Head group-modified sophorolipids: Synthesis of new ... - Springer Link

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Head Group-Modified Sophorolipids: Synthesis of New Cationic, Zwitterionic, and Anionic Surfactants Jonathan A. Zerkowski*, Daniel K.Y. Solaiman, Richard D. Ashby, and Thomas A. Foglia Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038

ABSTRACT: Amino acid–glycolipid conjugates were prepared using carbodiimide-mediated coupling methods. The amino acid units were multifunctional and possessed a para-aminobenzoic acid linker. The glycolipid used was a stearic sophoroside. The aim of preparing these modified sophorolipids was to increase their water solubility as well as to introduce sites at the polar head groups that permitted further chemical derivatization. After acidolytic or hydrogenolytic deprotection of the amino acid N-terminus or side chain, water-soluble compounds were obtained that displayed good surfactant properties. Critical micelle concentration values were clustered in the range of high 10−6 to low 10−5 M, and minimum surface tension values were below 40 mN m−1. Two of the compounds represented more complicated structural classes, namely, gemini and bolaform surfactants. Paper no. S1478 in JSD 9, 57–62 (Qtr. 1, 2006). KEY WORDS: Amino acids, bioconjugate, glycolipid, sophorolipid, surfactants.

Sophorolipids (SL, Fig. 1) are hydroxy fatty acid glycosides that can be produced in good yields, around 100 g/L, by microbial fermentation of a wide variety of feedstocks (1,2). Previous work has shown that agricultural by-products such as tallow or soy molasses can be converted to these interesting glycolipids (3). As produced, the material consists of a number of closely related variants, including a lactone and an open-chain form, each of which may bear up to two acetyl groups. Furthermore, depending on the feedstock, the predominantly C-18 lipid chain can be predominantly a stearic or oleic acid derivative or a mixture. The crude material does possess surfactant activity, and its ready availability recom*To whom correspondence should be addressed at Eastern Regional Research Center, ARS, USDA, 600 East Mermaid Lane, Wyndmoor, PA 19038. E-mail: [email protected] Abbreviations: Boc, butoxycarbonyl; Cbz, benzyloxycarbonyl; CMC, critical micelle concentration; DBU, 1,8-diazabicyclo[5.4.0]undec-7ene; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole hydrate; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography–mass spectrometry; Lys(Cbz), N-ε-benzyloxycarbonyl lysine; NMR, nuclear magnetic resonance; Paba, 4-aminobenzoic acid; SL, sophorolipids; THF, tetrahydrofuran; TLC, thin-layer chromatography. COPYRIGHT © 2006 BY AOCS PRESS

mends its study as an eco-friendly detergent. Its structural multiplicity, however, complicates attempts to correlate molecular shape with surfactant behavior. For example, the mode of aggregation, either at an interface or in a micelle or vesicle, of the compact lactone and the extended open-chain form would likely be quite different. Several groups of researchers have begun to elucidate the effect on surfactant properties of altering the carboxy terminus of the lipid chain through esterification or amidation (4–6). The moieties introduced were hydrophobic, consisting of aliphatic chains and aromatic groups. There have also been reports showing that SL can be selectively acylated at their 6′ or 6′′ hydroxy groups using lipases (7–9). The acyl groups introduced in these cases have been, for the most part, small units such as acetyl, propionyl, or acryloyl, although a succinoyl unit was also added. To our knowledge, more complex units exhibiting a range of functionality have not been appended to the head groups of SL. The goal of this work was to examine ways in which the fundamental sophorolipid skeleton could be chemically modified to increase its utility as a surfactant. The immediate concern was to increase the water solubility of SL with charged head groups. In particular, the aim was to append amino-acid derived groups: Through straightforward amide bond chemistry, a wide range of functionality could be introduced. Enzymes were not initially investigated for the preparation of these SL–amino acid conjugates. Lipases might attack ester groups in the amino acid units, for example, at the glutamic acid side-chain, and might be poorly tolerant of bulky carboxyl donors, although they have been used for benzoylation of 6′ groups of glucosides (10). As a result, the targeted modified SL are mixtures of isomers, and it was proposed this would have minimal impact on their surfactant properties.

