Isolation, structure elucidation and DFT study on two novel oligosaccharides from yak milk

Article abstract of DOI:10.1016/j.molstruc.2016.03.065

Two novel oligosaccharides were isolated from yak milk. The milk was processed by the method of Kobata and Ginsberg involving deproteination, centrifugation and lyophilization followed by gel filtrate chromatography acetylation and silica gel column chromatography of derivatized oligosaccharides while their homogeneity was confirmed by HPLC. The structures of these isolated oligosaccharides were elucidated by chemical transformation, chemical degradation, ¹H, ¹³C NMR, 2D NMR (COSY, TOCSY and HSQC) and mass spectrometry. The geometry of compound A (Bosiose) and B (Bovisose) have been optimized at B3LYP method and 6-311 + G(d,p) basis set. The difference between the energies of A and B is 1.269 a.u. or 796.309 kcal/mol.

Full text of DOI:10.1016/j.molstruc.2016.03.065

Accepted Manuscript
Isolation, structure elucidation and DFT study on two novel oligosaccharides from yak
milk
Meenakshi Singh, Alok Kumar, Gaurav Srivastava, Desh Deepak, M.P.V.V. Singh
PII:
S0022-2860(16)30261-7
10.1016/j.molstruc.2016.03.065
MOLSTR 22376
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 31 December 2014
Revised Date: 15 March 2016
Accepted Date: 20 March 2016
Please cite this article as: M. Singh, A. Kumar, G. Srivastava, D. Deepak, M.P.V.V. Singh, Isolation,
structure elucidation and DFT study on two novel oligosaccharides from yak milk, Journal of Molecular
Structure (2016), doi: 10.1016/j.molstruc.2016.03.065.
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ACCEPTED MANUSCRIPT
(Bosiose)
(Bovisose)
OH
OH
OH
OH
OH
O
O
O
O
O
S -5
OH
O
O
HO
HO
S-3
S-2
S-1
HO
OH
OH
O
OH
NHAc
OH
OH
O
S -4
HO
OH
(Yakalose)

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Isolation, Structure elucidation and DFT Study on Two novel
oligosaccharides from yak milk
a, b
b
a
a
c
Meenakshi Singh , Alok Kumar , Gaurav Srivastava , Desh Deepak * and M.P.V.V. Singh
a
Department of Chemistry, University of Lucknow, Lucknow 226007, India
b
Department of Chemistry, BBD, University, Faizabad Road, Lucknow, Uttar Pradesh 226028
c
Department of Botany, Shri Jai Narain Post Graduate (KKC) College, Lucknow 226001
Corresponding author, Email: singhmeenakshi144@gmail.com
1

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Abstract
Two novel oligosaccharides were isolated from yak milk. The milk was processed by the method
of Kobata and Ginsberg involving deproteination, centrifugation and lyophilization followed by
gel filtrate chromatography acetylation and silica gel column chromatography of derivatized
oligosaccharides while their homogeneity was confirmed by HPLC. The structures of these
1
isolated oligosaccharides were elucidated by chemical transformation, chemical degradation, H,
1
3
C NMR, 2D NMR (COSY, TOCSY and HSQC) and mass spectrometry. The geometry of
compound A (Bosiose) and B (Bovisose) have been optimized at B3LYP method and 6-
11+G(d,p) basis set. The difference between the energies of A and B is 1.269 a.u. or 796.309
3
kcal/mol.
Keywords-Oligosaccharides, Yak milk, Bosiose (A), Bovisose (B), HPLC
2

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1. Introduction
Milk has co-evolved with mammals and mankind to nourish their offspring
and is a biological fluid of unique complexity and richness [1]. It contains all necessary
nutrients for the growth and development of the newborn [2]. The development of
carbohydrates and glycoconjugates as therapeutics continues to gain interest when
biological relevance of these biopolymers were further revealed and understood [3].
Oligosaccharides have established themselves as an effective class of organic
biomolecules impacting various physiological and pathological processes such as
molecular recognition, signal transaction, differentiation and developmental events and
exhibit varied biological activities such as antitumor [4], immunostimulant [5],
anticancer [6], anticomplementry, anticoagulant, antiinflamatory,
antiviral & immunological activities [7].
hypoglycemic,
Oligosaccharides isolated from various milk sources are categorized in two
classes i.e. sialylated & nonsialylated. Both these class of oligosaccharide have been
tested for their varied biological activities [8]. Human milk oligosaccharides (HMOs) are
important parts of these functional ingredients [9, 10]. Numbers of biologically active
oligosaccharides have been isolated from human, buffalo, donkey, cow, mare, sheep and
goat milk [11]. The milk oligosaccharides recognize cancer associated antigens, used as
antimicrobial agents; tumor associated antigens and has physiological significance in
infants [12]. Buffalo and Donkey milk oligosaccharides have shown promising
immunostimulant activity [1]. Glycosylation of human milk lactoferrin exhibits dynamic
changes during early lactation enhancing its role in pathogenic bacteria-host interactions.
Oligosaccharides are the third most abundant component in human milk [13, 8]. Human
milk oligosaccharides (HMOs) containing α1, 2-linked fucose inhibits the stable toxin-
producing Escherichia coli in vitro and its toxin-induced secretary diarrhea in vitro and
3

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in vivo [14, 15]. Recently it was found that the content of 2-linked fucosyl
oligosaccharides in human milk is significantly associated with lower risk of diarrhea in
breastfed infants, suggesting a major role for these oligosaccharides in immunity [16].
Functions of HMOs are not fully understood but it has been postulated that HMOs plays
an important role in the development of immune system, the prevention of pathogenic
infection and, in the modulation of infant GI tract to bifidogenic microbiota [17-19].
Milk is not only food for neonates; it also contains many nutritional as well as medicinal
values [20, 21].
People living on the Tibetan Plateau rely for survival upon the yak, the
region’s native cattle [22]. The yak milk was used in Tibetan medicinal system for the
treatment of enema therapy, in which yak milk was used as enema solution along with
other drugs [23]. It has been seen in the literature survey that the amino acid, calcium
and vitamin A in yak milk are comparatively higher than in cow’s milk and yak milk
caseins could become a resource to generate antihypertensive peptides and be used as
multifunctional active ingredients for many value-added functional foods as well as a
traditional food protein [24-26]. The China Nutrition society claimed that the amino
acid, calcium and vitamin A in yak milk are comparatively higher than in cow’s milk
[
27].
In view of above facts and observations, therefore isolation, structure
elucidation, chemical degradation, chemical transformation and spectroscopic
characterization of new three oligosaccharides from yak (Bos grunniens) milk have been
presented in the present paper. The completed analysis of structure has been evaluated
1
3
using spectroscopic technique (2D (COSY, TOCSY, and HSQC) and C NMR data.
Other techniques like deacetylation, methylation, hydrolysis, chemical degradation and
ESI MS (mass spectrometry) have also been used for structure elucidation of
4

