Báo cáo khoa học: Mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively odd-numbered oligosaccharides

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

1 Glycoscience and Glycoengineering Laboratory, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China 2 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

from polysaccharides

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-carra- geenan 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 frag- ments from its hydrolytic products. Detailed sequence analysis of the prod- ucts 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 oligo- saccharides composed of ordered disaccharide repeats.

(Received 6 November 2008, revised 30 December 2008, accepted 4 February 2009)

doi:10.1111/j.1742-4658.2009.06947.x

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 papillo- mavirus [7,8], and hence they have become of consider- able biomedical interest, owing to their antiviral activities and therapeutic potential.

The diverse oligosaccharide sequences present in poly- saccharides, glycoproteins, glycolipids and proteogly- cans serve multiple functions. Acidic polysaccharide glycosaminoglycans (GAGs) are ubiquitous in verte- brate 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-

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.

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-[3-linked

the

specificity

and mechanism of

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 4- linked galactose, and the differing sulfate contents and substitutions; for example, j-carrageenan, i-carrageenan and k-carrageenan have different disaccharide building 4-O-sulfated-b-d-galactopyranose blocks: (G4S)-A]n-, -[G4S-4-linked 2-O-sulfated-a-d-3,6-anhy- drogalactose (A2S)]n-, and -[3-linked 2-O-sulfated-b-d- galactose (G2S)-4-linked 2,6-O-sulfated-a-d-galactose (D2S6S)]n-, respectively [9].

Unexpectedly, we found that the j-carrageenan hydro- lysis products are exclusively odd-numbered oligosac- charides, 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 com- posed of highly ordered disaccharide repeats. Under- the standing hydrolysis process is key to its application, as this knowledge will help us to determine the oligosaccha- ride sequences obtained from partial depolymerization, in order to deduce the overall structure of the parent polysaccharides and to derive structure–function rela- tionships for biological function studies, such as in the investigation of their potent inhibitory antiviral prop- erties [5–8]. In this article, we report our detailed inves- tigations on the mechanism of acid hydrolysis of the 3,6-anGal-containing galactans.

Results and Discussion

Identification of a complete series of odd-numbered oligosaccharides resulting from mild acid hydrolysis of j-carrageenan

reported

Detailed knowledge of these polysaccharide struc- tures is necessary for an in-depth understanding of their biological roles. However, their structural com- in sequence plexity causes considerable difficulties analysis and assignment of structure–function relation- ships. 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 cleav- age at selected glycosidic bonds without the risk of modification of the native structures. However, suit- able enzymes are not always available for all polysac- charides. 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-contain- ing carrageenans (e.g. j-carrageenan and i-carra- geenan), mild acid hydrolysis has been used, and is considered to cleave the 1,3-linkages, producing even- numbered 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 -(G- A)n-, without affecting 1,4-linkages. Similar results were obtained with 3,6-anGal-containing neutral galac- tan polysaccharide agarose, which has a similar linear chain, -(G-A)n-, although unit A has an l-configura- tion rather than a d-configuration. However, the mech- anism 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.

Polysaccharide j-carrageenan was hydrolyzed under mild acid conditions using 0.1 m H2SO4 at 60 (cid:2)C for 1.5 h. The hydrolytic product was fractionated by gel filtration chromatography. As shown in Fig. 1A, a ser- ies 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 polymeriza- tion (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, indi- cating 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 iden- tified was a trisaccharide, and not the expected disac- [15–17,20]. The previously charide as molecular mass of fraction K2 was the adjacent 386 Da higher than that of K1, suggesting a pentasac- charide 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 odd- numbered j-carra-oligosaccharides were corroborated collision-induced dissociation by negative-ion ESI

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Odd-numbered oligosaccharides from galactan polysaccharides

A

their

alditols.

agaro-oligosaccharides

(A)

resulting

alditols

4 K

2 K

6 K

5 K

3 K

1 K

8 K

7 K

Fig. 1. Gel filtration chromatography of j-carra-oligosaccharides and j-Carra- and (B) j-Carra- oligosaccharides resulting from mild acid hydrolysis. oligosaccharide hydrolysis. from reductive (C) Agaro-oligosaccharides resulting from mild acid hydrolysis. (D) Agaro-oligosaccharide alditols resulting from reductive hydro- lysis. (E) i-Carra-oligosaccharides resulting from mild acid hydrolysis (-S represents desulfated product).

