Why Is Direct Glycosylation with N-Acetylglucosamine Donors Such a Poor Reaction and What Can Be Done about It?

Article abstract of DOI:10.1021/acs.joc.6b02305

The monosaccharide N-acetyl-d-glucosamine (GlcNAc) is an abundant building block in naturally occurring oligosaccharides, but its incorporation by chemical glycosylation is challenging since direct reactions are low yielding. This issue, generally agreed upon to be caused by an intermediate 1,2-oxazoline, is often bypassed by introducing extra synthetic steps to avoid the presence of the NHAc functional group during glycosylation. The present paper describes new fundamental mechanistic insights into the inherent challenges of performing direct glycosylation with GlcNAc. These results show that controlling the balance of oxazoline formation and glycosylation is key to achieving acceptable chemical yields. By applying this line of reasoning to direct glycosylation with a traditional thioglycoside donor of GlcNAc, which otherwise affords poor glycosylation yields, one may obtain useful glycosylation results.

Full text of DOI:10.1021/acs.joc.6b02305

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Article
Why is Direct Glycosylation with N-Acetylglucosamine Donors
Such a Poor Reaction and What Can Be Done About It?
Mikkel H. S. Marqvorsen, Martin J Pedersen, Michelle R. Rasmussen,
Steffan K. Kristensen, Rasmus Dahl-Lassen, and Henrik Helligsø Jensen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02305 • Publication Date (Web): 05 Dec 2016
Downloaded from http://pubs.acs.org on December 5, 2016
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Why is Direct Glycosylation with NꢀAcetylglucosamine Donors
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Such a Poor Reaction and What Can Be Done About It?
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Mikkel H. S. Marqvorsen, Martin J. Pedersen, Michelle R. Rasmussen, Steffan K. Kristensen,
Rasmus DahlꢀLassen and Henrik H. Jensen*
Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C,
Denmark
hhj@chem.au.dk
Graphical Abstract:
Abstract
The monosaccharide NꢀacetylꢀDꢀglucosamine (GlcNAc) is an abundant building block in
naturally occurring oligosaccharides, but its incorporation by chemical glycosylation is
challenging since direct reactions are lowꢀyielding. This issue, generally agreed upon to be
caused by an intermediate 1,2ꢀoxazoline, is often bypassed by introducing extra synthetic
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steps to avoid the presence of the NHAc functional group during glycosylation. The present
paper describes new fundamental mechanistic insights into the inherent challenges of
performing direct glycosylation with GlcNAc. These results show that controlling the balance
of oxazoline formation and glycosylation is key to achieving acceptable chemical yields. By
applying this line of reasoning to direct glycosylation with a traditional thioglycoside donor
of GlcNAc, which otherwise affords poor glycosylation yields, one may obtain useful
glycosylation results.
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Introduction
Glycosylation chemistry has become increasingly important since the emergence of
glycomics in cellularꢀ and molecular biology as an important field of study.1 Indeed, the vast
majority of functional proteins have been shown to be heavily and diversely glycosylated in
vivo. The role of these abundant postꢀtranslational modifications in such matters as protein
localization, function regulation and stability towards degradation has been probed only
superficially even today. Access via synthetic organic chemistry to analytically pure samples
of relevant oligosaccharides is required for further inꢀdepth investigation of the roles of such
carbohydrates.
NꢀAcetylꢀDꢀglucosamine (GlcNAc) is the most abundant monosaccharide building block
in mammalian glycoconjugates,2 and their incorporation into synthetic oligosaccharides for
biological studies is therefore a necessity. Chemical manipulation of this particular unit is,
however, far from trivial due to solubility issues and to the fact that GlcNAcꢀderivatives
function poorly both as glycosyl donors3,4 and acceptors.5,6,7 A wellꢀaccepted general
presumption is that an intermediate oxazoline (Scheme 1)8,9 is the cause of this poor donor
reactivity. Many protecting group strategies prevent the formation of such oxazolines by
ensuring the lack of a proton on the nitrogen (e.g. N3, NAc2, NPhth). Thus, even if
neighbouring group participation is possible during glycosylation, no stable intermediate can
be formed (Scheme 2A).
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Scheme 1. General scheme for direct glycosylation with GlcNAc with oxazolines as the presumed critical
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5
intermediate during the reaction.
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9
Other well studied and high yielding protecting group strategies, however, preserve the NH
functionality in the glycosyl donor (e.g. NHTroc and NHC(O)CH2Cl), thus maintaining the
possibility of forming the oxazoline (or a derivative thereof, see Scheme 2B). In fact, the
existence and stability of such oxazolines has been investigated and established for both
NHTroc10 and NHC(O)CH2Cl11 protected derivatives of GlcNAc.
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Scheme 2. Nꢀprotecting groups typically participate in glycosylation through oxazoline/oxazolinium ion
formation during glycosylation.
We12 and others13,14 have recently addressed the issue of performing direct glycosylation
reactions using GlcNAc donors (GlcNAcꢀylation) in the presence of the 2ꢀacetamido
functionality. Through investigations of the catalytic activation of tetraꢀOꢀacetylꢀβꢀGlcNAc
with various Lewis acidic promotors, we found that the βꢀanomeric acetyl ester was a suitable
leaving group in such systems to give acceptable glycosylation yields (Scheme 3A).12
Similarly, we have recently demonstrated that βꢀconfigured anomeric pivalates (Scheme 3B),
which can be readily synthesized from GlcNAc,15,16 are good donors of Nꢀacetylꢀ
Dꢀ
galactosamine (GalNAc).17
In an attempt to increase the reaction rate for GlcNAcꢀylation by exchanging the donor
functionality to either 1ꢀSPh or 1ꢀOꢀpentenyl, we found the donor fully activated instantly
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regardless of the arming/disarming character of the Oꢀprotecting groups. This system,
however, only returned diminished yields of glycosylation products (Scheme 3C).18
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Scheme 3. Overview of previous direct and catalytic GlyNAcꢀylation. For references, see: A: ref. 12; B: ref. 17;
C: ref. 18.
Given the results listed in Scheme 3, we speculated that balancing the rate of the donor
activation (k1, Scheme 1) with that of the reaction between the oxazoline intermediate and the
acceptor alcohol (k2, Scheme 1) could lead to an improved overall reaction outcome. In this
article, we report on the effect of having arming benzyl protection groups on the donor whilst
maintaining the slowly activating anomeric βꢀacetate (donor 1, Scheme 3D), thereby tuning
the reaction rates (small k1, large k2) in a way that keeps the oxazoline concentration low in
solution. Our studies have led to fundamental insights into the reasons for the disappointing
results found with conventional GlcNAc donors.
Results and Discussion
We synthesized donor 1 from commercially available
DꢀGlcNAc using a modified
diastereoselective acetylation of 2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ
D
ꢀglucopyranose to
give donor 1 with a satisfying crude α/βꢀratio of 6:94 (Scheme 4A).19
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Scheme 4. Formation of donor 1 and glycosylation with allyl alcohol.
In accordance with previous work a range of Lewis acids was screened to establish the
behaviour of the newly synthesized donor in chemical glycosylation. The most efficient
catalysts with respect to reaction time were Bi(OTf)3, Fe(OTf)3 and TfOH. By using the
metal catalyst in combination with the base triꢀtertꢀbutyl pyrimidine (TTBP)20 or as their
21
dimethylsulfoxide (DMSO) complexes led to a significant deterioration in overall reaction
rate, the magnitude of which for TTBPꢀadded reactions is in contrast to that reported by Beau
and coꢀworkers, where only a slight rate decrease with TTBP was observed.13
Table 1. Catalytic activation of donor 1 according to Scheme 4B.a
Entry Catalyst
Loadingb Timec
Yieldd
83%
1
Bi(OTf)3
15%
15%
15%
20 min
2
Bi(OTf)3 + TTBP
Bi(OTf)3·7.8DMSO
> 6 h 30 min 87%
3
40 min
4 h 30 min
25 min
50 min
5 h 20 min
20 min
35 min
45 min
4 h
74%
91%
88%
86%
88%
74%
70%
83%
87%
86%
82%
90%
87%
4
Bi(OTf)3·7.8DMSO + TTBP 15%
5e
6
Fe(OTf)3
15%
15%
Fe(OTf)3·6.2DMSO
7
Fe(OTf)3·6.2DMSO + TTBP 15%
8
Cu(OTf)2
Yb(OTf)3
FeCl3
15%
15%
15%
15%
15%
15%
5%
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10
11
12
13
14
15
BiCl3
CuCl2
6 h
TfOH
30 min
40 min
60 min
Bi(OTf)3
Fe(OTf)3
5%
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16
Sc(OTf)3
Cu(OTf)2
Yb(OTf)3
Bi(OTf)3
Bi(OTf)3
5%
60 min
70 min
115 min
110 min
18 h
88%
86%
91%
83%
73%
17
5%
18
5%
19f
20g
15%
15%
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aAll reactions performed in CH2Cl2 at reflux with 3 equivalents of allyl alcohol as the acceptor with or without 2 equivalents
b
c
of TTBP; Catalyst loading relative to donor; Time required for full conversion of donor as determined by TLC analysis;
f
dIsolated yield after chromatography; eCatalyst added in a glove box due to catalyst hygroscopicity; TetraꢀOꢀacetyl βꢀ
GlcNAc with benzyl alcohol12; g1ꢀepimer of donor 1 (1α) used.
Going to only 5 mol% catalyst loading still provided a fast turnover and produced allyl
glycoside 2 in a useful timespan (Entries 14ꢀ18, Table 1). As expected, reaction times were
considerably shorter for donor 1 than for its αꢀanomer (Entry 20, Table 1) and for the
corresponding tetraꢀOꢀacetylꢀβꢀGlcNAc (Entry 19, Table 1), which we had previously
reported.
