Development of oleanane-type triterpenes as a new class of HCV entry inhibitors

Article abstract of DOI:10.1021/jm301910a

Development of hepatitis C virus (HCV) entry inhibitors represents an emerging approach that satisfies a tandem mechanism for use with other inhibitors in a multifaceted cocktail. By screening Chinese herbal extracts, oleanolic acid (OA) was found to display weak potency to inhibit HCV entry with an IC₅₀ of 10 μM. Chemical exploration of this triterpene compound revealed its pharmacophore requirement for blocking HCV entry, rings A, B, and E, are conserved while ring D is tolerant of some modifications. Hydroxylation at C-16 significantly enhanced its potency for inhibiting HCV entry with IC ₅₀ at 1.4 μM. Further modification by conjugation of this new lead with a disaccharide at 28-COOH removed the undesired hemolytic effect and, more importantly, increased its potency by ～5-fold (54a, IC₅₀ 0.3 μM). Formation of a triterpene dimer via a linker bearing triazole (70) dramatically increased its potency with IC₅₀ at ～10 nM. Mechanistically, such functional triterpenes interrupt the interaction between HCV envelope protein E2 and its receptor CD81 via binding to E2, thus blocking virus and host cell recognition. This study establishes the importance of triterpene natural products as new leads for the development of potential HCV entry inhibitors.

Full text of DOI:10.1021/jm301910a

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Article
Development of Oleanane-Type Triterpenes
as a New Class of HCV Entry Inhibitors
Fei Yu, Qi Wang, Zhen Zhang, Yi-yun Peng, Yun-yan Qiu, Yong-ying Shi, Yong-xiang
Zheng, Su-Long Xiao, Han Wang, Xiao-xi Huang, Lingyi Zhu, Kun-bo Chen, Chuan-
ke Zhao, Chuan-ling Zhang, Mao-rong Yu, Di-an Sun, Li-he Zhang, and De-min Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301910a • Publication Date (Web): 10 May 2013
Downloaded from http://pubs.acs.org on May 21, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Development of Oleanane-Type Triterpenes as a New Class of HCV Entry
Inhibitors
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Fei Yu,†,§ Qi Wang,†,‡,§ Zhen Zhang,†,§ Yiyun Peng,† Yongyan Qiu,† Yongying Shi,†
Yongxiang Zheng,† Sulong Xiao,†,* Han Wang,† Xiaoxi Huang,† Lingyi Zhu,† Kunbo
Chen,† Chuanke Zhao,† Chuanling Zhang,† Maorong Yu,† Dian Sun,⊥ Lihe Zhang,†
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and Demin Zhou †,*
† State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
‡ Department of Molecular and Cellular Pharmacology, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
⊥ Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing 100094, China
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ABSTRACT
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Development of hepatitis C virus (HCV) entry inhibitors represents an emerging
approach that satisfies a tandem mechanism for use with other inhibitors in a
multifaceted cocktail. By screening Chinese herbal extracts, oleanolic acid (OA) was
found displaying weak potency to inhibit HCV entry with IC50 at 10 ꢀM. Chemical
exploration of this triterpene compound revealed its pharmacophore requirement for
blocking HCV entry ꢀ ring A, B and E are conserved while ring D is tolerant to some
modifications. Hydroxylation at Cꢀ16 significantly enhanced its potency for inhibiting
HCV entry with IC50 at 1.4 ꢀM. Further modification by conjugation of this new lead
with a disaccharide at 28ꢀCOOH removed the undesired hemolytic effect, and more
importantly increased its potency by ~5ꢀfold (54a, IC50 0.3 ꢀM). Formation of a
triterpene dimer via a linker bearing triazole (70) dramatically increased its potency
with IC50 at ~10 nM. Mechanistically, such functional triterpenes interrupt the
interaction between HCV envelope protein E2 and its receptor CD81 via binding to
E2, thus blocking virus and host cell recognition. This study establishes the
importance of triterpene natural products as new leads for the development of
potential HCV entry inhibitors.
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Keywords:
Triterpene; Oleanolic acid; Echinocystic acid; HCV entry inhibitor; SAR
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INTRODUCTION
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Hepatitis C virus (HCV) is the leading cause of liver fibrosis and cirrhosis that
eventually lead to liver carcinomas.1 Treatment of HCV infection with
ribavirin/interferon has been used for almost 30 years. The recently approved
Telaprevir and Boceprevir targeting HCV replication represent the beginning of a new
era in the control of HCV infection.2 However, resistance to individual antiviral drug
is likely to appear and a combination of drugs targeting different stages of HCV life
cycle is required. Inhibition of virus entry into HCVꢀpermissive cells represents an
emerging field for the prevention and reduction of infection. Development of HCV
entry inhibitors could satisfy the tandem use with other inhibitors of viral replication,
leading to a multifaceted approach to control HCV infection more effectively.
Pentacyclic triterpenes are secondary plant metabolites found in different plant
organs, with a few species containing up to 30% of their dry weight.3 These
triterpenes have been proposed possessing defense activities due to their capability to
prevent host from various pathogen and herbivore infections.4 Betulinic acid, a
lupaneꢀtype triterpene, has been confirmed by many studies displaying significant
inhibiting activity against HIV entry and virus mature/release. One derivative,
bevirimat (PAꢀ457), is already in clinical trial.5 Moronic acid and maslinic acid, two
oleananeꢀtype triterpenes, also display antiꢀHIV activity in vitro.6 Other diverse and
promising biological activities of triterpenes, including antiꢀinflammatory,
hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory
and tonic effects, warrant further pharmaceutical development and even clinical
investigation.7
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Recently, a variety of compounds have been shown to exhibit antiꢀHCV entry
activity with their IC50 at ꢀM to even subꢀꢀM level, depending on the sensitivity of
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the assay in different laboratories.8 Here, we report oleanolic acid (OA) and
echinocystic acid (EA), two naturally occurring oleananeꢀtype triterpenes (Figure 1),
and their derivatives displaying substantial activity to inhibit HCV entry with one
derivative (70) showing IC50 at ~10 nM. Further studies showed that these compounds
represented a group of specific antiꢀHCVpp agents targeting viral entry process. The
putative mechanism underlying the blocking of HCV entry by EA is its strong
binding to E2, one of the envelope proteins of HCV, which blocks the interaction
between E2 and CD81, a wellꢀidentified receptor for HCV entry. This study
establishes the importance of triterpene natural products as leads for the development
of potential HCV entry inhibitors.
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RESULTS AND DISCUSSION
Discovery of Oleanolic Acid (OA) as a HCV-entry inhibitor. A library of Chinese
herbal extracts was screened using a HCV pseudo particle (HCVpp) entry assay.8b, 9
Vesicular stomtatis virus G protein pseudo particle (VSVpp) was also tested in
parallel to determine the specificity and toxicity as previously reported.8b, 9 The
nꢀbutanol/H2O crude extract of Dipsacus asperoides, a traditional Chinese herbal
medicine, displayed specific antiꢀHCV entry activity with 50% of viral entry being
inhibited at the concentration of 50 ꢀg/mL (S Figure 1). Subsequent
bioactivityꢀguided phytochemical studies indicated the major active constituents in
the extract were saponin due to their strong polarity and weak UV absorption at 254
nm (S Figure 2). One major aglycone isolated from the saponin was OA (Figure 1), a
relatively nonꢀtoxic triterpene natural product exhibiting attractive hepatoprotective,
antitumor and antiviral properties.10 Biological assays indicated that OA displayed
substantial activity on blocking HCV entry, with IC50 at ~10 ꢀM (Figure 1), but was
far less potent than their parental saponin mixture, which was more potent following
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increase of their polarity (S Figure 2).
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Identification of the Pharmacophore of OA. To assess the antiꢀHCV potential of
structurally related triterpene compounds and determine the structureꢀactivity
relationship, a series of commercially available triterpenes were acquired from
ChromaDex and Sigma. In addition, a variety of chemical modifications were carried
out at the 3ꢀOH, 28ꢀCOOH positions of OA to identify the structural requirements. As
shown in Figure 1, initial exploration of OA indicated that 3ꢀOH, the chemically
modifiable group at ring A, was functionally important since its oxidation (1),
acetylation (2) and glucosylation (3) all significantly decreased or even eliminated the
activity. Further exploration at ring A indicated that not only 3ꢀOH but also other
positions were also less tolerant to some modifications: hydroxylation at Cꢀ2 (4),
Cꢀ23 (5) or both (6) reduced the activity greatly. The potency of 5 was totally lost
upon further oxidation of the 23ꢀCH2OH group into 23ꢀCHO (7) or 23ꢀCOOH (8), or
glucosidation at 3ꢀOH (9). In addition to ring A, we found ring B was unmodifiable
since hydroxylation at Cꢀ6 (10) completely abolished the potency of OA. These data
suggested the left part of OA, including ring A and B, was conserved and any
modification might significantly decrease its activity.
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We subsequently explored the structureꢀactivity relationship of ring C, in
particular the allyl skeleton, and found that a substituent hydroxyl group at either the
methylene (11) or vinyl skeleton (12) had almost no or just marginal effect on the
potency of OA. In addition, introduction of a keto at the allyl skeleton (13) also had
no obvious effect, suggesting ring C is a modifiable site. Continued exploration of the
ring D of OA indicated that hydroxylation at Cꢀ16 (EA) remarkably enhanced the
antiꢀHCV activity and the resulting EA showed an increase of IC50 by almost 8ꢀfold
(1.4 ꢀM). This result suggested ring D was a modifiable part and more potent leads
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might be prepared by modifying ring D. In addition, we synthesized a series of OA
derivatives and found ester or amide substitutions at 28ꢀCOOH (14, 15) maintained or
even enhanced the potency, suggesting carboxyl acid is not absolutely required and
can be modified.
