Chromatographic determination of optical configuration of 3-hydroxy fatty acids composing microbial surfactants

FEMS Microbiology Letters 108 (1993) 99-102 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier 99 FEMSLE 05349 Yoji Nakagawa a and Tohey Matsuyama b a Division of Chemistry, General Education Department, Niigata University, and o Department of Bacteriology, Niigata Unieersity School of Medicine, Niigata, Japan (Received 20 October 1992; revision received 8 January 1993; accepted 11 January 1993) Abstract: The chromatographic determination of the optical configuration of 3-hydroxy fatty acids of microbial surfactants was achieved in chiral high pressure liquid chromatography (HPLC) by injecting 3,5-dinitroaniline-derivatives of crude hydrolysates (less than 1 rag). Serrawettin W2, a surface-active cyclodepsipeptide of Serratia marcescens, was shown to contain D-3-hydroxydecanoic acid. Rubiwettin R1 and RG1, surface active glycolipid and linked fatty acids of Serratia rubidaea, were shown to contain D-3-hydroxytetradecanoic acid and D-3-hydroxydecanoic acid. The new method does not require purified sample or authentic optical isomers, and could be useful in the structural analysis of microbial lipids. Key words: 3-Hydroxy fatty acid; Optical configuration; Chiral chromatography; Serratia marcescens; Biosurfactants Introduction W e have n o t e d a w e t t i n g activity of Serratia spp. [1] a n d i d e n t i f i e d specific surface-active exolipids n a m e d s e r r a w e t t i n s a n d r u b i w e t t i n s [2,3]. D e t a i l s o f c h e m i c a l s t r u c t u r e s o f t h e s e lipids have b e e n d e s c r i b e d previously [4,5]. 3-Hydroxy fatty acids of C10-C16 chain l e n g t h w e r e shown to be c o m m o n s t r u c t u r a l m o i e t i e s of t h e s e c o m p l e x amp h i p h i l i c exolipids, as has b e e n o b s e r v e d in o t h e r b i o s u r f a c t a n t s , e.g., Bacillus subtilis surfactin o r r h a m n o l i p i d s of Pseudomonas aeruginosa [6]. W e t r i e d to d e t e r m i n e the optical c o n f i g u r a t i o n s o f t h e s e 3-hydroxy fatty acids by c h r o m a t o g r a p h i c resolution. H o w e v e r , in c o n t r a s t to 2-hydroxy fatty acids which have an a s y m m e t r i c c a r b o n a t o m next to a carboxyl g r o u p , t h e r e have b e e n no r e p o r t s of successful r e s o l u t i o n o f 3-hydroxy fatty acids with m e d i u m c h a i n length by c h r o m a t o g r a p h i c m e t h o d s . H e r e we r e p o r t on the s e p a r a t i o n of d e r i v a t i z e d 3-hydroxy fatty acids using chiral stationary phase column and a convenient method a p p l i c a b l e to small a m o u n t s of c r u d e lipid hydrolysate. Materials and Methods Correspondence to: Tohey Matsuyama, Department of Bacteriology, Niigata University School of Medicine, Niigata 951, Japan. Bacteria and exolipids S. marcescens strains N S 38 a n d NS 25 a n d a S. rubidaea strain A T C C 27593 w e r e d e s c r i b e d Chromatographic determination of optical configuration of 3-hydroxy fatty acids composing microbial surfactants 100 previously [2]. The bacteria were grown at 30°C for 3 days on peptone glycerol agar [3]. Preparation of serrawettin W1 (from strain NS 38), serrawettin W2 (from strain NS 25), and rubiwettin R1 and RG1 (from strain ATCC 27593) were carried out as described previously [2,4]. Rhamnolipid is a gift from Y. Ishigami, National Chemical Laboratory for Industry, Tsukuba, Japan. Bacterial