Masked thiol sugars: Chemical behavior and synthetic applications of S-glycopyranosyl-N-monoalkyl dithiocarbamates

Article abstract of DOI:10.1002/asia.201301270

The chemical behavior of S-glycopyranosyl-N-monoalkyl dithiocarbamates (DTCs) as masked 1-glycosyl thiols, easily prepared by the nucleophilic displacement of 1-halo sugars with dithiocarbamate salts of primary amines, has been studied and synthetically exploited. This behavior relies on the abstraction of the proton of the carbamate functionality that allows controlled access to thiolate sugar intermediates. The basic character of the DTC salts used as reagents leads to thiolates that evolve in situ to symmetrical diglycosyldisulfides (DGDSs) when long reaction times are allowed. Alternatively, controlled unmasking of the thiolate function can be efficiently attained by treatment with an external base of isolated anomeric glycosyl DTCs, the formation of which is prevalent when using short reaction times. In this manner, a second methodology for the preparation of symmetrical DGDSs and a chemical protocol for the S-glycosylation of any electrophilic substrate are established. The applications of this last strategy for the preparation of thioglycosyl vinyl sulfones, thiodisaccharides, and S-linked homo- and heterodivalent neoglycoconjugates are described as a proof-of-concept of the great potential of the sugar DTCs in any chemical scenario in which the covalent attachment of a thiol sugar is required. The evaluation of the biological functionality of some divalent sulfurated sugar systems is also described. Copyright

Full text of DOI:10.1002/asia.201301270

FULL PAPER
DOI: 10.1002/asia.201301270
Masked Thiol Sugars: Chemical Behavior and Synthetic Applications of S-
Glycopyranosyl-N-monoalkyl Dithiocarbamates
Alicia Megia-Fernandez,[a] Diego de la Torre-Gonzalez,[a] Jose Parada-Aliste,[b]
Francisco Javier Lopez-Jaramillo,[a] Fernando Hernandez-Mateo,[a] and
Francisco Santoyo-Gonzalez*[a]
Abstract: The chemical behavior of S-
glycopyranosyl-N-monoalkyl dithiocar-
bamates (DTCs) as masked 1-glycosyl
thiols, easily prepared by the nucleo-
philic displacement of 1-halo sugars
with dithiocarbamate salts of primary
amines, has been studied and syntheti-
cally exploited. This behavior relies on
the abstraction of the proton of the car-
bamate functionality that allows con-
trolled access to thiolate sugar inter-
mediates. The basic character of the
DTC salts used as reagents leads to thi-
olates that evolve in situ to symmetri-
cal diglycosyldisulfides (DGDSs) when
long reaction times are allowed. Alter-
natively, controlled unmasking of the
thiolate function can be efficiently at-
tained by treatment with an external
base of isolated anomeric glycosyl
DTCs, the formation of which is preva-
lent when using short reaction times. In
this manner, a second methodology for
the preparation of symmetrical DGDSs
and a chemical protocol for the S-gly-
cosylation of any electrophilic substrate
are established. The applications of this
last strategy for the preparation of thi-
oglycosyl vinyl sulfones, thiodisacchar-
ides, and S-linked homo- and heterodi-
valent neoglycoconjugates are de-
scribed as a proof-of-concept of the
great potential of the sugar DTCs in
any chemical scenario in which the co-
valent attachment of a thiol sugar is re-
quired. The evaluation of the biological
functionality of some divalent sulfurat-
ed sugar systems is also described.
Keywords: carbohydrates · dithio-
carbamates
·
glycoconjugates
·
glycopeptides · glycoproteins
Introduction
the biochemical stability and enzymatic resistance toward
glycosidases and to better target specificities of the resulting
glycomimetics. For those reasons, these sulfur-containing
carbohydrates have become popular tools in glycobiology
and medicinal research.[6] Likewise, carbohydrate derivatives
that contain sulfurated functionalities at the anomeric posi-
tion benefit from the rich chemistry of sulfur that has made
them ideal for the development of new glycosylation and
carbohydrate linking strategies and for their implementation
in some rediscovered synthetic methodologies, such as thiol–
ene[7] and thiol–yne[8] coupling.
In this context, S-glycosyl-N-disubstituted dithiocarba-
mates (DTCs; in particular, N,N’-dialkyl derivatives) are of
interest because of the ability of the dithiocarbamoyl func-
tional group to form stable complexes with divalent metals
and free-radical species and also for the known fungicidal,
insecticidal, and anticarcinogenic properties of related dia-
lkyldithiocarbamates.[1,9] The most widely used methodology
for accessing these carbohydrate DTCs relies on the nucleo-
philic displacement by dialkyl dithiocarbamate salts of gly-
cosyl derivatives that bear halides and, less frequently, tosy-
lates and pyridinium derivatives at the anomeric position.[1]
The reaction of glycosyl bromides with carbon disulfide and
amines under solvent-free conditions[10] and of epoxyglycal
derivatives[11] are also included in the list of employed reac-
tions. Antibacterial, antifungal, and antimicrobial activities
as well as in vivo inhibition of nitrosamine-induced DNA
Carbohydrate derivatives that bear S-linked functionalities
at the anomeric position have attracted attention because of
the intrinsic fungicidal, insecticidal, and anticarcinogenic
properties exhibited by some of these compounds.[1] Addi-
tionally, these compounds are of interest because of their
utility for the preparation of thiooligosaccharides, S-linked
bioconjugates (glycopeptides and glycoproteins),[2] and some
innovative thioglycosylated systems and architectures (gly-
copolymers,[3] glycodendrimers,[4] and glyconanoparticles[5]).
The primary goal that underpins the replacement of a native
O-glycosidic linkage by an S-glycosidic bond is to enhance
[a] Dr. A. Megia-Fernandez, D. de la Torre-Gonzalez,
Prof. F. J. Lopez-Jaramillo, Prof. F. Hernandez-Mateo,
Prof. F. Santoyo-Gonzalez
Departmento de Q. Organica, Facultad de Ciencias
Insitituto de Biotecnologꢀa, Universidad de Granada
18071 Granada (Spain)
Fax : (+34)958248437
E-mail: fsantoyo@ugr.es
[b] Prof. J. Parada-Aliste
Departmento de Q. Inorganica y Analitica
Facultad de Ciencias Quimicas y Farmaceuticas
Universidad de Chile (Chile)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201301270.
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Francisco Santoyo-Gonzalez et al.
damage have been reported as appealing properties for
some of these sugar-linked DTCs.[9,12]
of S-glycopyranosyl-N-monoalkyl DTCs, compounds not
previously exploited despite their easy preparation by the
classic nucleophilic substitution methodology of 1-halo
sugars with N-alkyl dithiocarbamate salts. Our findings
prove that S-glycopyranosyl-N-monoalkyl DTCs behave as
masked thiols that can be liberated in a controlled manner
by abstraction of the proton of the carbamate functionality.
This characteristic makes these DTC sugar derivatives ideal
for the development of two novel and efficient chemical ap-
proaches for the synthesis of DGDSs and also enables them
to be thiol equivalents in any chemical scenario in which the
covalent attachment of a thiol sugar is required. This is dem-
onstrated by the preparation of structurally diverse thiogly-
cosyl vinyl sulfones, thiodisaccharides, and S-linked homo-
and heterodivalent neoglycoconjugates. The biological func-
tionality (i.e., recognition by plant lectins) of some of those
divalent sulfurated sugar systems and the structural factors
that govern their activity are also reported as a proof-of-
concept of the potential and versatility of S-glycopyranosyl-
N-alkyl DTCs in glycobiology.
On the other hand, the use of the disulfide motif to con-
nect two identical or different sugar moieties to lead to sym-
metrical or unsymmetrical diglycosyldisulfides (DGDSs) has
also been the object of numerous studies. Advantages of-
fered by the use of this function as an interglycosidic bridge
are a higher conformational flexibility, increased distance
between the two glycosidic components, and the possibility
to introduce structural variability on the glycose residues of
these divalent systems. DGDSs have been investigated in
light of their utility as useful tools in glycobiology and their
biological potential (e.g., good affinity towards lectins,[13]
promising properties against tumor cells,[14] inhibitory activi-
ties against Trypanosoma cruzi,[15] and the discovery of bio-
active compounds through the generation of dynamic librar-
ies[14b,16]).
Several interesting state-of-the-art methods exist to ach-
ieve the synthesis of symmetrical as well as unsymmetrical
DGDSs. Hence, symmetrical DGDSs are commonly pre-
pared by the clear self-oxidation of 1-thiosugars. These last
important nucleophilic compounds are frequently accessed
by the mild hydrolysis of some sulfur-containing functionali-
ties (e.g., thioesters, xanthates, thiouroium salts, or iso-
thiouronium salts) placed at the anomeric position by means
of nucleophilic substitutions on glycosyl halides or 1-O-
acetyl sugars,[4,14a] whereas other methods perform the direct
thiolation of reducing sugars using Lawessonꢂs reagent[17] or
the Birch reaction reduction of anomeric thiobenzyl deriva-
tives.[18] Formation of the disulfide linkage of DGDSs has
also been widely performed by nucleophilic attack of glyco-
syl thiols or the corresponding thiolates on a glycosyl sulfe-
nyl derivative that contains divalent electrophilic sulfur,
such as alkylthiosulfonate esters.[19] Alternatively, glycosyl
thiols also allow access to symmetrical DGDSs by means of
oxidation with diethyl azodicarboxylate (DEAD)[20] or
iodine.[21] Other methodologies make use of glycosyl halides
as starting material by their treatment with tetrathiomolyb-
date reagents.[22] The more challenging preparation of un-
symmetrical nonreducing disaccharides that contain intergly-
cosidic (1!1’) disulfide linkages by the efficient coupling of
two different oligosaccharide moieties versus the formation
of the corresponding homodimers has been currently per-
formed by means of the glycosulfenyl-transfer methodology
using either glycosyl methanethiosulfonates[19] and S-glyco-
syl-N-acyl sulfenamides.[23] In addition, glycosyl thiols are at
the base of a one-pot methodology that uses 1-chlorobenzo-
triazole as an in situ trapping/oxidizing agent[24] and also in
a more elaborate three-step procedure in which sulfenic
acids are generated from suitable sulfonyl intermediates.[25]
Finally, mixtures of structurally different glycosyl thiols can
be coupled through a disulfide bond by an adaptation of the
DEAD-based oxidation methodology discussed above[20]
and also by using 2,3-dichloro-5,6-dicyanobenzoquinone
(DDQ) as an oxidant.[21]
Results and Discussion
Synthesis and Chemical Behavior
In an ongoing project of our laboratory aimed at the devel-
opment of new methodologies for the preparation of silica-
based hybrid glycomaterials, the synthesis of S-glycopyrano-
syl alkyloxysilane DTC derivatives to be used as silanization
reagents was needed. To this aim, the synthesis of such com-
pounds was envisaged by the reaction of 1-halo sugars with
the sodium salt of N-alkyl dithiocarbamates (DTCꢀNa+),
a classic synthetic methodology for the preparation of com-
parable S-glycosyl-N-disubstituted dithiocarbamates. Sur-
prisingly, when the reaction of per-O-acetylated-a-bromo
glucose 1A with the N-alkyloxysilane DTCꢀNa+ salt 2a was
performed under mild reaction conditions (acetone, RT,
6 h), the expected S-glucopyranosyl-N-[3-(triethoxysilyl)-
propyl] DTC 3Aa was not obtained. Instead, structural char-
acterization of the isolated compound reveals the formation
of the symmetrical 1,1’-diglucosyl disulfide 4A in a moderate
yield (Table 1, entry 1).
