Synthesis and X-ray crystallographic investigation of N-(β-d-glycosyl) butanamides derived from GlcNAc and chitobiose as analogs of the conserved chitobiosylasparagine linkage of N-glycoproteins

Article abstract of DOI:10.1016/j.carres.2013.07.004

The linkage region, GlcNAcβAsn, is conserved in all eukaryotic N-glycoproteins. As a logical extension of a research endeavor aimed at understanding the structural significance of GlcNAc and Asn as the linkage region constituents, the newer analogs GlcNAcβNHBu and (GlcNAcβ(1-4) GlcNAc)alkanamides have been synthesized to assess the influence of aglycon as well as additional GlcNAc on the linkage region. X-ray crystallographic analysis of the GlcNAcβNHBu and (GlcNAcβ(1-4)GlcNAc)βNHBu is described. Comparative analysis of these structures with those of reported models and analogs shows that the deviation in N-glycosidic torsion, φ_N among the GlcNAc alkanamides is negligible (<2) whereas (GlcNAcβ(1-4) GlcNAc)βNHBu deviates by ～15 as compared to GlcNAcβNHBu. Under the influence of the molecular packing, the conformation around the C1′-C2′ bond deviates from anti to gauche in (GlcNAcβ(1-4) GlcNAcβNHBu. Interestingly, C2-acetamido group in (GlcNAcβ(1-4)GlcNAc) NHBu orients differently as compared to GlcNAc alkanamides and this orientation was found to be almost similar to β-N,N′-diacetylchitobiose trihydrate. The bifurcated anti-parallel pattern involving N-HO and C-HO hydrogen bonds, a hallmark feature of the N-glycoprotein models, GlcNAcβNHAc and GlcNAcβAsn, is absent in both the title alkanamides. This is the first report on the crystal structure analysis of chitobiosyl alkanamide as analog of the N-glycoprotein linkage region, (GlcNAcβ(1-4) GlcNAc)βAsn.

Full text of DOI:10.1016/j.carres.2013.07.004

Carbohydrate Research 380 (2013) 37–44
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and X-ray crystallographic investigation of N-(b-D-glyco-
syl)butanamides derived from GlcNAc and chitobiose as analogs of
the conserved chitobiosylasparagine linkage of N-glycoproteins
a
b
c,
Manoharan Mathiselvam a, , Amrita Srivastava , Babu Varghese , Serge Pérez
,
⇑
⇑
Duraikkannu Loganathan a,
a Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India
b Sophisticated Analytical Instrumentation Facility, Indian Institute of Technology Madras, Chennai 600036, India
c Centre de Recherches sur les Macromolécules Végétales, CERMAV, CNRS, Grenoble, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2013
Received in revised form 7 July 2013
Accepted 10 July 2013
Available online 19 July 2013
The linkage region, GlcNAcbAsn, is conserved in all eukaryotic N-glycoproteins. As a logical extension of a
research endeavor aimed at understanding the structural significance of GlcNAc and Asn as the linkage
region constituents, the newer analogs GlcNAcbNHBu and (GlcNAcb(1–4)GlcNAc)alkanamides have been
synthesized to assess the influence of aglycon as well as additional GlcNAc on the linkage region. X-ray
crystallographic analysis of the GlcNAcbNHBu and (GlcNAcb(1–4)GlcNAc)bNHBu is described. Compara-
tive analysis of these structures with those of reported models and analogs shows that the deviation in
N-glycosidic torsion, /N among the GlcNAc alkanamides is negligible (<2°) whereas (GlcNAcb(1–4)
GlcNAc)bNHBu deviates by ꢀ15° as compared to GlcNAcbNHBu. Under the influence of the molecular
packing, the conformation around the C10–C20 bond deviates from anti to gauche in (GlcNAcb(1–4)Glc-
NAcbNHBu. Interestingly, C2-acetamido group in (GlcNAcb(1–4)GlcNAc)NHBu orients differently as
compared to GlcNAc alkanamides and this orientation was found to be almost similar to b-N,N0-diacetyl-
chitobiose trihydrate. The bifurcated anti-parallel pattern involving N–Hꢁ ꢁ ꢁO and C–Hꢁ ꢁ ꢁO hydrogen
bonds, a hallmark feature of the N-glycoprotein models, GlcNAcbNHAc and GlcNAcbAsn, is absent in both
the title alkanamides. This is the first report on the crystal structure analysis of chitobiosyl alkanamide as
analog of the N-glycoprotein linkage region, (GlcNAcb(1–4)GlcNAc)bAsn.
Keywords:
N-Glycoprotein
Linkage region
Analogs
X-ray and molecular assembly
Ó 2013 Published by Elsevier Ltd.
1. Introduction
covalently linked to 2-deoxy-2-acetamido-b-D-glucopyranose (Glc-
NAc). Such a motif is a pillar to which are attached more complex
branched carbohydrates which are involved in recognition events
to other biomacromolecules. Elucidation of the structure and con-
formation of the linkage region of N-glycoproteins is of importance
to understand the presentation and dynamics of the carbohydrate
chain at the protein/cell surface. Detailed studies of the impact of
carbohydrate on glycoprotein structure have been limited due to
the flexibility of the carbohydrate, which complicates the crystallo-
graphic analysis of glycoprotein. Besides, oligosaccharides, either
in their free form and when parts of glycoconjugates are inherently
difficult to crystallize and structural data from X-ray studies are
sparse.
To understand the structural significance of the N-glycoprotein
linkage region, we have studied previously X-ray crystal structure
of models and various analogs. These analyses revealed that the
key torsion, /N, is influenced to a greater extent by the structural
variation of the glycan part than that of the aglycon group.4,5 It is
mainly influenced by neighboring group such as C2-acetamido
and environmental factors particularly inter and intramolecular
Glycosylation, the attachment of sugar moieties to proteins, is
thought to be the most complex post-translational modification
(PTM), that provides greater proteomic diversity than other PTM.
Glycosylation is critical for a wide range of biological processes,
including cell attachment to the extracellular matrix and pro-
tein–ligand interactions in the cell. This PTM is characterized by
various glycosidic linkages, including N-, O-, and C-linked glycosyl-
ation, glypiation (GPI anchor attachment), and phosphoglycosyla-
tion.1–3 While it is estimated that over 50% of all eukaryotic
proteins are glycosylated only around 5% of the three-dimensional
structures deposited in the structural data bases include protein
with N- or O-linked carbohydrates. N-Glycosylation is one of the
major classes of glycosylation, where the L-asparagine (Asn) is
⇑
Corresponding authors. Tel.: +91 44 22575221; fax: +91 44 22574202 (M.M.).
