A straightforward synthetic access to symmetrical glycosyl disulfides and biological evaluation thereof

Article abstract of DOI:10.1039/c1ob05619k

Symmetrical glycosyl disulfides can be prepared within a few hours from per-O-acetylated precursors via a sequential approach entailing short reactions and no purification of any intermediate. The final thiolate-to-disulfide oxidation step is noticeably accelerated by low amounts of phenyl diselenide under air. Applicability of the strategy to non-saccharidic symmetrical alkyl disulfides has also been examined. A preliminary assay of the cytotoxic activity of symmetrical 1,1′- disulfides was performed on two human tumor cell lines, and a noteworthy activity was recorded for a range of these synthetic compounds.

Full text of DOI:10.1039/c1ob05619k

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PAPER
A straightforward synthetic access to symmetrical glycosyl disulfides and
biological evaluation thereof†
Matteo Adinolfi,a Domenica Capasso,*b Sonia Di Gaetano,c Alfonso Iadonisi,*a Loredana Leonea and
Antonello Pastorea
Received 18th April 2011, Accepted 9th June 2011
DOI: 10.1039/c1ob05619k
Symmetrical glycosyl disulfides can be prepared within a few hours from per-O-acetylated precursors
via a sequential approach entailing short reactions and no purification of any intermediate. The final
thiolate-to-disulfide oxidation step is noticeably accelerated by low amounts of phenyl diselenide under
air. Applicability of the strategy to non-saccharidic symmetrical alkyl disulfides has also been
examined. A preliminary assay of the cytotoxic activity of symmetrical 1,1¢- disulfides was performed
on two human tumor cell lines, and a noteworthy activity was recorded for a range of these synthetic
compounds.
procedures purified glycosyl thiols are needed and a variety of
Introduction
S-activators have been described for the purpose.6
Over recent years glycosyl disulfides have been attracting an
In this paper we wish to report a more streamlined approach
increasing interest in view of their biological potential. For
where symmetrical glycosyl disulfides are efficiently accessed
example, some members of this class of compounds exhibit
from cheap per-O-acetylated precursors without isolation of any
good affinity towards lectin,1 and display promising properties
intermediate, glycosyl thiols included. All the steps of the scheme
against tumor cells.2 Indeed, the reversible formation of disulfide
linkages provides interesting opportunities for finding bioactive
compounds through the generation of dynamic libraries from a
set of symmetrical disulfides.3
are fast enough so that the targets can be accessed in a matter
of a few hours. Furthermore, we also report the noteworthy in
vitro cytotoxicity of some of these compounds against two human
tumor cell lines.
Synthesis of symmetrical 1,1¢-glycosyl disulfides is commonly
achieved by oxidation of the corresponding glycosyl thiols which
can be in turn obtained by elaboration of some sulfurated func-
tionalities (thioesters, xanthates, or thiouronium salts)1,4 installed
Results and discussion
Synthesis of symmetrical glycosyl disulfides
at the anomeric positions via substitution steps conducted on
glycosyl bromides or, less frequently, 1-O-acetyl sugars. These pro-
cedures require isolation and purification of at least a sulfurated
saccharidic intermediate (the glycosyl thiol or/and one of its above
mentioned precursors). On the other hand, glycosyl disulfides can
also be directly accessed from glycosyl bromides by a prolonged
treatment (about 24 h) with tetrathiomolybdate reagents.5 Synthe-
sis of symmetrical glycosyl disulfides has also been occasionally
achieved by procedures extensible to the generation of disulfides
bearing different saccharidic moieties at either sulfur: also in these
The approach proposed here has been inspired by a very effi-
cient protocol for the synthesis of alkyl thioglycosides that we
have recently described.7 This protocol takes advantage of the
sequential steps illustrated in Scheme 1 in which a per-O-acetylated
pyranoside is initially converted into the corresponding glycosyl
iodide 1 by short exposure to a slight stoichiometric excess of
I2 and Et3SiH in refluxing DCM.8 Crude 1, obtained by a simple
extractive work-up, is then converted into the corresponding isoth-
iouronium derivative 2 by treatment with thiourea in acetonitrile at
60 ◦C.9 Addition of a base as mild as TEA and a suitable alkylating
agent results in the rapid generation of the corresponding S-alkyl
thioglycoside (Scheme 1, path A).9
Morerecently,10 this scheme was extended to the straightforward
synthesis of S-(phenylselenyl)thioglycosides11 as precursors of
thioglycosylated indoles which in turn were converted into soluble
eumelanin-like polymers. In particular, in the last step of the
sequence stoichiometric amounts of phenyl diselenide were added
together with TEA to build the S–Se linkage. On performing
the synthesis of (phenylselenyl)thio-galactoside 3 (Scheme 1, path
aDipartimento di Chimica Organica e Biochimica, Universita` “Federico II”
di Napoli, via Cinthia 4, I-80126, Naples, Italy. E-mail: iadonisi@unina.it;
Fax: +39 81 674393; Tel: +39 81 674154
bFacolta` di Scienze Biotecnologiche, Universita` di Napoli “Federico II”,
via Mezzocannone 16, 80134, Naples, Italy. E-mail: domenica.capasso@
unina.it
cIstituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134,
Naples, Italy
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of all the synthesized disulfides. See DOI: 10.1039/c1ob05619k
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Table 1 Synthesis of glycosyl disulfides from per-O-acetylated precursors
Entry Reagent
1
Product
Yield (dr)a (%)
66 (bb/ba 9)
Scheme 1 Synthesis of thioalkyl- and thio(phenylselenyl) galactosides via
glycosyl iodides.
