CHIRALITY 3 :268-276 (1991)
Absolute Configuration for Peptidomimetic Residues in Bioactive Peptides TOSHIMASA YAMAZAKI AND MURRAY GOODMAN Department of Chemistry, 0343, University of California, San Diego, La Jolla, California 92093-0343
ABSTRACT
A modern method is reported for the assignment of absolute configuration for peptidomimetics in bioactive peptides by use of 'H-NMR parameters in solution. Four peptide systems incorporating either retro-inverso modifications or 2-aminocyclopentanecarboxylicacid (2-Ac5c)a s a peptidomimetic for proline are d i s c u s s e d . (1) Two 1 4 - m e m b e r e d c y c lic d e r m o r p h i n a n a l o g s T y r c[D-A,bu-Phe-gPhe-(S and R)-mLeu] with a reverse amide bond between gPhe and mLeu residues where gPhe denotes a gem-diamino analog of Phe and mLeu refers to a malonyl analog of Leu. (2) Two cyclic hexapeptides related to somatostatin, with a reverse amide bond bec[gSar'-(S and R)-mPhe7-~-Trps-Lysg-Thr'o-Phe1'], tween the gSar and mPhe residues where the gSar and mPhe denote the gemdiamino and malonyl analogs of the Sar and Phe residues, respectively. The superscript numbers refer to positions in native somatostatin. (3) Cyclic hexapeptide s o m a t o s t a t i n an alo g s co n tain in g 2-Ac5c [trans-(1S,2S)-2-Ac5c, trans(1R,2R)-2-Ac5c,cis-(1R,2S)-2-Ac5c,and cis-(1S,2R)-2-Ac5c]in place of proline c[(2A~~~)~-Phe~-~-Trp~-Lys~-Thr~~-Phe"l. (4) Morphiceptin related analogs incorpoThe methodrating a cis-2-Ac5cresidue as shown in Tyr-cis-2-Ac5c-Phe-Val-NH,. ology described in this investigation could be applied to a wide variety of peptide systems.
KEY WORDS: absolute configuration, peptidomimetics, retro-inverso modifications, 2-aminocyclopentanecarboxylicacid, nuclear magnetic resonance (NMR), nuclear Overhauser effect, bioactive peptides INTRODUCTION
The biological activity of peptides requires proper spatial arrangements of functional groups to interact with receptors. Although most functional groups exist at the chain termini and at the peptide side chains, the main chain conformation is quite important in recognition and to orient functional groups in the correct arrays. In a n attempt to evaluate conformational requirements for biological activity, we have applied a stereoisomeric approach and incorporation of peptidomimetics to various bioactive peptide systems. For the biological evaluation of the analogs and the development of structure-bioactivity relationships, the assignment of the absolute configuration for peptidomimetic residues in bioactive peptide analogs is of particular importance because diastereomers display different biological activity profiles from one another. Absolute configuration of peptidomimetic residues could be assigned by X-ray diffraction studies, if suitable crystals are obtained. However, most times it is difficult to prepare crystals of biological active peptides suitable for X-ray diffraction. In this paper, we present a modern method for the assignment of the absolute configuration for peptidomimetic residues in biologically active peptides by use of 'H-NMR parameters, such as nuclear Overhauser effects (NOES) and vicinal 0 1991 Wiley-Liss, Inc.
proton-proton coupling constants for H-N-C-H groupings, observed in solution. Four typical peptide systems, for which we have proved the structurebioactivity relationships and the absolute configuration for peptidomimetic residues, are discussed. 1. Two 14-membered cyclic dermorphin analogs Tyr-cb-A,bu-Phe-gPhe-(S and R)-mLeu1. 2. Two cyclic hexapeptides related to somatostatin c[gSar6-(S and R)-mPhe7-~-Trp8-Lysg-Thr'o-Phe11] (the superscript numbers refer to the location of the residues in native somatostatin). 3. Cyclic hexapeptide somatostatin analogs cI(2-
A~~c)~-Phe~-~-Trp~-Lys~-Thr~~-Phe~'l. 4. Linear analogs of morphiceptin containing valine at the fourth position and a cis-2-Ac5c residue as shown in Tyr-cis-2-Ac5c-Phe-Val-NH,. The first two peptide systems incorporate partial retroinverso modifications. The remaining two systems contain 2-aminocyclopentanecarboxylic acid (2-Ac5c)as a peptidomimetic for proline. Received for publication February 10,1991; accepted March 22,1991. Address reprint requests to Murray Goodman at the address given above. Dedicated to the memory of Piero Pino, a very good friend and a great scientist.
269
ABSOLUTE CONFIGURATIONS FOR PEPTIDOMIMETICS
Schematic representations of th e partial retroinverso modifications are shown in Figure 1.The retroinverso modification reverses only the direction of the peptide bond and involves essentially isosteric structures as compared to the parent molecules. Therefore, the modification provides information concerning the importance of the carbonyl and amide groups for specific intramolecular hydrogen bonds, and backbone and side chain conformations while maintaining amide geometry. In addition, this modification typically increases resistance to enzymatic cleavage and degradation of the parent peptides. Synthesis of retro-inverso modified peptide analogs required the development of new methodology. Key steps include the preparation of the dipeptidomimetic structures unit gem-diaminoalkyl-2-alkylmalonyl which is incorporated into larger fragments. The gemdiaminoalkyl residues can be obtained from peptide amides or acylazides through molecular rearrangements that follow nitrene mechanisms. These reactions preserve optical purity. The 2-alkylmalonyl residues are incorporated into the peptide chains as a racemic residue. The resulting diastereomeric products could be often separated at the conclusion of the synthetic scheme by reverse-phase HPLC. We have successfully assigned the absolute configuration of 2-alkylmalonyl residues by use of NMR observations as will be shown below. The in vitro biological studies of the 14-membered cyclic dermorphin analogs with a reverse amide bond PRINCIPLESOF THE PARTIAL RETRWNVERSO MODIFICATION
between residues 4 and 5, Tyr-c[D-A,bu-Phe-gPhe-(S and R)-mLeu1, clearly show the effects of the chirality of the mLeu residues on the biological activity and receptor selectivity of these opioids.' The S-mLeu containing analog is superactive a t both the p- and 6-opioid receptors. The R-mLeu containing analog, on the other hand, is superactive at the preceptor but displays reduced potency at the &receptor as compared to the native enkephalin Tyr-Gly-Gly-Phe-Leu-OH. The biological activities of the cyclic hexapeptides related to somatostatin, c[gSar6-(S a n d R)mPhe7-~-Trp8-Lysg-Thr'o-Phe''l, are also dependent on the chirality of the mPhe residue. The S-mPhe containing analog displays activity on inhibition of the release of growth hormone in vitro while the R-mPhe analog is inactive in the same assay.' The in vivo studies on inhibition of pentagastrin-stimulated acid release in dogs show that the S-mPhe analog is superactive, but the R-mPhe analog is i n a ~ t i v e . ~ The incorporation of 2-aminocyclopentanecarboxylic acid (2-Ac5c)also offers a n attractive main chain modification. The structure of 2-Ac5c is unique as a n amino acid isostere in which it contains two chiral centers associated with the peptide backbone (Fig. 2). It has been also reported that the 2-Ac5cresidue tends to form p-turn structures in a n analogous fashion to proline when incorporated into a peptide chain.4 The 2-Ac5c is a p amino acid, thus four configurational isomers a r e probable, i.e., two cis isomers [(1R,2S)-2-Ac5cand (1S,2R)-2-Ac5cland two trans isomers [(1S,2S)-2-Ac5cand (1R,2R)-2-Ac5cl.The synthesis of 2-Ac5c was undertaken employing two methodologies which specifically generated either cis or trans configurations and these were utilized in separate syntheses in order to obtain the all-cis or all-trans isomers. The cis isomers were obtained a s a racemic mixture according to the method of Plieninger and Schneider by Hofmann degradation of cis-2-carbamoylcyclopentanecarboxylic a ~ i d . The ~ . ~trans isomers were prepared as a racemic mixture by the Michael addition of ammonia ~-~ the to 1-cyclopentenecarboxylic a ~ i d . Therefore, syntheses of peptide analogs containing 2-Ac5c were performed as a diastereomeric mixtures with either cis or trans isomers, and then separated at the final stage by reverse-phase HPLC. We have successfully assigned the absolute configuration of the four possible isomers of 2-Ac5c using 'H-NMR parameters.
co
NH
Fig. 1. Schematic representation of partially retro-inverso modifications of a parent peptide (A). The painvise retro-inverso modification (B) involves two adjacent amino acid residues containing side chains R, and R,, by which the gem-diaminoalkyl and 2-alkylmalonyl peptidomimetic structures are incorporated. In the extended retroinverso modification (C), the altered residues containing side chains R, and R, are moved apart so that an amino acid with reversed direction and chirality separates the gem-diaminoalkyl and 2alkylmalony residues. In (D) a reversed dipeptide is inserted between the peptidomimetic residues.
\ 6 / C4H2 Fig. 2. Schematic illustration of 2-aminocyclopentanecarboxylic acid (2-Ac5c).
