Synthesis of a series of maltotriose phosphates with an evaluation of the utility of a fluorous phosphate protecting group

Article abstract of DOI:10.1016/j.carres.2012.12.015

A series of methyl maltotrioside phosphates were synthesized for application in the determination of the actual molecular substrate of the Lafora enzyme involved in Lafora disease. Several different synthetic routes were applied for the successful synthesis of six methyl maltotrioside phosphate regioisomers. The utility of a new fluorous phosphate protecting group was also evaluated, but its utility was found to be limited in this particular late stage introduction.

Full text of DOI:10.1016/j.carres.2012.12.015

Carbohydrate Research 369 (2013) 14–24
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a series of maltotriose phosphates with an evaluation
of the utility of a fluorous phosphate protecting group
Lin Liu a, Nicola L. B. Pohl a,b,
⇑
a Department of Chemistry, Department of Chemical and Biological Engineering, and the Plant Sciences Institute, Hach Hall, Iowa State University, Ames, IA 50011-3111, USA
b Department of Chemistry, Simon Hall, Indiana University, Bloomington, IN 47405, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 October 2012
Received in revised form 13 December 2012
Accepted 15 December 2012
Available online 24 December 2012
A series of methyl maltotrioside phosphates were synthesized for application in the determination of the
actual molecular substrate of the Lafora enzyme involved in Lafora disease. Several different synthetic
routes were applied for the successful synthesis of six methyl maltotrioside phosphate regioisomers.
The utility of a new fluorous phosphate protecting group was also evaluated, but its utility was found
to be limited in this particular late stage introduction.
Ó 2013 Published by Elsevier Ltd.
Keywords:
Maltotriose
Phosphate
Glycosylation
Lafora disease
Fluorous assisted synthesis
1. Introduction
Although the synthesis of a-glucans has been extensively stud-
ied and reviewed,10 the synthesis of maltose analogs with phos-
phate at the 2-or 3-hydroxyl of a sugar ring remains relatively
a-Glucans, including starch and glycogen, are important in bio-
logical systems functioning as energy storage.1 A series of specific
enzymes are involved in the synthesis of these glucan polymers
and determine their structures and properties.2 Mistakes in this
polymer biosynthesis can lead to problems such as Lafora disease,3
or Lafora progressive myoclonic epilepsy—a fatal disease with no
known cure or treatment.4 Hyperphosphorylation of glycogen is
the cause of Lafora disease and it is related to mutations in the
genes that code for the protein laforin. In the synthesis of glycogen,
phosphates are added to the 2- or 3-position hydroxyls of glucose
at a rate of one phosphate per every 10,000 glucose residues.5,6
Laforin should remove those phosphate groups subsequently.7
However, with the mutated laforin, this dephosphorylation reac-
tion cannot take place and eventually Lafora disease sets in.8,9 To
date, the actual molecular mechanism of the laforin enzyme is still
unknown. In order to gain a detailed understanding of enzyme
function, especially its positional requirements for phosphoryla-
tion of a glucose residue, molecules with well-defined chemical
structures were needed as substrates. To this end, a series of mal-
totriose analogs with phosphate groups at one of either the 2- or 3-
position hydroxyl was designed and synthesized as substrates for
the dephosphorylation enzyme (Fig. 1).
understudied. The desired 1,4-a-glycosidic linkage generally re-
quires non-participating protecting groups at the 2-position of
the donor, which would need to be cleaved selectively later to in-
stall the phosphate at the 2-position. The temporary protecting
group at the 3-position should also be chosen carefully, since the
protecting group could also affect the glycosylation reaction itself.
Ideally, the synthesis could be achieved via a set of orthogonal
protecting groups, preferably using six protecting groups that
could be cleaved under conditions that do not affect any of the
other groups. This would allow us to synthesize the six desired
compounds from a single precursor, thus greatly simplifying the
synthesis. However, the limited choice of non-participating pro-
tecting groups is a major challenge of that task when material is
needed in a short time frame. The other difficulty in using orthog-
onal protecting groups is the lack of convenient methods to selec-
tively install an ether protecting group at the 2-position. In
thinking about an efficient strategy to form all six desired regioi-
somers under these constrains, we also considered the possible
use of our recently developed fluorous protecting group for phos-
phate groups.11 This group can easily be added to a phosphate
and then be used as a tag to separate the sugar chain from other
reagents using fluorous solid-phase extraction (FSPE).12,13 In our
previous work, this approach was used for the synthesis of a disac-
charide from Leishmania and had the advantage of easy purification
and deprotection under mild conditions. However, in the current
⇑
Corresponding author.
E-mail address: npohl@indiana.edu (N.L.B. Pohl).
0008-6215/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.carres.2012.12.015

L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
15
OH
phosphate group: phosphates at the reducing end, phosphates at
the non-reducing end, and phosphates in the middle saccharides.
We started the synthesis of the analogs with phosphates at the
reducing end. In this case, the temporary protecting group for fu-
ture installation of phosphates only exists in the acceptor, simpli-
fying the glycosylation reaction.
Initially, a p-methoxybenzyl (PMB) group was chosen as the
temporary protecting group on the reducing end. However,
although the survival of PMB groups under HCl/ether/NaBH3CN
conditions used for selective benzylidene opening has been re-
ported,16 we experienced extensive decomposition in the benzyli-
dene opening reactions using either TfOH/Et3SiH17 or NaBH3CN/
HCl18 conditions. A milder condition using DIBALH in CH2Cl219 only
resulted in modest yields of the desired product.
O
HO
OH
R1O
R2O
O
O
OH
R3O
R4O
O
O
R5O
R6O
OMe
R1 = PO32-, R2 = R3 = R4 = R5 = R6 = H
R
R
2 = PO32-, R1 = R3 = R4 = R5 = R6 = H
3 = PO32-, R1 = R2 = R4 = R5 = R6 = H
R4 = PO32-, R1 = R2 = R3 = R5 = R6 = H
R
R
5 = PO32-, R1 = R2 = R3 = R4 = R6 = H
6 = PO32-, R1 = R2 = R3 = R4 = R5 = H
Figure 1. Six methyl maltotrioside phosphates as potential substrates for the
laforin protein involved in Lafora disease.
To circumvent this problem, we attempted to use benzoyl
groups instead of the acid-labile PMB groups as the temporary pro-
tecting group at the reducing end of maltotriose. Known acceptor
520 was synthesized by a modified route (Scheme 1).
case, it was unclear whether, first, introduction of the group so
close to the anomeric center might negatively affect the glycosyla-
tion and, second, whether a relatively late stage introduction of the
fluorous group would actually significantly facilitate the synthesis
of the various methyl maltotrioside phosphate regioisomers. In
many cases of the synthesis of a phosphate bearing compound,
the phosphate group is installed in the last few steps.14 We rea-
soned that if the purification of the non-fluorous phosphate
group-bearing compound was problematic, it might be still useful
to perform a late stage fluorous phosphate installation; otherwise
the practicality of such a late stage installation would be question-
able. As opposed to a late stage installation strategy, the other pos-
sibility is to perform an early stage installation of the fluorous
phosphate group and to then carry the fluorous group throughout
the synthesis. In this way, the fluorous group functions as a tag. In
the previous cases of fluorous tag-assisted synthesis, most tags
were attached at the anomeric position of the reducing end of
the oligosaccharide, so the tag is remote from the reaction center.
In the synthesis of the desired maltose phosphates, however, the
fluorous phosphate group would inevitably be closer to the reac-
tion site. Although fluorous groups could be used as a convenient
method of purification, if the stereoselectivity is not ideal in the
When designing the maltose donor, we decide to use the ben-
zylidene-protected donors. The perbenzylated donors sometimes
proved too armed and will decompose too quickly upon treatment
of promoter. Also, the solubility of the benzylidene-protected do-
nors is generally better than perbenzylated donors in relatively
more polar solvents like ether. The maltose disaccharide donors
7 and 8 (Scheme 2) were synthesized with modifications on the
known procedure starting from maltose 6. One pot acetylation
and bromination of maltose afforded the maltose bromide, which
was converted to a thioglycoside under phase-transfer condi-
tions.21 Deacetylation, followed by benzylidene acetal formation
and benzylation yielded the previously reported fully protected
maltose thioglycoside donor 7.22
The glycosylation reaction was performed using the thioglycoside
donor 7 first (Scheme 2). In the presence of NIS and TMSOTf at 0 °C in
ether/dichloromethane (1:1), the reaction proceeded well to give a
mixture of
a/b glycosides in the ratio of 2:3, and the a glycoside
was separated using silica gel chromatography in a 22% isolated yield.
To try to improve the stereoselectivity of this glycosylation reaction,
we carried out the glycosylation of benzoylated acceptors with malt-
ose imidate donors 8 in light of Motawia’s work on the ‘blockwise
three-stage glycosylation strategy’23 by converting the thiophenyl
group to a trichloroacetimidate using standard chemistry. In this
case, in a mixture of ether/dichloromethane, the glycosylation be-
synthesis of the
a-glycosidic linkage, it might be difficult to sepa-
rate the /b mixture, thus diminishing the usefulness of the fluor-
a
ous-assisted synthesis. Since our initial report, Van der Marel
reported another fluorous phosphate protecting group earlier this
year to be used in the synthesis of carbohydrates.15 The reaction
utilized in their study did not involve the glycosidic bond forma-
tion, so the fluorous tag proved to be very efficient in their synthe-
sis. Herein we probe how the fluorous tag would perform in a
situation when the selectivity of the reaction might not be ideal,
but in which normal chromatography could then be used for that
particular step alone.
tween the imidate donor and the acceptor gave the desired
a product
9 in 45% yield with no b anomer detected.
To remove the 3-position benzoyl group in trisaccharide 9 for
installation of the phosphate, various conditions were screened.
Interestingly, routine conditions, including using sodium methox-
ide in methanol or using NaOH in water/THF could only remove
the benzoyl group slowly to give 10, probably due to steric hin-
drance. However, a combination of cesium hydroxide and sodium
hydroxide together could speed up the reaction greatly and
provide a better result for the deprotection of the benzoyl ester,
possibly due to the cesium effect.21 Proton NMR was used to deter-
mine when the deprotection reaction had finished. The doublet
peak from the benzoyl group moves slightly down field, indicating
2. Results and discussion
The desired six methyl maltotrioside phosphates (Fig. 1) can be
broken into three categories depending on the position of the
OBn
O
Ph
O
Ph
BnBr, TBAI
NaBH3CN,
O
O
O
O
O
HO
HO
R1O
BzO
NaOH, CH2Cl2
H2O, 24 h
HCl, ether,
89%
HO
R2O
BnO
OMe
OMe
OMe
1
2 R1 = Bn, R2 = H, 20%
3 R1 = H, R2 = Bn, 43%
5
BzCl, py
4 R1 = Bz, R2 = Bn, 90% (from 3)
Scheme 1. The synthesis of benzoyl-protected reducing end building block of methyl maltotriose and the benzylidene acetal opening reaction.