Table 2. Glycosylation of various alcohols with donor 1.a
Entry Acceptor
Product
Catalystb Tempc
Timed
Yielde
5%
1
Reflux 50 h
80% (16)
Bi(OTf)3
3
8
9
5%
2
Reflux 48 h
90%
Bi(OTf)3
4
5%
3
Reflux 50 h
71% (22)
Bi(OTf)3
5
10
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2.5%
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Fe(OTf)3
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10
45 ºC
16 h
87%
·6.2DMSO
5%
Reflux 48 h
84% (16)
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Bi(OTf)3
6
11
11
5%
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7
6
6
80 ºCf 1.5 h
Reflux 43 h
50% (39)
98%
Bi(OTf)3
2.5%
11
11
Bi(OTf)3
2.5%
Fe(OTf)3
8
6
45 ºC
16 h
88%
·6.2DMSO
2.5%
9
Reflux 6 days 29% (71)
Bi(OTf)3
12
12
7
7
2.5%
Fe(OTf)3
10
45 ºC
72 h
37% (58)
·6.2DMSO
aReactions were conducted in CH2Cl2 with donor and acceptor in a 2:1 ratio; Catalyst loading with respect to donor; All
b
c
d
reactions were conducted in sealed vials except where marked “Reflux”; No further conversion as determined by TLC
analysis; eIsolated yield; in brackets reꢀisolation of acceptor in percent; fReaction carried out under microwave irradiation.
A series of more complex and relevant glycosyl acceptors were explored as substrates to
establish the scope of this new glycosylation system. Of the most timeꢀefficient catalysts, we
carried on with the easily handled Bi(OTf)3. In addition, we tested Fe(OTf)3·6.2DMSO,
which has been reported as a catalyst in similar glycosylation reactions13 with the advantage
of a lower hygroscopicity than Fe(OTf)3 itself.21 Both excellent glycosylation yields, shorter
reaction times and stoichiometry were found with primary acceptor alcohols compared to
previous studies (Entries 1ꢀ8, Table 2).12,13 The secondary acceptor 7 (Entries 9 and 10, Table
2) posed more of a challenge, as has often been the case in earlier studies. The yield could be
optimised to 37% with reꢀisolation of most (58%) of the unreacted acceptor. This resulted is
an improvement of our earlier findings with the corresponding tetraꢀOꢀacetylated donor (21%
yield, 15 mol% Sc(OTf)3), where four equivalents of glycosyl donor were used.12 In general,
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reactions with challenging acceptors were found to be improved by using less forcing
conditions by changing to a milder or less catalyst. This is in accordance with our previous
observations for GalNAcꢀylation (Scheme 3B).17
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The extended reaction time needed for reaction with the secondary acceptor alcohol 7
resulted in significant amounts of the unexpected benzyl βꢀglycoside 13 (23%, Scheme 5A),
which was also formed in the absence of acceptor (Scheme 5B). This surprising sideꢀreaction
will be discussed further below (Scheme 9).
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Scheme 5. Reaction of donor 1 over prolonged reaction times with acceptor 5 or in the absence of acceptor.
Detailed reaction monitoring
We considered that a more detailed knowledge about the course of these glycosylation
reactions might result in important fundamental insights necessary for future developments in
the field. Therefore, representative glycosylation reactions were performed in an NMRꢀtube
at 40 °C with CDCl3 as the solvent and 1,3,5ꢀtris(trifluoromethyl)benzene as internal
reference for quantification (Scheme 6).
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OBn
O
ROH
OBn
OBn
O
R = All, iPr or L-Menth
BnO
BnO
O
BnO
BnO
BnO
BnO
via
OAc
OR
O
TfOH or Bi(OTf)3 (cat.)
CDCl3
AcHN
AcHN
N
H
1
CF3
F3C
7
8
(1 eq)
CF3
9
Scheme 6: Reaction conditions for GlcNAcꢀylation monitored by 1HꢀNMR analysis (see Figure 1 and Figure 2)
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Figure 1 and Figure 2 contain normalized integral values as a function of reaction time for
selected experiments. Glycosylation with donor 1, AllOH or iPrOH (3 eq.) and 2.5 mol%
Bi(OTf)3 at 40 °C both went cleanly, forming product and consuming donor in a synchronous
manner across the course of the reaction. A similar result, albeit with a considerably longer
reaction time, was obtained under similar conditions using 1 equivalent of the acceptor
(ꢀ)ꢀLꢀmenthol (Figure 1 and Scheme 7). In none of these reactions did the signals from an
oxazolinium species appear integratable in the obtained spectra.
The results changed considerably on going from 2.5 mol% to 15 mol% catalyst (Figure 2).
Under these more forcing conditions, (ꢀ)ꢀLꢀmenthol consumption and product formation
occurred more slowly than the donor activation and oxazolinium ion was in this case
observable throughout the reaction to a level corresponding to the catalyst loading. At the 33
min. data point 15% of donor is in actual fact unaccounted for. The same overall observation
was made using 15 mol% TfOH as the catalyst.
Figure 1. GlcNAcꢀylation reaction course according to Scheme 6 by 1HꢀNMR integrals of donor 1 and product
in glycosylation of AllOH or iPrOH (2.5 mol% Bi(OTf)3) (left). Closed symbols: Donor; Open symbols:
Product. Circles: iPrOH; Squares: AllOH; and (ꢀ)ꢀ
Lꢀmenthol (2.5 mol% Bi(OTf)3) (right). The integrals were
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normalized according to the internal standard 1,3,5ꢀtris(trifluoromethyl)benzene. Closed symbols: Donor; Open
symbols: product.
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Figure 2GlcNAcꢀylation reaction course according to Scheme 6 by 1HꢀNMR integrals of donor 1 and product in
glycosylation of (ꢀ)ꢀLꢀmenthol (15 mol% Bi(OTf)3, left; 15 mol% TfOH, right). Closed symbols: Donor; Open
symbols: Product. Dotted symbols: Oxazolinium. The integrals were normalized according to the internal
standard 1,3,5ꢀtris(trifluoromethyl)benzene.
Based on these observations, reaction yields might be expected to decrease upon going from
low to high catalyst loading. Indeed, with a fixed reaction time of 16 hours, we observed a
significantly higher yield of the corresponding reaction using (ꢀ)ꢀLꢀmenthol when the catalyst
loading was lowered from 15 mol% to 2.5 mol% (Scheme 7A). Yields could also be
optimized with acceptors 5 and 6 as shown in Table 2.
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A
5
6
7
8
HO
(L-Menthol)
OBn
OBn
O
Yield*
O
BnO
BnO
BnO
BnO
OAc
O
Bi(OTf)3 (cat)*, CH2Cl2
40 oC, 16 h
52%
78%
15 mol%:
2.5 mol%:
NHAc
NHAc
15
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B
Bi(OTf)3 (2.5 mol%)
L-Menthol (1 eq)
OBn
O
OBn
O
Yield
BnO
BnO
BnO
BnO
SPh
O
<44 %
79 %
Direct:
16 h:
NIS (1.5 eq.)
CH2Cl2, 40 C
NHAc
NHAc
16
15
OH
O
BnO
BnO
OBn
O
BnO
BnO
BnO
6
OBn
O
SPh
NHAc
Yield (corrected)
OMe
O
0.5 eq.
AcHN
BnO
BnO
Direct: 28 %
16 h:
24 h:
O
Bi(OTf)3 (2.5 mol%)
NIS (1.5 eq.)
CH2Cl2, 40 C
BnO
BnO
63 %
71 %
BnO
OMe
16
11
1
Scheme 7. Isolated yields of A: Reactions corresponding to the HꢀNMR time course experiments with (ꢀ)ꢀ
L
ꢀ
menthol as the acceptor using high (15 mol%) or low (2.5 mol%) catalyst loading. B: Reactions with
conventional thioglycoside donor 16 and (ꢀ)ꢀLꢀmenthol or 6 as the acceptor adding NIS either directly or over
extended periods of time.
As shown by NMR, glycosylation with 15 mol% TfOH or Bi(OTf)3, as compared to 2.5
mol%, clearly resulted in the presence of oxazolinium ion in increased concentrations.
1
Furthermore, separate HꢀNMR experiments indicated that one equivalent of TfOH would
convert the acetate donor quantitatively to the oxazolinium species in a few minutes (data not
shown), and this species (or the oxazoline) was readily observable as a single spot by TLC
analysis. These and the above observations led to the hypothesis that, as a consequence of
high catalyst loading, a high concentration of oxazoline during reaction would lead to
formation of sideꢀproducts and diminished yields.
We hereupon turned to a conventional donor type, thioglycoside 16 (Scheme 7B). TLC
analysis revealed the fact that with this donor, activation was in all cases complete after a few
minutes when introducing 2.5 mol% Bi(OTf)3, acceptor and 1.5 eq. NIS in CH2Cl2 at 40 °C.
After this time, oxazoline could be observed on TLC analysis for the remaining reaction time.
As shown in the above, this is in stark contrast to the acetate 1, which activates slowly and in
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an acceptorꢀdependent fashion (Scheme 7A). We speculate that the reason for the generally
observed poor performance of this donor in 2ꢀacetamido systems is that activation of these
thioglycosides is catalytic and acceptorꢀindependent (Scheme 8A). This would lead to an
extreme case of the highꢀcatalystꢀscenario from above. During activation of the
thioglycoside, formation of the relatively acidic oxazolinium species could activate another
equivalent of thioglycoside (Scheme 8A). Oxazolinium, however, would not constitute a
strong enough acid to activate the less labile acetate donor (Scheme 8B). This donor requires
glycosylation of the acceptor to regenerate the acid catalyst for continuation of the activation
cycle.
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A
OBn
O
OBn
O
O
BnO
BnO
BnO
BnO
H
(cat)
OR
ROH
NHAc
O
N
I
N
H
(cat)
Glycosylation not required
for donor activation
O
Oxazolinium ion strong
enough to activate SPh donor 10
in combination with NIS
H
OBn
O
O
BnO
BnO
N
I
O
N
O
H
OBn
O
BnO
BnO
OBn
O
SPh
I2
PhSSPh
I
NHAc
BnO
BnO
SPh
O
NHAc
NH
O
B
OBn
O
OAc
NHAc
OBn
O
OBn
O
BnO
BnO
O
H
BnO
BnO
BnO
BnO
H
(cat)
OAc
O
NHAc
NHAc
Does not occur!
