Mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively odd-numbered oligosaccharides Bo Yang1, Guangli Yu1, Xia Zhao1, Guangling Jiao1, Sumei Ren1 and Wengang Chai1,2 Glycoscience and Glycoengineering Laboratory, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China Glycosciences Laboratory, Faculty of Medicine, Imperial College London, Harrow, UK Keywords acid hydrolysis; agarose; carrageenan; mass spectrometry; polysaccharide Correspondence W Chai, Glycosciences Laboratory, Faculty of Medicine, Imperial College London, Northwick Park & St Mark’s Campus, Harrow, Middlesex HA1 3UJ, UK Fax: +44 20 8869 3455 Tel: +44 20 8869 3255 E-mail: w.chai@imperial.ac.uk G Yu Glycoscience and Glycoengineering Laboratory, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China Fax: +86 532 8203 3054 Tel: +86 532 8203 1560 E-mail: glyu@ouc.edu.cn (Received November 2008, revised 30 December 2008, accepted February 2009) Sulfated galactan j-carrageenan is a linear polysaccharide with a repeating disaccharide sequence of alternating 4-sulfated 3-linked galactose and 4-linked 3,6-anhydrogalactose units In contrast to many examples of chemical hydrolysis of polysaccharides, mild acid treatment of j-carrageenan resulted in facile and highly specific cleavage In this article, we report the identification, by various MS and chromatographic techniques, of an unexpected series of exclusively odd-numbered oligosaccharide fragments from its hydrolytic products Detailed sequence analysis of the products indicated that all the oligosaccharide fragments have the 4-sulfated 3-linked galactose residues at both the reducing and the nonreducing ends Further detailed investigation and analysis suggested that these odd-numbered oligosaccharides were derived from two-step cleavages of the glycosidic bonds on either sides of the 3,6-anhydrogalactose residues Neutral galactan agarose also contains 3,6-anhydrogalactose and has a similar backbone sequence, and exhibited similar results upon mild acid hydrolysis It is highly unusual to obtain exclusively odd-numbered oligosaccharides from polysaccharides composed of ordered disaccharide repeats doi:10.1111/j.1742-4658.2009.06947.x The diverse oligosaccharide sequences present in polysaccharides, glycoproteins, glycolipids and proteoglycans serve multiple functions Acidic polysaccharide glycosaminoglycans (GAGs) are ubiquitous in vertebrate tissues, and have important biological functions through binding to various proteins Marine-derived polysaccharides are often of an anionic nature, and these GAG-like molecules have been exploited for their antiviral, antioxidant, anticoagulant and other signal- ing activities [1–4] Recent studies have shown that marine polysaccharide carrageenans can inhibit the attachment of several pathogenic viruses, e.g herpes simplex virus [5], dengus virus [6], and human papillomavirus [7,8], and hence they have become of considerable biomedical interest, owing to their antiviral activities and therapeutic potential Carrageenans are highly sulfated galactans isolated from marine red algae, with linear repeating sequences Abbreviations A, 4-linked a-3,6-anhydrogalactose; A2S, 4-linked 2-O-sulfated-a-D-3,6-anhydrogalactose; anGal, 3,6-anhydrogalactose; CID, collision-induced dissociation; CTMS, chlorotrimethylsilane; D, 4-linked a-D-galactopyranose; DP, degree of polymerization; ELSD, evaporative light scattering detector; Gal, D-galactopyranose; G, 3-linked b-D-galactopyranose; GAG, glycosaminoglycan; G2S, 3-linked 2-O-sulfated-beta-D-galactopyranose; G4S, 3-linked 4-O-sulfated-b-D-galactopyranose; 5-HMF, 5-hydroxymethyl-furfural; MMB, 4-methylmorpholine borane; TFA, trifluoroacetic acid FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS 2125 Odd-numbered oligosaccharides from galactan polysaccharides B Yang et al of alternating 3-linked b-d-galactopyranose (b-Gal, unit G) and 4-linked a-d-galactopyranose (a-Gal, unit D), with unit D often occurring as its 3,6-anhydro form (anGal, unit A) The classification of carrageenans is based on the presence of a D or A form of the 4linked galactose, and the differing sulfate contents and substitutions; for example, j-carrageenan, i-carrageenan and k-carrageenan have different disaccharide building blocks: -[3-linked 4-O-sulfated-b-d-galactopyranose (G4S)-A]n-, -[G4S-4-linked 2-O-sulfated-a-d-3,6-anhydrogalactose (A2S)]n-, and -[3-linked 2-O-sulfated-b-dgalactose (G2S)-4-linked 2,6-O-sulfated-a-d-galactose (D2S6S)]n-, respectively [9] Detailed knowledge of these polysaccharide structures is necessary for an in-depth understanding of their biological roles However, their structural complexity causes considerable difficulties in sequence analysis and assignment of structure–function relationships Partial depolymerization by either chemical or enzymatic means to obtain a range of oligosaccharide fragments is a common strategy for detailed structural analysis and for use in activity assays Enzymatic depolymerization