Enzymatic monoacylation of fructose by two procedures

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UTTERWORTH I N E M

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Enzymatic monoacylation of fructose by two procedures Christian Scheckermann,* Andrea Schlotterbeck,” Michael Schmidt,’ Victor Wray* and Siegmund Lang* *Institut fur Biochemie und Biotechnologie, Technische Universitat. Braunschweig, ‘Unilever Research Lab., Vlaardingen, The Netherlands ‘Gesellschaft fur Biotechnologische Forschung. Braunschweig, Germany

Germany

Two methods for the lipase-catalyzed monoacylation of fructose with long-chain fatty acids (e.g.. palmitic, stearic) in hexane or 2-methyl-2-butanol are described. Using method A the enzymatic monoacylation offructosa was carried out at a molar ratio up to 15:l of fatty acid to fructose in 2-methyl-2-butanol at 55°C using Lipozyme” The reaction of method B using Lipozyme” or the lipase of Candida antarctica in n-hexane at 60°C was favored by a I :3:4.5 molar ratio of fatty acid to fructose to phenylboronic acid; phenylboronic acid served us a solubilizing agent for the sugar in hexane. Method A gave a mixture of the C-l and C-6 monoacylated fructose fatty acid esters, whereas for method B only the C-l monoester was found. in each case pyranose and furunose isomeric forms of the sugar were apparent in solution. Typically, yields of about 4O?k were obtained. Adding fructose fatty acid esters to water lowered the surface tension from 72 to a minimum of 27 mn m-t ar critical micelle concentrations of IO-’ to 10m3 mol 1-’ at 3PC. Keywords:

Fructose fatty acid esters; lipase; synthesis in organic solvents; phenylboronic

I ntreduction E,mymatic

approaches

to the monoacylation

of carbohy-

dw.es with fatty acids have received much attention in recent years. One reason is that the competition to chemical nteihods was unsatisfying until now; another reason is that Em: nonionic surfactants with definite molecular structures could be of interest as adjuvants for pharmaceutic purposes. hlost reports showed that nearly anhydrous conditions and nonpolar solvents or solvent-free systems were necessary fi)r a lipase-catalyzed process. The solubility of both subs:rates, fatty acids and carbohydrates, created difficulties in tlte case of the more hydrophilic reactants. ‘To solve this problem Therisod and Klibanov’ used pyr dine and activated fatty acids. Bjorkling et al.’ and Mutua and Akoh3 used prealkylated glucose (e.g., ethyl-o-Dglucose), and Fregapane et al., 4 isopropylidene derivatives of :;ugars. Each group succeeded in producing a direct 6-tr.onoacylation of glucose. The acylation of the 2- and _.This artide is dedicated to the 65th birthday of Prof. Dr. Fritz Wagner. Irstiut fur Biochemie und Biotechnologie, Technische Universitlt, BraunSChweig, Germany Addl.ess reprint requests to Dr. Lang at the Institut fur Biochemie und Biot&nologie. Technische Universit& Spielmannstr. 7, D-38106 Braunscha,eig, Germany R’eceived 1 February 1994; revised 13 June 1994; accepted 8 July 1994

Enzvme and Microbial Technology 17:157-162, 1995 Q 1!395 by Elsevier Science Inc. 6!j5 Avenue of the Americas, New York, NY 10010

acid

3-positions of 4,6-o-0-benzylidene glycopyranoside was reported by Panza et a1.5 Further progress was reported by Khaled et a1.6 and Guillardeau et al.’ using a very high excess of oleic acid or caprylic acid for the monoacylation of fructose. However, the presence of more than one isomerit structure in solution gave rise to several fructose monoester products. Using phenylboronic acid as solubilizing agent for fructose in n-hexane, Schlotterbeck et al.* and Oguntimein et a1.9 found monoacylation of only one single primary hydroxyl group. Applying the latter method, Ikeda and Klibanov” succeeded in the 6-acylation of glucose with vinylacrylate on a preparative scale. The present article describes our investigations of the method of Khaled et a1.6 for the production of a number of glycolipids, and further studies with respect to the method using phenylboronic acid as a solubilizing agent for hydrophilic substrates in unpolar solvents. In addition, an elucidation of the molecular structures and physicochemical properties of the enzymatically derived compounds is presented.

Materials and methods (Biochemicals) Lipozyme” IM 20 was purchased from Novo Industries (Mainz, Germany); the lipase SP 435 of Candida antarctica was a gift of Novo (Bagsvaerd, Denmark).