EXPERIMENTAL PROCEDURES General. SL were prepared as previously reported, using stearic acid and glucose as carbon sources (3). Protected amino acids were obtained commercially from Advanced Chemtech (Louisville, KY) and Novabiochem (EMD Biosciences, San Diego, CA). All solvents and reagents were used as received. Silica gel used for column chromatography was JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

58 J.A. ZERKOWSKI ET AL.

FIG. 1. Outline of the synthesis of molecule 1, showing the sophorolipid (SL) structure and the para-aminobenzoate (Paba) linker discussed in this work. Abbreviations: PG, protecting group; AA, amino acid. Reaction conditions: “a,” water-soluble carbodiimide and hydroxybenzotriazole (see Experimental Procedures section); R1, either PG-AA (compounds 1a, 1b, and 1g) or acyl-AA(PG) (compounds 1d, 1e, and 1f; see Scheme 1). Compound 1c is a hybrid of these two arrangements, since it possesses PG both at the N-terminus and on its side chain, and it is attached to the Paba through its γ-carboxylate. Although the benzoate is shown here attached to a specific OH, molecules 1a–g and 2a–g are in fact mixtures of isomers that differ in the point of attachment of the amino acid–benzoate conjugate to the SL. In the lactone version of SL, the carboxylic acid of the lipid chain is condensed with the 4′′ OH.

obtained from Fisher Scientific (Fairlawn, NJ). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Associates Gemini 200 MHz instrument, and liquid chromatography–mass spectrometry (LC-MS) data were recorded on a Waters/Micromass ZMD instrument with electrospray or atmospheric pressure chemical ionization. SL ethyl and benzyl esters. The crude SL (2 g) obtained from Candida bombicola fermentation was added to a solution of 0.6 g KOH in 50 mL ethanol and stirred at room temperature overnight. A complete solution was not obtained, but the mixture remained a pale yellow suspension. The reaction was acidified to an apparent pH of 4 with 2 M HCl, and ethanol was removed on a rotary evaporator. The solid was washed with 2 × 10 mL water to remove acetic acid (produced from hydrolysis of the acetyl groups on the crude SL), air-dried, and purified by column chromatography on silica gel with 80:20:1 chloroform/methanol/water (referred to hereafter as solution A). Two products were obtained: SL ethyl ester (1.3 g) and SL free acid (0.4 g). The free acid (0.4 g, 0.64 mmol) was dissolved in 10 mL 3:1 chloroform/dimethylformamide (DMF). To this solution were added benzyl alcohol (2 mL, 19.3 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 122 mg, 0.8 mmol), and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 153 JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

mg, 0.8 mmol). The mixture was stirred magnetically under N2 for 36 h. DMF and CHCl3 were removed with the rotary evaporator, and the material was purified with column chromatography on silica gel, first in 5% methanol/chloroform to remove the benzyl alcohol, then with solution A to afford SL benzyl ester (373 mg, 82%). 1H NMR [d6-dimethylsulfoxide (DMSO) + D2O]: 1.11 d (3H), 1.20 br s (26H), 1.51 m (2H), 2.32 t (2H), 2.92–3.28, m (7H), 3.30–3.50 m (3H), 3.55–3.73 m (3H), 4.30 d (1H), 4.41 d (1H), 5.05 s (2H), 7.34 br s (5H). Synthesis of amino acid-derivatized SL 1a–g. A representative procedure is as follows: A solution of propionic anhydride was prepared in situ by reacting propionic acid (0.48 g, 6.5 mmol) with EDC (614 mg, 3.2 mmol) in acetonitrile for 15 min at room temperature. This solution was then added over the course of 5 min to a magnetically stirred solution of N-ε-benzyloxycarbonyl lysine [Lys(Cbz)] (1 g, 3.6 mmol) in 36 mL 0.1 M NaOH and 20 mL 2-propanol. A fine white precipitate formed, another 36 mL 0.1 M NaOH was added, and stirring was continued overnight. The reaction mixture was then concentrated in vacuo to remove the organic solvents and was acidified to a pH of 2.5 with 1 M HCl. During acidification, the white precipitate dissolved. The aqueous solution was extracted with chloroform and then ethyl acetate. These organic solvents were combined and concentrated on the rotary evaporator to yield

