 ppm
13
(dd, 1H, J = 3.2 Hz, J = 10.0 Hz, H3); C-NMR (CDCl3) : δ = 20.63, 20.68, 20.71, 20.84 (4CH3);
50.32 (CH2N3); 62.42 (C6); 65.96 (C4); 67.02 (CH2CH2N3); 68.82 (C5 and C3); 69.36 (C2); 97.71 (C1);
169.73, 169.78, 169.98, 170.59 ppm (4CO); MS (ESI) m/z: 440.12 [M+Na]+.
2'-Azidoethyl-α-D-mannopyranoside (3): Compound 2 (16.0 g, 38.36 mmol, 1 eq.) and NaOMe (2.07 g,
38.36 mmol, 1 eq.) were added to methanol (100 mL). After 30 min stirring at RT, the mixture was
neutralized with Amberlite IRC-50 H+ resins, filtered and concentrated in vacuo. Purification by
chromatography on silica gel (CH2Cl2/MeOH 9:1) gave a white powder (65%). Rf = 0.40
1
(CH2Cl2/MeOH 4:1); ሾߙሿଶ଴ = + 54.9 (c = 1.00 in chloroform); H-NMR (CD3OD): δ = 3.41 (t, 2H,
஽
J = 5.0 Hz, CH2N3); 3.60 (m, 3H, H3, H5 and CH2CH2N3); 3.71 (m, 2H, H4 and H6a); 3.85 (m, 2H, H2
and H6b); 3.92 (m, 1H, CH2CH2N3); 4.81 ppm (d, 1H, J = 1.2 Hz, H1); MS (ESI) m/z: 272.11 [M+Na]+,
288.02 [M+K]+, 521.19 [2M+Na]+.
2'-Azidoethyl-2,3-O-isopropylidene-α-D-mannopyranoside (4): A solution of compound 3 (9.5 g,
38.15 mmol, 1 eq.), 2,2-dimethoxypropane (23.4 mL, 190.76 mmol, 5 eq.) and para-toluenesulfonic acid
(362 mg, 1.90 mmol, 0.05 eq.) in acetone (40 mL) was stirred for 4 h at RT. The para-toluenesulfonic
acid was neutralized with 5% aq NaHCO3. Acetone was removed in vacuo and the aqueous phase was
washed with petroleum ether to remove the diisopropylidene species. This organic layer was dried
(Na2SO4) and concentrated in vacuo. Then the aqueous layer containing the monoisopropylidene was
lyophilized. The diisopropylidene compound (7.2 g, 21.88 mmol, 1 eq.) was stirred in a solution of
acetic acid/water 8:2 (60 mL) at 35 °C. After 2 h, solvents were evaporated, and then coevaporated
with toluene several times. The crude product obtained was purified by chromatography on silica gel
(petroleum ether/EtOAc 2:3) to give a yellow oil (85% monoisopropylidene compound, over two steps).

Molecules 2014, 19
1132
Diisopropylidene derivative: Rf = 0.63 (EtOAc/petroleum ether 1:1); 1H-NMR (acetone-d6):
δ = 1.31, 1.32 (2s, 6H, 2CH3); 1.47, 1.48 (2s, 6H, 2CH3); 3.50 (t, 2H, J = 4.8 Hz, CH2N3); 3.53 (m, 1H,
H5); 3.72 (m, 3H, H6a, H4 and CH2CH2N3); 3.82 (dd, 1H, J = 5.8 Hz, J = 10.6 Hz, H6b); 3.93 (qt, 1H,
J = 5.2 Hz, CH2CH2N3); 4.03 (dd, 1H, J = 5.6 Hz, J = 8.0 Hz, H3); 4.18 (d, 1H, J = 5.6 Hz, H2);
5.09 (s, 1H, H1); 13C-NMR (acetone-d6): δ = 20.11, 29.38, 27.45, 30.50 (4CH3); 52.18 (CH2N3); 63.48,
63.53 (C5andC6); 68.17 (CH2CH2N3); 74.47 (C4); 76.78 (C3); 77.83 (C2); 99.68 (C1); 100.10,109.76
ppm (2C(CH3)2); MS (ESI) m/z: 352.20 [M+Na]+, 368.02 [M+K]+.
1
Monoisopropylidene derivative: Rf = 0.26 (EtOAc/petroleum ether 3/2); H-NMR (acetone-d6 + D2O):
δ = 1.27, 1.41 (2s, 6H, 2CH3); 3.45 (t, 2H, J = 5.0 Hz, CH2N3); 3.52 (m, 2H, H4andH5); 3.62 (dd, 1H,
J = 5.2 Hz, J = 11.6 Hz, H6a); 3.67 (m, 1H, CH2CH2N3); 3.80 (m, 1H, H6b); 3.93 (m, 1H, CH2CH2N3);
4.02 (m, 1H, H3); 4.09 (d, 1H, J = 5.6 Hz, H2); 5.03 (s, 1H, H1); 13C-NMR (acetone-d6 + D2O):
δ = 20.34, 29.11 (2CH3); 51.95 (CH2N3); 62.97 (C6); 67.74 (CH2CH2N3); 70.41, 72.62 (C4 and C5);
77.30 (C2); 80.42 (C3); 98.60 (C1); 110.60 (C(CH3)2); MS (ESI) m/z: 312.12 [M+Na]+, 328.15 [M+K]+,
324.12 [M+Cl]−.
2'-Azidoethyl-2,3-O-isopropylidene-α-D-mannopyranoside-4,6-cyclic sulfate (5): Compound 4 (100 mg,
0.35 mmol, 1 eq.) and Et3N (144 µL, 1.04 mmol, 3 eq.) in CH2Cl2 (2 mL) were stirred for 5 min at
0 °C. Then SOCl2 (27 µL, 0.38 mmol, 1.1 eq.) was added dropwise to the mixture. After 10 min, the
solution was filtered. Impurities were removed with water and the organic layer was washed with 1N
HCl, dried (Na2SO4) and concentrated in vacuo to give a brown solid. The crude sulfite obtained was
then reacted with NaIO4 (81 mg, 0.38 mmol, 1.1 eq.), water (0.5 mL) and RuCl3 (1.38.10−3 mmol,
0.004 eq.) in CH2Cl2/CH3CN 1:1 (2 mL). After 1h at RT, the solution was filtered before adding water.
After extraction, the organic layer was dried (Na2SO4) and concentrated in vacuo. Filtration on silica
gel and washes with CH2Cl2 gave a white solid (66%). Rf = 0.58 (EtOAc/petroleum ether 1:1); mp:
1
80–82 °C; H-NMR (acetone-d6): δ = 1.37, 1.52 (2s, 6H, 2CH3); 3.55 (m, 2H, CH2N3); 3.80 (m, 1H,
CH2CH2N3); 4.29 (m, 1H, CH2CH2N3); 4.26 (td, 1H, J = 10.7 Hz, J = 5.5 Hz, H5); 4.36 (d, 1H, J = 6.0 Hz,
H2); 4.43 (dd, 1H, J = 5.6 Hz, J = 8.0 Hz, H3); 4.6 (dd, 1H, J = 7.6 Hz, J = 10.8 Hz, H4); 4.63 (t, 1H,
J = 10.8 Hz, H6a); 4.84 (dd, 1H, J = 5.6 Hz, J = 10.4 Hz, H6b); 5.28 ppm (s, 1H, H1); 13C-NMR
(acetone-d6): δ = 27.16, 29.13 (2CH3); 52.06 (CH2N3); 60.35 (C5); 68.75 (CH2CH2N3); 74.34 (C6);
74.95 (C3); 77.88 (C2); 86.65 (C4); 99.70 (C1); 112.07 ppm (C(CH3)2); MS (ESI) m/z: 374.13 [M+Na]+,
386.08 [M+Cl]−.
3,6,9,12,15,18-Hexaoxatricos-22-en-1-ol (6): A mixture of 50% aqueous sodium hydroxide (1.93 mL,
24.18 mmol, 1.1 eq.) and hexa(ethylene glycol) (25 g, 88.55 mmol, 4.12 eq.) was stirred for 30 min at
100 °C, before adding 5-bromopent-1-ene (2.55 mL, 21.50 mmol, 1 eq.). After 15 min, the reaction
mixture was cooled, diluted in CH2Cl2 and washed with water. The organic phase was dried (Na2SO4),
filtered and concentrated in vacuo. Purification by chromatography on silica gel (EtOAc/petroleum
ether 9:1 to EtOAc/MeOH 9:1) gave a yellow oil (99%). Rf = 0.14 (AcOEt/petroleum ether 5:5);
ሾߙሿଶ଴ = + 54.9 (c = 1.00 in chloroform); 1H-NMR (CDCl3): δ = 1.68 (m, 2H, CH2CH2CH=CH2); 2.09
஽
(m, 2H, CH2CH=CH2); 3.46 (t, 2H, J = 6.6 Hz, CH2CH2CH2CH=CH2); 3.56–3.73 (m, 24 h, 12CH2O);
4.99 (m, 2H, CH=CH2); 5.81 ppm (m, 2H, CH=CH2); 13C-NMR (CDCl3): δ = 28.66 (CH2CH2CH=CH2);

Molecules 2014, 19
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30.12 (CH2CH=CH2); 61.51–72.58 (13CH2O); 114.59 (CH=CH2); 138.18 ppm (CH=CH2); MS (ESI)
m/z: 373.27 [M+Na]+, 389.20 [M+K]+.
S-(23-Hydroxy-6,9,12,15,18,21-hexaoxatricos-1-yl)ethane-thioate (7):
A
solution containing
compound 6 (3.1 g, 8.85 mmol, 1 eq.), thiolacetic acid (3.17 mL, 44.28 mmol, 5 eq.) and AIBN
(100 mg) in anhydrous THF (12 mL) was refluxed for 1 h under nitrogen. The mixture was diluted
with EtOAc, washed with a saturated solution of NaHCO3. The organic layer was dried (Na2SO4),
filtered and reduced in vacuo. Purification by chromatography on silica gel (EtOAc/petroleum ether
1
9:1 to EtOAc/MeOH 9:1) gave a yellow oil (71%). Rf = 0.27 (EtOAc/MeOH 9:1); H-NMR (CDCl3):
δ = 1.40 (m, 2H, CH2(CH2)2S); 1.58 (m, 4 h, CH2(CH2)3S and CH2CH2S); 1.83 (s, 1H, OH); 2.32 (s,
3H, CH3); 2.86 (t, 2H, J = 7.2 Hz, CH2S); 3.44 (t, 2H, J = 6.6 Hz, CH2(CH2)4S); 3.56–3.73 ppm (m, 24 h,
12CH2O); 13C-NMR (CDCl3): δ = 25.25 (CH2(CH2)2S); 28.90 (CH2S); 28.99, 29.24 (CH2(CH2)3S and
CH2CH2S); 30.52 (CH3); 61.55–72.43 (13CH2O); 195.84 ppm (CO); MS (ESI) m/z: 449.26 [M+Na]+,
461.17 [M+Cl]−.
1-(Methoxytritylthio)-8,11,14,17,20,23-hexaoxa-2-thiapentacosan-25-ol (8): Compound 7 (2.6 g,
6.1 mmol, 1 eq.) and a concentrated solution of HCl (3 mL) were stirred in EtOH (65 mL). After 20 h
reaction at 60 °C, the mixture was neutralized with ammonia then reduced under pressure. The
obtained solution was diluted with EtOAc, and the organic layer was quickly washed with water, dried
(Na2SO4), and concentrated in vacuo. The crude product was directly put in reaction with MeOTrCl
(2.83 g, 9.15 mmol, 1.5 eq.) in anhydrous THF (60 mL). After 24 h stirring at RT, the solution was
concentrated in vacuo and purified by chromatography on silica gel (EtOAc/MeOH 9:1) to give a
1
yellow oil (91%): Rf = 0.40 (EtOAc/MeOH 7:3); H-NMR (acetone-d6): δ = 1.31 (m, 2H,
CH2(CH2)2S); 1.40 (m, 4 h, CH2(CH2)3S and CH2CH2S); 2.17 (t, 2H, J = 7.4 Hz, CH2S); 2.87 (s, 1H,
OH); 3.35 (t, 2H, J = 6.4 Hz, CH2(CH2)4S); 3.47–3.63 (m, 24 h, 12CH2O); 3.79 (s, 3H, CH3);
13
6.86–7.42 ppm (m, 14 h, CHAr); C-NMR (acetone-d6): δ = 27.37, 30.16 (CH2CH2CH2CH2S); 33.54
(CH2S); 56.50 (CH3); 62.94–72.33 (13CH2O); 74.48 (SC); 114.86–132.54 (14CHAr); 138.81, 147.37
(3SCCAr); 160.12 ppm (COCH3); MS (ESI) m/z: 679.34 [M+Na]+.
1-(Methoxytritylthio)-8,11,14,17,20,23-hexaoxa-2-thiahexacos-25-yne (9): NaH (7.3 mg, 0.30 mmol,
2 eq.) and 2-bromopropyne (19 µL, 0.21 mmol, 1.4 eq.) were added to a solution containing compound
8 (100 mg, 0.15 mmol, 1 eq.) in anhydrous THF (3 mL) at 0 °C. After 18 h stirring at RT, the mixture
was concentrated then purified by chromatography on silica gel (EtOAc/petroleum ether 8:2) to give a
1
white oil (97%): Rf = 0.34 (EtOAc); H-NMR (CDCl3): δ = 1.28 (m, 2H, CH2(CH2)2S); 1.42 (m, 4 h,
CH2(CH2)3S and CH2CH2S); 2.14 (t, 2H, J = 7.4 Hz, CH2S); 2.43 (t, 1H, J = 2.4 Hz, CH); 3.36 (t, 2H,
J = 6.8 Hz, CH2(CH2)4S); 3.52–3.71 (m, 24 h, 12CH2O); 3.79 (s, 3H, CH3); 4.20 (d, 2H, J = 2.4 Hz,
CH2CCH); 6.79–7.40 ppm (m, 14 h, CHAr); 13C-NMR (CDCl3): δ = 25.59 (CH2(CH2)2S); 28.46, 29.19
(CH2(CH2)3S and CH2CH2S); 31.96 (CH2S); 55.20 (CH3); 58.39 (CH2CCH); 65.85 (CH); 69.10–71.17
(13CH2O); 74.51 (SC); 113.03–130.73 (14CHAr); 137.12, 145.32 (3SCCAr); 157.94 ppm (COCH3);
MS (ESI) m/z: 717.39 [M+Na]+.