is generally more specific, with cleavage at selected glycosidic bonds without the risk of modification of the native structures However, suitable enzymes are not always available for all polysaccharides Chemical hydrolysis is widely employed for depolymerization of various types of carrageenan Enzyme digestion cleaves the 1,4-linkages, resulting in even-numbered neocarra-oligosaccharides, -(A-G)n- or -(D-G)n-, with G at the reducing terminus and A or D at the non-reducing terminus [10–14] For A-containing carrageenans (e.g j-carrageenan and i-carrageenan), mild acid hydrolysis has been used, and is considered to cleave the 1,3-linkages, producing evennumbered carra-oligosaccharides, -(G-A)n-, with A at the reducing and G at the non-reducing terminus [15– 20] It was surprising that acid hydrolysis selectively cleaved the 1,3-linkage, giving oligosaccharides -(GA)n-, without affecting 1,4-linkages Similar results were obtained with 3,6-anGal-containing neutral galactan polysaccharide agarose, which has a similar linear chain, -(G-A)n-, although unit A has an l-configuration rather than a d-configuration However, the mechanism for the cleavage at the 1,3-linkage, the reducing side of the anGal residue, is not yet known This highly specific and facile cleavage of galactans by acid hydrolysis is unusual However, Yu et al have recently observed pentasaccharides, heptasaccharides and undecasaccharides among many other hydrolysis products of j-carrageenan isolated by anion exchange chromatography [21] This prompted us to carry out a detailed study of the acid hydrolysis of carrageenans Unexpectedly, we found that the j-carrageenan hydrolysis products are exclusively odd-numbered oligosaccharides, in contrast to the results of various previous studies, in which even-numbered oligosaccharides were found among the mild acid hydrolysis products of 3,6-anGal-containing galactans [15–17,20] This is a very unusual finding, as the polysaccharides are composed of highly ordered disaccharide repeats Understanding the specificity and mechanism of the hydrolysis process is key to its application, as this knowledge will help us to determine the oligosaccharide sequences obtained from partial depolymerization, in order to deduce the overall structure of the parent polysaccharides and to derive structure–function relationships for biological function studies, such as in the investigation of their potent inhibitory antiviral properties [5–8] In this article, we report our detailed investigations on the mechanism of acid hydrolysis of the 3,6-anGal-containing galactans 2126 Results and Discussion Identification of a complete series of odd-numbered oligosaccharides resulting from mild acid hydrolysis of j-carrageenan Polysaccharide j-carrageenan was hydrolyzed under mild acid conditions using 0.1 m H2SO4 at 60 °C for 1.5 h The hydrolytic product was fractionated by gel filtration chromatography As shown in Fig 1A, a series of well-separated peaks with a regular pattern was obtained Each of the eight pooled fractions, K1–K8, was analyzed by negative-ion ESI-MS The presence of multiple sulfates in each fraction gave rise to spectra in which multiply charged ions dominated, and from which the molecular mass and degree of polymerization (DP) of components were determined (Table 1) In the mass spectrum of the slowest-eluting fraction K1, the doubly charged ion at m ⁄ z 322.1 identified a trisaccharide with a molecular mass of 646.2 Da, indicating a composition of two G4S units and one A unit (Table 1), with a likely sequence of G4S-A-G4S It was surprising initially that the shortest fragment identified was a trisaccharide, and not the expected disaccharide as reported previously [15–17,20] The molecular mass of the adjacent fraction K2 was 386 Da higher than that of K1, suggesting a pentasaccharide with an additional A-G4S biose unit (Table 1) A similar regular increment of 386 Da was determined for each of the next six fractions, K3–K8, with DP7 to DP17, respectively The detailed sequences of oddnumbered j-carra-oligosaccharides were corroborated by negative-ion ESI collision-induced dissociation FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS B Yang et al Odd-numbered oligosaccharides from galactan polysaccharides Fig Gel filtration chromatography of j-carra-oligosaccharides and agaro-oligosaccharides and their alditols (A) j-Carraoligosaccharides resulting from mild acid hydrolysis (B) j-Carraoligosaccharide alditols resulting from reductive hydrolysis (C) Agaro-oligosaccharides resulting from mild acid hydrolysis (D) Agaro-oligosaccharide alditols resulting from reductive hydrolysis (E) i-Carra-oligosaccharides resulting from mild acid hydrolysis (-S represents desulfated product) K1 K2 K4 K3 K5 K8 K7 K6 A K R1 K R2 K R3 K R4 K R8 K R7 K R6 KR5 B A1 A2 A3 A4 A5 A8 A7 A6 C (CID) MS ⁄ MS Owing to the lability of the free acid forms of the sulfated molecules, singly charged molecular ions [M ) Na]) of the fully sodiated