Structure of Galactomannan from Seeds of Crotalaria alata

Article abstract of DOI:10.1007/s10600-015-1303-y

A galactomannan of MW 540 kDa was isolated from seeds of Crotalaria alata. Chemical and spectral (PMR and ¹³C NMR) methods established that the main chain of the galactomannan consisted of β-1,4-mannan with α-1,6-D-galactopyranose side chains.

Full text of DOI:10.1007/s10600-015-1303-y

DOI 10.1007/s10600-015-1303-y
Chemistry of Natural Compounds, Vol. 51, No. 3, May, 2015
STRUCTURE OF GALACTOMANNAN FROM SEEDS OF Crotalaria alata
1
2
1*
F. Kh. Kodiralieva, A. S. Shashkov, and R. K. Rakhmanberdyeva
A galactomannan of MW 540 kDa was isolated from seeds of Crotalaria alata. Chemical and spectral (PMR
13
and C NMR) methods established that the main chain of the galactomannan consisted of ꢀ-1,4-mannan
with ꢁ-1,6-D-galactopyranose side chains.
13
Keywords: polysaccharide, galactomannan, methylation, periodate oxidation, PMR and C NMR spectroscopy.
We reported previously that seeds of Crotalaria alata (Fabaceae) represent a source of a water-soluble galactomannan
polysaccharide [1, 2]. The isolation and structure elucidation of galactomannans from various species of the genus Crotalaria
were also reported [3–6]. Their contents varied from 6.6 to 20% with different monomer ratios. However, galactomannans
from C. alata introduced into Uzbekistan have not been investigated. The chain of galactomannans isolated from seeds of
various Crotalaria species consisted mainly of ꢀ-1,4 bonded mannans in which several mannose residues were replaced by
C-6 ꢁ-D-galactopyranose residues although individual galactose residues in C. medicaginea Lam. galactomannan were connected
to the main chain through an ꢁ-1,3-bond [6].
The goals of the present work were to isolate and elucidate the structure of galactomannan from C. alata seeds that
was obtained by extraction with cold water followed by purification using Fehling’s solution. The monosaccharide composition
of the purified polysaccharide included galactose and mannose in a 1:3 ratio. The polysaccharide was heterogeneous according
to gel chromatography and was fractionated by EtOH in order to produce four homogeneous fractions in yields of 15.0%
(fr. 1), 65.0% (fr. 2), 13.0% (fr. 3), and 3.0% (fr. 4). Total acid hydrolysis showed that all fractions consisted of galactose and
mannose and differed only in the quantitative monosaccharide contents and molecular weights (MW) (Table 1).
Table 1 shows that fr. 2 had the greatest yield, was homogeneous according to gel filtration, and was designated GMS by us.
22
GMS was an amorphous white powder with [ꢁ] +20° (c 0.1%, H O) and dissolved in H O to form viscous solutions
D
2
2
(ꢂ 49.86, c 0.75%). Its MW was calculated as 540 kDa using the sedimentation constant and the Doublier method [7]. The
rel
–1
IR spectrum of GMS contained characteristic absorption bands at 815 cm (pyranose ring), 872 (ꢀ-glycoside bond), and 725
(ꢁ-glycoside bond).
Structural features of GMS were elucidated using chromic and periodate oxidation and methylation. Smith oxidation
[8] of GMS consumed 0.93 mol of oxidant per mol of anhydro unit and released 0.11 mol of formic acid. Paper chromatography
(PC) of the hydrolysis products of the polyalcohol detected mainly erythritol, which was indicative of a 1,4-bond between
hexose residues in GMS, and glycerol, which provided evidence of a high degree of polymerization and the presence of 1,6-bonds
between hexose residues. PC also identified traces of mannose, which showed that the GMS chain was slightly branched.
Methylation of GMS by the Hakomori method [9] produced the completely methylated product in 63% yield with
[ꢁ] +27° (c 0.1%, CHCl ). The completeness of the methylation was confirmed by IR spectroscopy from the lack of absorption
D
3
–1
bands at 3200–3600 cm (OH). The permethylate of GMS was subjected sequentially to formolysis and hydrolysis. GC of
the hydrolysis products with known markers identified 2,3,4,6-O-Me -D-Gal (14.8%), 2,3,4,6-O-Me -D-Man (2.06%),
4
4
2,3,6-O-Me -D-Man (43.9%), and 2,3-O-Me -D-Man (11.5%). The presence of 2,3,6-O-Me -D-Man as the principal product
3
2
3
was consistent with 1,4-bonds between mannose residues in the main chain. The identification of 2,3,4,6-O-Me -D-Gal and
4
di-O-methyl-D-mannose confirmed the periodate-oxidation data regarding branching in the macromolecule chain where the
second galactose component was bonded to the main chain through a 1,6-bond. The formation of 2,3,4,6-O-Me -D-Man
4
showed that the GMS chain had mannopyranose residues on the non-reducing end.
1) S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan,
Tashkent, e-mail: rakhmanberdieva@mail.ru; 2) N. D. Zelinsky Institute of Organic Chemistry, RussianAcademy of Sciences,
Moscow, B-334, Leninsky Prosp., 47. Translated from Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2015, pp. 355–358.
Original article submitted October 20, 2014.
©
0009-3130/15/5103-0405 2015 Springer Science+Business Media New York
405