Molecules 2014, 19
1134
2-{4-[27-(4-Methoxyphenyl)-27,27-diphenyl-2,5,8,11,14,17,20-heptaoxa-26-thiaheptacos-1-yl]-2,3-
dihydro-1H-1,2,3-triazol-1-yl}ethyl-6-deoxy-2,3-O-(1-methylethylidene)-4,6-cyclic sulfate-α-D-manno-
pyranoside (10): Compounds 5 (40 mg, 0.11 mmol, 1 eq.) and 9 (87 mg, 0.13 mmol, 1.1 eq.) were
suspended in a mixture of t-BuOH/H2O 1:1 (4 mL). Cu(0) nanosize activated powder (4 mg,
0.06 mmol, 0.5 eq.) and NHEt3Cl (32 mg, 0.23 mmol, 2 eq.) were added, and the heterogeneous
mixture was stirred vigorously for 20 h at RT. The reaction mixture was diluted in EtOAc then washed
with water. The organic layer was dried, filtered and concentrated in vacuo. Purification by
chromatography on silica gel (CH2Cl2/MeOH 99:1 to 9:1) gave a colourless oil (60%): Rf = 0.40
1
(CH2Cl2/MeOH 9:1); ሾߙሿଶ଴ = + 36.0 (c = 1.00 in chloroform); H-NMR (CD3OD): δ = 1.36 (m, 6H,
஽
CH2CH2CH2CH2S); 1.34, 1.49 (2s, 6H, 2CCH3); 2.14 (t, 2H, J = 7.2 Hz, CH2S); 3.38 (t, 2H, J = 6.4 Hz,
CH2(CH2)4S); 3.44–3.66 (m, 25 h, H5 and 12CH2O); 3.78 (s, 3H, OCH3); 3.97 (m, 1H, CH2CH2N);
4.15 (m, 1H, CH2CH2N); 4.27 (m, 3H, H2, H3 and H6a); 4.50 (m, 2H, H6b and H4); 4.64 (m, 4 h, CH2N
and CH2C=CH); 5.12 (s, 1H, H1); 6.81–7.39 (m, 14 h, CHAr); 8.07 ppm (s, 1H, NCH); 13C-NMR
(CD3OD): δ = 26.34, 28.20 (2CCH3); 26.71 (CH2(CH2)2S); 29.62 (CH2CH2S); 30.23 (CH2(CH2)3S);
33.03 (SCH2); 51.19 (CH2N); 55.79 (OCH3); 59.79 (C5); 65.14 (CH2C=CH); 67.42 (CH2CH2N); 70.95,
71.19, 71.49, 71.58, 72.03 (13CH2O); 73.53 (C6); 74.46, 77.23 (C2 and C3); 85.66 (C4); 98.96 (C1);
108.26, 111.66 (SC and C(CH3)2); 114.11, 127.66, 128.86, 130.73, 132.02 (14CHAr); 126.04 (NCH);
138.40, 146.86 (3SCCAr and C=CH); 159.71 ppm (COCH3); MS (ESI) m/z: 1068.62 [M+Na]+,
1080.77 [M+Cl]−.
2-{4-[27-(4-Methoxyphenyl)-27,27-diphenyl-2,5,8,11,14,17,20-heptaoxa-26-thiaheptacos-1-yl]-2,3-
dihydro-1H-1,2,3,-triazol-1-yl}ethyl-6-deoxy-2,3-O-(1-methylethylidene)-4-sodiumsulfate-6-azido-α-D-
mannopyranoside (11): The procedure described for compound 2 was applied to 10 to give compound
11 as a yellow oil (62%). Rf = 0.15 (CH2Cl2/MeOH 8.5:1.5); ሾߙሿଶ଴ = +17.1 (c = 1.00 in chloroform);
஽
1H-NMR (CD3OD): δ = 1.37 (m, 6H, CH2CH2CH2CH2S); 1.30, 1.45 (2s, 6H, 2CCH3); 2.17 (t, 2H,
J = 7.2 Hz, CH2S); 3.37 (t, 2H, J = 6.6 Hz, CH2(CH2)4S); 3.42–3.67 (m, 27H, H2 and 13CH2O); 3.79
(s, 3H, OCH3); 3.96 (m, 1H, CH2CH2N); 4.12 (m, 1H, CH2CH2N); 4.07 (d, 1H, J = 6.0 Hz, H5); 4.25
(m, 1H, H3); 4.38 (t, 1H, J = 5.8 Hz, H4); 4.70 (m, 6H, H6, CH2N and CH2C=CH); 4.93 (s, 1H, H1);
13
6.87–7.41 (m, 14 h, CHAr); 9.19 ppm (s, 1H, NCH); C-NMR (CD3OD): δ = 27.20, 28.67 (2CCH3);
27.33, 30.16, 30.87 (CH2CH2CH2CH2S); 33.55 (CH2S); 51.66 (C6 and CH2(CH2)4S); 54.55 (CH2N);
56.53 (OCH3); 65.58 (CH2C=CH); 67.89 (CH2CH2N); 70.39–72.33 (12CH2O); 72.13 (C2); 73.88 (C3);
76.31 (C5); 77.37 (C4); 99.59 (C1); 110.79 (SC); 114.87–132.54 (14CHAr); 126.23 (NCH);
138.80–160.13 (3SCCAr and C=CH); 158.40 ppm (COCH3); MS(ESI) m/z:1134.58 [M+Na]+.
2-{4-[27-(4-Methoxyphenyl)-27,27-diphenyl-2,5,8,11,14,17,20-heptaoxa-26-thiaheptacos-1-yl]-2,3-
dihydro-1H-1,2,3,-triazol-1-yl}ethyl-6-deoxy-6-azido-α-D-mannopyranoside (12): Firstly, compound
11 (200 mg, 0.18 mmol, 1 eq.) and CAN (50 mg, 0.09 mmol, 0.5 eq.) were added to a mixture of
CH3CN/H2O 95:5 (4 mL). After 4 h stirring at 60 °C, the solution was diluted in CH2Cl2, washed
several times with water and the aqueous layer was lyophilized. Purification by chromatography on silica
gel (CH2Cl2/MeOH 9:1 to CH2Cl2/MeOH 8:2) gave the product as a colourless oil (72%). Secondly,
this intermediate was dissolved in a mixture of MeOH/THF 1:1 (6 mL) before adding Amberlyst H+
resins. After 24 h at RT, the resins were filtered, and the solution was neutralized with a saturated

Molecules 2014, 19
1135
solution of NaHCO3. Organic solvents were evaporated and water lyophilized. The crude product was
dissolved in methanol and the insoluble NaHCO3 was filtered. Purification by chromatography on silica
gel (CH2Cl2/MeOH 9:1) gave the product as a colourless oil (53%): Rf = 0.25 (CH2Cl2/MeOH 9:1);
1
ሾߙሿଶ଴ = −2.1 (c = 1.00 in chloroform); H-NMR (CD3OD): δ = 1.47 (m, 2H, CH2(CH2)2S); 1.60 (m,
஽
2H, CH2(CH2)3S); 1.71 (m, 2H, CH2CH2S); 2.70 (t, 2H, J = 7.2 Hz, CH2S); 3.48 (t, 2H, J = 6.2 Hz,
CH2(CH2)4S); 3.19–3.78 (m, 30H, H2-6 and 12CH2O); 3.88 (m, 1H, CH2CH2N); 4.13 (m, 1H,
13
CH2CH2N); 4.63 (m, 4 h, CH2N and CH2C=CH); 4.72 (s, 1H, H1); 8.03 ppm (s, 1H, NCH); C-NMR
(CD3OD): δ = 26.13 (CH2(CH2)3S); 30.07 (CH2CH2S); 30.36 (CH2(CH2)3S); 39.66 (CH2S); 51.34
(CH2N); 62.85 (C6); 65.05 (CH2C=CH); 66.79 (CH2CH2N); 68.38, 70.81, 71.24, 71.59, 71.93, 72.15,
72.51, 75.01 (C2-5 and 13CH2O); 101.70 (C1); 132.57 (NCH); 161.04 ppm (CH=C); MS (ESI) m/z:
798.62 [M+Na]+.
2-{4-[27-(4-Methoxyphenyl)-27,27-diphenyl-2,5,8,11,14,17,20-heptaoxa-26-thiaheptacos-1-yl]-2,3-
dihydro-1H-1,2,3,-triazol-1-yl}ethyl-6-deoxy-2,3-O-(1-methylethylidene)-4-sodium sulfate-6-cyano-α-
D-mannopyranoside (13): Sodium cyanide (15 mg, 0.31 mmol, 2 eq.) was added to a suspension of
compound 10 (160 mg, 0.15 mmol, 1 eq.) in DMF (1.5 mL). After 4 h stirring at RT, the mixture was
poured into brine and extracted with CH2Cl2. The organic layers were dried (Na2SO4) and concentrated
in vacuo. The residue was purified by chromatography on silica gel (CH2Cl2/MeOH 9:1) to give the
1
appropriated intermediate as a colourless oil (65%): Rf = 0.24 (CH2Cl2/MeOH 9:1); H-NMR
(CD3OD): δ = 1.31, 1.50 (2s, 6H, 2CCH3); 1.38 (m, 6H, CH2CH2CH2CH2S); 2.15 (t, 2H, J = 7.2 Hz,
CH2S); 2.70 (dd, 1H, J = 8.8 Hz, J = 17.2 Hz, H6a); 3.02 (dd, 1H, J = 3.0 Hz, J = 17.0 Hz, H6b); 3.38
(t, 2H, J = 6.4 Hz, CH2(CH2)4S); 3.48–3.66 (m, 26H, H4,5 and 12CH2O); 3.78 (s, 3H, OCH3); 3.93 (m,
1H, CH2CH2N); 4.10 (d, 1H, J = 4.8 Hz, H2); 4.19 (m, 2H, CH2CH2N and H3); 4.65 (m, 4 h, CH2N and
13
CH2C=CH); 5.00 (s, 1H, H1); 6.82–7.39 (m, 14 h, CHAr); 8.04 ppm (s, 1H, NCH); C-NMR (100.62
MHz, CD3OD): δ = 21.83 (C6); 26.60, 28.05 (2CCH3); 26.69 (CH2(CH2)2S); 29.62 (CH2CH2S); 30.17
(CH2(CH2)3S); 33.04 (CH2S); 51.17 (CH2N); 55.81 (OCH3); 64.87 (CH2C=CH); 66.66, 76.70 (C4 and
C5); 67.02 (CH2CH2N); 70.47–72.02 (13CH2O); 77.54 (C2); 77.92 (C3); 98.52 (C1); 110.98 (SC and
C(CH3)2); 118.96 (CH2CN); 114.12–132.01 (14CHAr); 125.87 (NCH); 138.38, 146.00, 146.84 (3SCCAr
and C=CH); 159.71 ppm (COCH3); MS (ESI) m/z: 1117.77 [M+Na]+,1071.63 [M-Na]−.
2-{4-[27-(4-Methoxyphenyl)-27,27-diphenyl-2,5,8,11,14,17,20-heptaoxa-26-thiaheptacos-1-yl]-2,3-
dihydro-1H-1,2,3,-triazol-1-yl}ethyl-6-deoxy-α-D-heptomannopyranouronic acid (14): Firstly, NaOH
(60 mg, 1.46 mmol, 8 eq.) was added to a solution of compound 13 (200 mg, 0.18 mmol, 1 eq.) and
H2O2 at 30% (1.5 mL). 1.5 mL of H2O2 at 30% and 60 mg of NaOH were added to the mixture after 12 h
stirring at RT and again after 24 h stirring at RT. After 48 h, the solution was neutralized with
Amberlite IRC-50 (H+) resin, filtered, and concentrated in vacuo. The crude product was purified by
chromatography on silica gel (CH2Cl2/MeOH 9:1 to NH4OH/iPrOH 1:1) to give a yellow oil (52%):
1
Rf = 0.15 (AcOEt/MeOH 5:5); ሾߙሿଶ଴ = −9.0 (c = 1.00 in chloroform); H-NMR (D2O): δ = 1.35, 1.52
஽
(2s, 6H, 2CCH3); 1.44 (m, 2H, CH2(CH2)2S); 1.60 (m, 2H, CH2(CH2)3S); 1.73 (m, 2H, CH2CH2S);
2.29 (dd, 1H, J = 10.6 Hz, J = 15.0Hz, H6a); 2.80 (dd, 1H, J = 2.0 Hz, J = 15.2 Hz, H6b); 2.89 (t, 2H,
J = 8.0 Hz, CH2S); 3.53 (t, 2H, J = 6.6 Hz, CH2(CH2)4S); 3.50–3.69 (m, 25 h, H5 and 12CH2O); 3.75
(s, 3H, OCH3); 3.88 (m, 1H, CH2CH2N); 4.17 (m, 2H, CH2CH2N and H4); 4.19 (d, 1H, J = 5.6 Hz,