E-mail addresses: mathi24@gmail.com (M. Mathiselvam), serge.perez@
cermav.cnrs, spsergeperez@gmail.com (S. Pérez).
Deceased on Feb 09, 2013.
0008-6215/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.carres.2013.07.004

38
M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
interactions involving hydrogen bonds and weak C–Hꢁ ꢁ ꢁO contacts.
The N-glycoprotein linkage region models GlcNAcbNHAc and Glc-
NAcbAsn showed the occurrence of a cooperative anti-parallel net-
work of bifurcated hydrogen bonds consisting of N–Hꢁ ꢁ ꢁO and C–
Hꢁ ꢁ ꢁO interactions. This unique molecular assembly motif involv-
ing C–Hꢁ ꢁ ꢁO hydrogen bonds was absent in the crystal structures
of all the N-glycoprotein linkage region analogs including the
propionamido derivative, GlcNAcbNHPr.6,7 Ab Initio Quantum
Chemical Calculations and X-ray crystallographic studies on propi-
onamido and chloroacetamido derivatives showed the critical role
2.2. Crystal structure of GlcNAcbNHBu (3) and (GlcNAcb(1–
4)GlcNAc)NHBu (6)
2.2.1. Structure description
Alkanamides 3 and 6 were crystallized from aqueous methanol.
Analog 3 crystallized as a monohydrate whereas 6 crystallized as a
trihydrate. Both belong to the space group P21. The crystal mosaic-
ity of alkanamides 6 was quite high as indicated from the
x-spread
of reflections. This led to poor observed/unique reflection ratio and
relatively higher residual factor. Repeated data collection at low
temperature (77.2 K) did not improve the quality of the data set.
ORTEP representations of the structures with atom numbering
are shown in Figure 2. All the atoms of the reducing end residue
of disaccharide are numbered with a suffix A whereas those of
non-reducing end residue are numbered with a suffix B. List of se-
lected bond lengths and bond angles is provided in Table 1.
Analog 3 and both the reducing and non-reducing rings of ana-
log 6 adopt 4C1 conformation. The aglycon part of analog 6 has dis-
order which leads to two different side chain conformations
namely N1–C10–C20–C30–C40 and N1–C10–C20–C3–C6. All C–C bond
lengths are close to 1.54 Å, which is observed in most of the sugar
derivatives. In analog 3, the endocyclic C–O bond distances, namely
C1–O5 is longer than C5–O5, similar trend was observed in Glc-
NAcbNHPr (2).5 The C1–N1 and C2–N2 bond distances are 1.43
and 1.45 Å, respectively. The C–N bond lengths of carbonyl carbon
and nitrogen (C10–N1 and C100–N2) are 1.34 and 1.32 Å, respec-
tively, due to partial double bond character. In analog 6 the endo-
cyclic C–O distances, namely C5A–O5A is longer than C1A–O5A
whereas C5B–O5B is shorter than C1B–O5B. The C1A–N1, C2A–
N2A, and C2B–N2B bond distances are 1.40, 1.47, and 1.49 Å,
respectively. The C–N bond lengths of carbonyl carbon and nitro-
gen (C10–N1, C1A0–N2A, and C1B0–N2B) are 1.29, 1.36, and
1.33 Å, respectively. The exocyclic bond angles, namely C4–C5–
C6 and O5–C5–C6, are comparable to each other and agree well
with those of other models and analogs reported earlier.14 The lat-
ter angle is found to be consistently smaller than the former, and
this is attributed to the effect of the lone pair of electrons causing
a bend at the oxygen on the O5–C5–C6 bond angle. Such a difference
in these bond angles has been observed in the crystal structures of
GlcNAcbAsn15 and several N-linked pyranoses.16 The N-glycosidic
valence angle, O5–C1–N1, is smaller than that of C2–C1–N1 for 3
and the same trend is consistently seen in analog 6.
of the C2-acetamido group of GlcNAc in controlling
v2 at the link-
age region of N-glycoproteins and conserving the extended side
chain conformation.8,9 As a logical extension of the above system-
atic investigation, the newer analogs GlcNAcbNHBu (3) and (Glc-
NAcb(1–4)GlcNAc)alkanamides (4–6) have been synthesized to
understand the influence of aglycon as well as additional GlcNAc
on the linkage region (Fig. 1). Herein we present details of the
chemical synthesis and subsequent structural investigation of Glc-
NAcbNHBu (3) and (GlcNAcb(1–4)GlcNAc)bNHBu (6).
2. Results and discussion
2.1. Synthesis of glycosyl alkanamides
The N-(2-deoxy-2-acetamido-b-
(3) was prepared from the 2-deoxy-2-acetamido-b-
nose (GlcNAc) by using reported procedure.10 Synthesis of N-[2-
deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-b- -glucopyrano-
syl)-b- -glucopyranosyl]alkanamides (4–6) was started with chitin
D-glucopyranosyl)butanamide
D
-glucopyra-
D
D
(Scheme 1). The powdered chitin (7) was subjected to acetolysis
using H2SO4 and acetic anhydride under sonication condition to
give the chitobiose octaacetate (8),11 which was de-O-acetylated
using catalytic amount of sodium methoxide in methanol to give
the GlcNAcb(1–4)GlcNAc (9). The chitobiose (9) was converted to
1,b-aminochitobiose by Kochekov amination, followed by reaction
with acetic anhydride to give (GlcNAcb(1–4)GlcNAc)NHAc (4)12 in
7% yield. In the case of GlcNAcb(1–4)GlcNAcbNHPr (5) and Glc-
NAcb(1–4)GlcNAcbNHBu (6) derivatives, amination of chitobiose
(9) was carried out with ammonium carbamate for 24 h at 37 °C
in dry methanol. The chitobiosamine-salt precipitated out as a
white solid, which was transformed into the corresponding b-chi-
tobiosamine after brief drying under high vacuum.13 The amine
was reacted with propionic and butyric anhydride to give the pro-
pionamide (5) and butanamide (6) derivatives, with respective
yield of 20% and 12% for two steps.
