result of the bulky t-butyl groups, the pK. value of H(thd) is much larger than the pK, value for either acetylacetone or benzoylacetone. The high distribution ratios that were observed can be attributed, to a large extent, to the highly substituted @-diketone that was used as reagent. Brown, Steinback, and Wagner (14) have attributed poor extraction (28% for neodymium, 49% for terbium, and 84% for ytterbium) with acetylacetone to the formation of hydrates of the lanthanide chelates which are not as highly extracted as the anhydrous chelates, nor as stable. They suggest that highly substituted P-diketones have additional bulky groups which sterically hinder the attachment of water to the chelate and, therefore, tend to increase the distribution coefficient of the chelate. This concept of blocking of coordination sites by the bulky t-butyl groups and prevention of hydrate formation was also used by Sievers (15) in his ligand shell concept to explain the formation of anhydrous dipivaloylmethane lanthanide chelates. This shield(14) W. B. Brown, J. F. Steinback, and W. F. Wagner, J. Inorg. Nucl. Chem., 13, 119 (1960). (15) R. E. Sievers, K. J. Eisentraut, C. S.Springer, and D. W. Meek, “Advances in Chemistry,” No. 71, R. F. Gould, Ed., American Chemical Society, Washington, 1967, p 141.
ing of the central metal ion leads to high stability constants as shown by Van Uitert and Fernelius (16) and together with the high pK. of H(thd) explains the stability constants of the complexes studied in this work. Using the KDpoovalues in Table I, separation factors for a number of element pairs were calculated. These are: Pr-Sm, 18; Sm-Eu, 1.5; Gd-Tb, 6; Eu-Tb, 6; Pr-Tb, 160; Sm-Tb, 9; Gd-Eu, 1. Although praseodymium, samarium, europium, gadolinium, and terbium cannot be quantitatively separated from each other by a one-stage extraction with this extraction system, it does appear, on the basis of the separation factors shown, that many of these metals could be separated very well by a multiple extraction technique. RECEIVED for review February 5 , 1968. Accepted June 25, 1968. Work supported by the Aerospace Research Laboratories, Office of Aerospace Research, U. S. Air Force, WrightPatterson Air Force Base, Ohio, under contract number AF33(615)-3441. Taken in part from the Ph.D. dissertation of Henry W. Parlett, The Ohio State University, Columbus, Ohio, 1967. (16) L. G. Van Uitert and W. C. Fernelius, J. Amer. Chem. SOC., 75, 3862 (1953).
Determination of Steric Pulrity and Configuration of Diketopiperazines by Gas-Liquid Chromatography, Thin-Layer Chromatography, and Nuclear Magnetic Resonance Spectrometry J. W. Westley, V. A, Close, D. N. Nitecki, and Berthold Halpern Department of Genetics, Stanford University School of Medicine, Palo Alto Calf. 94304 SEVERAL authors have reported the isolation of optically active diketopiperazines from microbiological sources (1). While the chemical identity of most of these compounds has been established by classical methods, the optical purity and the absolute configuration of some of the isolated diketopiperazines is still in doubt. Since new sensitive techniques [thin-layer chromatography (2), gas-liquid chromatography (3), and nuclear magnetic resonance ($1 for the determination of optical purity and absolute configuration of diastereoisomers are now available, we have examined the relative merits of these methods for the steric analysis of diketopiperazines. EXPERIMENTAL
The diketopiperazines were prepared from the respective t-Boc-dipeptide methyl esters by treatment with formic acid (1) L. A. Mitscher, M. P. Kuntsmann, J. H. Martin, W. W. Andres, R. H. Evans, Jr., K. J. Sax, and E. L. Patterson, Experientia, 23, 796 (1967). (2) D. E. Nitecki, B. Halpern, and J. W. Westley, J. Org. Chem., 33, 864 (1968). (3) M. Raban and K. Mislow in “Topics in Stereochemistry,” Vol. 2, N. L. Allinger and E. L. Eliel, Eds., Interscience, New York, 1967, p 199. (4) N. S. Wulfson, V. A. Puchkov, U. V. Denison, B. V. Rozynov, V. N. Bocharev, M. M. Shemyakin, U. A. Ovchiwnikov, B. K. Antonov, Khim. Geterotsikl. Soedin., Akad. Nauk. Latv. SSR, 4, 614 (1966). 1888
ANALYTICAL CHEMISTRY
