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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 5,Issue of March 10, pp.

1741-1745,
Printed in U.S.A.

1973

Asparaginase
STEREOCHEMISTRY SUBSTRATES* OF THE ACTIVE CESTER AS DETERMINED BY CIRCULAR DICHROISXI OF

(Received

for

publicat,ion,

July

7, 1972)

JOSEPH E. COLEMAN

AND ROBERT

E. ~ANDscHunIAcHER Biophysics and Biochemistry
and

From the Departments o;f Molecular New Haven, Connecticut 06510

Pharmacology,

Yale University,

SUMMARY Large Cotton effects associated with the r + T* and n + g* transitions of asparaginase substrates have been shown in substrates containing a 2nd asymmetrical carbon atom at the p position, diaminosuccinic acid monoamide and @-methylasparagine. Hydrolysis of the amide bond is accompanied by large changes in the rotatory strength of these transitions. These substrates have four possible isomers, L-L, D-D, L-D, and D-L. From the signs of the Cotton effects appearing on hydrolysis of mixtures of these isomers it can be determined which isomer is hydrolyzed. The enzyme has an almost absolute stereospecificity for the L-D isomer which defines the three-dimensional arrangement of the protein groups interacting with the substituents on the o( carbon and the catalytic groups adjacent to the P-amide within rather narrow limits. While the L-L isomer of diaminosuccinic acid monoamide is not detectably hydrolyzed, the L-L isomer of P-methylasparagine is attacked at a rate 0.2 to 0.3 % of that observed for L-asparagine, suggesting that a P-CH3 group can be accommodated in this configuration while a ,&NH2 cannot.

broad range of activity has been extended t’o substrates containing 2 asymmetrical carbon atoms which show large changes in optical activity accompanying hydrolysis of the amide. From the changes in the substrate Cotton effect during hydrolysis it has been possible to define the absolute stereochemical requirements of this enzyme in positions 2 and 3 of the substrate.
MATERIALS AND METHODS

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The enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been the object of increasing interest since the purified enzyme has been found useful in the treatment of acute lymphoblastic leukemia (l-4). Its clinical utility is attributed to the asparagine-dependent nature of these neoplastic cells in contrast to normal cells which can synthesize asparagine (4). The physicochemical properties of the crystalline enzymes from Escherichia coli B and Erwinia caratovora have been reported on extensively (5-7) _ The protein is a tetramer of molecular Tveight -133,000 (4, 5, 7). A variety of alternate substrates for the enzyme have been described including /3-cyanoalanine and the P-hydroxamate of Laspartic acid both of which are converted to L-aspartic acid (4). Catalytic decomposition of 5-diazo-4-oxo-L-norvaline as well as active site labeling by this reagent have been reported (8). This
* This work was supported by Grants AM-09070-08 and CA10748 from the National Institutes of Health and Grants IC-64L and PR-27 from the American Cancer Societv.

Xubstrates and Enzymes-L-Aspartic acid, L-asparagirre, and Lleucine were chromatographically pure (Schwarz-Mann, New York). L-Leucine amide was a gift of Professor Sofia Simmonds of Yale University. D-L, L-n-Diaminosuccinic acid monoamide and L-L, D-D-diaminosuccinic acid monoamide were synthesized by Dr. Pauline Chang, Yale University, and the syntheses will be reported in detail elsewhere (9). P-Methylasparagine (L-L, D-D) was also synthesized by Dr. Chang starting with commercial three-DL-fi-methylaspartic acid (Nutritional Biochemicals, Cleveland, 0.). Asparaginase (270 units per mg) from E. coli B was prepared by Merck and Co. and supplied as a lyophilized sample of an electrophoretically homogeneous preparation. One unit of asparaginase is the amount of protein required for the release of 1 pmole of ammonia per min from L-asparagine at pH 8.5, 37”. Optical Rotatory Dispersion and Circular Dichroismn-ORDr was recorded with a Cary 60 recording spectropolarimeter. CD leas recorded with a Cary 61 spcctropolarimeter. Slit widths were programmed to maintain constant light intensity. The CD instrument was calibrated with an aqueou:: solution of d-10camphorsulfonic acid (J. T. Baker Co.). en - Ed = 2.20 at 290 nm. All measurements were carried out at 25”. ORD is expressed as specific rotation, [a], or as molecular rotation, [M] = [a]M.W./lOO, in units of dcg cm2 per dccimole. CD is espressed in terms of molecular cllipticity, [e] = 2.303 (45OO:l7r) (EL - en) wit’h units of dcg cm2 per decimole.
RESULTS

