Design and synthesis of hyaluronan-mimetic gemini disaccharides

Article abstract of DOI:10.1016/S0040-4020(02)01567-3

An efficient strategy has been designed for the preparation of synthetic mimics of hyaluronan (HA, 1) and its dimerized (Gemini) disaccharides (2a,b) via n-pentenyl glycoside formation. Construction of the target molecules was achieved through a combination of protection/deprotection protocols, imidate glycosylation methodology followed by ozonolysis, and reductive amination. These tailored synthetic mimics could act as versatile building blocks with therapeutic applications in tissue engineering, treatment of cancer and as drug delivery agents.

Full text of DOI:10.1016/S0040-4020(02)01567-3

Tetrahedron 59 (2003) 631–638
Design and synthesis of hyaluronan-mimetic gemini disaccharides
Suri S. Iyer,a,b Shyam M. Rele,a,b S. Baskarana,b and Elliot L. Chaikofa,b
,
*
aDepartments of Surgery and Biomedical Engineering, Emory University, Atlanta, GA 30332, USA
bSchool of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
Received 27 September 2002; accepted 2 December 2002
Abstract—An efficient strategy has been designed for the preparation of synthetic mimics of hyaluronan (HA, 1) and its dimerized (Gemini)
disaccharides (2a,b) via n-pentenyl glycoside formation. Construction of the target molecules was achieved through a combination of
protection/deprotection protocols, imidate glycosylation methodology followed by ozonolysis, and reductive amination. These tailored
synthetic mimics could act as versatile building blocks with therapeutic applications in tissue engineering, treatment of cancer and as drug
delivery agents. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
In this report, we describe a linear strategy for the
development of HA-based oligomers consisting of (GlcA-
b-(1!3)-GlcNAc) separated by a flexible spacer ((Fig. 2)
2a and 2b). Previously reported syntheses of di-, tri- and
tetrasaccharides of HA afforded methyl glycosides or
4-methoxyphenyl glycosides with D-GlcA or D-GlcNAc
moieties at the reducing end.6–8 However, in all of these
reports, the glucuronic acid residue was generated after the
construction of the oligosaccharide backbone by selective
oxidation of the –CH2OH group (C-6) of a D-glucose
residue. This invariably led to low yields of the final
oxidized product. Consequently, in this series of investi-
gations we elected to use readily available acetobromo
D-glucuronic acid methyl ester as a precursor for the
synthesis of the trichloroacetimidate glycosyl donor.
Significantly, an n-pentenyl glycoside was designed as the
acceptor. Following the work of Fraser-Reid,9,10 we
envisioned that incorporating an n-pentenyl group at the
anomeric position would offer a number of potential
advantages. For example, apart from serving as a glycosidic
donor,9 using oxidation or radical chemistry,10 the olefinic
unit contained in the n-pentenyl glycoside could be readily
Glycosoaminoglycans (GAG) are complex biomolecules
which mediate a variety of events, including cell growth,
inflammatory and immunological responses, tumor
metastasis, as well as bacterial and viral recognition
processes.1 Of particular importance is hyaluronan2 (HA,
1) which is a high molecular weight, non-sulfated and linear
polysaccharide consisting of alternating D-glucuronic acid
(D-GlcA) and D-N-acetyl glucosamine (D-GlcNAc) moieties
linked by b-(1!3) and b-(1!4) linkages (Fig. 1). Both high
molecular weight and short oligomeric chains of hyaluronan
are important constituents of the extracellular matrix, the
synovial fluid of joints, and the scaffolding of cartilage. In
this context, HA acts as a signaling molecule via cellular
receptors, CD44 and RHAMM, to modulate inflammatory
and mesenchymal cell adhesion, migration, and phenotypic
differentiation.2–5 Thus, controlling and modulating HA
mediated biological events with selective and specific
small carbohydrate molecules will likely have an
important impact in the control of a number of pathological
processes.
Figure 1. Structure of natural hyaluronan (1) illustrating b-(1!3) and b-(1!4) linkages.
Keywords: hyaluronan; glycosoaminoglycans; carbohydrate–protein interactions; biomimetics; n-pentenyl glycoside.
*
Corresponding author. Address: 5105 Woodruff Memorial Research Building, 1639 Pierce Drive, Atlanta, GA 30322, USA. Tel.: þ1-404-727-8413; fax:
þ1-404-727-3660; e-mail: echaiko@emory.edu
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01567-3

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S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
Figure 2. Gemini disaccharides of HA (1).
retrosynthetic analysis of 2 (Fig. 3), an efficient strategy
was devised to obtain the desired monomeric building
blocks, i.e. the imidate donor (B) and the n-pentenyl
glycosyl acceptor (C) via a series of key intermediates on a
multigram scale.
We envisioned that incorporation of a versatile spacer arm
like n-pentenyl would facilitate direct access to well-defined
higher ordered glycoconjugates. As an initial assessment of
this approach, Gemini disaccharides were produced via
reductive amination with ethylenediamine, after conversion
of the terminal olefinic bond to an aldehyde (Scheme 1). Our
rationale for the selection of the ethylenediamine linker in
the glycomimetic design was based on the consideration that
it would provide a sufficiently flexible chain possessing
head-to-head HA-based disaccharides. Since, the ligand
affinity and specificity is often dependant upon the proper
spacing and orientation of the carbohydrate residues, we
anticipate that such compounds would then be able to
preorganize and fold into a conformation suitable for fitting
and complexing with the specific receptor binding site—a
fundamental requirement for bioactivity. Nonetheless,
generation of such flexible tethered compounds with distinct
carbohydrate recognition domains can easily be screened,
and the resulting bound antagonists/inhibitors identified.
The methodology for the synthesis of n-pentenyl terminated
glycoside acceptor 8 is summarized in Scheme 2. 2-Azido-
2-deoxy-3,4,6-tri-O-acetyl-D-glucopyranosyl acetate 4 was
synthesized from TfN3 and D-glucosamine hydrochloride.13
Although the synthesis of an n-pentenyl glycoside from the
mannose analog, 2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-
mannosepyranosyl acetate,14 using 4-penten-1-ol and
BF3·Et2O were successful, similar attempts using compound
4 in presence of BF3·Et2O, SnCl4, or TMSOTf as promoters
did not succeed. As an alternate route, selective hydrolysis
of the anomeric acetate to the hydroxyl group 5 was
performed using hydrazine acetate. The hydroxyl group was
then converted to the imidate derivative 6 in 74% yield. The
n-pentenyl group at the anomeric position was produced
using a catalytic amount of TMSOTf at 08C.
