Abstract

In search for more and more biologically active milk oligosaccharides, milk of different cow species was investigated for their oligosaccharide contents. It was found that the oligosaccharides isolated from this milk carry different stereoscopic structures which varied in their inter glycosidic linkages, configurations and conformations of the monosaccharides present therein. In our present study we have isolated a novel tri-saccharide Friuose from Friesian cow milk comprised of Glucose and Galactose with α Glycosidic linkage. The structure of oligosaccharide was elucidated with high resolution 800 MHz NMR experiments. The identification of monosaccharide contents of Friuose were not only done by traditional methods, Structure Reporter Group (SRG), but also justified by the splitting pattern of epicentre four of Glucose and Galactose. This is the first report for identification of monosaccharides based on J values of epicentre four of the monosaccharides present in a novel oligosaccharide. The structure of Friuose was established as under- (Image 1).

Keywords:Friesian Cow Milk; 1D NMR; 2DNMR (COSY, TOCSY, HSQC, HMBC) Experiments; Friuose

Introduction

Oligosaccharides are biologically active components of plants, bacteria and milk. Milk is a mammalian fluid essential for development of neonates. It also provides immunity and strength to new born. Besides the human milk, cow milk is the most efficacious and medicinally important intake for neonates which not only develops the immune system, it strengthens the cardiac muscles and brain gangliosides and it also improves eye muscles as well [1]. It is important to note that oligosaccharide content of cow milk depends on the fodder and geographical environment around them. In India a large number of cow species are found along with the cow of foreign origin. Oligosaccharides found in cow milk constitute Glucose, Galactose, GlcNHAc, GalNHAc etc which are linked together by α-and β-glycosidic linkages positioned at different ring OH groups of monosaccharide building blocks, resulting in straight and branched chain oligosaccharide structures [2]. Specific conformation and position of glycosidic linkages result in the specific biological activities. In literature of ancient medicinal systems [3], the cow milk is defined as ‘Amrita’ the panacea and is beneficial for development of bones, improves milk output in lactating women, more over cow milk is beneficial for gut health improvement, considering the medicinal efficacies of cow milk and the milk of Friesian cow was collected in bulk (10L) from Hardoi district of Uttar Pradesh in India and was processed by modified method of Kobata and Ginsburg, incorporating de-protonation micro-filteration and lyophilisation, as a result crude oligosaccharide mixture 260g was obtained. This oligosaccharide mixture was acetylated by acetic anhydride and pyridine and it was further purified on silica column chromatography which resulted in the isolation of a new Trisaccharide comprised of Glc, and GalNHAc. Besides the identification of Glc and GalNHAc by TLC/PC and position of anomeric proton/carbon with the literature value of Glc and GalNHAc (structure reporter group theory) [5], it was also identified by splitting pattern of epicentre4of Glc and GalNHAc. This is the first report of a trisaccharide Friuose in which the reducing Glc is attached to two GalNHAc moieties with α glycosidic linkages and the identification of Glc and GalNHAc by splitting pattern of 4^th epicentre of monosaccharide constituents. The Structure of the novel oligosaccharide was elucidated by combining the result obtain from the NMR experiments of ¹H, ¹³C and 2D NMR (HSQC, TOCSY, COSY, HMBC) [6-8], Mass spectrometry, chemical degradation, chemical transformation.

Material & Methods

General procedures were the same as given in our earlier communication [9], besides the NMR experiments were performed on 800MHz and 300MHz Bruker NMR machine.

General procedure for isolation of milk oligosaccharides [9]

10L of cow milk was collected in 4 days in normal milking condition from a single domestic cow of district Hardoi Uttar Pradesh, India. Milk was fixed immediately by addition of an equal amount of ethanol (10L). The preserved milk was filtered by a glass wool column and then it was centrifuged on C-25 centrifuging machine at 5000rpm at -4°C, after centrifugation, the solidified layer was removed by filtration through glass wool column. After filtration lipid layer was discarded and supernatant was precipitated by addition of a maximum of 68% ethanol which was again separated by centrifugation and removal of protein and lactose by centrifugation, supernatant was filtered through a micro filter (0.2μ) to remove remaining lactose. Further it was then lyophilized to obtain the oligosaccharide mixture (260gm).

Acetylation of oligosaccharide mixture [9]

Acetylation of oligosaccharide mixture 10g oligosaccharides mixture was acetylated with pyridine (10 ml) and acetic anhydride (10ml) at 600C and solution was stirred overnight. The mixture was evaporated under reduced pressure and viscous residue was taken in CHCl3 (3×100ml) and washed twice with ice cold water and was evaporated to dryness yielding 10.2g of acetylated mixture. The acetylation converted the free sugars into their nonpolar acetyl derivatives which resolved nicely on TLC, giving 6 spots i.e. a, b, c, d, e, and f. The six compounds were finally purified by silica column chromatography using varying proportions of chloroform and methanol as eluents (Image 2).