16
L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
OH
1. HBr/HOAc,Ac2O
1. NBS, Acetone/H2O
2. CCl3CN/Cs2CO3,
65% over 2 steps
O
HO
HO
2. PhSH, K2CO3,Bu4NHSO4, EtOAc
3. NaOCH3, CH3OH
OH
HO
7
8
O
O
4. PhCH(OMe)2, TsOH; NaH, BnBr
31% over 5 steps
HO
OH
HO
6
O
Ph
O
O
Ph
O
O
O
OBn
O
TMSOTf, ether
OBn
BnO
OBn
O
BnO
BnO
-20 °C
BnO
O
O
HO
OBn
O
O
BnO
BzO
45% with 8
BnO
BnO
BnO
R
22% with 7
BnO
O
OMe
R1O
5
7
8
R = SPh
R = OC(NH)CCl3
9 R1 = Bz
10 R1 = OH
CsOH, NaOH,
MeOH, 71%
BnO
OMe
1. tetrazole,
(BnO)2PNiPr2
2. tBuOOH
52%
OH
11 R1 = P(O)(OBn)2
O
HO
HO
OH
Pd/C, H2,
EtOH, 70%
HO
O
O
OH
HO
HO
O
O
O
HO
HO
P
OMe
O
HO
12
Scheme 2. Synthesis of the 3-position phosphorylated methyl maltotrioside via a benzoyl-protected acceptor.
the formation of benzoic acid. We envisioned that if there would be
any problem in the purification of trisaccharide 11, the fluorous
phosphate protecting group would be beneficial. After the benzoyl
deprotection, a phosphate group was installed on the resulting hy-
droxyl moiety using standard phosphoramidite chemistry24 to
yield 11, followed by hydrogenolysis to provide the desired phos-
phate 12. No problems were encountered in the purification of tri-
saccharide 11—a finding that eliminates the need for installing a
fluorous phosphate group at this late stage.
glycosylation outcomes. The size of the fluorous protecting group
and the change of the molecule’s polarity due to the attachment
of the fluorous portion might influence the reaction and the subse-
quent separation in a reaction that can give product mixtures such
as many glycosylation reactions. Not many reports focus on the sit-
uation when the reaction would lead to a mixture; however, if the
fluorous protecting group-modified product is inseparable from
undesired products by any means, a fluorous assisted-synthesis
would not prove efficient or practical.
Although late stage installation of the fluorous phosphate group
was not required for synthetic ease in this particular case, an early
stage installation could still be useful. Van der Marel recently re-
ported the synthesis of teichoic acids using a fluorous phosphate
group.15 In their synthesis, the fluorous protecting group is far
away from the reaction center, and no glycosylation was involved
in the synthesis. However, remote electron-withdrawing effects
and certainly neighboring protecting groups can readily affect
Alcohol 3 was chosen as a model compound to test the early
stage installation of the fluorous phosphate protecting group
(Scheme 3). Compound 3 was coupled with the fluorous phosphor-
amidate 13 followed by selective benzylidene-acetal opening to
provide glycosyl acceptor 14. However, the glycosylation between
14 and the perbenzylated donor 12 gave an inseparable
a/b mix-
ture in low yield. In our synthesis of the Leishmania saccharide,
we also witnessed a phenomenon with a change of stereochemical
Br
O
N
OBn
C8F17
tetrazole;
P
O
Ph
OBn
O
O
O
HO
1.
Br
C8F17
HO
O
t-BuOOH
O
BnO
BnO
P
OMe
OMe
O
2. TfOH, Et3SiH, 76%
two steps
BnO
3
14
OBn
O
BnO
BnO
OBn
BnO
O
CCl3
O
BnO
BnO
OBn
NH
BnO
O
O
Br
C8F17
O
O
BnO
P
OMe
O
BnO
15
Scheme 3. Early stage introduction of the fluorous phosphate protecting group.

L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
17
OBn
O
TfOH, Et3SiH
Ph
O
O
O
-78 oC,CH2Cl2
HO
R1O
R1O
R2O
R2O
OMe
OMe
2 R1 = Bn, R2 = H
3 R1 = H, R2 = Bn
18 R1 = Bn, R2 = P(O)(OBn)2, 81%
1. tetrazole,
(BnO)2PNiPr2
2. tBuOOH
19 R1 = P(O)(OBn)2, R2 = Bn, 71%
16 R1 = Bn, R2 = P(O)(OBn)2, 67%
17 R1 = P(O)(OBn)2, R2 = Bn, 56%
OH
O
Ph
O
O
O
HO
HO
OH
OBn
O
BnO
BnO
HO
O
O
O
TMSOTf, 8
Pd/C
EtOH, H2
OH
OBn
O
HO
BnO
ether, -20 o
C
HO
R1O
BnO
O
O
O
R1O
R2O
R2O
OMe
OMe
20 R1 = Bn, R2 = P(O)(OBn)2, 39%
11 R1 = P(O)(OBn)2, R2 = Bn, 65%
21 R1 = H, R2 = P(O)(OH)2, 61%
12 R1 = P(O)(OH)2, R2 = H, 70%
Scheme 4. Synthesis of methyl maltotrioside phosphates using phosphorylated acceptors.
outcomes. These results showed that even though fluorous pro-
tecting groups and tags can provide a convenient way of purifica-
tion, tags too closely attached to the sugar building block itself
can lead to subtle changes in the reaction that might render its
usage impractical.
Since using benzoyl as a temporary protecting group worked
well, and the early stage installation of the fluorous phosphate
group leads to a mixture, we wanted to further examine the possi-
bility of using phosphorylated acceptors directly. In this way, the
protection/deprotection could be omitted by using phosphate di-
rectly as the protecting group. The free hydroxyl in compounds 2
or 3 was directly protected as dibenzyl phosphates to give 16 or
17 (Scheme 4). The benzylidene acetal was opened smoothly to
provide the phosphorylated acceptor 18 or 19. The glycosylation
reaction between donor 8 and acceptor 18 or 19 in ether at
ꢀ20 °C also resulted in an anomeric mixture of products, but this
mixture was readily separable to isolate methyl maltotriose phos-
23 was studied under different conditions. Using Tf2O/PhS2O/TTBP
at ꢀ78 °C failed to give the desired glycosylation product 29, and
using NIS/TfOH as the promoter at ꢀ45 °C led to an inseparable
mixture of
a/b products in the ratio of 2:1. Changing the donor
from thioglycoside to imidate did not improve the stereoselectivity
of the glycosylation reaction. Fortunately the mixture became sep-
arable after the cleavage of the PMB group. It was found that the
deprotection of PMB on 29 using ceric ammonium nitrate (CAN)
gave a much cleaner reaction than using DDQ; however, if the reac-
tion using CAN took too long, the acidic conditions could cleave the
benzylidene. The desired a-linked product 31 was phosphorylated
to give 33, and hydrogenolysis gave the desired final product 35.
Compound 36 was then synthesized via an analogous route
(Scheme 7).
For the maltotriose with phosphates on the middle saccharide,
we surveyed the methods that could potentially lead to selective
protection and differentiation of hydroxyls on maltose, in the hope
of finding an easier way of installing the phosphates. However, the
selective protection of hydroxyls on disaccharides is far from
developed, so we decided to synthesize the desired maltotriose
via two glycosylation reactions.
phate isomers in an
a/b ratio of 3:1. Hydrogenolysis of 20 or 11
gave the desired compound 21 or 12, respectively (Scheme 4).
Encouraged by these results, we moved to synthesize the mal-
totriose with a phosphate at the non-reducing end. To prepare
the maltose acceptor, the thiophenyl group on the maltose donor
The glycosylation between the donor 27 with a PMB group at
7 was converted to an
a-methoxy group by reacting with methanol
the 2-position and acceptor 37 gave an inseparable mixture of a/
in the presence of N-chlorosuccinimide (Scheme 5).22,25 It was
found that the formation of methyl glycoside 22 required the pres-
ence of molecular sieves, otherwise large amounts of a side prod-
uct, presumably the NHS-substituted maltoside, would be
formed. We also found that using freshly recrystallized NCS would
slow the reaction down; the un-recrystallized NCS gave much bet-
ter results. Selective opening of the benzylidene acetal using TfOH/
Et3SiH afforded the acceptor 23 with a free hydroxyl group at the
4-position (Scheme 5).
b stereoisomers in the ratio of 3:1 (Scheme 8). As in the case of
the previous trisaccharide, the mixture became separable after
PMB cleavage. The free hydroxyl in 40 was phosphorylated to give
disaccharide 42, followed by selective benzylidene acetal opening
to provide disaccharide acceptor 44. The glycosylation between
acceptor 44 and donor 44 in ether gave the desired methyl malto-
trioside 20-phosphate 47, followed by hydrogenolysis to give 49.
The methyl maltotrioside 30-phosphate 50 was synthesized in a
similar straightforward fashion to successfully provide the last of
the desired phosphate regioisomers (Scheme 8).
To obtain the desired
a-linkage, a PMB-substituted donor was
used. Donors 27 and 28 with the desired protecting group patterns
were synthesized following the route shown in Scheme 6. Glyco-
sylation using the thioglycoside donor 27 and maltose acceptor
We have reported the first fluorous phosphate protecting group
previously11 and here show the limitations of using this particular
protecting group in the context of the synthesis of maltotrioside
OBn
O
Ph
O
Ph
O
O
O
O
O
HO
BnO
NCS, CH3OH
4Å MS, 85%
TfOH, Et3SiH
OBn
O
BnO
OBn
O
OBn
O
BnO
4Å MS, CH2Cl2
BnO
BnO
BnO
O
O
O
SPh
BnO
69%
BnO
BnO
BnO
BnO
BnO
OMe
OMe
7
22
23
Scheme 5. Preparation of the methyl maltoside acceptor.

18
L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
O
O
O
Ph
Ph
Ph
1. nBu2SnO, CH3OH
PMBCl or BnBr
NaH, DMF
O
O
O
O
O
O
SPh
SPh
SPh
R1O
R1O
R1O
2. BnBr or PMBCl, TBAB
HO
HO
R2O
PhCH3, 60 o
C
24
25 R1 = Bn, 63%
27 R1 = Bn , R2 = PMB, 84%
28 R1 = PMB, R2 = Bn, 87%
26 R1 = PMB, 70%
Scheme 6. Synthesis of the glucose donor.