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oligosaccharides. Furthermore, quantum chemical calculation has been performed to
determine the lower geometry structure and stability of the isolated and characterized
compounds.
2
Theoretical studies
All computations were performed using the Gaussian 09 program package [28].
The quantum chemical calculation have been performed on B3LYP functional and 6-
11+G(d,p) basis set. Geometries of compound A and B have been first optimized and
3
the presence of positive wavenumbers values for all the optimized geometry indicates
stability of the compounds [29].
2
.1 Experimental
General procedures
Optical rotations were measured using an AA-5 automatic polarimeter in a 1
dm tube for water solutions whose concentrations are expressed in g/100mL.
1
13
The H and C NMR spectra were recorded in CDCl and D O on Bruker
3
2
DRX-300 spectrometer at 300 and 400 MHz using TMS as an internal reference.
The DART Mass spectra were recorded on a MICROMASS QUATTRO II
triple quadruple mass spectrometer. The C, H and N analyses were recorded on
CARDO-ELBA 1108 elemental analyzer.
The detection of new spots for carbohydrates was monitored by thin layer
chromatography (TLC) using Silica Gel 60 F254 plates. TLC plates were visualized by
exposure to them in with 50% aq. H SO reagent.
2
4
A new spot for carbohydrates was also monitored by paper chromatography
(PC) with acetyl acetone and p-dimethyl amino benzaldehyde reagents. PC was
5

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performed on Whatman No.1 filter paper using ethylacetate-pyridine (2:1) saturated
with H O as solvent system. Authentic samples of TLC and PC were same and
2
purchased from Aldrich Chemicals (glucosamine (GlcN), galactosamine (GalN), glucose
(
Glc), galactose (Gal), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine
GalNAc)).
(
2
.2. Isolation of yak milk oligosaccharides by Kobata and Ginsberg method
0 liter milk was collected from a yak and was stored at -20°C until use. The
milk was processed by the method of Kobata and Ginsberg [29]. It was centrifuged for
5 min. at 6500 rpm at -4°C. The solidified lipid layer was removed by filtration through
1
1
glass wool column in cold atmospheric condition (0°C). Ethanol was added to the clear
filtrate (supernatant) to a final concentration of 68% for precipitating out the lactose and
proteins and the resulting solution was left overnight at 0°C. The white precipitate of
lactose and protein was formed and removed by centrifugation for 15 min. at 6500 rpm
at -2°C and washed twice with 68% ethanol. Further for complete remove of remaining
lactose the supernatant was passed through a microfilter (0.24 µm) and lyophilized to
get the crude oligosaccharide mixture (12.0 grams).
The lyophilized material responded positively to Morgan-Elson test [30] and
thiobarbituric-acid assay suggesting the presence of N-acetyl sugars and sialic acid in
oligosaccharide mixture. The detailed of the test has been given in supplementary file 1.
2
.3. Yak milk oligosaccharide mixture chromatographed over Sephadex G-25 (1.6x40
cm) column
The gel filtration was performed by Sephadex G-25 chromatography of crude
yak milk oligosaccharide mixture. The oligosaccharide mixture was packed in a column
1.6 x 40 cm) (void volume = 25 mL) equilibrated with glass double distilled water and
(
6

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left for 10-12 h to settle down. The 18.0 gm of the crude oligosaccharide mixture was
obtained from 10 liter milk. From which 200 mg material in 150 mL distilled water was
applied onto a Sephadex G-25 column and was eluted for separation of protein and
glycoprotein from oligosaccharide mixture (low molecular weight component). The flow
rate was adjusted to 3 mL / min. Then the eluent was reapplied on the same Sephadex
G25 column. The process was repeated twice for further purification. 1 ml fractions
were collected in all eluted fractions and tested for the presence of neutral sugars by
phenol-sulphuric acid test [31]. Same above procedure (approximately 88 x 2 times) was
applied for the remaining 16.0 gm of the crude oligosaccharide mixture to obtain 12.0
gm of pool I and pool II. The sephadex G-25 chromatography of yak milk
oligosaccharide mixture which was monitored by UV (absorbance 280 nm)
spectrophotometry showed four peaks i.e. I, II, III and IV. A substantial amount of
proteins, glycoproteins and serum albumin were eluted in the void volume which was
confirmed by positive coloration with p-dimethyl amino benzaldehyde reagent and
phenol-sulphuric acid reagent. Fractions under peaks II and III gave a positive phenol-
sulphuric acid test [31] which showed the presence of oligosaccharide mixture in yak
milk. They were pooled together and lyophilized.
2.4. Confirmation of homogeneity of yak milk oligosaccharide by reverse phase HPLC
Fraction II and III obtained from Sephadex G-25 column, containing
oligosaccharide mixture were qualitatively analyzed by reverse phase HPLC. The HPLC
system was equipped with Perkin-Elmer 250 solvent delivering system, 235 diode array
detector and G.P. 100 printer plotter. The column used for this purpose was C18
Purosphere 25 cm x 0.4 cm x 5 µm. A binary gradient system of acetonitrile: 0.5%
trifluro acetic acid (5:95) in distilled water to CH CN: 0.5% TFA (60:40) within 25 mm
3
at a flow rate of 1 mL / min was used. The eluants were detected at 220 nm. Eleven
7