lipids (800-300 Izg) were hydrolyzed in 1.0 ml of 6 M hydrochloric acid at 120°C for 90 min. The hydrolysates extracted with 2.0 ml of n-hexane were dried, dissolved in 100/zl of dioxane, then 100 /xl of dioxane containing 0.4% (w/v) N,N'-dicyclohexylcarbodiimide (Wako, Osaka, Japan) and 100 Izl of dioxane containing 0.4% (w/v) 3,5-dinitroaniline (DA) (Aldrich) were added. To introduce the 3,5-dinitroanilino group to 3-hydroxy fatty acids by forming amide linkage [7], the mixture (300 Izl) was incubated at room temperature for 4 h, then, heated at 60°C for 1 h. The reactants were passed through a disposable syringe filter DISMIC-3 (Advantec Toyo Co., Tokyo) to remove the dioxane-insoluble co-product, N,N'-dicyclohexylurea. For HPLC analysis, 10/zl of the filtrate containing 3,5-dinitroanilinederivatives (DA-derivatives) of the fatty acids was injected into the column. Authentic D-3-hydroxy fatty acids were prepared by fractional crystallization of cichonidine salts of DL-3-hydroxy fatty acids (Wako) and identified by measuring the melting points as described previously [8-11]. Such standard fatty acids (2.2 /zmol) were dissolved in 100 /zl of dioxane and treated by the same derivatization method for HPLC analysis. Results Resolution of 3-hydroxydecanoic acid oL-isomers After injection of DA-derivatives of DL-3-hydroxy-decanoic acid and elution with an eluent M, neighboring two peaks with equal size appeared in addition to the peaks corresponding to dioxane and reaction reagents (Fig. 1, upper chromatogram). This indicates a chromatographic resolution of optical isomers of 3-hydroxydecanoic acid; the paired peaks transpositioned in parallel, responding to the increased ethanol concentration in eluent N (Fig. 1, lower ehromatogram). In contrast, each DA-derivative of non-hydroxy fatty acid (C10, CI2 , Ct4 , and C16) gave a single peak preceding the elution of DA, irrespective of the different concentrations of ethanol in the two eluents (data not shown). E e-3 eq ¢9 a e', < HPLC analysis A commercially available chiral stationary phase (TSK gel Enantio P2 column, 4.6 mm i.d. × 250 mm l., 4 Izm particle size; TOSOH, Tokyo) [11-13] was used. The liquid chromatographic system consisted of a Shimadzu LC-6A pump, a Rheodyne 7413 injector with a 10 Izl loop, and a UV detector Shimadzu SPD-6AV equipped with a 0.3 lzl cell; the detector was set at 254 nm. Elution was performed with an eluent M or N, n-hexane-l,2-dichloroethane-ethanol (35 : 15 : 1, 0 20 40 Retention time ( rain ) Fig. 1. Elution profiles of DL-enantiomers derived from 3-hydroxydecanoic acid on chiral HPLC. Peaks: DL-CI0 , 3,5-dinitroaniline-derivative of DL-3-hydroxydecanoic acid; a, dioxane and unknown compounds; b, 3,5-dinitroaniline (DA). Eluent M (upper chromatogram) and eluent N (lower chromatogram) were used as a mobile phase. The details of the procedure are described under Materials and Methods. Derivatization of 3-hydroxy acids or 35 : 15 : 0.5, v / v / v , respectively), at a flow rate of 1.0 ml/min. 101 Determination of optical configuration u3 N j E C 40.28 49.70 Q 0 C £ O ,D 40.44 44.15 49.85 0 20 Retention 40 time (min) Fig. 3. Elution profiles of enantiomers derived from 3-hydroxy acids mixtures on chiral HPLC. (a) Mixture of authentic D-3-hydroxydecanoic acid, D-3-hydroxydodecanoicacid, and D-3-hydroxytetradecanoicacid. (b) Mixture of DL-3-hydroxy fatty acids (C10, C12, C14, and C16). (c) Hydrolysates of rubiwettin RI. (d) Hydrolysates of rubiwettin RG1. Peaks: D-C (...truncated)