Anal. Chem. 1005, 65, 1861-1867
1861
Conformational Effects in Reversed-Phase Liquid Chromatographic Separation of Diastereomers of Cyclic Dipeptides Noriaki Funasski,’ Sakae Hada, and Saburo Neya Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607, Japan
The capacity factors, k’, of 11 cyclic dipeptides (X-Y) including diastereomers have been determined on an RP-HPLC column in 30% and 50% methanol and lo%, 30%, and 50% acetonitrile solutions. These factors are roughly correlated with hydrophobic parameters, such as octanolwater partition coefficients estimated and k‘values for alcohols. For a pair of diastereomers of cyclic (L-X-L-Phe)and (L-X-D-Phe)derivatives k % L is larger than k’LD, and for cyclic (D-Ala-L-Trp) and (L-Ala-L-Trp)k’LL is smaller than k’DL, particularly in highly aqueous solutions. These elution orders can be well predicted by the holistic molecular surface area approachwhich takes into account the folded structuresof cyclic dipeptides. The present results will be useful for prediction of the log k’ values of larger peptides and the hydrophobicity and related properties of peptides.
INTRODUCTION Reversed-phase high-performance liquid chromatography (RP-HPLC) is often used to separate and analyze amino acids, peptides, and proteins.’-16 For RP-HPLC columns such as octadecyl silica columns, the hydrophobicity of a solute is the predominant factor in the retention mechanisms.11 The hydrophobicity and RP-HPLC retention time of a peptide, therefore, may be predicted by using the additivity of Rekker’s fragmental constants17 or the logarithms of octanol-water partition coefficients PlaJ9 of the amino acid residues which
constitute the peptide.lJpDJ0 Furthermore, Meek estimated the retention coefficient of each amino acid residue from the retention times of its homopeptides and predicted the retention times of peptides from these retention coefficients.6 Positional isomers, which are different from each other in the sequence order of amino acids, can be separated on RP-HPLC columns.14J6 For the diastereomers of linear dipeptides, the retention time of LL-form is generally shorter than that of the LD-isomer.2-s These results for the positional isomers and optical isomers cannot be predicted by the additivity rules mentioned above.EJ7-19 Some cyclic peptides have antibacterial action,20and others are regarded as models of enzymes and proteins.2l The cyclization of a linear peptide decreases two ionizable groups and the degree of freedom of molecular conformations. These will facillitate a detailed investigation of the relation between the chemical structure and the retention time for cyclic peptides, although few RP-HPLC data are available on these peptides.22 Cyclic dipeptides in crystals and solutions have been established to have some folded structureaa by X-ray,%% 1H-and 13C-NMR,am7and circular dichroismicanalysea.323’3 These folded structures themselves are interesting from the viewpoint of the applications of NMR. The hydrophobicity of hydrocarbons,% dialkyl ethers,* oligoethylene glycol dialkyl ethers,41and amino acids42 is well correlated with their cavity molecular surface areas, 5’.The
(19) Yunger, L. M.; Cramer, R. D., 111. Mol. Pharmacol. 1981,20,602608. (20) Ovchinnikov, Y. A.; Ivanov, V. T.; Shkrob, A. M. Membraneactiue Complexesones; Ekevier: Amsterdam, 1974. (21) Tanihara, M.; Imanishi, Y.; Higaehimura, T. Biopolymers 1977, 16,2217-2229. (22) Ueda, T.; Saito, M.; Kato, T.; Izumiya, N. Bull. Chem. SOC.Jpn. 1983,56,568-572. (23) Bose, A. K.; Manhas, M. S.; Tavares, R. F.; van der Veen, J. M.; (1) MoUar, I.; HorvAth, C. J. Chromatogr. 1977, 142, 623-640. Fujiwara, H. Heterocycles 1977, 7, 1227-1270. (2) Kroeff, E. P.; Pietrzyk, D. J. Anal. Chem. 1978,50,1353-1358. (3) Pietnyk,D.J.;Smith,R.L.;Cahill,W.R.,Jr.J.LiquidChromotogr. (24) Benedetti, E.; Corradini, P.; Pedone, C. J. Phys. Chem. 1969,73, 2891-2895. 1983,6,1645-1671. (25) Sletten, E. J. Am. Chem. SOC.1970, 92, 172-177. (4) Steinauer, R.; Chen, F. M. F.; Benoiton, N. L. J. Chromatogr. 1985, (26) Lin, C.-F.; Webb, L. E. J. Am. Chem. SOC.1973,95,68036811. 325,111-126. (5) Lundanea, E.; Greibrokk. T. J. Chromatogr. 1978, 149, 241-254. (27) Cotrait, M.; Ptak, M.; Busetta, B.; Heitz, A. J. Am. Chem. SOC. 1976,98,1073-1076. (6) Zou,H. F.; Zhang, Y. K.; Dong, L. F.; Lu, P. C. Chromatographia (28) Degeih, R.; Marsh, R. E. Acta Crystallogr. 1959,12,1007-1014. 1991,31,27-30. (29) Kopple, K. D.; Ohnishi, M. J. Am. Chem. SOC.1969,91,962-970. (7) OHare, M. J.; Nice, E. C. J. Chromatogr. 