B) under a variety of conditions, we observed that symmetrical
galactosyl disulfide 4 was the main by-product of the sequence
together with lower amounts of the corresponding glycosyl thiol
5. This outcome suggested the possibility of generating glycosyl
disulfides in high yields by adapting the above sequence and simply
adjusting the experimental conditions of the last step. In particular,
omitting the addition of phenyl diselenide at this stage of Scheme 1
(path B) was expected, under otherwise identical conditions, to
furnish glycosyl disulfides in high yields, logically via TEA-induced
generation of a glycosyl thiolate and in situ oxidation under air.
As a matter of fact this idea proved viable, but generation of
the desired product took prolonged times at room temperature
(overnight stirring). This result appeared rather surprising, as the
generation of the same product in acceptable yields (30–50%) took
a few minutes when a stoichiometric amount of phenyl diselenide
was present. This evidence suggested a possible catalytic role
played by phenyl diselenide in generating the symmetrical disulfide
4 through a mechanism in which the in situ generated SSe derivative
3 might further react with the glycosyl thiolate 6 as this latter is
gradually generated (Scheme 2).12
4
7
2
3
51 (bb/ba 9)
44 (aa/ab 1)b
8
4c
5
46 (aa/ab 1.5)b
65 (bb/ba 2.5)
9
6
48 (bb/ba 16)
10
a Isolated overall yield (diastereoisomeric ratio refers to the anomeric
configuration of the two saccharidic residues of the disulfide). b Inseparable
mixture also containing minor amounts (less than 10%) of the bb isomer.
c The last step was performed at rt for 24 h rather than at 50 ◦C for 1 h.
In order to optimize the yields of disulfides, the sequence was
repeated starting from per-O-acetylated galactose as the model
precursor and gradually reducing the amount of phenyl diselenide
in the last step, so to minimize the concomitant generation of
the (phenylselenyl) thioglycoside 3 while maintaining a faster
obtainment of the disulfide product than in the absence of the
diselenide catalyst. After several attempts, the best compromise in
terms of yield and reduction of experimental time was observed by
performing the last step at 50 ◦C for one hour and in the presence
of as low an amount of catalyst as 3%. Under these conditions
disulfide 4 was obtained in high overall yield (66% over three
steps, Table 1, entry 1).
Scheme 2 Hypothesized catalytic role of (PhSe)2 in the synthesis of
symmetric glycosyl disulfides.
According to this hypothesis, phenyl selenolate is generated as
the leaving group, and this intermediate can be quickly oxidized to
the corresponding diselenide performing the reaction under air.13
As previously suggested by Davis and co-workers,11 it is interesting
to recall that SSe intermediates are also involved in the synthesis
of disulfides catalyzed by seleno-enzymes.14
It is pertinent to note that under otherwise identical conditions,
but in the absence of the catalyst, disulfide 4 was obtained
in lower yields (less than 50%) within a more complex crude
mixture. Also remarkable is that the overall sequence takes shorter
experimental times than other described multi-step procedures
that often start from more advanced intermediates (glycosyl
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bromides rather than per-O-acetylated precursors), and that a
single purification procedure is required here. On the other hand,
minor amounts of the separable ab-linked disulfide were observed
in the resulting mixture.
(32%) mixtures (Scheme 4), to yield derivatives 17–22 in high yield
(over 90% in all cases after chromatographic purification).