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peaks were noticed in the sample (pooled fractions II and III) at the varied retention
times from 2.272 min. to 5.717 min. The peaks were numbered in increasing order of
retention time i.e. 2.272 (R1), 3.115 (R2), 3.637 (R3), 3.968 (R ), 4.331 (R ), 4.693 (R6),
4
5
5
.077 (R7), 5.227 (R8), 5.419 (R9), 5.557 (R ), 5.717 (R ).
10
11
2.5. Acetylation of oligosaccharide mixture
The pooled fraction (II and III) (12.0 g) which gave positive phenol-sulphuric acid
test [31] was acetylated with pyridine (15 mL) and acetic anhydride (15 mL) at 60°C
and the solution was stirred overnight. The mixture was evaporated under reduced
pressure and the viscous residue was taken in CHCl (250 mL) and washed in sequence
3
with 2N HCI (1x25 mL), ice cold 2N NaHCO (2x25 mL) and finally with H O (2x25
3
2
mL). The organic layer was dried over anhydrous Na SO , filtered, and evaporated to
2
4
dryness yielding the acetylated mixture. By acetylation the oligosaccharides were
converted into their non-polar acetyl derivatives which resolved very nicely on TLC.
2.6. Purification of acetylated milk oligosaccharide on silica gel column
Separation of the acetylated products (12.0 g) was carried over silica gel using
varying proportions of hexane: CHCl , CHCl and CHCl : MeOH as eluants. The
3
3
3
acetylated products was packed in a column (1.6 x 40 cm) (void volume = 25 mL)
equilibrated with CHCl and left for 10-12 h to settle down. Repeated column
3
chromatography (7 column) led to the isolation of three chromatographically pure
compounds A (858 mg), B (46 mg) and C (125 mg). The flow rate adjusted to 3 mL /
min and the detailed purification processes description of polarity ration of eluent and
volume of each fractions of compound A, and B is given in Supplementary material
Table S1-S7.
8

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2.7. Deacetylation of compounds
Compound A (52 mg) obtained from repeated column chromatography of acetylated
oligosaccharide mixture was dissolved in acetone (3 mL) and NH (3.5 mL) was added
3
and left overnight in a stoppered hydrolysis flask. Ammonia was removed under reduced
pressure and the product was washed with CHCl (3x3 mL) and was finally freeze dried
3
giving the deacetylated oligosaccharide 'A' (46 mg) [α] +62.4°(c, 0.2, H O). Compound
D
2
B (32 mg) was deacetylated by the same method as for compound A, resulting in
deacetylated compound B (24 mg) [α] -41.3° (c, 0.2, H O).
D
2
2.8. Description of compounds
The two compounds Compound 'A' (Bosiose) and Compound B (Bovisose) have been
isolated and characterized with the help of spectroscopic techniques.
2.8.1. Compound 'A' (Bosiose)
Compound 'A' C H O N , [α] +62.4° (c, 2.0, H O). For elemental
2
2
38 16
2
D
2
analysis, this compound was dried over P O at 100°C and 0.1 mm pressure for 8 h.
2
5
Elemental analysis: Calcd. C, 45.05; H, 6.48; N, 4.77; Found C, 45.01; H, 6.47 ; N, 4.76;
It gave purple-violet spot on PC for amino sugars when sprayed with acetyl-acetone
reagent I and heated, the dry strips were then sprayed with p-dimethyl amino
benzaldehyde reagent II and returned to the oven for a further short hot treatment.
Methyl glycosidation/Acid hydrolysis: Compound A (8 mg) was ref1uxed with
absolute MeOH (2 ml) at 70°C for 18 h in the presence of cation exchange IR-l20 (H)
resin. The reaction mixture was filtered while hot and filtrate was concentrated. In the
solution of methylglycoside of B, 1,4-dioxane (1 ml), and 0.1N H S0 (1 ml) was added
2
4
and the solution was warmed for 30 minutes at 50°C. The hydrolysis was complete after
2
4 h. The hydrolysate was neutralized with freshly prepared BaCO filtered and
3
9

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concentrated under reduced pressure to afford α-and β-methylglucosides along with the
Glc, GalNAc and GlcNAc. Their identification was confirmed by comparison with
authentic samples (TLC, PC).
Kiliani Hydrolysis: For Kiliani hydrolysis [32], the compound A (5 mg) was dissolved
in 2 ml Kiliani mixture (AcOH-H O-HCI, 7:11:2) and heated at 100°C for 1 h followed
2
by evaporation under reduced pressure. Then 2 ml of H O was added and extracted
2
twice with 3 ml CHCl . The aqueous residual solution was made neutral by addition of
3
1-2 drops of 2N NaOH and was evaporated under reduced pressure to afford Glc,
GalNAc and GlcNAc on comparison with authentic samples of Glc, GalNAc and
GlcNAc, respectively. This hydrolysis confirming the presence of Glc, GalNAc and
GlcNAc sugar units in it. The presence of these sugar units have been further confirmed
by NMR and Mass spectrometry given in supplementary file 1.
Mannich-Siewart hydrolysis: For Mannich-Siewart hydrolysis [33] 6 mg of the
compound A was dissolved in 2.5 ml acetone and conc. HCl (0.02 ml). The solution was
kept under carbon dioxide in dark room at room temperature.
After five days paper
chromatogram showed three spots, mobility of one spot was identical in mobility with
authentic sample of GlcNAc, and the other spot with lowest mobility was identical with
unreacted compound A. Further the compound with intermediate mobility may be the
disaccharide. After eleven days two new spots with faster mobility than disaccharide
were observed on TLC and PC which were found identical to authentic sample of
GalNAc and Glc. The hydrolysis was completed in eleven days showing three spots on
TLC which were found identical with Glc, GalNAc and GlcNAc (PC, TLC) on
comparison with authentic samples.
1
H NMR values of compound A in D O: 5.27[d, 1H, J=3.6 Hz, α-Glc(S-1) H-1],
2
4.57[d, 2H, J=8.0Hz, β-Glc(S-1) & β-GlcNAc(S-3)H-1], 4.50[d, 1H, J=8.0Hz,
1
0

