Challenging deprotection steps during the synthesis of tetra- and pentasaccharide fragments of the LeaLex tumor-associated hexasaccharide antigen

Article abstract of DOI:10.1021/jo301644w

We report the convergent synthesis of two novel tetrasaccharide and two novel pentasaccharide fragments of the Le^aLe^x TACA: the tetrasaccharides contain neither the galactose at the Le^a nonreducing end nor the fucose at the Le^x reducing end; the pentasaccharides only lack the galactose residue at the Le^a nonreducing end. Two of the analogues were prepared as hexyl glycosides to be used in NMR experiments and as soluble inhibitors in binding studies and two as 6-aminohexyl glycosides to be conjugated to carrier proteins. Our strategy relied on stepwise extensions using excess monosaccharide glycosyl donors (trichloroacetimidates and thioglycosides) in sequential glycosylation reactions. The protecting groups were chosen to limit the number of deprotection steps required to obtain the final derivatives. While this strategy ensured that all glycosylation reactions proceeded in very good yields (70-84%), deprotection of the oligosaccharide intermediates was challenging. Global deprotection using Birch metal dissolving conditions did not remove the tert-butyldiphenylsilyl group, which indeed was incompatible with such reaction conditions. Attempts to remove the TBDPS with tetrabutylammonium fluoride was unsuccessful and led to a complex mixture of compounds that could not be separated. The desired hexyl and aminohexyl tetrasaccharides were finally obtained after four- and five-step deprotection sequences, respectively. Deprotection of the pentasaccharide intermediate to give the hexyl and aminohexyl analogues also led to unexpected results. Indeed, during Zemplén deacylation, a chloroacetamide chlorine atom was displaced by methoxide ions leading to the corresponding methoxyacetamide. Once the chloroacetamide was fully reduced to an acetamide the pentasaccharides were obtained in four and five steps, respectively.

Full text of DOI:10.1021/jo301644w

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Featured Article
Challenging Deprotection Steps During the Synthesis
of Tetra- and Pentasaccharide Fragments of the
LeaLex Tumor-Associated Hexasaccharide Antigen
Mickael Guillemineau, and France-Isabelle Auzanneau
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 25 Sep 2012
Downloaded from http://pubs.acs.org on September 28, 2012
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Challenging Deprotection Steps During the Synthesis of Tetraꢀ and
a
x
Pentasaccharide Fragments of the Le Le TumorꢀAssociated
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Hexasaccharide Antigen
Mickaël Guillemineau, FranceꢀIsabelle Auzanneau*
Department of Chemistry, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.
fauzanne@uoguelph.ca
BnO
OBn
O
OBn
OTBDPS
OBn AcO
OBn
O
O
O
O
O
O
ClAcO
AcO
2 6
O(CH ) Cl
TCAHN
AcO
AcHN
HO
OH
OH
HO
O
HO
O
OH
O
OH
OH
O
OH
O
OH
O
O
HO
OH
O
O
OH
O
HO
OH
O
O
O(CH
2 6
) R
O
HO
AcHN
HO
O
O
O
NHAc
HO
O(CH
NHAc
2 6
) R
HO
OH
AcHN
OH
HO
4
5
steps (R = H)
2
2
+ 4 steps (R = H)
+ 5 steps (R = NH AcO )
+
-
steps (R = NH AcO )
+
-
3
3
Abstract
We report the convergent synthesis of two novel tetrasaccharide and two novel
a
x
pentasaccharide fragments of the Le Le TACA: the tetrasaccharides contain neither the
a
x
galactose at the Le nonꢀreducing end nor the fucose at the Le reducing end; the
a
pentasaccharides only lack the galactose residue at the Le nonꢀreducing end. Two of the
analogues were prepared as hexyl glycosides to be used in NMR experiments and as soluble
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5
5
6
inhibitors in binding studies, and two as 6ꢀaminohexyl glycosides to be conjugated to carrier
proteins. Our strategy relied on stepwise extensions using excess monosaccharide glycosyl
donors (trichloroacetimidates and thioglycosides) in sequential glycosylation reactions. The
protecting groups were chosen to limit the number of deprotection steps required to obtain the
final derivatives. While this strategy ensured that all glycosylation reactions proceeded in very
good yields (70–84%), deprotection of the oligosaccharide intermediates was challenging.
Global deprotection using Birch metal dissolving conditions did not remove the tꢀ
butyldiphenylsilyl group which indeed was incompatible with such reaction conditions. Attempts
to remove the TBDPS with tetrabutylammonium fluoride was unsuccessful and led to a complex
mixture of compounds that could not be separated. The desired hexyl and aminohexyl
tetrasaccharides were finally obtained after 4 and 5 steps deprotection sequences, respectively.
Deprotection of the pentasaccharide intermediate to give the hexyl and aminohexyl analogues
also led to unexpected results. Indeed, during Zemplén deacylation, a chloroacetamide chlorine
atom was displaced by methoxide ions leading to the corresponding methoxyacetamide. Once
the chloroacetamide was fully reduced to an acetamide the pentasaccharides were obtained in 4
and 5 steps, respectively.
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Introduction
Targeting tumorꢀassociated carbohydrate antigens (TACAs) to develop antiꢀcancer
vaccines is an area of intensive research and the advances made in this exciting field have been
1
a
x
recently summarized in multiple reviews. Even though the TACA Le Le hexasaccharide (1)
2
has long been associated with lung cancer and particularly squamous lung carcinoma, it has yet
to be used in the development of antiꢀcancer vaccines. In this context, our research aims at
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a
x
discovering a therapeutic antiꢀcancer vaccine based on the Le Le hexasaccharide. Unfortunately,
a
x
while the expression of the Le Le hexasaccharide is highly localized to squamous lung
a
carcinoma (SLC) cells, it has been wellꢀestablished that the Le trisaccharide expressed at the
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nonꢀreducing end of this large structure
HO
OH
was displayed at the surface of many nonꢀ
OH
C'
O
OH HO
O
O
OH
O
OH
O
2a,3
cancerous cells.
Thus, immunization
O
O
B
HO
A'
O
A
HO
HO
B'
OR
NHAc
a
x
O
HO
with the Le Le TACA will likely trigger
AcHN
O
OH
O
C
OH
a
an immune response against the Le
OH
HO
a
x
1
Le Le
trisaccharide and, in turn, lead to the
destruction of numerous healthy cells.
Most important to our program is the work
by Olsson et al. who after immunization
of mice with SLC cells cloned a
monoclonal antibody named 43ꢀ9F which
was shown to specifically recognize
HO
OH
OH
C'
O
HO
OH
O
OH
O
OH
O
HO
B
A'
O
O
O
HO
A
O(CH ) R
2 6
HO
2 R = H
3 R = NH AcO
AcHN
NHAc
+
-
3
HO
OH
OH
C'
O
OH HO
OH
O
OH
O
O
O
HO
B
A'
O
O
A
O(CH ) R
HO
O
2 6
a
x
AcHN
Le Le while it only weakly bound to the
O
NHAc
C
OH
a
2aꢀc
Le trisaccharide. Such finding supports
OH
HO
4
5
R = H
R = NH AcO
a
x
+
-
that the Le Le TACA displays internal
3
a
epitopes that do not involve the Le trisaccharide and that can therefore be targeted for antiꢀ
cancer vaccine development. In search of truncated structures that would retain internal epitopes
a
such as that recognized by mAb 43ꢀ9F but noꢀlonger carry the Le trisaccharide, we report here
the preparation of tetraꢀ and pentasaccharides 2–5. Tetrasaccharides 2 and 3 contain neither the
a
x
galactose moiety at the Le nonꢀreducing end nor the fucose moiety at the Le reducing end,
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a
while pentasaccharides 4 and 5 only lack the galactose residue at the Le nonꢀreducing end.
While analogues 2 and 4 were obtained as hexyl glycosides to be used in NMR experiments and
binding studies, derivatives 3 and 5 were prepared as the corresponding 6ꢀaminohexyl glycosides
that will be conjugated to carrier proteins such as Bovine Serum Albumin or Tetanus Toxoid.
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x 4
Relying on our recent success in preparing analogues of dimLe , we embarked on the synthesis
of fragments 2–5 following a stepwise approach that employed monosaccharide glycosyl donors
in sequential glycosylation reactions. The choice of protecting groups was made with the
intention to limit the number of deprotection steps required to obtain the final derivatives. We
4ꢀ5
intended to carry out a oneꢀstep deprotection in Birch metal dissolving conditions to obtain the
hexyl glycosides 2 and 4 and a twoꢀsteps deprotection involving first the reduction of the nonꢀ
reducing end trichloroacetamide followed by a deprotection in metal dissolving conditions to
prepare 6ꢀaminohexyl glycosides 3 and 5. As shown below, the use of a tꢀbutyldiphenylsilyl
ether (TBDPS) protecting group to selectively protect Oꢀ6B impeded these strategies and
dictated the sequence of the multiꢀstep deprotection sequences that were required to prepare
analogues 2–5.
Results and Discussion
The desired analogues were prepared
OTBDPS
O
AcO
ClAcO
Ph
O
O
O
AcO
using novel building blocks 6 and 7 and
AcO
TCAHN OC(NH)CCl3
OC(NH)CCl3
5
c
6
6
7
known monosaccharides
8
and 9 .
OBn
O
O
SEt
OBn
HO
ClAcO
Galactosyl donor 6 was prepared in 5 steps
O(CH ) Cl
2 6
OBn
BnO
AcHN
7
from
known
pꢀthiotolyl
βꢀDꢀ
9
8
8
galactopyranoside (Scheme 1). Selective silylation at Oꢀ6 gave the known triol 10 which was
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fully characterized. In turn, triol 10 was converted to the corresponding 3,4ꢀorthoacetate that was
acetylated in situ at position 2 and treated in mild acidic conditions to give alcohol 11 in
excellent yield. Chloroacetylation at Oꢀ3 gave monosaccharide 12 which was submitted to the
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hydrolysis of the anomeric thioglycoside
9
S
CHEME 1. Synthesis of building blocks 6, 7
to
give
hemiacetal
13.
Not
OTBDPS
O
STol
HO
0 (84%)
OTBDPS
4,10
HO
HO
1. MeC(OEt)3
AcO
RO
surprisingly,
such reaction led to
2. Ac O
O
2
STol
3
. AcOH/H O
2
alcohol 14 as the major product while the
desired hemiacetal 13 was only formed as
the minor product (13/14 ratio 0.6:1) and
obtained as an α/β mixture (ratio 1:0.5).