The optimized sequential procedure was applied to a variety
of per-O-acetylated mono- (Table 1, entries 2–5) and disaccharide
(Table 1, entry 6) building blocks and satisfying overall yields
were obtained in all cases by performing the last step of the
sequence under the same conditions as for the galacto precursor.
Only in the case of the manno precursor (Table 1, entry 3) was
the diastereoselectivity of the process poor, with the disulfide
products proving to be inseparable. Performing the last step of
the sequence at rt for 24 h (Table 1, entry 4) rather than at 50 ◦C
for 1 h (Table 1, entry 3) produced just a slight improvement in
the diastereoselectivity and a very similar yield. In all cases, the
expected predominance of 1,2-trans thioglycoside residues was
recorded, consistent with the behaviour observed on applying the
procedure to the synthesis of alkyl thioglycosides.7
In order to expand the scope of the sequential procedure,
structurally different substrates were examined. Due to the easier
anomeric functionalization of N-acetyl glucosamine with Cl15
rather than with I,16 chloride 1115 was reacted with thiourea in
acetone, according to previously reported conditions17 and the
resulting thiouronium intermediate was submitted to excess TEA
and catalytic (PhSe)2 as previously shown in Table 1. Disulfide 12
was obtained in good yield and excellent stereocontrol (Scheme 3).
Another interesting application of the protocol was examined
on the non-anomeric primary iodide 13 which proved much less
reactive than anomeric iodides towards substitution with thiourea,
so that a treatment at 135 ◦C in DMF was required for the first step;
subsequent exposure of the thiouronium intermediate to TEA and
phenyl diselenide (cat.) at 60 ◦C yielded disulfide 14 in high overall
yield (Scheme 3). The feasible generalization of the strategy to
non-saccharidic substrates was tested on 11-bromoundecan-1-ol
(15) and a satisfying yield of disulfide 16 was again obtained on
conducting the initial substitution step at high temperature in
DMF (Scheme 3).
Scheme 4 De-O-acetylation of symmetrical glycosyl 1,1¢-disulfides.
Biological evaluation
The effect of the deprotected symmetric disulfides 17–22 was
evaluated for in vitro cytotoxicity against two human cancer cell
lines (A375 and HepG2) (Fig. 1).
Fig. 1 Cytotoxic effect of 17–22 on A375 and HepG2 cell lines. The cells
were incubated in the presence of the compounds at 5 mM concentration
at 37 ◦C for 72 h. Statistical significance was analyzed using Student’s
t-test, paired, two-sided. All experiments were performed in triplicate and
repeated at least 3 times (* p < 0.05; ** p £ 0.005).
Scheme 3 Applications of the strategy to different substrates.
The results obtained showed that some of them highly inhibited
the proliferation of the selected cell lines. Experiments performed
at 5 mM concentration for 72 h, shown in Fig.1, indicated four
molecules (19–22) featuring an interesting cytotoxic activity, as
Symmetrical 1,1¢-disulfides 4, 7–10 and 12 were smoothly de-O-
acetylated in high yields upon exposure to methanol/aq.ammonia
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they induced a decrease of cell proliferation ranging from 20%
of 19 on HepG2 cells to 80% of 22 on A375 cells. All the active
compounds displayed a higher in vitro inhibitory activity on the
A375 cell line whereas 17 and 18 did not exhibit appreciable
activity on the cells tested. The observed cytotoxic effect was not
due to osmotic changes, as the parent mono- and disaccharides,
devoid of thioglycoside and disulfide linkages, had no effect on
cell proliferation at the same concentration (data not shown).
then phenyl diselenide (0.03 mmol, 9.4 mg) and TEA (0.55 mL,
4 mmol) were sequentially added. After 10 min the reaction vessel
was placed on an oil bath at 50 C and kept for an hour at this
temperature. The mixture was then concentrated in vacuo, the
residue dissolved in DCM, and the organic phase washed with
water. The aqueous phase was then re-extracted with DCM and
combined organic phases were dried and concentrated. Silica gel
flash-chromatography of the residue (eluent: hexane/ethyl acetate
6 : 4) yielded disulfide 4 as a foam (240 mg, 66% overall yield).