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βGalNAc(S2), 3.82[βGalNAc(S-2), H-4], 3.32[t, 1H, J=7.2 Hz, β-Glc(S-1), H-2], 2.05[s,
3
H, NHCOCH , β-GlcNAc(S-3), 2.04[s, 3H, NHCOCH , β-GalNAc(S-2)].
3
3
1
3
C NMR values of compound A in D O:
2
1
77.43[s,NHCOCH ], 173.71[s,NHCOCH ], 102.84[β-GlcNAc(S-3),C-1], 101.09[β-
3 3
GalNAc(S-2),C-1], 95.74[β-Glc(S-1),C-1], 88.79[α-Glc(S-1),C-1], 21.81[s,NHCOCH3],
21.25[s,NHCOCH3].
1
H NMR values of acetylated A in CDCl : 6.25 [d, IH, J=3.3 Hz, β-Glc (S-1), H-l],
3
5.67 [d,1H, J=8.1 Hz, β-Glc (S-1), H-1], 4.49 [d, 1H, J=8.1 Hz, β-GlcNAc (S-3), H-1],
4.45 [d, 1H, J=9.9 Hz, β-GalNAc (S-1), H-1], 3.87 [β-Glc (S-1), H-4], 3.84 [α-Glc (S-1),
H-4], 3.78 [β-GalNAc (S-2) H-6].
1
3
C NMR values of acetylated A in CDCI : 101.15 [β-GlcNAc (S-3), C-l], 100.87 [β -
3
GalNAc (S-2), C-1], 91.51 [β-Glc (S-l), C-1], 88.94 [α-Glc (S-1), C-1], 75.63 [α-Glc
(S-1), C-4], 73.47 [β-GalNAc (S-2), C-6].
+
ES Mass of compound A found 586[M]
2.8.2. Compound B (Bovisose)
Compound B, C H O N , [α] +41.3° (c, 2.0, H O). Elemental analysis, Calcd. C,
4
0
68 31
2
D
2
44.57; H, 6.34; N 2.61; Found C, 44.75; H, 6.33, N, 2.60; It gave purple-violet spot on
PC for amino sugars when sprayed with acetyl-acetone reagent I and heated, the dry
strips were then sprayed with p-dimethyl amino benzaldehyde reagent II and returned to
the oven for a further short hot treatment.
Methyl glycosidation/Acid hydrolysis: 8 mg of the compound was ref1uxed with
absolute MeOH (2 ml) at 70°C for 18 h. The similar procedure was opted for acid
hydrolysis like compound A. After neutralized, filtrate, concentrated under reduced
pressure of hydrolysate, afford α-and β-methylglucosides along with the Glc, Gal and
GlcNAc. Their identification was confirmed by comparison with authentic samples
1
1

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(TLC, PC).
Kiliani Hydrolysis
:
Compound B (5 mg) was used for Kiliani hydrolysis and the
hydrolysate was checked by paper chromatography with authentic samples which
inferred the presence of Glc, Gal and GlcNAc in compound B and further sugar units
have been confirmed by NMR and Mass spectrometry given in supplementary file 1.
Mannich-Siewart hydrolysis: For this hydrolysis compound B (5 mg) was dissolved in
2
ml acetone and conc. HCl (0.02 ml) was added. The solution was kept under carbon
dioxide in dark room at room temperature. After two days paper chromatogram showed
three spots, the compound with faster mobility was identical in mobility with authentic
sample of Gal while the spot with lowest mobility was identical with unreacted
compound C, further the compound with the intermediate mobility may be the
pentasaccharide. Further after five days two new spots were observed of which one was
identical in mobility with authentic sample of GlcNAc and other, which was faster in
mobility than the pentasaccharide, may be the tetrasaccharide. After seven days one
more spot was observed which was having faster mobility than the tetrasaccharide, so it
may be trisaccharide, which was formed by loss of another Gal moiety. Further after ten
days a new spot was observed which was faster in mobility than the trisaccharide and
having same mobility as the authentic sample of lactose and it was formed by the loss of
GlcNAc moiety. The hydrolysis was partially completed in twelve days and showed a
new spot, which was found identical with authentic sample of Glc on TLC and PC. Thus
the hydrolysis was completed in twelve days showing three spots on TLC which were
found identical with Glc, Gal, and GlcNAc (PC, TLC) on comparison with authentic
samples.
1
H NMR values of compound B in D O: 5.25 [d, 1H, J=3.6Hz, α-Glc (S-1) H-1], 4.69
2
[
d, 1H, J=8.0Hz, β-GlcNAc (S-3) H-1], 4.61 [d, 1H, J=7.6Hz, β-GlcNAc (S-5) H-l],
1
2

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4.59 [d, 1H, J=7.6Hz, β-Glc (S-1) H-1], 4.55 [d, 1H, J=8.0Hz, β−Gal (S-6) H-1], 4.48
[
d, 2H, J=8.0Hz, β-Gal (S-2)& β-Gal (S-4) H-1], 4.16 [β-Gal (S-2), H-4], 3.32 [t, 1H,
J=8. 1 Hz, β-Glc(S-l), H-2], 2.03 [s, 3H, NHCOCH3 β-GlcNAc (S-5)], 2.02 [s, 3H,
NHCOCH3 β-GlcNAc (S-3)]
C NMR values of compound B in D O: 177.38 [s, NHCOCH ], 173.66 [s,
,
,
1
3
2
3
NHCOCH ], 103.8 [β-Gal (S-6), C-1], 102.9 [β-Gal (S-4), C-1], 102.8 [β-Gal (S-2), C-
3
1], 101.1 [β-GlcNAc (S-5), C-1], 100.1 [β-GlcNAc (S-3), C-1], 95.8 [β−Glc (S-1), C-1],
9
1.7 [α-Glc (S-1), C-1], 21.9 [s, NHCOCH ], 21.3 [s, NHCOCH ]
3
3
1
H NMR values of acetylated B in CDCl : 6.27 [d, 1H, J=3.6Hz, α-Glc (S-1), H-1],
3
5
.68 [d, 1H, J=8.1Hz, β-Glc (S-1), H-1], 4.64 [d, 1H, J=7.8Hz, β-GlcNAc (S-3) H-l],
.62 [d, 1H, J=7.8Hz, β-GlcNAc (S-5), H-1], 4.40 [d, 1H. J=7.8 Hz, β-Gal (S-4), H-l],
.38 [d, 1H, J=7.5 Hz, β-Gal (S-2), H-1], 4.36 [d, 1H, J=9.0 Hz, β-Gal (S6), H-1], 3.99
4
4
[
β-Gal (S-2), H-3], 3.88 [β-Gal (S-4), H-6], 3.81 [β-Glc (S-1), H-4], 3.81[ β−GlcNAc
S-3), H-3], 3.75 [β-GlcNAc (S-5), H-3].
(
1
3
C NMR values of acetylated B in CDCl :103.6 [β-Gal (S-4), C-1], 102.5 [β-Gal (S-
3
2) & β-Gal (5-6) C-1], 101.1 [β-GlcNAc (S-5), C-1], 100.8 [β-GlcNAc (S-3), C-1], 91.7
[β-Glc (S-1), C-1], 89.0 [α-Glc (S-1), C-1].
ES Mass of compound B: 1073 [M+H]
3
Result and discussion
3.1 Stability of Molecular geometries of the isolated compounds
The geometry of compound A (Bosiose) and B (Bovisose) have been optimized at
B3LYP method and 6-311+G(d,p) basis set and given in Figure 1. As we know that
1
3