forms were selected as the precursors [22] As an example, the product-ion spectrum of trisaccharide K1, [M ) Na]) at m ⁄ z 667, is shown in Fig 2A A reducing or nonreducing terminal fragment ion was assigned on the basis of the product-ion spectrum of its alditol after reduction, in which the reducing terminal ions would have a Da increment [23] The intense B2 and C2 ions [22] clearly identified an internal A residue and two terminal G4S residues (Table 1) There were no coeluting even-numbered oligosaccharides detected as minor components in any fraction Therefore, a complete series of exclusively odd-numbered oligosaccharides was obtained from mild acid hydrolysis of j-carrageenan Identification of odd-numbered oligosaccharides resulting from mild acid hydrolysis of agarose AR1 AR2 AR3 AR8 AR7 AR6 AR5 AR4 D I1 I2 I3 I4 I5 I6 I8 I7 E -S -S To investigate whether the unusual finding of oddnumbered oligosaccharides obtained from mild acid hydrolysis was related to the presence of the 3,6-anGal residue, neutral galactan agarose was also subjected to hydrolysis under the same conditions Not surprisingly, gel filtration chromatography of the hydrolysate gave a very similar pattern (Fig 1C) Eight fractions, A1–A8, were collected, and the molecular masses and the DPs of these neutral oligosaccharides were determined by positive-ion MALDI-MS (Table 2) A trisaccharide was identified in fraction A1 with [M + Na]+ at m ⁄ z 509 Odd-numbered agaro-oligosaccharides were identified in fractions A2–A8, each having an additional agarobiose (A-G) with a mass increment of 306 Da (Table 2) Negative-ion ESI-CID-MS ⁄ MS was used to confirm their sequences, and the product-ion spectrum of agaropentasaccharide A2 ([M ) H]) at m ⁄ z 791) is shown in Fig 2B as an example Clearly, a complete series of odd-numbered oligosaccharide fragments was generated by mild acid hydrolysis from the nonsulfated 3,6-anGalcontaining galactan agarose, with the 3,6-anGal residue exclusively at internal positions FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS 2127 Odd-numbered oligosaccharides from galactan polysaccharides B Yang et al Table Negative-ion ESI-MS of j-carra-oligosaccharide fragments obtained from mild and reductive acid hydrolysis Assignment Fractions Ions founda (charge) Calculated molecular mass DP Sequences Theoretical molecular mass K1 K2 K3 K4 K5 K6 K7 K8 KR1 KR2 KR3 KR4 KR5 KR6 KR7 KR8 322.1 343.1 353.6 359.9 364.1 367.1 369.3 371.1 395.1 391.7 390.0 389.0 388.4 387.9 387.6 387.3 646.2 1032.2 1418.4 1804.5 2190.6 2576.7 2962.4 3348.9 792.2 1178.2 1564.2 1950.3 2336.4 2722.4 3108.8 3494.7 11 13 15 17 10 12 14 16 18 G4S-A-G4S G4S-A-G4S-A-G4S G4S-A-G4S-A-G4S-A-G4S G4S-A-G4S-A-G4S-A-G4S-A-G4S G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S G4S-A-G4S-Aol G4S-A-G4S-A-G4S-Aol G4S-A-G4S-A-G4S-A-G4S-Aol G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol 646.1 1032.1 1418.2 1804.2 2190.3 2576.3 2962.4 3348.4 792.1 1178.2 1564.2 1950.3 2336.3 2722.4 3108.4 3494.5 ()2) ()3) ()4) ()5) ()6) ()7) ()8) ()9) ()2) ()3) ()4) ()5) ()6) ()7) ()8) ()9) a Major ion detected; other ions with different charge states and sodiated ion species were all present at much lower intensities and are therefore not listed Analysis of monosaccharide degradation products It has been found in the past, during the investigation of conditions for monosaccharide composition analysis [24–30], that the hydrolyzed monosaccharide 3,6-anGal is not stable at high temperature under strong acidic conditions, e.g m trifluoroacetic acid (TFA) at 120 °C, typically used for complete hydrolysis of galactan into its constituent monosaccharides Upon release, it was readily destroyed and converted into 5-hydroxymethyl-furfural (5-HMF) However, the stability of a 3,6-anGal residue at the reducing terminal of an oligosaccharide under mild acid conditions is not known This prompted us to carry out further detailed analysis of the hydrolysate Normal-phase HPLC was used for analysis of the potential monosaccharide-related degradation products Gal, 3,6-anGal and 5-HMF, which coeluted with the large excess of salt in gel filtration chromatography (Fig 1), and the elution profiles of which are shown in Fig 3A Gal and 3,6-anGal not have a UV chromophore and can only be detected by an evaporative light scattering detector (ELSD), whereas the furancontaining 5-HMF is readily detectable by UV The hydrolysis products from j-carrageenan and agarose produced by 0.1 m TFA at various reaction time intervals were analyzed The reaction products at h are shown in Fig 3B, and those obtained at other time intervals were similar to this, although the relative 2128 intensities of the peaks were different The fraction with high absorption at 280 nm was collected and further analyzed by GC-MS It gave a GC peak at 15.65 (Fig 3D) and a mass spectrum (Fig 3E) identical to the library spectrum of 5-HMF The content of 5-HMF increased, whereas the content of 3,6-anGal decreased, with increasing