◦
Conclusions
Synthesis of symmetrical disulfides 12, 14, 16
In this paper we have developed a streamlined sequential approach
to symmetrical glycosyl disulfides which entails the generation of
an isothiouronium key intermediate and its direct elaboration in
the presence of a base as mild as TEA and minimal amounts
of (PhSe)2. Symmetrical 1,1¢-disulfides can be generally obtained
from cheap per-O-acetylated precursors in a few hours as all
the steps are fast enough (especially when intermediate glycosyl
iodides can be adopted) and no isolation of any intermediate
is required. Additionally, the overall procedure is not especially
demanding from an experimental point view, no inert atmosphere
being necessary at any stage. Some synthesized disulfides revealed
a notable cytotoxic activity against two human tumor cell lines. A
more thorough investigation aimed at establishing the mechanism
of this inhibitory effect is ongoing and the results will be reported
in due course.
The compounds were prepared following the same procedures as
above described, with differences being essentially in the solvents
used, in the temperatures at which the steps are performed and in
the corresponding reaction times. These parameters are indicated
in each example of Scheme 3.
Bis(2,3,4,6-tetra-O-acetyl-1-deoxy-1-thio-b-D-galactopyrano-
syl) 1,1¢-disulfide 4bb6c. Rf 0.4 (3 : 2, ethyl acetate/n-hexane);
[a]D -7.6 (c 0.90, CHCl3); Found: C, 46.45; H, 5.20. Calcd. for
1
C28H38O18S2: C, 46.28; H, 5.27%); H NMR (500 MHz, CDCl3):
d 5.45 (H-4, bd, J = 3.5 Hz, 1 H), 5.36 (H-2, t, J = 10.0 Hz, 1 H),
5.09 (H-3, dd, J = 3.5 and 10.0 Hz, 1 H), 4.58 (H-1, d, J = 10.0 Hz,
1 H), 4.23 (H-6a, dd, J = 6.5 and 11.5 Hz, 1 H), 4.11 (H-6b, dd, J =
6.5 and 11.5 Hz, 1 H), 4.05 (H-5, bt, J = 6.5 Hz, 1 H), 2.18, 2.09,
2.05, and 1.99 (4 ¥ –COCH3, 4¥ s, 12 H). 13C NMR (125 MHz,
CDCl3): d 169.9, 169.8, 169.6, and 168.9 (-COCH3), 87.7 (C-1),
74.3 (C-5), 71.4 (C-3), 67.2, 66.8 (C-2 and C-4), 60.6 (C-6); 20.2
(-COCH3). MALDI-MS [M + Na]+, found 749.10, calc. 749.15.
Experimental
General methods
1H and 13C NMR spectra were recorded in CDCl3 (internal
Bis(2,3,4,6-tetra-O-acetyl-1-deoxy-1-thio-b-D-glucopyranosyl)
1
standard, for H: CHCl3 at d 7.26; for 13C: CDCl3 at d 77.0)
1,1¢-disulfide 7bb4f,5
.
Rf 0.4 (3 : 2, ethyl acetate/n-hexane); [a]D
1
or D2O. H NMR assignments were based on homo-decoupling
-96.8 (c 1.49, CHCl3); Found: C, 46.40; H, 5.20. Calcd. for
C28H38O18S2: C, 46.28; H, 5.27%); 1H NMR (500 MHz, CDCl3): d
5.26 (H-3, t, J = 9.5 Hz, 1H), 5.19 (H-4, t, J = 9.8 Hz, 1H), 5.09
(H-2, t, J = 9.5 Hz, 1H), 4.65 (H-1, d, J = 9.5 Hz, 1H), 4.33 (H-6a,
d, J = 4.5 and 12.5 Hz, 1H), 4.22 (H-6b, d, J = 2.0 and 12.5 Hz,
1H), 3.78 (H-5, ddd, 1H), 2.13, 2.10, 2.03, and 2.00 (4¥ –COCH3,
4¥ s, 12H). 13C NMR (125 MHz, CDCl3): d 170.5, 169.8, 169.1
and 169.0 (-COCH3), 86.7 (C-1), 75.7 (C-5), 73.5 (C-3), 69.3 (C-
2), 67.6 (C-4), 61.3 (C-6); 20.4-20.2 (-COCH3). MALDI-MS [M +
Na]+, found 749.25, calc. 749.15.
experiments. MALDI-MS spectra were recorded in the positive
mode: compounds were dissolved in acetonitrile at a concentration
of 0.1 mg mL-1 and one microlitre of these solutions was
mixed with one microlitre of a 20 mg mL-1 solution of 2,5-
dihydroxybenzoic acid in 7 : 3 CH3CN/H2O. Analytical thin layer
chromatography (TLC) was performed on aluminium plates pre-
coated with Silica Gel 60 F254 as the adsorbent. The plates were
developed with 5% H2SO4 ethanolic solution and then heating
◦
to 130 C. Column chromatography was performed on silica gel
(63–200 mesh). [a]D values are given in 10-1 deg cm2 g-1.
