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The crystal structures of four dipeptides that contain the stereochemically constrained gamma-amino acid residue gabapentin (1-(aminomethyl) cyclohexaneacetic acid Gpn) are described. The molecular conformation of Piv-Pro-Gpn-OH (1), reveals a beta-turn mimetic conformation, stabilized by a ten atom C-H…O hydrogen bond between the Piv CO group and the pro S hydrogen of the Gpn CH2-CO group. The peptides Boc-Gly-Gpn-OH (2),Boc-Aib- Gpn-OH (3),and Boc-Aib-Gpn-OMe (4) form compact,folded structures,in which a distinct reversal of polypeptide chain direction is observed. In all cases, the Gpn residue adopts a gauche, gauche (g,g) conformation about the C alpha-C gamma (tita1) and C beta-C alpha (tita 2) bonds. Two distinct Gpn conformational families are observed. In peptides 1 and 3,the average backbone torsion angle values for the Gpn residue are 98, 1, 62, 2, 73 and 79,while in peptide 2 and 4 the average values are -103, -1, - 46, - 49 and - 92. In the case of 1 and 3,an intramolecular nine membered O-H…O hydrogen bond is formed between the C=O of the preceding residue and the terminal carboxylic acid OH group. All four alpha-gamma dipeptide sequences yield compact folded backbone conformations; this suggests that the Gpn residue may be employed successfully in the design of novel folded structures.

The possible occurrence of a novel helix terminating structural motif in proteins involving a stabilizing short C-H...O interaction has been examined using a dataset of 634 non-homologous protein structures (\leq 2.0 \AA). The search for this motif was prompted by the crystallographic characterization of a novel structural feature in crystals of a synthetic decapeptide in which extension of a Schellman motif led to the formation of a C-H...O hydrogen bond between the T-4 $C^ \alpha H$ and the T+1 C=O groups, where T is the helix terminator adopting a left handed $(\alpha_L)$ conformation. More than 100 such motifs with backbone conformation superposing well with the peptide examples were identified. In several examples, formation of this motif led to an approximately antiparallel arrangement of a helical segment with an extended \beta-strand. Careful examination of these examples suggested the possibility of registering antiparallel arrangement of helices and strands by means of backbone C-H...O interactions with a regular periodicity. Model building resulted in the generation of idealized \alpha \beta and \beta \alpha motifs, which can then be generalized to higher order repetitive structures. Inspection of the antiparallel \alpha \beta motif revealed a significant propensity for Ser, Glu, and Gln residues at the T-4 position resulting in further stabilization using an O...H-N side chain-backbone hydrogen bond. Modeling studies revealed ready accommodation of serine residues along the helix face that contacts the strand. The theoretically generated folds correspond to "open" polypeptide structures.

Recent progress in the design of beta-hairpin peptides[1] and beta-sheet models has been based on the ability to nucleate reverse turns of the appropriate stereochemistry. D-Pro-Gly[2] and to a lesser extent Asn-Gly[3] segments have been shown to facilitate formation of type I’ and II’ beta-turns, which are most often found at the site of sharp polypeptide chain reversal, that is, beta-hairpins in proteins.[4, 5] The prime turns, I’ and II’, can exert differing influences on the relative twist of the antiparallel strands. The I’ turn has the sense of twist that matches the twisting of adjacent beta-strands in proteins. In contrast, the II’ turn results in a more planar arrangement with the hairpin flattening to a considerable degree.[4a,e] The D-Pro-Xxx segment can in principle adopt both II’ and I’ turn conformations as si (D-Pro) values of +30 deg. and –120 deg. are energetically favorable.[1a, 5]

The insertion of alpha-hydroxy acids into peptide chains provides a convenient means for investigating the effects of hydrogen bond deletion on polypeptide secondary structures. The crystal structures of three oligopeptides containing L-lactic acid (Lac) residue have been determined. Peptide 1, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (Boc: tert-butyloxycarbonyl; Aib: alpha- aminoisobutyric acid; OMe: methyl ester), and peptide 2, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Leu-OMe, adopt completely helical conformations in the crystalline state with the Lac(6) residue comfortably accommodated in the center of a helix. The distance between the O atoms of Leu(3) CO group and the Lac(6) O (ester) in both the structures is 3.1-3.3 A. The NMR and CD studies of peptide 1 and its all-amide analogue 4, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe, provide firm evidence for a continuous helical conformation in solution in both the cases. In a 14-residue peptide 3, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Val-Ala-Leu-Aib-Val-Lac-Leu-OMe, residues Val(1)-Leu(10) adopt a helical conformation. Aib(11) is the site of chiral reversal resulting in helix termination by formation of a Schellman motif. Residues 12-14 adopt nonhelical conformations. The loss of the hydrogen bond near the C-terminus appears to facilitate the chiral reversal at Aib(11). Published 2001 John WileySons, Inc. Biopolymers 59: 276-289, 2001