TABLE 1. Principal Characteristics of C. alata Galactomannan Fractions
ꢂrel
Fraction
Precipitant volume, mL
Fraction yield, %
Gal–Man ratio
MW, kDa
(ñ 0.5%; Í2Î)
1
2
3
4
50
15.0
65.0
13.0
3.0
1:3.2
1:3.0
1:2.8
–
55.4
49.8
31.2
–
658
540
230
–
100
150
200
TABLE 2. Characteristics of GMS and GMSD
Parameter
Galactomannans
GMS
GMSD
MW, kDa
Gal–Man ratio
540
1.0:3.0
25
1.0:2.7
[ꢁ]2D2 (c 0.1%; H2O)
+20ꢄ
+24ꢄ
49.8
815, 872, 725
25.6
814, 872, 720
ꢂrel (ñ 0.75%; H2O)
IR spectra, cm–1
13
TABLE 3. Chemical Shifts in PMR and C NMR Spectra of C. alata Galactomannan (D O, TSP Standard, ꢃ 1.6 ppm,
H
2
C
ꢃ 0.0 ppm)
Ñ-6
Í-6, 6ꢅ
Ñ-1
Í-1
Ñ-2
Í-2
Ñ-3
Í-3
Ñ-4
Í-4
Ñ-5
Í-5
Residue
99.97
5.02
69.60
3.82
70.45
3.94
70.55
3.99
72.44
3.89
62.39
3.74
3.74
67.69
3.97
3.78
ꢁ-Galp-(1
ꢆ
6)
101.16,
101.33a
4.75
71.07
71.16a
4.12
72.52
3.80
77.93
78.05
78.20b
3.87
74.52
74.44b
3.89
ꢇ4)-ꢀ-D-Manp-(1ꢇ
4.74a
101.16
101.33a
4.75
71.07
71.16a
4.12
72.62
3.80
77.70
3.82
76.21
3.55
61.72
3.91
3.74
ꢇ4)-ꢀ-D-Manp-(1ꢇ
4.74a
______
a
b
Depending on the presence or absence of ꢁ-Galp in the glycosylated residue; depending on the through-space environment
of the residue.
–1
The positive specific rotation of GMS permethylate and an absorption band at 725 cm in the IR spectrum were
indicative of an ꢁ-glycoside bond for D-galactopyranose. This was confirmed by oxidation of fully acetylated GMS by
chromic anhydride [10].
The structure of galactomannan GMS was also elucidated using NMR spectroscopy. The spectrum of destroyed
GMS was also recorded.
As shown above, GMS formed a very viscous aqueous solution even at low concentrations. This degraded significantly
13
the resolution of the C NMR spectrum. The destroyed polysaccharide (GMSD) was a fragment of lower MW and the same
13
monosaccharide ratio [11] and was used to obtain well resolved PMR and C NMR spectra.
22
GMSD was an amorphous white powder with [ꢁ] +24° and MW 25 kDa. It dissolved completely in H O and had
D
2
ꢂ
25.6. The hydrolysate of GMSD contained galactose and mannose in a 1:2.7 ratio. The principal parameters of GMSD
rel
agreed with those of GMS. Differences were observed in the MW and viscosity (Table 2).
Anomeric atoms showed two main resonances in the PMR spectrum (Table 3) with chemical shifts ꢃ 5.02 and
13
4.74 ppm and a 1:2.3 ratio of integrated intensities. Resonances of other protons were located at ꢃ 3.4–4.2 ppm. The C NMR
spectrum (Table 3) had strong resonances for anomeric C atoms at ꢃ 101.33, 101.16, and 99.97 ppm in addition to minor
resonances at ꢃ 95.01 and 94.88 ppm. Resonances of C atoms bonded to a single O atom were found at ꢃ 61–79 ppm.
406

