Ind. Eng. Chem. Res. 2006, 45, 6619-6621
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Enzymatic O-Galactosylation of Protected Serine and Threonine by β-Galactosidase Employing High Lactose Concentrations Andreas Layer and Fabian Fischer* Biotechnology Unit, Life Technologies Institute, UniVersity of Applied Sciences Valais, Route du Rawyl 47, CH-1950 Sion, Switzerland
Trans-galactosylation of protected serine and threonine by β-galactosidase is the first rational step in the in vitro glycosylation of peptides and proteins. The experiments show the potential of trans-galactosylations with lactose for applications in which no product purification (such as that used in the food and life science industries) is needed. To examine these glycosylations systematically, the protected amino acids Boc-SerOMe, Boc-Thr-OMe, Cbz-Ser-OMe, and Cbz-Thr-OMe are used as substrates. The trans-mono-galactosylation of serine with an excess of lactose yields 28% of N-tert-butoxycarbonyl-1-O-β-D-galactopyranosyl-L-serinemethylester. The same transformational conditions, when applied to threonine, produced N-tert-butoxycarbonyl1-O-β-D-galactopyranosyl-L-threonine-methylester in lower quantities. Mono-galactosylated serine and threonine are further galactosylated in the examined experimental setup to yield bi-galactosylated products also, especially at 50 °C with completely dissolved lactose. Introduction
Results and Discussion
Glycoproteins and glycopeptides consist of amino acid moieties with glycan substituents, which are added during biosynthesis in plant and animal cells. In biological systems, oligosaccharide side chains are to be found as O- and N-glycans, linked to the hydroxy functionality of serine/threonine by a glycosidic bond or to the amine substituent of asparagine. In addition to N- and O-linked structures, uncommon glycan conjugates are known as well, including C-glycosides, Sglycosides, and carbohydrates linked via a phosphodiester bridge.2 The reaction is stereospecific, because of the fact that β-galactosidase3 forms only the β-isomer of the coupling product. The development of a high yielding glycosylation process is a challenge for applied biotechnology, because yields are typically low, because of product hydrolysis, which is a competing side reaction. The trans-glycosylation with lactose of various substrates in large-scale industrial production is applicable1 (Figure 1), based on the fact that (i) lactose is inexpensive and (ii) for many processes, byproducts such as water, glucose, and other sugars are not considered to be problematic waste and are therefore tolerable remaining components in final product mixtures. In the food industry, such a process seems to be particularly beneficial, but lactose-based transfer reactions are also welcome options in biotechnology, because byproducts may be used later in hole cell fermentations for enzyme generation. Nevertheless, other glycosyl transfer options exist. Lactose may be substitued by para-nitrophenyl glycosides which are applied in the same manner and nitrophenol remains as side product. Another approach is the reversed glycolysis with glycosidases in organic solvents where the equilibrium is shifted to the product side by a lack of water. The two latter solutions appear in many cases more expensive and are usually considered for high-value-added products such as pharmaceuticals. Nevertheless, the minimal water approach that uses organic solvents is an appealing option and is gaining more and more attention.