Purification of acetylated milk oligosaccharides on silica gel column

Separation and purification of the acetylated oligosaccharide mixture (10.0gm) was carried out over silica gel (500g) using varying proportion of solvent such as CHCl₃, CHCl₃: MeOH as eluents, collecting fraction of 250mL each. All these fractions were checked on TLC and those showing similar spots were taken together for further investigations. Repeated column chromatography of acetylated oligosaccharide mixture resulted in isolation of 62mg of compound ‘a’.

Deacetylation of compound ‘A’ [9]

Compound ‘a’ (30mg) was dissolved in acetone (3mL) and 3mL of NH₃ was added in it and was left overnight in a Stoppard hydrolysis flask. After 24h ammonia was removed under reduced pressure and the compound was dissolved in water and washed thrice with CHCl₃ (3×5mL) (to remove acetamide) and water layer was freeze dried providing the deacetylated natural oligosaccharide ‘A’ (24mg).

Methyl glycosidation/acid hydrolysis of Compound ‘A’:

Compound ‘A’ (10mg) was refluxed with absolute MeOH (2ml) at 65°C for 18hr in the presence of cation exchange IR-l20 (H) resin. The reaction mixture was filtered while hot and filtrate was concentrated. To this reaction mixture of ‘A’, 1, 4-dioxane (1ml) and 0.1N H₂S0₄ (1ml) was added, and the solution was warmed for 30 minutes at 50°C. The hydrolysis was complete after 24hrs. (TLC) The hydrolysate was neutralized with freshly prepared BaCO₃ and further filtered and concentrated under reduced pressure to afford α-and β-methyl glucosides along with the Gal and GalNHAc. Identification of monosaccharides in compound ‘A’ was confirmed by comparison with authentic samples (TLC, PC) of α-and β-methyl glucosides along with the Gal and GalNHAc.

Kiliani hydrolysis of compound A, friuose [9]

Compound ‘A’ (5mg) was dissolved in 2ml Kiliani mixture (AcOH-H₂O-HCI, 7:11:2) and heated at 100°C for 1hr followed by evaporation under reduced pressure. It was dissolved in 2ml of H₂O and extracted twice with 3ml CHCl₃. The aqueous residual solution was made neutral by addition of 1-2 drops of 2N NaOH and was evaporated under reduced pressure to afford Glc, GalNHAc and GalNHAc on comparison with authentic samples of Glc, and GalNHAc (Scheme 1).

Description of compound-A, Friuose:

Compound a (62mg) was obtained from fraction 61-75 of column chromatography-6. On deacetylation of 32 mg of substance with NH₃/ acetone it afforded substance ‘A’ (24.0mg). For experimental analysis, this compound was dried over P₂O₅ at 100oC and 0.1mm pressure for 8hr. It gave positive phenolsulphuric acid test [10], Feigl test [11], Morgan-Elson test [12] (Table 1).

i. ¹H NMR of Friuose acetate in CDCl₃ at 800MHz

δ6.25[d, 1H, J=3.1Hz, α-Glc(S-1) H-1)], 5.68[d, 1H, J=8.1Hz, β-Glc(S-1) H-1], 4.47 [d, 1H, J=2 Hz, α-GalNHAc (S-2), H-1)], 4.47[d, 1H, J=2 Hz, α -GalNHAc (S-3) H-1].δ3.80(J=3.4 and 3.2 Hz) [m, 1H, β-Glc(S-1) H-4], δ3.80 (J=3.4 and 3.2 Hz)[α-GalNHAc(S-2) H-3].value of epicentre -4 Glc (S1) H-4, 3.38 (J=6.4 and 6.2 Hz.

ii. ¹³C NMR of Friuose acetate in CDCl₃ at 200MHz

δ88.94[1C, α-Glc(S-1), C-1], 91.50[1C, β-Glc(S-1), C-1], 100.93[1C, α-GalNHAc (S-2), C-1], 101.19[1C, α -GalNHAc (S-3), C-1].

iii. ¹H NMR of Friuose in D₂O at 300MHz

δ5.61[d, 1H, J=3.1Hz, α-Glc(S-1) H-1], 5.38 [d,2H, J=8.1, βGlu (S-1)], 4.32[d, 1H, J=2Hz, α -GlcNHAc (S-2) H-1], 4.39[d, 1H, J=2Hz, α-GlcNHAc (S-3) H-1].

iv. ES-MS fragments of friuose

609[M+Na]+, 586[M]+, 577[609-CH₂H₂O], 548[586-2H₂O,2H+], 465[548-3H₂O, CHO], 406[465-3CH₂OH], 383[586-S3], 365[383- H₂O], 325[383-2CHO], 281[365-2CH₂CO], 222[281-3CH₂OH], 180[383-S3], 162[180-H₂O].

