Regioselective opening of unsymmetrical cyclic anhydrides: Synthesis of N-glycosylated isoasparagine and isoglutamine conjugates

Article abstract of DOI:10.1039/c4ra01234h

N-Glycopeptide mimetic with N-glycosylated isoasparagine and isoglutamine conjugates were synthesized by regioselective opening of unsymmetrical cyclic anhydride derivatives of l-aspartic acid and l-glutamic acid, using per-O-acetylated β-d-glycopyranosyl amine. The α-chloro derivative gave a mixture of asparagine and isoasparagine linked glycoconjugates, whereas the trifluoroacetamide derivatives gave predominantly the isoasparagine and isoglutamine linked glycoconjugates as the product. The X-ray crystal structure of the α-chloro isoasparagine linked glycoconjugate showed unique pattern of hydrogen bonding. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra01234h

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Regioselective opening of unsymmetrical cyclic
anhydrides: synthesis of N-glycosylated
Cite this: RSC Adv., 2014, 4, 22042
isoasparagine and isoglutamine conjugates†
*
Laxminarayan Sahoo, Anadi Singhamahapatra, Vankatachalam Ramkumar
and Duraikkannu Loganathan‡
N-Glycopeptide mimetic with N-glycosylated isoasparagine and isoglutamine conjugates were synthesized
by regioselective opening of unsymmetrical cyclic anhydride derivatives of L-aspartic acid and L-glutamic
acid, using per-O-acetylated b-D-glycopyranosyl amine. The a-chloro derivative gave a mixture of
asparagine and isoasparagine linked glycoconjugates, whereas the trifluoroacetamide derivatives gave
predominantly the isoasparagine and isoglutamine linked glycoconjugates as the product. The X-ray
crystal structure of the a-chloro isoasparagine linked glycoconjugate showed unique pattern of
hydrogen bonding.
Received 12th February 2014
Accepted 20th March 2014
DOI: 10.1039/c4ra01234h
www.rsc.org/advances
their therapeutic application. Though the naturally occurring
glycosyl asparagine and to some extent its one carbon higher
Introduction
homolog glutamine conjugate are synthesized and studied,
their regioisomers i.e. N-glycosylated isoasparagine and iso-
glutamine have not been explored in the literature of glyco-
peptide mimetics (Fig. 1).
Regioselective chemical ligation of biomolecules is regularly
used for the synthesis of bioconjugates with complex structures
useful for targeted biomedical applications. This method also
has the scope for synthesis of other possible isomers of the
bioconjugates potentially useful as a mimic of the natural
isomers with improved pharmacokinetic properties. N-Glyco-
sylation is a major post-translational or co-translational modi-
cation of proteins. The glycan part inuences the biological
signicance of the protein, in addition to controlling their
folding and solubility. The linkage region in N-linked glyco-
proteins is conserved as GlcNAc-b-Asn-Xaa-Ser/Thr, where D-
GlcNAc is covalently attached to the amide side chain of
asparagine by b-linkage, followed by any amino acid other than
proline (Xaa) and serine or threonine.1–4 Naturally occurring N-
linked glycoproteins are susceptible to hydrolysis by enzymes
such as the N-glycanase family, thus decreasing their bioavail-
ability and reducing their therapeutic application. In recent
years there have been several approaches to improve the
proteolytic stability and bioavailability of N-linked glycoproteins
and utilize their wide range of bioactivity in drug design. With
small modication in structure of the natural glycoproteins,
these N-linked glycoprotein mimics are facilitated with better
proteolytic stability and increased bioavailability required for
Asparagine to isoasparagine conversion is not a rare
phenomenon in naturally occurring peptides and other bio-
conjugates. The formation of an isoasparagine linkage inserts
an extra methylene group in the peptide backbone resulting in
changes in the conformation of the peptide as well as their
biological activities. Since some of the asparagine to iso-
asparagine conversions are related to the immunological
disorder in living organism there are reports on the use of iso-
asparagine in peptide mimetics and evaluation of their thera-
peutic importance.5–17 But in the literature of N-linked
glycoprotein mimetics there are few reports on the synthesis of
glycosyl isoasparagine conjugates where a mixture of both
glycosyl asparagine and isoasparagine was obtained in almost
1 : 1 ratio.18,19 The substituent on the amine part of asparagine
has less signicance in controlling the conformation of the
linkage region.20 In this present work we have synthesized
Department of Chemistry, Indian Institute of Technology Madras, Chennai, 600036,
India. E-mail: laxminarayanchem@gmail.com; Fax: +91-44-22570509; Tel: +91-44-
22575221; +91-9380820326
† Electronic supplementary information (ESI) available. CCDC 938353. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ra01234h
Fig.
