Macromolecules zyxwvu 1990,23, 5059-5064 5059 Sequence Distribution of /3-Hydroxyalkanoate Units with Higher Alkyl Groups in Bacterial Copolyesters Albert0 Ballistreri and Giorgio Montaudo Dipartimento di Scienze Chimiche, Universith di Catania, Viale zyxwvutsrq A. Doria 6, 95125 Catania, Italy Giuseppe Impallomeni Istituto per la Chimica e la Tecnologia dei Materiali Polimerici, Consiglio Nazionale delle Ricerche, Viala A. Doria 6, 95125 Catania, Italy Robert W. Lenz,’*+ Young B. Kim,+and R. Clinton Fulled Polymer Science and Engineering Department and Biochemistry Department, University zy of Massachusetts, Amherst, Massachusetts 01003 Received February 22, 1990 ABSTRACT Two different samples of copolyesters produced by Pseudomonas oleouorans when grown either on nonanoic acid alone or on an equimolar mixture of nonanoic and octanoic acids were evaluated for sequence distribution by controlled methanolysisto their oligomers, separation of the oligomers by HPLC, and analysis of the fractions so obtained by fast atom bombardment mass spectrometry (FAB-MS). The copolyesters contained primarily 0-hydroxyheptanoate and 0-hydroxynonanoate units, which showed no observable differences in their zyxwvuts lH and *3C NMR spectra with changes in composition, so those analyses could not be applied. The observed oligomer composition and that calculated on the basis of Bernoullian statistics for sequence distributions in random copolymers of the compositions studied were in good agreement. Introduction A wide variety of bacteria produce aliphatic polyesters as an energy reserve storage material. The polymers, which are produced in the form of small granules within the cell, have the general structure r 01 in which R is an n-alkyl group varying in size from CH3 to C12H25 and higher, depending on the bacterium. In many cases copolymers are formed, and both ‘H and 13C NMR spectroscopy can be used to determine the sequence distributions of the copolymers in which R is CH3 and CzH5, that is, for copolyesters containing both @-hydroxy- butyrate (HB) and P-hydroxyvalerate (HV) Some bacteria produce copolyesters containing units with much higher n-alkyl groups than methyl or ethyl, and, for these copolymers, NMR analysis cannot be used because, unlike the HB-HV copolymers, there are no detectable differences in the chemical shifts of the units with such longer n-alkyl groups. One family of copolyesters in which this limitation applies is that produced by the aerobic bacterium Pseudomonas oleouorans, in which R is generally an n-pentyl group or higher.6-7 In this report we describe how such copolyesters can be analyzed for sequence distribution by fast atom bombard- ment mass spectrometry (FAB-MS) of the oligomers formed by partial methanolysis of the copolymers. We have previously verified the use of this procedure for the analysis of HB-HV copolymers,8 but the copolyesters in the present case contained mixtures of units with higher n-alkyl pendant groups, which are identified in the t Polymer Science and Engineering Department. Biochemistry Department. following discussion by the following abbreviations: HC, 0-hydroxycaproate (R = C3H7); HH, 0-hydroxyhep- tanoate (R = C4H9); HO, 0-hydroxyoctanoate (R = CsH11); HN, P-hydroxynonanoate (R = CGH13); HD, 0-hydroxy- decanoate (R = C7H15); HU, /3-hydroxyundecanoate (R = Two different samples of poly(0-hydroxyalkanoates) (PHAs) produced by P. oleouorans grown on n-nonanoic acid were investigated for sequence distributions. The compositions of these samples, as determined by gas chromatography of the methyl esters of the @-hydroxy- alkanoic acids obtained after total methanolysis, are given in Table I. Sample 1 was obtained from a culture in which n-nonanoic acid was the only carbon source available to the bacteria, while sample 2 was obtained from bacteria grown on an equimolar mixture of n-nonanoic acid and n-octanoic acid. CEH17). Results and Discussion The procedure used for the partial methanolysis of the two PHA samples studied and for the HPLC fractionation of the oligomers so formed was similar to that previously reported.8 The methanolysis time was optimized to produce a high proportion of oligomers with molecular masses below the detection limit of the mass spectrometer used of up to ca. 2000 daltons. The repeating-unit compositions of the two samples studied are given in Table I. Sample 1. The HPLC trace of the partial methanol- ysis products from sample 1 of Table I is shown in Figure 1. The peaks are well separated, which permitted the collection of fractions corresponding to each peak in Figure 1 up to peak 19. All of the HPLC fractions collected from a single injection of 20 pL of an acetonitrile solution were lyophilized and analyzed by FAB-MS. Each mass spectrum for peaks 1-13 obtained from the HPLC trace showed the presence of a single oligomer, as evidenced by the molecular ions MH+ and MNa+ in the 0024-9297/90/2223-5059$02.50/0 0 1990 American Chemical Society