The conformational analysis of two synthetic octapeptides, Boc–Leu–Val–Val–D-Pro–L-Ala–Leu–Val–Val–OMe (1) and Boc–Leu–Val–Val–D-Pro–D-Ala–Leu–Val–Val–OMe (2) has been carried out in order to investigate the effect of beta-turn stereochemistry on designed beta-hairpin structures. Five hundred megahertz 1HNMR studies establish that both peptides 1 and 2 adopt predominantly beta-hairpin conformations in methanol solution. Specific nuclear Overhauser effects provide evidence for a type II’ beta-turn conformation for the D-Pro–L-Ala segment in 1, while the NMR data suggest that the type I’ D-Pro–D-Ala b-turn conformation predominates in peptide 2. Evidence for a minor conformation in peptide 2, in slow exchange on the NMR time scale, is also presented. Interstrand registry is demonstrated in both peptides 1 and 2. The crystal structure of 1 reveals two independent molecules in the crystallographic asymmetric unit, both of which adopt beta-hairpin conformations nucleated by D-Pro–Lala type II’ beta-turns and are stabilized by three cross-strand hydrogen bonds. CD spectra for peptides and 2 show marked differences, presumably as a consequence of the superposition of spectral bands arising from both beta-turn and beta-strand conformations.

The conformational analysis of peptides containing a single alpha,alpha-di-n-propylglycine (Dpg) residue incorporated into valine-rich sequences has been undertaken in order to delineate the possible role of sequence effects in stabilizing fully extended (C5) or local helical conformations at this residue. The three peptides Boc–Val–Dpg–Val–OMe (3), Boc–Val–Val–Dpg–Val–OMe (4), Boc–Val–Val–Dpg–Val–Val–OMe (5), have been studied by 1H-nmr methods in chloroform (CDCl3) and dimethylsulfoxide (DMSO) solutions. Even in a relatively poorly solvating medium like CDCl3, all the valine NH groups appear to be solvent-exposed, suggesting an absence of folded beta-turn conformations. However, in both CDCl3 and DMSO the Dpg NH groups in all the three peptides appear to behave like apparently solvent-inaccessible groups. In fully extended C5 conformations, the proximity of the NH and CO groups of Dpg may preclude effective solvation due to a combination of stereoelectronic factors. Nuclear Overhauser effects provide support for the largely extended backbones. The crystal structure of peptide 3 reveals an extended conformation at Dpg (2) with f5 2176°, c 5 180°. A correlation between the crystallographically observed backbone conformation and solution nmr parameters in DMSO has been attempted using available data. Dpg residues placed in poor helix stabilizing environments may be expected to favor a local C5 conformation.

The Schellman motif is a widely observed helix terminating structural motif in proteins, which is generated when the C-terminus residue adopts a left-handed helical (aL) conformation. The resulting hydrogen-bonding pattern involves the formation of an intramolecular 6 - 1 interaction. This helix terminating motif is readily mimicked in synthetic helical peptides by placing an achiral residue at the penultimate position of the sequence. Thus far, the Schellman motif has been characterized crystallographically only in peptide helices of length 7 residues or greater. The structure of the hexapeptide Boc–Pro–Aib–Gly–Leu–Aib–Leu–OMe in crystals reveal a short helical stretch terminated by a Schellman motif, with the formation of 6 - 1 C-terminus hydrogen bond. The crystals are in the space group P212121 with a = 18.155(3) Å, b = 18.864(8) Å, c 5 11.834(4) Å, and Z = 4 . The final R1 and wR2 values are 7.68 and 14.6%, respectively , for 1524 observed reflections [Fo >- 3(Fo)]. A 6 - 1 hydrogen bond between Pro(1)CO . . . Leu(6)NH and a 5 - 2 hydrogen bond between Aib(2)CO . . . Aib(5)NH are observed. An analysis of the available oligopeptides having an achiral Aib residue at the penultimate position suggests that chain length and sequence effects may be the other determining factors in formation of Schellman motifs.