270
YAMAZAKI AND GOODMAN
The existence of proline residue in a peptide chain allows for the possibility of cisltrans isomerization about the amide linkage. Therefore, it is difficult to obtain a consistent explanation for biological activity of proline containing peptides. Since the 2-Ac5cresidue contains a normal amide, the possibility of cisltrans isomerization about the amide bond is eliminated. Replacement of proline residue with 2-Ac5c residue provides information regarding the effect of cisltrans isomerization for biological activity, and moreover the conformational features around the peptide fragment neighboring the proline (or 2-Ac5c) residue. Utilizing this modification to a cyclic hexapeptide somatostatin analog c[Pr~~-Phe~-~-Trp~-Lys'-Thr'~-Phe''], we have proved that the p-VI turn structure around the Thr'OPhe"-Pro6-Phe7 bridging region with a cis amide bond between the Phe"-Pro6 residues is required for the biological activity of the parent ana10g.~. The morphiceptin analogs incorporating 2-Ac5c at t h e second position i n place of proline, Tyrshow 2-Ac5c-Phe-X-NH, [X = Pro and (L and ~)-Vall, interesting configurational dependence of biological activity and conformational preference.',' Among the four possible configurational isomers, only the analogs containing (1S,2R)-2-Ac5cshow opioid activity. The biological profiles of the active ( 1S,2R)-2-Ac5canalogs are approximately as same as that of the parent morphiceptin. The conformational studies using the 'HNMR spectroscopy and computer simulations indicate that the preferred conformations for the (1S,2R)-2-Ac5c analogs have considerable topological similarity with the morphiceptin in which the conformer adopts the cis conformation about the Tyr-Pro amide bond.' We have, therefore, concluded that the cis form about the amide bond joining these two residues is required for the biological activity of the morphiceptin-related analogs containing the proline a t the second position. THEORETICAL ASPECTS FOR 'H-NMR PARAMETERS The nuclear Overhauser effect (NOE) is a measure of the dipole-dipole relaxation between different nuclear spins which are close in space. The magnitude of the NOE can be related to the spin-spin distance r by an equation of the general form: NOE a r - 6 AT,)
(1)
where the second term ~ T T , ) is a function of the corre, account for the influence of the lation time T ~ which motional averaging processes on the observed NOE. Because of the r-6 dependence, the NOE intensity is very large for spins in close spatial proximity (-2 A), but rapidly becomes smaller as the distance is increased. Depending on the signal-to-noise ratio in the NOE experiment, an upper limit for NOE observable 'H-lH distance may be of the order 3.5-4.5 A. Several methods have been proposed for a quantative evaluation of NOE data in terms of 'H-lH distances. One of the simplest approach estimating unknown 'H-
'H distance rv from NOE data can be applied by use of the expression: r,, = [rk;
(zkl/Ivl-6
(2)
where rkl is a known distance, and Ikl and I, are NOE intensities. A useful standard for rki is the distance between two geminal methylene protons, which is calculated to be 1.76 A using the bond length of 1.10 A for C-H and the bond angle of 106.1' for H-C-H.lO,'l This equation includes two assumptions: (1)the correlation times for the different parts of the molecule are the same, and (2) the NOE intensities are proportional to the cross-relaxation rates. The latter assumption will hold true only at short mixing times where spin diffusion and other relaxation effects are negligible. Therefore, in these studies, short mixing times (20200 msec) should be employed. In the present investigation, NOEs were measured using the two-dimensional rotating frame experiments (ROESY)" with a spin locking field of 2.5 kHz, on a General Electric GN-500 spectrometer. The two dimensional spectra were obtained using 2 K data points in the f, domain and 256 points in the fl domain. Applying the zero filling procedure to the fl domain resulted in a final matrix of 2 K x 2 K data points. Gaussian multiplication was used to enhance the spectra. The NOEs, observed in the ROESY spectra with mixing times of 50-200 msec, were classified as strong, medium, or weak according to their intensities. The corresponding upper distance constraints could be calibrated to be 2.5,3.0, or 3.5 A by comparing NOEs with the one for the methylene protons (1.76 A). The backbone conformation of peptides is defined by $, and w, which reprethree kinds of torsion angle sent the rotational states around the skeletal single bonds NH-C"H, C"H-CO, and CO-NH, respectively. Because of the partially double bond character, only the planar structure in either trans or cis conformation is probable for the amide bond. The cis conformation allows closer contact between a-protons of adjacent residues (-2 A), while the distance between these two protons is larger than 4 A in the trans conformation. Therefore, the cis conformation can be easily distinguished from the trans conformation by the strong sequential NOE between the a-protons of the adjacent residues. The sequential NOE between the NH proton and the a-proton of the preceding residue provides information regarding the rotational state ($) around the C"H-CO bond. The maximum (3.58 A) and minimum (2.15 A) values of the sequential distance daN between these two protons of L-amino acids are calculated for $ = - 60' and + 120', respectively. A strong NOE is expected only for a Jr range from 60" to 180", in which a value of daN is less than 2.5 A. The intraresidue 'H-'H distance dNuand thus NOE between vicinal protons for the H-N-C"-H moiety can be related to the torsion angle The distance dNa varies between approximately 2.38 and 3.06 A. For Lamino acids, the maximum and minimum values are
+,
+.
ABSOLUTE CONFIGURATIONS FOR PEFTIDOMIMETICS
+
calculated for = - 120" and + 60°, respectively. The 4 angle dependence of the intraresidue distance dNa is smaller than the angle dependence of the sequential distance duN. The vicinal 'H-'H coupling constant JNH-CH provides the same information concerning the torsion angle via the Karplus-type equation.
only when the mLeu residue has a n S-configuration. on the other For Tyr-C[D-A,bu-Phe-gPhe-R-mLeu1, hand, the strong NOEs between Phe Ha-gPhe NH and gPhe NH-gPhe H", and the weak NOE between gPhe H"-gPhe N*H suggest that the NH and N*H protons of the gPhe residue are projecting downward and upward, respectively, relative to the 14-membered ring plane. In this orientation, the strong NOE observed from the gPhe N*H to the mLeu H" requires a n R-configuration a t the mLeu C" center (Fig. 3b).
+
+
J,,
CH =
A
C O S ~Ic$ -
60"l B cos I+ - 60"l + C (for L-amino acids). -
(3)
c[gSa$-(S and R)-mPhe7-~-Trp8-Lys9-Thr'o-Phd11
In the literature, several sets of coefficients (A, B , C ) have been reported for Eq. (3). Among the (A, B , C) sets, those given by Bystrov et al.13 (8.6, 1.0, 0.4) and Cung et al.I4 (8.6, 2.9, 0.0) are typical. The vicinal coupling constant JNH-CH displays a different angle dependence as compared with the intraresidue distance dNa.Therefore, measurements of JNH-CH present valuable data for studies of the backbone conformation of peptides, in addition to NOE data.
Unlike the 14-membered cyclic dermorphin analogs
Tyr-c[D-A,bu-Phe-gPhe-(Sand R)-mLeu1 discussed above, cyclic h e x a p e p tid e s c[gSar6-(S a n d R)-
+
mPhe7-~-Trp8-Lyss-Thr10-Phe11] contain a n achiral
RESULTS AND DISCUSSION
Tyr-c[D-A,bu-Phe-gPhe-(Sand R)-mLeul The NOEs used for the assignment of the configuration about mLeu residues in 14-membered cyclic derand R)morphin analogs Tyr-C[D-A,bu-Phe-gPhe-(S mLeu], i n which t h e g P h e resid u e h a s a n Sconfiguration, are shown in Table l.15 In the table, gPhe NH refers to the NH originally present in the Phe residue as opposed to gPhe N*H, which is formed as a result of the rearrangement in the synthesis of the gem-diaminoalkyl moiety. Because of the nature of the 14-membered cyclic molecule, both a-protons of the Phe and gPhe residues are projecting downward with respect to the plane of the 14-membered backbone ring. The weak NOEs between Phe Ha-gPhe NH and gPhe N H - g P h e H" observed for Tyr-c[D-A,buPhe-gPhe-S-mLeu] indicate that the NH proton of the gPhe residue is projecting upward relative to the 14membered ring plane while the strong NOE between gPhe Ha-gPhe N*H indicates that the N*H proton must be projecting downward (Fig. 3a). The strong NOE observed from gPhe N*H to mLeu H" is expected TABLE 1. Nuclear Overhauser effects (NOEs) and vicinal 'H-'H coupling constants (.I,,,-) used for assignment of absolute configuration for 2-isobutylmalonyl residues (mLeu) in 14-membered cyclic dermorphin analogs Tyr-c[D-Abu-Phe-gPhe-(Sand R)-mLeu1" S-mLeu
R-mLeu ~
NOE(Phe a-gPhe NH) NOE(gPhe NH-gPhe a) NOE(gPhe a-gPhe N*H) NOE(gPhe N*H-mLeu a) JNH-CH JNH-CH
(gPhe NH-a)(Hz) (gPhe N*H-a)(Hz)
_
_
_
_
W
S
W
S
S
W
S
S
8.63 7.21
_
6.47 7.52
"The observed NOES are qualitatively classified according t o their intensities; s, strong; m, medium; w, weak.
271
_
gem-diaminoalkyl residue gSar. For the assignment of the configuration about the 2-alkylmalonyl residue mPhe, therefore, the structure of the tetrapeptide sequence Phe"-gSar6-mPhe7-~-Trp8must be examined. The 'H-NMR parameters required for the configurational assignment are summarized in Table 2.16 With the exception of the two sequential NOEs between gSar N*H-mPhe H" and mPhe Ha-~-TrpNH which are key NOEs for the configurational assignment, the remaining NMR parameters observed for both the diastereomers are quite similar. The structures around the Phel1-gSar6-mPhe7-~-Trp8 part consistent with the observed data are illustrated in Figure 4. The gSar N*H and D - T NH ~ protons are projecting in opposite directions from one another with respect to the cyclic hexapeptide ring in both the diastereomers. In this structure, the strong NOE observed from mPhe H" to D-Trp NH is expected only when the mPhe residue has a n S-configuration as shown in Figure 4a. On the other hand, the medium NOE observed from gSar N*H to mPhe Ha requires a n R-configuration at the mPhe C" center (Fig. 4b). The constrained nature of the cyclic hexapeptides allows u s to assign t h e pro-chiralities of t h e two a-protons of the gSar residue. Both the diastereomers adopt a cis conformation about the Phe-gSar amide bond, which is deduced from the strong sequential NOEs observed between Phe H" and gSar Hal. The absence of NOEs between Phe H" and gSar NMe protons expected for a trans conformation also supports the assignment of cis conformation. Medium NOEs were observed from gSar NMe protons to only gSar Ha', i.e., no NOEs were measured between gSar NMegSar Hal. The NOEs between Phe Ha-gSar Hal and gSar NMe-gSar H"', together with the cis arrangement about the Phe-gSar amide bond, lead to the assignment of H"l as pro-S H" and Ha2 as pro-R H" (Fig. 4c). The opposite assignment makes the molecules very high in energy becuse of steric interactions between the gSar C"H, and Thr CO groups.
6 ( 2-Ac?~)~-Phe~-~-Trp~-Lys~-Th~~-Phd~I
The NOEs and vicinal JNH-CH coupling constants, used for the configurational assignment of the 2-Ac5c
272
YAMAZAKI AND GOODMAN
(a) S-mLeu
(b)
R-mLeu
Fig. 3. Schematic illustrations for the structures around the Phe-gPhe-mLeuresidues of 14-membered cyclic dermorphin analogs (R3 = R4 = benzyl and R, = isobutyl): (a) Tyr-C[D-A,bu-Phe-gPhe-S-mhu] and (b) Tyr-c[c-A,bu-Phe-gPhe-R-mhul. The NOES observed for atomic groupings are shown in the figures; s, strong; m, medium; and w, weak.
