Turn induction by N-aminoproline Comparison of the Gly-Pro-Gly and Glyψ[CO-NH-N]Pro-Gly sequences

The folded structure induced by the N-aminoproline residue (the hydrazino analogue of proline, denoted hPro) in the Boc-Gly¹-hPro²-Gly³-NHiPr hydrazino tripeptide has been characterized in the solid state by X-ray diffraction, and compared to the usual βII-turn structure in the Boc-Gly¹-Pro²-Gly³⁶-NHiPr cognate tripeptide. It is stabilized by a bifurcated hydrogen bond in which (Gly³)NH interacts with both (Gly¹)CO and (hPro²)N^x. This conformation is retained in CH₂Cl₂ and CHC1₃ solutions, and allows an overall folded conformation of the hydrazino tripeptide in which (iPr)NH is hydrogen-bonded to (Boc)CO. The hPro α-hydrazino acid residue appears to promote a local folded structure, and might behave as a β-turn mimic. © Munksgaard 1994.     

A crystal-state,solution and theoretical study of the preferred conformation of linear Cα,α-diphenylglycine derivatives and dipeptides with potential anticonvulsant activity

Several linear molecules containing the C^α,α-diphenylglycine residue were prepared as potential anticonvulsants. The conformational preferences of the C^α,α-diphenylglycine residue were assessed in these synthetic derivatives and dipeptides by X-ray diffraction, FTIR absorption and ¹H NMR techniques, and by conformational energy computations. Five (out of six) derivatives adopt the fully extended C₅ conformation in the crystal state. This intramolecularly H-bonded form is largely populated in chloroform solution in all the derivatives investigated. Conformational energy computations in vacuo support the view that the intramolecularly H-bonded C₇-ring form is the most stable structure for these compounds. Only one linear derivative exhibits a (modest) anticonvulsant activity.     

Stabilization of β-turn conformations in enkephalins

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α-aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly. ¹H n.m.r. studies of Tyr-Aib-Gly-Phe-Met-NH₂, Tyr-Aib-Aib-Phe-Met-NH₂ and Tyr-Gly-Aib-Phe-Met-NH₂ demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t-butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β-turn centred at positions 2 and 3 is proposed for the Aib²-Gly³analog. In the Gly²-Aib³analog, the β-turn has Aib³-Phe⁴as the corner residues. The Aib²-Aib³analog adopts a consecutive β-turn or 3₁₀ helical conformation. High in vivo biological activity is observed for the Aib²and Aib²-Aib³analogs, while the Aib³peptide is significantly less active.     

Bioorganic stereochemistry. A study of the peptide oxazolones from Z-(Aib)n-OH (n = 2–4) in the solid state*

An investigation of the preferred conformations and modes of self-association of the peptide oxazolones from Z(-Aib-)_n-OH (n = 2–4) in the solid state has been performed by infrared absorption. More detailed information on the peptide oxazolone from Z(-Aib-)₃ OH (2) has been obtained using X-ray diffraction. In this compound the conformations of the first two Aib residues differ substantially, only the N -terminal one being found in the usual 3_10- (or α-) helical region of the Ramachandran map. The C=N-bond of the oxazolone group is not conjugated with the lactone moiety. A very weak intermolecular interaction occurs between the urethane N-H and the carbonyl group of the oxazolone ring.     

Conformations of peptides containing 1-aminocyclohexanecarboxylic acid (Acc6). Crystal structures of two model peptides

The crystal structures of two peptides containing 1-aminocyclohexanecarboxylic acid (Acc⁶) are described. Boc-Aib-Acc⁶-NHMe · H₂O adopts a β-turn conformation in the solid state, stabilized by an intramolecular 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The backbone conformational angles (φ_Aib = – 50.3°, ψ_Aib = – 45.8°; φ_Acc6 = – 68.4°, ψ_Acc6 = – 15°) lie in between the values expected for ideal Type I or III β-turns. In Boc-Aib-Acc⁶-OMe, the Aib residue adopts a partially extended conformation (φ_Aib = – 62.2°, ψ_Aib = 143°) while the Acc⁶residue maintains a helical conformation (φ_Acc6 = 48°, ψ_Acc⁶= 42.6°). ¹H n.m.r. studies in CDCl₃ and (CD₃)₂SO suggest that Boc-Aib-Acc⁶-NHMe maintains the β-turn conformation in solution.     