Bis(2,3,4,6-tetra-O-acetyl-1-deoxy-1-thio-D-mannopyranosyl)
1,1¢-disulfide 8 (aa/ab ca 1 : 1). Rf 0.45 (3 : 2, ethyl acetate/n-
hexane); 1H NMR (500 MHz, CDCl3): significant signals of 8aa
anomer at d 5.40 (H-2, dd, J = 2.0 and 3.0 Hz, 1 H), 5.26 (H-1, d,
J = 2.0 Hz, 1 H), 5.25 (H-4, t, J = 10.0 Hz, 1 H), 5.18 (H-3, dd,
J = 3.0 and 10.0 Hz, 1 H), 2.09, 2.04, 1.99, 1.94 (4¥ –COCH3, 4¥ s,
12 H). Significant signals of 8ab anomer at d 5.51 (H-2 b, bd, J =
3.5 Hz, 1H), 5.42 (H-2 a, bd, J = 1.5 and 3.0 Hz, 1H), 5.33 (H-1 a,
d, J = 1.5 Hz, 1 H), 5.11 (H-3 a, dd, J = 3.5 and 10.0 Hz, 1 H), 5.01
(H-3 b, dd, J = 3.5 and 10.0 Hz, 1 H), 4.93 (H-1 b, bs, 1 H), 2.12,
2.08, 2.05, 2.02, 1.99, 1.97, 1.92 and 1.90 (8¥ –COCH3, 8¥ s, 24
H). 13C NMR (125 MHz, CDCl3): significant signals of 8aa at d
87.2 (C-1), 70.7, 69.4, 68.6 (C-2, C-3, C-5), 65.6 (C-4), 61.7 (C-6);
20.6-20.3 (-COCH3). Significant signals of 8ab at d 89.4, 89.1 (C-1
a and b), 76.6 (C-5 b), 71.4, 70.3, 69.5 (¥2), 68.9 (C-5 a, C-2 and
C-3 a and b), 65.9, 64.9 (C-4 a and b), 62.2, and 61.8 (C-6 a and
b). MALDI-MS [M + Na]+, found 749.30, calc. 749.15.
General procedure for preparation of symmetrical disulfides 4, 7–10
To a solution of penta-O-acetyl galactose (390 mg, 1 mmol) in
anhydrous DCM (2 mL) was added I2 (356 mg, 1.4 mmol), and
Et3SiH (225 mL, 1.4 mmol) (caution: exothermic reaction). The
system was refluxed until complete consumption of the starting
material (5–10 min as monitored by TLC). The mixture was
then diluted with DCM and the organic phase washed with aq.
sodium carbonate containing sodium thiosulfate (this latter is
added portionwise until consumption of residual iodine in the
organic phase). The organic phase was then washed with water,
dried, and concentrated. Thiourea (114 mg, 1.5 mmol) was added
to the crude residue and the mixture suspended in acetonitrile
(2 mL), and then heated to 60 ◦C. After 15–30 min TLC analysis
evidenced complete consumption of the glycosyl iodide and the
generation of a polar product. The vessel was cooled to rt and
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Bis(2,3,4,6-tetra-O-acetyl-1-deoxy-1-thio-b-L-fucopyranosyl)
1,1¢-disulfide 9bb. Rf 0.55 (3 : 2, ethyl acetate/n-hexane); [a]D
-15.9 (c 1.93, CHCl3); Found: C, 47.45; H, 5.70. Calcd. for
H), 4.34 (H-4, dd, J = 1.5 and 7.5 Hz, 1 H), 4.30 (H-2, dd, J = 2.5
and 4.5 Hz, 1 H), 4.06 (H-5, dt, J = 1.5 and 7.0 Hz, 1 H), 2.92 (H2-
6, d, J = 7.0 Hz, 2 H), 1.55, 1.43, 1.33, and 1.32 (acetonide -CH3,
4¥ s, 12H). 13C NMR (125 MHz, CDCl3): d 109.3 and 108.8 (2¥
-C(CH3)2), 96.6 (C-1), 71.5 (C-4), 70.9 (C-3), 70.5 (C-2), 66.7 (C-
5), 38.2 (C-6); 26.0, 25.9, 25.0 and 24.4 (-C(CH3)2). MALDI-MS
[M + Na]+, found 573.25, calc. 573.35.