0141-0229/95/$10.00 SSDI 0141-0229(94)00005-C

Papers Carbohydrates and 2-methyl-2-butanol were purchased from Merck (Darmstadt, Germany), fatty acids from Sigma (Deisenhofen, Germany), and hexane from Biomol (Hamburg, Germany).

Experimental method A: Bioconversion, product purification, analysis, and elucidation of molecular structures

on RP-8-F,,,s plates (($-reversed phase silica gel). For development, methanol:water = 80:20 (vol:vol); for detection, an a-naphthohsulphuric acid spray reagent (10.5 ml of 15% ethanolic a-naphthol solution, 6.5 ml sulphuric acid, concentrated, 40.5 ml ethanol and 4 ml water, distilled) was used. After drying at 100°C

for 3 min. a densitometer (CD 60 Desaga, Heidelberg, Germany) served for quantitative measurements. Structure elucidation was performed by ‘H- (1D and 2D

The enzymatic reaction mixture contained 17.6660 mm01 palmitic acid, 4.4 mmol fructose, and 100 mg Lipozyme’” IM 20 in 100 ml 2-methyl-2-butanol. The reaction was carried out with shaking in capped glass flasks at 55°C and 250 rpm for 24 to 55 h. Periodically, samples were taken and analyzed by high-performance liquid chromatography, as described in the analytic section. At the end of the bioconversion, the biocatalyst was removed by filtration and the solvent was evaporated. The fatty acids and products were dissolved by adding chloroform. After filtration of the nonreacted fructose, the products were isolated by mediumpressure liquid chromatography (MPLC) on silica gel, mesh 23& 400, grade 60 (Merck) in a 330-ml-vol column (Labomatic. Erkerode, Germany). Initially, we used chloroform; later, we used chlorofotmmethanol mixtures from 99:1 to 90: 10 (vol:vol) for elution. For further purification, preparative thin-layer chromatography was carried out using silica gel 60. 2 mm (Merck) with (vol:vol: chloroform:methanol:water = 60: 15:2 vol) as a mobile phase. Finally, the product was filtered through a 0.25~Frn PTFE filter. For analysis, the screening of substrates among carbohydrates for enzymatic monoacylation was monitored by qualitative thinlayer chromatography (TLC) using silica gel plates 60 F,,, (Merck) and chloroform, methanol, and 20% acetic acid (65:15:2, vol:vol:vol) as a developing system. A mixture of anisaldehyde, acetic acid, and sulphuric acid (1:100:2, vol:vol:vol) served as a detecting agent. For quantitative analysis, samples of the reaction mixture were withdrawn and centrifuged, and l&20 l.~lof the supematant was analyzed by HPLC (Pharmacia, Uppsala, Sweden). As a stationary phase a 250 . 4.6 mm HS C,,-reversed-phase Adsorbosphere column (Alltech, Unterhaching, Germany) was used, equilibrated with a mobile phase (methanol:acetonitrile:water, 60:35: 5, vol:vol:vol) at 40°C. The analysis was carried out isocratically at 1 ml min and ultraviolet detection at 205 nm. The retention time of fructose palmitate was 6.05 min. All NMR spectra were recorded at ambient temperature on a Bruker WM 400 NMR spectrometer locked to the major deuterium resonance of the solvent, CD,OD.

COSY) and 13C-NMR spectroscopy.

Results and discussion Preliminary experiments used to test both methods for the lipase-catalyzed monoacylation of carbohydrates with palmitic or stearic acid led to the results shown in Table I. After qualitative TLC, including sugar- and lipid-specific detecting reagents, in most cases only one single spot for the desired conversion was observed. In general, pentoses and hexoses seemed to be suitable as acyl acceptors, whereas disaccharides were not acylated. Fructose was chosen for further studies, as this gave the highest yields compared with the other sugars tested. Further details of the biotechnologic studies of both methods are described subsequently.