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Shift of Cꢀ29ꢀmethyl group from Cꢀ20 to adjacent Cꢀ19 (16) at ring E decreased
the antiꢀHCV activity by more than 50%. Switch of 28ꢀCOOH with 29ꢀmethyl group
plus oxidation at Cꢀ11 (17), a rather tolerant site, also decreased the antiꢀHCV
potency. However, replacement of ring E, a 6ꢀmember ring, with a 5ꢀmember ring by
switching the linker Cꢀ21ꢀCꢀ20 to Cꢀ21ꢀCꢀ19 (betulinic acid, 18) significantly
decreased the report readings in both HCVpp and VSVpp infected cells. This
observation might reflect the cellular toxicity rather than potency enhancement by the
replacement of oleanolic skeleton with betulinic acid, a relatively cytotoxic triterpene
being developed as an antiꢀcancer drug.8 In this study, the VSVpp entry assay,
performed in parallel with HCVpp entry, was originally used to monitor the inhibitory
specificity towards different viruses. Later, we found VSVpp assay is also useful to
monitor chemical toxicity since the VSVpp, unlike HCVpp which infects only liver
cell (Huh7), has broad host range and infects almost all cell lines. When a chemical is
cytotoxic, the expression of luciferase reporter, delivered by VSVpp, in host cells will
be inhibited. We found the VSVpp assay is even more sensitive to chemical toxicity
than alamarBlue assay. Overall, OA SAR data provide positional requirements and
their pharmacophore contributions on the antiviral activity, suggesting part of OA
right side could be modified. Further modification at this region, just like EA, might
enhance its antiꢀHCV potency.
Pharmacophore confirmation via chemical modification of EA. EA is an analog of
OA, with a hydroxyl group at Cꢀ16 (Figure 1). Such a small variation significantly
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differentiated EA from OA with almost 10ꢀfold potency gain for EA. Therefore, EA
was chosen as a new lead for SAR exploration and the same conclusion was obtained.
As shown in Table 1, modifications of 3ꢀOH at ring A, including sulfonation (19),
amination (20), benzylation (21), oxidation (22), methylation (23), elimination (24)
and other modifications (25, 26), made these compounds less potent than EA.
Hydroxylation at other sites of ring A, including at Cꢀ1 (27) or Cꢀ23 (28), also
significantly decreased potency. Further oxidation of 23ꢀCH2OH into 23ꢀCHO (29)
and then 23ꢀCOOH (30) completely abolished the potency of EA. Similarly,
modification of ring B, including hydroxylation at Cꢀ6 (31) or Cꢀ7 (32) via
biotransformation approach,11 abolished the antiviral activity of EA. Therefore, the
left side of EA, including ring A and B, is functionally conserved and some
modification at this side might remarkably attenuate its potency.
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Modification of ring C indicated that a keto (33) or a hydroxyl (34) substitute at
the vinyl skeleton had almost no effect or only marginal effect on the potency of EA,
similar to the case of OA that ring C is tolerant for chemical modifications.
Explorations of ring D (35-45) indicated that the potency gained by the introduction
of a hydroxyl group at Cꢀ16 was lost upon acetylation or elimination of 16ꢀOH.
However, oxidation of 16ꢀOH seemed to bring less effect on the potency. Furthermore,
46, a glycoside conjugate at both 3ꢀOH and 28ꢀCOOH, also displayed remarkable
potency with >90% HCV entry being inhibited at 10 ꢀM (Table 1). The significant
inhibition of HCV entry by glucose substitution at the conserved 3ꢀOH (47) implied
that the potency of EA can be further enhanced if the glycoside was kept at 28ꢀCOOH,
consistent with the initial observation that the more polar the saponin extract the more
potent the virus entry blocking activity. An independent biotransformation experiment
indicated that the ring E of EA was not a potential modifiable site since hydroxylation
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at Cꢀ21 and Cꢀ29 (48, 49) all significantly decreased its potency on blocking HCV
entry. 11 Apparently, intact rings A, B and E were common structural features that
were required for the antiꢀHCV entry activity.
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Development of more potent HCV entry inhibitors. Pentacyclic triterpenes, such as
EA and OA, are hydrophobic, which may affect their potential pharmacokinetic
properties. To increase their solubility, a variety of carbohydrate moieties, including
monoꢀ, diꢀ and even triꢀsaccharides, were conjugated to EA via 28ꢀCOOH, a
modifiable site within ring D. As outlined in Scheme 1, direct esterification of EA
with 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀglucosyl bromide afforded 50a, which was then
hydrolyzed in the presence of CH3ONa to give 50b.12 In the same fashion,
conjugations of other saccharide with EA (51a-55b, Table 1) were carried out.
The HCV entry assay indicated that the EAꢀoligosaccharide conjugates, either
carrying protective acetyl groups or not, exhibited more potent effects than EA
(Table 1). Among them, acetylated galactose displayed the highest potency followed
by acetyl protected glucose and ribose. For disaccharide conjugates, the terminally
acetylated galactose (54a) showed the most promising inhibitory activity with IC50 at
0.3 ꢀM, 5ꢀfold more potent than EA (Figure 2). However, deacetylation of EA
glycoconjudates reduced the potency, even though the solubility was significantly
increased, especially in the case of 54a versus 54b. This result might be due to the
reduction of hydrophobicity, which decreased the affinity of EAꢀconjugates to the
protein targets that is usually via hydrophobic interactions.
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A parallel experiment was carried out by conjugating EA with the same
carbohydrate moieties through a triazole linker rather than an ester bond. Such
alteration might theoretically enhance the stability of EA glycoconjugates (58a-68b,
Table 1). The synthesis was based on a Cu(I) azideꢀalkyne cycloaddition reaction
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(CuAAC)
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Biological evaluations indicated that almost all such conjugates with acetyl groups in
the carbohydrate moieties exhibited comparable potency as their counterparts (Table
1). Deprotection of EA conjugates remarkably decreased their activities, independent
of a monoꢀ, diꢀ or trisaccharide conjugate, just like the cases in the ester bond
conjugates (50aꢀ55b). In addition, we found that EA glycoconjugates with monoꢀ, diꢀ
or trisaccharide display almost the identical activity, suggesting introduction of a
monosaccharide is sufficient to enhance its biological activity.
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Alternatively, combination of two pharmacophores together is one potential way
to further increase the activity of EA. Here, two types of EA dimer, 69 and 70, were
synthesized as shown in Scheme 3. We found that 69, the EA dimer linked by
1,3ꢀpropanediamine, displayed almost the same activity as EA. Unexpectedly, 70, the
EA dimer bearing a triazole linker, exhibited significantly higher potency with IC50 at
10.3 nM (Figure 2), almost two orders of magnitudes more potent than EA (1.4 ꢀM).
To our knowledge, this is the most potent compound based on HCVpp entry assay.
Further explorations of this new lead are ongoing to obtain promising HCV entry
inhibitors.
Mechanistic investigation of EA-mediated HCV entry inhibition. It is clear that
intact ring A, B and E are common structural features among EA and their active
derivatives and analogs, which leads us to hypothesize that they are the
pharmacophore for blocking HCV entry, either via interacting with HCVpp, host
Huh7 cells or both. To clarify this hypothesis, five different assay conditions were set
up as previously reported.14 In the first condition, the coꢀtreatment assay, EA was
present in the culture medium during the entire viral entry process. Briefly, cells were
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infected with HCVpp or VSVpp in the presence of 10 ꢀM EA and incubated for 72 h
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at 37 °C to allow virus entry. In the second condition, the pretreatment assay, cells
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compound and then exposed to viruses at 37 C for 72 h. In the third condition, the
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prebinding assay, cells were exposed to viruses in the presence of EA at 4 oC for 3 h,
washed to remove unbound viruses and compound and then cultured at 37 oC for 72 h.
In the fourth condition, the postꢀbinding and preꢀentry assay, cells were first incubated
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with viruses at 4 C for 3 h, washed to remove unbound virus and then treated with
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EA at 37 C for 72 h. In the fifth condition, the postꢀentry assay, cells were first
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treated with viruses at 37 C for 6 h to allow the virus entry into cells. After washed
unbound viruses, infected cells were treated with the compound at 37 oC for 72 h.
Under all five conditions, CD81 antibody was utilized as a positive control
because it blocks HCV virus entry via binding to CD81 receptors. IM2865 was an
unrelated compounds used as another control and 0.5% DMSO (final concentration)
was used for normalization in each condition.14 As compared to the coꢀtreatment
assay, a short pretreatment of the cells with the compound prior to virus infection
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(pretreatment) or coꢀtreatment of cells with viruses and the compound at 4 C
(prebinding) resulted in very weak, if any, activity (Figure 3), suggesting that EA
exerts its inhibitory activity post virus binding. This is remarkably different from
CD81 antibody which interferes with virus attachment to the target cells by
interacting with the surface receptors. In the postꢀbinding or preꢀentry condition, a
high activity of EA was noted, suggesting that EA interferes with the subsequent step
following virus attachment to target cells, presumably the step of viral envelopeꢀcell
membrane fusion. Under the postꢀentry condition, antiviral activity was not observed
at all, indicating that EA did not interfere with the multiple processes after viral entry.