5,5-Dimethylthiazolidine-4-carboxylic acid (DTC) as a proline analog with restricted conformation

Spectroscopic evidence is presented for the lack of intramolecular hydrogen bonding in a simple peptide derivative of 5,5-dimethylthiazolidine-4-carboxylic acid (Dtc). The infrared spectrum of Boc-Pro-Ile-OMe 1 in nonpolar solvents displays two N-H stretching bands at 3419 and 3330 cm^-1 in CCl₄ and one at 3417 and 3328cin^-1 in CHCl₃. The low frequency band at 3328–3330cm^-1 may be assigned to conformations with an intramolecular hydrogen bond between the Ile N-H and Boc C=O. The band at 3417-3419 cm^-1 is the normal Ile N-H stretch. In the polar solvent CH₃ CN only one NH stretching band at 3365 cm^-1 is observed. The IR spectrum of Boc-Dtc-Ile-OMe 2, on the other hand, displays one N-H stretching band at 3423cm^-1 in CCI, and one at 3418cm^-1 in CHCI₃. The IR spectrum of 2 does not display the N-H stretching band that would arise from intramolecular hydrogen bonding between the Boc C=O and Ile N-H. The lack of intramolecular hydrogen bonding for Boc-Dtc-Ile-OMe 2 was evident also in the NMR spectra in nonpolar solvents. The ¹H-NMR spectrum of the Pro dipeptide 1 in 50% CDCl₃/C₆D₆ at 20° displayed two Ile-NH signals at 6.58 and 7.74 ppm. The latter signal corresponds to the intramolecularly hydrogen bonded Ile-NH in the trans-Boc isomer of 1 (60% of the total population), while the former signal corresponds to the nonhydrogen bonded Ile-NH in the cis-Boc isomer. The ¹H-NMR spectrum of the Dtc dipeptide 2 displayed two slowly exchanging cis- and trans-Boc amide isomers as well, but both amide proton resonances were observed upfield at 6.67 and 6.74 ppm, which correspond only to a nonhydrogen bonded Ile N-H. The X-ray crystal structure of Boc-Dtc displays only a cis-Boc-Dtc urethane amide group and two conformations for the Dtc ring, one in which the beta carbon atom is anti to the carboxyl group and the other in which the gamma sulfur atom is anti to the carboxyl group. Conformational analysis of Ac-Dtc-NHMe suggests that in the hydrogen bonded C7 conformation steric interaction between the syn-beta methyl group and carbonyl group of Dtc adds nonbonded and angle strain energies to counteract the stabilizing coulombic interaction between the Boc C=O and terminal amide N-H. Whereas the C7 conformation is a prominent conformation for peptide derivatives of proline, other conformations are favored in peptide derivatives of Dtc (ψ -ñ 110-150° or ñ 320-360°). These results suggest that, in peptides where substitution of Pro appears to maintain or enhance biological activity, the substitution of Dtc for Pro may test the functional importance of the C7 conformation in that position of the peptide sequence.     

Turn conformations in peptides containing the -Xaa-Ser- sequence

The conformations of the protected dipeptides Boc-L-Pro-L-Ser-NHMe, Boc-L-Pro-D-Ser-NHMe, Boc-L-val-L-Ser-NHMe and Boc-L-Val-D-Ser-NHMe have been explored through interpretation of their infrared spectra in CH₂Cl₂, DMSO and D₂O solution. In CH₂Cl₂ solution the formation of a ten-membered ring (β-turn) for each compound is signaled by characteristic shifts in both the urethane C=O and the terminal NH stretching frequencies. For each peptide, differences in the amide I absorption patterns for LL and LD isomers are consistent with the formation of type I and type II β-turns respectively in CH₂C1₂ solution. The amide I absorptions suggest substantial disruption of intramolecular hydrogen bonding in DMSO, and no intermolecular hydrogen bonding whatsoever in aqueous solution. In CH₂C1₂ solution the OH stretching vibration is consistent with the formation of a hydrogen bond to the C=O of the serine group; however, two additional absorptions at frequencies characteristic of‘free’OH groups also appear in all spectra. Implications regarding the serine in stabilizing the β-turn are discussed. © Munksgaard 1996.     

Peptides from chiral Cα,α-disubstituted glycines

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral C^α,α-disubstituted glycine Cα-methyl, Cα-isopropylglycine [(αMe)Val] have been determined by X-ray diffraction. The derivative is mClAc-l -(α Me)Val-OH, and the peptides are Z-l -(αMe)Val-(l -Ala)₂-OMe monohydrate, Z-Aib-L-(αMe)Val-(Aib)₂-OtBu, and Ac-(Aib)₂-l -(αMe)Val-(Aib)₂OtBu acetonitrile solvate. The tripeptide adopts a type-I β-turn conformation stabilized by a 1 ← 4N-H . O=C intramolecular H-bond. The tetra- and pentapeptides are folded in regular right-handed 3₁₀-helices. All four L-(αMe)Val residues prefer φ, Ψ angles in the right-handed helical region of the conformational map. The results indicate that: (i) the (αMe)Val residue is a strong type-I/III β-turn and helix former, and (ii) the relationship between (αMe)Val chirality and helix screw sense is the same as that of Cα-monosubstituted protein amino-acids. The implications for the use of the (αMe)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.     