1
C24H34O14S2: C, 47.21; H, 5.61%; H NMR (500 MHz, CDCl3):
d 5.29 (H-2, t, J = 10.0 Hz, 1 H), 5.24 (H-4, bd, J = 3.0 Hz, 1 H),
5.04 (H-3 dd, J = 3.5 and 10.0 Hz, 1 H), 4.60 (H-1, d, J = 10.0 Hz,
1 H), 3.87 (H-5, bq, J = 6.0 Hz, 1 H), 2.16, 2.06, 1.96 (3¥ –COCH3,
3¥ s, 9 H), 1.21 (H3-6, J = 6.0 Hz, 3 H).13C NMR (50 MHz, CDCl3):
d 170.5, 170.0, 169.4 (-COCH3), 89.5 (C-1), 73.6 (C-5), 72.2 (C-3),
70.2, 67.6 (C-2 and C-4); 20.7, 20.6, and 20.5 (-COCH3), 16.3 (C-
6). MALDI-MS [M + Na]+, found 633.30, calc. 633.14. Significant
data of 9ab: 1H NMR (400 MHz, CDCl3) at d 5.78 (H-1 a, d, J =
5.6 Hz, 1 H), 5.30–5.25 (overlapped signals, 2H), 5.23 (H-4b, bd,
J = 3.2 Hz, 1 H), 5.20-5.14 (overlapped signals, 2 H), 5.01 (H-3 b
dd, J = 2.8 and 9.8 Hz, 1 H), 4.53 (H-1 b, d, J = 10.0 Hz, 1 H),
4.32 (H-5 a, bq, J = 6.4 Hz, 1 H), 4.32 (H-5 b, bq, J = 6.4 Hz, 1
H), 2.16 (¥2), 2.07, 2.05, 1.98, 1.96 (6¥ –COCH3, 5¥ s, 18 H), 1.22
and 1.18 (H3-6 a and b, 2¥ d, J = 6.4 Hz, 6 H). NMR (100 MHz,
CDCl3): d 170.5, 170.4, 170.0, 169.83, 169.78, 169.6; 90.8 and 88.9
(C-1 a and b), 73.3 (C-5 b), 72.0 (C-3 b), 70.6, 70.1, 68.2, 68.1,
67.3, 66.2 (C-2 and C-4 a and b, C-3 and C-5 a); 20.7, 20.6, 20.5
(-COCH3).
Bis(11-hydroxyundecanyl) disulfide 16. Rf 0.35 (3 : 2, n-
hexane/ethyl acetate); Found: C, 65.10; H, 11.570. Calcd. for
1
C22H46O2S2: C, 64.97; H, 11.40%; H NMR (200 MHz, CDCl3):
d 3.62 (t, J = 6.4 Hz, 2H), 2.67 (t, J = 6.4 Hz, 2H), 1.80–1.10
(overlapped signals, 18H). 13C NMR (50 MHz, CDCl3): d 63.0
(C-1), 39.2 (C-11), 32.7, 29.5, 29.4 (¥2), 29.3, 29.2 (¥2), 28.5, 25.7
(from C-2 to C-10). MALDI-MS [M + Na]+, found 429.20, calc.
429.28.
General procedure for de-O-acetylation of symmetrical
1,1¢-disulfides
O-Acetylated 1,1¢-disulfides were dissolved at rt in 3 : 1 MeOH/32
aq. ammonia (1.0–1.5 mL/10 mg of substrate). After 3–4 h TLC
analysis (eluent AcOEt/MeOH mixtures) typically displayed com-
pletion of the reaction and the solvent was removed in vacuo. For
convenience, some deprotections were occasionally left overnight.
The residue was purified by silica gel flash-chromatography (eluent
AcOEt/MeOH mixtures, HPLC grade solvents) to yield pure
disulfides 17–22.