OBn
O
O
Ph
O
O
HO
OBn
R1O
OBn
O
BnO
R2O
BnO
O
Ph
O
O
O
O
O
OBn
BnO
BnO
SPh
R1O
BnO
BnO
O
R2O
O
OMe
BnO
BnO
27 R1 = Bn , R2 = PMB
28 R1 = PMB, R2 = Bn
23
29 R1 = Bn , R2 = PMB, 59%
30 R1 = PMB, R2 = Bn, 57%
OMe
OH
O
Ph
O
O
O
HO
OBn
OH
R1O
R1O
R2O
R2O
CAN
O
H2, Pd/C
O
O
O
OBn
OH
BnO
HO
BnO
HO
O
O
O
O
BnO
HO
BnO
HO
35 R1 = H, R2 = P(O)(OH)2, 83%
36 R1 = P(O)(OH)2, R2 = H, 50%
OMe
OMe
31 R1 = Bn , R2 = H, 15%
32 R1 = H, R2 = Bn, 37%
1. tetrazole,
(BnO)2PNiPr2
2. tBuOOH
33 R1 = Bn, R2 = P(O)(OBn)2, 52%
34 R1 = P(O)(OBn)2, R2 = Bn, 60%
Scheme 7. Synthesis of methyl maltotrioside 200 and 300-phosphates.
phosphate isomers. In the studies of Leishmania-associated carbo-
hydrates, we reported the synthesis of a phosphorylated disaccha-
ride utilizing our fluorous phosphate protecting group. However,
our current attempts to explore the scope of this protecting group
alerted us to an interesting result. In the glycosylation coupling be-
tween the donor and a per-acetylated galactose donor, a mixture of
in the synthesis of maltotriose. Different protecting groups and
glycosylations can easily alter the outcome of the glycosylations.
Although fluorous tags and protecting groups can provide a con-
venient method of purification, this work demonstrates that the
tag can introduce some subtle changes in reactions at nearby
sites and provide little advantage in some cases when introduced
at a late stage of a synthesis. Until better separation methods on
normal phase silica gel are found for fluorous-containing com-
pound mixtures, the fluorous protecting groups show more
promise when used with sufficient distance from a reactive site.
Future studies include testing of these synthetic methyl maltotri-
oside phosphate isomers to probe the activity of the Laforin en-
zyme and thereby determining the most likely in vivo substrate
for this enzyme.
a/b isomers was formed instead of the desired b product, which
was not readily separable from the mixture. Interestingly, most
of the previously reported fluorous-assisted synthesis focused on
fairly straightforward reactions, so the formation of a mixture with
a fluorous tag is minimized. For example, in our automated solu-
tion-phase synthesis of oligosaccharides,26,27 the fluorous tag is at-
tached at the anomeric position of the reducing end, so the tag
remains far away from the reaction site and is also mediated by
a longer linker region.
3. Experimental
In this study, a series of methyl maltotrioside phosphate regioi-
somers were synthesized via a modular synthesis. Different strate-
gies were used to introduce the phosphate groups to the
maltotriose. Performing glycosylations on phosphorylated accep-
tors would greatly simplify the transformations needed at the tri-
saccharide stage. We found out that the glycosylation reactions
were not affected when phosphate esters were present at either
2 or 3 position of the acceptor, so the 2-, 3-, 20-, and 30-phosphor-
ylated maltotriose were synthesized using phosphorylated mono-
saccharides or disaccharides as acceptors. However, due to the
participating nature of the phosphate group, using a pre-phosphor-
ylated donor with a phosphate at the 2-position could not generate
3.1. General experimental methods
Reactions were performed using flame-dried glassware under
argon using anhydrous solvents unless otherwise noted. Thin layer
chromatography (TLC) was performed using glass-backed silica gel
plates w/UV254. Visualization of TLC plates was performed by UV
light and 5% sulfuric acid/ethanol. Silica gel flash chromatography
(SGC) was performed using silica gel (60 Å, 40–63
lm) from ZEO-
Chem AG. NMR spectra were recorded on a 400 MHz for 1H
(101 MHz for 13C, 162 MHz for 31P) spectrometer or on
a
600 MHz for 1H (150 MHz for 13C, 243 MHz for 31P). 1H NMR and
13C NMR taken in CDCl3 spectra were referenced to the solvent
peak at 7.260 ppm (1H) and 77.0 ppm (13C). 31P NMR was not ref-
erenced. High resolution mass spectra (HRMS, ESI mode) were ob-
tained using a Q-TOF LC/MS.
the desired a-glycosidic linkage. Therefore, in the synthesis of the
200 phosphorylated maltotriose, the post-phosphorylation strategy
was used.
The formation of an
a-glucosidic bond is still a significant
challenge when new protecting groups are introduced, as shown

L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
19
OBn
O
O
NIS, TfOH
Et2O/CH2Cl2
Ph
O
O
O
Ph
OBn
O
HO
R1O
O
O
BnO
R2O
SPh
R1O
BnO
O
R2O
OMe
BnO
BnO
OMe
27 R1 = Bn , R2 = PMB
28 R1 = PMB, R2 = Bn
37
38 R1 = Bn , R2 = PMB
39 R1 = PMB, R2 = Bn
O
Ph
BnO
HO
R1O
O
O
O
OBn
O
OBn
R1O
TfOH, Et3SiH
CH2Cl2
R2O
R2O
CAN
O
O
O
BnO
BnO
BnO
BnO
OMe
OMe
40 R1 = Bn , R2 = H, 32%
41 R1 = H, R2 = Bn, 50%
1. tetrazole,
(BnO)2PNiPr2
2. tBuOOH
44 R1 = Bn, R2 = P(O)(OBn)2, 63%
45 R1 = P(O)(OBn)2, R2 = Bn, 75%
42 R1 = Bn, R2 = P(O)(OBn)2, 85%
43 R1 = P(O)(OBn)2, R2 = Bn, 88%
OBn
BnO
O
BnO
O
BnO
OBn
BnO
BnO
BnO
O
O
BnO
OC(NH)CCl3
OBn
O
46
R1O
R2O
O
BnO
BnO
OMe
47 R1 = Bn, R2 = P(O)(OBn)2, 60%
48 R1 = P(O)(OBn)2, R2 = Bn, 72%
OH
O
HO
HO
OH
HO
Pd/C, EtOH, H2
O
O
OH
R1O
R2O
O
O
HO
HO
OMe
49 R1 = H, R2 = P(O)(OH)2, 48%
50 R1 = P(O)(OH)2, R2 = H, 67%
Scheme 8. Synthesis of methyl maltotrioside 20 and 30 phosphate.
3.2. General procedure for selective benzylidene opening
reaction
at ambient temperature until TLC indicated the conversion had
finished. The reaction was diluted with EtOAc, washed with
water and NaHCO3 (aq), dried, concentrated and purified via
SGC.
The substrate (1 equiv) was dissolved in CH2Cl2 (0.1 M), and
ASW-3000 (powder, 100 mg/mol) was added. The reaction was
cooled to ꢀ78 °C, and Et3SiH (3 equiv) was added followed by TfOH
(1.5 equiv). The reaction was stirred at ꢀ78 °C until TLC indicated
the conversion was complete. Methanol and Et3N were added,
and the reaction was filtered, concentrated under reduced pres-
sure, and subject to SGC for purification.
3.5. General procedure for glycosylation using thioglycoside
donor
The acceptor (1 equiv) and donor (1.5 equiv) were co-evapo-
rated with toluene for 3 times and dissolved in Et2O/CH2Cl2 (3:1,
0.05 M). ASW-3000 (powder, 100 mg/mmol) was added, and the
reaction was cooled to ꢀ78 °C. NIS (1.8 equiv) was added, and
the reaction was stirred at ꢀ78 °C for 20 min then warmed up to
ꢀ45 °C. TfOH (1.8 equiv) was added and the reaction was stirred
at ꢀ45 °C until TLC indicated the conversion had finished. The reac-
tion was quenched by adding Et3N, concentrated, and purified via
SGC.
3.3. General procedure for thioglycoside removal
The substrate (1 equiv) was dissolved in acetone/water (9:1,
0.1 M). N-Bromosuccinimide (NBS, 2 equiv) was added and the
reaction was stirred until TLC indicated the conversion had fin-
ished. The reaction was quenched with adding solid NaHCO3, con-
centrated under reduced pressure, diluted with EtOAc, washed
with Na2S2O3, NaHCO3, brine, and dried. The resulting organic layer
was concentrated under reduced pressure and the crude mixture
was purified via SGC.
3.6. General procedure for trichloroacetimidate formation
The substrate (1 equiv) was dissolved in CH2Cl2 (0.1 M),
Cs2CO3 (0.5 equiv) was added, followed by CCl3CN (3 equiv).
The reaction was stirred at ambient temperature until TLC indi-
cated the conversion was complete. The reaction was filtered
through Celite, and concentrated under reduced pressure to give
the crude imidate.
3.4. General procedure for PMB removal
The substrate (1 equiv) was dissolved in CH3CN/H2O (9:1
0.05 M), and CAN (4 equiv) was added. The reaction was stirred

20
L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
3.7. General procedure for glycosylation using
trichloroacetimidate donor
The reaction was stirred for 16 h, concentrated, extracted with
EtOAc, and purified with SGC (hexanes/EtOAc 3:1) to give 10
(15 mg, 0.012 mmol, 71%) as a syrup. 1H NMR (400 MHz, CDCl3) d
7.61–7.04 (m, 40H, PhH), 5.69 (d, J = 3.83 Hz, 1H, H-10), 5.55 (s,
1H, PhCH), 5.00–4.79 (m, 5H, H-100), 4.79–4.66 (m, 4H, 4 ꢁ OCH
HPh), 4.66–4.52 (m, 5H, 4 ꢁ OCH HPh, H-1), 4.51–4.41 (m, 2H,
2 ꢁ OCH HPh), 4.35 (d, J = 11.79 Hz, 1H, OCHHPh), 4.15 (dd,
J = 5.23, 10.96 Hz, 1H, H-600a), 4.12–4.03 (m, 2H, H-5, H-400), 4.02–
3.90 (m, 2H, H-3, H-4), 3.88–3.80 (m, 1H, H-500), 3.78–3.42 (m,
10H, H-30, H-200, H-300, H-40, H-600b, H-6a, H-20, H-6b, H-60a, H-60b),
3.37 (s, 3H, OCH3); 13C NMR (101 MHz, CDCl3) d 138.6, 138.6,
138.2, 137.9, 137.7, 137.6, 136.8, 128.9, 128.8, 128.6, 128.4,
128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.0, 127.9, 127.8,
127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 127.2, 126.5, 126.0,
101.2, 100.4, 98.4, 97.6, 82.3, 82.1, 82.1, 80.3, 78.7, 78.7, 78.0,
77.3, 75.3, 74.3, 74.3, 74.0, 73.4, 73.3, 73.1, 71.9, 70.8, 69.0, 68.8,
68.6, 63.4, 55.2; HRMS (ESI) calcd for C75H80O16Na [M+Na]+:
1259.5339, found 1259.5326.
The acceptor (1 equiv) and donor (1.5 equiv) were co-evapo-
rated with toluene 3 times and dissolved in Et2O (0.05 M). The
reaction was cooled to ꢀ20 °C, and TMSOTf (0.1 equiv, 0.0268 M
in CH2Cl2) was added; the reaction was stirred at ꢀ20 °C until
TLC indicated the conversion was complete. The reaction was
quenched by adding Et3N, concentrated under reduced pressure,
and purified via SGC.