59 HEAD GROUP-MODIFIED SOPHOROLIPID SURFACTANTS

a colorless oil, which was used without purification in the next step. The crude propionyl-Lys(Cbz) thus obtained was dissolved in 10 mL 1:1 tetrahydrofuran (THF)/acetonitrile, and to this solution was added HOBt (460 mg, 3 mmol) and EDC (573 mg, 3 mmol). The solution was swirled at room temperature for 15 min and was then added over the course of 5 min to a solution of 4-aminobenzoic acid (Paba, 550 mg, 4 mmol) in 20 mL THF/acetonitrile. After addition, the reaction mixture was stirred overnight. The solvent was removed by rotary evaporation to yield a yellow semisolid, which was then stirred with 50 mL 1 M HCl. The liquid was decanted and the residue was purified by column chromatography on silica gel with solution A. The product, N-α-propionyl-Lys(Cbz)-Paba, had an Rf of 0.5 in this solvent system (510 mg, 35% based on initial use of propionic acid). Yields for the analogous amino acid–Paba adducts are as follows: N-α-Cbz-alaninePaba (where Cbz is benzyloxycarbonyl), used to make 1a and 1g, 73% based on Cbz-alanine; N-α-Boc-4-hydroxyprolinePaba, used to make 1b, 68% based on Boc-hydroxyproline; N-α-Boc-α-butyl-glutamic acid-Paba, used to make 1c, 54% based on Boc-glutamic acid α-butyl ester; N-α-palmitoylLys(Cbz)-Paba, used to make 1e, 51% based on palmitic acid; N-α-propionyl-γ-benzyl-glutamic acid-Paba, used to make 1f, 63% based on propionic acid. The propionyl-Lys(Cbz)-Paba (455 mg, 1 mmol) was dissolved along with SL ethyl ester (900 mg, 1.38 mmol) in 6 mL 3:1 chloroform/DMF. To this solution was added HOBt (168 mg, 1.1 mmol), EDC (210 mg, 1.1 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 149 µL, 1 mmol). Preliminary results had indicated that a strong base enhances the rate of this reaction. The reaction mixture was heated under nitrogen to 50–55°C and stirred magnetically for 36 h. It was then concentrated on a rotary evaporator and purified by column chromatography on silica gel with solution A to afford compound 1d (0.56 g, 51%). Yields for compounds 1a–g were as follows: 1a, 55%; 1b, 50%; 1c, 42%; 1e, 35%; 1f, 45%; 1g, 52%. Preparation of compounds 2a–g. For the surfactants requiring acid deprotection (1b and 1c), a weighed amount of the compound was treated with 2 mL trifluoroacetic acid for 1 h, after which 2 mL CH2Cl2 was added and the reaction was continued for another hour. No starting material remained by thin-layer chromatography (TLC). The solvents were then removed under a stream of N2 and rotary evaporated twice out of acetonitrile, then dried on a vacuum line overnight. The deprotected compounds were then dissolved in buffer for use in surface tension measurements. For the other surfactants, which required hydrogenolytic deprotection, the compound was dissolved in 50 mL N2sparged 2:1 THF/ethanol and 5% Pd/C was added (the ratio of the weight of catalyst to the weight of compound 1 was approximately 1:2 in all cases). N2 was passed through the flask for 10 min, followed by H2 at a rate of about 1 bubble/s as monitored with an oil bubbler. Progress of the reaction was monitored by TLC in solution A; H2 bubbling was continued