Substrate and Optical Properties-The primary substrate employed iu this study was the monoamide of diaminosuccinic acid (Scheme I). Since there are 2 asymmetrical carbon atoms the compound can exist as four different stereoisomers,
1 The abbreviations CD, circular dichroism. IIsed are: ORD, optical

rotatory

dispersion;

1741

and the meso form (DL = LD). or a single isomer with two asymmetrical centers (of opposite configuration) which make equal contributions to the rotation.164 indicate the ranges of a solution untreated with 25”. It would have the advantages of a direct spcctrophotometric assay without the addition of other enzymes as in the assay coupled to the reduction of pyridine nucltotidc (1 I) or the analytical problems of mcnsuring ammonia release. Deduction of the sterrochemistry of the hydrolyzed and unhydrolyzed isomers from the sign of this Cotton cffcct requires both an assignment of this transition and a knomlcdge of its sign in cnvironmcnts of known This can readily be deduced from the CD of some symmetry.05 nr Tris .nm Cotton effect is directly related to enzymatic hydrolysis of the substrate. This number is the same order of nlagnitude as the turnover lrumbcrs reported for L-asl)umgine as substrate under similar conditions (4.he hydrolysis of L-asparagine is 8.0. 1) confirming that the product must contain either tlvo isomers showing equal and opposite rotations. For convenience in comparing this corn pound to asparagine the carbon adjacent to the carboxyl is designated as the (Y carbon and that carrying the amide as the fi carbon. D SCHEME LL. reduce the precision of the slope over that of determinations taken at longer pen periods with less random noise. Since hydrolysis of only one of the isomers is expected to be catalyzed by the enzyme.84 of X IOP RI) after 24 hollrs incrlbation with 10 The WTOT bars on the axis mg per ml). I). This suggests that only 50% of the substrate is hydrolyzed as has been confirmed by determining ammonia release by Nesslerization (9). at a substrate concentration of 1 X 1OP nr.2. however. and DL. ~-L-P-D and (Y-D-@-L (Scheme I). Since the first two isomers can be readily separated from the meso form.85 mM solution (1 mg per ml) observed over a l-cm path yields a change in observed ellipticity of -0. as assayed by the coupled spectrophotomctric assay (5). H D I. LD. model compounds. DD. The highest maximum velocity rcportcd for t. 2011 6 i 210 5 220 230 x. Appearance of the 222. Once maximum rotation is achieved the CD spectrum remains the same indefinitely suggesting that the other isomer is completely resistant to hydrolysis. pH 8. Stereochemistry of Isomer of Diaminosuccinic Acid Monoanzide Hydrolyzed by Aspclraginase-The Cotton effect gcncrated b> 50 y0 hydrolysis of the misturc of mcsoisomers of diaminosuccinic acid monoamidc is negative (Fig. Complete enzymatic hydrolysis of a 6. The substrate inhibition may relate to the prcscncc of the unhydrolyzcd isomer and it is possible that with a pure isomer this CD assay could be devclopcd into one satisfactory for a complet~c kinetic analysis.05 M Tris. complete enzymatic hydrolysis should be accompallied by the appearance of optical activity at the wave length of the absorption bands of the unhydrolyzed amide.org by guest.