Synthesis and mass spectrometric analysis of disaccharides from methanolysis of heparan sulfate

Article abstract of DOI:10.1039/c8ob02225a

The quantification of heparan sulfate (HS) in biological matrices, e.g., urine, cerebrospinal fluid, tissue samples etc., is of great importance for the diagnosis and prognosis of several of the mucopolysaccharidosis (MPS) disorders, which are lysosomal storage diseases of impaired glycosaminoglycan metabolism. The development of suitable assays for this purpose is challenging due to the high molecular weight and complexity of HS. Recent efforts towards this goal include the acid catalysed methanolysis of HS, which desulfates the polymer and results in the formation of disaccharide cleavage products which can be detected and quantified by LC-MS/MS. We have synthesized a library of 12 HS-derived disaccharides as methanolysis standards via the stereoselective 1,2-cis glycosylation of suitably protected GlcA and IdoA acceptors with a 2-deoxy-2-azido thioglucoside donor. This facilitated identification of the major peaks in the LC-MS/MS chromatograms, and potentially will allow the monitoring of specific metabolites as surrogate markers for genotype. This work also paves the way towards a fully quantitative LC-MS/MS assay for HS via the preparation of a suitably labelled derivative.

Full text of DOI:10.1039/c8ob02225a

Organic &
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PAPER
Synthesis and mass spectrometric analysis of
disaccharides from methanolysis of heparan
sulfate†
Cite this: Org. Biomol. Chem., 2018,
16, 8791
a
Qi Qi He,a Paul J. Trim,b Marten F. Snel,b John J. Hopwoodb and Vito Ferro
*
The quantification of heparan sulfate (HS) in biological matrices, e.g., urine, cerebrospinal fluid, tissue
samples etc., is of great importance for the diagnosis and prognosis of several of the mucopolysacchari-
dosis (MPS) disorders, which are lysosomal storage diseases of impaired glycosaminoglycan metabolism.
The development of suitable assays for this purpose is challenging due to the high molecular weight and
complexity of HS. Recent efforts towards this goal include the acid catalysed methanolysis of HS, which
desulfates the polymer and results in the formation of disaccharide cleavage products which can be
detected and quantified by LC-MS/MS. We have synthesized a library of 12 HS-derived disaccharides as
methanolysis standards via the stereoselective 1,2-cis glycosylation of suitably protected GlcA and IdoA
acceptors with a 2-deoxy-2-azido thioglucoside donor. This facilitated identification of the major peaks
in the LC-MS/MS chromatograms, and potentially will allow the monitoring of specific metabolites as sur-
rogate markers for genotype. This work also paves the way towards a fully quantitative LC-MS/MS assay
for HS via the preparation of a suitably labelled derivative.
Received 9th September 2018,
Accepted 30th October 2018
DOI: 10.1039/c8ob02225a
rsc.li/obc
(GlcA) or α-L-iduronic acid (IdoA), (1→4)-linked to
Introduction
D-glucosamine (GlcN) (Fig. 1).3,4 The high molecular weight
Heparan sulfate (HS) is a highly complex polysaccharide of the and remarkable degree of structural heterogeneity of HS
glycosaminoglycan (GAG) family that is a marker for a number makes its analysis and quantification extremely challenging.
of lysosomal storage diseases, most notably several of the In order to simplify the analytical problem, a number of de-
mucopolysaccharidoses (MPS), including MPS types I, II, polymerisation methods using enzymatic5,6 and chemical
IIIA–D and VII.1 The MPS disorders are rare diseases that methods7 have been employed. Of particular note is the devel-
affect approximately 1 in 25 000 people and are characterized opment of a simple method for methanolysis of HS in urine
by genetic defects in GAG-degrading enzymes. This results in and cerebrospinal fluid samples of MPS patients,8–11 and its
impaired GAG degradation and lysosomal accumulation of recent adaptation for use with solid tissue samples.12 Recently,
GAGs with associated abnormalities in multiple organ systems this method has been used to monitor the effectiveness of
and reduced life expectancy. As a direct result of disease, there enzyme replacement therapies in animal models of MPS
are higher levels of HS in biological samples from MPS disorders.13–15 Methanolysis degrades HS into simple de-
patients, such as urine, blood and cerebrospinal fluid.2 The sulfated and deacetylated disaccharides (Scheme 1), which can
quantification of HS concentrations in such samples is thus then be analysed by liquid chromatography/tandem mass
very important for diagnosis and prognosis of MPS disorders spectrometry (LC-MS/MS).
and for monitoring the efficacy of new therapies.
HS is a complex, linear polysaccharide composed of repeat-
ing disaccharide subunits of uronic acid, β-D-glucuronic acid
aSchool of Chemistry and Molecular Biosciences, The University of Queensland,
Brisbane, QLD 4072, Australia. E-mail: v.ferro@uq.edu.au
bHopwood Centre for Neurobiology, South Australian Health and Medical Research
Institute, Adelaide, SA 5000, Australia
†Electronic supplementary information (ESI) available: Experimental and
characterization details for compounds 13, 17–24, 29, 37, 42, 44, 46. Copies of
1H and 13C NMR spectra for compounds 1–14, 17–30, 32, 34–48. LC-MS/MS chro-
matograms for compounds 1–12. See DOI: 10.1039/c8ob02225a
Fig. 1 The structure of heparan sulfate (HS) depicting the major non-
sulfated and variant disaccharide sequences.
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charides are of “type I” where cleavage has occurred at the
more labile β-D- or α-L-glycosidic bonds. Under the conditions
of the methanolysis reaction (3 N methanolic HCl, reflux) the
newly formed methyl glycosidic bonds are expected to be pre-
dominantly α. However, as the formation of minor amounts of
β-linked products cannot be ruled out, these anomers were
also targeted for synthesis. Similarly, while previous MS/MS
analyses indicated that N-acetyl groups are cleaved under the
reaction conditions, the presence of minor N-acetylated pro-
ducts could not be ruled out. Such compounds should in any
case be readily accessible via simple N-acetylation of the major
disaccharides. Disaccharides of “type II” were only expected as
minor products, if present at all. Therefore, only the more
readily available, and likely more abundant, α-methyl glyco-
sides were prepared.
Scheme 1 A. Methanolysis of HS to give desulfated, methylated disac-
charides resulting from cleavage of uronic acid glycosidic bonds (type I)
or GlcN glycosidic bonds (type II); B. MS/MS of the major peak observed
from the methanolysis of heparan sulfate.
The methanolysis of HS greatly reduces the complexity of the
sample to be analysed, however, it can yield several isobaric and
isomeric disaccharide products. Previous MS/MS studies12 indi-
cate that the GlcN-(1→4)-uronic acid disaccharides (Scheme 1,
type I) are the major reaction products of HS methanolysis of
Synthesis of methyl GlcN-GlcA disaccharides
HS, however, the identities of the minor disaccharide products Synthetic access to the target disaccharides requires the use of
are unknown. The identities of these minor products could suitable glycosyl donors and acceptors with the correct combi-
provide additional disease specific information and are thus of nation of protecting groups to ensure stereocontrol of the key
great interest. The lack of authentic disaccharide standards has glycosylation reaction. The readily available16,17 thioglycoside
also hampered attempts to develop a quantitative assay and to 13 was converted into the glycosyl donor 14 in 80% yield by
control assay variability. Therefore, the aims of the current study treatment with benzyl bromide and sodium hydride. Donor 14
were to synthesize and fully characterize a series of methylated has a non-participating 2-azido group to facilitate formation of
HS disaccharide standards to (i) verify the identity of the major the desired 1,2-cis glycosidic linkage and ready installation of
peaks in the LC-MS/MS chromatograms from the methanolysis the desired 2-amino or acetamido groups. The α-methyl GlcA
assay, (ii) to determine the structures of the products corres- acceptor18 23 and the β-methyl GlcA acceptor19 24 were both
ponding to the minor peaks, and (iii) to facilitate HS quanti- prepared, respectively, via a similar sequence of reactions from
tation by the methanolysis digestion followed by LC-MS/MS ana- commercially available methyl α-D-glucopyranoside 15 and
lysis with the addition of known internal standards.
methyl β-D-glucopyranoside 16, respectively, as shown in
Scheme 2. 4,6-O-p-Methoxybenzylidenation,20 followed by ben-
zylation21 and acid hydrolysis22 gave the 4,6-diols 21 and 22 in
good yield (91–94%). Next, TEMPO oxidation of the primary
alcohol in the presence of excess BAIB as co-oxidant,23,24 fol-
Results and discussion
The disaccharides targeted for synthesis (1–12) are shown in lowed by base-promoted esterification25 gave the target accep-
Fig. 2. A flexible synthetic strategy was developed in order to tors 23 and 24 in acceptable overall yield (52–80%).
maximise the number of possible disaccharides obtained from
The preparation of the target methyl GlcN-GlcA disacchar-
common intermediates. As discussed above, the major disac- ides was carried out as illustrated in Scheme 3. The NIS/TfOH-
Scheme 2 Preparation of glycosyl donor 14 and α- and β-methyl GlcN
acceptors 23 and 24. Reagents and conditions: (a) NaH, BnBr, DMF, r.t.,
3 h, 98–100%; (b) anisaldehyde dimethyl acetal, CSA, DMF, r.t., 18 h,
39–52%; (c) TFA/H2O 1 : 1, r.t., 18 h, 91–94%; (d) i. TEMPO, BAIB, DCM/
Fig. 2 Structures of target disaccharides from methanolysis of HS.
8792 | Org. Biomol. Chem., 2018, 16, 8791–8803
H2O 3 : 1, r.t., 2 h; ii. MeI, KHCO3, DMF, r.t., 18 h, 52–80%.
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Scheme 4 Preparation of methyl IdoA acceptors 37 and 38. Reagents
and conditions: (a) NaH, BnBr, DMF, r.t., 4 h, 82%; (b) MeOH, NIS/TfOH,
DCM, −78 → −20/−10 °C, 2–3 h, 98% (32), 68% (33); (c) DCM/TFA/H2O
100 : 10 : 1, r.t., 30 min, 91–100% (35-α, 36); or 80% HOAc/H2O, 100 °C,
24 h, 51% (34-β) (d) i. TEMPO, BAIB, DCM/H2O 3 : 1, r.t., 3 h; ii. MeI,
KHCO3, DMF, r.t., 24 h, 46–67% (2 steps).
Scheme 3 Synthesis of methyl GlcN-GlcA disaccharides 1–4. Reagents
and conditions: (a) NIS/TfOH, DCM, −78 → −20 °C, 2 h, 94–95%; (b) H2,
10% Pd/C, MeOH, r.t., 24 h, 51%; (c) Ac2O, Et3N, MeOH, r.t., 90 min,
100% (2), 62% (4, 2 steps); (d) H2, 20% Pd(OH)2/C, MeOH, conc. HCl, r.t.,
21 h, 60%.
or L-IdoA derivatives was confirmed by 1D NOESY spec-
troscopy: all β-L-configured glycosides, which favour the 1C4
chair conformation in solution, show an NOE signal from H-1
to H-5, while the corresponding α-L-glycosides do not.29
Interestingly, the anomeric configuration had a marked effect
on the conformation of the product (Table 1). The β-anomer
activated glycosylation26,27 of thioglycoside donor 14 and
α-methyl GlcA acceptor 23 proceeded in excellent yield (94%)
but with moderate selectivity, giving a ∼2 : 1 mixture of
α:β-linked GlcN-GlcA disaccharides (25-α, 26-β). Following
careful flash chromatography, the desired28 α-isomer 25 (J1′,2′
3.7 Hz) was obtained in 49% yield along with 26 (24%, J1′,2′
=
=
1
(32-β) was shown to favour the C4 conformation in solution,
2
while the α-anomer (32-α) favoured the So skew-boat confor-
7.9 Hz) and a mixed fraction (21%). The structure of 25 was
confirmed by comparison of the NMR and MS data with the
literature.28 Next, 25 was subjected to hydrogenolysis with 10%
Pd/C catalyst to cleave all the benzyl protecting groups and
simultaneously reduce the azide to the free amine, to furnish
the target disaccharide 1 in moderate yield (51%). A sample of
amine 1 was converted into the acetamide 2 in quantitative
yield by treatment with acetic anhydride and Et3N in
methanol.
The β-methyl disaccharides 3 and 4 were similarly prepared
(Scheme 3). Glycosylation of thioglycoside 14 with acceptor 24
promoted by NIS and TfOH also proceeded in excellent yield
(95%) but with significantly improved selectivity (α : β = 9 : 1).