3.8. General procedure for phosphorylation
The substrate (1 equiv) was dissolved in CH2Cl2 (0.1 M). Diben-
zyl N,N-di isopropylphosphoramidite (2 equiv) was added followed
by 1-H tetrazole (3 equiv, 0.45 M in CH3CN). The reaction was stir-
red at ambient temperature until TLC indicated the conversion was
complete. t-BuOOH (5 equiv, 5–6 M in decane) was added, and the
reaction was stirred for 1 h. The mixture was concentrated under
reduced pressure, and purified via SGC.
3.10.3. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-
a-D-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl- -glucopyranosyl-
a
-D
3.9. General procedure for global deprotection
(1?4)-2,6-di-O-benzyl-3-dibenzylphosphate-a-D-
glucopyranoside (11)
The substrate was dissolved in EtOH, and Pd/C was added. The
reaction was stirred under 800 psi of H2 for 8 h, filtered, and con-
centrated to give the product.
Compound 10 (15 mg, 0.012 mmol) was subjected to the gen-
eral procedure for phosphorylation described above and purified
via SGC (hexanes/EtOAc 3:2) to give 11 (9.3 mg, 0.006 mmol,
52%) as a syrup. 1H NMR (400 MHz, CDCl3) d 7.59–6.97 (m, 50H,
PhH), 5.86 (d, J = 3.45 Hz, 1H, H-1), 5.76 (d, J = 3.89 Hz, 1H, H-100),
5.53 (s, 1H, PhCH), 5.05–4.76 (m, 9H, 9 ꢁ OCHHPh), 4.70 (s, 2H,
2 ꢁ OCHHPh), 4.68–4.60 (m, 4H, 4 ꢁ OCHHPh), 4.58–4.47 (m, 5H,
4 ꢁ OCHHPh H-10), 4.43 (d, J = 12.19 Hz, 2H, 2 ꢁ OCHHPh), 4.24
(d, J = 9.54 Hz, 2H, 2 ꢁ OCHHPh), 4.17 (t, J = 9.07 Hz, 1H, H-4),
4.10 (dd, J = 4.68, 10.17 Hz, 1H, H-50), 4.01 (t, J = 8.84 Hz, 1H, H-
3), 3.93 (t, J = 9.27 Hz, 1H, H-300), 3.90–3.74 (m, 4H, H-400, H-5, H-
6a, H-30), 3.72–3.56 (m, 5H, H-60a, H-60b, H-6b, H-20, H-600a),
3.55-3.45 (m, 3H, H-2, H-200, H-600b), 3.28 (s, 3H, OCH3); 13C NMR
(101 MHz, CDCl3) d 138.8, 138.7, 138.3, 138.2, 137.8, 137.6,
137.5, 136.0, 128.8, 128.5, 128.5, 128.5, 128.4, 128.4, 128.3,
128.3, 1281.3, 128.2, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0,
127.9, 127.8, 127.7, 127.6, 127.6, 127.5, 127.5, 127.4, 127.4,
127.4, 127.3, 127.1, 127.0, 126.7, 126.11, 101.1, 97.4, 96.8, 93.8,
82.3, 81.9, 81.8, 81.3, 79.8, 79.1, 78.7, 78.7, 75.0, 73.8, 73.5, 73.3,
73.0, 72.3, 71.1, 70.4, 69.9, 69.8, 69.8, 69.3, 69.2, 68.9, 68.8, 68.7,
68.1, 65.4, 63.1, 55.1; 31P NMR (162 MHz, CDCl3) d ꢀ2.96; HRMS
3.10. Synthetic procedures
3.10.1. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl- -glucopyranosyl-
(1?4)-2,6-di-O-benzyl-3-benzoyl- -glucopyranoside (9)
a-D-
a-D
a-D
Compound 7 (500 mg, 0.513 mmol) was subjected to the condi-
tions in the general method for thiolphenol removal to give the
hemiacetal (320 mg, 0.367 mmol). The hemiacetal was treated with
CCl3CN and Cs2CO3 as described in the general procedure for tri-
chloroacetimidate formation to give 8 as a yellow foam (350 mg,
0.34 mmol, 67% for two steps) and used without further purifica-
tion. Donor
8 (98 mg, 0.98 mmol) and acceptor 5 (28 mg,
0.058 mmol) were glycosylated using the general glycosylation
method and purified via SGC (hexanes/EtOAc 3:1) to give 9
(35 mg, 0.026 mmol, 45%) as a syrup. 1H NMR (400 MHz, CDCl3) d
8.09–7.94 (m, 2H, PhH), 7.54–7.00 (m, 43H, PhH), 5.88 (t,
J = 9.58 Hz, 1H, H-3), 5.73 (d, J = 3.87 Hz, 1H, H-10), 5.53 (s, 1H,
PhCH), 4.92 (d, J = 3.24 Hz, 1H, H-100), 4.87 (d, J = 11.20 Hz, 1H,
OCHHPh), 4.81–4.75 (m, 2H, 2 ꢁ OCHHPh), 4.75–4.63 (m, 2H,
2 ꢁ OCHHPh), 4.63–4.53 (m, 6H, 6 ꢁ OCHHPh), 4.49 (d,
J = 11.79 Hz, 1H, OCHHPh), 4.42 (dd, J = 5.65, 12.06 Hz, 2H,
2 ꢁ OCHHPh), 4.19 (d, J = 12.25 Hz, 1H, H-600a), 4.16–4.05 (m, 3H,
H-5, H-400, H-6a), 4.05–3.90 (m, 5H, H-3, H-4, H-60a, H-300), 3.91–
3.76 (m, 3H, H-50, H-600b), 3.72–3.70 (m, 2H, H-200, H-40), 3.67–3.55
(m, 3H, H-20, H-30, H-60b), 3.55–3.46 (m, 2H, H-500, H-6b), 3.43 (s,
3H, OCH3), 3.29–3.27 (m, 1H, H-2); 13C NMR (101 MHz, CDCl3) d
165.5, 138.9, 138.7, 138.1, 137.9, 137.9, 137.9, 137.7, 137.6, 132.7,
130.7, 129.8, 128.8, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2,
128.2, 128.2, 128.1, 128.0, 128.0, 127.8, 127.8, 127.8, 127.7, 127.6,
127.6, 127.5, 127.4, 127.4, 127.0, 126.5, 126.1, 101.1, 97.8, 97.7,
96.9, 82.2, 81.3, 79.7, 78.9, 78.7, 76.9, 75.5, 75.2, 73.8, 73.5, 73.4,
73.3, 73.2, 72.8, 72.4, 71.2, 70.6, 69.9, 68.7, 63.2, 55.3; HRMS (ESI)
calcd for C82H84O17Na [M+Na]+: 1363.5601, found 1363.5561.
(ESI) calcd for
1519.5949.
C
89H93O19PNa [M+Na]+: 1519.5914, found
3.10.4. Methyl
a-
D-glucopyranosyl-(1?4)-a-D-glucopyranosyl-
(1?4)-3-phosphate-
a
-D
-glucopyranoside (12)
Hydrogenolysis of 11 (12 mg, 0.007 mmol) according to the
general method for global deprotection gave 12 (3 mg,
0.005 mmol, 71%) as a syrup. 1H NMR (400 MHz, CD3OD) d 5.44
(d, J = 3.4 Hz, 1H, H-10), 5.15 (d, J = 3.7 Hz,1H, H-100), 4.69 (d,
J = 4.4 Hz,1H, H-1), 4.55–4.47 (m, 1H, H-3), 3.90–3.74 (m, 5H, H-
6a, H-60a, H-60b, H-600a, H-600b), 3.73–3.56 (m, 5H, H-2, H-30, H-6b,
H-40, H-400), 3.57–3.47 (m, 3H, H-300, H-4, H-5), 3.42–3.34 (m, 4H,
H-20, H-50, H-200), 3.34–3.15 (m, 4H, OCH3); 13C NMR (101 MHz,
CD3OD) d 101.5, 99.4, 99.1, 79.9, 78.2, 75.3, 73.6, 73.3, 72.9, 71.8,
70.7, 70.1, 61.3, 60.8, 60.7, 54.1, 46.4; 31P NMR (162 MHz, CD3OD)
d 1.57; HRMS (ESI) calcd for C19H34O19P [MꢀH]ꢀ: 597.1437, found
597.1447.
3.10.2. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl- -glucopyranosyl-
(1?4)-2,6-tri-O-benzyl- -glucopyranoside (10)
a-D-
a-D
3.10.5. Methyl 2-dibenzylphosphate-3-O-benzyl-4,6-O-
benzylidene-a-D-glucopyranoside (16)
a-D
Compound 9 (23 mg, 0.017 mmol) was dissolved in CH3OH
Compound 2 (76 mg, 0.20 mmol) was subjected to the general
(3 mL). CsOH–H2O (50 mg) was added, followed by Na (23 mg).