1979, 171, 209-226. (30) Gawne, G.; Kenner, G. W.; Rogers, N. H.; Sheppard, R. C.; (8) Meek, J. L. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 1632-1636. Titlestad, K. In Peptides; Bricas, E., Ed.; North-Holland Amsterdam, (9) Wilson, K. J.; Honegger, A.; Stbtzel, R. P.; Hughes, G. J. Biochem. 1968; pp 28-39, J. 1987, 299, 31-41. (31) Vicar, J.; Piriou, F.; Fromageot, B.; B l h , K.; Fermadjian, S. (10) Sasagawa, T.; Okuyama, T.; Teller, D.C. J. Chromatogr. 1982, Collect. Czech. Chem. Commun.1980,45,482-490. 240,329-340. (11) Krstulovic,A. M.;Brown, P. R. Reversed-PhaseHigh-Performonce (32) Tanihara. M.: Him. T.: Imanishi.Y.: Bull. Chem. . ,Hinashimura.T. ” SOC. j p n . i983,56, i m - i i 6 6 . Liquid Chromatography. Theory,Practice and Biomedical Applications; (33) Kopple, K. D.; Marr, D. H. J . Am. Chem. SOC. 1967,89, 6193Wiley: New York, 1982. (12) Teller, D. C. Nature 1976,260, 729-731. 6200. (13)Mant,C.T.;Burke,T.W.L.;Zhou,N.E.;Parker,J.M.R.;Hodges, (34) Davies, D. B.; Khaled, Md. A. J.Chem. SOC.Perkin II 1976,187196. R. S. J. Chromutogr. 1989,485, 365-382. (35) Davies, D. B.; Khaled, Md. A. J. Chem. SOC.Perkin II 1976,1238(14) Wehr, C. T.; Correia, L.; Abbott, S. R. J.Chromatogr. Sci. 1982, 20.114-1 IR. 1244. --- ---(36) Deslauriers, R.; Grzonka, Z.; Schaumburg, K.; Shiba, T.; Walter, (15) Guo,D.; Mant, C.; Taneja, A. K.; Parker, J. M. R.; Hodges, R. S. J. Chromatogr. 1986, 359,499-518. R. J . Am. Chem. SOC.1975,97,5093-5100. (37) Shiba, T.; Nunai,K. Tetrahedron Lett. 1974, 509-512. (16) Fong, G. W.-K.; Grushka, E. Anal. Chem. 1978,50, 1154-1161. (38) Hooker, T. M., Jr.; Bayley, P. M.; Radding, W.; Schellman, J. A. Fragmental Constant; Elsevier: (17) Rekker, R. F. The Hydrophobic . . Biopolymers 1974, 13, 549-566. Amsterdam, 1977; p 301. (18) FauchBre, J.-L.; Pliska, V. Eur. J.Med. Chem. 1983,18,369-375. (39)Hermann, R. B. J. Phys. Chem. 1972, 76, 2754-2759. ~~
~
~
--I
0003-2700/93/0365-1861$04.00/0
0 1993 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
Table I. Elementary Analyses and Melting Points ("C) of Cyclic Dipeptides mp ("C) calcd ( % ) no. C-DP obsd literature C H 1
Ala-Ala
287-288
2
Ala-D-Ala
277-278
3
Ala-Phe
291-293
4
Ala-D-Phe
269-271
5
Val-Phe
270-272
6 7
Val-D-Phe Leu-Phe
287-289 271-272
8 9 10 11
Leu-D-Phe Ala-Trp D-Ala-Trp Val-Trp
265-267 295-298 263-266 290-291
272-275" 288-290' 276-280' 277-27gb 256-259' 276-280" 295-300b 290-291d 253-255' 265-266* 266-267" 271-272e 267-268' 263-264d 253-256d 282-28g 265-267f
found ( % ) N
C
H
N
50.69
7.09
19.71
50.42
7.13
19.43
50.69
7.09
19.71
50.31
7.09
19.15
66.03
6.47
12.84
65.75
6.55
12.53
66.03
6.47
12.84
65.86
6.45
12.60
68.27
7.37
11.37
67.93
7.23
11.21
68.27 69.20
7.37 7.74
11.37 10.76
67.97 69.04
7.25 7.95
11.11 10.93
69.20 65.36 65.36 67.35
7.74 5.87 5.87 6.72
10.76 16.33 16.33 14.73
69.29 65.34 65.26 67.41
7.60 5.92 5.83 6.67
11.26 16.12 16.21 14.68
Pickenhagen,W.; Dietrich, P.; Keil, B.; Polansky,J.; Nouaille, F.; Lederer, E. Helu. Chim. Acta 1975,58, 1078-1086. Znd. 1969, 1092-1092. Reference 22. Reference 45. e Reference 36. f Reference 46. RP-HPLC retention times for hydrophobic substances are also correlated with S.43944 This is termed a holistic molecular surface area approach.44 The term holistic refers to a particular fixed conformation for the complete molecule and does not necessarily imply that the total area for the whole molecule is more than the sum of its parts. Since the holistic area depends on the molecular conformation, it is not generally equal to the sum of the group or atomic areas. For ethers and alcohols, including positional and stereo isomers, the logarithms of capacity factors show a linear correlation with holistic areas.@,43The differencebetween the retention times of the diastereomer of a linear dipeptide is qualitatively explained in terms of their molecular areas.24 In general, a linear peptide can have a number of probable conformations and a negative, positive, or zwitterionic charge. These complications prevent definite interpretations of such retention time data. In this work we investigate the quantitative correlation between the RP-HPLC retention data and molecular conformations of 11 cyclic dipeptides. These cyclic dipeptides, including five diastereomeric pairs [c-(L-X-L-Y)and c-(L-XD-Y)], are synthesized. The folded structures of the cyclic dipeptides influence retention behavior and lead to the separation of diastereomers on an RP-HPLC column. These chromatograms are well simulated by the holistic molecular area approach.