Lipase catalysis in 2-methyl-2-butanol excess of fatty acids (method A)

at a high

Qualitative determination of the acylation of fructose with palmitic, stearic, myristic, lauric, capric, and oleic acid was performed. In the following, however, we have focused our attention on the C-16 acid, palmitic acid. The acylation products (I-IV) are shown in Figure 1. Downstream processing of the reaction led to a single spot in TLC, although the NMR studies, detailed subsequently, indicated that both the C-l and C-6 monoacylated fructose esters were formed. No acylation of the solvent 2-methyl-2-butanol was observed; in general, secondary al-

Table 1 Carbohydrates tested monoacylation using two methods

for

the

lipase-catalyzed

Reaction

Experimental method B: Bioconversion, product purification, analysis, and elucidation of molecular structures

Carbohydrate

For bioconversion, 1 g of Lipozyme” IM 20 or lipase SP 435 was added to a solution of 1 mm01 fatty acid, 3 mmol fructose, and 4.5 mm01 phenylboronic acid in 100 ml n-hexane. The mixture was shaken at 60°C and 120 rpm for 24 h. After reaction the biocatalyst was separated and the filtrate evaporated to half the original volume. After cooling it to - 20°C we filtered off the excess of substrates that precipitated. After total evaporation of the solvent, the residue was usually purified by column chromatography (LiChroprep@ C,-reversed-phase silica gel, RP-8; Merck). The elution with methanol:water, 80:20 (vol: vol) effected both the hydrolysis of phenylboronic acid complexes and the separation of compounds. For fructose-palmitate and stearate, crystallization also occurred*; in these cases the hydrolysis was completed in an acetone:water mixture under reflux for 15 min. The extent of conversion was determined by quantitative TLC

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Arabinose Ribose Xylose Fructose Glucose Galactose Mannose Sucrose Lactose Maltose

Method Aa -kd + + ++++ + + + _ _ _

Method Bb _c ++ + ++++ +++ +++ ++ _ _ _

aMethod A: High excess of fatty acid in 2-methyl-2-butanol: 0.64-44 mmol palmitic acid, 0.064-4.4 mmol carbohydrates, 100 ml 2-methyl-2-butanol; 55”C, 250 rpm, 24 h. bMethod B: Sugars solubilized in hexane by phenylboronic acid: See Materials and methods; exception: In the case of arabinose, ribose, and xylose (where there are only four free hydroxyl groups) 3 mmol phenylboronic acid were used.

Monoacylation

of fructose:

CONVERSION

C. Scheckermann

et al.

WTER CONTENT (lb)

I%)

2

50 *ml~?R

-!bCONVLRIlON

40

n

CONTINT

a 1,s

30

a Figure 1 Reaction products of the enzymatic monoacylations of fructose with an excess of palmitic acid in 2-methyl-2-butanol (I-IV) and of fructose with stearic acid after sugar solubilization in hexane by phenylboronic acid (III and IV)

coholic groups are minor acyl acceptors for this lipase. Substrate solubility was restricted in 2-methyl-2-butanol such that approximately 10 g 1- ’ fructose and approximately 170 g 1-l palmitic acid could be dissolved at the reaction temperature of 55°C. Based on these data we varied the palmitic acid:fructose ratio from 4: 1 to 15: 1. Figure 2 shows the time course of the reaction up to 60 h, indicating the increase of fructose palmitate production at a higher excess of fatty acid. TLC studies proved the absence of dior polyacylated carbohydrate. The optimum enzyme concentration to obtain high yields at a given time was found by varying the Lipozyme concentration up to 50 g 1-l. The

CONVERSION

FP (o/l) 7

MOLAR RATIO MLMITIC t

6-

-%-

1S:l

+

1O:l

1

20

0,s 10

\

0

0

,

40

30

20 LIPOZYME

--z--

(g/I)

Figure 3 Influence of the enzyme content on the fructose palmitate production after 24 h. Reaction mixture contained 44 mmol palmitic acid, 4.4 mmol fructose, and 0.05-5 g Lipozyme” in 100 ml 2-methyl-2-butanol at 55°C

(%L) CONVERSION 501-

ACID : FRUCTOSE

*

I

10

,

t%)

3:l

6:l

5-

4-

3-

20 -

20

30

40

so

oi 0

60

’

’

’

’

I

’

0,l

0,2

0,3

0.4

0.6

06

I

’

0,s

09

1 1

1.1

PEA (mm00

TIME (hour81 Figure 2 Influence of the molar substrate ratios on the enzymatic fructose palmitate production. Reaction mixture contained n .4.4 mmol palmitic acid, 4.4 mmol fructose, and 100 mg Lipozyme” in 100 ml 2-methyl-2-butanol at 55°C. FP, Fructose palmitate

’ 0,7

Figure 4 Effect of Lipozyme.’ preincubation with phenylboronic acid on conversion. Tested in the following standard reaction: 100 mg Lipozyme-, 0.1 mmol stearic acid, 0.3 mmol fructose, and 0.39 mmol phenylboronic acid in 15 ml n-hexane at 60°C and 120 rpm for 24 h

