Choice of the coupling method

The coupling method should be gentle so as to preserve the enzymic and biological activity of the enzymes subjected to polysialylation, as well as acid labile

structures, for instance the CA internal glycosidic linkages. An ideal coupling method would also take advantage of a single, specifically activated site for coupling. This would avoid multipoint attachment of CA and thus intermolecular cross-linkage leading to the formation of very high molecular weight products that would probably be insoluble in water, immunogenic and difficult to characterize. Since a-(2->8) internal linkages are not sensitive to periodate (Lifely et al., 1986), a free terminal aldehyde group can be introduced at the non-reducing end of CA by controlled periodate oxidation (see Fig. 1.5 and 1.9). The resultant polysaccharide has essentially a single terminal aldehyde group (Jennings & Lugowski, 1981) that can then be coupled to the amino groups of the protein molecule. It should be noted however that, in solution, the NeuSAc residue at the reducing end of CA exists in a keto-enol equilibrium, as shown in Fig 1.8. The keto form, although in a much smaller proportion (Jennings & Lugowski, 1981), can potentially also react with the proteins.

HO—C—COOH

ÇH2

O CHOH I HCNHAc CH HÇOH HCOH CH2OH COOH I

c=o

I ÇH2 CHOH I HCNHAc HOCH I HÇOH HCOH CH2OH

Fig. 1.8 Keto-enol equilibrium o f N-acetylneuraminic acid (NeuSAc). The open form exists in solution in much smaller quantities than the cyclic form (Jennings & Lugowski, 1981), as indicated by the length o f the arrows. The NeuSAc residue at the terminal reducing end o f CA is also in equilibrium with its keto form (carbon 2 holds the reducing keto group) and can also react with the primary amino groups o f the proteins. Ac= acetate.

1.7.3.1 Polysialylation o f proteins by reductive amination

The reductive amination of proteins and saccharides was initially developed by Gray et al. (1978) as a one-step method for the coupling of reducing mono and disaccharides to proteins. It was subsequently applied to the covalent linkage of PSAs to tetanus toxoid and albumin (Jennings & Lugowski, 1981) which were used as carrier proteins in an attempt to boost the immunological response to these polysaccharides. The method of reductive amination is also useful for the specific labelling of sialylglycoproteins at the terminal non-reducing sialic acid by the attachment of radioactive compounds containing an amino group (Murray et al., 1989).

The method of Jennings & Lugowski (1981) was adopted for the grafting of CA to the proteins.The coupling reaction, as summarized in Fig. 1.9, can be divided into two steps. The first (Fig. 1.9A) involves the introduction of a free aldehyde group at the non-reducing end of CA (Carbon 7) (see also Fig. 1.5) by means of periodate oxidation. In the second step (Fig. 1.9B), the recently introduced aldehyde group reacts, under alkaline conditions, with the primary amino groups of the protein (mostly the e-amino groups of the lysine residues (Glazer et al., 1976) although the terminal amino group can also be involved). The imine or Schiff base formed is then readily reduced by sodium cyanoborohydride (NaCNBHg) to a secondary amine. Unlike sodium borohydride, NaCNBHg (under the alkaline conditions of the reaction) does not reduce free aldehyde groups (Borch et al., 1971). The high selectivity of NaCNBHg thus allows it to be present throughout the reaction (even in the presence of the carbonyl compound) and the rapid reduction of the unstable intermediate imine (which is in equilibrium with the initial reagents, i.e. protein and CA), shifts the equilibrium to the right (note arrows in Fig. 1.9B) thus increasing the yield of conjugate.

OH OH CA I-C H -C H -C H2OH 7 8 9 NaI0 4 O CA \ - C — H 7

B

Prot—NH2 + O CA - C —H pH 9.0^ 3 5-40'C Prot - N = C H — CA NaCNBHg Prot -N H -C H2— CA

Fig. 1.9 Overall reaction scheme for the coupling o f colominic acid to proteins. A. introduction o f a free aldehyde group at the terminal non-reducing end o f CA by oxidation with periodate and B. reductive amination o f the activated CA with the protein, forming an intermediate Schiff base. In the presence o f NaCNBHg this unstable intermediate is readily reduced to a secondary amine. Only the groups involved in the covalent coupling are depicted. The backbones o f the protein and CA are represented by Prot and CA respectively.

Advantages of this conjugation method include its selectivity for the primary amino groups of the proteins and the formation of a stable linkage (Gray et al., 1978) between the PSA and the protein. Moreover, no spacer is needed for attachment, which is a further advantage if we consider that spacers can give rise to immunological problems (Francis et al., 1992; Delgado et al., 1992)

The loss of enzyme activity observed upon enzyme modification can be due, among other causes, to the modification of amino acid residues in the enzyme’s active site that are vital to its catalytic function. A common strategy (Poznansky, 1986; Cao et al., 1990) to protect the active site from modification, is to perform the covalent coupling reaction in the presence of excess substrate. Since catalase is rapidly inactivated in the presence of high concentrations (>100 mM) of substrate (Aebi,

1983), its use during reductive amination was precluded. The use of asparagine, the substrate for asparaginase, during the polysialylation of this enzyme was also ruled out in view of the probable competition of the asparagine primary amino groups (see Eq.2) for the formation of Schiff s bases with CA.

The following chapters describe methodology (Chapter 2) and results obtained during the polysialylation of the proteins mentioned in 1.7.2. The optimization of the coupling method with a model protein (poly-L-lysine) was the goal of the initial phase of the work (Chapter 3). Chapter 4 deals with the synthesis and in vitro characterization of the polysialyted therapeutic enzymes (catalase and asparaginase) and Chapters 5 and

6 with the in vivo behaviour of the two neoglycoproteins, in intact and pre-immunised

animals. Chapter 6 also describes the immunological evaluation of the polysialylated

asparaginase and catalase conjugates.

In document Polysialylation of proteins: an approach to improve their therapeutic efficacy (Page 65-70)