The desired disaccharide 27 was separated by flash chromato-
graphy and subjected to global deprotection by hydrogenolysis
to give disaccharide 3 in 60%, while a sample was acetylated to
give disaccharide 4 in 62% overall yield. The target methyl
GlcN-GlcA disaccharides 1–4 were fully characterized via 1D
and 2D NMR spectroscopy and HRMS experiments.
mation,30,31 based on its unusual coupling constants (J2,3
=
9.8 Hz, J3,4 = 4.7 Hz)32 and diagnostic NOE signal between
H-2 and H-5.33 After acid catalysed hydrolysis of the iso-
propylidene protecting group, the product 35-α favoured the
1C4 chair conformation (J2,3 = 3.8 Hz) in solution. This route
provided insufficient material to continue through to the
desired α-linked acceptor, however, similar acid catalysed
hydrolysis of 32-β followed by TEMPO/BAIB oxidation and
base-promoted esterification gave the desired β-methyl IdoA
acceptor 37,34 in 67% yield.
To access an α-methyl IdoA acceptor, donor 31 was glycosy-
lated with MeOH using NIS/TfOH as the promoter to give a
2 : 1 mixture of α- and β-methyl glycosides 33 in 68% yield.29
Although 31 has a participating benzoyl group at C-2 which
favours formation of the α-L-glycoside, recent studies29 have
shown that neighbouring group participation is of lesser
importance in glycosylations between Ido-configured donors
and primary alcohol acceptors. The products 33-α and -β were
separated by flash chromatography, and 33-α was taken
Synthesis of methyl GlcN-IdoA disaccharides
Table 1 Coupling constants (J, Hz) measured from 1H NMR spectra
Initially it was decided to attempt to prepare both the
required α- and β-L-IdoA acceptors from a common intermedi- (CDCl3 or CD3OD) for the L-idose or L-iduronic acid rings of selected
compounds, with indicative conformations in parentheses
ate, i.e., a glycosyl donor with a non-participating group at
C-2 to favour formation of an anomeric mixture of methyl gly-
32-α
(2So)
32-β
(1C4)
35-α
(1C4)
39
7
8
cosides (Scheme 4). Thus, donor 30, prepared by benzylation
of the alcohol 29,17 was glycosylated with MeOH using NIS/
TfOH as the promoter to furnish 32 as a 1 : 4 mixture of
α/β-anomers which were separated by flash chromatography.
Throughout this study, the anomeric configuration of L-idose
(2So/1C4)
(1C4)
(1C4)
J1,2 4.9
J2,3 9.8
J3,4 4.7
1.5
3.9
2.1
2.1
1.4
3.8
—
3.1
7.3
7.3
4.5
1.6
3.4
3.4
2.0
1.6
3.3
3.3
2.3
J4,5
—
—
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Fig. 3 Structures of intermediates for the synthesis of methyl
IdoA-GlcN and GlcA-GlcN disaccharides.
Scheme 5 Synthesis of target GlcN-IdoA disaccharides 5–8. Reagents
and conditions: (a) NIS/TfOH, DCM, −78 → −20 °C, 2 h, 52% (39), 72%
(40); (b) H2, 20% Pd(OH)2/C, +/−conc. HCl, MeOH, r.t., 24 h, 98–100%;
(c) Ac2O, Et3N, MeOH, r.t., 90 min, 39–72; (d) NaOMe/MeOH, r.t., 5 h,
82%.
to give 45 in excellent yield (93%). It was recognized that methyl
thioglycoside 45 could be converted into a more efficient donor
(e.g., a trichloroacetimidate), however, given the small quantities
of final product required, it was decided to utilise it directly in
the next step. Thus, glycosylation of donor 45 with the readily
available acceptor 46 17,29 with NIS and TMSOTf as the promoter
gave the β-linked disaccharide 47 in a modest but acceptable
31% yield (J1′,2′ = 7.9 Hz). Debenzoylation followed by hydro-
genolysis (with Pearlman’s catalyst) in the presence of HCl or
Ac2O as described above for 42 gave the target disaccharides 11
and 12 in 90% and 48% yield, respectively. The structures were
confirmed by 1D and 2D NMR spectroscopy and HRMS.
through the following synthetic steps. Acid catalysed hydrolysis
of the isopropylidene group to give the diol 36 (91%) was fol-
lowed by regioselective TEMPO/BAIB oxidation of the primary
alcohol and base-promoted esterification with iodomethane to
furnish α-methyl IdoA acceptor 38 35 in 46% yield.
The GlcN-IdoA disaccharides were then prepared as shown
in Scheme 5. Unlike the glycosylations with gluco-configured
acceptors, the NIS/TfOH-activated coupling of the thioglyco-
side 14 with acceptors 37 and 38 proceeded with excellent ster-
ocontrol and gave exclusively the α-linked disaccharide pro-
ducts (J1′,2′ = 3.5–3.6 Hz) 39 and 40 in acceptable yield
(52–72%). The disaccharides were deprotected and acetylated
in a similar fashion to the GlcN-GlcA disaccharides in good
overall yields to furnish the target disaccharides 5–8, which
were fully characterized by 1D and 2D NMR spectroscopy and
HRMS. Once again, interesting conformational effects were
evident in the NMR spectra. The conformations of the α-L-IdoA
series are consistent with 1C4 chairs for the IdoA ring,
LC-MS/MS analyses
The pure synthesized disaccharides 1–12 were each subjected to
LC-MS/MS analysis (see ESI†) which demonstrated their purity.
They were then spiked into a sample of HS that had been sub-
jected to methanolysis according to the published protocol12 in
order to identify the individual peaks in the chromatogram of
the latter. The results are summarized in Fig. 4. Interestingly, the
major peak at 1.56 min corresponds to disaccharide 7 with a
1
however, the protected β-L-IdoA ring of 39 is in between a C4
2
and So conformation,30 as indicated by a large coupling con-
stant for J2,3 = J3,4 = 7.3 Hz, while there are small coupling con-
stants for J1,2 = 3.1 Hz and J4,5 = 4.5 Hz (Table 1). Upon de-
protection to 7 and 8 the IdoA rings are once again observed
in the 1C4 chair conformation.
Synthesis of methyl IdoA-GlcN disaccharides
The target methyl IdoA-GlcN disaccharides were readily pre-
pared from disaccharide 42 17 via removal of the benzoate pro-
tecting groups under Zemplén conditions followed by hydroge-
nolysis (Pd/C) in methanol, either in the presence of conc. HCl
to give 9 (79%), or Ac2O to give 10 (58%), respectively (Fig. 3).
The structures of 9 and 10 were confirmed by 1D and 2D NMR
spectroscopy and HRMS.
Fig. 4 Identification of disaccharides by LC-MS/MS produced from the
methanolysis of heparan sulfate (solid red trace, MRM m/z 384–162)
compared to an internal standard of d6 deuterated heparan sulfate
digest (dotted blue trace, MRM m/z 390–162). Peaks have been ident-
ified by retention time comparison of the synthesised disaccharides
Synthesis of methyl GlcA-GlcN disaccharides
For the preparation of the methyl GlcA-GlcN disaccharides, the
thioglycoside 44 36 was benzoylated under standard conditions spiked with the d6 internal standard.
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β-linked methyl IdoA at the reducing end. The other major peak solvent peaks (CDCl3: δH 7.26 and δC 77.0; CD3OD: δH 3.3 and
at 1.13 min corresponds to disaccharide 5. Disaccharide 1, con- δC 49.0) served as internal standards. Coupling constants in
taining an α-linked methyl GlcA, was expected to be a major Hz were measured from one-dimensional spectra. The analyses
1
component due to the anomeric effect, yet it is only present in of H and 13C NMR spectra were assisted by COSY, HSQC ad
minor amounts (small peak at 1.40 min). Disaccharide identifi- NOE experiments. Analytical LRMS and HRMS were performed
cations were confirmed by performing an additional experiment. in a positive or negative ion ESI mode on a Bruker HCT
HS (10 µg) was digested using the methanolysis method and ana- spectrometer and a Bruker micrOTOFQ spectrometer, respect-
lysed. It was then spiked with disaccharides 1, 5 or 7 to a final ively. All reagents and solvents were obtained from Sigma-
concentration of 50 ng mL−1 and the peaks observed from the Aldrich, Australia, and were used without further purification,
digestion increased in area with each respective disaccharide except EtOAc, n-hexane, MeOH and DCM which were distilled
spiking experiment, further confirming the identification (see prior to use. Reactions were monitored by analytical thin layer
ESI, p. S37†). Given that GlcA is known to be the major uronic chromatography (TLC) on silica gel 60 F254 plates and visual-
acid component of HS,3 these results suggest that the glycosidic ized by charring with 10% sulfuric acid in ethanol. Flash
bond of IdoA is more labile than that of GlcA. The results also chromatography was performed on silica gel under positive
indicate that the disaccharides have not equilibrated fully to the pressure with specified solvent systems. Compounds 13, 29,
more stable α-methyl glycosides during the methanolysis reac- 31, 42, 44, 46 were gifts from Dr Mike West, Alchemia Ltd.
tion. In fact, they are largely in agreement with a recent study37 Preparation of deuterated heparan sulfate digested internal
during which the time course of HS methanolysis was deter- standard was made using a previously published method.12
mined. The amount of disaccharides produced during the reac- LC-MS/MS analyses were performed on an AP14000 QTrap
tion continually rose over a 24 hour period, not reaching a (Sciex, Concord, Ontario, Canada) coupled to a Waters Acquity
maximum, and was at lower levels compared with the disacchar- UPLC system (Milford, MA, USA) equipped with a BEH C18
ides produced at higher temperature via butanolysis.37 column (2.1 mm × 50 mm). Data were obtained in positive
N-Acetylated disaccharides were not present in significant electrospray ionization multiple reaction monitoring (MRM)
amounts. As expected, the four synthesized methyl uronic acid- mode. Transitions were monitored based on product/ion pairs
(1→4)-GlcN disaccharides 9–12 were not detected in the chroma- for the measurement of disaccharides. LC-MS/MS chromato-
tograms, confirming that glycosidic bond cleavage during metha- grams were selected based on the MRM transitions for
nolysis occurs preferentially at the uronic acid glycosidic bond.
amino disaccharides and N-acetate disaccharides, and they
are noted in figure captions. All disaccharides were made
up to 1 μg mL−1, injection volume of 10 μL was used.
Chromatographic separation was by means of a 8.5 min gradi-
ent going from 1% B to 5% B or 2 min, followed by a 0.01 min
Conclusions
In conclusion, acid catalysed methanolysis of HS results in step to 20% B followed by a gradient to 25% B over 3 min, a
exhaustive desulfation and cleavage of the polymer into a 2.5 min wash step of 99% B followed by a re-equilibration for
mixture of methylated disaccharides, which can be analysed 1 min at 1% B (mobile phase A = 0.1% formic acid (aq.) and
by LC-MS/MS. This process forms the basis of an assay for the mobile phase B = acetonitrile 0.1% formic acid).
quantitation of HS in biological samples with utility for the
4′-Methylphenyl 2-azido-3,4,6-tri-O-benzyl-2-deoxy-1-thio-β-D-
glucoside (14)
diagnosis and prognosis of MPS disorders and for monitoring
the efficacy of new therapies. We have synthesized a library of
HS-based disaccharides predicted to be the likely major pro- Thioglycoside 13 (1.0 g, 3.2 mmol) was dissolved in anhydrous
ducts of the methanolysis reaction. The key step in the syn- DMF (10 mL). A suspension of NaH (462 mg, 11.6 mmol, pre-
thesis of the library was the stereoselective 1,2-cis glycosylation washed with n-hexane (3 × 20 mL)) in anhydrous DMF (10 mL)
of GlcA or IdoA acceptors with a 2-deoxy-2-azido thioglucoside was added. The mixture was stirred at r.t. for 10 min, and then
donor, promoted by NIS/TMSOTf (or TfOH). The disaccharides benzyl bromide (1.4 mL, 11.6 mmol) was added dropwise
were used to identify the major peaks in the LC-MS/MS chro- under ice cooling. The stirring was continued for 10 min
matograms from methanolysis of HS and labelled versions under ice cooling, and for an additional 1 h at r.t. under N2.
may be suitable as internal standards for HS quantitation.