OBn
O
OR
NHAc
OBn
O
BnO
BnO
ROH
BnO
BnO
O
N
AcOH
H
(Not strong enough for
donor activation)
H
(cat)
Catalyst regenerated
upon glycosylation!
Scheme 8. A: Activation of GlcNAc thioglycosides are acceptorꢀindependent; B: Activation of GlcNAc 1ꢀOAc
donors are glycosylation dependent for regeneration of the acid catalyst.
For the above hypothesis to hold true, in accordance with the NMRꢀstudies above, the yields
of the glycosylation reactions would be expected to increase if activation of the donor could
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be slowed down. Accordingly, we performed glycosylation reactions under modified
conditions as shown in Scheme 7B. To control the activation of the thioglycoside, NIS was
added directly to the reaction at the start in one portion or gradually over the 16 hours
reaction time via syringe pump. Scheme 7B reveals that slow activation of the thioglycoside
16 does indeed increase the glycosylation yield of 1 equivalent (ꢀ)ꢀLꢀmenthol from below
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44% to 79%. Furthermore, the reaction became cleaner with slow activation, allowing the
isolation of pure product. This trend extends to the carbohydrateꢀbased acceptor 6; when
subjected to similar reaction conditions, the glycosylation yield increased from the rather
poor 28% to a more pleasing 63%. Increasing the addition time from 16 hours to 24 hours led
to a further enhanced yield of 71%.
Given the above observations, it is interesting to note that when subjected to 2.5 mol%
Bi(OTf)3 and 1.5 eq. NIS in CH2Cl2 at 40 °C without acceptor, an analogous Troc protected
thioglycoside activated immediately, much like the observations we had just made with
thioglycoside 16 (data not shown). We attribute the high yields usually obtained with this
donor type in spite of this to a higher glycosylation rate (k2).
Donor degradation
Keen on understanding what becomes of the donor in situations like the one in Figure 2
where the activation (k1) and glycosylation (k2) rates are mismatched, we set out to identify
some of the donor derived byꢀproducts. Under glycosylation conditions with no acceptor
present three major products could be identified (Scheme 5B). One dominating side reaction
was found to be donor anomerization, which will lower the yield of the overall reaction since
1α is a less reactive donor (Entry 20, Table 1). Lactol is a commonly formed byꢀproduct in
glycosylation reactions22 and could therefore also be expected. More puzzling was the
discovery of benzyl glycoside 13 as a major byꢀproduct, the same compound as observed in
the glycosylation of acceptor 7 in the above (Scheme 5A). As no source of benzyl alcohol had
been added, the donor (1) must itself have been the source. This should lead to the
realization, that in a glycosylation like the one in Scheme 5, not only has 23% of the donor 1
been converted to benzyl glycoside 13, but another unknown quantity of 1 will have
presumably provided the benzyl alcohol, making this a very important donor degradation
pathway indeed. In order to investigate the origin of this benzyl alcohol, we synthesized
three deuteriumꢀlabelled analogues 17, 18 and 19 (Scheme 9A) including an alloꢀepimer (19),
and subjected them to glycosylation conditions (Scheme 9B). Each reaction was monitored
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using HRMS and the product distributions of nonꢀlabelled (light) and labelled (heavy)
glycosides were determined for each donor (Scheme 9B).19
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Scheme 9. A: Deuterium labelled glycosyl donors to study the origin of benzyl alcohol in formation of
glycoside 13. B: Experimental conditions used to study product distributions by HRMS.
Assuming equal ionizability of these monoꢀ, bisꢀ and trisꢀlabelled benzyl glycosides, HRMS
indicates the source of the anomeric benzyl group of 13. We were surprised to find that the
combined results indicated that the 3ꢀOBn and the 4ꢀOBn account for roughly 40% of the
liberated benzyl alcohol each.19 We speculate that the Ferrierꢀtype rearrangements reported by
Lichtenthaler et al.23,24 in similar systems might at least partially explain these findings
(Scheme 10). Participation of the NHAc was contraindicated by experiments with the alloꢀ
configured analogue 19, which yielded the benzyl glycosides in roughly the same ratio as did
the corresponding glucoꢀconfigured 17 (1.6:1 and 1.5:1, respectively).19
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Scheme 10. Proposed mechanism for the formation of BnOH from donor 1 per analogy to related observations
by Lichtenthaler and coꢀworkers.23,24
Conclusion
We have shown that donor 1 performs excellently as a glycosyl donor in direct GlcNAcꢀ
ylation with primary alcohol acceptors, and that this is an improvement compared to previous
donor types investigated in terms of reaction time, catalyst loading and excess of donor used.
Compared to existing methodology the glycosylation yield has been significantly improved,
but for reactions with a secondary carbohydrate based acceptor yields remain moderate.
More importantly, this study demonstrates the power of balancing the rate of oxazoline
formation (k1, Scheme 1) controlled by e.g. catalyst loading/Lewis acidity and glycosylation
(k2, Scheme 1) influenced by the nature of the acceptor nucleophile. Time course experiments
showed a correlation between the synchronous nature of the glycosylation reaction and the
success of a direct GlcNAcꢀylation. The gained insight led to the controlled activation of a
GlcNAc thioglycoside secured by slow addition of NIS to a reaction mixture containing
glycosyl donor, acceptor and Lewis acid catalyst. In the case of a carbohydrate based
acceptor the yield could be rescued from 28% under conditions with direct addition to 71%
with slow addition over 24 hours, approaching the yield obtained with the slowly activating
acetate donor function.
We have furthermore found that a significant sideꢀreaction of the glycosyl donor is loss of
benzyl alcohol, and that this does not proceed through neighbouring group participation since
the analogous AllNAc donor behaved similarly as the GlcNAc donor.
We strongly believe we have shed much needed scientific light upon paramount fundamental
issues concerning GlyNAcꢀderived donors. It is our hope to see these insights kindle a
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renewed interest in GlyNAcꢀchemistry. The question remains whether direct GlyNAcꢀylation
can indeed be optimized significantly beyond the results disclosed herein.
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Experimental Section
General remarks
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Airꢀ and moisture sensitive reactions were conducted in flameꢀdried glassware under an
atmosphere of Ar or N2. Anhydrous solvents were dried over aluminum oxide and dispensed
from a solvent purification system. DMF and dioxane were purchased as anhydrous. Solvents
were removed under reduced pressure at 40 °C. Flash column chromatography was
performed using silica gel (230ꢀ400 Mesh) unless otherwise noted. TLC analysis was
conducted on silica gel coated aluminum foil (Kieselgel 60 F254) and observed under UVꢀ
light or visualized by staining in 10 % H2SO4 with orcinol followed by vigorous heating.
NMRꢀspectra were recorded on a 400 spectrometer. Chemical shifts (δ) are reported in ppm
relative to the residual signals (1HꢀNMR: CDCl3, 7.26; CD3OD, 4.87; DMSOꢀd6, 2.50 and
13CꢀNMR: CDCl3, 77.2; CD3OD, 49.0; DMSOꢀd6, 39.5). Hꢀ and 13CꢀNMR spectra were
1
interpreted on the basis of gCOSY, gHMQC and DEPTꢀ135 techniques and where necessary
1
1Dꢀselective HꢀTOCSYꢀ, proton coupled 13Cꢀ, or HMBCꢀspectra. Mass spectra were
recorded on a TOFꢀQ High Performance MS system. Optical rotations were measured in
concentrations reported in g/100mL on a polarimeter and reported in units of deg·cm2·gꢀ1.
Sonication of reactions was carried out in a ultrasonic bath. Microwave experiments were
carried out in a Biotage Initiator (external sensor type). Reaction times listed in these cases
refer to ‘hold time’ at the specified temperature.
General procedure for glycosylation
2ꢀAcetamidoꢀ1ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀDꢀglucopyranose (1) (2 eq.) and
acceptor (1 eq.) were dissolved in dry CH2Cl2 to an acceptor concentration of 0.10 M. Lewis
acid (2.5 or 5 mol% with respect to donor according to Table 2.) was added and the reaction
mixture stirred at 45 °C. The reaction was monitored by TLC analysis until there was no
further indication of reaction development. The reaction mixture was poured into a separation
funnel, diluted with CH2Cl2 and washed with water. The aqueous layer was extracted twice
with CH2Cl2, and the combined organic layers were dried over MgSO4, filtered, concentrated
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under reduced pressure and purified using flash column chromatography which afforded the
desired glycoside products (Table 2.).
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1HꢀNMR time course experiments (Figure 1 and Figure 2)
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2ꢀAcetamidoꢀ1ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀ
D
ꢀglucopyranose (1) (48 mg, 0.09
mmol, 1 eq.), acceptor (3 eq. for AllOH and iPrOH, 1 eq. for (ꢀ)ꢀ
Lꢀmenthol) and the internal
standard 1,3,5ꢀtris(trifluoromethyl)benzene (16.8 µL, 1 eq.) were dissolved in dry CDCl3
(600 µL) and transferred to a NMRꢀtube. Lewis acid (Bi(OTf)3 or TfOH, 2.5 or 15 mol%)
1
was added whereupon the sample was heated to 40 °C while obtaining HꢀNMR spectra at
selected time points (See SI for stacked spectra). Integral values from each spectrum
corresponding to baseline separated signals from donor and product (and oxazolinium where
relevant) were normalized using the internal standard and plotted against time in Figure 1 and
Figure 2 in the main article.
Glycosylation according to Scheme 7A
Two reactions were set up. 2ꢀAcetamidoꢀ1ꢀOꢀacetylꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀβꢀ
Dꢀ
glucopyranose (1) (48 mg, 0.09 mmol, 1 eq.), (ꢀ)ꢀ ꢀmenthol (14.1 mg, 0.09 mmol, 1 eq.) and
L
Bi(OTf)3 (2.5 or 15 mol%) were dissolved in dry CH2Cl2 (0.6 mL) and heated to 40 °C for 16
hours. The reactions were then evaporated onto Celite® and subjected to flash column
chromatography (5→10% EtOAc in toluene) which afforded the desired product 15.