reaction time (data not shown) It is highly likely, therefore, that a 3,6-anGal residue at the reducing terminal of an oligosaccharide is unstable even under the mild acid condition Following mild acid hydrolysis, even-numbered oligosaccharides with Gal at the nonreducing terminus and 3,6-anGal at the reducing terminus were initially obtained As the reducing terminal 3,6-anGal is unstable, we believe that it is immediately hydrolyzed from the even-numbered oligosaccharides originally formed, resulting in odd-numbered oligosaccharides, and the cleaved monosaccharide 3,6-anGal is further degraded to 5-HMF [25] Complete series of even-numbered oligosaccharide fragments resulting from reductive hydrolysis Various methods have been developed previously to prevent the degradation of the unstable monosaccharide 3,6-anGal during monosaccharide composition analysis [31] Conversion of monosaccharides into their alditols by reduction has been conventionally used for FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS B Yang et al Odd-numbered oligosaccharides from galactan polysaccharides % relative intensity A % relative intensity B % relative intensity C Fig Negative-ion ESI-CID-MS ⁄ MS product-ion spectra (A) j-Carra-trisaccharide (B) Agaro-pentasaccharide (C) j-Carra-tetrasaccharide alditol (D) Agaro-hexasaccharide alditol % relative intensity D FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS 2129 Odd-numbered oligosaccharides from galactan polysaccharides B Yang et al Table Positive-ion MALDI-MS of agaro-oligosaccharides obtained by mild and reductive acid hydrolysis Assignment Fractions Found MNa+ DP Sequences Theoretical MNa+ A1 A2 A3 A4 A5 A6 A7 A8 AR1 AR2 AR3 AR4 AR5 AR6 AR7 AR8 509.4 815.2 1121.3 1427.5 1733.4 2039.4 2345.4 2651.2 655.2 961.3 1267.3 1573.4 1879.5 2185.0 2491.0 2797.2 11 13 15 17 10 12 14 16 18 G-A-G G-A-G-A-G G-A-G-A-G-A-G G-A-G-A-G-A-G-A-G G-A-G-A-G-A-G-A-G-A-G G-A-G-A-G-A-G-A-G-A-G-A-G G-A-G-A-G-A-G-A-G-A-G-A-G-A-G G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G G-A-G-Aol G-A-G-A-G-Aol G-A-G-A-G-A-G-Aol G-A-G-A-G-A-G-A-G-Aol G-A-G-A-G-A-G-A-G-A-G-Aol G-A-G-A-G-A-G-A-G-A-G-A-G-Aol G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-Aol G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-Aol 509.1 815.2 1121.3 1427.4 1733.5 2039.6 2345.7 2651.8 655.2 961.3 1267.4 1573.5 1879.6 2185.7 2491.8 2797.9 this purpose [25,32] We attempted a similar procedure to stabilize oligosaccharides with 3,6-anGal residues at the reducing termini Hydrolysis was carried out in the presence of the reducing agents sodium borohydride or 4-methylmorpholine borane (MMB); both reducing agents gave identical results (Fig S1) The hydrolysis products from j-carrageenan were analyzed by PAGE; the reductive hydrolysate gave a series of discrete bands (Fig 4A, lane 1) with different mobilities from those of the bands obtained from the nonreductive hydrolysate (Fig 4A, lane 3) Similar results were obtained with HP-TLC, in which the bands of the reductive hydrolysate (Fig 4B, lane 2) had different mobilities from those of the nonreductive hydrolysate (Fig 4B, lane 1) The products from reductive hydrolysis were fractionated by gel filtration chromatography, and eight fractions, KR1–KR8, were pooled (Fig 1B) The retention times of the fractions were clearly different from those of the fractions obtained from nonreductive hydrolysis (Fig 1A) The molecular masses and DPs determined by negative-ion ESI-MS (Table 1) unambiguously identified a complete series of even-numbered alditols Negative-ion ESI-CID-MS ⁄ MS of the j-carra-tetrasaccharide alditol KR2, using its sodiated ion (m ⁄ z 813) as the precursor, indicated the predicted sequence of G4SA-G4S-Aol (Fig 2C) No reducing terminal monosaccharide anGal or its degradation product 5-HMF (and their reduced forms) was detected with HPLC analysis (Fig 3C) A complete series of even-numbered oligosaccharide alditols was similarly obtained from 3,6-anGal-containing agarose The reductive acid hydrolysate of agarose also showed different mobilities from those of the 2130 nonreductive hydrolysate on HP-TLC (Fig 4B, lanes and 4) The identities of oligosaccharide fragments (fractions AR1–AR8, Fig 1D), following their fractionation by gel filtration chromatography, were confirmed by positive-ion MALDI-MS (Table 2) The sequences were unambiguously identified by ES-CIDMS ⁄ MS, as illustrated by the product-ion spectrum of agaro-hexasaccharide AR2 ([M ) H]- at m ⁄ z 937) The almost full set of sequence ions clearly indicated a hexasaccharide G-A-G-A-G-Aol, with a reduced terminal 3,6-anGalol (Fig 2D) The results indicated that the reducing terminal 3,6anGal is labile but can be stabilized by reduction Therefore, the primary acid hydrolysis products, evennumbered oligosaccharides, can be preserved as alditols It is interesting to note that reduction can be carried out with both MMB and sodium borohydride The former is acid-stable, whereas the latter decomposes in