MeOH (10 mL) was added to quench the reaction. The result-
ing mixture was then diluted with EtOAc, washed with H2O,
1 M HCl, dried (MgSO4), and concentrated. It was then purified
by flash chromatography (EtOAc/n-hexane, 1 : 9) to give the tri-
benzyl ether 14 as colourless oil (1.50 g, 80%); Rf = 0.56
(EtOAc/n-hexane, 1 : 4); [α]D −53.6 (c 9.4, CHCl3; lit.21 −65.94,
Experimental section
General methods
Melting points were determined on a DigiMelt MSRS appar- c 1.0, CHCl3). The NMR data of 14 were in accord with the lit-
atus. Optical rotations were determined on a JASCO P-2000 erature.21 1H NMR (500 MHz, CDCl3): δ 7.50–7.02 (m, 19H, Ar),
polarimeter at ambient temperature and are given in units of 4.84, 4.81 (ABq, 2H, JAB = 10.5 Hz, CH2Ph), 4.76, 4.57 (ABq, 2H,
10−1 deg cm2 g−1. 1H and 13C NMR spectra were recorded on a JAB = 10.9 Hz, CH2Ph), 4.60, 4.52 (ABq, 2H, JAB = 11.9 Hz,
Bruker Avance 500 MHz spectrometer at 20 °C. The residual CH2Ph), 4.34 (d, 1H, J1,2 = 10.0 Hz, H-1), 3.76 (dd, 1H, A part of
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ABX, J5,6a = 2.2 Hz, J6a,6b = 11.0 Hz, H-6a), 3.73 (dd, 1H, B part (ABq, 2H, JA,B = 12.1 Hz, CH2Ph), 4.73, 4.47 (ABq, 2H, JA,B
=
of ABX, J5,6b = 3.9 Hz, H-6b), 3.57 (dd, 1H, J3,4 = J4,5 = 9.3 Hz, 11.0 Hz, CH2Ph), 4.57, 4.40 (ABq, 2H, JA,B = 12.1 Hz, CH2Ph),
H-4), 3.49 (dd, 1H, J2,3 = J3,4 = 9.3 Hz, H-3), 3.46–3.43 (m, 1H, 4.55 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.16 (d, 1H, J4,5 = 9.3 Hz, H-5),
H-5), 3.28 (dd, 1H, H-2), 2.31 (s, 3H, CH3); 13C NMR (100 MHz, 4.03–4.00 (m, 2H, H-3, H-4), 3.84 (dd, 1H, J2′,3′ = 10.3 Hz, J3′,4′
=
CDCl3): δ 138.7, 138.2, 137.8, 137.6, 134.3, 129.7, 128.5, 128.4, 9.0 Hz, H-3′), 3.73–3.69 (m, 2H, H-4′, H-6′a), 3.65 (s, 3H,
128.3, 128.2, 128.0, 127.8, 127.8, 127.5, 126.9 (Ar), 85.8 (C-1), CO2CH3), 3.57–3.55 (m, 2H, H-6′b, H-2), 3.43 (ddd, 1H, J5′,6′a
=
85.0 (C-3), 79.3 (C-5), 77.5 (C-4), 75.9, 75.0, 73.4 (3 × CH2Ph), 2.1 Hz, J5′,6′b = J5′,4′ = 10.0 Hz, H-5′), 3.37 (s, 3H, OCH3), 3.29
68.7 (C-6), 64.8 (C-2), 21.1 (CH3); LRMS: m/z = 604.3 [M + Na]+, (dd, 1H, H-2′). 13C NMR (125 MHz, CDCl3): δ 169.9 (CvO),
620.3 [M + K]+.
138.3, 138.1, 137.8, 137.7, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4 (Ph), 98.5 (C-1),
97.8 (C-1′), 81.1 (C-3), 79.9 (C-3′), 79.5 (C-2), 77.8 (C-4′), 75.3
(C-4), 75.4, 74.9, 74.7, 73.6, 73.5 (5 × CH2Ph), 71.1 (C-5′), 70.0
4′-Methylphenyl 2,3-di-O-benzyl-4,6-O-isopropylidene-
α-L-idoside (30)
The alcohol 29 (2.0 g, 4.8 mmol) was benzylated as described (C-5), 67.6 (C-6′), 63.2 (C-2′), 55.8 (OCH3), 52.6 (CO2CH3);
for compound 14. Following work-up, the residue was purified LRMS: m/z = 877.3 [M + NH4]+, 882.3 [M + Na]+, 898.3 [M + K]+;
by flash chromatography (EtOAc/n-hexane, 1 : 9) to give the HRMS: m/z calcd for C49H53N3O11Na [M + Na]+: 882.3572,
benzyl ether 30 as a pale yellow oil (2.0 g, 82%); Rf = 0.56 found: 882.3568; β-isomer 26: Rf = 0.17 (EtOAc/n-hexane, 1 : 4);
(EtOAc/n-hexane, 1 : 4); [α]D −60.3 (c 0.1, CHCl3); 1H NMR [α]D +7.4 (c 0.1, CHCl3; lit.28 +0.6, c 0.8, CHCl3); 1H NMR
(500 MHz, CDCl3): δ 7.38–7.27 (m, 12 H, Ar), 7.12–7.05 (m, 2H, (500 MHz, CDCl3): δ 7.33–7.10 (m, 25H, Ph), 5.02, 4.77 (ABq,
Ar), 5.50 (d, 1H, J1,2 = 4.5 Hz, H-1), 4.73, 4.66 (ABq, 4H, JA,B
=
2H, JA,B = 11.4 Hz, CH2Ph), 4.81, 4.76 (ABq, 2H, JA,B = 10.8 Hz,
11.8 Hz, 2 × CH2Ph), 4.24 (ddd, 1H, J4,5 = 3.0 Hz, J5,6a = 4.4 Hz, CH2Ph), 4.73, 4.51 (ABq, 2H, JA,B = 11.0 Hz, CH2Ph), 4.72, 4.55
J5,6b = 4.0 Hz, H-5), 4.03 (dd, A part of ABX, 1H, J6a,6b = 12.4 (ABq, 2H, JA,B = 12.0 Hz, CH2Ph), 4.54 (d, 1H, J1,2 = 3.6 Hz,
Hz, H-6a), 4.00 (dd, 1H, J3,4 = 4.1 Hz, H-4), 3.80 (dd, 1H, B part H-1), 4.34, 4.30 (ABq, 2H, JA,B = 12.0 Hz, CH2Ph), 4.31 (d, 1H,
of ABX, H-6b), 3.76 (dd, 1H, J2,3 = 6.6 Hz, H-3), 3.65 (dd, 1H, J1′,2′ = 7.9 Hz, H-1′), 4.21 (d, 1H, J4,5 = 9.8 Hz, H-5), 4.03 (dd,
H-2), 2.31 (s, 3H, ArCH3), 1.41 (s, 3H, CH3), 1.43 (s, 3H, CH3); 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 3.90 (dd, 1H, J2,3 = J3,4 = 9.4 Hz,
13C NMR (100 MHz, CDCl3): δ 138.1, 138.0, 137.0, 132.0, 131.2, H-3), 3.79 (s, 3H, CO2CH3), 3.62 (dd, 1H, J2′,3′ = 9.7 Hz, J3′,4′
=
129.6, 128.3, 128.2, 127.8, 127.7, 127.6 (C–Ar), 98.9 (C(CH3)2), 8.7 Hz, H-3′), 3.56 (dd, 1H, J5′,6′a = 2.1 Hz, J6a′, 6b′ = 10.9 Hz,
87.5 (C-1), 78.3 (C-3), 77.2 (C-2), 73.4 (CH2Ph), 73.1 (CH2Ph), H-6′a), 3.50 (dd, 1H, J6b′,5 = 4.1 Hz, J6b′,6a′ = 10.9 Hz, H-6′b),
70.0 (C-4), 63.1 (C-5), 61.6 (C-6), 27.5 (CH3), 21.0 (ArCH3), 20.2 3.48 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.5 Hz, H-2), 3.39 (s, 3H,
(CH3); LRMS: m/z = 524.2 [M + NH4]+, 529.2 [M + Na]+, 545.2 OCH3), 3.35 (dd, 1H, J3′,4′ = J4′,5′ = 8.7 Hz, H-4′), 3.29 (dd, 1H,
[M + K]+; HRMS: m/z calcd for C30H34O5SNa [M + Na]+: H-2′), 3.30–3.27 (m, 1H, H-5′); 13C NMR (125 MHz, CDCl3):
529.2019; found: 529.2004.
δ 170.1 (CvO), 139.2, 138.1, 137.9, 137.8, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4, 127.1
(Ph), 101.7 (C-1′), 98.8 (C-1), 83.2 (C-4′), 79.7 (C-3), 79.1 (C-4),
78.6 (C-2), 77.6 (C-3′), 75.3 (C-5′), 75.4, 75.2, 74.8, 73.7, 73.2
(5 × CH2Ph), 69.8 (C-5), 68.4 (C-6′), 66.8 (C-2′), 55.7 (OCH3),
Methyl(2-azido-3,4,6,-tri-O-benzyl-2-deoxy-D-glucopyranosyl)-
(1→4)-(methyl 2,3-di-O-benzyl-α-D-glucopyranosid)uronate
(25, 26)
A solution of donor 14 (943 mg, 1.62 mmol) and acceptor 23 52.7 (CO2CH3).
(544 mg, 1.35 mmol) and freshly dried AW300 mol sieves
(1.5 g) in anhydrous DCM (15 mL) was stirred at r.t. for 30 min
under N2. NIS (426 mg, 1.89 mmol) was added to the solution.
Methyl(2-amino-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl
α-D-glucopyranosid)uronate (1)
The solution was then stirred at −78 °C for 10 min. TfOH The protected disaccharide 25 (108 mg, 0.13 mmol) was dis-
(24 μL, 0.27 mmol) was added at −78 °C, and the mixture was solved in anhydrous MeOH (5 mL) and 10% Pd/C (65 mg) was
slowly warmed up to −20 °C over 2 h under N2. The reaction added. The resulting mixture was stirred at r.t. under an atmo-
was then quenched by addition of Et3N (5 mL), sat. aq. sphere of H2 for 24 h. The catalyst was removed by filtration
NaHCO3 (7 mL) and 10% Na2S2O3 in H2O (7 mL) to pH 8. The (Celite), and the filtrate was concentrated. The residue was
resulting solution was filtered through Celite and the filter then purified by flash chromatography (NH4OH/i-PrOH, 1 : 4)
cake was washed with DCM. The filtrate was then washed with to give the disaccharide 1 as white foam (25 mg, 51%); Rf = 0.14
10% Na2S2O3 in H2O solution, dried (MgSO4), filtered and con- (NH4OH/i-PrOH, 1 : 4); [α]D +132.9 (c 0.5, MeOH); 1H NMR
centrated. The residue was then purified by flash chromato- (500 MHz, CD3OD): δ 5.28 (d, 1H, J1′,2′ = 3.6 Hz, H-1′), 4.70 (d,
graphy (EtOAc/n-hexane, 1 : 9) to give the α-linked disaccharide 1H, J1,2 = 3.7 Hz, H-1), 4.09 (d, 1H, J4,5 = 9.6 Hz, H-5), 3.83 (dd,
25 as a colourless oil (576 mg), the β-linked isomer 26 as a col- 1H, J2,3 = J3,4 = 9.3 Hz, H-3), 3.78 (dd, 1H, J3,4 = J4,5 = 9.6 Hz,
ourless oil (275 mg) and an α/β-mixture (25, 26) as yellow oil H-4), 3.77 (s, 3H, CO2CH3), 3.72–3.70 (m, 2H, H-6′), 3.46 (dd,
(248 mg). In total, it was a 2 : 1 α/β mixture in 94% yield. 1H, H-2), 3.42–3.40 (m, 2H, H-3′, H-5′), 3.41 (s, 3H, OCH3),
α-Isomer 25: Rf = 0.18 (EtOAc/n-hexane, 1 : 4); [α]D +32.6 (c 1.1, 3.33 (dd, 1H, J3′4′ = J4′,5′ = 8.9 Hz, H-4′), 2.65 (dd, J2′,3′ = 9.9 Hz,
CHCl3; lit.28 +32.0); 1H NMR (500 MHz, CDCl3): δ 7.34–7.08 H-2′); 13C NMR (125 MHz, CD3OD): δ 171.3 (CvO), 101.7 (C-1),
(m, 25H, Ph), 5.57 (d, 1H, J1′,2′ = 3.7 Hz, H-1′), 5.01, 4.85 (ABq, 101.3 (C-1′), 80.1 (C-4), 74.8 (C-3′), 74.5 (C-3), 74.4 (C-5′), 72.8
2H, JA,B = 10.6 Hz, CH2Ph), 4.83 (s, 2H, CH2Ph), 4.75, 4.58 (C-2), 71.8 (C-5), 71.2 (C-4′), 61.9 (C-6′), 57.0 (C-2′), 56.1 (OCH3),
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53.2 (CO2CH3); LRMS: m/z = 384.1 [M + H]+; HRMS: m/z calcd 2H, JA,B = 11.0 Hz, CH2Ph), 4.78, 4.62 (ABq, 2H, JA,B = 11.0 Hz,
for C14H26NO11 [M + H]+: 384.1500, found: 384.1493.