Molecules 2014, 19
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H2); 4.30 (m, 1H, H3); 4.68 (m, 4 h, CH2N and CH2C=CH); 4.95 (s, 1H, H1); 6.80–7.37 (m, 14 h,
13
CHAr); 8.12 ppm (s, 1H, NCH); C-NMR (D2O): δ = 23.80 (CH2CH2S); 24.24 (CH2(CH2)2S); 25.45,
26.80 (2CCH3); 28.06 (CH2(CH2)3S); 38.92 (C6); 49.99 (CH2N); 50.89 (CH2S); 55.86 (OCH3); 62.95
(CH2C=CH); 65.47 (CH2CH2N); 66.41 (C5); 66.54, 68.69, 69.07, 69.40, 69.53, 70.70 (13CH2O); 75.21
(C2); 76.00 (C3); 78.30 (C4); 95.99 (C1); 110.39 (SC and C(CH3)2); 114.14–132.02 (14CHAr); 125.55
(NCH); 143.85 (C=CH); 138.38, 146.00, 146.84 (3SCCAr); 159.71 (COCH3);177.75 (CO2H); MS (ESI)
m/z: 1037.34 [M+Na]+.
Next, the procedure described for compound 12 was applied to the preceeding intermediate to give
compound 14 as a colourless oil (80%). Rf = 0.18 (EtOAc/MeOH 5:5); ሾߙሿଶ଴= +10.2 (c = 1.00 in
஽
chloroform); 1H-NMR (CD3OD): δ = 1.49 (m, 2H, CH2(CH2)2S); 1.61 (m, 2H, CH2(CH2)3S); 1.80 (m,
2H, CH2CH2S); 2.41 (dd, 1H, J = 10.2 Hz, J = 16.2 Hz, H6a); 2.84 (m, 3H, CH2S and H6b); 3.49 (t, 2H,
J = 6.4 Hz, CH2(CH2)4S); 3.40–3.79 (m, 28 h, H2-5 and 12 CH2O); 3.92 (m, 1H, CH2CH2N); 4.22 (m,
1H, CH2CH2N); 4.71 (d, 1H, J = 1.2 Hz, H1); 4.87 (m, 2H, CH2N); 4.92 (m, 2H, CH2C=CH); 8.65 (s,
13
1H, NCH); C-NMR (D2O): δ = 23.79 (CH2CH2S); 24.23 (CH2(CH2)2S); 28.06 (CH2(CH2)3S); 36.51
(C6); 50.67 (CH2N); 50.90 (CH2S); 62.55 (CH2C=CH); 65.29 (CH2N); 67.88, 69.08, 69.54, 70.70
(13CH2O); 52.32, 69.36, 69.82, 70.16 (C2-5); 99.48 (C1); 109.39 (C=CH); 146.74 (NCH); 175.27 ppm
(CO2H); MS (ESI) m/z: 765.86 [M-3H+3Na]+.
Methyl-2,3-O-isopropylidene-4,6-cyclic sulfate-α-D-mannopyranoside (15): Firstly, the procedure
described for compound 4 was applied to methyl α-D-mannopyranoside to give the appropriate
1
intermediate as a white solid (63%): Rf = 0.53 (EtOAc); mp: 103–105 °C; H-NMR (acetone-d6):
δ = 1.28, 1.42 (2s, 6H,2CCH3); 3.35 (s, 3H, OCH3); 3.46 (ddd, 1H, J = 2.6 Hz, J = 5.6 Hz, J = 10.2 Hz,
H5); 3.52 (dd, 1H,J = 6.9 Hz, J = 10.2 Hz, H4); 3.65 (dd, 1H, J = 5.8 Hz, J = 12.0 Hz, H6a); 3.81 (dd,
1H, J = 2.6 Hz, J = 11.9Hz, H6b); 4.01 (dd, 1H, J = 5.7 Hz, J = 6.9 Hz, H3); 4.06 (dd, 1H, J = 0.8 Hz,
13
J = 5.7 Hz, H2); 5.01 ppm (s, 1H,H1); C-NMR (acetone-d6+D2O): δ = 26.50, 28.31 (2CCH3), 55.57
(OCH3), 62.79 (C6), 69.74, 70.02 (C4,C5), 75.97 (C2), 78.83 (C3), 98.80 (C1), 110.07 ppm (C(CH3)2);
MS (ESI): m/z [M+Na]+ calcd. for C10H18O6 Na: 257.10, found: 257.21.
Next, the procedure described for compound 5 was applied to the preceeding intermediate to give
compound 15 as a white solid (85%). mp: 80–82 °C; Rf = 0.48 (EtOAc/petroleum ether 3:7);
1H-NMR (acetone-d6): δ = 1.38, 1.53 (2s, 6H, 2CCH3); 3.46 (s, 3H, OCH3); 4.17 (td, 1H, J = 5.5 Hz,
J = 10.6 Hz, H5); 4.32 (dd, 1H, J = 0.4 Hz, J = 5.6 Hz, H2); 4.42 (dd, 1H, J = 5.6 Hz, J = 7.7 Hz, H3);
4.59 (dd, 1H, J = 7.8 Hz, J = 10.4 Hz, H4); 4.64 (t, 1H, J = 10.7 Hz, H6a); 4.87 (dd, 1H, J = 5.5 Hz,
13
J = 10.5 Hz, H6b); 5.01 (d, 1H, J = 0.5 Hz, H1); C-NMR (CDCl3): δ = 26.44, 28.45 (2CCH3); 56.14
(OCH3); 58.94 (C5); 72.32 (C6); 76.37 (C2); 73.61 (C3); 84.67 (C4); 99.42 (C1); 111.08 ppm (C(CH3)2);
MS (ESI) m/z: 297.37 [M+H]+, 319.32 [M+Na]+.
Methyl-6-deoxy-6-azido-2,3,4-tri-O-acetyl-α-D-mannopyranoside (16): First, the procedures described
for compounds 2 and 4 were applied to 15 to give 16 as a white solid: Rf = 0.50 (CH2Cl2/MeOH 9:1);
ሾߙሿଶ଴ = +54.8 (c = 1.00 in methanol); 1H-NMR (D2O): δ = 3.40 (s, 3H, OCH3); 3.54 (dd, 1H, J = 6.2 Hz,
஽
J = 13.3 Hz, H6a); 3.60–3.73 (m, 4 h, H6b, H5, H4 and H3); 3.91 (dd, 1H, J = 3.3 Hz, J = 1.7 Hz, H2);
13
4.73 (d, 1H, J = 1.6 Hz, H1); C-NMR (D2O): δ = 51.4 (C6); 55.2 (OCH3); 67.8 (C5); 70.2 (C2); 70.7
(C3); 71.6 (C4); 101.4 ppm (C1); MS(ESI) m/z:242.31 [M+Na]+, 218.14 [M-H]−.

