Divergent Synthesis of Heparan Sulfate Oligosaccharides

Article abstract of DOI:10.1021/acs.joc.5b02172

Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis.

Full text of DOI:10.1021/acs.joc.5b02172

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Article
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Divergent Synthesis of Heparan Sulfate Oligosaccharides
Steven B. Dulaney,† Yongmei Xu,‡ Peng Wang,† Gopinath Tiruchinapally,† Zhen Wang,† Jolian Kathawa,†
Mohammad H. El-Dakdouki,†,§ Bo Yang,† Jian Liu,*,‡ and Xuefei Huang*,†
†Department of Chemistry, Michigan State University, 578 S. Shaw Lane, East Lansing, Michigan 48824, United States
‡Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina,
Chapel Hill, North Carolina 27599, United States
§Department of Chemistry, Beirut Arab University, P.O. Box 11-5020, Riad El Solh 11072809, Beirut, Lebanon
S
* Supporting Information
ABSTRACT: Heparan sulfates are implicated in a wide range
of biological processes. A major challenge in deciphering their
structure and activity relationship is the synthetic difficulties to
access diverse heparan sulfate oligosaccharides with well-
defined sulfation patterns. In order to expedite the synthesis, a
divergent synthetic strategy was developed. By integrating
chemical synthesis and two types of O-sulfo transferases, seven
different hexasaccharides were obtained from a single hexa-
saccharide precursor. This approach combined the flexibility of
chemical synthesis with the selectivity of enzyme-catalyzed
sulfations, thus simplifying the overall synthetic operations.
In an attempt to establish structure activity relationships of
heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and
their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches
expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light
on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in
biosynthesis.
Recently, enzymatic synthesis of HS has emerged as a
synthetic tool,23,27,44,45 which proved highly efficient for certain
targets without the need for selective protection/deprotection.
However, one limitation is that some HS sequences are not
accessible through the enzymatic approach due to substrate
specificities of the enzymes.
Herein, we report the development of a synthetic approach by
combining the flexibility of chemical synthesis and the
regioselectivities of HS biosynthetic enzymes. The iduronic
acid containing HS backbones were chemically prepared, and a
selective chemical sulfation strategy was developed to create
multiple HS sequences. To further diversify HS structures,
enzymatic sulfations were carried out using 2-O-sulfotransferase
(2-OST) and 6-O-sulfotransferase (6-OST). The synthetic HS
oligosaccharides were subsequently immobilized on a carbohy-
drate microarray to analyze the structural requirements for HS
binding with fibroblast growth factor-2 (FGF-2).
INTRODUCTION
■
Heparan sulfate (HS) is a class of highly charged polysaccharides,
which play important roles in a variety of biological events such as
cell proliferation, viral infection, and cancer development.1−4 HS
is made of disaccharide units of glucosamine α-1,4-linked with a
uronic acid.5 In nature, the backbone of HS can be extensively
sulfated by a variety of enzymes.6 For example, the glucosamine
residue can bear sulfates on its amine, 3-OH or 6-OH, while the
uronic acid including both glucuronic acid and iduronic acid
can be 2-O sulfated. As the enzymatic reactions are often not
complete, natural sources of HS are highly heterogeneous.7
The structural diversity bestows HS the abilities to interact with a
wide range of biological targets.4 To better understand its
structure activity relationship, synthesis of well-defined HS
structures becomes crucial to avoid structural heterogeneities of
naturally existing HS.
Tremendous advances have been made in HS oligosaccharide
synthesis during the past two decades.5,8−11 Chemical synthesis
of HS relies on stepwise construction of the backbone and
strategic protection of the hydroxyl groups that will be ultimately
sulfated. Although complex HS structures have been con-
structed,12−39 chemical synthesis is still highly challenging and
unexpected obstacles in stereoselectivity and reactivity can rise.40,41
Thus, continual efforts are needed to expedite the synthesis and
enable the creation of diverse HS structures.17,28,31,34,42,43
RESULTS AND DISCUSSION
■
Chemical Synthesis of HS Backbones. Our synthesis
commenced from the construction of HS backbones with
disaccharide donor 1 as a key building block. 1 can serve as the
Received: September 15, 2015
© XXXX American Chemical Society
A
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Scheme 1
nonreducing end of HS. At the same time, it can be readily
transformed to the bifunctional module 2 for backbone
elongation, as well as disaccharide 3 with a functional linker at
the reducing terminal of HS.
tetrasaccharide 9 in 81% yield. The 4 + 2 glycosylation between 9
and disaccharide 3 generated the fully protected HS hexa-
saccharide backbone 10 (61% yield) (Scheme 2a). Analogously,
tetrasaccharide 11 was prepared from the reaction of 1 with 3
(Scheme 2b). In order to improve the synthetic efficiency, one-
pot synthesis of hexasaccharide 10 was tested (Scheme 2c).
Upon preactivation of 1 by p-TolSCl/AgOTf at −78 °C,
acceptor 2 was added. The reaction temperature was warmed
up to −30 °C over 2 h when TLC analysis showed complete
consumption of acceptor 2. Subsequently, acceptor 3 was added
to the reaction, followed by p-TolSCl/AgOTf, which led to the
formation of hexasaccharide 10 in 67% yield without the need to
purify the tetrasaccharide intermediate 9.
Challenges in Deprotection and Chemical Sulfation of
HS Hexasaccharide. To produce HS oligosaccharides, 10 was
subjected to deprotection and chemical sulfation. Since idose was
utilized as an iduronic acid surrogate in backbone formation, the
first step in the deprotection was to convert the idosyl units
to iduronic acids. The 6-O Lev esters in 10 were removed
selectively with hydrazine, followed by bis(acetoxy)iodobenzene
(BAIB) assisted 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO)
oxidation (Scheme 3).50 The newly formed carboxylic acids
were protected as benzyl esters with phenyl diazomethane,51
forming 12 for the ease of purification and characterization.
In order to install sulfates, the temporary acyl protective
groups needed to be removed. This was accomplished by treating
12 with lithium hydroxide and hydrogen peroxide, followed by
sodium hydroxide (Scheme 3). Staudinger reduction of the
azides provided hexasaccharide bearing free alcohols and amines.
However, sulfation of this newly formed hexasaccharide failed to
generate the desired hexasaccharide with several undersulfated
and unidentified side products found in the reaction mixture.
Addition of excess sulfation agents (up to 1 M), exploration of
different sulfation conditions including SO3·NEt3 in DMF,
prolonging the reaction time, and raising the reaction temperatures
did not lead to the anticipated fully N- and O-sulfated hexa-
saccharide.
The preparation of disaccharide 1 began from the reaction of
glucosamine derivative 417 and idoside 517 (Scheme 1a).
Preactivation46 of donor 4 with p-TolSCl and AgOTf at
−78 °C, followed by the addition of acceptor 5 and 2,4,5-tri-
tert-butylpyrimidine (TTBP)47 as the base, led to the α-linked
disaccharide 6 in 85% yield as the sole anomer isolated. The
stereochemistry of the newly formed glycosyl linkage was
3
confirmed by NMR analysis with JH1B−H2B = 3.7 Hz and
1JC1B−H1B = 171 Hz.48 As the 6-O-p-methoxylbenzyl (PMB)
moiety on a glycosyl donor tends to participate during
glycosylation, forming 1,6-anhydro glycan,49 the 6-O-PMB
group on disaccharide 6 was replaced with levuniloyl (Lev),
producing the key building block 1. Direct glycosylation of 6-O-
Lev-containing idoside acceptor by donor 4 failed to give
disaccharide 1 in high yield, presumably because Lev was more
electron-withdrawing than PMB, leading to lower nucleophilicity
of the 6-O-Lev-containing acceptor. Removal of the tert-
butyldimethylsilyl (TBS) moiety from 1 generated disaccharide
acceptor 2 in 98% yield (Scheme 1a). Glycosylation of alcohol
7
17 by 1 with subsequent TBS removal produced disaccharide 3
in 77% overall yield (Scheme 1b).
In order to overcome the difficulty in sulfation, we explored
the alternative of sulfating carboxylic ester containing sub-
strate.29,36 Methyl ester 14 was prepared, and its acyl protective
groups were removed under trans-esterification conditions
With disaccharide building blocks 1−3 in hand, glycosylation
was performed to elongate the chain length (Scheme 2).
Glycosylation of acceptor 2 by disaccharide donor 1 produced
B
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Scheme 2. Synthesis of Heparan Sulfate Backbones 10 and 11
Scheme 3
Scheme 4
with sodium methoxide, producing hexa-ol 15 in 91% yield
(Scheme 4). However, Staudinger reduction of the azides in 15
led to several products due to backbone cleavage. This is
consistent with the previous observation that HS backbones
bearing uronic esters were not stable under the Staudinger re-
duction conditions.25 Instead, the azides in 15 were transformed
to amines with 1,3-propanedithiol29 in 91% yield and the
resulting amino alcohol was sulfated with SO3·pyridine. The
subsequent removal of TBS turned out to be very chal-
lenging. HF·pyridine only partially cleaved TBS, while leading
to the loss of sulfate groups from the molecule. Addition
of pyridine to reduce the acidity of the reaction or perform-
ing the reaction at lower temperatures did not improve the
result. The use of other fluoride sources such as NaF or
C
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Scheme 5
tetrabutylammonium fluoride (TBAF) was unsuccessful with
the recovery of starting terial.
fully deprotected HS hexasaccharide 20. Alternatively, the
amines were acylated with acetic anhydride after sulfation of
19. Subsequent hydrogenolysis and ester cleavage generated the
acetamide-containing hexasaccharide 21. In order to prepare the
N-sulfated sequence, 19 was treated with 600 mM of SO3·
pyridine, which was followed by catalytic hydrogenolysis
and mild base hydrolysis, producing the N- and O-sulfated
hexasaccharide 22 with an overall yield of 63% from 19
(Scheme 5d).
The dichotomy in the outcome of sulfation prompted further
investigation. It should be pointed out that HS hexasaccharides
or longer bearing free uronic acids have been successfully
sulfated.12−20 However, in those cases, at most, two sulfates
were installed per disaccharide unit, while, for 13, three sulfates
would need to be introduced on each disaccharide. To better
understand the structural requirements for chemical sulfation,
the free uronic acid bearing tetrasaccharide 23 was synthesized
from deprotection of tetrasaccharide 11, followed by sulfation
(Scheme 6a). The successful generation of 23 combined with
prior observations12−20 suggests that, for extensive sulfations
(i.e., introduction of 3 sulfates including N- and O-sulfates per
disaccharide) of HS oligosaccharides longer than tetrasac-
charides, it is crucial that the uronic acids are protected as
carboxylic esters.29,36
The difficulty in TBS deprotection from the highly sulfated
hexasaccharide suggested that this transformation should be
carried out earlier. Treatment of hexasaccharide 14 with
HF·pyridine in pyridine removed the TBS group smoothly in
94% yield (Scheme 5a). The hydroxyl group freed needed to be
protected to avoid its sulfation. To differentiate this group from
the hydroxyl groups to be sulfated, benzyl ether was used as
the protective group. Benzylation of 17 under acidic conditions
with benzyl 2,2,2-trichloroacetimidate52 was tested first. Despite
multiple trials with several acid catalysts and reaction solvents, no
desired benzylated hexasaccharide 18 was obtained. Next, the
basic benzylation conditions with benzyl bromide and a host of
bases were screened. Strong bases such as NaH and NaHMDS53
led to multiple products due to acyl migration. Finally, the
hydroxyl group was successfully masked as a benzyl ether using
benzyl bromide promoted by freshly prepared silver oxide,
leading to hexasaccharide 18 in 50% yield (62% based on
recovered starting material) (Scheme 5a).
With the newly benzylated backbone 18, deprotection and
sulfation were performed. The acyl protective groups in 18 were
removed with sodium methoxide, which was followed by azide
reduction with 1,3-propanedithiol (Scheme 5b). The resulting
hexasaccharide 19 was subjected to sulfation. Interestingly,
by controlling the amount of the sulfation agents, divergent
sulfation could be achieved. Treatment of 19 with SO3·pyridine
(120 mM, 24 equiv) at 55 °C for 24 h installed six O-sulfates
without any N-sulfates (Scheme 5c). This was presumably
due to the protonation of the free amines under the slightly
acidic reaction conditions, reducing their nucleophilicities.
The O-sulfated hexasaccharide was hydrogenated to remove all
benzyl ethers, and the methyl esters were cleaved, producing the
Enzymatic Sulfation. To increase the sequence diversity that
can be generated, hexasaccharide 24 bearing only N-sulfation was
prepared from hexasaccharide 18 (Scheme 6b). Enzymatic
sulfations of hexasaccharide 24 were explored with two main
O-sulfotransferases, namely, 2-OST and 6-OST. The enzymatic
sulfation reactions were found to be sensitive to substrate
concentration. Trials on backbone 24 using a combination
of 6-OST-1 and 6-OST-3 in the presence of 3′-phosphoadeno-
sine 5′-phosphosulfate (PAPS) as the sulfate donor proceeded
D
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Scheme 6
Scheme 7
with very low yields when performed at a substrate concentration
of 100 μg/mL.54 Diluting the reaction to a concentration of
50 μg/mL while maintaining the concentrations of enzymes,
PAPS and buffer capacity enabled successful reaction (67%
isolated yield), producing hexasaccharide 25 bearing three
O-sulfates, indicating full 6-O sulfation (Scheme 7). Performing
the reaction at a lower concentration presumably overcame
product inhibition of enzymatic activities. Interestingly, when the
reaction was stopped prior to completion, only hexasaccharides
24 and 25 were observed with no partially O-sulfated hexa-
saccharides in the reaction mixture based on mass spectrometry
analysis. This is possibly because, once one O-sulfate was added,
the subsequent sulfation reactions proceeded more readily.
To install the 2-O sulfates, 24 was treated with 2-OST and
PAPS, which led to hexasaccharide 26 containing two additional
sulfates in 82% yield. NMR analysis was performed on 26.