CH2Ph), 4.73, 4.51 (ABq, 2H, JA,B = 11.0 Hz, CH2Ph), 4.37 (d,
1H, J1,2 = 7.6 Hz, H-1), 4.36, 4.28 (ABq, 2H, JA,B = 12.1 Hz,
Methyl(2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→4)-
(methyl α-D-glucopyranosid)uronate (2)
CH2Ph), 4.35 (d, 1H, J1′,2′ = 7.9 Hz, H-1′), 4.15 (dd, 1H, J4,5
=
9.5 Hz, J3,4 = 8.8 Hz, H-4), 3.95 (d, 1H, H-5), 3.81 (s, 3H,
To a solution of amine 1 (5.8 mg, 0.02 mmol) in anhydrous CO2CH3), 3.64 (dd, 1H, J3′,4′ = J2′,3′ = 9.1 Hz, H-3′), 3.62 (dd, 1H,
MeOH (0.5 mL) at 0 °C under N2 was added Et3N (4.2 μL, J3,4 = J2,3 = 8.8 Hz, H-3), 3.57–3.52 (m, 2H, H-6′), 3.52 (s, 3H,
0.03 mmol) and Ac2O (21 μL, 0.23 mmol). The mixture was OCH3), 3.41 (dd, 1H, H-2), 3.34 (dd, 1H, J4′,5′ = 9.9 Hz, H-4′),
stirred at r.t. for 90 min, and then concentrated in vacuo to 3.31 (dd, 1H, H-2′), 3.31–3.28 (m, 1H, H-5′); 13C NMR
give the amide 2 as a colourless oil (6.4 mg, 100%); Rf
=
(125 MHz, CDCl3): δ 169.1 (CvO), 139.0, 138.3, 138.1, 137.9,
0.5 (NH4OH/i-PrOH, 1 : 4); [α]D +116.2 (c 0.32, MeOH); 1H NMR 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.6,
(500 MHz, CD3OD): δ 5.20 (d, 1H, J1′,2′ = 3.6 Hz, H-1′), 4.68 (d, 127.5, 127.2 (Ph), 104.9 (C-1), 101.7 (C-1′), 83.1 (C-4′), 82.0
1H, J1,2 = 3.8 Hz, H-1), 4.09 (d, 1H, J4,5 = 9.1 Hz, H-5), 3.86 (dd, (C-3), 81.2 (C-2), 78.9 (C-4), 77.6 (C-3′), 75.5, 75.0, 74.9, 74.8,
1H, J2′,3′ = 10.6 Hz, H-2′), 3.81–3.78 (m, 2H, H-3, H-4), 3.77 (s, 73.3 (5 × CH2Ph), 75.1 (C-2′), 74.3 (C-5), 68.4 (C-6′), 66.8 (C-5′),
3H, CO2CH3), 3.72–3.71 (m, 2H, H-6′), 3.56 (dd, 1H, J3′,4′
=
57.4 (OCH3), 52.7 (CO2CH3).
8.5 Hz, H-3′), 3.47–3.42 (m, 3H, H-2, H-4′, H-5′), 3.41 (s, 3H,
OCH3), 1.98 (s, 3H, COCH3); 13C NMR (125 MHz, CD3OD):
δ 173.9, 171.2 (2 × CvO), 101.7 (C-1), 100.2 (C-1′), 79.8 (C-4),
Methyl(2-amino-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl
β-D-glucopyranosid)uronate (3)
74.31 (C-5′), 74.29 (C-3), 73.1 (C-2), 72.9 (C-3′), 72.1 (C-5), 71.4 The disaccharide 27 (159 mg, 0.19 mmol) was dissolved in
(C-4′), 61.8 (C-6′), 56.1 (OCH3), 55.5 (C-2′), 53.2 (CO2CH3), 22.7 anhydrous MeOH (10 mL), 20% Pd(OH)2/C (160 mg) and one
(COCH3); LRMS: m/z = 448.1 [M + Na]+, 464.1 [M + K]+; HRMS: drop of conc. HCl were added. The solution was stirred at r.t.
m/z calcd for C16H27NO12 [M + H]+: 448.1425, found: 448.1413.
under an atmosphere of H2 for 21 h. The catalyst was then
removed by filtration over Celite, and the filtrate was concen-
trated. The residue was then purified by flash chromatography
(NH4OH/i-PrOH, 1 : 4) to give disaccharide 3 as white foam
(42 mg, 60%); Rf = 0.11 (NH4OH/i-PrOH, 1 : 4); [α]D −11.4
Methyl(2-azido-3,4,6,-tri-O-benzyl-2-deoxy-D-glucopyranosyl)-
(1→4)-(methyl 2,3-di-O-benzyl-β-D-glucopyranosid)uronate
(27, 28)
The donor 14 (702 mg, 1.21 mmol) and acceptor 24 (405 mg, (c 1.1, MeOH); 1H NMR (500 MHz, CD3OD): δ 5.27 (d, 1H,
1.01 mmol) were glycosylated as described for compounds 25 J1′,2′ = 3.7 Hz, H-1′), 4.24 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.96 (d, 1H,
and 26. Following work-up, the residue was purified by flash J4,5 = 9.6 Hz, H-5), 3.78 (dd, 1H, J3,4 = 9.1 Hz, H-4), 3.78 (s, 3H,
chromatography (EtOAc/n-hexane, 1 : 9) to give the α-linked CO2CH3), 3.71–3.70 (m, 2H, H-6′), 3.62 (dd, 1H, J2,3 = J3,4
disaccharide 27 as colourless oil (558 mg), β-isomer 28 as col- 9.1 Hz, H-3), 3.49 (s, 3H, OCH3), 3.43–3.35 (m, 3H, H-3′, H-5′,
ourless oil (66 mg) and α,β-mixture (27, 28) as yellow oil H-4′), 3.23 (dd, 1H, H-2), 2.64 (dd, 1H, J2′,3′ = 10.1 Hz, H-2′); 13
=
C
(197 mg). In total, it was a 9 : 1 α/β mixture in 95% yield. NMR (125 MHz, CD3OD): δ 170.7 (CvO), 105.7 (C-1), 101.7
α-Isomer 27: Rf = 0.18 (EtOAc/n-hexane, 1 : 4); [α]D +23.7 (c 8.0, (C-1′), 80.0 (C-4), 77.4 (C-3), 75.9 (C-5), 75.1 (C-3′), 74.4 (C-5′),
1
CHCl3); H NMR (500 MHz, CDCl3): δ 7.33–7.22 (m, 23 H, Ph), 74.3 (C-2), 71.2 (C-4′), 61.9 (C-6′), 57.5 (C-2′), 57.0 (OCH3), 53.2
7.13–7.11 (m, 2H, Ph), 5.57 (d, 1H, J1′,2′ = 3.8 Hz, H-1′), 4.99, (CO2CH3); LRMS: m/z = 384.1 [M + H]+, 406.1 [M + Na]+;
4.80 (ABq, 2H, JA,B = 10.7 Hz, CH2Ph), 4.88, 4.75 (ABq, 2H, HRMS: m/z calcd for C14H25NO11Na [M + Na]+: 406.1320,
JA,B = 11.1 Hz, CH2Ph), 4.84, 4.82 (ABq, 2H, JA,B = 10.7 Hz, found: 406.1319.
CH2Ph), 4.66, 4.49 (ABq, 2H, JA,B = 11.1 Hz, CH2Ph), 4.60, 4.43
(ABq, 2H, JA,B = 12.1 Hz, CH2Ph), 4.37 (d, 1H, J1,2 = 7.6 Hz, H-1),
4.12 (dd, 1H, J3,4 = 9.1 Hz, J4,5 = 9.6 Hz, H-4), 3.93 (d, 1H, H-5),
Methyl(2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→4)-
(methyl β-D-glucopyranosid)uronate (4)
3.84 (dd, 1H, J3′,4′ = 9.0 Hz, J2′,3′ = 10.3 Hz, H-3′), 3.76–3.70 (m, The disaccharide 27 (79 mg, 0.09 mmol) was hydrogenated as
3H, H-3, H-4′, H-6′a), 3.65 (s, 3H, CO2CH3), 3.59 (dd, 1H, J5′,6′b
=
described for compound 1. The crude amine was then dis-
2.1 Hz, J6′a, 6′b = 10.8 Hz, H-6′b), 3.54 (s, 3H, OCH3), 3.49 (dd, solved in anhydrous MeOH (5 mL) and Ac2O (195 µL,
1H, J2,3 = 9.0 Hz, H-2), 3.40 (ddd, 1H, J5′,6′a = J4′,5′ = 10.0 Hz, 2.07 mmol) and Et3N (586 µL, 4.1 mmol) were added. The
H-5′), 3.27 (dd, 1H, H-2′); 13C NMR (125 MHz, CDCl3): δ 168.9 solution was stirred at r.t. for 24 h and was then concentrated.
(CvO), 138.1, 138.0, 137.8, 137.7, 128.4, 128.3, 128.2, 128.0, The residue was purified by flash chromatography (NH4OH/
127.9, 127.8, 127.7, 127.6, 127.5, 127.4 (Ph), 104.8 (C-1), 97.7 i-PrOH, 1 : 4) to give the amide 4 as a colourless oil (24 mg,
(C-1′), 83.8 (C-3), 81.8 (C-2), 79.8 (C-3′), 77.8 (C-4′), 75.3, 75.2, 62%); Rf = 0.55 (NH4OH/i-PrOH, 2 : 3); [α]D +8.0 (c 0.4, MeOH);
74.7, 73.6 (5 × CH2Ph), 74.5 (C-4), 74.3 (C-5), 71.1 (C-5′), 67.6 1H NMR (500 MHz, CD3OD): δ 5.21 (d, 1H, J1′,2′ = 3.6 Hz, H-1′),
(C-6′), 63.1 (C-2′), 57.3 (OCH3), 52.5 (CO2CH3); LRMS: m/z = 4.23 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.96 (d, 1H, J4,5 = 9.6 Hz, H-5),
877.2 [M + NH4]+, 882.2 [M + Na]+, 898.2 [M + K]+; HRMS: m/z 3.86 (dd, 1H, J2′,3′ = 10.7 Hz, H-2′), 3.79 (dd, 1H, J3,4 = 9.1 Hz,
calcd for C49H53N3O11Na [M + Na]+: 882.3572, found: 882.3571. H-4), 3.78 (s, 3H, CO2CH3), 3.73–3.72 (m, 2H, H-6′), 3.60 (dd,
β-Isomer 28: Rf = 0.17 (EtOAc/n-hexane, 1 : 4); [α]D −3.2 (c 5.8, 1H, J3,4 = J2,3 = 9.1 Hz, H-3), 3.58 (dd, 1H, J3′,4′ = 8.4 Hz, H-3′),
1
CHCl3); H NMR (500 MHz, CDCl3): δ 7.32–7.11 (m, 25H, Ph), 3.48 (s, 3H, OCH3), 3.45–3.40 (m, 2H, H-4′, H-5′), 3.21 (dd, 1H,
5.00, 4.72 (ABq, 2H, JA,B = 11.6 Hz, CH2Ph), 4.81, 4.75 (ABq, H-2), 1.99 (s, 3H, COCH3); 13C NMR (125 MHz, CD3OD):
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δ 173.9, 170.6 (2 × CvO), 105.7 (C-1), 100.2 (C-1′), 79.6 (C-4), (dd, 1H, J2,3 = J3,4 = 3.3 Hz, H-3), 3.63 (dd, 1H, J4,5 = 1.5 Hz,
77.1 (C-3), 62.2 (C-5), 74.9 (C-2), 74.2 (C-5′), 72.8 (C-3′), 71.3 H-4), 3.61 (dd, 1H, H-2), 3.56 (s, 3H, OCH3); 13C NMR
(C-4′), 61.8 (C-6′), 57.6 (OCH3), 55.4 (C-2′), 53.2 (CO2CH3), 23.0 (125 MHz, CDCl3): δ 137.4, 137.3, 128.5, 128.4, 128.1, 128.0,
(COCH3); LRMS: m/z = 426.1 [M + H]+, 448.1 [M + Na]+, 464.1 127.6 (Ph), 100.9 (C-1), 75.6 (C-3), 75.2 (C-5), 74.5 (C-2), 74.2,
[M + K]+; HRMS: m/z calcd for C16H27NO12Na [M + Na]+: 72.2 (2
× CH2Ph), 67.2 (C-4), 62.7 (C-6), 57.1 (OCH3);
448.1425, found: 448.1422.