The crystal structure of the model tripeptide Boc-Aib-Gly-Leu-OMe (1) reveals two independent molecules in the asymmetric unit that adopt ‘‘enantiomeric’’ type I and type I’ beta-turn conformations with the Aib and Gly residues occupying the corner ( i / 1 and I / 2) positions. 13C cross polarization and magic angle sample spinning spectra in the solid state also support the coexistence of two conformational species. 13C-nmr in CDCl3 establishes the presence of a single species or rapid exchange between conformations. 400 MHz 1H-nmr provides evidence for conformational exchange involving a major and minor species, with beta-turn conformations supported by the low solvent exposure of Leu(3) NH and the observation of NiH - Ni/1H nuclear Overhauser effects. CD bands in the region 190–230 nm are positive, supporting a major population of type I’ beta-turns. The isomeric peptide, Boc-Gly-Leu-Aib-Ome (2) , adopts an ‘‘open’’ type II’ b-turn conformation in crystals. Solid state and solution nmr support population of a single conformational species. Chiral perturbation introduced outside the folded region of peptides may provide a means of modulating screw sense in achiral sequences.

A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (10%) have little effect on the overall folded conformation of lysozyme as seen from 1H NMR chemical shift dispersion. At increasing DMSO concentrations (10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by approximately 50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain-side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel beta-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of non-aqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins.

In the last 30 years, since the early work of Goodman and co-workers introducing trifluoroethanol as a solvent in peptide conformational analysis,1,2 the ability of fluoroalcohols to stabilize secondary structures in peptides in aqueous solution is well documented.3–7 However, the detailed molecular mechanisms remain to be understood. 3,4,8–12 2,2,2-Trifluoroethanol (TFE) has been the most widely used fluoroalcohol additive to aqueous solutions of peptides and proteins.4,13–18 We describe in this communication the superior structure-stabilization properties of hexafluoroacetone trihydrate (HFA, a covalent hydrate, of the ketone, a gem diol, hexafluoropropan-2,2- diol) 19–21 and provide a rationale for the induction of intramolecularly hydrogen-bonded conformations in peptides, in aqueous fluoroalcohol solutions.

The crystal structures of two helical peptides Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (VALU-7) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe (VALU-8) have been determined to a resolution of 1.0 and 0.9 A, respectively. Both the seven and eight residue peptides crystallize with two conformers per asymmetric unit. The VALUS conformers are completely helical and differ only at the C-terminus by a sign reversal of the \phi, \psi angles of the last residue. One of the VALU-7 conformers occurs as a normal \alpha-helix, whereas in the other, the N(7)=O(3) \alpha-type hydrogen bond is ruptured by the entry of a water molecule (W) into the helix, which in turn makes hydrogen bonds N(7)…W = 2.97 A and W…O(3) = 2.77 A. The other side of the water molecule is surrounded by a hydrophobic pocket. These two conformers give a static representation of a step in a possible helix unwinding or folding process. In the VALU-8 crystal the helices aggregate in a parallel mode, whereas the aggregation is anti-parallel in the VALU-7 crystal. The crystal parameters are VALU-7, $P2_1$, a = 10.203 (3) A, b = 19.744 (6) A, c = 22.561 (6) A, \beta = 96.76 deg, Z = 4, $C_{38}H_{69}N_7O_{10}$.0.5 $H_2O$, R = 6.65% for 3674 reflections observed >3\sigma(F); and VALU-8, $P2_1$ a = 10.593 (4) A, b = 27.57 (6) A, c = 17.745 (5) A, \beta = 95.76 (3) deg, Z = 4, $C_{42}H_{76}N_8O_{11}$.0.25 $CH_3OH$, R = 6.63% for 4701 reflections observed >3\sigma(F).