Compared to 24, the chemical shifts of H1 and H2 of the
reducing end iduronic acid of 26 were changed little with H1
appearing at 4.78 ppm (vs 4.83 ppm in 24) and H2 at 3.58 ppm
(3.59 ppm for 24). The chemical shifts of the other two iduronic
acid residues were significantly altered. Chemical shifts of H-1s
were shifted from 4.90 ppm in 24 to 5.16 ppm in 26 presumably
due to the installation of electron-withdrawing O-sulfates. H 2s
were more deshielded and both moved downfield from 3.64 to
4.22 ppm. On the basis of these observations, the structure of
hexasaccharide 26 was assigned to contain 2-O sulfates on the
nonreducing end and internal disaccharides. The fact that the
reducing end iduronic acid is not modified in 26 suggests that
2-OST requires additional glycans at the reducing end of the
iduronic acid to be sulfated.54
Further elaborations of 25 and 26 were carried out. Treatment
of the 6-O sulfate bearing hexasaccharide 25 with 2-OST only
furnished the starting material, indicating that it is a poor
substrate for 2-OST. When the 2-OST product 26 was treated
with 6-OST and PAPS, the resulting hexasaccharide product 26
was found to contain two additional sulfates. Through NMR
E
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Figure 1. (a) A representative image of HS microarray upon incubation with FGF-2, which was detected by an anti-FGF2 IgG antibody, followed by a
FITC labeled secondary antibody. Columns 1, 2, 3, and 4 represented spots printed with 400, 80, 16, and 3.2 nM of glycans, respectively.
(b) Quantification of the fluorescence signals from the microarray. Additional images are presented in the Supporting Information.
analysis, the H-6 protons of the glucosamine in the nonreducing
and internal disaccharides of the product 27 were found to
appear at 4.15 and 4.30 ppm, respectively. In contrast, those
belonging to the reducing end disaccharide had chemical shifts of
3.60 and 3.72 ppm. Therefore, the 6-O sulfates of 27 were
determined to be located on the nonreducing and internal
disaccharide units. The 2-O sulfations in 26 directed the 6-OST
to selectively modify the disaccharides already carrying 2-O-
sulfation. The results from these enzymatic sulfations suggest
that, in biosynthesis of naturally existing HS, 2-O sulfation most
likely precedes the installation of 6-O sulfates on the same
disaccharide unit. This is consistent with the observations from
enzymatic modification of HS polysaccharides.55
Hexasaccharides 24−27 contain three consecutive iduronic
acid bearing disaccharides. This type of backbone structure is
inaccessible through the current enzymatic synthesis strategy.
At the same time, it should be pointed out that the disaccharide
units in hexasaccharides 26 and 27 are not uniformly sulfated.
To prepare these compounds via a pure chemical approach, a
new disaccharide module with the potential O-sulfation sites
blocked by protective groups different from those in disaccharide
1 must be prepared, which would increase the total number of
synthetic steps. Thus, the combination of chemical synthesis
with enzymatic modification improves the overall synthetic
efficiencies.
Higher Sulfation and Longer Backbone Enhance HS
Binding with FGF-2. In order to probe the effects of length and
sulfation pattern of synthetic HS oligosaccharides on their
biological properties, their bindings with FGF-2 were inves-
tigated. FGF-2 is an important protein involved in angiogenesis,
cell proliferation, and tumor development.56,57 Through direct
FGF-2 binding, HS and its highly sulfated form heparin are
known to play a central role in regulating FGF-2 activities.58
To increase the speed of analysis of FGF-2 binding, the glycan
microarray technology was utilized,59−62 which is a powerful
technique for analyzing carbohydrate−protein interactions
including HS studies.19,63,64 As all synthetic HS glycans bear
amino moieties at their respective reducing ends, the glycans
were printed onto an N-hydroxysuccinimide (NHS) ester
functionalized glass slide to covalently immobilize the glycans
through amide bonds. Furthermore, serial dilutions of each
HS oligosaccharide (from 400 to 3.2 nM) were printed onto
the microarray for semiquantitative analysis of the affinity.
The unreacted NHS esters were quenched with ethanolamine.
HS disaccharide 28, heparin, and chondroitin sulfate A (CS-A)
polysaccharides were also added to the slides at the same
concentrations.
The HS slides were incubated with a solution of FGF-2,
followed by washing to remove the unbound protein. The slides
were subsequently treated with a fluorescein isothiocyanate
(FITC) labeled anti-FGF-2 antibody. The binding of FGF-2 with
an HS oligosaccharide would enable the immobilization of the
anti FGF-2 antibody on the array and allow its detection by
fluorescence. Slides from multiple sources were examined, and
those from Xantec Bioanalytics were found to give the highest
signal-to-noise ratio and most reproducible results in our hands.
As shown in Figure 1, the spots with immobilized heparin
polysaccharide exhibited intense signals, indicating strong
binding between heparin and FGF-2. In contrast, despite the
presence of multiple sulfates, CS-A polysaccharide gave little
signals, suggesting that the nonspecific electrostatic interactions
between a cluster of negative charges on the microarray surface
and FGF-2 most likely do not play important roles in FGF-2
binding to array components. Similar phenomena have been
observed previously.62
Comparisons of the fluorescence signals from the oligosac-
charides on the array revealed HS structural impact on binding.
Disaccharide 28, tetrasaccharide 23, and hexasaccharide 22 all
bear full N-, 2-O, and 6-O sulfations. Whereas disaccharide 28 did
not bind much with FGF-2, tetrasaccharide 23 and hexasa-
ccharide 22 exhibited strong binding, with the signal intensities
of 22 approaching those of heparin polysaccharides (Figure 1b).
This suggested that tetrasaccharide is the minimum length for
strong binding in this assay.65 Previously, an HS disaccharide
similar to 28 was shown to bind with FGF-2,19 which was most
likely due to the higher glycan concentrations (16 μM to 2 mM)
utilized in that study.
The number of sulfates and backbone sequence are impor-
tant factors in FGF-2 binding. The seven HS hexasaccharides
(20−22, 24−27) contain small variations in the number of
sulfates. However, based on the array signals, 22 exhibited
the strongest binding to FGF-2, suggesting that full 2-O, 6-O, and
F
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(about 5 min at −78 °C), a solution of acceptor (54 μmol) in CH2Cl2
(1 mL) along with 1 equiv of TTBP was slowly added dropwise along
the walls of the flask. This was done to allow the acceptor solution to
cool before mixing with the activated donor. The reaction mixture was
warmed to 0 °C under stirring in around 2 h. The mixture was diluted
with CH2Cl2 and filtered through Celite. After washing the Celite with
CH2Cl2 until all organic compounds were removed, as verified by TLC,
the CH2Cl2 fractions were combined and washed twice with a saturated
aqueous solution of NaHCO3 (20 mL), and twice with water (10 mL).
The organic layer was collected and dried over Na2SO4. After removal of
the solvent, the product was purified by silica gel flash chromatography
unless noted.
General Procedure for TBS Removal. The TBS-containing
oligosaccharide (0.54 mmol) was transferred to a 50 mL plastic
centrifuge tube by three portions of 3.33 mL of pyridine. While stirring,
the pyridine solution was cooled to 0 °C. Then, 5 mL of HF·pyridine was
added dropwise to the stirring solution. The reaction was then allowed
to warm to room temperature and kept overnight. After verifying that
the reaction was complete by TLC, the reaction was diluted with
CH2Cl2 and washed sequentially with sat. CuSO4, sat. NaHCO3, and
10% HCl. The organic layer was dried over Na2SO4, concentrated, and
purified by silica gel flash chromatography.
General Procedure for Benzylation. The oligosaccharide to be
protected (15 μmol) was dissolved in 5 mL of CH2Cl2. To this solution
were added TBAI (1 equiv), benzyl bromide (40 equiv), and freshly
prepared Ag2O (20 equiv). The reaction was stirred at room tem-
perature until TLC indicated that the reaction was no longer progressing
(30 min). The reaction was quenched by diluting with CH2Cl2 and
filtering through Celite to remove Ag2O. The reaction was concentrated
and purified by silica gel chromatography.
General Procedure for Levulinoyl Ester Formation. A mixture
of the oligosaccharide (1 mmol), 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide hydrochloride (EDC.HCl 3.3 equiv per OH), and
N,N-dimethylaminopyridine (DMAP, 0.1 equiv per OH) was dissolved
in dichloromethane (DCM, 30 mL). To this solution was added
levulinic acid (3 equiv per OH), and the reaction was stirred at room
temperature overnight. The mixture was then diluted with DCM,
washed with sat. NaHCO3, dried over Na2SO4, concentrated, and
purified by flash silica gel chromatography.
General Procedure for Removal of Levulinoyl Esters. A
solution of the oligosaccharide containing Lev esters (56 μmol) in
2.4 mL of pyridine and 1.6 mL of glacial acetic acid was cooled to 0 °C.
To this was added 27 μL of hydrazine hydrate (560 μmol or 5 equiv per
Lev ester). The reaction was stirred at 0 °C for 3 h or until TLC shows
that the reaction is complete. To quench the reaction, 1 mL of acetone
was added and the reaction was stirred at room temperature for 30 min.
The reaction mixture was then diluted with ethyl acetate and washed
with 25 mL each of the following solutions: sat. NaHCO3, 10% HCl,
H2O, and brine. The resulting organic layer was then dried over Na2SO4,
concentrated, and purified by silica gel flash chromatography.
General Procedure for Oxidation of 6-OH. The desired
compound to be oxidized (45 μmol) was dissolved in a solution of
2 mL of DCM, 2 mL of tBuOH, and 0.5 mL of H2O. To this solution was
added TEMPO (26.5 μmol or 0.3 equiv per 6-OH), followed by BAIB
(221 μmol or 2.5 equiv per 6-OH). The reaction was then stirred at
room temperature overnight. After ensuring that the reaction was
complete by TLC (1% acetic acid in ethyl acetate), the reaction was
quenched by addition of 2 mL of Na2S2O3 solution and allowed to stir at
room temperature for 15 min. The mixture was then diluted with 10 mL
of DCM and 3 mL of H2O and separated. The aqueous layer was
acidified with 1 M HCl solution and extracted three times with DCM.
The organic layers were combined, dried over Na2SO4, and concen-
trated. The crude product could then be protected as a benzyl or methyl
ester.
N-sulfations are important with the lack of any sulfation, leading
to significant reduction in binding. It is interesting that, although
27 contains the full structure of tetrasaccharide 23, its binding
with FGF-2 was much weaker. This indicates that the reducing
end disaccharide without any O-sulfations in 27 was detrimental
to binding. The knowledge gained through the microarray
studies can be helpful to guide future design of HS oligo-
saccharide based probes to modulate FGF-2 activities.
CONCLUSIONS
■
We report a divergent methodology allowing the access to seven
HS hexasaccharides from a single common hexasaccharide
precursor. An efficient chemical glycosylation strategy was
developed to prepare the HS tetra- and hexasaccharide
backbones. Difficulties were encountered in chemical sulfation
and deprotection of the hexasaccharide. The substrate struc-
ture and concentration of the sulfation agent were found to be
crucial for successful sulfations. To enhance sequence diversity,
chemically synthesized HS hexasaccharide backbones were
enzymatically sulfated. Besides synthetic utilities, the well-
defined oligosaccharide structures helped shine light on the
fine substrate specificities of the 6-OST and 2-OST and confirm
the potential sequence of enzymatic reactions in HS biosynthesis.
The synthetic HS oligosaccharides were then immobilized onto
an HS oligosaccharide microarray, which was used to decipher
the impacts of HS structures on FGF-2 binding. Both high
sulfation and longer sequences were found to enhance the affinity
with FGF-2. Further studies are ongoing to expand this divergent
strategy in order to access a wide range of HS structures.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reactions were performed
under a nitrogen atmosphere with anhydrous solvents. Solvents were
dried using a solvent purification system. Glycosylation reactions were
performed with 4 Å molecular sieves that were flamed-dried under high
vacuum. Chemicals used were reagent grade unless noted. Reactions
were visualized by UV light (254 nm) and by staining with either
Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24·4H2O (24.0 g) in 6%
H2SO4 (500 mL), 5% H2SO4 in EtOH, or, for deprotected
oligosaccharides, 0.2 g of 1,3-dihyroxynaphthalene in 50 mL of 5%
H2SO4 in EtOH. Flash chromatography was performed on silica gel 601
(230−400 Mesh). NMR spectra were referenced using residual CHCl3
(δ H NMR 7.26 PPM 13C NMR 77.0 PPM). Peak and coupling
1
constants assignments are based on 1H NMR, 1H−1H gCOSY, 1H and
1H−1H TOCSY, H−1H NOESY, H−13C gHMQC/1H−13C HSQC,
and 1H−13C gHMBC. For NMR assignments, the glycosyl units in an
oligosaccharide were designated as A, B, C, D, E, and F sequentially
where necessary from the reducing end to the nonreducing end.
Characterization of Anomeric Stereochemistry. The stereo-
chemistries of newly formed glycosidic bonds for idose and glucosamine
1
1
3
1
1
were determined by JH1,H2 through H NMR and/or JC1,H1 through
1
3
gHMQC 2-D NMR (without H decoupling). Smaller JH1,H2 (3 Hz)
indicate α linkages, and larger 3JH1,H2 (7 Hz or larger) indicate β linkages.
1JC1,H1 coupling constants around 170 Hz suggest α linkages, whereas
values around 160 Hz imply β linkages.48
General Procedure for Preactivation Based Glycosylation. A
solution of donor (60 μmol) and freshly activated 4 Å molecular sieves
(200 mg) in CH2Cl2 was stirred at room temperature for 30 min and
then cooled to −78 °C. AgOTf (31 mg, 120 μmol) dissolved in Et2O was
added directly to the solution making sure the solution did not touch
the walls of the flask. After 5 min, orange-colored p-TolSCl (9.5 μL,
60 μmol) was added via a microsyringe directly to the flask, as the
reaction temperature was lower than the freezing point of p-TolSCl and
it would freeze on the walls of the flask. The color of p-TolSCl
disappeared rapidly, indicating the consumption of p-TolSCl.
After the donor was completely consumed, as verified by TLC analysis
General Procedure for Benzyl Ester Formation after
Oxidation. The crude product from oxidation was dissolved in 5 mL
of DCM. To this was added phenyl diazomethane until a deep red color
persisted.66 The reaction was allowed to stir overnight. After TLC
indicated that the reaction was complete, the mixture was concentrated
and purified by column chromatography.
G
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General Procedure for Methyl Ester Formation after
Oxidation. The crude product from oxidation was dissolved in DMF
(2 mL for 15 μmol). To this solution was added K2CO3 (5 equiv per
COOH), followed by CH3I (2.5 equiv per COOH), and the reaction
was allowed to stir overnight at room temperature. After verifying that
the reaction was complete by TLC, the reaction was diluted with ethyl
acetate and water. The mixture was then washed with 0.1 M HCl,
followed by sat. NaHCO3, dried over Na2SO4, concentrated, and
purified by flash silica gel chromatography.