LRMS: m/z = 397.2 [M + Na]+; HRMS: m/z calcd for C21H26O6Na
[M + Na]+: 397.1622, found: 397.1632.
Methyl 2,3-di-O-benzyl-4,6-O-isopropylidene-L-idopyranoside
(32)
Methyl 2,3-di-O-benzyl-α-L-idopyranoside (35)
The donor 30 (217 mg, 0.4 mmol) and anhydrous MeOH
(87 μL, 2.1 mmol) were glycosylated as described for com-
pounds 25 and 26. Following work-up the residue was purified
by flash chromatography (EtOAc/n-hexane, 1 : 9) to give 32-α as
colourless oil (33 mg) and 32-β as colourless oil (141 mg). In
total, it was a 1 : 4 α/β mixture in 98% yield. α-Isomer (32-α):
Rf = 0.55 (EtOAc/n-hexane, 2 : 3); [α]D −30.5 (c 2.9, CHCl3); H
NMR (500 MHz, CDCl3): δ 7.33–7.24 (m, 10H, Ph), 4.74–4.67
(m, 5H, 2 × CH2Ph, H-1), 3.98–3.94 (m, 2H, H-4, H-6a),
The isopropylidene 32-α (14 mg, 0.03 mmol) was dissolved in
a mixture of DCM/TFA/H2O (100 : 10 : 1, 3 mL) and the solution
stirred at r.t. for 30 min. The solution was then concentrated
and the residue purified by flash chromatography (EtOAc/
n-hexane, 1 : 1) to give the diol 35 as a colourless oil (12.6 mg,
100%); H NMR (500 MHz, CDCl3): δ 7.34–7.23 (m, 10 H, Ph),
4.80 (app s, 1H, H-1), 4.59, 4.52 (ABq, 2H, JA,B = 12.2 Hz,
CH2Ph), 4.53, 4.51 (ABq, 2H, JA,B = 11.8 Hz, CH2Ph), 4.12–4.10
1
1
(m, 1H, H-5), 3.93 (dd, 1H, A part of ABX, J5,6a = 6.3 Hz, J6a,6b
=
3.88–3.86 (m, 1H, H-5), 3.73 (dd, 1H, B part of ABX, J5,6b
4.8 Hz, J6a,6b = 12.1 Hz, H-6b), 3.69 (dd, 1H, J2,3 = 9.8 Hz, J3,4
=
=
11.8 Hz, H-6a), 3.78 (dd, 1H, B part of ABX, J5,6b = 3.9 Hz,
H-6b), 3.69–3.68 (m, 2H, H-3, H-4), 3.54 (dd, 1H, J1,2 = 1.4 Hz,
J2,3 = 3.8 Hz, H-2), 3.41 (s, 3H, OCH3), 3.28 (br. s, 1H, OH-4);
13C NMR (125 MHz, CDCl3): δ 137.7, 136.7, 128.6, 128.5, 128.2,
127.9, 127.7 (Ph), 99.8 (C-1), 73.6 (C-2), 73.5 (C-3), 72.5, 71.8
(2 × CH2Ph), 68.2 (C-4), 66.9 (C-5), 63.7 (C-6), 55.5 (OCH3);
LRMS: m/z = 397.2 [M + Na]+; HRMS: m/z calcd for C21H26O6Na
[M + Na]+: 397.1622, found: 397.1621.
4.7 Hz, H-3), 3.49 (dd, 1H, J1,2 = 4.9 Hz, H-2), 3.37 (s, 3H,
OCH3), 1.39 (s, 6H, 2 × CH3); 13C NMR (125 MHz, CDCl3):
δ 138.6, 138.5, 128.3, 128.2, 127.8, 127.7, 127.5 (Ph), 103.5
(C-1), 99.1 (CH(CH3)2), 80.2 (C-3), 79.4 (C-2), 73.6, 73.5 (2 ×
CH2Ph), 72.7 (C-4), 64.0 (C-5), 61.0 (C-6), 55.3 (OCH3), 26.9
(CH3), 20.9 (CH3); β-isomer (32-β): Rf = 0.37 (EtOAc/n-hexane,
2 : 3); [α]D +55.1 (c 1.7, CHCl3); 1H NMR (500 MHz, CDCl3):
δ 7.38–7.20 (m, 10H, Ph), 4.78, 4.64 (ABq, 2H, JA,B = 12.2 Hz,
CH2Ph), 4.63 (d, 1H, J1,2 = 1.5 Hz, H-1), 4.55, 4.53 (ABq, 2H,
Methyl(2-azido-3,4,6,-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1→4)-(methyl 2,3-di-O-benzyl-β-L-idopyranosid)uronate (39)
JA,B = 11.8 Hz, CH2Ph), 4.03 (dd, 1H, A part of ABX, J5,6a
=
2.7 Hz, J6a,6b = 12.7 Hz, H-6a), 3.96 (dd, 1H, B part of ABX, The donor 14 (819 mg, 1.41 mmol) and acceptor 37 (472 mg,
J5,6b = 2.1 Hz, H-6b), 3.87 (dd, 1H, J3,4 = J4,5 = 2.1 Hz, H-4), 3.76 1.17 mmol) were glycosylated as described for compounds 25
(dd, 1H, J2,3 = 3.9 Hz, H-3), 3.59–3.58 (m, 1H, H-5), 3.55 (dd, 1H, and 26. Following work-up the residue was purified by flash
H-2), 3.51 (s, 3H, OCH3), 1.59 (s, 3H, CH3), 1.43 (s, 3H, CH3); chromatography (EtOAc/n-hexane, 1 : 9) to give the disaccharide
13C NMR (125 MHz, CDCl3): δ 138.8, 137.7, 128.4, 128.0, 127.9, 39 as colourless oil (520 mg, 52%); Rf = 0.2 (EtOAc/n-hexane,
127.6, 127.5, 127.1 (Ph), 100.3 (C-1), 98.3 (CH(CH3)2), 77.3 (C-3), 1 : 4); [α]D +40.9 (c 1.1, CHCl3); 1H NMR (500 MHz, CDCl3):
73.8 (C-2), 72.6, 72.4 (2 × CH2Ph), 67.0 (C-4), 66.6 (C-5), 62.7 δ 7.36–7.11 (m, 25H, Ph), 5.12 (d, 1H, J1′,2′ = 3.6 Hz, H-1′), 4.85,
(C-6), 56.5 (OCH3), 28.9 (CH3), 18.8 (CH3). LRMS: m/z = 432.1 4.77 (ABq, 2H, JA,B = 10.8 Hz, CH2Ph), 4.73, 4.47 (ABq, 2H,
[M + NH4]+, 437.1 [M + Na]+, 453.1 [M + K]+; HRMS: m/z calcd JA,B = 11.1 Hz, CH2Ph), 4.74, 4.73 (ABq, 2H, JA,B = 11.7 Hz,
for C24H30O6Na [M + Na]+: 437.1935, found: 437.1948.
CH2Ph), 4.72, 4.63 (ABq, 2H, JA,B = 12.1 Hz, CH2Ph), 4.55, 4.43
(ABq, 2H, JA,B = 12.1 Hz, CH2Ph), 4.51 (d, 1H, J1,2 = 3.1 Hz,
Methyl 2,3-di-O-benzyl-β-L-idopyranoside (34)
H-1), 4.36 (d, 1H, J4,5 = 4.5 Hz, H-5), 4.34 (dd, 1H, J2,3 = J3,4
=
The isopropylidene 32-β (2.24 g, 5.4 mmol) was dissolved in 7.3 Hz, H-3), 3.88 (dd, 1H, H-4), 3.84 (dd, 1H, J2′,3′ = 10.3 Hz,
80% AcOH in H2O solution (30 mL) and heated at 100 °C for J3′,4′ = 8.8 Hz, H-3′), 3.74–3.72 (m, 1H, H-5′), 3.70 (s, 3H,
24 h. The mixture was then cooled to r.t., neutralised with CO2CH3), 3.67 (dd, 1H, A part of ABX, J5′,6a′ = 3.4 Hz, J6a′,6b′
=
solid NaHCO3 and diluted with a mixture of EtOAc/H2O (1 : 1, 10.6 Hz, H-6a′), 3.63 (dd, 1H, J4′,5′ = 9.9 Hz, H-4′), 3.54 (dd, 1H,
30 mL). The organic layer was separated, dried (MgSO4), fil- B part of ABX, J5′,6b′ = 1.9 Hz, H-6b′), 3.47 (dd, 1H, H-2), 3.40 (s,
tered and concentrated. The residue was then purified by flash 3H, OCH3), 3.32 (dd, 1H, H-2′); 13C NMR (125 MHz,
chromatography (EtOAc/n-hexane, 1 : 1) to give the diol 34 as CDCl3): δ 169.4 (CvO), 138.3, 138.2, 138.0, 137.9, 137.8, 128.4,
colourless oil (1.0 g, 50%); Rf = 0.22 (EtOAc/n-hexane, 7 : 3); 128.3, 128.0, 127.9, 127.8, 127.7, 127.6 (Ph), 100.9 (C-1),
[α]D +51.9 (c 4.5, CHCl3), lit.34 +86 (c 1.2, CHCl3); 1H NMR 99.5 (C-1′), 79.8 (C-3′), 78.1 (C-4′), 77.2 (C-2), 76.2 (C-4), 76.1
(500 MHz, CDCl3): δ 7.29–7.20 (m, 10H, Ph), 4.83, 4.60 (ABq, (C-3), 75.2, 74.9, 74.5, 73.6, 73.5 (5 × CH2Ph), 72.0 (C-5), 71.5
2H, JA,B = 12.1 Hz, CH2Ph), 4.68 (d, 1H, J1,2 = 1.1 Hz, H-1), 4.49, (C-5′), 67.9 (C-6′), 63.5 (C-2′), 57.4 (OCH3), 52.0 (CO2CH3);
4.45 (ABq, 2H, JA,B = 11.8 Hz, CH2Ph), 3.98 (dd, 1H, A part of LRMS: m/z = 877.3 [M + NH4]+, 882.2 [M + Na]+, 898.2 [M + K]+;
ABX, J6a,6b = 11.3 Hz, J5,6a = 7.2 Hz, H-6a), 3.93–3.91 (m, 1H, HRMS: m/z calcd for C49H53O11N3Na [M + Na]+: 882.3572,
H-5), 3.79 (dd, 1H, B part of ABX, J5,6b = 4.1 Hz, H-6b), 3.75 found: 882.3576.
8798 | Org. Biomol. Chem., 2018, 16, 8791–8803
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Paper
Methyl(2-amino-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl
β-L-idopyranosid)uronate (7)
HRMS: m/z calcd for C21H24O7Na [M + Na]+: 411.1414; found:
411.1420.