To explain the observed result, and considering the impor-
tance of DGDSs, as commented above, we decided to study
in detail the scope of the reaction of 1-halo sugars with
DTC salts. For this purpose the N-sec-butyl DTCꢀNa+ salt
2b was first chosen as a non-silyl-containing compound and
treated with 1A under equivalent conditions. In this case,
a slightly improved isolation of DGDS 4A was observed but
concomitant formation of a minor amount of the corre-
sponding S-glucopyranosyl-N-(sec-butyl) DTC 3Ab was also
detected (Table 1, entry 2). To direct the reaction towards
yielding the DTC derivative instead of the DGDS one, the
reaction was carried out using the minimal time required for
the consumption of the starting material as determined by
thin layer chromatography (2 h) followed by careful neutral-
ization of the reaction media. Under these conditions, it was
As part of ongoing projects in our laboratories, herein we
report on the chemical behavior and synthetic applications
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Francisco Santoyo-Gonzalez et al.
Table 1. Optimization of the conditions for the reaction of Glc-Br (1A) with DTCꢀM+ (2a–d).
Entry
DTCꢀ
R1, R2
H, (CH2)3Si(EtO)3
H, CHMe(Et)
H, CHMe(Et)
H, CHMe(Et)
H, CH2Ph
M+
T
Equiv
t [h]
3A
G
4A [%]
5A [%]
1
2
3
4
5
6
7
8
9
2a
2b
2b
2b
2c
2c
2c
2c
2d
G
Na+
Na+
RT
RT
RT
reflux
RT
RT
RT
RT
reflux
2.0
2.0
2.0
1.5
2.0
1.0
2.0
2.0
1.5
6
5
67
74
–
–
3Ab (9)
3Ab (71)
3Ab (47)
3Ac (80)
3Ac (66)
3Ac (8)
–
Na+
2[a]
1
4A/5A (1:0.4) (27)
4A/5A (1:1) (49)
Na+
Na+
2[a]
48
24[a]
8
12
15
74
74
–
–
–
–
–
–
H, CH2Ph
H, CH2Ph
H, CH2Ph
Et, Et
Na+
Cs+
Et3NH+
Na+
1
3Ad (99)
[a] Neutralization with 5% HCl aqueous solution.
observed that the formation of the glycosyl DTC 3Ab is fa-
vored over that of DGDS 4A, a compound that is obtained
together with tiny amounts of the corresponding thio sugar
5A as an inseparable mixture (Table 1, entry 3). In a further
step, the influence of the temperature on the course of the
reaction was analyzed by performing the reaction under
reflux conditions. This increase in temperature accelerated
the reaction as expected (1 h) but also facilitated formation
of the sulfur-linked bis-sugars 4A and 5 over the glycosyl
DTC counterpart 3Ab (Table 1, entry 4).
To gain further insight into the reaction, the influence of
the stoichiometry of the reagents was next studied by per-
forming the reactions using two different proportions of the
DTCꢀNa+ salts of N-benzylamine 2c towards 1A (2:1 and
1:1 molar ratio, respectively). The results obtained demon-
strate that the higher molar ratio of the DTCꢀNa+ salt in
combination with shorter reaction times followed by neu-
tralization of the reaction media are optimal conditions to
favor the formation of the glycosyl DTC 3Ac (Table 1,
entry 5). By contrast, when equimolecular amounts of the
reagents were used, the reaction time had to be extended
for the complete transformation of the glucosyl bromide
1A, and this caused concurrent formation of the DGDS 4A
in detriment to DTC 3Ac, albeit with similar overall yield
in both reactions (Table 1, entry 6). Benzylamine was also
used to test the effect of the nature of the counterion of the
DTCꢀM+ salt (M=Na, Cs, Et3NH). For that purpose, the
reactions of the Cs+ and Et3NH+ salts were performed
using long reaction times (Table 1, entries 7 and 8). Interest-
ingly, these conditions favored the formation of the 1,1’-di-
glucosyl disulfide 4A as was the case when using the
DTCꢀNa+ salt (Table 1, entry 2).
and was treated with 1A under the reaction conditions that
have been demonstrated to maximize the formation of the
sulfur-linked bis-sugars 4A and 5A over the glycosyl DTC
counterpart, that is, acetone heated at reflux (Table 1,
entry 4). In this case, and as expected, the reaction leads to
the exclusive and almost quantitative formation of the S-glu-
cosyl-N-diethyl DTC 3Ad (Table 1, entry 9). This result is in
accordance with previous reported preparations of carbohy-
drate derivatives that bear S-linked N-dialkyldithiocarba-
mate functionalities at the anomeric position and indicated
that the observed competitive formation of sulfur-linked bis-
sugars is characteristic of DTC salts derived from primary
amines.[1]
On the basis of this last observation, and from the assem-
bly of the outcomes obtained from the assays with DTC
salts derived from primary amines, a general mechanism
was elaborated to explain the special behavior of these com-
pounds in their reaction with 1-halo sugars (I) as depicted in
Scheme 1. Formation of S-glycosyl-N-alkyl DTCs (II) by nu-
Scheme 1. Proposed mechanism for the chemical behavior of S-glycopyr-
anosyl-N-alkyl DTCs as masked thiol sugars.
Once the scope of the reaction of anomeric glycosyl hal-
ides with DTC salts derived from primary amines such as
sec-butylamine and benzylamine was studied, it was consid-
ered important to corroborate whether the DTC salts ob-
tained from secondary amines previously described in the
literature exhibited a similar behavior. The easily accessible
sodium salt of diethylamine 2d was selected for this goal
cleophilic displacement of the halogen atom by the nucleo-
philic DTC salts must be the first event. However, the basic
character of these compounds determines abstraction of the
proton of the carbamate functionality of the sugar DTCs to
lead to the in situ formation of the corresponding 1-thiolate
sugars (III). This intermediate evolves by undergoing self-
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Francisco Santoyo-Gonzalez et al.
oxidation to give access to 1,1’-diglycosyl disulfide (IV) or
alternatively acts as a nucleophile with already unreacted
glycosyl halides I to afford bis-thiosugars (V). The proposed
mechanism means that sugar DTCs of type II are in fact
acting as masked thiol sugars, which are liberated under ad-
equate conditions. Actually, formation of the hypothesized
1-thiolate sugars III is influenced by the time, stoichiometry,
temperature, and pH of the reaction media, which are criti-
cal factors that determine the course of the reaction. Ac-
cordingly, the formation of DTC sugars is favored by short
reaction times, stoichiometries higher of the equimolecular
ratio, room temperature, and immediate neutralization of
the media after completion of the reaction (see Table 1, en-
tries 3 and 5). Under these reaction conditions the formation
of sugar DTCs (II) is accelerated, whereas proton abstrac-
tion and the concomitant formation of the 1-thiolate-sugars
III is minimized. Conversely, longer reaction times (see
Table 1, entries 2 and 6) and reflux temperatures (Table 1,
entry 4) favor the generation of the thiolate intermediate
and the subsequent formation of the sulfur-linked sugars
(IV and V).
With a view to the acquired understanding of the factors
and the mechanistic insights that govern the chemical be-
havior of S-glycosyl-N-monoalkyl DTCs, it appears clear
that two novel and complementary methodologies can be as-
serted for the synthesis of bis-glycosyl disulfides by exploit-
ing the potential of such compounds to act as masked thiol
sugars and a kinetic control as depicted in Scheme 3. Direct
Scheme 3. Alternative methodologies (direct: path I; stepwise: paths II–
III) for the synthesis of DGDSs from glycosyl halides.
To gather further experimental evidence that lends addi-
tional credence to our hypothesis, a set of new experiments
was designed with structurally diverse DTC sugars that were
subjected to a purpose fully basic treatment with a weak or-
ganic base such as triethylamine (TEA; Scheme 2). In the
access to DGDSs from glycosyl halides can be attained by
the reaction of glycosyl halides with DTCꢀM+ salts of pri-
mary amines by using prolonged reaction times (Scheme 3,
path I). Instead, a two-step protocol based on the use of
short reaction times that favors the formation and isolation
of the corresponding glycosyl DTCs followed by subsequent
treatment with TEA should alternatively allow the prepara-
tion of DGDSs (Scheme 3, path II and III).
To establish the generality of these methodologies, the de-
lineated transformations were performed in a variety of gly-
cosyl halides that differed in the nature of their sugar
ꢀ
ꢀ
moiety (e.g., per-O-acetylated derivatives of glucose Glc ,
ꢀ
ꢀ
ꢀ
ꢀ
N-acetylglucosamine GlcNAc , galactose Gal , and lac-
Scheme 2. Basic treatment of S-glycopyranosyl-N-monoalkyl DTC (3Ab),
S-glycopyranosyl-N,N-dialkyl DTC (3Ad), and nonanomeric S-sugar-N-
alkyl DTC (6).