TABLE 2. Nuclear Overhauser effects (NOES)and vicinal ring takes the highest possible value in order to bring 'H-'H coupling constants (JNHxH)used for assignment of the NH and CO groups as equatorial as possible. absolute configuration for 2-benzylmalonyl residues The structures around the Phe"-(2-Ac5c)'-Phe7 parts (mPhe) in cyclic hexapaptides related to somatostatin i n t h e f o u r s t e r e o i s o m e r s of c [ ( ~ - A c ~ c ) ~ clgSar6-(Sand R)-mPhe7-~-Trps-Lysg-Thr'o-Phe1'la Phe7-~-Trp8-Lysg-Thr10-Phe111, consistent with the obS-mPhe R-mPhe served NMR data, are illustrated in Figure 5. An unambiguous assignment of the absolute configuration at the p-carbon of the 2-Ac5c residue, to which the NH NOE(gSar N*H-mPhe a) group is bonded, could be achieved by use of the seNOE(pTrp NH-mPhe a) quential NOES between Phe" NH and 2-Ac5c NH and NOE(pTrp N H - D - T ~a)~ NOE(Phe a-gSar a,) between Phe" H" and 2-Ac5c NH. For all of the steNOE(Phe a-gSar NMe) reoisomers, no NOES or a weak NOE were observed for NOE(Phe a-gSar a,) the H-N-CP-H groupings of the 2-Ac5c residues, inNOE(gSar NMe-gSar a,) dicating a nearly trans orientation for the NH and HP NOE(gSar NMe-gSar a2) protons. In these arrangements, the torsion angle of NOE(gSar N*H-gSar a,) t h e 2-Ac5c residue is restricted -120" for a n RNOE(gSar N*H-gSar a2) is approxiconfiguration at the CP carbon while JNH-CH (gSar N*H-al)(Hz) 3.75 7.44 mately - 120" for a n S-configuration. According to the JNH-CH (gSar N*H-a2)(Hz) 8.34 4.50 nature of the cyclic molecules with this ring size (19JNH-CH (D-Tv NH*)(Hz) 6.85 6.60 membered ring), the Phe" side chain and cyclopentane ring of the 2-Ac5c residue exist outside of th e 19T h e observed NOES are qualitatively classified according t o their membered ring. In these structures, the Phe" H" and intensities; s, strong; m, medium; w, weak; -, absent. 2-Ac5c protons are projecting downward and upward, respectively, relative to the 19-membered ring when r e s i d u e s i n cyclic h e x a p e p t i d e s c[(2-Ac5cI6- the CP carbon has a n R-configuration (Fig. 5b and d). Phe7-~-Trp8-Lysg-Thr10-Phe"l, are summarized in Ta- Because the angle of the 2-Ac5c residues with a n ble 3.7 The trans-2-Ac5c and cis-2-Ac5cresidues can be R-configuration a t the CP center is restricted to values clearly distinguished from one another by the NOES around 120" as mentioned above, the 2-Ac5c NH proton between the 2-Ac5c a- and p-protons and between the is projecting downward and close to the Phe" H" pro2-Ac5c NH and a-protons. For the cis-2-Ac5c residues, ton. As a result, a strong NOE exists between the Phel' i.e. (1R,2S)-2-Ac5cand (1S,2R)-2-Ac5cresidues, strong H" and the NH protons of the 2-Ac5c residues with a n NOES were observed between the H" and HP protons R-configuration a t the CP carbon, i.e., one of the transsince these two protons exist on the same side with 2-Ac5c [(1R,2R)-2-Ac5c] a n d one of t h e cis-2-Ac5c respect to the cyclopentane ring. On the other hand, no [(1S,2R)-2-Ac5clresidues. In the remaining two stereoisomers containing NOES were measured between the H" and HP protons of the truns-2-Ac5c residues, i.e. (1S,2S)-2-Ac5c and 2-Ac5c with a n S-configuration at the CP carbon, i.e., (1R,2R)-2-Ac5c residues. Instead of the NOE(2-Ac5c another trans-2-Ac5c [(1S,2S)-2-Ac5cland another cisa-2-Ac5c p), strong NOES were observed between the 2-Ac5c [(1R,2S)-2-Ac5clresidues, both of the Phe" H" 2-Ac5c NH and H" protons for the trans-2-Ac5c resi- and 2-Ac5c HP protons are projecting downward reladues. For both the trans forms, the lack of NOE(2-Ac5c tive to the 19-membered ring (Fig. 5a and c). The a-2-Ac5c p) and the strong NOE(2-Ac5c NH-2-Ac5c a ) angles (- - 120") for the 2-Ac5c residues with a n Sindicate that the C"H and CPH protons are oriented in configuration at the CP carbon orient the NH proton of a nearly trans arrangement and that the internal ro- this residues upward with respect to the 19-membered tation angle about the c"-CP bond in the cyclopentane ring. In these orientations, the Phe" H" and 2-Ac5cNH
+
+
+
+
273
ABSOLUTE CONFIGURATIONS FOR PEPTIDOMIMETICS
(a) S-mPhe I
(c) Ia 1= Dro-S, a2spro-R
I
I
3
(bl R-mPhe I
I
Fig. 4. Schematic illustrations for the structures around the gSar6-mPhe7-~-Trpa residues of cyclic indolylmethyl): (a) c[gSar6-S-mPhe7-~hexapeptides related to somatostatin (R, = benzyl and R The structure, with a cis conTrps-Lys9-Thr'o-Phe''l and (b) ~[gSar~-R-mPhe~-~-Trp~-Lys~-~r~~-Phe"]. formation about the Phe-gSar amide bond, observed for both the diastereomers is shown in (c). The NOES observed for the atomic groupings are included in the figures; s, strong; m, medium; and w, weak.
protons are distant from each other, and the NOE between these two protons should be weak. In addition, the Phe" NH and 2-Ac5c NH protons are close enough to expect a n NOE between them. These predictions are in agreement with the experimental results shown in Table 3, where a weak NOE(Phe" Ha-2-Ac5c NH) and a medium NOE(Phel' NH-2-Ac5c NH) are observed only for the cyclic hexapeptides containing the 2-Ac5c residues with a n S-configuration at the Cp carbon, i.e., truns-(1S,2S)-2-Ac5cand cis-(1R,2S)-2-Ac5cresidues. A combination of the results for the trans and cis configurational assignment and the results of the chirality assignment a t the C p carbon obtained above allows us to draw a definite conclusion about the absolute configuration for the four possible isomers of 2-Ac5c residues.
1. In the molecule where the 2-Ac5c residue displays me d iu m NOE(Phe" NH-2-Ac5c N H ), w eak NOE(Phe" a-2-Ac5c NH), and strong NOE(2-Ac5c NH-2-Ac5c a),the trans-(lS,BS)-configuration has been assigned (Fig. 5a). 2. In the molecule where the 2-Ac5c residue exhibits strong NOE(Phe'' a-2-Ac5c NH) a n d s t rong NO E (2 -A c 5 c N H -2 -A c 5 c a), t h e ( 1 R , 2 R ) configuration has been assigned (Fig. 5b). 3. In the molecule where the 2-Ac5c residue shows med i u m NOE(Phe'' NH-2-Ac5c NH), weak NOE(Phe'' a-2-Ac5c NH), and strong NOE(2-Ac5c a-2-Ac5c p), the cis-(lR,2S)-configuration has been assigned (Fig. 5c). 4. In the molecule where the 2-Ac5c residue gives a 'H-NMR s p e c t r u m w i t h s t r o n g NOE(Phe"
TABLE 3. Nuclear Overhauser effects (NOES) and vicinal 'H-'H coupling constants ( J N H x H ) used for assignment of absolute configuration for 2-aminocyclopentanecarboxylicacids (2-Ac5c)in cyclic hexapeptides related to somatostatin 1' ' c[ (2-A~~c)~-Phe'-~-Trp~-Lys~-Thr'~-Phe' trans -2-Ac5c
(1S,2S)-2-Ac5c
(Phe" NH-a)(Hz) (2-Ac5c NH+)(Hz) (Phe7 NH-cl)(Hz)
(1R,2R)-2-Ac5c
(1R,2S)-2-Ac5c
(1S,2R)-2-Ac5c
7.83
8.09
m
NOE(Phe" NH-Phe" a) NOE(2-Ac5c NH-Phe" NH) NOE(2-Ac5c NH-Phe" a) NOE(2-Ac5c NH-2-Ac5c p) NOE(2-Ac5c NH-2-Ac5c a) NOE(2-Ac5c a-2-Ac5c p) NOE(Phe7 NH-2-Ac5c p) NOE(Phe7 NH-2-Ac5c a) NOE(Phe7 NH-Phe7 a) JNH-CH JNH-CH JNH-CH
cis-2-Ac5c
S
W
S
s s m
6.68 5.83 8.00
3.98 8.61 8.51
b
5.73
T h e observed NOES are qualitatively classified according to their intensities; s, strong; m, medium; w, weak; -, absent. bA value could not be determined because of severe overlap of the NH proton signal with the aromatic proton peaks.