Enzyme

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159

Papers CONVERSION

Lipase-catalyzed monoacylation of fructose solubilized in n-hexane by phenylboronic acid (method B)

(W)

60[ 0

LIPOZYME

m

SP 435

50 -

40 -

30 -

20 -

10 -

Oc-12

c-14

C-16

C-16

Figure 5 Influence of the fatty-acid chain length on the conversion to fructose fatty-acid monoesters by use of Lipozyme and lipase SP 435. C-12, Laurie acid; C-14, myristic acid; C-16, palmitic acid; C-18, stearic acid. For reaction conditions, see Materials and methods

for an incubation time of 24 h presented in Figure 3 show a maximum conversion, 39% (related to the minor substrate), at 10 g 1- ’ to 15 g 1- ’ of Lipozyme . Higher amounts of immobilized enzyme led to a reduction in yields, which was probably caused by the high water content measured after 20 min, associated with the catalyst. results

By using this method fructose was acylated at the C-l position (Figure 1; structures III and IV). NMR data of the structures were reported recently.8 In the present study we found that a 1:3:4.5 ratio of stearic acid:fructose:phenylboronic acid was necessary for optimum conversion (40%). As this maximum value is not particularly good, we investigated the possible inhibition of phenylboronic acid of the enzyme activity. Thus, Lipozyme was preincubated in n-hexane for 12 h in the presence of different quantities of phenylboronic acid. After this preconditioning the biocatalyst was filtered off, washed with n-hexane, and added to a standard reaction mixture of fatty acid, fructose, and phenylboronic acid in n-hexane. The conversion values presented in Figure 4 show that without phenylboronic acid during the preincubation phase the yield of fructose monostearate was comparable to experiments without preincubation of the enzyme. With an increase in the concentration of solubilizing agent during the preincubation phase, the conversion decreased from 40% to 10% at 1.04 mmol phenylboronic acid. From these results, it can be assumed that the enzyme is inhibited. Using the usual standard reaction procedure, other fatty acids and another immobilized lipase, SP 435 from C. antarctica, were tested. The results presented in Figure 5 show a decrease in the conversion ofvabout 20% to 40% compared with the values of Lipozyme . For both enzymes, higher yields of glycolipids were observed at lower fatty acid chain lengths.

Elucidation of molecular structures of glycolipids The structure of the fructose monopalmitates obtained from the two methods were elucidated in solution from ‘H- (1D

Figure 6 13C-NMR spectrum in the sugar region of fructose palmitate produced by method A. The signal assignments are denoted as follows: 0, 6-p-fructofuranose palmitate; x, l-6-fructopyranose palmitate; Cl, 6-a-fructofuranose palmitate; & l-6-fructofuranose palmitate

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Monoacylation Table 2 13C chemical shifts in CD,OD for the various fructose palmitate isomers produced by method A

I

II

Ill

IV

64.40 103.37 77.40 77.03 80.11 66.69

64.92 105.97 84.06 79.03 80.47 65.43

66.57 98.12 69.72 71.38 70.86 64.63

65.51 101.44 78.40 76.35 83.31 63.92

Carbon compounda C-l c-2 c-3 c-4 c-5 C-6

aThe shifts of the fatty acid carbons are identical in all compounds except for C-l’ and are as follows: 175.39 (I, C-l’); 175.30 (Ill, C-l’); 175.33, 174.94 (II and IV, C-l’); 34.86 (C-2’); 25.86 (C-3’); 30.69-30.15 (C-4,-C-13’); 32.98 (C-14’); 23.61 (C15’); and 14.37 (C-16’).

and 2D COSY) and 13C-NMR data measured at 400 and 100 MHz, respectively. in deuteriomethanol-d, at ambient temperature. Under these conditions an equilibrium mixture of the two furanose forms for the 6-acylated compound and both furanose and pyranose forms for the 1-acylated derivatives were observed. We have previously* shown by ‘HNMR spectroscopy that method B exclusively produces the l-fructose ester that exists in solution as two major isomers, l-P-fructopyranose and 1-fructofuranose isomers. This was confirmed here, and the 13C shifts of the latter were found to be compatible with the l-P-fructofuranose isomer. A comparison of the ‘H-NMR spectrum of the product from method A with that of method B clearly indicates that the