Analytical samples of 13 and 14 were
isolated for characterization purposes and
the remaining mixture of 13 and 14 was
AcO
1 R = H (93%)
2 R = ClAc (quant)
1
1
1
ClAcCl
OTBDPS
O
OTBDPS
NIS
TfOH
AcO
AcO
ClAcO
O
MeCN ClAcO
H O
2
AcO OH
HO
14
OAc
1
3
(0.6:1) 86%
Cl3CCN DBU
6 (83%)
Ph
O
Ph
O
BnNH2
O
O
O
O
AcO
TCAHN
17
RO
TCAHN
4
,10
OH
engaged in the next step. As expected,
OR
1
1
5 R = H
6 R = Ac (97%)
(
80%)
Ac O
2
under treatment with DBU in the presence
of trichloroacetonitrile, the anomeric
Cl CCN DBU
3
7
(73%)
acetate in monosaccharide 14 underwent migration to Oꢀ2 and the desired trichloroacetimidate
donor 6 was formed from both compounds 13 and 14 and isolated in excellent yield as the α
anomer (JHꢀ1, Hꢀ2 = 3.6 Hz). Glucosamine glycosyl donor 7 was prepared in 3 steps from the
11
known benzylidene 15. Thus diol 15 was acetylated giving diꢀacetate 16, which was submitted
to selective deacetylation at the anomeric position with benzylamine leading to the α/β anomeric
mixture (ratio 1:0.25) of hemiacetal 17 in excellent yield. In turn, treatment of this anomeric
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mixture with DBU and trichloroacetonitrile gave the desired glucosyl donor 7 as its αꢀanomer
(JHꢀ1, Hꢀ2 = 3.7 Hz).
S
CHEME 2. Synthesis of tetrasaccharide 23 and
pentasaccharide 25
The stepwise synthesis of
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linear tetrasaccharide 23 and
subsequent pentasaccharide 25 is
outlined on Scheme 2 and began by
the coupling of acceptor 8 with
galactosyl donor 6. This coupling
was performed under the conditions
BF .OEt
3
2
40 °C
AcO OTBDPS
OBn
O
O
2
R O
O
1
R O
AcHN
O(CH ) Cl
2 6
AcO
1
2
18 R , R = ClAc (71%)
(H
2 2
N) CS
1
2
19 R , R = H (8%)
1
2
2
0 R = ClAc, R = H (66%)
TESOTf
rt
7
1
2
developed in our group to promote
efficient glycosylation at Oꢀ4 of Nꢀ
acetylglucosamine acceptors. Thus,
2
AcO OTBDPS
R O
1
O
O
OBn
O
R O
AcO
B
A'
O
O
ClAcO
A
AcO
O(CH ) Cl
2 6
TCAHN
AcHN
1 R ,R = CHPh (70%)
1
2
2
HCl/Et O
NaCNBH3
2
1
2
22 R = H, R = Bn (91%)
acceptor
8
was reacted with
1
. MeOTf, 9 2. 25% AcOH/Ac O
2
BnOOBn
galactosyl donor 6 (4 equiv) in the
OBn
C'
O
AcO OTBDPS
OBn
presence of an excess of BF ꢁOEt (2
3
2
OBn
O
O
O
O
A'
B
O
RO
AcO
O
A
equiv) in CH Cl at 40 °C and the
O(CH
)
Cl
2
2
2
6
TCAHN
N) CS
AcO
AcHN
2
2
3 R = ClAc (82%)
4 R = H (73%)
desired disaccharide 18 was isolated
in good yield. Selective removal of
the Oꢀ3’ chloroacetate with thiourea
was carried out using similar
conditions to those that we have
(H
2
2
1
. MeOTf, 9 2. 25% AcOH/Ac O
2
BnO
OBn
OBn
C'
O
AcO OTBDPS
OBn
OBn
O
O
O
O
B
A'
O
O
AcO
A
O
O(CH ) Cl
2 6
TCAHN
AcO
NHAc
OBn
O
C
5
b,c
BnOOBn
5 (84%)
previously described.
A small
2
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excess of thiourea (1.2 equiv) in a 2:1 pyridine/EtOH mixture at 55 °C led to the disappearance
followed by TLC of diꢀchloroacetate 18 within 4.5 h and gave only a small amount of diol 19
(8%) while desired alcohol 20 was isolated in 66% yield. The position of the free hydroxyl group
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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1
in acceptor 20 was confirmed by H NMR comparing the chemical shifts of the nonꢀreducing
end Hꢀ3’ in product 20 (δHꢀ3’ = 3.57 ppm) to the same signal in starting material 18 (δHꢀ3’ = 4.84
ppm). Glycosylation of acceptor 20 with donor 7 was then performed at room temperature in
1
1
CH Cl under activation with two equiv of TESOTf as recommended by Jacquinet to prevent
2
2
the formation of the undesired donor oxazoline. Under these conditions the desired trisaccharide
1 was obtained in good yield as the expected β anomer (JHꢀ1A’, Hꢀ2A’ = 7.9 Hz). Regioselective
reductive opening of the 4,6ꢀOꢀbenzylidene in trisaccharide 21 (NaCNBH ꢀHCl/Et O, THF, rt)
2
3
2
1
provided trisaccharide acceptor 22 free at Oꢀ4A’ as confirmed by H NMR which showed the
presence of a doublet corresponding to OHꢀ4A’ (JOHꢀ4A’, Hꢀ4A’ = 2.9 Hz) at 3.25 ppm.
Trisaccharide acceptor 22 was engaged in a glycosylation reaction (CH Cl ) with an excess of
2
2
6
known fucosyl donor 9 (2 equiv) activated by MeOTf (5 equiv) at room temperature. After
13
treatment of the crude mixture with 25% AcOH in Ac O to convert any expected methyl
2
imidate formed into Nꢀacetyl group, the desired tetrasaccharide 23 was isolated in excellent
1
yield. As the anomeric signal for Hꢀ1C’ was not resolved in the H NMR spectrum, the
stereochemistry of the newly formed fucosidic bond was established by measuring the coupling
constant between Cꢀ1C’ and Hꢀ1C’ in a coupled HSQC experiment. This coupling constant
14
(
JHꢀ1C’, Cꢀ1C’ = 170 Hz) supported an expected α configuration for the newly introduced fucosyl
unit. A fraction of tetrasaccharide 23 was set aside to prepare deprotected analogues 2 and 3
Scheme 3), while the remaining tetrasaccharide was engaged in the synthesis of pentasaccharide
(
2
5. Thus, tetrasaccharide 23 was converted to acceptor 24 free at Oꢀ3A in good yield by
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treatment with thiourea (10 equiv) in a 2:1 pyridine/EtOH at 65 °C. MeOTfꢀpromoted coupling
of tetrasaccharide acceptor 24 and fucosyl donor 9 was then achieved using the same (two steps)
reaction conditions as those described above for the synthesis of tetrasaccharide 23. The desired
pentasaccharide 25 was obtained in excellent yield and the α configuration of the newly
introduced fucosidic bond at Oꢀ3A was confirmed by measuring the coupling constant between
Hꢀ1 and Hꢀ2 of the fucosyl unit (JHꢀ1C’, Hꢀ2C’ = 3.6 Hz).
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With tetraꢀ and pentasaccharides 23 and 25 in hands, we investigated deprotection
reactions to prepare the desired analogues 2–5. Focusing on the deprotection of tetrasaccharide
23 to obtain analogue 2, we first attempted its global deprotection in metal dissolving conditions
o
4ꢀ5
(
NH /Na, THF) at –78 C. While it is known that in such conditions benzyl ethers, acetates,
3(l)
and trichloroacetamides will be removed and the chlorohexyl chain reduced to the hexyl chain,
the behavior of the TBDPS group at Oꢀ6B was unknown. After quenching the reaction with
MeOH and acetylation in situ of the expected Cꢀ2A’ free amine the poorly water soluble crude
product was analyzed by TLC and NMR. It was found to be a complex mixture of compounds
containing ethylenic protons which likely came from the partial reduction of the aromatic rings
in the TBDPS group. Concluding that the silyl group at Oꢀ6B could not be removed in metal
dissolving conditions, we then attempted to remove it first with TBAF in THF at room
temperature. However, while the silyl group was removed in these conditions a complex mixture
of compounds was isolated. Since it has been well established that trichloroacetamides are
removed in Zemplén conditions occasionally leading to the formation of undesired methyl
1
5
carbamates, we then engaged trisaccharide 23 in a deprotection strategy (Scheme 3) which first
involved the reduction of the trichloroacetamide prior to Zemplén deacetylation. Reduction of
the Cꢀ2A’ trichloroacetamide was first attempted using an excess of freshly activated zinc
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powder (100 equiv) in AcOH at 50 °C for 20 h. While TLC showed disappearance of the starting
material, two new products were formed and isolated together in 79% yield. Analytical reverse
phase HPLC showed that these compounds were present in a 35:65 ratio. An analytical sample of
the minor compound could be
10
11
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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50
51
52
53
54
55
56
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59
60
S
CHEME 3. Deprotection steps to analogues 2 and 3
2
3
isolated and was identified as
chloroacetamide 26 (estimated yield
27%), while the major product
(estimated yield 52%) was identified
as acetamide 27. In both compounds
the chloroacetate at Oꢀ3A had been
reduced to an acetate. Since
acetamide 27 was the major product
of this reaction, we optimized its
Zn/AcOH
BnO
OBn
OBn
C'
O
AcO OTBDPS
OBn
O
OBn
O
O
O
A'
B
O
O
AcO
AcO
A
1
O(CH ) R
2
2 6
R HN
AcO
AcHN
1
2
2
2
2
6 R = Cl, R = ClAc
1
2
7 R = Cl, R = Ac (80%)
NaN
3
1
2
8 R = N , R = Ac (94%)
3
MeONa
BnO
OBn
OBn
C'
O
OR2
OBn HO
OBn
O
O
O
O
O
HO
A'
B
O
HO
A
O(CH )
R1
2 6
AcHN
HO
AcHN
formation
by
increasing
the
1
2
29 R = Cl, R = TBDPS (quant)
temperature of reaction to 65 °C.
Indeed, after 5 h at this temperature,
tetrasaccharide 27 was isolated in an
excellent (80%) yield. Before
1
2
3
3
3
0 R = N , R = TBDPS (quant)
3
TBAF
1
2
1 R = Cl, R = H (59%)
TBAF
1
2
2 R = N , R = H (72%)
3
Na/NH (l)
3
2
3
(50% from 29)
(38% from 30)
engaging tetrasaccharide 27 into further deprotection steps towards analogue 2 a fraction of this
intermediate was converted to the 6ꢀazidohexyl tetrasaccharide 28 via nucleophilic displacement
of the chlorine atom with sodium azide (DMF, 80 °C). In turn, the 6ꢀchlorohexyl and 6ꢀ
azidohexyl tetrasaccharides 27 and 28 were engaged into the final deprotection steps to lead to
the desired analogues 2 and 3, respectively. Zemplén deacetylation (MeONa/MeOH, rt) first led
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1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
quantitatively to triols 29 and 30 which were treated with TBAF in THF at room temperature to
give the 6ꢀchlorohexyl tetrasaccharide 31 and 6ꢀazidohexyl tetrasaccharide 32, in 59 and 72%
yield, respectively. These rather moderate yields resulted from the challenging purifications
required to obtain the desired tetrasaccharides free of tetrabutylammonium salts. Thus, we
minimized purification steps and attempted in subsequent reactions to engage tetrasaccharides 31
and 32 still contaminated with tetrabutylammonium salts into the final deprotection step in metal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
dissolving conditions (NH /Na, THF, –78 C). These reactions were quenched with MeOH,
3
(l)
neutralized with AcOH and the final compounds were purified by size exclusion chromatography
®
on Biogel P2 eluted with water for hexyl glycoside 2 and 0.05 M aqueous ammonium acetate
for aminohexyl glycoside 3. Following these last two deprotection steps, the desired final
compounds 2 and 3 were obtained pure in 50 and 38% yield from protected tetrasaccharide 29
+
1
and 30, respectively. Mass spectrometry ([M + Na] = 839.3668) along with H NMR showing a
triplet at 0.86 ppm corresponding to the terminal methyl group, coming from the reduction of the
chlorine, confirmed the structure of tetrasaccharide 2. Similarly, the structure of tetrasaccharide 3
+
was confirmed by mass spectrometry ([M + H] = 832.3906) and by the presence of a triplet at
1
+
2.97 ppm in H NMR corresponding to the terminal methylene group (CH NH ).