1
Rf 0.56 (20% EtOAc in toluene). HꢀNMR (400 MHz, CDCl3):
δH 7.36ꢀ7.20 (m, 15H, ArH),
5.70 (d, J2,NH 7.9 Hz, 1H, NH), 4.96 (d, J1,2 8.2 Hz, 1H, H1), 4.85 (d, Jgem 11.5 Hz, 1H,
OCHHPh), 4.81 (d, Jgem 10.8 Hz, 1H, OCHHPh), 4.68 (d, 1H, OCHHPh), 4.64 (d, 1H,
OCHHPh), 4.63 (d, Jgem 12.9 Hz, 1H, OCHHPh), 4.55 (d, 1H, OCHHPh), 4.33 (t, J2,3 = J3,4
9,5 Hz, 1H, H3), 3.75 (dd, Jgem 11.0, J5,6a 4.1 Hz, 1H, H6a), 3.69 (dd, J5,6b 1.9 Hz, 1H, H6b),
3.63 (t, J3,4 = J4,5 9,2 Hz, 1H, H4), 3.52 (ddd, 1H, H5), 3.42 (dt, J 4.3 Hz, J 10.7 Hz, 1H,
CHOGlcNAc), 3.15 (dt, 1H, H2), 2.32 (d septet, J 2.5 Hz, J 7.1 Hz, 1H, CH(CH3)2), 1.93 (dt,
J 3.9 Hz, J 12.2 Hz, 1H) 1.84 (s, 3H, NHC(O)CH3), 1.68ꢀ1ꢀ58 (m, 2H), 1.40ꢀ1.25 (m, 1H),
1.24ꢀ1.14 (m, 1H), 1.06ꢀ0.72 (m, 3H), 0.89 (d, J 6.8 Hz, 6H, CH3CHCH3), 0.80 (d, J 7.0 Hz,
13
3H, CH3). CꢀNMR (100 MHz, CDCl3) δC 170.5 (C=O), 138.8, 138.4, 138.3, 128.5, 128.4,
128.03, 127.99, 127.8, 127.72, 127.70, 127.6 (Ar), 97.5 (C1), 80.5 (C3), 79.2 (C4), 78.2,
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74.81 (C5, OCH2Ph), 74.78, 73.76 (OCH2Ph), 69.3 (C6), 58.6 (C2), 47.9, 41.0, 34.4, 31.4,
25.1, 23.7 (NHC(O)CH3), 23.1, 22.4 (CH3), 21.2 (CH3), 15.8 (CH3). HRMS(ES): Calcd. for
C39H51NO6H+ 630.3789; found 630.3866.
7
8
9
The NMR data were in accordance with those previously published.18
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Glycosylation according to Scheme 7B
Two identical reactions were set up in parallel. Phenyl 2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀ
ꢀglucopyranoside18 (16) (53 mg, 0.09 mmol, 1 eq.), (ꢀ)ꢀ
benzylꢀ1ꢀthioꢀβꢀD Lꢀmenthol (14.1 mg,
0.09 mmol, 1 eq.) and Bi(OTf)3 (1.5 mg, 2.3 µmol, 2.5 mol%) were dissolved in dry CH2Cl2
(0.6 mL) and heated to 40 °C. NIS (30 mg, 0.14 mmol, 1.5 eq.) was added as a solution in dry
dioxane (0.5 mL) either directly or over 16 hours via syringe pump (0.52 µL/min). TLC
analysis (20% EtOAc in toluene) indicated full conversion of donor after 30 min. with direct
addition of NIS. After 16 hours TLC also indicated full conversion of donor with slow
addition of NIS. Both reactions were evaporated onto Celite® and subjected to flash column
chromatography (0→5→10% EtOAc in toluene) which afforded the desired product 15.
The glycosylation of methyl 2,3,4ꢀtriꢀOꢀbenzylꢀβꢀ
Dꢀglucopyranoside (6) with phenyl 2ꢀ
ꢀglucopyranoside (16)18 was carried out like
acetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ1ꢀthioꢀβꢀ
D
described above with addition of NIS as described in the scheme.
Isolation of donorꢀderived byꢀproducts according to Scheme 5B
2ꢀAcetamidoꢀ1ꢀOꢀacetylꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀβꢀDꢀglucopyranose (1) (86 mg, 0.16
mmol, 1 eq.) and Bi(OTf)3 (5 mg, 8.1 µmol, 5 mol%) were dissolved in dry CH2Cl2 (1.1 mL)
and heated to 40 °C for 7 days. The resulting mixture was diluted, washed with half saturated
NaHCO3 (aq) which was backꢀextracted thrice with CH2Cl2. The combined organics were
dried over MgSO4, filtered and evaporated to give a crude which was subjected to flash
column chromatography (2:1→1:1→2:3→1:2 pentane:EtOAc, then EtOAc:CH2Cl2 9:1)
which afforded benzyl 2ꢀacetamidoꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀ
Dꢀglucopyranoside (13)
(15.8 mg, 17%), 2ꢀacetamidoꢀ1ꢀOꢀacetylꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀ
D
ꢀglucopyranose (1α)
(13.2 mg, 15%) and 2ꢀacetamidoꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀ
D
ꢀglucopyranose (14) (18.5 mg,
24%).
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2ꢀacetamidoꢀ1ꢀOꢀacetylꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀ
OꢀbenzylꢀβꢀDꢀglucopyranose (1)
7
8
2ꢀAcetamidoꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀ
D
ꢀglucopyranose (12) (14.3 g, 29.0 mmol, 1 eq.)
9
was dissolved in dry pyridine to a concentration of 0.17 M. Pyridinium paraꢀtoluenesulfonate
(PPTS) (11.0 g, 43.6 mmol, 1.5 eq.) was added and the reaction mixture was heated to 100 °C
for 15 minutes under stirring. Ac2O (8.2 mL, 87 mmol, 3 eq.) was added as a solution in dry
pyridine (1.4 M) to the reaction over 15 minutes using syringe pump. The reaction mixture
was quenched with CH3OH (2 mL) and concentrated under reduced pressure to dryness. The
resulting crude was dissolved in CH2Cl2, washed with 1 M HCl (aq), sat. NaHCO3 (aq), and
H2O. The combined aqueous layers were extracted twice with CH2Cl2 which was washed
with 1 M HCl (aq), sat. NaHCO3 (aq), and H2O. The combined organic layers were dried
over anhydrous MgSO4, filtered and concentrated under reduced pressure to give a crude
(16.4 g) with an α/β ratio according to NMR of 7:93. The crude was recrystallized from
EtOAc, filtered and washed with cold Et2O to afford the desired compound 1 (9.85 g, 64%)
as a white solid.
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Rf 0.28 (EtOAc/toluene 1:1). [α]D20 +28.0 (c 1, CHCl3). Lit. +34 (c 0.5, CHCl3).25a Mp 148.1ꢀ
148.8 °C (EtOAc). Lit. 159ꢀ162 °C (EtOAc).25a 1HꢀNMR (400 MHz, CDCl3): δH 7.37ꢀ7.27
(m, 13H, ArH), 7.19 (dd, J 2.2 Hz, J 7.2 Hz, 2H, ArH), 5.69 (d, J1,2 7.8 Hz, 1H, H1), 5.19 (d,
JNH,2 8.8 Hz, 1H, NH), 4.80 (d, Jgem 11.7 Hz, 1H, OCHHPh), 4.76 (d, Jgem 10.5 Hz, 1H,
OCHHPh), 4.65 (d, 1H, OCHHPh), 4.61 (d, Jgem 12.0 Hz, 1H, OCHHPh), 4.58 (d, 1H,
OCHHPh), 4.50 (d, 1H, OCHHPh), 4.03 (dt, J2,3 9,2 Hz, 1H, H2), 3.79 (t, J3,4 = J4,5 8.3 Hz,
1H, H4), 3.75ꢀ3.72 (m, 2H, H6a, H6b), 3.68 (dd, 1H, H3), 3.66 (td, 1H, J5,6 3.5 Hz, H5), 2.06
13
(s, 3H, OCOCH3), 1.79 (s, 3H, NHCOCH3). CꢀNMR (100 MHz, CDCl3) δC 170.1, 169.8
(C=O), 138.1, 137.9, 137.8 (ipsoꢀArC), 128.5, 128.5, 128.4, 128.2, 128.0, 127.9, 127.9, 127.7
(Ar), 92.8 (C1), 80.7 (C4), 77.5 (C3), 75.6, 74.6, 74.3 (OCH2Ph), 73.5 (C5), 68.5 (C6), 53.6
(C2), 23.4 (NHC(O)CH3), 21.1 (OC(O)CH3). HRMS(ES): Calcd. for C31H35NO7Na+
556.2306; found 556.2312.
The NMR data were in accordance with those previously published.11,25
2ꢀAcetamidoꢀ1ꢀOꢀacetylꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀglucopyranose (1α)
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2ꢀAcetamidoꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀ
D
ꢀglucopyranose (12) (380 mg, 0.77 mmol, 1 eq.)
was dissolved in dry pyridine (3.8 mL) before Ac2O (220 ꢁL, 2.3 mmol, 3 eq.) was added to
the mixture and stirred for 20 h. The reaction mixture was poured into iceꢀwater before
extracting with EtOAc and washing with 1 M HCl (aq), sat. NaHCO3 (aq) and brine. The
organic layer was dried over MgSO4, filtered, concentrated under reduced pressure and
purified by flash column chromatography (EtOAc/CH2Cl2 1:5→1:4→1:3) to separate the
anomeric mixture and afford the desired compound 1α (106 mg, 26%) as a colorless
amorphous solid, which was recrystallized in EtOAc to afford colorless crystals (59 mg).