an acidic medium The fact that borohydride can be used as an effective reducing agent under mild acidic conditions despite its instability highlights the fast rate of reduction Effect of acidity on acid hydrolysis Acid hydrolysis of polysaccharides is conventionally carried out in strong mineral acid and, as described above, j-carrageenan and agarose can be readily cleaved under mild conditions by H2SO4 (pKa: )3) We further examined acid hydrolysis under the same mild conditions with weaker organic acids, including TFA (pKa: 0.23), oxalic acid (pKa1: 1.23), maleic acid (pKa1: 1.83), phthalic acid (pKa1: 2.89), citric acid FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS B Yang et al Odd-numbered oligosaccharides from galactan polysaccharides A B C D Fig Analysis of monosaccharide degradation products (A) HPLC of standard 5-HMF, 3,6-anGal and Gal detected by UV (280 nm) and ELSDs, respectively (B) Hydrolysate obtained from j-carrageenan with 0.1 M TFA at 60 °C for h (C) Reductive hydrolysate obtained from j-carrageenan with 0.1 M TFA at 60 °C for h (D) GC-MS total ion current chromatogram of the HPLC fraction of j-carrageenan hydrolysate with high absorption at UV 280 nm (E) Mass spectrum of the fraction at 15.65 E (pKa1: 3.13), formic acid (pKa: 3.75), succinic acid (pKa1: 4.16), and acetic acid (pKa: 4.75) The hydrolysates from j-carrageenan were analyzed by PAGE (Fig 5), and selected fractions were analyzed by ESIMS (not shown) Clearly, even with these weaker organic acids, the same odd-numbered j-carrageenan oligosaccharides were obtained, indicating the uniquely facile nature of the hydrolysis Effect of the 3,6-anGal form on the hydrolysis of galactan As the initial glycosidic cleavage by mild acid hydrolysis of galactan j-carrageenan and agarose takes place at the reducing side, and subsequent cleavage at the nonreducing side, of the 3,6-anGal residue to give oddnumbered oligosaccharide fragments, it is highly likely that the 3,6-anGal form of the galactose has a major effect on the specificity of the hydrolysis It is important to compare directly, and investigate in detail, a pair of polysaccharides or oligosaccharides with a similar galactan sequence but with Gal substituting for the 3,6-anGal residues Unfortunately, such a pair is not available We selected k-carrageenan and carried out desulfation to prepare a polysaccharide with a nonsulfated sequence of -(4Gala1-3Galb1)n-, similar to that of agarose, -[4(3,6-anGal)a1-3Galb1]n-, apart from the anhydro form in the latter The structure of the FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS 2131 Odd-numbered oligosaccharides from galactan polysaccharides A B Yang et al B DP’ DP DP 18 16 14 17 15 11 10 8 6 13 12 DP DP’ DP 7 1 3 Fig Analyses of acid hydrolysis products obtained from j-carrageenan and agarose (A) PAGE analysis: lane 1, reductive hydrolysate of j-carrageenan; lane 2, a mixture of reductive and nonreductive hydrolysates of j-carrageenan; lane 3, nonreductive mild acid hydrolysate of j-carrageenan (B) HP-TLC analysis: lane 1, mild acid hydrolysate of j-carrageenan; lane 2, reductive hydrolysate of j-carrageenan; lane 3, mild acid hydrolysate of agarose; lane 4, reductive hydrolysate of agarose Arrow indicates the origin DP DP 19 18 17 16 15 14 13 12 11 19 17 15 13 11 10 5 3 10 Fig PAGE analysis of acid hydrolysis products obtained from j-carrageenan with various acids Lane 1: H2SO4 (pKa: ) 3) Lane 2: H2SO4 under reducing conditions Lane 3: acetic acid (pKa: 4.76) Lane 4: succinic acid (pKa1: 4.16) Lane 5: formic acid (pKa: 3.77) Lane 6: citric acid (pKa1: 3.13) Lane 7: fumaric acid (pKa1: 3.03) Lane 8: phthalic acid (pKa1: 2.89) Lane 9: maleic acid (pKa1: 1.83) Lane 10: oxalic acid (pKa1: 1.23) Lane 11: TFA (pKa: 0.23) Arrow indicates the origin desulfated k-carrageenan was confirmed by 13C-NMR and GC-MS (Figs S2 and S3, respectively) Mild acid hydrolysis was carried out with the nonsulfated anGal-lacking k-carrageenan, without success 2132 10 11 Fig HP-TLC analyses of acid hydrolysis products of agarose and nonsulfated k-carrageenan polysaccharides and heptasaccharides Lane 1: agarose treated with 0.1 M H2SO4 at 60 °C for 1.5 h Lane 2: agaro-heptasaccharide Lanes 3–5: agaro-heptasaccharide treated with 0.1 M H2SO4 at 60 °C for 15, 30 and 45 min, respectively Lane 6: nonsulfated k-carrageenan treated with 0.1 M H2SO4 at 80 °C for h Lane 7: nonsulfated k-carrageenan heptasaccharide Lane 8: nonsulfated k-carrageenan heptasaccharide treated with 0.1 M H2SO4 at 60 °C for h Lanes 9–11: nonsulfated k-carrageenan heptasaccharide treated with 0.1 M H2SO4 at 80 °C for 2, and h, respectively Arrow indicates the origin Only at a higher temperature and with a longer reaction time (e.g 80 °C, h) was the polysaccharide hydrolyzed and both odd-numbered and evennumbered oligosaccharides obtained, as identified by HP-TLC (Fig 6, lane 6) and MS (data not shown) However, the anGal-containing agarose was readily hydrolyzed under the mild