EXPERIMENTAL SECTION Reagents. Eleven cyclic dipeptides [c-DP; c-(X-Y)] shown in Table I were synthesized as follows. All starting materials for c-DP synthesis were purchased from Kokusan Chemical Works (Tokyo,Japan). Cyclicdipeptides were synthesized via coupling of amino group protected tert-butyloxycarbonyl (Boc-) or benzyloxycarbonyl (Z-) amino acid and the methyl ester of amino acid HC1 or p-toluenesulfonate (Tos) using N,N-dicyclohexylcarbodiimide (DCC).4S$4B The methyl ester of amino acid Tos (40) Funasaki, N.; Hada, S.; Neya, S.J . Phys. Chem. 1985,89, 30463049. ... .
(41) Funasaki, N.; Hada, S.;Neya, S.; Machida, K. J . Phys. Chem. 1984,88, 5786-5790. (42) Leodidis, E. B.; Hatton, A. J. Phys. Chem. 1990,94, 6400-6411. (43) Funasaki, N.; Hada, S.; Neya, S.J. Chromatogr. 1986,361,33-45. (44) Eng, G.; Johannesen, R. B.; Tierney, E. J.; Bellama, J. M.; Brinckman, F. E. J . Chrornatogr. 1987, 403, 1-9. (45) Nitecki, D. E.; Halpern, B.; Westley, J. W. J . Org. Chem. 1968, 33, 864-866.
b
Slater, G. P. Chem.
was synthesized by refluxing an amino acid and excess p-toluenesulfonic acid in methanol and was crystallized by washing with the product with dry ether:"
Z (or Boc)-X-OH + H-Y-OCH, HCl (or Tos)
-
DCC -Ha0
Z (or Boc)-X-Y-OCH, In the case of phenylalanyl derivatives 3-8, a Boc-X-Y-OCH3 was treated with formic acid to remove the Boc group, and dipeptide methyl ester formate was cyclized by refluxing in a mixture of toluene and 2-butano1:G
-
HCGQH
BOC-X-Y-OCH3
-
reflux
401,-(CHshC=CH2
H-X-Y-OCH,
c-(X-Y)
-CHIOH
For c-DPs 1, 2, and 9-11, a Z-X-Y-OCH3 was dissolved in methanol, and the Z-group was removed by catalytic hydrogenation with palladium black. The product was cyclized by the treatment with ammonia-saturated methanol.@ Z-X-Y-OCH,
HdPd +
H-X-Y-OCH,
-COa, -CP,HSCHS
"s -+
-CHaOH
c-(X-Y)
The product recrystallized from appropriate solvent was subject to thin-layer chromatography45 and RP-HPLC. Some of the c-DPs were checked by mass spectrometry. The purities of all c-DPs were estimated to be 98% pure or more by RP-HPLC. The results of elementary analyses and the melting points are shown in Table I. Alcohols were obtained from Tokyo Kasei Organic Chemicals (Tokyo, Japan). Chromatography. All chromatograms were obtained using a Shimadzu LC-3A liquid chromatograph (Kyoto, Japan) equipped with a Shimadzu spectrophotometric detector SPD2A (at a wavelength of 210 nm) and a Shodex refractive index detector SE-51 (Tokyo,Japan). A DuPont Zorbax ODS column (25 cm X 4.6 mm i.d.) was used. Isocratic elution was carried out with 30% and 50% (v/v) mixtures of methanol (MeOH) and water or 10 % , 3 0 % ,and 50% (v/v)mixtures of acetonitrile (ACN) and water at 45 "C. The flow rate was 1.0 mL/min. Methanol (HPLC grade) was obtained from Wako Chemicals (Osaka, Japan), and the ion-exchanged water was twice distilled before use. The mobile phase holdup time t o was measured by injecting sodium nitrate. The retention times for all c-DPswere measured three times and used their average values for further analysis. The simulation of chromatograms was performed on the basis of plate theory.@ (46) Nakashima, R.; Slater,G . P. Can. J . Chem. 1969,47,2069-2074. (47) Bodanszky, M. Int. J. Peptide Protein Res. 1984, 23, 111-111.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
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Table 11. Observed log Jc’ Values (30% and 60% MeOH), Estimated log P Values, and Estimated log k‘ Values for Cyclic Dipeptides log k‘ 50% 30% log log log log no. c-DP MeOH MeOH P f h b k ’ d k’ad -0.444 -0.211 0.62 1.06 -0.444 -1.034 1 Ala-Ala 2 Ala-D- Ala -0.455 -0.219 0.62 1.06 -0.444 -1.034 0.621 2.10 2.77 0.692 -0.258 3 Ala-Phe 0.043 4 Ala-D-Phe -0.010 0.513 2.10 2.77 0.692 -0.258 1.313 3.01 3.70 0.757 0.189 0.502 5 Val-Phe 0.275 0.938 3.01 3.70 0.757 0.189 6 Val-DPhe 0.700 1.593 3.49 4.23 0.898 0.483 7 Leu-Phe 1.394 3.49 4.23 0.898 0.483 8 Leu-D-Phe 0.574 -0.086 0.448 2.56 2.84 0.743 -0.398 9 Ala-Trp 0.459 2.56 2.84 0.743 -0.398 10 DAla-Trp -0.073 ~~
11
0
5
t
(mid
10
15
Val-Trp
~
0.282
1.057 3.47 3.77
0.801
0.049