b
5.51
274
YAMAZAKI AND GOODMAN
of the i + l t h residue to a-proton of the ith residue were observed (i = 1,2, or 3). These strong sequential NOES indicate that a torsion angle J? about the C"-CO bond of the ith residue is restricted in a range from 60" to 180" when t h e ith residue h a s a n L- or Sconfiguration while in a range from - 60" to - 180" for a D- or R-configuration. Relatively large values of JNH-CH and weak intraresidue NOES between the NH and CH protons of the H-N-C-H moieties observed for the cis-2-Ac5c, Phe, and Val residues of both the diastereomers indicate a nearly trans orientation of ( d ) (IS, 2R)- 2-Ac5c (b) (IR,2R)-2-Ac5c these two protons (+ --120" for a n L- or Sconfiguration a n d - 120" for a D- a n d Rconfiguration). Model building with these and J? values clearly indicates that the NOES between Tyr H"-Val HY and Tyr +z,6-Val HY are expected only when t h e cis-2-Ac5c residue h a s t h e (1S,2R)configuration. To confirm the assignment of the configuration for the cis-2-Ac5c residue, all of the NOES observed for Fig. 5. Structures around the Phe"-(2-Ac5c)'-Phe7 residues of cy- Tyr-cis-(1S,2R)-2-Ac5c-Phe-Val-NH, were used as conclic hexapeptides related to somatostatin ~ [ ( 2 - A c ~ c ) ~ - P h e ~ - ~ - T p * straints for the molecular dynamics simulations (10 Lysg-Thr'o-Phe"l: (a) trans-(1S,2S)-2-Ac5c,(b) trans-(lR,2R)-2-Ac5c, (c) cis-(1R,2S)-2-Ac5c,and (d) cis-(1S,2R)-2-Ac5c.The NOES observed psec) on both the (1R,2S)-2-Ac5cand (1S,2R)-2-Ac5c for the atomic groupings are given in the figures; s, strong; m, me- containing analogs. The calculations were carried out dium; and w, weak. in vacuo employing the DISCOVER force field program,17modified to allow for NOE constraints. The a-2-Ac5c NH) and strong NOE(2-Ac5c a--2-Ac5cp), NOE constraints were applied using the following the cis-(lS,2R)-configurationhas been assigned function: (Fig. 5d). ENOE = Kp(rk - rd2 (rij > ro) Tyr-cis-2-Ac5c-Phe-Val-NH, (0)
(IS,2S)-2-Ac5c
( c ) (I R , 2 S)- Z-AC'C
+
ENOE= 0
+
(TI,
c ro)
(4)
As described above, the absolute configuration of peptidomimetic residues in cyclic peptides could be de- where ri, represents a distance between two specific termined by examining the local structures around the protons. Force constants K , = 15,10,5 kcal mol-' A-2 peptidomimetic residues only with intraresidue and se- and target separations ro = 2.5, 3.0, 3.5 A were asquential NOEs. In contrast to cyclic molecules, a con- sumed for strong, medium, and weak NOEs, respecsideration of overall conformations of the molecules is tively. The potential and forcing energies resulted from the necessary for the assignment of configuration in the case of linear molecules. The long-range NOES provide application of the NOE constraints were consistently useful information concerning the overall conforma- higher for the analog containing the (1R,2S)-2-Ac5c tions, in addition to the intraresidue and sequential residue than for the analog containing the (1S,2R)NOEs. Here we describe an unambiguous assignment 2-Ac5c residue. In addition, structures taken from the of the chiralities of the cis-2-Ac5c residue in the dia- dynamics at 1psec intervals were minimized with and s t e r e o m e r i c p a i r of T y r - c i s - ( 1R,2S)-2-Ac5c- without the NOE constraints. All of the structures for P h e - V a l - N H , a n d T y r - c i s - (1S , 2 R ) - 2 - A c 5 c - the (1R,2S)-2-Ac5ccontaining analog with the NOES Phe-Val-NH,. This peptide system represents a specific applied were very high in energy. The structures for case in which we determine the absolute configuration this analog obtained from the energy minimizations without the NOE constraints showed large conformaby use of the long-range NOEs. tional changes from the initial structures, and thereThe NOES for Tyr-cis-(1R,2S)-2-Ac5c-Phe-Val-NH, and Tyr-cis-(1S,2R)-2-Ac5c-Phe-Val-NH, measured in fore are not consistent with the long-range NOEs, i.e., HYand H". On the DMSO-d, are shown in Figure 6.' The observed values Tyr Ha-Val HYand Tyr +,,,Val of vicinal 'H-lH coupling constants are summarized in other hand, the minimum energy conformations for the Table 4." The long-range NOES between Tyr Ha-Val (1S,2R)-2-Ac5ccontaining analog are in agreement HY,Tyr +,,,Val HY,and Tyr +z,,Val H" observed for w i t h a l l t h e NMR d a t a o b s e r v e d for T y r Tyr-cis-(1S,2R)-2-Ac5c-Phe-Val-NH, are especially in- cis-(1S,2R)-2-Ac5c-Phe-Val-NH,. T h e lowest energy conformations for Tyrformative. Except for these long-range NOEs, the remaining NMR parameters observed €or both the dia- cis-(1R,2S)-2-Ac5c-Phe-Val-NH, and Tyrstereomers are quite similar. For both the diaste- cis-(1S,2R)-2-Ac5c-Phe-Val-NHz are shown in Figure reomers, strong sequential NOEs from the NH proton 7a and b, respectively.' For each conformation the
275
ABSOLUTE CONFIGURATIONS FOR PEFTIDOMIMETICS Tyr-(1R.2S)-A~c-Phe-Val-NH2
2-AcSc N
~
P
Phe Y
E
aN P
Val
O
/ N
a
S
P
y
mlh
Y
a
Val
N
Wl
O P
S
a
Phe
N E
Y
j
P
2-Ac'c
a N W
m
$2.6
P
s
W
a
Tyr
N
Tyr-(1S.2R)-Ac5c-Phe-Val-NH2
Fig. 6. Observed NOES for morphiceptin analogs 'Tyr-cis-2-Ac5c-Phe-Val-NH, measured in dimethylsulfoxide-$ using the ROESY experiments. The two protons of the CH, groups and the two y-methyl groups of the Val residues are distinguished by appending a superscripth for the one at higher field and 1 for that at lower field. The NOES are qualitatively classified according to their intensities;s, strong; m, medium; and w, weak.
TABLE 4. Experimental values of vicinal 'H-'H coupling are expected for Tyr-cis-(1S,2R)-2-Ac'c-Phe-Val-NH, constants (.INHxH) observed for H-N-C-H groupings in between these atomic groupings. The conformational morphiceptin related analogs Tyr-cis-2-Ac5c-Phe-Val-NH, analysis supports the assignment of the diastereomer for which the longof Tyr-cis-2-Ac'c-Phe-Val-NH2, range NOES between Tyr Ha-Val HYand Tyr +2,6-Val HY a n d Ha a r e observed, to t h e s tr u c tu r e Tyrcis-(1S,2R)-2-Ac'c-Phe-Val-NH,.The other diastereomer, for which no long-range NOES are observed between the Tyr and Val residues, has been assigned as Tyr-cis -(1R,2S)-2-Ac'c-Phe-Val-NH,. shortest distances between Tyr Ha-Val HY and Tyr CONCLUSION +2,6Val HYwere calculated. These distances are quite We have launched a program to study structurelarge for t h e st r u ctu re i n Fig u re 7a, i.e., Tyrcis-(1R,2S)-2-Ac5c-Phe-Val-NH, (-8.9 A). It should be bioactivity relationships of various peptide systems noted that this minimum energy structure is consistent utilizing a stereoisomeric approach and peptidomimetwith all the NMR data observed for the analog contain- ics. This program requires the unambiguous assigning the (1R,2S)-2-Ac5cresidue (Fig. 6 and Table 4). In ment of the absolute configuration for peptidomimetic the structure shown in Figure 7b, on the other hand, residues in the analogs since the biological recognition t h e distances between Ty r Ha-Val HY and Tyr is certainly stereospecific. Studies by X-ray diffraction +2,6Val HYare approximately 2.6 A, and thus NOES are one of the most powerful methods for the configu-
276
YAMAZAKI AND GOODMAN
.
I
\ Fig. 7. The lowest energy conformations for (a) Tyr-cis-(1R,2S)-2-Ac5c-Phe-Val-NH, and (b) Tyr-cis(1S,2R)-2-Ac5c-Phe-Val-NH,. The shortest distances between Tyr-Ha-Val H' and Tyr &-Val H ' are given for each conformation.
rational assignment. However, the utilization of X-ray diffraction in the analysis of peptides is often limited by the fact that many peptidic systems are not crystalline. In this investigation, we presented a modern method for the assignment of the absolute configuration for peptidomimetic residues in biologically active peptides based on the use of NOEs and vicinal 'H-IH coupling constants for H-N-C-H groupings (JNH-CHS). Four peptide systems incorporating either retro-inverso modifications or 2-aminocyclopentanecarboxylicacid (2-Ac5c) as a peptidomimetic for proline were discussed. The absolute configuration for peptidomimetic residues in the cyclic peptides was assigned by examining the local structures around the peptidomimetic residues with intraresidue and sequential NOEs. In contrast to the cyclic molecules, the overall conformations of the molecules must be examined in the case of linear peptides. For this purpose, long-range NOEs are useful in conjunction with intraresidue and sequential NOEs. Both of the retro-inverso modified peptide systems Tyr-c[D-A,bu-Phe-gPhe-(S and R)-mLeu1 and c[gSar-(S for which the chiraland R)-mPhe-D-Trp-Lys-Thr-Phe], ities of the malonyl residues were assigned, include pairwise retro-inverso modifications (Fig. lb). A similar consideration could be applied to extended retroinverso modified peptide systems in which reversed amino acids a r e i n s e r t e d between t h e g e m diaminoalkyl and 2-alkylmalonyl residues (Fig. l c and d). The present method using NOEs and JNH-CHS is powerful for the configurational assignment and applicable to many retro-inverso modified peptides and peptides containing a wide variety of peptidomimetic residues. ACKNOWLEDGMENTS The authors would like to acknowledge the support of this research through grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK 15410) and the National Institute of Drug Abuse
(DA 06254). We gratefully acknowledge Drs. Odile E. Said-Nejad, Christian Pattaroni, and Albert Probstl for the syntheses of Tyr-c[D-A,bu-Phe-gPhe-(Sand R)mLeu1, c[gSar-(S and R)-mPhe-D-Trp-Lys-Thr-Phel, a n d t h e 2-Ac5c c o n t a i n i n g m o l e c u l e s (1212Ac5c-Phe7-~-Trp-Lys-Thr-Phe1'1 and Tyrcis-2-Ac5c-Phe-Val-NH,), respectively. We would like to thank Dr. Dale F. Mierke and Ziwei Huang for helpful discussions and measurements. LITERATURE CITED 1 Mierke, D.F., Said-Nejad, O.E., Yamazaki, T., Felder, E.R., Schiller, P.W., Goodman, M. In: Peptides: Proceedings ofthe Eleventh American Peptide Symposium. Revier, J.E., Marshall, G.R., eds. 1990:348-350. 2. Pattaroni, C., Lucietto, P., Goodman, M., Yamamoto, G., Vale, W., Moroder, L., Gazerro, L., Gohring, W., Schmied, B., Wunsch, E. Int. J. Peptide Protein Res. 36:401-417, 1990. 3. Beglinger, L., Gyr, K. Personal communication. 4. Plieninger, H., Schneider, K. Chem. Ber. 92:1594-1599, 1959. 5. Bernath, G., Lang, K.L., Gondos, G., Marai, P., Kovacs, K. Acta Chim. Acad. Sci. Hung. 74:479-497, 1972. 6. Conners, T.A., Ross, W.C.J. J . Chem. SOC.2119-2132, 1960. 7. Yamazaki, T., Huang, Z., Probstl, A., Goodman, M. Peptide 1990: Proceedings of the 21th European Peptide Symposium, Giralt, E., Andreu, D., eds. 389-392,1991. 8. Mierke, D.F., NoRner, G., Schiller, P.W., Goodman, M. Int. J. Peptide Protein Res. 3535-45, 1990. 9. Yamazaki, T., Probstl, A., Schiller, P.W., Goodman, M. Int. J. Peptide Protein Res. 37:364-381, 1991. 10. Lide D.R., Jr. J. Chem. Phys. 33:1514-1518, 1960. 11. Iijima, T. Bull. Chem. Soc. Jpn. 45:1291-1293, 1972. 12. Bothner-By, A.A., Steppens, R.L., Lee, J., Warren, C.D., Jeanloz, R.W. J . Am. Chem. SOC. 106:811-813,1984. 13. Bystrov, V.F., Ivanov, V.T., Portnova, S.L., Balashova, T.A., Ovchinnikov, Yu. A. Tetrahedron 29873-877, 1973. 14. Cung, M.T., Marraud, M., Neel, J. Macromolecules 7:606-613, 1974. 15. Yamazaki, T., Mierke, D.F., Said-Nejad, O.E., Felder, E.R., Goodman, M. In preparation. 16. Mierke, D.F., Pattaroni, C., Delaet, N., Toy, A., Goodman, M., Tancredi, T., Motta, A., Temussi, P.A., Moroder, L., Bovermann, G., Wunsch, E. Int. J. Peptide Protein Res. 36:418-432,1990. 17. Hagler, A.T. In: The Peptides, Vol. 7.Udenfriends, S., Meienhofer, J., Hruby, V.J., Eds., Orlando, FL: Academic Press, 1985214-296.