2.2.2. Molecular conformation
The conformation of the N-glycosidic linkage is defined by the
torsion angles fNB, xB, / w0, / wN, v2, and v1
x0B, xA, x0A, fNA,
The 1H NMR spectrum of chitobiosyl alkanamides (4–6) dis-
played two doublets corresponding to anomeric protons. The first
one is around 5.04 ppm with a coupling constant of ꢀ8.8 Hz corre-
sponding to H-1a proton and it shows the b-orientation of N-glyco-
sidic linkage. The second one, around 4.58 ppm with coupling
constant of ꢀ8.0 Hz, corresponds to H-1b proton of the non-reduc-
ing sugar. Three carbonyl carbon signals were found in 13C NMR
spectrum. The anomeric carbon signal of proximal GlcNAc ap-
peared around 78.7 ppm and that of distal GlcNAc residue around
101.3 ppm; they are also consistent with the b-configuration of the
(1–4) disaccharides.
0, N,
(Fig. 3). Molecular conformation is compared with that of mono-
saccharide alkanamide (GlcNAc) derivatives 1, 2, and 3. Selected
torsion angles of compounds 3 and 6 are presented in Table 2.
The /N value of the chitobiosyl analog 6 is ꢂ106.6° which is devi-
ating by 15.1° as compared to that of GlcNAc analog 3 (Table 2)
whereas the deviation among the monosaccharide GlcNAc alkana-
mides 1, 2, and 3 is negligible. The wN values of 3 and 6 are com-
parable and taken together with the /N values reveal the Z-anti
conformation of the amide aglycon moiety. The Z-anti conforma-
tion of the amide linkage has been observed in 1, 2, and
OH
OH
NHAc
O
H
N
O
H
N
HO
HO
HO
HO
O
R
R
HO
O
NHAc
NHAc
OH
O
O
GlcNAcβNHAc (1) R = CH3
(GlcNAcβ(1-4)GlcNAc)βNHAc (4) R = CH3
GlcNAcβNHPr (2) R = CH2CH3
(GlcNAcβ(1-4)GlcNAc)βNHPr (5) R = CH2CH3
(GlcNAcβ(1-4)GlcNAc)βNHBu (6) R = CH2CH2CH3
GlcNAcβNHBu (3) R = CH2CH2CH3
Figure 1. N-Glycoprotein linkage region models (1 and 4) and analogs (2, 3, 5, and 6).

M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
39
OAc
OH
NHAc
O
NHAc
O
O
AcO
AcO
i
HO
O
n
O
O
HO
O
AcO
O
AcHN
NHAc
OH
OAc
OAc
7
8 (6%)
ii
OH
OH
O
NHAc
NHAc
O
H
N
NHAc
iii/iv
O
HO
HO
HO
HO
O
R
O
HO
HO
O
OH
NHAc
O
OH
4 R = CH3 (7%) 5 R = CH2CH3 (20%)
6 R = CH2CH2CH3 (12%)
OH
9
Scheme 1. Synthesis of glycosyl alkanamides. Reagents and conditions: (i) Ac2O, H2SO4, 50 °C, 3 h; (ii) NaOMe/MeOH, 0 °C; (iii) (a) aq NH4HCO3, 5 days, (b) acetic anhydride,
MeOH, 0 °C, 12 h; (iv) (a) NH4COOꢂNH2+, MeOH, 37 °C, High vacuum, (b) Propionic/butyric anhydride, MeOH, 0 °C.
GlcNAcbAsn.15 The
v2 value of the analog 3 is 174.5° which corre-
sponds to an anti conformation around C10–C20 bond (Fig. 2). The
same conformation was observed in the analog GlcNAcNHPr (2)5
and GlcNAcbAsn. The v2 value of the disaccharide analog 6 turns
out to be 136.1° in one disordered form (N1–C10–C20–C30) which
takes up a conformation around the C10–C20 bond in between anti
and gauche but it adopts gauche conformation (72°) in another
form (N1-C10-C20-C3) due to differences in the molecular packing
(Table 2 and Fig. 2). The additional torsion angle C10–C20–C30–C40
(
v
1) was found to be ꢂ176.3° and thus the alkyl chain takes up
an extended conformation in analog 3. In the case of analog 6,
the torsion angle, v1 was found to be ꢂ51° for C10–C20–C30–C40
and 54.2° in another disordered form (C10–C20–C3–C6). The orien-
tation of C2-acetamido groups (fNA) is nearly same (Z-anti) among
GlcNAc alkanamides 1, 2, and
3 whereas GlcNAcb(1–4)Glc-
NAcbNHBu (6) showed deviation of 30.3° relative to that of mono-
saccharide model GlcNAcbNHAc (1). Orientation of C2-acetamido
groups (fNA and fNB) of analog 6 is comparable to that of b-N,N0-
diacetylchitobiose trihydrate16 (Table 2). The hydroxymethyl
group in analog 3 is in gg conformation whereas in the case of ana-
log 6, the non-reducing ring adopts gt and reducing ring adopts gg
conformation. These hydroxymethyl groups play an important role
in hydrogen bonding. Therefore, the preference for anyone of the
conformations gt and gg that predominates in mono- and disaccha-
ride is most likely to be determined by several interrelated geo-
metric requirements associated with molecular conformation and
packing.17,18 The inter glycosidic torsion angles /0 and
6 are ꢂ89.8° and ꢂ153.6°, respectively, which are comparable to
w0 of analog
Figure 2. ORTEP representation (with 30% probability level) of (a) GlcNAcbNHBu
(3); (a) (GlcNAcb(1–4)GlcNAc)bNHBu (6).