conditions (Fig 6, lane 1) To compare further the difference in the hydrolysis, heptasaccharide pairs derived from agarose and nonsulfated k-carrageenan were prepared (Fig 6, lanes and 7, respectively) and used for hydrolysis Under the mild conditions, agaro-heptasaccharide was readily hydrolyzed (Fig 6, lane 3) Further incubation did not result in even-numbered oligosaccharides, but only increased the content of monosaccharides and trisaccharides (Fig 6, lanes and 5) However, under these conditions, no hydrolysis product was observed for the desulfated k-carra-heptasaccharide (Fig 6, lane 8) Only under forcing conditions can hydrolysis of the desulfated k-carra-heptasaccharide take place and both odd-numbered and even-numbered oligosaccharides be generated (Fig 6, lanes 9–11) The identities of the hydrolytic products dL2–dL6, together with that of the parent heptasaccharide dL7, were confirmed by MS analysis (Table 3) As the cleavage took place in a random fashion, and the b1–4 and a1–3 linkages could be similarly cleaved, both odd-numbered and even- FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS B Yang et al Odd-numbered oligosaccharides from galactan polysaccharides Table Negative-ion ESI-MS of mild acid hydrolysis products obtained from a heptasaccharide of desulfated k-carrageenan Assignment Fractions Found [M ) H]) DP dL2 dL3 dL4 dL5 dL6 dL7 341.1 503.1 665.1 827.2 989.2 1151.4 G-D G-D-G G-D-G-D G-D-G-D-G G-D-G-D-G-D G-D-G-D-G-D-G Theoretical [M ) H]) Sequences numbered oligosaccharides were obtained with each series in comparable amounts (Fig 6, lanes 6, and 10) The results clearly demonstrated that the 3,6-anGal residue has a profound effect on the hydrolysis of galactan polysaccharides, and that the high specificity of cleavage by acid hydrolysis is due to the 3,6-anGal residue Effect of 2-O-sulfate on the stability of 3,6-anGal We investigated the effect of sulfate substitution on the 2-OH of 3,6-anGal (the only free hydroxyl group of the residue) on the stability of the glycosidic bond at the nonreducing side and, therefore, the effect on the release of the reducing terminal 3,6-anGal residue The 2-O-sulfated 3,6-anGal occurs widely in carrageenans as the i-carrabiose unit -(G4S-A2S)- The difference between j-carrageenan and i-carrageenan is the additional 2-O-sulfation of 3,6-anGal in the latter Degradation of i-carrageenan under mild acid conditions required a longer time [25,29] (3 h) than that for j-carrageenan (1.5 h) The hydrolysate was fractionated by gel filtration chromatography (Fig 1E) The oligosaccharide fractions (I1–I8) were analyzed by ESI-MS and MS ⁄ MS (Fig S4) The molecular masses and sequences obtained were in agreement with those of even-numbered i-carrageenan oligosaccharides of the carra-series [-(G4S-A2S)n-]; for example, I1 was a disaccharide, I2 a tetrasaccharide, and I8 a hexadecasaccharide Some minor desulfation products were also detected, owing to the effect of the prolonged reaction time It is well known that 2-O-sulfation stabilizes its glycosidic bond (at the reducing side), so that stronger conditions are required for its hydrolysis [25,29] However, the results with i-carrageenan indicated that 2-Osulfation of 3,6-anGal also has a major stabilizing effect on the glycosidic bond at the nonreducing side The 2-O-sulfated 3,6-anGal residue at the reducing termini produced by the acid hydrolysis was stable, and could not be released by cleavage of the glycosidic D-G D-G-D D-G-D-G D-G-D-G-D D-G-D-G-D-G D-G-D-G-D-G-D 341.3 503.4 665.6 827.7 989.9 1152.0 bond at its nonreducing side to give odd-numbered oligosaccharides The effect is very similar to that of reduction Conclusions Acid hydrolysis is a classic method for depolymerization of polysaccharides Generally, for a polysaccharide with highly ordered disaccharide repeats (such as the GAGs), if selective cleavage takes place, even-numbered oligosaccharides are normally produced In the case of random and nonspecific cleavage, both oddnumbered and even-numbered oligosaccharides with the two different residues at both termini are generated There has been no report that a complete series of exclusively odd-numbered oligosaccharide fragments can be produced from such a polysaccharide, and it is highly unusual for this to happen We have proposed a two-step cleavage for the mild acid hydrolysis of 3,6anGal-containing galactans (Scheme 1): initial cleavage of the a1–3 glycosidic bond at the reducing side of the 3,6-anGal residue to give even-numbered oligosaccharides with a Gal at the nonreducing terminus and 3,6anGal at the reducing terminus, followed by immediate removal of the newly created unstable reducing terminal 3,6-anGal at the b1–4 bond to give odd-numbered oligosaccharides with Gal at both termini The labile reducing terminal 3,6-anGal can be stabilized by conversion into alditol via reduction and by 2-O-sulfation Clearly, the 3,6-anGal residue has a profound effect on the hydrolysis of galactan