ꢀ
ꢀ
tose Lac ) to demonstrate substrate versatility. The results
are summarized in Tables 2 and 3 and clearly show the val-
idity of both approaches. Remarkably, all the transforma-
tions allow the isolation of the reaction products DTCs or
DGDSs in yields that range from good to excellent. Like-
wise, stereoselective formation of anomerically pure b-
anomers is achieved in all cases. Furthermore, controlled
access to either DGDSs or DTCs is easily attainable but
controlled unmasking of thiol sugars is also feasible.
first instance, a solution in acetone of the previously isolated
S-glucosyl-N-monoalkyl and S-glucosyl-N-dialkyl DTCs,
3Ab and 3Ad, respectively, were separately treated with
TEA. As expected, full consumption of the DTC sugar and
transformation into the bis-glucosyl disulfide 4A took place
only for the N-monoalkyl DTC sugar 3Ab, whereas in the
case of the N-dialkyl DTC sugar 3Ad the starting material
was recovered intact. On the other hand, the xylofuranose
DTC derivative 6 previously reported by us[26] that bears the
S-linked N-monoalkyl dithiocarbamate functionality at the
non-anomeric 5-position of the sugar was also exposed to
TEA. Similarly to the case of compound 3Ad, the starting
material was recovered intact. The results obtained in these
experiments convincingly prove that the claimed behavior
of sugar DTC derivatives as masked thiols is characteristic
of S-glycosyl-N-monoalkyl DTCs at the anomeric position
and is also in accordance with the already known stability of
N-dialkyl DTCs in basic media.
Synthetic Applications
The last assertion opens the door to explore the synthetic
applicability of S-glycosyl-N-monoalkyl DTCs as thiol
equivalents in any chemical scenario in which the covalent
attachment of a thiol sugars is required. As a first proof-of-
concept to validate this hypothesis, an equimolecular mix-
ture of two structurally different sugar DTCs, namely, 3Ac
and 3Cc, was treated with TEA to yield an inseparable mix-
ture of the corresponding homoglycosyl (4A and 4C) and
heteroglycosyl disulfides (Glc-S-S-Gal, 8) in high yield, as
demonstrated by the NMR spectroscopic data.
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Francisco Santoyo-Gonzalez et al.
Table 2. Synthesis of sugar-DTCs and DGDSs from sugar halides.[a]
On the basis of this predictable result, the con-
trolled release of a thiolate sugar from a S-glycosyl-
N-monoalkyl DTC was next attempted for its trap-
ping by a suitable substrate that contains an electro-
philic functionality. In line with our continuing ac-
tivities on the exploration of the synthetic utilities
of vinyl sulfone[27] and cyclic sulfates,[28] we decided
to exploit the high electrophilic reactivity of those
functions for our purposes.
Entry
Sugar-X
DTCꢀM+
t [h]
Sugar-DTC [%]
DGDS [%]
1
2
3
4
5
6
7
8
9
1A(Br)
1A(Br)
1B(Cl)
1C(Br)
1C(Br)
1D(Br)
1A(Br)
1B(Cl)
1C(Br)
1D(Br)
2b(Na)
2c(Na)
2c(Na)
2b(Na)
2c(Na)
2c(Na)
2c(Cs)
2c(Cs)
2c(Cs)
2c(Cs)
2
2
2
2
2
3Ab (71)
3Ac (80)
3Bc (73)
3Cb (81)
3Cc (98)
3Dc (72)
3Ac (8)
3Bc (5)
–
4A (16)
4A (12)
4B (3)
–
Vinyl sulfones[29] as sulfonyl-containing com-
pounds readily undergo conjugate additions as ex-
cellent Michael acceptors because of the electron-
poor nature of their double bond on account of the
electron-withdrawing capability of the sulfone that
makes them good electrophiles. Carbon nucleo-
philes as well as heteroatomic nucleophiles that in-
volve nitrogen, sulfur, and oxygen can participate
efficiently in those conjugate additions; they share
a similar reaction pattern by addition at the b-posi-
tion of the sulfone and, on this basis, these reactions
are a well-established method of creating b-hetero-
–
2
4D (10)
4A (74)
4B (70)
4C (91)
4D (71)
16
16
16
16
10
3Dc (9)
[a] Sugar A=Glc (R1 =R2 =OAc; R3 =H); B=GlcNAc (R1 =NHAc, R2 =OAc; R3 =
H); C=Gal (R1 =R3 =OAc; R2 =H); D=Lac (R1 =OAc; R2 =bGlc
X=Br, Cl; for b, R4 =CH(Me)Et; for c, R4 =CH2Ph.
(OAc); R3 =H);
ACHTUNGTRENNUNG
substituted sulfones. In the particular cases of thiols, weak
bases are often used in their thio-Michael-type addition with
vinyl sulfones to deprotonate them.[30] Considering these
facts, divinyl sulfone (DVS) was chosen as a model reagent
and treated with the full series of sugar DTCs derived from
benzyl amine 3(A–D)b (Scheme 4). The successful conver-
sion of those compounds into the corresponding homodiva-
lent neoglycoconjugates 9A–D was easily accomplished in
high yields (71-90%) by subjecting a 2.2:1 DTC/DVS molar
mixture to the optimized reaction conditions.
To further extend the synthetic scope of this transforma-
tion, the bifunctional nature of DVS was exploited in a dif-
ferent approach. The controlled reaction of DTCs 3ACHTUNGTRENNUNG(A–D)b
with DVS (3 equivs) led to the thioglycosyl vinyl sulfones
11A–D in short reaction times with high efficiency
(Scheme 4). In this manner, a new methodology for access-
ing glycosyl vinyl sulfones by using DTC sugars was estab-
lished. These compounds have been previously synthesized
by a protocol based on the use of glycosyl thiols as starting
Table 3. Synthesis of per-O-acetylated and unprotected DGDSs from
sugar-DTCs.[a]
Entry
Sugar-DTC
DGDS [%]
DGDS(OH) [%]
1
2
3
4
5
6
3Ab
3Ac
3Bc
3Cb
3Cc
3Dc
4A (98)
4A (90)
4B (71)
4C (92)
4C (88)
4D (81)
7A(OH) (91)
7B(OH) (99)
7C(OH) (95)
7D(OH) (90)
[a] Sugar A=Glc (R1 =R2 =OAc; R3 =H); B=GlcNAc (R1 =NHAc,
R2 =OAc; R3 =H); C=Gal (R1 =R3 =OAc; R2 =H); D=Lac (R1 =
ACHTUNGTRENNUNG
OAc; R2 =bGlc(OAc); R3 =H); sugar(OH) A=Glc(OH) (R1 =R2 =OH;
R3 =H); B=GlcNAc(OH) (R1 =NHAc, R2 =OH; R3 =H), C=Gal(OH)
R1 =R3 =OH; R2 =H; D=Lac(OH) (R1 =OH; R2 =bGlc(OH); R3 =H);
for b, R4 =CH(Me)Et; for c, R4 =CH2Ph.
Scheme 4. Synthesis of thioglycosyl vinylsulfones (11A–D) and S-linked homo- (9A–D) and heterodivalent neoglycoconjugates (12) from S-glycosyl-N-
alkyl DTCs. A=Glc (R1 =R2 =OAc; R3 =H); B=GlcNAc (R1 =NHAc, R2 =OAc; R3 =H); C=Gal (R1 =R3 =OAc; R2 =H); D=Lac (R1 =OAc; R2 =
bGlcACHTUNGTRENNUNG
(OAc); R3 =H).
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Francisco Santoyo-Gonzalez et al.
materials, a contribution that has demonstrated the great
potential of such saccharide vinyl sulfones.[27b,31]
efficient methodologies in which analogous intermediates
that play the role of masked thiol sugars are directly used
without any purification, such as the case of glycosyl thio-
uronium derivatives easily obtained from glycosyl hal-
ides.[4,34] Isolation of stable S-glycosyl-N-monoalkyl DTCs
made their storage as well as the use of the required exact
amounts of these reagents feasible.
.
The introduction of this functionalization at the anomeric
carbon on different carbohydrates is highly feasible and
allows the development of procedures for the chemical gly-
cosylation of proteins as well as the exploration of protein–
carbohydrate interactions in the context of glycoscien-
ce.[27b,31] Herein, the applicability of thioglycosyl vinyl sul-
fones has been further extended to the preparation of heter-
odivalent neoglycoconjugates. The reaction of 11D with the
GlcNAc-containing DTC 3Bb furnished the sugar divalent
compound 12 in a very efficient and facile manner by a mod-
ular strategy (Scheme 4). In fact, this approach globally im-
plies the sequential incorporation of two structurally differ-
ent DTC sugars into a complementary reactive scaffold
under mild basic conditions
On the other hand, cyclic sulfates are versatile electrophil-
ic synthons that have emerged as important intermediates in
organic synthesis.[32] They show a particular enhanced reac-
tivity toward nucleophilic reagents, and their applications
have gained importance since the development of efficient
methods for their preparation. For these reasons they were
chosen to further demonstrate the synthetic possibilities of
S-glycosyl-N-monoalkyl DTCs. In particular, the novel com-
pounds 3B,Cc were treated with the cyclic sulfate derived
from glucose 14[33] (Scheme 5). The selection of this com-
Biological Assays
As stated above, S-glycopyranosyl-N-alkyl DTCs, when be-
having as masked thiol sugars, can be used as precursor for
the synthesis of divalent neoglycoconjugates with different
spacers that may have biological applications. Thus, DGDSs
have shown promising properties against tumor cells,[14a] and
the disulfide linkage yields increased flexibility and an ex-
tended length relative to the O-glycosidic bond[19] without
invalidating them as the target of lectins.[13b,14b,35] However,
the nature of the linker might influence the biological func-
tion of the divalent neoglycoconjugates as was demonstrated
when the unexpected strong cooperative inhibitory effect of
diglucosyl and digalactosyl disulfides against concanavalin A
(ConA) was found to be a consequence of an interference
with the calcium binding (i.e., the interaction of ConA with
carbohydrates is dependent on the binding of calcium and
a transition metal) and not with the carbohydrate binding
site.[35a] This result also illus-
trates the importance of the se-
lection of suitable lectins to
rule out false positives since it
is well established that ConA
does not interact with galac-
tose.