followed by cyclization of the intermediate formate salt in boiling toluene and sec butanol (4 :1) as described previously (2). All compounds were characterized by mass spectrometry and all diastereoisomers were examined using a Varian Aerograph gas chromatograph coupled to a Finnigan 1015 Quadrupole mass spectrometer. All compounds gave a molecular ion and the fragmentation pattern was in agreement with published data (4). The gas chromatographic conditions used for the separation of diastereoisomers are summarized in Table I. Thin-layer chromatograms (Table 11) were run on silica gel G plates using the solvent systems A (isopropyl ether-chloroform-acetic acid 6 :3 :1) and B (chloroform-methanol-acetic acid 14 :2 :1). Nuclear magnetic resonance spectra were determined on a Varian A-60 spectrometer and the results are summarized in Table 111. The racemization of echinulin (5) was achieved by refluxing XVII ( 5 mg.) in ethanol (0.5 ml.) and triethylamine (0.5 ml.) for 2 to 3 days under anhydrous conditions. The course of the reaction was followed by TLC on silica gel G plates using the solvent system described in Table 11. Final confirmation of the chemical identity of the more mobile product with echinulin (XVII) was made by eluting the material from a preparative TLC with chloroform and examining the residue by mass spectrometry (A.E.I., MS-9). The new product and XVII both showed a molecular ion at mle = 461 (6%) and a base peak at m/e = 334 (5) C. Cardani and F. Piozzi, Gazz. Chim. Ital., 86,211 (1956).
Table I. Gas Chromatographic Separation of Diastereoisomeric Diketopiperazines Retention time Ratio of retention time Temp., "C DL (trans) LL (cis) Columna Cyclo-dipeptide 1.16 240 8.0 9.25 Cyclo-alanyl-cyclohexylglycine(I) A 1.10 A 240 8.4 9.3 Cyclo-alanyl-phenylglycine(11) 1.10 240 14.5 15.9 C yclo-leucyl-phenylalanine(111) A 1.10 A 240 8.4 9.3 C yclo-alanyl-phenylalanine(IV) 1.07 A 200 8.9 9.5 C yclo-valyl-valine (V) 1.08 B 210 12.1 13.1 1.04 Cyclo-leucyl-valine(VI) A 200 13.4 13.9 1.08 B 210 14.2 15.3 1.00* Cyclo-leucyl-leucine (VII) A 200 19.9 19.9 1.04 B 210 19.7 20.6 1.006 Cyclo-alanyl-cyclohexylalanine(VIII) A 240 9.8 9.8 1.00* Cyclo-alanyl-valine (IX) A 200 7.1 7.1 1 .w Cyclo-alanyl-alanine (X) A 200 5.5 5.5 0.93 B 210 19.5 18.2 0.91 Cyclo-prolyl-proline (XI) A 240 5.5 5.0 0.87 B 210 10.7 9.3 1.18 Cyclo-prolyl-alanine (XII) A 240 3.3 3.9 0.76 B 210 10.7 8.1 Cyclo-prolyl-leucine (XIII) A 240 5.4 4.6 0.85 B 210 11.3 8.6 0.76 Column A : 5-foot X l/s-inch column packed with 5 % QF-1 on Aeropak eo. Nitrogen flow 30 ml/min. Column B : 5-foot X '/*-inch column packed with 3 EGS on Gas Chrom Q. Nitrogen flow 30 ml/min. a Diastereoisomers not resolved under these conditions. 0
1
Table 11. R j Values of Diastereoisomeric Diketopiperazines. R , value DL
A
4 similar experiment was run with cyclo-L-alanyl-L-tryptophan (XVI) as a control. Hydrolysis of naturally occurring diketopiperazines was carried out in glacial acetic acid-25 % hydrochloric acid (4 : 1) for 48 hours at 90'. RESULTS AND DISCUSSION
The results of the gas chromatographic analysis of diastereoisomeric diketopiperazines are given in Table I. The compounds are listed according to the degree of resolution which is measured as a ratio of retention times (CY). With the exception of two alanyl derivatives, (VIII) and (IX), all diastereoisomers could be separated using an ethylene glycol succinate column (EGS) or a trifluoropropyl methyl silicone column (OF-1). Apart from cyclo-alanyl-alanine (X) and all proline containing compounds, the LL (cis) diastereoisomers always had the longer retention time than the DL (trans) isomer. This is in agreement with the results we have observed with the N-TFA-L-prolyl-DL-amino acid esters where the DLdipeptide always has a shorter retention time than the LLisomer (6). We believe that this is due to the smaller molecular volume of the m-dipeptides (7) which gives rise to greater volatility and lower interaction with the stationary phase. However, in the diketopiperazines, both isomers are cyclic and must have similar molecular volumes and, therefore, the (6) B. Halpern and J. W. Westley, Biochem. Biophys. Res. Comm., 19,361 (1965); B. Halpern and J. W. Westley, Tetrahedron Lett., 1966,p 2283. (7) T. Wieland and E. Bende, Chem. Ber., 98 504 (1965). (8) D. E. Nitecki and J. W. Goodman, Biochemistry, 5,665 (1966). (9) J. C. MacDonald, Biochem. J., 96,533 (1965).