until the starting material was gone. Reaction times ranged from 2 to 4 h. N2 was bubbled through, and the mixture was filtered first through Celite® 545, then through two layers of Whatman #1 filter paper. Solvent was removed on the rotary evaporator and the solid was dried on the vacuum line overnight. Weighed portions of the dried solid were dissolved in buffer for use in surface tension measurements. Surface tension measurements. Measurements were performed with the Wilhelmy plate method on a DataPhysics Instruments DCAT-11 tensiometer at ambient temperature, 21–22°C. To avoid any effects that might arise from a change in counterions, the buffer used was 10 mM acetate/borate/phosphate, since this combination can be adjusted to a wide range of pH by addition of NaOH. The buffer used to dissolve compound 2 was used as the blank into which the surfactant solution was titrated. Solutions were filtered through a 1.2-µm nylon syringe filter prior to use.

RESULTS AND DISCUSSION Selection of targets. The design of molecules 2a–g was the result of several considerations. First, the use of a saturated stearic derivative of SL was chosen rather than the oleic version, which has received more attention in the literature. Although it might be argued that the saturated chain would lead to more compact packing in a micelle core, hence a lower critical micelle concentration (CMC), than would the kinked oleic version, use of the saturated version had a less conceptual and more practical motivation. It was necessary to demonstrate the utility of benzyl protection schemes for functionality at the head group, but hydrogenolysis could also lead to reduction of the oleic C–C double bond. The stearic derivative was used to avoid that complication. Second was the choice of the carboxyl ester at the distal end of the lipid chain. There was no strong reason to prefer the ethyl ester; it was easy to prepare and work with, but other small alkyl esters would presumably also be effective. It did afford a convenient pattern in the NMR spectra. The preparation of this ethyl ester was accomplished by a slight modification of the route found in the literature (7), namely, by replacing sodium ethoxide with KOH in ethanol as the means for opening the macrolactone. Finally, there was the choice of head group modifications themselves. The key feature was the use of a benzoate moiety (Paba) to link polyfunctional amino acids to the carbohydrate. There were two reasons for this: First, we observed that the acetyl groups of the naturally occurring SL mixture were easily removed in protic solvents when only small amounts of base were present. Similarly, a simple acyl group such as glycine protected with a t-Boc group was cleaved off the SL to the extent of about 25% overnight in methanol with 1 equivalent of triethylamine, according to semiquantitative high-performance liquid chromatography (HPLC) data. Decomposition of this sort might curtail the use of head group-modified SL surfactants in basic solutions if the modification consists of an aliphatic acyl group. The benzoate linker appears to negate JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006


SCHEME 1. Sophorolipid (SL) derivatives prepared in this work (see Fig. 1). Cbz, benzyloxycarbonyl; Boc, butoxycarbonyl.