Molecules 2014, 19
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Secondly, Ac2O (1.72 mL, 18.26 mmol, 5 eq.) and DMAP (134 mg, 1.10 mmol, 0.3 eq.) were
added to a solution of pyridine (15 mL) and methyl 6-azido-6-deoxy-α-D-mannopyranoside (800 mg,
3.65 mmol, 1 eq.). After 4 h stirring, the mixture was diluted in ethyl acetate and washed with a
solution of HCl 2N (until pH = 1), a solution of NaHCO3 5%, water (until pH = 7) and with a saturated
solution of NaCl. The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The
residue was purified by chromatography on silica gel (EtOAc/petroleum ether 2:3) to give a yellow
powder (97%). Rf = 0.62 (EtOAc/petroleum ether 1:1); mp: 98–100 °C (lit. 99–100 °C); ሾߙሿଶ଴ = +65.7
஽
1
(c = 1.00 in chloroform); H-NMR (CDCl3): δ = 1.97, 2.05, 2.13 (3s, 9H, COCH3); 3.16 (dd, 1H,
J = 8.8 Hz, J = 10.8 Hz, H6a); 3.29 (dd, 1H, J = 2.6 Hz, J = 11.0 Hz, H6b); 3.46 (s, 3H, OCH3); 3.78 (td,
1H, J = 2.4 Hz, J = 9.2 Hz, H5); 4.71 (s, 1H, J = 1.2 Hz, H1); 5.09 (t, 1H, J = 9.8 Hz, H4); 5.20 (m, 1H,
13
H2); 5.29 ppm (dd, 1H, J = 3.6 Hz, J = 10.0 Hz, H3); C-NMR (CDCl3): δ = 3.85 (C6); 20.60, 20.73,
20.80 (3COCH3); 55.49 (OCH3); 68.60 (C3); 69.52 (C2); 69.90 (C4); 70.07 (C5); 98.44 (C1); 169.77,
169.80, 169.95 ppm (3C=O); MS (ESI) m/z: 368.24 [M+Na]+.
(6-Deoxy-6-azido-1,2,3,4-tetra-O-acetyl-α-D-mannopyranose 17): Compound 16 (500 mg, 1.45 mmol,
1 eq.) dissolved in acetic anhydride (10 mL) was added dropwise to a solution of Ac2O/AcOH/
H2SO4 5:4:1 (12.5 mL) at 0 °C. After 4 h at RT, the mixture was diluted with EtOAc then ice was
added slowly. The obtained organic layer was washed with a solution of NaHCO3 5% then water, dried
(Na2SO4), filtered and concentrated in vacuo. The beige oil was used without purification (83%):
1
Rf = 0.83 (CH2Cl2/MeOH 9:1); ሾߙሿଶ଴ = −42.7 (c = 1.01 in chloroform); H-NMR (CDCl3): δ = 1.98,
஽
2.03, 2.14, 2.15 (4s, 12H, 4CH3); 3.28 (dd, 1H, J = 5.6 Hz, J = 13.6 Hz, H6a); 3.37 (dd, 1H, J = 2.4 Hz,
J = 13.2 Hz, H6b); 3.97 (m, 1H, H5); 5.22 (s, 1H, H2); 5.31 (m, 2H, H3 and H4); 6.06 ppm (d, 1H,
13
J = 1.6 Hz, H1); C-NMR (CDCl3): δ = 20.53, 20.56, 20.63, 20.72 (4CH3); 50.55 (C6); 66.30, 68.43
(C3 and C4); 68.14 (C2); 71.70 (C5); 90.19 (C1); 167.96, 169.49, 169.68, 169.92 ppm (4C=O);
MS (ESI) m/z: 396.22 [M+Na]+, 408.35 [M-Cl]−.
4-Bromobut-2-en-1-yl-6-deoxy-6-azido-2,3,4-tri-O-acetyl-α-D-mannopyranoside (18): The procedure
described for compound 1 was applied to 17 and 4-bromo-but-2-en-1-ol to give compound 18 as a
1
beige oil (78%): Rf = 0.59 (EtOAc/petroleum ether 1:1); H-NMR (CDCl3): δ = 1.97, 2.03, 2.13 (3s,
9H, 3CH3); 3.25 (dd, 1H, J = 5.6 Hz, J = 13.5 Hz, H6a); 3.32 (dd, 1H, J = 2.4 Hz, J = 13.5 Hz, H6b);
3.99 (m, 1H, H5); 4.01 (d, 2H, J = 8.4 Hz, CH2Br); 4.68 (m, 2H, CH2O); 4.96 (d, 1H, J = 1.6 Hz, H1);
5.45 (m, 2H, H3 and H4); 5.62 (s, 1H, H2); 5.71 (m, 1H, CHCH2O); 5.93 ppm (m, 1H, CHCH2Br);
13C-NMR (CDCl3): δ = 20.51, 20.54, 20.62 (3CH3); 25.66 (CH2Br); 50.54 (C6); 59.12 (CH2O); 66.33,
68.46 (C3and C4); 68.34 (C2); 71.71 (C5); 90.23 (C1); 128.10 (CHCH2O); 129.75 (CHCH2Br); 167.97,
169.52, 169.70 ppm (3C=O); MS (ESI) m/z: 487.45 [M+Na]+.
Methyl 6-deoxy-2,3-O-(1-methylethylidene)-4-O-sodiumsulfate-α-D-heptomanno-pyranosiduronic acid
(19): Firstly, the procedure described for compound 13 was applied to 15 to give the appropriate
intermediate as a yellow solid (quantitative): Rf = 0.49 (CH2Cl2/MeOH 8.5:1.5); ሾߙሿଶ଴ = + 37.7
஽
(c = 1.00 in chloroform); 1H-NMR (acetone-d6): δ = 1.24, 1.41 (2s, 6H, 2CCH3); 2.76 (dd, 1H, J = 9.3 Hz,
J = 17.3 Hz, H6a); 3.18 (dd, 1H, J = 2.8 Hz, J = 17.3 Hz, H6b); 3.46 (s, 3H, OCH3); 3.86 (td, 1H,
J = 9.6 Hz, J = 2.8 Hz, H5); 4.15 (d, 1H, J = 7.4 Hz, H2); 4.21 (dd, 1H, J = 9.9 Hz, J = 7.0 Hz, H4);

Molecules 2014, 19
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4.44 (m, 1H, H3); 4.93 ppm (s, 1H, H1); 13C-NMR (acetone-d6): δ = 20.60 (C6); 25.5, 27.10 (2CCH3);
54.5 (OCH3); 64.90 (C5); 75.62 (C2); 76.34 (C4); 76.90 (C3); 98.17 (C1); 109.88 (C(CH3)2); 118.13
ppm (CN); MS (ESI) m/z: 384.23 [M+Na]+, 322.42 [M-Na]−.
Secondly, the procedure described for compound 14 was applied to the precedent intermediate to
give compound 19 as a colourless oil (quantitative): Rf = 0.61 (EtOAc/MeOH 1:1); ሾߙሿଶ଴ = +17.23
஽
(c = 1.00 in chloroform); 1H-NMR (CD3OD): δ = 1.33, 1.53 (2s, 6H, 2CCH3); 2.40 (dd, 1H, J = 9.8 Hz,
J = 15.8 Hz, H6a); 3.09 (dd, 1H, J = 2.2 Hz, J = 16.2 Hz, H6b); 3.41 (s, 3H, OCH3); 4.09 (m, 2H, H2
and H5); 4.21 (m, 2H, H3 and H4); 4.81 ppm (s, 1H, H1); 13C-NMR (CD3OD): δ = 26.57, 28.12
(2CCH3); 38.32 (C6); 55.76 (OCH3); 66.91 (C2); 77.19, 78.07, 79.05 (C3, C4 and C5); 99.42 (C1);
110.75 (C(CH3)2); 175.20 ppm (CO2H); MS (ESI) m/z: 387.99 [M+Na]+, 363.12 [M-H]−.
Methyl 6-deoxy-2,3-4-tri-O-acetyl-α-D-heptomannopyranosiduronic acid (20): Firstly, the procedure
described for compound 4 was applied to 19 to give the appropriate intermediate as a colourless oil
(78%): Rf = 0.25 (i-PrOH/NH4OH 8.5:1.5); 1H-NMR (D2O): δ = 2.86 (dd, 1H, J = 7.4 Hz, J = 17.3 Hz,
H6a); 3.04 (dd, 1H, J = 3.6 Hz, J = 17.3 Hz, H6b); 3.44 (s, 3H, OCH3); 3.60 (t, 1H, J = 9.7 Hz, H4); 3.76
(dd, 1H, J = 9.6 Hz, J = 3.4 Hz, H3); 3.84 (m, 1H, H5); 3.96 (dd, 1H, J = 3.4 Hz, J = 1.7 Hz, H2); 4.78
13
ppm (d, 1H, J = 1.5 Hz, H1); C-NMR (D2O): δ = 51.44 (C6); 55.20 (OCH3); 67.89 (C5); 70.27 (C2);
70.76 (C3); 71.60 (C4); 101.42 (C1); 176.01 ppm (CO2H); MS (ESI) m/z: 245.56 [M+Na]+, 221.03 [M-H]−.
Secondly, the procedure described for compound 16 was applied to the preceeding intermediate to
1
give compound 20 as a white powder (92%): Rf = 0.71 (EtOAc/petroleum ether 1:1); H-NMR
(CDCl3): δ = 1.98, 2.03, 2.10 (3s, 9H, 3COCH3); 3.40 (s, 3H, OCH3); 3.96 (m, 1H, H5); 4.11 (dd, 1H,
J = 2.4 Hz, J = 12.4 Hz, H6a); 4.28 (dd, 1H, J = 5.4 Hz, J = 12.2 Hz, H6b); 4.71 (d, 1H, J = 1.6 Hz, H1);
5.23 (m, 1H, H2); 5.27 (t, 1H, J = 9.8 Hz, H4); 5.32 ppm (dd, 1H, J = 3.2 Hz, J = 10.0 Hz, H3); 13C-NMR
(CDCl3): δ = 20.67, 20.72, 20.87 (3COCH3); 55.28 (OCH3); 62.46 (C6); 66.08 (C4); 68.32 (C5); 69.00
(C3); 69.45 (C2); 98.54 (C1); 169.88, 170.04, 170.66 (3C=O); 175.89 ppm (CO2H); MS (ESI) m/z:
371.59 [M+Na]+.
6-Deoxy-1-2,3-4-tetra-O-acetyl-α-D-heptomannopyranosiduronic acid (21): The procedure described for
compound 17 was applied to 20 to give compound 21 as a beige oil (83%): Rf = 0.48 (EtOAc/petroleum
1
ether 1:1); H-NMR (CDCl3): δ = 1.95, 2.06, 2.12, 2.15 (4s, 12H, 4CH3); 3.91 (m, 1H, H5); 4.17 (dd,
1H, J = 2.7 Hz, J = 12.6 Hz, H6a); 4.32 (dd, 1H, J = 5.4 Hz, J = 12.2 Hz, H6b); 5.30 (t, 1H, J = 9.9 Hz,
13
H4); 5.34 (m, 1H, H2); 5.35 (dd, 1H, J = 3.2 Hz, J = 10.0 Hz, H3); 5.98 ppm (s, 1H, H1); C-NMR
(CDCl3): δ = 20.67, 20.72, 20.87, 20.90 (4CH3); 62.44 (C6); 66.12 (C4); 68.36 (C5); 68.95 (C3); 69.43
(C2); 90.53 (C1); 169.72, 169.88, 170.04, 170.66 (4C=O); 176.08 ppm (CO2H); MS (ESI) m/z: 399.89
[M+Na]+.
4-Bromobut-2-en-1-yl-6-deoxy-2,3,4-tri-O-acetyl-α-D-heptomannopyranosiduronic acid (22): The
procedure described for compound 18 was applied to 21 and 4-bromo-but-2-en-1-ol to give
1
compound 22 as a beige oil (78%): Rf = 0.23 (EtOAc/petroleum ether 1:1); H-NMR (CDCl3):
δ = 1.95, 2.06, 2.12 (3s, 9H, 3CH3); 3.91 (m, 1H, H5); 4.08 (d, 2H, J = 8.6 Hz, CH2Br); 4.17 (dd, 1H,
J = 2.7 Hz, J = 12.6 Hz, H6a); 4.32 (dd, 1H, J = 5.4 Hz, J = 12.2 Hz, H6b); 4.70 (m, 2H, CH2O); 5.30
(t, 1H, J = 9.9 Hz, H4); 5.34 (m, 1H, H2); 5.35 (dd, 1H, J = 3.2 Hz, J = 10.0 Hz, H3); 5.77 (m, 1H,