Figure 3. General retrosynthetic strategy for synthesis of dimers possessing
b-(1!3) HA dissacharides (P1–P5¼protecting groups).
attached to spacer functionalities, a carrier protein11 or a
polymer backbone providing direct access to glyco-
dendrimers and/or glycopolymers.12
2. Results and discussion
The success of a synthetic strategy involving GAGs depends
on a number of factors, including the preparation of a
suitable glycosyl acceptor and donor as building blocks, use
of selective protecting/deprotecting strategies, appropriate
glycosylation or glycosidic bond formation, as well as facile
removal of protecting groups with appropriate incorporation
of acetamido or sulfate groups. On the basis of a
Despite variations in temperature, promoter, and solvents to
increase the selectivity ratio, a/b isomers were obtained in a
2:3 ratio. Zemplen conditions15 performed on the mixture
Scheme 1. Conceived strategy for further manipulation of alkene functionality in the n-pentenyl glycoside to obtain hyaluronan mimetic dimers.

S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
633
Scheme 2. Reagents and conditions: (a) (i) TfN3, MeOH, DMAP, 258C, 18 h, (ii) Ac2O, pyridine, 08C, 10 h, yield¼75%; (b) H2NNH2·AcOH, DMF, 0–258C,
˚
45 min, yield¼70%; (c) anhydrous K2CO3, CCl3CN, dry CH2Cl2, 258C, 48 h, yield¼74%; (d) (i) 4 A molecular sieves, TMSOTf, 4-pentene-1-ol, dry CH2Cl2,
08C, 1 h, (ii) MeONa, MeOH, 0–258C, 6.0 h, yield¼80% over two steps; (e) camphorsulfonic acid, dry THF, C6H5CH(OMe)2, reflux, 6 h, yield¼75%.
Scheme 3. (a) CdCO3, H2O (2 equiv.), CH3CN, 758C, 4 h, yield¼75%; (b) CCl3CN, 1,2-dichloroethane, 1,8-DBU, 210 to 08C, 1 h, yield¼60%.
subsequently realized the triol in 80% yield, which was
easily converted to the acceptor 8 in 75% yield over two
steps. The a- and b- isomers were separated using silica
column chromatography using hexane/ethylacetate (70:30)
as the eluant.
converted to the a-imidate derivative 11 using trichloro-
acetonitrile and 1,8-diazabicyclo [5,4,0] undec-7-ene.
Glycosylation of the n-pentenyl glycoside acceptor 8b,
carried out separately for both a- and b- isomers, with the
trichloroacetimidate donor 11 in the presence of a Lewis
catalyst TMSOTf afforded the b-(1!3) linked disaccharide
12b in 78% yield (Scheme 4).
The glucuronic acid imidate donor was synthesized in 50%
yield in two steps from the commercially available
acetobromo-a-D-glucuronic acid methyl ester 9 (Scheme
3). In turn, 9 was converted to its corresponding free
hemiacetal 10 using CdCO3/H2O, which was subsequently
Characterization of compound 12b by NMR and FAB-MS
spectrum was in agreement with the expected structure.
Scheme 4. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0–258C, 3.5 h, yield¼78%; (b) CH3COSH, 258C, 24 h, yield¼70%; (c) 3 ml of TFA/H2O (2:1),
CH2Cl2, 08C, 1 h, yield¼86%; (d) 3 M NaOH, 9:1 MeOH/H2O, 258C, 2 h, yield¼86%; (e) O3 (2788C), then add Me2S, 2788C to 258C, 24 h, yield¼85%.

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S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
Scheme 5. Reagents and conditions: (a) MeOH, ethylenediamine (0.5 equiv.), stir for 3 h, add NaCNBH3 (2 equiv.), 258C, 3 h, yield¼80%.
Thus, for the b-isomer 12b, the anomeric protons (H-1 and
H-10) were observed as a doublet at d 4.78 (J¼8.0 Hz) and
4.35 (J¼8.0 Hz) ppm characteristic of the 1,2-trans system.
Reduction of the azido group to the acetamido functionality
using standard Staudinger reaction16 conditions (PPh3/
CH2Cl2/Ac2O) led to very low yields. Alternatively,
conversion of the azido functionality to the acetamido
(–NHAc) group was performed using thioacetic acid,17
column using water as the eluant and was lyophilized to
yield 2b as white foam (Scheme 5).
Computational modeling procedures18 have suggested that
the intrinsic binding affinity of oligosaccharide mimetics to
target proteins depends upon the distance and appropriate
orientation of the sugar epitopes, as well as the carbohydrate
conformational flexibility.19 Moreover, small and subtle
differences in configuration, conformation and functionality
can have a profound influence on the biological activity of
glycoconjugates. To this end, while the b-linked neoglyco-
conjugate 2b resembles the native b-(1!3) linked HA
structure, compounds based on a-linked disaccharides may
exhibit different receptor binding affinities. With this
background, the reductive amination procedure was
repeated with a-acceptor (8a) to obtain the homodimeric
a-product 2a. Thus, starting with the acceptor 8a/8b the
final gemini homodimers 2a/2b were obtained in overall
yield of 23% over six steps. Complete spectral character-
ization of the two target compounds using NMR (1H,13C)
and mass spectral (FAB-HRMS) analysis is provided in
Section 4.
1
which furnished 13b in appreciable yield (70%). The H
NMR spectrum of 13b showed a variable doublet between d
5.8–6.3 (J¼6.8–9.2 Hz) ppm attributed to the –NH proton
of the acetamido group in addition to the –NHCOCH3 peak
at 1.94 ppm. The benzylidene protecting group was then
removed using aqueous TFA to give 14b, which was
subsequently saponified using 3 M NaOH in MeOH/water
(9:1) mixture to yield the completely deprotected disacchar-
ide 15b in 86% yield. The crude product was purified using
Sephadex LH-20 with MeOH as the eluant. The synthesized
disaccharide fragment obtained is similar to the native
b-(1!3) linked region of hyaluronan (1) with a pendant
n-pentenyl functionality as a spacer arm at the anomeric
position of the GlcNAc moiety. The presence of character-
istic signals in the 13C NMR spectrum at d 173.0 (–COOH),
171.0 (–NHCO–) along with resonance signals at d 138.2
(vCH), 114.1 (vCH2), 103.8 and 101.1 (C-1 and C-10) and
22.0 (–COCH3) ppm further corroborated the formation of
compound 15b.