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Rf (EtOAc/toluene 1:1) 0.19. [α]D20 +94.7 (c 1.0, CHCl3). Lit. +149 (c 0.29, MeOH).26 1Hꢀ
NMR (400 MHz, CDCl3): δH 7.40ꢀ7.27 (m, 13H, ArH), 7.22ꢀ7.19 (m, 2H, ArH), 6.14 (d, J1,2
3.5 Hz, 1H, H1), 4.95ꢀ4.88 (m, 1H, NH), 4.89 (d, Jgem 11.8 Hz, 1H, OCHHPh), 4.82 (d, Jgem
10.6 Hz, 1H, OCHHPh), 4.65 (d, 1H, OCHHPh), 4.64 (d, 1H, Jgem 12.1 Hz, OCHHPh), 4.59
(d, 1H, OCHHPh), 4.51 (d, 1H, OCHHPh), 4.32 (ddd, J2,NH 8.6 Hz, J2,3 10.7 Hz, 1H, H2),
3.87 (dd, J3,4 8.7 Hz, J4,5 9.7 Hz, 1H, H4), 3.83ꢀ3.76 (m, 2H, H5, H6a), 3.71 (dd, 1H, H3),
3.69ꢀ3.64 (m, 1H, H6b), 2.07 (s, 3H, OCOCH3), 1.79 (s, 3H, NHCOCH3). 13CꢀNMR (100
MHz, CDCl3): δC 169.9, 169.0 (C=O), 138.3, 138.0, 137.8 (ipsoꢀArC), 128.7, 128.6, 128.5,
128.3, 128.1, 128.0, 127.9, 127.7 (Ar), 91.7 (C1), 79.0 (C3), 77.9 (C4), 75.2, 74.6, 73.6
(OCH2Ph), 73.4 (C5), 68.2 (C6), 51.5 (C2), 23.3 (NHCOCH3), 21.0 (OCOCH3). HRMS(ES):
Calcd. for C31H35NO7Na+ 556.2306; found 556.2309.
Allyl 2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀ
From Allyl 2ꢀacetamidoꢀ2ꢀdeoxyꢀ ꢀglucopyranoside (20):
Allyl 2ꢀacetamidoꢀ2ꢀdeoxyꢀ ꢀglucopyranoside (12.7 g, 48.6 mmol, 1 eq.) was dissolved in
OꢀbenzylꢀβꢀDꢀglucopyranoside (2)
β
ꢀD
β
ꢀD
dry DMF (130 mL). 60 % NaH on mineral oil (7.58 g, 190 mmol, 3.9 eq.) was added slowly
under vigorous stirring before BnBr (19.1 mL, 160 mmol, 3.3 eq.) was added in small
portions over a total of 30 min, after which the mixture was left stirring overnight at room
temperature. The mixture was poured onto ice/water and the precipitate was filtered off and
the mother liquor extracted with EtOAc (3 x 100 mL). The precipitate and the combined
organics were coꢀevaporated to dryness with toluene and then purified by flash column
chromatography (0 → 25% EtOAc in CHCl3) to give the desired product 2 (14.1 g, 54%) as a
colorless solid.
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From 2ꢀacetamidoꢀ1ꢀOꢀacetylꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀβꢀDꢀglucopyranose (1):
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8
Donor 1 (1 eq.) was dissolved in dry CH2Cl2 to a concentration of 0.15 M. Acceptor (3 eq.)
and Lewis acid (according to Table 1) were added and the resulting mixture stirred under
reflux. The reaction was monitored by TLC analysis and upon completion, the mixture was
evaporated onto Celite® and purified by column chromatography to afford the desired allyl
glycoside 2 in yields according to Table 1.
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Rf (EtOAc/CH2Cl2 1:5) 0.26. Mp 144.5ꢀ145.9 °C (CH2Cl2). Lit. 146.5ꢀ149 °C (MeOH).27 1Hꢀ
NMR (400 MHz, CDCl3): δH 7.35ꢀ7.27 (m, 13H, ArH), 7.22ꢀ7.19 (m, 2H, ArH), 5.88 (ddt,
Jtrans 16.9 Hz, Jcis 10.5 Hz, Jvic 5.5 Hz, 1H, CH2CH=CH2), 5.56 (d, JNH,2 7.8 Hz, 1H, NH),
5.25 (dq, Jgem=4J 1.4 Hz, 1H, CH=CHH), 5.16 (dq, 1H, CH=CHH), 4.85 (d, J1,2 7.7 Hz, 1H,
H1), 4.81 (d, Jgem 11.6 Hz, 1H, OCHHPh), 4.78 (d, Jgem 11.1 Hz, 1H, OCHHPh), 4.67 (d, 1H,
OCHHPh), 4.61 (d, Jgem 12.3 Hz, 1H, OCHHPh), 4.59 (d, 1H, OCHHPh), 4.55 (d, 1H,
OCHHPh), 4.33 (ddt, Jgem 13.0 Hz, 1H, OCHHCH=CH2), 4.11 (dd, J2,3 9.4 Hz, J3,4 8.0 Hz,
1H, H3), 4.07 (ddt, 1H, OCHHCH), 3.77 (dd, Jgem 10.7 Hz, J5,6a 2.6 Hz, 1H, H6a), 3.72 (dd,
J5,6b 4.3 Hz, 1H, H6b), 3.64 (dd, J4,5 9.1 Hz, 1H, H4), 3.60 (ddd, 1H, H5), 3.46 (dt, 1H, H2),
1.85 (s, 3H, NHCOCH3). 13CꢀNMR (100 MHz, CDCl3): δC 170.4 (C=O), 138.5, 138.2, 138.1
(ipsoꢀArC), 134.1 (CH2CH=CH2), 128.3, 128.3, 128.3, 127.9, 127.8, 127.7, 127.6, 127.5
(Ar), 117.0 (CH=CH2), 99.4 (C1), 80.9 (C4), 78.5 (C3), 74.8, 74.5 (OCH2Ph), 73.3 (C5), 69.6
(OCH2CH), 69.0 (C6), 56.3 (C2), 23.5 (NHCOCH3). LRMS(ES): calcd. for C32H37NO6Na+
554.3; found 553.9.
The NMR data were in nearꢀcomplete accordance with those previously published,28 although
we disagree with a few of the assignments.
N
ꢀ(9ꢀFluorenylmethoxycarbonyl)ꢀ
Oꢀ(2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀOꢀbenzylꢀβꢀDꢀ
glucopyranosyl)ꢀ ꢀserine methyl ester (8)
L
The general glycosylation procedure was followed using donor 1 (211 mg, 0.40 mmol),
Bi(OTf)3 (5 mol%) and Nꢀ(9ꢀfluorenylmethoxycarbonyl)ꢀ ꢀserine methyl ester 3 (68.4 mg,
L
0.20 mmol). TLC analysis indicated reaction cessation after 50 hours. Flash column
chromatography afforded the desired product 8 (131 mg, 80%) as a clear colorless glass and
reꢀisolated Nꢀ(9ꢀfluorenylmethoxycarbonyl)ꢀLꢀserine methyl ester (3) (11 mg, 16%).
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Rf (Et2O/CH2Cl2 2:5) 0.32. Mp 166.8ꢀ167.8 °C. HꢀNMR (CDCl3, 400 MHz): δH 7.74 (d, J
7.5 Hz, 2H, ArH), 7.68–7.60 (m, 2H, ArH), 7.38 (t, J 7.5 Hz, 2H, ArH), 7.34–7.25 (m, 15H,
ArH), 7.21–7.16 (m, 2H, ArH), 5.98 (d, JNHSer,Hα 8.2 Hz, 1H, NHSer), 5.50 (d, JNH,2 6.6 Hz,
1H, NH), 4.87 (d, J1,2 7.7 Hz, 1H, H1), 4.81 (d, Jgem 11.6 Hz, 1H, OCHHPh), 4.77 (d, Jgem
10.9 Hz, 1H, OCHHPh), 4.64 (d, 1H, OCHHPh), 4.59 (d, Jgem 12.1 Hz, 1H, OCHHPh), 4.57
(d, 1H, OCHHPh), 4.51 (d, 1H, OCHHPh), 4.48–4.40 (m, 2H, Hα, H9), 4.33 (dd, Jgem 10.5
Hz , JHα,Hβ 7.5 Hz, 1H, Hβ), 4.29ꢀ4.21 (m, 2H, H10), 3.98 (t, J2,3 8.8 Hz, 1H, H3), 3.83 (dd,
1H, J 3.1 Hz, Hβ’), 3.74–3.68 (m, 5H, CH3, H6), 3.64 (t, J3,4 8.7 Hz, 1H, H4), 3.54 (dt, J4,5
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9.2 Hz , J5,6 3.1 Hz, 1H, H5), 3.39 (broad q, 1H, H2), 1.81 (s, 3H, NHC(O)CH3). CꢀNMR
(CDCl3, 100 MHz) δ 170.9, 170.5 (C=O), 156.3 (NHC(O)O), 144.0, 143.8, 141.3 (ArC),
138.4, 138.0 (ipsoꢀArC), 128.5, 128.4, 128.4, 128.0, 127.8, 127.7, 127.7, 127.1, 125.3, 125.3,
120.0 (Ar), 100.4 (C1), 80.6 (C4), 78.4 (C3), 75.0 (C5), 74.7, 74.6, 73.5 (OCH2Ph), 68.8
(C6), 68.7 (Cβ), 67.1 (C10), 56.5 (C2), 54.5 (Cα), 52.6 (CH3), 47.2 (C9), 23.4 (NHCOCH3).
HRMS(ES): Calcd. for C48H50N2O10Na+ 837.3363; found 837.3381.
Methyl
Oꢀ(2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀ
OꢀbenzylꢀβꢀDꢀglucopyranosyl)ꢀ(1→6)ꢀ2,3,4ꢀtriꢀ
O
ꢀbenzoylꢀαꢀD
ꢀglucopyranoside (9)
The general glycosylation procedure was followed using donor 1 (213 mg, 0.40 mmol),
Bi(OTf)3 (5 mol%) and methyl 2,3,4ꢀtriꢀOꢀbenzoylꢀαꢀ
ꢀglucopyranoside 429 (102 mg, 0.20
D
mmol). TLC analysis indicated reaction cessation after 48 hours. Flash column
chromatography afforded the desired disaccharide 9 (178 mg, 90%) as an amorphous solid.