Trans-galactosylation with protected serine and lactose demonstrates that the transformation is kinetically more favored, with 28% yield versus 2% yield for the threonine/lactose couple, under identical conditions (Figure 2). The threonine side chain differs only slightly from serine and, in addition, carries a methyl group that replaces the H atom in the R-position with the primary hydroxy substituent of the side chain. The small structural difference proves to be the determining reason for the lower reactivity of threonine. Cantacuzene and Attal4 reported that yields for the serine/lactose transformation are usually in the lower range (15%). The most productive protocol (35%) that was developed5 uses β-glactosidase from the digestive juice of an African snail (Achatina achatina). Because of the fact that yields seem to be dependent mostly on the enzyme source, we were interested in changing reaction conditions to increase yields through the use of relatively inexpensive commercially available enzymes, which may be less active or specific. β-Galactosidase from E. coli is such an enzyme, with which we obtained yields of up to 28%. The approach to use an excess of lactose shifted the equilibrium to the product side. The generation of a multitude of side products is well-known and seems inevitable.6 Side products include free galactose and glucose that originates from lactose hydrolysis, galactosylated lactose, galactose dimmers, doubly galactosylated7 BOC-Ser-OMe (Figure 3), and presumably traces of oligomers from further conversions of the desired product. Although β-galactosidase is soluble in aqueous solutions, all employed substrates are poorly miscible in water. Boc-Ser-OMe and Boc-Thr-OMe are viscous liquids at room temperature and have the tendency to form separate phases. Cbz-Ser-OMe and Cbz-Thr-OMe are insoluble powders at room temperature and dissolve in a phosphate buffer, but then form equally separate phases. Small lactose quantities dissolve well in aqueous solutions but, at high concentrations, form white suspensions. Adding suspended lactose to protected serine or threonine while vigorously shaking allows well-mixed but still white emulsions to be obtained. Because lactose dissolves at 20 °C with ∼16% (wt/wt) in deionized water,8 concentrated biphasic aqueous
* To whom correspondence should be addressed. E-mail:
[email protected].
10.1021/ie060308k CCC: $33.50 © 2006 American Chemical Society Published on Web 08/10/2006
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Ind. Eng. Chem. Res., Vol. 45, No. 19, 2006
Figure 1. General description of trans-galactosylation with alcohol and lactose, forming a galactose aglycon and glucose, including water as a side product.
Figure 2. Description of the trans-galactosylation of protected serine and threonine.
Figure 3. Description of double trans-galactosylation, utilizing lactose in excess.
Figure 4. Trans-galactosylations executed with Cbz-Serine-OMe. Solid circles (b) represent data recorded at 50 °C in dissolved lactose solutions and solid triangles (2) represent data recorded at 30 °C in dissolved lactose solutions, whereas the solid squares (9) show transformations at 50 °C in lactose suspensions and the solid diamonds ([) show transformations at 30°C in lactose suspensions. The lengths of the vertical bars indicate the amount of doubly galactosylated products.
lactose solutions were heated to 99 °C, where they became transparent, and were subsequently cooled to 50 and 30 °C, respectively.
With this pretreatment, it is possible to maintain a considerable excess of lactose in reaction mixtures, which may double the productivity of the transformation and N-tert-butoxycarbonyl-1-O-β-D-galactopyranosyl-L-serine-methylester (28% yield). Although the changed conditions improved the yields by 7%10%, on average, for serine-glycan, applying the improved reaction conditions to protected threonine did not give yields higher than that observed previously (2%). The impressive difference in the reactivity of these rather similar amino acids is related to the aforementioned methyl substituent in threonine. In addition, the reactivity is also dependent on other factors, such as the chosen protection group or reaction temperature. The carbazol protection group slightly reduces the rate of conversion, in comparison with the BOC group (see Figures 4 and 5). An elevated incubation temperature (50 °C) initially provides a reaction speed that is at least twice as fast; however, with prolonged reaction time, side product formation (including further transformation of the desired product) becomes visible. The decreasing concentration of the product is not only related to side product formation but also to the fact that lactose concentration decreases and, because of this, hyrdolysis is competing with the trans-galactosylation. To study the influence
Figure 5. Trans-galactosylations executed with Boc-Serine-OMe. The transformation at 50 °C with dissolved lactose (represented by solid circles, b) gives the best yields, but the influence of double galactosylation and hydrolysis reduce the yield faster than in the other three transformations. The same reaction at 30 °C (denoted by solid triangles, 2) produces less side products. The use of lactose suspensions at 50 °C (denoted by solid squares, 9) and at 30 °C (denoted by solid diamonds, [) leads to product formation at a slower rate.
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of the temperature on the productivity, the experiments were conducted at 30 and 50 °C. When the best yields are analyzed (see Figure 5), then we find that, with an incubation at 50 °C during 8-48 h, the overall yields of mono-glycosylated product decrease from initially 28% after 8 h to