3. Conclusions
In summary, an efficient strategy that provides well-defined
HA disaccharide analogues with a b-(1!3) linkage
between D-GlcA and D-GlcNAc units is described. Further,
step-wise transformations afforded dimerized HA oligomers
via an n-pentenyl disaccharide intermediate. Investigations
of carbohydrate–protein binding interactions are ongoing
and will be reported in due course. Addressing these
challenges will ultimately provide a foundation for the
design and synthesis of small molecule glycomics for
modulating cellular interactions relevant to the design of
novel biomaterials, therapeutics and diagnostic agents.
The presence of an n-pentenyl group in compound 15b
potentially serves as a versatile handle by transformation of
the terminal olefin to aldehyde, carboxylic acid, ester,
thioether, thioester or hydroxyl group.10 To this end, the
n-pentenyl disaccharide 15b was subjected to reductive
ozonolysis which afforded a terminal aldehyde glycoside
intermediate 16b in 85% yield (Scheme 4). The appearance
of the aldehyde signal at d 9.63 and 203.8 ppm in the 1H and
13C NMR spectrum, respectively, along with the dis-
appearance of the olefinic double bond peaks at d 5.81 (dc
138.2) and 5.1–4.9 (dc 114.1) from the parent compound
confirmed the formation of 16b. Further extension of the
aliphatic glycosyl aldehyde by reductive amination of 16b
with the aglycone linker ethylenediamine resulted in the
formation of a homodimerized b-(1!3) linked HA
neoglycoconjugate analogue 2b (yield 80%). The crude
product was purified using Sephadex G-10 gel filtration
4. Experimental
4.1. General methods
All chemicals were reagent grade and used as supplied
unless otherwise noted. Solvents were purchased from

S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
635
Aldrich and dried and distilled before use. Molecular sieves
were activated at 3508C for 3 h in vacuo. Dichloromethane
1H NMR (CDCl3) d 6.30 (d, 1H, H-1a, J¼3.6 Hz), 5.55 (d,
1H, H-1b, J¼8.7 Hz), 5.43 (t, 1H, J¼8.0 Hz), 5.18–5.04
(m, 3H), 4.34–4.27 (m, 2H), 4.11–4.05 (m, 4H), 3.68–3.64
(m, 2H), 2.19 (s, 6H) 2.11 (s, 3H), 2.10 (s, 3H), 2.08 (s, 6H),
2.05 (s, 3H), 2.02 (s, 3H).
˚
was distilled from CaH2 and stored over 4 A molecular
sieves. All of the reactions were performed with oven-dried
glassware under argon atmosphere and monitored by TLC
on 250-mm Whatmann aluminum pre-coated silica gel
plates. Detection was performed by examination under UV
light (254 nm) and by charring with 10% sulfuric acid in
water. Flash chromatography was performed on silica gel
(Fischer, mesh 200–425) with the appropriate solvents. Size
exclusion chromatography was performed on Sephadex
G-10 and LH-20 (Sigma) with water or methanol,
respectively, as eluant. Extracts were concentrated under
4.1.2. 2-Azido-2-deoxy-3,4,6 tri-O-acetyl a,b D-gluco-
pyranoside (5). To an ice cooled solution of 4 (0.0241 mol,
9.0 g) dissolved in 100 ml of dry DMF, solid hydrazine
acetate (2.5 equiv., 0.06 mol, 5.5 g) was added under argon
atmosphere. The ice-bath was then removed and the
reaction mixture was allowed to stir for 45 min. On
completion (TLC), the reaction mixture was quenched
using ethyl acetate (50 ml) and water (50 ml). The organic
layer was the separated and the aqueous layer extracted with
EtOAc (3£25 ml). Finally, the combined organic layers
were washed with saturated NaHCO3, brine and dried over
MgSO4. Concentration of the solvent under vacuo gave a
light yellow colored gel which on flash column chromato-
graphy (EtOAc/hexane 80:20) furnished 5 as a white solid.
Yield¼5.9 g (70%). 1H NMR (CDCl3) d 5.35 (t, 2H,
J¼10.4, 9.6 Hz), 5.23 (d, 1H, H-1a, J¼3.2 Hz), 4.88 (m,
2H), 4.58 (d, 1H, H-1b, J¼8.0 Hz), 4.12 (m, 2H), 3.94 (m,
2H), 3.80 (br s, 2H, OH), 3.35–3.28 (m, 2H), 3.20 (s, 2H),
2.09 (s, 6H), 2.08 (s, 6H), 2.08 (s, 6H); 13C NMR (CDCl3) d
171.3, 171.2, 170.5, 170.4, 170.2, 170.0, 96.1, 91.9, 72.5,
71.7, 70.5, 68.7, 68.5, 67.2, 64.8, 62.2, 61.4, 20.7, 20.6.
1
reduced pressure at ,458C (water bath). H NMR and 13C
NMR spectra were recorded on a Varian Mercury 300 MHz
or INOVA 400 MHz or on 600 MHz NMR spectrometer.
1
For H NMR and 13C NMR spectra recorded in CDCl3,
CD3OD, D2O and DMSO chemical shifts (d) are given in
1
ppm relative to solvent peaks (for CDCl3, H d¼7.26, 13C
1
d¼77.3; for CD3OD H d¼4.87 and 3.32, 13C d¼47.8; for
CD3SOCD3 1H d¼2.5, 13C d¼39.8; for D2O 1H d¼4.85) as
internal standard. Coupling constants (J) are reported in
Hertz (Hz). Hetero-nuclear multiple quantum coherence
(HMQC) experiments were carried out on compounds
15a/b, 16a/b and 2a/b. Optical rotations were measured on a
Perkin–Elmer 241 MC polarimeter using a 1 cm cell, and
[a]D values are given in units of 10218 cm3 g21 at 258C.
High-resolution fast atom bombardment mass spectrometry
(FAB-HRMS) was recorded on JEOL SX102 at 6kV-XE
using 3-nitrobenzylalcohol, in some cases with addition of
LiI as a matrix.