1 N-Glycoprotein with GlcNAc–asparagine (natural) and
‡ Deceased on 9th February, 2013.
GlcNAc–isoasparagine (unnatural) linkage.
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N-glycosylated isoasparagine and isoglutamine conjugates by
regioselective opening of cyclic aspartic or glutamic acid anhy-
drides using per-O-acetylated glycopyranosyl amine.
Results and discussion
In addition to the importance of halogenated bio-molecules in
medicinal chemistry, considering the higher electronegativity of
halogens regioselective reaction of cyclic anhydrides of aspartic
or glutamic acid were planned to be achieved using halogenated
derivatives of the amino acids. The reaction of per-O-acetylated
b-D-glucopyranosyl amine (derived from the reduction of the
corresponding azide) with 2-chlorosuccinic anhydride gave
mixture of two regioisomers (Scheme 1). Both the isoasparagine
(3) and asparagine (4) derivatives were obtained in 90% overall
yield with 2 : 1 ratio of the two regioisomers. Purication of the
crude reaction mixture by column chromatography followed by
recrystallization gave the chlorine substituted isoasparagine
precursor 3 in 50% yield. The rest of the product was obtained
as a mixture of compounds 3 and 4.
Fig. 2 Conformers A and B in the crystal structure of molecule 3.
In conformer B the ring oxygen and the C-2 carbon have a
torsion angle of ꢀ134.57ꢁ (O5B–C1B–N1B–C10B) and 106.19ꢁ
(C2B–C1B–N1B–C10B) with the amide bond, respectively. The N-
glycosylated amide bond and the free acid group in compound 3
has a torsion angle of 173.42ꢁ (C10–C20–C30–C40) and 62.59ꢁ
(C10B–C20B–C30B–C40B) in conformer A and B, respectively. The
chlorine has a torsion angle of 143.55ꢁ (N1A–C10–C20–Cl1A) and
72.69ꢁ (N1B–C10B–C20B–Cl1B) with the amide bond for
conformer A and B, respectively; whereas the angle between the
chlorine and the free acid is ꢀ88.82ꢁ (Cl1A–C20–C30–C40) and
ꢀ178.11ꢁ (Cl1B–C20B–C30B–C40B) in A and B, respectively.
The two conformers (A and B) of molecule 3 not only differ in
their conformation, but also in their participation in hydrogen
bonding in the crystal structure. The bond parameters of three
different types of hydrogen bonds in molecule 3 are listed in
Table 2.
All the molecules with conformation A are connected to each
other by regular H-bonds O20–H2O/O8A in zigzag style, where
the acid groups connected to anomeric positions are H-bonded
to C-3 acetate carbonyls (Fig. 3).
On the other hand, all the molecules with conformation B
are connected to each other by an innite chain of H-bonds
N1B–H1NB/O10B, where the N–H protons of the anomeric
amide groups are connected to CO oxygen of anomeric amide of
the next molecule. This innite chain of hydrogen bonding
arranges the molecules (conformer B) in anti-parallel orienta-
tion (Fig. 4).
In the 1H NMR spectrum of compound 3 the anomeric
proton appeared as a doublet at 5.22 ppm with coupling
constant of 9.2 Hz, whereas the amido proton (NH) appeared at
7.45 ppm with a coupling constant of 8.8 Hz. The alpha proton
of the chlorine functionalized glycoamino acid (–CHCl)
appeared as a multiplet at 4.64–4.61 ppm which was correlated
to the methylene protons which appeared as a multiplet in the
range of 3.22–3.04 ppm. In the 13C NMR spectrum (100 MHz,
CDCl3), the anomeric carbon appeared at 78.6 ppm. The alpha
carbon of the chlorine functionalized glycoamino acid (–CHCl)
appeared at 53.1 ppm and the methylene carbon of the chlor-
oamino acid appeared at 38.8 ppm. The molecular ion peak [M
+ H]+ of compound appeared at 482.1049 in the ESI-MS HRMS
spectrum.