Absolute Configuration for Peptidomimetic Residues in Bioactive Peptides TOSHIMASA YAMAZAKI AND MURRAY GOODMAN Department of Chemistry, 0343, University of California, San Diego, La Jolla, California 92093-0343
ABSTRACT
A modern method is reported for the assignment of absolute configuration for peptidomimetics in bioactive peptides by use of 'H-NMR parameters in solution. Four peptide systems incorporating either retro-inverso modifications or 2-aminocyclopentanecarboxylicacid (2-Ac5c)a s a peptidomimetic for proline are d i s c u s s e d . (1) Two 1 4 - m e m b e r e d c y c lic d e r m o r p h i n a n a l o g s T y r c[D-A,bu-Phe-gPhe-(S and R)-mLeu] with a reverse amide bond between gPhe and mLeu residues where gPhe denotes a gem-diamino analog of Phe and mLeu refers to a malonyl analog of Leu. (2) Two cyclic hexapeptides related to somatostatin, with a reverse amide bond bec[gSar'-(S and R)-mPhe7-~-Trps-Lysg-Thr'o-Phe1'], tween the gSar and mPhe residues where the gSar and mPhe denote the gemdiamino and malonyl analogs of the Sar and Phe residues, respectively. The superscript numbers refer to positions in native somatostatin. (3) Cyclic hexapeptide s o m a t o s t a t i n an alo g s co n tain in g 2-Ac5c [trans-(1S,2S)-2-Ac5c, trans(1R,2R)-2-Ac5c,cis-(1R,2S)-2-Ac5c,and cis-(1S,2R)-2-Ac5c]in place of proline c[(2A~~~)~-Phe~-~-Trp~-Lys~-Thr~~-Phe"l. (4) Morphiceptin related analogs incorpoThe methodrating a cis-2-Ac5cresidue as shown in Tyr-cis-2-Ac5c-Phe-Val-NH,. ology described in this investigation could be applied to a wide variety of peptide systems.
KEY WORDS: absolute configuration, peptidomimetics, retro-inverso modifications, 2-aminocyclopentanecarboxylicacid, nuclear magnetic resonance (NMR), nuclear Overhauser effect, bioactive peptides INTRODUCTION
The biological activity of peptides requires proper spatial arrangements of functional groups to interact with receptors. Although most functional groups exist at the chain termini and at the peptide side chains, the main chain conformation is quite important in recognition and to orient functional groups in the correct arrays. In a n attempt to evaluate conformational requirements for biological activity, we have applied a stereoisomeric approach and incorporation of peptidomimetics to various bioactive peptide systems. For the biological evaluation of the analogs and the development of structure-bioactivity relationships, the assignment of the absolute configuration for peptidomimetic residues in bioactive peptide analogs is of particular importance because diastereomers display different biological activity profiles from one another. Absolute configuration of peptidomimetic residues could be assigned by X-ray diffraction studies, if suitable crystals are obtained. However, most times it is difficult to prepare crystals of biological active peptides suitable for X-ray diffraction. In this paper, we present a modern method for the assignment of the absolute configuration for peptidomimetic residues in biologically active peptides by use of 'H-NMR parameters, such as nuclear Overhauser effects (NOES) and vicinal 0 1991 Wiley-Liss, Inc.
proton-proton coupling constants for H-N-C-H groupings, observed in solution. Four typical peptide systems, for which we have proved the structurebioactivity relationships and the absolute configuration for peptidomimetic residues, are discussed. 1. Two 14-membered cyclic dermorphin analogs Tyr-cb-A,bu-Phe-gPhe-(S and R)-mLeu1. 2. Two cyclic hexapeptides related to somatostatin c[gSar6-(S and R)-mPhe7-~-Trp8-Lysg-Thr'o-Phe11] (the superscript numbers refer to the location of the residues in native somatostatin). 3. Cyclic hexapeptide somatostatin analogs cI(2-
A~~c)~-Phe~-~-Trp~-Lys~-Thr~~-Phe~'l. 4. Linear analogs of morphiceptin containing valine at the fourth position and a cis-2-Ac5c residue as shown in Tyr-cis-2-Ac5c-Phe-Val-NH,. The first two peptide systems incorporate partial retroinverso modifications. The remaining two systems contain 2-aminocyclopentanecarboxylic acid (2-Ac5c)as a peptidomimetic for proline. Received for publication February 10,1991; accepted March 22,1991. Address reprint requests to Murray Goodman at the address given above. Dedicated to the memory of Piero Pino, a very good friend and a great scientist.
269
ABSOLUTE CONFIGURATIONS FOR PEPTIDOMIMETICS
Schematic representations of th e partial retroinverso modifications are shown in Figure 1.The retroinverso modification reverses only the direction of the peptide bond and involves essentially isosteric structures as compared to the parent molecules. Therefore, the modification provides information concerning the importance of the carbonyl and amide groups for specific intramolecular hydrogen bonds, and backbone and side chain conformations while maintaining amide geometry. In addition, this modification typically increases resistance to enzymatic cleavage and degradation of the parent peptides. Synthesis of retro-inverso modified peptide analogs required the development of new methodology. Key steps include the preparation of the dipeptidomimetic structures unit gem-diaminoalkyl-2-alkylmalonyl which is incorporated into larger fragments. The gemdiaminoalkyl residues can be obtained from peptide amides or acylazides through molecular rearrangements that follow nitrene mechanisms. These reactions preserve optical purity. The 2-alkylmalonyl residues are incorporated into the peptide chains as a racemic residue. The resulting diastereomeric products could be often separated at the conclusion of the synthetic scheme by reverse-phase HPLC. We have successfully assigned the absolute configuration of 2-alkylmalonyl residues by use of NMR observations as will be shown below. The in vitro biological studies of the 14-membered cyclic dermorphin analogs with a reverse amide bond PRINCIPLESOF THE PARTIAL RETRWNVERSO MODIFICATION
between residues 4 and 5, Tyr-c[D-A,bu-Phe-gPhe-(S and R)-mLeu1, clearly show the effects of the chirality of the mLeu residues on the biological activity and receptor selectivity of these opioids.' The S-mLeu containing analog is superactive a t both the p- and 6-opioid receptors. The R-mLeu containing analog, on the other hand, is superactive at the preceptor but displays reduced potency at the &receptor as compared to the native enkephalin Tyr-Gly-Gly-Phe-Leu-OH. The biological activities of the cyclic hexapeptides related to somatostatin, c[gSar6-(S a n d R)mPhe7-~-Trp8-Lysg-Thr'o-Phe''l, are also dependent on the chirality of the mPhe residue. The S-mPhe containing analog displays activity on inhibition of the release of growth hormone in vitro while the R-mPhe analog is inactive in the same assay.' The in vivo studies on inhibition of pentagastrin-stimulated acid release in dogs show that the S-mPhe analog is superactive, but the R-mPhe analog is i n a ~ t i v e . ~ The incorporation of 2-aminocyclopentanecarboxylic acid (2-Ac5c)also offers a n attractive main chain modification. The structure of 2-Ac5c is unique as a n amino acid isostere in which it contains two chiral centers associated with the peptide backbone (Fig. 2). It has been also reported that the 2-Ac5cresidue tends to form p-turn structures in a n analogous fashion to proline when incorporated into a peptide chain.4 The 2-Ac5c is a p amino acid, thus four configurational isomers a r e probable, i.e., two cis isomers [(1R,2S)-2-Ac5cand (1S,2R)-2-Ac5cland two trans isomers [(1S,2S)-2-Ac5cand (1R,2R)-2-Ac5cl.The synthesis of 2-Ac5c was undertaken employing two methodologies which specifically generated either cis or trans configurations and these were utilized in separate syntheses in order to obtain the all-cis or all-trans isomers. The cis isomers were obtained a s a racemic mixture according to the method of Plieninger and Schneider by Hofmann degradation of cis-2-carbamoylcyclopentanecarboxylic a ~ i d . The ~ . ~trans isomers were prepared as a racemic mixture by the Michael addition of ammonia ~-~ the to 1-cyclopentenecarboxylic a ~ i d . Therefore, syntheses of peptide analogs containing 2-Ac5c were performed as a diastereomeric mixtures with either cis or trans isomers, and then separated at the final stage by reverse-phase HPLC. We have successfully assigned the absolute configuration of the four possible isomers of 2-Ac5c using 'H-NMR parameters.
co
NH
Fig. 1. Schematic representation of partially retro-inverso modifications of a parent peptide (A). The painvise retro-inverso modification (B) involves two adjacent amino acid residues containing side chains R, and R,, by which the gem-diaminoalkyl and 2-alkylmalonyl peptidomimetic structures are incorporated. In the extended retroinverso modification (C), the altered residues containing side chains R, and R, are moved apart so that an amino acid with reversed direction and chirality separates the gem-diaminoalkyl and 2alkylmalony residues. In (D) a reversed dipeptide is inserted between the peptidomimetic residues.
\ 6 / C4H2 Fig. 2. Schematic illustration of 2-aminocyclopentanecarboxylic acid (2-Ac5c).