4.1.3. 2-Azido 2-deoxy-3,4,6 tri-O-acetyl a,b D-gluco-
pyranosyl trichloroacetimidate (6). To the reaction flask
containing compound 5 (0.024 mol, 8 g) dissolved in dry
CH2Cl2 (35 ml), anhydrous K2CO3 (0.06 mol, 8.33 g) was
added. This was followed by the dropwise addition of
Cl3CCN (0.144 mol, 14.5 ml) after which the reaction
mixture was stirred for 48 h under argon atmosphere. On
completion (TLC), the reaction mixture was cooled to 08C,
diluted with 25 ml of CH2Cl2 and quenched with 20 ml of
ice cold water. The organic layer was separated and the
aqueous layer extracted with CH2Cl2 (2£20 ml). The
combined organic layers were washed with brine, dried
over Na2SO4 and concentrated to yield a light yellow
colored solid. Purification of the crude mixture by column
chromatography (EtOAc/hexane 60:40) gave compound 6
4.1.1. 2-Azido 2-deoxy-3,4,6 tri-O-acetyl a,b-D-gluco-
pyranosyl acetate (4). To a round-bottomed flask contain-
ing NaN3 (0.43 mol, 28 g) and equipped with an argon
balloon, water (80 ml) and CH2Cl2 (75 ml) was added to it.
The emulsion was stirred and cooled to 08C followed by
dropwise addition of dry Tf2O (0.088 mol, 15 ml) via
syringe. The reaction mixture was stirred at 08C for 2 h at
258C for 15 min. The organic layer was separated and the
aqueous layer was extracted with CH2Cl2 (2£25 ml). The
combined organic layers were neutralized with a saturated
solution of NaHCO3, washed with water, dried over MgSO4
and filtered. This stock solution contains ca. 0.1 mol of
TfN3.
1
(off-white solid). Yield¼8.5 g (74 %). H NMR (CDCl3) d
8.81 (s, 1H, NH), 8.79 (s, 1H, NH), 6.43 (d, 1H, H-1a,
J¼3.6 Hz), 5.68 (d, 1H, H-1b, J¼7.8 Hz), 5.55 (dd, 2H,
J¼9.6, 9.6 Hz), 5.11 (t, 1H, J¼10.0 Hz), 5.09 (m, 1H), 4.23
(m, 4H), 4.05 (m, 2H), 3.76 (dd, 2H, J¼3.6 Hz), 2.05 (s,
3H), 2.04 (s, 3H), 2.01 (s, 3H), 2.0 (s, 3H), 1.99 (s, 3H), 1.96
(s, 3H); 13C (CDCl3) d 170.7, 170.6, 170.0, 169.8, 169.7,
160.6, 96.5, 94.1, 90.6, 72.8, 72.7, 70.8, 70.2, 68.1, 63.4,
61.5, 60.7, 20.8, 20.7.
In a separate 3-necked flask equipped with an argon inlet
containing D-glucosamine hydrochloride (3, 0.042 mol, 9 g)
dissolved in methanol (250 ml), NaOMe (80 ml of 0.5 M
solution in MeOH) was added and stirred for 30 min.
DMAP (5 g) was added to it and the reaction mixture was
diluted with additional 100 ml of MeOH. The freshly
prepared solution of TfN3 was added to this reaction mixture
via cannula under positive pressure of argon and left to stir
at room temperature for 18 h. The solvent was removed in
vacuo to give a sticky yellow paste. The residue obtained
was then acetylated at 0–48C for 10 h using anhydrous
pyridine (250 ml) and dry acetic anhydride (120 ml). On
completion of the reaction, the reaction mixture was
co-evaporated with toluene (3£50 ml) to give a light
brown colored sticky residue. Flash chromatography
(EtOAc/hexane 60:40) of the crude reaction mixture using
silica gel gave the desired product 4. Yield¼11.5 g (75%).
4.1.4. Pent-4-enyl 2-azido 2-deoxy- a,b D-glucopyrano-
˚
side (7). To a oven-dried flask containing activated 4 A
sieves and 6 (0.016 mol, 7.5 g) dissolved in anhydrous
CH2Cl2 (25 ml), 4-penten-1-ol (0.0195 mol, 2.0 ml) was
added via syringe. The reaction mixture and was initially
stirred for 10 min at room temperature and then cooled to
08C under argon atmosphere. Freshly prepared solution of
TMSOTf (1.6 mmol, 7.4 ml of 0.22 M solution in
anhydrous CH2Cl2) was added dropwise through a syringe
to the reaction mixture and stirred at 08C. On completion

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S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
(TLC), the reaction mixture was quenched with Et3N,
filtered through a pad of celite and concentrated under
reduced pressure to give a semicrystalline material. Flash
chromatography (EtOAc/hexane 50:50) afforded a light
yellow semi-crystalline material, which was directly used
for the next step. (The compound obtained is contaminated
with trace amounts of 4-penten-1-ol.) The above mixture
was dissolved in anhydrous MeOH followed by addition of
NaOMe (10 ml of 0.5 M in MeOH, 0.05 mol) at 08C under
argon atmosphere. The reaction mixture was stirred at room
temperature for 6 h (monitored by TLC), and quenched with
DOWEX 50W X 8-200 (Hþ) ion-exchange resin at 08C. The
resin was filtered and on evaporation of the solvent gave a
light brown colored syrup, which on flash chromatography
(EtOAc/hexane 50:50 to MeOH/CHCl3 10:90) furnished 7
as a light yellow colored compound. Yield over two steps:
3.56 g (80%). 1H NMR (CDCl3) d 5.81 (m, 1H, CHvCH2),
5.02–4.92 (m, 2H, CHvCH2), 4.87 (d, 0.3H, H-1a,
J¼3.2 Hz), 4.6 (br s, 3H, OH), 4.34 (d, 0.7H, H-1b,
J¼7.6 Hz), 3.98–3.74 (m, 3H), 3.68–3.31 (m, 4H), 3.28–
3.20 (m, 1H), 2.11–2.05 (m, 2H, CH2–CHvCH2), 1.80–
1.65 (m, 2H, –CH2); 13C NMR (CDCl3) d 138.7, 115.1,
102.2, 98.1, 75.2, 74.6, 71.4, 70.1, 70.05, 69.5, 67.7, 66.0,
62.6, 61.4, 61.1, 30.3, 30.1, 28.8, 28.6.
J¼10.0 Hz), 3.56–3.48 (m, 3H), 3.45–3.38 (m, 2H), 2.65
(br s, 1H, OH) 2.17–2.15 (m, 2H), 1.76–1.73 (m, 2H); 13C
NMR (CDCl3) d 138.1, 137.1, 129.6, 128.6, 126.5, 115.4,
102.7, 102.1, 80.8, 72.0, 70.1, 68.7, 66.7, 66.3, 30.22, 28.94.