General Procedure for Saponification. The mixture of
compound (for 100 mg of compound, 1 equiv), THF (2.5 mL), and
1 M LiOH (13 equiv per COOBn) was cooled to 0 °C, followed by
addition of H2O2 (150 equiv per COOBn, 30%). The mixture was
stirred at room temperature for 16 h, and then methanol (6 mL) and
3 M potassium hydroxide (80 equiv per COOBn) were added to the
solution. The mixture was stirred for another 24 h, then acidified with
10% HCl, and concentrated to dryness. The resulting residue was
purified by quickly passing through a short silica gel column (4:1,
DCM:MeOH).
General Procedure for Transesterification. The methyl ester
containing oligosaccharide (10 μmol) was dissolved in 2 mL of dry
DCM and 2 mL of anhydrous methanol. The two solvents were dried
over 4 Å molecular sieves for 24 h. A sodium methoxide solution was
made by adding sodium to a portion of anhydrous methanol. This fresh
sodium methoxide solution was added to the oligosaccharide solution
until the pH reached 12. The reaction was maintained at pH = 12 and
stirred at room temperature for 2 h. After the reaction was confirmed
complete by TLC, it was quenched to pH = 7 by a 1 M acetic acid
solution in dry methanol. The quenched reaction was concentrated and
purified by silica gel chromatography.
General Procedure for Staudinger Reduction. 1 M PMe3
solution in THF (5 equiv per N3), 0.1 M aqueous solution of NaOH
(3 equiv per N3), and H2O (2 mL) were added consecutively to a
solution of azide-containing compound (for 50 mg of compound,
1 equiv) in THF (7 mL). The mixture was stirred at room temperature
overnight and neutralized with 0.1 M HCl until pH = 7. The mixture was
concentrated to dryness, and the resulting residue was purified with
Sephadex LH-20 (50/50 DCM/MeOH).
General Procedure for 1,3-Dithiopropane Mediated Azide
Reduction. The starting oligosaccharide was dissolved in anhydrous
MeOH (dried over 4 Å molecular sieves) and protected from light.
To this solution were added triethylamine (6.7 equiv per N3) and
1,3-dithiopropane (6.7 equiv per N3), and the reaction was stirred at
room temperature for 24 h. After 1 day, additional portions of
triethylamine and 1,3-dithiopropane (6.7 equiv per N3 of each) were
added, and the reaction was stirred for another 72 h. The reaction was
diluted with a 1:1 mixture of DCM:MeOH and was layered onto a
Sephadex LH-20 column and eluted with 1:1 DCM:MeOH.
General Procedure for O-Sulfation. The mixture of OH-
containing compound (for 5 mg of compound, 1 equiv), DMF (1 mL
dried over 4 Å molecular sieves), and SO3·NEt3 (5 equiv per OH) was
stirred at 55 °C for 24 h. The mixture was quenched by adding
NEt3 (0.2 mL) and then diluted with DCM/MeOH (1 mL: 1 mL).
The resulting solution was layered on the top of a Sephadex LH-20
chromatography column that was eluted with DCM/MeOH (1:1).
General Procedure for N-Sulfation. A mixture of NH2-containing
compound (for 5 mg of compound, 1 equiv), pyridine (1 mL dried over
4 Å molecular sieves), Et3N (0.2 mL), and SO3·pyridine (5 equiv per
NH2) was stirred at room temperature for 3 h. The mixture was diluted
with DCM/MeOH (1 mL/1 mL), and the resulting solution was layered
on the top of a Sephadex LH-20 chromatography column that was
eluted with DCM/MeOH (1/1).
exclusion chromatography (G-15) and, for final compounds, then eluted
from a column of Dowex 50WX4-Na+ to convert the compound into the
sodium salt form.
General Procedure for Selective O-Sulfation. A compound
(8 mg or 4 μmol) containing both free OH and NH2 groups was
dissolved in 1 mL of dry pyridine (dried over 4 Å molecular sieves).
To this mixture was added 20 mg of SO3·pyridine (120 mM). The
sulfating agent had been previously washed with H2O, MeOH, and
DCM and dried under vacuum. The reaction was protected from light
and stirred for 24 h at 55 °C. The reaction was diluted with 1:1
DCM:MeOH and eluted from a Sephadex LH-20 column, ensuring that
all pyridine was removed. The fractions containing sugar were con-
centrated and further purified by prep TLC (3:1:1 EtOAC:MeOH:H2O
1% AcOH).
General Procedure for Simultaneous O,N-Sulfation. A
compound (8 mg or 4 μmol) containing both free OH and NH2
groups was dissolved in 1 mL of dry pyridine (dried over 4 Å molecular
sieves). To this mixture was added 100 mg of SO3·pyridine (600 mM).
The sulfating agent had been previously washed with H2O, MeOH, and
DCM and dried under vacuum. The reaction was protected from light
and stirred for 24 h at 55 °C. The reaction was diluted with 1:1
DCM:MeOH and eluted from a Sephadex LH-20 column, ensuring that
all pyridine was removed. The fractions containing sugar were con-
centrated and further purified by prep TLC (3:1:1 EtOAC:MeOH:H2O
1% AcOH).
General Procedure for Methyl Ester Saponification. The
compound to be saponified was dissolved in H2O (1 mL for 5 mg), and
1 M LiOH (15 equiv per ester) was added. The mixture was cooled to
0 °C. This was followed by addition of H2O2 (150 equiv per ester, 30%),
and the reaction was allowed to warm to room temperature and stir
overnight. The reaction was neutralized with 1 M AcOH and eluted
from a Sephadex G-15 column with H2O. To simplify mass
spectrometry analysis, the product was then eluted from a column of
Dowex 50WX4-Na+ to convert the compound into the sodium salt form.
General Procedure for Enzymatic Sulfation. The oligosacchar-
ide to be sulfated (500 μg or 0.4 μmol) was mixed with 1 mg of the
needed enzyme(s) in 12.5 mL of solution. This solution had a
concentration of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES)
and 0.05 mg/mL of PAPS. This reaction was stirred at 37 °C overnight.
Another 1 mg of the needed enzyme(s) was added, and the reaction was
diluted to 25 mL, keeping the concentration of MES at 20 mM and
PAPS at 0.05 mg/mL, respectively. After another 24 h at 37 °C, the
reaction was stopped. It was concentrated by utilizing a Q-Sepharose
Fast Flow column. The mixture was passed through the column, which
was then washed with 20 mL of 25 mM NaOAc. The product was then
eluted from the column with a solution of 1 M NaCl and 25 mM
NaOAc. The product eluted within the first 2 mL, and the column was
further washed with 10 mL of the elution solution and 25 mL of the
25 mM NaOAc solution. The fractions containing sugar were
lyophilized and loaded onto a P-2 column (2m × 0.75 cm diameter)
with 1 mL of 0.1 M NH4HCO3. Additional NH4HCO3 was added until
the loading solution was neutralized. An indicator, Phenol red (5 μL),
was added to monitor the column, and the product was eluted with
0.1 M NH4HCO3. Tubes containing product were lyophilized at least
3 times to remove any residual NH4HCO3 to allow for mass
spectrometry analysis.
General Procedure for Microarray Preparation. All solutions
were prepared with nanopure water. Recombinant human basic
Fibroblast Growth Factor (FGF-2) and rabbit antihuman FGF-2 were
purchased from PeproTech (Rocky Hill, NJ), and Cy5 conjugated goat
ant-rabbit IgG (H+L) was purchased from Life Technologies (Grand
Island, NY). NHS coated slides (SL HCX) were purchased from Xantec
Bioanalytics GmbH (Germany). Microarrays were produced using a
PixSys 5500 robotic printer (Cartesian Technologies Inc., California).
Oligosaccharides were dissolved in 50 mM sodium phosphate buffer
(pH = 9) and mechanically printed onto the NHS coated slides at
50% relative humidity and room temperature. After printing, slides
were incubated at 75% humidity and room temperature overnight.
Oligosaccharides were printed in four concentrations (400, 80, 16, and
3.2 nM), and each spot was replicated four times. Two natural sources
General Procedure for Global Debenzylation. A mixture of the
Bn-containing compound (for 6 mg of compound, 1 equiv), MeOH/
H2O (4 mL/2 mL), and Pd(OH)2 on carbon (100 mg) was stirred
under H2 at room temperature overnight and then filtered. The filtrate
was concentrated to dryness under vacuum and then diluted with
H2O (15 mL). The aqueous phase was further washed with CH2Cl2
(3 × 5 mL) and EtOAc (3 × 5 mL), and then the aqueous phase was
dried under vacuum. The crude product was further purified by size
H
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were printed alongside the synthesized oligosaccharides. Heparin (HP,
sodium salt, average molecular weight 18 kDa, 177 USP unit/mg) and
Chondroitin sulfate A (sodium salt from bovine trachea average
molecular weight 50 kDa) were both purchased from Sigma-Aldrich and
printed in the same concentration as synthesized oligosaccharides using
their average molecular weights. Slides were washed three times with
water. To quench unreacted NHS groups, the slides were then incubated
in a preheated 50 °C solution of 100 nM ethanolamine in sodium
phosphate buffer (50 mM, pH = 9) for 1 h. After quenching, the slides
were washed three times with water, dried by centrifugation (2000 rpm
for 2 min), and stored in a desiccator at −5 °C until use. For all protein
incubations, Lifterslips from Thermo Scientific were used in concert
with 20 μL of solution. Analysis of slides was done on an Agilent
G2565AA Array Scanner.
General Procedure for Microarray Binding Assay. Slides to be
used were warmed to room temperature before removing from the
desiccator. Protein solutions were prepared by diluting stock solutions
to concentrations of 8 μg/mL with PBS buffer (10 mM pH = 7.5)
containing 1% BSA. An assay was run as follows. Slides were incubated
with 20 μL of FGF-2 solution (placed between Lifterslip and slide) and
incubated in a microarray cassette at room temperature protected from
light for 1 h. After 1 h, the slide was washed once with a solution of PBS
(10 mM pH = 7.5) with 1% Tween-20 and 0.1% BSA and twice with
water. The slide was dried by centrifugation, then incubated with 20 μL
of rabbit anti-Human FGF-2 for 1 h as done previously. The slide was
then washed in the same way and finally incubated with 20 μL of the
secondary antibody Cy5 goat antirabbit IgG for 1 h and washed. After
drying by centrifugation, the slide was imaged on an Agilent G2565AA
Array Scanner. The intensities of the bands were quantified using ImageJ
software.
3.32−3.23 (2 H, m, H-2B, H-3B), 2.79−2.67 (2 H, m, CH2 Lev), 2.64−
2.53 (2 H, m, CH2 Lev), 2.33 (3 H, s, S-Ph-CH3), 2.16 (3 H, s), 2.04
(3 H, s), 0.86 (9 H, s, (CH3)3CSi), −0.03 (3 H, s, CH3Si), −0.14 (3 H, s,
CH3Si).13C NMR δC (125 MHz, CDCl3) 206.2, 172.2, 170.6, 165.6,
137.8, 137.6, 137.2, 133.2, 132.0, 131.9, 130.0, 129.8, 129.7, 128.5, 128.4,
128.3, 128.1, 128.0, 127.2, 126.9, 99.2, 86.3, 80.6, 75.9, 74.4, 72.6, 71.6,
71.2, 71.0, 69.7, 66.2, 64.6, 63.8, 63.0, 60.3, 37.8, 29.8, 27.8, 25.9, 21.1,
21.0, 20.8, 18.0, 14.2, −3.8, −4.9. HRMS [M + H]+ C53H66N3O13SSi+
calcd. 1012.4080, obsd. 1012.4083.
p-Tolyl 6-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-1-thio-α-L-
idopyranoside (2). Compound 2 (0.300 g, 0.33 mmol) was prepared
from 1 by following the general procedure for TBS removal and
subsequent purification by silica gel chromatography (2:1 hexanes:E-
tOAc then 1:1 hexanes:EtOAc), providing 0.260 g of 2 in 98% yield.