polysaccharides, leading to highly specific and facile cleavage Experimental procedures Materials The polysaccharides j-carrageenan (type III, from Eucheuma cottonii), i-carrageenan (type V, Eucheuma spinosa), and k-carrageenan (type IV, from Gigartina aciculaire), and agarose (type I, low electroendosmosis), MMB, NaBH4, FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS 2133 Odd-numbered oligosaccharides from galactan polysaccharides O O OH O O OH Polysaccharides O O O OR OH O O OH O O O O OR OR' OR' OR' O B Yang et al OH O OR O OH OR' (R=H, SO3Na; R'=SO3Na) H+ (R=H, SO3Na) OH O HO OH O O O O OH O OH O O O O OH OR H+ OH OR' OH O HO OH O O O O OH O O OH CH2OH OR OR' OH O O OR' O OH OR OR' (R=H) H O OH O O Even-numbered oligosaccharide alditols OH OR (R=SO3Na) + O O BH4– OR' OR' OR' OH O O O O O OSO3Na OH O OH O O O OH OSO3Na O O CHO O Even-numbered oligosaccharides OH OH H+ OR' OR' OH O O OH O O O OH O O OH O O OH + O CHO HO OH Odd-numbered oligosaccharides 3,6-anGal (furanose) HO-CH2 O HO O OH OH 3,6-anGal (pyranose) CHO 5-HMF Scheme Proposed mechanism for the mild acid hydrolysis of galactan polysaccharides 5-HMF, galactose, 3,6-anhydrogalactose, and ion exchange resin Amberlite IR 120 (H+ form), were purchased from Sigma-Aldrich (Shanghai, China) Chlorotrimethylsilane (CTMS) was from J&K Chemical (Beijing, China) The Superdex 30 column (30 lm, 1.6 · 60 cm), Superdex Peptide HR column (10 · 300 mm) and Q-Sepharose Fast Flow ion exchange resin were from Pharmacia Bioscience (Uppsala, Sweden) The Aminex HPX-87H column (300 · 7.8 mm, lm) and AG50W-X8 ion exchange resin were obtained from Bio-Rad Laboratories (Hemel Hempstead, UK) Fused-silica capillary columns HP-5MS (30 m · 0.32 mm, internal diameter 0.25 mm) and DB-225MS (30 m · 0.32 mm, internal diameter 0.25 mm) were purchased from J&W Scientific (Folsom, CA, USA) Aluminum-backed silica gel 60 HP-TLC plates were from Merck (Darmstadt, Germany) All other reagents and solvents used were of analytical grade Mild acid hydrolysis Large-scale (100 mg) acid hydrolyses of the polysaccharides j-carrageenan and agarose were carried out typically with 0.1 m H2SO4 (10 mgỈmL)1) at 60 °C for 1.5 h, 2134 similarly to the published procedure using HCl [21] Hydrolysis of i-carrageenan was extended to h Smallscale (10 mg) hydrolysis of j-carrageenan was also carried out with various organic acids, including TFA, oxalic acid, maleic acid, phthalic acid, fumaric acid, citric acid, formic acid, succinic acid, and acetic acid, at 60 °C for 1.5 h Reductive hydrolysis of j-carrageenan and agarose was carried out on a large scale with addition of 0.2 m MMB at 60 °C for 1.5 h or 0.2 m NaBH4 at 60 °C for h The reaction was terminated by neutralization with m NaOH before analysis Analysis and preparation of oligosaccharides For HP-TLC analysis, aliquots ( 0.4 lL) of samples were applied to a TLC plate and developed in n-butanol ⁄ formic acid ⁄ water (4 : : 1, v ⁄ v ⁄ v) Plates were stained by dipping them in diphenylamine ⁄ aniline ⁄ phosphoric acid reagent for s, and then heating them at 105 °C for for color development, as described previously [33] For PAGE, continuous gel with 22% polyacrylamide was used, and PAGE was performed on a vertical slab (0.1 · · 10 cm) gel system The gel was loaded with FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS B Yang et al Odd-numbered oligosaccharides from galactan polysaccharides 20–50 lg of sample, and subjected to electrophoresis at 200 V for h The gel was stained with Alcian blue (0.5% in 2% AcOH) and destained with 2% AcOH [21,34,35] For oligosaccharide preparation, the hydrolysates were concentrated by lyophilization and subjected to gel ltration ă chromatography using the AKTA-FPLC (Pharmacia Biotech, Sweden) system with a Superdex 30 column, as previously described [15] Elution was carried out with 0.1 m NH4HCO3 for both acidic and neutral oligosaccharides, at a flow rate of 0.2 mLỈmin)1, and detected by a refractive index detector Fractions were pooled, and the volatile buffer was removed by repeated lyophilization with water The nonsulfated k-carrageenan was then hydrolyzed with 0.1 m H2SO4 (10 mgỈmL)1) at 80 °C for h The oligosaccharide products were fractionated and purified with a Superdex 30 and a Superdex Peptide column, respectively Hydrolysis of heptasaccharides (250 lg) derived from nonsulfated k-carrageenan and agarose was carried out with 0.1 m H2SO4 (10 lgỈlL)1) at 60 °C and 80 °C, respectively The hydrolysis products were analyzed by HP-TLC Following sample application with a Linomat V TLC applicator (Camag Scientific, Switzerland), the TLC plate was developed and stained as described above MS Detection and characterization of monosaccharide degradation products The hydrolysate (10 lL) was analyzed by HPLC (LC-10Ai; Shimadzu, Kyoto, Japan) on an Aminex HPX-87H column [36], with elution by 40% aqueous CH3CN containing 0.01 m TFA, at a flow rate of 0.6 mLỈmin)1 Detection was by UV (280 nm) and ELSDs in series The