ACCEPTED MANUSCRIPT
molecular geometry and conformational analysis play a very important role in
determining the structure-activity relationship [34-39]. The optimized geometries of two
compounds show positive wavenumbers [34-39] values indicate the stability of the
compound A and B. The optimized geometry of ground state structure of A and B are
shown in Figure 1. The compound B is lower in energy as compared to compound A
and C. The conformer of compound A and B has energies -1878.730 and -1879.999 a.u.
at room temperature. The difference between the energies of A and B is 1.269 a.u. or
7
96.309 kcal/mol. The Molecular structures of all three conformers possess C point
1
group symmetry.
3.2 Structure elucidations of the isolated yak milk oligosaccharide
TLC of acetylated oligosaccharide mixture at different polarity proportions is
given in Supplementary Figure S2. The Sephadex G-25 chromatography of yak milk
oligosaccharide mixture showed four peaks i.e. I, II, III and IV as shown in Figure 2 and
collected fractions is shown in supplementary Table S1. A positive phenol-sulphuric
acid test for sugars has been given by fractions under peaks II and III which indicate the
presence of oligosaccharide in yak milk. These fractions were pooled and lyophilized.
Pooled fractions (peaks II and III) obtained after Sephadex G-25 column were further
qualitatively examined by reverse phase HPLC as shown in supplementary Figure S2.
The reverse phase HPLC shows eleven peaks at varied retention times from 2.272 min.
to 5.717 min as shown in Table 1.
3.2.1 NMR spectroscopy
The isolated compounds have been identified with the help of 1H, 13C NMR,
D NMR (COSY, TOCSY and HSQC) and mass spectrometry. The experimental
2
1
4

ACCEPTED MANUSCRIPT
spectra of the isolated compounds have been given in Figure S3-S17 of supplementary
file 1. Most of milk oligosaccharides are made up of basic core units which are
comprised of D-Glucose, D-Galactose, D-GlcNAc, D-GalNAc, Fucose and Sialic acid.
The structure of milk oligosaccharides were elucidation by chemical degradation and
spectroscopic techniques (NMR and ES-MS). Keeping above mentioned basic core units
in mind, the structures of various milk oligosaccharides were established by comparing
the chemical shift data (1H and 13C NMR) of anomeric signals and other important
signals of unknown milk oligosaccharides with the chemical shifts of known milk
oligosaccharides. In the present study analogies between chemical shift of certain
‘structural reporter group resonances’ (SRG) [40-52] were used to make proton
resonance assignments as well as structural assignments of the oligosaccharides. All
chemical shifts of anomeric proton signals of milk oligosaccharides were further
confirmed by 2D (COSY, TOCSY and HSQC) NMR experiments, which were earlier
assigned with the help of 1H and 13C NMR data. Other techniques like deacetylation,
methylation, hydrolysis, chemical degradation and mass spectrometry were also used in
the structural elucidation of the oligosaccharides.
Compound A (Bosiose) C H O N [α] +62.4° (c, 0.2, H O) and B
22
38 16
2
D
2
(
Bovisose) C H O N [α] +41.3° (c, 0.2, H O) gave positive phenol-sulphuric acid
40 68 31 2 D 2
test [31], Fiegl test [53] and Morgon-Elson [30] test showing the presence of normal and
amino sugar moiety in the compound A. The HSQC spectrum of acetylated compound A
and B given in supplementary file 1 show the presence of four and seven cross peaks of
respective protons and carbons in the anomeric region at δ(6.25x88.94, 5.67x91.51,
4.49x100.89, 4.45x101.15) and δ(6.27x89.0, 5.68x91.7, 4.64x100.8, 4.62x101.1,
4.40x103.6, 4.38x103.5, 4.36x103.5), respectively, suggested the presence of four and
seven anomeric protons. The presence of four and seven anomeric protons were
1
5

ACCEPTED MANUSCRIPT
confirmed by the presence of doublets at δ{6.25 (1H), 5.67(1H), 4.49(1H), 4.45(1H)},{
δ 6.27 (J=3.6 Hz), 5.68 (J=8.1 Hz), 4.64 (J=7.8 Hz), 4.62 (J=7.8 Hz), 4.40 (J=7.5 Hz),
1
4
.38 (J=7.5 Hz), 4.36 (J=9.0 Hz)} in the H NMR spectrum of acetylated A and B in
CDCl respectively. Again presence of four and seven anomeric carbons were confirmed
3
by the presence of four and seven anomeric signals at {δ101.15, 100.89, 91.51 and
1
3
8
8.94} and {δ 104.35(2C), 104.16(1C), 99.95(1C), 91.51(1C), 89.19(1C)} in the C
NMR spectrum of acetylated A and B respectively. These four anomeric protons could
be interpreted for the presence of a trisaccharide in its reducing form. The reducing
nature of compound A and B were confirmed by the methyl glycosylation of compound
+
A and B by MeOH/H followed by its acid hydrolysis which led to the isolation of α and
β methyl glucosides (3) which suggested the presence of glucose at the reducing end as
seen in Scheme 1 and 2. The three and six monosaccharide unit have been present in
compound A and B and designated as (S , S and S ) and (S , S , S , S , S S )
1
2
3
1
2
3
4
5, 6,
respectively. The monosaccharide constituents in compound A and B were confirmed by
Kiliani hydrolysis [32] under strong acidic conditions, followed by PC and TLC. In this
hydrolysis three spots were found on PC and TLC which were identical with (Glc,
GalNAc and GlcNAc) in compound A and (Glc, Gal and GlcNAc) in compound B by
co-chromatography with authentic samples.
Thus the compound A (trisaccharide) and compound B (hexasaccharide) contained three
types of sugar moieties i.e. (Glc, GalNAc, GlcNAc) and (Glc, Gal and GlcNAc) in it,
respectively, and given in Scheme 1 and 2.
The monosaccharide constituents in compound A and B have been further
confirmed by the hydrolyzing the compounds in under mild acidic conditions (Mannich-
Siewart hydrolysis [33]) followed by PC and TLC. As shown in Scheme A and B after
five days paper chromatogram showed three spots, mobility of one spot was identical in
1
6