Table 1
Selected bond lengths (Å) and bond angles (°) of compounds 3 and 6
Parameter
(3)
Parameter
(6) Proximal
Parameter
(6) Distal
C1–O5
C5–O5
C6–O6
1.4283(19)
1.420(2)
1.415(2)
1.428(2)
1.345(2)
1.224(2)
1.447(2)
1.324(2)
114.72(14)
107.30(15)
107.66(13)
112.56(14)
122.43(17)
115.61(19)
112.8(2)
C1A–O5A
C5A–O5A
C6A–O6A
1.426(11)
1.428(11)
1.430(13)
1.397(11)
1.286(14)
1.236(14)
1.467(11)
1.364(13)
115.0(9)
108.2(9)
106.8(9)
110.0(9)
123.4(12)
119.9(12)
113.9(17)
C1B–O5B
C5B–O5B
C6B–O6B
—
—
—
C2B–N2B
C1B0–N2B
C4B–C5B–C6B
O5B–C5B–C6B
—
—
—
—
—
1.433(10)
1.422(11)
1.437(10)
—
—
—
1.485(12)
1.327(14)
110.7(9)
105.5(9)
—
—
—
—
—
C1–N1
C1A–N1
C10–N1
C10–N1
C10–O10
C10–O10
C2–N2
C100–N2
C2A–N2A
C1A0–N2A
C4A–C5A–C6A
O5A–C5A–C6A
O5A–C1A–N1
C2A–C1A–N1
N1–C10–O10
C10–C20–C30
C20–C30–C40
C4–C5–C6
O5–C5–C6
O5–C1–N1
C2–C1–N1
N1–C10–O10
C10–C20–C30
C20–C30–C40

40
M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
H3C
C1B'
O
O6A
OH
ωA
H
N2B
C6A
C4A
ζNB
N
O5A
O
ψ0
C1B
HψΝ
HO
φΝ
χ2
O
C5A C2A
C4'
CH3
C5B C2B
φ0
O4A
C2'
HO
C1'
N
HO
O
O5B
N1
C4B
C3'
C1A
ζNA
ωB
χ1
N2A
C6B
N
O
OH
O
O6B
C1A'
H
CH3
Figure 3. Schematic representation of the (GlcNAcb(1–4)GlcNAc)bNHBu (6) together with various torsion angles of interest fNB = C1B–C2B–N2B–C1B0,
O6B, x0B = C4B–C5B–C6B–O6B, A = O5A–C5A–C6A–O6A, x0A = C4A–C5A–C6A–O6A, fNA
O = C4A–O4A–C1B–O5B, O = C1B–O4A–C4A–C5A,
N = O5A–C1A–N1–C10, N = C1A–N1–C10–C20, 2 = N1–C10–C20–C30 and 1 = C10–C20–C30–C40.
x
B = O5B–C5B–C6B–
/
w
x
=
C1A–C2A–N2A–C1A0,
/
w
v
v
Table 2
Selected torsion angles of compounds 1, 2, 3, and 6
Torsion angle
GlcNAcb NHAcꢁH2O (1)
—
—
—
—
—
ꢂ66.4(2)
55.4(3)
124.8
ꢂ89.8(1)
174.2(2)
—
GlcNAcb NHPrꢁH2O (2)
—
—
—
—
—
ꢂ66.2(4)
55.5(5)
126.9
ꢂ91.0(5)
172.5(5)
172.4(6)
—
GlcNAcb NHBuꢁH2O (3)
—
—
—
—
—
ꢂ65.69(19)
56.4(2)
122.87(19)
ꢂ91.5(2)
176.59(17)
174.5(2)
176.3(2)
Chitob NHBuꢁ3H2O (6)
99.2(12)
b-Chitobioseꢁ3H2O16
C1B-C2B-N2B-C1B0 (fNB
)
)
)
113.7
58.6
178.8
ꢂ90.3
ꢂ162.3
ꢂ60.6
60.8
100.4
—
O5B-C5B-C6B-O6B (xB
C4B-C5B-C6B-O6B (x0B
C4A-O4A-C1B-O5B (/O)
C1B-O4A-C4A-C5A (wO
O5A-C5A-C6A-O6A (xA
60.7(12)
ꢂ178.8(10)
ꢂ89.8(9)
ꢂ153.6(9)
ꢂ71.8(12)
50.4(13)
)
)
)
)
C4A-C5A-C6A-O6A (x0A
C1A-C2A-N2A-C1A0 (fNA
O5A-C1A-N1-C10 (/N)
94.5(12)
ꢂ106.6(12)
177.2(10)
136.1(14)
ꢂ51 (2)
C1A-N1-C10-C20
N1-C10-C20-C30
C10-C20-C30-C40
(
v2
wN
)
)
)
—
—
—
(
(v1
—
the values of ꢂ90.3° and ꢂ162.3° observed for b-N,N0-diacetylchi-
and 4. The characteristic molecular assembly feature of the linkage
region models GlcNAcbAsn and GlcNAcbNHAc (1) is the antiparal-
lel double-pillared network of bifurcated hydrogen bonds. This
network consists of N1 and C20 acting as donors for O10 in one
direction and C1 and N2 acting as donors for O100 (C2-amide car-
bonyl oxygen) in the opposite direction. Inspection of hydrogen
bonds features in the analog 3 (Table 3, Fig. 4) revealed the
tobiose trihydrate.
2.2.3. Molecular packing
Detailed analysis of molecular packing in the crystal structures
of the title compounds was then undertaken. The various hydrogen
bonding descriptors of compounds 3 and 6 are listed in the Tables 3
Table 3
Hydrogen bond parameters for compounds 3 and 6
0
0
D–Hꢁ ꢁ ꢁA
D–Hꢁ ꢁ ꢁA (°)
Symmetry
Hꢁ ꢁ ꢁA (AÅ)
Dꢁ ꢁ ꢁA (ÅA)
GlcNAcbNHBuꢁH2O (3)
N1–H1Nꢁ ꢁ ꢁO10
2.205(11)
1.941(13)
2.00(3)
1.99(3)
2.03(3)
3.025(2)
2.719(2)
2.7570(19)
2.740(2)
2.771(2)
2.7297(19)
2.715(2)
166.9(18)
154(2)
164(3)
161(3)
158(2)
162(3)
175(3)
x+1,y,z
N2–H2Nꢁ ꢁ ꢁO100
xꢂ1, y, z
O3–H3Oꢁ ꢁ ꢁO6
x,yꢂ1,z
O4–H4Oꢁ ꢁ ꢁO1W
O6–H6Oꢁ ꢁ ꢁO1W
O1W–H1Wꢁ ꢁ ꢁO3
O1W–H1Wꢁ ꢁ ꢁO4
ꢂx+1,y+1/2,ꢂz+1
ꢂx+1,y+1/2,ꢂz+1
xꢂ1,y,z
1.914(13)
1.867(11)
—
ChitobNHBuꢁ3H2O (6)
N2B–H2NBꢁ ꢁ ꢁO1B0
N1–H1Nꢁ ꢁ ꢁO10
2.09
2.06
1.98
2.00
1.86
2.05
1.93
1.88(3)
1.88(2)
1.90(3)
1.86(2)
1.87(4)
1.98(6)
2.908(13)
2.802(13)
2.782(10)
2.795(11)
2.661(6)
159.3
144.3
167.7
164.8
164.1
153.4
165.8
162(8)
170(5)
167(7)
174(6)
160(12)
151(10)
x, y+1, z
x, y+1, z
x, y, z
x, y, z
O3B–H3OBꢁ ꢁ ꢁO2W
O4B–H4OBꢁ ꢁ ꢁO3W
O6B–H6OBꢁ ꢁ ꢁO6B
O3A–H3OAꢁ ꢁ ꢁO5B
O6A–H6OAꢁ ꢁ ꢁO1W
O2W–H2W2ꢁ ꢁ ꢁO3B
O2W–H1W2ꢁ ꢁ ꢁO1A0
O3W–H1W3ꢁ ꢁ ꢁO2W
O3W–H2W3ꢁ ꢁ ꢁO3W
O1W–H2W1ꢁ ꢁ ꢁO1W
O1W–H1W1ꢁ ꢁ ꢁO10
ꢂx+1, yꢂ1/2, ꢂz
2.809(9)
x, y, z
2.733(14)
2.773(11)
2.781(10)
2.774(11)
2.766(8)
ꢂx+1, y+1/2,ꢂz+1
x, y+1, z
x+1, y+1, z
ꢂx+2, yꢂ1/2, ꢂz
ꢂx+2, y+1/2, ꢂz
ꢂx+1, y+1/2, ꢂz+1
ꢂx+1, y+1/2, ꢂz+1
2.749(10)
2.811(14)

M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
41
Table 4
bifurcated hydrogen bonding pattern present in model compound
(1) is completely absent. In contrast, in 6, there is a direct hydrogen
bond between N1 and O10 accompanied by a similar one involving
N2B and O1B0 (of distal residue) leading to a parallel double-pil-
lared hydrogen bonding along the crystallographic b axis (Fig. 6).