2
3
The deprotection of pentasaccharide 25 to yield the desired final pentasaccharides 4 and 5
is outlined in Scheme 4 and followed the strategy that we had established for the conversion of
tetrasaccharide 23 to the final compounds 2 and 3. Thus, the first step in this sequential
deprotection was the reduction of the Cꢀ2A’ trichloroacetamide with zinc in acetic acid.
1
6
However, since fucosyl residues at Oꢀ3 of glucosamine units are known to be unstable when
treated in acidic conditions, this reduction was left to proceed at room temperature rather than
o
heated to 50 or 60 C. After 7 h of reaction at this temperature, TLC showed complete
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disappearance of the starting material and formation of one major and one minor product. The
major product was identified as chloroacetamide 33 (79%), and the minor product was
characterized as the fully reduced
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
acetamide 34 isolated in 11% yield.
Since we expected the
S
CHEME 4. Deprotection steps to analogues 4 and 5
2
5
Zn/AcOH
chloroacetamide to be easily reduced
BnO
OBn
OBn
to the acetamide in the last step
C'
1
O
R O
OTBDPS
O
OBn
O
OBn
O
4
O
(Na/NH ) and unaffected by the
A'
B
3(l)
1
O
R O
O
A
1
O
O(CH
NHAc
OBn
) Cl
2 6
2
R O
R HN
subsequent reactions, we engaged
pentasaccharide 33 in a Zemplén
deacetylation step in 0.25 M sodium
methoxide in methanol. This
reaction was first left to proceed at
room temperature for 2 h at which
time TLC showed the formation of a
major product. However, work up of
O
C
OBn
BnO
1
2
3
3
3
3 R = Ac, R = ClAc (79%)
1
2
4 R , R = Ac (11% to 91%)
MeONa
1
2
5 R = H, R = MeOAc (54%)
MeONa
1
2
36 R = H, R = Ac (73%)
TBAF
BnO
OBn
OBn
C'
O
HO
O
OH
O
OBn
O
OBn
O
O
A'
B
O
HO
A
O(CH ) R
O
2 6
AcHN
HO
NHAc
OBn
O
C
1
OBn
BnO
the reaction and H NMR of the
3
7 R = Cl (82%)
NaN3
38 R = N (95%)
crude product showed that the Oꢀ4B
acetyl group had not been removed
(δHꢀ4B = 5.47 ppm, bd). Therefore,
3
Na/NH (l)
3
4
5
(70%)
(70%)
this crude product was reꢀengaged in Zemplén deacetylation conditions and the reaction was left
to proceed at room temperature for 18 h, at which time TLC showed the formation of a new
major compound. Surprisingly, this new compound was isolated in 54% yield and characterized
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
by NMR as being the methoxyacetamide 35 (Scheme 4). The first indication that the
chloroacetamide had been modified in these conditions was given by the downfield shift
1
observed for the NHA’ signal found at almost 7 ppm in the H NMR spectrum while the same
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
signal in chloroacetamides 26 and 33 was found at 6.3 ppm. In addition, the chloroacetamide
1
3
methylene carbon signal found at 42.7 ppm in the C NMR spectra of chloroacetamide
derivatives 26 and 33 was replaced by a methylene signal found at 72 ppm in pentasaccharide
35. We also observed the presence of an additional Oꢀmethyl group in pentasaccharide 35 which
1
13
gave signals at 3.33 ppm and 59.3 ppm in the H and C NMR spectra, respectively. An HMBC
experiment showed correlations between the methylene hydrogens (~3.8 ppm) and this Oꢀmethyl
carbon (59.3 ppm) as well as a correlation between the methyl hydrogens (3.33 ppm) and the
methylene carbon (72 ppm). These correlations confirmed that the methylene and methyl group
were linked to the same oxygen and suggested that during this Zemplén deacetylation, the
chloroacetamide chlorine atom in pentasaccharide 33 had undergone nucleophilic displacement
by a methoxide ion leading to the unexpected methoxyacetamide pentasaccharide 35. The
structure of pentasaccharide 35 was eventually confirmed by HRMS. While trichloroacetamides
are sensitive to Zemplén conditions, the nucleophilic displacement of a chloroacetamide chlorine
atom by methoxide ions has, to our knowledge, not been previously observed in such conditions.
1
7
Indeed, Zemplén deacetylations of chloroacetamide intermediates have been reported. In our
case, the extended reaction time that was required to deacetylate Oꢀ4B led to the formation of
pentasaccharide 35 that could not be prevented. Thus, we decided to explore reactions conditions
that would lead to the total reduction of the trichloroacetamide to an acetamide in
pentasaccharide 25 prior to engaging the product into the Zemplén deacetylation. Running the
reaction (Zn/AcOH) at 65 °C for 3 h increased the yield of desired acetamide 34 but it was only
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isolated in 23% yield while chloroacetamide 33 was obtained in 69% yield. Thus, we attempted
1
8
o
to perform the reaction under sonication and left it to proceed for 4 h at 65 C. At this time,
TLC showed that all starting material had been consumed and that acetamide 34 had become the
major product formed. Additional zinc (100 equiv) was added, the reaction was allowed to
proceed at 65 °C under sonication during an additional 3 h and the desired acetamide 34 was
isolated in an excellent 91% yield. In turn, Zemplén deacetylation (MeONa/MeOH, rt) of
acetamide 34 gave the desired triol 36 in good yield after purification by RPꢀHPLC
(CH CN/H O). Rather than engaging a portion of 6ꢀchlorohexyl pentasaccharide 36 into a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
2
nucleophilic displacement with sodium azide prior to the removal of the TBDPS group at Oꢀ6B
as was done for the preparation of tetrasaccharide 3), we decided to first treat 36 with TBAF
(
(THF, rt) and prepare pentasaccharide 37. Fortunately, even though pentasaccharides 36 and 37
coꢀmigrated on TLC, sufficient reaction time (16 h) provided the desired pentasaccharide 37 pure
and in very good yield despite requiring two successive purification by silica gel
chromatography and RP HPLC. At this time, a fraction of the 6ꢀchlorohexyl pentasaccharide 37
o
was converted to the 6ꢀazidohexyl pentasaccharide 38 (NaN /DMF, 80 C) which was obtained
3
in excellent yield. Both pentasaccharide 37 and 38 were then treated in metal dissolving
o
conditions (NH /Na, THF, –78 C) to give the desired hexyl and 6ꢀaminohexyl
3
(l)
pentasaccharides 4 and 5, both in 70% yield after purification by size exclusion column
®
+
1
chromatography on Biogel P2 . Mass spectrometry ([M + H] = 963.4435) along with H NMR
showing a triplet at 0.85 ppm corresponding to the terminal methyl group confirmed the structure
of pentasaccharide 4. The structure of the pentasaccharide 5 was confirmed by mass
+
1
spectrometry ([M + H] = 978.4522) and by the presence of a triplet at 2.97 ppm in H NMR
+
3
corresponding to the terminal CH NH .
2
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Conclusion
In conclusion, we report here the convergent synthesis of two novel tetrasaccharide and
a
x
two novel pentasaccharide fragments of the Le Le TACA. Our strategy relied on stepwise
monosaccharide extensions using excess equivalents (up to 4 equiv) of the relatively cheap
monosaccharide glycosyl donors. This strategy ensured that all glycosylation reactions
proceeded in very good yields (70–84%) even when preparing the largest structures. Thus the
protected tetraꢀ and pentasaccharide intermediates 23 and 25 were obtained in 25% yield over 6
steps and 15% yield over 8 steps, respectively. In contrast, the deprotection of the tetraꢀ and
pentasaccharide intermediates turned out to be challenging as we met numerous unforeseen
difficulties. Indeed, working on tetrasaccharide 23 to prepare the 6ꢀhexyl derivative 3 gave us the
first indication that such deprotection steps were not as straightforward as the protecting groups
chosen would have led us to believe. Investigating whether a global deprotection strategy using
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4,5b,c
Birch metal dissolving conditions
would be applicable here revealed that the TBDPS group
was not removed and incompatible with such reaction conditions. Our results subsequently
indicated that that the removal of this silyl group with fluoride ions led to partial migration of the
Oꢀ4B acetyl group as well as to the formation of other uncharacterized products. Once the
trichloroacetamide was reduced to an acetamide, deprotection steps involving TBAF and
MeONa proceeded in good yields giving the desired tetrasaccharides 2 and 3 in 40% (4 steps)
and 28% (5 steps) yield, respectively, from tetrasaccharide 23. Evidence of the reactivity of
halogenated acetamides with nucleophiles was obtained when chloroacetamide pentasaccharide
33 was submitted to Zemplén deacetylation conditions. As a result of the extended reaction time
required to remove all acetates, we observed the unexpected nucleophilic displacement of the
chloroacetamide chlorine atom by methoxide ions leading to the methoxyacetamide 35. Full
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reduction of the chloroacetamide to an acetamide in pentasaccharide 34 allowed for efficient
subsequent deprotection steps leading to pentasaccharide 4 in 38% yield (4 steps from 34) and
aminohexyl analogue 5 in 36% yield (5 steps from 34). To conclude, the combined presence of
the TBDPS at Oꢀ6B and trichloroacetamide at Cꢀ2A’ in the protected intermediates were not
compatible with efficient deprotection steps.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental Section
pꢀTolyl 6ꢀOꢀtertꢀbutyldiphenylsilylꢀβꢀ1ꢀthioꢀ
D
ꢀgalactopyranoside (10). Imidazole (6.5 g,
7
95 mmol, 2.5 equiv) was added to a solution of known βꢀthiotolyl galactopyranoside (10.9 g, 38
mmol) in anhyd DMF (93 mL) at rt under N . TBDPSCl (12 mL, 46 mmol, 1.2 equiv) was then
2
slowly added and the reaction was left under stirring for 22 h at rt. The solvent was evaporated
and the residue was dissolved in CH Cl (500 mL) and washed with HCl 2 N (3 × 500 mL). The
2
2
aq layer was reꢀextracted with CH Cl (5 × 200 mL) and the combined organic layers were dried
2
2
8
and concentrated. Column chromatography (EtOAc/hexanes, 6:4–7:3) gave the known silylated
1
intermediate 10 (16.8 g, 84%) pure as a white amorphous foam. [α] –24.5 (c 1.0, MeOH). H
D
NMR (400 MHz, CDCl , 296 K): δ 7.73–7.65 (m, 4 H, Ar), 7.45–7.33 (m, 8 H, Ar), 7.03 (d, J =
3
H
8.0 Hz, 2 H, Ar), 4.43 (d, J = 9.6 Hz, 1 H, Hꢀ1), 4.07 (m, 1 H, Hꢀ4), 3.96–3.87 (m, 2 H, Hꢀ6a, Hꢀ
6b), 3.59–3.50 (m, 2 H, Hꢀ3, Hꢀ5), 3.37 (d, J = 5.1 Hz, 1 H, OHꢀ3), 3.05 (d, J = 3.6 Hz, 1 H, OHꢀ
13
4
), 2.98 (br s, 1 H, OHꢀ2), 2.28 (s, 3 H, CH tolyl), 1.04 (s, 9 H, C(CH ) ). C NMR (100 MHz,
3
3 3
CDCl , 296 K): δ 138.0 (quat Ar), 135.6, 135.5, 132.8 (Ar), 132.7 (quat Ar), 129.9, 129.7 (Ar),
3
C
128.5 (quat Ar), 127.8 (Ar), 88.8 (Cꢀ1), 78.1 (Cꢀ5), 74.9 (Cꢀ3), 69.8 (Cꢀ2), 69.4 (Cꢀ4), 63.7 (Cꢀ
6
), 26.7 (C(CH ) ), 21.1 (CH tolyl), 19.1 (C(CH ) ). HRMS (ESIꢀTOF) m/z: calcd for
3 3
3
3 3
+
C H O SSiNa [M+Na] 547.1950, found 547.1978.