ACCEPTED MANUSCRIPT
mobility with authentic sample of GlcNAc, and the other spot with lowest mobility was
identical with unreacted compound 'A' (I). Further the compound with intermediate
mobility may be the disaccharide (II). After eleven days two new spots with faster
mobility than disaccharide were observed on TLC and PC which were found identical to
authentic sample of GalNAc and Glc.
The Mannich-Siewart hydrolysis [33] of compound B supported the
1
monosaccharide sequence deduced by H NMR. In this hydrolysis after two days paper
chromatogram showed three spots, the compound with faster mobility was identical in
mobility with authentic sample of Gal while the spot with lowest mobility was identical
with unreacted compound B(I), further the compound with the intermediate mobility
may be the pentasaccharide (II). Further after five days two new spots were observed
which was identical in mobility with authentic sample of GlcNAc and other, and faster
in mobility than the pentasaccharide, may be the tetrasaccharide (III). After seven days
one more spot was observed which was having faster mobility than the tetrasaccharide,
so it may be trisaccharide (IV), which was formed by loss of another Gal moiety.
Further after ten days a new spot was observed which was faster in mobility than the
trisaccharide and having same mobility as the authentic sample of lactose (V) and it was
formed by the loss of GlcNAc moiety. The hydrolysis was partially completed in twelve
days and showed a new spot, which was found identical with authentic sample of Glc on
TLC and PC.
The reducing nature of glucose was confirmed by the presence of two
doublets at δ5.27 (J=3.6Hz) , 4.57 (J=8.0Hz) and 5.25(J=3.6Hz), 4.59 (J=7.6Hz) for α
1
and β glucose [54, 55] respectively, in the H NMR spectrum of compound A and B in
D O solvent, respectively, showing the presence of glucose at the reducing end. Another
2
anomeric proton signal which appeared as doublet at δ4.50 along with a singlet of amide
1
7

ACCEPTED MANUSCRIPT
1
methyl at δ 2.04 in the H NMR spectrum of compound 'A' was due to the presence of
1
GalNAc moiety [56]. Since βGlc H-2 signal appeared as a triplet at δ3.32 in the H
NMR spectrum of compound A, it indicated that the equatorially oriented hydroxyl
group at C-4 of the reducing Glc was involved in glycosidation and the linkage was
1
ꢀ4 between S and S (SRG) [40-52] showing the presence of a lactose type of
2 1
structure with addition al signal of NHAc group of GalNHAc at 2.04. This linkage was
further supported by the presence of β-Glc H-4 resonance which appeared at δ3.87 in the
1
H NMR spectrum of acetylated A.
3.4 Mass Spectrometry
Additional supports for the isolated structures have been obtained by mass
spectrometry. The fragmentation pattern of compound A and B is shown in Figure 3
and 4. The appearance of molecular ion peak in compound A at m/z = 586 which was
+
°
due to M C H O N [α] +62.4 . Further the mass fragments were formed by
2
2
38 16
2
D
repeated H transfer in the oligosaccharide and was accompanied by the elimination of
terminal sugar less water which gave a fragment at m/z = 383 for disaccharide
fragment. On further fragmentation the disaccharide fragment at m/z = 383 gave a
monosaccharide fragment at m/z = 180 which attributed to the loss of GalNAc (S2).
This confirmed that trisaccharide was comprised of one Glc, one GalNAc and one
GlcNAc moieties and the second sugar in sequence from the non-reducing end of
trisaccharide was GalNAc and glucose was present at the reducing end. Thus mass
spectrometry not only confirmed the assigned structure but also confirmed the sequence
of monosaccharide in oligosaccharide. Thus on the basis of all the above data the
structure of isolated trisaccharide, 'A' was deduced as- GlcNAc-
(1ꢀ6)GalNAc(1ꢀ4)Glc and shown in Figure 5.
In compound B observed molecular ion peak at m/z = 1073 due to [M+H],
1
8

ACCEPTED MANUSCRIPT
confirmed the molecular weight of compound as 1072. The hexasaccharide m/z = 1072
on fragmentation gave pentasaccharide at m/z = 910(I), which was due to loss of S-6
sugar unit i.e. Gal (S-6) sugar unit linked to the S-5 of hexasaccharide. The peak m/z =
910 further fragmented to give a fragment peak at m/z = 707(II), corresponding to
tetrasaccharide unit, which was due to loss of S-5 sugar unit i.e. Gl cNAc(S-5) sugar
unit linked to the S-4 of pentasaccharide. The tetrasaccharide on fragmentation gave a
fragment ion peak at m/z = 545(III), which was due to loss S-4 sugar unit i.e. Gal(S-4)
sugar unit linked to the S-3 of tetrasaccharide unit. The trisaccharide on fragmentation
gave fragment peak at m/z = 342(IV), which was due to loss of S-3 sugar unit i.e.
GlcNAc(S-3) sugar unit linked to the S-2 of trisaccharide unit. This disaccharide on
further fragmentation gave a fragment peak at m/z = 180(V), which was due to loss of
S-2 sugar unit i.e. Gal(S-2) sugar unit linked to the S-l unit of disaccharide. Thus based
on the above data the structure of compound B was deduced and shown in Figure 6.
4.
Conclusion
The geometries of compound A (Bosiose) GlcNAc-(1ꢀ6)GalNAc(1ꢀ4)Glc
and B (Bovisose) Gal-β(l 3)GlcNAc-β(1 6)Gal-β(1 3)GlcNAc-β(1 3)Gal-β
1ꢀ4)Glc have been optimized at B3LYP method and 6-311+G(d,p) basis set. The
ꢀ
ꢀ
ꢀ
ꢀ
(
optimized geometries of two compounds show positive wavenumbers values indicated
the stability of the compound A and B. The difference between the energies of A and B
is 1.269 a.u. or 796.309 kcal/mol. The two novel oligosaccharides have been isolated
by yak milk. The isolated compound A (Bosiose) and B (Bovisose) have been
1
13
characterized with the help of H, C, 2D NMR spectroscopy, further the molecular ion
peaks in compound A and B have been confirmed by the analysis of mass spectrometry.
The isolated compound may possess the variety of biological activity. The testing of
1
9