Crystal and molecular structure of two geometrically restricted chemotactic tripeptides,analogues of formyl-methionine-leucine-phenylalanine*

The crystal structures of HCO-Met-Leu-Phe-OC(CH₃)₃, (CH₂₅H₃₉N₃O⁵S), fMLP-OtBu, and HCO-Metψ[CSNH]-Leu-Phe-OCH₃, (C₃₃H₃₃N₃O₄S₂), fM^SLP-OMe, have been determined by single crystal X-ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP-OtBu are monoclinic, space group P2₁, a = 12.027(4), b = 9.492(3), c = 12.660(4) Å, β= 101.99(3)°, Z = 2; those of fM^SLP-OMe are orthorhombic, space group P2₁2₁2₁, a = 7.130(1), b = 12.097(2), c = 31.060(5) Å, Z = 4. The first compounds fMLP-OtBu is the t-butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fM^SLP-OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X-ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy-minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C⁷_eqconformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C⁷_eq conformations for fMLP is made. The resulting conformers are compared to previously active structures of these chemotactic agents.     

Incorporation of vinylogous scaffolds in the C‐terminal tripeptide of substance P

Abstract: Glycine‐9 and leucine‐10 of substance P (SP) are critical for (NK)‐1 receptor recognition and agonist activity. Proψ(Z)‐CH=CH(CH₃)‐CONH)Leu (or Met) and Proψ((E)‐CH=CH(CH₃)‐CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C‐terminal tripeptide, Gly‐Leu‐Met‐NH₂, of SP. Proψ((Z)‐CH=C(CH₂CH(CH₃)₂)‐CONH)Met‐NH₂, with an isobutyl substituent to mimic the Leu side‐chain, was also incorporated in place of the C‐terminal tripeptide. The substituted‐SP analogs were tested for their affinity to human NK‐1 receptor specific binding sites (NK‐1M and NK‐1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK‐1 receptor. The most potent SP analogs [Pro⁹ψ((Z)CH=C(CH₃)CONH)Leu¹⁰]SP and [Pro⁹ψ ((E)CH=C(CH₃)CONH)Leu¹⁰]SP, are about 100‐fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)‐ or (E)‐CH=C(CH₃)‐ or (Z)‐CH=C(CH₂CH(CH₃)₂) moieties hamper the correct positioning of the C‐terminal tripeptide of SP within both the NK‐1M‐ and NK‐1m‐specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.     

Conformation and activity of +H2-Pro-Leu-Gly-NH2 and its analogues modified at the leucyl residue

In order to investigate the relation between the conformation and the ability to modulate dopamine receptors, conformational free-energy calculations using an empirical potential (ECEPP/3) and hydration shell model were carried out on the tripeptide ⁺H₂-Pro-Leu-Gly-NH₂ (PLG) and its analogues in which the Leu residue is replaced with Phe, Ahx (L-2-aminohexanoic acid), Ile, Abu and Ala residues in the unhydrated and hydrated states. PLG and two tripeptides possessing Phe and Ahx residues show dopamine receptor modulating activity, while the other tripeptides do not. Irrespective of activities, PLG and its analogues are found to have the high probabilities to form β-bends of types II and I in the unhydrated state. However, in the hydrated state, the β-bend probabilities of the PLG analogues decrease significantly compared with those in the unhydrated state. These results indicate that the β-bend structure is a necessary factor for the PLG analogues to be active, but not a sufficient factor, and that the interactions of water molecules with the backbone may force the tripeptides to be more distorted or extended. The size and the orientation of the hydrophobic moiety of the second residue seem to be of consequence in eliciting the activity of the tripeptides. © Munksgaard 1995.     