270
YAMAZAKI AND GOODMAN
The existence of proline residue in a peptide chain allows for the possibility of cisltrans isomerization about the amide linkage. Therefore, it is difficult to obtain a consistent explanation for biological activity of proline containing peptides. Since the 2-Ac5cresidue contains a normal amide, the possibility of cisltrans isomerization about the amide bond is eliminated. Replacement of proline residue with 2-Ac5c residue provides information regarding the effect of cisltrans isomerization for biological activity, and moreover the conformational features around the peptide fragment neighboring the proline (or 2-Ac5c) residue. Utilizing this modification to a cyclic hexapeptide somatostatin analog c[Pr~~-Phe~-~-Trp~-Lys'-Thr'~-Phe''], we have proved that the p-VI turn structure around the Thr'OPhe"-Pro6-Phe7 bridging region with a cis amide bond between the Phe"-Pro6 residues is required for the biological activity of the parent ana10g.~. The morphiceptin analogs incorporating 2-Ac5c at t h e second position i n place of proline, Tyrshow 2-Ac5c-Phe-X-NH, [X = Pro and (L and ~)-Vall, interesting configurational dependence of biological activity and conformational preference.',' Among the four possible configurational isomers, only the analogs containing (1S,2R)-2-Ac5cshow opioid activity. The biological profiles of the active ( 1S,2R)-2-Ac5canalogs are approximately as same as that of the parent morphiceptin. The conformational studies using the 'HNMR spectroscopy and computer simulations indicate that the preferred conformations for the (1S,2R)-2-Ac5c analogs have considerable topological similarity with the morphiceptin in which the conformer adopts the cis conformation about the Tyr-Pro amide bond.' We have, therefore, concluded that the cis form about the amide bond joining these two residues is required for the biological activity of the morphiceptin-related analogs containing the proline a t the second position. THEORETICAL ASPECTS FOR 'H-NMR PARAMETERS The nuclear Overhauser effect (NOE) is a measure of the dipole-dipole relaxation between different nuclear spins which are close in space. The magnitude of the NOE can be related to the spin-spin distance r by an equation of the general form: NOE a r - 6 AT,)
(1)
where the second term ~ T T , ) is a function of the corre, account for the influence of the lation time T ~ which motional averaging processes on the observed NOE. Because of the r-6 dependence, the NOE intensity is very large for spins in close spatial proximity (-2 A), but rapidly becomes smaller as the distance is increased. Depending on the signal-to-noise ratio in the NOE experiment, an upper limit for NOE observable 'H-lH distance may be of the order 3.5-4.5 A. Several methods have been proposed for a quantative evaluation of NOE data in terms of 'H-lH distances. One of the simplest approach estimating unknown 'H-
'H distance rv from NOE data can be applied by use of the expression: r,, = [rk;
(zkl/Ivl-6
(2)
where rkl is a known distance, and Ikl and I, are NOE intensities. A useful standard for rki is the distance between two geminal methylene protons, which is calculated to be 1.76 A using the bond length of 1.10 A for C-H and the bond angle of 106.1' for H-C-H.lO,'l This equation includes two assumptions: (1)the correlation times for the different parts of the molecule are the same, and (2) the NOE intensities are proportional to the cross-relaxation rates. The latter assumption will hold true only at short mixing times where spin diffusion and other relaxation effects are negligible. Therefore, in these studies, short mixing times (20200 msec) should be employed. In the present investigation, NOEs were measured using the two-dimensional rotating frame experiments (ROESY)" with a spin locking field of 2.5 kHz, on a General Electric GN-500 spectrometer. The two dimensional spectra were obtained using 2 K data points in the f, domain and 256 points in the fl domain. Applying the zero filling procedure to the fl domain resulted in a final matrix of 2 K x 2 K data points. Gaussian multiplication was used to enhance the spectra. The NOEs, observed in the ROESY spectra with mixing times of 50-200 msec, were classified as strong, medium, or weak according to their intensities. The corresponding upper distance constraints could be calibrated to be 2.5,3.0, or 3.5 A by comparing NOEs with the one for the methylene protons (1.76 A). The backbone conformation of peptides is defined by $, and w, which reprethree kinds of torsion angle sent the rotational states around the skeletal single bonds NH-C"H, C"H-CO, and CO-NH, respectively. Because of the partially double bond character, only the planar structure in either trans or cis conformation is probable for the amide bond. The cis conformation allows closer contact between a-protons of adjacent residues (-2 A), while the distance between these two protons is larger than 4 A in the trans conformation. Therefore, the cis conformation can be easily distinguished from the trans conformation by the strong sequential NOE between the a-protons of the adjacent residues. The sequential NOE between the NH proton and the a-proton of the preceding residue provides information regarding the rotational state ($) around the C"H-CO bond. The maximum (3.58 A) and minimum (2.15 A) values of the sequential distance daN between these two protons of L-amino acids are calculated for $ = - 60' and + 120', respectively. A strong NOE is expected only for a Jr range from 60" to 180", in which a value of daN is less than 2.5 A. The intraresidue 'H-'H distance dNuand thus NOE between vicinal protons for the H-N-C"-H moiety can be related to the torsion angle The distance dNa varies between approximately 2.38 and 3.06 A. For Lamino acids, the maximum and minimum values are
+,
+.
ABSOLUTE CONFIGURATIONS FOR PEFTIDOMIMETICS
+
calculated for = - 120" and + 60°, respectively. The 4 angle dependence of the intraresidue distance dNa is smaller than the angle dependence of the sequential distance duN. The vicinal 'H-'H coupling constant JNH-CH provides the same information concerning the torsion angle via the Karplus-type equation.
only when the mLeu residue has a n S-configuration. on the other For Tyr-C[D-A,bu-Phe-gPhe-R-mLeu1, hand, the strong NOEs between Phe Ha-gPhe NH and gPhe NH-gPhe H", and the weak NOE between gPhe H"-gPhe N*H suggest that the NH and N*H protons of the gPhe residue are projecting downward and upward, respectively, relative to the 14-membered ring plane. In this orientation, the strong NOE observed from the gPhe N*H to the mLeu H" requires a n R-configuration a t the mLeu C" center (Fig. 3b).
+
+
J,,
CH =
A
C O S ~Ic$ -
60"l B cos I+ - 60"l + C (for L-amino acids). -
(3)
c[gSa$-(S and R)-mPhe7-~-Trp8-Lys9-Thr'o-Phd11
In the literature, several sets of coefficients (A, B , C ) have been reported for Eq. (3). Among the (A, B , C) sets, those given by Bystrov et al.13 (8.6, 1.0, 0.4) and Cung et al.I4 (8.6, 2.9, 0.0) are typical. The vicinal coupling constant JNH-CH displays a different angle dependence as compared with the intraresidue distance dNa.Therefore, measurements of JNH-CH present valuable data for studies of the backbone conformation of peptides, in addition to NOE data.
Unlike the 14-membered cyclic dermorphin analogs
Tyr-c[D-A,bu-Phe-gPhe-(Sand R)-mLeu1 discussed above, cyclic h e x a p e p tid e s c[gSar6-(S a n d R)-
+
mPhe7-~-Trp8-Lyss-Thr10-Phe11] contain a n achiral
RESULTS AND DISCUSSION
Tyr-c[D-A,bu-Phe-gPhe-(Sand R)-mLeul The NOEs used for the assignment of the configuration about mLeu residues in 14-membered cyclic derand R)morphin analogs Tyr-C[D-A,bu-Phe-gPhe-(S mLeu], i n which t h e g P h e resid u e h a s a n Sconfiguration, are shown in Table l.15 In the table, gPhe NH refers to the NH originally present in the Phe residue as opposed to gPhe N*H, which is formed as a result of the rearrangement in the synthesis of the gem-diaminoalkyl moiety. Because of the nature of the 14-membered cyclic molecule, both a-protons of the Phe and gPhe residues are projecting downward with respect to the plane of the 14-membered backbone ring. The weak NOEs between Phe Ha-gPhe NH and gPhe N H - g P h e H" observed for Tyr-c[D-A,buPhe-gPhe-S-mLeu] indicate that the NH proton of the gPhe residue is projecting upward relative to the 14membered ring plane while the strong NOE between gPhe Ha-gPhe N*H indicates that the N*H proton must be projecting downward (Fig. 3a). The strong NOE observed from gPhe N*H to mLeu H" is expected TABLE 1. Nuclear Overhauser effects (NOEs) and vicinal 'H-'H coupling constants (.I,,,-) used for assignment of absolute configuration for 2-isobutylmalonyl residues (mLeu) in 14-membered cyclic dermorphin analogs Tyr-c[D-Abu-Phe-gPhe-(Sand R)-mLeu1" S-mLeu
R-mLeu ~
NOE(Phe a-gPhe NH) NOE(gPhe NH-gPhe a) NOE(gPhe a-gPhe N*H) NOE(gPhe N*H-mLeu a) JNH-CH JNH-CH
(gPhe NH-a)(Hz) (gPhe N*H-a)(Hz)
_
_
_
_
W
S
W
S
S
W
S
S
8.63 7.21
_
6.47 7.52
"The observed NOES are qualitatively classified according t o their intensities; s, strong; m, medium; w, weak.
271
_
gem-diaminoalkyl residue gSar. For the assignment of the configuration about the 2-alkylmalonyl residue mPhe, therefore, the structure of the tetrapeptide sequence Phe"-gSar6-mPhe7-~-Trp8must be examined. The 'H-NMR parameters required for the configurational assignment are summarized in Table 2.16 With the exception of the two sequential NOEs between gSar N*H-mPhe H" and mPhe Ha-~-TrpNH which are key NOEs for the configurational assignment, the remaining NMR parameters observed for both the diastereomers are quite similar. The structures around the Phel1-gSar6-mPhe7-~-Trp8 part consistent with the observed data are illustrated in Figure 4. The gSar N*H and D - T NH ~ protons are projecting in opposite directions from one another with respect to the cyclic hexapeptide ring in both the diastereomers. In this structure, the strong NOE observed from mPhe H" to D-Trp NH is expected only when the mPhe residue has a n S-configuration as shown in Figure 4a. On the other hand, the medium NOE observed from gSar N*H to mPhe Ha requires a n R-configuration at the mPhe C" center (Fig. 4b). The constrained nature of the cyclic hexapeptides allows u s to assign t h e pro-chiralities of t h e two a-protons of the gSar residue. Both the diastereomers adopt a cis conformation about the Phe-gSar amide bond, which is deduced from the strong sequential NOEs observed between Phe H" and gSar Hal. The absence of NOEs between Phe H" and gSar NMe protons expected for a trans conformation also supports the assignment of cis conformation. Medium NOEs were observed from gSar NMe protons to only gSar Ha', i.e., no NOEs were measured between gSar NMegSar Hal. The NOEs between Phe Ha-gSar Hal and gSar NMe-gSar H"', together with the cis arrangement about the Phe-gSar amide bond, lead to the assignment of H"l as pro-S H" and Ha2 as pro-R H" (Fig. 4c). The opposite assignment makes the molecules very high in energy becuse of steric interactions between the gSar C"H, and Thr CO groups.
6 ( 2-Ac?~)~-Phe~-~-Trp~-Lys~-Th~~-Phd~I
The NOEs and vicinal JNH-CH coupling constants, used for the configurational assignment of the 2-Ac5c
272
YAMAZAKI AND GOODMAN
(a) S-mLeu
(b)
R-mLeu
Fig. 3. Schematic illustrations for the structures around the Phe-gPhe-mLeuresidues of 14-membered cyclic dermorphin analogs (R3 = R4 = benzyl and R, = isobutyl): (a) Tyr-C[D-A,bu-Phe-gPhe-S-mhu] and (b) Tyr-c[c-A,bu-Phe-gPhe-R-mhul. The NOES observed for atomic groupings are shown in the figures; s, strong; m, medium; and w, weak.