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In conclusion, these data suggested that EA exerts its inhibitory activity at the stage of
postꢀvirus binding but before virus entry, presumably interfering with virus
envelopeꢀhost cell membrane fusion.
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EA-mediated disruption of CD81 and E2 interaction. In order to determine which
target EA potentially binds to and thereby blocks HCV entry, a biotinꢀlabeled EA
conjugate (73) was synthesized (Scheme 4)15 and then utilized as a probe to
pullꢀdown the targets in HCVpp packaging cell lysate. Conjugation of the biotin
molecule at its 28ꢀCOOH through CuAAC had no detectable effect on the potency of
EA (Table 1). As shown in Figure 4, HCV envelope protein E2 was selectively
pulled down by streptavidin (SA) immobilized agrose. In contrast, no protein was
captured in a parallel pullꢀdown experiment wherein free biotin or EA was utilized,
replacing 73 as the probe. Furthermore, competitive experiments indicated that no E2
was pull down when extra free EA was added into the cell lysate, and such
competition was verified by an alternative assay ꢀ surface plasmon resonance (SPR)
experiments wherein the binding of E2 to biotinꢀlabeling EA chip was decreased by
free EA in a significant doseꢀdependence manner (S Figure 3). Therefore, both
supported the direct binding of EA to E2 protein. To confirm the binding is indeed
due to the interaction between EA and E2, we then selected another set of
experiments where SAꢀbiotin interaction was used for the immobilization of E2 on the
SA sensor tip in BIAcore assay. The sensor was then treated with samples of different
concentrations of CD81 and CD81 together with EA. The sensor immobilized E2 on
superstreptavidin (SSA) was subjected to treatment with sample EA.16 A series of
BIAcore assays indicated that the KD between E2 and EA is around 24 ꢀM, about
1000ꢀfold lower than E2 and CD81 (KD = 21 nM), and no binding was detected at all
for EA to CD81 (Figure 5). Characterization of E2 and CD81 binding in the presence
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of 10 ꢀM EA indicated that their KD decreased to 100 nM, ~5ꢀfold lower than that
without EA. Based on data from the biological and affinity assays, we proposed a
tentative mechanism for EAꢀmediated blocking of HCV entry (Figure 6): EA binds
to HCV envelope protein E2 and thus disrupts its interaction with CD81 receptor;
such disruption does not block virusꢀhost cell attachment but the followed fusion of
the virus envelope and host cell membrane.
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Hemolytic and cytotoxic studies of EA and its derivatives. A series of studies
have demonstrated that the aglycon of triterpenoids possess crucial influence on the
hemolytic properties, one of the wellꢀknown characteristics for saponins.17 Compared
with OA which possesses mild hemolytic activity, EA gains substantial hemolytic
side effect with CC50 at 15 ꢀM. Such hemolytic property may restrict these EA
derivatives used as potential antiꢀHCV entry inhibitors. Here, we found that almost all
the EA derivatives depleted the hemolytic activity upon modification of Cꢀ28
carboxyl group (S Figure 4). In addition, the cytotoxicity of the most representative
compounds in this study, EA, 54a and 70, towards HepG2 (a liver cancer cell), Hela
(a cervical cancer cell line) and 293T (a derived kidney normal cell) has been tested
based on alamarBlue assay and no toxicity was detected even at concentration high as
50 ꢀM (S Figure 5). Furthermore, data from the VSVpp entry, a useful assay
reflecting the cytotoxicity of screen chemicals, also support that most EA derivatives
display far less cytotoxicity (Table 1).
CONCLUSION
We identified oleananeꢀtype triterpenes, oleanolic acid (OA) and echinocystic
acid (EA), as new scaffolds for blocking HCV entry. Guided by SAR studies, a new
class of HCV inhibitors with IC50 from ꢀM to nM was prepared. Mechanistic studies
indicated that such functional triterpenes bind to HCV envelope protein E2 and thus
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interrupt the interaction between E2 and CD81 receptor. Such disruption does not
block virus and host cell attachment but the followed virusꢀhost fusion (Figure 6).
This study establishes the importance of triterpene natural products as new leads for
the development of potential HCV entry inhibitors.
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EXPERIMENTAL SECTION
Chemistry. High Resolution Mass Spectra (HRMS) were obtained with an APEX IV
FT_MS (7.0 T) spectrometer (Bruker) in positive ESI mode. NMR spectra were
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recorded on a Bruker DRX 400 spectrometer at ambient temperature. H NMR
chemical shifts were referenced to the internal standard TMS (δ = 0.00) or the
H
solvent signal (δ H = 3.31 for the central line of MeOD). 13C NMR chemical shifts are
referenced to the solvent signal (δ C = 77.00 for the central line of CDCl3, δ C = 49.00
for the central line of MeOD). Reactions were monitored by thinꢀlayer
chromatography (TLC) on a preꢀcoated silica gel 60 F254 plate (layer thickness 0.2
mm; E. Merck, Darmstadt, Germany) and detected by staining with a yellow solution
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containing Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24 4H2O (24.0 g) in 6 % H2SO4
(500 mL) followed by heating. Flash column chromatography was performed on silica
gel 60 (200 ꢀ 300 mesh, Qingdao Haiyang Chemical Co. Ltd). The method employed
in determining the purity of synthesized compounds was HPLC using a Waters e2695
instrument and Waters 2998 photodiode array detector, with chromatography
performed on a Diamonsil 250 × 4.6 mm, 5 ꢀm C18(2) column, and eluted with
CH3OHꢀH2O (80:20 to 100:0) at a flow rate of 1.0 mL/min.
General procedure A for the synthesis of EA glycoconjugates (50a-55a). To the
glycosyl bromide (0.8 mmol) and EA (189 mg, 0.4 mmol) stirring in CH2Cl2/H2O
(10:1, 22 mL) was added K2CO3 (138 mg, 1 mmol) and Bu4NBr (64 mg, 0.2 mmol).
The mixture was refluxed under nitrogen atmosphere. After completion (TLC) the
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reaction mixture was diluted with CH2Cl2 (20 mL) and washed with water (10 mL ×
3), brine and dried over Na2SO4. The crude product was purified by column
chromatography.
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General procedure B for the O-deacetylation of EA glycoconjugates (50b-55b,
58b-68b). To the Oꢀpentacetylated compound stirring in methanol was added sodium
methoxide (cat.). The mixture was stirred at rt. After completion (TLC) the reaction
mixture was neutralized with HCl (1 M). Water was added and the resulting
suspension was filtered. Crude product was purified by column chromatography.
General procedure C for the CuAAC “click” reaction (58a-68a). To a solution of
alkyne (0.16 mmol) and azide (0.19 mmol) in CH2Cl2 (3 mL) and H2O (3 mL), was
added CuSO4 (30 mg, 0.19 mmol) and NaꢀLꢀascorbate (75 mg, 0.38 mmol). The
resulting solution was stirred vigorously for 12 h at rt. The reaction mixture was
diluted with H2O (10 mL), then extracted with CH2Cl2 (10 mL × 3). The combined
organic layer was dried over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography.
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Compound 50a. Prepared from EA (189 mg, 0.4 mmol) and acetylꢀglucosyl
bromide (329 mg, 0.8 mmol) according to General procedure A. The residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 50a as a
white solid (252 mg, 80%). 1H NMR (400 MHz, CDCl3): δ 0.70, 0.75, 0.88, 0.89, 0.92,
0.96, 1.32 (7 × CH3), 1.99, 2.00, 2.00, 2.05 (4 × CH3CO), 0.72ꢀ2.16 (m, other aliphatic
ring protons), 2.97 (dd, 1H, J = 4.0, 14.3 Hz), 3.19 (dd, 1H, J = 4.2, 10.6 Hz), 3.74ꢀ3.78
(m, 1H), 4.02 (dd, 1H, J = 2.1, 12.4 Hz), 4.25 (dd, 1H, J = 4.4, 12.4 Hz), 4.39 (brs, 1H),
5.08ꢀ5.24 (m, 3H), 5.39 (t, 1H, J = 3.2 Hz), 5.54 (d, 1H, J = 8.2 Hz). 13C NMR (100
MHz, CDCl3): δ 15.4, 15.6, 17.0, 18.2, 20.5 (3C), 20.6, 23.2, 24.5, 26.8, 27.1, 28.0,
30.2 (2C), 32.6, 33.0, 35.1, 35.5, 36.9, 38.5, 38.7, 39.5, 40.4, 41.3, 46.0, 46.6, 48.8,
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55.2, 61.4, 67.9, 69.9, 72.4, 72.7, 74.2, 78.8, 91.6, 123.2, 141.9, 169.1, 169.4, 170.0,
170.5, 174.7. ESIꢀHRMS (m/z): [M+Na]+calcd for C44H66NaO13, 825.4396, found
825.4387.
