Specific degradation of pectins via a carbodiimide-mediated Lossen rearrangement of methyl esterified galacturonic acid residues

Abstract

A specific, chemical degradation of the methyl esterified galacturonic acid residues of pectins is described. These residues are converted, with hydroxylamine, to hydroxamic acids, and then, with a carbodiimide, to isoureas; the latter undergo a Lossen rearrangement on alkaline hydrolysis. The isocyanates formed are hydrolysed to 5-aminoarabinopyranose derivatives, which spontaneously ring open to give 1,5-dialdehydes. The latter are reduced, in situ, to avoid peeling reactions, with sodium borohydride to give substituted arabitol residues. Thus, overall, partially esterified pectins are specifically cleaved to generate a series of oligogalacturonic acids bearing an arabitol residue as aglycone. Analysis of oligomers so generated discloses the pattern of contiguous nonesterification in a variety of pectins of differing degrees of esterification. Other potential applications are described.

Introduction

Specific chemical degradation of polysaccharides has been a useful tool in structural determination.1., 2., 3. The selective derivatisation and cleavage of uronic acid residues, and in particular of galacturonic acid residues, has received much attention, as these sugars are a major constituent of pectins that occur in plant cell walls. Pectins are important in cell wall assembly,⁴ and in isolated form have proved useful in the food industry. An improved knowledge of their structure and its relationship to their physical properties would be of considerable application.

Selective cleavage of galacturonic acid residues in pectins allows, in particular, two problems to be addressed. Firstly, if the methyl galacturonic acid esters are selectively cleaved, the arrangement of unesterified residues can be determined, both in native pectins and in pectin methyl esterase-treated samples. This distribution is important, as it is thought to determine the gelling properties of the pectin.⁵ Secondly, selective cleavage of unesterified galacturonic acid residues allows sidechains, known to be composed predominately of arabinose and/or galactose, to be released from the molecule.

In principle, three properties of galacturonic acid residues might be exploited to selectively cleave pectins: (a) the resistance to hydrolysis of their glycosidic linkages compared with neutral sugars; (b) the propensity of esterified galacturonic acid residues to β-eliminate their 4-substituents; (c) their ability to undergo derivatisation selectively at their carboxyl groups. In principle, such derivatisation could promote ring opening and attendant glycosidic cleavage.

All three approaches have been applied to pectins. Mort⁵ reduced the methyl esterified residues of pectins to galactose, which were then selectively removed by hydrolysis with anhydrous hydrogen fluoride (HF) at −20 °C to produce unesterified blocks of galacturonic acid, terminated by galactose. Analysis of the resulting mixtures gave the distribution of non-esterified residues. Although highly effective, the method has several disadvantages. Firstly, HF is a highly corrosive material which requires specialised, stringent handling procedures, and thus use of the method has been highly restricted. Secondly, the selectivity of the method is kinetic in nature; low levels of nonspecific galacturonic acid cleavage might be expected in long, acidic blocks, such as might be produced in studies of enzyme hydrolysates. Lastly, long sequences of α-(1→4)-linked galactose are rather insoluble.

Selective β-elimination of uronyl ester-containing molecules has been much studied and reviewed.⁶ Unfortunately, ester hydrolysis competes under the basic conditions required and, with methyl esters, β-elimination is never quantitative (although conditions to maximise it have been developed⁷). Thus, repeated cycles of reesterification and hydrolysis are needed. β-Elimination is thus unsuited to determinations of unesterified residue distribution, but has been used, with some success, to liberate sidechains.⁸

So far, the most successful degradation, based on targeting the carboxyl groups of underivatised pectins, is a reductive cleavage using lithium in ethylenediamine. This was used to liberate sidechains from rhamnogalacturonan I (RG-1).⁹ If esterified galacturonic residues were first reduced to galactosyl residues,⁵ lithium in ethylenediamine would liberate blocks of galactose corresponding to the esterified regions of the pectin. However Lau et al.¹⁰ demonstrated, using model compounds, that up to 10% of neutral glycosidic linkages are also cleaved by lithium in ethylenediamine, and a method of block distribution based on this scheme has not been reported.

At present, no enzymes are available to specifically degrade all of the galacturonic acid residues, either esterified or unesterified, in a pectin. For example, rhamnogalacturonases require the prior degradation of sidechains,¹¹ and the binding requirements of pectin and pectic lyases and galacturonases¹² preclude a direct assessment of acid-block distribution. However, a method for the characterisation of non-esterified galacturonic acid sequences, which uses endopolygalacturonase, has recently been described.¹³ But as not all non-esterified residues are removed by the enzyme, and because the degradation products obtained reflect the binding propensity of the surrounding regions, interpretation is complicated.

A truly specific degradation of galacturonic acid residues was thus clearly a desirable tool. A Lossen rearrangement14., 15. seemed attractive (i.e., a chemical degradation of type (c) above), as it would, in principle, require no prior protection of the pectin. (Hydroxyl protection, usually by methylation or acetylation, is essential to the majority of classical chemical degradations of polysaccharides.1., 2., 3.) Hoare et al.¹⁶ reported the quantitative conversion of simple hydroxamic acids, via a Lossen rearrangement, to amines under mild conditions. This facility suggested that the procedure might be used to selectively degrade acidic polymers such as pectins.

We envisaged generation of contiguous unesterified acidic blocks from pectins as follows (Scheme 1). Methyl esters would be converted into hydroxamic acids (Step A, Scheme 1). Simple methyl esters¹⁷ and methyl esterified pectins¹⁸ have been reported to give hydroxamic acids when treated with hydroxylamine under alkaline conditions. We anticipated (correctly, see below) that the latter would promote extensive β-elimination of pectins, and at least some deesterification (both processes are base-catalysed⁸). However, we felt that successful substitution might be possible at, or near, neutral pH, with concentrated hydroxylamine solutions and extended reaction times, for two reasons. Firstly, uronyl esters are activated, by electron withdrawal by O-5, to nucleophilic attack. (For example they are reduced by sodium borohydride; the latter is incapable of reducing unactivated esters.¹⁹) Secondly, hydroxylamine is a powerful nucleophile, activated by an α-effect.²⁰

After hydroxamic acid formation, we would remove excess hydroxylamine by dialysis. (This would prevent destruction of added carbodiimide in the next stage,²¹ and undesired conversion of unesterified galacturonic acid residues to hydroxamic acids or O-acylhydroxylamines.¹⁶) Treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) would, we anticipated, lead to an isourea derivative (Step B, Scheme 1), which would undergo Lossen rearrangement to an isocyanate (Step C, Scheme 1). Hydrolysis would be expected to cleave the latter to a hemiaminal (Step D, Scheme 1), which should spontaneously ring open and release the aglycone, to yield a 1,5-dialdehyde (Step E, Scheme 1). The 1,5-dialdehyde could then be protected from base degradation by reduction, in situ, with borohydride (or borodeuteride; Step F, Scheme 1).