TABLE 2. Nuclear Overhauser effects (NOES)and vicinal ring takes the highest possible value in order to bring 'H-'H coupling constants (JNHxH)used for assignment of the NH and CO groups as equatorial as possible. absolute configuration for 2-benzylmalonyl residues The structures around the Phe"-(2-Ac5c)'-Phe7 parts (mPhe) in cyclic hexapaptides related to somatostatin i n t h e f o u r s t e r e o i s o m e r s of c [ ( ~ - A c ~ c ) ~ clgSar6-(Sand R)-mPhe7-~-Trps-Lysg-Thr'o-Phe1'la Phe7-~-Trp8-Lysg-Thr10-Phe111, consistent with the obS-mPhe R-mPhe served NMR data, are illustrated in Figure 5. An unambiguous assignment of the absolute configuration at the p-carbon of the 2-Ac5c residue, to which the NH NOE(gSar N*H-mPhe a) group is bonded, could be achieved by use of the seNOE(pTrp NH-mPhe a) quential NOES between Phe" NH and 2-Ac5c NH and NOE(pTrp N H - D - T ~a)~ NOE(Phe a-gSar a,) between Phe" H" and 2-Ac5c NH. For all of the steNOE(Phe a-gSar NMe) reoisomers, no NOES or a weak NOE were observed for NOE(Phe a-gSar a,) the H-N-CP-H groupings of the 2-Ac5c residues, inNOE(gSar NMe-gSar a,) dicating a nearly trans orientation for the NH and HP NOE(gSar NMe-gSar a2) protons. In these arrangements, the torsion angle of NOE(gSar N*H-gSar a,) t h e 2-Ac5c residue is restricted -120" for a n RNOE(gSar N*H-gSar a2) is approxiconfiguration at the CP carbon while JNH-CH (gSar N*H-al)(Hz) 3.75 7.44 mately - 120" for a n S-configuration. According to the JNH-CH (gSar N*H-a2)(Hz) 8.34 4.50 nature of the cyclic molecules with this ring size (19JNH-CH (D-Tv NH*)(Hz) 6.85 6.60 membered ring), the Phe" side chain and cyclopentane ring of the 2-Ac5c residue exist outside of th e 19T h e observed NOES are qualitatively classified according t o their membered ring. In these structures, the Phe" H" and intensities; s, strong; m, medium; w, weak; -, absent. 2-Ac5c protons are projecting downward and upward, respectively, relative to the 19-membered ring when r e s i d u e s i n cyclic h e x a p e p t i d e s c[(2-Ac5cI6- the CP carbon has a n R-configuration (Fig. 5b and d). Phe7-~-Trp8-Lysg-Thr10-Phe"l, are summarized in Ta- Because the angle of the 2-Ac5c residues with a n ble 3.7 The trans-2-Ac5c and cis-2-Ac5cresidues can be R-configuration a t the CP center is restricted to values clearly distinguished from one another by the NOES around 120" as mentioned above, the 2-Ac5c NH proton between the 2-Ac5c a- and p-protons and between the is projecting downward and close to the Phe" H" pro2-Ac5c NH and a-protons. For the cis-2-Ac5c residues, ton. As a result, a strong NOE exists between the Phel' i.e. (1R,2S)-2-Ac5cand (1S,2R)-2-Ac5cresidues, strong H" and the NH protons of the 2-Ac5c residues with a n NOES were observed between the H" and HP protons R-configuration a t the CP carbon, i.e., one of the transsince these two protons exist on the same side with 2-Ac5c [(1R,2R)-2-Ac5c] a n d one of t h e cis-2-Ac5c respect to the cyclopentane ring. On the other hand, no [(1S,2R)-2-Ac5clresidues. In the remaining two stereoisomers containing NOES were measured between the H" and HP protons of the truns-2-Ac5c residues, i.e. (1S,2S)-2-Ac5c and 2-Ac5c with a n S-configuration at the CP carbon, i.e., (1R,2R)-2-Ac5c residues. Instead of the NOE(2-Ac5c another trans-2-Ac5c [(1S,2S)-2-Ac5cland another cisa-2-Ac5c p), strong NOES were observed between the 2-Ac5c [(1R,2S)-2-Ac5clresidues, both of the Phe" H" 2-Ac5c NH and H" protons for the trans-2-Ac5c resi- and 2-Ac5c HP protons are projecting downward reladues. For both the trans forms, the lack of NOE(2-Ac5c tive to the 19-membered ring (Fig. 5a and c). The a-2-Ac5c p) and the strong NOE(2-Ac5c NH-2-Ac5c a ) angles (- - 120") for the 2-Ac5c residues with a n Sindicate that the C"H and CPH protons are oriented in configuration at the CP carbon orient the NH proton of a nearly trans arrangement and that the internal ro- this residues upward with respect to the 19-membered tation angle about the c"-CP bond in the cyclopentane ring. In these orientations, the Phe" H" and 2-Ac5cNH
+
+
+
+
273
ABSOLUTE CONFIGURATIONS FOR PEPTIDOMIMETICS
(a) S-mPhe I
(c) Ia 1= Dro-S, a2spro-R
I
I
3
(bl R-mPhe I
I
Fig. 4. Schematic illustrations for the structures around the gSar6-mPhe7-~-Trpa residues of cyclic indolylmethyl): (a) c[gSar6-S-mPhe7-~hexapeptides related to somatostatin (R, = benzyl and R The structure, with a cis conTrps-Lys9-Thr'o-Phe''l and (b) ~[gSar~-R-mPhe~-~-Trp~-Lys~-~r~~-Phe"]. formation about the Phe-gSar amide bond, observed for both the diastereomers is shown in (c). The NOES observed for the atomic groupings are included in the figures; s, strong; m, medium; and w, weak.
protons are distant from each other, and the NOE between these two protons should be weak. In addition, the Phe" NH and 2-Ac5c NH protons are close enough to expect a n NOE between them. These predictions are in agreement with the experimental results shown in Table 3, where a weak NOE(Phe" Ha-2-Ac5c NH) and a medium NOE(Phel' NH-2-Ac5c NH) are observed only for the cyclic hexapeptides containing the 2-Ac5c residues with a n S-configuration at the Cp carbon, i.e., truns-(1S,2S)-2-Ac5cand cis-(1R,2S)-2-Ac5cresidues. A combination of the results for the trans and cis configurational assignment and the results of the chirality assignment a t the C p carbon obtained above allows us to draw a definite conclusion about the absolute configuration for the four possible isomers of 2-Ac5c residues.
1. In the molecule where the 2-Ac5c residue displays me d iu m NOE(Phe" NH-2-Ac5c N H ), w eak NOE(Phe" a-2-Ac5c NH), and strong NOE(2-Ac5c NH-2-Ac5c a),the trans-(lS,BS)-configuration has been assigned (Fig. 5a). 2. In the molecule where the 2-Ac5c residue exhibits strong NOE(Phe'' a-2-Ac5c NH) a n d s t rong NO E (2 -A c 5 c N H -2 -A c 5 c a), t h e ( 1 R , 2 R ) configuration has been assigned (Fig. 5b). 3. In the molecule where the 2-Ac5c residue shows med i u m NOE(Phe'' NH-2-Ac5c NH), weak NOE(Phe'' a-2-Ac5c NH), and strong NOE(2-Ac5c a-2-Ac5c p), the cis-(lR,2S)-configuration has been assigned (Fig. 5c). 4. In the molecule where the 2-Ac5c residue gives a 'H-NMR s p e c t r u m w i t h s t r o n g NOE(Phe"
TABLE 3. Nuclear Overhauser effects (NOES) and vicinal 'H-'H coupling constants ( J N H x H ) used for assignment of absolute configuration for 2-aminocyclopentanecarboxylicacids (2-Ac5c)in cyclic hexapeptides related to somatostatin 1' ' c[ (2-A~~c)~-Phe'-~-Trp~-Lys~-Thr'~-Phe' trans -2-Ac5c
(1S,2S)-2-Ac5c
(Phe" NH-a)(Hz) (2-Ac5c NH+)(Hz) (Phe7 NH-cl)(Hz)
(1R,2R)-2-Ac5c
(1R,2S)-2-Ac5c
(1S,2R)-2-Ac5c
7.83
8.09
m
NOE(Phe" NH-Phe" a) NOE(2-Ac5c NH-Phe" NH) NOE(2-Ac5c NH-Phe" a) NOE(2-Ac5c NH-2-Ac5c p) NOE(2-Ac5c NH-2-Ac5c a) NOE(2-Ac5c a-2-Ac5c p) NOE(Phe7 NH-2-Ac5c p) NOE(Phe7 NH-2-Ac5c a) NOE(Phe7 NH-Phe7 a) JNH-CH JNH-CH JNH-CH
cis-2-Ac5c
S
W
S
s s m
6.68 5.83 8.00
3.98 8.61 8.51
b
5.73
T h e observed NOES are qualitatively classified according to their intensities; s, strong; m, medium; w, weak; -, absent. bA value could not be determined because of severe overlap of the NH proton signal with the aromatic proton peaks.