this problem; decomposition of compound 1 or 2 was not observed under similar conditions. A further advantage is that benzoates are less susceptible to migration between carbohydrate hydroxyl groups. The benzoate unit also allows further derivatization, and future work will investigate diaminobenzoic acid as a multipoint linker for the attachment of multiple charged groups. Second, and more important, steric hindrance of any α-substituted amino acid was believed to significantly inhibit acylation at the carbohydrate hydroxy groups. Preliminary results (data not shown) indicated that acylating SL with Cbz-alanine under the same conditions used for the amino acid–Paba units proceeded in yields around 10% after 72 h, whereas Boc–leucine gave no product even when 4-dimethylaminopyridine (DMAP) was present as the catalyst. The amino acids chosen for this study were intended to provide a range of polar groups. Molecules 2a, 2b, 2d, and 2e are all cationic but with slight structural differences. The alanine and hydroxyproline derivatives 2a and 2b differed in that the latter had the added polar hydroxy group. The lysine derivative 2d had the charge extended away from the head group. The palmitoyl lysine derivative 2e had the added feature of a second hydrophobic chain. This molecule fits the definition of a gemini surfactant (11): The palmitoyl lysine unit represents one surfactant, the SL a second surfactant, and the spacer unit connecting the two is the Paba moiety. An anionic variant was provided by the propionyl glutamic acid derivative 2f, whereas the unsubstituted glutamic derivative 2c afforded a zwitterion. Finally, a different zwitterionic arrangement was provided by removal of the Cbz and benzyl groups of 1g, giving the bolaform amphiphile 2g. Synthesis. The preparation of 1a–g relied on classical carbodiimide-mediated coupling reactions for both amide and ester formation, in conjunction with the standard Boc, Cbz, t-butyl, and benzyl protection schemes (see Scheme 1). The SL benzyl ester used to prepare 1g was obtained by reacting SL free acid (obtained as a by-product of ethanolysis during the preparation of the SL ethyl ester) with benzyl alcohol, EDC, and HOBt. Linkage of the amino acids to the Paba spacer was accomplished similarly with the same EDC/HOBt coupling methods. The amino acid structures that we affixed

JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

to SL through the Paba linker came in two basic varieties. The simpler kind were amino acids with protecting groups at their α-nitrogens, used in variants 1a, 1b, and 1g. The other kind were acylated amino acids with protecting groups on their side chains, e.g., the propionyl and palmitoyl lysine and glutamic acid in 1d–f. These variants were prepared from symmetrical anhydrides of the acyl groups. Compound 1c was somewhat more complicated in that it possessed two protecting groups, one on the α-nitrogen and one on the α-carboxylate. The more difficult step was attaching the compound amino acid–Paba units to the sophorose head group. There were seven hydroxy groups on SL, and in principle all of them could react with the benzoate, although of course the primary 6′ and 6′′ hydroxy groups should react preferentially. An excess of SL ester was used to avoid formation of doubleacylation products, although in a few instances when those were observed to be formed in very low amounts, they could be separated out easily by column chromatography. Preliminary experiments were performed to determine the reaction conditions that yielded the best selectivity, but optimization could very likely continue further. When DMAP was added as a catalyst, no selectivity was observed: seven isomeric compounds of 1a were observed by LC-MS. The solvent system settled on was chloroform/DMF, although DMSO, dioxane, and trifluoroethanol were also investigated. The conditions reported in the Experimental Procedures section are those that gave the fewest number of isomers, two roughly equal in intensity and one minor one, 10–20% of the intensity of the two major peaks. Whether the two major isomers were the 6′ and 6′′ adducts is not known, but that is our working hypothesis in the absence of evidence to the contrary. At the very least, it is clear that these two major isomers had the amino acid–Paba unit on different glucose rings, based on MS data (Fig. 2). Only one isomer showed a strong peak at M-163, corresponding to a loss of glucose (C6H11O5); this fragmentation should occur only for the isomer with the amino acid–Paba unit attached to the “internal” glucose ring, i.e., at the 6′ position. The other major isomer did not show an M-163 peak. Conditions that permitted separation of these

61 HEAD GROUP-MODIFIED SOPHOROLIPID SURFACTANTS TABLE 1 Tensiometry Data for Compounds 2a–ga Compound Stearic SL ethyl ester, unbuffered 2a, pH = 5.8 2b, pH = 5.8 2b, pH = 9.1 2c, unbuffered 2c, pH = 2.5 2c, pH = 5.8 2c, pH = 9.1 2d, pH = 5.8 2e, pH = 5.8 2f, pH = 2.5 2f, pH = 5.8 2g, pH = 5.8

CMC (µM)

Γmin

90 8 6 24 13 17 14 17 15 6 110 51 85

48 39 37 42 40 41 39 40 39 47 49 43 40

a

Measurements were performed in duplicate. SL, sophorolipid.