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Compound 50b. Prepared from 50a (50 mg, 0.06 mmol) according to General
procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH,
10/1 v/v) to afford 50b as a white solid (12.4 mg, 31%). 1H NMR (400 MHz, MeOD):
δ 0.77, 0.79, 0.89, 0.96, 0.97, 1.37 (7 × CH3), 0.74ꢀ1.96 (m, other aliphatic ring
protons), 2.29 (t, 1H, J = 13.3 Hz), 2.99 (dd, 1H, J = 4.0, 14.2 Hz), 3.15 (dd, 1H, J = 5.0,
11.4 Hz), 3.27ꢀ3.34 (m, 4H), 3.67 (dd, 1H, J = 4.3, 12.0 Hz), 3.82 (d, 1H, J = 11.1 Hz),
4.53 (brs, 1H), 5.32 (t, 1H, J = 3.4 Hz), 5.35 (d, 1H, J = 8.1 Hz). 13C NMR (100 MHz,
MeOD): δ 16.1, 16.3, 17.8, 19.5, 24.5, 25.0, 27.3, 27.9, 28.7, 31.3, 31.7, 33.3, 34.2,
36.3, 36.4, 38.2, 39.8, 40.0, 40.8, 42.1, 42.7, 47.8, 48.2, 50.0, 56.9, 62.4, 71.1, 74.0,
74.9, 78.3, 78.7, 79.7, 95.7, 123.6, 144.6, 177.2. ESIꢀHRMS (m/z) [M+Na]+calcd for
C36H58NaO9, 657.3973, found 657.3987.
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Compound 51a. Prepared from EA (189 mg, 0.4 mmol) and acetylꢀxylosyl
bromide (270 mg, 0.8 mmol) according to General procedure A. The residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 51a as a
white solid (202 mg, 67%). 1H NMR (400 MHz, CDCl3): δ 0.69, 0.74, 0.86, 0.88, 0.91,
0.94, 1.31 (7 × CH3), 2.01, 2.01, 2.02 (3 × CH3CO), 0.79ꢀ2.15 (m, other aliphatic ring
protons), 2.97 (dd, 1H, J = 4.1, 14.1 Hz), 3.17 (dd, 1H, J = 4.2, 10.4 Hz), 3.45 (dd, 1H,
J = 8.5, 12.0 Hz), 4.05ꢀ4.10 (m, 1H), 4.38 (brs, 1H), 4.89ꢀ5.00(m, 2H), 5.16 (t, 1H, J =
8.2 Hz), 5.37 (t, 1H, J = 3.3 Hz), 5.56 (d, 1H, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3):
δ 15.5, 15.6, 17.0, 18.2, 20.6, 20.6, 23.3, 24.6, 26.8, 28.0, 30.2, 32.6, 33.0, 35.1, 35.4,
36.9, 38.5, 38.7, 39.5, 40.4, 41.3, 46.1, 46.6, 48.9, 55.2, 62.5, 68.2, 69.4, 70.9, 74.1,
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76.7, 77.0, 77.4, 78.8, 91.9, 123.1, 142.1, 169.2, 169.7, 169.8, 174.7. ESIꢀHRMS (m/z)
[M+Na]+calcd for C41H62NaO11, 753.4184, found 753.4199.
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Compound 51b. Prepared from 51a (90 mg, 0.12 mmol) according to General
procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH,
10/1 v/v) to afford 51b as a white solid (58.6 mg, 78%). 1H NMR (400 MHz, MeOD):
δ 0.78, 0.89, 0.95, 0.96, 0.97, 1.37 (7 × CH3), 0.74ꢀ1.93 (m, other aliphatic ring
protons), 2.28 (t, 1H, J = 13.3 Hz), 3.02 (dd, 1H, J = 4.0, 14.2 Hz), 3.15 (dd, 1H, J = 5.0,
11.4 Hz), 3.25ꢀ3.31 (m, 2H), 3.38 (t, 1H, J = 8.4 Hz), 3.47ꢀ3.53 (m, 1H), 3.88 (dd, 1H,
J = 5.0, 11.5 Hz), 4.51 (brs, 1H), 5.31ꢀ5.33 (m, 2H). 13C NMR (100 MHz, MeOD): δ
16.1, 16.3, 17.8, 19.5, 24.5, 25.1, 27.3, 27.9, 28.8, 31.3, 31.9, 33.4, 34.3, 36.3, 36.4,
38.2, 39.8, 40.0, 40.8, 42.1, 42.7, 47.7, 48.2, 50.1, 56.9, 67.2, 70.8, 73.5, 74.9, 77.3,
79.7, 96.2, 123.7, 144.6, 177.3. ESIꢀHRMS (m/z) [M+Na]+ calcd for C35H56NaO8,
627.3867, found 627.3881.
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Compound 52a. Prepared from EA (189 mg, 0.4 mmol) and acetylꢀarabinosyl
bromide (270 mg, 0.8 mmol) according to General procedure A. The residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 52a as a
white solid (341 mg, 82%). 1H NMR (400 MHz, CDCl3): δ 0.72, 0.75, 0.87, 0.89, 0.92,
0.96, 1.31 (10 × CH3), 2.02, 2.04, 2.11 ( 3 × CH3CO), 0.69ꢀ2.14 (m, other aliphatic ring
protons), 3.00 (dd, 1H, J = 4.0, 14.3 Hz), 3.19 (dd, 1H, J = 4.2, 10.6 Hz), 3.70 (dd, 1H,
J = 1.8, 12.9 Hz), 3.97 (dd, 1H, J = 3.8, 13.0 Hz), 4.38 (t, 1H, J = 3.4 Hz), 5.10 (dd, 1H,
J = 3.5, 8.9 Hz), 5.19ꢀ5.26 (m, 2H), 5.41 (t, 1H, J = 3.2 Hz), 5.51 (d, 1H, J = 6.8 Hz).
13C NMR (100 MHz, CDCl3): δ 15.5 (2C), 17.0, 18.2, 20.6, 20.7, 20.8, 23.2, 24.8, 26.8,
27.1, 28.0, 29.8, 30.2, 32.6, 33.0, 35.0, 35.5, 36.9, 38.5, 38.7, 39.5, 40.5, 41.4, 46.1,
46.6, 49.0, 55.2, 63.5, 67.0, 68.0, 69.6, 73.9, 78.8, 91.9, 123.1, 141.9, 169.3, 169.8,
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170.1, 174.6. ESIꢀHRMS (m/z) [M+Na]+ calcd for C41H62NaO11, 753.4184, found
753.4204.
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Compound 52b. Prepared from 52a (50 mg, 0.07 mmol) according to General
procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH,
10/1 v/v) to afford 52b as a white solid (38.6 mg, 93%). 1H NMR (400 MHz, MeOD):
δ 0.76, 0.77, 0.88, 0.94, 0.96, 1.36 (7 × CH3), 0.73ꢀ1.93 (m, other aliphatic ring
protons), 2.27 (t, 1H, J = 13.3 Hz), 3.05 (dd, 1H, J = 4.1, 14.3 Hz), 3.14 (dd, 1H, J =
5.0, 11.4 Hz), 3.54 (dd, 1H, J = 9.7 Hz), 3.64ꢀ3.65 (m, 2H), 3.86ꢀ3.90 (m, 2H), 4.51
(brs 1H), 5.33 (t, 1H, J = 3.2 Hz), 5.37ꢀ5.41 (m, 1H). 13C NMR (100 MHz, MeOD): δ
16.1, 16.3, 17.8, 19.5, 24.5, 25.2, 27.3, 27.9, 28.7, 31.3, 31.8, 33.4, 34.2, 36.2, 36.3,
38.1, 39.8, 39.9, 40.8, 42.0, 42.6, 47.6, 48.2, 50.2, 56.8, 66.1, 68.2, 71.2, 73.5, 74.8,
79.7, 95.7, 123.7, 144.6, 177.2. ESIꢀHRMS (m/z) [M+Na]+ calcd for C35H56NaO8,
627.3867, found 627.3880.
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Compound 53a. Prepared from EA (189 mg, 0.4 mmol) and acetylꢀgalactosyl
bromide (329 mg, 0.8 mmol) according to General procedure A. The residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 53a as a
white solid (98.5 mg, 31%). 1H NMR (400 MHz, CDCl3): δ 0.75, 0.78, 0.91, 0.92, 0.95,
0.99, 1.34 (7 × CH3), 1.99, 2.02, 2.04, 2.17 (4 × CH3CO), 0.73ꢀ2.14 (m, other aliphatic
ring protons), 2.99ꢀ3.01 (brs, 1H, J = 10.6 Hz), 3.22 (dd, 1H, J = 4.1, 10.4 Hz), 4.00 (t,
1H, J = 6.7 Hz), 4.10ꢀ4.15 (m, 2H), 4.39 (brs, 1H), 5.07 (dd, 1H, J = 3.4, 10.4 Hz), 5.31
(t, 1H, J = 10.3 Hz), 5.40ꢀ5.44 (m, 2H), 5.54 (d, 1H, J = 8.4 Hz). 13C NMR (100 MHz,
CDCl3): δ 15.5, 15.6, 17.1, 18.2, 20.4, 20.6 (2C), 20.6, 23.3, 24.7, 26.8, 27.1, 28.0, 29.8,
30.2, 32.6, 33.0, 35.1, 35.5, 37.0, 38.5, 38.7, 39.5, 40.5, 41.4, 46.1, 46.6, 49.0, 55.2,
60.7, 66.7, 67.6, 70.7, 71.4, 74.1, 78.8, 92.0, 123.1, 141.9, 169.3, 169.8, 170.1, 170.2,
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174.6 ESIꢀHRMS (m/z) [M+Na]+ calcd for C44H66NaO13, 825.4396, found 825.4387;
[M+NH4]+ calcd for C44H70NO13, 820.4842, found 820.4839.