13
1
1
Resonances in PMR and C NMR spectra were assigned using 2D homonuclear H– H COSY, TOCSY, and ROESY
1
13
and heteronuclear H– C HSQC and HMBC. An analysis of COSY, TOCSY, and ROESY spectra revealed ꢀ-mannopyranose
(ꢀ-Manp) and ꢁ-galactopyranose (ꢁ-Galp) residues in the polymer. The HSQC spectrum showed that all ꢁ-Galp residues
were terminal whereas all ꢀ-Manp residues were substituted in the 4-positions and a part of them also in the 6-positions
(Table 3). ROESY and HMBC spectra exhibited trans-glycoside correlation peaks for H-1 (ꢁ-Galp)/C-6 (ꢀ-Manp) and H-1
(ꢀ-Manp)/C-4 (ꢀ-Manp). This indicated unambiguously that the polymer chain included ꢇ4)-ꢀ-Manp-(1ꢇ moieties where
some of the residues were substituted at the C-6 hydroxyl by ꢁ-Galp residues.
Splitting of C-4 resonances of disubstituted ꢀ-Manp residues indicated indirectly that residues unsubstituted and
substituted at C-6 were statistically (not block) distributed. This could be explained by the effect of the through-space
environment, i.e., different combinations of C-6-substituted and unsubstituted residues as a glycosylating and glycosylated
pyranose for each actual residue.
ꢉꢉꢉꢉꢉꢉꢉꢉꢁ-Galp-(1 ꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢁ-Galp-(1
ꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢆꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢆ
ꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢊꢋꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢊꢋ
ꢇ4)-ꢀ-D-Manp-(1ꢇ4)-ꢀ-D-Manp-(1ꢇ4)-ꢀ-D-Manp-(1ꢇ4)-ꢀ-D-Manp-(1ꢇ
The minor resonances at ꢃ 5.02 and 4.74 ppm and ꢃ 95.01 and 94.88 ppm, which were characteristic of ꢁ- and
H
C
ꢀ-Manp residues at the reducing end of the chain, allowed the average chain length ꢇ4)-ꢀ-Manp-(1ꢇ to be determined as
approximately 30 units.
The chemical and spectral investigations found that galactomannan from seeds of C. alata consisted of ꢀ-1,4-bonded
D-mannopyranose residues, a part of which was substituted in the O-6-position by individual ꢁ-D-galactopyranose residues.
A comparison of these data with the literature on Crotalaria plant galactomannans enabled the structure of the C. alata
polysaccharide to be identified as the most common type of galactomannan (mannan) in the family Fabaceae. However,
C. alata GMS differed from those of other Crotalaria species with respect to content, viscosity, specific rotation, ratio of
monosaccharide residues, molecular weight, and degree of polymerization. The mannopyranose chain structure had a statistical
distribution of terminal galactopyranose residues.
EXPERIMENTAL
We studied seeds of C. alata that were collected in F. N. Rusanov Botanical Garden during ripening (Oct. 15, 2011).
TLC was carried out on Silufol UV
and KSK silica gel (LS-5/40 mm) plates using solvent systems (v/v) CHCl –MeOH
254
3
(9:1, 1), CHCl –Me CO (30:1, 2), and MEK–NH OH (1%) (30:1, 3). PC used Filtrak FN 18 paper and 1-BuOH–Py–H O
3
2
4
2
(6:4:3, 1). Spots were identified by anilinium biphthalate (1) and KIO –KMnO –benzidine (2).
4
4
Specific rotation of polysaccharides and their methyl derivatives was measured at 20–23°C on a Zeiss polarimeter in
1-dm tubes of volume 10 mL and in 0.5-dm tubes of volume 1 mL. IR spectra of galactomannans in pressed KBr pellets were
recorded on a PerkinElmer Model 2000 IR-Fourier spectrometer using 100 scans. GC analysis was performed on a Chrom-5
chromatograph with a flame-ionization detector and a stainless-steel column (200 ꢈ 0.3 cm) of XE-60 Silicone (5%) on
Chromaton NAW (0.200–0.250 mm) at 210°C with N carrier gas at flow rate 60 mL/min. Molecular weights of the
2
galactomannans were determined as before [7].
13
PMR and C NMR spectra were taken in D O at 313 K on a Bruker AV-600 instrument using sodium
2
3-(trimethylsilyl)propionate (TSP) with ꢃ 0.0 ppm and ꢃ 1.6 ppm as a standard. 2D spectra were taken and processed using
H
C
standard Bruker methods.
The monosaccharide composition of the galactomannans was determined by total acid hydrolysis in H SO (1 N) at
2
4
+
100°C for 8 h. The hydrolysates were neutralized by BaCO , de-ionized by KU-2 (H ) cation exchanger, and evaporated.
3
Monosaccharides were identified by PC (system 1, detector 1) and GC (as aldononitrile acetates) [12].
Isolation of Water-soluble Polysaccharide (WSPS). Seeds (100 g) of C. alata were grounded and treated with
refluxing CHCl :MeOH (1:1) in a 1:3 ratio on a water bath for 1 h. WSPS were extracted three times by H O (1:5 ratio) with
3
2
stirring at room temperature. Pulp was separated from the aqueous extract by centrifugation. The aqueous extracts were
combined and precipitated by EtOH in a 1:3 ratio. The resulting precipitate was separated, rinsed with EtOH of increasing
concentration, and dried. Then, the precipitate was dissolved in H O and purified by adding Fehling’s reagent according to the
2
literature method [8]. The final yield of purified GMS was 15% of the air-dried raw material.
407