With this body of knowledge
Scheme 5. Synthesis of thiodisaccharides from S-glycosyl-N-alkyl DTCs and cyclic sulfate sugars.
as background, we decided to
evaluate the biological func-
pound as a representative example of the cyclic sulfate
family was based on the well-established value of cyclic sul-
fates derived from saccharides as building blocks for the
preparation of a wide variety of sugar-related compounds
on account of the combination of their straightforward and
efficient formation and their high chemical stability and re-
activity.[28] By performing the indicated reactions, the corre-
sponding heterodivalent neoglycoconjugates 15–16 were ob-
tained in a regiospecific manner by the nucleophilic ring
attack of the unmasked thiolates generated from the basic
treatment of the sugar DTCs on the cyclic sulfate ring at the
less hindered carbon atom. This reactivity is in accordance
with the usual chemical reactivity reported for such cyclic
sulfates. On the basis of this result, it can be asserted that
a new synthetic methodology for the preparation of thiodi-
saccharides is established.
tionality (i.e., recognition by lectins) of some of the divalent
sulfurated glycosylated compounds obtained from S-glyco-
pyranosyl-N-alkyl DTCs, namely, the DGDSs 7B–D(OH)
and the homo- 10B,C(OH) and heterodivalent neoglycocon-
jugates 13(OH). To test their ability to interact simultane-
ously with two molecules of lectin, different concentrations
of the divalent neoglycoconjugates that bear GlcNAc, Gal,
or Lac were first incubated in enzyme-linked immunosorb-
ent assay (ELISA) wells coated with peanut agglutinin
(PNA) or wheat germ agglutinin (WGA), respectively, and
then with a PNA or WGA conjugated with horseradish per-
oxidase (HRP; Figure 1). Thus, only those wells in which
the neoglycoconjugate interacted with both the adsorbed
lectin and the lectin-HRP will yield peroxidase activity. As
shown in Figure 2, regardless of the spacer, all divalent
homo neoglycoconjugates 7B–D(OH) and 10B,C(OH) in-
From the results reported herein it can be stated that the
developed methodology expands the current repertory of
thioglyosylation reactions. It should be mentioned that the
required chromatographic purification of S-glycosyl-N-mon-
oalkyl DTCs could offer some advantages relative to other
teracted with the lectin in a concentration-dependent
manner. This result supports their biological functionality
and is consistent with the reported interaction of dithiodiga-
lactose with human galectins and with the plant lectin from
Viscum album.[13b]
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Francisco Santoyo-Gonzalez et al.
Figure 1. Analysis of the biological functionality of the compounds syn-
thesized from S-glycopyranosyl-N-alkyl DTCs. The interaction of the two
sugar moieties (rhombus and circle) with two different lectins is explored
simultaneously. A first lectin is adsorbed onto an ELISA well and incu-
bated with the divalent neoglycoconjugate. The unbound divalent neogly-
coconjugate is washed, and then a second lectin (lectin–HRP) is incubat-
ed. Peroxidase activity (i.e., color) is detected only when the divalent ne-
oglyconjugate is recognized by both immobilized lectin and lectin–HRP.
Additionally, the reaction of S-glycopyranosyl-N-mono-
alkyl DTCs is a straightforward strategy to synthesize heter-
odivalent neoglycoconjugates. The analysis of compound
13(OH) reveals that it is recognized by both WGA and
PNA lectin (Figure 3) in a pattern similar to that of the ho-
modivalent neoglycoconjugates. The profile of the curves is
dependent on the lectin adsorbed on the well and on the
lectin–HRP used to detect the interaction; it cannot be ra-
tionalized by the different peroxidase activity of PNA–HRP
and WGA–HRP. In fact, when the WGA is the immobilized
lectin and the peroxidase activity is a consequence of PNA–
HRP, a plateau is reached at 1 mm of 13(OH), whereas a ten-
fold concentration does not saturate the inverse arrange-
ment. Moreover, the fact that 1 mm of 13(OH) saturates the
carbohydrate binding sites of the adsorbed WGA seems to
be contradictory when it is compared with the results ob-
tained with the same enzyme and experiment for the homo-
divalent N-acetylglucosamine (Figure 2A). This result can
be explained by taking into account that some of the several
carbohydrate binding sites proposed for WGA[36] and dis-
tributed over each subunit are inaccessible when the lectin
is adsorbed on the surface of the well.
Figure 2. Analysis of the biological functionality of the homo diglycosyl
conjugates linked by a disulfide bond or by a sulfonyl bisACTHNUTRGNE(UNG ethyl) linker.
A) Interaction of GlcNAc conjugates (7B(OH), squares; 10B(OH), tri-
angles) with WGA. B) Interaction of Gal conjugates with PNA
(7C(OH), triangles; 10C(OH), squares; and 7D(OH), circles; interaction
of dithiodilactosyl disulfide with PNA).
Conclusion
In summary, the results reported herein clearly prove the
synthetic possibilities of S-glycopyranosyl-N-monoalkyl
DTCs on the basis of their demonstrated behavior as
masked thiols on account of the presence of the proton of
the carbamate functionality of these sugar DTCs that make
Figure 3. Analysis of the biological functionality of the hetero-divalent
neoglycoconjugate 13(OH) by immobilizing PNA and revealing with
WGA-HPR (squares) and by immobilizing WGA and revealing with
PNA-HPR (circles).
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Francisco Santoyo-Gonzalez et al.
under vacuum. The corresponding DTC sodium salts 2b[40] (3.14 g, 54%),
2c[41] (4.54 g, 78%), and 2d[42] (2.63 g, 69%) were isolated and showed
physical and spectroscopic data identical to those previously reported.
The cesium and triethylammonium benzyl DTC salts (2c(Cs) and 2c-
AHCTUNTGREG(NNNU Et3NH)) were obtained following an identical procedure by using
Cs2CO3 (10 mmol) or Et3N (20 mmol) instead of NaOH in 39 (2.45 g)
and 54% (2.92 g) yield, respectively.
them suitable for the efficient and controlled formation of
the corresponding 1-thiolate sugars. In situ formation of
these intermediates is feasible due to the basic character of
the DTCꢀM+ salts of primary amines salts that are used as
reagents and thus allows a novel synthetic strategy for direct
access to DGDSs from the starting glycosyl halides when
long reaction times are used. This behavior does not invalid-
ate the approach for the prevalent formation and isolation
of S-glycosyl-N-alkyl DTCs that takes place when short re-
action times are used. By this method, these compounds can
be used in turn for a controlled unmasking of the thiol
sugars by their treatment with an external base to allow for
an alternatively preparative methodology for the aforemen-
tioned symmetrical DGDSs or instead the chemical S-glyco-
sylation of any electrophilic substrate that acts as a thio ac-
ceptor. These applications of S-glycosyl-N-alkyl DTCs as
thiol equivalents expand in an easy manner the actual reper-
tory of methodologies for the preparation not only of
DGDSs but also for the synthesis of thiodisaccharides, thio-
glycosyl vinyl sulfones, and S-linked homo- and heterodiva-
lent neoglycoconjugates. The evaluation of the biological
functionality of some of the prepared divalent sulfurated
sugar systems also proves the utility of the reported strat-
egies for easy access to carbohydrate-containing bioactive
compounds.
General Procedure for the Reactions of Glycosyl Halides with DTCꢀM+
Salts
A mixture of the corresponding per-O-acetylated bromo (1A, 1C, 1D)
or chloro sugar (1B) (1.0 mmol) and the DTC salt ACHTNUTRGNEUNG(2a–d) (2.0 mmol) in
anhydrous acetone (15 mL) was magnetically stirred at RT until complete
disappearance of the starting material was observed by TLC (1–6 h).
Aqueous 5% HCl was then added until reaching neutral pH and the or-
ganic solvent was removed under reduced pressure. Water (20 mL) was
added, and the resulting mixture was extracted with dichloromethane
(2ꢃ30 mL). The combined organic extracts were dried (Na2SO4), and the
solvent was removed under reduced pressure to give a crude that was pu-
rified by column chromatography. First the corresponding S-glycosyl
DTC 3ACTHNUGRTENNUG(A–D)ACHTUNTGREN(NUGN b–c) was eluted, and, secondly, the related DGDS 4A–D
was eluted in the isolated yields reflected in Tables 1 and 2. Compounds
4A–D showed spectroscopic data identical to those reported previously
in the literature[14a] (see below).
S-(2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl)-sec-butyl Dithiocarbamate
(3Ab)
Column chromatography (EtOAc/hexane 1:2) of the reaction mixture
gave 3Ab (340 mg, 71%) as
a syrup. [a]D = +4.0 (c=1, CHCl3);
1H NMR (CDCl3, 400 MHz) (diastereomeric mixture): d=7.22 (brs, 1H;
NH), 5.66 (t, J=9.9 Hz, 1H; H-1), 5.31 (t, J=9.3 Hz, 1H; H-3), 5.18 (td,
J=9.9, 4.5 Hz, 1H; H-2), 5.08 (t, J=9.6 Hz, 1H; H-4), 4.51 (m, 1H;
CHN), 4.25 (dd, J=12.4, 4.6 Hz, 1H; H-6), 4.09 (d, J=12.4 Hz, 1H; H-
6’), 3.84 (m, 1H; H-5), 2.04 (s, 3H), 2.00 (2 s, 6H), 1.98 (s, 3H), 1.69–1.50
(m, 2H; CH2CH3), 1.22 (d, J=5.9 Hz, 3H; CHCH3), 0.91 ppm (t, J=
7.2 Hz, 3H; CH2CH3); 13C NMR (CDCl3, 100 MHz) (diastereomeric mix-
ture): d=191.2, 191.1, 170.8, 170.1, 169.7, 169.6, 86.1, 86.1, 76.5, 74.1,
68.7, 68.7, 68.2, 61.9, 54.7, 54.7, 29.4, 28.8, 20.9, 20.8, 20.7, 19.0, 19.0, 10.4,
10.4 ppm; IR (film): n˜max =3292, 2969, 2877, 1754, 1520, 1374, 1225, 1044,
Experimental Section
General Methods
Unless otherwise noted, commercially available reagents (including the
halo sugar derivatives 1A and 1C) and solvents were used as purchased
without further purification. The starting materials per-O-acetylated-a-
bromo lactose 1D,[37] per-O-acetylated-N-acetyl-a-chloro glucosamine
1B,[38] carbamodithioic acid N-[3-(triethoxysilyl)propyl] sodium salt 2a,[39]
S-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-N,N-diethyldithiocarbamate
3Ad,[12a] dithiocarbamate 6,[26] and 1,2-O-isopropylidene-a-d-xylofura-
nose 3,5-cyclic sulfate 14[33] were prepared according to reported proce-
dures in the literature. TLC was performed with Merck silica gel 60 F254
aluminum sheets. Reagents used for developing plates include sulfuric
acid (5% v/v in ethanol), potassium permanganate (1% w/v), ninhydrin
(0.3% w/v) in ethanol, and UV light when applicable. Flash column chro-
matography was performed with silica gel (230–400 mesh, ASTM). Melt-
ing points were measured with a melting point apparatus and are uncor-
rected. Optical rotations were recorded on a polarimeter at room temper-
ature. IR spectra were recorded with a FTIR spectrometer. 1H and
13C NMR spectra were recorded at room temperature on a 300, 400, or
500 MHz spectrometer. Chemical shifts are given in ppm and referenced
to internal CDCl3. Coupling constant (J) values are given in Hz. Electro-
spray ionization (ESI) mass spectra were recorded with an LCT Premier
spectrometer (TOF). Biological assays were carried out in ELISA plates
using commercial lectins. The peroxidase activity was determined with
a sunrise absorbance reader using o-phenylenediamine dihydrochloride
as substrate.