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Compound 53b. Prepared from 53a (50 mg, 0.06 mmol) according to General
procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH,
10/1 v/v) to afford 53b as a white solid (47 mg, 99%). 1H NMR (400 MHz, MeOD): δ
0.77, 0.78, 0.89, 0.96, 0.97, 1.37 (7 × CH3), 0.74ꢀ1.96 (m, other aliphatic ring protons),
2.29 (t, 1H, J = 13.3 Hz), 3.00 (dd, 1H, J = 3.8, 14.2 Hz), 3.15 (dd, 1H, J = 4.9, 11.4 Hz),
3.50 (dd, 1H, J = 3.2, 9.7 Hz), 3.58ꢀ3.65 (m, 2H), 3.69ꢀ3.71 (m, 2H), 3.88 (d, 1H, J =
3.0 Hz), 4.55 (brs, 1H), 5.31ꢀ5.33 (m, 2H). 13C NMR (100 MHz, MeOD): δ 16.1, 16.3,
17.8, 19.5, 24.5, 25.0, 27.3, 27.9, 28.7, 31.3, 31.8, 33.4, 34.2, 36.3, 36.4, 38.2, 39.8,
40.0, 40.8, 42.1, 42.6, 47.8, 48.2, 50.0, 56.9, 62.0, 70.0, 71.3, 74.9, 75.2, 77.4, 79.7,
96.2, 123.6, 144.6, 177.3. ESIꢀHRMS (m/z) [M+NH4]+calcd for C36H62NO9, 652.4419,
found 652.4415.
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Compound 54a. Prepared from EA (189 mg, 0.4 mmol) and acetylꢀlactosyl
bromide (559 mg, 0.8 mmol) according to General procedure A. The residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 54a as a
white solid (234 mg, 59%). 1H NMR (400 MHz, CDCl3): δ 0.71, 0.78, 0.90, 0.92, 0.94,
0.98, 1.34 (7 × CH3), 1.96, 2.02, 2.03, 2.05, 2.07, 2.10, 2.15 (7 × CH3CO), 0.74ꢀ2.17
(m, other aliphatic ring protons), 2.98 (dd, 1H, J = 3.9, 14.4 Hz), 3.21 (dd, 1H, J = 3.8,
10.6 Hz), 3.69ꢀ3.72 (m, 1H), 3.83ꢀ3.88 (m, 2H), 4.05ꢀ4.15 (m, 3H), 4.36ꢀ4.39 (m, 2H),
4.47 (d, 1H, J = 7.9 Hz), 4.95 (dd, 1H, J = 3.4, 10.4 Hz), 5.05ꢀ5.12 (m, 2H), 5.23 (t, 1H,
J = 9.2 Hz), 5.35 (d, 1H, J = 3.2 Hz), 5.39 (brs, 1H), 5.53 (d, 1H, J = 8.3 Hz). 13C NMR
(100 MHz, CDCl3): δ 15.4, 15.5, 17.0, 18.2, 20.4, 20.5 (2C), 20.6 (2C), 20.7, 20.7, 23.2,
24.5, 26.8, 27.1, 28.0, 30.0, 30.2, 32.6, 33.0, 35.1, 35.4, 36.9, 38.5, 38.7, 39.5, 40.4,
41.3, 46.0, 46.6, 48.8, 55.2, 60.8, 61.6, 66.6, 69.0, 70.3, 70.7, 70.9, 72.6, 73.3, 74.2,
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75.7, 78.8, 91.4, 100.9, 123.2, 141.9, 168.9, 169.4, 169.5, 170.0, 170.1, 170.2, 170.3,
174.6. ESIꢀHRMS (m/z) [M+Na]+calcd for C56H82NaO21, 1113.5241, found 1113.5238;
[M+NH4]+ calcd for C56H86NO21, 1108.5687, found 1108.5698.
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Compound 54b. Prepared from 54a (140 mg, 0.13) according to General
procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH,
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7/1 v/v) to afford 54b as a white solid (92.6 mg, 91%). H NMR (400 MHz,
Pyridineꢀd5): δ 0.99, 1.03, 1.06, 1.08, 1.17, 1.23, 1.85 (7 × CH3), 0.90ꢀ2.61 (m, other
aliphatic ring protons), 2.83 (t, 1H, J = 13.4 Hz), 3.46ꢀ3.56 (m, 2H), 3.98ꢀ4.00 (m, 1H),
4.12ꢀ4.17 (m, 3H), 4.29ꢀ4.57 (m, 7H), 5.12 (d, 1H, J = 7.8 Hz), 5.31 (brs, 1H), 5.65 (brs
1H), 6.27 (d, 1H, J = 8.2 Hz), 6.43 (d, 1H, J = 3.5 Hz). 13C NMR (100 MHz,
Pyridineꢀd5): δ 16.3, 17.1, 18.1, 19.4, 24.4, 25.1, 27.8, 28.7, 29.3, 31.4, 32.7, 33.7,
34.1, 36.5, 36.6, 38.0, 39.6, 39.9, 40.7, 41.9, 42.6, 47.7, 47.8, 49.7, 56.5, 62.2, 62.5,
70.6, 73.0, 74.2, 74.9, 75.7, 77.5, 77.7, 77.8, 78.7, 82.0, 95.9, 106.3, 123.3, 144.9,
176.4. ESIꢀHRMS (m/z) [M+Na]+ calcd for C42H68NaO14, 819.4501, found 819.4492.
Compound 55a. Prepared from EA (189 mg, 0.4 mmol) and acetylꢀmaltosyl
bromide (559 mg, 0.8 mmol) according to General procedure A. The residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 55a as a
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white solid (83 mg, 19%). H NMR (400 MHz, CDCl3): δ 0.72, 0.78, 0.90, 0.92, 0.94,
0.98, 1.34 (7 × CH3), 2.00, 2.02, 2.02, 2.05, 2.10, 2.12 (7 × CH3CO), 0.74ꢀ2.17 (m,
other aliphatic ring protons), 2.98 (dd, 1H, J = 3.9, 14.2 Hz), 3.21(dd, 1H, J = 3.9, 10.5
Hz), 3.76ꢀ3.80 (m, 1H), 3.91ꢀ3.94 (m, 1H), 4.00ꢀ4.05 (m, 2H), 4.20ꢀ4.26 (m, 2H),
4.36ꢀ4.40 (m, 2H), 4.84 (dd, 1H, J = 4.0, 10.5 Hz), 4.98ꢀ5.07 (m, 2H), 5.26ꢀ5.40 (m,
4H), 5.59 (d, 1H, J = 8.2 Hz). 13C NMR (100 MHz, CDCl3): δ 15.4, 15.5, 17.0, 18.2,
20.5, 20.5 (3C), 20.6, 20.7, 20.8, 23.2, 24.5, 26.8, 27.1, 28.0, 30.1, 30.2, 32.6, 33.0,
35.2, 35.4, 36.9, 38.5, 38.7, 39.5, 40.4, 41.3, 46.1, 46.6, 48.8, 55.2, 61.4, 62.5, 68.0,
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68.5, 69.2, 70.0, 70.7, 72.6, 72.8, 74.3, 75.2, 78.8, 91.2, 95.6, 123.2, 141.9, 169.4 (2C),
169.8, 170.0, 170.3, 170.4, 170.5, 174.5. ESIꢀHRMS (m/z) [M+Na]+ calcd for
C56H82NaO21, 1113.5241, found 1113.5247.
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Compound 55b. Prepared from 55a (41 mg, 0.04 mmol) according to General
procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH,
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7/1 v/v) to afford 55b as a white solid (25 mg, 83%). H NMR (400 MHz, MeOD): δ
0.77, 0.79, 0.89, 0.95, 0.97, 1.37 (7 × CH3), 0.74ꢀ1.96 (m, other aliphatic ring protons),
2.28 (t, 1H, J = 13.4 Hz), 3.00 (dd, 1H, J = 3.9, 14.2 Hz), 3.15 (dd, 1H, J = 5.0, 11.3 Hz),
3.23ꢀ3.28 (m, 1H), 3.34ꢀ3.38 (m, 1H), 3.43ꢀ3.46 (m, 2H), 3.58ꢀ3.72 (m, 5H), 3.82ꢀ3.84
(m, 3H), 4.53 (brs, 1H), 5.18 (d, 1H, J = 3.8 Hz), 5.32(brs, 1H), 5.37 (d, 1H, J = 8.2 Hz).
13C NMR (100 MHz, MeOD): δ 16.1, 16.3, 17.8, 19.5, 24.5, 25.1, 27.3, 27.9, 28.7, 31.2,
31.7, 33.3, 34.2, 36.4 (2C), 38.2, 39.8, 40.0, 40.8, 42.1, 42.7, 47.8, 48.2, 50.1, 56.9,
61.8, 62.8, 71.6, 73.6, 74.1, 74.8, 74.9, 75.1, 77.4, 78.1, 79.7, 80.5, 95.6, 102.8, 123.7,
144.6, 177.2. ESIꢀHRMS (m/z) [M+Na]+ calcd for C42H68NaO14, 819.4501, found
819.4491.