1
Rf (EtOAc/CH2Cl2 1:5) 0.42. Mp 153.0ꢀ153.5 °C. HꢀNMR (CDCl3, 400 MHz): δ 7.99–7.95
(m, 2H, ArH), 7.92 (dd, J 8.3 Hz, J 1.2 Hz, 2H, ArH), 7.82 (dd, J 8.4 Hz, J 1.3 Hz, 2H, ArH),
7.56–7.26 (m, 22H, ArH), 7.18 (dd, J 7.4 Hz, J 2.1 Hz, 2H, ArH), 6.13 (t, J2,3 = J3,4 9.4 Hz,
1H, H3), 5.95 (d, JNH,2 8.1 Hz, 1H, NH), 5.61 (t, J3,4 = J4,5 9.8 Hz, 1H, H4), 5.26–5.20 (m, 2H,
H1, H2), 4.81 (d, Jgem 11.5 Hz, 1H, OCHHPh), 4.78 (d, Jgem 11.1 Hz, 1H, OCHHPh), 4.71 (d,
1H, OCHHPh), 4.56 (d, Jgem 12.2 Hz, 1H, OCHHPh), 4.55 (d, 1H, OCHHPh), 4.50 (d, J1’,2’
8.0 Hz, 1H, H1'), 4.50 (d, 1H, OCHHPh), 4.21–4.11 (m, 2H, H5, H6a), 3.88 (dd, J3’,4’ 9.6 Hz,
J2’,3’ 8.5 Hz, 1H, H3'), 3.81–3.73 (m, 1H, H2'), 3.71 (dd, J6’a,6’b 10.8 Hz, J5’,6’a 2.4 Hz, 1H,
H6'a), 3.68–3.59 (m, 2H, H6'b, H4'), 3.55 (dd, Jgem 11.7 Hz, J5,6b 4.6 Hz, 1H, H6b), 3.49
(ddd, J4’,5’ 9.3 Hz, J5’,6’b 4.7 Hz, 1H, H5'), 3.44 (s, 3H, OCH3), 1.94 (s, 3H, OC(O)CH3). 13Cꢀ
NMR (CDCl3, 100 MHz): δ 170.4 (C=O), 165.8, 165.7, 165.7 (OC(O)Ph), 138.5, 138.3,
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138.1 (ipsoꢀArC, Bz), 133.7, 133.4, 133.1 (ipsoꢀArC, Bn), 130.0, 129.9, 129.6, 129.3, 129.2,
128.9, 128.6, 128.5, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8, 127.7, 127.6 (Ar), 101.4 (C1'),
97.0 (C1), 81.8 (C4'), 78.4 (C3'), 75.3 (C5'), 74.8 (C4), 73.4, 72.0 (double intensity)
(OCH2Ph), 70.8, 69.3, 69.1, 68.5, 67.9 (C6', C6, C5, C3, C2), 56.2 (C2'), 55.6 (OCH3), 23.6
(OC(O)CH3). LRMS(ES): Calcd. for C57H57NO14Na+ 1002.4; found 1001.8.
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The NMR data were in accordance with those previously published.18
O
ꢀ(2ꢀAcetamidoꢀ3,4,6ꢀtriꢀ
Oꢀbenzylꢀ2ꢀdeoxyꢀβꢀDꢀglucopyranosyl)ꢀ(1→6)ꢀ1,2:3,4ꢀdiꢀOꢀ
isopropylideneꢀ ꢀgalactopyranose (10)
α
ꢀD
The general glycosylation procedure was followed using donor 1 (150 mg, 0.28 mmol),
Fe(OTf)3·6.2 DMSO (7 mg, 2.5 mol%) and 1,2:3,4ꢀdiꢀOꢀisopropylideneꢀ
αꢀDꢀ
galactopyranose (5) (37 mg, 0.14 mmol). TLC analysis indicated full conversion of acceptor
after 16 hours. Flash column chromatography (EtOAc/pentane 1:1→1.5:1) afforded the
desired disaccharide 10 (90 mg, 87%) as a clear colorless syrup.
1
Rf (CH3OH/CH2Cl2 1:10) 0.71. HꢀNMR (CDCl3, 400 MHz): δ 7.36–7.26 (m, 13H, ArH),
7.18 (dd, J 7.4Hz , J 1.9 Hz, 2H, ArH), 5.55 (d, JNH,2' 7.9 Hz, 1H, NH), 5.50 (d, J1,2 5.0 Hz,
1H, H1), 4.80 (d, Jgem 11.5 Hz, 1H, OCHHPh), 4.78 (d, Jgem 10.9 Hz, 1H, OCHHPh), 4.69 (d,
J1’,2’ 7.9 Hz, 1H, H1'), 4.68 (d, 1H, OCHHPh), 4.62 (d, Jgem 12.1 Hz, 1H, OCHHPh), 4.58–
4.51 (m, 3H, OCH2Ph, H6a), 4.28 (dd, J2,3 2.4 Hz, 1H, H2), 4.16 (dd, Jgem 7.9 Hz, J5,6b 1.8
Hz, 1H, H6b), 4.00 (dd, Jgem 11.3 Hz, J5’,6’a 3.5 Hz, 1H, H6'a), 3.97–3.90 (m, 2H, H3, H6'b),
3.75–3.61 (m, 5H, H2', H3', H4', H4, H5), 3.54 (dt, J4’,5’=J5’,6’b 9.4 Hz, 1H, H5'), 1.89 (s, 3H,
NHC(O)CH3), 1.51 (s, 3H, CCH3), 1.42 (s, 3H, CCH3), 1.31 (s, 3H, CCH3), 1.30 (s, 3H,
CCH3). 13CꢀNMR (CDCl3, 100 MHz): δ 170.5 (C=O), 138.6, 138.3, 138.3 (ipsoꢀArC), 128.5,
128.4, 128.1, 127.9, 127.8, 127.8, 127.7, 127.6 (Ar), 109.4, 108.7 (C(CH3)2), 101.3 (C1'),
96.4 (C1), 81.4 (C4'), 78.5 (C3'), 75.1, 74.7, 74.5 (OCH2Ph), 73.6, 71.3, 70.8, 70.5, 69.1 (C2,
C3, C4, C5', C5), 68.9, 67.9 (C6', C6), 56.2 (C2'), 26.2, 26.1, 25.1, 24.5, 23.7 (NHC(O)CH3,
C(CH3)2). LRMS(ES): Calcd. for C41H51NO11Na+ 756.3; found 755.9.
The NMR data were in accordance with those previously published.18
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Methyl
O
ꢀ(2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀ
OꢀbenzylꢀβꢀDꢀglucopyranosyl)ꢀ(1→6)ꢀ2,3,4ꢀtriꢀ
O
ꢀbenzylꢀαꢀD
ꢀglucopyranoside (11)
7
8
9
The general glycosylation procedure was followed using donor 1 (231 mg, 0.43 mmol),
Bi(OTf)3 (2.5 mol%) and methyl 2,3,4ꢀtriꢀOꢀbenzylꢀ ꢀglucopyranoside (6) (100 mg, 0.22
αꢀD
10
11
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59
60
mmol). TLC analysis indicated reaction cessation after 43 hours. Flash column
chromatography (EtOAc/CH2Cl2 1:5) afforded the desired disaccharide 11 (198 mg, 98%) as
an amorphous solid.
1
Rf (EtOAc/CH2Cl2 1:5) 0.39. Mp 203.7ꢀ205.2 °C. HꢀNMR (CDCl3, 400 MHz) δH 7.38–7.18
(m, 30H, ArH), 5.43 (d, JNH,2’ 7.8 Hz, 1H, NH), 4.98 (d, Jgem 10.9 Hz, 1H, OCHHPh), 4.84
(d, J1’,2’ 8.3 Hz, 1H, H1'), 4.84–4.75 (m, 5H, OCH2Ph), 4.65 (d, Jgem 12.2 Hz, 1H, OCHHPh),
4.63 (d, Jgem 11.6 Hz, 1H, OCHHPh), 4.60 (d, J1,2 2.9 Hz, 1H, H1), 4.58 (d, Jgem 10.3 Hz, 1H,
OCHHPh), 4.57 (d, Jgem 10.8 Hz, 1H, OCHHPh), 4.56 (d, Jgem 12.3 Hz, 1H, OCHHPh), 4.51
(d, 1H, OCHHPh), 4.14–4.11 (m, 1H, H4), 4.08 (dd, Jgem 10.4 Hz, J5,6a 1.5 Hz, 1H, H6a),
3.98 (t, J2,3=J3,4 9.3 Hz, 1H, H3), 3.77–3.54 (m, 7H, H3', H4', H5', H5, H6'a, H6'b, H6b), 3.51
(dd, 1H, H2), 3.44 (dt, J2’,3’ 9.4 Hz, 1H, H2'), 3.35 (s, 3H, OCH3), 1.71 (s, 3H, NHC(O)CH3).
13CꢀNMR (CDCl3, 100 MHz): δC 170.2 (C=O), 138.9, 138.6, 138.4, 138.4, 138.3, 138.1
(ipsoꢀAr), 128.6, 128.6, 128.5, 128.5, 128.4, 128.3, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9,
127.9, 127.8, 127.7, 127.6 (Ar), 99.9 (C1'), 98.2 (C1), 82.2 (C3), 80.3 (C4'), 79.8 (C2), 78.8
(C3'), 77.7, 75.9 (C4, C5'), 75.1, 74.9, 74.7, 74.6, 73.4 (double intensity) (OCH2Ph), 69.7,
69.2, 67.6 (C5, C6, C6'), 56.9 (C2'), 55.2 (OCH3), 23.7 (NHC(O)CH3).
LRMS(ES): Calcd. for C57H63NO11Na+ 960.4; found 959.9.