4.1.8. Synthesis of imidate donor methyl-(2,3,4-tri-O-
acetyl-a-D-glucopyranosyl trichloacetimidate) gluco-
ronate (11). Acetobromo-a-D-glucuronic acid methyl
ester (9, 0.025 mol, 10 g) was added to a flame dried
three-necked flask containing CdCO3 (0.026 mol, 4.5 g),
CH3CN (150 ml) and 0.8–1.0 ml of degassed H2O. The
reaction mixture was stirred and heated to 708C for a period
of 2 h under argon, filtered through celite, washed with
50 ml of CH3CN and concentrated in vacuo. Purification of
the crude residue by flash chromatography (EtOAc/hexane
50:50) afforded the hydrolyzed product 10 (white solid),
which was subsequently dissolved in 200 ml of (ClCH2)2,
Cl3CCN (10 equiv., 0.142 mol, 13.6 ml) was added to it
under argon. After cooling to 08C, 1,8-diazabicyclo[5.4.0]-
undec-7-ene (1,8-DBU) (0.28 equiv., 3.96 mmol, 595 ml)
was added in a dropwise manner. The reaction mixture was
allowed to stir for 1 h and the mixture was concentrated to
afford a sticky dark brown residue. Subsequent flash column
chromatography of the crude extract (EtOAc/hexane 40:60,
1
1% Et3N) furnished an off-white compound 11 (60%). H
4.1.5. Pent-4-enyl 2-azido 2-deoxy-4,6-O-benzylidene
a,b D-glucopyranoside (8). To a solution of compound 7
(0.011 mol, 3.2 g) dissolved in anhydrous THF (25 ml),
200 mg of (þ) camphorsulfonic acid as a catalyst was added
followed by dropwise addition of benzaldehyde dimethyl
acetal (0.0197 mol, 2.95 ml). The reaction mixture was
refluxed under argon atmosphere for 6 h (monitored by the
complete disappearance of starting material), cooled to
room temperature, diluted with 50 ml of EtOAc and
quenched with 10 ml of saturated solution of NaHCO3.
The organic layer was separated and the aqueous layer was
extracted with 3£25 ml of EtOAc. The organic layers were
combined and washed with brine, dried over Na2SO4 and
concentrated to yield a light yellow colored solid.
Purification of the crude product by flash chromatography
(EtOAc/hexane 5:95 to EtOAc/hexane 30:70) gave 8 as a
mixture of a and b isomers. Yield¼3.17 g (75 %). The a
(8a) and b (8b) isomers were separated by silica gel column
chromatography using hexane/ethylacetate mixture (70:30).
NMR (CDCl3, 400 MHz) 8.71 (s, 1H, NH), 6.58 (d, 1H,
H-1, J¼3.6 Hz), 5.62 (t, 1H, H-4, J¼10 Hz), 5.26 (t, 1H,
H-?, J¼10 Hz), 5.11 (dd, 1H, H-2, J¼3.2 Hz), 4.43 (d, 1H,
H-5, J¼10.2 Hz), 3.74 (s, 3H, COOMe), 2.05 (s, 6H, OAc),
2.02 (s, 3H, OAc).
4.1.9. Pent-4-enyl 2-azido 4,6-O-benzylidene 2-deoxy-3-
O-(methyl 2,3,4-tri-O-acetyl-b-D-glucopyranosyl uro-
nate)-a/b-D-glucopyranoside (12a/12b). The acceptor
8a/8b (1.8 mmol, 0.65 g) was dissolved in dry CH2Cl2
and stirred at 2158C under argon atmosphere. To this
freshly prepared solution of TMSOTf (0.9 mmol, 0.41 ml of
0.22 M) was added dropwise over a period of 5 min via
syringe. The imidate donor 11 (2.3 mmol, 1.12 g) dissolved
in dry CH2Cl2 was then added to the reaction mixture via
cannula and the reaction mixture was stirred for 2 h at 08C
and warmed to room temperature over a period of 30 min.
On completion (TLC), the reaction mixture was quenched
with N,N-diisopropylamine, concentrated and subjected to
column chromatography (EtOAc/hexane 30:70) to give the
desired disaccharide 12a/12b as an off-white solid.
Yield¼1.2 g (78 %). For 12a: [a]2D5¼þ42.9 (c¼2.9,
4.1.6. Pent-4-enyl 2-azido 2-deoxy-4,6-O-benzylidene
a-D-glucopyranoside (8a). [a]2D5¼237.31 (c¼0.98,
1
1
CHCl3); H NMR (CDCl3) d 7.50–7.45 (m, 2H, C6H5),
CHCl3); H NMR (CDCl3) d 7.45–7.30 (m, 5H, C6H5),
7.39–7.27 (m, 3H, C6H5), 5.81 (m, 1H, CHvCH2), 5.59 (s,
1H, CHPh), 5.08–4.99 (m, 2H, CHvCH2), 4.89 (d, 1H,
H-1a, J¼3.6 Hz), 4.28–4.21 (m, 2H), 3.87–3.83 (m, 1H),
3.75–3.70 (m, 2H), 3.53–3.47 (m, 2H), 3.24 (dd, 1H, J¼4,
3.6 Hz), 2.80 (br s, 1H, OH), 2.19–2.16 (m, 2H), 1.77–1.73
(m, 2H); 13C NMR (CDCl3) d 138.1, 137.1, 129.6, 128.6,
126.5, 115.3, 102.3, 98.6, 82.1, 69.0, 68.8, 68.1, 63.2, 62.6,
30.4, 28.5.