Compound 56. To EA (944 mg, 2 mmol) and TBTU (963 mg, 3 mmol) stirring
in 20 mL THF was added DIEA (0.5 mL, 3 mmol). The mixture was stirred at room
temperature overnight. After completion (TLC) the reaction mixture was filtered, and
the filtration was concentrated. The crude was purified by column chromatography
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(petroleum ether/AcOEt, 4/1 v/v) to give 56 as a white solid (1.01 g, 86%). H NMR
(400 MHz, CDCl3): δ 0.79, 0.83, 0.92, 0.98, 1.01, 1.04, 1.45 (7 × CH3), 0.75ꢀ2.35 (m,
other aliphatic ring protons), 3.16 (dd, 1H, J = 4.2, 14.2 Hz), 3.23 (dd, 1H, J = 3.8,
10.7 Hz), 4.85 (brs, 1H), 5.49 (t, 1H, J = 3.2 Hz), 7.34 (d, 1H, J = 8.3 Hz), 7.39ꢀ7.55
(m, 2H), 8.05 (d, 1H, J = 8.4 Hz). 13C NMR (100 MHz, CDCl3): δ 15.5, 15.6, 17.3,
18.2, 23.3, 24.4, 26.9, 27.1, 28.1, 30.4, 31.1, 32.6, 33.0, 35.1, 36.0, 37.0, 38.5, 38.7,
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39.6, 41.0, 41.5, 46.2, 46.5, 49.3, 55.2, 74.2, 78.9, 108.0, 120.5, 124.2, 124.7, 128.6,
128.6, 141.4, 143.4, 172.7. ESIꢀHRMS (m/z) [M+H]+ calcd for C36H52N3O4, 590.3952,
found 590.3949.
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Compound 57. To 56 (589 mg, 1 mmol) and Na2CO3 (106 mg, 1 mmol) stirring
in 8 mL DMF was added 2ꢀpropynylamine (0.1 mL, 1.5 mmol). The mixture was
stirred at room temperature for 20 min. After completion (TLC) the solvent was
removed under reduced pressure. The mixture was resolved in AcOEt and washed
with water and brine twice. The organic layer was dried over MgSO4, then filtered
and concentrated. The crude was purified by column chromatography (petroleum
ether/AcOEt, 2/1 v/v) to give 57 as a white solid (473 mg, 93%). 1H NMR (400 MHz,
CDCl3): δ 0.79, 0.81, 0.91, 0.93, 0.95, 1.00, 1.38 (7 × CH3), 0.73ꢀ2.30 (m, other
aliphatic ring protons), 2.21 (t, 1H, J = 2.6 Hz), 2.70 (dd, 1H, J = 3.8, 13.7 Hz), 3.22
(dd, 1H, J = 3.7, 10.8 Hz), 3.86ꢀ4.00 (m, 2H), 4.40 (brs, 1H), 5.54 (t, 1H, J = 3.4 Hz),
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6.42 (t, 1H, J = 4.8 Hz). C NMR (100 MHz, CDCl3): δ 15.6 (2C), 17.1, 18.2, 23.4,
24.9, 26.7, 27.1, 28.0, 29.4, 29.6, 30.2, 32.5, 32.5, 35.1, 35.2, 36.9, 38.6, 38.7, 39.6,
41.6, 41.8, 46.6, 46.9, 49.0, 55.1, 71.6, 75.3, 78.8, 79.4, 123.4, 144.0, 177.5.
ESIꢀHRMS (m/z) [M+H]+ calcd for C33H52NO3, 510.3942, found 510.3941.
Compound 58a. Prepared from 57 (82 mg, 0.16 mmol), acetylꢀglucosyl azide
(71 mg, 0.19 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 58a as a white solid
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(117 mg, 83%). H NMR (400 MHz, CDCl3): δ 0.71, 0.78, 0.90, 0.91, 0.99, 1.36 (7 ×
CH3), 1.87, 2.03, 2.07, 2.09 (4 × CH3CO), 0.76ꢀ2.38 (m, other aliphatic ring protons),
2.75ꢀ2.76 (m, 1H, J = 10.9 Hz), 3.22 (dd, 1H, J = 2.9, 10.1 Hz) 4.00ꢀ4.03 (m, 1H), 4.15
(d, 1H, J = 12.6 Hz), 4.26ꢀ4.32 (m, 3H), 4.37 (brs, 1H), 4.55 (dd, 1H, J = 5.8, 15.2 Hz),
5.23ꢀ5.27 (m, 1H), 5.40ꢀ5.47 (m, 2H), 5.55 (brs, 1H), 5.86 (d, 1H, J = 8.6Hz), 6.92 (t,
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1H, J = 4.6 Hz), 7.79 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 15.5, 15.6, 16.8, 18.2,
20.1, 20.5 (2C), 20.6, 23.3, 25.2, 26.8, 27.1, 28.0, 29.0, 30.1, 32.4, 32.5, 35.0, 35.1 (2C),
36.9, 38.5, 38.7, 39.6, 41.6, 41.7, 46.7, 46.8, 49.1, 55.1, 61.5, 67.6, 70.3, 72.6, 75.0,
75.1, 78.8, 85.6, 120.9, 123.5, 143.5, 145.2, 168.7, 169.3, 169.9, 170.5, 178.0.
ESIꢀHRMS (m/z) [M+H]+ calcd for C47H71N4O12, 883.5063, found 883.5053.
Compound 58b. Prepared from 58a (80 mg, 0.09 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 58b as a white solid (61 mg, 95%). 1H NMR (400 MHz, MeOD):
δ 0.66, 0.78, 0.89, 0.94, 0.96, 0.97, 1.37 (7 × CH3), 0.76ꢀ2.00 (m, other aliphatic ring
protons), 2.35 (t, 1H, J = 13.2 Hz), 2.87 (dd, 1H, J = 3.2, 13.6 Hz), 3.15 (dd, 1H, J = 4.9,
11.4 Hz), 3.47ꢀ3.58 (m, 3H), 3.71 (dd, 1H, J = 5.3, 12.1 Hz), 3.83ꢀ3.89 (m, 2H), 4.31 (d,
1H, J = 15.2 Hz), 4.35 (brs, 1H), 4.43 (d, 1H, J = 15.2 Hz), 5.47 (brs, 1H), 5.57 (d, 1H,
J = 9.2 Hz), 7.98 (s, 1H). 13C NMR (100 MHz, MeOD): δ 16.2, 16.3, 17.7, 19.5, 24.5,
25.4, 27.3, 27.9, 28.7, 31.2, 31.7, 33.3, 33.9, 36.0 (2C), 36.3, 38.1, 39.8, 39.9, 40.8,
42.3, 42.8, 48.0, 48.2, 50.0, 56.8, 62.4, 70.9, 74.0, 75.6, 78.4, 79.7, 81.1, 89.5, 123.5,
124.3, 144.9, 145.9, 180.2. ESIꢀHRMS (m/z) [M+H]+ calcd for C39H63N4O8, 715.4640,
found 715.4647.
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Compound 59a. Prepared from 57 (89 mg, 0.17 mmol), acetylꢀxylosyl azide (57
mg, 0.19 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 59a as a white solid
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(106 mg, 78%). H NMR (400 MHz, CDCl3): δ 0.67, 0.78, 0.90, 0.91, 0.91, 0.99, 1.36
(7 × CH3), 1.88, 2.05, 2.08 (3 × CH3CO), 0.63ꢀ2.25 (m, other aliphatic ring protons),
2.74 (dd, 1H, J = 3.3, 13.5 Hz), 3.22 (dd, 1H, J = 3.5, 10.4 Hz), 3.60 (t, 1H, J = 10.8 Hz),
4.26ꢀ4.31 (m, 2H), 4.38 (brs, 1H), 4.54 (dd, 1H, J = 5.7, 15.2 Hz), 5.14ꢀ5.18 (m, 1H),
5.40ꢀ5.43 (m, 2H), 5.53 (brs, 1H), 5.76 (d, 1H, J = 8.6 Hz), 6.90 (t, 1H, J = 5.1 Hz), 7.77
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(s, 1H). 13C NMR (100 MHz, CDCl3): δ 15.5 (2C), 16.7, 18.1, 20.1, 20.5 (2C), 23.3,
25.1, 26.7, 27.0, 27.9, 29.2, 30.1, 32.4, 32.5, 35.0 (2C), 35.1, 36.8, 38.5, 38.6, 39.5,
41.5, 41.7, 46.6, 46.7, 48.9, 55.1, 65.3, 68.3, 70.3, 71.9, 75.0, 78.7, 86.1, 120.9, 123.3,
143.5, 145.1, 168.7, 169.6, 169.8, 177.9. ESIꢀHRMS (m/z) [M+H]+ calcd for
C44H67N4O10, 811.4852, found 811.4858.
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Compound 59b. Prepared from 59a (55 mg, 0.07 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 59b as a white solid (45 mg, 97%). 1H NMR (400 MHz, MeOD):
δ 0.52, 0.76, 0.88, 0.91, 1.34, 0.95 (7 × CH3), 0.70ꢀ2.01 (m, other aliphatic ring protons),
2.32 (t, 1H, J = 13.2 Hz), 2.85ꢀ2.88 (m, 1H,), 3.15 (dd, 1H, J = 4.8, 11.0 Hz), 3.45ꢀ3.55
(m, 2H), 3.66ꢀ3.72 (m, 1H), 3.88 (t, 1H, J = 9.0), 4.00 (dd, 1H, J = 5.3, 11.2 Hz), 4.35
(brs, 1H), 4.39 (brs, 2H), 5.45 (brs, 1H), 5.50 (d, 1H, J = 9.1 Hz), 7.97 (s, 1H). 13C
NMR (100 MHz, MeOD): δ 16.1, 16.3, 17.6, 19.3, 24.4, 25.3, 27.3, 27.7, 28.7, 31.1,
31.7, 33.2, 33.8, 35.7, 35.8, 36.3, 38.0, 39.7, 39.9, 40.6, 42.2, 42.6, 48.0 (2C), 49.9, 56.7,
69.6, 70.6, 73.7, 75.5, 78.4, 79.7, 89.9, 123.7, 124.2, 144.8, 145.8, 180.1. ESIꢀHRMS
(m/z) [M+H]+ calcd for C38H61N4O7, 685.4535, found 685.4526.