B

5 R K

3 R K

7 R K

6 R K

2 R K

8 R K

4 R K

1 R K

(CID) MS ⁄ MS. Owing to the lability of the free acid forms of the sulfated molecules, singly charged molec- ular 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 nonre- ducing terminal fragment ion was assigned on the basis of the product-ion spectrum of its alditol after reduc- tion, in which the reducing terminal ions would have a 2 Da increment [23]. The intense B2 and C2 ions [22] clearly identified an internal A residue and two termi- nal G4S residues (Table 1).

C

5 A

4 A

2 A

3 A

8 A

7 A

6 A

1 A

There were no coeluting even-numbered oligosaccha- rides detected as minor components in any fraction. Therefore, a complete series of exclusively odd-num- bered oligosaccharides was obtained from mild acid hydrolysis of j-carrageenan.

Identification of odd-numbered oligosaccharides resulting from mild acid hydrolysis of agarose

D

8 R A

2 R A

5 R A

4 R A

7 R A

6 R A

1 R A

3 R A

E

3 I

5 I

4 I

2 I

6 I

7 I

8 I

1 I

-S

-S

To investigate whether the unusual finding of odd- numbered 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 agaro- biose (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 agaro- pentasaccharide 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-anGal- containing galactan agarose, with the 3,6-anGal residue exclusively at internal positions.

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Table 1. 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

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

K1 K2 K3 K4 K5 K6 K7 K8 KR1 KR2 KR3 KR4 KR5 KR6 KR7 KR8

322.1 ()2) 343.1 ()3) 353.6 ()4) 359.9 ()5) 364.1 ()6) 367.1 ()7) 369.3 ()8) 371.1 ()9) 395.1 ()2) 391.7 ()3) 390.0 ()4) 389.0 ()5) 388.4 ()6) 387.9 ()7) 387.6 ()8) 387.3 ()9)

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

3 5 7 9 11 13 15 17 4 6 8 10 12 14 16 18

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

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

intensities of the peaks were different. The fraction with high absorption at 280 nm was collected and fur- ther analyzed by GC-MS. It gave a GC peak at 15.65 min (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 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. 2 m trifluoroacetic acid (TFA) at 120 (cid:2)C, typically used for complete hydrolysis of galac- tan into its constituent monosaccharides. Upon release, it was readily destroyed and converted into 5-hydrox- ymethyl-furfural (5-HMF). However, the stability of a 3,6-anGal residue at the reducing terminal of an oligo- saccharide under mild acid conditions is not known. This prompted us to carry out further detailed analysis of the hydrolysate.

and the

is

It is highly likely, therefore, that a 3,6-anGal residue at the reducing terminal of an oligosaccharide is unsta- ble 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 is unstable, we believe reducing terminal 3,6-anGal that it is immediately hydrolyzed from the even-num- bered oligosaccharides originally formed, resulting in odd-numbered oligosaccharides, cleaved monosaccharide 3,6-anGal further degraded to 5-HMF [25].

Complete series of even-numbered oligosaccharide fragments resulting from reductive hydrolysis

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 do not have a UV chro- mophore and can only be detected by an evaporative light scattering detector (ELSD), whereas the furan- containing 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 inter- vals were analyzed. The reaction products at 2 h are shown in Fig. 3B, and those obtained at other time intervals were similar to this, although the relative

Various methods have been developed previously to prevent the degradation of the unstable monosaccha- ride 3,6-anGal during monosaccharide composition analysis [31]. Conversion of monosaccharides into their alditols by reduction has been conventionally used for

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A

l

y t i s n e t n i e v i t a e r

%

B

l

y t i s n e t n i e v i t a e r

%

C

l

y t i s n e t n i e v i t a e r

%

D

l

y t i s n e t n i e v i t a e r

%

Fig. 2. Negative-ion ESI-CID-MS ⁄ MS prod- uct-ion spectra. (A) j-Carra-trisaccharide. (B) Agaro-pentasaccharide. (C) j-Carra-tetrasac- charide alditol. (D) Agaro-hexasaccharide alditol.

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Table 2. Positive-ion MALDI-MS of agaro-oligosaccharides obtained by mild and reductive acid hydrolysis.

