Assembly of a pentasaccharide repeating unit corresponding to
extracellular polysaccharide S-88
Having achieved success in synthesizing L-glycosyl fluoride from the
corresponding D-sugars, our focus now shifts towards the application of
this method in the synthesis of intricate oligosaccharides obtained fromPseudomonas ATCC 31554 extracellular polysaccharide (S-88). S-88
is a representative gellan which shows potential in food, chemical, and
pharmaceutical industries due to its functional
characteristics.[22] Furthermore, recent
discoveries have revealed the probiotic potential of gellan
oligosaccharides derived from the hydrolysis of S-88, as they have the
capability to modulate gut flora in a manner that promotes human
health.[23] S-88 is composed of a pentasaccharide
repeating unit made up of a backbone of →4)-α-L-mannopyranosyl
(L-Manp )-(1→3)-β-D-glucopyranosyl
(Glcp )-(1→4)-β-D-glucuronic acid
(Glcp A)-(1→4)-β-D-Glcp -(1→ and a branch of
α-L-rhamnopyranosyl (Rhap ) appended to C3-OH of D-Glcp at
the reducing end.[24] The occurrence of
L-Manp moiety and of D-Glcp A residue linked to the
sterically hindered C4-OH of D-Glcp makes S-88 an attractive, but
synthetically challenging target. The pursuit of comprehending the
intricate structure and functionalities of S-88 has driven our
motivation to engage in chemical syntheses of S-88 oligosaccharides.
Herein, we report the synthesis of a pentasaccharide fragment
(7 ) corresponding to the repeating unit of extracellular
polysaccharide S-88. The synthesis features the successful construction
of challenging glycosidic linkage by which D-Glcp A is appended to
D-Glcp and the efficient installation of L-mannosyl moiety with
L-mannosyl fluoride as the glycosyl donor. L-mannosyl fluoride was
prepared from D-galactose via a head-to-tail inversion strategy.
Highly efficient incorporation of uronic acid residues is crucial for
assembly of oligosaccharides containing such units. Typically, two
strategies are employed to accomplish this
objective.[25] The first approach involves the
postglycosylation oxidation strategy, wherein an oligosaccharide
backbone is constructed before converting a sugar into its uronic acid
form through oxidation. Despite additional protecting group
manipulations required, the advantages of this approach lie in the
higher reactivity of non-oxidized building blocks than the corresponding
carboxylate counterparts, deceased side reactions associated with
epimerization α to carboxylate, and β-elimination leading to the
formation of 4-deoxy-hex-4-enopyranuronic
acid.[26] The second is preglycosylation oxidation
strategy, that is, directly using a uronic acid-based building block as
glycosylating donor and acceptor. Its strength is to prevent oxidation
event on a complex setting. However, the presence of carboxylate
substituent imposes challenges to glycosylation reaction due to the
withdrawing-electron effect of carboxylate resulting in decreased
reactivity of the building blocks as glycosyl donor and
acceptor.[27] Mindful of these considerations, we
designed two retrosynthetic plans for the target pentasaccharide7 . As depicted in Scheme 3, we envisioned that the target
molecule 7 could be obtained from the fully protected
pentasaccharide 8 by a global deprotection. Glycan 8in turn was planned to assemble by glycosylation of L-mannopyranosyl
fluoride 9 with tetrasaccharide either 10 or11 . Compound 9 could be traced back to readily
available 1-phenyl-2-(β-D-C -galactossyl) ethanone12 [18]. Fluoride 9 is designed
to possess a benzoyl at C2-OH to ensure the formation of
1,2-trans
Scheme 3 Retrosynthetic analysis of pentasaccharide fragment
(7 ) of extracellular polysaccharide S-88
Scheme 4 Synthesis of L-mannosyl fluoride 9
Reagents and conditions: (a) NaBH4, MeOH, ice
bath; (b) 2,6-lutidine, Tf2O,
CH2Cl2, 70% over two steps; (c) MeONa,
MeOH; (d) PhCH(OMe)2, CSA, 40°C, CH3CN;
(e) BnBr, KOH, 18-Crown-6, THF, 72% over three steps; (f)
OsO4, 2,6-lutidine, NaIO4,
1,4-dioxane/H2O; (g) NaBH4, MeOH, 73%
over two steps; (h) BzCl, DMAP, pyridine; (i)p -TsOH·H2O,
CH2Cl2/MeOH, 79% over two steps; (j)
TBSCl, DMAP, pyridine; (k) BzCl, DMAP, pyridine; (l) 70% HF·pyridine,
CH3CN, 76% for three steps; (m) TEMPO, BAIB,
CH2Cl2/H2O, 93%; (n)
Selectfluor, KF·2H2O,
Ag2CO3, acetone/H2O,
88%. CSA = Camphorsulfonic acid; THF = Tetrahydrofuran; TBSCl =tert -Butyldimethylsilyl chloride.
glycosidic bonds through neighboring group participation. Given the
strength and weakness of post- or preglycosylation oxidation strategy,
tetrasaccharides either 10 or 11 embedded by
GlcAp residue could be constructed by coupling reaction of
disaccharide acceptor 13 with either uronic acid-based
disaccharide donor 14 or the corresponding non-oxidized donors15 . These disaccharides 13 , 14 , and15 could be disconnected to D-glucosyl thioglycoside16 and L-rhamnosyl trichloroacetimidate (TCAI) 17 .