Cyclo-dipeptideb Solventc LL (cis) (trans) Cyclo-alanyl-cyclohexylglycine(I) A 0.29 0.50 Cyclo-alanyl-phenylglycine(11) A 0.23 0.34 Cyclo-valyl-valine (V) A 0.44 0.60 Cyclo-alanyl-cyclohexylalanine(VIII) A 0.39 0.59 Cyclo-alanyl-alanine (X) A 0.10 0.10d Cyclo-prolyl-alanine (XII) A 0.15 0.1Y Cyclo-prolyl-leucine(XIJ1) A 0.27 0.20 Cyclo-alanyl-tyrosine(XV) B 0.77 0.87 B 0.65 0.75 Cyclo-alanyl-tryptophan (XVI) Echinulin (XVII) A 0.64 0.79 a The chromatograms developed by chlorination technique (8). * R , values reported previously for cyclo-leucyl-leucine (9), cyclo-alanyl-phenylalanine, cyclo-leucyl-phenylalanine,cyclo-leucylvaline, and cyclo-phenylalanyl-phenylalanine(2). Solvent A: Isopropylether-chloroform-acetic acid (6 :3 :1). Solvent B: Chloroform-methanol-acetic acid (14:2: 1). Not resolved by TLC. e Produced by racemization. 0
Table 111. Proton Resonancesa of Alanyl Methyl Groups in Alanyl Diketopiperazines Alanyl P-methyl resonance* LL
DL
(Ycia-
Cyclo-dipeptide
(cis) (trans) vtrans) Cyclo-alanyl-phenylglycine (11) 106.5 106.5 0 Cyclo-alanyl-phenylalanine(IV) 52.5 86.5 -34 Cyclo-alanyl-cyclohexylalanine(VIII) 103.5 99.5 +4 Cyclo-alanyl-valine(IX) 104.5 101.5 +3 103.5 Cyclo-alanyl-alanine (X) 101.5 +2 97.5 Cyclo-alanyl-proline (XII) 100.5 -3 Cyclo-alanyl-tyrosine (XV) 90.5 -30 60.5 Cyclo-alanyl-tryptophan (XVI) 43.5 82.5 -39 = Hz below Me&i at 60 MHz. Spectra all measured in trifluoracetic acid. * J,, = 7.0 H z for all compounds.