a The odanol-water partition coefficients estimated from Fauchhre’s data.18 b The octanol-water partition coefficients estimated from Rekker’sdata.“ C The capacityfactors estimated from Meek’s data.8 d The sum of the capacity factors of the corresponding alcohols, namely, log k’ (RI-OH)plua log k’ (R2-OH)for 50% MeOH.
Flgwe 1. Observed chromatograms of (a)and (b) the cycllc dlpeptldes labeled In Table I and (c) alcohols: A l , methanol;A2,2-propanoI; A3, 2-methyl-1-propanol; A4, benzyl alcohol; A5, lndde-3-methanol.
Molecular Surface Area Measurements. A CPK molecular model of a solute is constructed; Styrofoam balle representing water molecules (radius 0.1 nm) are glued onto the model and packed aa tightly aa possible. The Styrofoam balls are counted, and the number is multipliedby 3.99 to convert absolute surface area S. The S value for a molecule in water is defined aa the area of the surface traced out by the center of a water molecule rolling over the van der Waals surface of the solute molecule.’0 For a solute,the reported S value is the average of four or five values.
RESULTS Retention Times of Cyclic Dipeptides. T w o instances of the chromatograms obtained for cyclic dipeptides are displayed in Figure 1. The capacity factor, k’,for a peptide was calculated from the observed retention times of the peptide ( t ) and sodium nitrate (to):
I
10
30
50
%bCN
Flgurr 2. Effects of acetonitrlle volume percent on the log k’values for (a) the Trp derhratlves and (b) the Phe derhratlves.
(1) The logarithms of capacity factors for 11 cyclic peptides in MeOH solutions are shown in Table 11, and the log k’ values in ACN solutions are shown in Figure 2. From comparison of the k’ values for L-alanylderivatives (1,3,and 91, the elution order for L-amino acids is Phe > Trp > Ala. This order holds for L-valyl derivatives (5 and 11). The elution order of linear LL-dipeptides is Leu > Phe > Trp > Val > Ala on a phenyl column and Trp > Phe > Leu > Val > Ala on an ODS column.5 The elution order for L-amino acids is Trp > Phe > Leu > Val > Ala on several reversed-phase ~0lumns.15~15 Various parameters are proposed as measures of the hydrophobicityof substances.S.11J7-*@From the octanol-water partition coefficientsP of amino acids derivatives, the log P values for peptides can be estimated using Rekker’s“ and FauchBre’s approaches.’* The total molecular surface area S is also a hydrophobicparameter.& Meek proposed retention coefficientsfor amino acid residues on the basis of the capacity factors of homopeptides on an RP-HPLC column.* From any of these four parameters for L-amino acids, the order of hydrophobicity is Trp > Phe > Leu > Val > Ala. Based on our data and literature data on linear and cyclic peptides, we can conclude that the elution order of amino acids on RPHPLC column is determined by the hydrophobicity of
peptides (main factor), the kind of columns (ODs or phenyl column), the kind of the mobile phase, the pH of the mobile phase (for peptides containing ionizable groups), the degree of silaniition,6 the sequence of amino acids, and the molecular conformations of peptides (which will be discussed below). Aa shown in Table 11 and Figures 1 and 2, four pairs of diastereomers for cyclic dipeptides can be separated on the ODS column, without using any chiral column.49 This result is not only important in the applications of RP-HPLC but also interesting from the theoretical viewpoint. The elution order of diastereomers for cyclic dipeptides depends on the kinds of amino acid and the mobile phase: it is u > LD for dialanyls (1 and 2) and phenylalanyl derivatives (3 and 4,5 and 6, and 7 and 8) and LL < DL for alanyltryptophyls (9 and 10) in the MeOH solutions and 10%ACN. In 30% and 50% ACN solutions, the elution order of diastereomers is LL < LD for dialanyls (1 and 2) and phenylalanyl derivatives (3and 4 and 7 and 8) and u < DL for alanyltryptophyls (9 and 10). To our knowledge, the elution order of a pair of diastereomers for linear dipeptides is LL < LD without exception.= This result is explained qualitatively in terms of molecular surface areas S of the peptides;u the side chains of LL- and DD-forms are in the trans position with respect to the C-N bond and those of LD- and DL-forms are in the cis position50 and SLL = SDD < SDL= Sm.u
(48) Fundi,N.; Hada, 5.;Neya, S. Bull. Chem. SOC.Jpn. 1989,62, 380-386.