Molecules 2014, 19
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CHCH2O); 5.90 (m, 1H, CHCH2Br); 5.98 ppm (s, 1H, H1); 13C-NMR (CDCl3): δ = 20.65, 20.70, 20.80
(3CH3); 25.62 (CH2Br); 59.26 (CH2O); 62.48 (C6); 66.18 (C4); 68.39 (C5); 69.00 (C3); 69.40 (C2);
90.51 (C1); 128.15 (CHCH2O); 129.77 (CHCH2Br); 169.67, 169.83, 170.06 (3C=O); 176.09 ppm
(CO2H); MS (ESI) m/z: 490.05 [M+Na]+.
(2E)-1-Bromo-3,7-dimethylocta-2,6-diene (23): PBr3 (1.1 mL, 9.51 mmol, 0.33 eq.) was added
dropwise at 0 °C to geraniol (5 mL, 28.83 mmol, 1 eq.) dissolved in CH2Cl2 (50 mL). After 1 h at 0 °C,
the mixture was diluted in CH2Cl2 and ice-cubes were added.The obtained organic layer was washed
with a solution of NaHCO3 5% and water, dried (Na2SO4), filtered and concentrated in vacuo. Purification
by chromatography on silica gel (EtOAc/petroleum ether 1:1) gave a yellow oil (93%): Rf = 0.80
1
(EtOAc/petroleum ether 1:1); H-NMR (CDCl3): δ = 1.59, 1.66, 1.67 (2s, 9H, 3CH3); 2.01,2.09 (2m,
4 h, CH2CH2); 4.13 (d, 2H, J = 7.2 Hz, CH2Br); 5.08 (m, 1H, CHC=C(CH3)2); 5.40 ppm (m, 1H,
CHCH2Br); 13C-NMR (CDCl3): δ = 16.20, 17.62,25.62 (3CH3); 26.31,39.48 (CH2CH2); 59.29
(CH2Br); 123.28 (CHCH2Br); 123.83 (CHC=C(CH3)2); 131.68, 139.61 ppm (2C=CH); MS (ESI) m/z:
241.67 [M+Na]+.
{[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]sulfonyl}benzene (24): Compound 23 (6 g, 27.52 mmol, 1 eq.)
and NaSO2Ph (9 g, 55.05 mmol, 2eq.) were stirred in DMF (12 mL) for 2 h at RT. Then the solution
was concentrated and the crude product was purified by chromatography on silica gel (EtOAc/petroleum
1
ether 1:1) to give a yellow oil (93%): Rf = 0.45 (EtOAc /petroleum ether 3:7); H-NMR (CDCl3):
δ = 1.24, 1.52, 1.62 (3s, 9H, 3CH3); 1.93 (s, 4 h, CH2CH2); 3.74 (d, 2H, J = 8.0 Hz, CH2SO2); 4.96 (m,
1H, CHC=C(CH3)2); 5.12 (t, 1H, J = 7.8 Hz, CHCH2SO2); 7.46 (t, 2H, J = 7.8 Hz, CHAr); 7.57 (t, 1H,
13
J = 7.4 Hz, CHAr); 7.80 ppm (d, 2H, J = 8.0 Hz, CHAr); C-NMR (CDCl3): δ = 16.10, 17.65, 25.66
(3CH3); 26.13, 39.63 (CH2CH2); 56.04 (CH2SO2); 110.25 (CHCH2SO2); 123.39 (CHC=C(CH3)2);
128.52, 128.89, 133.49 (CHAr); 132.04, 138.56, 146.34 ppm (2C=CH and CSO2); MS (ESI) m/z:
301.17 [M+Na]+.
(2E,6E)-2,6-Dimethyl-8-(phenylsulfonyl)octa-2,6-dien-1-al (25): Under argon and in the dark, SeO2
(8 mg, 0.07 mmol, 0.1 eq.), t-BuOOH (233 mg, 2.59 mmol, 3.6 eq.) and salicylic acid (4-hydroxybenzoic
acid) (10 mg, 0.07 mmol, 0.1 eq.) were dissolved in CH2Cl2 (1 mL). A solution of compound 24
(200 mg, 0.72 mmol, 1 eq.) in CH2Cl2 (10 mL) was added at 0 °C. The ice bath was removed after
10 min and stirring was left for 24 h. The mixture was diluted in CH2Cl2, washed with a saturated
solution of NaHCO3 then with water to neutralize tBuOOH and to eliminate HSeCO3. The organic
layer was dried, filtered and concentrated in vacuo. Purification by chromatography on silica gel
(EtOAc/petroleum ether 3:7) gave the aldehyde and the alcohol (50:50): Rf = 0.50 (EtOAc/petroleum
1
ether 3:7); H-NMR (CDCl3): δ = 1.39, 1.70 (2s, 6H, 2CH3); 2.18 (t, 2H, J = 7.4 Hz, CH2CH2); 2.38
(q, 2H, J = 7.4 Hz, J = 15.0 Hz, CH2CH2); 3.81 (d, 2H, J = 8.0 Hz, CH2SO2); 5.22 (td, 1H, J = 1.2 Hz,
J = 8.0 Hz, CHCH2SO2); 6.38 (td, 1H, J = 1.2 Hz, J = 7.2 Hz, CH=CCHO); 7.52 (t, 2H, J = 7.8 Hz,
CHAr); 7.63 (t, 1H, J = 7.6 Hz, CHAr); 7.84 (d, 2H, J = 7.2 Hz, CHAr); 9.36 ppm (s, 1H, CHO);
13C-NMR (CDCl3): δ = 9.16, 16.10 (2CH3); 26.78 (CH2CH2); 37.85 (CH2CH2); 55.82 (CH2SO2); 111.31
(CHCH2SO2); 128.26,129.00,133.62 (3CHAr); 152.87 (CH=CCHO); 138.64, 139.62, 144.75 (2C=CH and
CSO2); 194.97 ppm (CHO); MS (ESI) m/z: 315.17 [M+Na]+.

Molecules 2014, 19
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(2E,6E)-2,6-Dimethyl-8-(phenylsulfonyl)octa-2,6-dien-1-ol (26): To a solution containing compound
25 (8 g, 27.40 mmol, 1 eq.) in EtOH (80 mL) NaBH4 (1.04 g, 27.40 mmol, 1 eq.) was added in several
portions. After 10 min at 0 °C, the mixture was diluted with CH2Cl2 and then washed with water. The
organic layer was dried (Na2SO4), filtered and reduced under pressure. Purification by chromatography
on silica gel (EtOAc/petroleum ether 1:1) gave a colourless oil (63% in alcohol, 2 steps): Rf = 0.30
1
(EtOAc/petroleum ether 3:7); H-NMR (CDCl3): δ = 1.37, 1.66 (2s, 6H, 2CH3); 2.08 (m, 4 h,
CH2CH2); 3.80 (d, 2H, J = 7.6 Hz, CH2SO2); 3.99 (s, 2H, CH2OH); 5.19 (t, 1H, J = 7.4 Hz,
CHCH2SO2); 5.33 (t, 1H, J = 6.0 Hz, CH=CCH2OH); 7.54 (t, 2H, J = 7.8 Hz, CHAr); 7.64 (t, 1H,
13
J = 7.4 Hz, CHAr); 7.87 ppm (d, 2H, J = 7.6 Hz, CHAr); C-NMR (CDCl3): δ = 13.68, 16.13 (2CH3);
25.39 (CH2CH2); 39.17 (CH2CH2); 56.00 (CH2SO2); 68.70 (CH2OH); 110.34 (CHCH2SO2); 124.63
(CH=CCH2OH); 128.37 (CHAr); 129.02 (CHAr); 133.58 (CHAr); 135.54, 138.81, 146.11 ppm
(2C=CH and CSO2); MS (ESI) m/z: 317.19 [M+Na]+.
S-(2E,6E)-2,6-Dimethyl-8-(phenylsulfonyl)octa-2,6-dien-1-yl ethanethioate (27): Firstly, compound
26 (4 g, 13.60 mmol, 1 eq.), CBr4 (5.41 g, 16.33 mmol, 1.2 eq.) and PPh3 (5 g, 19.05 mmol, 1.4 eq.)
were reacted in CH2Cl2 (7 mL) at RT. After 2h, the mixture was reduced under pressure then purified
by chromatography on silica gel (EtOAc/petroleum ether 2:3) to give a yellow oil (quantitative):
Rf = 0.66 (EtOAc/petroleum ether5:5); 1H-NMR (CDCl3): δ = 1.33,1.73 (2s, 6H, 2CH3); 2.05 (m, 4 h,
CH2CH2); 3.81 (d, 2H, J = 8.0 Hz, CH2SO2); 3.95 (s, 2H, CH2Br); 5.19 (t, 1H, J = 8.0 Hz,
CHCH2SO2); 5.50 (m, 1H, CH=CCH2Br); 7.54 (t, 2H, J = 7.6 Hz, CHAr); 7.64 (t, 1H, J = 7.4 Hz,
CHAr); 7.86 ppm (d, 2H, J = 7.2 Hz, CHAr); 13C-NMR (CDCl3): δ = 14.65, 16.13 (2CH3); 26.29
(CH2CH2); 38.68 (CH2CH2); 41.42 (CH2Br); 55.95 (CH2SO2); 110.78 (CHCH2SO2); 128.35 (CHAr);
128.98 (CHAr); 129.99 (CH2Br); 133.56 (CHAr); 132.63, 138.63, 145.57 ppm (2C=CH and CSO2); MS
(ESI) m/z: 379.04 and 381.09 [M+Na]+, 394.94 and 397.06 [M+K]+.
Next, to a solution of the preceeding intermediate (5 g, 14.00 mmol, 1 eq.) in DMF (50 mL) KSAc
(3.2 g, 28.00 mmol, 2 eq.) was added. After 1h at RT, the solution was diluted in CH2Cl2 and washed
with water. The organic layer was dried, filtered and reduced under pressure and purification by
chromatography on silica gel (EtOAc/petroleum ether 1:1) gave a brown oil (quantitative):
Rf = 0.66 (EtOAc/petroleum ether 1:1); 1H-NMR (CDCl3): δ = 1.30, 1.60 (2s, 6H, 2CH3); 2.01 (m, 4 h,
CH2CH2); 2.32 (s, 3H, COCH3); 3.51 (s, 2H, CH2S); 3.80 (d, 2H, J = 8.0 Hz, CH2SO2); 5.17 (t, 1H,
J = 8.4 Hz, CHCH2SO2); 5.31 (m, 1H, CH=CH2S); 7.53 (t, 2H, J = 7.6 Hz, CHAr); 7.63 (t, 1H, J = 7.4 Hz,
CHAr); 7.86 ppm (d, 2H, J = 7.2 Hz, CHAr); 13C-NMR (CDCl3): δ = 15.14, 16.12 (2CH3); 26.18
(CH2CH2); 30.48 (COCH3); 38.00 (CH2S); 39.09 (CH2CH2); 55.99 (CH2SO2); 110.59 (CHCH2SO2);
127.77 (CH=CCH2S); 128.47 (CHAr); 128.94 (CHAr); 133.52 (CHAr); 131.01, 138.62, 145.85 ppm
(2C=CH and CSO2); 195.55 (CO); MS (ESI) m/z: 375.67 [M+Na]+.
(2Z,6E,10E)-12-(Acetylthio)-7,11-dimethyl-5-(phenylsulfonyl)dodeca-2,6,10-trien-1-yl
2,3,4-tri-O-
acetyl-6-deoxy-6-azido-α-D-mannopyranoside (28): Under argon, the solution of compound 27
(200 mg, 0.57 mmol, 1.3 eq.) dissolved in anhydrous THF (4 mL) was cooled to −78 °C and LiHMDS
(550 µL) was slowly added. After 10 min, a solution containing compound 18 (200 mg, 0.43 mmol, 1 eq.)
was introduced dropwise. The reaction was left at −78 °C under for 4 h then 18 h at RT. The mixture
was diluted in CH2Cl2 then washed with water. The organic layer was dried, filtered and reduced under