Assignment

Sequences

DP

Theoretical MNa+

Fractions

Found MNa+

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

509.4 815.2 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

3 5 7 9 11 13 15 17 4 6 8 10 12 14 16 18

A1 A2 A3 A4 A5 A6 A7 A8 AR1 AR2 AR3 AR4 AR5 AR6 AR7 AR8

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

Fig. 1D),

following

full

nonreductive hydrolysate on HP-TLC (Fig. 4B, lanes 3 and 4). The identities of oligosaccharide fragments (fractions AR1–AR8, their fractionation by gel filtration chromatography, were confirmed by positive-ion MALDI-MS (Table 2). The sequences were unambiguously identified by ES-CID- MS ⁄ MS, as illustrated by the product-ion spectrum of agaro-hexasaccharide AR2 ([M ) H]- at m ⁄ z 937). The almost 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 3,6- anGal is labile but can be stabilized by reduction. Therefore, the primary acid hydrolysis products, even- numbered 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 decom- poses 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

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 hydroly- sate (Fig. 4B, lane 2) had different mobilities from those of the nonreductive hydrolysate (Fig. 4B, lane 1). The products from reductive hydrolysis were fraction- ated by gel filtration chromatography, and eight frac- tions, 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-tetrasac- charide alditol KR2, using its sodiated ion (m ⁄ z 813) as the precursor, indicated the predicted sequence of G4S- A-G4S-Aol (Fig. 2C). No reducing terminal monosac- charide 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-contain- ing agarose. The reductive acid hydrolysate of agarose the also showed different mobilities from those of

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

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A

B

C

D

E

Fig. 3. Analysis of monosaccharide degrada- tion 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 (cid:2)C for 2 h. (C) Reductive hydrolysate obtained from j-carrageenan with 0.1 M TFA at 60 (cid:2)C for 2 h. (D) GC-MS total ion current chromatogram of the HPLC fraction of j-car- rageenan hydrolysate with high absorption at UV 280 nm. (E) Mass spectrum of the fraction at 15.65 min.

(pKa1: 3.13), formic acid (pKa: 3.75), succinic acid (pKa1: 4.16), and acetic acid (pKa: 4.75). The hydroly- sates from j-carrageenan were analyzed by PAGE (Fig. 5), and selected fractions were analyzed by ESI- MS (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 hydroly- sis 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 odd- numbered 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 impor- tant to compare directly, and investigate in detail, a pair of polysaccharides or oligosaccharides with a simi- lar 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 nonsulf- ated sequence of -(4Gala1-3Galb1)n-, similar to that of agarose, -[4(3,6-anGal)a1-3Galb1]n-, apart from the the anhydro form in the latter. The structure of

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B

A

DP’

DP

1

DP

2 3

3

DP 3 4

17

DP’ 3 4

15

5 6

DP 18 16 14

5

13

5 6

12

4 5 6 7

11

7

7 8

10

7 8

9

8

7

6

5

3

5

6

8

9

10

1

2

4

7

11

3

1

2

3

1

2

4

lane 2, a mixture of

Fig. 4. Analyses of acid hydrolysis products obtained from j-carra- geenan and agarose. (A) PAGE analysis: lane 1, reductive hydroly- reductive and sate of j-carrageenan; 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 hydroly- sate of j-carrageenan; lane 3, mild acid hydrolysate of agarose; lane 4, reductive hydrolysate of agarose. Arrow indicates the origin.

Fig. 6. 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 (cid:2)C for 1.5 h. Lane 2: agaro-heptasaccharide. Lanes 3–5: agaro-heptasaccharide treated with 0.1 M H2SO4 at 60 (cid:2)C for 15, 30 and 45 min, respec- tively. Lane 6: nonsulfated k-carrageenan treated with 0.1 M H2SO4 at 80 (cid:2)C for 6 h. Lane 7: nonsulfated k-carrageenan heptasaccha- ride. Lane 8: nonsulfated k-carrageenan heptasaccharide treated with 0.1 M H2SO4 at 60 (cid:2)C for 2 h. Lanes 9–11: nonsulfated k-carra- geenan heptasaccharide treated with 0.1 M H2SO4 at 80 (cid:2)C for 2, 4 and 6 h, respectively. Arrow indicates the origin.

and both

odd-numbered

DP DP

3

5

6

8

9

10

1

2

4

7

19 17 15 18 16 14 13 12 11 19 17 15 13 11 10 9 9 8 7 7 6 5 5 4 3

(Fig. 6,

Fig. 5. 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).