The aggregation behavior of the chemotactic peptide analogs, Formyl-Met-Leu-Phe-OMe (1) and Formyl-Met-AibPhe-OMe ${(2)}_1$, has been studied in chloroform and dimethylsulfoxide over the concentration range of 0.2-110 mM by 'H-nmr spectroscopy. Both peptides associate in $CD{Cl}_3$, at concentrations \geq 2 mM, while there is no evidence for aggregation in ${({CD}_3)}_2SO$. Analog 1 adopts an extended conformation in both solvents favoring association to form \beta-sheet structures. A folded, \gamma-turn conformation involving a 3 \rightarrow 1 hydrogen bond between Met CO and Phe NH is supported by 1H-, 13Gnmr, and ir studies of analog 2. The influence of backbone conformation on the ease of peptide aggregation is demonstrated by ir studies in $CH{Cl}_3$ and CD studies in dioxane.

Pivaloyl-L-Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in ${({CD}_3)}_2SO$ solution, by 'H-nmr methods, suggesting the existence of \beta-turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II \beta-turn conformations are about 2 kcal $mol^{-1}$ more stable than Type III structures. A crystallographic study has established the Type II \beta-turn in the solid state. The molecule crystallizes in the space group ${P2}_1$ with a = 5.865 A, b = 11.421 A, c = 12.966 A, \beta = 97.55 deg, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II \beta-turn conformation is stabilized by an intramolecular 4 \rightarrow 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are ${\phi}_{pro}$ = -57.8 deg, ${\psi}_{pro}$ = 139.3 deg, ${\phi}_{Aib}$ = 61.4 deg, and ${\psi}_{Aib}$ = 25.1 deg. The Type II \beta-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.

A second example of insertion of a water molecule into the helical backbone of an apolar peptide is presented here and compared to a similar occurrence in a longer peptide with the same type of sequence of residues, i.e., $Boc-Aib-{(Ala-Leu-fib)}_3-Ome$. The backbone of the title compound assumes an approximate $3_{10}$-helical form with three 4 \rightarrow 1 hydrogen bonds. In the place of a fourth 4 \rightarrow 1 hydrogen bond, a water molecule is inserJed between 0(1) and N(4), and acts as a bridge by forming hydrogen bonds N(4) . . . W(1) (2.95 A) and W(l) . . . O(1) (2.81 A). The water molecule participates in a third hydrogen bond with a neighboring peptide molecule, W(1) . . . O(4) (2.91 A). The insertion of the water molecule causes the apolar peptide to mimic an amphiphilic helix. Crystals grown from ethyl acetate/petroleum ether (reported here)p from methanol/water solution are in space group ${P2}_12_12_1$ with a = 12.024(4) A, b = 15.714(6) A, c = 21.411(7) A, Z = 4 and $d_{calc}$ = 1.124 g/$cm^3$ for $C_{32}H_{58}N_6O_9$ . $H_2O$. The overall agreement factor R is 6.3% for 2707 reflections observed with intensities > 3 \sigma (F) and the resolution is 0.90 A.

The ideal \gamma-turn conformation in peptides is stabilized by the formation of two intramolecular hydrogen bonds. These are between the NH of residue i and the C = O of residue i + 2 (1 \rightarrow 3, $C_{11}$) and the C = O of residue i and the NH of residue i + 2 (3 \rightarrow 1, $C_7$). While this reverse-turn structural feature has been observed in proteins, unambiguous characterization of this conformation has yet to be realized in small peptides. Several examples of a single 3 \rightarrow 1 $(C_7)$ hydrogen bond have been reported in crystal structures of cyclic peptides and inferred from spectroscopic studies in apolar solvents. We wish to describe the spectroscopic characterization of a \gamma-turn conformation in a protected tripeptide, stabilized by formation of a disulfide crosslink.

The CD spectra of the peptides $Boc-X-{(Aib-X)}_n-OMe$ (n = 1,2,3) and $Boc-{(Aib-X)}_5-OMe$, where X = L-Ala or L-Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole-dipole interactions.

The decapeptides $Boc-Aib-L-Val-Aib-Aib-{(L-Val)}_3-Aib-L-Val-Aib-OMe$ and $Boc-Aib-L-Leu- Aib-Aib-{(L-Leu)}_3-Aib-L-Leu-Aib-OMe$ have been studied in $CD{Cl}_3$ and ${({CD}_3)}_2SO$ solutions by 270-MHz' H-nmr. In $CD{Cl}_3$ the presence of eight intramolecularly hydrogen-bonded NH groups has been established, consistent with a $3_{10}$-helical conformation, for both decapeptides. In ${({CD}_3)}_2SO$ only seven solvent-shielded NH groups are observed, supporting either an \alpha-helical conformation or a partially unfolded $3_{10}$-helix. Ir studies provided supporting evidence for intramolecularly hydrogen-bonded structures in $CH{Cl}_3$, while CD studies suggest helical conformation in both decapeptides in various solvents. CD studies also support helical folding in the C-terminal hexapeptides. The central triplet of L-amino acids appears to destabilize $3_{10}$-helical conformations in polar solvents like ${({CD}_3)}_2SO$.