The chlorinated N-linked glycoamino acid 3 was recrystal-
lized from a mixture of ethyl acetate and hexane at room
temperature by slow evaporation method and the structure was
solved in the monoclinic space group P21. The X-ray structure
conrmed the isoaspargine linkage in the molecule. But there
are two different conformations of the molecule in the crystal
structure, depicted as A and B in Fig. 2. The conformation of the
chlorinated aglycon part of the molecule differs in two different
conformers (A and B) of the molecule in the crystal structure.
The torsion angles in both the conformers of the molecule are
listed in Table 1. The amide bond has a torsion angle of
ꢀ125.06ꢁ (O5A–C1A–N1A–C10) and 114.38ꢁ (C2A–C1A–N1A–C10)
with the ring oxygen and C-2 carbon of glucose in conformer A.
The molecules with conformation A are connected to mole-
cules with conformation B by regular H-bonds O30B–H3O/
O9A, where the C-4 acetate CO group in conformer A are H-
bonded to an acid functionality at the anomeric position of
conformer B. These three different types of regular H-bonds
Table 1 Selected torsion angles in molecule 3
Conformer A
Conformer B
Selected atoms
Angle (ꢁ)
Selected atoms
Angle (ꢁ)
O5A–C1A–N1A–C10
C2A–C1A–N1A–C10
N1A–C10–C20–Cl1A
O10–C10–C20–Cl1A
C10–C20–C30–C40
Cl1A–C20–C30–C40
C20–C30–C40–O20
C20–C30–C40–O30
ꢀ125.06
114.38
143.55
ꢀ37.75
173.42
ꢀ88.82
ꢀ8.41
O5B–C1B–N1B–C10B
C2B–C1B–N1B–C10B
N1B–C10B–C20B–Cl1B
O10B–C10B–C20B–Cl1B
C10B–C20B–C30B–C40B
Cl1B–C20B–C30B–C40B
C20B–C30B–C40B–O20B
C20B–C30B–C40B–O30B
ꢀ134.57
106.19
72.69
ꢀ106.60
62.59
ꢀ178.11
20.04
ꢀ160.05
Scheme 1 Synthesis of a-chloro N-glycosylated derivative of aspartic
acid.
174.58
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Table 2 H-bond parameters
D–H/A
protons which appeared in the range of 2.98–2.79 ppm. In the
13C NMR spectrum the anomeric carbon appeared at 78.4 ppm.
The formation of the product 7 was further conrmed by
appearance of the molecular ion peak [M + Na]+ at 580.1379 in
the high resolution ESI-MS spectrum.
Angle D–H/A (ꢁ)
˚
D–A (A)
N1B–H1NB/O10B
O20–H2O/O8A
O30B–H3O/O9A
2.861
2.685
2.763
165.11
165.66
175.81
Aer synthesis of the glycosyl isoasparagine conjugate by
reaction of per-O-acetylated glycopyranosyl amine and 2-
substituted succinic anhydride, the methodology was extended
to the synthesis of glycosyl glutamine conjugates. The synthesis
of glycosyl glutamine conjugate was carried out under identical
reaction conditions involving conversion of L-glutamic acid to
anhydride (9) by reaction with triuoroacetic anhydride followed
by reaction of the vacuum dried anhydride with 2-deoxy-2-acet-
amido-3,4,6-tri-O-acetyl-b-D-glucopyranosyl amine (Scheme 3).
The 1H NMR and 13C NMR spectra of the column puried
product showed formation of single product. The 1H NMR
spectrum of compound 11 showed the anomeric proton
appearing as multiplet along with H-3 in the range of 5.27–5.22
ppm. The alpha proton of the glycoamino acid appeared along
with H-6a as a multiplet in the range of 4.30–4.22 ppm, which
was correlated to the methylene protons, which appeared as
three different multiplets in the range of 2.50–1.90 ppm. The
absence of peaks corresponding to the triuoroacetamido group
(NHCOCF3) in the 13C NMR spectrum suggested the formation
of cyclic glycoamino acid 11 which was further conrmed by the
appearance of a molecular ion peak at 480.1587 in the ESI-MS
HRMS spectrum.