procedure for phosphorylation described above to and purified

L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
21
via SGC (hexanes/EtOAc 2:1) give 16 (85 mg, 0.134 mmol, 67%) as a
3.10.9. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-
a-D-
white foam. 1H NMR (400 MHz, CDCl3) d 7.57–7.14 (m, 20H, PhH),
5.57 (s, 1H, PhCH), 5.04 (d, J = 7.51 Hz, 2H, OCH2Ph), 5.02–4.94 (m,
3H, OCH2Ph, H-1), 4.91 (d, J = 11.35 Hz, 1H, OCHHPh), 4.73 (d,
J = 11.32 Hz, 1H, OCHHPh), 4.41 (ddd, J = 3.81, 7.70, 9.40 Hz, 1H,
H-2), 4.30 (dd, J = 4.66, 10.12 Hz, 1H, H-6a), 4.06 (t, J = 9.28 Hz,
1H, H-3), 3.87 (td, J = 4.66, 9.90 Hz, 1H, H-5), 3.76 (t, J = 10.25 Hz,
1H, H-6b), 3.67 (t, J = 9.34 Hz, 1H, H-4), 3.37 (s, 3H, OCH3); 13C
NMR (101 MHz, CDCl3) d 138.2, 137.2, 135.7, 129.0, 128.5, 128.5,
128.5, 128.3, 128.2, 128.2, 127.9, 127.8, 127.7, 127.6, 126.0,
101.3, 98.6, 82.0, 82.0, 75.0, 69.4, 69.3, 69.3, 69.2, 68.9, 62.2,
55.4; 31P NMR (162 MHz, CDCl3) d ꢀ1.77; HRMS (ESI) calcd for
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl- -glucopyranosyl-
(1?4)-3-dibenzylphosphate-,6-di-O-benzyl-a-D-
glucopyranoside (20)
a-D
Donor
8 (72 mg, 0.071 mmol) and acceptor 18 (30 mg,
0.047 mmol) were subjected to the conditions in the general meth-
od of glycosylation and purified via SGC (hexanes/EtOAc 2:1) to
give 20 (27 mg, 0.018 mmol, 39%) as a syrup.1H NMR (400 MHz,
CDCl3) d 7.73–6.85 (m, 50H, PhH), 5.65 (d, J = 3.52 Hz, 1H, H-10),
5.50 (s, 1H, PhCH), 5.41 (d, J = 2.90 Hz, 1H, H-100), 4.99–4.91 (m,
4H, 4 ꢁ OCHHPh), 4.90–4.76 (m, 5H, 4 ꢁ OCHHPh, H-1), 4.73–
4.57 (m, 3H, 3x4 ꢁ OCHHPh), 4.57–4.33 (m, 7H, 6x4 ꢁ OCHHPh,
H-2), 4.16–3.84 (m, 7H, H-40, H-300, H-400, H-600a, H-500, H-3, H-4),
3.82––3.74 (m, 2H, H-600, H-50), 3.72–3.51 (m, 5H, H-6a, H-6b, H-
60a, H-5, H-30), 3.53–3.39 (m, 3H, H-20, H-200, H-60b), 3.31 (s, 3H,
OCH3); 13C NMR (101 MHz, CDCl3) d 138.3, 138.1, 138.0, 137.9,
137.6, 137.6, 137.5, 137.2, 137.1, 135.3, 135.2, 135.1, 135.1,
128.4, 128.0, 128.0, 128.0, 128.0, 127.9, 127.8, 127.8, 127.8,
127.7, 127.6, 127.5, 127.4, 127.4, 127.4, 127.3, 127.3, 127.2,
127.2, 127.2, 127.1, 127.1, 127.1, 127.1, 127.0, 127.0, 127.0,
126.7, 126.7, 126.6, 126.3, 126.2, 126.1, 125.6, 100.7, 97.3, 97.1,
96.8, 96.3, 91.4, 81.8, 81.2, 80.2, 80.1, 79.9, 79.8, 79.1, 78.4, 78.2,
74.7, 73.5, 73.5, 73.3, 73.0, 73.0, 72.9, 72.7, 72.6, 71.5, 70.9, 70.5,
70.1, 69.4, 68.9, 68.8, 68.8, 68.7, 68.7, 68.5, 68.2, 68.0, 62.7, 54.8,
54.7; 31P NMR (162 MHz, CDCl3) d ꢀ1.74; HRMS (ESI) calcd for
C
35H38O9P [M+H]+: 633.2248, found 633.2244.
3.10.6. Methyl 2-O-benzyl-3-dibenzylphosphate-4,6-O-
benzylidene- -glucopyranoside (17)
a
-D
Compound 3 (220 mg, 0.59 mmol) was subjected to the general
procedure for phosphorylation described above and purified via
SGC (hexanes/EtOAc 3:2) to give 17 (210 mg, 0.33 mmol, 56%) as
a white foam. 1H NMR (600 MHz, CDCl3) d 7.73–6.95 (m, 20H,
PhH), 5.52 (s, 1H, PhCH), 5.08–4.88 (m, 5H, 4 ꢁ OCHHPh, H-3),
4.80 (d, J = 12.19 Hz, 1H, OCHHPh), 4.73–4.55 (m, 2H, OCHHPh,
H-1), 4.30 (dd, J = 4.86, 10.30 Hz, 1H, H-6a), 3.90 (td, J = 4.79,
9.89 Hz, 1H, H-6b), 3.83–3.62 (m, 3H, H-4, H-2, H-5), 3.40 (s, 3H,
OCH3); 13C NMR (151 MHz, CDCl3) d 137.8, 136.9, 136.2, 136.2,
136.2, 129.2, 128.5, 128.3, 128.3, 128.3, 128.3, 128.1, 128.1,
128.0, 128.0, 127.5, 127.4, 126.4, 102.0, 99.0, 80.2, 80.2, 78.4,
78.4, 76.9, 76.9, 73.4, 69.1, 69.0, 69.0, 69.0, 69.0, 62.2, 55.4; 31P
NMR (243 MHz, CDCl3) d 15.82; HRMS (ESI) calcd for C35H38O9P
[M+H]+: 633.2248, found 633.2257.
C
89H93O19PNa [M+Na]+: 1519.5914, found 1519.5934.
3.10.10. Methyl
a-
D-glucopyranosyl-(1?4)-a-D-glucopyranosyl-
(1?4)-2-phosphate-
a
-D
-gluco pyranoside (21)
Hydrogenation of 20 (5 mg, 0.003 mmol) according to the gen-
eral method for global deprotection gave 12 (1 mg, 0.002 mmol,
1
3.10.7. Methyl 2-dibenzylphosphate-3,6-di-O-benzyl-
a-
D-
61%) as a syrup. H NMR (400 MHz, CD3OD) d 5.15 (s, 1H, H-100),
glucopyranoside (18)
5.11 (d, J = 3.14 Hz, 1H, H-10), 4.63–4.55 (m, 1H, H-1), 4.06–3.92
(m, 2H), 3.87–3.70 (m, 7H), 3.70–3.51 (m, 5H), 3.51–3.41 (m,
2H), 3.37 (s, 3H); 13C NMR (101 MHz, CD3OD) d 101.5, 101.1,
97.9, 79.8, 79.5, 73.6, 73.5, 73.3, 72.8, 72.4, 72.0, 71.9, 70.6, 70.2,
70.1, 61.3, 60.7, 60.5, 54.3, 54.2; 31P NMR (162 MHz, CD3OD) d
ꢀ0.43, HRMS (ESI) calcd for C19H34O19P [MꢀH]ꢀ: 597.1437, found
597.1433.
Compound 16 (104 mg, 0.165 mmol) was subjected to the con-
ditions described in the general procedure for selective benzyli-
dene opening and purified via SGC (hexanes/EtOAc 2:1) to give
18 (84 mg, 0.133 mmol, 81%) as a syrup. 1H NMR (400 MHz, CDCl3)
d 7.56–7.16 (m, 20H, PhH), 5.05 (d, J = 7.64 Hz, 2H, OCH2Ph), 5.03–
4.96 (m, 3H, OCH2Ph, H-1), 4.87 (d, J = 11.45 Hz, 1H, OCHHPh), 4.71
(d, J = 11.37 Hz, 1H, OCHHPh), 4.64–4.52 (m, 2H, OCH2Ph), 4.36
(ddd, J = 3.67, 7.22, 9.60 Hz, 1H, H-2), 3.84 (t, J = 9.14 Hz, 1H, H-
3), 3.80–3.61 (m, 4H, H-4, H-6ab, H-5), 3.35 (s, 3H, OCH3), 2.56
(d, J = 2.75 Hz, 1H, OH); 13C NMR (101 MHz, CDCl3) d 138.4,
137.9, 135.8, 135.7, 135.6, 128.6, 128.5, 128.5, 128.5, 128.5,
128.4, 128.4, 127.9, 127.9, 127.8, 127.8, 127.7, 127.6, 97.9, 80.2,
80.1, 77.0, 75.2, 73.6, 71.2, 71.2, 69.8, 69.6, 69.4, 69.4, 69.3, 69.2,
55.3; 31P NMR (162 MHz, CDCl3) d ꢀ1.59; HRMS (ESI) calcd for
3.10.11. Methyl 2,3,6-tri-O-benzyl-a-D-glucopyranosyl-(1?4)-
2,3,6-tri-O-benzyl- -glucopyranoside (23)
D
Compound 22 (59 mg, 0.066 mol) was subjected to the condi-
tions according to the general procedure for selective benzylidene
opening to and purified via SGC (hexanes/EtOAc 3:1) afford 23
(41 mg, 0.046 mmol, 69%) as white foam. 1H NMR (400 MHz,
CDCl3) d 7.47–7.06 (m, 30H, PhH), 5.72 (d, J = 3.59 Hz, 1H, H-1),
5.07 (d, J = 11.62 Hz, 1H, OCHHPh), 4.91 (d, J = 11.29 Hz, 1H,
OCHHPh), 4.82 (d, J = 11.70 Hz, 1H, OCHHPh), 4.77–4.68 (m, 2H,
2 ꢁ OCHHPh), 4.66–4.41 (m, 7H, H-10, 6 ꢁ OCHHPh), 4.35 (d,
J = 12.09 Hz, 1H, OCHHPh), 4.10 (d, J = 8.80 Hz, 2H, H-30, H-50),
3.96–3.81 (m, 2H, H-60a), 3.81–3.42 (m, 8H, H-60b, H-3, H-6a, H-
4, H-6b, H-20, H-40, H-2), 3.40 (s, 3H, OCH3), 2.52 (d, J = 2.43 Hz,
1H, OH); 13C NMR (101 MHz, CDCl3) d 138.9, 138.8, 138.2, 137.9,
137.9, 137.9, 128.5, 128.4, 128.3, 128.3, 128.3, 128.2, 127.9,
127.9, 127.7, 127.7, 127.7, 127.6, 127.4, 127.3, 127.1, 126.7, 97.7,
96.5, 82.0, 81.3, 80.2, 79.0, 75.3, 74.4, 73.5, 73.4, 73.2, 73.1, 72.3,
71.5, 70.5, 69.8, 69.5, 69.0, 55.2; HRMS(ESI): calcd for C55H61O11
[M+H]+: 897.4206, found 897.4208.
C
35H39O9PNa [M+Na]+: 657.2224, found 657.2209.
3.10.8. Methyl 2,6-di-O-benzyl-3-dibenzylphosphate-a-D-
glucopyranoside (19)
Compound 17 (91 mg, 0.143 mmol) was subjected to the condi-
tions described in the general procedure for selective benzylidene
opening and purified via SGC (hexanes/EtOAc 3:2) to give 19
(64 mg, 0.101 mmol, 71%) as a syrup. 1H NMR (600 MHz, CDCl3) d
7.35 (m, 20H, PhH), 5.25–5.00 (m, 4H, 4 ꢁ OCHHPh), 4.81–4.69
(m, 3H, OCHHPh, H-1, H-3), 4.69–4.57 (m, 3H, 3 ꢁ OCHHPh),
3.88–3.72 (m, 4H, H-4, H-5, H-6a, H-6b), 3.59 (dd, J = 4.03, 10.10,
Hz, 1H, H-2), 3.40 (s, 3H, OCH3); 13C NMR (151 MHz, CDCl3) d
138.2, 137.7, 135.6, 135.6, 128.7, 128.7, 128.6, 128.6, 128.6,
128.5, 128.5, 128.5, 128.4, 128.3, 128.1, 128.0, 128.0, 128.0,
127.9, 127.6, 127.6, 127.6, 97.8, 82.0, 81.9, 73.6, 73.2, 70.3, 70.0,
70.0, 69.9, 69.8, 69.7, 68.9, 55.2; 31P NMR (243 MHz, CDCl3) d
18.83; HRMS (ESI) calcd for C35H40O9P [M+H]+: 635.2404, found
635.2411.