(49)Allenmark, S. Chromatographic Enantioseparation; Ellis H o r w d Chichester, England, 1988.
k’ = (t - to)/to
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
Table 111. Correlation Parameters for the Equation: log k’ = aX + b 30% MeOH 50% MeOH X 1% PR 1% PF log k ‘ a b lgo k ‘ M
10% ACN
U
b
ro
a
b
r
a
b
r
0.530 0.544 1.134 1.009
-0.871 -0.619 0.255 0.160
0.970 0.933 0.984 0.829
0.333 0.338 0.721 0.619
-0.880 -0.712 0.249 -0.224
0.957 0.911 0.983 0.797
0.683 0.714 1.431 1.344
-0.936 -0.643 0.525 0.366
0.988 0.969 0.983 0.875
The correlation coefficient. * The sum of the capacity factors of the corresponding alcohols, namely, log k’(R1-OH) plus log k’(R2-OH) for
50% MeOH.
7
0
Flgure 3. Correlation between the log k’values (50 % MeOH) and log PR vaiue for cyclic dipeptides. The solid line shows the best-fit relationship.
Flguro 4. Correlation between the log k’vaiues for cyclic dipeptides and alcohols In 50 % MeOH. The solid line shows the best fit relationshlp and the dashed line stands for the relationship having a unit slope through the data point of 1.
Quantitative Correlations with Hydrophobic Parameters. It has been recognized that quantitative correlations exist between the logarithms of the capacity factors and various physical parameters X:
the whole molecule by chemical bonds. For instance, the log k’ value for an alcohol ROH may be written as
+
log k’ = aX b (2) The use of such a relation has allowed us to predict the retention times of desired compounds and to identify unknown compounds from experimentally determined retention times. Assuming the simple additivity of group properties, we calculated the log P values from Rekker’s fragmental constants,” those from FauchBre’s T R values,ls and the log k’ values from Meek’s retention coefficientss for all peptides shown in Table 11. In Figure 3 the observed log k’ values for 11cyclic peptides in 50 ?6 MeOH are plotted against Rekker’s lot P values. For this relation the correlation coefficient, r , is 0.958. Fauchdre’s log P values and Meek’s log k’ values are not as well correlated with the observed log k’values, as shown in Table 111. These three parameters cannot distinguish between the diastereomers and are inconsistent with the elution order of the Trp and Phe groups. The same tendency is observed for the data of all mobile phases used. In 50% MeOH, we determined the retention times for alcohols possessing the same side chains with amino acids, as shown in Figure IC. For instance, since valine has the isopropylgroup as the side chain, it corresponds to 2-propanol. The elution order for the alcohols is benzylalcohol> 2-methyl1-propanol > indole-3-methanol > 2-propanol > ethanol > methanol. This order is consistent with that for cyclic peptides. This result suggests that the stationaryand mobile phases are important factors for determining the elution order of homologs. If there is not intramolecular interaction among side chains, the log k’ value for a compound is often assumed to be equal to the sum of the log k’ values for all groups which constitute (50) Lemieux, R.U.;Barton, M. A. Can. J. Chem. 1971,49, 767-776.