Glucosyl thioglycoside 16 was designed to allow for direct
incorporation of rhamnosyl residue at C3-OH while
4,6-O -benzylidene would facilitate the incorporation of
carboxylate group and the glycosylation of C4-OH by the hydrolysis of
acetal functionality and subsequent differentiation between the primary
alcohol and the secondary one. The presence of 2-O -benzoyl (Bz)
substituent in 16 could enable anchimerically assisted
glycosylation leading to β-glucosidic linkage formation. The
incorporation of a spacer 6-amino-hexanoxyl at the reducing end would
provide feasibility for conjugation of 7 with biomolecules such
as carrier protein. Orthogonal levulinoyl (Lev) group on the
disaccharide fragment either 14 or 15 allows for its
chemoselective removal and ensuing glycosylation at that site.
Our synthesis began with the preparation of rare L-mannosyl fluoride9 following a head-to-tail switch strategy (Scheme 4). Similar
to the synthesis of 6a and 6b , C -galactoside12 was converted to vinyl C -glycoside 18 in
70% yield over two steps. To differentiate the 4-OH and the 6-OH from
the 2,3-diol for late-stage equipment of benzoyl group at the C4-OH and
the formation of carboxylic acid, 18 was converted into19 in 72% yield over three steps involving deacetylation, the
benzylidene protection of 4,6-diol, and the benzylation of 2,3-diol. The
oxidative cleavage of C═C double bond in 19 followed by the
reduction of aldehyde group led to the introduction of hydroxymethyl at
the anomeric position, providing 73% of 20 over two steps.
Benzoyl protection of 20 coupled with hydrolysis of benzylidene
produced diol 21 in 79% overall yield. Selective benzoyl
protection of C4-OH was smoothly realized leading to 22 in 76%
overall yield through TBS protection of the primary hydroxy group, the
benzoylation of the remaining secondary hydroxy group, and the cleavage
of TBS ether with hydrogen fluoride-pyridine complex. Uronic acid23 , prepared by oxidation of 22 in 93% yield, was
exposed to Ag2CO3-mediated
decarboxylative fluorination to give rise to the required α-L-mannosyl
fluoride 9 in 88% yield as the sole product.
Having successfully prepared the L-mannosyl fluoride 9 , we
turned to constructing the tetrasaccharide 10 . To this end,
disaccharide acceptor 13 was first made. As shown in Scheme 5a,
Scheme 5 Synthesis of disaccharide acceptor 13 as well
as donors 14 and 28
Reagents and conditions: (a) TMSOTf, -40 °C,
CH2Cl2, 4 Å MS, 87%; (b)
Et3SiH, TFA, 5 Å MS,
CH2Cl2, 91%, (c)
HO(CH2)6N3, NIS, AgOTf,
4 Å MS, -20 °C, CH2Cl2, 70%; (d)
BF3·Et2O, -40 °C,
CH2Cl2, 4 Å MS, 77%; (e) TCCA,
H2O, ice bath, acetone; (f) o -hexynylbenzoic
acid, DCC, DMAP, THF, 70% over two steps. TMSOTf = Trimethylsilyl
trifluoromethanesulfonate; MS = Molecular sieve; NIS =N -Iodosuccimide; AgOTf = Silver triflate; TCCA =
Trichloroisocyanuric acid; DCC = Dicyclohexylcarbodiimide; n -Bu =n -butyl.
Glucose-derived alcohol 16 [27] was
glycosylated with Bz-masked L-rhamnosyl TCAI17 [28]. The coupling reaction proceeded
smoothly and delivered the desired disaccharide 24 in 81%
yield in CH2Cl2 at -40oC under the catalysis of 0.1 equiv of TMSOTf.
Reductive ring- opening of the benzylidene in 24 with TFA and
Et3SiH in the presence of 5 Å molecular sieves resulted
in the formation of 25 with C4’-OH free in 91% yield.
Reactivity-based chemoselective glycosylation of
6-azido-1-hexanol[29] with 25 produced
disaccharide glycoside 13 in 70% yield under the combined
promotion of NIS and AgOTf. The reaction left C4’-OH intact, which is
ready for sugar chain elongation at this site.
With 13 in hand, the application of preglycosylation oxidation
strategy was first explored in construction of tetrasaccharide10 . We therefore embarked on the preparation of uronic
acid-based disaccharide 14 . As outlined in Scheme 5b,
BF3·Et2O-catalyzed glycosylation of26 [30] with27 [31] supplied 77% of disaccharide14 in CH2Cl2 in the presence of
4 Å MS. At this stage, the coupling reaction of 14 and13 was explored. As tabulated in Table 1, we were disappointed
to find that the reaction under the promotion of NIS with either TfOH or
Lewis acids such as AgOTf and TBSOTf supplied the desired
tetrasaccharide 10 in the best yield up to 23% (Table 1,
entries 1–5). Gold-catalyzed glycosylation of challenging nucleophiles
with glycosyl ortho -alkynylbenzoate donors has
Table 1 Optimization of glycosylation reactions of acceptor13 with donor 14 or28