3.10.12. Methyl 3-O-benzyl-4,6-O-benzylidene-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl- -glucopyranosyl-
(1?4)-2,3,6-tri-O-benzyl- -glucopyranoside (31)
a-D-
a
-D
D
Donor 27 (19 mg, 0.033 mmol) and acceptor 23 (22 mg,
0.023 mmol) were glycosylated using the general glycosylation

22
L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
method for thioglycosides to give a 3:1
a/b mixture of 29 (17.5 mg,
0.040 mmol, 52%). Compound 30 (22 mg, 0.016 mmol) was treated
0.013 mmol). Compound 29 was treated with DDQ according to
the conditions in the general method for PMB removal, and puri-
fied by SGC (hexanes/ethyl acetate 3:1) to give 31 (6 mg,
0.005 mmol, 15% for two steps) as a white foam. 1H NMR
(400 MHz, CDCl3) d 7.64–6.95 (m, 40H, PhH), 5.69 (d, J = 3.59 Hz,
1H, H-100), 5.54 (s, 1H, PhCH), 5.19 (d, J = 3.53 Hz, 1H, H-10), 5.06
(dd, J = 11.33, 22.77 Hz, 2H, 2 ꢁ OCHHPh), 4.84–4.66 (m, 5H,
5 ꢁ OCHHPh), 4.66–4.31 (m, 11H, 10 ꢁ OCHHPh, H-1), 4.13–4.02
(m, 3H, H-600a, H-3, H-40), 4.00–3.81 (m, 4H, H-600b, H-400, H-4, H-
50), 3.80–3.42 (m, 10H, H-5, H-200, H-300, H-6a, H-6b, H-60a, H-60b,
H-2, H-20), 3.39 (s, 3H, OCH3); 13C NMR (101 MHz, CDCl3) d
137.8, 137.6, 137.0, 136.9, 136.9, 136.6, 136.4, 136.4, 127.9,
127.4, 127.4, 127.3, 127.3, 127.3, 127.3, 127.2, 127.2, 127.2,
127.0, 126.9, 126.8, 126.8, 126.8, 126.7, 126.7, 126.6, 126.5,
126.5, 126.4, 126.4, 126.3, 126.1, 125.7, 125.6, 125.0, 100.1,
100.0, 96.7, 95.0, 80.9, 80.4, 79.3, 79.2, 78.8, 78.3, 74.1, 73.6, 73.4,
72.6, 72.4, 72.3, 72.2, 71.8, 71.3, 70.0, 68.6, 67.9, 67.3, 62.8, 54.2;
HRMS (ESI) calcd for C75H80O16Na [M+Na]+: 1259.5339, found
1559.5337.
with DDQ according to the conditions in the general method for
PMB removal, and purified by SGC (hexanes/EtOAc 3:1) to give
32 (8 mg, 0.006 mmol, 37%) as a syrup. 1H NMR (400 MHz, CDCl3)
d 7.55–6.82 (m, 40H, PhH), 5.68 (d, J = 3.76 Hz, 1H, H-100), 5.55 (d,
J = 3.52 Hz, 1H, H-10), 5.41 (s, 1H, PhCH), 4.98 (d, J = 11.65 Hz, 1H,
OCHHPh), 4.91 (d, J = 11.78 Hz, 1H, OCHHPh), 4.73 (dd, J = 9.98,
10.69 Hz, 2H, 2 ꢁ OCHHPh), 4.68–4.57 (m, 1H, OCHHPh), 4.57–
4.45 (m, 4H, H-1), 4.45–4.26 (m, 5H, 5 ꢁ OCHHPh), 4.15–3.89 (m,
6H, H-600a, H-600b, H-400, H-3, H-40, H-5), 3.90–3.64 (m, 5H, H-50,
H-200, H-300), 3.59–3.49 (m, 3H, H-60a, H-60b, H-2), 3.49–3.36 (m,
3H, H-6a, H-6b, H-20), 3.30 (s, 3H, OCH3); 13C NMR (151 MHz,
CDCl3) d 138.8, 138.3, 138.1, 138.0, 137.8, 137.6, 137.3, 129.1,
128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.3, 128.2, 128.0,
127.9, 127.8, 127.7, 127.7, 127.5, 127.4, 127.4, 127.3, 127.1,
126.8, 126.6, 126.4, 101.8, 97.8, 96.4, 96.0, 81.9, 81.8, 81.3, 80.1,
79.7, 79.2, 74.4, 73.9, 73.4, 73.2, 72.9, 72.6, 71.4, 70.5, 70.2, 69.6,
68.9, 68.8, 68.6, 62.9, 55.2, 29.7; HRMS (ESI) calcd for C75H80O16Na
[M+Na]+: 1259.5339, found 1559.5332.
3.10.16. Methyl 2-O-benzyl-3-dibenzylphosphate-4,6-O-
3.10.13. Methyl 2-dibenzylphosphate-3-O-benzyl-4,6-O-
benzylidene-a-D-glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-a-D-
benzylidene-
a
-D
-glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-
a-
D-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-D-glucopyranoside
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-
a-D
-glucopyranoside
(34)
(33)
Compound 32 (12 mg, 0.01 mmol) was subjected to the general
procedure for phosphorylation described above and purified by
SGC (hexanes/EtOAc 2:1) to give 34 (8 mg, 0.006 mmol, 60%) as a
syrup. 1H NMR (600 MHz, CDCl3) d 7.55–7.06 (m, 50H, PhH), 5.80
(d, J = 3.80 Hz, 1H, H-100), 5.67 (d, J = 3.48 Hz, 1H, H-10),5.08–4.87
(m, 6H, 5 ꢁ OCHHPh, H-300), 4.86–4.69 (m, 3H, ꢁ OCHHPh), 4.71–
4.60 (m, 3H, H-1, 2 ꢁ OCHHPh), 4.60–4.38 (m, 8H, 8 ꢁ OCHHPh),
4.20–4.09 (m, 4H, H-400, H-500, H-600a, H-300), 4.09–4.03 (m, 1H, H-
30), 3.97–3.77 (m, 5H, H-50, H-4, H-600b, H-3, H-40), 3.77–3.66 (m,
3H, H-6a, H-6a), 3.63–3.48 (m, 4H, H-2, H-20, H-60b, H-6b), 3.42
(s, 3H, OCH3); 13C NMR (151 MHz, CDCl3) d 139.0, 138.9, 138.3,
138.2, 138.0, 137.6, 137.1, 135.8, 135.8, 129.1, 128.7, 128.6,
128.6, 128.5, 128.5, 128.5, 128.4, 128.3, 128.3, 128.3, 128.2,
128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.6,
127.5, 127.5, 127.4, 127.4, 127.3, 127.3, 127.3, 127.3, 127.1,
127.1, 126.8, 126.5, 126.5, 126.4, 101.9, 97.8, 96.8, 96.0, 82.0,
81.7, 80.1, 79.7, 78.0, 75.0, 74.4, 73.8, 73.4, 73.3, 73.1, 72.9, 72.5,
71.7, 70.4, 69.6, 69.3, 69.3, 69.0, 68.9, 68.7, 68.5, 63.0, 55.2; 31P
NMR (243 MHz, CDCl3) d 16.09; HRMS (ESI) calcd for C89H93O19PNa
[M+Na]+: 1519.5914, found 1519.5944.
Compound 31 (13 mg, 0.011 mmol) was subjected to the gen-
eral procedure for phosphorylation described above and purified
by SGC (hexanes/ethyl acetate 4:1) to give 33 (8.6 mg,
0.0055 mmol, 52%) as a syrup. 1H NMR (400 MHz, CDCl3) d
7.61–6.98 (m, 50H, PhH), 5.82 (d, J = 3.69 Hz, 1H, H-100), 5.62 (d,
J = 3.49 Hz, 1H, H-10), 5.55 (s, 1H, PhCH), 5.06–4.97 (m, 2H,
2 ꢁ OCHHPh), 4.97–4.80 (m, 6H, 6 ꢁ OCHHPh), 4.81–4.63 (m,
6H, 6 ꢁ OCHHPh), 4.63–4.37 (m, 8H, 6 ꢁ OCHHPh, H-1, H-200),
4.19–4.11 (m, 2H, H-400, H-500), 4.09–3.94 (m, 5H, H-30, H-300,
H-600a, H-5), 3.95–3.75 (m, 4H, H-3 H-40, H-600b, H-50, H-4),
3.74–3.55 (m, 4H, H-2, H-6a, H-6b, H-60a), 3.54–3.39 (m, 2H,
H-20, H-60b), 3.37 (s, 3H, OCH3); 13C NMR (101 MHz, CDCl3) d
138.9, 138.5, 138.3, 138.1, 138.0, 137.9, 137.4, 128.9, 128.6,
128.5, 128.5, 128.4, 128.4, 128.4, 128.4, 128.3, 128.2, 128.2,
128.2, 127.9, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6, 127.6,
127.6, 127.5, 127.5, 127.4, 127.4, 127.3, 127.3, 127.1, 127.0,
126.7, 126.7, 126.0, 101.2, 97.8, 96.0, 82.2, 82.0, 81.0, 80.1, 79.6,
77.2, 76.8, 74.8, 74.3, 74.0, 73.4, 73.3, 73.1, 73.0, 72.1, 71.5,
70.5, 69.5, 69.3, 68.8, 68.6, 68.6, 63.0, 55.2; 31P NMR (162 MHz,
CDCl3) d ꢀ1.19; HRMS (ESI) calcd for C89H93O19PNa [M+Na]+:
1519.5914, found 1519.5942.
3.10.17. Methyl 3-phosphate-
a-D-glucopyranosyl-(1?4)-a-D-
glucopyranosyl-(1?4)- -gluco pyranoside (36)
a
-D
3.10.14. Methyl 2-phosphate-
a
-
D
-glucopyranosyl-(1?4)-
a
-
D
-
Hydrogenation of 34 (10 mg, 0.006 mmol) according to the gen-
eral method for global deprotection gave 36 (2.5 mg, 0.003 mmol,
50%) as a syrup.1H NMR (600 MHz, MeOD) d 5.23 (d, J = 3.40 Hz,
1H, H-10), 5.18 (d, J = 3.71 Hz, 1H, H-100), 4.70 (d, J = 3.69 Hz, 1H,
H-1), 3.36–3.30 (m, 8H), 3.91–3.78 (m, 9H), 5.12–5.10 (m, 1H),
3.70–3.55 (m, 5H), 3.53 (m, 4H); 13C NMR (101 MHz, MeOD) d
101.3, 101.2, 99.7, 89.7, 80.2, 80.1, 73.5, 73.0, 72.3, 71.8, 71.7,
70.7, 61.0, 60.7, 54.2, 52.6; 31P NMR (162 MHz, MeOD) d 1.25;
glucopyranosyl-(1?4)- -glucopyranoside (35)
D
Compound 33 (8.6 mg, 0.0055 mmol) was subjected to the gen-
eral procedure for global deprotection described above to give 35
(2.7 mg, 0.0045 mmol, 83%) as a syrup.