+
(3) log k’ (R-OH) = log k’ (R) log k’ (OH) Since a cyclic dipeptide consists of two side chains (R1 and R2) and the diketopiperazine ring (DKP),its log &‘valuemay be written as log k’ (R,-DKP-R,) = log k’ (R,) + log k’ (R,) + log k’ (DKP) (4) A combination of eqs 3 and 4 yields
+
log k’ (R1-DKP-R,) = log k’ (RI-OH) + log k’ (R2-OH) log k’ (DKP) - 2 log k’ (OH) (5) According to eq 5, the log k‘ value for a cyclic dipeptide in 50% MeOH is plotted against the sum of the log k’ values for the corresponding alcohols in Figure 4. From the linear regression analysis of these data, we obtained a slope of 0.721 and a correlation coefficient of 0.983. As Table I11 shows, this correlation is better than that for any of Rekker’s, FauchBre’s, and Meek’s values. The reasons for this better correlation are that the k’ values for alcohols and cyclic dipeptides were determined under the same experimental conditions and that the elution order of the Trp and Phe groups is correctly predicted. For all predictors, the correlation is improved with increasingwater contenta in the mobile phase. Unexpected from eq 5, however, the slope (the solid line in Figure 4) is smaller than unity (the dashed line). This result suggests that the hydrophobicity of cyclic dipeptides is decreased as the result of intramolecular interactions among their side chains. Furthermore, any of the four approaches described above cannot explain the difference between the retention times for the diastereomers. These two results will be considered below on the basis of the folding of cyclic peptides.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
Table IV. Predominant Molecular Conformations of Cyclic Dipeptides, Defined in Fiqure 4 chain 1 chain 2 DKP no. C-DP rinp 8-C 7-C 8-C 1
Ala-Ala
2 3 4 6 6
Ala-PAla Ala-Phe Ala-D-Phe Val-Phe Val-D-Phe Leu-Phe Leu-D-Phe Ala-Trp D-Ala-Trp Val-np Val-% Leu-Trp D- Ala-Phe Gly-Gly Leu-Leu
7 8
9 10 11 12 13 14 16 16
7-C
1886
methodb
q-eq
ax+eqc lower F 1121 F [121 F+U [16Ic F+U [16Ic
eq ax eq ax
eq
ax q-eq
F F
F 1121 F
F F
d
F+Uc
F F F F F F F F
ax
F+UC
a The number in bracket refers to the cyclic dipeptide used for the estimation of the conformation. b The number shown as the superscript denotes the reference. X = X-ray; N 3: nuclear magnetic resonance; and C = circular dichroism. The plus stands for an equimolar mixture of two conformers. d Unknown.
twist-boat forms.a*30 For the planar diketopiperazine ring, the @-carbonatom of an L-amino acid is located on the upper side, as shown by A of Figure 5a and that of a D-amino acid is on the lower side. For the boat form, the @-carbonatom of an amino acid is in the axial (ax) position (B in Figure 5a) and that of a qu amino acid is in the equatorial (eq)position. For the bowsprit-boat form, the @-carbonatom of an L-amino acid is in the equatorial position (C in Figure 5a) and that of a D-amino acid is in the axial position. For the twist-boat form, the @-carbon atom of an L-amino acid is in the [AI Planar [BI Boat quasiequatorial (q-eq) position (D in Figure 5a) and that of a D-amino acid is in the quasiaxial (q-ax) position. When the second side chain is an aromatic group (the Phe group in Figure 5a), the peaks assigned to the a-and @-protonsof the first side chain shift to the higher field by the ring current effect, depending on the degree of folding of the aromatic group. This shift, therefore, provides some information about the conformations of the diketopiperazine ringso*%(14) and [CI Bowsprikboat tD1 Twist-boat the y-carbon atommpmJ4 (3, and 12-14). The ring conformation of 13 is estimated from the proton-proton vicinal (b) coupling constant 3 J (CaH-NH).s7 The carbon-carbon gem0 H inal coupling constant 2 J (C'-Cg) may also depend on the ring conformation.31 Figure 5b, where the planar diketopiperazine ring is N-C' presumed, shows two predominant conformations of the y-carbon atom of chain 2 (the phenylalanyl group in this figure) for a cyclic LL-phenylalanyl dipeptide. As Table IV shows, most of the cyclic dipeptides have the folded structure (F),and only a few dipeptides are almost equimolar mixtures IF1 Folded IU1 Unfolded of the unfolded (U)and F conformers. The conformation of Flguro 6. (a) Four conformations of the diketopiperazine rlng for a the y-carbon atom is estimated from "43, 12-14, and 16) cycllc u-phenylalanyl dipeptide and (b) two conformations of the and W-NMR (131% chemicalshifta,the proton-protonvicinal y-carbon atom of chain 2 (the phenylalanyl group). For the sake of couplingconstant3J(CaH-CgH),91,52 the carbon-carbon vicinal simpilcity the planar diketopiperazine ring is presumed in Figure 5b. coupling constant (16) 3 J (C'C,), and the carbon-carbon geminal coupling constant 2 5 (C'Cg).31 The information about Molecular Conformationsand Surfaces Areas. The the molecular symmetry (the overall molecular conformation) three-dimensional structures of cyclic dipeptides in crystals can be obtained from the CD s p e ~ t r u m . ~ ~ ~ ~ are determined by X-ray analysis,U-B and those in solutions The solution structure38 of c-Ala-Ala 1 may be different are estimated from chemical shifts and coupling constants of from its crystal structure.26 Although any report on the ring 1H- and W-NMR spectra.29-7 Circular dichroism (CD) data also provide some information on solution structure~.3~*98 conformations of compounds 4-1 1 is unavailable, they may be guessed from those of the analogous compounds 3 and Table IV summarizes the predominant conformations of 16 12-14. The ring conformations of compounds 9-11 are cyclic dipeptides. These conformations are governed mainly assumed to be the same with c-Leu-Trp 13. The ring by the kind of the side chain. conformations of compounds 4,6, and 8 are estimated from As shown in Figure 5a, the diketopiperazine ring adopts that of c-D-Na-Phe 14. Here it should be noted that the four main conformations; planar, boat, bowsprit-boat, and
&-!@
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
Table V. Correlation Parameters for the Equation log k’ = aS+ b c-(X-Phe)a c- (X-Trp)b mobile a b r a b r phase
30% 50% 10% 30% 50%
MeOH MeOH ACN ACN ACN
2.064 1.368 2.475 1.260 0.609
-5.720 0.992 -4.150 0.992 -6.629 0.994 -3.842 0.970 -2.129 0.913
2.228 -6.810 1.336 -4.435 2.742 -8.029 1.259 -4.126 0.558 -2.130
0.999 0.999 0.999 1.OOO
0.994
The number of data is 6. The number of data is 3.
