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yield over 15 steps. The purification of polymannosides longer
than 12mers (5, 6, 7, and 8) was more challenging owing to
their changing solubility. As the crude products dissolved only
in chlorinated solvents, reverse-phase, normal phase, and size
exclusion HPLC became very challenging.
A cap-and-tag strategy was adopted to facilitate polysac-
charide purification. Peptides, oligonucleotides, and oligosac-
charides have been purified using affinity chromatography
that relies on labels such as biotin[10] and oligohistidine.[11] This
strategy cannot be adopted for the purification of protected
oligomannosides since aqueous solvents are required as
eluents. Fluorous tags have helped to separate oligonucleo-
tides up to 100mers[12] using fluorous solid-phase extraction
and liquid–liquid extraction.[13] Solubility issues associated
with fluorous labels rendered them useless in the context of
the isolation of longer a-(1,6)-oligomannosides. Therefore,
we considered capture–release techniques[14] that rely on the
covalent attachment of the labeled target molecule to a solid
support to separate the desired oligosaccharide from any
deletion sequences. A capping step was included in the
synthesis cycle to block any unreacted hydroxy groups prior
to the following glycosylation reaction. After completing the
oligosaccharide sequence, the tag was attached to the C6
hydroxy group, thus allowing facile separation of the desired
product from the deletion sequences and byproducts.
A successful catch–release approach applied to auto-
mated solid phase oligosaccharide synthesis requires a fast
and efficient capping reaction. The caps introduced after each
glycosylation have to be stable during subsequent synthetic
steps and the tag has to contain a unique handle for facile
separation of the product. Based on these considerations,
acetylation was used for capping and the full length oligo-
saccharide was tagged as a 6-amino caproic acid ester. The
unique amino group facilitates isolation of the desired
oligosaccharide by attachment to magnetic beads decorated
with carboxylic acid moieties.
This cap–tag strategy was first evaluated in the context of
the automated synthesis of a-(1,6)-dimannoside 13
(Scheme 2). Utilizing the automated procedure, a capping
step employing acetic anhydride in pyridine was incorporated
into the synthetic cycle. Following completion of the auto-
mated synthesis, building block 10, which is equipped with an
amino caproic ester at the C6 position, was used. The
disaccharide product was cleaved from the resin in continuous
flow by exposure to UV light before the crude product
mixture was reacted with magnetic beads functionalized with
NHS-activated carboxylic acid.[15]
Scheme 2. Catch–release purification applied to the automated solid-
phase synthesis of a-(1,6)-dimannoside 13. Reactions and conditions:
a) glycosylation: 1 or 10, TMSOTf, CH2Cl2, À158C (45 min)–08C
(15 min); b) capping: Ac2O, pyridine 258C (60 min); c) Fmoc depro-
tection: piperidine, DMF, 258C (5 min); d) cleavage from solid sup-
port: hn, CH2Cl2; e) immobilization on magnetic beads: 1. NEt3,
CH2Cl2, 258C; 2. washing with CH2Cl2 to remove deletion sequences
of the type X and Y; f) release from magnetic beads: NaOMe, MeOH;
g) Pd/C, H2, H2O. Ac=acetyl.
The unique amine group on the disaccharide resulted in
covalent attachment to the beads before washing with
dichloromethane and methanol. Treatment of the magnetic
beads with sodium methoxide in methanol released depro-
tected disaccharide 12 that was purified by gel filtration
chromatography to afford 12 in 22% yield over nine steps.
Cleavage of the benzyl carbamate by hydrogenation provided
disaccharide 13 in 74% yield.
To improve the coupling efficiency of the target saccha-
ride to the activated magnetic beads and thus the overall
yield, a second coupling step was introduced. Any oligosac-
charides remaining after the first coupling were reacted with
the magnetic beads from the first “release” in the presence of
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate and N,N-diisopropylethylamine (Scheme 3).
The automated synthesis of the dodecamanoside pro-
vided, after gel filtration chromatography, deprotected 12mer
16 in 13% overall yield without the need for HPLC
purification. Removal of the Cbz protecting group by hydro-
genolysis on Pd/C in water provided a-(1,6)-dodecamanno-
side 18 in 62% yield.
The automated synthesis of an a-(1,6)-oligomannoside
30mer presented the next challenge as this polysaccharide is
2
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!
Calin, Oliviana