Only at a higher temperature and with a longer reac- tion time (e.g. 80 (cid:2)C, 6 h) was the polysaccharide hydrolyzed and even- numbered 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 non- sulfated k-carrageenan were prepared (Fig. 6, lanes 2 and 7, respectively) and used for hydrolysis. Under the readily mild conditions, agaro-heptasaccharide was hydrolyzed (Fig. 6, lane 3). Further incubation did not result in even-numbered oligosaccharides, but only increased the content of monosaccharides and trisac- charides (Fig. 6, lanes 4 and 5). However, under these conditions, no hydrolysis product was observed for the desulfated k-carra-heptasaccharide 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-

Mild acid hydrolysis was carried out with the non- sulfated anGal-lacking k-carrageenan, without success.

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Table 3. Negative-ion ESI-MS of mild acid hydrolysis products obtained from a heptasaccharide of desulfated k-carrageenan.

Assignment

DP

Sequences

Theoretical [M ) H])

Fractions

Found [M ) H])

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

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

341.1 503.1 665.1 827.2 989.2 1151.4

2 3 4 5 6 7

dL2 dL3 dL4 dL5 dL6 dL7

341.3 503.4 665.6 827.7 989.9 1152.0

numbered oligosaccharides were obtained with each series in comparable amounts (Fig. 6, lanes 6, 9 and 10).

bond at its nonreducing side to give odd-numbered oligosaccharides. The effect is very similar to that of reduction.

Conclusions

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 carrageen- ans as the i-carrabiose unit -(G4S-A2S)-. The differ- ence between j-carrageenan and i-carrageenan is the additional 2-O-sulfation of 3,6-anGal in the latter.

Acid hydrolysis is a classic method for depolymeriza- tion of polysaccharides. Generally, for a polysaccha- ride with highly ordered disaccharide repeats (such as the GAGs), if selective cleavage takes place, even-num- bered oligosaccharides are normally produced. In the case of random and nonspecific cleavage, both odd- numbered and even-numbered oligosaccharides with the two different residues at both termini are gener- ated. 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 3,6- anGal-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 oligosaccha- rides with a Gal at the nonreducing terminus and 3,6- anGal at the reducing terminus, followed by immediate removal of the newly created unstable reducing termi- nal 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 con- version 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 Euche- i-carrageenan (type V, Eucheuma spinosa), uma cottonii), and k-carrageenan (type IV, from Gigartina aciculaire), and low electroendosmosis), MMB, NaBH4, agarose (type I,

Degradation of i-carrageenan under mild acid condi- tions required a longer time [25,29] (3 h) than that for j-carrageenan (1.5 h). The hydrolysate was fraction- ated 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 hexadeca- saccharide. 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]. How- ever, the results with i-carrageenan indicated that 2-O- sulfation 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

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Polysaccharides (R=H, SO3 Na; R'=SO3 Na)

(R=H, SO3Na)

BH4–

Even-numbered oligosaccharide alditols

H+ (R=SO3Na)

Even-numbered oligosaccharides

OR' OR' OR' OH O O O OH O O OH O O O O O O O O O O O OH OH OR OH OR OR OR' OR' OH O O OH OH O O CH2 OH O H + O O O O OH OH OR OR OR' OR' OH O O O O OH O O OH O O O HO OR OH OH OR OR' OR' OH O O (R=H) H + OH O O O O O O O O OH OH OH OSO3 Na OR' OR' OSO3 Na OH O O CHO O O OH O O O O O HO OH OH OH

H + OR' OR' OH O O O O O OH O CHO O HO O OH OH + O HO O O OH OH OH

3,6-anGal (furanose)

Odd-numbered oligosaccharides

CHO

HO-CH 2

O

5-HMF

Scheme 1. Proposed mechanism for the mild acid hydrolysis of galactan polysaccharides.

column

(30 lm,

1.6 · 60 cm),

similarly to the published procedure using HCl [21]. Hydrolysis of i-carrageenan was extended to 3 h. Small- scale (10 mg) hydrolysis of j-carrageenan was also carried including TFA, oxalic out with various organic acids, acid, maleic acid, phthalic acid, fumaric acid, citric acid, formic acid, succinic acid, and acetic acid, at 60 (cid:2)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 (cid:2)C for 1.5 h or 0.2 m NaBH4 at 60 (cid:2)C for 3 h. The reaction was terminated by neutralization with 2 m NaOH before analysis.