The NMR data were in accordance with those previously published.18
Methyl
Oꢀ(2ꢀacetamidoꢀ2ꢀdeoxyꢀ3,4,6ꢀtriꢀ
OꢀbenzylꢀβꢀDꢀglucopyranosyl)ꢀ(1→4)ꢀ2,3,6ꢀtriꢀ
O
ꢀbenzylꢀαꢀD
ꢀglucopyranoside (12)
The general glycosylation procedure was followed using donor 1 (150 mg, 0.28 mmol),
Fe(OTf)3·6.2 DMSO (7 mg, 2.5 mol%) and methyl 2,3,6ꢀtriꢀOꢀbenzylꢀ ꢀglucopyranoside
αꢀD
(7)30 (65 mg, 0.14 mmol). TLC analysis indicated reaction cessation after 72 hours. Flash
column chromatography (EtOAc/CH2Cl2 1:8→1:5) afforded benzyl 2ꢀacetamidoꢀ2ꢀdeoxyꢀ
3,4,6ꢀtriꢀOꢀbenzylꢀ
β
ꢀDꢀglucopyranoside (13) (17 mg), a 1:1 mixture of 13 and the desired
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disaccharide 12 (36 mg) and the desired disaccharide 12 (27 mg, 21%) as an amorphous
solid. Unreacted methyl 2,3,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀglucopyranoside (7) was reꢀisolated (23 mg,
6
7
55%). The product was recrystallized from acetone to afford colorless grained crystals. The
8
9
total corrected yield of the desired product 12was 37%.
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Rf (CH3OH/CH2Cl2 1:10) 0.74. Mp 184.1ꢀ185.3 °C. HꢀNMR (400 MHz, CDCl3): δH 7.38ꢀ
7.26 (m, 15H, ArH), 7.25ꢀ7.16 (m, 15H, ArH), 5.00 (d, Jgem 11.6 Hz, 1H, OCHHPh), 4.84 (d,
JNH,2’ 8.3 Hz, 1H, NH), 4.80 (d, 1H, Jgem 11.7 Hz, OCHHPh), 4.78 (d, Jgem 11.4 Hz, 1H,
OCHHPh) 4.75ꢀ4.40 (m, 10H, OCH2Ph, H1, H1'), 4.32 (d, J 12.2 Hz, 1H, OCHHPh), 3.91ꢀ
3.39 (m, 11H, H2, H2', H3, H3', H4, H5, H5', H6a, H6a', H6b, H6b'), 3.35 (s, 3H, OCH3),
13
3.34ꢀ3.30 (m, 1H, H4'), 1.68 (s, 3H, NHC(O)CH3). CꢀNMR (CDCl3, 100 MHz) δC 170.1
(C=O), 139.8, 138.7, 138.6, 138.4, 138.3, 138.2 (ipsoꢀAr), 128.7, 128.6, 128.5, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.1 (Ar), 99.8 (C1'), 98.5
(C1), 81.5 (C3), 80.4 (C4'), 79.4 (C2), 78.9 (C3'), 76.5 (C5'), 75.2 (double intensity) (C5,
OCH2Ph), 74.8, 74.7, 73.7, 73.5, 73.5 (OCH2Ph), 69.8 (C4), 69.0, 68.4 (C6, C6'), 57.5, 55.4
(C2', OCH3), 23.7 (NHC(O)CH3). HRMS(ES): Calcd. for C57H63NO11Na+ 960.4299; found
960.4310.
Benzyl 2ꢀacetamidoꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀDꢀglucopyranoside (13)
The general glycosylation procedure was followed using donor 1 (109 mg, 0.20 mmol),
Bi(OTf)3 (5 mol%) and benzyl alcohol (22 µL 0.21 mmol). After 3 hours, TLC analysis
indicated reaction completion. Flash column chromatography followed by recrystallization
from CH3OH afforded the desired compound 13 as colorless crystals, (100 mg, 84%).
1
Rf (EtOAc/CH2Cl2 1:5) 0.41. Mp 156.2ꢀ157.7 °C. HꢀNMR (CDCl3, 400 MHz): δH 7.37–7.26
(m, 18H, ArH), 7.20 (dd, J 7.4 Hz, J 2.0 Hz, 2H, ArH), 5.45 (d, JNH,2 7.7 Hz, 1H, NH), 4.89
(d, Jgem 12.0 Hz, 1H, OCHHPh), 4.83 (d, J1,2 7.7 Hz, 1H, H1), 4.80 (d, Jgem 11.5 Hz, 1H,
OCHHPh), 4.78 (d, 1H, Jgem 10.9 Hz, OCHHPh), 4.65 (d, 1H, OCHHPh), 4.63 (d, Jgem 12.1
Hz, 1H, OCHHPh), 4.59 (d, 1H, OCHHPh), 4.57 (d, 1H, OCHHPh), 4.55 (d, 1H, OCHHPh),
4.04 (dd, J2,3 9.5 Hz, J3,4 8.2 Hz, 1H, H3), 3.79 (dd, Jgem 10.7 Hz, J5,6a 2.8 Hz, 1H, H6a), 3.74
(dd, J5,6b 4.3 Hz, 1H, H6b), 3.67 (dd, J4,5 9.1Hz, 1H, H4), 3.60 (ddd, 1H, H5), 3.55 (dt, 1H,
H2), 1.81 (s, 3H, NHC(O)CH3). 13CꢀNMR (CDCl3, 100 MHz): δC 170.3 (C=O), 138.5, 138.3,
138.2, 137.7 (ipsoꢀArC), 128.5, 128.5, 128.5, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7 (Ar),
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99.4 (C1), 80.5 (C3), 78.6 (C4), 75.0 (C5), 74.6, 74.5, 73.6, 70.8 (OCH2Ph), 69.2 (C6), 56.5
(C2), 23.6 (NHC(O)CH3). LRMS(ES): Calcd. for C36H39NO6Na+ 604.3; found 604.1.
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The data were in accordance with the previously published values.31
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2ꢀAcetamidoꢀ1ꢀ
Oꢀacetylꢀ4,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ3ꢀOꢀ(2,3,4,5,6ꢀpentadeuteriobenzyl)ꢀβꢀ
Dꢀglucopyranose (17)
In a glove box as previously described, allyl 2ꢀacetamidoꢀ4,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ3ꢀOꢀ
(2,3,4,5,6ꢀpentadeuteriobenzyl)ꢀ ꢀglucopyranoside (21) (329 mg, 0.61 mmol, 1 eq.) was
β
ꢀD
dissolved in dry dioxane (1.7 mL). Pd(dba)2 (17.6 mg, 31 µmol, 5 mol%), P(tBu)3 (6.2 mg,
31 µmol, 5 mol%) and COgen (CAS: 1072315ꢀ89ꢀ9) (7.4 mg, 31 µmol, 5 mol%) were mixed
separately in dry dioxane (0.3 mL) and subsequently added to the solution containing the
allyl glycoside. The reaction vessel was sealed, removed from the glove box and heated to 80
1
°C. After 24 hours of heating, a crude HꢀNMR spectrum indicated full conversion. The
mixture was concentrated to dryness and the resulting residue was reꢀdissolved in a 1:9
mixture of 1M HCl (aq) and acetone (5 mL total volume) and heated to 55 °C for 6 hours.
The mixture was coꢀevaporated with toluene to dryness and taken up in dry pyridine (3.7
mL). The resulting mixture pyridinium pꢀtoluenesulfonate (225 mg, 0.90 mmol, 1.5 eq.) was
added before heated to 100 °C. A mixture of Ac2O (172 µL, 1.8 mmol, 3 eq.) in dry pyridine
(7.5 mL) was added by syringe pump over 15 min, whereupon the reaction mixture was
concentrated to dryness and purified by flash column chromatography (10→40% EtOAc in
toluene). This afforded the desired compound 17 (120 mg, 40%) as a white solid.
1
Rf 0.28 (EtOAc/toluene 1:1). HꢀNMR (400 MHz, CDCl3):
δH 7.36ꢀ7.25 (m, 8H, ArH), 7.22ꢀ
7.16 (m, 2H, ArH), 5.69 (d, J1,2 7.9 Hz, 1H, H1), 5.39 (d, JNH,2 9.1 Hz, 1H, NH), 4.82 (d, Jgem
11.7 Hz, 1H, OCHHPh), 4.77 (d, Jgem 10.9 Hz, 1H, OCHHPh), 4.67 (d, 1H, OCHHPh), 4.61
(d, Jgem 12.1 Hz, 1H, OCHHPh), 4.57 (d, 1H, OCHHPh), 4.50 (d, 1H, OCHHPh), 4.03 (dt,
J1,2=J2,3 9.2 Hz 1H, H2), 3.79 (dd, J3,4 8.4 Hz, J4,5 8.6 Hz, 1H, H4), 3.74 (d, J5,6 3.4 Hz, 2H,
H6), 3.70 (dd, 1H, H3), 3.66 (td, 1H, H5), 2.06 (s, 3H, OC(O)CH3), 1.81 (s, 3H,
NHC(O)CH3). 13CꢀNMR (100 MHz, CDCl3): δC 170.2, 169.9 (C=O), 138.03, 138.00, 137.9
(ipsoꢀArC), 128.6, 128.5, 128.1, 127.99, 127.96, 127.8 (Ar), 92.9 (C1), 80.7 (C4), 77.6 (C3),
75.7, 74.7, 74.3 (OCH2Ph), 73.6 (C5), 68.6 (C6), 53.7 (C2), 23.4 (NHC(O)CH3), 21.1
(OC(O)CH3). HRMS(ES): Calcd. for C31H30D5NO7Na+ 561.2620; found 561.2626.
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The NMR data were in agreement with those of the unlabeled congener.