824 cmꢀ1
;
HRMS (ESI): m/z calcd for C19H29NO9S2Na [M+Na]+:
502.1181; found: 502.1189.
S-(2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl)-N-benzyl Dithiocarbamate
(3Ac)
Column chromatography (EtOAc/hexane 1:2) of the reaction mixture
gave 3Ac (410 mg, 80%) as
a syrup. [a]D = +12.2 (c=1, CHCl3);
1H NMR (CDCl3, 400 MHz): d=7.67 (t, J=5.1 Hz, 1H; NH), 7.39–7.26
(m, 5H),5.67 (d, J=10.5 Hz, 1H), 5.31 (t, J=9.3 Hz, 1H), 5.18 (t, J=
10.1 Hz, 1H), 5.06 (t, J=9.7 Hz, 1H), 4.86 (d, J=5.0 Hz, 2H), 4.20 (dd,
J=12.5, 4.8 Hz, 1H), 4.04 (dd, J=12.7, 2.3 Hz, 1H), 3.82 (ddd, J=10.1,
4.7, 2.2 Hz, 1H), 2.01, 2.00, 2.00, 1.99 ppm (4 s, 12H); 13C NMR (CDCl3,
100 MHz): d=192.9, 170.8, 170.2, 169.7, 169.6, 135.8, 129.1, 128.5, 128.5,
86.2, 76.5, 74.1, 68.8, 68.2, 61.8, 51.6, 20.9, 20.8, 20.7 ppm; IR (film):
n˜max =3291, 2942, 1753, 1512, 1372, 1227, 1046, 914 cmꢀ1; HRMS (ESI):
m/z calcd for C22H27NO9S2Na [M+Na]+: 536.1019; found: 536.1025.
S-(2-Acetamido-3,4,6-tri-O-acetyl-b-d-glucopyranosyl)-N-benzyl
Dithiocarbamate (3Bc)
Column chromatography (hexane/EtOAc 1:3) of the reaction mixture
gave 3Bc (373 mg, 73%) as a solid. M.p. 65–668C; [a]D = +28.7 (c=1,
CHCl3); 1H NMR (CDCl3,400 MHz): d=8.42 (brs, 1H), 7.30 (m, 5H),
6.28 (d, J=9.5 Hz, 1H), 5.74 (d, J=10.7 Hz, 1H), 5.19 (t, J=9.6 Hz, 1H),
5.08 (t, J=9.5 Hz, 1H), 4.85 (brs, 2H), 4.36 (q, J=9.8 Hz, 1H), 4.19 (dd,
J=12.4, 4.6 Hz, 1H), 4.05 (d, J=12.1 Hz, 1H), 3.81 (m, 1H), 2.01, 2.00,
1.98, 1.83 ppm (4s, 12H); 13C NMR (CDCl3, 100 MHz): d=194.3, 171.3,
170.9, 170.7, 169.5, 136.0, 129.0, 128.4, 128.2, 87.5, 76.6, 74.3, 68.2, 62.1,
52.2, 51.5, 23.3, 20.9, 20.8, 20.8 ppm; IR (film): n˜max =3290, 3029, 2935,
General Procedure for the Synthesis of DTCꢀM+ Salts (2b–d)
CS2 (1.2 mL, 20 mmol) was slowly added to a cooled (ice-bath) aqueous
solution (10 mL) of the corresponding amine (sec-butylamine, benzyla-
mine, or diethylamine) (20 mmol) that contained NaOH (0.8 g, 20 mmol)
under magnetic stirring. The reaction mixture was allowed to reach RT
for 1 h. After this time, most of the solvent was removed under reduced
pressure. The resulting precipitate was collected by filtration and dried
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Francisco Santoyo-Gonzalez et al.
1748, 1667, 1526, 1236, 1047 cmꢀ1
;
HRMS (ESI): m/z calcd for
Bis(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl) Disulfide 4(C)[14a]
C22H28N2O8S2Na [M+Na]+: 535.1185; found: 535.1179.
Column chromatography (EtOAc/hexane 1:1) of the reaction mixture
gave 4C (667 mg, 92% from 3Cb, 638 mg, 88% from 3Cc) as a syrup.
[a]D =ꢀ33.1 (c=1, CHCl3).
S-(2,3,4,6-Tetra-O-acetyl-b-d-galactopyranosyl)-sec-butyl Dithiocarbamate
(3Cb)
Bis(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl-(1!4)-2,3,4,6-tetra-O-
acetyl-b-d-glucopyranosyl) Disulfide (4D)[14a]
Column chromatography (EtOAc/hexane 1:2) of the reaction mixture
gave 3Cb (387 mg, 81%) as
a syrup. [a]D = +40.7 (c=1, CHCl3);
1H NMR (CDCl3, 400 MHz) (diastereomeric mixture): d=7.22 and 7.15
(2d, J=7.8 Hz, 1H; NH), 5.55–5.46 (m, 2H), 5.41 (t, J=10.0 Hz, 1H),
5.18–5.13 (m, 1H), 4.54 (m, 1H), 4.17–4.05 (m, 3H), 2.15 (s, 3H), 2.04
(brs, 6H), 1.98 (s, 3H), 1.73–1.53 (m, 2H), 1.27–1.23 (m, 3H), 0.95 ppm
(t, J=7.5 Hz, 3H); 13C NMR (CDCl3, 100 MHz) (diastereomeric mix-
ture): d=191.6, 191.6, 170.5, 170.3, 170.0, 169.6, 86.5, 86.4, 75.5, 72.0,
72.0, 67.5, 66.2, 66.1, 61.5, 61.5, 54.6, 54.6, 29.0, 20.9 (ꢃ3), 20.8, 20.7, 19.2,
19.1, 10.4 ppm; IR (film): n˜max =3295, 2970, 1751, 1522, 1371, 1226, 1084,
Column chromatography (EtOAc/hexane 2:1) of the reaction mixture
gave 4D (1.04 g, 81% from 3Cb) as a syrup. [a]D =ꢀ70.1 (c=1, CHCl3).
Reaction of Glycosyl DTCs 3Ac and 3Cc
An equimolecular mixture of the S-glycosyl DTCs 3Ac and 3Cc
(1 mmol) was dissolved in anhydrous acetone (15 mL) and treated with
Et3N (2 mmol). The reaction mixture was magnetically stirred at RT until
complete disappearance of the starting material was observed by TLC
(18 h). Evaporation of the solvent gave a crude that was purified by
column chromatography on silica gel to yield an inseparable mixture of
the corresponding homo-DGDSs 4A and 4C as well as the hetero-
DGDS Glc-S-S-Gal (8) (86%), which were identified by their 1H and
13C NMR spectroscopic data.
1056 cmꢀ1
;
HRMS (ESI): m/z calcd for C19H29NO9S2Na [M+Na]+:
502.1181; found: 502.1205.
S-(2,3,4,6-Tetra-O-acetyl-b-d-galactopyranosyl)-N-benzyl Dithiocarbamate
(3Cc)
Column chromatography (EtOAc/hexane 1:2) of the reaction mixture
gave 3Cc (501 mg, 98%) as
a syrup. [a]D = +19.8 (c=1, CHCl3);
General Procedure for the Synthesis of Homodivalent Neoglycoconjugates
1H NMR (CDCl3, 400 MHz): d=7.80 (brs, 1H), 7.38–7.24 (m, 5H),5.57
(d, J=10.4 Hz, 1H), 5.44 (d, J=3.3 Hz, 1H), 5.39 (t, J=10.1 Hz, 1H),
5.14 (dd, J=9.9, 3.4 Hz, 1H), 4.89 (m, 2H), 4.12–4.01 (m, 3H), 2.05, 2.02,
1.98, 1.96 ppm (4 s, 12H); 13C NMR (CDCl3, 100 MHz): d=193.1, 170.5,
170.2, 170.0, 169.8, 135.9, 129.1, 128.4, 128.2, 86.4, 75.4, 72.0, 67.4, 66.2,
61.4, 51.3, 20.9, 20.8, 20.7, 20.6 ppm; IR (film): n˜max =3291, 2973, 1749,
1510, 1369, 1223, 1082, 1055, 933 cmꢀ1; HRMS (ESI): m/z calcd for
C22H26NO9S2 [MꢀH]ꢀ: 512.1049; found: 512.1050.
9A–D
Divinyl sulfone (1 mmol) and Et3N (4 mmol) were added to a solution of
the corresponding S-glycosyl DTC 3
acetone (15 mL) for 3(A,C,D)b or anhydrous DMF (5 mL) for 3Bb. The
reaction mixture was magnetically stirred at RT overnight. Evaporation
of the solvent in the case of the reactions of 3(A,C,D)b gave a crude that
was purified by column chromatography on silica gel to yield the homo-
divalent neoglycoconjugates 9A,C,D. In the case of 3Bb, diethyl ether
(50 mL) was added to the reaction mixture, and the solid that precipitat-
ed was filtrated to yield 9B.