-P-D-diiLmirlosI1ccinic acid monoamide (1. The product of this synthesis is not optically active (Fig. the meso form of diaminosuccinic acid has been used for synthesis of the amide substrate. on March 9. Time courses for the increase in negative ellipticity at 222 nm at three different enzyme concentrations are shown. Aliphatic amino acids of the L configuration iucluding r. I nn.1742 0 NH.jbc. Rate of Change of Ellipticity as Function of Enzyme Concentration-Enzymatic hydrolysis of 50% of the mixture of mesoisomers of diaminosuccinic acid monoamide yields a product which has a combined molar ellipt’icity at the minimum of the CD spectrum of -6. enzyme activity. since the rate of appearance of this Cotton effect is directly proportional t’o enzyme concentration (Fig. The product should contain two meso-type isomers of diaminosuccinic acid monoamide.HCI. Thus the change in cllipticity can be adapted as a satisfactory assay of 13 4 Downloaded from www.95 x 104 moles per min per mole of enzyme. Since the amide chromophore will not make the same contribution to rotation as the carboxyl chromophore (see below).0. The resulting large random noise does not. 1).75 X 10” moles of substrate hydrolyzed per min per molt of enzyme in 0. The maximum turnover observed for diaminosuccinic acid monoamide n-as 4.-aspnrtic and L-glutamic acids have positive Cotton .H 0 -o-c-c-c-c Ii I I I I NH2 L or II \ NH. nm 240 250 260 FIG. ~1 of nsparaqinase (0. Condilions: 0. base-line Cl> sprctrmn taken on the same enzyme.0445” at 222 nm. A solution of the diaminosuccinic acid amide treated with asparaginase for 24 hours shows a large negative Cotton effect centered near 222 nm (Fig. ~-D-&I- 1.cm path length. pH 8. 5). The parent compound. 2. 1). This inhibition prevented a complete kinetic analysis. 0. The pen period on the Cary 61 was set at 1 s to insure rapid pen response. l-llfortunately in the caec of the mixture of a-L-&Dand ol-1)-P-L-diamirlosuccinic acid monoamide the1 c is severe subst~rate inhibition at substrate concentrations >5 x 1OW X.3 X lo2 deg cm2 per decimole (Fig. ORI1 (CWW 1) and CD (Curve 2) spectra of asolution and LY-r. 2). diaminosuccinic acid exists as three stercoisomcrs LL. Enzymatic hydrolysis results in the release of only 50% as much ammonia nitrogen as does complete acid hydrolysis of the substrate (9). The rate function over a 6-fold range of enzyme concentration is shown in the inset to Fig. the first alternative appears more likely and suggests the presence of the two isomers in equal concentrations as expected from the synthesis.