ACCEPTED MANUSCRIPT
biological activity of these isolated compounds has been continued in one laboratory.
Acknowledgments
Authors acknowledge CDRI for spectral recording and Department of Chemistry for
computational help.
2
0

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References
1
) B. Casado, M. Affolter, M. Kussmann, J. Proteomics 73 (2009) 196–208
) S. Divya, B. S. Subramonian, Intern. J. Engin. Trends Tech. 2(2013) 86-89.
2
3
3
) J. C. Gildersleeve, S. M. Muthana, C. T. Campbell, ACS Chem. Biol. 7(2012)
1–43
4
) M. Schwonzen, R. Schmits, S. E. Baldus, M. Vierbuchen, F. G. Hanish, Br J
Cancer. 65(1992) 559–565.
5
1
) K. Abe, J. M. J. Mckibbin, S. Hakomori, Eur J Biochem Bio Chem 258(1983)
1793–11797.
6) J N Fang, H. Proksch, H. Wagner, Phytochemistry 24(1985) 2619-22.
7) R Srivastava, D. K. Kulshretha, Phytochem 28(1989) 2877-2883.
8) A. Ferdouse, Md. Monirujjaman, Nova J. Med. Bio. Sci. 2(2014) 1-7
9
1
) R. Peterson, Wai Y. Cheah, J. Grinyer, N. Packer, Glycobiology 23(2013) 1425–
438
1
1
0) L. R. Ruhaak, J. Huang, B. Lönnerdal, J. B. German, Mol Cell Proteomics.
1(2012) 015248.
1
1) W.L. Claeys, C. Verraes, S. Cardoen, J. De Block, A. Huyghebaert, K. Raes, K.
Dewettinck, L. Herman, Food Control 42 (2014) 188-201
1
2) Kei E Fujimura, Nicole A Slusher, Michael D Cabana, and Susan V Lynch,
Expert Rev Anti Infect Ther. 8(2010) 435–454
1
3) R. Mehra, D. Barile, M. Marotta, C. B. Lebrilla, C. Chu, J. B. German, PLoS
One. 9(2014) 96040-96047.
1
4) M. Guillermo, R. Palacios, L. E. Cervantes, P. Ramos, B. Chavez-Munguia, D.
S. Newburg, J. Biol. Chem. 278(2003) 14112-14120.
1
5) L.E. Cervantes, D.S. Newburg, G.M. Ruiz-Palacios, Pediatr. Res. 37(1995) 171-
78.
1
2
1

ACCEPTED MANUSCRIPT
1
6) Maciej Chichlowski, J. Bruce German, Carlito B. Lebrilla, David A. Mills,
Annu. Rev. Food Sci. Technol. 2(2011) 331-351
1
1
7) C. Kunz, S. Rudloff, Adv. Exp. Med. Biol. 606(2008) 455-65.
8) A. M. Zivkovic, J. B. German, C. B. Lebrilla, D. A. Mills, Proc Natl Acad Sci U
S A. 108(2011) 4653-4658.
1
9) F. Lara-Villoslada, E. Debras, A. Nieto, A. Concha, J. Galvez, E. Lopez-
Huertas, J. Boza, C. Obled, Xaus, Clin Nutr. 25(2006) 477-88.
2
0) R. Saksena, D. Deepak, A. Khare, R. Sahai, L. M. Tripathi, Biophy Acta,
428(1999) 433-445
1
2
1) T. Urashima, S. Asakuma, F. Leo, K. Fukuda, M. Messer, Oftedal, T. Olav
Amer. Soc. Nut. Adv. Nutr. 3(2012) 473S–482S
2
9
2) D.d Rhode, D. B. Madsen, P. J. Brantingham, T. Dargye, Develop. Quat. Sci.
(2007) 1571-0866
2
3) Y. W. Park, Bioactive Components in Milk and Dairy Products, John Wiley &
Sons, Ltd., Georgia, Athens (2009).
2
4) J. Jiang, S. Chen, F. Ren, Z. Luo, S. S. Zeng, J. Dairy Res. 74(2007) 18−25
2
5) S. Kumar, V. S. Chouhan, A. Sanghi, U. V.S. Teotia, Veterinary World, 6(2013)
2
231-0916
2
6) S. Kumar, U. V. S. Teotia, A. P. S. Aswal, Int. J. Livest. Res. 3(2013) 130-134
7) E. Muehlhof, A. Bennett, D. McMahon, Milk and dairy products in human
2
nutrition, (2013), Nutrition Division, Food and Agriculture Organization of the
United Nations (FAO), Viale delle Terme di Caracalla, 00153 Rome, Italy
2
2