ACHTUNGTNER(NUNG A–D)b (2.2 mmol) in anhydrous
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
S-(2,3,4,6-Tetra-O-acetyl-b-d-galactopyranosyl-(1!4)-2,3,4,6-tetra-O-
acetyl-b-d-glucopyranosyl)-N-benzyl Dithiocarbamate (3Dc)
Column chromatography (EtOAc/hexane 1:1) of the reaction mixture
Bis[(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)thioethyl]sulfone (9A)
gave 3Dc (576 mg, 72%) as
a
syrup. [a]D =ꢀ16.9 (c=1, CHCl3);
Column chromatography (EtOAc/hexane 2:1) of the reaction mixture
gave 9A as a solid (642 mg, 76%). M.p. 182–1838C (desc); [a]D =ꢀ29.8
(c=1, CHCl3); 1H NMR (CDCl3, 400 MHz): d=5.23 (t, J=9.2 Hz, 2H),
5.06 (t, J=9.9 Hz, 2H), 5.04 (t, J=9.8 Hz, 2H), 4.58 (d, J=9.9 Hz, 2H),
4.21 (t, J=12.1 Hz, 2H), 4.17 (dd, J=12.3, 4.5 Hz, 2H), 3.74 (d, J=
7.6 Hz, 2H), 3.39 (m, 4H), 3.18 (m, 2H), 3.02 (m, 2H), 2.11, 2.05, 2.03,
2.00 ppm (4s, 24H); 13C NMR (CDCl3, 75 MHz): d=170.9, 170.2, 169.6,
169.5, 84.0, 76.5, 73.7, 69.7, 68.3, 61.9, 54.6, 22.7, 21.0, 20.8, 20.7 ppm; IR
(KBr): n˜max =2948, 1744, 1372, 1227, 1039, 912 cmꢀ1; HRMS (ESI): m/z
calcd for C32H46O20S3Na [M+Na]+: 869.1642; found: 869.1647.
1H NMR (CDCl3, 400 MHz): d=7.61 (brt, J=5.1 Hz, 1H; NH), 7.39–
7.25 (m, 5H), 5.59 (d, J=10.4 Hz, 1H), 5.33 (d, J=3.4 Hz, 1H), 5.28 (t,
J=8.7 Hz, 1H), 5.13–5.05 (m, 2H), 4.93 (dd, J=10.4, 3.4 Hz, 1H), 4.85
(d, J=4.9 Hz, 1H), 4.44 (d, J=7.8 Hz, 1H), 4.37 (d, J=11.0 Hz, 1H),
4.14–4.02 (m, 3H), 3.86 (t, J=6.7 Hz, 1H), 3.82–3.69 (m, 2H), 2.13, 2.04,
2.03, 2.03, 2.02, 2.02, 2.00 ppm (7s, 21H); 13C NMR (CDCl3, 100 MHz):
d=192.7, 170.3, 170.3, 170.1, 170.0, 169.7, 169.5, 169.0, 135.7, 128.9, 100.9,
85.6, 77.2, 75.8, 73.6, 70.9, 70.7, 69.0, 68.9, 66.6, 61.8, 60.9, 51.3, 20.8, 20.7,
20.6, 20.6, 20.5 ppm; IR (film): n˜max =3295, 2942, 1751, 1370, 1229, 1053,
913, 733 cmꢀ1; HRMS (ESI): m/z calcd for C34H43NO17S2Na [M+Na]+:
824.1870; found: 824.1877.
Bis[(2-acetamido-3,4,6-tri-O-acetyl-1-b-d-glucopyranosyl)thioethyl]
Sulfone (9B)
General Procedure for the Synthesis of GDSDs (4A–D) from S-Glycosyl
DTCs 3
ACHTUNGTRENNUNG(A–D)ACHTUNGTRENNUNG(b,c)
Filtration of the formed solid gave 9B (633 mg, 75%). M.p. 2938C
(desc); [a]D =ꢀ26.4 (c=0.5, DMSO); 1H NMR ([D6]DMSO, 400 MHz):
d=7.99 (d, J=9.4 Hz, 2H; NH), 5.06 (t, J=9.8 Hz, 2H), 4.84 (t, J=
9.7 Hz, 2H), 4.78 (d, J=10.4 Hz, 2H), 4.11 (dd, J=12.3, 5.3 Hz, 2H),
4.04 (dd, J=12.2, 1.9 Hz, 2H), 3.92–3.86 (m, 4H), 3.43 (t, J=7.9 Hz,
4H), 3.06–2.97 (m, 2H), 2.94–2.86 (m, 2H), 2.01, 1.96, 1.91, 1.76 ppm (4s,
24H); 13C NMR ([D6]DMSO, 100 MHz): d=170.1, 169.6, 169.2, 169.2,
83.4, 74.6, 73.4, 68.4, 62.0, 53.0, 51.8, 22.6, 21.9, 20.5, 20.4, 20.3 ppm; IR
Et3N (2 mmol) was added to a solution of the corresponding glycosyl di-
thiocarbamate 3(A–D)(b,c) (1 mmol) in anhydrous acetone (15 mL). The
A
ACHTUNGTRENNUNG
reaction mixture was magnetically stirred at RT until complete disappear-
ance of the starting material was observed by TLC (6–12 h). Evaporation
of the solvent gave a crude that was purified by column chromatography
on silica gel to yield glycosyl disulfides 4A–D, which showed spectro-
scopic data identical to those previously reported in the literature.[14a] Iso-
lated yields are reflected in Table 3.
(KBr): n˜max =3291, 2942, 2878, 1746, 1657, 1542, 1227, 1110 ,1036 cmꢀ1
;
HRMS (ESI): m/z calcd for C32H49N2O18S3 [M+H]+: 845.2143; found:
Bis(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl) Disulfide (4A)[14a]
845.2137.
Bis[(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl)thioethyl] Sulfone (9C)
Column chromatography (EtOAc/hexane 1:2) of the reaction mixture
gave 4A (711 mg, 98% from 3Ab, 653 mg, 90% from 3Ac) as a syrup.
[a]D =ꢀ135.5 (c=1, CHCl3).
Column chromatography (EtOAc/hexane 2:1) of the reaction mixture
gave 9C as a syrup (600 mg, 71%). [a]D =ꢀ6.9 (c=1, CHCl3); 1H NMR
(CDCl3, 400 MHz): d=5.42 (d, J=3.0 Hz, 2H), 5.23 (t, J=9.9 Hz, 2H),
5.04 (dd, J=10.0, 3.3 Hz, 2H), 4.55 (d, J=9.9 Hz, 2H), 4.17–4.05 (m,
4H), 3.95 (t, J=6.3 Hz, 2H), 3.39 (t, J=7.8 Hz, 2H), 3.20 (m, 2H), 3.01
(m, 2H), 2.15, 2.14, 2.05, 1.97 ppm (4s, 24H); 13C NMR (CDCl3,
75 MHz): d=170.6, 170.3, 170.1, 169.7, 84.4, 75.1, 71.8, 67.5, 66.9, 61.9,
Bis(2-acetamido-3,4,6-tri-O-acetyl-b-d-glucopyranosyl) Disulfide (4B)[14a]
Column chromatography (EtOAc!EtOAc/MeOH 20:1) of the reaction
mixture gave 4B (515 mg, 71% from 3Bc) as a solid. M.p. 227–2288C;
[a]D =ꢀ166.6 (c=1, CHCl3).
Chem. Asian J. 2014, 9, 620 – 631
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Francisco Santoyo-Gonzalez et al.
54.6, 22.7, 20.9, 20.9, 20.8, 20.7 ppm; IR (KBr): n˜max =2932, 1749, 1369,
1220, 1036, 915, 730 cmꢀ1; HRMS (ESI): m/z calcd for C32H46O20S3Na
[M+Na]+: 869.1642; found: 869.1633.
1369, 1241, 1053, 735 cmꢀ1; HRMS (ESI): m/z calcd for C18H26O11S2Na
[M+Na]+: 505.0814; found: 505.0805.
[2-(Ethenesulfonyl)ethyl]sulfanyl(2,2’,3,3’,4,4’,6,6’-octa-O-acetyl-b-d-
lactosyde (11D)
Bis[(2,2’,3,3’,4,4’,6,6’-octa-O-acetyl-b-lactosyl)thioethyl] Sulfone (9D)
Column chromatography (EtOAc/hexane 2:1) of the reaction mixture
Column chromatography (EtOAc/hexane 1:1!2:1) of the reaction mix-
ture gave 9D as a syrup (455 mg, 32%). [a]D =ꢀ38.9 (c=1, CHCl3);
1H NMR (CDCl3, 400 MHz): d=5.42 (d, J=3.0 Hz, 2H), 5.23 (t, J=
9.9 Hz, 2H), 5.04 (dd, J=10.0, 3.3 Hz, 2H), 4.55 (d, J=9.9 Hz, 2H),
4.17–4.05 (m, 4H), 3.95 (t, J=6.3 Hz, 2H), 3.39 (t, J=7.8 Hz, 2H), 3.20
(m, 2H), 3.01 (m, 2H), 2.15, 2.14, 2.05, 1.97 ppm (4s, 24H); 13C NMR
(CDCl3, 75 MHz): d=170.6, 170.3, 170.1, 169.7, 84.4, 75.1, 71.8, 67.5, 66.9,
61.9, 54.6, 22.7, 20.9, 20.9, 20.8, 20.7 ppm; IR (KBr): n˜max =2928, 1751,
gave 11D as
a
syrup (546 mg, 71%). [a]D =ꢀ17.4 (c=1, CHCl3);
1H NMR (CDCl3, 400 MHz): d=6.68 (dd, J=16.6, 9.9 Hz, 1H), 6.45 (d,
J=16.6 Hz, 1H), 6.21 (d, J=9.9 Hz, 1H), 5.34 (d, J=2.9 Hz, 1H), 5.20 (t,
J=9.1 Hz, 1H), 5.10 (dd, J=10.3, 7.9 Hz, 1H), 4.96 (dd, J=10.4, 3.4 Hz,
1H), 4.93 (t, J=9.6 Hz, 1H), 4.56–4.48 (m, 3H), 4.14–4.02 (m, 3H), 3.88
(t, J=6.4 Hz, 1H), 3.76 (t, J=9.4 Hz, 1H), 3.63 (m, 1H), 3.37–3.26 (m,
2H), 3.06 (m, 1H), 2.89 (m, 1H), 2.14, 2.05, 2.03, 2.03, 1.95 ppm (several
s, 21H); 13C NMR (CDCl3, 100 MHz): d=170.5, 170.4, 170.2, 170.1,
169.7, 169.7, 169.2, 136.1, 131.3, 101.2, 83.7, 77.2, 76.1, 73.5, 71.1, 70.9,
69.9, 69.2, 66.8, 61.9, 60.9, 55.3, 22.8, 21.0, 20.9, 20.8, 20.7, 20.6 ppm; IR
(film): n˜max =2928, 1749, 1429, 1370, 1229, 1050, 913 cmꢀ1; HRMS (ESI):
m/z calcd for C30H42O19S2Na [M+Na]+: 793.1659; found: 793.1665.