1H NMR δH (500 MHz, CDCl3) 8.20−8.17 (2 H, m), 7.53−7.25 (13 H,
m), 7.19 (2 H, dd, J 9.1, 2.6), 7.15 (2 H, d, J 8.1), 5.60 (1 H, s, H-1A),
5.41 (1 H, s, H-2A), 5.02−4.97 (2 H, m, Bn, H-5), 4.78 (1 H, d, J 11.7,
Bn), 4.60 (1 H, d, J 3.8, H-1B), 4.50 (1 H, dd, J 12.4, 4.4, H-6B), 4.42
(1 H, dd, J 11.6, 8.0, H-6A), 4.37 (1 H, d, J 10.6, Bn), 4.34 (1 H, dd,
J 11.6, 4.4, H-6A), 4.22 (1 H, dt, J 6.0, 3.0, H-6B), 4.19 (1 H, t, J 2.4,
H-3A), 4.07 (1 H, d, J 10.6, Bn), 3.86 (1 H, ddd, J 10.0, 4.2, 2.0, H-5B),
3.67 (1 H, t, J 2.3, H-4A), 3.47 (1 H, dd, J 10.1, 9.0, H-3B), 3.35 (1 H, t, J
9.5, H-4B), 3.24 (1 H, dd, J 10.1, 3.8, H-2B), 3.02 (1H, br, OH), 2.75
(2 H, t, J 6.6, CH2 Lev), 2.60 (2 H, t, J 6.6, CH2 Lev), 2.36 (3 H, s, S-Ph-
CH3), 2.17 (3 H, s, CH3, Lev), 2.08 (3 H, s, Ac).13C NMR δC (125 MHz,
CDCl3) 206.7, 172.5, 171.8, 165.7, 137.8, 137.7, 137.2, 133.3, 132.2,
131.8, 129.94, 129.86, 129.7, 128.6, 128.48, 128.46, 128.33, 128.26,
128.12, 128.10, 127.9, 98.9, 86.4, 80.1, 75.3, 75.1, 72.6, 71.3, 71.2, 70.5,
69.5, 66.1, 63.8, 63.3, 63.0, 37.9, 29.8, 27.8, 21.2, 20.8, 14.2. HRMS
[M + Na]+ C47H51N3NaO13S+ calcd. 920.3040, obsd. 920.3034.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-
3-O-benzyl-6-levulinoyl-α-L-idopyranoside (3). Compound 3 was
prepared from 8 (549 mg, 0.46 mmol) using the general proce-
dure for TBS removal, followed by silica gel chromatography (1:1
hexanes:EtOAc) to yield 95% of compound 3 (470 mg). 1H NMR δH
(500 MHz, CDCl3) 8.17 (2 H, d, J 7.1), 7.51 (1 H, t, J 7.3), 7.45 (2 H, t,
J 7.5), 7.41−7.14 (20 H, m), 5.22−5.12 (3 H, m, H-2A, CH2-CBz), 4.96
(1 H, d, H-1A), 4.85 (1 H, t, J 10.6, Bn), 4.77−4.72 (1 H, m, Bn), 4.71
(1 H, d, J 3.5, H-1B), 4.56−4.45 (4 H, m, H-6A, H-6B, 2Bn), 4.44−
4,37(2 H, d, J 9.5, H-5A, Bn), 4.32−4.22 (3 H, m, H-6B, H-6A, Bn), 4.10
(1 H, s, J 10.4, H-3A), 3.86 (1 H, s, H-5B), 3.79 (1 H, s, H-linker), 3.71
(1 H, s, H-4A), 3.59 (1 H, t, J 9.5, H-3B), 3.56−3.32 (4 H, m, H-4B,
H-linker, CH2-linker), 3.24 (1 H, dd, J 10.1, 3.5, H-2B), 2.73 (2 H, s,
CH2 Lev), 2.58 (2 H, d, J 13.9, CH2 Lev), 2.17 (3 H, s, CH3 Lev), 2.07
(3 H, s, Ac), 1.89 (2 H, d, J 23.2, CH2-linker). 13C NMR δC (125 MHz,
CDCl3) 206.6, 172.4, 171.7, 165.6, 156.7, 156.1, 137.9, 137.8, 137.6,
136.7, 133.3, 129.9, 129.8, 128.61, 128.56, 128.53, 128.45, 128.3, 128.2,
128.1, 128.0, 127.92, 127.86, 127.78, 127.5, 127.3, 98.4, 98.3, 80.0, 75.1,
74.5, 72.4, 72.3, 71.2, 70.7, 68.8, 67.2, 65.6, 63.6, 63.2, 63.0, 60.4, 51.0,
p-Tolyl 6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethyl-
silyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-
levulinoyl-1-thio-α-L-idopyranoside (1). Compound 1 was synthesized
from 6 in two steps. First, 6 (1.982 g, 1.89 mmol) was dissolved in
144 mL of DCM and 16 mL of water and cooled to 0 °C. To this
solution was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ,
0.645 g, 2.84 mmol), and the reaction as allowed to warm to room
temperature and stir overnight. The reaction was then diluted with
DCM and washed with sat. NaHCO3 and water until the wash was
colorless. After concentration and purification by silica gel chromatog-
raphy (4:1:1 hexanes:DCM:ethyl acetate (EtOAC)), 1.695 g of p-tolyl
6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-1-thio-α-L-idopyrano-
1
side was isolated in 98% yield. H NMR δH (500 MHz, CDCl3) 8.07
(2 H, dd, J 7.1, 1.3), 7.45−7.12 (14 H, m), 7.06 (2 H, d, J 7.9), 7.00 (2 H,
d, J 7.5), 5.52 (1 H, s, H-1A), 5.31 (1 H, d, J 1.3, H-2A), 4.92 (1 H, d,
J 11.8, Bn), 4.78 (1 H, t, J 6.8, H-5A), 4.69 (1 H, d, J 11.8, Bn), 4.48 (1 H,
d, J 1.8, H-1B), 4.33 (1 H, dd, J 11.8, 1.7), 4.10 (1 H, s, H-3A), 4.08−3.99
(2 H, m), 3.91 (1 H, dd, J 11.8, 6.6), 3.88−3.78 (2 H, m, H-6A, H-4B),
3.74 (2 H, d, J 11.2, H-6A), 3.62 (1 H, s, H-4A), 3.39−3.31 (1 H, m,
H-3B), 3.19 (2 H, d, J 4.8, H-2B), 2.26 (3 H, s, S-Ph-CH3), 2.02 (3 H, s,
Ac), 0.80 (9 H, s, (CH3)3CSi), −0.08 (3 H, s, CH3Si), −0.21 (3 H, s,
CH3Si). 13C NMR δC (125 MHz, CDCl3) 170.6, 165.6, 137.9, 137.7,
137.3, 133.2, 132.4, 131.7, 130.2, 129.83, 129.79, 128.5, 128.4, 128.3,
128.05, 128.03, 127.3, 126.9, 99.5, 86.5, 80.6, 76.2, 74.4, 72.6, 71.8, 71.3,
71.2, 69.9, 68.0, 64.6, 63.4, 61.5, 25.93, 25.88, 21.1, 20.7, 18.0, 14.2, −3.8,
−4.8. HRMS [M + H]+ C48H60N3O11SSi+ calcd. 914.3712, obsd.
914.3800. 1.695 g (1.85 mmol) of p-tolyl 6-O-acetyl-2-azido-3-O-
benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-
2-O-benzoyl-3-O-benzyl-1-thio-α-L-idopyranoside was then protected
using the general procedure for levlinoyl ester protection and purified by
silica gel chromatography (2:1 hexanes:EtOAc), providing 1.747 g of
the product 1 in 92% yield (88% over the two steps from compound 6).
1H NMR δH (500 MHz, CDCl3) 8.13 (2 H, d, J 7.3), 7.48 (4 H, d, J 8.0),
7.45−7.20 (9 H, m), 7.13 (2 H, d, J 7.8), 7.09 (2 H, d, J 7.7), 5.58 (1 H, s,
H-1A), 5.35 (1 H, s, H-2A), 4.97 (1 H, d, J 11.7, Bn), 4.95 (1H, m,
H-5A), 4.76 (1 H, d, J 11.7, Bn), 4.56 (1 H, d, J 3.3, H-1B), 4.45−4.39
(1 H, m, H-6A), 4.35 (2 H, d, J 11.9, H-6A, H-6B), 4.16 (2 H, d, J 11.4, Bn,
H-3A), 4.03 (1 H, dd, J 12.0, 5.3, H-6B), 3.90 (1 H, d, J 11.5, Bn), 3.84−
3.80 (1 H, m, H-5B), 3.63 (1 H, s, H-4A), 3.48 (1 H, t, J 8.5, H-4B),
50.8, 44.9, 43.9, 37.8, 30.9, 29.8, 28.4, 27.8, 21.0, 20.8, 14.2. 1JC+1BH1B
=
1
171.5 Hz, JC1AH1A = 170 Hz. HRMS [M + H]+ C58H65N4O16 calcd.
1073.4390, obsd. 1073.4394.
p-Tolyl 6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethyl-
silyl-2-deoxy-1-thio-β-D-glucopyranoside (4). Compound 4 was
prepared as previously described.17 1H NMR δH (500 MHz, CDCl3)
7.50 (2 H, m), 7.36 (4 H, m), 7.30 (1 H, m), 7.15 (2 H, m), 4.91 (1 H, d,
J 11.0, Bn), 4.79 (1 H, d, J 11.0, Bn), 4.50 (1 H, dd, J 11.8, 2.3, H-6), 4.42
(1 H, m, J 9.9, H-1), 4.08 (1 H, dd, J 11.8, 5.6, H-6), 3.57 (1 H, m, H-4),
3.46 (1 H, ddd, J 9.4, 5.6, 2.3, H-5), 3.32 (2 H, m, H-2, H-3), 2.38 (3 H, s,
SPhCH3), 2.11 (3 H, s, Ac), 0.92 (9 H, s, J 7.8, (CH3)3CSi), 0.05 (3 H, s,
CH3Si), 0.04 (3 H, s, CH3Si). 13C NMR (125 MHz, CDCl3): 170.8,
138.9, 138.1, 134.3, 130.0, 128.6, 128.0, 127.8, 127.5, 86.6, 85.4, 78.7,
76.0, 70.7, 65.7, 63.3, 26.1, 21.5, 21.2, 18.2, −3.4, −4.6. HRMS [M + H]+
C28H40N3O5SSi+ calcd. 558.2452, obsd. 558.2461.
p-Tolyl 2-O-Benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-α-
L-idopyranoside (5). Compound 5 was prepared as previously
described.17 1H NMR δH (500 MHz, CDCl3) 8.02 (2 H, m), 7.59
(1 H, m), 7.39 (11 H, m), 7.06 (2 H, m), 6.91 (2 H, m), 5.56 (1 H, s, H-1),
I
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5.52 (1 H, dt, J 2.4, 1.0, H-2), 5.00 (1 H, t, J 4.9, H-5), 4.93 (1 H, d, J 11.8,
Bn), 4.69 (1 H, d, J 11.9, Bn), 4.56 (2 H, q, J 11.5, Bn), 3.90 (1 H, td,
J 2.9, 1.3, H-3), 3.83 (6 H, m, H-4, H-6, OCH3), 2.85 (1 H, d, J 9.7, OH),
2.33 (3 H, s, SPhCH3). 13C NMR (125 MHz, CDCl3): δ 164.9, 159.1,
137.5, 137.3, 133.4, 132.4, 131.9, 130.0, 129.6, 113.6, 129.5, 129.2, 129.1,
128.4, 128.3, 127.8, 127.6, 86.9, 74.0, 73.0, 72.1, 69.9, 69.7, 67.9, 67.1,
60.2, 55.1, 21.0,14.0. HRMS [M + H]+ C35H37O7S+ calcd. 601.2255,
obsd. 601.2260.
5.51 (1 H, s, H-2A), 5.28 (1 H, t, J 4.1, H-2C), 5.21 (1 H, d, J 3.0, H-1C),
5.10 (1 H, d, J 11.7, Bn), 5.08−5.02 (1 H, m, H-5A), 4.95 (1 H, d, J 2.4,
H-1D), 4.91 (3 H, t, J 11.0, 3Bn), 4.69 (1 H, d, J 3.4, H-1B), 4.65 (2 H, t,
J 9.9, Bn), 4.58−4.51 (2 H, m, Bn), 4.50−4.42 (4 H, m, H-6A), 4.42−4.34
(2 H, m, H-6A, H-6D), 4.30−4.27 (1 H, m, H-3A), 4.26−4.16 (3 H, m,
H-3C, H-6D), 3.98 (1 H, d, J 8.9, H-5B), 3.88 (3 H, d, J 10.3, Bn, H-5D),
3.81 (1 H, t, J 9.4, H-4B), 3.75 (1 H, s, H-4A), 3.71 (1 H, t, J 9.0, H-4D),
3.62 (2 H, dt, J 12.2, 9.5, H-3B, H-3D), 3.39 (2 H, td, J 9.8, 2.8, H-2B,
H-2D), 2.90−2.60 (8 H, m, 4-CH2 Lev), 2.48 (3 H, s, S-Ph-CH3), 2.28
(3 H, s, CH3 Lev), 2.26 (3 H, s, CH3 Lev), 2.16 (3 H, s, Ac), 2.14 (3 H, s,
Ac), 1.03 (9 H, s, (CH3)3CSi), 0.15 (3 H, s, CH3Si), 0.08 (3 H, s,
CH3Si). 13C NMR δC (125 MHz, CDCl3) 206.2, 172.17, 172.16, 170.64,
170.58, 165.7, 165.4, 137.8, 137.7, 137.32, 137.26, 133.4, 133.3, 132.2,
131.9, 129.9, 129.8, 129.7, 129.6, 128.7, 128.6, 128.45, 128.40, 128.29,
128.26, 128.15, 128.10, 128.05, 128.01, 127.98, 127.5, 127.4, 127.1, 98.9,
98.6, 97.7, 86.4, 80.3, 79.0, 75.8, 75.5, 74.8, 74.7, 74.6, 73.6, 72.6, 71.5,
71.4, 71.1, 70.8, 70.3, 69.6, 68.2, 66.0, 64.2, 63.9, 63.5, 62.8, 62.4, 62.0,
37.9, 37.8, 29.78, 29.77, 27.8, 25.9, 21.1, 20.74, 20.71, 18.0, −3.7, −4.9.
p-Tolyl 6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethyl-
silyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-
O-p-methoxybenzyl-1-thio-α-L-idopyranoside (6). Compound 6
was synthesized from donor 4 (0.45 g, 0.81 mmol) and 5 (0.533 g,
0.9 mmol) in 85% yield (0.719 g of 6) following the general procedure of
a single-step glycosylation with one alteration. The reaction solvent used
contained more than 50% Et2O at all times. The lower fraction of Et2O
led to the activation of product and the formation of a 1,6-anhydro
glycan.17,49 Purification was performed by silica gel chromatography
1
(6:1:1 hexanes:DCM:EtOAc). H NMR (500 MHz, CDCl3) δH 8.14
(2 H, dd, J 8.4, 1.3), 7.52−7.22 (15 H, m), 7.15 (2 H, d, J 6.9), 7.07−7.03
(2 H, m), 6.93−6.89 (2 H, m), 5.58 (1 H, s, H-1A), 5.38 (1 H, t, J 2.1,
H-2A), 5.00−4.94 (2 H, m, H-5A, Bn), 4.76 (1 H, d, J 11.7,Bn), 4.69 (1 H,
d, J 3.7, H-1B), 4.57−4.49 (2 H, m, Bn), 4.31−4.22 (2 H, m, H-6B, Bn),
4.18 (1 H, t, J 3.2, H-3A), 4.08 (1 H, d, J 11.3, Bn), 4.06−4.02 (1 H, m,
H-6B), 3.87−3.80 (5 H, m, PhOCH3, H-5B,H-6A) 3.77 (1 H, dd, J 10.2,
5.0, H-6A), 3.71 (1 H, s, H-4A), 3.55 (1 H, t, J 9.1, H-4B), 3.40−3.35
(1 H, m, H-3B), 3.29 (1 H, dd, J 10.2, 3.7, H-2B), 2.33 (3 H, s, S-Ph-
CH3), 2.06 (3 H, s, Ac), 0.90 (9 H, s, (CH3)3CSi), −0.00 (3 H, s CH3Si),
−0.10 (3 H, s, CH3Si). 13C NMR (125 MHz, CDCl3) δC 170.8, 165.9,
159.5, 138.0, 137.8, 137.6, 133.4, 132.7, 132.0, 130.4, 130.2, 130.1, 129.8,
129.5, 128.7, 128.6, 128.4, 128.3, 128.2, 127.5, 127.3, 114.0, 98.7, 86.7,
80.8, 75.2, 74.8, 73.2, 72.9, 72.2, 71.6, 71.2, 70.4, 69.4, 67.6, 64.9,
63.2, 55.5, 26.2, 21.4, 21.1, 18.2, −3.5, −4.7. gHSQCAD (without
1H decoupling) 1JC1BH1B = 171 Hz, 1JC1AH1A = 167.5 Hz; HRMS [M + Li]+
C56H67LiN3O12SSi+ calcd. 1040.4369, obsd. 1040.4373.