12
0
I
2.5
I
3.0
I
I
3.5
s (m2) Flgure 8. Correlation between the k’value and the S value for cycllc dipeptldes In 50 % MeOH (0)and 10 % ACN (0). The solld and dashed llnes show the best-fit linear relationshipsfor the Phe and Trpdertvatkes, respectively. former compounds are the LD-form and the latter is the DLform. The holistic molecular surface areas S of compounds 1-11 were measured and are plotted against the observed capacity factors (50 5% MeOH and 10% ACN) in Figure 6. The S values for the two conformers of 1 shown Table IV are almost the same. The positions of the phenyl and indole groups for compounds 3-1 1 are uniquely defined because of the steric hindrance of these groups. For compounds 7 and 8, one of the leucyl &carbonatoms is assumed to be in the trans position to the a-carbon atom and the other is to be in the gauche position. As Figure 6 shows, the S values for compounds 7 and 8 depend on the conformation of the leucyl group, namely, F or U. The average S value is used for further analysis. As Figures 2 and 6 and Table I1 show, the capacity factor and the surface area for c-Ala-Ala 1 are close to those for c-Ala-D-Ala2, respectively. The reason for this result is due to the smallness of the side chains. Therefore, the intramolecular interaction between the side chains is negligible. The dashed line in Figure 4 is drawn through the data point of 1 and has a unit slope according to eq 5. As Figure 4 shows, the observed capacity factors of cyclic dipeptides 3-11 are smaller than the dashed line predicted by the additivity of the group capacity factors of alcohols. This decreased hydrophobicity is consistent with the folded structures of the cyclic dipeptides shown in Table IV. The results shown in Figure 6 may be regarded to consist of three classes; the Ala-Ala’s, the Phe derivatives, and the Trp derivatives. The solid line shows the best-fit linear relationship for the Phe derivatives. Indeed, regardless of the diastereomers (3 and 4,5 and 6, and 7 and 8) and the kind of the aliphatic group, all Phe derivatives fall roughly on a straight line. As the dashed line shows, the same tendency is observed for the Trp derivatives (9-11). Thus, the separation of the diastereomers shown in Figure 1is explicable by the holistic molecular surface area approach. The slopes and intercepts of the solid and dashed lines are summarized in Table V, together with the correlation coefficients r. As Table V shows, the correlation is very good for 30% and 50% MeOH and 10% ACN. It is however, worse for the Phe derivatives in 30% and 50% ACN, probably since for these phases the hydrophobicity of the peptides plays a less important role in the retention mechanism. Simulation of Elution Profiles. Based on the correlations of the capacity factor with the hydrophobic parameters shown in Table 111, we can predict the capacity factor of a cyclic dipeptide. In plate theory for the c h r ~ m a t o g r a mthe ,~~
?lo I
I
0
(b)
OP ,I
11
5
10
15
t (min) Flgure 7. Simulated chromatogramsof cyclicdlpeptldes In 50 % MeOH predicted from the surface area values (the dashed llnes) and the log PR values (the solid lines) for (a) the Phe derivatives and (b) the Trp derivatives.
height and width of each peak can be taken into consideration in terms of the number of plate. This number can be calculated from the experimental result shown in Figure 1for each cyclic peptide and are used for the simulation. The mobile phase holdup time to or the void volume can be obtained from the observed peak or sodium nitrate. In Figure 7 , the simulated chromatograms based on the molecular area approach are shown by the dashed lines for the Phe derivatives (corresponding to the solid line for 50% MeOH in Figure 6) and for the Trp derivatives (corresponding to the dashed line for 50%MeOH in Figure 6). The agreement between experiment and the dashed lines is very good. Particularly, the separation of the diastereomers is well simulated. The solid lines in Figure 7 are based on the solid line in Figure 3 (Rekker’s partition Coefficients). By this predictor, the observedseparation of the diastereomers cannot be reproduced, though this parameter has the advantage of a wider applicability over the surface area approach. Meek’s retention coefficient ( k ’ ~ )Fauchbre’s , partition coefficient (PF),and the parameter ( k ’ a ) based on alcohols, shown in Table 111, predict chromatograms similar to the solid lines shown in Figure 7 ,although no data are shown herein. Similar simulations based on the molecular surface area approach were performed for the other mobile phases shown in Table V. Agreement between the simulated and observed chromatograms is better for the mobile phase in which the correlation coefficient is larger (data not shown).