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pressure and purification by chromatography on silica gel (EtOAc/petroleum ether 1:1) gave a
colourless oil (15%): Rf = 0.33 (EtOAc/petroleum ether 1:1); ሾߙሿଶ଴ = +10.9 (c = 1.00 in chloroform);
஽
1H-NMR (CDCl3): δ = 1.29, 1.59 (2s, 6H, 2CH=CCH3); 2.00 (m, 4 h, CH2CH2); 2.01, 2.09, 2.14, 2.31
(4s, 12H, 4COCH3); 3.50 (s, 2H, CH2S); 3.78 (m, 1H, CHSO2); 3.97 (m, 4 h, H6 and CH2CHSO2);
4.11 (m, 3H, H5 and CH2O); 4.64 (d, 1H, J = 1.2 Hz, H1); 4.74 (m, 2H, OCH2CH=CH); 5.11 (t, 1H,
J = 10.2 Hz, H4); 5.20 (m, 1H, CHCHSO2); 5.30 (m, 2H, H2 and CH=CCH2S); 5.35 (dd, 1H, J = 10.4 Hz,
J = 3.6 Hz, H3); 7.53 (m, 2H, CHAr); 7.64 (m, 1H, CHAr); 7.84 ppm (d, 2H, J = 7.2 Hz, CHAr); 13C-NMR
(CDCl3): δ = 15.11, 16.09 (2CH=CCH3); 20.58, 20.72, 20.79, 30.44 (4COCH3); 26.15, 39.05
(CH2CH2); 37.96 (CH2S); 55.53 (CHSO2); 55.96, 59.43 (C6 and CHCHSO2); 62.24 (CH2O); 63.25
(C5); 66.10 (C3); 66.82 (C4); 70.31 (C2); 98.34, 98.65 (OCH2CH=CH); 99.35 (C1); 110.57
(CHCHSO2); 127.73 (CH=CCH2S); 128.43 (CHAr); 128.91 (CHAr); 131.96 (CHAr); 133.49 (CHAr);
130.97, 138.61, 145.81 (2C=CH and CSO2); 169.63, 169.73, 170.78 ppm (4C=O); MS (ESI) m/z:
737.01 [M+H]+, 758.98 [M+Na]+.
(2Z,6E,10E)-12-(Acetylthio)-7,11-dimethyl-5-(phenylsulfonyl)dodeca-2,6,10-trien-1-yl
2,3,4-tri-O-
acetyl-6-deoxy-α-D-heptomannopyranosiduronic acid (29): The procedure described for compound 28
was applied to 22 and 27 to give compound 29 as a beige oil (17%): Rf = 0.16 (EtOAc/petroleum ether
1
1:1); ሾߙሿଶ଴ = +9.4 (c = 1.00 in chloroform); H-NMR (CDCl3): δ = 1.29, 1.58 (2s, 6H, 2CH=CCH3);
஽
1.99 (m, 4 h, CH2CH2); 2.04, 2.07, 2.15, 2.31 (4s, 12H, 4COCH3); 3.50 (s, 2H, CH2S); 3.78 (m, 1H,
CHSO2); 4.05 (m, 5 h, H5, CH2O and CH2CHSO2); 4.12 (dd, 1H, J = 2.4 Hz, J = 14.4 Hz, H6a); 4.27
(dd, 1H, J = 4.8 Hz, J = 12.4 Hz, H6b); 5.15 (t, 4 h, J = 8.0 Hz, CH=CH, 2CH=CCH3); 5.25 (m, 1H,
H2); 5.32 (m, 2H, H3 and H4); 6.08 (d, 1H, J = 1.6 Hz, H1); 7.52 (t, 2H, J = 7.6 Hz, CHAr); 7.63 (m, 1H,
CHAr); 7.84 ppm (d, 2H, J = 7.6 Hz, CHAr); 13C-NMR (CDCl3): δ = 15.02, 15.99 (2CH=CCH3); 20.50,
20.60, 20.70 (4COCH3); 26.05, 38.95 (CH2CH2); 37.86 (CH2S); 55.85 (CSO2); 61.92 (C6, CH2O and
CH2CHSO2); 65.33, 68.57 (C3 and C4); 68.16 (C2); 70.42 (C5); 90.41 (C1); 110.49, 126.98
(2CH=CCH3); 110.81 (CH=CH); 127.44–133.42 (5CHAr); 138.48, 145.70, 146.84 (2C=CH and
CSO2); 169.36, 169.56, 168.80, 170.44 (4C=O); 195.37 ppm (CO2H); MS (ESI) m/z: 740.05 [M+H]+,
761.99 [M+Na]+.
(2Z,6E,10E)-12-Mercapto-7,11-dimethyl-5-(phenylsulfonyl)dodeca-2,6,10-trien-1-yl-6-deoxy-6-azido-
α-D-mannopyranoside (30): The procedure described for compound 3 was applied to 2 to give
compound 30 as a white oil (97%): Rf = 0.34 (CH2Cl2/MeOH 4:1); 1H-NMR (CDCl3):
δ = 1.61, 1.66 (2s, 6H, 2CH3); 2.02, 2.11 (2m, 4 h, CH2CH2); 3.49 (m, 1H, H5); 3.61 (t, 1H, J = 9.4 Hz,
H4); 3.67 (dd, 1H, J = 3.2 Hz, J = 9.2 Hz, H3); 3.72 (dd, 1H, J = 5.6 Hz, J = 12.0 Hz, H6a); 3.79 (q, 1H,
J = 1.6 Hz, H2); 3.83 (dd, 1H, J = 2.4 Hz, J = 12.0 Hz, H6b); 4.08 (d, 4 h, J = 6.4 Hz, CH2O and CH2S);
13
4.64 (d, 1H, J = 1.6 Hz, H1); 5.11 (m, 3H, 3CH=C); 5.35 ppm (m, 3H, 3CH=C); C-NMR (CDCl3):
δ = 16.27, 25.89 (2CH3); 27.52 (CH2CH2); 40.72 (CH2CH2); 59.45 (CH2O and CH2S); 62.83 (C6);
68.57 (C4); 72.09 (C2); 72.59 (C3); 74.42 (C5); 102.71 (C1); 124.92, 125.14, 129.95, 130.07, 133.05,
133.15 (6CH=C); 132.44, 139.40 ppm (2C=CH); MS (ESI) m/z: 426.12 [M+H]+, 448.24 [M+Na]+.
(2Z,6E,10E)-12-Mercapto-7,11-dimethyl-5-(phenylsulfonyl)dodeca-2,6,10-trien-1-yl-6-deoxy-α-D-
heptomannopyranosiduronic acid (31): The procedure described for compound 3 was applied to 29 to

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give compound 31 as a white oil (97%): Rf = 0.50 (i-PrOH/NH4OH 1:1); ሾߙሿଶ଴ = +4.4 (c = 1.00 in
஽
1
chloroform); H-NMR (CDCl3): δ = 1.30, 1.55 (2s, 6H, 2CH3); 1.99 (m, 4 h, CH2CH2); 3.50 (s, 2H,
CH2S); 3.76 (m, 1H, CH=CH); 4.03 (m, 5 h, H5, CH2O and CH=CH); 4.12 (dd, 1H, J = 2.4 Hz,
J = 14.4 Hz, H6a); 4.27 (dd, 1H, J = 4.8 Hz, J = 12.4 Hz, H6b); 5.15 (t, 4 h, J = 8.0 Hz, 2CH=CH,
13
2CH=C); 5.25 (m, 1H, H2); 5.32 (m, 2H, H3 and H4); 6.08 ppm (d, 1H, J = 1.6 Hz, H1); C-NMR
(CDCl3): δ = 15.02, 15.99 (2CH3); 26.05, 38.95 (CH2CH2); 37.86 (CH2S); 55.85 (CH=CH); 61.92 (C6,
CH2O andCH=CH); 65.33, 68.57 (C3 and C4); 68.16 (C2); 70.42 (C5); 90.41 (C1); 110.49, 126.98
(2CH=C); 110.81 (2CH=CH); 138.48, 145.70 (2C=CH); 195.37 ppm (CO2H); MS (ESI) m/z: 471.76
[M+H]+, 493.78 [M+Na]+.
6-Deoxy-6-azido-2,3,4-tri-O-acetyl-α-D-mannopyranosyl bromide (32): A solution of hydrobromic
acid (5.7 M in acetic acid, 2.35 mL,13.40 mmol, 25 eq.) was added to a solution of acetic anhydride
(1 mL) and compound 17 (200 mg, 0.54 mmol, 1 eq.). After 16 h at RT, the mixture was diluted in
CH2Cl2 and washed with a saturated solution of NaHCO3 until basic pH. The aqueous phase was
extracted with CH2Cl2 (3 times). The organic layers were assembled, then washed with a saturated
solution of NaCl, dried, filtered and concentrated in vacuo. The obtained colourless oil was used
without purification (quantitative): Rf = 0.56 (petroleum ether/Et2O 3:7); ሾߙሿଶ଴ = +96.1 (c = 1.00 in
஽
chloroform); 1H-NMR (CDCl3): δ = 2.01, 2.10, 2.17 (3s, 9H, 3CH3); 3.21 (dd, 1H, J = 6.6 Hz, J = 11.4 Hz,
H6a); 3.35 (dd, 1H, J = 2.9 Hz, J = 11.4 Hz, H6b); 3.94–3.99 (m, 1H, H5); 5.27 (t, 1H, J = 10.0 Hz, H4);
5.41 (dd, 1H, J = 1.4 Hz, J = 3.3 Hz, H2); 5.71 (dd, 1H, J = 3.4 Hz, J = 10.0 Hz, H3); 6.3 ppm (d, 1H,
J = 1.1 Hz, H1); 13C-NMR (CDCl3): δ = 2.46 (C6); 20.60, 20.73, 20.78 (3CH3); 67.66 (C3); 69.49 (C4);
72.21 (C2); 73.45 (C5); 82.46 (C1); 169.58, 169.64, 169.72 (3C=O); MS (ESI) m/z: 393.98, 395.40
[M+H]+.
6-Deoxy-6-azido-2,3,4-tri-O-acetyl-1-thio-β-D-mannopyranose (34): A solution of compound 32 (100 mg,
0.25 mmol, 1 eq.) and thiourea (25 mg, 0.33 mmol) in acetone (2 mL) was stirred under reflux for
20 h. The reaction was cooled to room temperature. The solvent was removed under reduced pressure
to give the isothiouronium salt as a white solid. K2S2O5 (85 mg, 0.38 mmol) was added to a suspension
of this salt in CHCl3/H2O (1/1 v/v) (3 mL). After stirring under reflux for 5 h, the solution was cooled
to RT, the CHCl3 layer was separated and the aqueous layer was extracted with CH2Cl2. The combined
organic layers were dried and concentrated under reduced pressure. Purification by chromatography
onsilica gel (EtOAc/petroleum ether 1:1) gave a colourless oil (50%): Rf = 0.56 (CH2Cl2/Et2O 8:2);
1H- NMR (CDCl3): δ = 2.00, 2.06, 2.19 (3s, 9H, 3CH3); 3.32 (m, 2H, H6); 4.17 (ddd, 1H, J = 4.1 Hz,
J = 5.4 Hz, J = 9.5 Hz, H5); 4.56 (d, 1H, J = 4.1 Hz, H1); 5.22 (m, 2H, H2 et H4); 5.39 ppm (dd, 1H,
13
J = 3.2 Hz, J = 10.0 Hz, H3); C-NMR (CDCl3): δ = 20.6, 20.6, 20.8 (3CH3); 51.1 (C6); 67.2 (C4);
68.6 (C3); 69.5 (C5); 70.1 (C2); 91.8 (C1); 169.9, 170.1, 170.3 (3C=O); MS (ESI) m/z: 370.44
[M+Na]+.
6-Deoxy-2,3,4-tri-O-acetyl-α-D-heptomannopyranuronosyl bromide (33): The procedure described for
compound 32 was applied to 21 to give compound 33 as a white oil (quant): Rf = 0.22 (petroleum
ether/EtOAc 1:1); 1H-NMR (CDCl3): δ = 1.97, 2.05, 2.12 (3s, 9H, CH3); 1.96–2.21 (m, 2H, H6); 3.91
(td, 1H, J = 2.6 Hz, J = 10.0 Hz, H5); 5.13 (t, 1H, J = 10.0 Hz, H4); 5.20 (dd, 1H, J = 1.9 Hz, J = 3.5 Hz,