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2ꢀAcetamidoꢀ1ꢀ
Oꢀacetylꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀ3,4ꢀdiꢀOꢀ(2,3,4,5,6ꢀpentadeuteriobenzyl)ꢀβꢀ
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Dꢀglucopyranose (18)
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In a glove box as previously described, allyl 2ꢀacetamidoꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀ3,4ꢀdiꢀOꢀ
(2,3,4,5,6ꢀpentadeuteriobenzyl)ꢀβꢀ ꢀglucopyranoside 22 (219 mg, 0.40 mmol, 1 eq.) was
D
dissolved in dry dioxane (0.8 mL). Pd(dba)2 (11.6 mg, 20 µmol, 5 mol%), P(tBu)3 (4.1 mg,
20 µmol, 5 mol%) and COgen (CAS: 1072315ꢀ89ꢀ9) (4.9 mg, 20 µmol, 5 mol%) were mixed
separately in dry dioxane (0.3 mL) and subsequently added to the solution containing the
allyl glycoside. The reaction vessel was sealed, removed from the glove box and heated to 80
1
°C. After 29 hours of heating, a crude HꢀNMR spectrum indicated full conversion. The
mixture was reduced to dryness and the resulting residue was redissolved in a 1:9 mixture of
1M HCl (aq) and acetone (5 mL total volume) and heated to 55 °C for 6 hours. The mixture
was coevaporated with toluene to dryness and redissolved in dry pyridine (2.4 mL). The
resulting mixture was added pyridinium pꢀtoluenesulfonate (152 mg, 0.60 mmol, 1.5 eq.) and
heated to 100 °C. A mixture of Ac2O (114 µL, 1.2 mmol, 3 eq.) in dry pyridine (0.85 mL)
was added by syringe pump over 15 min, whereupon the mixture was evaporated to dryness
and purified by flash column chromatography (10→40% EtOAc in toluene) which afforded
the desired compound 18 (47.5 mg, 24%) as a white solid.
1
Rf 0.28 (EtOAc/toluene 1:1). HꢀNMR (400 MHz, CDCl3): δH 7.39ꢀ7.25 (m, 5H, ArH) 5.69
(d, J1,2 7.9 Hz, 1H, H1), 5.28 (d, JNH,2 9.1 Hz, 1H, NH), 4.82 (d, Jgem 11.7 Hz, 1H, OCHHPh),
4.77 (d, Jgem 10.9 Hz, 1H, OCHHPh), 4.66 (d, 1H, OCHHPh), 4.61 (d, Jgem 12.1 Hz, 1H,
OCHHPh), 4.58 (d, 1H, OCHHPh), 4.50 (d, 1H, OCHHPh), 4.04 (dt, J1,2=J2,3 9.2 Hz 1H,
H2), 3.79 (dd, J3,4 8.4 Hz, J4,5 8.5 Hz, 1H, H4), 3.74 (d, J5,6 3.5 Hz, 2H, H6), 3.69 (dd, 1H,
H3), 3.66 (td, 1H, H5), 2.05 (s, 3H, OC(O)CH3), 1.79 (s, 3H, NHC(O)CH3). 13CꢀNMR (100
MHz, CDCl3): δC 170.1, 169.9 (C=O), 138.0, 137.7 (ipsoꢀArC), 128.5, 128.0, 127.5 (Ar),
92.9 (C1), 80.5 (C4), 77.6 (C3), 75.7, 74.6, 74.2 (OCH2Ph), 73.6 (C5), 68.6 (C6), 53.7 (C2),
23.5 (NHC(O)CH3), 21.2 (OC(O)CH3). HRMS(ES): Calcd. for C31H25D10NO7Na+ 566.2933;
found 566.2944.
The NMR data were in agreement with those of the unlabeled congener.
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2ꢀAcetamidoꢀ1ꢀ
Oꢀacetylꢀ4,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ3ꢀOꢀ(2,3,4,5,6ꢀpentadeuteriobenzyl)ꢀβꢀ
Dꢀallopyranose (19)
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(1,5ꢀCyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (3 mg, 3
µmol, 3 mol%) was dissolved in dry THF (2 mL), bubbled through with H2 for 15 min. and
then degassed with a flow of argon under sonication for 10 min. Allyl 2ꢀacetamidoꢀ4,6ꢀdiꢀOꢀ
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benzylꢀ2ꢀdeoxyꢀ3ꢀOꢀ(2,3,4,5,6ꢀpentadeuteriobenzyl)ꢀβꢀDꢀallopyranoside (24) (61.7 mg, 0.12
mmol, 1 eq.) was dissolved in dry THF (3 mL) and added to the catalyst containing solution.
The resulting yellow solution was stirred under an atmosphere of argon at room temperature.
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After 24 hours, a crude HꢀNMR spectrum indicated incomplete conversion. Another 3 mg
catalyst was activated in a separate vial and transferred to the reaction mixture. After three
days, the mixture was diluted with acetone (9 mL) and a 1 M solution hydrochloric acid and
heated to reflux for two hours. TLC analysis indicated full conversion, and the mixture was
coꢀevaporated with toluene to dryness. The resulting crude was reꢀdissolved in dry pyridine
(0.5 mL), added pyridinium pꢀtoluenesulfonate (45 mg, 0.18 mmol, 1.5 eq.) and heated to
100 °C. A solution of Ac2O (33 µL, 0.35 mmol, 3 eq.) in dry pyridine (0.25 mL) was added
by means of syringe pump during the course of 10 min. and the resulting mixture was left
overnight at room temperature. After 16 hours, the excess Ac2O was quenched with MeOH
(0.5 mL). The mixture was evaporated to dryness under reduced pressure, reꢀdissolved in
CHCl3 and washed with 1 M HCl (aq) followed by sat. aq. NaHCO3, in both cases extracting
thrice with CHCl3. The combined organic layers were dried over MgSO4, filtered and
evaporated to dryness. Purification by repeated flash column chromatography (30% EtOAc in
toluene, then 10% EtOAc in CH2Cl2) afforded the desired compound 19 (19.5 mg, 31%) as a
white solid.
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Rf (30% EtOAc in toluene) 0.35. HꢀNMR (400 MHz, CDCl3):
δH 7.38ꢀ7.23 (m, 10H, ArH),
5.83 (d, J1,2 8.1 Hz, 1H, H1), 5.74 (d, JNH,2 9.6 Hz, 1H, NH), 4.91 (d, Jgem 11.8 Hz, 1H,
OCHHPh), 4.64 (d, Jgem 11.4 Hz, 1H, OCHHPh), 4.63 (d, Jgem 12.1 Hz, 1H, OCHHPh), 4.55
(d, 1H, OCHHPh), 4.50 (d, 1H, OCHHPh), 4.47 (d, 1H, OCHHPh), 4.21ꢀ4.11 (m, 2H, H2,
H5), 4.02 (t, J2,3=J3,4 2.7 Hz, 1H, H3), 3.79 (dd, J4,5 8.9 Hz, 1H, H4), 3.73 (d, J5,6 3.3 Hz, 2H,
H3), 2.05 (s, 3H, OC(O)CH3), 1.71 (s, 3H, NHC(O)CH3). 13CꢀNMR (100 MHz, CDCl3): δC
170.1, 169.9 (C=O), 138.13, 138.09, 137.7 (ipsoꢀArC), 128.7, 128.5, 128.2, 128.1, 128.0,
127.8 (Ar), 92.4 (C1), 75.8 (C4), 74.8 (C3), 74.5 (OCH2Ph), 74.0 (C5), 73.7, 72.8 (OCH2Ph),
68.9 (C6), 51.1 (C2), 23.2 (NHC(O)CH3), 21.2 (OC(O)CH3). HRMS(ES): Calcd. for
C31H30D5NO7Na+ 561.2620; found 561.2622.
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Dꢀglucopyranoside (20)
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NꢀAcetylꢀ ꢀglucosamine (10.0 g, 45.1 mmol, 1 eq.) was dissolved in a mixture of dry
D
pyridine (25 mL) and dry CH2Cl2 (50 mL) in a preꢀdried flask under an atmosphere of
nitrogen. The mixture was left stirring overnight at room temperature. The mixture was
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cooled to ꢀ10 °C and pivaloyl chloride (17 mL, 138 mmol, 3 eq.) was added. The mixture was
allowed to reach room temperature and was left stirring for 2 days before acetic anhydride
(21.3 mL, 226 mmol, 5 eq.) was added and the mixture stirred for another 3 hours. The
reaction mixture was then washed several times with hydrochloric acid (1 M) until the
aqueous phase stayed acidic according to pH paper test. The combined organic layers were
then washed with water and brine before dried over Na2SO4 and filtered. The mixture was
concentrated to give a crude mixture, which was used in the next step without further
purification.
The crude mixture was dissolved in dry CH2Cl2 and triflic acid (1.80 mL, 20.3 mmol, 45
mol%) was added using a glass syringe. AllOH (9.2 mL, 135 mmol, 3 eq.) was added and the
reaction mixture heated to 40 °C overnight before washed with water and aq. NaHCO3. The
organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The
resulting crude glycoside mixture was reꢀdissolved in dry methanol, and sodium (1.5 g, 65.2
mmol, 1.4 eq.) was added. The reaction mixture was heated to 50 °C for 24 hours and then
concentrated onto Celite® and purified before purified by flash column chromatography
(water/i-propanol/EtOAc 1:4:16) to give the desired compound 20 (8.15 g, 69%) as a
colorless solid.
Mp: 161.4ꢀ162.8 °C. Lit. 160ꢀ163 ° δH 5.89 (ddt, Jtrans 17.3
C.32 1HꢀNMR (400 MHz, CD3OD):
Hz, Jcis 10.6 Hz, Jvic 5.3 Hz, 1H, CH=CH2), 5.27 (dd, Jgem 1.7 Hz, 1H, CH=CHH), 5.13 (dd,
1H, CH=CHH), 4.44 (d, J1,2 8.4 Hz, 1H, H1), 4.34 (dd, Jgem 13.3 Hz, 1H, OCHHCH), 4.07
(dd, 1H, OCHHCH), 3.88 (dd, Jgem 11.9 Hz, J5,6a 2.1 Hz, 1H, H6a), 3.72 – 3.63 (m, 2H, H2,
H6b), 3.45 (dd, J 10.2 Hz, J 8.6 Hz, 1H, H3), 3.38 – 3.21 (m, 3H, H4, H5, NH), 1.97 (s, 3H,
NHCOCH3). 13CꢀNMR (100 MHz, CD3OD) δC 173.8 (C=O), 135.6 (CH2CH=CH2), 116.9
(CH=CH2), 101.9 (C1), 78.0 (C5), 76.1 (C3), 72.2 (C4), 70.7 (OCH2CH), 62.8 (C6), 57.3
(C2), 22.9 (NHCOCH3). HRMS (ES): Calcd. for C11H19O6NH+ 262.1285; found 262.1290.
The NMR data were in accordance with those previously published.33
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