capillary

0.25 mm)

diameter

internal

OH 3,6-anGal (pyranose)

Analysis and preparation of oligosaccharides

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 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, 9 lm) and AG50W-X8 ion exchange resin were obtained from Bio-Rad Laboratories (Hemel Hemp- columns HP-5MS stead, UK). Fused-silica and (30 m · 0.32 mm, 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

For HP-TLC analysis, aliquots ((cid:2) 0.4 lL) of samples were applied to a TLC plate and developed in n-butanol ⁄ formic acid ⁄ water (4 : 6 : 1, v ⁄ v ⁄ v). Plates were stained by dipping them in diphenylamine ⁄ aniline ⁄ phosphoric acid reagent for 3 s, and then heating them at 105 (cid:2)C for 5 min 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 · 8 · 10 cm) gel system. The gel was loaded with

Large-scale (100 mg) acid hydrolyses of the polysaccha- rides j-carrageenan and agarose were carried out typically (10 mgÆmL)1) at 60 (cid:2)C for 1.5 h, with 0.1 m H2SO4

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20–50 lg of sample, and subjected to electrophoresis at 200 V for 2 h. The gel was stained with Alcian blue (0.5% in 2% AcOH) and destained with 2% AcOH [21,34,35].

The nonsulfated k-carrageenan was then hydrolyzed with 0.1 m H2SO4 (10 mgÆmL)1) at 80 (cid:2)C for 6 h. The oligo- saccharide products were fractionated and purified with a Superdex 30 and a Superdex Peptide column, respectively.

using

Hydrolysis of heptasaccharides (250 lg) derived from nonsulfated k-carrageenan and agarose was carried out with 0.1 m H2SO4 (10 lgÆlL)1) at 60 (cid:2)C and 80 (cid:2)C, respec- tively. The hydrolysis products were analyzed by HP-TLC. Following sample application with a Linomat V TLC appli- cator (Camag Scientific, Switzerland), the TLC plate was developed and stained as described above.

For oligosaccharide preparation, the hydrolysates were concentrated by lyophilization and subjected to gel filtration the A¨ KTA-FPLC (Pharmacia chromatography Biotech, Sweden) system with a Superdex 30 column, as pre- viously described [15]. Elution was carried out with 0.1 m NH4HCO3 for both acidic and neutral oligosaccharides, at a flow rate of 0.2 mLÆmin)1, and detected by a refractive index detector. Fractions were pooled, and the volatile buffer was removed by repeated lyophilization with water.

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 mLÆmin)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.

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 LÆh)1 and 15 LÆh)1, respectively. The source temperature was 80 (cid:2)C, and the desolvation temperature was 150 (cid:2)C. Samples were dissolved in CH3CN ⁄ 2 mm NH4HCO3 (1 : 1, v ⁄ v), typically at a con- centration of 5–10 pmolÆlL)1, of which 5 lL was loop- injected. The mobile phase (CH3CN ⁄ 2 mm NH4HCO3, 1 : 1, v ⁄ v) was delivered by a syringe pump at a flow rate of 5 lLÆmin)1. The capillary voltage was maintained at 3 kV and the cone voltage was 50–120 V, depending on the size of oligosaccharides.

The identity of 5-HMF was confirmed by GC-MS analy- sis following its collection from HPLC [37]. An Agi- lent 6980 system equipped with an HP-5MS fused-silica capillary column was used. The injector temperature was set at 220 (cid:2)C. Helium was used as carrier gas, at a flow rate of 1.0 mLÆmin)1. The oven temperature was initially kept at 50 (cid:2)C for 4 min, then increased to 250 (cid:2)C at a rate of 8 (cid:2)CÆmin)1, and finally kept at 250 (cid:2)C for 10 min. The ion source temperature at 280 (cid:2)C, and the ionization energy was 80 eV. The mass spectrum acquired was compared with the NIST library spectrum.

For CID-MS ⁄ MS product-ion scanning, argon was used as the collision gas at a pressure of 1.7 bar, and the colli- sion energy was adjusted between 17 and 100 eV for opti- mal 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

Desulfation of k-carrageenan and hydrolysis of the desulfated product

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).

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