Result and Discussion of Compound A, Friuose

Compound A, Friuose (C22H38O16N2), gave positive phenolsulphuric acid test [10], Feigl test [11] and Morgan-Elson test [12] showing the presence of normal and amino sugars moieties in the compound A. The Oligosaccharide mixture of Friesian cow milk which was acetylated and chromatographed by silica gel column chromatography resulted in isolation of compound ‘a’ which was later deacetylated with NH3 and acetone to obtain natural oligosaccharide ‘A’ The name of the compound Friuose originated from the source i.e.; Friesian cow and designated as ‘A’ while its acetylated derivative was named as ‘a’.

The HSQC spectrum of acetylated Friuose showed the presence of four cross peaks (Figure 1) of anomeric protons and carbons in their respective region at δ6.25× 88.94, δ5.68×91.50, δ4.47×100.93, δ4.43×101.19 suggesting the presence of four anomeric protons and carbons into it. Further the 1H NMR of acetylated Friuose at 300 MHz showed four doublets of one proton i.e. δ6.25(1H), 5.68 (1H), δ4.47 (1H), δ4.43(1H) (Figure 2). The trisaccharide nature of friuose was further confirmed by the presence of four anomeric carbons peaks at δ88.94(1C), 91.50(1C), 100.93(1C), 101.19(1C), in the acetylated spectrum of Friuose at 75MHz (Figure 4). The 1H NMR of natural friuose at 300 MHz in D₂O showed four anomeric proton signals at δ5.61, 5.38, 4.32, 4.39, (Figure 3) along with two, three proton singlet at 1.91 and 1.81 confirming the presence of three monosaccharide units in oligosaccharide in their reducing form, out of which two of the monosaccharides had -NHAc group in them. Further the anomeric proton doublet presents at δ6.25 and δ5.68 in 1H NMR of acetylated Friuose showed downfield shifted α and β anomeric protons confirming that the oligosaccharides was present in its reducing form and also suggested that compound Friuose was a trisaccharide in reducing form. Thus ¹H and ¹³C NMR spectra justify the four anomeric signals for trisaccharide with total integral intensity of three anomeric proton carbons.

f1 234

The reducing nature of compound was further confirmed by its methyl glycosylation (MeOH/H+) followed by its acid hydrolysis, which led to isolation of α- and β- methyl glucosides, along with GalNHAc suggesting the presence of glucose at the reducing end, along with GalNHAc unit. For convenience the monosaccharides present in oligosaccharide ‘A’ were designated as S-1, S-2and S-3. The monosaccharides present in the compound ‘A’ were also confirmed by its Kiliani hydrolysis under strong acidic conditions which were monitored on paper chromatography (PC) and TLC. On completion of Kiliani hydrolysis it gave two spots of Glc, and GalNHAc, which were identified by their comparison with the authentic samples of Glc and GalNHAc confirming that the oligosaccharide Friuose was composed of two types of monosaccharide units. Further, the anomeric proton signals present at δ6.25 and 5.68 in ¹H NMR of Friuose acetate was assigned to α- and β-anomeric protons of Friuose acetate ‘a’ at 800 MHz in CDCl3.The HSQC spectrum of ‘a’ showed a cross peak at δ5.68× 91.50 for monosaccharide (S-1) which resembled with the literature value of Glc [13] hence the S-1 was reconfirmed as Glucose.