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difference in retention times is probably due to different polar interactions with the stationary phase. This view is supported by the higher a-values observed on the polar ester phase relative to the silicone phase. Any polar interactions must involve the amide groups of the diketopiperazine ring and as the ring is more accessiblein the case of the LL (cis) isomer, this explains its higher retention time. Cyclo-alanyl-alanine (X) is the first exception to this rule and it is also exceptional in having a longer retention time than compounds of higher molecular weight such as V, VI, and IX on the polar EGS phase. This stronger retention implies an enhanced polar interaction between both diastereoisomers and the polyester phase, resulting from the small bulk of the alanyl methyl groups. Under these conditions the trans isomer apparently is retained more strongly than the cis. The latter conclusion also holds in the case of the prolyl diketopiperazines (XI-XIII) where the LL isomer consistently has the shorter retention time. In these compounds the diketopiperazine exists as part of a bicyclic system which confers even greater rigidity to the system resulting in the higher degree of resolution. The results of thin-layer chromatography (Table 11) indicate that of the 14 pairs of diastereoisomers resolved, all except cyclo-prolyl-leucine (XIII) exhibited a lower R, for the LL (cis) isomer than the LD. This indicates that with the exception of the bicyclic prolyl diketopiperazines, there was greater interaction between the LL (cis) diastereoisomer with the absorbent than was the case for the LD isomer. Note that all the diastereoisomeric diketopiperazines could be resolved by either GLC, TLC, or both techniques. In addition to the two chromatographic techniques, the diketopiperazines containing alanine were also examined using nuclear magnetic resonance. The alanine derivatives were selected for this study because of the ease with which the alanine methyl resonance can be assigned in the spectrum, In all cases this resonance existed as a doublet due to coupling with the alanine a-proton (J = 7 cps). It can be seen from the results (Table 111) that in all cases except cyclo-alanylphenylglycine (11), the two diastereoisomeric forms of each diketopiperazine could be distinguished by NMR. In the case of compounds not containing an aromatic side chain, the LL (cis) isomer had a methyl resonance at lower field than the LD (trans) isomer, whereas in the case of compounds containing an arylmethyl side chain, and of cyclo-alanyl-proline (XII), the reverse is true. In addition, the difference in chemical shift (v,is-vtrans) is much greater in the aromatic compounds (30 to 40 Hz) than the aliphatic (2 to 4 Hz.). This must be due to the preferred conformation of the aryl-methyl side chain, in which the aromatic ring faces the diketopiperazine ring (IO). This folded conformation will give rise to shielding of the methyl groups of the alanyl residue. The cis isomer will be affected to a much greater extent which explains the up-field shift in the case of the LL isomers. In the case of cyclo-alanyl-phenylglycine,this folding is not possible because of absence of methylene group between the aromatic system and the diketopiperazine ring. (10) K. D. Kopple and D. H. Marr, J. Amer. Chem. SOC.,89,6193 (1967).
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ANALYTICAL CHEMISTRY
Finally, we have examined three naturally occurring diketopiperazines, 3-benzyl-6-isobutyl-2,5-diketopiperazine (I]), 3,6-dibenzyl-2,5-diketopiperazine (albonoursin) (12, IS) and echinulin (XVII) (5).
~H*-cH=
c(cH,), XVII
The compounds were first compared by TLC with compounds of known configuration. In this manner, the first two natural products were shown to be identical to cyclo-L-leucylL-phenylalanine (111) and cyclo-L-phenylalanyl-phenylalanine (XIV), respectively, and this was confirmed by gas chromatography. In the case of echinulin, it was necessary to racemize some of the natural product to measure the R I value of the diastereoisomeric form. By running a control experiment with cyclo-L-alanyl-L-tryptophan, it was shown that the newly formed diastereoisomer in both cases had a higher R, value, This implies that echinulin must have either the LL or DD configuration. Note that none of the three techniques described here distinguishes DL from LD or DD from LL diastereoisomers. For final confirmation it was, therefore, necessary to hydrolyze all three natural products and to determine the configuration of the resulting amino acids by the N-TFA-L-prolyl chloride technique (9. In all cases, the amino acids were found to have the L configuration. In the case of echinulin, the tryptophan moiety is destroyed by hydrolysis, but the fact that the alanine is L, which had been established previously (3, means that echinulin, like the other two natural products belonged to the LL configuration. This conclusion is contrary to the published ORD assignment ( 1 4 , in which the tryptophan moiety was given the D-configuration, but we understand that this will be corrected in a forthcoming publication (15). ACKNOWLEDGMENT
The authors thank A. B. Manger for useful discussions concerning the GLC of diketopiperazines and a gift of cyclovalyl-valine. We also thank G. P. Slater for a generous gift of echinulin and Rachel Brown for gifts of 3-benzyl-6isobutyl-2,5-diketopiperazine and 3,6-dibenzyl-2,5-diketopiperazine isolated from Szrepfornyces noursei var. 5286. RECEIVED for review June 6, 1968. Accepted July 15, 1968. This investigation was supported by National Aeronautics and Space Administration Grant NsG 81. (11) C . Kelley and R. Brown, Experientia, 22,721 (1966). (12) R. Brown, C. Kelley, and S. E. Wiberley, J. Org. Chem., 30, 277 (1965). (13) A. S. Khokhlov and G. B. Lokshin, Tetrahedron Lett., 27, 1881 (1963). (14) R. Nakashima and G . P. Slater, ibid., 45, 4433 (1967). (15) G . P. Slater, National Research Council, Saskatoon, Sask., Canada (May 1, 1968).