The disaccharide 39 (66 mg, 0.08 mmol) was hydrogenated as
described for compound 3 to give the disaccharide 7 as a col-
ourless oil (29 mg, 98%); Rf = 0.08 (NH4OH/i-PrOH, 1 : 4);
Methyl(methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosid)
uronate (38)
1
The diol 36 (456 mg, 1.2 mmol) was dissolved in a mixture of
DCM/H2O (3 : 1, 16 mL), and to this mixture, TEMPO (37 mg,
0.23 mmol) and BAIB (757 mg, 2.3 mmol) were added. The
resulting mixture was stirred vigorously at r.t. for 3 h. The solu-
tion was washed with sat. aq. Na2S2O3 solution, dried (MgSO4),
filtered and concentrated. The residue was dissolved in anhy-
drous DMF (12 mL), and to this mixture, iodomethane
(0.11 mL, 1.76 mmol) and KHCO3 (235 mg, 2.35 mmol) were
added. The resulting mixture was stirred at r.t. for 24 h in the
dark under argon. The mixture was then concentrated and the
residue purified by flash chromatography (EtOAc/n-hexane,
1 : 1) to give the ester 38 as yellow oil (223 mg, 46%); Rf = 0.23
(EtOAc/n-hexane, 1 : 1); [α]D −10.5 (c 0.4, CHCl3), lit.35 −19
[α]D +63.7 (c 0.6, MeOH); H NMR (500 MHz, CD3OD): δ 5.31
(d, 1H, J1′,2′ = 3.7 Hz, H-1′), 4.71 (d, 1H, J4,5 = 2.0 Hz, H-5), 4.69
(d, 1H, J1,2 = 1.6 Hz, H-1), 4.24 (dd, 1H, J2,3 = J3,4 = 3.4 Hz, H-3),
4.02 (dd, 1H, H-4), 3.79 (s, 3H, CO2CH3), 3.75–3.68 (m, 3H,
H-6′, H-3′), 3.65 (dd, 1H, H-2), 3.58 (s, 3H, OCH3), 3.37–3.36
(m, 2H, H-4′, H-5′), 3.09 (dd, 1H, J2′,3′ = 10.6 Hz, H-2′);
13C NMR (125 MHz, CD3OD): δ 171.0 (CvO), 101.8 (C-1), 93.8
(C-1′), 74.7 (C-5′), 74.0 (C-5), 73.4 (C-4), 71.1 (C-4′), 70.8 (C-3′),
70.4 (C-2), 66.6 (C-3), 61.8 (C-6′), 57.4 (OCH3), 55.9 (C-2′), 52.9
(CO2CH3); LRMS: m/z = 384.1 [M + H]+, 406.1 [M + Na]+, 422.0
[M + K]+; HRMS: m/z calcd for C14H26O11N [M + Na]+:
384.1500, found: 384.1492.
1
(c 1.0, CHCl3); H NMR (500 MHz, CDCl3): δ 8.01–7.94 (m, 2H,
Methyl(2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→4)-
(methyl β-L-idopyranosid)uronate (8)
Ph), 7.58–7.24 (m, 10H, Ph), 5.18 (dd, 1H, J1,2 = 1.1 Hz, J2,3
2.4 Hz, H-2), 4.99 (d, 1H, H-1), 4.88 (d, 1H, J4,5 = 1.5 Hz, H-5),
4.82, 4.66 (ABq, 2H, JA,B = 12.0 Hz, CH2Ph), 4.06 (dd, 1H, J4,5
J3,4 = 1.5 Hz, H-4), 3.82 (dd, 1H, H-3), 3.81 (s, 3H, CO2CH3),
3.49 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3): δ 169.9, 164.9
(2 × CvO), 137.4, 133.7, 129.7, 128.9, 128.6, 128.4, 127.9, 127.7
(Ph), 99.9 (C-1), 74.1 (C-3), 71.9 (CH2Ph), 67.9 (C-4), 67.4 (C-2,
C-5), 56.4 (OCH3), 52.4 (CO2CH3); LRMS: m/z = 439.1 [M + Na]+;
HRMS: m/z calcd for C22H24O8Na [M + Na]+: 439.1363; found:
439.1371.
=
The amine 7 (5.2 mg, 0.014 mmol) was acetylated as described
for compound 2. The crude product was purified by flash
chromatography (NH4OH/i-PrOH, 1 : 4) to give the amide 8 as a
white foam (4.1 mg, 72%); Rf = 0.44 (NH4OH/i-PrOH, 1 : 4); [α]D
+97.6 (c 0.1, MeOH); 1H NMR (500 MHz, CD3OD): δ 4.97 (d,
1H, J1′,2′ = 3.7 Hz, H-1′), 4.68 (d, 1H, J1,2 = 1.6 Hz, H-1), 4.66 (d,
1H, J4,5 = 2.3 Hz, H-5), 4.11 (dd, 1H, J2,3 = J3,4 = 3.3 Hz, H-3),
3.91–3.89 (m, 2H, H-4, H-2′), 3.78 (s, 3H, CO2CH3), 3.73–3.72
(m, 2H, H-6′), 3.60 (dd, H-2), 3.57 (s, 3H, OCH3), 3.54 (dd, 1H,
=
J2′,3′ = 10.6 Hz, J3′,4′ = 8.4 Hz, H-3′), 3.41–3.36 (m, 2H, H-4′, Methyl(2-azido-3,4,6,-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
H-5′), 1.96 (s, 3H, COCH3); 13C NMR (125 MHz, CD3OD): (1→4)-(methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosid)
δ 174.1, 171.2 (2 × CvO), 101.9 (C-1), 97.1 (C-1′), 74.5 (C-5), uronate (40)
74.4 (C-4), 74.2 (C-5′), 73.1 (C-3′), 71.6 (C-4′), 70.7 (C-2), 67.6
The donor 14 (188 mg, 0.32 mmol) and acceptor 38 (112 mg,
(C-3), 62.3 (C-6′), 57.3 (OCH3), 55.1 (C-2′), 52.8 (CO2CH3), 22.8
0.27 mmol) were glycosylated as described for compounds 25
(COCH3); LRMS: m/z = 448.1 [M + Na]+, 464.1 [M + K]+; HRMS:
and 26. Following work-up the residue was purified by flash
m/z calcd for C16H27O12NNa [M + Na]+: 448.1425; found:
chromatography (EtOAc/n-hexane, 1 : 9) to give the disaccharide
448.1443.
40 as yellow oil (170 mg, 72%); Rf = 0.08 (EtOAc/n-hexane,
1 : 4); [α]D −47.5 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3):
Methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranoside (36)
δ 8.09–8.05 (m, 2H, Ph), 7.44–7.06 (m, 23H, Ph), 5.09 (app t,
The isopropylidene 33-α [5] (500 mg, 1.17 mmol) was hydro- 1H, J2,3 = 2.5 Hz, H-2), 5.04 (app. s, 1H, H-1), 4.89, 4.73 (ABq,
lysed as described for compound 35. The crude product was 2H, JA,B = 11.7 Hz, CH2Ph), 4.80 (d, 1H, J4,5 = 2.5 Hz, H-5), 4.67
purified by flash chromatography (EtOAc/n-hexane, 1 : 1) to (d, 1H, J1′,2′ = 3.5 Hz, H-1′), 4.63, 4.46 (ABq, 2H, JA,B = 10.9 Hz,
give the diol 36 as colourless oil (411 mg, 91%); Rf = 0.19 CH2Ph), 4.56, 4.43 (ABq, 2H, JA,B = 12.2 Hz, CH2Ph), 4.12, 3.90
(EtOAc/n-hexane, 1 : 1); [α]D −24.9 (c 0.4, CHCl3); 1H NMR (ABq, 2H, JA,B = 10.5 Hz, CH2Ph), 4.08 (dd, 1H, J2,3 = J3,4
(500 MHz, CDCl3): δ 8.01–7.97 (m, 2H, Ph), 7.61–7.24 (m, 10H, 2.5 Hz, H-3), 3.99 (dd, 1H, H-4), 3.88 (ddd, 1H, J4′,5′ = J5′,6b′
Ph), 5.20 (dd, 1H, J1,2 = 1.2 Hz, J2,3 = 2.4 Hz, H-2), 4.86 (app. s, 9.6 Hz, J5′,6a′ = 2.4 Hz, H-5′), 3.78 (dd, 1H, A part of ABX, J6a′,6b′
=
=
=
1H, H-1), 4.82, 4.62 (ABq, 2H, JA,B = 12.0 Hz, CH2Ph), 4.27 10.9 Hz, H-6a′), 3.71 (s, 3H, CO2CH3), 3.64 (dd, 1H, B part of
(ddd, 1H, J5,6a = 6.2 Hz, J5,6b = 4.2 Hz, J4,5 = 1.0 Hz, H-5), 3.95 ABX, H-6b′), 3.63 (dd, 1H, J4′,5′ = J3′,4′ = 9.6 Hz, H-4′), 3.49 (dd,
(ddd, 1H, J6a,6b = 11.9 Hz, J6a,OH = 4.6 Hz, H-6a), 3.85 (ddd, 1H, 1H, J3′,4′ = 9.6 Hz, J2′,3′ = 10.2 Hz, H-3′), 3.46 (s, 3H, OCH3), 3.18
J6a,OH = 7.8 Hz, H-6b), 3.78–3.74 (m, 2H, H-3, H-4), 3.46 (s, 3H, (dd, 1H, H-2′); 13C NMR (125 MHz, CDCl3): δ 169.6, 165.5 (2 ×
OCH3), 2.71 (d, 1H, J4,OH = 9.3 Hz, OH-4), 2.09 (dd, 1H, OH-6); CvO), 138.3, 137.8, 137.4, 133.1, 129.9, 129.6, 128.6, 128.4,
13C NMR (125 MHz, CDCl3): δ 165.0 (CvO), 137.6, 133.5, 128.3, 128.2, 128.1, 128.0, 127.7, 127.6 (Ph), 100.3 (C-1), 99.9
129.7, 129.2, 128.6, 128.4, 127.9, 127.7 (Ph), 99.8 (C-1), 74.9 (C-1′), 80.0 (C-3′), 77.6 (C-4′), 76.0 (C-4), 74.7 (CH2Ph), 74.6
(C-3), 71.9 (CH2Ph), 68.2 (C-4), 68.1 (C-2), 66.8 (C-5), 63.4 (C-6), (CH2Ph), 73.5 (CH2Ph), 72.9 (C-3), 72.4 (CH2Ph), 71.6 (C-5′),
55.7 (OCH3); LRMS m/z = 411.1 [M + Na]+, 427.1 [M + K]+; 67.8 (C-6′, C-2), 67.0 (C-5), 63.7 (C-2′), 56.2 (OCH3), 52.2
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(CO2CH3); LRMS: m/z = 896.3 [M + Na]+, 912.3 [M + K]+; HRMS: HRMS: m/z calcd for C14H25O11NNa [M + Na]+: 406.1320;
m/z calcd for C49H51O12N3Na [M + Na]+: 896.3365; found: found: 406.1326.
896.3365.
Methyl(2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→4)-
Methyl(2-azido-3,4,6,-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl)-
(1→4)-(methyl 3-O-benzyl-α-L-idopyranosid)uronate (41)
(methyl α-L-idopyranosid)uronate (6)
The amine 5 (5.5 mg, 0.014 mmol) was acetylated as described
The benzoate 40 (15 mg, 0.02 mmol) was dissolved in anhy- for compound 2. The crude product was purified by flash
drous MeOH (3 mL), and a solution of 1 M NaOMe in MeOH chromatography (NH4OH/i-PrOH, 1 : 4) to give the amide 6 as a
(2 mL) was added. The resulting solution was stirred at r.t. for colourless oil (2.4 mg, 39%); Rf = 0.28 (NH4OH/i-PrOH, 1 : 4);
1
5 h. It was then neutralized (Amberlite IR-120, H+ form, pre- [α]D +8.5 (c 0.05, MeOH); H NMR (500 MHz, CD3OD): δ 5.04
washed with MeOH (3 × 20 mL) before use) in small portions (d, 1H, J1′,2′ = 3.6 Hz, H-1′), 4.79 (d, 1H, J4,5 = 2.9 Hz, H-5), 4.74
to pH 7. The mixture was then filtered and the filtrate concen- (d, 1H, J1,2 = 2.7 Hz, H-1), 3.99 (dd, 1H, J3,4 = 3.3 Hz, H-4),
trated under reduced pressure. The residue was then purified 3.92–3.89 (m, 2H, H-3, H-2′), 3.79 (s, 3H, CO2CH3), 3.76 (dd,
by flash chromatography (EtOAc/n-hexane, 1 : 9) to give the 1H, A part of ABX, J5′,6a′ = 2.3 Hz, J6a′,6b′ = 11.6 Hz, H-6a′), 3.71
alcohol 41 as colourless oil (10.8 mg, 82%); Rf = 0.14 (EtOAc/ (dd, 1H, B part of ABX, J5′,6b′ = 4.1 Hz, H-6b′), 3.55–3.51 (m,
1
n-hexane, 3 : 7); [α]D −16.0 (c 0.1, CHCl3); H NMR (500 MHz, 2H, H-3′, H-2), 3.42–3.39 (m, 2H, H-5′, H-4′), 3.40 (s, 3H,
CDCl3): δ 7.34–7.10 (m, 20 H, Ph), 4.95 (d, 1H, J1′,2′ = 3.7 Hz, OCH3), 1.95 (s, 3H, COCH3); 13C NMR (125 MHz, CD3OD):
H-1′), 4.93 (app. s, 1H, H-1), 4.85 (d, 1H, J4,5 = 1.7 Hz, H-5), δ 174.0 (CvO), 171.7 (CvO), 104.1 (C-1), 97.2 (C-1′), 74.9 (C-4),
4.78 (s, 2H, CH2Ph), 4.75, 4.48 (ABq, 2H, JA,B = 11.4 Hz, 74.3 (C-5′), 73.1 (C-3′), 71.6 (C-4′), 70.7 (C-2), 68.7 (C-5), 68.5
CH2Ph), 4.71, 4.56 (ABq, 2H, JA,B = 11.9 Hz, CH2Ph), 4.58, 4.42 (C-3), 62.4 (C-6′), 56.3 (OCH3), 55.1 (C-2′), 52.9 (CO2CH3), 22.7
(ABq, 2H, JA,B = 12.0 Hz, CH2Ph), 4.15 (dd, 1H, J3,4 = 2.8 Hz, (NAc); LRMS: m/z = 448.1 [M + Na]+, 464.1 [M + K]+; HRMS: m/z
H-4), 3.84 (dd, 1H, J2,3 = 2.4 Hz, H-3), 3.79 (dd, 1H, J2′,3′
9.1 Hz, J3′,4′ = 8.0 Hz, H-3′), 3.74 (ddd, 1H, J2,OH = 11.8 Hz, J2,3
2.4 Hz, J1,2 = 1.1 Hz, H-2), 4.72 (s, 3H, CO2CH3), 3.71–3.68 (m,
2H, H-4′, H-6a′), 3.61–3.54 (m, 3H, H-6b′, H-2′, H-5′), 3.53 (d,
=
=
calcd for C16H27O12NNa [M + Na]+: 448.1425; found: 448.1420.