. 3. Bobs per min as a function of asparaginasc concentration. 3. signed to the n ---) 7r* transition which undergoes a pronounced red shift in amides (10). significant amount of ammonia from the mixture of C&L and a~-/?D isomers over this time period.2HBr (4. This is illustrated in Fig..64 units of a-~-p-~nsyaraginasc (0.76. since it is due to the vicinal effect of the asymmetrical center locat’ed 2 carbon atoms away. The Cotton effects appearing upon treating a mixture of (Y-L-~-L. CD spectra of L-leucine (---).&L. I I\ I I --+3 0 c-o0 II C-NH2 Downloaded from www.o the unhydrolyzcd amide and that amide is of the L configuration The (Fig. The free acid shows the typical positive Cotton effect at 198 nm. Hydrolysis of . 3). Bobs. 4 by the CD spectra of L-leucine and L-leucine amide.05 M Tris. The change in cllipticity from the amide to the acid is relatively small. L-xspartic acid (--).1743 effects near 200 nm due largely to the overlapping r + 7r* and n + r* transitions of the carboql chromophore at the cy position (Fig.2-cm path. 25”.org by guest. 5cc 100 FIG. Changes in observed ellipticity. relationship of this finding to the stereochemistry of the active site of the enzyme will be discussed below (SW discussion). 200 225 X.nm IGo.85. hence the L-D isomer must be the one hydrolyzed. and 2.h optical rotation (Fig. If a mixture of ~-L-. 2011 200 TIME. but a prominent negaOive The latter can be asband appears at 227 nm in the amide. the n ---) T* transition 220 nm with a resultant change in the CD pattern in the region of 220 nm. Thus in the case of the diaminosuccinic acid monoamide hydrolyzed by asparaginase. diaminosuccinic acid monoamide.05 M Tris.jbc. .75 X 10M3 M amino acid. To insure adequate pen response the time constant on the Gary 61 was set at I s. The relatively small change in optical rotatory strength and the low wave length of the change near 210 nm. If the p. pH 8. Furthermore.S X 10-a M amino acid.-).2-cm path. 5A).or y-carboxyl is changed to an amide chromophore as in L-asparagine there is a small but significant change in the rotation (Fig. 0.L-Methylasparagine-Although no hydrolysis of the a-~-p-~ and (Y-D-@-D mixture of diaminosuccinic acid amide was detected. This mixture of isomers can be prepared by amidation of the commercially available three-nL&methylaspartic acid. 3. 0.).and a-D-fl-n-diaminosuccinic acid monoamide ‘2HBr (2. 2 n b x G I 0 t 227 -I x5 200 225 250 1 I I I I I I I I I L 2 50 X. a very slow hydrolysis of the ~-L-P-L and ~-D&D isomers of fl-methylasparagine was observed. CD spectra of L-asparagine (.05 M Tris. Inset. 25”. l). 4.164 mg per ml) to a solution of WI-B-D-. 3). However. Conditions: 0. at 222 nm as a function of time afler additions of 0. 1. 2. Conditions: 0. 3).nm (---) and L-leucine amide FIG.8 X 1OP M) contained in 0.44 unit/l0 ~1. on March 9.10 mgperml of asparaginase no optical rotatory changes are observed over a t’he enzyme did not release a period of 72 hours. the Cotton effect at 222 nm is due t. 58. The n --j P* Cotton effect is negative when the amide is adjacent to a carbon in the L configuration.0.. 25”.and ac-D-fl-D-P-methylasparagine with asparaginase are shown in Fig.7 mg per ml) is treated with up to 0.. A large negative ellipticity band appears at 204 nm and an apparent smaller negative elipticity band at ~220 nm (Fig. and L-aspnragine after complete hydrolysis by asparaginase (. make it unsatisfactory to follow asparagine hydrolysis wit. The n + r* transitiou of the cnrbosyl group is usually near 204 nm (10). 0. if an amide is substituted for shifts to the region of the cu-carboxyl.