b
5.51
274
YAMAZAKI AND GOODMAN
of the i + l t h residue to a-proton of the ith residue were observed (i = 1,2, or 3). These strong sequential NOES indicate that a torsion angle J? about the C"-CO bond of the ith residue is restricted in a range from 60" to 180" when t h e ith residue h a s a n L- or Sconfiguration while in a range from - 60" to - 180" for a D- or R-configuration. Relatively large values of JNH-CH and weak intraresidue NOES between the NH and CH protons of the H-N-C-H moieties observed for the cis-2-Ac5c, Phe, and Val residues of both the diastereomers indicate a nearly trans orientation of ( d ) (IS, 2R)- 2-Ac5c (b) (IR,2R)-2-Ac5c these two protons (+ --120" for a n L- or Sconfiguration a n d - 120" for a D- a n d Rconfiguration). Model building with these and J? values clearly indicates that the NOES between Tyr H"-Val HY and Tyr +z,6-Val HY are expected only when t h e cis-2-Ac5c residue h a s t h e (1S,2R)configuration. To confirm the assignment of the configuration for the cis-2-Ac5c residue, all of the NOES observed for Fig. 5. Structures around the Phe"-(2-Ac5c)'-Phe7 residues of cy- Tyr-cis-(1S,2R)-2-Ac5c-Phe-Val-NH, were used as conclic hexapeptides related to somatostatin ~ [ ( 2 - A c ~ c ) ~ - P h e ~ - ~ - T p * straints for the molecular dynamics simulations (10 Lysg-Thr'o-Phe"l: (a) trans-(1S,2S)-2-Ac5c,(b) trans-(lR,2R)-2-Ac5c, (c) cis-(1R,2S)-2-Ac5c,and (d) cis-(1S,2R)-2-Ac5c.The NOES observed psec) on both the (1R,2S)-2-Ac5cand (1S,2R)-2-Ac5c for the atomic groupings are given in the figures; s, strong; m, me- containing analogs. The calculations were carried out dium; and w, weak. in vacuo employing the DISCOVER force field program,17modified to allow for NOE constraints. The a-2-Ac5c NH) and strong NOE(2-Ac5c a--2-Ac5cp), NOE constraints were applied using the following the cis-(lS,2R)-configurationhas been assigned function: (Fig. 5d). ENOE = Kp(rk - rd2 (rij > ro) Tyr-cis-2-Ac5c-Phe-Val-NH, (0)
(IS,2S)-2-Ac5c
( c ) (I R , 2 S)- Z-AC'C
+
ENOE= 0
+
(TI,
c ro)
(4)
As described above, the absolute configuration of peptidomimetic residues in cyclic peptides could be de- where ri, represents a distance between two specific termined by examining the local structures around the protons. Force constants K , = 15,10,5 kcal mol-' A-2 peptidomimetic residues only with intraresidue and se- and target separations ro = 2.5, 3.0, 3.5 A were asquential NOEs. In contrast to cyclic molecules, a con- sumed for strong, medium, and weak NOEs, respecsideration of overall conformations of the molecules is tively. The potential and forcing energies resulted from the necessary for the assignment of configuration in the case of linear molecules. The long-range NOES provide application of the NOE constraints were consistently useful information concerning the overall conforma- higher for the analog containing the (1R,2S)-2-Ac5c tions, in addition to the intraresidue and sequential residue than for the analog containing the (1S,2R)NOEs. Here we describe an unambiguous assignment 2-Ac5c residue. In addition, structures taken from the of the chiralities of the cis-2-Ac5c residue in the dia- dynamics at 1psec intervals were minimized with and s t e r e o m e r i c p a i r of T y r - c i s - ( 1R,2S)-2-Ac5c- without the NOE constraints. All of the structures for P h e - V a l - N H , a n d T y r - c i s - (1S , 2 R ) - 2 - A c 5 c - the (1R,2S)-2-Ac5ccontaining analog with the NOES Phe-Val-NH,. This peptide system represents a specific applied were very high in energy. The structures for case in which we determine the absolute configuration this analog obtained from the energy minimizations without the NOE constraints showed large conformaby use of the long-range NOEs. tional changes from the initial structures, and thereThe NOES for Tyr-cis-(1R,2S)-2-Ac5c-Phe-Val-NH, and Tyr-cis-(1S,2R)-2-Ac5c-Phe-Val-NH, measured in fore are not consistent with the long-range NOEs, i.e., HYand H". On the DMSO-d, are shown in Figure 6.' The observed values Tyr Ha-Val HYand Tyr +,,,Val of vicinal 'H-lH coupling constants are summarized in other hand, the minimum energy conformations for the Table 4." The long-range NOES between Tyr Ha-Val (1S,2R)-2-Ac5ccontaining analog are in agreement HY,Tyr +,,,Val HY,and Tyr +z,,Val H" observed for w i t h a l l t h e NMR d a t a o b s e r v e d for T y r Tyr-cis-(1S,2R)-2-Ac5c-Phe-Val-NH, are especially in- cis-(1S,2R)-2-Ac5c-Phe-Val-NH,. T h e lowest energy conformations for Tyrformative. Except for these long-range NOEs, the remaining NMR parameters observed €or both the dia- cis-(1R,2S)-2-Ac5c-Phe-Val-NH, and Tyrstereomers are quite similar. For both the diaste- cis-(1S,2R)-2-Ac5c-Phe-Val-NHz are shown in Figure reomers, strong sequential NOEs from the NH proton 7a and b, respectively.' For each conformation the
275
ABSOLUTE CONFIGURATIONS FOR PEFTIDOMIMETICS Tyr-(1R.2S)-A~c-Phe-Val-NH2
2-AcSc N
~
P
Phe Y
E
aN P
Val
O
/ N
a
S
P
y
mlh
Y
a
Val
N
Wl
O P
S
a
Phe
N E
Y
j
P
2-Ac'c
a N W
m
$2.6
P
s
W
a
Tyr
N
Tyr-(1S.2R)-Ac5c-Phe-Val-NH2
Fig. 6. Observed NOES for morphiceptin analogs 'Tyr-cis-2-Ac5c-Phe-Val-NH, measured in dimethylsulfoxide-$ using the ROESY experiments. The two protons of the CH, groups and the two y-methyl groups of the Val residues are distinguished by appending a superscripth for the one at higher field and 1 for that at lower field. The NOES are qualitatively classified according to their intensities;s, strong; m, medium; and w, weak.
TABLE 4. Experimental values of vicinal 'H-'H coupling are expected for Tyr-cis-(1S,2R)-2-Ac'c-Phe-Val-NH, constants (.INHxH) observed for H-N-C-H groupings in between these atomic groupings. The conformational morphiceptin related analogs Tyr-cis-2-Ac5c-Phe-Val-NH, analysis supports the assignment of the diastereomer for which the longof Tyr-cis-2-Ac'c-Phe-Val-NH2, range NOES between Tyr Ha-Val HYand Tyr +2,6-Val HY a n d Ha a r e observed, to t h e s tr u c tu r e Tyrcis-(1S,2R)-2-Ac'c-Phe-Val-NH,.The other diastereomer, for which no long-range NOES are observed between the Tyr and Val residues, has been assigned as Tyr-cis -(1R,2S)-2-Ac'c-Phe-Val-NH,. shortest distances between Tyr Ha-Val HY and Tyr CONCLUSION +2,6Val HYwere calculated. These distances are quite We have launched a program to study structurelarge for t h e st r u ctu re i n Fig u re 7a, i.e., Tyrcis-(1R,2S)-2-Ac5c-Phe-Val-NH, (-8.9 A). It should be bioactivity relationships of various peptide systems noted that this minimum energy structure is consistent utilizing a stereoisomeric approach and peptidomimetwith all the NMR data observed for the analog contain- ics. This program requires the unambiguous assigning the (1R,2S)-2-Ac5cresidue (Fig. 6 and Table 4). In ment of the absolute configuration for peptidomimetic the structure shown in Figure 7b, on the other hand, residues in the analogs since the biological recognition t h e distances between Ty r Ha-Val HY and Tyr is certainly stereospecific. Studies by X-ray diffraction +2,6Val HYare approximately 2.6 A, and thus NOES are one of the most powerful methods for the configu-
276
YAMAZAKI AND GOODMAN
.
I
\ Fig. 7. The lowest energy conformations for (a) Tyr-cis-(1R,2S)-2-Ac5c-Phe-Val-NH, and (b) Tyr-cis(1S,2R)-2-Ac5c-Phe-Val-NH,. The shortest distances between Tyr-Ha-Val H' and Tyr &-Val H ' are given for each conformation.
rational assignment. However, the utilization of X-ray diffraction in the analysis of peptides is often limited by the fact that many peptidic systems are not crystalline. In this investigation, we presented a modern method for the assignment of the absolute configuration for peptidomimetic residues in biologically active peptides based on the use of NOEs and vicinal 'H-IH coupling constants for H-N-C-H groupings (JNH-CHS). Four peptide systems incorporating either retro-inverso modifications or 2-aminocyclopentanecarboxylicacid (2-Ac5c) as a peptidomimetic for proline were discussed. The absolute configuration for peptidomimetic residues in the cyclic peptides was assigned by examining the local structures around the peptidomimetic residues with intraresidue and sequential NOEs. In contrast to the cyclic molecules, the overall conformations of the molecules must be examined in the case of linear peptides. For this purpose, long-range NOEs are useful in conjunction with intraresidue and sequential NOEs. Both of the retro-inverso modified peptide systems Tyr-c[D-A,bu-Phe-gPhe-(S and R)-mLeu1 and c[gSar-(S for which the chiraland R)-mPhe-D-Trp-Lys-Thr-Phe], ities of the malonyl residues were assigned, include pairwise retro-inverso modifications (Fig. lb). A similar consideration could be applied to extended retroinverso modified peptide systems in which reversed amino acids a r e i n s e r t e d between t h e g e m diaminoalkyl and 2-alkylmalonyl residues (Fig. l c and d). The present method using NOEs and JNH-CHS is powerful for the configurational assignment and applicable to many retro-inverso modified peptides and peptides containing a wide variety of peptidomimetic residues. ACKNOWLEDGMENTS The authors would like to acknowledge the support of this research through grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK 15410) and the National Institute of Drug Abuse
(DA 06254). We gratefully acknowledge Drs. Odile E. Said-Nejad, Christian Pattaroni, and Albert Probstl for the syntheses of Tyr-c[D-A,bu-Phe-gPhe-(Sand R)mLeu1, c[gSar-(S and R)-mPhe-D-Trp-Lys-Thr-Phel, a n d t h e 2-Ac5c c o n t a i n i n g m o l e c u l e s (1212Ac5c-Phe7-~-Trp-Lys-Thr-Phe1'1 and Tyrcis-2-Ac5c-Phe-Val-NH,), respectively. We would like to thank Dr. Dale F. Mierke and Ziwei Huang for helpful discussions and measurements. LITERATURE CITED 1 Mierke, D.F., Said-Nejad, O.E., Yamazaki, T., Felder, E.R., Schiller, P.W., Goodman, M. In: Peptides: Proceedings ofthe Eleventh American Peptide Symposium. Revier, J.E., Marshall, G.R., eds. 1990:348-350. 2. Pattaroni, C., Lucietto, P., Goodman, M., Yamamoto, G., Vale, W., Moroder, L., Gazerro, L., Gohring, W., Schmied, B., Wunsch, E. Int. J. Peptide Protein Res. 36:401-417, 1990. 3. Beglinger, L., Gyr, K. Personal communication. 4. Plieninger, H., Schneider, K. Chem. Ber. 92:1594-1599, 1959. 5. Bernath, G., Lang, K.L., Gondos, G., Marai, P., Kovacs, K. Acta Chim. Acad. Sci. Hung. 74:479-497, 1972. 6. Conners, T.A., Ross, W.C.J. J . Chem. SOC.2119-2132, 1960. 7. Yamazaki, T., Huang, Z., Probstl, A., Goodman, M. Peptide 1990: Proceedings of the 21th European Peptide Symposium, Giralt, E., Andreu, D., eds. 389-392,1991. 8. Mierke, D.F., NoRner, G., Schiller, P.W., Goodman, M. Int. J. Peptide Protein Res. 3535-45, 1990. 9. Yamazaki, T., Probstl, A., Schiller, P.W., Goodman, M. Int. J. Peptide Protein Res. 37:364-381, 1991. 10. Lide D.R., Jr. J. Chem. Phys. 33:1514-1518, 1960. 11. Iijima, T. Bull. Chem. Soc. Jpn. 45:1291-1293, 1972. 12. Bothner-By, A.A., Steppens, R.L., Lee, J., Warren, C.D., Jeanloz, R.W. J . Am. Chem. SOC. 106:811-813,1984. 13. Bystrov, V.F., Ivanov, V.T., Portnova, S.L., Balashova, T.A., Ovchinnikov, Yu. A. Tetrahedron 29873-877, 1973. 14. Cung, M.T., Marraud, M., Neel, J. Macromolecules 7:606-613, 1974. 15. Yamazaki, T., Mierke, D.F., Said-Nejad, O.E., Felder, E.R., Goodman, M. In preparation. 16. Mierke, D.F., Pattaroni, C., Delaet, N., Toy, A., Goodman, M., Tancredi, T., Motta, A., Temussi, P.A., Moroder, L., Bovermann, G., Wunsch, E. Int. J. Peptide Protein Res. 36:418-432,1990. 17. Hagler, A.T. In: The Peptides, Vol. 7.Udenfriends, S., Meienhofer, J., Hruby, V.J., Eds., Orlando, FL: Academic Press, 1985214-296.