There is no accompanying C–Hꢁ ꢁ ꢁO interaction involving C20. On
the other hand, the C2B amide carbonyl oxygen O1B0 acts as a
bifurcated acceptor for C2B0–H as well as N2B–H.
C–Hꢁ ꢁ ꢁO interaction pattern of compounds 3 and 6
0
0
D–Hꢁ ꢁ ꢁA
D–Hꢁ ꢁ ꢁA (°)
Symmetry
Hꢁ ꢁ ꢁA (AÅ)
Dꢁ ꢁ ꢁA (AÅ)
GlcNAcbNHBuꢁH2O (3)
C1–H1ꢁ ꢁ ꢁO100
2.581
2.707
3.266
3.196
124
122
ꢂ1+x, y, z
x, ꢂ1+y, z
C200–H00Bꢁ ꢁ ꢁO5
ChitobNHBuꢁ3H2O (6)
C2B0–H1B0ꢁ ꢁ ꢁO1B0
C4–H5ꢁ ꢁ ꢁO3B
2.714
2.608
3.482
3.401
137
140
1ꢂx, 1/2+y, 1ꢂz
ꢂ1+x, ꢂ1+y, z
Several finite and infinite chains of hydrogen bonds strengthen
the molecular packing of 6. There are three finite chains involving
O3A–H3OAꢁ ꢁ ꢁO5B, O6A–H6OAꢁ ꢁ ꢁO1W–H1W1ꢁ ꢁ ꢁO10, and O4B–
H4OBꢁ ꢁ ꢁO3W–H1W3ꢁ ꢁ ꢁO2W–H1W2ꢁ ꢁ ꢁO1A0. Among these, O3A–
H3OAꢁ ꢁ ꢁO5B is the inter-residue intramolecular hydrogen bond
stabilizing the up-side down conformation of the non-reducing
end. In addition, There are four different infinite chains of hydro-
gen bonds; among these two infinite chains are propagating only
through water molecule. The water molecules O1W and O3W are
donors as well as acceptors in the respective chains. The third infi-
nite chain is propagating through O3B and O2W which act as do-
nors as well as acceptors. In the fourth one O6B is acting as
donor as well as an acceptor and the hydrogen bond advancing
along crystallographic b axis (Fig. 6). Two C–Hꢁ ꢁ ꢁO interactions (Ta-
ble 4) are also present in this crystal. The O3B and O1B0 are accep-
tors for C4–H5 and C2B0–H1B0, respectively. These interactions
combined with hydrogen bonds O2W–H2W2ꢁ ꢁ ꢁO3B and N2B–
H2NBꢁ ꢁ ꢁO1B0, respectively, result in the formation of bifurcated
hydrogen bonds (C4–H5ꢁ ꢁ ꢁO3Bꢁ ꢁ ꢁH2W2–O2W and N2B–
H2NBꢁ ꢁ ꢁO1B0ꢁ ꢁ ꢁH3B0–C2B0). The analog 6 has two hydrophobic
Hꢁ ꢁ ꢁH (van der Waals) contacts (Fig. 7) which involve the pairs
C4A–H4Aꢁ ꢁ ꢁC5A–H5A (2.31 Å) and C2B–H2Bꢁ ꢁ ꢁC3B–H3B (2.16 Å).
These interactions may help in the stacking of sugar ring one above
the other. All the above noncovalent interactions for butanamide
(6) molecule as well as three molecules of water are creating
hydrophilic and hydrophobic regions leading to a compact packing
(Fig. 8). This compact packing is influencing the glycosidic torsion
Figure 4. Molecular packing of GlcNAcbNHBuꢁH2O (3).
presence of direct hydrogen bond between N1 and O10 (N1–
H1Nꢁ ꢁ ꢁO10) as well as a bifurcated hydrogen bonding pattern
involving C1 and N2 as donors and O100 as acceptor (C1–
H1ꢁ ꢁ ꢁO100ꢁ ꢁ ꢁH2N–N2). The distinctive C20–Hꢁ ꢁ ꢁO10 interaction is
clearly missing. Similar hydrogen bonding pattern was observed
for GlcNAcbNHPr.6 Packing of analog 3 is further stabilized by
two infinite chains of hydrogen bonds. The first scheme involves
the water molecule and oxygen atoms O3 and O6 which propa-
gates along the b-axis in the form of helix. In the second infinite
chain, water molecule, acts as a bifurcated donor, forms hydrogen
bonds with its neighboring O4 facilitating the formation of a homo-
dromic cycle of hydrogen bonded network. The above molecular
packing features were also observed in other GlcNAc alkanamides
(1 and 2) reported earlier.4,5 Two C–Hꢁ ꢁ ꢁO interactions are also
present. First one involves C1 as a donor and O100 as an acceptor
(C1–O100ꢁ ꢁ ꢁH1) and the second one is involving C200 as a donor
and O5 as acceptor (C200–H00Bꢁ ꢁ ꢁO5). Weak van der Waals contact
in aglycon part involving C200-H00Cꢁ ꢁ ꢁH40C–C40 atoms with distance
of 2.32 Å facilitates the interaction of hydrocarbon chain (Fig. 5).