Compound 60a. Prepared from 57 (79 mg, 0.15 mmol), acetylꢀarabinosyl azide
(60 mg, 0.2 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 60a as a white solid
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(111 mg, 92%). H NMR (400 MHz,CDCl3): δ 0.69, 0.78, 0.91, 0.99, 1.36 (7 × CH3),
1.89, 2.04, 2.23 (3 × CH3CO), 0.72ꢀ2.30 (m, other aliphatic ring protons), 2.75 (dd, 1H,
J = 2.5, 13.0 Hz), 3.22 (dd, 1H, J = 2.0, 10.0 Hz), 3.95 (d, 1H, J = 13.3 Hz), 4.18 (d, 1H,
J = 13.4 Hz), 4.27 (dd, 1H, J = 4.4, 15.0 Hz), 4.36 (brs, 1H), 4.56 (dd, 1H, J = 5.8, 15.1
Hz), 5.25 (dd, 1H, J = 3.3, 10.0 Hz), 5.43 (brs, 1H), 5.54ꢀ5.60 (m, 2H), 5.72 (d, 1H, J =
9.1 Hz), 6.90 (t, 1H, J = 4.7 Hz), 7.82 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 15.5 (2C),
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16.8, 18.2, 20.2, 20.5, 20.9, 23.3, 25.2, 26.8, 27.1, 28.0, 29.0, 30.1, 32.4, 32.5, 35.0,
35.1 (2C), 36.9, 38.5, 38.7, 39.5, 41.5, 41.7, 46.7, 46.8, 49.0, 55.1, 67.1, 67.6, 68.1, 70.4,
75.1, 78.8, 86.6, 120.9, 123.4, 143.5, 145.0, 168.9, 169.8, 170.1, 177.9. ESIꢀHRMS
(m/z) [M+H]+ calcd for C44H67N4O10, 811.4852, found 811.4859.
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Compound 60b. Prepared from 60a (77 mg, 0.9 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 60b as a white solid (64 mg, 99%). 1H NMR (400 MHz, MeOD):
δ 0.59, 0.77, 0.89, 0.92, 0.96, 0.97, 1.36 (7 × CH3), 0.72ꢀ1.99 (m, other aliphatic ring
protons), 2.36 (t, 1H, J = 13.2 Hz), 2.87 (dd, 1H, J = 3.2, 13.5 Hz), 3.15 (dd, 1H, J = 4.8,
11.2 Hz), 3.71 (dd, 1H, J = 3.3, 9.4 Hz), 3.85 (d, 1H, J = 12.4 Hz), 3.95 (brs, 1H), 4.01
(dd, 1H, J = 1.6, 12.7 Hz), 4.14 (t, 1H, J = 9.3 Hz), 4.32ꢀ4.45 (m, 3H),5.46 (brs, 1H),
5.46 (d, 1H, J = 9.0 Hz), 7.59 (t, 1H, J = 5.3 Hz), 8.00 (s, 1H). 13C NMR (100 MHz,
MeOD): δ 16.1, 16.4, 17.6, 19.4, 24.4, 25.4, 27.3, 27.9, 28.7, 31.2, 31.9, 33.3, 33.9,
35.9, 36.1, 36.3, 38.0, 39.8, 39.9, 40.7, 42.3, 42.7, 48.0, 48.1, 49.9, 56.7, 70.2, 70.7,
71.3, 74.8, 75.6, 79.6, 90.4, 123.0, 124.3, 144.9, 146.0, 180. ESIꢀHRMS (m/z) [M+H]+
calcd for C38H61N4O7, 685.4535, found 685.4540.
Compound 61a. Prepared from 57 (80 mg, 0.16 mmol), acetylꢀmannosyl azide
(71 mg, 0.19 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 61a as a white solid
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(112 mg, 80%). H NMR (400 MHz, CDCl3): δ 0.51, 0.79, 0.90, 0.90, 0.92, 0.98, 1.35
(7 × CH3), 2.00, 2.10, 2.10, 2.15 (4 × CH3CO), 0.70ꢀ2.27 (m, other aliphatic ring
protons), 2.72 (dd, 1H, J = 3.2, 13.4 Hz), 3.21 (dd, 1H, J = 3.5, 10.3 Hz), 3.93ꢀ3.97 (m,
1H), 4.20 (dd, 1H, J = 1.8, 12.4 Hz), 4.28ꢀ4.46 (m, 4H), 5.25ꢀ5.39 (m, 2H), 5.54 (brs,
1H), 5.66 (d, 1H, J = 2.2 Hz), 6.14 (brs, 1H), 6.90 (t, 1H, J = 5.3 Hz), 7.76 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ 15.5, 15.6, 16.6, 18.1, 20.4, 20.6 (2C), 20.7, 23.3, 24.9,
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26.8, 27.1, 28.0, 29.7, 30.2, 32.4, 32.5, 35.0, 35.1, 35.3, 36.9, 38.5, 38.7, 39.5, 41.5,
41.7, 46.6, 46.9, 48.9, 55.2, 62.1, 64.8, 68.7, 70.8, 75.5, 75.6, 78.9, 84.6, 121.5, 123.7,
143.5, 144.6, 169.2, 169.5, 169.8, 170.5, 178.0. ESIꢀHRMS (m/z) [M+H]+ calcd for
C47H71N4O12, 883.5063, found 883.5065.
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Compound 61b. Prepared from 61a (70 mg, 0.08 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 61b as a white solid (53 mg, 93%). 1H NMR (400 MHz, MeOD):
δ 0.62, 0.78, 0.89, 0.94, 0.96, 0.97, 1.37 (7 × CH3), 0.73ꢀ1.97 (m, other aliphatic ring
protons), 2.35 (t, 1H, J = 13.1 Hz), 2.87 (dd, 1H, J = 3.0, 13.5 Hz), 3.15 (dd, 1H, J =
4.9, 11.3 Hz), 3.52ꢀ3.54 (m, 1H), 3.73ꢀ3.80 (m, 3H), 3.92 (dd, 1H, J = 1.8, 12.1 Hz),
4.09 (brs, 1H), 4.31 (d, 1H, J = 14.6 Hz), 4.35 (brs, 1H), 4.42 (d, 1H, J = 15.1 Hz),
5.47 (brs, 1H), 5.98 (brs, 1H), 8.10 (s, 1H). 13C NMR (100 MHz, MeOD): δ 16.2, 16.3,
17.6, 19.4, 24.5, 25.4, 27.3, 27.9, 28.7, 31.2, 31.8, 33.3, 33.9, 36.0 (2C), 36.3, 38.1, 39.8,
39.9, 40.7, 42.3, 42.8, 48.0, 48.2, 49.9, 56.8, 62.6, 67.8, 72.3, 75.0, 75.6, 79.6, 81.5,
88.2, 124.1, 124.3, 144.9, 145.3, 180.1. ESIꢀHRMS (m/z) [M+H]+ calcd for
C39H63N4O8, 715. 4640, found 715.4637.
Compound 62a. Prepared from 57 (95 mg, 0.19 mmol), acetylꢀgalactosyl azide
(82 mg, 0.22 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 62a as a white solid
(160 mg, 98%). 1H NMR (400 MHz, CDCl3): δ 0.72, 0.78, 0.90, 0.92, 0.99, 1.36 (7 ×
CH3), 1.88, 2.01, 2.05, 2.24 (4 × CH3CO), 0.76ꢀ2.21 (m, other aliphatic ring protons),
2.74 (dd, 1H, J = 3.5, 13.6 Hz) 3.22ꢀ3.24 (m, 1H), 4.11ꢀ4.30 (m, 4H), 4.36 (brs, 1H),
4.56 (dd, 1H, J = 6.0, 15.2 Hz), 5.25 (dd, 1H, J = 3.3, 10.2 Hz), 5.51ꢀ5.56 (m, 3H),
5.80 (d, 1H, J = 9.3 Hz), 6.90 (t, 1H, J = 5.2 Hz), 7.83 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 15.5, 15.6, 16.8, 18.2, 20.2, 20.4, 20.6 (2C), 23.3, 25.2, 26.9, 27.1, 28.0, 28.9,
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30.1, 32.4, 32.5, 35.0, 35.1 (2C), 36.9, 38.5, 38.7, 39.6, 41.7, 41.8, 46.7, 46.8, 49.1, 55.1,
61.1, 66.7, 67.9, 70.7, 73.9, 75.2, 78.8, 86.2, 121.0, 123.5, 143.5, 145.1, 168.9, 169.8,
170.0, 170.3, 177.9. ESIꢀHRMS (m/z) [M+H]+ calcd for C47H71N4O12, 883.5063, found
883.5062.