5.81 (m, 1H, CHvCH2), 5.51 (s, 1H, CHPh), 5.25–5.08 (m,
3H), 5.06–4.96 (m, 2H), 4.88 (d, 1H, H-1, J¼3.6 Hz), 4.80
(d, 1H, H-10, J¼7.8 Hz), 4.25–4.18 (m, 2H), 3.86–3.70 (m,
5H), 3.55 (s, 3H), 3.49 (m, 1H), 3.26 (dd, 1H, J¼3.6,
3.6 Hz), 2.22–2.12 (m, 2H), 2.08 (s, 3H), 1.99 (s, 3H), 1.97
(s, 3H), 1.82–1.76 (m, 2H); 13C NMR (CDCl3) d 170.4,
170.0, 169.6, 167.1, 137.9, 137.4, 129.1, 128.4, 126.2,
115.5, 101.8, 101.1, 98.6, 80.4, 72.7, 72.3, 72.0, 69.5, 69.1,
67.8, 63.1, 52.8, 30.4, 28.7, 20.8; HRMS (FAB) calcd for
C31H39O14N3 (MþþLi) 684.2592, found 684.2597. For
4.1.7. Pent-4-enyl 2-azido 2-deoxy-4,6-O-benzylidene
b-D-glucopyranoside (8b). [a]D25¼þ33.6 (c¼1.5, CHCl3);
1H NMR (CDCl3) d 7.48–7.45 (m, 2H, Ph), 7.38–7.26 (m,
3H, Ph), 5.83–5.79 (m, 1H, CHvCH2), 5.56 (s, 1H, CHPh),
5.06–4.97 (m, 2H, CHvCH2), 4.33 (d, 1H, H-1b,
J¼7.8 Hz), 4.29 (m, 1H), 3.89 (m, 1H), 3.73 (t, 1H,
1
12b: [a]2D5¼229.7 (c¼2.4, CHCl3); H NMR (CDCl3) d
7.42–7.28 (m, 5H, C6H5), 5.82 (m, 1H, CHvCH2), 5.49 (s,
1H, CHPh), 5.25–5.10 (m, 2H), 5.07–4.97 (m, 3H), 4.78 (d,
1H, H-10, J¼8.0 Hz), 4.35 (d, 1H, H-1, J¼8.0 Hz), 4.31–
4.28 (dd, 1H, J¼3.6 Hz), 3.92 (m, 1H), 3.82–3.60 (m, 5H),

S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
637
3.58 (s, 3H), 3.42–3.34 (m, 2H) 2.09–2.07 (m, 2H), 2.06 (s,
3H), 1.99 (s, 3H), 1.97 (s, 3H), 1.78–1.74 (m, 2H); 13C
NMR (CDCl3) d 170.4, 169.10, 169.06, 166.55, 137.49,
136.64, 129.1, 128.77, 125.64, 114.90, 102.34, 101.03,
100.71, 79.62, 79.15, 72.4, 72.1, 71.4, 69.8, 69.3, 68.4, 66.2,
65.9, 52.7, 29.9, 28.6, 20.5; HRMS (FAB) calcd for
C31H39O14N3 (MþþLi) 684.2592, found 684.2567.
5.61 (d, 1H, J¼9.0 Hz, NH), 5.25–5.21 (m, 2H), 5.08–4.94
(m, 3H), 4.72 (d, 1H, H-1, J¼3.6 Hz), 4.69 (d, 1H, H-10,
J¼7.8 Hz), 4.28–4.23 (m, 1H), 4.13–4.09 (m, 1H), 3.92–
3.79 (m, 1H), 3.75 (s, 3H), 3.73–3.56 (m, 5H), 3.42–3.36
(m, 1H), 2.18–2.09 (m, 2H), 2.08 (s, 3H), 2.03 (s, 3H), 2.02
(s, 3H), 2.00 (s, 3H), 1.76–1.64 (m, 2H); 13C NMR (CDCl3)
d 170.4, 169.6, 167.2, 138.8, 115.4, 100.8, 97.4, 84.3, 71.8,
71.4, 69.9, 69.1, 67.4, 63.1, 53.5, 51.6, 30.5, 28.4, 23.6,
20.8; HRMS (FAB) calcd for C26H39O15N (MþþLi)
612.2480, found 612.2490. For 14b: [a]2D5¼23.0 (c¼0.51,
CHCl3); 1H NMR (CDCl3) d 5.76 (m, 1H, CHvCH2), 5.64
(d, 1H, J¼7.2 Hz, NH), 5.29–5.16 (m, 2H, CHvCH2),
5.03–4.97 (m, 3H), 4.86 (d, 1H, H-10, J¼8.4 Hz), 4.66 (d,
1H, H-1, J¼7.6 Hz), 4.41 (dd, 1H, J¼8.4, 8.4 Hz), 4.11–
4.05 (m, 2H), 3.95–3.78 (m, 3H), 3.75 (s, 3H), 3.52–3.42
(m, 3H), 3.03 (m, 1H), 2.09–2.06 (m, 2H), 2.05 (s, 3H), 2.03
(s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.62 (m, 2H); 13C NMR
(CDCl3) d 170.1, 169.9, 167.2, 138.7, 115.0, 100.3, 99.0,
83.1, 75.0, 72.1, 71.7, 71.5, 70.6, 69.2, 68.6, 63.1, 57.5,
53.2, 30.2, 29.9, 28.9, 23.9, 20.7; HRMS (FAB) calcd for
C26H39O15N (MþþLi) 612.2480, found 612.2493.
4.1.10. Pent-4-enyl 4,6-O-benzylidene 2-deoxy 2-N-acetyl
3-O-(methyl 2,3,4-tri-O-acetyl b-D-gluco pyranosyluro-
nate)-a/b-D-glucopyranoside (13a/13b). In a dry flame
dried flask compound 12a/12b (1.5 mmol, 1.0 g) was
dissolved in 5 ml of thioacetic acid (CH3COSH). The
reaction mixture was then allowed to stir for 24 h under
argon atmosphere. On completion of the reaction, the excess
thioacetic acid was removed in vacuo and the reaction
mixture was subjected to column chromatography (CHCl3/
CH3OH 100:0 to CHCl3/CH3OH 2.5:95) to give the
N-acetylated product 13a/13b as a white crystalline
material. Yield¼0.71 g (70%). For 13a: [a]2D5¼þ19.5
1
(c¼3.3, CHCl3); H NMR (CDCl3) d 7.45–7.30 (m, 5H,
C6H5), 6.20 (d, 1H, J¼9.0 Hz, NH), 5.70 (m, 1H,
CHvCH2), 5.51 (1H, s, CHPh), 5.17 (m, 2H), 5.01–4.92
(m, 3H), 4.88 (d, 1H, H-10, J¼7.8 Hz), 4.78 (d, 1H, H-1,
J¼3.6 Hz), 4.32–4.19 (m, 2H), 4.02–3.96 (m, 1H), 3.79–
3.58 (m, 5H), 3.55 (s, 3H), 3.45–3.34 (m, 1H), 2.18–2.10
(m, 2H), 2.01 (s, 3H), 1.99 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H),
1.84–1.78 (m, 2H); 13C NMR (CDCl3) d 170.4, 170.0,
169.8, 169.7, 167.2, 137.9, 137.4, 129.2, 128.5, 126.3,
115.4, 101.5, 100.3, 98.1, 80.5, 72.7, 72.4, 72.0, 69.6, 69.1,
67.8, 63.1, 52.8, 30.5, 28.7, 23.4, 20.8; HRMS (FAB) calcd
for C33H43O15N (MþþLi) 700.2793, found 700.2795. For
4.1.12. Pent-4-enyl 2-deoxy 2-N-acetyl 3-O-(b-D-gluco-
(15a/15b).