he rotatory change near 225 nm on hydrolysis as well as moving the change to the more specific n ---) rr* transition of the amide (Fig.6 units (0. 3 and 4). 1). Conditions for CD as in I .KD-NH~ was attempted. 2011 i 200 225 X. 5B. II). I). ellipticity Curve contributed !Z is after “by 110 min the protein since at this concentration of enzyme the ultraviolet Cotton effects of the of incubation at 25”. Fig. pH 8. Such a procedure may be applicable to the substrates of other enzymes where the development of assays based on change in optical activity are desirable. This was accomplished with a Dowes IX2 column and the CD spectra of the resolved compounds are conshown in Fig. The significance of the D configuration at the amide in the rapidly hydrolyzed substrate was not apparent until enzymatic hydrolysis of t. on March 9. 2) where no clear separation of the 7r + QT* and n + r* transitions is present. 5.3% as fast as the a-L-.l-cm path. This greatly enhances t. Since the p carbon is not asymmetrical the amide can assume all positions of the D or L configuration of the /3 carbon. nm I 230 I 240 I 250 FIG. giving rise to large negative Cotton effects. The bond hydrolyzed in the case of the natural substrate for asparaginase is part of a chromophore located 2 carbon atoms away from the asymmetrical center.&r-diaminosuccinic acid monoamide as shown by the time course for dcvclopment of the Cotton effects in Fig. it was necessary to separate the product acid from the unhydrolyzed amide. Curve 4 after 24 or 48 hours. -6 UNHYDROLYZED D-D B-methyl AMIDE asparagine -12 6 I 190 I 200 I 210 I 220 1 .he alternate pair of isomers. Fig. however. 1). This is in keeping with the analogy to asparagine where the configuration around the carbon carrying the carboxyl must be L.nm I I I I PRODUCT 6 250 1 I ACID aspartic acid I L-L \ \ \ B -methyl \ \ \ ‘: ox s 0 . Curve 1.jbc.-) and a-D-p-D-p-methylasparagine (-). the c~-L-P-D-NH~ configuration is hydrolyzed (Fig. Hence t.50 due to the enzyme. Ko significant hydrolysis was detected and thus the CGL-/!-L-NH~ isomer does not meet the requirements. This is significantly faster. In the case of fl-methylasparagine no clear n * @ amide transition is separated out between 220 and 230 nm as in the cnsc of the diaminosuccinic arid amide (Fig. Curve 3 after 140 min.he catalytic region of the enzyme interacting with the . The unhydrolyzed amide is of the WD+-D figuration.. ultraviolet CD of cr-L-&L&methylaspartic acid (.&L isomer of diaminosuccinic acid amide. O. CD changes as a function of time during the hydrolysis of a mixture of ~-L-P-Land ol-u-p-D-(3-methylasparagine by 0. 5A) is due to a decrease in magnitude and slight blue shift of the positive Cotton effects for the a-L-&L-acid compared to the oc-r-/-L-amide. When the mixture of isomers is of the oc-u-/-L-NH2 and ~~-L-P-D-NH~ configurations.54 X IOP M asparaginase. Thus in order to be certain of the stereochemistry of the isomer hydrolyzed. DISCUSSION Downloaded from www. The mixture was then applied to a column of Dowex I-X2 to separate the product acid from the unhvdrolvzed amide. The diaminosuccinic acid derivative maintains the substrate requirements for asparaginasc. Conditions: @-methylasparagine. the enzyme itself makes a significant contribution to the CD spectrum and the ellipticity at 220 nm has a large contribution from the enzyme.org by guest. The turnover number calculated from this data is only 150 moles of substrate hydrolyzed per min per mole of enzyme. 58. 5A. B. Since the natural substrate for the enzyme is L-asparagine and D-asparagine is poorly hydrolyzed (4. 1. than the hydrolysis of the ~-L-. is largely I A 1 I I I I I I 1. A. and hence the rotatory changes accompanying this hydrolysis are qualitatively less significant than if the amide bond hydrolyzed involved a carbon adjacent to an asymmetrical center (Figs. giving rise to large positive Cotton effects.0. The net increase in negative ellipticity observed on hydrolysis (Fig. stereospecificity is conferred on the fl carbon. LY-L-P-L-NH~ and o(-D-. and thus the enzyme active site might accept only one of the /3 carbon configurations.1744 At the enzyme concentration required to observe significant hydrolysis. The error bars on the axis indicate the ranges ot a base-line CD spectrum taken on the same solution untreated with enzyme. These shifts in CD between the acid and the amide are more like those occurring on hydrolysis of asparagine (Fig. 7. A4 mixture of a-L-~-Land a-D-p-D-p-methylasparagine was treated with aspnraginase until complete hydrolysis had taken place (509” ammonia release). while the hydrolyzed isomer is of the cy-L-~-L configuration. In the case of diaminosuccinic acid amide. holr-ever. Whether there is a strict stereospecificity for the P-amide once the substrate is in place on the enzyme surface cannot be determined.05 M Tris. the proper relationship between t’he enzyme-binding groups of the substrate and the catalytic centers on the enzyme resulting in the hydrolysis of the P-amide presumably requires the L-configuration about the cz carbon. but introduces an asymmetrical carbon adjacent to the carbon of the hydrolyzed amide bond. 5A.028 mg) if aspaiaginase per ml. The ~-L-&L isomer of P-methylasparagine is hydrolyzed only 0. Curve 1 taken immediatelv after addition of the enzyme-is protein are largely detected.