Molecules 2014, 19
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H2); 5.27 (dd,1H, J = 3.5 Hz, J = 10.0 Hz, H3); 5.94 ppm (d, 1H, J = 1.8 Hz, H1); 13C-NMR (CDCl3):
δ = 20.60, 20.75 (3CH3); 30.02 (C6); 68.47 (C2); 68.74 (C3); 68.97 (C4); 69.24 (C5); 90.11 (C1);
168.09, 169.00, 169.33, 169.75 (3C=O et CO2H); MS (ESI) m/z: 419.67, 421.28 [M+Na]+.
6-Deoxy-2,3,4-tri-O-acetyl-1-thio-β-D-heptomannopyranuronic acid (35): The procedure described for
compound 34 was applied to 33 to give compound 35 as a white oil (48%): Rf = 0.20 (CH2Cl2/MeOH
1
4:1); H-NMR (CDCl3): δ = 1.99, 2.06, 2.14 (3s, 9H, CH3); 1.95–2.26 (m, 2H, H6); 4.01 (td, 1H,
J = 2.6 Hz, J = 10.0 Hz, H5); 5.11 (t, 1H, J = 10.0 Hz, H4); 5.12 (d, 1H, J = 3.2 Hz, H1); 5.23 (dd, 1H,
J = 1.7 Hz, J = 3.4 Hz, H2); 5.35 ppm (dd,1H, J = 3.4 Hz, J = 10.0 Hz, H3); 13C-NMR (CDCl3):
δ = 20.72, 20.88, 20.93 (3CH3); 30.32 (C6); 67.46 (C5); 68.88 (C3); 69.46 (C4); 70.11 (C2); 92.03 (C1);
169.40, 167.55, 170.08, 170.13 (3C=O et CO2H); MS (ESI) m/z: 373.28 [M+Na]+.
2-{2-[2-(Allyloxy)ethoxy]ethoxy}ethanol (36): The procedure described for compound 6 was applied to
triethylene glycol and 3-bromopropene to give compound 36 as a red-orange oil (85%): Rf = 0.34
(CH2Cl2/MeOH 9:1); 1H-NMR (CDCl3): δ = 3.57–3.71 (m, 12H, 6CH2O); 4.00 (m, 2H, CH2CH=CH2);
5.16 (dd, 1H, J = 1.6 Hz, J = 10.4 Hz, CH2 = CH); 5.25 (dd, 1H, J = 1.6 Hz, J = 17.2 Hz, CH2=CH);
5.89 ppm (m, 1H, CH=CH2); 13C-NMR (CDCl3): δ = 61.66–72.57 (7CH2O); 117.30 (CH2=CH); 134.59
ppm (CH=CH2); MS (ESI) m/z: 213.22 [M+Na]+, 229.17 [M+K]+.
3-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}propyl2,3,4-tri-O-acetyl-6-deoxy-6-azido-1-thio-β-D-manno-
pyranoside (37): Under argon, compound 36 (36 mg, 0.19 mmol) and AIBN (0.29 mmol,
1.5 eq.) were added to a solution of compound 34 (200 mg, 0.58 mmol) in dioxane degassed under
argon (12 mL). After 3 h under ultrasound (amplitude of 20%, pulse on 0.2 s, pulse off 0.2 s), the
mixture was reduced under pressure then purified by chromatography on silica gel (EtOAc/petroleum
ether 7:3 to 10:0) to give a colourless oil (79%) Rf = 0.38 (EtOAc); ሾߙሿଶ଴ = +43.8 (c = 1.00 in
஽
chloroform); 1H-NMR (CDCl3): δ = 1.98 (s, 3H, CH3); 2.03 (m, 5 h, CH2CH2S and CH3); 2.14 (s, 3H,
CH3); 3.27 (m, 5 h, H6a, CH2S and CH2(CH2)2S); 3.35 (dd, 1H, J = 6.4 Hz, J = 13.2 Hz, H6b); 3.59–3.72
(m, 12H, 6CH2O); 4.01 (m, 1H, H5); 4.87 (d, 1H, J = 7.4 Hz, H1); 5.22 (t, 1H, J = 10.0 Hz, H4); 5.25
(dd, 1H, J = 1.6 Hz, J = 3.6 Hz, H2); 5.35 ppm (dd, 1H, J = 3.2 Hz, J = 10.0 Hz, H3); 13C-NMR
(CDCl3): δ = 20.65, 20.69, 20.83 (3CH3); 27.98 (CH2S); 35.59 (CH2CH2S); 58.73 (CH2(CH2)2S);
51.05 (C6); 61.62–72.51 (CH2O); 67.16 (C4); 68.80 (C3); 69.48 (C2); 69.93 (C5); 97.42 (C1); 169.82,
169.95, 170.08 ppm (3C=O); MS(ESI) m/z: 560.12 [M+Na]+.
3-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}propyl2,3,4-tri-O-ace-tyl-6-deoxy-6-azido-1-thio-β-D-hepto-
mannopyranosiduronic acid (38): The procedure described for compound 37 was applied
to 35 and 36 to give compound 38 as a beige oil (80%): Rf = 0.08 (EtOAc); ሾߙሿଶ଴ = +40.0 (c = 1.00 in
஽
1
chloroform); H-NMR (CDCl3): δ = 1.87 (qt, 2H, J = 6.8 Hz, CH2CH2S); 1.95, 2.06, 2.12 (3s, 12H,
4CH3); 2.69 (m, 2H, CH2S); 3.51 (t, 2H, J = 6.0 Hz, CH2(CH2)2S); 3.54–3.69 (m, 12H, 6CH2O); 4.05
(dd, 1H, J = 2.2 Hz, J = 12.2 Hz, H6a); 4.27 (dd, 1H, J = 5.4 Hz, J = 12.2 Hz, H6b); 4.34 (m, 1H, H5);
4.80 (d, 1H, J = 7.4 Hz, H1); 5.23 (m, 2H, H3 et H4); 5.30 ppm (dd, 1H, J = 1.6 Hz, J = 3.2 Hz, H2);
13C-NMR (CDCl3): δ = 19.62, 19.69, 19.91 (3CH3); 27.19 (CH2S); 28.41 (CH2CH2S); 60.69
(CH2(CH2)2S); 61.41 (C6); 65.31 (C3); 67.96 (C5); 68.45 (C4); 68.26–71.53 (7CH2O); 70.14 (C2); 81.64
(C1); 168.72, 168.80, 168.99, 169.63 ppm (3C=O et CO2H); MS (ESI) m/z: 563.76 [M+Na]+.

Molecules 2014, 19
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3-{2-[2-(2-Mercaptoethoxy)ethoxy]ethoxy}propyl 6-deoxy-6-azido-1-thio--D-mannopyranoside (39):
The procedures described for compounds 27 then 3 were applied to 37 to give compound 39
1
as a beige oil: Rf = 0.21 (CH2Cl2/MeOH 4:1); ሾߙሿଶ଴ = +40.0 (c = 1.00 in chloroform); H-NMR
஽
(CD3OD): δ = 1.95 (m, 2H, CH2CH2SC); 2.88 (t, 2H, J = 6.6 Hz, CH2SH); 3.26 (m, 4 h, CH2SC and
CH2(CH2)2SC); 3.40 (dd, 1H, J = 7.0 Hz, J = 13.0 Hz, H6a); 3.55 (m, 2H, H5 andH6a); 3.58–3.69 (m,
10H, H3,4 and 4CH2O); 3.72 (t, 2H, J = 6.4 Hz, CH2CH2SH); 3.80 (dd, 1H, J = 1.6 Hz, J = 3.2 Hz, H2);
4.77 ppm (d, 1H, J = 1.6 Hz, H1); 13C-NMR (CD3OD): δ = 28.02 (CH2SC); 37.42 (CH2CH2SC); 39.51
(CH2SH); 57.92 (CH2(CH2)2SC); 53.02 (C6); 62.55–71.65 (5CH2O); 69.52, 72.01, 72.36 (C3, C4
andC5); 73.86 (C2); 101.87 ppm (C1); MS (ESI) m/z: 428.73 [M+H]+, 460.57 [M+Na]+.
3-{2-[2-(2-Mercaptoethoxy)ethoxy]ethoxy}propyl 6-deoxy-1-thio-β-D-heptomannopyranosiduronic acid
(40): The procedures described for compounds 27 then 3 were applied to 38 to give compound 40 as a
beige oil: Rf = 0.18 (i-PrOH/NH4OH 1:1); ሾߙሿଶ଴ = +32.0 (c = 1.05 in chloroform); 1H-NMR (CD3OD):
஽
δ = 1.92 (qt, 2H, J = 6.4 Hz, CH2CH2SC); 2.73 (m, 2H, CH2SC); 3.09 (t, 2H, J = 6.6 Hz,
CH2(CH2)2SC); 3.53 (t, 2H, J = 6.0 Hz, CH2SH); 3.55–3.62 (m, 10H, 5CH2O); 4.08 (dd, 1H, J = 2.2 Hz,
J = 12.2 Hz, H6a); 4.30 (dd, 1H, J = 5.4 Hz, J = 12.2 Hz, H6b); 4.37 (m, 1H, H5); 5.21
13
(m, 2H, H1, H3 and H4); 5.31 (dd, 1H, J = 1.6 Hz, J = 3.2 Hz, H2); C-NMR (CD3OD): δ = 27.22
(CH2SC); 27.82 (CH2CH2SC); 28.48 (CH2(CH2)2SC); 61.41 (C6); 65.32 (C3); 67.96 (C5); 68.44 (C4);
68.28–69.59 (5CH2O and CH2SH); 70.14 (C2); 81.65 (C1);168.90 (CO2H); MS (ESI) m/z: 431.59
[M+H]+, 463.64 [M+Na]+.
3.2. Preparation of Citrate-Reduced Gold Nanoparticles
Hydrogen tetrachloroaurate trihydrate (60 mg, 0.15 mmol) was dissolved in water (250 mL) to give
a pale-yellow solution. A second solution with sodium citrate (150 mg, 0.58 mmol) dissolved in water
(10 mL) was prepared. Both solutions were heated to 60 °C for 10 min then the sodium citrate solution
was rapidly added to the gold solution. The temperature was then increased to 120 °C with continuous
stirring for 2 h 30 min. A deep-red solution was formed. The solution was warmed to RT and each
thiol-derivatized carbohydrate (50 mg) dissolved in methanol (1 mL) was added to freshly prepared
citrate-reduced gold nanoparticles. Self-assembly was facilitated by leaving the solution under stirring
for 48 h. To remove any unbound carbohydrate, the solution was diluted with brine to precipitate
nano-objects and left to rest over night. Then the supernatant was removed and nanoparticles were
resuspended in water. They were then centrifuged for 30 min at 14,000 rpm. The centrifugation step
was repeated three times to ensure complete removal of any unbound carbohydrate.
3.3. CAM Assays
Paper discs were saturated with a phosphate buffer saline dispersion of coated AuNPs (60 mg/mL)
in PBS or a control (phosphate buffer saline) and then deposited on chorioallantoic membranes
of 7-day-old chicken embryos for 4 days in ovo at 38 °C. Sutent® (sunitinib, a non-proteic inhibitor)
and ECGS (endothelial cell growth supplement) were used at 60 mg/mL as negative and positive
stimuli, respectively. Quantification of the angiogenic response was carried out by measuring the area

Molecules 2014, 19
1145
of neo-vascularization on each particular membrane (Figure 2). The vascularization was evaluated
using Image J software, and are given in pixels compared to phosphate buffer saline (PBS, control).
Figure 2. (A) CAM activity in percentage compared to PBS (control); (B) CAM activity in
pixels (Image J) compared to PBS (control).
(A)
(B)
PBS: 25790.5+/−2534, n = 4 (100%); Sutent®: 12765+/−4913.5, p = 0.00003, n = 10; NP1: 14944.2+/−3348.2,
p = 0.00044, n = 5; NP2: 15169+/−2527.2, p = 0.00027, n = 5; NP3: 16813+/−7274.4, p = 0.01444, n = 6;
NP4: 22276.2+/−1549.8, p = 0.0309, n = 5; NP5: 22463.2+/−2210.6, p = 0.0416, n = 5; NP6: 22657.3+/−1215,
p = 0.04239, n = 4; Uronic (methyl 6,7-dideoxy-α-D-mannopyranoside) acid (MeCOOH): 31758+/−4919.8,
p = 0.0449, n = 4; 6-deoxy-6-azido-α-D-methylmannopyranoside (MeN3): 32348+/−1805.5, p = 0.00350,
n = 4; ECGS: 44455.8+/−11955.3, p = 0.00138, n = 8.
4. Conclusions
We have reported the synthesis of a series of gold glyconanoparticles bearing diverse M6P
neoglycoconjugates. These M6P analogues were synthesized either by Huisgen cycloaddition, by the
Julia olefination, or by thiol-ene coupling. The thiol-ene reaction strategy under ultrasound activation
proved to be the most efficient in terms of yields and ease of implementation. The angiogenic activities
of the AuNPs have been tested by the CAM assay, and all possess angiogenic activities via the M6P
receptor with no apparent toxicity. The results of this study have allowed us: (a) to demonstrate that
the activity is not dependent of the structure of the linker between the nanoparticles and the
carbohydrate and (b) to identify the inhibitory multivalent effect of M6P derivatives on gold surfaces
compared with the corresponding monomeric activators. Although our biological results are obviously
in a preliminary stage, the work described herein is valuable in that it provides synthetic access to
some potentially useful multi-functional and biologically active systems.
Acknowledgments
This work was supported by grants from the Ministère de la Recherche et de l’Enseignement
Supérieur and by the Ligue contre le Cancer Comité du Languedoc Roussillon. We thank also Mélanie
Sabadotto (Laboratoires Pierre Fabre Dermocosmetique, Toulouse) for useful discussion.