Further, the anomeric proton presents at δ 5.68, showed three cross peaks at δ5.68× (3.80, 5.05, 5.50,) in TOCSY spectrum of Friuose acetate (Figure 5), which were later confirmed as (H2 5.05, H3 5.50, H4 3.80) of S-1 by COSY spectrum of Friuose acetate (Figure 6). The chemical shift of H-4 of S-1 at δ3.80 suggested that the position of H-4 of S-1 was available for glycosidic linkage by the next monosaccharide unit. Further, the signal for H-4- of S-1 at δ3.80 gave a cross peak with C1 of S-2 at δ3.80×100.93, in the HMBC spectrum (Figure 7) of Friuose acetate confirming a 1→4 glycosidic linkage between S-2 and S-1. The anomeric carbon signal at δ100.93 gave a cross peak at δ100.93 × 4.47 in the δ100.93×4.47 resemble with the literature value of anomeric proton/carbon value of GalNHAc [13]. Hence, the monosaccharide S-2 was confirmed as GalNHAc. The anomeric proton doublet at δ4.47(J= 2Hz) showed that glycosidic linkage between S2→S1 was a ᾳ-glycosidic linkage, it is also confirmed that GalNHAc was present in its 4C1 L conformation more over the splitting pattern of H4 of S1(J=6.4 and 6.2 Hz) also confirmed that Glc, S1 was Glucose. Further, the anomeric proton signal present at δ4.47 in the TOCSY spectrum of Friuose acetate ‘a’ exhibited three cross peaks at (δ4.47×3.80, 4.47×4.10, 4.47×5.30). These peaks were later identified by COSY spectrum as (H2 4.10, H3 3.80, and H4 5.30) (Figure 6). The chemical shift of H3 at δ3.80 suggested that H3 of (S-2) was available for glycosidic linkage with the next monosaccharide unit. Further the signal present at δ3.80 gave a cross peak at δ3.80×101.19 in the HMBC Spectrum of Friuose acetate which was between H3 of S-2 and C1 of S-3 confirming a (1→3) linkage between S-2 and S-3. The HSQC spectrum of Friuose acetate at 800 MHz in CDCl₃ gave a cross peak between H1 of (S-3) and C1 of (S-3) at δ4.43 ×101.19, confirming the anomeric proton/carbon value of S-3 unit at δ4.43 ×101.19. The anomeric carbon/proton value at δ101.19 and δ4.43 resembled with literature value of GalNHAc [13] confirmed that S-3 was a GalNHAc. The anomeric proton doublet for GalNHAc S-3 present at δ4.43 showed a coupling constant of 2 Hz and hence the configuration of GalNHAc (S-3) was α. It also confirmed that GalNHAc, S3 had a ⁴C₁ L conformation. Besides this splitting pattern of H4 of S-2 J= 3.2 and 3.4 Hz also confirm the sugar S2 was GalNHAc. The anomeric proton signal at δ4.43 showed three cross peaks at (5.10, 5.30, 5.55) in TOCSY spectrum of Friuose acetate. These were later confirmed by the COSY spectrum as (H2 5.10, H3 5.50, and H4 5.55). The chemical shift of Ring Proton values of monosaccharide S3 confirmed that none of its ring protons were involved in glycosidic linkage by next monosaccharide unit and hence the monosaccharide S3 was present at the non-reducing end of Friuose.

All the ¹H NMR assignments for ring protons of monosaccharide units of Friuose were confirmed by COSY and TOCSY experiments. The positions of glycosidation in the oligosaccharide were confirmed by position of anomeric signals, Structure Reporter Groups (S.R.G.) and comparing the signals in ¹H and ¹³C NMR of acetylated and deacetylated oligosaccharide. The glycosidic linkages in Friuose were assigned by the cross peaks for glycosidically linked carbons with their protons in the HSQC and HMBC spectrum of acetylated Friuose which were in confirmity with the assigned structure and their position were confirmed by 2D NMR. All signals obtained in ¹H and ¹³C NMR of compound Friuose were in confirmity with the assigned structure and their positions were confirmed by 2D NMR viz. COSY, TOCSY, HSQC and HMBC experiments. Thus, based on the pattern of chemical shifts of ¹H NMR, ¹³C NMR, COSY, TOCSY, HSQC and HMBC experiments it was inferred that Compound ‘A’ was a trisaccharide having following structure -

The ESI Mass spectrometry data of Friuose (Figure 8) not only confirmed the derived structure but also confirmed the sequence of monosaccharides in Friuose [14]. The highest mass ion peaks were recorded at m/z 609 and 586 which were due to [M+Na]⁺ and [M]+ respectively, confirming the molecular weight of Friuose as 586 in agreement with its molecular formula C₂₂H₃₈O₁₆N₂. Further, the mass fragments were formed by repeated H transfer with elimination of terminal sugar less water. The triasaccharide, m/z 586 (I) fragmented to give mass ion at m/z 383 (II), this fragment arose due to the loss of terminal GalNHAc (S-3) moiety from trisaccharide indicating the presence of GalNHAc (S-3) at the non-reducing end. This disaccharide further fragmented to give mass ion peak at m/z

180 (III), which arose due to the loss of β-GalNHAc (S-2) moiety from disaccharide. The other fragmentation pathway in ESI-MSspectrum of Compound A, m/z 586 showed mass ion peak at 609[M+Na]⁺, 586[M]⁺, 577[609-CH₂H₂O], 548[586-2H₂O,2H⁺], 465[548-3H₂O,CHO], 406[465-3CH₂OH], 383[586-S3], 365[383- H₂O], 325[383-2CHO], 281[365-2CH₂CO], 222[281-3CH₂OH], 180[383-S3], 162[180-H₂O]

Conclusion

With a view to isolate medicinally important oligosaccharides from cow milk, a novel triasaccharide ‘Friuose’ was isolated from Friesian cow milk consisting of Glc, and GalNHAc with 1→4 and 1→3 glycosidic linkages having α and β configurations.

Acknowledgement

Authors are thankful to Head Department of Chemistry, University of Lucknow, Director CBMR-SGPGI, Lucknow for advanced NMR facilities and CSIR-CDRI, Lucknow for Mass Spectrometry.

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