70

SURFACE TENSION -- - ~~--___-~---.--

fmN/m)

of fructose:

C. Scheckermann

et al.

major components were different. The low field shifts of the methylene protons belonging to C-6 in the product from method A (4.34 and 4.16 ppm; both signals are double doublets with couplings of 11.513.5 and 11.5j7.0 Hz, respectively) indicated that this component was the 6-fructose palmitate. The 13C spectrum of the sugar region of this same product (Figure 6) unambiguously showed the presence of four components, which from the signal intensities, were in the ratio of 3.5:2: 1: 1 (1I:III:I:IV; Figure I). Comparison of these data with the 13C chemical shift data for l-methyl fructosides from the literature’ ’ allowed the assignments shown in Table 2, and indicated that the product from method A was a mixture of the 6-fructose palmitate [occurring in solution as the o- and p-furanose forms (Figure I, II and I, respectively), in a ratio of 3.5: 1] and the 1-fructose palmitate [occurring in solution as the @-pyranose and p-furanose forms (Figure I, III and IV, respectively), in a ratio of 2: 11. The latter are identical with the product from method B. Physicochemical

properties

of fructose

fatty

acid esters The results of surface tension measurements in aqueous systems are shown in Figure 7. The surface tension of water at 37°C was reduced from 72 mN m- ’ to about 27 KIN m- * The shorter the fatty-acid chain length, the higher were the critical micelle concentrations. Corresponding values for fructose stearate produced by method B were in the range 47 mN m-’ and 4 mg 1-l at 25°C. With these results all nonionic glycolipids except the last one showed similar good surfactant behavior such as detergent mixtures from chemical synthesis. Based on the above results, it is clear that the monoacylated fructose esters are readily produced enzymatically and offer a viable alternative to the usual chemically derived alternatives (mixtures of carbohydrate esters). Their application and approval in the field of cosmetics, cleaning, food processing, and pharmacy await future testing.

Acknowledgments We thank Unilever Research Lab., Vlaardingen (The Netherlands) and the German Ministry for Research and Technology, Bonn (Project no. 0319450 B) for financial support.

References 1 2

3

4

5 Figure 7 Influence of the fatty-acid chain length of fructose esters on the lowering of surface tension of water at 37°C

Therisod, M. and Klibanov, A. M. Facile enzymatic preparation of monoacylated sugars in pyridine. J. Am. Sot. 1986,166,5638-5640 Bjiirkling, F., Godtfredsen, S. E. and Kirk, 0. A highly selective enzyme-catalysed esterification of simple glucosides. /. Chem. Sot. Chem. Commun. 1989, 934-935 Mutua, L. N. and Akoh, C. C. Synthesis of alkyl glucoside fatty acid esters in non-aqueous media by Candida sp. lipase. J. Am. Oil Chemisr’s SOC. 1993. 70, 43346 Fregapane, G., Samey, D. B. and Vulfson, E. N. Enzymatic solvent-free synthesis of sugar acetal fatty acid esters. Enzyme Microbiol. Tecknol. 1991, 13, 796-800 Panza, L., Luisetti, M., Crociati, E. and Riva, S. Selective acylation of 4,6-0-benzylidene glycopyranosides by enzymatic catalysis. J. Carbohydrate Ckem. 1993. 12, 125-130

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Kbaled, N., Montet, D., Pina, M. and Graille, J. Fructose oleate synthesis in a fixed catalyst bed reactor. Biorechnol. Let?. 1991, 13, 167-172 Guillardeau, L., Montet, D., Khaled, N., Pma, M. and Graille, J. Fructose caprylate biosynthesis in a solvent-free medium. Tenside Surf. Det. 1992, 29, 342-344 Schlotterbeck, A., Lang, S., Wray, V. and Wagner, F. Lipasecatalyzed monoacylation of fructose. Biorechnol. Len. 1993, 15, 61-64

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Oguntimein, G. B., Erdmann, H. and S&mid, R. D. Lipase catalysed synthesis of sugar ester in organic solvents. Biorechnol. Lett. 1993, 15, 175-180 Ikeda, I. and Klibanov, A. M. Lipase-catalyzed acylation of sugars solubilized in hydrophobic solvents by complexation. Biorechnol. Biuengin. 1993, 42, 788-791 Bock, K. and Thogersen, H. Nuclear magnetic resonance spectroscopy in the study of mono- and oligosaccharides. Ann. Rep. NMR Spectroscopy 1982, 13, l-57.