FIG. 2. Mass spectra of the two major isomers of compound 2b. The peak at 722 in the top spectrum indicates loss of one glucose unit from the sophorose head group. The peak at 557 occurred from loss of the ethyl hydroxystearate side chain.

three isomers from each other using silica gel column chromatography were not found, and preparative HPLC would be required. However, compounds 1a–g were readily purified from all extraneous compounds, such as unreacted SL ester, unreacted amino acid–Paba, and the coupling reagents, so the material used to prepare the actual surfactants 2a–g was free from contaminants. Deprotection of molecules 1b and 1c with trifluoroacetic acid caused no damage to the glycoside linkage, that is, cleavage of the ethyl 17-hydroxystearate was not observed. Compounds 2a–g had good solubility in water. Molecules 2b and 2d, for example, were readily soluble at 500 mg/100 mL at room temperature (higher concentrations should be achievable but were not tested due to the limited amount of material that had been synthesized). By contrast, the ethyl ester of the stearic variant of SL was soluble only at about 18 mg/100 mL at room temperature. Using these SL derivatives 2a–g, that have good water solubility, we expect to be able to test surfactant efficiency as a function of the length of the fatty acid ester’s alkyl chain without encountering solubility problems at room temperature or even below. Surface tension measurements. Figure 3 and Table 1 show that molecules 2a–g behaved as surfactants. Several features are worthy of comment. First, the CMC and Γmin values for the zwitterionic 2c appeared to vary little with pH. This kind of behavior has been widely noted for zwitterionic surfactants and is frequently listed as a feature in favor of their use in various applications, particularly in consumer-care products (12). In the absence of potentiometric and/or spectroscopic data, it is not possible to say whether the amino and carboxylic groups of the glutamic unit of 2c were both ionized over the pH range from 2.5 to 9.1. We suspected that the zwitterion predominated in the range examined, since a

monomeric α-amino acid should have pKa values in the range of 2.1–2.3 and 9.5–9.7. Nonetheless, ionization in an aggregated state such as a micelle is complicated by the proximity of neighboring charges and is difficult to predict. To quote from an authoritative reference, “the acid–base chemistry of micellar surfactants . . . cannot be described by using a single value for the pKa” (13). At any rate, no decrease in the water solubility of 2c was observed over this pH range. CMC and Γmin values also remained roughly the same for 2c in unbuffered solution. This latter feature was gratifying, since it suggests that any specific ion effects attributable to the presence of a trifluoroacetate anion from the deprotection of a Boc–amino acid were negligible or, again, shielded by other charges in the aggregated state, or at the very least, could readily be counteracted in 10 mM buffer. Second, the bolaform amphiphile 2g was not expected to form a classical micelle. It should, however, be able to assemble in a head-totail fashion into a vesicle. This difference in self-assembly may be responsible for the higher CMC value observed for this molecule. Future work will involve electron microscopy to investigate the morphology of aggregates that 2g forms. Finally, the CMC and Γmin values for the parent nonionic stearic SL

FIG. 3. Representative surfactant behavior for several compounds. Solutions of 2a–c were buffered at 10 mM and pH 5.8 with a temperature of 21–22°C, whereas the stearic ethyl ester solution was unbuffered. For abbreviation see Figure 1.



ethyl ester showed that the charged groups appended to the head group had a significant impact on the surface tensionlowering properties in most cases. To what extent does the composition of compounds 2a–g as mixtures of isomers affect the CMC values of these materials? We hypothesize that differences in surfactant behavior of the 6′ and 6′′ adducts for each compound may have been minimal, especially after noting that all the charged SL variants prepared in this work have CMC values and Γmin values that differ little from each other. Although it is true that the shape of the molecule at the hydrophilic head group is different for these two major isomers (the former has a branched shape, whereas the latter is more linear)—and as a result, aggregation in a micelle or vesicle would be expected to differ—we propose that the dynamic nature of these aggregates, the extensive hydrogen bonding that should occur for such carbohydrate surfactants in all isomers, the consistent shape of the hydrophobic portion, and the possibilities for rotamers at the glucose–glucose bond all contributed to minimizing the orientation or electrostatic effects associated with the different isomers. For example, although structurally the comparison is far from precise, the α and β anomers of several ethyl acylglucopyranosides differed by a factor of less than 2 in CMC and by 2 or 3 mN/m in Γmin, variations which may well lie within experimental error (14). Nonetheless, preparative-scale HPLC work is underway to separate the isomers for one or two examples of compound 2 and investigate this matter further.

ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Bun-Hong Lai (chromatography and tensiometry), Marshall Reed (microbiology), Loida Cruz-Bass, and Dr. Alberto Nunez (MS). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

2. Cavalero, D.A., and D.G. Cooper, The Effect of Medium Composition on the Structure and Physical State of Sophorolipids Produced by Candida bombicola ATCC 22214, J. Biotechnol. 103:31 (2003). 3. Solaiman, D.K.Y., R.D. Ashby, A. Nunez, and T.A. Foglia, Production of Sophorolipids by Candida bombicola Grown on Soy Molasses as Substrate, Biotech. Lett. 26:1241 (2004). 4. Lang, S., A. Brakemeier, R. Heckmann, S. Spockner, and U. Rau, Production of Native and Modified Sophorose Lipids, Chim. Oggi 18:76 (2000). 5. Singh, S.K., A.P. Felse, A. Nunez, T.A. Foglia, and R.A. Gross, Regioselective Enzyme-Catalyzed Synthesis of Sophorolipid Esters, Amides, and Multifunctional Monomers, J. Org. Chem. 68:5466 (2003). 6. Zhang, L., P. Somasundaran, S.K. Singh, A.P. Felse, and R. Gross, Synthesis and Interfacial Properties of Sophorolipid Derivatives, Coll. Surf. A: Physicochem. Eng. Aspects 240:75 (2004). 7. Bisht, K.S., R.A. Gross, and D.L. Kaplan, Enzyme-Mediated Regioselective Acylations of Sophorolipids, J. Org. Chem. 64:780 (1999). 8. Bisht, K.S., W. Gao, and R.A. Gross, Glycolipids from Candida bombicola: Polymerization of a 6-O-Acryloyl Sophorolipid Derivative, Macromolecules 33:6208 (2000). 9. Carr, J.A., and K.S. Bisht, Enzyme-Catalyzed Regioselective Transesterification of Peracylated Sophorolipids, Tetrahedron 59:7713 (2003). 10. Enaud, E., C. Humeau, B. Piffaut, and M. Girardin, Enzymatic Synthesis of New Aromatic Esters of Phloridzin, J. Mol. Catal. B: Enzymatic 27:1 (2003). 11. Chevalier, Y., New Surfactants: New Chemical Functions and Molecular Architectures, Curr. Opin. Coll. Interface Sci. 7:3 (2002). 12. Tsubone, K., N. Uchida, H. Niwase, and K. Honda, Syntheses of Sodium 2-(N-Alkyl-N-methylamino)ethanephosphates and Their Physicochemical Properties, J. Am. Oil Chem. Soc. 66:829 (1989). 13. Laughlin, R.G., Fundamentals of the Zwitterionic Hydrophilic Group, Langmuir 7:842 (1991). 14. Adelhorst, K., F. Bjorkling, S.E. Godtfredsen, and O. Kirk, Enzyme Catalysed Preparation of 6-O-Acylglucopyranosides, Synthesis:112 (1990).

REFERENCES 1. Asmer, H.-J., S. Lang, F. Wagner, and V. Wray, Microbial Production, Structure Elucidation and Bioconversion of Sophorose Lipids, J. Am. Oil Chem. Soc. 65:1460 (1988).


[Received February 2, 2005; accepted August 21, 2005]