1JC1AH1A = 168 Hz, 1JC1BH1B = 171.5 Hz, 1JC1CH1C = 169.5 Hz, 1JC1DH1D
=
173 Hz. HRMS [M + H]+ C93H109N6O26SSi+ calcd. 1785.6876, obsd.
1785.6873.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-α-L-
idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-1-thio-
α-L-idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-α-
L-idopyranoside (10). Compound 10 was synthesized in 61% yield from
donor 9 (346 mg, 190 μmol) and acceptor 3 (208 mg, 190 μmol)
following the general procedure for glycosylation and was purified by
silica gel flash chromatography (1:1:1 hexanes:DCM:EtOAc), providing
384 mg of 10 (72% yield). 1H NMR δH (600 MHz, CDCl3) 8.09 (2 H, d,
J 7.1), 8.06 (4 H, ddd, J 8.5, 2.2, 1.3), 7.50−7.07 (49 H, m), 5.15−5.09
(4 H, m), 5.06 (1 H, d, J 3.9), 5.03 (1 H, s), 5.02 (1 H, d, J 4.0), 4.88 (1 H,
m), 4.81−4.70 (8 H, m), 4.68 (1 H, s), 4.58 (2 H, dd, J 12.9, 7.0), 4.49−
4.39 (3 H, m), 4.39−4.27 (9 H, m), 4.26−4.22 (2 H, m), 4.20−4.12
(4 H, m), 4.08−3.96 (6 H, m), 3.90 (1 H, d, J 10.2), 3.81−3.76 (1 H, m),
3.75−3.67 (5 H, m), 3.67−3.63 (2 H, m), 3.62−3.56 (2 H, m), 3.52
(2 H, dd, J 18.7, 9.6), 3.44 (1 H, dd, J 10.0, 8.6), 3.41−3.26 (2 H, m),
3.24−3.19 (3 H, m), 2.69−2.34 (12 H, m, 4-CH2 Lev), 2.10 (3 H, s, CH3
Lev), 2.09 (3 H, s, CH3 Lev), 2.07 (3 H, s, CH3 Lev), 1.98 (3 H, s, Ac),
1.97 (3 H, s, Ac), 1.97 (3 H, s, Ac), 1.88−1.76 (2 H, m), 0.86 (9 H, s,
(CH3)3CSi), −0.03 (3 H, s, CH3Si), −0.10 (3 H, s, CH3Si). 13C NMR δC
(125 MHz, CDCl3) 206.5, 172.5, 172.43, 172.39, 170.9, 170.8, 165.9,
165.67, 165.65, 138.1, 138.0, 137.9, 137.6, 137.5, 133.6, 130.07, 130.04,
129.78, 129.75, 128.87, 128.84, 128.75, 128.63, 128.59, 128.55, 128.39,
128.36, 128.34, 128.29, 128.25, 128.15, 128.1, 127.9, 127.8, 127.6, 127.5,
127.3, 98.8, 98.6, 98.1, 98.0, 80.6, 79.2, 79.0, 75.6, 75.2, 75.1, 75.0, 74.8,
74.5, 73.7, 72.5, 71.7, 71.3, 70.7, 70.4, 68.9, 68.2, 67.4, 65.6, 64.5, 64.0,
63.9, 63.6, 63.1, 62.4, 62.2, 38.02, 37.99, 30.03, 30.01, 28.04, 28.00, 26.2,
21.3, 21.0, 18.3, 14.5, −3.5, −4.7. 1JC1AH1A = 170.4 Hz, 1JC1BH1B = 169.2
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-α-L-idopyra-
noside (8). Compound 8 was prepared by glycosylation of donor 1
(307 mg, 0.3 mmol) with 725 (118 mg, 0.394 mmol) in 81% yield
following the procedure for glycosylation. Purification was performed by
silica gel chromatography (2 fractions 2:1 hexanes:EtOAC, then 1:1
1
hexanes:EtOAC), providing 290 mg of 8. H NMR δH (500 MHz,
CDCl3) 8.14 (2 H, d, J 7.2), 7.52−7.08 (23 H, m), 5.17 (2 H, d, J 8.5,
CH2-Cbz), 5.11 (1 H, s, H-2A), 4.99 (0.5 H due to restricted rotation
around N, bs, H-1A), 4.94 (0.5 H due to restricted rotation around N,
bs, H-1A), 4.87 (0.5 H, d, J 11.4, Bn), 4.83 (0.5 H, d, J 11.4, Bn), 4.74
(1 H, d, J 11.4, Bn), 4.67 (1 H, d, J 3.5, H-1B), 4.56−4.40 (4 H, m, H-5A,
H-6A, CH2-Bn), 4.40−4.34 (2 H, m, H-6B, Bn), 4.30 (1 H, s, H-6A),
4.14 (1 H, d, J 9.0, Bn), 4.11−4.04 (2 H, m, H-3A, H-6B), 3.89−3.75
(2 H, m, H-5B, H-linker), 3.68 (1 H, s, H-4A), 3.57−3.46 (2 H, m, H-4B,
H-linker), 3.45−3.32 (3 H, m, J 10.1, H-3B, CH2-linker), 3.27 (1 H, dd,
J 10.1, 3.5, H-2B), 2.74 (2 H, s, CH2 Lev), 2.60 (2 H, d, J 16.9, CH2 Lev),
2.17 (3 H, s, CH3 Lev), 2.06 (3 H, s, Ac), 1.89 (2 H, d, J 21.0, CH2-
linker), 0.90 (9 H, s, (CH3)3CSi), 0.01 (1 H, s, CH3Si), −0.08 (2 H, s,
CH3Si). 13C NMR δC (125 MHz, CDCl3) 206.2, 172.3, 170.6, 165.6,
156.6, 156.1, 138.0, 137.8, 137.6, 133.2, 130.0, 129.8, 128.51, 128.48,
128.3, 128.09, 128.06, 127.90, 127.85, 127.78, 127.3, 127.0, 98.6, 98.3,
80.5, 75.0, 74.6, 72.5, 72.3, 71.5, 71.1, 68.9, 67.1, 65.7, 64.5, 63.6, 63.0,
60.4, 50. 8, 44.9, 43.9, 37.8, 29.8, 28.4, 27.8, 25.9, 21.0, 20.8, 18.0, 14.2,
−3.8, −4.9. HRMS [M + H]+ C64H79N4O16Si+ calcd. 1187.5255, obsd.
1187.5254.
1
1
1
Hz, JC1CH1C = 168.4 Hz, JC1DH1D = 171.6 Hz, JC1EH1E = 172.8 Hz,
1JC1FH1F = 168.6 Hz. HRMS [M + H]+ C144H165N10O42Si+ calcd.
2735.0880, obsd. 2735.0883.
Procedure for One-Pot Synthesis of Hexasaccharide 10. A solution
of donor 1 (60 μmol) and freshly activated 4 Å molecular sieves
(200 mg) in CH2Cl2 (1.5 mL) was stirred at room temperature for
10 min, and cooled to −78 °C, which was followed by addition of AgOTf
(47 mg, 180 μmol) dissolved in MeCN (47 μL) without touching the
wall of the flask. After 20 min, orange-colored p-TolSCl (9.5 μL,
60 μmol) was added to the solution through a microsyringe. Since the
reaction temperature was lower than the freezing point of p-TolSCl,
p-TolSCl was added directly into the reaction mixture to prevent it from
freezing on the flask wall. The characteristic yellow color of p-TolSCl in
the reaction solution dissipated within a few seconds, indicating
depletion of p-TolSCl. After the donor was completely consumed,
according to TLC analysis (about 5 min at −78 °C), a solution of
acceptor 2 (42 μmol) in CH2Cl2 (1 mL) was slowly added along the wall
by using a syringe. The reaction mixture was warmed to −30 °C under
p-Tolyl 6-O-Acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethyl-
silyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-
levulinoyl-α-L-idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-
2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levu-
linoyl-1-thio-α-L-idopyranoside (9). Compound 9 was synthesized
according to the general procedure of glycosylation with donor 1
(200 mg, 197 μmol) and acceptor 2 (177 mg, 197 μmol) in 81% yield
and purified by silica gel chromatography (1:1 hexanes:EtOAc),
1
providing 285 mg of 9. H NMR δH (500 MHz, CDCl3) 8.31−8.26
(2 H, m), 8.22 (2 H, t, J 9.0), 7.66−7.25 (30 H, m), 5.70 (1 H, s, H-1A),
J
DOI: 10.1021/acs.joc.5b02172
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
stirring in 2 h and then room temperature. The second acceptor 3
(30 μmol) in CH2Cl2 (1 mL) was added, and the mixture was stirred for
5 min at room temperature. The mixture was then cooled to −78 °C,
followed by addition of AgOTf (37 mg, 144 μmol) in MeCN (37 μL).
The mixture was stirred for 20 min, and then p-TolSCl (6.7 μL,
42 μmol) was added to the solution. The reaction mixture was warmed
to −30 °C under stirring in 2 h. Then, the mixture was diluted with
CH2Cl2 (20 mL) and filtered over Celite. The Celite was further washed
with CH2Cl2 until no organic compounds were observed in the filtrate
by TLC analysis. All solutions in CH2Cl2 were combined and washed
twice with a saturated aqueous solution of NaHCO3 (20 mL) and twice
with water (10 mL). The organic layer was collected and dried over
Na2SO4. After removal of the solvent, the desired oligosaccharide 10 was
purified from the reaction mixture in a yield of 67% by silica gel flash
chromatography.
tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzo-
yl-3-O-benzyl-α-L-idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-ben-
zyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-
idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranoside
(27 mg, 11 μmol) was oxidized, and the resulting carboxylates were
protected as benzyl esters according to the general procedures to
produce 27 mg of compound 12 in 80% yield after silica gel
chromatography (3:1 hexanes:EtOAc). 1H NMR δH (500 MHz,
CDCl3) 8.30−8.22 (6 H, m), 7.74−7.23 (64 H, m), 5.69 (1 H, d,
J 5.3), 5.62 (1 H, d, J 5.4), 5.39−5.32 (3 H, m), 5.31−5.11 (10 H, m),
5.03−4.77 (11 H, m), 4.63−4.36 (11 H, m), 4.34−4.30 (2 H, m), 4.27−
4.20 (4 H, m), 4.10 (2 H, t, J 5.6), 4.05 (1 H, d, J 9.9), 4.02−3.98 (3 H,
m), 3.93−3.81 (3 H, m), 3.78 (1 H, t, J 9.1), 3.66−3.61 (1 H, m), 3.61−
3.53 (3 H, m), 3.53−3.41 (2 H, m), 3.38 (1 H, dd, J 10.2, 3.7), 3.33 (1 H,
dd, J 10.2, 3.7), 3.28 (1 H, dd, J 10.2, 3.6), 2.20 (4 H, s, J 0.7), 2.18 (3 H,
s, J 5.0), 2.14 (3 H, s), 2.00−1.87 (2 H, m), 1.04 (9 H, s), 0.17 (3 H, s),
0.10 (3 H, s). 13C NMR (150 MHz, CDCl3) δ 172.0, 171.3, 171.0, 170.8,
169.2, 165.6, 165.4, 165.4, 138.1, 138.1, 138.0, 137.5, 137.4, 135.4, 135.3,
135.0, 133.9, 133.8, 130.23, 130.19, 130.1, 129.5, 129.4, 129.1, 129.04,
129.01, 128.98, 128.95, 128.94, 128.91, 128.90, 128.87, 128.84, 128.82,
128.78, 128.72, 128.71, 128.66, 128.63, 128.61, 128.59, 128.58, 128.5,
128.39, 128.37, 128.34, 128.30, 128.21, 128.17, 128.14, 128.13, 128.1,
128.01, 128.00, 127.8, 127.6, 100.1, 99.5, 99.2, 98.6, 98.5, 79.4, 78.5,
78.4, 76.2, 76.0, 75.3, 75.1, 75.0, 74.47, 74.45, 74.3, 73.4, 72.6, 71.8, 71.4,
71.3, 70.7, 70.2, 69.9, 68.1, 67.9, 67.2, 63.7, 63.4, 63.1, 62.7, 62.1, 60.6,
21.3, 21.0, 14.4. MALDI [M + Na]+ C129H146N10NaO36Si+ calcd. 2775.0,
obsd. 2775.3.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-methyl 2-O-Benzoyl-3-O-benzyl-α-L-idopyrano-
syluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-methyl 2-O-Benzoyl-3-O-benzyl-α-L-
idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-
deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-Benzoyl-3-O-benzyl-
α-L-idopyranosyluronate (14). Compound 14 was prepared from
30 mg (12 μmol) of N-(benzyl)-benzyloxycarbonyl-3-aminopropyl
6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranoside-
(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-
(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranoside-(1→4)-6-O-acetyl-
2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-
3-O-benzyl-α-L-idopyranoside using the general procedure for oxidation
and methyl ester formation. Compound 14 was isolated in 77% yield
(24 mg) after silica gel chromatography (3:2 hexanes:EtOAc). 1H NMR
δH (500 MHz, CDCl3) 8.21−8.07 (6 H, m), 7.61−7.05 (49 H, m), 5.57
(1 H, d, J 5.9), 5.47 (1 H, d, J 5.7), 5.24 (1 H, t, J 6.3), 5.19 (1 H, t, J 6.1),
5.15 (2 H, s), 5.07 (3 H, d, J 3.5), 4.92 (1 H, d, J 3.6), 4.87−4.76 (5 H,
m), 4.73 (4 H, dd, J 10.8, 7.3), 4.69−4.60 (4 H, m), 4.51−4.33 (7 H, m),
4.27 (3 H, dt, J 16.7, 11.4), 4.19−4.13 (2 H, m), 4.12−4.05 (4 H, m),
3.98 (1 H, s), 3.96−3.92 (1 H, m), 3.90 (2 H, d, J 9.1), 3.87−3.83 (1 H,
m), 3.79 (4 H, dd, J 10.7, 7.4), 3.71 (3 H, d, J 5.4), 3.70−3.65 (1 H, m),
3.61−3.55 (4 H, m), 3.53−3.49 (1 H, m), 3.47−3.41 (4 H, m), 3.33−
3.26 (3 H, m), 3.21 (1 H, dd, J 10.2, 3.5), 2.12 (3 H, s, J 3.3), 2.11 (3 H, s),
2.03 (3 H, s), 1.82 (2 H, d), 0.92 (9 H, s), 0.03 (3 H, s), −0.00 (3 H, s).