In the case of analog 6, molecules are interconnected through
an extensive network of hydrogen bonds (Table 3). Double pillared
/
N
as well as v2.
3. Conclusions
As planned, GlcNAcbNHBu (3) and GlcNAcb(1–4)GlcNAc alkana-
mides (4–6) have been synthesized. Hitherto novel analogs Glc-
NAcb(1–4)GlcNAcbNHPr (5) and GlcNAcb(1–4)GlcNAcbNHBu (6)
were completely characterized based on 2D NMR and HR-MS data.
Comparison of crystal structures of N-acetylglucosaminyl alkana-
mides (1, 2 and 3) with chitobiosyl butanamide (6) revealed that
the glycosidic torsion /N and v2 are influenced by C2-acetamido
group as well as molecular packing. In the case of monosaccharide
alkanamides (GlcNAc), the influence of molecular packing on link-
age region conformation is relatively less. Thus the steric effect of
C2-acetamido group is controlling the N-glycosidic torsion values
Figure 5. van der Waals contacts in GlcNAcbNHBuꢁH2O (3).

42
M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
controlling conformation which leads to noticeable deviation in
values and large deviation in 2. In chitobiosyl butanamide
crystal structure (6), disorder in butyl group leads to two different
2 values but similar /N value in spite of dominant hydrogen bond
network. This could be due to the steric effect of C2-acetamido
group in controlling the /N value being more comparable to 2 val-
/
N
v
v
v
ues. Detailed analysis of molecular packing of GlcNAcbNHBu (3)
and GlcNAcb(1–4)GlcNAcNHBuꢁ3H2O (6) showed that molecular
self assembly is governed by intensive network of non-covalent
interactions like hydrogen bonds, C–Hꢁ ꢁ ꢁO, and hydrophobic
interactions.
These
interactions
between
GlcNAcb(1–
4)GlcNAcNHBuꢁ3H2O (6) and three molecule of water are creating
hydrophilic and hydrophilic regions and leading to compact
molecular assembly. Crystal structures of GlcNAcbNHBu (3) and
GlcNAcb(1–4)GlcNAcNHBuꢁ3H2O (6) showed that the direct hydro-
gen bond between N–H and carbonyl oxygen facilitates the mole-
cules to stack parallel one over the another.
The present work is the first report on the crystal structure
analysis of chitobiosyl alkanamide as analog of the N-glycoprotein
linkage region, GlcNAcb(1–4)GlcNAc)bAsn. Parts of the accurate
structural data that have gathered bring structural information
which are relevant to the N-glycosidic linkage. These are the con-
formations at the b1-4) glycosidic linkage between the two contig-
uous GlcNAc residues and the relative orientation of the
disaccharide with respect to the Asn N-linked residue, which deter-
mines the presentation of the glycan at protein surface. Other
structural data pertain to the area of carbohydrate crystallography,
such as the relative orientations of the molecules in the crystal lat-
tice, or the occurrence of intermolecular hydrogen bonds. One full
value of the present work arises for the possibility to combine the
determined features with those recently obtained from low-tem-
perature neutron diffraction structure of a series of three N-glyco-
protein linkage models which provided highly accurate
determination of hydrogen atoms.7 The results of such an integra-
tion will be therefore of interest for testing and improving force
fields in order to develop better tools for the modeling of N-
glycoproteins.
Figure 6. Molecular packing of GlcNAcb(1–4)GlcNAcbNHBuꢁ3H2O (6).
Figure 7. van der Waals contacts in GlcNAcb(1–4)GlcNAcbNHBuꢁ3H2O (6).
4. Experimental section
4.1. General information
Thin-layer chromatogram was performed on 25 mm E. Merck
silica gel plates (60F-254). Detection was done by spraying the
plates with 10% sulfuric acid in ethanol and heating on a hot plate.
Optical rotations were measured at 30 °C on a JASCO-DIP 200 dig-
ital polarimeter using a cell of 10 mm length. NMR spectra were re-
corded on a Bruker AV400 spectrometer. ESI-MS spectra were
measured on a Micromass Q-Tof mass spectrometer. N-Acetyl glu-
cosamine was purchased from Carbosynth UK.
Crystal structures solution and refinement. Crystallization of the
compounds 3 and 6 was done in aqueous methanol by the slow
evaporation method at room temperature. X-ray data collection
was performed with a Bruker AXS Kappa Apex II CCD diffractome-
ter equipped with graphite monochromated Mo (Ka) (k = 0.7107 Å)
radiation. Crystal fixed at the tip of the glass fiber was mounted on
the goniometer head with the aid of a video microscope. The auto-
matic cell determination routine, with 32 frames at three different
orientations of the detector, was employed to collect reflections,
and the program APEX2-SAINT19 was used for finding the unit cell
parameters. Fourfold redundancy per reflection was utilized for
achieving good absorption correction using multiscan procedure.
Besides absorption, Lorentz, polarization, and decay correction
were applied during data reduction. The program SADABS20 was
used for absorption correction using multiscan procedure. The
Figure 8. Hydrophilic and hydrophobic regions in GlcNAcb(1–4)GlcNAcbNHBu.