1430, 1370, 1229, 1052, 913 cmꢀ1
; HRMS (ESI): m/z calcd for
C56H78O36S3Na [M+Na]+: 1445.3333; found: 1445.3357.
General Procedure for the Synthesis of Thioglycosyl Vinylsulfones (11A–
D)
Divinyl sulfone (3 mmol) and Et3N (2 mmol) were added to a solution of
the corresponding S-glycosyl DTC 3ACTHNUGRTNEUNG(A–D)b (1 mmol) in anhydrous ace-
Synthesis of Heterodivalent Neoglycoconjugate ({2-[2-(2-Acetamido-3,4,6-
tetra-O-acetyl-b-d-
tone (15 mL). The reaction mixture was magnetically stirred at RT until
complete disappearance of the starting material was observed by TLC
(1 h). Evaporation of the solvent gave a crude that was purified by
column chromatography on silica gel to yield the corresponding thiogly-
cosyl sulfones 11A–D.
glucopyranosylsulfanyl)ethanesulfonyl]ethyl}sulfanyl)2,2’,3,3’,4,4’,6,6’-octa-
O-acetyl-b-lactoside (12)
S-GlcNAc DTC 3Bb (1.0 mmol), Et3N (2 mmol), and tributylphosphine
(0.1 mmol) were added to an Ar-degassed solution of the corresponding
lactose–vinyl sulfone 11D (1.5 mmol) in anhydrous acetone (15 mL). The
reaction mixture was magnetically stirred at RT for 4 h. Evaporation of
the solvent gave a crude that was purified by column chromatography
(EtOAc/hexane 3:1!EtOAc) on silica gel to yield the heterodivalent ne-
oglycoconjugate 12 as a solid (0.926 g, 82%): M.p. 107–1098C; [a]D =
ꢀ22.8 (c=1, CHCl3); 1H NMR (CDCl3, 400 MHz): d=5.85 (m, 1H;
NH), 5.35 (d, J=3.0 Hz, 1H), 5.21 (t, J=9.2 Hz, 1H), 5.17 (t, J=9.6 Hz,
1H), 5.12–5.05 (m, 2H), 4.98 (dd, J=10.4, 3.2 Hz, 1H), 4.94 (t, J=
9.6 Hz, 1H), 4.69 (d, J=10.4 Hz, 1H), 4.63–4.50 (m, 3H), 4.25–4.05 (m,
6H), 3.90 (t, J=6.8 Hz, 1H), 3.81 (t, J=9.5 Hz, 1H), 3.74 (m, 1H), 3.66
(m, 1H), 3.48–3.32 (m, 4H), 3.25–3.08 (m, 2H), 3.07–2.94 (m, 2H), 2.15,
2.10, 2.05, 2.04, 2.03, 1.96, 1.95 (several s, 30H), 1.79 ppm (s, 3H);
13C NMR (CDCl3, 75 MHz): d=171.1, 170.9, 170.8, 170.5, 170.4, 170.3,
170.2, 169.8, 169.8, 169.4, 169.3, 101.2, 85.2, 83.8, 77.4, 76.3, 76.1, 73.7,
71.1, 70.9, 70.0, 69.3, 68.4, 66.8, 62.2, 61.8, 60.9, 54.7, 54.4, 53.2, 23.4, 23.0,
22.7, 21.1, 20.9, 20.9, 20.8 (ꢃ3), 20.7 ppm; IR (film): n˜max =3277, 2933,
[2-(Ethenesulfonyl)ethyl]sulfanyl(2,3,4,6-tetra-O-acetyl-b-d-
glucopyranoside (11A)
Column chromatography (EtOAc/hexane 1:1!EtOAc) of the reaction
mixture gave 11A as a syrup (361 mg, 75%). [a]D =ꢀ22.5 (c=1, CHCl3);
1H NMR (CDCl3, 400 MHz): d=6.67 (dd, J=16.6, 9.8 Hz, 1H), 6.45 (d,
J=16.6 Hz, 1H), 6.21 (d, J=9.9 Hz, 1H), 5.21 (t, J=9.4 Hz, 1H), 5.03 (t,
J=9.8 Hz, 1H), 5.00 (t, J=9.7 Hz, 1H), 4.54 (d, J=10.0 Hz, 1H), 4.16 (d,
J=3.7 Hz, 2H), 3.72 (dt, J=10.1, 3.8 Hz, 1H), 3.40–3.27 (m, 2H), 3.07
(m, 1H), 2.91 (m, 1H), 2.09, 2.03, 2.01, 1.99 ppm (4s, 12H); 13C NMR
(CDCl3, 100 MHz): d=170.8, 170.2, 169.5, 136.1, 131.4, 84.0, 76.3, 73.6,
69.6, 68.3, 62.1, 55.3, 23.0, 20.8, 20.8, 20.7, 20.7 ppm; IR (film): n˜max
=
2943, 1752, 1431, 1374, 1226, 1136, 1039, 914 cmꢀ1; HRMS (ESI): m/z
calcd for C18H26O11S2Na [M+Na]+: 505.0814; found: 505.0819.
[2-(Ethenesulfonyl)ethyl]sulfanyl(2-acetamido-3,4,6-tri-O-acetyl-b-d-
glucopyranoside (11B)
1748, 1671, 1370, 1229, 1045 cmꢀ1
; HRMS (ESI): m/z calcd for
C44H64NO27S3 [M+H]+: 1134.2828; found: 1134.2821.
Column chromatography (EtOAc/hexane 3:1!EtOAc/MeOH 10:1) of
the reaction mixture gave 11B as a syrup (428 mg, 89%). [a]D =ꢀ32.4
(c=1, CHCl3); 1H NMR ([D6]DMSO, 400 MHz): d=7.98 (d, J=9.6 Hz,
1H; NH), 6.97 (dd, J=16.6, 10.0 Hz, 1H), 6.27 (d, J=9.5 Hz, 1H), 6.25
(d, J=16.4 Hz, 1H), 5.04 (t, J=9.7 Hz, 1H), 4.84 (t, J=9.8 Hz, 1H), 4.75
(d, J=10.4 Hz, 1H), 4.06 (m, 2H), 3.88 (m, 2H), 3.43 (m, 2H), 2.92 (m,
1H), 2.78 (m, 1H), 2.01, 1.96, 1.90, 1.75 ppm (4s, 12H); 13C NMR
([D6]DMSO, 75 MHz): d=170.0, 169.6, 169.2, 169.2, 136.5, 130.3, 83.4,
74.6, 73.4, 68.4, 62.0, 53.9, 51.8, 22.6, 22.3, 20.5, 20.4, 20.3 ppm; IR (film):
n˜max =3296, 2942, 1745, 1656, 1542, 1217, 1117, 1036 cmꢀ1; HRMS (ESI):
m/z calcd for C18H28NO10S2 [M+H]+: 482.1155; found: 482.1152.
Synthesis of Thiodisaccharides 15 and 16
Cyclic sulfate 14 (1 mmol) and triethylamine (2 mmol) were added to
a solution of the corresponding S-glycosyl DTC 3Bb and 3Cc (1 mmol)
in anhydrous acetone (10 mL). The reaction mixture was magnetically
stirred at RT for 24 h. Evaporation of the solvent gave a crude product
that was purified by column chromatography with silica gel to yield thio-
disaccharides 15 and 16.
(6-Deoxy-1,2-O-isopropilidene-3-O-methyl-5-O-sulfateglucofuranose-6-
yl)-2-acetamido-3,4,6-tri-O-acetyl-1-thio-b-d-glucopyranoside (15)
Column chromatography (EtOAc/MeOH 10:1) of the reaction mixture
[2-(Ethenesulfonyl)ethyl]sulfanyl(2,3,4,6-tetra-O-acetyl-b-d-
galactopyranoside (11C)
gave 15 as
a
syrup (428 mg, 65%). [a]D =ꢀ56.2 (c=0.5, MeOH);
1H NMR (CD3OD, 400 MHz): d=5.85 (d, J=3.8 Hz, 1H; H-1), 5.40 (d,
J=3.2 Hz, 1H; H-4’), 5.12 (dd, J=7.1, 2.7 Hz, 1H; H-3’), 5.10–5.02 (m,
2H; H-1’ and H-2’), 4.86 (m, 1H; H-5), 4.66 (d, J=3.8 Hz, 1H; H-2),
4.38 (dd, J=8.7, 2.8 Hz, 1H; H-4), 4.17–4.05 (m, 3H; H-6a, H-6b, H-5’),
3.78 (d, J=2.9 Hz, 1H; H-3), 3.44 (s, 3H; OCH3), 3.36 (dd, J=15.2,
3.7 Hz, 1H; H-6’a), 3.09 (dd, J=15.2, 2.7 Hz, 1H; H-6’b), 2.14, 2.04, 2.03,
Column chromatography (EtOAc/hexane 1:1) of the reaction mixture
gave 11C as a syrup (433 mg, 90%). [a]D =ꢀ5.1 (c=1, CHCl3); H NMR
1
(CDCl3, 400 MHz): d=6.65 (dd, J=16.6, 9.8 Hz, 1H; SO2CH=), 6.43 (d,
J=16.6 Hz, 1H; =CH2), 6.19 (d, J=9.9 Hz, 1H; =CH2), 5.40 (d, J=
2.4 Hz, 1H; H-4), 5.20 (t, J=9.9 Hz, 1H; H-2), 5.02 (dd, J=10.0, 3.4 Hz,
1H; H-3), 4.52 (d, J=9.9 Hz, 1H; H-1), 4.12–4.04 (m, 2H; H6–6’), 3.93
(t, J=5.9 Hz, 1H; H-5),3.39–3.30 (m, 2H; CH2SO2), 3.09 (ddd, J=14.4,
10.2, 5.9 Hz, 1H; SCH), 2.91 (ddd, J=14.0, 10.0, 6.4 Hz, 1H; SCH’), 2.13,
2.04, 2.03, 1.95 ppm (4 s, 12H; COOCH3); 13C NMR (CDCl3,100 MHz):
d=170.6, 170.2, 170.0, 169.7, 136.1, 131.3, 84.3, 75.0, 71.7, 67.4, 66.9, 61.9,
55.3, 23.0, 20.9, 20.8, 20.8, 20.7 ppm; IR (film): n˜max =3059, 2970, 1750,
1.93 (4s, 12H; COCH3), 1.53, 1.33 ppm (2s, 6H; OCACTHNUTRGNEUNG
(CH3)2); 13C NMR
(CD3OD, 100 MHz): d=172.4, 172.2, 171.6, 113.1, 106.5, 84.5, 84.3, 83.1,
79.6, 75.5, 74.5, 73.5, 69.9, 69.4, 62.8, 58.5, 32.2, 27.2, 26.4, 21.0, 20.8, 20.7,
20.6 ppm; IR (film): n˜max =3475, 2986, 2935, 1751, 1638, 1372, 1226,
1020 cmꢀ1; HRMS (ESI): m/z calcd for C24H35O17S2 [MꢀH]ꢀ: 659.1316;
found: 659.1315.