Fractionation of Polysaccharides. WSPS (1 g) from C. alata were dissolved in H O (100 mL), stirred vigorously,
2
and treated dropwise with EtOH (50 mL, 96°). The resulting white precipitate (fraction 1) was separated by centrifugation,
rinsed with EtOH, and dehydrated with Me CO. Yield, 0.15 g. The supernatant liquid was treated with another portion
2
of EtOH (50 mL). The resulting finely dispersed precipitate was separated and worked up analogously. Yield of fraction 2
(GMS), 0.65 g. Fraction 3 was obtained by adding another portion of EtOH (50 mL) to the supernatant. The yield was
0.13 g. The supernatant solution was evaporated to half the volume and treated with another portion of EtOH (50 mL). Yield
of fraction 4, 0.03 g.
Periodate Oxidation of GMS. GMS (0.05 g) were dissolved in H O (24.9 mL), treated with NaIO solution
2
4
(5.1 mL, 0.25 M), and left at +5°C. Aliquots (1 mL) were taken every day and titrated with sodium thiosulfate solution
(0.01 N). The consumption of NaIO was 0.93 mol after 15 d. The yield of HCOOH was 0.11 mol. The oxidized product was
4
dialyzed (after destroying periodate with ethyleneglycol), treated with NaBH (0.1 g), and left overnight. The solution was
4
+
treated with KU-2 (H ) cation exchanger. The filtrate was evaporated with MeOH. The dry solid was hydrolyzed by H SO
2
4
(5 mL, 1 N) for 8 h at 100°C. PC (system 1, detectors 1 and 2) of the hydrolysis products detected mainly erythritol and
glycerol.
Methylation of GMS. Galactomannan (0.1 g) was methylated by the Hakomori method [9]. The yield of GMS
permethylate was 0.063 g. GMS permethylate (0.05 g) was subjected to formolysis by HCOOH (2 mL, 85%) for 1 h, cooled,
and evaporated. Then, the precipitate was hydrolyzed by H SO (0.5 N) for 8 h at 100°C. The hydrolysate was worked up as
2
4
usual. TLC (systems 1-3, detector 1) detected 2,3,4,6-tetra-O-Me-D-mannose, 2,3,4,6-tetra-O-Me-D-galactose, 2,3,6-tri-O-
Me-D-mannose, and di-O-Me-D-hexose.
Oxidation of GMS by Chromic Anhydride [10]. GMS (0.05 g) was dissolved in formamide (5 mL), treated with
anhydrous Py (2.5 mL) and dropwise with acetic anhydride (2.5 mL), and heated for 1 h on a water bath at 50°C. GMS acetate
was precipitated by icy distilled H O. The precipitate was separated and rinsed with MeOH and Me CO to afford GMS
2
2
–1
peracetate (0.056 g). The IR spectrum lacked an OH absorption band (3200–3600 cm ) and showed clearly resolved absorption
–1
bands at 1720 and 1240 cm .
GMS peracetate (0.05 g) was treated with CrO (0.02 g) in glacial AcOH (2 mL), heated for 3 h at 50°C, diluted with
3
H O, and extracted with CHCl . The extract was dried over anhydrous Na SO and evaporated to dryness. The solid was
2
3
2
4
dissolved in H SO solution (1 mL, 1 N) and hydrolyzed for 8 h on a boiling-water bath. The hydrolysate was worked up as
2
4
usual. PC (system 1, detector 1) detected galactose.
Depolymerization of GMS. GMS (0.5 g) was dissolved in TFA (50 mL, 0.1 M) and hydrolyzed at 100°C on a water
bath for 45 min. The hydrolysate was cooled to room temperature and precipitated by EtOH (150 mL). The resulting precipitate
was separated by centrifugation. The solid was rinsed with EtOH and dehydrated by Me CO. Yield of depolymerized GMS,
2
0.28 g.
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