ACCEPTED MANUSCRIPT
2
2
1
3
8) M.J. Frisch et al,. Gaussian 09, (2009) Gaussian, Inc., Pittsburgh.
9) E. Taufik, K. Fukuda, A. Senda, T. Saito, C. Williams, C. Tilden, Glycocon. J.
(2012) 012-037
0) J. A. Cifonelli, D. Albert, Biol. Chem. 231(1958) 11-18.
3
1) M. Dubois, K.A. Gilles, J. K. Hamilton, P. A. Rebers, F. Smith, Analytical
Chemistry. 28(1956) 350-356.
3
2) H. Kiliani, U. D. Verum. Ber. Deutsch Chem.Ges. 63(1930) 2866-2869.
3
3
1
3
1
3
3
3
4
3
3) C. Mannich, G. Siewert, Ber. Deutsch. Chem. Ges. 75(1942) 737-750.
4) R.N. Singh, P. Rawat, S. Sahu Spectrochimica Acta Part A 135 (2015) 1162–
168
5) R.N. Singh, A. Kumar, R.K. Tiwari, P. Rawat Spectrochimica Acta Part A
13(2013) 378–385
6) P. Rawat, R.N. Singh, Spectrochimica Acta Part A 140(2015) 344–355
7) P. Rawat, R. N. Singh, J. Mol. Struc. 1082(2015) 118–130
8) R.N. Singh, A. Kumar, P. Rawat, A. Srivastsva, J. Mol. Struc. 1049(2013) 419–
28
9) P. Rawat, R.N. Singh, J. Mol. Struc. 1081(2015) 293–303
3
4) V. K. Dua, C. A. Bush, Analy. Biochem.133(1983) 1-8.
35) I.M.V. Nieuwenhof, W.E. Schiphorst, D.H.V. Eijden, FEBS Letters. 459(1999)
3
77-380.
3
2
6) G. Strecker, S. Fievre, J. Micheal, J. Montreuil, Carbohydrate Research.
26(1992) 1-14.
2
3

ACCEPTED MANUSCRIPT
3
7) Y. Uemura, S. Asakuma, L. Yon, T. Saito, K. Fuuda, I. Arai, T. Urashima,
Comp. Biochem. Phy. Part A Mol. Int. Phy. 145(2006) 468-478.
4
4) T. Urashima, T. Nakamura, K. Teramoto, I. Arai, T. Saito, T. Komatsu, Comp.
Biochem. Phy. Part A Mol. Int. Phy. 139(2004) 587-595.
4
4
4
5) V.K. Dua, K. Goso, V.E. Dube, C.A. Bush, J. Chromat. 259(1985) 259-269.
6) P. Chaturvedi, C.B. Sharma, Biophysica Acta. 967(1988) 115-121
7) M.T. Tarrago, K.H. Tucker, H.V. Halbeak, D.V. Smith, Archieves Biochem.
Biophys. 207(1988) 352-362.
4
1
8) D. Viverge, L. Grimmonprez, M. Solere, Biochimica et Biophysica Acta
336(1997) 157-164.
4
1
3) T. Urashima, T. Saito, K. Ohmisya, K. Shimazak, Biochim. Biophys. Acta.
073(1991) 295-300.
5
0) T. Urashima, M. Kobayashi, S. Asakuma, Y. Uemura, I. Arai, Comp. Biochem.
Phy. Part B: Biochem. Mol. Bio. 146(2007) 153-159.
5
1) G. Gunnar, P. Lipniunas, T. Lundgren, F. Lindh, N. Bo, Archives Biochem.
Biophy. 278(1990) 546-555
5
2) T. Urashima, T. Nakamura, K. Teramoto, T. Mizukami, I. Arai, T. Saito, Comp.
Biochem. Phy. Part B: Biochem. Mol. Bio. 135(2003) 439-448.
5
3) F. Fiegl, Spots tests in organic analysis. Elsvier Publication, Amsterdam, (1975)
pp. 337.
2
4

ACCEPTED MANUSCRIPT
5
4) F. Dorland, B. L. Schut, J.F.G. Vliegenthart, G. Strecker, B. Fournet, G. Spik, J.
Montreuil, Eur. J. Biochem. 73(1977) 93-97.
5
5
5) P. Chaturvedi, B. C. Sharma, Carbohydrate Research. 203(1990) 91-101
6) Y. Uemura, S. Asakuma, T. Nakamura, I. Arai, M. Taki, T. Urashima,
Biochimica et Biophysica Acta. 1725(2005) 290-297.
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Captions
Scheme 1 The trisaccharide contained three types of sugar moieties Glc(4),
GalNAc(5) and GlcNAc (6)
Scheme 2 The hexasacharride contained three types of sugar moieties Glc(4), Gal (5)
and GlcNAc (6)
Scheme-A Mannich-Siewart hydrolysis of compound A (Bosiose)
Scheme-B Mannich-Siewart hydrolysis of compound B (Bovisose)
Figure 1. The optimized geometries of A (Bosiose) and B (Bovisose)
Figure 2. Spehadex G-25 chromatography of yak milk oligosaccharides detected
by phenol sulphuric acid method. Elution was made with distilled water
Figure 3. Mass fragmentation of oligosaccharide 'A' (Bosiose) (a) by repeated H
transfer (b) ES-MS mass fragments
Figure 4. ES Mass fragmentation of oligosaccharide B (Bovisose) (a) by repeated H
transfer (b) ES-MS mass fragments
Figure 5. The elucidate structure of isolated compound A (Bosiose)
Figure 6. The elucidate structure of isolated compound B (Bovisose)
Table 1: Reverse phase HPLC of yak milk oligosaccharides
Supplementary material
Figure S1. TLC (a) acetylated oligosaccharide mixture at different polarity
proportions and (b) Isolated acetylated oligosaccharide at CHCl :MeOH (95:5).
3
Figure S2. Reverse phase HPLC
Figure S3-S17. NMR and Mass Spectral data of compound A and B
Table S1. Combined fractions (200 mg (88 x 2 = 18.09 gm) of yak milk
oligosaccharide mixture chromatographed over Sephadex G-25 chromatography.
Table S2-S7. Purification of acetylated milk oligosaccharides on silica gel column
2
6

ACCEPTED MANUSCRIPT
Table 1: Reverse phase HPLC of yak milk oligosaccharides
S.No Retention time
Area
9822
40360
13282
542
Area (%)
0.67
Height Height (%)
1
2
3
4
5
6
7
8
9
0
1
2.272
3.115
3.637
3.968
4.331
4.693
5.077
5.227
5.491
5.557
5.717
1688
4827
1551
91
7.17
20.51
6.57
0.39
5.90
0.44
0.22
0.17
0.22
0.23
0.27
2.74
0.90
0.04
16671
746
1.13
1388
104
52
0.05
312
0.02
170
0.01
40
122
0.01
52
1
1
206
0.02
54
284
0.02
64

ACCEPTED MANUSCRIPT
A
B
Figure 1 The optimized geometries of A (Bosiose) and B (Bovisose)