1H NMR (400 MHz, CD3OD) d 5.52 (d, J = 3.50 Hz, 1H, H-100), 5.15
(d, J = 3.20 Hz, 1H, H-10), 4.66 (d, J = 3.20 Hz, 1H, H-1), 4.07–3.92
(m, 2H), 3.92–3.72 (m, 11H), 3.72–3.56 (m, 2H), 3.56–3.34 (m,
6H); 13C NMR (101 MHz, CD3OD) d 101.1, 99.7, 98.7, 80.3, 74.8,
73.5, 73.2, 72.7, 72.3, 71.7, 70.8, 70.5, 61.4, 60.7, 54.2, 45.9; 31P
HRMS (ESI) calcd for
597.1425.
C
19H34O19
P
[MꢀH]ꢀ: 597.1437, found
NMR (162 MHz, CD3OD) d 3.43; HRMS (ESI) calcd for C19H34O19
P
[MꢀH]ꢀ: 597.1437, found 597.1455.
3.10.18. Methyl 2-O-benzyl-4,6-O-benzylidene-a-D-
glucopyranosyl-(1?4)-2,3,6-tri-O–benzyl-a-D-glucopyranoside
3.10.15. Methyl 2-O-benzyl-4,6-O-benzylidene-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl- -glucopyranosyl-
(1?4)-2,3,6-tri-O-benzyl- -glucopyranoside (32)
Donor 28 (72 mg, 0.126 mmol) and acceptor 23 (63 mg,
0.078 mmol) were glycosylated using the general glycosylation
a
-
D
-
(41)
a-D
Donor 28 (82 mg, 0.144 mmol) and acceptor 37 (38 mg,
0.082 mmol) were subjected to the conditions in the general meth-
od of glycosylation to give 39 as a mixture, followed by PMB re-
moval and purified by SGC (hexanes/EtOAc 4:1) to give 41
(33 mg, 0.041 mmol, 50%) as a white foam. 1H NMR (400 MHz,
a-D
method for thioglycoside to give a 3:1
a/b mixture of 30 (56 mg,

L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
23
CDCl3) d 7.59–7.16 (m, 25H), 5.80 (d, J = 3.69 Hz, 1H, H-10), 5.50 (s,
1H, PhCH), 5.12 (d, J = 11.73 Hz, 1H, OCHHPh), 4.78 (d, J = 11.80 Hz,
1H, OCHHPh), 4.71 (d, J = 12.11 Hz, 2H, OCH2Ph), 4.68–4.45 (m, 5H,
H-1, 4 ꢁ OCHHPh), 4.21–4.05 (m, 4H, H-3, H-30, H-60a, H-40), 3.93–
3.78 (m, 3H, H-60b, H-4, H-50), 3.74–3.57 (m, 3H, H-2, H-6a, H-6b),
3.48 (t, J = 9.46 Hz, 1H, H-5), 3.44–3.28 (m, 4H, H-20, OCH3), 2.48 (br
s, 1H, OH); 13C NMR (101 MHz, CDCl3) d 138.9, 138.1, 137.9, 137.6,
137.2, 129.1, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.0, 127.9,
127.9, 127.5, 127.4, 127.2, 126.7, 126.3, 101.8, 97.7, 96.5, 82.1,
81.3, 80.3, 79.1, 74.2, 73.4, 73.3, 73.0, 71.5, 70.2, 69.4, 68.9, 68.7,
63.0, 55.2; HRMS (ESI) calcd for C48H56NO11 [M+NH4]+: 822.3848,
found 822.3834.
(12 mg, 0.015 mmol, 32%) as a syrup. 1H NMR (400 MHz, CDCl3) d
7.54–7.21 (m, 25H, PhH), 5.52 (s, 1H, PhCH), 5.26 (d, J = 3.47 Hz,
1H, H-10), 5.09 (d, J = 10.91 Hz, 1H, OCHHPh), 4.81–4.73 (m, 2H,
2 ꢁ OCHHPh), 4.73–4.51 (m, 6H, 5 ꢁ OCHHPh, H-1), 4.13 (dd,
J = 4.84, 10.27 Hz, 1H, H-60a), 3.99 (t, J = 9.29 Hz, 1H, H-3), 3.95–
3.80 (m, 3H, H-5, H-4’, H-50), 3.77–3.51 (m, 6H, H-4, H-2’, H-60b,
H-2, H-6a, H-6b), 3.46–3.30 (m, 4H, H-30, OCH3); 13C NMR
(101 MHz, CDCl3) d 138.6, 137.8, 137.8, 137.7, 137.4, 128.9,
128.5, 128.4, 128.3, 128.2, 128.1, 127.9, 127.7, 127.6, 127.5,
126.0, 101.2, 100.9, 97.8, 81.5, 80.7, 80.3, 79.2, 77.2, 76.8, 75.3,
74.7, 73.5, 73.4, 73.2, 69.9, 68.9, 68.5, 63.7, 55.3; HRMS (ESI) calcd
for C48H52O11Na [M+Na]+: 827.3402, found 827.3402.
3.10.19. Methyl 2-O-benzyl-3-dibenzylphosphate-4,6-O-
3.10.22. Methyl 2-dibenzylphosphate-3-O-benzyl-4,6-O-
benzylidene-
a
-D
-gluco pyranosyl-(1?4)-2,3,6-tri-O-benzyl-
a-
D-
benzylidene-a-D-glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-a-D-
glucopyranoside (43)
glucopyranoside (42)
Compound 41 (33 mg, 0.041 mmol) was subjected to the gen-
eral procedure for phosphorylation described above and purified
by SGC (hexanes/EtOAc 4:1) to give 43 (38 mg, 0.036 mmol, 88%)
as a white foam. 1H NMR (600 MHz, CDCl3) d 7.60–6.99 (m, 35H,
PhH), 5.84 (d, J = 3.67 Hz, 1H, H-10), 5.50 (s, 1H, PhCH), 5.18–5.04
(m, 5H, 5 ꢁ OCHHPh), 5.04–4.91 (m, 2H, OCHHPh, H-30), 4.86 (dd,
J = 7.69, 12.12 Hz, 1H, OCHHPh), 4.75–4.67 (m, 3H, H-1,
2 ꢁ OCHHPh), 4.66–4.57 (m, 2H, 2 ꢁ OCHHPh), 4.54 (d, J =
11.69 Hz, 1H, OCHHPh), 4.23–4.11 (m, 3H, H-60a, H-4, H-5),
3.98–3.88 (m, 3H, H-60b, H-3, H-50), 3.72–3.65 (m, 3H, H-2, H-6a,
H-6b), 3.61 (dd, J = 3.76, 9.57 Hz, 1H, H-20), 3.45 (s, 3H, OCH3);
13C NMR (151 MHz, CDCl3) d 139.0, 138.2, 137.9, 137.5, 137.1,
136.3, 136.2, 136.1, 135.6, 135.6, 129.1, 128.7, 128.7, 128.6,
128.5, 128.3, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1,
128.1, 128.0, 128.0, 127.6, 127.6, 127.5, 127.4, 127.3, 127.1,
126.6, 126.5, 101.9, 97.7, 96.9, 82.0, 80.4, 80.2, 78.1, 74.1, 73.4,
73.3, 72.9, 71.7, 69.4, 69.1, 69.0, 69.0, 69.0, 68.9, 68.8, 67.3, 67.3,
63.2, 55.3; 31P NMR (243 MHz, CDCl3) d 16.02; HRMS (ESI) calcd
for C62H65O14PK [M+K]+: 1103.3744, found 1103.3756.
Compound 40 (11 mg, 0.015 mmol) was subjected to the gen-
eral procedure for phosphorylation described above and purified
by SGC (hexanes/EtOAc 3:1) to give 42 (13 mg, 0.013 mmol, 85%)
as a syrup. 1H NMR (600 MHz, CDCl3) d 7.47–7.20 (m, 35H, PhH),
5.91 (d, J = 3.81 Hz, 1H, H-10), 5.59 (s, 1H, PhCH), 5.16–4.84 (m,
7H, 7 ꢁ OCHHPh), 4.80–4.67 (m, 3H, OCH2Ph), 4.64 (d, J = 3.56 Hz,
1H, H-1), 4.59 (dd, J = 6.56, 12.17 Hz, 2H, OCH2Ph), 4.51–4.44 (m,
1H, H-20),4.21 (dd, J = 4.84, 10.31 Hz, 1H, H-60a), 4.16 (t,
J = 9.15 Hz, 1H, H-3), 4.10 (t, J = 9.05 Hz, 1H, H-5), 4.05 (t,
J = 9.34 Hz, 1H, H-30), 3.96 (dd, J = 5.43, 9.92, 1H, H-50), 3.90–3.81
(m, 2H, H-60b, H-4), 3.77–3.67 (m, 3H, H-40, H-6a, H-6b), 3.59
(dd, J = 3.46, 8.60 Hz, 1H, H-2), 3.40 (s, 3H, OCH3); 13C NMR
(151 MHz, CDCl3) d 138.7, 138.4, 138.1, 138.0, 137.4, 137.2,
137.1, 128.7, 128.7, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3,
128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.7,
127.6, 127.5, 127.5, 101.8, 97.9, 96.1, 82.2, 81.4, 80.4, 76.6, 76.6,
74.9, 74.5, 73.5, 73.4, 71.7, 69.5, 69.4, 68.9, 68.8, 67.3, 67.3, 67.2,
67.2, 63.1, 55.3; 31P NMR (243 MHz, CDCl3) d 12.20; HRMS (ESI)
calcd for C62H65O14PNa [M+Na]+: 1087.4004, found 1087.3988.
3.10.20. Methyl 2,6-di-O-benzyl-3-dibenzylphosphate-
a-
D-
3.10.23. Methyl 3-dibenzylphosphate-2,6-di-O-benzyl-a-D-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-
a-
D-glucopyranoside
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-a-D-glucopyranoside
(45)
(44)
Compound 43 (38 mg, 0.036 mmol) was subjected to the condi-
tions according to the general procedure for selective benzylidene
opening and purified by SGC (hexanes/EtOAc 3:2) to afford 45
(29 mg, 0.027 mmol, 75%) as a syrup. 1H NMR (600 MHz, CDCl3) d
7.55–7.09 (m, 35H, PhH), 5.77 (d, J = 3.68 Hz, 1H, H-10), 5.18–4.95
(m, 7H, 7 ꢁ OCHHPh), 4.75 (d, J = 11.86 Hz, 1H, OCHHPh), 4.72 (d,
J = 12.33 Hz, 1H, OCHHPh), 4.68–4.57 (m, 4H, H-1, H-30, OCHHPh,
H-40), 4.58–4.44 (m, 5H, 5 ꢁ OCHHPh), 4.12–4.03 (m, 2H, H-3, H-
4), 3.86–3.81 (m, 3H, H-5, H-50, H-6a), 3.75 (dd, J = 3.62,
10.72 Hz, 1H, H-60a), 3.70–3.58 (m, 3H, H-6b, H-2, H-60a), 3.52
(dd, J = 2.52, 10.88 Hz, 1H, H-60b), 3.49 (dd, J = 3.58, 9.60 Hz, 1H,
H-20), 3.44 (s, 3H, OCH3); 13C NMR (151 MHz, CDCl3) d 139.0,
138.2, 138.2, 138.0, 137.6, 135.6, 135.6, 135.6, 135.5, 128.7,
128.7, 128.6, 128.5, 128.5, 128.3, 128.3, 128.3, 128.2, 128.2,
128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.6, 127.6, 127.5,
127.4, 127.4, 127.1, 126.6, 97.7, 96.5, 82.0, 82.0, 81.9, 80.2, 74.3,
73.6, 73.3, 73.2, 73.0, 72.8, 71.1, 69.9, 69.8, 69.7, 69.7, 69.5, 69.0,
68.6, 67.3, 67.3, 55.3; 31P NMR (243 MHz, CDCl3) d 18.69; HRMS
(APCI) calcd for C62H68O14P [M+H]+: 1067.4341, found 1067.4342.