since data on l*O exchange (12) and on the formation of fi-aspartohydroxamic acid catalyzed by the enzyme (12. 246. Chem. We are indebted to Dr. perhaps assisted by donation of a proton from a protein group. MASHBURK. L. P. 106. J. MARLBOROUGH. 172-174 12. (1971) Enzymes 4.c during hydrolysis by asparaginase. 2011 FIG. Chem. a methyl group can be accommodated in this position. D.. the enzyme-substrate dissociation constant apprars to be relatively small and binding could force some changes from the extended configuration.. (1962) Theoru and Abdications of havioiet Spectroscopy p. MILIKIN. J. L. CAMMACK. P. The ~-L-/~-II isomer is shown in the extended form. CEDAR.. BURCK. 98. 3716-3724 7.&n isomer in Fig.... JAFFE. 451-452 4.org by guest. J. although the rate of hydrolysis is much decreased over the Steric factors rate observed if a hydrogen occupies this position. Jesse &hilling of the Department of Molecular Biophysics and Biochemistry for programming the stereodrawings. to the amide. L.. AND Ho.. would appear to be primarily responsible for the decreasing rate of hydrolysis in the order H > CHB > NH2 when these groups in the These steric factors p position are placed in the L-configuration. J. P. R. A..1745 relationships for t. AND JONES. E. 6.. PEKAR. FORM Downloaded from www.he (Y-L-/-U. attacking the carbonyl carbon. natively if binding requires that the substituents on the cx carbon be in the L configuration and that the amino group of the 0 carbon be in the position it occupies in the a-~-p-~ isomer.. 6. REFERENCES 1. Biol. The isomers are both shown in an extended form with the --NH2 groups on opposite sides of the C-2-C-3 bond and occupying the same positions in the ~-L-P-D and ~-L-@-L configurations. 126. The catalytic groups are represented in purely hypothetical fashion as a base. 6. AH. (1970) Biochemistry 9. have been implicated as having binding interactions with the protein in the case of asparaginase from E. E. 6. Such rotation does not change the general features of substrate-enzyme stereochemistry pictured in Fig. H.. (1972) Biochem. ~ I ‘ < New York 11. B.in press 10. D. amide group and the site on the protein accepting the binding groups around the Q: carbon arc located in the configuration shown Both the cu-carboxyl and a-amino groups by the a-L&n-isomer. (1970) J.. may influence the binding step or alter the relationship between the catalytic groups and the substrate once the latter is bound. D. then t’he placement of the P-amino group in this same position for the cyL-@-L isomer results in rotation of the amide away from the site These that must contain the catalytic groups on the protein. A. JACKSON. carotovora (12). (1972) Biochem. It appears that the L configuration of substituents at. 6 by rotation about the a-P C--C bond. B. WRISTON.J. (1972) J. C. C. K. K.. since this is the likely form in solution with the amino groups on opposite sides of the C-2-C-3 bond. FRANK. Gaumond is much appreciated. This site may accommoXterdate a hydrogen in this position but not an amino group. (1970) J. E. T. 88-94 . C. (1971) J.. M. Biophys. 1020-1030 13.. Med. A. FRANK. 247.jbc. Acknowledgments-The technical assistance of Miss Judith We Pascale and Miss Celeste 9. IZ. K. Arch. AND WRISTON. the fl carbon interferes with the proper positioning of the substrate if the binding groups at the OLcarbon are confined to the L configuraDion. HANDSCHUMACHER. Ho. This results in the susceptible amide bond pointing away from the catalytic groups in the ~-L-@-L isomer. B. AKD CHANG. R.. AND ORCHIN. Biol. KIDD. L-D. J. H. on March 9. G.. Res. AND SCHWARTZ. EHRMAN. P. (1964) Biophys. R. P. Ho.ic groups (/l and U) attacking the amide bond is then as shown by the a-~-. P. H. H. A specific nucleophilic attack is pictured. 3708-3715 6. P. P. The hypothetical catalytic groups of the enzyme are represented as a Base B and an acidic group AH. (1961) Nature 191. 246. J. 13) have suggested but not proved the formation of an acyl enzyme intermediat. HOWARD. Biochem.. L. AND HANDSCHUMACHER. lb).. L. Stereodrawing of the conformations of the ~-L-P-D (A) and ~-L-O-L (B) isomers of diaminosuccinic acid monoamide.C. fK. M. W. While the p-amino group in the C+L-P-L isomer of diaminosuccinic acid amide appears to interfere with the proper positioning of the substrate. F. Ada 177. AND MILLER. B.. thank Miss Phyllis Salzman for separating the isomers of flmethylasparagine. VI&OS. 246. This stereochemistry is not absolutely fixed in the sense that the configuration of the a-L-p-n isomer can change from that shown in Fig. R. If the amide group of the (Y-L-/-L isomer is to be placed in the same position as that of the effectively hydrolyzed ~-L-@-D isomer. S. A. BOBBITT. H. kiophys. The relationship of the binding points on the protein receiving t’he groups around the cycarbon (carboxyl and amino) and the catalyt. J. 178. John Wiley and Sons.. H. 565-605 2. Chem. J. Chem. I. (1969) Biochim. AND CARPENTER. H.. Cor&zun. J. Biol. Exp. K.LK. 3585-3590 9. 1114-1115 3. (1953) J. Biol. GRIXN.~. Although extensive deviation from the extended form dots not appear likely. AND SQUIRES.. a-~-p-~ pair are illustrated in the stereodrawing in Fig. then the p-amino group occupies the position occupied by the P-hydrogen in the (Y-L-P-D isomer. 101-121 5. BROO~SE. BOECI~. g. 361-379 8. GAUMOND.