former was for the detection of 5-HMF, and the latter for the detection of 3,6-anGal The identity of 5-HMF was confirmed by GC-MS analysis following its collection from HPLC [37] An Agilent 6980 system equipped with an HP-5MS fused-silica capillary column was used The injector temperature was set at 220 °C Helium was used as carrier gas, at a flow rate of 1.0 mLỈmin)1 The oven temperature was initially kept at 50 °C for min, then increased to 250 °C at a rate of °CỈmin)1, and finally kept at 250 °C for 10 The ion source temperature at 280 °C, and the ionization energy was 80 eV The mass spectrum acquired was compared with the NIST library spectrum Desulfation of k-carrageenan and hydrolysis of the desulfated product Desulfation was performed essentially as previously described [38,39], with some modifications In brief, k-carrageenan (100 mg) was converted into its pyridinium salt by cation exchange, and desulfation of the pyridinium salt of k-carrageenan was carried out in anhydrous pyridine with CTMS (CTMS ⁄ sulfate molar ratio 400 : 1) in a sealed reaction vial at 100 °C for h The excess CTMS was destroyed by hydrolysis with water, and the desulfated product was recovered by dialysis The desulfated product was purified by anion exchange chromatography with an ă AKTA-FPLC system on a Q-Sepharose Fast Flow column (1.5 · cm) The column was eluted with 50 mL of H2O at a flow rate of 0.5 mLỈmin)1 The eluant was lyophilized to give a nonsulfated k-carrageenan, and its structure was confirmed by 13C-NMR and GC-MS [40] to have the repeating disaccharide sequence -(4Gala1-3Galb1)n- Negative-ion ESI-MS was performed on Micromass Q-Tof or Q-Tof Ultima instruments (Waters, Manchester, UK) for the sulfated oligosaccharides, as previously described [41] Nitrogen was used as the desolvation and nebulizer gas, at flow rates of 250 LỈh)1 and 15 LỈh)1, respectively The source temperature was 80 °C, and the desolvation temperature was 150 °C Samples were dissolved in CH3CN ⁄ mm NH4HCO3 (1 : 1, v ⁄ v), typically at a concentration of 5–10 pmolỈlL)1, of which lL was loopinjected The mobile phase (CH3CN ⁄ mm NH4HCO3, : 1, v ⁄ v) was delivered by a syringe pump at a flow rate of lLỈmin)1 The capillary voltage was maintained at kV and the cone voltage was 50–120 V, depending on the size of oligosaccharides For CID-MS ⁄ MS product-ion scanning, argon was used as the collision gas at a pressure of 1.7 bar, and the collision energy was adjusted between 17 and 100 eV for optimal sequence information Neutral agaro-oligosaccharides were analyzed by positive-ion MALDI-MS with a Tof Spec 2E instrument (Waters, Manchester, UK), with 1,2-diamino-4,5-methylene dioxybenzene as the matrix Acknowledgements This study was supported in part by the International Science and Technology Cooperation Program of China (2007DFA30980), the National Basic Research Program of China (2003CB716401), the OUC Luka Program (1405-814147), and a UK Medical Research Council research grant (G0600512) References Mourao PA & Pereira MS (1999) Searching for alternatives to heparin: sulfated fucans from marine invertebrates Trends Cardiovasc Med 9, 225– 232 McCarthy B (2007) Antivirals – an increasingly healthy investment Nat Biotechnol 25, 1390–1393 FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 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Desulfation of sulfated galactans with chlorotrimethylsilane Characterization of beta-carrageenan by 1H NMR spectroscopy Carbohydr Res 339, 1619–1629 40 Ciucanu I & Costello CE (2003) Elimination of oxidative degradation during the per-O-methylation of carbohydrates J Am Chem Soc 125, 16213–16219 41 Chai W, Luo J, Lim CK & Lawson AM (1998) Characterization of heparin oligosaccharide mixtures as ammonium salts using electrospray mass spectrometry Anal Chem 70, 2060–2066 Supporting information The following supplementary material is available: Fig S1 PAGE analysis of reductive acid hydrolysis products from j-carrageenan Fig S2 13C-NMR spectrum of desulfated k-carraeenan Fig S3 GC-MS analysis of desulfated k-carraeenan Fig S4 Negative-ion ESI-CID-MS ⁄ MS product-ion spectrum of i-carra-tetrasaccharide This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS 2137 ... G -A- G G -A- G -A- G G -A- G -A- G -A- G G -A- G -A- G -A- G -A- G G -A- G -A- G -A- G -A- G -A- G G -A- G -A- G -A- G -A- G -A- G -A- G G -A- G -A- G -A- G -A- G -A- G -A- G -A- G G -A- G -A- G -A- G -A- G -A- G -A- G -A- G -A- G G -A- G-Aol G -A- G -A- G-Aol G -A- G -A- G -A- G-Aol... G -A- G -A- G-Aol G -A- G -A- G -A- G-Aol G -A- G -A- G -A- G -A- G-Aol G -A- G -A- G -A- G -A- G -A- G-Aol G -A- G -A- G -A- G -A- G -A- G -A- G-Aol G -A- G -A- G -A- G -A- G -A- G -A- G -A- G-Aol G -A- G -A- G -A- G -A- G -A- G -A- G -A- G -A- G-Aol 509.1 815.2 1121.3... TFA, oxalic acid, maleic acid, phthalic acid, fumaric acid, citric acid, formic acid, succinic acid, and acetic acid, at 60 °C for 1.5 h Reductive hydrolysis of j-carrageenan and agarose was carried