Compound 42 (8 mg, 0.008 mmol) was subjected to the condi-
tions according to the general procedure for selective benzylidene
opening and purified by SGC (hexanes/EtOAc 3:1) to afford 44
(5 mg, 0.005 mmol, 63%) as a syrup. 1H NMR (400 MHz, CDCl3) d
7.34–6.99 (m, 35H), 5.69 (d, J = 3.57 Hz, 1H, H-10), 4.98–4.70 (m,
7H, 7 ꢁ OCHHPh), 4.70–4.55 (m, 2H, 2 ꢁ OCHHPh), 4.54–4.42 (m,
3H, 2 ꢁ OCHHPh, H-1), 4.36 (d, J = 12.00 Hz, 1H, OCHHPh),
4.32–4.23 (m, 2H, H-20, OCHHPh), 4.02–3.91 (m, 2H, H-3, H-50),
3.84–3.68 (m, 4H, H-30, H-60a, H-5), 3.68–3.54 (m, 3H, H-40, H-
60b, H-6a), 3.48-3.42 (m, 2H, H-2, H-6b), 3.28 (s, 3H, OCH3), 2.53
(d, J = 2.51 Hz, 1H, OH); 13C NMR (151 MHz, CDCl3) d 138.9,
138.5, 138.1, 137.9, 135.8, 135.7, 128.7, 128.6, 128.4, 128.4,
128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9,
127.8, 127.8, 127.7, 127.6, 127.5, 127.3, 97.9, 95.7, 81.4, 80.2,
79.6, 75.0, 74.5, 73.6, 73.4, 73.3, 72.5, 72.1, 70.3, 70.0, 69.6, 69.4,
69.3, 68.9, 67.3, 55.3; 31P NMR (162 MHz, CDCl3) d ꢀ0.99; HRMS
(ESI) calcd for C62H68O14P [M+H]+: 1067.4341, found 1067.4319.
3.10.24. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1?4)-3-dibenzyl phosphate-2,6-di-O-benzyl-
a-D-
3.10.21. Methyl 3-O-benzyl-4,6-O-benzylidene-
a-
D-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-a-D-glucopyranoside
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-
a-
D-glucopyranoside
(48)
(40)
Donor 46 (29 mg, 0.042 mmol) and acceptor 45 (15 mg,
0.014 mmol) were subjected to the conditions according to the gly-
cosylation procedure and purified by SGC (hexanes/EtOAc 4:1) to
give 48 (16 mg, 0.010 mmol, 72%) as a syrup. 1H NMR (600 MHz,
CDCl3) d 7.50–6.98 (m, 55H, PhH), 5.83 (d, J = 3.58 Hz, 1H, H-100),
Donor 27 (50 mg, 0.088 mmol) and acceptor 37 (22 mg,
0.047 mmol) were subjected to the conditions in the general meth-
od of glycosylation to give 38 as a mixture, followed by PMB re-
moval and purified by SGC (hexanes/EtOAc 4:1) to give 40

24
L. Liu, N. L. B. Pohl / Carbohydrate Research 369 (2013) 14–24
5.66 (d, J = 3.54 Hz, 1H, H-10), 5.05–4.95 (m, 3H, H-30, 2 ꢁ OCHHPh),
4.95–4.89 (m, 1H, OCHHPh), 4.89–4.83 (m, 5H, 5 ꢁ OCHHPh), 4.83–
4.79 (m, 2H, 2 ꢁ OCHHPh), 4.77 (d, J = 11.12 Hz, 1H, OCHHPh),
4.75–4.68 (m, 1H, OCHHPh), 4.66–4.58 (m, 6H, H-1, 5 ꢁ OCHHPh),
4.56–4.38 (m, 8H, 8 ꢁ OCHHPh), 4.32 (dd, J = 12.11, 16.95 Hz, 2H,
2 ꢁ OCHHPh), 4.27 (dd, J = 6.52, 9.76 Hz, 1H, H-40), 4.06–3.99 (m,
2H, H-400, H-3), 3.87–3.81 (m, 2H, H-300, H-4), 3.79–3.74 (m, 2H,
H-5), 3.74–3.66 (m, 3H, H-600a, H-600b), 3.66–3.51 (m, 5H, H-20, H-
6a, H-2, H-6b, H-60a), 3.51–3.42 (m, 5H, H-500, H-50, H-200, OCH3,
H-60b); 13C NMR (151 MHz, CDCl3) d 138.9, 138.8, 138.7, 138.4,
138.2, 138.2, 138.1, 138.0, 137.9, 128.5, 128.5, 128.4, 128.4,
128.3, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0,
127.9, 127.9, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 127.4,
127.4, 127.4, 127.3, 127.2, 126.9, 97.8, 95.2, 94.5, 82.0, 81.7, 80.2,
80.1, 75.4, 74.9, 74.3, 73.4, 73.3, 73.3, 72.9, 72.6, 72.3, 71.8, 71.0,
70.4, 69.5, 69.4, 69.4, 69.2, 69.2, 68.8, 68.7, 68.5, 55.3; 31P NMR
(243 MHz, CDCl3) d 15.13; HRMS (ESI) calcd for C96H101O19PNa
[M+Na]+: 1611.6567, found 1611.6524.
3.10.27. Methyl
glucopyranosyl-(1?4)-
a
-
D
-glucopyranosyl-(1?4)-3-phosphate-
a-D-
a
-D
-gluco pyranoside (50)
Hydrogenation of 48 (10 mg, 0.006 mmol) according to the gen-
eral method for global deprotection gave 50 (3 mg, 0.004 mmol,
67%) as a syrup. 1H NMR (600 MHz, MeOD) d 5.49 (br s, 1H, H-
10), 5.19 (br s, 1H, H-100), 4.60 (br s, 1H, H-1), 4.07–3.76 (m, 4H),
3.76–3.58 (m, 5H), 3.58–3.36 (m, 6H), 3.24–3.14 (m, 6H); 13C
NMR (151 MHz, MeOD) d 99.7, 96.8, 92.6, 76.7, 76.6, 74.9, 73.5,
73.0, 72.5, 71.6, 71.4, 70.5, 70.4, 61.5, 61.4, 60.8, 54.2; 31P NMR
(162 MHz, MeOD)
d 2.20; HRMS (ESI) calcd for C19H34O19P
[MꢀH]ꢀ: 597.1437, found 597.1436.
Acknowledgments
This work was supported in part by the National Institutes of
Health (1R01GM090280). N.L.B.P. also acknowledges the Wilkin-
son Professorship in Interdisciplinary Engineering.
Supplementary data
3.10.25. Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1?4)-2-dibenzylphosphate-3,6-di-O-benzyl-
glucopyranosyl-(1?4)-2,3,6-tri-O-benzyl-a-D-glucopyranoside
(47)
a-D-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.
12.015.
Donor 46 (10 mg, 0.012 mmol) and acceptor 44 (5 mg,
0.005 mmol) were subjected to the conditions according to the
glycosylation procedure and purified by SGC (PhCH3/EtOAc 8:1)
References
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(600 MHz, CDCl3) 7.65–7.00 (m, 50H, PhH), 5.73 (d,
a
syrup. 1H NMR
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J = 3.51 Hz, 1H, H-10), 5.59 (d, J = 3.63 Hz, 1H, H-100), 5.09–4.98
(m, 2H, 2 ꢁ OCHHPh), 4.98–4.88 (m, 2H, 2 ꢁ OCHHPh), 4.88–
4.77 (m, 6H, 6 ꢁ OCHHPh), 4.77–4.68 (m, 3H, 3 ꢁ OCHHPh),
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2H, H-3, H-5), 4.02–3.94 (m, 1H, H-40), 3.94–3.87 (m, 2H, H-600a,
H-300), 3.80 (d, J = 7.13 Hz, 1H, H-4), 3.79–3.73 (m, 2H, H-600b, H-
50), 3.72–3.66 (m, 2H, H-60a, H-400), 3.57 (m, 2H, H-60b, H-6a),
3.54–3.47 (m, 2H, H-200, H-2), 3.41 (dd, J = 2.00, 10.75 Hz, 1H, H-
6b), 3.38 (s, 3H, OCH3); 13C NMR (151 MHz, CDCl3) d 139.0,
138.7, 138.4, 138.2, 138.0, 129.0, 128.6, 128.4, 128.3, 128.3,
128.2, 128.2, 128.2, 128.1, 128.0, 127.8, 127.8, 127.7, 127.7,
127.6, 127.6, 127.5, 127.5, 127.4, 127.4, 127.2, 127.1, 126.7,
97.9, 97.1, 95.7, 82.1, 81.2, 80.0, 79.5, 77.6, 75.4, 74.9, 74.7,
73.7, 73.5, 73.3, 73.1, 71.1, 71.1, 69.7, 69.2, 68.8, 68.2, 55.3; 31P
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d
16.68; HRMS (ESI) calcd for
96H101O19PNa [M+Na]+: 1611.6567, found 1611.6565.
3.10.26. Methyl
a-
D-glucopyranosyl-(1?4)-2-phosphate-
a-D-
glucopyranosyl-(1?4)-
a-D
-gluco pyranoside (49)
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Hydrogenation of 48 (3 mg, 0.0018 mmol) according to the gen-
eral method for global deprotection gave 12 (0.5 mg, 0.001 mmol,
48%) as a syrup.1H NMR (700 MHz, MeOD) d 5.75 (m, 1H, H-10),
5.24 (m, 1H, H-100), 4.73 (m, 1H, H-1), 4.20–4.02 (m, 3H), 3.99–
3.81 (m, 5H), 3.79–3.58 (m, 6H), 3.54–3.14 (m, 6H); 13C NMR
(176 MHz, MeOD) d 104.1, 102.3, 99.0, 82.1, 80.8, 80.4, 78.4, 76.5,
76.3, 76.1, 75.5, 74.4, 74.1, 73.0, 72.9, 72.8, 64.0, 63.6, 63.3, 56.9;
31P NMR (243 MHz, MeOD)
d 3.20; HRMS (ESI) calcd for
C
19H34O19P [MꢀH]ꢀ: 597.1437, found 597.1435.