29
36
5
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pꢀTolyl 2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀβꢀ1ꢀthioꢀDꢀgalactopyranoside (11).
Triethylorthoacetate (33 mL, 181 mmol, 4 equiv) and CSA (842 mg, 3.62 mmol, 0.08 equiv)
were added to a solution of triol 10 (23.8 g, 45 mmol) in anhyd MeCN (650 mL) under N . The
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solution was stirred at rt for 15 min, anhyd pyridine (183 mL, 2.26 mol, 50 equiv) and Ac O (107
2
mL, 1.13 mol, 25 equiv) were then added, and the mixture was heated to 50 °C for 1.5 h. The
mixture was coꢀconcentrated with toluene (4 × 150 mL), and the resulting oily residue was left
under high vacuum overnight. It was dissolved in a mixture of AcOH and H O (8:2, 200 mL),
2
stirred for 10 min, then diluted with CH Cl (300 mL) and washed sequentially with satd aq
2
2
NaHCO (2 × 500 mL) and HCl 2 N (2 × 500 mL). The aq layers were reꢀextracted with CH Cl
2
3
2
(
2 × 100 mL) and the combined organic layers were dried and concentrated. Column
chromatography (EtOAc/hexanes, 4:6) gave alcohol 11 (25.7 g, 93%) pure as a white amorphous
1
glass. [α] +8.0 (c 1.0, MeOH). H NMR (400 MHz, CDCl , 295 K): δ 7.64–7.58 (m, 4 H, Ar),
D
3
H
7
9
3
.45–7.32 (m, 8 H, Ar), 7.03 (d, J = 7.9 Hz, 2 H, Ar), 5.44 (d, J = 3.3 Hz, 1 H, Hꢀ4), 4.95 (t, J =
.8 Hz, 1 H, Hꢀ2), 4.58 (d, J = 10.0 Hz, 1 H, Hꢀ1), 3.85 (ddd, J = 3.5, 5.3, 9.1 Hz, 1 H, Hꢀ3),
.74 (dd, J = 4.5, 8.0 Hz, 1 H, Hꢀ6a), 3.71–3.61 (m, 2 H, Hꢀ5, Hꢀ6b), 2.43 (d, J = 5.3 Hz, 1 H,
13
OHꢀ3), 2.29 (s, 3 H, CH tolyl), 2.14, 2.00 (2 s, 6 H, 2 OCOCH ), 1.01 (s, 9 H, C(CH ) ). C
3
3
3 3
NMR (100 MHz, CDCl , 295 K): δ 171.2, 170.7 (C=O), 138 (quat Ar), 135.6 (Ar), 133.0 (quat
3
C
Ar), 132.7, 129.9, 129.8, 129.6 (Ar), 129.2 (quat Ar), 127.8, 127.7 (Ar), 86.8 (Cꢀ1), 77.4 (Cꢀ5),
72.9 (Cꢀ3), 70.9 (Cꢀ2), 70.0 (Cꢀ4), 61.8 (Cꢀ6), 26.7 (C(CH ) ), 21.1, 21.0, 20.7 (CH tolyl,
3
3
3
+
OCOCH ), 19.1 (C(CH ) ). HRMS (ESIꢀTOF) m/z calcd for C H O SSiNa [M+Na] 631.2162,
3
3 3
33 40
7
found 631.2162.
pꢀTolyl
2,4ꢀdiꢀOꢀacetylꢀ3ꢀOꢀchloroacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀ1ꢀthioꢀβꢀDꢀ
galactopyranoside (12). Chloroacetylchloride (6.7 mL, 84 mmol, 2 equiv) was slowly added to
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a solution of alcohol 11 (25.7 g, 42 mmol) in anhyd CH Cl (420 mL) containing anhyd pyridine
2
2
(
17 mL, 211 mmol, 5 equiv) at rt under N . The reaction mixture was stirred for 15 min then
2
diluted with CH Cl (50 mL) and washed sequentially with HCl 2 N (2 × 500 mL) and satd aq
2
2
10
11
12
13
14
15
16
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60
NaHCO (2 × 500 mL). The organic layer was dried and concentrated to give chloroacetate 12
3
1
(29 g, quant) pure as a yellow amorphous glass. [α] +12.8 (c 1.0, CH Cl ). H NMR (400 MHz,
D
2
2
CDCl , 296 K): δ 7.62–7.55 (m, 4 H, Ar), 7.45–7.32 (m, 8 H, Ar), 7.05 (d, J = 8.0 Hz, 2 H, Ar),
3
H
5.54 (d, J = 3.0 Hz, 1 H, Hꢀ4), 5.18 (t, J = 9.9 Hz, 1 H, Hꢀ2), 5.10 (dd, J = 3.2, 9.9 Hz, 1 H, Hꢀ3),
4.63 (d, J = 9.8 Hz, 1 H, Hꢀ1), 3.94 (s, 2 H, COCH Cl), 3.80–3.73 (m, 2 H, Hꢀ5, Hꢀ6a), 3.61 (dd,
2
J = 9.4, 11.8 Hz, 1 H, Hꢀ6b), 2.30 (s, 3 H, CH tolyl), 2.07, 1.96 (2 s, 6 H, 2 OCOCH ), 1.00 (s, 9
3
3
13
H, C(CH ) ). C NMR (100 MHz, CDCl , 296 K): δ 170.2, 169.4, 166.6 (C=O), 138.3 (quat
3 3
3
C
Ar), 135.6, 132.8 (Ar), 132.8 (quat Ar), 132.7 (quat Ar), 129.9, 129.8, 129.7 (Ar), 128.8 (quat
Ar), 127.8 (Ar), 87.1 (Cꢀ1), 77.0 (Cꢀ5), 74.2 (Cꢀ3), 67.2, 67.0 (Cꢀ2, Cꢀ4), 61.2 (Cꢀ6), 40.5
(COCH Cl), 26.7 (C(CH ) ), 21.1, 20.8, 20.6 (CH tolyl, OCOCH ), 19.0 (C(CH ) ). HRMS
2 3 3 3 3 3 3
+
(
ESIꢀTOF) m/z calcd for C H ClO SSiNa [M+Na] 707.1878, found 707.1937 and for
3
5
41
8
+
C H ClO SSiK [M+K] 723.1617, found 723.1650.
35
41
8
2,4ꢀDiꢀOꢀacetylꢀ3ꢀOꢀchloroacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀα,βꢀ
Dꢀgalactopyranose
(13)
and acetyl 4ꢀOꢀacetylꢀ3ꢀOꢀchloroacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀαꢀDꢀ
galactopyranose (14). NIS (11 g, 46 mmol, 1.1 equiv) and TfOH (374 ꢂL, 4.2 mmol, 0.1 equiv)
were added to a solution of chloroacetate 12 (29 g, 42 mmol) in a mixture of MeCN (650 mL)
and H O (6.50 mL) at rt. The reaction mixture was stirred for 5 min at rt, then quenched with
2
NEt (1.5 mL, 11 mmol, 0.25 equiv) and concentrated. The residue was dissolved in CH Cl (500
3
2
2
mL) and was washed with a 20% w/w solution of aq Na S O (400 mL). The aq layer was reꢀ
2
2
3
extracted with CH Cl (2 × 100 mL) and the combined organic layers were dried and
2
2
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concentrated. Column chromatography (EtOAc/hexanes, 4:6) of the residue gave a 0.6:1 mixture
of alcohols 13 and 14 (21 g, 86%). For analytical purpose, a small amount of the mixture
containing 13 and 14 was submitted to further chromatography to afford analytical samples of
0
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0
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13(α,β) and 14.
1
Analytical data for 13: Yellow foam, α/β ratio (1:0.5), H NMR (400 MHz, CDCl , 297 K): δ
3
H
7.61–7.55 (m, 6 H, Ar), 7.44–7.33 (m, 9 H, Ar), 5.62 (dd, J = 1.0, 3.2 Hz, 1 H, Hꢀ4α), 5.58 (d, J
=
3.3 Hz, 0.5 H, Hꢀ4β), 5.48 (dd, J = 3.3, 10.8 Hz, 1 H, Hꢀ3α), 5.40 (d, J = 2.9 Hz, 1 H, Hꢀ1α),
5.15–5.10 (m, 1.5 H, Hꢀ2α, Hꢀ3β), 5.03 (dd, J = 7.8, 10.4 Hz, 0.5 H, Hꢀ2β), 4.60 (t, J = 7.4 Hz,
.5 H, Hꢀ1β), 4.32 (t, J = 7.5 Hz, 1 H, Hꢀ5α), 3.97 (s, 3 H, COCH Cl), 3.80–3.71 (m, 1 H, Hꢀ5β,
0
2
Hꢀ6aβ), 3.68–3.56 (m, 2.5 H, Hꢀ6aα, Hꢀ6bα, Hꢀ6bβ), 3.43 (br s, 0.5 H, OHꢀ1β), 2.85 (br s, 1 H,
1
3
OHꢀ1α), 2.07, 2.06, 2.00, 1.99 (4 s, 9 H, 4 OCOCH ), 1.00 (s, 13.5 H, C(CH ) ). C NMR (100
3
3 3
MHz, CDCl , 297 K): δ 171.2, 170.3 170.2, 166.6, 166.5 (C=O), 135.6, 135.5 (Ar), 132.9,
3
C
1
32.8, 132.6 (quat Ar), 129.9, 129.8, 127.8, 127.7 (Ar), 95.8 (Cꢀ1β), 90.6 (Cꢀ1α), 73.2 (Cꢀ5β),
2.4 (Cꢀ2α), 71.1 (Cꢀ2β), 69.5 (Cꢀ3α), 68.5 (Cꢀ3β), 68.3 (Cꢀ5α), 67.8 (Cꢀ4α), 66.7 (Cꢀ4β), 61.2
7
(
Cꢀ6α), 60.7 (Cꢀ6β), 40.5 (COCH Clα), 40.4 (COCH Clβ), 26.7 (C(CH ) ), 20.8, 20.6
2 2 3 3
+
(OCOCH ), 19.0 (C(CH ) ). HRMS (ESIꢀTOF) m/z calcd for C H ClO SiNa [M+Na]
3
3 3
28 35
9
601.1637, found 601.1614.