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
DISCUSSION The most significant result in this work is to have demonstrated that the separation of the diastereomers of cyclic dipeptides on an RP-HPLC column is quantitatively explicable by the holistic molecular surface area approach. This is enabled since a detailed knowledge about the conformations of the cyclic dipeptides is accumulated or predictable. The separation of some diastereomers of linear peptides is qualitatively interpreted in terms of molecular surface areas,u but further quantitative analysis was hampered by many possible conformations of these linear molecules. Since cyclic dipeptides have more or less folded structures, the hydrophobicity of these molecules is smaller than the sum of the hydrophobicities of two fragmentary residues, and is, therefore, unpredictable by any additivity rule. This effect is also important for long peptides and proteins. Several researchers have noted that peptides larger than 15-20 residues tended to be eluted more rapidly than predicted from hydrophobic considerations alone.9J”16 The retention times of proteins are well correlated with the twothird powers of their molecular weights which are proportional to their surface areas.12 Furthermore, we have shown that the folded or crowded structures of some ethers and alcohols tend to decrease RP-HPLC retention times on ODS and phenyl columns.40@ In most cases of RP-HPLC, the retention time of the Trp derivative is longer than that of the Phe derivative. In our case, thii elution order is reverse (comparethe Phe derivatives 3,6,and A4 with the Trp derivatives 9,11, and A5 in Figure 1 and Table 11). The same reverse order is also observed for the amino acids a t pH 6.5 on a phenyl-Sil-X ~0l~1nn.6 This order, therefore, may depend on the kind of column and eluent. The dependenceof the capacityfactor on solventcompositions (61) Schulz, G.E.;Schrimer, R. H. Principles of Protein Structure; Springer: Berlin, 1979. (62) Wuthrich, K. NMR of Proteine and Nucleic Acids; John Wiley: New York, 1986. (63) Kaliszan, R. Anal. Chern. 1992,64,619A-631A. (54) Funaeaki, N.; Hada, S.;Neya, 5.;Machida, K. J.Colloid Interface Sci. ISM, 106,256268. (55) Funasaki,N.; Hada, S. J . Colloid Interface Sci. 1978,64,464-460. (56)Hada, S.;Neya, S.;Funasaki, N.J. Med. Chern. 1983,26,686-693. (57) Tanford, C. The Hydrophobic Effect; Wiley-Interscience: New York, 1980. (58) Chothia, C. Nature 1974,248, 338-339.
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(Tables 11,111,and V and Figure 2) can offer basic data for gradient elution of cyclic dipeptides. In Figure 6, the Phe derivatives and the Trp derivatives fall on different lines. The reason for this discrepancy is closely related with the above interpretation of the elution order. The aliphatic dipeptides 1 and 2 deviate from the aromatic dipeptides. The same tendency is observed in the solubilities of hydrocarbons in water39 and in the capacity factors of alcohols on an RP-HPLC column.43 These results show that the totalsurface area must be fragmented into the group areas of different surface properties. The strength of the holistic surface area approach is to give a physical basis of the hydrophobicityof a molecule. However, since the molecular area depends on the molecular conformation, this approach requires a detailed conformational knowledge. The determination of the conformations of large peptides becomes much easier as the result of rapid advances in energy calculation5’ and in NMR techniques.62 Thus, our approach can be extended to explain chromatographic behavior of such large peptides. Holistic molecular surface area and solvent-accessible surface area53 are actually the same. The importance of these areas is being widely recognized for chromatographic identification of unknown compounds, optimization of separation conditionsP3 prediction of chromatographic separation of diastereomers, estimation of hydrophobicity (e.g., octanol-water partition coefficient,4@41 aqueous solubility,” and critical micellization concentration55), prediction of relative biological activity,M prediction of molecular conformations,3w1and understanding of the structure and function of biological membranes,67 peptides,42 and proteins.51~”
ACKNOWLEDGMENT Thanks are due to Misses Masako Takizawa, Mayumi Morinaga, Noriko Ito, Mayumi Ueno, Yayoi Okuda, and Youko Kat0 and Mrs. Toshio Abe, Koshyu Hosoyamada, Yoshihiro Fujii, and Katsuhito Matsushita for the syntheses of some cyclic dipeptides. RECEIVEDfor review November 3, 1992. Accepted March 30, 1993.