Bis[2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2,3,6-
tri-O-acetyl-1-deoxy-1-thio-b-D-glucopyranosyl)] 1,1¢-disulfide
10bb4d. Rf 0.45 (4 : 1, ethyl acetate/n-hexane); [a]D -41.4 (c 1.52,
CHCl3); Found: C, 47.75; H, 5.50. Calcd. for C52H70O34S2: C,
47.92; H, 5.41%); 1H NMR (400 MHz, CDCl3): d 5.33 (H-4¢, bd,
J = 2.8 Hz, 1H), 5.23 (H-3, t, J = 9.2 Hz, 1H), 5.11 (H-2¢, dd, J =
8.4 and 10.4 Hz, 1H), 5.05 (H-2, t, J = 10.0 Hz, 1H), 4.96 (H-3¢,
dd, J = 2.8 and 10.4 Hz, 1H), 4.61 (H-6a, bd, J = 12.0 Hz, 1H),
4.57 (H-1¢, d, J = 8.0 Hz, 1H), 4.54 (H-1, d, J = 10.0 Hz, 1H),
4.20–4.00 (H-6b and H2-6, overlapped signals, 3H), 3.89 (H-5¢,
bt, J = 6.8 Hz, 1H), 3.69 (H-5, m, 1H), 2.16, 2.14, 2.05, 2.04
(¥2), 2.03, 1.96 (7¥ –COCH3, 6¥ s, 21H). 13C NMR (100 MHz,
CDCl3): d 170.2, 170. 1 (¥2), 170.0, 169.6, 169.3, 169.0 (-COCH3),
100.7 (C-1), 86.6 (C-1¢), 76.9 (C-5), 75.6 (C-4), 73.7 (C-3), 70.9,
70.5, 70.0, 69.0 (C-2¢, C-3¢, C-5 and C-2), 66.5 (C-4¢), 61.5,
60.5 (C-6 and C-6¢); 20.8, 20.7, 20.6, 20.5 (¥3), 20.4 (-COCH3).
MALDI-MS [M + Na]+, found 1325.45, calc. 1325.32.
Bis(1-deoxy-1-thio-b-D-galactopyranosyl) 1,1¢-disulfide 173. Rf
0.15 (3 : 2, ethyl acetate/methanol); 1H NMR (500 MHz, D2O): d
4.57 (H-1, d, J = 9.0 Hz, 1 H), 4.00 (H-4, d, J = 3.0 Hz, 1 H), 3.85
(H-4, t, J = 9.0 Hz, 1 H), 3.85–3.75 (overlapped signals, 3H), 3.70
(H-3, dd, J = 3.0 and 9.0 Hz, 1 H). 13C NMR (100 MHz, D2O):
d 89.9 (C-1), 79.6 (C-5), 73.9 (C-3), 68.8, 68.6 (C-2 and C-4), 61.1
(C-6). MALDI-MS [M + Na]+, found 412.98, calc. 413.06.
Bis(1-deoxy-1-thio-b-D-glucopyranosyl) 1,1¢-disulfide 183. Rf
0.15 (3 : 2, ethyl acetate/methanol); 1H NMR (400 MHz, D2O): d
4.61 (H-1, d, J = 10.0 Hz, 1 H), 3.93 (H-6a, dd, J = 2.0 and 12.0 Hz,
1 H), 3.75 (H-6b, dd, J = 2.0 and 6.0 Hz, 1 H), 3.62 (t, J = 9.6 Hz,
1 H), 3.55 (t, J = 10.0 Hz, 1 H), 3.52 (H-5, m, 1 H), 3.44 (t, J =
9.6 Hz, 1 H). 13C NMR (100 MHz, D2O) d 89.4 (C-1), 80.2 (C-5),
77.0, 71.1, 69.1 (C-3, C-2 and C-4), 60.8 (C-6). MALDI-MS [M +
Na]+, found 412.95, calc. 413.06.
Bis(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-1-deoxy-1-thio-b-
glucopyranosyl)
1,1¢-disulfide
12bb4d. Rf
0.45
(92 : 8,
dichloromethane/methanol); [a]D -72.7 (c 1.0, CHCl3); Found:
C, 46.55; H, 5.45. Calcd. for C28H40N2O16S2: C, 46.40; H, 5.56%;
1H NMR (500 MHz, CDCl3): d 6.24 (NH-2, d, J = 9.0 Hz, 1 H),
5.39 (H-3, t, J = 10.0 Hz, 1 H), 5.00 (H-4, t, J = 10.0 Hz, 1 H), 4.87
(H-1, d, J = 10.5 Hz, 1 H), 4.43 (H-6a, dd, J = 5.5 and 12.5 Hz,
1 H), 4.07 (H-6b, dd, J = 1.5 and 12.5 Hz, 1 H), 4.00 (H-2, m,
1 H), 3.77 (H-5, m, 1 H); 2.12, 2.02 (¥2), 2.00 (4¥ –COCH3,
4¥ s, 12H). 13C NMR (125 MHz, CDCl3): d 170.9, 170.6 (¥2)
and 169.5 (-COCH3), 88.7 (C-1), 76.0 (C-5), 72.9 (C-3), 68.4
(C-4), 62.0 (C-6), 53.3 (C-2); 23.3, 20.9, and 20.6 (¥2) (-COCH3).