1
Analytical data for 14: Yellow foam, [α] +68.5 (c 1.0, CH Cl ). H NMR (400 MHz, CDCl ,
D
2
2
3
296 K): δ 7.60–7.54 (m, 4 H, Ar), 7.44–7.32 (m, 6 H, Ar), 6.22 (d, J = 3.9 Hz, 1 H, Hꢀ1), 5.62
H
(dd, J = 1.1, 3.1 Hz, 1 H, Hꢀ4), 5.24 (dd, J = 3.2, 10.5 Hz, 1 H, Hꢀ3), 4.12 (ddd, J = 3.9, 7.7, 11.1
Hz, 1 H, Hꢀ2), 4.09–4.03 (m, 3 H, Hꢀ5, COCH Cl), 3.66 (dd, J = 5.7, 10.0 Hz, 1 H, Hꢀ6a), 3.57
2
(
dd, J = 8.5, 9.9 Hz, 1 H, Hꢀ6b), 2.14, 1.98 (2 s, 6 H, 2 OCOCH ), 2.08 (d, J = 7.7 Hz, 1 H, OHꢀ
3
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), 1.00 (s, 9 H, C(CH ) ). C NMR (100 MHz, CDCl , 296 K): δ 170.1, 169.4, 167.1 (C=O),
3 3 3 C
35.6 (Ar), 132.8, 132.7 (quat Ar), 129.9, 127.8 (Ar), 91.8 (Cꢀ1), 72.7 (Cꢀ3), 71.0 (Cꢀ5), 67.1 (Cꢀ
), 66.0 (Cꢀ2), 60.8 (Cꢀ6), 40.7 (COCH Cl), 26.7 (C(CH ) ), 21.0, 20.5 (OCOCH ), 19.0
2
3 3
3
10
11
12
13
14
15
16
17
18
19
20
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23
24
25
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
(
C(CH ) ). HRMS (ESIꢀTOF) m/z calcd for C H ClO SiNa [M+Na] 601.1637, found
3 3 28 35 9
601.1597.
2
,4ꢀDiꢀOꢀacetylꢀ3ꢀOꢀchloroacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀαꢀDꢀgalactopyranosyl
trichloroacetimidate (6). Trichloroacetonitrile (11 mL, 109 mmol, 3 equiv) was added to a
solution of alcohols 13 and 14 (21.0 g, 36.3 mmol) in anhyd CH Cl (350 mL) at rt under N .
2
2
2
DBU (1.4 mL, 9.08 mmol, 0.25 equiv) was then slowly added to the mixture, which was stirred
at rt for 3 h. The reaction mixture was then concentrated and column chromatography of the
residue (EtOAc/hexanes, 3:7 with 0.1% NEt ) gave trichloroacetimidate 6 (21.8 g, 83%) pure as
3
1
a slightly yellowish amorphous foam. [α] –50.2 (c 1.0, CH Cl ). H NMR (400 MHz, CDCl ,
D
2
2
3
2
96 K): δ 8.60 (s, 1 H, NH), 7.60–7.54 (m, 4 H, Ar), 7.44–7.32 (m, 6 H, Ar), 6.54 (d, J = 3.6
H
Hz, 1 H, Hꢀ1), 5.71 (d, J = 3.1 Hz, 1 H, Hꢀ4), 5.51 (dd, J = 3.2, 10.8 Hz, 1 H, Hꢀ3), 5.35 (dd, J =
.7, 10.8 Hz, 1 H, Hꢀ2), 4.29 (t, J = 7.2 Hz, 1 H, Hꢀ5), 4.00 (s, 2 H, COCH Cl), 4.20 (dd, J =
3
2
6.0, 10.1 Hz, 1 H, Hꢀ6a), 4.12 (dd, J = 8.0, 10.1 Hz, 1 H, Hꢀ6b), 2.03, 2.00 (2 s, 6 H, 2
1
3
OCOCH ), 0.98 (s, 9 H, C(CH ) ). C NMR (100 MHz, CDCl , 296 K): δ 170.2, 170.0, 166.5
3
3 3
3
C
(
C=O), 160.9 (C=N), 135.5 (Ar), 132.8, 132.7 (quat Ar), 129.9, 127.8, 127.7 (Ar), 93.5 (Cꢀ1),
71.3 (Cꢀ5), 69.7 (Cꢀ3), 67.1 (Cꢀ4), 67.0 (Cꢀ2), 61.0 (Cꢀ6), 40.5 (COCH Cl), 26.6 (C(CH ) ),
2
3 3
2
0.6, 20.5 (OCOCH ), 19.0 (C(CH ) ). HRMS (ESIꢀTOF) m/z calcd for C H Cl NO SiNa
3 3 3 30 35 4 9
+
[
M+Na] 744.0733, found 744.0737.
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,3ꢀDiꢀOꢀacetylꢀ4,6ꢀOꢀbenzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀα,βꢀDꢀ
1
1
glucopyranoside (16). Known diol 15 (9.07 g, 21.71 mmol) was treated with a mixture of
pyridine and Ac O (1:1, 90 mL) at 50 °C for 2 h. The reaction mixture was coꢀconcentrated with
2
0
1
2
3
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0
1
2
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9
0
toluene (4 × 100 mL) and the residue was dissolved in CH Cl (200 mL) and washed
2
2
sequentially with HCl (2 N, 2 × 200 mL) and satd aq NaHCO (2 × 200 mL). The aqueous layers
3
were reꢀextracted with CH Cl (2 × 50 mL) and the combined organic layers were dried and
2
2
concentrated. Column chromatography (CH Cl /MeOH, 50:1) gave diacetate 16 (10.55 g, 97%)
2
2
1
pure (α/β ratio, 1:0.21) as white amorphous powder. H NMR (600 MHz, CDCl , 295 K): δ
3
H
7
.53 (d, J = 9.7 Hz, 0.21 H, NHβ), 7.46–7.41 (m, 2.42 H, Ar), 7.37–7.32 (m, 3.63 H, Ar), 7.09
(
d, J = 8.4 Hz, 1 H, NHα), 6.24 (d, J = 3.9 Hz, 1 H, Hꢀ1α), 5.80 (d, J = 8.7 Hz, 0.21 H, Hꢀ1β),
5.57–5.53 (m, 1,21 H, Hꢀ3β, >PhCHα), 5.51–5.46 (m, 1.21 H, Hꢀ3α, >PhCHβ), 4.36–4.29 (m, 2
H, Hꢀ2α, Hꢀ6aα), 4.26 (m, 0.21 H, Hꢀ2β), 3.96 (td, J = 4.9, 9.9 Hz, 1 H, Hꢀ5α), 3.88 (dd, J = 4.9,
1
0.3 Hz, 0.21 H, Hꢀ6aβ), 3.82 (t, J = 9.7 Hz, 1 H, Hꢀ4α), 3.78 (t, J = 10.4 Hz, 1 H, Hꢀ6bα), 3.72
(
t, J = 9.5 Hz, 0.21 H, Hꢀ4β), 3.62 (t, J = 10.1 Hz, 0.21 H, Hꢀ6bβ), 3.54 (td, J = 4.9, 9.6 Hz,
13
0
.21 H, Hꢀ5β), 2.09, 2.05 (2 s, 1.26 H, 2 OCOCH β), 2.08, 2.04 (2 s, 6 H, 2 COCH α). C NMR
3
3
(125 MHz, CDCl , 295 K): δ 171.8, 168.6, 162.1 (C=Oα), 171.2, 168.9, 162.5 (C=Oβ), 136.6
3
C
(
quat Ar), 129.3, 128.3, 126.1 (Ar), 101.7 (>PhCHαβ), 92.5 (Cꢀ1β), 92.3 (CCl β), 91.8 (CCl α),
3 3
9
0.0 (Cꢀ1α), 78.1 (Cꢀ4α, Cꢀ4β), 71.0 (Cꢀ3β), 69.2 (Cꢀ3α), 68.4 (Cꢀ6α), 67.9 (Cꢀ6β), 67.3 (Cꢀ5β),
64.9 (Cꢀ5α), 55.2 (Cꢀ2β), 53.8 (Cꢀ2α), 20.8, 20.7, 20.5 (OCOCH ). HRMS (ESIꢀTOF) m/z calcd
3
+
for C H Cl NO Na [M+Na] 518.0152, found 518.0170.
1
9
20
3
8
3
ꢀOꢀAcetylꢀ4,6ꢀOꢀbenzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀα,βꢀ
Dꢀglucopyranose
(
17). To a solution of the diacetate 16 (2.38 g, 4.74 mmol) in THF (70 mL) was added BnNH (1
2
mL, 9.49 mmol, 2 equiv) at rt and the mixture was left under stirring for 66 h. The mixture was
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poured into water (70 mL), extracted with CH Cl (5 × 40 mL) and the combined organic layers
2
2
were dried and concentrated. Column chromatography (toluene/EtOAc, 5:1) gave hemiacetal 17
1
(
1.74 g, 80%) pure (α/β ratio, 1:0.25) as a white amorphous glass. H NMR (600 MHz, CDCl ,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
95 K): δ 7.46–7.41 (m, 2.50 H, NHβ, Ar), 7.38–7.32 (m, 4 H, Ar), 7.11 (d, J = 9.1 Hz, 1 H,
H
NHα), 5.53 (m, 1.25 H, >PhCHα, >PhCHβ), 5.48 (t, J = 10.1 Hz, 1 H, Hꢀ3α), 5.37 (t, J = 3.5 Hz,
1
H, Hꢀ1α) , 5.26 (t, J = 10.0 Hz, 0.25 H, Hꢀ3β), 4.79 (t, J = 8.6 Hz, 0.25 H, Hꢀ1β), 4.38 (dd, J =
.0, 10.5 Hz, 0.25 H, Hꢀ6aβ), 4.28 (dd, J = 4.9, 10.3 Hz, 1 H, Hꢀ6aα), 4.22 (ddd, J = 3.5, 9.1,
5
10.3 Hz, 1 H, Hꢀ2α), 4.16 (td, J = 4.9, 10.0 Hz, 1 H, Hꢀ5α), 4.03 (d, J = 9.0 Hz, 0.25 H, OHꢀ1β),
3
.93 (m, 0.25 H, Hꢀ2β), 3.83 (t, J = 10.3 Hz, 0.25 H, Hꢀ6bβ), 3.80–3.73 (m, 2.25 H, Hꢀ4α, Hꢀ
bα, Hꢀ4β), 3.50 (td, J = 4.9, 9.7 Hz, 0.25 H, Hꢀ5β), 2.95 (d, J = 1.9 Hz, 1 H, OHꢀ1α), 2.09 (s,
6
1
3
0.75 H, OCOCH β), 2.05 (s, 3 H, COCH α). C NMR (125 MHz, CDCl , 295 K): δ 172.3,
3
3
3
C
1
64.3 (C=Oβ), 171.4, 162.2 (C=Oα), 136.8 (quat Ar α), 136.8 (quat Ar β), 129.2, 128.3, 126.2
(Ar α), 129.3, 128.3, 126.1 (Ar β), 101.7 (>PhCHα), 101.6 (>PhCHβ), 97.2 (Cꢀ1β), 92.0 (CCl α),
3
91.7 (Cꢀ1α), 78.7 (Cꢀ4α), 78.2 (Cꢀ4β), 70.8 (Cꢀ3β), 69.3 (Cꢀ3α), 68.7 (Cꢀ6α), 68.4 (Cꢀ6β), 66.8
(Cꢀ5β), 62.9 (Cꢀ5α), 59.0 (Cꢀ2β), 54.8 (Cꢀ2α), 20.8 (OCOCH ). HRMS (ESIꢀTOF) m/z calcd for
3
+
C H Cl NO Na [M+Na] 476.0047, found 476.0042.