Methyl(methyl 3-O-benzyl-α-L-idopyranosyluronate)-(1→4)-2-
azido-3-O-benzyl-2-deoxy-α-D-glucopyranoside (43)
1H, J2,OH = 11.8 Hz, OH-2), 3.45 (s, 3H, OCH3); 13C NMR The disaccharide 42 (500 mg, 0.62 mmol) was deprotected as
(125 MHz, CDCl3): δ 170.1 (CvO), 138.1, 137.7, 137.5, 137.3, described for compound 41. The crude product was purified
128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, by flash chromatography (EtOAc/n-hexane, 3 : 1) to give the
127.2 (Ph), 102.9 (C-1), 94.9 (C-1′), 81.0 (C-3′), 77.5 (C-4′), 75.8, triol 43 as white solid (325 mg, 88%); Rf = 0.64 (EtOAc/MeOH,
74.6, 73.5, 71.9 (4 × CH2Ph), 71.5 (C-5′), 71.4 (C-3, C-4), 67.7 9 : 1); m.p. 77–80 °C; [α]D +24.1 (c 3.0, CHCl3); 1H NMR
(C-6′), 66.7 (C-5), 66.3 (C-2), 63.6 (C-2′), 56.2 (OCH3), 52.4 (500 MHz, CD3OD): δ 7.45–7.19 (m, 10H, Ph), 5.12 (d, 1H,
(CO2CH3); LRMS: m/z = 792.3 [M + Na]+, 808.3 [M + K]+; HRMS: J1′,2′ = 2.5 Hz, H-1′), 4.81 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.74, 4.66
m/z calcd for C42H47O11N3Na [M + Na]+: 792.3103; found: (ABq, 2H, JA,B = 11.1 Hz, CH2Ph), 4.71 (d, 1H, J4′,5′ = 2.7 Hz,
792.3105.
H-5′), 4.68, 4.51 (ABq, 2H, JA,B = 11.2 Hz, CH2Ph), 3.92–3.88 (m,
2H, H-4, H-4′), 3.84–3.81 (m, 2H, H-2′, H-6a), 3.78 (dd, 1H, B
part of ABX, J5,6b = 3.8 Hz, J6a,6b = 12.2 Hz, H-6b), 3.73 (dd, 1H,
J2,3 = J3,4 = 10.3 Hz, H-3), 3.72 (dd, 1H, J2′,3′ = J3′,4′ = 3.5 Hz,
Methyl(2-amino-2-deoxy-α-D-glucopyranosyl)-(1→4)-(methyl
α-L-idopyranosid)uronate (5)
The disaccharide 41 (19 mg, 0.025 mmol) was dissolved in H-3′), 3.68–3.65 (m, 1H, H-5), 3.42 (s, 3H, CO2CH3), 3.40 (dd,
anhydrous MeOH (5 mL) and 20% Pd(OH)2/C (120 mg) was 1H, H-2), 3.38 (s, 3H, OCH3); 13C NMR (100 MHz, CD3OD):
added. The mixture was then stirred under an atmosphere of δ 171.9 (CvO), 139.6, 139.5, 129.4, 129.3, 129.0, 128.9, 128.3,
H2 at r.t. for 24 h. The catalyst was removed by filtration over 128.2 (Ph), 102.1 (C-1′), 100.0 (C-1), 79.7 (C-3), 78.0 (C-3′), 75.8
Celite, the filtrate was concentrated to give the disaccharide 5 (C-4′), 75.1 (CH2Ph), 73.3 (CH2Ph), 73.3 (C-5), 70.8 (C-5′), 70.1
as a colourless oil (9.5 mg, 100%); Rf = 0.06 (NH4OH/i-PrOH = (C-4), 68.5 (C-2′), 64.9 (C-2), 61.6 (C-6), 55.5 (CO2CH3), 52.2
1 : 4); [α]D +11.6 (c 0.3, MeOH); 1H NMR (500 MHz, CD3OD): (OCH3); LRMS: m/z = 607.2 [M + NH4]+, 612.2 [M + Na]+, 628.2
δ 5.30 (d, 1H, J1′,2′ = 3.6 Hz, H-1′), 4.82 (d, 1H, J4,5 = 2.7 Hz, [M + K]+; HRMS: m/z calcd for C29H35N3O11 [M + Na]+:
H-5), 4.74 (d, 1H, J1,2 = 2.4 Hz, H-1), 4.06 (dd, 1H, J4,5 = 2.7 Hz, 612.2164; found: 612.2166.
J3,4 = 4.0 Hz, H-4), 4.03 (dd, 1H, J3,4 = J2,3 = 4.0 Hz, H-3),
Methyl(methyl α-L-idopyranosyluronate)-(1→4)-2-amino-2-
deoxy-α-D-glucopyranoside (9)
3.79 (s, 3H, CO2CH3), 3.77 (dd, 1H, A part of ABX, J5′,6a′
=
2.4 Hz, J6a′,6b′ = 11.9 Hz, H-6a′), 3.71 (dd, 1H, B part of ABX,
J5′,6b′ = 4.5 Hz, H-6b′), 3.61 (dd, 1H, J2′,3′ = 10.5 Hz, J3′,4′
=
The disaccharide 43 (50 mg, 0.085 mmol) was hydrogenated as
8.9 Hz, H-3′), 3.56 (dd, 1H, H-2), 3.44 (ddd, 1H, J4′,5′ = 8.9 Hz, described for compound 3 to give the amine 9 as yellow oil
H-5′), 3.40 (s, 3H, OCH3), 3.36 (dd, 1H, H-4′), 3.02 (dd, (25.7 mg, 79%); Rf = 0.36 (i-PrOH/NH4OH, 3 : 1); [α]D +14.6
1H, H-2′); 13C NMR (125 MHz, CD3OD): δ 171.6 (CvO), (c 2.2, MeOH); 1H NMR (500 MHz, CD3OD): δ 5.04 (d, 1H,
104.1 (C-1), 95.3 (C-1′), 74.7 (C-5′), 74.4 (C-4), 71.7 (C-3′), 71.2 J1′,2′ = 3.8 Hz, H-1′), 4.98 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.91 (d, 1H,
(C-4′), 70.5 (C-2), 68.6 (C-5), 67.8 (C-3), 61.9 (C-6′), 56.3 (OCH3), J4′,5′ = 3.5 Hz, H-5′), 3.90 (dd, 1H, J3′,4′ = 4.9 Hz, H-4′), 3.85–3.78
56.0 (C-2′), 52.9 (CO2CH3); LRMS: m/z = 406.1 [M + Na]+; (m, 4H, H-3, H-6a, H-6b, H-3′), 3.75 (s, 3H, CO2CH3), 3.66–3.65
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(m, 2H, H-4, H-5), 3.52 (dd, 1H, J2′,3′ 5.7 Hz, H-2′), 3.45 (s, 3H, Methyl(methyl 2,3,4-tri-O-benzoyl-β-D-glucopyranosyluronate)-
OCH3), 3.22 (dd, 1H, J2,3 = 10.4 Hz, H-2); 13C NMR (100 MHz, (1→4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-α-D-
CD3OD): δ 172.1 (CvO), 103.4 (C-1′), 97.6 (C-1), 79.3 (C-4), 72.7 glucopyranoside (47)
(C-5), 72.4 (C-3′), 71.9 (C-5′), 71.8 (C-4′), 71.7 (C-2′), 70.3 (C-3),
The glycosyl acceptor 46 (250 mg, 0.6 mmol) and thioglycoside
61.3 (C-6), 55.8 (C-2), 55.7 (OCH3), 52.6 (CO2CH3); LRMS: m/z =
donor 45 (333 mg, 0.6 mmol) were dissolved in anhydrous
384.1 [M + H]+; HRMS: m/z calcd for C14H25NO11 [M + H]+:
DCM (10 mL), and then activated 3 Å mol sieves (200 mg) were
added. The mixture was stirred at r.t. for 30 min under argon.
384.1500; found: 384.1511.
The mixture was then cooled to −78 °C, and NIS (135 mg,
Methyl(methyl α-L-idopyranosyluronate)-(1→4)-2-acetamido-2-
deoxy-α-D-glucopyranoside (10)
0.6 mmol) and TMSOTf (108 μL, 0.6 mmol) were added. The
resulting mixture was stirred at −78 °C under argon for 17 h.
Sat. aq. Na2S2O3 was added to quench the reaction, and the
purple colour faded. The mixture was then filtered through
Celite and the filter cake was washed with DCM. The com-
bined filtrate and washings were washed with H2O, NaHCO3,
brine, dried (MgSO4), filtered and concentrated. The residue
was then purified by flash chromatography (EtOAc/n-hexane,
3 : 7) to give the disaccharide 47 as a white solid (172 mg, 31%);
Rf = 0.4 (EtOAc/n-hexane, 1 : 3); m.p. 211 °C; [α]D +46.83 (c 1.16,
The disaccharide 43 (50 mg, 0.085 mmol) was dissolved in
anhydrous MeOH (5 mL) and 10% Pd/C (50 mg) and Ac2O
(0.1 mL, 1.7 mmol) were added. The mixture was then stirred
at r.t. under an atmosphere of H2 for 24 h. The catalyst was
removed by filtration over Celite, and the filtrate was concen-
trated. The residue was then purified by flash chromatography
(EtOAc/MeOH/H2O, 7 : 2 : 1) to give the amide 10 as a colourless
oil (20.8 mg, 58%); Rf = 0.4 (EtOAc/MeOH/H2O, 6 : 3 : 1); [α]D
1
1
CHCl3); H NMR (500 MHz, CDCl3): δ 7.95–7.80 (m, 8H, Ph),
+26.48 (c 1.67, MeOH); H NMR (500 MHz, CD3OD): δ 5.03 (d,
7.59–7.26 (m, 17H, Ph), 5.83 (dd, 1H, J3′,4′ = J2′,3′ = 9.6 Hz, H-3′),
1H, J1′,2′ = 3.6 Hz, H-1′), 4.94 (d, 1H, J4′,5′ = 3.4 Hz, H-5′), 4.64
(d, 1H, J1,2 = 3.6 Hz, H-1), 3.93 (dd, 1H, J2,3 = 10.6 Hz, H-2),
3.87 (dd, 1H, J4′,5′ = 3.4 Hz, J3′,4′ = 4.9 Hz, H-4′), 3.81–3.76 (m,
5.64 (dd, 1H, J4′,5′ = J3′,4′ = 9.6 Hz, H-4′), 5.61 (dd, 1H, J1′,2′
=
7.9 Hz, H-2′), 5.24, 4.82 (ABq, 2H, JA,B = 11.2 Hz, CH2Ph), 5.08
(d, 1H, H-1′), 4.72 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.53 (dd, 1H, A
part of ABX, J5,6a = 1.9 Hz, J6a,6b = 12.1 Hz, H-6a), 4.37 (dd, 1H,
B part of ABX, J5,6b = 4.1 Hz, H-6b), 4.13 (d, 1H, H-5′),
4.00–3.97 (m, 2H, H-3, H-4), 3.82–3.80 (m, 1H, H-5), 3.49 (s,
3H, CO2CH3), 3.37 (dd, 1H, J2,3 = 10.2 Hz, H-2), 3.35 (s, 3H,
OCH3); 13C NMR (100 MHz, CDCl3): δ 166.6, 165.7, 165.4,
165.0, 164.8 (4 × CvO), 138.2, 133.4, 133.3, 133.2, 129.7, 129.6,
129.5, 128.6, 128.5, 128.4, 128.3, 127.8, 127.5 (Ph), 101.1 (C-1′),
98.5 (C-1), 78.6 (C-4), 78.2 (C-3), 75.4 (CH2Ph), 73.1 (C-5′), 72.1
(C-3′), 71.9 (C-2′), 70.1 (C-4′), 68.5 (C-5), 63.1 (C-2), 61.9 (C-6),
55.4 (OCH3), 52.7 (CO2CH3); LRMS: m/z = 933.2 [M + NH4]+,
938.2 [M + Na]+, 954.2 [M + K]+; HRMS: m/z calcd for
C49H45N3O15 [M + Na]+: 938.2743; found: 938.2709.