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Compound 62b. Prepared from 62a (70 mg, 0.08 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 62b as a white solid (53 mg, 93%). 1H NMR (400 MHz, MeOD):
δ 0.65, 0.78, 0.89, 0.94, 0.96, 0.97, 1.37 (7 × CH3), 0.73ꢀ1.98 (m, other aliphatic ring
protons), 2.36 (t, 1H, J = 13.2 Hz), 2.87 (dd, 1H, J = 3.3, 13.4 Hz), 3.15 (dd, 1H, J = 4.9,
11.4 Hz), 3.69 (dd, 1H, J = 3.2, 9.5 Hz), 3.75 (d, 2H, J = 6.0 Hz), 3.81ꢀ3.84 (m, 1H),
3.99 (d, 1H, J = 2.9 Hz), 4.14 (t, 1H, J = 9.4 Hz), 4.32 (dd, 1H, J = 5.2, 15.6 Hz), 4.36
(brs, 1H), 4.44 (dd, 1H, J = 5.3, 15.2 Hz), 5.48 (brs, 1H), 5.54 (d, 1H, J = 9.2 Hz), 7.60
(t, 1H, J = 5.2 Hz), 8.02 (s, 1H). 13C NMR (100 MHz, MeOD): δ 16.2, 16.3, 17.6, 19.4,
24.5, 25.4, 27.3, 27.9, 28.7, 31.2, 31.8, 33.3, 33.9, 36.0, 36.1, 36.3, 38.1, 39.8, 39.9,
40.7, 42.3, 42.8, 48.0, 48.2, 50.0, 56.8, 62.2, 70.2, 71.3, 75.3, 75.6, 79.7, 79.8, 90.1,
123.1, 124.3, 144.9, 146.0, 180.3. ESIꢀHRMS (m/z) [M+H]+ calcd for C39H63N4O8,
715.4640, found 715.4646.
Compound 63a. Prepared from 57 (77 mg, 0.15 mmol), acetylꢀrhamnosyl azide
(60 mg, 0.19 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 63a as a white solid
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(109 mg, 89%). H NMR (400 MHz, CDCl3): δ 0.57, 0.78, 0.89, 0.91, 0.99, 1.36 (7 ×
CH3), 1.99, 2.10, 2.12 (3 × CH3CO), 1.35 (d, 3H, J = 6.2 Hz), 0.71ꢀ2.46 (m, other
aliphatic ring protons), 2.70 (dd, 1H, J = 3.3, 13.5 Hz), 3.21 (dd, 1H, J = 3.6, 10.2 Hz),
3.79ꢀ3.86 (m, 1H), 4.34 (dd, 1H, J = 5.4, 15.0 Hz), 4.42ꢀ4.47 (m, 2H), 5.15ꢀ5.20 (m,
1H), 5.24 (dd, 1H, J = 3.1, 10.2 Hz), 5.51 (brs, 1H), 5.66 (dd, 1H, J = 1.0, 2.9 Hz), 6.12
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(d, 1H, J = 0.8 Hz), 6.90 (t, 1H, J = 10.5 Hz), 7.74 (s, 1H). 13C NMR (100 MHz, CDCl3):
δ 15.4, 15.5, 16.6, 17.4, 18.1, 20.4, 20.5, 20.6, 23.2, 24.8, 26.7, 27.0, 28.0, 29.8, 30.1,
32.5 (2C), 34.9, 35.0, 35.2, 36.8, 38.4, 38.6, 39.4, 41.4, 41.6, 46.6, 46.8, 48.7, 55.1, 69.0,
69.5, 70.7, 73.8, 75.2, 78.7, 84.6, 121.4, 123.5, 143.4, 144.4, 169.1, 169.7, 169.8, 177.9.
ESIꢀHRMS (m/z) [M+H]+ calcd for C45H69N4O10, 825.5008, found 825.5018.
Compound 63b. Prepared from 63a (70 mg, 0.08 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 63b as a white solid (56 mg, 95%). 1H NMR (400 MHz, MeOD):
δ 0.51, 0.77, 0.89, 0.91, 0.96 (6 × CH3), 1.36ꢀ1.38 (2 × CH3), 0.72ꢀ1.99 (m, other
aliphatic ring protons), 2.37 (t, J = 13.1 Hz), 2.84 (dd, 1H, J = 3.0, 13.3 Hz), 3.14 (dd,
1H, J = 4.5, 11.0 Hz), 3.51 (brs, 2H), 3.67 (brs, 1H), 4.06 (brs, 1H), 4.31ꢀ4.44 (m, 3H),
5.45 (brs, 1H), 5.93 (brs, 1H), 8.01 (s, 1H). 13C NMR (100 MHz, MeOD): δ 16.2, 16.4,
17.6, 18.2, 19.5, 24.4, 25.3, 27.3, 27.9, 28.7, 31.3, 32.0, 33.3, 33.9, 35.9, 36.0, 36.4,
38.1, 39.8, 39.9, 40.7, 42.3, 42.8, 48.1, 48.1, 49.9, 56.7, 72.3, 73.1, 74.7, 75.6, 76.9,
79.6, 88.2, 124.0, 124.3, 144.9, 145.3, 180.1. ESIꢀHRMS (m/z) [M+H]+ calcd for
C39H63N4O7, 699.4691, found 699.4697.
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Compound 64a. Prepared from 57 (88 mg, 0.17 mmol), acetylꢀlyxosyl azide (57
mg, 0.19 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 64a as a white solid
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(87 mg, 64%). H NMR (400 MHz, CDCl3): δ 0.52, 0.79, 0.90, 0.92, 0.99, 1.35 (7 ×
CH3), 2.02, 2.09, 2.13 (3 × CH3), 0.70ꢀ2.27 (m, other aliphatic ring protons), 2.71 (dd,
1H, J = 3.6, 13.6 Hz), 3.21 (dd, 1H, J = 3.9, 10.4 Hz), 3.54 (dd, 1H, J = 10.0, 11.1 Hz),
4.29ꢀ4.46 (m, 4H), 5.25ꢀ5.36 (m, 2H), 5.53 (brs, 1H), 5.67 (dd, 1H, J = 1.1, 2.8 Hz),
6.05 (d, 1H, J = 0.84 Hz), 6.88 (t, 1H, J = 5.4 Hz), 7.72 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 15.5 (2C), 16.6, 18.1, 20.5 (2C), 20.7, 23.3, 24.9, 26.8, 27.1, 28.0, 29.8, 30.2,
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32.4, 32.5, 35.0, 35.1, 35.3, 36.9, 38.5, 38.7, 39.5, 41.5, 41.7, 46.6, 46.9, 48.9, 55.2,
65.4, 65.8, 68.8, 70.3, 75.4, 78.9, 85.1, 121.4, 123.6, 143.5, 144.5, 169.1, 169.8, 169.9,
177.9. ESIꢀHRMS (m/z) [M+H]+ calcd for C44H67N4O10, 811.4852, found 811.4862.
Compound 64b. Prepared from 64a (40 mg, 0.05 mmol) according to General
procedure B. The crude product was washed with water three times without further
purification to afford 64b as a white solid (32 mg, 95%). 1H NMR (400 MHz, MeOD):
δ 0.56, 0.77, 0.88, 0.93, 0.96, 0.97, 1.36 (7 × CH3), 0.72ꢀ1.97 (m, other aliphatic ring
protons), 2.35 (t, 1H, J = 13.2 Hz), 2.88 (dd, 1H, J = 3.4, 13.6 Hz), 3.15 (dd, 1H, J =
4.9, 11.4 Hz), 3.39 (t, 1H, J = 10.7 Hz), 3.66 (dd, 1H, J = 2.9, 9.2 Hz), 3.92ꢀ3.98 (m,
1H), 4.03ꢀ4.07 (m, 2H), 4.32ꢀ4.43 (m, 3H), 5.45 (brs, 1H), 5.89 (brs, 1H), 8.03 (s, 1H).
13C NMR (100 MHz, MeOD): δ 16.1, 16.3, 17.6, 19.4, 24.4, 25.4, 27.3, 27.9, 28.7, 31.2,
31.9, 33.3, 33.9, 36.0 (2C), 36.3, 38.1, 39.8, 39.9, 40.7, 42.3, 42.8, 48.0, 48.2, 49.9, 56.8,
67.3, 69.8, 72.0, 75.0, 75.6, 79.7, 88.8, 124.0, 124.2, 144.9, 145.5, 180.0. ESIꢀHRMS
(m/z) [M+H]+ calcd for C38H61N4O7, 685.4535, found 685.4527.
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Compound 65a. Prepared from 57 (60 mg, 0.12 mmol), acetylꢀlactosyl azide
(126 mg, 0.19 mmol) according to General procedure C. The residue was purified by
column chromatography (petroleum ether/AcOEt, 1/2 v/v) to give 65a as a white solid
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(120 mg, 86%). H NMR (400 MHz, CDCl3): δ 0.69, 0.78, 0.91, 0.99, 1.36 (7 × CH3),
1.86, 2.00, 2.02, 2.05, 2.11, 2.12, (7 × CH3CO), 0.72ꢀ2.38 (m, other aliphatic ring
protons), 2.71ꢀ2.74 (m, 1H), 3.21ꢀ3.23 (m, 1H), 3.70ꢀ3.71 (m, 1H), 3.93ꢀ3.97 (m, 2H),
4.06ꢀ4.15 (m, 2H), 4.27 (dd, 1H, J = 4.6, 15.2 Hz), 4.35 (brs, 1H), 4.40 (dd, 1H, J = 4.2,
12.5 Hz), 4,50ꢀ4.58 (m, 3H), 4.93ꢀ4.97 (m, 1H), 5.06ꢀ5.20 (m, 2H), 5.38ꢀ5.40 (m, 2H),
5.54 (brs, 1H), 5.75ꢀ5.82 (m, 1H), 6.93 (t, 1H, J = 5.1 Hz), 7.73 (s, 1H). 13C NMR (100
MHz, CDCl3): δ 15.5 (2C), 16.7, 18.1, 20.1, 20.3, 20.4 (3C), 20.6, 20.7, 23.3, 25.1, 26.8,
27.0, 27.9, 28.9, 30.1, 32.3, 32.5, 35.0, 35.1 (2C), 36.8, 38.5, 38.6, 39.5, 41.6, 41.7, 46.6,
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