17
18
3
7
3
ꢀOꢀAcetylꢀ4,6ꢀOꢀbenzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀαꢀDꢀglucopyranosyl
trichloroacetimidate (7). A solution of hemiacetal 17 (6.38 g, 13.9 mmol) in anhyd CH Cl2
2
(175 mL) under N was cooled to 0 °C, Cl CCN (7 mL, 69.4 mmol, 5 equiv) was added to the
2
3
solution, followed by the dropwise addition of DBU (520 ꢂL, 3.47 mmol, 0.25 equiv) over a
period of 3 min. The reaction was left at 0 °C for 3 h and the solvent was evaporated. Column
chromatography (EtOAc/hexanes, 25:75 with 0.1 % NEt ) gave trichloroacetimidate 7 (6.09 g,
3
1
7
3%) pure as a slightly yellowish foam. [α] +53.2 (c 1.0, CH Cl ). H NMR (400 MHz, CDCl ,
D
2
2
3
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5
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6
2
96 K): δ 7.47–7.43 (m, 2 H, Ar), 7.38–7.34 (m, 3 H, Ar), 7.11 (d, J = 8.4 Hz, 1 H, NH), 6.46
H
(
d, J = 3.7 Hz, 1 H, Hꢀ1), 5.57 (s, 1 H, >PhCH), 5.53 (t, J = 10.2 Hz, 1 H, Hꢀ3), 4.42 (ddd, J =
.7, 8.5, 10.6 Hz, 1 H, Hꢀ2), 4.36 (dd, J = 4.9, 10.5 Hz, 1 H, Hꢀ6a), 4.08 (td, J = 4.9, 9.9 Hz, 1
H, Hꢀ5), 3.88 (t, J = 9.7 Hz, 1 H, Hꢀ4), 3.82 (t, J = 10.3 Hz, 1 H, Hꢀ6b), 2.09 (s, 3 H,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
OCOCH ). C NMR (100 MHz, CDCl , 296 K): δ 171.5, 162.2 (C=O), 160.3 (C=N), 136.5
3
3
C
(quat Ar), 129.3, 128.3, 126.1 (Ar), 101.7 (>PhCH), 94.1 (Cꢀ1), 91.7, 90.5 (CCl ), 78.0 (Cꢀ4),
3
69.0 (Cꢀ3), 68.4 (Cꢀ6), 65.5 (Cꢀ5), 54.6 (Cꢀ2), 20.8 (OCOCH ). HRMS (ESIꢀTOF) m/z calcd for
3
+
C H Cl N O Na [M+Na] 618.9143, found 618.9172.
19
18
6
2
7
6
ꢀChlorohexyl
2ꢀacetamidoꢀ4ꢀOꢀ(2,4ꢀdiꢀOꢀacetylꢀ3ꢀOꢀchloroacetylꢀ6ꢀOꢀtertꢀ
ꢀgalactopyranosyl)ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ
butyldiphenylsilylꢀβꢀ
D
Dꢀ
5
c
glucopyranoside (18). A stirred solution of known alcohol 8 (1.86 g, 3.67 mmol) and
galactosyl trichloroacetimidate 6 (10.61 g, 14.67 mmol, 4 equiv) in anhyd CH Cl (95 mL) was
2
2
heated to 40 °C under N . Freshly distilled BF ꢁOEt (921 ꢂL, 7.33 mmol, 2 equiv) was added to
2
3
2
the mixture, which was stirred for 1 h at 40 °C. The reaction was quenched with NEt (1.2 mL,
3
8.80 mmol, 2.4 equiv), the mixture diluted with CH Cl (100 mL) and washed with satd aq
2 2
NaHCO (200 mL). The aq layer was reꢀextracted with CH Cl (3 × 50 mL) and the combined
3
2
2
organic layers were dried and concentrated. Column chromatography (EtOAc/hexanes, 4:6–5:5)
gave disaccharide 18 (2.77 g, 71%) pure as a slightly yellowish amorphous foam. [α] –4.8 (c
D
1
1.0, CH Cl ). H NMR (400 MHz, CDCl , 297 K): δ 7.61–7.52 (m, 4 H, Ar), 7.46–7.30 (m, 11
2
2
3
H
H, Ar), 5.57 (d, J = 9.3 Hz, 1 H, NHA), 5.40 (d, J = 3.0 Hz, 1 H, Hꢀ4B), 5.03 (dd, J = 8.8, 10.1
Hz, 1 H, Hꢀ3A), 4.91 (dd, J = 7.7, 10.3 Hz, 1 H, Hꢀ2B), 4.84 (dd, J = 3.3, 10.4 Hz, 1 H, Hꢀ3B),
4.74 (d, J = 12.0 Hz, 1 H, CHHPh), 4.44–4.40 (m, 2 H, Hꢀ1, CHHPh), 4.31 (d, J = 7.7 Hz, 1 H,
Hꢀ1B), 3.96–3.61 (m, 10 H, Hꢀ2A, Hꢀ4A, Hꢀ6Aa, Hꢀ6Ab, Hꢀ6Ba, 2 COCH Cl , OCHHCH ),
2
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.53–3.36 (m, 6 H, Hꢀ5A, Hꢀ5B, Hꢀ6Bb, CH Cl, OCHH(CH ), 1.97, 1.93 (2 s, 6 H, 2 OCOCH ),
2
2
3
1
.90 (s, 3 H, NHCOCH ), 1.73 (m, 2 H, O(CH ) CH CH Cl), 1.55 (m, 2 H,
3
2 4
2
2
OCH CH (CH ) CH Cl), 1.40 (m,
2
H, O(CH ) CH (CH ) Cl), 1.32 (m,
2
H,
2
2
2 3
2
2 3
2
2 2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
26
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49
50
51
52
53
54
55
56
57
58
59
60
13
O(CH ) CH (CH ) Cl), 1.02 (s, 9 H, C(CH ) ). C NMR (100 MHz, CDCl , 297 K): δ 170.3,
2
2
2
2 3
3 3
3
C
170.1, 168.9, 167.2, 166.6 (C=O), 137.7 (quat Ar), 135.5 (Ar), 132.5, 132.4 (quat Ar), 130.1,
1
28.6, 128.2, 128.1, 127.9 (Ar), 100.9 (Cꢀ1A), 100.0 (Cꢀ1B), 74.5 (Cꢀ3A), 74.4 (Cꢀ4A), 74.3 (Cꢀ
5
A), 73.6 (CH Ph), 72.9 (Cꢀ3B), 72.7 (Cꢀ5B), 69.3 (OCH CH ), 69.1 (Cꢀ2B), 67.3 (Cꢀ6A), 66.4
2
2
2
(Cꢀ4B), 60.6 (Cꢀ6B), 53.4 (Cꢀ2A), 45.0 (CH Cl), 40.7, 40.5 (COCH Cl), 32.4
2
2
(
O(CH ) CH CH Cl),
29.2
(OCH CH (CH ) CH Cl),
26.7
(C(CH3)3),
26.5
2
4
2
2
2
2
2 3
2
(
O(CH ) CH (CH ) Cl), 25.2 (O(CH ) CH (CH ) Cl), 23.3 (NHCOCH ), 20.7, 20.6 (OCOCH ),
2 3
2
2 2
2 2
2
2 3
3
3
+
19.0 (C(CH ) ). HRMS (ESIꢀTOF) m/z calcd for C H Cl NO Si [M+H] 1066.336, found
3
3
51 67
3
15
1066.326.
6ꢀChlorohexyl
galactopyranosyl)ꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀ
acetamidoꢀ4ꢀOꢀ(2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀβꢀ
benzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (20). Disaccharide 18 (2.27 g, 2.13
2ꢀacetamidoꢀ4ꢀOꢀ(2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀβꢀ
ꢀglucopyranoside (19) and 6ꢀChlorohexyl 2ꢀ
ꢀgalactopyranosyl)ꢀ6ꢀOꢀ
Dꢀ
D
D
D
mmol) was dissolved in a mixture of pyridine and EtOH (2:1, 54 mL), thiourea (195 mg, 2.56
mmol, 1.2 equiv) was added and the solution was heated to 55 °C for 4.5 h, then allowed to cool
to rt. The reaction mixture was diluted with CHCl (100 mL) and washed with HCl 2 N (200
3
mL). The aq layer was reꢀextracted with CHCl (5 × 100 mL) and the combined organic layers
3
were dried and concentrated. Column chromatography (CHCl /MeOH, 30:1) gave pure alcohol
3
1
9 (2.12 g, 66%) and pure diol 20 (155 mg, 8%).
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5
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6
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Analytical data for 19: White foam, [α] –15.6 (c 1.0, CH Cl ). H NMR (400 MHz, CDCl ,
D
2
2
3
2
97 K): δ 7.61–7.55 (m, 4 H, Ar), 7.45–7.28 (m, 11 H, Ar), 5.49 (d, J = 7.8 Hz, 1 H, NHA),
H
5
.33 (d, J = 3.2 Hz, 1 H, Hꢀ4B), 4.90 (dd, J = 8.1, 9.8 Hz, 1 H, Hꢀ2B), 4.74 (d, J = 8.2 Hz, 1 H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hꢀ1A), 4.67 (d, J = 12.1 Hz, 1 H, CHHPh), 4.50 (d, J = 12.1 Hz, 1 H, CHHPh), 4.42 (d, J = 8.0
Hz, 1 H, Hꢀ1B), 4.02 (d, J = 1.2 Hz, 1 H, OHꢀ3A), 3.96 (t, J = 9.5 Hz, 1 H, Hꢀ3A), 3.82 (m, 1 H,
OCHHCH ), 3.77–3.38 (m, 11 H, Hꢀ3B, Hꢀ4A, Hꢀ5A, Hꢀ5B, Hꢀ6Aa, Hꢀ6Ab, Hꢀ6Ba, Hꢀ6Bb,
2
CH Cl, OCHHCH ), 3.32 (m, 1 H, Hꢀ2A), 2.63 (d, J = 5.1 Hz, 1 H, OHꢀ3B), 2.03, 1.99 (2 s, 6 H,
2
2
2 OCOCH ), 1.87 (s, 3 H, NHCOCH ), 1.74 (m, 2 H, O(CH ) CH CH Cl), 1.55 (m, 2 H,
3
3
2 4
2
2
OCH CH (CH ) CH Cl), 1.42 (m,
2
H, O(CH ) CH (CH ) Cl), 1.34 (m,
2
H,
2
2
2 3
2
2 3
2
2 2
13
O(CH ) CH (CH ) Cl), 1.02 (s, 9 H, C(CH ) ). C NMR (100 MHz, CDCl , 297 K): δ 171.1,
2 2
2
2 3
3 3
3
C
170.7, 170.3 (C=O), 138.2 (quat Ar), 135.6, 135.5 (Ar), 132.7, 132.4 (quat Ar), 130.0, 129.9,
1
28.4, 127.9, 127.7, 127.6 (Ar), 100.9 (Cꢀ1B), 100.1 (Cꢀ1A), 80.8 (Cꢀ4A), 74.0, 73.8 (Cꢀ5A, Cꢀ
B), 73.5 (CH Ph), 72.6 (Cꢀ2B), 71.4 (Cꢀ3B), 71.3 (Cꢀ3A), 69.4 (Cꢀ4B), 69.3 (OCH CH ), 68.3
5
2
2
2
(
Cꢀ6A), 61.3 (Cꢀ6B), 57.0 (Cꢀ2A), 45.0 (CH Cl), 32.5 (O(CH ) CH CH Cl), 29.3
2
2 4
2
2
(
OCH CH (CH ) CH Cl),
26.7
(C(CH3)3),
26.5
(O(CH ) CH (CH ) Cl),
25.2
2
2
2 3
2
2 3
2
2 2
(O(CH ) CH (CH ) Cl), 23.6 (NHCOCH ), 20.9, 20.7 (OCOCH ), 19.0 (C(CH ) ). HRMS (ESIꢀ
2
2
2
2 3
3
3
3 3
+
TOF) m/z calcd for C H ClNO SiNa [M+Na] 936.3733, found 936.3698.