3H2O (6)
and leading to negligible deviation in N-glycosidic torsion /N and
v
2 values among GlcNAc alkanamides (1, 2, and 3). In disaccharide
crystal structure (6), due to the intensive network of hydrogen
bonding, molecular packing is becoming a dominant force for

M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
43
Yield (6.1%); mp 300 °C, (Lit.11 301–303 °C); ½a 3D0
ꢄ
ꢂ19.6 (c 1,
Table 5
Data collection and refinement statistics for compounds 3 and 6
MeOH); IR (KBr, cmꢂ1): 3349, 3067, 2948, 2882, 1746, 1662,
1532, 1438, 1375, 1234, 1135, 1043, 940, 910, 678, 599, 554; 1H
NMR (400 MHz, CDCl3): d 6.32 (d, 1H, J = 9.2 Hz, H-N2b), 6.10, (d,
1H, J = 3.6 Hz, H-1a), 5.89 (d, 1H, J = 8.8 Hz, H-N2a), 5.23 (t, 1H,
J = 9.6 Hz, H-3a), 5.14 (t, 1H, J = 9.6 Hz, H-3b), 5.06 (t, 1H,
J = 9.6 Hz, H-4b), 4.49 (d, 1H, J = 8.4 Hz, H-1b), 4.46–4.32 (m, 3H,
H-2a, H-6a1, H-6b1), 4.22, (d, 1H, J = 11.2 Hz, H-6a2), 4.14–3.86
(m, 3H, H-6b2, H-5b, H-2b), 3.77 (t, 1H, J = 9.6 Hz, H-4a), 3.72–
3.60 (m, 1H, H-5a), 2.19, 2.15, 2.09, 2.06, 2.02, 2.01, 1.96, 1.93
(8s, 24H, 8 ꢃ –COCH3); 13C NMR (100 MHz, CDCl3): d 171.5,
171.4, 170.9, 170.6, 170.5, 169.4, 169.1 (8 ꢃ COCH3), 101.8 (C-
1B), 90.6 (C-1A), 75.9, 72.7, 72.0, 70.8, 68.2, 61.9 (C-6A and C-
6B), 54.4, 51.2, 29.7, 23.2, 23.0, 21.0, 20.7 (8 ꢃ –COCH3); ESI-MS:
Calcd for C28H41N2O17: 677.2405. [M+H]+. Found: 677.2427.
Parameter
GlcNAcbNHBuꢁH2O
(3)
ChitobNHBuꢁ3H2O
(6)
Empirical formula
Formula weight
Wavelength
C
12H24 N2O7
C20H41N3O14
549.57
0.71073 Å
308.33
0.71073 Å
Crystal system
Space group
Monoclinic
P21
Monoclinic
P21
Cell dimensions
a = 4.8960(2) Å
b = 7.8976(4) Å
c = 20.2724(11) Å
a = 14.494(7) Å
b = 4.8581(4) Å
c = 19.089(6) Å
a
= 90°
b = 90.447 (2)°
= 90°
783.84(7)
a
= 90°
b = 98.87(1)°
c = 90°
c
Volume (Å3)
1327.9(10)
2, 1.374
0.116
Z, calculated density (mg/m3) 2, 1.306
Absorption coefficient
(mmꢂ1
F(000)
0.107
)
4.3. N-[2-Deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-b-
glucopyranosyl)-b- -glucopyranosyl]acetamide (4)
D-
332
592
D
Crystal size (mm)
Theta range (°)
Index ranges
0.20 ꢃ 0.16 ꢃ 0.12
2.01 to 24.99
ꢂ5<=h<=4,
ꢂ9<=k<=9,
ꢂ24<=l<=24
7629/2682
[R(int) = 0.0228]
2682/5/239
1.035
0.25 ꢃ 0.22 ꢃ 0.15
2.16 to 25.0
ꢂ17<=h<=16,
ꢂ5<=k<=4,
In a 50 mL round bottom flask, 2-deoxy-2-acetamido-4-O-(2-
deoxy-2-acetamido-3,4,6-tri-O-acetyl-b- -glucopyranosyl)-1,3,6-
tri-O-acetyl- -glucopyranose (8) (545 mg, 0.8 mmol) was
D
ꢂ22<=l<=22
7569/3818
[R(int) = 0.1483]
3818/42/368
0.961
a-D
Reflections collected/unique
dissolved in dry MeOH (2 mL) and cooled to 10 °C. To this solution,
freshly prepared NaOMe (1 M, 0.5 mL) was added and stirred for
4 h. The reaction mixture was then treated with Ambererlite
IR-120 ion-exchange resin. After filtration of the resin, the filtrate
was concentrated to give chitobiose (9) as a solid. The solid chito-
biose (9) was stirred with saturated ammonium bicarbonate solu-
tion [1% (w/v)] for 5 days at room temperature. Solid ammonium
bicarbonate was added during course of the reaction to ensure
the saturation. After 5 days, the solution was lyophilized twice. Ex-
cess ammonium bicarbonate was removed by extracting the crude
chitobiosylamine in dry methanol followed by filtration. Concen-
tration of the methanolic solution to dryness gave the chitobiosyl-
amine as a solid.12 This chitobiosylamine was dissolved in dry
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I >2sigma (I)] R1 = 0.0297,
wR2 = 0.0712
R1 = 0.0827,
wR2 = 0.1371
R1 = 0.2603,
wR2 = 0.1967
R indices (all data)
R1 = 0.0356,
wR2 = 0.0743
structures were solved by direct methods using SIR92 (WinGX) and
refined by full-matrix least-squares techniques using the SHELXL-
9721 computer program. All hydrogen atoms except those of nitro-
gen were fixed at chemically meaningful positions and riding mod-
el refinement was applied. The nitrogen hydrogen was located
through difference Fourier map and refined with isotropic thermal
parameters. Molecular graphics were drawn using ORTEP3222 and
Mercury programs.23 The crystal data with refinement details are
summarized in Table 5. Complete structural data of the 3 and 6
have been deposited at the Cambridge Crystallographic Data Cen-
tre (CCDC 939935–939936), and can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif (or from the Director,
Cambridge Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK; fax: +44 1223 336033; or email:
deposit@ccdc.cam.ac.uk).
methanol (2 mL), acetic anhydride (123 lL, 1.2 mmol) was added,
in portions, and the reaction mixture was stirred for 2 h at 0 °C, fol-
lowed by stirring at room temperature overnight. The solid sepa-
rated out from the solution was filtered, washed with cold
methanol to give 25 mg of the product as white solid (4).
Yield (25 mg, 7%); mp 300–305 °C, Lit.12 303–304 °C; ½a D30
ꢄ
1.8 (c
1, H2O), Lit.9 ½a D30
ꢄ
2.5° (c 1, D2O); IR (KBr, cmꢂ1): 3400, 3286, 3106,
2939, 2890, 1654, 1557, 1426, 1374, 1310, 1162, 1114, 1073, 1037,
953, 899, 687, 607, 571; 1H NMR (400 MHz, D2O): d 5.04 (d, 1H,
J = 8.0 Hz, H-1a), 4.59 (d, 1H, J = 6.8 Hz, H-1b), 3.95–3.70 (m, 6H),
3.68–3.43 (m, 6H), 2.06, 2.00, 1.99 (3s, 9H, 3 ꢃ –COCH3); 13C
NMR (100 MHz, D2O): d 174.9, 174.7, 174.6 (3 ꢃ –COCH3), 101.3,
78.7, 78.2, 76.1, 75.9, 73.4, 72.8, 69.7, 60.5, 59.9, 55.5, 53.7, 22.1,
4.2. 2-Deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-30,40,60-
tri-O-acetyl-b-
(8)
D
-glucopyranosyl)-1,3,6-tri-O-acetyl-
a
-
D
-glucopyranose
22.0, 21.9 (3 ꢃ –COCH3); ESI-MS: Calcd for C18H32N3O11
:
466.1964. [M+H]+: Found: 466.1964.