13C NMR δC (125 MHz, CDCl3) 170.7, 170.64, 170.56, 169.7, 169.5,
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-α-L-
idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-levulinoyl-1-thio-
α-L-idopyranoside (11). Compound 11 was synthesized in 75% yield
from 403 mg (0.4 mmol) of donor 1 and 403 mg (0.38 mmol) of
acceptor 3 following the general procedure for glycosylation and was
purified by silica gel flash chromatography (1:1:1 hexanes:DCM:E-
tOAc). This provided 548 mg of 11. 1H NMR δH (500 MHz, CDCl3)
8.15−8.11 (4 H, m), 7.39−7.32 (36 H, m), 5.17 (3 H, d, J 4.2), 5.13
(2 H, s), 4.97−4.93 (1 H, m), 4.79−4.71 (7 H, m), 4.51 (3 H, t, J 12.1),
4.42−4.36 (7 H, m), 4.27−4.23 (2 H, m), 4.15−4.09 (4 H, m), 3.98
(1 H, d, J 10.2), 3.89−3.83 (1 H, m), 3.78−7.73 (3 H, m), 3.66 (1 H, s),
3.60 (2 H, td, J 9.6, 5.0), 3.51 (2 H, t, J 9.3), 3.38−3.34 (3 H, m), 3.31−
3.25 (2 H, m), 2.72−2.68 (4 H, m), 2.55−2.50 (4 H, m), 2.14 (6 H, s),
2.05 (3 H, s), 2.02 (3 H, s), 1.91−1.88 (2 H, m), 0.92 (9 H, s), 0.04
(3 H, s), −0.03 (3 H, s). 13C NMR δC (125 MHz, CDCl3) 206.3, 172.24,
172.17, 170.68, 170.65, 165.6, 165.4, 156.6, 156.1, 137.92, 137.88,
137.77, 137.76, 137.6, 137.2, 136.8, 136.7, 133.4, 129.8, 129.5, 128.6,
128.5, 128.39, 128.35, 128.30, 128.13, 128.11, 128.03, 128.00, 127.9,
127.83, 127.79, 127.6, 127.4, 127.3, 127.1, 98.6, 98.3, 98.2, 97.7, 80.3,
79.0, 75.4, 75.0, 74.85, 74.76, 74.6, 74.3, 73.5, 72.3, 72.2, 71.4, 71.0, 70.5,
70.1, 68.6, 67.9, 67.1, 65.6, 65.4, 65.3, 64.2, 63.8, 63.4, 62.8, 62.3, 62.1,
51.0, 50.7, 44.8, 43.8, 37.8, 29.82, 29.80, 28.3, 27.8, 27.7, 25.9, 20.8, 20.7,
1
1
1
18.0, −3.8, −5.0. JC1AH1A = 169 Hz, JC1BH1B = 170.5 Hz, JC1CH1C
=
171.5 Hz, 1JC1DH1D = 174 Hz. HRMS [M + H]+ C104H122N7O29Si+ calcd.
1961.8084, obsd. 1961.8092.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-benzyl 2-O-Benzoyl-3-O-benzyl-α-L-idopyrano-
syluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-benzyl 2-O-Benzoyl-3-O-benzyl-α-L-
idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-
deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-Benzoyl-3-O-benzyl-
α-L-idopyranosyluronate (12). Compound 12 was prepared in two
steps starting by treating compound 10 (300 mg, 110 μmol) to remove
levulinoyl esters using the general procedure. This provided 239 mg of
N-(benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3-O-
benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-
2-O-benzoyl-3-O-benzyl-α-L-idopyranoside-(1→4)-6-O-acetyl-2-azido-
3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-ben-
zyl-α-L-idopyranoside-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-
α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranoside
in 87% yield after silica gel chromatography (1:1:2 hexanes:DCM:E-
165.6, 165.2, 165.1, 137.8, 137.7, 137.34, 137.30, 133.7, 133.6, 133.4,
130.0, 129.9, 129.8, 129.2, 129.1, 128.8, 128.7, 128.52, 128.45, 128.36,
128.3, 128.2, 128.01, 127.98, 127.92, 127.88, 127.84, 127.76, 127.7,
127.6, 127.4, 127.2, 99.0, 98.72, 98.66, 98.1, 98.0, 80.2, 78.2, 78.1, 76.5,
76.2, 76.0, 75.0, 74.9, 74.7, 74.6, 74.5, 74.4, 74.1, 72.6, 72.2, 72.0, 71.6,
71.4, 71.2, 71.1, 70.6, 69.7, 69.6, 68.0, 67.5, 67.2, 63.8, 63.4, 63.0, 62.4,
61.6, 52.1, 51.9, 51.7, 25.9, 20.81, 20.78, 18.0, −3.7, −5.1. HRMS
[M + H]+ C132H147N10O39Si+ calcd. 2524.9624, obsd. 2524.9628.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-Azido-3-O-ben-
zyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4)-
methyl 3-O-Benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-
benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-Benzyl-α-L-
idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-methyl 3-O-Benzyl-α-L-idopyranosyluronate
(15). Compound 15 was prepared from 24 mg of 14 (9.5 μmol) using
1
tOAC then 1:1:3 hexanes:DCM:EtOAC). H NMR δH (600 MHz,
CDCl3) 8.15−8.09 (6 H, m), 7.48−7.12 (49 H, m), 5.19−5.12 (4 H, m),
5.09 (1 H, d, J 14.8), 5.07−5.04 (2 H, m), 5.03−4.91 (1 H, m), 4.90−
4.80 (3 H, m), 4.72 (5 H, ddd, J 12.7, 11.5, 7.3), 4.57 (1 H, m), 4.53−
4.27 (9 H, m), 4.27−4.19 (3 H, m), 4.19−4.13 (3 H, m), 4.07−4.03
(3 H, m), 4.02−3.73 (9 H, m), 3.72−3.47 (11 H, m), 3.45−3.40 (1 H, m),
3.26 (2 H, dd, J 10.1, 3.7), 3.23 (2 H, dd, J 11.3, 5.3), 3.23 (2 H, dd, J
11.3, 5.3), 2.03 (3 H, s), 2.02 (3 H, s), 2.01 (3 H, s), 1.95−1.80 (2 H, m),
0.87 (9 H, s), −0.01 (3 H, s), −0.09 (3 H, s). HRMS [M + H]+
C129H147N10O36Si+ calcd. 2440.9777, obsd. 2440.9768. N-(Benzyl)-
benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3-O-benzyl-4-O-
K
DOI: 10.1021/acs.joc.5b02172
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The Journal of Organic Chemistry
Article
the general procedure for transesterification, and 18 mg of 15 was
isolated (91% yield) after elution from a Sephadex LH-20 column (1:1
DCM:MeOH). 1H NMR δH (500 MHz, CDCl3) 7.45−7.09 (40 H, m),
5.28 (1 H, s), 5.25 (1 H, d, J 1.7), 5.17 (2 H, s), 5.04−4.95 (4 H, m),
4.90−4.78 (6 H, m), 4.78−4.68 (5 H, m), 4.62 (3 H, dd, J 11.2, 2.7),
4.60−4.49 (4 H, m), 4.49−4.43 (2 H, m), 4.15 (1 H, s), 4.04 (2 H, d,
J 2.9), 3.96−3.83 (6 H, m), 3.83−3.70 (12 H, m), 3.68−3.48 (10 H, m),
3.46 (1 H, dd, J 9.8, 3.7), 3.42 (3 H, s), 3.40−3.35 (2 H, m), 1.89−1.80
(2 H, m), 0.87 (9 H, s), 0.07 (3 H, s), −0.03 (3 H, s). 13C NMR δC
(125 MHz, CDCl3) 169.6, 169.4, 137.8, 137.6, 137.04, 137.01, 128.72,
128.69, 128.5, 128.45, 128.39, 128.35, 128.27, 128.26, 128.2, 128.1,
128.0, 127.8, 127.7, 127.4, 127.3, 127.24, 127.23, 127.1, 101.8, 100.92,
100.88, 95.6, 95.4, 80.8, 79.3, 79.1, 75.5, 75.1, 75.0, 74.2, 74.0, 73.4, 73.1,
73.0, 72.81, 72.75, 72.6, 72.5, 72.2, 71.8, 70.4, 68.1, 67.9, 67.7, 67.4,
67.25, 66.20, 64.4, 64.2, 64.0, 61.3, 61.2, 61.0, 52.4, 52.13, 52.07, 25.8,
17.9, −3.8, −4.8. HRMS [M + H]+ C105H129N10O33Si+ calcd. 2086.8521,
obsd. 2086.8518.
74.56, 74.3, 74.1, 72.7, 72.2, 71.4, 70.9, 70.85, 70.5, 70.1, 69.7, 69.6, 68.0,
67.5, 67.3, 64.3, 63.4, 63.37, 63.1, 62.2, 61.7, 52.1, 52.0, 51.7, 20.8, 20.75.
HRMS [M + H]+ C133H139N10O39+ calcd. 2500.9229, obsd. 2500.9230.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-Amino-3,4-di-O-
benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-Benzyl-α-L-
idopyranosyluronate-(1→4)-2-amino-3-O-benzyl-2-deoxy-α-D-
glucopyranosyl-(1→4)-methyl 3-O-Benzyl-α-L-idopyrano-
syluronate-(1→4)-2-amino-3-O-benzyl-2-deoxy-α-D-gluco-
pyranosyl-(1→4)-methyl 3-O-Benzyl-α-L-idopyranosyluronate (19).
Compound 19 was prepared by treating compound 18 (12 mg, 4.8 μmol)
with the procedures for transesterification and 1,3-dithiopropane
reduction. This furnished 7 mg of compound 19, a 76% yield over
two steps. The compound was purified via silica gel chromatography
(DCM:MeOH = 8:1 with 5% triethylamine) after1,3-dithiopropane
reduction. 1H NMR δH (500 MHz, CDCl3) 7.38−7.17 (44 H, m), 7.13−
7.10 (1 H, m), 5.29 (2 H, dd, J 8, 3.5), 5.15 (2 H, s), 4.99 (1 H, d, J 12),
4.97−4.92 (5 H, m), 4.91−4.87 (3 H, m), 4.86 (1 H, d, J 3), 4.81 (2 H, d,
J 11.5), 4.72 (2H, dd, J 11.5, 3.5), 4.66 (4 H, s), 4.64 (2 H, d, J 11), 4.58
(2 H, d, J 11.5), 4.54−4.43 (4 H, m), 4.41 (2 H, d, J 11.5), 4.22−4.15
(3 H, m), 4.02−3.96 (2 H, m), 3.94 (2 H, s), 3.91−3.82 (4 H, m), 3.82−
3.75 (5 H, m), 3.74 (3 H, s), 3.73−3.70 (2 H, m), 3.68−3.65 (2 H, m),
3.55 (3 H, s), 3.53 (3 H, s), 3.51−3.34 (10 H, m), 3.32−3.24 (2 H, m),
2.90−2.83 (2 H, m), 1.88−1.78 (2 H, m). 13C NMR (125 MHz, CDCl3)
δ 170.2, 170.14, 170.11, 156.7, 156.2, 141.1, 138.49, 138.43, 138.2,
138.0, 137.8, 137.7, 137.6, 137.40, 137.38, 136.8, 136.6, 128.6, 128.54,
128.51, 128.50, 128.48, 128.46, 128.41, 128.30, 128.28, 128.03, 127.99,
127.95, 127.90, 127.83, 127.81, 127.78, 127.70, 127.64, 127.58, 127.47,
127.3, 127.1, 126.96, 126.95, 126.93, 101.9, 101.4, 101.3, 97.0, 96.8, 82.8,
81.3, 78.0, 75.8, 75.3, 75.00, 74.96, 74.5, 72.7, 72.6, 72.21, 72.16, 71.7,
70.9, 69.8, 69.7, 69.2, 67.5, 67.2, 66.6, 66.1, 65.0, 61.3, 60.8, 55.3, 55.1,
52.4, 52.0, 51.9, 50.8, 50.5, 44.6, 43.8, 27.9. HRMS [M + H]+
C106H127N4O33+ calcd. 1984.8411, obsd. 1984.8417.