pyranosyluronate)-a/b-D-glucopyranoside
Compound 14a/14b (1.3 mmol, 0.8 g) was dissolved in
9:1 MeOH/H2O mixture (3 ml) and stirred at 08C. 3 M
NaOH solution was added to it and the reaction mixture was
allowed to stir at room temperature for 5 h (monitor by
TLC). The reaction was then quenched using strongly acidic
Dowex Hþ ion exchange resin. The resin was filtered,
filtrate concentrated and the product was then purified using
gel filtration Sephadex LH-20 column with methanol as
eluant. Removal of solvent followed by subsequent
lyophilization gave the saponified product as a white solid
15a/15b. Yield¼86%. For 15a: [a]2D5¼þ30.6 (c¼2.7,
1
13b: [a]2D5¼29.8 (c¼0.50, CHCl3); H NMR (CDCl3) d
7.40–7.32 (m, 5H, C6H5), 5.91 (d, 1H, J¼6.8 Hz, NH), 5.80
(m, 1H, CHvCH2), 5.46 (s, 1H, CHPh), 5.16–5.08 (m, 2H),
5.03–4.91 (m, 3H), 4.79 (d, 1H, H-10, J¼7.6 Hz), 4.70 (t,
1H, J¼9.6, 9.6 Hz), 4.29 (dd, 1H, J¼5.1, 4.8 Hz), 3.85–
3.49 (m, 6H), 3.60 (s, 3H), 3.03 (m, 1H), 2.09–2.06 (m, 2H),
1.97 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H), 1.62 (m,
2H); 13C NMR (CDCl3) d 170.8, 170.2, 169.6, 138.0, 137.3,
129.2, 128.4, 128.3, 126.0, 115.0, 101.6, 99.3, 80.9, 72.1,
71.7, 71.6, 69.5, 69.1, 68.8, 65.8, 58.4, 52.8, 30.1, 28.9,
23.6, 20.7; HRMS (FAB) calcd for C33H43O15N (MþþLi)
700.2793, found 700.2787.
CH3OH); 1H NMR (D2O)
d 5.71–5.68 (m, 1H,
CHvCH2), 4.90–4.79 (m, 2H, CHvCH2), 4.34 (d, 1H,
H-10, J¼8.0 Hz), 3.90 (dd, 1H, J¼3.3, 3.6 Hz), 3.77 (d, 1H,
J¼7.2 Hz), 3.68–3.60 (m, 3H), 3.55–3.49 (m, 2H), 3.42–
3.25 (m, 2H), 3.15 (t, 1H, J¼9.0, 8.1 Hz), 2.01–1.93 (m,
2H), 1.88 (s, 3H), 1.53–1.48 (m, 2H); 13C NMR (DMSO) d
170.8, 170.4, 138.9, 115.7, 103.2, 97.4, 80.2, 76.6, 76.1,
73.5, 73.3, 72.0, 69.4, 66.9, 61.2, 53.2, 30.4, 28.7, 23.2;
HRMS (FAB) calcd for C19H30O12N (Mþþ2Li–H)
478.2088, found 478.2083. For 15b: [a]2D5¼254.4
(c¼0.98, CH3OH); 1H NMR (CD3OD) d 5.81 (m, 1H,
CHvCH2), 5.1–4.91 (m, 2H, CHvCH2), 4.45 (d, 1H, H-10,
J¼8.0 Hz), 4.37 (d, 1H, H-1, J¼8.0 Hz), 3.88 (m, 3H),
3.72–3.63 (m, 3H), 3.62–3.42 (m, 2H), 3.41–3.38 (m, 2H),
3.29 (br s, 2H), 2.09–2.01 (m, 2H), 1.95 (s, 3H), 1.62–1.49
(m, 2H); 13C NMR (CD3OD) d 173.0, 171.0, 138.20, 114.1,
103.8, 101.1, 84.3, 76.2, 75.9, 74.8, 73.0, 71.7, 69.2, 68.7,
61.4, 55.0, 30.0, 28.7, 22.0; HRMS (FAB) calcd for
C19H30O12N (Mþþ2Li–H) 478.2088, found 478.2068.
4.1.11. Pent-4-enyl 2-deoxy 2-N-acetyl 3-O-(methyl 2,3,4
tri-O-acetylb-D-glucopyranosyluronate)-a/b-D-gluco-
pyranoside (14a/14b). Compound 13a/13b (0.014 mmol,
1.0 g) was dissolved in 10 ml of anhydrous CH2Cl2 and
cooled to 08C. 3.6 ml of TFA/H2O (2.5:1.1) was then added
to the reaction mixture in a dropwise manner. The reaction
mixture was stirred at room temperature for 5 h and on
completion of the reaction (TLC) was cooled to 08C and
quenched with a saturated solution of NaHCO3. The organic
layer was separated and the aqueous layer extracted with
CH2Cl2 (3£10 ml). The combined organic layers were
washed with brine and the organic layer was separated,
dried over Na2SOz4. Evaporation of the solvent gave a light
yellow colored gel which when subjected to column
chromatography (EtOAc/hexane 20:80 to CHCl3/MeOH
95:5) afforded the debenzylidenated product 14a/14b.
Yield¼0.85 g, 86%. For 14a: [a]2D5¼þ17.3 (c¼2.5,
CHCl3); 1H NMR (CDCl3) d 5.81 (m, 1H, CHvCH2),
4.1.13. 3-Formylpropyl-2-deoxy-2-N-acetyl-O-(b-D-glu-
copyranosyluronate)-a/b-D-glucopyranoside (16a/16b).
Compound 15a/15b (10 mmol, 500 mg) was dissolved in
3 ml of dry methanol and cooled to 2788C. Ozone was
bubbled through the light yellow solution for 1.5 h. The pale
yellow solution appeared to turn pale blue. To this, 3 ml of
dimethyl sulfide was added at 2788C and the reaction was
stirred and allowed to warm up to room temperature

638
S. S. Iyer et al. / Tetrahedron 59 (2003) 631–638
(c) Varki, A. Glycobiology 1993, 3, 97–130. (d) Sears, P.;
Wong, C. H. Cell Mol. Life Sci. 1998, 54, 223–252.