Powdered chitin (15 g) was taken in a 500 mL round bottom
4.4. N-[2-Deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-b-
D-
flask to which 120 mL acetic anhydride and 15 mL H2SO4 were
added and the mixture was sonicated for 3 h. The resulting suspen-
sion was stirred for 3 h at 55 °C and then poured onto ice water; pH
was adjusted to 7 with powdered NaOAc (12 g), and finally filtered
through Buchner funnel. The filtrate was extracted with CHCl3
(4 ꢃ 50 mL). The combined extract was washed with water
(250 mL), saturated NaHCO3 (250 mL), water (250 mL), dried over
Na2SO4 (20 g), filtered, and concentrated under reduced pressure
to obtain an orange solid, which was recrystallized from MeOH
(20 mL) to give the title compound as a white solid (370 mg).
The mother liquor was concentrated and subjected to column
chromatography over silica gel (100–200 mesh). Elution of the col-
umn with a mixture of MeOH and CH2Cl2 (1:99) gave pure product
as a white solid (545 mg).
glucopyranosyl)-b- -glucopyranosyl]alkanamides (5 and 6)
D
Chitobiose (140 mg, 0.33 mmol) was dissolved in methanol
(2 mL) and ammonium carbamate (013 mg, 1.32 mmol) was added
to this. The reaction mixture was stirred for 16 h at 37 °C and then
at 0 °C for 1 h. The fluffy white precipitate of glycosylammonium
carbamate formed was filtered, washed twice with cold methanol
(2 mL), and subjected to high vacuum for no more than 10 s. The
glycosylamine thus obtained as a colorless solid was dissolved in
dry MeOH (2 mL) and cooled to 0 °C. Propionic/butyric anhydrides
(0.5 mmol) were then added and the mixture was stirred overnight
at room temperature. The reaction mixture was concentrated on a
rotavaporator to remove excess anhydrides to obtain the propiona-
mide (5) and butanamide (6) in pure form.

44
M. Mathiselvam et al. / Carbohydrate Research 380 (2013) 37–44
4.4.1. N-[2-Deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-b-
glucopyranosyl)-b-D-glucopyranosyl]propionamide (5)
D
-
entific and Industrial Research (CSIR), New Delhi, for award of a
Senior Research Fellowship. We also thank Cambridge Crystallo-
graphic Data Centre (CCDC), United Kingdom, for making the pro-
gram Mercury 3.0 available for use.
Yield (32 mg, 20%); mp 300–305 °C (decomposes); ½a D30
ꢄ
2.2 (c 1,
H2O); IR (KBr, cmꢂ1): 3304, 3102, 2979, 2926, 2881, 1654, 1551,
1426, 1374, 1312, 1274, 1236, 1160, 1077, 948, 901, 675, 622; 1H
NMR (400 MHz, D2O): d 5.01 (d, 1H, J = 8.8 Hz, H-1a), 4.55 (d, 1H,
J = 8.0 Hz, H-1b), 3.94–3.66 (m, 6H), 3.65–3.36 (m, 6H), 2.21 (q,
2H, J = 7.6 Hz, –CH2CH3), 2.02, 1.95 (2s, 6H, 2 ꢃ –COCH3), 1.03 (t,
3H, J = 6.4 Hz, –CH2CH3); 13C NMR (D2O, 100 MHz): d 178.7,
174.6, 174.5 (3 ꢃ –COCH3), 101.3, 78.7, 78.2, 76.1, 75.8, 73.3,
72.6, 69.6, 60.4, 59.8, 55.5, 53.7, 29.1 (–CH2CH3), 22.0, 21.8 (2 ꢃ –
COCH3), 9.2 (–CH2CH3); ESI-MS: Calcd for C19H34N3O11: 480.2193.
[M+H]+: Found: 480.2196.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.
07.004.
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4.4.2. N-[2-Deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-b-D-
glucopyranosyl)-b-D-glucopyranosyl]butanamide (6)
Yield (20 mg, 12%); mp 300–305 °C (decomposes); ½a D30
ꢄ
2.6 (c 1,
H2O); IR (KBr, cmꢂ1): 3416, 3288, 3099, 2929, 2875, 2677, 1651,
1553, 1427, 1375, 1314, 1262, 1161, 1110, 1056, 952, 894, 682,
616; 1H NMR (400 MHz, D2O): d 5.07 (d, 1H, J = 9.4 Hz, H-1a),
4.61 (d, 1H, J = 8.1 Hz, H-1b), 4.00–3.72 (m, 6H), 3.72–3.43 (m,
6H), 2.25 (t, 2H, J = 6.8 Hz, –CH2CH2CH3), 2.08, 2.00 (2s, 6H, 2 ꢃ –
COCH3), 1.00–0.84 (m, 2H, –CH2CH2CH3), 0.89 (q, 3H, J = 7.1 Hz, –
CH2CH2CH3); 13C NMR (100 MHz, D2O): d 177.9 (–COCH3), 174.6
(2 ꢃ –COCH3), 101.4 (C1-B), 78.8 (C-1A), 78.2, 76.2, 75.9, 73.4,
72.8, 69.7, 60.5, 59.9, 55.6, 53.7, 37.7 (–CH2CH2CH3), 22.1, 22.0
(2 ꢃ –COCH3), 18.8 (–CH2CH2CH3), 12.5 (–CH2CH2CH3); ESI-MS:
Calcd for C20H36N3O11: 494.2350. [M+H]+. Found: 494.2335.
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Acknowledgments
We are grateful to the Indo-French Centre for Promotion of Ad-
vanced Research (IFCPAR), New Delhi, for the financial support and
encouragement. Funding provided by the Department of Science
and Technology, New Delhi, for the purchase of the 400 MHz
NMR under IRHPA Scheme and ESI-MS under the FIST program
to the Department of Chemistry, IIT Madras is gratefully acknowl-
edged. The authors thank the single crystal X-ray facility, Chemis-
try Department, IIT Madras, for the X-ray data collection. We are
thankful to Mr. V. Ram Kumar for X-ray data collection and coop-
eration. Two of us (M.M. and A.S.) are thankful to the Council of Sci-
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