3-Aminopropyl 2-Deoxy-2-amino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate-(1→4)-2-
deoxy-2-amino-6-O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-sul-
fonate-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-amino-6-O-sul-
fonate-α-D-glucopyranosyl-(1→4)-2-O-sulfonate-α-L-idopyrano-
syluronate Salt (20). Compound 20 was prepared from compound 19
(6 mg, 2.4 μmol) in 3 steps. Treatment with the general procedures for
selective O-sulfation, global debenzylation, and methyl ester saponifi-
cation provided 3 mg of 20 in 72% yield from compound 19. NMR
analysis showed that the anomeric carbons of the three glucosamine
units of 25 gave chemical shifts of 93.0 ppm (3 carbons), which suggests
that the glucosamines were not sulfated. These values were consistent
with literature reports,67,68 where anomeric carbons of unsulfated
glucosamines in heparin resonate around 94 ppm, while those of
N-sulfated glucosamines typically appear above 100 ppm. 1H NMR δH
(500 MHz, D2O) 5.37−5.29 (3 H, m, H-1B, H-1D, H-1F), 5.15 (1 H, s,
H-1A), 5.13 (1H, s, H-1C)5.05 (1 H, s, H-1E), 4.84 (2 H, dd, J 7.4, 1.3),
4.46 (1 H, d, J 1.5), 4.31−4.25 (5 H, m, H-2A, H2C, H-3E), 4.21 (4 H,
s), 4.18−4.12 (2 H, m, H-2E), 4.11−4.07 (3 H, m, H-3A, H-3C), 3.97−
3.91 (2 H, m), 3.90−3.85 (3 H, m, H-3B, H-3D), 3.84−3.75 (3 H, m,
H-3F), 3.74−3.68 (2 H, m), 3.66−3.61 (1 H, m), 3.48 (1 H, t, J 9.7),
3.35−3.25 (3 H, m, H-2B, H-2D, H-2F), 3.12−3.03 (2 H, m), 1.96−1.87
(2 H, m). δC (values obtained from F1 dimension of HMQC spectrum)
δ 100.7 (C-1C), 100.5 (C-1A), 99.8 (C-1E), 93.0 (C-1B, C-1D, C-1F),
77.7, 77.3, 77.2, 74.6, 72.2, 72.1, 71.4, 71.2, 70.6, 70.5, 70.4, 70.1, 69.0,
68.6, 68.1, 68.0, 64.5, 56.2, 55.9, 55.0, 40.2, 27.9; HRMS [M]3−
C39H63N4O49S63− calcd. 521.0301, obsd. 521.0304.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-
Benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-
Benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-
Benzoyl-3-O-benzyl-α-L-idopyranosyluronate (17). Compound 17
was prepared from compound 14 (211 mg, 83.6 μmol) by removing
the TBS protecting group according to the general procedure furnishing
179 mg of 17, a 94% yield after silica gel chromatography (1:1
1
hexanes:EtOAc). H NMR δH (500 MHz, CDCl3) 8.17−8.10 (6 H),
7.60−7.09 (49 H, m), 5.52 (1 H, d, J 5.3), 5.47 (1 H, d, J 5.5), 5.24−5.21
(1 H, m), 5.21−5.17 (1 H, m), 5.15 (2 H, s), 5.07 (2 H, s), 5.01 (1 H, d,
J 3.5), 4.92 (1 H, d, J 3.6), 4.84−4.61 (11 H, m), 4.59−4.53 (2 H, m),
4.48−4.39 (5 H, m), 4.32−4.23 (3 H, m), 4.21−4.06 (7 H, m), 4.00−
3.84 (6 H, m), 3.83−3.76 (3 H, m), 3.65 (3 H, s), 3.62−3.55 (5 H, m),
3.51−3.41 (6 H, m), 3.31 (2 H, dd, J 10.3, 3.6), 3.22 (2 H, ddd, J 10.3,
3.5, 1.6), 2.94 (1 H, s), 2.11 (3 H, s), 2.10 (3 H, s), 2.08 (3 H, s), 1.84
(2 H, bs). 13C NMR δC (125 MHz, CDCl3) 171.9, 170.8, 170.7, 169.6,
169.5, 165.6, 165.2, 137.8, 137.73, 137.70, 137.3, 137.2, 133.7, 133.6,
133.4, 130.0, 129.9, 129.6, 129.2, 129.1, 128.8, 128.7, 128.6, 128.5,
128.39, 128.37, 128.3, 128.2, 128.08, 128.06, 128.02, 127.99, 127.97,
127.95, 127.9, 127.71, 127.67, 127.4, 127.3, 99.3, 99.0, 98.6, 98.2, 98.1,
78.9, 78.3, 78.2, 76.2, 76.0, 75.9, 75.7, 75.0, 74.9, 74.7, 74.62, 74.57, 74.3,
74.1, 72.7, 72.2, 71.4, 71.1, 71.0, 70.4, 69.8, 69.6, 68.0, 67.5, 67.2, 63.4,
63.1, 62.9, 62.5, 61.74, 61.66, 60.4, 52.1, 52.0, 51.7, 29.7, 20.80, 20.78,
+
20.77. HRMS [M + H]+ C126H133N10O39 calcd. 2410.8759, obsd.
2410.8751.
N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-Acetyl-2-
azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl
2-O-Benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-ace-
tyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl
2-O-Benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-ace-
tyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl
2-O-Benzoyl-3-O-benzyl-α-L-idopyranosyluronate (18). Compound
18 was prepared from compound 17 (18 mg, 7.5 μmol) using the
general procedure for benzylation to provide 10 mg of 18 in 50% yield
(66% BRSM with 3 mg of 17 recovered) after silica gel chromatography
1
(3:2 hexanes:EtOAc). H NMR δH (500 MHz, CDCl3) 8.14−8.08
(6 H), 7.58−7.07 (54 H, m), 5.49 (1 H, d, J 4.8), 5.45 (1 H, d, J 5.5), 5.17
(2 H, ddd, J 8.8, 5.9, 2.5), 5.13 (2 H, d, J 3.0), 5.04 (2 H, s), 4.96 (1 H, d,
J 3.5), 4.89 (1 H, d, J 3.6), 4.83−4.75 (6 H, m), 4.72 (3 H, dd, J 17.9, 7.0),
4.66 (2 H, dd, J 6.2, 4.2), 4.57 (1 H, d, J 10.9), 4.48 (1 H, d, J 10.6), 4.41
(5 H, dd, J 18.2, 7.9), 4.32−4.18 (6 H, m), 4.16−4.11 (2 H, m), 4.06
(3 H, ddd, J 8.0, 5.8, 2.7), 3.98−3.92 (3 H, m), 3.88 (2 H, t, J 9.5), 3.81−
3.72 (4 H, m), 3.69−3.62 (4 H, m), 3.61−3.54 (4 H, m), 3.54−3.49
(1 H, m), 3.48−3.42 (5 H, m), 3.37−3.26 (3 H, m), 3.24 (1 H, dd, J 10.3,
3.5), 3.20 (1 H, dd, J 10.2, 3.6), 2.12−2.06 (6 H, m), 1.99−1.95 (3 H,
m), 1.85−1.76 (2 H, m). 13C NMR (125 MHz, CDCl3) δ 170.63,
170.58, 170.4, 169.5, 169.45, 165.5, 165.2, 165.1, 137.8, 137.6, 137.5,
137.3, 137.2, 133.6, 133.4, 130.0, 129.9, 129.6, 129.2, 129.1, 128.8, 128.7,
128.5, 128.45, 128.4, 128.36, 128.3, 128.2, 128.1, 128.01, 127.99, 127.96,
127.9, 127.88, 127.86, 127.7, 127.4, 127.2, 99.1, 99.0, 98.6, 98.3, 98.1,
80.0, 78.3, 78.26, 77.4, 76.0, 75.9, 75.7, 75.5, 75.0, 74.99, 74.8, 74.6,
3-Aminopropyl 2-Deoxy-2-acetamido-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate-(1→4)-2-
deoxy-2-acetamido-6-O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-
sulfonate-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-acetamido-6-
O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-sulfonate-α-L-
idopyranosyluronate (21). Compound 21 was prepared from 19 in
four steps. 19 was first selectively sulfated using the procedure for
selective O-sulfation, which was followed by acetylation. Next, 5 mg of
the sulfated product (2 μmol) was dissolved in 2 mL of methanol.
To this was added 30 μL of triethylamine and 30 equiv of acetic
anhydride (10 equiv per NH2 and 6 μL total). This was stirred at room
temperature for 5 h and was diluted with 1:1 DCM:MeOH and eluted
L
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The Journal of Organic Chemistry
Article
from a Sephadex LH-20 column. The product of acetylation was further
treated with global debenzylation and methyl ester saponification
conditions to produce 2 mg of 21 in 54% yield over 4 steps from 19.
NMR analysis showed that the anomeric carbons of the three
glucosamine units of 21 gave chemical shifts of 95.6 ppm (3 carbons),
which suggests that the glucosamines were not sulfated. These values
were consistent with literature reports,67,68 where anomeric carbons of
unsulfated glucosamines in heparin resonate around 94 ppm, while
those of N-sulfated glucosamines typically appear above 100 ppm.
1H NMR δH (500 MHz, D2O) 5.09 (2 H, s, H-1A, H-1C), 5.05 (3 H, t,
J = 4.1, H-1B, H-1D, H-1F), 5.00 (1 H, s, H-1E), 4.83 (2 H, s), 4.43 (1 H,
s), 4.28−4.18 (10 H, m), 4.14 (2 H, d, J 10.9), 4.00−3.90 (8 H, m), 3.89
(1 H, d, J 3.5), 3.88−3.80 (1 H, m), 3.71−3.58 (6 H, m), 3.50−3.46
(1 H, m), 3.14−3.05 (2 H, m), 1.98−1.95 (11 H, m, 3 Ac, CH2-linker).
δC (values obtained from F1 dimension of HMQC spectrum) δ 101.2
(C-1A, C-1C), 100.4 (C-1E), 95.6 (C-1B, C-1D, C-1F), 78.4, 75.8, 73.0,
71.8, 71.3, 68.5, 66.2, 62.7, 59.2, 55.3, 27.2, 26.9 (CH3); HRMS [M]3−
C45H66N4Na3O52S63− calcd. 585.0226, obsd. 585.0218.
79.1, 78.5, 76.9, 76.89, 76.88, 76.87, 76.8, 76.1, 75.8, 75.0, 74.9, 74.5,
74.1, 73.1, 72.4, 71.5, 71.2, 71.1, 70.5, 69.9, 67.9, 67.7, 67.2, 67.1, 63.6,
62.9, 62.4, 61.8, 60.4, 29.7, 21.0, 20.9, 20.8, 14.2. HRMS [M + H]+
+
C102H03N7O27 calcd. 1858.6935 obsd. 1868.6931. By treating 9 mg
(4.8 μmol) of N-(benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-
acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl
2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-
2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-
benzoyl-3-O-benzyl-α-L-idopyranosyluronate with the general proce-
dures for benzylation, 1,3-dithiopropane mediated azide reduction,
N,O-sulfation, and finally global debenzylation and saponification
1
produced 3 mg of 23 in 49% yield (34% from 11). H NMR δH
(500 MHz, D2O) 5.35 (1 H, d, J 3.1), 5.28 (1 H, d, J 3.2), 5.09 (1 H, s),
4.96−4.92 (1 H, m), 4.38 (1 H, d, J 3.0), 4.29−4.18 (3 H, m), 4.18−4.13
(2 H, m), 4.09−4.05 (3 H, m), 4.03−3.95 (2 H, m), 3.95−3.81 (3 H, m),
3.73−3.56 (3 H, m), 3.55−3.50 (1 H, m), 3.45 (1 H, t, J 9.4), 3.23 (1 H,
d, J 0.7), 3.16 (1 H, dd, J 10.5, 2.7), 3.12 (1 H, dd, J 9.8, 3.8), 3.09−3.00
(2 H, m), 1.92−1.84 (2 H, m). δC (values obtained from F1 dimension
of HSQC spectrum) 101.0, 100.9, 98.6, 97.8, 79.3, 77.7, 77.6, 72.6,
71.60, 71.55, 71.3, 71.0, 70.9, 70.8, 68.3, 68.2, 68.1, 59.66, 59.61, 40.0,
27.8. HRMS C27H41N3O39Na3S63− calcd. 430.6450 obsd. 430.6460.
3-Aminopropyl 2-Deoxy-2-sulfoamino-α-D-glucopyranosyl-(1→
4)-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-α-D-
glucopyranosyl-(1→4)-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-
sulfoamino-α-D-glucopyranosyl-(1→4)-2-O-sulfonate-α-L-
idopyranosyluronate (24). Compound 24 was prepared from
compound 18 (43 mg, 15.7 μmol). Treatment of 18 with the general
procedure for saponification, followed by Staudinger reduction,
provided 28 mg of a hexasaccharide intermediate in a 95% yield over
3-Aminopropyl 2-Deoxy-2-sulfoamino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate-(1→4)-2-
deoxy-2-sulfoamino-6-O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-
sulfonate-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-
6-O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-sulfonate-α-L-
idopyranosyluronate (22). Treatment of compound 19 (5 mg,
1.85 μmol) with the procedures for global debenzylation and methyl
ester saponification provided 2.4 mg of 22 in 63% yield from 19. NMR
analysis showed that the anomeric carbons of 22 gave chemical shifts of
101.8 (1 carbon), 101.3 (2 carbons), 100.8 (1 carbon), 100.0 (1 carbon)
and 99.5 (1 carbon) ppm, respectively, which suggests that the
glucosamines were N-sulfated. These values were consistent with
literature reports,67,68 where anomeric carbons of unsulfated glucos-
amines in heparin resonate around 94 ppm, while those of N-sulfated
glucosamines typically appear above 100 ppm. 1H NMR δH (500 MHz,
D2O) 5.38−5.26 (2 H, m), 5.21 (1 H, d, J 3.3), 5.13 (1 H, d, J 3.7), 5.10
(1 H, s), 5.07−5.02 (1 H, m), 4.83 (1 H, d, J 3.1), 4.38 (1 H, d, J 16.7),
4.29−4.16 (8 H, m), 4.14−4.11 (1 H, m), 4.08 (1 H, s), 4.04 (1 H, s),
3.88−3.83 (1 H, m), 3.80−3.73 (7 H, m), 3.72−3.65 (2 H, m), 3.61
(3 H, t, J 9.4), 3.55−3.50 (2 H, m), 3.49−3.43 (2 H, m), 3.16 (2 H, dd,
J 10.3, 3.2), 3.12 (1 H, dd, J 10.2, 3.3), 3.09−3.05 (1 H, m), 1.97−1.86 (2
H, m). δC (values obtained from F1 dimension of HMQC spectrum) δ
101.8, 101.3, 100.8, 100.0, 99.5, 78.2, 78.0, 76.0, 71.2, 71.1, 70.7, 69.2,
−1
the two steps. HRMS [M − H]−1 C103H119N4O33 calcd. 1940.7796
obsd. 1940.7793. The hexasaccharide intermediate (4.3 mg, 2.3 μmol)
was then dissolved in 0.5 mL of MeOH and cooled to 0 °C. The pH of
the solution was brought to 9.5 by addition of 1 M aqueous solution of
NaOH. Next, 6.3 mg of sulfur trioxide triethylamine complex (5 equiv
per amine) was added, and pH was maintained at 9.5 by addition of
more 1 M NaOH as needed. The reaction was allowed to warm to room
temperature and stirred overnight. By TLC (3:1:1 EtOAc:MeOH:H2O
1%AcOH), the reaction was incomplete. An additional 2.5 mg of sulfur
trioxide triethylamine was added, and the reaction was stirred for an
additional 12 h. The reaction was diluted with 1:1 DCM:MeOH and
eluted from a Sephadex LH-20 column with the same mixture. The
product of selective N-sulfation was fully deprotected by global
debenzylation, providing 2 mg of 24 in 70% yield over the two steps.