4
7
64
13
1
Analytical data for 20: White foam, [α] –12.4 (c 1.0, CH Cl ). H NMR (400 MHz, CDCl ,
D
2
2
3
297 K): δ 7.62–7.55 (m, 4 H, Ar), 7.45–7.28 (m, 11 H, Ar), 5.72 (d, J = 9.2 Hz, 1 H, NHA),
H
5
.29 (d, J = 3.3 Hz, 1 H, Hꢀ4B), 5.06 (t, J = 9.9 Hz, 1 H, Hꢀ3A), 4.75–4.66 (m, 2 H, Hꢀ2B,
CHHPh), 4.49–4.40 (m, 2 H, Hꢀ1A, CHHPh), 4.28 (d, J = 7.9 Hz, 1 H, Hꢀ1B), 3.96–3.70 (m, 7
H, Hꢀ2A, Hꢀ4A, Hꢀ6Aa, Hꢀ6Ab, COCH Cl, OCHHCH ), 3.66–3.33 (m, 8 H, Hꢀ3B, Hꢀ5A, Hꢀ
2
2
5
B, Hꢀ6Ba, Hꢀ6Bb, CH Cl, OCHHCH ), 2.61 (br s, 1 H, OHꢀ3B), 2.02, 2.00 (2 s, 6 H, 2
2
2
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OCOCH ), 1.91 (s, 3 H, NHCOCH ), 1.73 (m, 2 H, O(CH ) CH CH Cl), 1.55 (m, 2 H,
3
3
2 4
2
2
OCH CH (CH ) CH Cl), 1.40 (m,
2
H, O(CH ) CH (CH ) Cl), 1.33 (m,
2
H,
2
2
2 3
2
2 3
2
2 2
13
O(CH ) CH (CH ) Cl), 1.04 (s, 9 H, C(CH ) ). C NMR (100 MHz, CDCl , 297 K): δ 171.1,
2
2
2
2 3
3 3
3
C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
70.9, 170.4, 167.3 (C=O), 137.8 (quat Ar), 135.5 (Ar), 132.7, 132.6 (quat Ar), 130.1, 130.0,
128.5, 127.9 (Ar), 100.9 (Cꢀ1A), 100.0 (Cꢀ1B), 74.5 (Cꢀ3A, Cꢀ4A), 74.4 (Cꢀ5A), 73.6 (CH Ph),
2
7
3.1 (Cꢀ2B), 73.0 (Cꢀ5B), 71.5 (Cꢀ3B), 69.4 (Cꢀ4B), 69.3 (OCH CH ), 67.5 (Cꢀ6A), 61.1 (Cꢀ
2 2
6
B), 53.4 (Cꢀ2A), 45.0 (CH Cl), 40.8 (COCH Cl), 32.4 (O(CH ) CH CH Cl), 29.2
2
2
2 4
2
2
(OCH CH (CH ) CH Cl),
26.7
(C(CH3)3),
26.5
(O(CH ) CH (CH ) Cl),
25.2
2
2
2 3
2
2 3
2
2 2
(O(CH ) CH (CH ) Cl), 23.2 (NHCOCH ), 21.0, 20.7 (OCOCH ), 19.1 (C(CH ) ). HRMS (ESIꢀ
2 2 2 2 3 3 3 3 3
+
TOF) m/z calcd for C H Cl NO Si [M+H] 990.3630, found 990.3546.
49 66
2
14
6
ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀ3ꢀOꢀ(3ꢀOꢀ
acetylꢀ4,6ꢀOꢀbenzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ ꢀglucopyranosyl)ꢀβꢀ
galactopyranosyl]ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside
D
Dꢀ
D
(21).
Disaccharide acceptor 20 (1.94 g, 1.96 mmol) and glycosyl donor 7 (3.54 g, 5.87 mmol, 3 equiv)
were dissolved in anhyd CH Cl (100 mL) under N . Freshly distilled TESOTf (885 ꢂL, 3.91
2
2
2
mmol, 2 equiv) was added to the reaction mixture, which was stirred at rt for 45 min. The
reaction was quenched with NEt (655 ꢂL, 4.70 mmol, 2.4 equiv) and the mixture was diluted
3
with CH Cl (50 mL) and washed with satd aq NaHCO (150 mL). The aq layer was reꢀextracted
2
2
3
with CH Cl (3 × 40 mL) and the combined organic layers were dried and concentrated. Column
2
2
chromatography (EtOAc/hexanes, 45:55–6:4) gave trisaccharide 21 (1.99 g, 70%) pure as a
1
yellowish amorphous foam. [α] –21.8 (c 1.0, CH Cl ). H NMR (600 MHz, CDCl , 295 K): δ
D
2
2
3
H
7
.63–7.57 (m, 4 H, Ar), 7.46–7.25 (m, 16 H, Ar), 7.05 (d, J = 9.8 Hz, 1 H, NHA’), 5.78 (d, J =
.4 Hz, 1 H, NHA), 5.51 (s, 1 H, >CHPh), 5.39 (t, J = 10.0 Hz, 1 H, Hꢀ3A’), 5.35 (d, J = 3.4 Hz,
9
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4
H, Hꢀ4B), 4.99 (t, J = 8.3 Hz, 1 H, Hꢀ3A), 4.84 (dd, J = 8.1, 9.8 Hz, 1 H, Hꢀ2B), 4.70 (d, J =
2.1 Hz, 1 H, CHHPh), 4.57 (d, J = 7.9 Hz, 1 H, Hꢀ1A’), 4.50 (d, J = 12.0 Hz, 1 H, CHHPh),
.37 (d, J = 7.2 Hz, 1 H, Hꢀ1A), 4.32 (d, J = 8.0 Hz, 1 H, Hꢀ1B), 4.27 (dd, J = 4.9, 10.4 Hz, 1 H,
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
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8
9
0
1
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5
6
7
8
9
0
Hꢀ6Aa’), 4.03 (m, 1 H, Hꢀ2A), 3.91–3.61 (m, 10 H, Hꢀ4A, Hꢀ6Aab, Hꢀ6Ba, Hꢀ2A’, Hꢀ4A’, Hꢀ
6Ab’, COCH Cl, OCHHCH ), 3.56–3.47 (m, 5 H, Hꢀ3B, Hꢀ6Bb, Hꢀ5A’, CH Cl), 3.47–3.41 (m,
2
2
2
2
H, Hꢀ5A, Hꢀ5B), 3.38 (m, 1 H, OCHHCH ), 2.02, 2.01, 1.92 (3 s, 9 H, 3 OCOCH ), 1.91 (s, 3
2 3
H, NHCOCH ), 1.72 (m, 2 H, O(CH ) CH CH Cl), 1.54 (m, 2 H, OCH CH (CH ) CH Cl), 1.40
3
2 4
2
2
2
2
2 3
2
(m, 2 H, O(CH ) CH (CH ) Cl), 1.32 (m, 2 H, O(CH ) CH (CH ) Cl), 1.06 (s, 9 H, C(CH ) ).
2
3
2
2 2
2 2
2
2 3
3 3
13
C NMR (125 MHz, CDCl , 295 K): δ 171.3, 170.0, 169.8, 168.6, 167.1, 161.9 (C=O), 137.9,
3
C
136.9 (quat Ar), 135.6, 135.5 (Ar), 132.8, 132.7 (quat Ar), 130.0, 129.1, 128.4, 128.3, 128.0,
127.9, 127.8, 126.0 (Ar), 101.1 (>CHPh) 100.9 (Cꢀ1A), 100.4 (Cꢀ1A’), 100.2 (Cꢀ1B), 92.3
(CCl ), 78.3 (Cꢀ4A’), 75.2 (Cꢀ3B), 74.6 (Cꢀ5A), 74.1 (Cꢀ3A), 73.8 (Cꢀ4A), 73.7 (CH Ph), 73.6
3
2
(Cꢀ5B), 71.2 (Cꢀ2B), 70.8 (Cꢀ3A’), 69.2 (OCH CH ), 68.6 (Cꢀ4B), 68.3 (Cꢀ6A’), 67.6 (Cꢀ6A),
2 2
6
6.1 (Cꢀ5A’), 61.1 (Cꢀ6B), 56.5 (Cꢀ2A’), 52.2 (Cꢀ2A), 45.0 (CH Cl), 40.7 (COCH Cl), 32.4
2
2
(O(CH ) CH CH Cl),
29.2
(OCH CH (CH ) CH Cl),
26.8
(C(CH3)3),
26.5
2
4
2
2
2
2
2 3
2
(O(CH ) CH (CH ) Cl), 25.2 (O(CH ) CH (CH ) Cl), 23.2 (NHCOCH ), 21.0, 20.8, 20.7
2
3
2
2 2
2 2
2
2 3
3
+
(OCOCH ), 19.1 (C(CH ) ). HRMS (ESIꢀTOF) m/z calcd for C H Cl N O Si [M+H]
3 3 3 66 82 5 2 20
1425.367, found 1425.367.