The mechanism of formation of the unnatural cyclic gluta-
mine conjugate can be explained as follows. When glutamine is
treated with triuoroacetic anhydride, the two acid groups form
a mixed anhydride with triuoroacetic anhydride. The amine
reacts with the g-amino acid anhydride to get the ve membered
ring containing cyclic amide compound (9) whereas the a-acid
group remains as the mixed anhydride which upon reaction with
2-deoxy-2-acetamido-3,4,6-tri-O-acetyl-b-D-glucopyranosyl amine
forms the single cyclic glutamine conjugate (11).
arranged the molecules in a three-pillared structure, where two
pillars of conformer B arranged in an anti-parallel way are
connected to molecules with conformation A (Fig. 5 and 6).
Since the reaction with chlorinated cyclic anhydride gave a
mixture of asparagine and isoasparagine derivatives we
concentrated on improving the regioselectivity of the reaction.
Compared to literature reported methods when the amino group
of the aspartic acid was replaced by a more electronegative group
such as chlorine, the unnatural glycosyl asparagine precursor
was obtained as the major product. Reports on the use of a larger
group such as –cbz in place of chlorine showed lack of regiose-
lectivity in the reaction (1 : 1).19 These results suggested that
electronic factors, rather than steric factors, play a major role in
controlling the regioselectivity of the reaction. Keeping this in
mind, the amino group of aspartic acid was converted to its
triuoroacetamido derivative by reacting aspartic acid with tri-
uoroacetic anhydride to give 2-triuoroacetamido succinic
anhydride (6). 2-Deoxy-2-acetamido-3,4,6-tri-O-acetyl-b-D-gluco-
pyranosyl amine synthesized by catalytic hydrogenation of 2-
deoxy-2-acetamido-3,4,6-tri-O-acetyl-b-D-glucopyranosyl azide
(5) was reacted with 2-triuoroacetamido succinic anhydride in
dry acetonitrile (Scheme 2) to give the unnatural glycosyl iso-
asparagine (7) as the major product along with a small amount
of compound 8 (ratio 9 : 1). The crude reaction mixture was
puried by column chromatography to give the unnatural
glycosyl isoasparagine derivative 7 in 80% overall yield.
The anomeric proton of compound 7 appeared as multiplet
in the range of 5.08–5.04 ppm with H-4 in the 1H NMR spectrum
of compound. The alpha proton of the triuoroacetamido
functionalized glycoamino acid (–CHNHCOCF3) appeared as a
triplet at 4.71 ppm which was correlated to the methylene
Aer synthesis of the GlcNAc–IsoGln derivative (11), the
methodology was further veried with D-glucose and the cor-
responding cyclic glutamine conjugate (10) was found to be the
only product based on their NMR and ESI-MS HRMS
Fig. 3 Infinite chain of H-bonding in compound 3 (conformer A).
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Fig. 4 Infinite chain of hydrogen bonding in compound 3 (conformer B).
spectroscopic analysis aer column purication. Compared to present work is not known in the literature and can be used in
aspartic acid in the case of glutamic acid the presence of an the area of peptidomimetic and glycopeptide synthesis.
additional methylene group in the side chain facilitates the Although the chlorinated derivative gave a mixture of aspara-
formation of N-glycosylated cyclic amide derivatives of gluta- gine and isoasparagine linked products, the X-ray structural
mine (10 and 11).
study of the isoaspargine derivative (4) showed a unique pattern
of hydrogen bonding. The regioselective reaction of tri-
uoroacetamide functionalized cyclic anhydrides gave the cor-
responding isoasparagine derivative as the major product and
the cyclic isoglutamine derivatives as the only product. The
replacement of the methylene group from the N-glycosylated
side chain in natural GlcNAc–Asn linkage to the peptide back-
bone in isoasparagine will increase the conformational exi-
bility and proteolytic stability. The application of these
Summary and conclusion
In conclusion, chlorinated as well as triuoroacetamido deriv-
ative of N-glycosylated isoasparagine and cyclic isoglutamine
conjugates were synthesized from the reaction of sugar amine
and corresponding cyclic anhydrides. The synthesis of cyclic
amides from amino acid using simple methodology done in this
Fig. 5 Three-pillared hydrogen bonding in compound 3 (conformer A and B).