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Francisco Santoyo-Gonzalez et al.
(6-Deoxy-1,2-O-isopropilidene-3-O-methyl-5-O-sulfateglucofuranose-6-
1053 cmꢀ1
;
HRMS (ESI): m/z calcd for C20H37N2O12S3 [M+H]+:
yl)-3,4,6-tri-O-acetyl-1-thio-b-d-galactopyranoside (16)
593.1509; found: 593.1504.
Column chromatography (EtOAc/MeOH 5:1) of the reaction mixture
gave 16 as a syrup (342 mg, 52%). [a]D =ꢀ27.1 (c=1, MeOH); H NMR
Bis[(b-d-galactopyranosyl)thioethyl] Sulfone 10C(OH)
1
Column chromatography (acetonitrile/H2O 5:1) of the reaction mixture
gave 10C(OH) as a solid (489 mg, 96%). M.p. 181–1828C (desc); [a]D =
ꢀ10.3 (c=1, H2O); 1H NMR (D2O, 400 MHz): d=4.61 (d, J=9.7 Hz,
2H), 4.02 (d, J=3.2 Hz, 2H), 3.82–3.74 (m, 6H), 3.73–3.68 (m, 3H), 3.62
(t, J=9.6 Hz, 2H), 3.26 (m, 2H), 3.20 ppm (m, 2H); 13C NMR (D2O,
125 MHz): d=86.0, 79.0, 73.8, 69.4, 68.7, 61.1, 53.5, 22.1 ppm; IR (KBr):
n˜max =3336, 2923, 1652, 1560, 1416, 1259, 1086 cmꢀ1; HRMS (ESI): m/z
calcd for C16H30O12S3Na [M+Na]+: 533.0797; found: 533.0795.
(CD3OD, 400 MHz): d=5.81 (d, J=3.7 Hz, 1H; H-1), 5.15 (t, J=9.6 Hz,
1H; H-4’), 5.03 (d, J=10.6 Hz, 1H; H-1’), 4.98 (t, J=9.7 Hz, 1H; H-3’),
4.84 (m, 1H; H-5), 4.62 (d, J=3.8 Hz, 1H; H-2), 4.49 (dd, J=6.4, 3.1 Hz,
1H; H-4), 4.27 (dd, J=12.3, 4.6 Hz, 1H; H-6’a), 4.13 (m, 1H; H-6’b),
3.92 (t, J=10.3 Hz, 1H; H-2’), 3.85 (m, 1H; H-5’), 3.82 (d, J=3.1 Hz,
1H; H-3), 3.42 (s, 3H), 3.18 (dd, J=15.1, 3.1 Hz, 1H; H-6a), 2.99 (dd, J=
15.1, 6.1 Hz, 1H; H-6b), 2.06, 1.99, 1.96, 1.92 (4s, 12H), 1.47, 1.31 ppm
(2s, 6H); 13C NMR (CD3OD, 100 MHz): d=173.6, 172.7, 172.0, 171.5,
113.2, 106.5, 85.5, 85.0, 82.8, 81.6, 77.8, 76.8, 76.0, 70.3, 63.6, 58.2, 55.0,
32.9, 27.3, 26.7, 23.0, 20.9, 20.7, 20.7 ppm; IR (film): n˜max =3496, 3317,
2987, 2937, 1746, 1666, 1238, 1041, 736 cmꢀ1; HRMS (ESI): m/z calcd for
C24H36NO16S2 [MꢀH]ꢀ: 658.1476; found: 658.1472.
({2-[2-Acetamido-(b-d-
glucopyranosylsulfanyl)ethanesulfonyl]ethyl}sulfanyl)b-lactose 13(OH)
Column chromatography (acetonitrile/H2O 4:1) of the reaction mixture
gave 13 as a solid (706 mg, 99%). M.p. 182–1848C; [a]D =ꢀ22.5 (c=1,
CHCl3); 1H NMR (D2O, 400 MHz): d=4.60 (d, J=10.5 Hz, 1H), 4.54 (d,
J=10.0 Hz, 1H), 4.35 (d, J=7.8 Hz, 1H), 3.86 (dd, J=12.6, 1.4 Hz, 1H),
3.85–3.80 (m, 2H), 3.71 (dd, J=4.9, 2.1 Hz, 1H), 3.69–3.60 (m, 4H),
3.61–3.49 (m, 9H), 3.48–3.41 (m, 2H), 3.40–3.36 (m, 2H), 3.30 (t, J=
9.1 Hz, 1H), 3.10 (m, 2H), 3.02 (m, 2H), 1.94 ppm (s, 3H); 13C NMR
(D2O, 125 MHz): d=174.5, 102.8, 85.4, 84.6, 79.9, 78.7, 78.0, 75.6, 75.3,
74.9, 72.5, 71.8, 70.9, 69.7, 68.5, 61.0, 60.8, 60.1, 54.4, 53.5, 53.4, 22.3, 22.1,
General Procedure for De-O-acetylation
The per-O-acetylated compounds 4A–D, 9A–D, and 12 (1 mmol) were
dissolved at RT in 3:1 MeOH/aqueous ammonia (1.0–1.5 mL/10 mg of
substrate). The reaction mixture was kept at RT until complete disap-
pearance of the starting material was observed by TLC (1–8 h). Evapora-
tion of the solvent gave a residue that was purified by silica gel flash-
chromatography to yield the corresponding de-O-acetylated compounds
7A–D(OH), which showed spectroscopic data identical to those reported
previously in the literature,[14a] 10A–D(OH) and 13(OH).
22.0 ppm; IR (KBr): n˜max =3408, 2923, 1655, 1551, 1108, 1072, 1032 cmꢀ1
;
HRMS (ESI): m/z calcd for C24H44NO17S3 [M+H]+: 714.1771; found:
714.1785.
Bis(b-d-glucopyranosyl) Disulfide 7A(OH)[14a]
Biological Assays
Column chromatography (acetonitrile/H2O 5:1) of the reaction mixture
gave 7A(OH) as a solid (354 mg, 91%). M.p. 115–1168C; [a]D =ꢀ109.2
(c=1, H2O).
A solution (200 mL) of the suitable lectin (10 mgmLꢀ1) in carbonate
buffer (0.1m, pH 9.6) was incubated at 48C overnight. Wells were washed
with phosphate buffer ((PBST; 300 mL, 20 mm, pH 7.4), NaCl (154 mm),
Tween 20 (0.05% v/v)) (3ꢃ3 min) and then incubated for 6 h at 378C
with different concentrations of the divalent neoglyconjugates 7B–
D(OH), 10B,C(OH), or 13(OH). Afterwards, they were washed with
PBST (300 mL, 3ꢃ3 min) to remove the unbound divalent neoglycoconju-
gates and then incubated with either WGA–HRP (0.5 mg/10 mL PBST)
or PNA–HRP (0.5 mg/10 mL PBST) at 378C for 1 h. After a new washing
with PBST (3ꢃ3 min), the wells were revealed by incubation at 378C
with 200 mL of citrate-phosphate (Na2HPO4, 100 mm; citrate pH 5,
50 mm), o-phenylenediamine dihydrochloride (0.04% w/v), and H2O2
(0.05%, v/v)) for 30 min for the homodivalent neoglycoconjugates (7B–
D(OH) and 10B,C(OH)) and for 1 h for the heterodivalent neoglyconju-
gates (13(OH)). The peroxidase activity was stopped by the addition of
H2SO4 (100 mL, 1.5m), and the resulting optical density was measured at
495 nm with an absorbance reader.
Bis(2-acetamido-b-d-glucopyranosyl) Disulfide 7B(OH)[14a]
Column chromatography (acetonitrile/H2O 5:1) of the reaction mixture
gave 7B(OH) as a solid (467 mg, 99%). M.p. 218–2198C; [a]D: ꢀ194.4
(c=1, H2O).
Bis(b-d-galactopyranosyl) Disulfide 7C(OH)[14a]
Column chromatography (acetonitrile/H2O 5:1) of the reaction mixture
gave 7C(OH) as a solid (370 mg, 95%). M.p. 166–1678C; [a]D =ꢀ52.5
(c=1, H2O).
Bis(b-lactosyl) Disulfide 7D(OH)[14a]
Column chromatography (acetonitrile/H2O 3:1) of the reaction mixture
gave 7D(OH) as a solid (642 mg, 90%). M.p. 205–2078C (desc); [a]D =
ꢀ50.4 (c=0.5, H2O).
Bis[(b-d-glucopyranosyl)thioethyl] Sulfone 10A(OH)
Column chromatography (acetonitrile/H2O 5:1) of the reaction mixture
gave 10A(OH) as a solid (504 mg, 99%). M.p. 176–1778C (desc); [a]D =
ꢀ33.2 (c=1, H2O); 1H NMR (D2O, 400 MHz): d=4.65 (d, J=9.9 Hz,
2H), 3.94 (dd, J=12.4, 2.1 Hz, 2H), 3.74 (t, J=5.9 Hz, 2H), 3.72–3.67
(m, 4H), 3.55–3.49 (m, 4H), 3.45 (d, J=9.2 Hz, 2H), 3.38 (t, J=9.3 Hz,
2H), 3.25 (m, 2H), 3.18 ppm (m, 2H); 13C NMR (D2O, 100 MHz): d=
86.7, 81.1, 78.2, 73.2, 70.6, 62.0, 54.7, 23.2 ppm; IR (KBr): n˜max =3364,
2923, 1653, 1561, 1420, 1283, 1040 cmꢀ1; HRMS (ESI): m/z calcd for
C16H30O12S3Na [M+Na]+: 533.0797; found: 533.0795.
Acknowledgements
The authors acknowledge the Ministerio de Ciencia and Innovacion for
financial support (grant no. CTQ2011-29299-CO2-01).
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Received: September 17, 2013
Published online: November 26, 2013
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