Molecules 2014, 19
1146
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Kornfeld, S. Structure and function of the mannose 6-phosphate insulin-like growth factor-ii
receptors. Annu. Rev. Biochem. 1992, 61, 307–330.
2. Kang, J.K.; Li, Y.; Leaf, A. Retinoic acid alters the intracellular trafficking of the
mannose-6-phosphate insulin-like growth factor II receptor and lysosomal enzymes. Proc. Natl.
Acad. Sci. USA 1998, 95, 13671–13676.
3. Volpert, A.; Jackson, D.; Bouck, N.; Linzer, D.I.H. The insulin-like growth factor II mannose
6-phosphate receptor is required for proliferin-induced angiogenesis. Endocrinology 1996, 137,
3871–3876.
4. Barragan-Montero, V.; Awwad, A.; Combemale, S.; de Santa Barbara, P.; Jover, B.; Molès, J.-P.;
Montero, J.-L. Synthesis of Mannose-6-Phosphate analogues and their utility as angiogenesis
regulators. Chem. Med. Chem. 2011, 6, 1771–1774.
5. Clavel, C.; de Santa Barbara, P.; Jover, B.; Moles, J.-P.; Montero, J.-L.; Montero, V. Novel Uses
of D-Mannopyranose Derivatives. U.S. Patent WO 2009138601 A2, 19 November 2009.
6. Awwad, A.; Combemale, S.; de Santa Barbara, P.; Jover, B.; Molès, J.-P.; Montero, J.-L.;
Barragan-Montero, V. Nouveaux Derives de Mannopyranoside Ayant une Activite Anticancereuse.
EP 2499150 A1, 19 September 2012.
7. De Santa Barbara, P.; Jover, B.; Moles, J.-P.; Montero, J.-L.; Montero, V. Nouvelles Utilisations de
Derives de D-Mannopyranose Activateurs de l’angiogenese. EP 2285817 A2, 23 February 2011.
8. Kitov, P.I.; Bundle, D.R. Carbohydrate-Based Drug Discovery; Wong, C.-H., Ed.; Wiley-VCH:
Weinheim, Germany, 2003; Volume 2, pp. 541–574.
9. Kiessling, L.L.; Pontrello, J.K.; Schuster, M.C. Carbohydrate-Based Drug Discovery;
Wong, C.-H., Ed.; Wiley-VCH: Weinheim, Germany, 2003; Volume 2, pp. 575–608.
10. Kiessling, L.; Gestwicki, J.E.; Strong, L.E. Synthetic multivalent ligands as probes of signal
transduction. Angew. Chem. Int. Ed. 2006, 45, 2348–2368.
11. Lee, Y.; Lee, C.R.T. Synthetic multivalent ligands as probes of signal transduction. Acc. Chem.
Res. 1995, 28, 321–327.
12. Lis, H.; Sharon, N. Lectins: Carbohydrate-specific proteins that mediate cellular recognition.
Chem. Rev. 1998, 98, 637–674.
13. Lundquist, J.J.; Toone, E.J. The cluster glycoside effect. Chem. Rev. 2002, 102, 555–578.
14. De La Fuente, J.M.; Barrientos, A.G.; Rojas, T.C.; Rojo, J.; Cañada, J.; Fernández, A.; Penadés, S.
Gold glyconanoparticles as water-soluble polyvalent models to study carbohydrate interactions.
Angew. Chem. Int. Ed. 2001, 40, 2257–2261.
15. Huisgen, R. 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.; Wiley-VCH: Weinheim,
Germany, 1984; Volume 1, pp. 1–176.
16. Kolb, H.C.; Finn, M.G.; Sharpless, K.B. Click chemistry: Diverse chemical function from a few
good reactions. Angew. Chem. Int. Ed. 2001, 40, 2004–2021.

Molecules 2014, 19
1147
17. Kolb, H.C.; Sharpless, K.B. The growing impact of click chemistry on drug discovery.
Drug Discov. Today 2003, 8, 1128–1137.
18. Li, N.; Binder, W.H. Click-chemistry for nanoparticle-modification. Mater. Chem. 2011, 21,
16717–16734.
19. Bastide, J.; Henri-Rousseau, O. The Chemistry of the Carbon-Carbon Triple Bond; Patai, S., Ed.;
Interscience Publishers: London, UK, 1978; pp. 447–552.
20. Tornøe, W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by
regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.
J. Org. Chem. 2002, 67, 3057–3064.
21. Davis, M. Chemotherapy of schistosomiasis. V. Cholesteryl and choloyl derivatives of
4-amino-2-methoxyphenyl ethers. J. Chem. Soc. 1962, 1962, 178–181.
22. Zemplén, G. Decomposition of reducing disaccharides, VII Determination of the constitution of
maltose. Berich. Deutschen Chem. Gesellsch. 1927, 7, 1555–1564.
23. Khanjin, N.A.; Montero, J.-L. Synthesis of mannose-6-phosphate analogs: Large-scale preparation
of isosteric mannose-6-phosphonate via cyclic sulfate precursor. Tetrahedron Lett. 2002, 43,
4017–4020.
24. Kim, B.M.; Sharpless, K.B. Cyclic sulfates containing acid-sensitive groups and chemoselective
hydrolysis of sulfate esters. Tetrahedron Lett. 1989, 30, 655–658.
25. Gibson, T. Phase-transfer synthesis of mono-alkyl ethers of oligoethylene glycols. J. Org. Chem.
1980, 45, 1095–1098.
26. Cunneen, J.I. The addition of thio-compounds to olefins. II. Reactions of thiolacetic and
mono-chlorothiolacetic, di-chlorothiolacetic, and tri-chlorothiolacetic acids. J. Chem. Soc. 1947,
1947, 134–141.
27. Ciavatta, L.; Ferri, D.; Palombari, R. On the equilibrium Cu2++Cu(s) reversible 2Cu+. J. Inorg.
Nucl. Chem. 1980, 42, 593–598.
28. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A stepwise Huisgen cycloaddition
process: Copper(I)-Catalyzed regioselective ligation of azides and terminal alkynes. Angew.
Chem. Int. Ed. 2002, 41, 2596–2599.
29. Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V.V.; Noodleman, L.; Sharpless, K.B.; Fokin, V.V.
Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and
intermediates. J. Am. Chem. Soc. 2005, 127, 210–216.
30. Bock, V.D.; Hiemstra, H.; van Maarseveen, J.H. Cu-I-catalyzed alkyne-azide “click” cycloadditions
from a mechanistic and synthetic perspective. Eur. J. Org. Chem. 2006, 2006, 51–68.
31. Rodionov, V.O.; Fokin, V.V.; Finn, M.G. Mechanism of the ligand-free CuI-catalyzed azide-alkyne
cycloaddition reaction. Angew. Chem. Int. Ed. 2005, 44, 2210–2215.
32. Meldal, M.; Tornøe, C.W. Cu-catalyzed azide-alkyne cycloaddition. Chem. Rev. 2008, 108,
2952–3015.
33. Bertrand, P.; Gesson, J.-P. Click chemistry with O-dimethylpropargylcarbamate for preparation of
pH-sensitive functional groups. A case study. J. Org. Chem. 2007, 72, 3596–3599.
34. Orgueira, H.A.; Fokas, D.; Isome, Y.; Chan, P.C.; Baldino, C.M. Regioselective synthesis of
[1,2,3]-triazoles catalyzed by Cu(I) generated in situ from Cu(0) nanosize activated powder and
amine hydrochloride salts. Tetrahedron Lett. 2005, 46, 2911–2914.

Molecules 2014, 19
1148
35. Smith, G.F.; Sullivan, V.R. Frank, hexanitrato ammonium cerate as a proposed reference standard
in oxidimetry G. Ind. Eng. Chem. Anal. Ed. 1936, 8, 449–451.
36. Jarowicki, K.; Kocienski, P. Protecting groups. J. Chem. Soc. Perkin Trans. 2001, 18, 2109–2135.
37. Wittig, G.; Geissler, G. Zur reaktionsweise des pentaphenyl-phosphors und einiger derivate.
Liebigs Ann. Chem. 1953, 580, 44–57.
38. Wittig, G.; Schöllkopf, U. Uber triphenyl-phosphinmethylene als olefinbildende reagenzien.
Chem. Ber. 1954, 87, 1318–1330.
39. Maryanoff, B.E.; Reitz, A.B. The Wittig olefination reaction and modifications involving
phosphoryl-stabilized carbanions-stereochemistry, mechanism, and selected synthetic aspects.
Chem. Rev. 1989, 89, 863–927.
40. Horner, L.; Hoffmann, H.; Wippel, H.G. Phosphororganische Verbindungen, XII. Phosphinoxyde
als Olefinierungsreagenzien. Chem. Ber. 1958, 91, 61–63.
41. Horner, L.; Hoffmann, H.; Wippel, H.G.; Klahre, G. Phosphororganische verbindungen, XX.
phosphinoxyde als olefinierungsreagenzien. Chem. Ber. 1959, 92, 2499–2505.
42. Wadsworth, W.S.; Emmons, W.D. Utility of phosphonate carbanions in olefin synthesis. J. Am.
Chem. Soc. 1961, 83, 1733–1738.
43. Wadsworth, W.S. Synthetic applications ofphosphoryl-stabilized anions. Org. React. 1977, 25,
73–253.
44. Peterson, D.J. A carbonyl olefination reaction using silyl-subsituted organometallic compounds.
J. Org. Chem. 1968, 33, 780–784.
45. Van Staden, L.F.; Gravestock, D.; Ager, D.J. New developments in the Peterson olefination reaction.
Chem. Soc. Rev. 2002, 31, 195–200.
46. Ager, D.J. The Peterson olefination reaction. Org. React. 1990, 38, 1–223.
47. Johnson, C.R.; Shanklin, J.R.; Kirchhoff, R.A. Olefin synthesis by reductive elimination of
β-hydroxysulfoximines-methylenation of carbonyl-compounds. J. Am. Chem. Soc. 1973, 95,
6462–6463.
48. Julia, M.; Paris, J.-M. Syntheses using sulfones V(+). Method for general synthesis of double
bonds. Tetrahedron Lett. 1973, 14, 4833–4836.
49. Baudin, J.B.; Hareau, G.; Julia, S.A.; Ruel, O. A direct synthesis of olefins by reaction of
carbonyl-compounds with lithio derivatives of 2-[alkyl-sulfonyl or (2'-alkenyl)-sulfonyl or
benzyl-sulfonyl]-benzothiazoles. Tetrahedron Lett. 1991, 32, 1175–1178.
50. Baudin, J.B.; Hareau, G.; Julia, S.A.; Lorne, R.; Ruel, O. Stereochemistry of the olefin formation
from anti and syn heterocyclic beta-hydroxy-sulfones. Bull. Soc. Chim. Fr. 1993, 130, 336–357.
51. Blakemore, P.R. The modified Julia olefination: Alkene synthesis via the condensation of
metallated heteroarylalkylsulfones with carbonyl compounds. J. Chem. Soc. Perkin Trans. 2002,
2002, 2563–2585.
52. Umbreit, M.A.; Sharpless, K.B. Allylic oxidation of olefins by catalytic and stoichiometric
selenium dioxide with tert-butyl hydroperoxide. J. Am. Chem. Soc. 1977, 99, 5526–5528.
53. Stevens, M.P. Polymer Chemistry, 3rd ed.; Oxford University Press: Oxford, NY, USA, 1999;
pp. 265–267.
54. Mayo, F.R.; Walling, C. The peroxide effect in the addition of reagents to unsaturated compounds
and in rearrangement reactions. Chem. Rev. 1940, 27, 351–412.

Molecules 2014, 19
1149
55. Zard, S.Z. Radical Reactions in Organic Synthesis, 1st ed.; Oxford University Press: Oxford, UK,
2003.
56. Posner, T. Information on unsaturated compounds II. The addition of mercaptan to unsaturated
hydrocarbon. Berich. Deutschen Chem. Gesellsch. 1905, 38, 646–657.
57. Katz, E.; Willner, I. Integrated nanoparticle-biomolecule hybrid systems: Synthesis, properties,
and applications. Angew. Chem. Int. Ed. 2004, 43, 6042–6108.
58. Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D.J.; Whyman, R. Synthesis of thiol-derivatized gold
nanoparticles in a 2-phase liquid-liquid system. J. Chem. Soc. Chem. Commun. 1994, 7, 801–802.
59. Turkevich, J.; Stevenson, P.C.; Hillier, J.A. A study of the nucleation and growth processes in the
synthesis of colloidal gold. Disc. Far. Soc. 1951, 11, 55–75.
60. Frens, G. Controlled nucleation for regulation of particle-size in monodisperse gold suspensions.
Nat. Phys. Sci. 1973, 241, 20–22.
61. Slot, J.W.; Gueuze, H.J. Sizing of protein a-colloidal gold probes for immunoelectron microscopy.
J. Cell Biol. 1981, 90, 533–536.
62. Chow, M.K.; Zukoski, C.F. Gold sol formation mechanisms-role of colloidal stability. J. Colloid
Interf. Sci. 1994, 165, 97–109.
63. Kimling, J.; Maier, M.; Okenve, B.; Kotaidis, V.; Ballot, H.; Plech, A. Turkevich method for gold
nanoparticle synthesis revisited. J. Phys. Chem. B 2006, 110, 15700–15707.
64. Ribatti, D. Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis.
Int. Rev. Cell Mol. Biol. 2008, 270, 181–224.
65. Richardson, M.; Singh, G. Observations on the use of the avian chorioallantoic membrane (CAM)
model in investigations into angiogenesis. Curr. Drug Targets Cardiovasc. Haematol. Disord.
2003, 3, 155–185.
66. Lokman, N.A.; Elder, A.S.F.; Ricciardelli, C.; Oehler, M.K. Chick chorioallantoic membrane
(CAM) Assay as an in vivo model to study the effect of newly identified molecules on ovarian
cancer invasion and metastasis. Int. J. Mol. Sci. 2012, 13, 9959–9970.
67. Perrin, D.D.; Armarego, W.L.F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon Press:
Oxford, UK, 1998.
Sample Availability: Samples of the compounds are available from the authors.
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