The crystal and molecular structure of N-benzyloxycarbonyl-\alpha-aminoisobutyryl-L-prolyl methylamide, the amino terminal dipeptide fragment of alamethicin, has been determined using direct methods. The compound crystallizes in the orthorhombic system with the space group ${P2}_12_12_1$. Cell dimensions are a = 7.705 A, b = 11.365 A, and c = 21.904 A. The structure has been refined using conventional procedures to a final R factor of 0.054. The molecular structure possesses a 4 \rightarrow 1 intramolecular N-H- - -0 hydrogen bond formed between the CO group of the urethane moiety and the NH group of the methylamide function. The peptide backbone adopts the type III \beta-turn conformation, with ${\phi}_2$ = -51.0 deg, ${\psi}_2$ = -39.7 deg, ${\phi}_3$ = -65.0 deg, ${\psi}_3$ = -25.4 deg. An unusual feature is the occurrence of the proline residue at position 3 of the \beta-turn. The observed structure supports the view that Aib residues initiate the formation of type III \beta-turn conformations. The pyrrolidine ring is puckered in $C^{\gamma}-exo$ fashion.

The conformational analysis of a protected homodipeptide of 1-aminocyclopentanecarboxylic acid $({Acc}^5)$ has been carried out. 'H-nmr studies establish a \beta-turn conformation for $Boc-{Acc}^5-{Acc}^5-NHMe$ in chloroform and dimethylsulfoxide solutions involving the methylamide NH in an intramolecular hydrogen bond. Supportive evidence for the formation of an intramolecular hydrogen bond is obtained from ir studies. X-ray diffraction studies reveal a type III \beta-turn conformation in the solid state stabilized by a 4 \rightarrow 1 hydrogen bond between the Boc CO and methylamide NH groups. The \phi, \psi values for both ${Acc}^5$ residues are close to those expected for an ideal $3_{10}$-helical conformation (\phi = \pm 60 deg, \psi = \pm 30 deg).

Peptide NH chemical shifts and their temperature dependences have been monitored as a function of concentration for the decapeptide, Boc-Aib-Pro-Val-Aib-Val-Ala- Aib-Ala-Aib-Aib-OMe in $CD{Cl}_3$ (0.001-0.06M) and ${({CD}_3)}_2SO$ (0.001-0.03M). The chemical shifts and temperature coefficients for all nine NH groups show no significant concentration dependence in ${({CD}_3)}_2SO$. Seven NH groups yield low values of temperature coefficients over the entire range, while one yields an intermediate value. In $CD{Cl}_3$, the Aib(1) NH group shows a large concentration dependence of both chemical shift and temperature coefficient, in contrast to the other eight NH groups. The data suggest that in ${({CD}_3)}_2SO$, the peptide adopts a $3_{10}$ helical conformation and is monomeric over the entire concentration range. In $CD{Cl}_3$, the $3_{10}$ helical peptide associates at a concentration of 0.01M, with the Aib(1) NH involved in an intermolecular hydrogen bond. Association does not disrupt the intramolecular hydrogen-bonding pattern in the decapeptide.

1H-nmr is one of the most widely used techniques for the study of intramolecular hydrogen-bonded conformations of peptides in solution. The parameters used to delineate solvent-shielded amide NH groups include rates of hydrogen-deuterium exchange: solvent shifts paramagnetic radical-induced broadening: transfer of saturation from exchangeable solvent protons, and temperature coefficients (d\delta / dT) of NH chemical shifts, in strongly hydrogen-bond-accepting solvents like ${({CD}_3)}_2SO$. Of these, the use of temperature coefficients is probably the most widespread. In general, values 0.004 ppm/deg C have been assigned to exposed groups. The model compound, N-methylacetamide ${CH}_3CONH{CH}_3$ yields a value of 0.006 ppm/deg C in ${({CD}_3)}_2SO$. The large temperature coefficients for exposed NH protons presumably arise by the breaking of solute-solvent hydrogen bonds on increasing the temperature. Peptides dissolved in $CD{Cl}_3$ show low temperature coefficients for both solvent-shielded and exposed amide hydrogens, a reflection of the poor hydrogen-bonding capability of this solvent. We describe in this report the use of temperature coefficients of peptide NH groups in benzene $(C_6D_6)$, as a sensitive parameter for the determination of the degree of solvent exposure.