3H, H-6a, H-6b, H-3′), 3.74 (s, 3H, CO2CH3), 3.71 (dd, 1H, J3,4
=
8.6 Hz, J2,3 = 10.6 Hz, H-3), 3.64–3.59 (m, 2H, H-4, H-5), 3.50
(dd, 1H, J2′,3′ = 5.3 Hz, H-2′), 3.34 (s, 3H, OCH3), 1.96 (s, 3H,
COCH3); 13C NMR (100 MHz, CD3OD): δ 173.6, 172.1 (2 ×
CvO), 103.3 (C-1′), 99.6 (C-1), 80.3 (C-4), 72.3 (C-5), 72.2 (C-3′),
71.9 (C-5′), 71.8 (C-4′), 71.7 (C-2′), 71.4 (C-3), 61.8 (C-6), 55.5
(C-2), 55.4 (OCH3), 52.4 (CO2CH3), 22.5 (COCH3); LRMS: m/z =
426.1 [M + H]+, 448.1 [M + Na]+, 464.1 [M + K]+; HRMS: m/z
calcd for C16H27NO12 [M + Na]+: 448.1425; found: 448.1417.
Methyl(methyl 2,3,4-tri-O-benzoyl-1-thio-β-D-glucopyranosid)
uronate (45)
The triol 44 (1 g, 4.2 mmol) was dissolved in anhydrous pyri-
dine (10 mL) and BzCl (1.76 mL, 15.1 mmol) was added drop-
wise to the solution at 0 °C under N2 over 10 min. The ice bath
was removed and the reaction mixture was stirred at r.t. for 2 h
Methyl(methyl β-D-glucopyranosyluronate)-(1→4)-2-azido-3-O-
benzyl-2-deoxy-α-D-glucopyranoside (48)
under N2. The mixture was then diluted with CHCl3 (10 mL), The disaccharide 47 (126 mg, 0.14 mmol) was deprotected as
washed with H2O (10 mL), 0.1 M HCl (10 mL), sat. aq. NaHCO3 described for compound 41. The crude product was purified
(10 mL) and dried (MgSO4), filtered and concentrated. The by flash chromatography (EtOAc/MeOH, 4 : 1) to give the tetrol
residue was then recrystallized from EtOAc/n-hexane to give 48 as a colourless oil (60.6 mg, 88%); Rf = 0.44 (EtOAc/MeOH,
1
the tribenzoate 45 as colourless crystals (2.13 g, 93%); Rf = 0.68 4 : 1); [α]D +1.36 (c 0.44, CHCl3); H NMR (500 MHz, CD3OD):
(EtOAc/n-hexane, 1 : 1); m.p. 164 °C; [α]D +4.0 (c 0.2, CHCl3); 1H δ 7.45–7.21 (m, 5H, Ph), 5.13, 4.68 (ABq, 2H, JA,B = 10.9 Hz,
NMR (500 MHz, CDCl3): δ 7.96–7.91 (m, 4H, Ph), 7.84–7.81 (m, CH2Ph), 4.77 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.59 (d, 1H, J1′,2′
2H, Ph), 7.54–7.25 (m, 9H, Ph), 5.94 (dd, 1H, J2,3 = J3,4 = 9.7 Hz, 7.8 Hz, H-1′), 3.98–3.93 (m, 2H, H-4, H-6a), 3.90 (dd, 1H, J2,3
=
=
H-3), 5.67 (dd, 1H, J3,4 = J4,5 = 9.7 Hz, H-4), 5.61 (dd, 1H, J1,2
J2,3 = 9.7 Hz, H-2), 4.74 (d, 1H, H-1), 4.36 (d, 1H, H-5), 3.70 (s, J6a,6b = 12.3 Hz, H-6b), 3.68 (ddd, 1H, J5,6a = 3.5 Hz, J4,5
3H, CO2CH3), 2.29 (s, 3H, SCH3); 13C NMR (100 MHz, CDCl3): 9.8 Hz, H-5), 3.58 (d, 1H, J4′,5′ = 9.6 Hz, H-5′), 3.50 (dd, 1H,
δ 167.0, 165.6, 165.1, 165.0 (4 × CvO), 133.4, 133.3, 133.2, J3′,4′ = 9.0 Hz, H-4′), 3.39 (s, 3H, CO2CH3), 3.37 (dd, 1H, J2′,3′
=
J3,4 = 8.9 Hz, H-3), 3.82 (dd, 1H, B part of ABX, J5,6b = 2.0 Hz,
=
=
129.9, 129.8, 129.7, 128.9, 128.7, 128.6, 128.4, 128.3, 128.2 J3′,4′ = 9.0 Hz, H-3′), 3.33 (s, 3H, OCH3), 3.31 (dd, 1H, H-2), 3.26
(Ph), 83.5 (C-1), 76.6 (C-5), 73.3 (C-3), 70.1 (C-4), 69.3 (C-2), (dd, 1H, H-2′); 13C NMR (125 MHz, CD3OD): δ 172.1 (CvO),
52.8 (OCH3), 11.6 (SCH3); LRMS: m/z = 568.1 [M + NH4]+, 139.8, 129.5, 129.2, 128.5 (Ph), 104.1 (C-1′), 100.1 (C-1), 79.6
573.1 [M + Na]+, 589.1 [M + K]+; HRMS: m/z calcd for (C-3), 77.7 (C-4), 77.6 (C-3′), 75.7 (C-5′), 75.6 (C-2′, OCH2), 73.5
C29H32O9S [M + Na]+: 573.1190; found: 573.1208.
(C-4′), 72.9 (C-5), 64.5 (C-2), 61.7 (C-6), 55.4 (CO2CH3), 49.6
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(OCH3); LRMS: m/z = 522.2 [M + Na]+; HRMS: m/z calcd for
Acknowledgements
C21H29N3O11 [M + Na]+: 522.1694; found: 522.1695.
We thank the National MPS Society (USA) and the Australian
Research Council (DP170104431 to VF) for funding. QQH
gratefully acknowledges the University of Queensland for a UQ
International PhD Scholarship and Arlo D. Harris Travel
Scholarship.
Methyl(methyl β-D-glucopyranosyluronate)-(1→4)-2-amino-2-
deoxy-α-D-glucopyranoside (11)
The disaccharide 48 (20.3 mg, 0.04 mmol) was hydrogenated
as described for compound 3. The crude product was purified
by flash chromatography (EtOAc/MeOH, 1 : 1) to give the amine
11 as a colourless oil (14.5 mg, 90%); Rf = 0.28 (EtOAc/MeOH,
References
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1 : 1); [α]D +17.29 (c 1.67, MeOH); H NMR (500 MHz, CD3OD):
δ 4.88 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.48 (d, 1H, J1′,2′ = 7.9 Hz,
H-1′), 3.94 (d, 1H, J4′,5′ = 9.6 Hz, H-5′), 3.90 (dd, 1H, A part of
1 S. Tomatsu, T. Fujii, M. Fukushi, T. Oguma, T. Shimada,
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ABX, J5,6a = 3.7 Hz, J6a,6b = 12.2 Hz, H-6a), 3.85 (dd, 1H, J3,4
8.5, J2,3 = 10.4 Hz, H-3), 3.83 (dd, 1H, B part of ABX, J5,6b
=
=
1.7 Hz, H-6b), 3.76 (s, 3H, CO2CH3), 3.68–3.65 (m, 1H, H-5),
3.61 (dd, 1H, J4,5 = 9.8 Hz, H-4), 3.49 (dd, 1H, J3′,4′ = 9.1 Hz,
H-4′), 3.44 (s, 3H, OCH3), 3.41 (dd, 1H, J2′,3′ = J3′,4′ = 9.1 Hz,
H-3′), 3.25 (dd, 1H, H-2′), 3.15 (dd, 1H, 1H, H-2); 13C NMR
(125 MHz, CD3OD): δ 171.2 (CvO), 104.6 (C-1′), 97.8 (C-1), 80.4
(C-4), 77.1 (C-3′), 76.3 (C-5′), 74.6 (C-2′), 73.0 (C-4′), 72.3 (C-5),
70.3 (C-3), 61.0 (C-6), 55.8 (OCH3), 55.6 (C-2), 52.9 (CO2CH3);
LRMS: m/z = 384.0 [M + H]+; HRMS: m/z calcd for C14H25NO11
[M + H]+: 384.1500; found: 384.1500.
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Methyl(methyl β-D-glucopyranosyluronate)-(1→4)-2-acetamido-
2-deoxy-α-D-glucopyranoside (12)
The disaccharide 48 (20 mg, 0.04 mmol) was dissolved in
anhydrous MeOH (3 mL) and 20% Pd(OH)2/C (20 mg) and
Ac2O (75 μL, 0.4 mmol) were added. The resulting solution was
stirred at r.t. under an atmosphere of H2 for 24 h. The catalyst
was removed by filtration through Celite, and the filtrate was
concentrated. The residue was then purified by flash chrom-
atography (EtOAc/MeOH, 4 : 1) to give the amide 12 as a syrup
(8.1 mg, 48%); Rf = 0.52 (EtOAc/MeOH, 1 : 1); [α]D +13.09
(c 0.53, MeOH); 1H NMR (500 MHz, CD3OD): δ 4.64 (d, 1H,
J1,2 = 3.5 Hz, H-1), 4.48 (d, 1H, J1′,2′ = 7.9 Hz, H-1′), 3.94 (d, 1H,
J4′,5′ = 9.8 Hz, H-5′), 3.91 (dd, 1H, J2,3 = 10.7 Hz, H-2), 3.86 (dd,
1H, A part of ABX, J5,6a = 3.9 Hz, J6a,6b = 12.2 Hz, H-6a), 3.81
(dd, 1H, B part of ABX, J5,6b = 2.4 Hz, H-6b), 3.78 (dd, 1H, J3,4
=
8.5 Hz, H-3), 3.76 (s, 3H, CO2CH3), 3.66–3.63 (m, 1H, H-5), 3.57
(dd, 1H, J4,5 = 9.9 Hz, H-4), 3.49 (dd, 1H, J3′,4′ = 9.2 Hz, H-4′),
3.40 (dd, 1H, J2′,3′ = J3′,4′ = 9.2 Hz, H-3′), 3.35 (s, 3H, OCH3),
3.25 (dd, 1H, H-2′), 1.96 (s, 3H, COCH3); 13C NMR (125 MHz,
CD3OD): δ 173.5, 171.0 (2 × CvO), 104.7 (C-1′), 99.5 (C-1), 81.6
(C-4), 77.2 (C-3′), 76.4 (C-5′), 74.6 (C-2′), 73.0 (C-4′), 71.9 (C-5),
71.0 (C-3), 61.7 (C-6), 55.6 (OCH3), 55.0 (C-2), 52.9 (CO2CH3),
22.5 (COCH3); LRMS: m/z = 426.1 [M + H]+, 448.1 [M + Na]+,
464.0 [M + K]+; HRMS: m/z calcd for C16H27NO12 [M + Na]+:
448.1425; found: 448.1420.
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Conflicts of interest
There are no conflicts to declare.
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