Weak dependence of octapeptide solubility in organic solvents on the amino acid sequence*

The six kinds of the octapeptides 1-6 to be conceivable from permutation of three peptide fragments, i.e. Gln-Val-Gly, Asn-Ala-Ile, and Phe-Leu, were prepared to confirm that their solubility was not strongly dependent on their amino acid sequences. The solubility of the octapeptides 1-6 resembled each other and they were insoluble in MeOH, EtOH, THF, and CH₂Cl₂, and soluble in HMPA and DMSO . I.r. absorption spectroscopic analysis of the octapeptides 1-6 indicated that they mainly had a β-sheet structure in the solid state. The results of the solubility and conformation of the octapeptides 1-6 strongly suggested the adequacy of the hypothesis that their solubility and their potential for β-sheet formation in the solid state were not strongly dependent on their amino acid sequences but on their amino acid compositions.     

Conformation of cyclo-(D-phenylalanyl-trans-4-fluoro-D-prolyl)

cyclo(D-Phenylalanyl-trans-4-fluoro-D-prolyl), c(D-Phe-D-FPro), was synthesized and its conformation determined both in solution and in the solid state by ¹H NMR and X-ray analysis, respectively. In the crystals the 2,5-diketopiperazine (DKP) ring assumes the uncommon conformation, for cyclodipeptides containing Pro residue, of a flattened chair, which seemingly results from a compromise between, on the one hand, the DKP-aromatic intramolecular ring-ring attraction (folding), requiring the C^α-C^β bond of the Phe to be axial, and, on the other hand, the intrinsic tendency of the Pro residue to have its C^α-C^β bond equatorial. Unlike the solid state, the ¹H NMR data in CDCl₃ and C₆D₆ demonstrate that in both solutions the DKP ring assumes a boat-like shape, typical for the Pro-containing cyclodipeptides, with the equatorial C^α-C^β bonds in both amino acid residues, which preclude ring-ring folding. A similar conformation was encountered in the closest analog of c(D-Phe-D-FPro), viz. in c(Phe-Pro), both in solution (21, 22, 26) and in the solid state (12). A subtle interplay of intramolecular interactions introduced into a cyclodipeptide by a Pro-type and a Phe-type residue is emphasized.     

Non coded Cα,α-disubstituted amino acids

The crystal and molecular structure of the fully protected dipeptide Boc-Val-(S)-α-MeSer-OMe has been determined by X-ray diffraction techniques. Crystals grown from ethyl acetate/n-pentane mixtures are tetragonal, space group 14₁, with cell parameters at 295 K of a= 15.307(2), c= 18.937(10)Å, V = 4437.1 ų, M.W. = 332.40, Z = 8, D_m= 0.99 g/cm³ and D_x= 0.995 g/cm³. The structure was solved by application of direct methods and refined to an R value of 0.028 for 1773 reflections with I≥3σ(I) collected on a CAD-4 diffractometer. Both chiral centers have the (S) configuration. The dipeptide assumes in the solid state an S shape. The urethane moiety is in the cis conformation, while the amide bond is in the common trans conformation. The conformational angles φ₁, ψ₁ of the Val and φ₂, and ψ₂ of the (S)-αMeSer fall in the F region of the φ-ψ map. The isopropyl side chain of the Val residue has the (t, g^?) conformation, while the Ser side chain has a g⁺ conformation. The hydrogen bond donor groups are all involved in intermolecular H-bond interactions. Along the quaternary axis the dipeptide molecules are linked to each other with the formation of infinite rows.     

Methanol-mediated,modular self-assembly of antiparallel β-dimers

The backbone-modified tripeptide Bz-Aib-NHCOCO-Aib-OMe ( l ), in which the central Cα has been replaced with CO, self-assembles in the solid state into highly ordered two-dimensional arrays through MeOH mediated intermolecular stacking of dimeric ‘disk’ modules formed by an antiparallel β-sheet-type arrangement of tripeptide molecules. The Cα(C′O)N(C′O) (C′O)NC^α segment is nearly coplanar (average deviation from a mean least-square plane is ± 0.037 Å). The oxalyl moiety forms two pseudo-C₅ intramolecular hydrogen bonds. Crystal parameters are as follows: C₁₈H₂₃N₃O⁶CH₃OH, triclinic space group P1 a = 9.796(4), b = 10.348(3), c = 11.836(4) Å, α= 78.28(3), β= 73.72(3), γ= 69.45(3), R = 0.082 for 1747 reflections measured with ‖Fo‖3σ(F).     

Fate and disposition of [ 14Ia1 methylene chloride in the rat.