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Fig. 6 Hydrogen bonding pattern in the crystal structure of compound 3 (conformer A and B).
from commercial sources were used without any purication.
NMR spectra were recorded on 400 MHz NMR spectrometer.
1
The assignment of H NMR spectra was done with the help of
1H–1H COSY spectra. All mass spectra were recorded in Q-TOF
electrospray ionization spectrometer. Column chromatography
was performed over 100–200 mesh silica with ethyl acetate and
hexane as the eluent.
1. Synthesis of glycopyranosyl asparagine analogs by reac-
tion of per-O-acetylated glycopyranosyl amine and 2-substituted
succinic anhydride (3 and 7). Per-O-acetylated glycopyranosyl
azide (1 or 5; 1 mmol) was reduced to the corresponding amine
using Pd/C (10 wt%) in dry dichloromethane (5 mL) under
hydrogen atmosphere. Aer the reduction was over, indicated
Scheme 2 Synthesis of a-N-trifluroacetamido N-glycosylated deriv-
ative of aspartic acid.
ꢁ
by TLC, the solution was cooled to 0 C. To this a solution 2-
substituted succinic anhydride (2 or 6, 1 mmol in 5 mL dry
acetonitrile) was added dropwise. The reaction was continued
for 24 h at room temperature. Acetonitrile (50 mL) was added to
the reaction mixture and the contents were ltered through a
lter paper. The ltrate was concentrated to dryness and the
crude product was puried by column chromatography.
ꢁ
Compound 3. Yield 60%, m.p.: 155–159 C, [a]D: ꢀ18.8ꢁ (c ¼
Scheme 3 Synthesis of N-glycosylated lactam derivative of L-glutamic
acid.
0.5, MeOH, 25 ꢁC), 1H NMR (CDCl3, 400 MHz): d 7.45 (d, 1H, J ¼
8.8 Hz, NH), 5.33 (t, 1H, J ¼ 9.2 Hz, H-3), 5.22 (t, 1H, J ¼ 9.2 Hz,
H-1), 5.09 (t, 1H, J ¼ 9.6 Hz, H-4), 5.02 (t, 1H, J ¼ 9.2 Hz, H-2),
4.64–4.61 (m, 1H, Cl–CH–CH2), 4.31–4.28 (dd, 1H, H-6a), 4.11–
4.08 (dd, 1H, H-6b), 3.85–3.83 (m, 1H, H-5), 3.22–3.04 (dq, 2H,
Cl–CH–CH2), 2.09, 2.06, 2.03 (ꢂ2) (4s, 12H, COCH3) ppm; 13C
NMR (CDCl3, 100 MHz): d 171.1, 170.7, 170.0, 169.5, 168.5 (–CO–
CH3), 78.6, 73.7, 72.6, 70.2, 68.1, 61.5, 53.1, 38.8, 20.7, 20.5 ppm;
ESI-MS HRMS: observed 482.1049 for [M + H]+ calculated
482.1065 for C18H25NO12Cl.
isoaspargine and isoglutamine conjugates in the synthesis of
glycopeptide mimic has enormous scope in biomedical
research yet to be explored.
Experimental section
General methods
All the solvents were used aer distillation and dry solvents
Compound 7. Yield 80%, 1H NMR (400 MHz, MeOD + CDCl3):
were prepared using standard methods. All reagents purchased d 5.14 (t, 1H, J ¼ 9.2 Hz, H-3), 5.08–5.04 (m, 2H, H-1 & H-4), 4.71
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(t, 1H, J ¼ 9.6 Hz, NH–CH–CH2), 4.29–4.24 (dd, 1H, H-6a), 4.13– Delhi and AS thanks CSIR, New Delhi for research fellowships.
4.09 (dd, 2H, H-2 & H-6b), 3.81–3.78 (m, 1H, H-5), 2.98–2.79 (dq, The authors are thankful to Prof. S. Sankararaman, Department
2H, NH–CH–CH2), 2.08, 2.04, 2.03, 1.93 (4s, 12H, COCH3) ppm; of Chemistry, Indian Institute of Technology Madras, and Dr.