MALDI-MS [M + Na]+, found 747.30, calc. 747.18.
Bis(1-deoxy-1-thio-D-mannopyranosyl) 1,1¢-disulfide 19 (aa/ab
ca 1 : 1)3. Rf 0.15 (3 : 2, ethyl acetate/methanol); Isomer 19
aa,3 1H NMR (400 MHz, D2O): d 4.20 (H-2, bs, 1 H), 3.95–
3.85 (m, 2 H), 3.85–3.70 (m, 3 H). Significant signals of the ab
diastereoisomer at d 5.45 (H-1 a, bs, 1H), 5.07 (H-1 b, bs, 1H).
13C NMR (100 MHz, CDCl3) d 90.0 (C-1), 74.6, 70.8 (¥2) (C-2,
C-3 and C-5), 66.8 (C-4), 60.6 (C-6). Significant signals of the ab
diastereoisomer at d 92.4, 90.6 (C-1 b and a). MALDI-MS [M +
Na]+, found 412.96, calc. 413.06.
Bis(1-deoxy-1-thio-b-L-fucopyranosyl) 1,1¢-disulfide 20. Rf
0.45 (65 : 35, ethyl acetate/methanol); Found: C, 40.35; H, 6.10.
Calcd. for C12H22O8S2: C, 40.21; H, 6.19%; 1H NMR (400 MHz,
D2O): d 4.50 (H-1, d, J = 9.6 Hz, 1 H), 3.81 (H-5, bq, J = 6.4 Hz,
1 H), 3.75 (H-4, bs, 1 H), 3.71 (H-2, t, J = 9.6 Hz, 1 H), 3.64 (H-3,
bd, J = 9.6 Hz, 1 H), 1.23 (H3-6, J = 6.0 Hz, 3 H). 13C NMR
Bis(6-deoxy-6-thio-1,2;3,4-di-O-isopropylidene-D-galactopyra-
nose) 6,6¢-disulfide 1418. Rf 0.40 (3 : 2, n-hexane/ethyl acetate);
[a]D -75.3 (c 1, CHCl3); Found: C, 52.45; H, 6.85. Calcd. for
C24H38O10S2: C, 52.35; H, 6.96%); 1H NMR (500 MHz, CDCl3): d
5.51 (H-1, d, J = 4.5 Hz, 1 H), 4.61 (H-3, dd, J = 2.5 and 7.5 Hz, 1
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(100 MHz, D2O): d 90.0 (C-1), 75.5 (C-5), 73.8 (C-3), 71.4, 68.3
(C-2 and C-4), 15.6 (C-6). MALDI-MS [M + Na]+, found 380.98,
calc. 381.07.
University (“Programma FARO 2009”). NMR and MS fa-
cilities of the “Centro Interdipartimentale di Metodolo-
gie Chimico-Fisiche dell’Universita` di Napoli” (CIMCF) are
acknowledged.
Bis[b-D-galactopyranosyl-(1→4)-1-deoxy-1-thio-b-D-glucopyra-
nosyl)] 1,1¢-disulfide 2119. Rf 0.20 (3 : 2, methanol/ethyl acetate);
1H NMR (400 MHz, D2O): d 4.63 (H-1¢, d, J = 8.8 Hz, 1 H), 4.49
(H-1, d, J = 7.8 Hz, 1 H), 3.99 (H-6a, bd, J = 11.2 Hz, 1 H), 3.94
(H-4¢, d, J = 3.2 Hz, 1 H), 3.85–3.70 (overlapped signals, 9H), 3.56
(H-2¢, dd, J = 9.6 and 7.8 Hz, 1 H). 13C NMR (100 MHz, D2O): d
102.8 (C-1¢), 89.2 (C-1), 79.0, 77.6, 75.5, 75.3, 72.5, 70.9 (¥2) (C-2¢,
C-3¢, C-5¢, C-2, C-3, C-4 and C-5), 68.5 (C-4¢), 61.0, 60.0 (C-6 and
C-6¢). MALDI-MS [M + Na]+, found 737.55, calc. 737.70.
Notes and references
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Acknowledgements
This work was supported by a grant from Italian Ministry
of University (PRIN 2008 project) and a grant from Naples
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