[¹⁴C]Methylene chloride (¹⁴CH₂Cl₂) was administered ip to Sprague-Dawley rats at doses ranging from 412 to 930 mg/kg. Animals were sacrificed 2, 8, and 24 hr after dosing. CH₂Cl₂ was largely eliminated in the breath unchanged during the first 2 hr. A fraction of the original dose was metabolized to carbon monoxide (CO), carbon dioxide (CO₂), and an uncharacterized metabolite. At 24 hr 91.5% of the dose was eliminated in the breath unchanged, 2% was eliminated as CO, 3% as CO₂, 1.5% as an uncharacterized metabolite, 1% was excreted in urine, and 2% remained in the carcass. Although the dissemination of radioactivity was widespread in rat tissue, the overall tissue uptake of ¹⁴C was relatively small. The highest tissue specific activities were found in the liver, kidneys, and adrenal glands. There was no significant accumulation of radioactivity in the fat. In a separate study serum and tissue formaldehyde concentrations (CH₂O) were measured in rats dosed with CH₂Cl₂. A substantial increase in serum CH₂O (62%) and a decrease in liver CH₂O (64%) were noted in rats treated with CH₂Cl₂. Formaldehyde concentrations in other tissues remained unchanged. ¹⁴CH₂O was not found in the breath, serum, or tissues of rats treated with ¹⁴CH₂Cl₂. Apparently the changes in serum and liver CH₂O were physiologically induced by CH₂Cl₂. There was no evidence from these experiments that CH₂Cl₂ was metabolized to CH₂O.     

Structure and conformation of linear peptides

The crystal structure of a dipeptide tert-butyloxycarbonyl-l -alanylglycine monohydrate (C₁₀H₁₈N₂O₅·H₂O), molecular weight 264, has been determined. The crystals are monoclinic, space group P2₁, with a= 10.767(1), b= 6.317(1), c= 10.981(2) Å, β= 109.15(2)°, and Z= 2, D_c= 1.24 g cm^?3. The structure was solved by direct methods and refined to 3 final R-index of 0.045 for 856 reflections (sin θ/λ < 0.55 Å^?1) with I > 2 σ. The N-terminus of the molecule blocked with the t-Boc group is uncharged and the C-terminus exists in an unionized state. The peptide unit is trans and shows slight deviations from planarity. (Δω= 3.1°). The peptide backbone is folded, with torsion angles of φ₁= -76.0(5), ψ₁= 164.3(4), ω₁= 176.9(5), φ₂= 116.1(5), ψ₂₁= - 2.8(7) and ψ₂₂= 177.8(4)°. The conformation about the urethane bond (C5–N1) is trans. The urethane group is essentially planar. The conformation of the boc group is trans–trans.     

A physiological model for the pharmacokinetics of methylene chloride in B6C3F1 mice following i.v. administrations

A physiologic mathematical model was developed to describe the time course of¹⁴C-methylene chloride (¹⁴CH₂Cl₂) distribution and elimination in mice following single i.v. administrations of 10 and 50mg/kg. A whole-body model was used to simulate¹⁴CH₂Cl₂ concentrations in blood and tissues, pulmonary clearance of unchanged¹⁴CH₂Cl₂, and metabolic conversion to¹⁴CO₂ and¹⁴CO as monitored by the appearances of these metabolites in expired breath. This diffusion-limited model was identified via a sequential optimization scheme using hybrid models for each compartment. Pulmonary elimination of unchanged¹⁴CH₂Cl₂ was modeled as a linear process while hepatic metabolism of¹⁴CH₂Cl₂ to the compounds¹⁴CO₂and¹⁴CO was described by a saturable metabolic rate term. The model adequately described the dose dependence in methylene chloride distribution and metabolism when simulations were compared to experimental data.     

Conformation of reduced glutathione in aqueous solution by 1H and 13C n.m.r.

We report on the conformation of reduced glutathione in solutions at low and physiological pH, examined with ¹H and ¹³C n.m.r. spectroscopy. The tripeptide in ¹H₂O was shown to interconvert rapidly between an array of conformers; in addition, the carbon backbone of the glutamyl was more rigid than anticipated if the residue were freely mobile. This restricted motion results from interaction of the α-amino and α-carboxyl groups on the glutamyl, with the γ-Glu-Cys peptide-carbonyl and amino, respectively. Our results support theoretical predictions of the conformation but they are at variance with previous ultraviolet spectroscopic and lower field n.m.r. studies.     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and ¹H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II ß-turn in the solid state with Met and Aib as the corner residues (øMet =- 51.8^o, øMet = 139.5^o, øAib = 58.1^o, øAib = 37.0^o). A single, weak 4 -> 1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N—O 3.25 Å, N-H—O 128.4^o). ¹Hn.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagneti radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCI₃ and (CD₃)₂SO. Nuclear Overhauser effects observed between Met Cα H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II ß-turns in these solvents.