2. For excellent review see: Lapcik, Jr. L.; Lapcik, L.; Smedt, S.;
Demeester, J.; Chabrecek, P. Chem. Rev. 1998, 98, 663–2684.
3. (a) Entwistle, J.; Hall, C. I.; Turley, E. A. J. Cell Biochem.
1996, 61, 569–577. (b) Evanko, S. P.; Wight, T. N.
J. Histochem. Cytochem. 1999, 47, 1331–1342. (c) Travis,
J. A.; Hughes, M. G.; Wong, J. M.; Wagner, W. D.; Geary,
R. L. Circ. Res. 2001, 88, 77–83.
overnight. The reaction mixture was concentrated and the
crude product was purified using Sephadex LH-20 with
methanol as eluant. The pure compound obtained was then
lyophilized to give an off white solid. Yield¼85%. For 16a:
1H NMR (DMSO) d 9.67 (1H, CHO), 7.58 (d, 1H, NH,
J¼7.8 Hz), 4.67 (d, 1H, H-1, J¼3.3 Hz), 4.43 (d, 1H, H-10,
J¼7.8 Hz), 3.74–3.58 (m, 2H), 3.61–3.47 (m, 7H), 3.38–
3.14 (m, 8H), 3.03 (m, 1H), 1.79 (s, 3H, NHCOCH3), 1.70–
1.40 (br m, 4H); 13C NMR (DMSO) d 204.1, 171.0, 170.5,
103.2, 97.3, 80.5, 76.7, 75.4, 73.4, 72.2, 69.5, 66.9, 61.2,
58.3, 53.0, 23.2, 22.4; HRMS (FAB) calcd for C18H29O13N
4. (a) Underhill, C. B. The Biology of Hyaluronan;
C. Foundation; John Wiley: Chichester, UK, 1989; pp 87–
106. (b) Underhill, C. J. Cell Sci. 1992, 103, 293–298.
(c) Savani, R. C.; Turley, E. A. Int. J. Tissue React. 1995, 17,
141–151.
1
(MþþLi) 474.2556, found 474.2552. For 16b: H NMR
(DMSO) d 9.63 (s, 1H, CHO), 7.78 (d, 1H, NH, J¼8.1 Hz),
5.15 (br s, 1H), 4.64–4.57 (m, 2H), 4.35 (d, 1H, H-1,
J¼7.8 Hz), 4.27 (d, 1H, H-10, J¼7.8 Hz), 3.72–3.64 (m,
4H), 3.63–3.40 (br m, 6H), 3.32–3.16 (m, 4H), 3.10–3.07
5. Laurent, T. C. The Chemistry Biology and Medical
Applications of Hyaluronan and its Derivatives; Portland:
London, 1998.
(m, 1H), 1.77 (s, 3H, NHCOCH3), 1.70–1.46 (m, 4H); 13
C
NMR (CD3OD) d 203.8, 173.1, 170.9, 104.5, 103.8, 101.1,
84.4, 76.2, 75.9, 74.8, 73.1, 71.7, 69.3, 69.1, 61.5, 52.3,
29.1, 24.5, 22.6; HRMS (FAB) calcd for C18H29O13N
(MþþLi) 474.2556, found 474.2550.
6. (a) Carter, M. B.; Petillo, P. A.; Anderson, L.; Lerner, L. E.
Carbohydr. Res. 1994, 258, 299–306. (b) Slaghek, T. M.;
Nakahara, Y.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F.
G. Carbohydr. Res. 1994, 255, 61–85. (c) Slaghek, T. M.;
Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1992, 33,
4971–4974.
4.1.14. Synthesis of 2a/2b. Compound 16a/16b
(0.043 mmol, 20 mg) was dissolved in 0.5 ml dry methanol
and ethylenediamine (0.021 mmol, 1.4 ml) was added to it
in a dropwise manner. There was an immediate precipitation
of a white solid. The reaction mixture was stirred for 1 h at
room temperature and NaBH3CN (0.11 mmol, 6.9 mg) was
then added to it and the reaction mixture was allowed to stir
overnight. Removal of the solvent gave a residue, which
was purified using Sephadex G-25 with water as eluant and
subsequently lyophilized to give a white powder (2a/2b).
Yield¼16 mg (80%). For 2a: 1H NMR (DMSO) d 7.95 (br,
1H, NH), 7.84 (d, 1H, J¼8 Hz, NH), 4.74 (d, 1H, H-10,
J¼2.7 Hz), 4.70 (d, 1H, H-10, J¼2.7 Hz), 4.46 (d, 1H, H-1,
J¼7.8 Hz), 4.32 (br m, 2H), 3.9–3.55 (m, 13H, ring
protons), 3.48–3.18 (m, 8H, ring protons), 3.12–2.85 (m,
10H), 1.81 (s, 6H, COCH3), 1.60–1.40 (m, 8H); 13C NMR
(DMSO) d 170.8, 170.7, 104.5, 103.0, 97.7, 97.0, 80.5, 77.1,
74.0, 73.5, 73.3, 72.9, 72.7, 69.8, 69.4, 67.2, 61.4, 61.2,
53.2, 53.1, 29.4, 27.2, 24.8, 23.3; HRMS (FAB) calcd for
C38H67O24N4 (MþþH) 963.3493, found 963.4137. For 2b:
1H NMR (DMSO) d 7.70 (br, 2H, NH), 4.36 (d, 1H,
J¼7.8 Hz), 4.31 (br m, 2H), 4.18 (d, 1H, J¼7.2 Hz), 4.10
(m, 2H), 3.88–3.28 (m, 14H), 3.22–3.08 (br m, 10H),
2.98–2.82 (m, 8H), 1.75 (s, 6H, COCH3), 1.60–1.20 (br m,
8H); 13C NMR (DMSO) d 170.5, 167.7, 132.3, 129.4, 104.3,
104.0, 101.2, 101.1, 85.0, 77.2, 76.6, 74.5, 73.7, 72.3, 69.5,
68.8, 68.1, 61.5, 54.8, 52.9, 37.4, 36.7, 34.6, 32.2, 30.5,
29.2, 29.0, 24.9, 23.9, 23.1; HRMS (FAB) calcd for
C38H67O24N4 (MþþH) 963.3493, found 963.4139.
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