6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀ3ꢀOꢀ(3ꢀOꢀ
acetylꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ ꢀglucopyranosyl)ꢀβꢀ
galactopyranosyl]ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside A
D
Dꢀ
D
(22).
solution of the benzylidene acetal 21 (962 mg, 0.672 mmol) in anhyd THF (17 mL) containing
freshly activated molecular sieves 3 Å (2.55 g), NaCNBH (633 mg, 10.1 mmol, 15 equiv) and
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methyl orange indicator (2 mg) was stirred under N for 45 min at rt and cooled to 0 °C. A 2 M
2
solution of HCl in Et O (5 mL, 10.1 mmol, 15 equiv) was added dropwise to the reaction mixture
2
at 0 °C until the methyl orange indicator turned pink, remained as such for 10 min, and H (g)
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
®
was no longer generated. The reaction was stirred at rt for 1 h, then filtered over Celite . The
solids were washed with THF (2 × 50 mL) and the combined filtrate and washings were
concentrated. The residue was dissolved in CH Cl (100 mL) and washed with satd aq NaHCO
3
2
2
(
100 mL). The aq layer was reꢀextracted with CH Cl (3 × 30 mL) and the combined organic
2 2
layers were dried and concentrated. Column chromatography (CH Cl /MeOH, 100:1–30:1) gave
2
2
1
alcohol 22 (877 mg, 91%) pure as an amorphous white foam. [α] –16.0 (c 1.0, CH Cl ). H
D
2
2
NMR (600 MHz, CDCl , 295 K): δ 7.61–7.55 (m, 4 H, Ar), 7.44–7.24 (m, 16 H, Ar), 6.84 (d, J
3
H
= 8.7 Hz, 1 H, NHA’), 5.76 (d, J = 9.4 Hz, 1 H, NHA), 5.41 (d, J = 3.5 Hz, 1 H, Hꢀ4B), 5.14 (dd,
J = 9.0, 10.8 Hz, 1 H, Hꢀ3A’), 4.98 (t, J = 8.8 Hz, 1 H, Hꢀ3A), 4.83 (dd, J = 8.1, 9.9 Hz, 1 H, Hꢀ
2
B), 4.68 (d, J = 12.1 Hz, 1 H, CHHPh), 4.64 (d, J = 8.0 Hz, 1 H, Hꢀ1A’), 4.57 (d, J = 11.8 Hz, 1
H, CHHPh), 4.51 (d, J = 11.8 Hz, 1 H, CHHPh), 4.45 (d, J = 12.1 Hz, 1 H, CHHPh), 4.37 (d, J =
.4 Hz, 1 H, Hꢀ1A), 4.20 (d, J = 8.0 Hz, 1 H, Hꢀ1B), 3.97 (m, 1 H, Hꢀ2A), 3.88–3.51 (m, 14 H,
Hꢀ4A, Hꢀ6Aab, Hꢀ3B, Hꢀ6Bab, Hꢀ2A’, Hꢀ4A’, Hꢀ5A’, Hꢀ6Aab’, COCH Cl, OCHHCH ), 3.49
7
2
2
(
t, J = 6.7 Hz, 2 H, CH Cl), 3.45–3.36 (m, 3 H, Hꢀ5A, Hꢀ5B, OCHHCH ), 3.25 (d, J = 2.9 Hz,
2
2
OHꢀ4A’), 2.04, 1.97, 1.94 (3 s, 9 H, 3 OCOCH ), 1.90 (s, 3 H, NHCOCH ), 1.72 (m, 2 H,
3
3
O(CH ) CH CH Cl), 1.53 (m,
2
H, OCH CH (CH ) CH Cl), 1.40 (m,
2
H,
2
4
2
2
2
2
2 3
2
13
O(CH ) CH (CH ) Cl), 1.31 (m, 2 H, O(CH ) CH (CH ) Cl), 1.03 (s, 9 H, C(CH ) ). C NMR
2
3
2
2 2
2 2
2
2 3
3 3
(
(
(
125 MHz, CDCl , 295 K): δ 171.8, 170.1, 169.8, 168.9, 167.2, 161.7 (C=O), 137.8, 137.3
3 C
quat Ar), 135.6, 135.5 (Ar), 132.7, 132.6 (quat Ar), 130.0, 128.5, 128.0, 127.9, 127.8, 127.7
Ar), 100.9 (Cꢀ1A), 100.2 (Cꢀ1B), 99.7 (Cꢀ1A’), 92.3 (CCl ), 75.2 (Cꢀ3B), 74.4 (Cꢀ5A), 74.1 (Cꢀ
3
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Page 28 of 57
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A), 74.0 (Cꢀ5A’), 73.9 (Cꢀ4A, CH Ph), 73.7 (Cꢀ3A’), 73.6 (Cꢀ5B, CH Ph), 71.3 (Cꢀ2B), 71.0
2
2
(Cꢀ4A’), 70.7 (Cꢀ6A’), 69.2 (OCH CH ), 68.4 (Cꢀ4B), 67.5 (Cꢀ6A), 61.3 (Cꢀ6B), 56.2 (Cꢀ2A’),
2 2
52.5 (Cꢀ2A), 45.0 (CH Cl), 40.7 (COCH Cl), 32.4 (O(CH ) CH CH Cl), 29.2
2 2 2 4 2 2
0
1
2
3
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0
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0
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0
(OCH CH (CH ) CH Cl),
26.8
(C(CH3)3),
26.5
(O(CH ) CH (CH ) Cl),
25.2
2
2
2 3
2
2 3
2
2 2
(O(CH ) CH (CH ) Cl), 23.2 (NHCOCH ), 21.0, 20.9, 20.6 (OCOCH ), 19.0 (C(CH ) ). HRMS
2
2
2
2 3
3
3
3 3
+
(ESIꢀTOF) m/z calcd for C H Cl N O Si [M+H] 1427.384, found 1427.383.
66 84 5 2 20
6
ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀ3ꢀOꢀ[4ꢀOꢀ
(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ ꢀfucopyranosyl)ꢀ3ꢀOꢀacetylꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀ2ꢀ
trichloroacetamidoꢀβꢀ ꢀglucopyranosyl]ꢀβꢀ ꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ
ꢀglucopyranoside (23). A solution of glycosyl acceptor 22 (550 mg,
L
D
D
chloroacetylꢀ2ꢀdeoxyꢀβꢀ
D
6
3
.84 mmol) and known thioglycoside donor 9 (367 mg, 7.67 mmol, 2 equiv) in anhyd CH Cl
2
2
(19 mL) containing freshly activated molecular sieves 4 Å (1.9 g) was stirred under N for 3 h at
2
rt. MeOTf (217 ꢂL, 1.92 mmol, 5 equiv) was added to the reaction mixture, which was stirred at
rt for 30 min. The reaction was quenched with NEt (321 ꢂL, 2.30 mmol, 6 equiv) and filtered
3
®
over Celite . The solids were washed with CH Cl (2 × 30 mL) and the combined filtrate and
2
2
washings were washed with satd aq NaHCO (80 mL). The aq layer was reꢀextracted with
3
CH Cl (3 × 30 mL) and the combined organic layers were dried and concentrated. The resulting
2
2
residue was dissolved in a mixture of Ac O and AcOH (3:1, 36 mL) and the solution was stirred
2
at rt for 18 h, then coꢀconcentrated with toluene (3 × 25 mL). Column chromatography
(EtOAc/hexanes, 45:55–1:1) gave tetrasaccharide 23 (585 mg, 82%) pure as white amorphous
1
glass. [α] –37.1 (c 1.0, CH Cl ). H NMR (600 MHz, CDCl , 295 K): δ 7.61–7.55 (m, 4 H,
D
2
2
3
H
Ar), 7.43–7.15 (m, 31 H, Ar), 6.55 (d, J = 8.8 Hz, 1 H, NHA’), 5.71 (d, J = 9.4 Hz, 1 H, NHA),
.47 (d, J = 3.5 Hz, 1 H, Hꢀ4B), 5.12 (dd, J = 7.7, 10.6 Hz, 1 H, Hꢀ3A’), 4.97–4.89 (m, 3 H, Hꢀ
5
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A, Hꢀ1C’, CHHPh), 4.82–4.66 (m, 5 H, Hꢀ2B, 4 CHHPh), 4.63–4.58 (m, 2 H, 2 CHHPh), 4.56
(
d, J = 7.9 Hz, 1 H, Hꢀ1A’), 4.43 (d, J = 12.2 Hz, 1 H, CHHPh), 4.38–4.33 (m, 2 H, Hꢀ1A,
CHHPh), 4.27 (d, J = 11.9 Hz, 1 H, CHHPh), 4.15–4.11 (m, 2 H, Hꢀ1B, Hꢀ6Aa’), 4.02–3.94 (m,
H, Hꢀ2A, Hꢀ2C’), 3.85–3.76 (m, 5 H, Hꢀ4A, Hꢀ3C’, Hꢀ5C’, COCHHCl, OCHHCH ), 3.74–
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2
2
3.56 (m, 10 H, Hꢀ6Aab, Hꢀ3B, Hꢀ6Ba, Hꢀ2A’, Hꢀ4A’, Hꢀ5A’, Hꢀ6Ab’, Hꢀ4C’, COCHHCl), 3.54
dd, J = 7.3, 10.0 Hz, 1 H, Hꢀ6Bb), 3.50 (t, J = 6.7 Hz, 2 H, CH Cl), 3.45–3.35 (m, 3 H, Hꢀ5A,
(
2
Hꢀ5B, OCHHCH ), 1.98 (2 s, 6 H, 2 OCOCH ), 1.90 (2 s, 6 H, NHCOCH OCOCH ), 1.73 (m,
2
3
3,
3
2 H, O(CH ) CH CH Cl), 1.53 (m, 2 H, OCH CH (CH ) CH Cl), 1.40 (m, 2 H,
2
4
2
2
2
2
2 3
2
O(CH ) CH (CH ) Cl), 1.32 (m, 2 H, O(CH ) CH (CH ) Cl), 1.06ꢀ1.00 (m, 12 H, Hꢀ6C’,
2
3
2
2 2
2 2
2
2 3
13
C(CH ) ). C NMR (125 MHz, CDCl , 295 K): δ 171.6, 170.0, 169.8, 168.7, 167.1, 161.6
3 3
3
C
(C=O), 138.6, 138.5, 138.4, 138.3, 137.7 (quat Ar), 135.6, 135.5 (Ar), 132.7, 132.6 (quat Ar),
1
1
4
30.0, 129.9, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 127.4 (Ar), 100.9 (Cꢀ
A), 100.4 (Cꢀ1C’), 100.1 (Cꢀ1B), 99.6 (Cꢀ1A’), 92.2 (CCl ), 79.0 (Cꢀ3C’), 77.5 (Cꢀ4A’, Cꢀ
3
C’), 76.5 (Cꢀ2C’), 75.4 (Cꢀ5A’), 74.9 (CH Ph), 74.3 (Cꢀ5A, Cꢀ3B), 74.1 (Cꢀ3A), 73.9, 73.7
2
(
CH Ph), 73.6 (Cꢀ4A, Cꢀ5B), 73.4 (CH Ph), 73.0 (Cꢀ3A’), 72.8 (CH Ph), 71.4 (Cꢀ2B), 70.0 (Cꢀ
2 2 2
6A’), 69.2 (OCH CH ), 68.7 (Cꢀ4B), 67.8 (Cꢀ5C’), 67.4 (Cꢀ6A), 61.3 (Cꢀ6B), 56.5 (Cꢀ2A’), 52.4
2
2
(
Cꢀ2A),
45.0
(CH Cl),
40.6
26.8
(COCH Cl),
32.4
26.5
(O(CH ) CH CH Cl),
29.2
25.2
2
2
2 4
2
2
(OCH CH (CH ) CH Cl),
(C(CH3)3),
(O(CH ) CH (CH ) Cl),
2 3 2 2 2
2
2
2 3
2
(O(CH ) CH (CH ) Cl), 23.2 (NHCOCH ), 21.2, 21.0, 20.7 (OCOCH ), 19.0 (C(CH ) ), 16.3
2
2
2
2 3
3
3
3 3
+
(
Cꢀ6C’). HRMS (ESIꢀTOF) m/z calcd for C H Cl N O Si [M+H] 1843.582, found 1843.585.
93 112 5 2 24
6
ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldiphenylsilylꢀ3ꢀOꢀ[4ꢀOꢀ
2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ ꢀfucopyranosyl)ꢀ3ꢀOꢀacetylꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀ2ꢀ
ꢀglucopyranosyl]ꢀβꢀ ꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀ
(
L
trichloroacetamidoꢀβꢀ
D
D
Dꢀ
29
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