The conformation of an acyclic dehydrophenylalanine (delta Z-Phe) containing hexapeptide, Boc-Phe-delta Z-Phe-Val-Phe-delta Z-Phe-Val-OMe, has been investigated in CDCl3 and (CD3)2SO by 270-MHz 1H-nmr. Studies of NH group solvent accessibility and observation of interresidue nuclear Overhauser effects (NOEs) suggest a significant solvent-dependent conformational variability. In CDCl3, a population of folded helical conformations is supported by the inaccessibility to solvent of the NH groups of residues 3-6 and the detection of several NiH----Ni + 1H NOEs. Evidence is also obtained for conformational heterogeneity from the detection of some Ci alpha H----Ni + 1H NOEs characteristic of extended strands. In (CD3)2SO, the peptide largely favors an extended conformation, characterized by five solvent-exposed NH groups and successive Ci alpha H----Ni + 1H NOEs for the L-residues and Ci beta H----Ni + 1H NOEs for the delta Z-Phe residues. The results suggest that delta Z-Phe residues do not provide compelling conformational constraints.

The synthesis of the tetrapeptide $benzyloxycarbonyl{(\alpha-aminoisobutyryl-L-prolyl)}_2-methyl ester$ $(Z-{(Aib-Pro)}_2-OMe)$ and an analysis of its conformation in solution and the solid state are reported. Stepwise synthesis using dicyclohexylcarbodiimide leads to racemization at Pro(2). Evidence for the presence of diastereomeric tetrapeptides is obtained from 270-${MHz}^1H-nmr$ and 67.89-MHz 13C-nmr. The all-L tetrapeptide is obtained by fractional crystallization from ethyl acetate. The NH of Aib(3) is shown to be involved in an intramolecular hydrogen bond by variable-temperature 1H-nmr and the solvent dependence of NH chemical shifts. The results are consistent with a \beta-turn conformation with Aib(1) and Pro(2) at the corners stabilized by a 4 \rightarrow 1 hydrogen bond. The molecule crystallizes in the space group ${P2}_12_12_1$, with a = 8.839, b = 14.938, and c = 22.015 A. The structure has been refined to an R value of 0.051. The peptide backbone is all-trans, and a 4 \rightarrow 1 hydrogen bond, between the CO group of the urethane moiety and Aib(3) NH, is observed. Aib(1) and Pro(2) occupy the corner positions of a type I \beta-turn with \phi = -55.4 deg, \psi = -31.3 deg for Aib(1) and \phi = -71.6 deg, \psi = -38 deg for Pro(2). The tertiary amide unit linking Pro(2) and Aib(3) is significantly distorted from planarity (${\Delta}_{\omega}$ = 14.3 deg).

The crystal and molecular structures of two \alpha-aminoisohutyric acid (Aib)-containing diketopiperazines, cyclo(Aib-Aib) 1 and cyclo(Aib- L-Ile) 2, are reported. Cyclo(Aib-Aib) crystallizes in the space group P1 with a = 5.649(3), b = 5.865(2), c = 8.363(1), \alpha = 69.89(6), \beta = 113.04(8), \gamma = 116.0(3), and Z = 1, while 2 occurs in the space group ${P2}_12_12_1$ with a = 6.177(1), b = 10.791(1), c = 16.676(1), and Z = 4. The structures of 1 and 2 have been refined to final R factors of 0.085 and 0.086, respectively. In both structures the diketopiperazine ring shows small but significant deviation from planarity. A very flat chair conformation is adopted by 1, in which the $C^{\alpha}$ atoms are displaced by 0.07 A on each side of the mean plane, passing through the other four atoms of the ring. Cyclo(Aib-Ile) favors a slight boat conformation, with Aib $C^{\alpha}$ and Ile $C^{\alpha}$ atoms displaced by 0.11 and 0.05 A, on the same side of the mean plane formed by the other ring atoms. Structural features in these two molecules are compared with other related diketopiperazines.