1H NMR δH (900 MHz, D2O) 5.26 (1 H, d, J 3.5, H-1F), 5.25 (1 H, d,
J 3.5, H-1D), 5.20 (1 H, d, J 3.6, H-1B), 4.87 (4 H, d, J 7.9, H-1C, H-1E,
H-5C, H-5E), 4.80 (1 H, s, H-1A), 4.44 (1 H, s, H-5A), 4.07 (1 H, t, J 2.9,
H-3A), 4.05−4.01 (2 H, m, H-3C, H-3E), 3.98−3.94 (2 H, m, H-4C, H-
4E), 3.93 (1 H, s, H-4A), 3.78 (1 H, ddd, J 10.0, 7.9, 4.7, H-linker), 3.72−
3.55 (16 H, m, H-2A, H-4B, H-5B, H-6B, H-2C, H-4D, H-5D, H-6D,
H-2E, H-5F, H-6F, H-linker), 3.55−3.52 (1 H, m, H-3D), 3.49 (1 H, t,
J 9.8, H-3F), 3.38 (1 H, t, J 9.6, H-4F), 3.14−3.10 (2 H, m, H-2B, H-2D),
3.09 (1 H, dd, J 10.4, 3.5, H-2F), 3.06−3.02 (2 H, m, CH2-linker), 1.92−
1.84 (2 H, m, CH2-linker). 13C NMR (225 MHz, D2O) δ 176.3, 175.7,
103.0 (C-1C), 102.9 (C-1E), 101.9 (C-1A), 97.4 (C-1D, C-1F), 97.3
(C-1B), 78.5, 78.4, 76.2, 76.0, 75.9, 73.4, 72.7, 72.66, 72.5, 71.2, 71.1,
71.0, 70.3, 70.2, 69.9, 69.5, 69.3, 69.0, 68.4, 68.0, 61.6, 61.2, 61.1, 59.5,
4−
67.9, 59.6, 54.9, 54.7, 40.2, 27.8. HRMS [M]4− C39H55N4Na7O58S9
calcd. 488.9526, obsd. 488.9573.
3-Aminopropyl 2-Deoxy-2-sulfoamino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate-(1→4)-2-
deoxy-2-sulfoamino-6-O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-
sulfonate-α-L-idopyranosyluronate (23). Compound 23 was prepared
from 11 in 6 steps. First, 99 mg (50 μmol) of 11 was treated with the
general procedures for levulinoyl ester removal, 6-O oxidation, benzyl
ester formation, and, lastly, TBS removal. This provided 65 mg of
product, N-(benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-
azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-ben-
zoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-
O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-
1
benzyl-α-L-idopyranosyluronate, in a 69% yield from 11. H NMR
(500 MHz, CDCl3) δ 8.23−8.02 (m, 5H), 7.58−7.10 (m, 45H), 5.55 (d,
J = 5.4 Hz, 1H, H-1B), 5.24 (t, J = 5.3 Hz, 1H, H-2C), 5.17−5.12 (m,
4H), 5.09−5.05 (m, 3H, H-1A), 4.94 (d, J = 3.7 Hz, 1H, H-2D), 4.81−
4.79 (m, 2H), 4.72−4.68 (m, 1H), 4.66 (d, J = 3.4 Hz, 1H, H-5C), 4.64
(d, J = 4.9 Hz, 1H, H-1B), 4.50 (t, J = 10.9 Hz, 2H), 4.47−4.38 (m, 5H),
4.37−4.33 (m, 1H), 4.19−4.16 (m, 1H, H-3C), 4.15−4.11 (m, 3H,
H-2A), 4.09−4.07 (m, 1H, H-4C), 3.99−3.93 (m, 2H), 3.88−3.83 (m,
2H), 3.80−3.71 (m, 2H), 3.53−3.45 (m, 3H), 3.45−3.37 (m, 2H),
3.35−3.26 (m, 2H), 3.19 (t, J = 3.4 Hz, 1H, H-2B), 3.17 (t, J = 3.7 Hz,
1H, H-2D), 2.76 (d, J = 4.6 Hz, 1H, OH), 2.13 (s, 3H), 2.06 (s, 3H),
1.86−1.77 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 171.8, 171.1, 170.8,
160.0, 168.8, 165.4, 165.3, 137.9, 137.8, 137.2, 135.2, 134.9, 133.6, 130.1,
129.9, 129.6, 129.3, 128.83, 128.78, 128.74, 128.69, 128.67, 128.63,
128.58, 128.51, 128.49, 128.45, 128.44, 128.41, 128.37, 128.36, 128.3,
128.2, 128.14, 128.10, 128.0, 127.95, 127.87, 127.85, 127.8, 127.5,
127.33, 127.32, 127.29, 127.27, 127.26, 127.24, 127.23, 99.9, 99.4, 98.5,
3−
59.46, 59.3, 57.0, 39.8, 27.7. HRMS [M]3− C39H63N4O40S3 calcd.
441.0732, obsd. 441.0717.
3-Aminopropyl 2-Deoxy-2-sulfoamino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfo-
amino-6-O-sulfonate-α-D-glucopyranosyl-(1→4)-α-L-idopyrano-
syluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-α-L-idopyranosyluronate (25). Compound 25 was
prepared from 0.5 mg of 24 using the general procedure for enzymatic
sulfation and 6-OST-1 and 6-OST-3 as the enzymes. This produced 25
in 67% yield after purification. 1H NMR δH (900 MHz, D2O) 5.23 (1 H,
d, J 3.6, H-1B), 5.22 (1 H, d, J 3.7, H-1D), 5.20 (1 H, d, J 3.5, H-1F), 4.98
(1 H, s, H-1C), 4.95 (1 H, s, H-1E), 4.83−4.75 (2 H, m, H-1A), 4.36 (1
H, s, H-5A), 4.26−4.20 (3 H, m, H-6B, H-6D, H-6F), 4.11−4.07 (2 H,
m, H-6B, H-6D), 4.07−4.03 (2 H, m, H-3A, H-6F), 4.03−4.01 (1 H, m),
3.97−3.91 (3 H, m, H-5B, H-5D, H-5F), 3.91−3.88 (1 H, m, H-4A),
M
DOI: 10.1021/acs.joc.5b02172
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
3.83−3.80 (1 H, m, H-4B), 3.80−3.75 (2 H, m), 3.74−3.69 (3 H, m,
H-2C, H-2E), 3.65−3.62 (2 H, m), 3.62−3.54 (5 H, m, H-2A), 3.54−
3.51 (1 H, m, H-3B), 3.47−3.44 (1 H, m, H-2B), 3.15−3.09 (3 H, m,
H-2B, H-2D, H-2F), 3.05−3.00 (2 H, m, CH2-linker), 1.90−1.85 (2 H, m,
CH2-linker). δC (values obtained from F1 dimension of HSQC spec-
trum) 103.1 (C-1C, C-1E), 101.7 (C-1A), 96.7 (C-1B, C-1D, C-1F), 78.7,
75.7, 75.6, 72.2, 71.1, 70.1, 70.0, 69.6, 69.3, 69.2, 68.9, 68.3, 67.8, 58.8,
δC (values obtained from F1 dimension of HMQC spectrum) 100.5
(C-1A), 98.4 (C-1B), 78.1 (C-2A), 77.6 (C-4A), 72.7 (C-3B), 71.8
(C-5B), 70.9 (C-4B), 70.6 (C-5A), 70.5 (C-3A), 68.3 (C-linker), 68.2
(C-6B), 59.7 (C-2B), 40.2 (C-linker), 27.8 (C-linker). HRMS [M]+
C15H25N2O20Na4S3+ calcd. 740.9748 obsd. 740.9734.
ASSOCIATED CONTENT
3−
27.8. HRMS [M]3− C39H60N4Na3O49S6 calcd. 543.0120, obsd.
■
S
* Supporting Information
543.0130.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b02172.
3-Aminopropyl 2-Deoxy-2-sulfoamino-α-D-glucopyranosyl-(1→
4)-2-O-sulfonate-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfo-
amino-α-D-glucopyranosyl-(1→4)-2-O-sulfonate-α-L-idopyrano-
syluronate-(1→4)-2-deoxy-2-sulfoamino-α-D-glucopyranosyl-(1→
4)-2-O-sulfonate-α-L-idopyranosyluronate (26). Compound 26 was
prepared from 500 μg of compound 24 following the general procedure
for enzymatic sulfation using 2-OST as the enzyme. This provided 26 in
82% yield after purification. 1H NMR δH (900 MHz, D2O) 5.22 (1 H, d,
J 3.6, H-1F), 5.19 (2 H, d, J 3.4, H-1B, H-1D), 5.16 (2 H, m, H-1C,
H-1E), 5.15 (1 H, s), 4.78 (1 H, s, H-1A), 4.77−4.74 (4 H, m, H-5B,
H-5D, H-6B, H-6D), 4.36−4.34 (1 H, m, H-5A), 4.22 (2 H, s, H-2C, H-2E),
4.13 (2 H, dd, J 7.7, 4.2, H-3C, H-3E), 4.06−4.04 (1 H, m, H-3A), 3.93
(3 H, d, J 10.2, H-4A, H-4C, H-4E), 3.81−3.65 (10 H, m, H-5B, H-5D),
3.62−3.57 (4 H, m, H-2A, H-3F), 3.54 (2H, t, J 9.8, H-3B, H-3D) 3.35
(2 H, t, J 9.8, H-4B, H-4D), 3.13 (1 H, dd, J 10.3, 3.1, H-2F), 3.12−3.08
(2 H, m, H-2B, H-2D), 3.04−3.00 (2 H, m, linker CH2), 1.91−1.86
(2 H, m, linker CH2). δC (values obtained from F1 dimension of HMQC
spectrum) 102.5 (C-1A), 101.0 (C-1C, C-1E), 99.5 (C-1B, C−D), 97.7
(C-1F), 79.2, 77.6, 76.7, 73.4, 73.1, 72.2. 70.5, 69.8, 69.1, 61.4, 60.0, 39.8,
Additional glycan microarray images and selected NMR
spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: liuj@email.unc.edu. Tel: 919-843-6511. Fax: 919-843-
6511 (J.L.).
*E-mail: xuefei@chemistry.msu.edu. Tel: 517-355-9715, ext.
329. Fax: 517-353-1793 (X.H.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
3−
27.8. HRMS [M]3− C39H59N4Na4O46S5 calcd. 523.6870, obsd.
This work was supported by the National Science Foundation
(CHE 1507226) and the National Institute of General Medical
Sciences, NIH (R01GM072667, U01GM116262, and
U01GM102137). We are grateful for the helpful suggestions
523.6890.
3-Aminopropyl 2-Deoxy-2-sulfoamino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate-(1→4)-2-
deoxy-2-sulfoamino-6-O-sulfonate-α-D-glucopyranosyl-(1→4)-2-O-
sulfonate-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-
α-D-glucopyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate
(27). Compound 27 was prepared from 0.5 mg of compound 26 using
the procedure for enzymatic sulfation. Utilizing enzymes 6-OST-1 and
6-OST-3 provided a 39% yield of 27 after purification. δH (900 MHz,
D2O) 5.30 (1 H, d, J 3.1, H-1F), 5.22 (1 H, d, J 3.8, H-1D), 5.20 (1 H, d, J
3.3, H-1B), 5.18 (1 H, s, H-1C), 5.14 (1 H, s, H-1E), 4.78 (1 H, d, J 2.2,
H-1A), 4.77 (1H, s, H-5C) 4.74 (1 H, s, H-5E), 4.35 (1 H, d, J 2.1,
H-5A), 4.30 (2 H, d, J 11.3, H-6D, H-6F), 4.26−4.21 (2 H, m, H-2C, H-2E),
4.15 (2 H, d, J 10.6, H-6D, H-6F), 4.13−4.08 (2 H, m, H-3C, H-3E),
4.06−4.04 (1 H, m, H-3A), 3.99 (1 H, d, J 2.3, H-4E), 3.96−3.94 (1 H,
m, H-4C), 3.93−3.92 (1 H, m, H-4A), 3.91−3.87 (2 H, m, H-5D, H-5F),
3.82−3.76 (2 H, m, H-5B, linker), 3.73−3.71 (2 H, m, H-4B, H-6B),
3.70−3.66 (1 H, m, H-4D), 3.63−3.56 (5 H, m, H-2A, H-3B, H-3D,
H-6B, H-linker), 3.53 (1 H, t, J 9.9, H-3F), 3.46 (1 H, t, J 9.6, H-4F), 3.17
(1 H, dd, J 10.3, 3.0, H-2D), 3.13 (1 H, dd, J 10.4, 3.3, H-2F), 3.10 (1 H,
dd, J 9.5, 3.3, H-1B), 3.05−3.00 (2 H, m, CH2-linker), 1.91−1.86 (2 H,
m, CH2-linker). δC (values obtained from F1 dimension of HMQC
spectrum) 102.2 (C-1A), 100.9 (C-1C, C-1E), 99.2 (C-1B, C-1D), 97.5
(C−F), 77.8, 77.5, 77.4, 76.5, 72.8, 71.5, 71.4, 71.2, 71.1, 70.8, 70.5, 70.3,
70.1, 69.8, 69.1, 68.1, 61.7, 59.6, 40.2, 27.8. HRMS [M]3−
C39H59N4Na4O52S73− calcd. 576.9916, obsd. 576.9904.
3-Aminopropyl 2-Deoxy-2-sulfoamino-6-O-sulfonate-α-D-gluco-
pyranosyl-(1→4)-2-O-sulfonate-α-L-idopyranosyluronate (28).
Compound 28 was prepared from 3. 3 (47 mg, 46 μmol) was treated
with the conditions for benzylation, followed by the general procedure
to remove levulinoyl esters. The product was then oxidized and
protected as a benzyl ester according to the procedures for oxida-
tion and benzyl ester formation. Treatment of the oxidized product
sequentially with the general procedures for saponification, Staudinger
reduction, O-sulfation, N-sulfation, and finally global debenzylation
provided 9 mg of 28 in 34% yield from 3. 1H NMR δH (600 MHz, D2O)
5.29 (1 H, d, J 3.6, H-1B), 4.99 (1 H, d, J 3.1, H-1A), 4.37 (1 H, d, J 2.8,
H-5A), 4.24−4.20 (1 H, m, H-6B), 4.16−4.11 (1 H, m, H-2A), 4.11−
4.05 (2 H, m, H-6B, H-3A), 4.00−3.95 (1 H, m, H-4A), 3.88−3.76 (2 H,
m, linker-H, H-5B), 3.62−3.55 (1 H, m, linker-H), 3.52 (1 H, dd, J 10.1,
9.3, H-3B), 3.44 (1 H, dd, J 10.1, 9.3, H-4B), 3.13 (1 H, dd, J 10.3, 3.5,
H-2B), 3.06−3.02 (2 H, m, linker-CH2), 1.91−1.85 (2 H, m, linker-CH2);
from Dr. David Bonnaffe.
́
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