13C NMR (100 MHz, CDCl3): d 171.1, 170.0, 169.6, 169.5, 169.3, Satyanarayan Sahoo, Department of Chemistry, Berhampur
156.3, 156.0, 117.0 (JC–F ¼ 291.9 Hz, CF3), 78.4, 73.0, 72.3, 68.3, University, for their valuable input during the manuscript
61.6, 51.8, 50.1, 34.9, 22.3, 20.4, 20.2 ppm; ESI-MS HRMS: preparation.
observed 580.1379 for [M+ + Na] calculated 580.1366 for
C
20H26N3O12NaF3.
2. Synthesis of glycopyranosyl glutamine analogs (10, 11).
References
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Per-O-acetylated glycopyranosyl azide (1 or 5; 1 mmol) was
reduced to the corresponding amine using Pd/C (10 wt%) in dry
dichloromethane (5 mL) under hydrogen atmosphere. Aer the
reduction was over, indicated by TLC, the solution was cooled to
0 ꢁC. Glutamic acid (1 mmol) was taken in a 100 mL two necked
RB ask tted to a reux condenser. To this triuoroacetic
anhydride (5ꢁ mL) was added and the reaction mixture was
stirred at 40 C for 30 minutes. Aer that, the condenser was
removed and excess triuoroacetic anhydride was removed
under vacuum. To the syrupy material (9) obtained, dry aceto-
nitrile (5 mL) was added and the total content was added to the
cooled solution of per-O-acetylated glycopyranosyl amine
synthesized following procedure described in 1. The reaction
was continued for 24 h at room temperature. Acetonitrile (50
mL) was added to the reaction mixture and the contents were
ltered through a lter paper. The ltrate was concentrated to
dryness and puried by column chromatography.
Compound 10. Yield 60%, [a]D: +14.3ꢁ (c ¼ 0.7, MeOH, 25 ꢁC),
1H NMR (400 MHz, DMSO-d6): d 8.78 (d, 1H, J ¼ 9.2 Hz, NH),
7.83 (s, 1H, NH), 5.40–5.30 (m, 2H, H-1 & H-3), 4.94–4.87 (m, 2H,
H-2 & H-4), 4.19–3.95 (3m, 4H, H-6a, H-6b, H-5 & CH–CH2–CH2),
2.37–2.05 (2m, 3H, CH–CH2–CH2), 1.99, 1.98, 1.94, 1.92 (4s,
12H, COCH3), 1.85–1.75 (m, 1H, CH–CH2–CH2) ppm; 13C NMR
(100 MHz, DMSO-d6): d 177.4, 173.1, 170.0, 169.5, 169.3, 169.1,
76.9, 72.7, 72.1, 70.5, 67.7, 61.6, 55.7, 28.9, 25.1, 20.4 ppm; ESI-
MS HRMS: observed 481.1430 for [M+ + Na] calculated 481.1434
for C19H26N2O11Na.
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Compound 11. Yield 60%, m.p.: 175–178 ꢁC; [a]D: ꢀ35.8ꢁ (c ¼
0.2, MeOH, 25 ꢁC), 1H NMR (400 MHz, D2O): d 5.27–5.22 (m, 2H,
H-1 & H-3), 5.00 (t, 1H, J ¼ 9.6 Hz, H-4), 4.30–4.22 (m, 2H, –CH–
CH2–CH2– & H-6a), 4.10–4.05 (m, 2H, H-2 & H-6b), 4.01–3.98 (m,
1H, H-5), 2.50–2.45 (m, 1H, CH–CH2–CH2), 2.31 (t, 2H, J ¼ 8.0
Hz, CH–CH2–CH2), 2.02, 2.00, 1.97, 1.85 (4s, 13H, CH–CH2–CH2
& COCH3) ppm; 13C NMR (100 MHz, D2O): d 175.8, 174.8, 173.3,
172.8 (ꢂ2), 78.0, 73.7, 73.0, 68.4, 62.0, 56.9, 52.2, 29.0, 25.3, 21.8,
20.1, 20.0 ppm; ESI-MS HRMS: observed 480.1587 for [M+ + Na]
calculated 480.1594 for C19H27N3O10Na.
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Acknowledgements
The authors are thankful to Indian Institute of Technology 20 J. M. Risley, D. H. Huang, J. J. Kaylor, J. J. Malik, Y. Q. Xia and
Madras for instrumental facilities. LNS is thankful to UGC, New
W. M. York, Arch. Biochem. Biophys., 2001, 391, 165.
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