Conformational investigation of α, β-dehydropeptides. VIII.*N-Acetyl-α, β-dehydroamino acid N'-methylamides: conformation and electron density perturbation from infrared and theoretical studies

The Fourier transform infrared spectra are analyzed in the regions of v_s(N-H), amide I, amide II and v_s(C^α=C^β) bands for a series of Ac-ΔXaa-NHMe, where ΔXaa =ΔAla, (Z)-ΔAbu, (Z)-ΔLeu, (Z)-ΔPhe and AVal, to determine the predominant solution conformation of these α, β-dehydropeptide-related molecules and the electron distribution perturbation in their amide bonds. The measurements were performed in dichloromethane (DCM). To confirm and rationalize the assignments, the spectra of the respective series of saturated Ac-Xaa-NHMe, recorded in DCM, and the spectra of these two series of unsaturated and saturated compounds, recorded in acetonitrile, were examined. To help interpret the spectroscopic results, the equilibrium geometrical parameters for some selected amides were used. These were optimized with ab initio methods in the 6-31G** basis set. Each of the dehydroamides studied adopted a C₅ structure, which in Ac-ΔAla-NHMe is fully extended and accompanied by the strong C₅ hydrogen bond. Interaction with the C^α=C^β bond lessened the amidic resonance within each of the flanking amide groups. The N-terminal C=O bond was noticeably shorter, both amide bonds were longer than the corresponding bonds in the saturated entities and the N-terminal amide system was distorted. Ac-ΔAla-NHMe constituted an exception. Its C-terminal amide bond was shorter than the standard one and both amide systems were prototypically planar.     

Conformational investigation of α,β-dehydropeptides VII*. Conformation of Ac-Pro-ΔAla-NHCH3 and Ac-Pro-(E)-ΔAbu-NHCH3: comparison with (Z)-substituted α,β-dehydropeptides†

The crystal structure and solution conformation of Ac-Pro-ΔAla-NHCH₃ and the solution conformation of Ac-Pro-(E)-ΔAbu-NHCH₃ were investigated by X-ray diffraction method and NMR, FTIR and CD spectroscopies. Ac-Pro-ΔAla-NHCH, adopts an extended-coil conformation in the crystalline state, with all-trans peptide bonds and the ΔAla residue being in a C₅ form, φ₁=– 71.4(4), ψ₁=– 16.8(4), φ₂=– 178.4(3) and ψ₂= 172.4(3)°. In inert solvents the peptide also assumes the C₅ conformation, but a γ-turn on the Pro residue cannot be ruled out. In these solvents Ac-Pro-(E)- ΔAbu-NHCH₃ accommodates a βII-turn, but a minor conformer with a nearly planar disposition of the CO—NH and C=C bonds (φ₂～0°) is also present. Previous spectroscopic studies of the (Z)-substituted dehydropeptides Ac-Pro-(Z)- ΔAbu-NHCH, and Ac-Pro-ΔVal-NHCH₃ reveal that both peptides prefer a βII-turn in solution. Comparison of conformations in the family of four Ac-Pro-ΔXaa-NHCH₃ peptides let us formulate the following order of their tendency to adopt a β-turn in solution: (Z)- ΔAbu > (E)- δAbu > ΔVal; ΔAla does not. None of the folded structures formed by the four compounds is stable in strongly solvating media. © Munksgaard 1996.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Conformational investigation of α,β-dehydropeptides

Conformations of three series of model peptides: homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHCH₃ (Xaa=Phe, Val, Leu. Abu. Ala) as ivell as α,β-dehydro Ac-Pro-ΔXaa-NHCH_s [ΔXaa = (Z)-ΔPhe, ΔVal. (Z)-ΔLeu, (Z)-ΔAbu] were investigated by CD spectroscopy in 2 % dichloromethanecyclohexane, trifluoroethanol. water. and occasionally in other solvents. The spectra of homochiral peptides show a significant solvent dependence. Folded structures are present in 2% dichloromethane-cyclohexane and unordered ones occur in water. The folded conformers are of the inverse γ-turn type for all the peptides but Ac-Pro-L-Phe-NHCH₃ for which the type-I β-turn is preferred. The changes in the spectra of the heterochiral peptides are limited. The compounds adopt the typc-II β–turn in 2% dichloromethanecyclohexane, represented by class B spectra, and retain this conformation in water as well as in fluorinated alcohols but not always to a full extent. The CD spectra of the unsaturated peptides in 2%, dichloromethanecyclohexane, although they cannot be assigned to any common spectral class, must be attributed to the βII-turn conformation as determined for these coinpounds by NMR and IR spectroscopy. The CD spectra of dehydropeptides exhibit a considerable solvent dependence and suggest unordered structures in water.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.     

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.     

Structural versatility of peptides from Cα,α-disubstituted glycines: crystal-state conformational analysis of peptides from Cα-methylhomophenylalanine, (αMe)Hph

The molecular and crystal structures of the C^α-tetrasubstituted, δ-branched α-amino acid C^α-methyl-homophenylalanine, H-d -(αMe)Hph-OH, and three peptides (to the pentamer level), including the homotripeptide, have been determined by X-ray diffraction. The peptides are Z-l -(αMe)Hph-(l -Ala)₂-OMe, pBrBz-[d -(αMe)Hph]₃-OtBu and Ac-(Aib)₂-l -(αMe)Hph-(Aib)₂-OtBu. All the (αMe)Hph residues prefer φ,ψ torsion angles in the helical region of the conformational map. The two terminally blocked tripeptides adopt a β-bend conformation stabilized by a 1→4 C = O?H-N intramolecular H-bond. The terminally blocked pentapeptide is folded in a regular 3₁₀-helix. In general, the relationship between (αMe)Hph α-carbon chirality and helix handedness is the same as that exhibited by protein amino acids. A comparison is also made with the conclusions extracted from published work on peptides from other types of C^α-alkylated aromatic α-amino acids. © Munksgaard 1996.     

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.     

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.     

Design of peptides withα,β-dehydro-residues: synthesis,and crystal and molecular structure of a310-helical tetrapeptide Boc-l-Val-ΔPhe-ΔPhe-l-Ile-OCH3

Abstract: The peptide Boc-l -Val-ΔPhe-ΔPhe-l -Ile-OCH₃ was synthesized using the azlactone method in the solution phase, and its crystal and molecular structures were determined by X-ray diffraction. Single crystals were grown by slow evaporation from solution in methanol at 25°C. The crystals belong to an orthorhombic space group P2₁2₁2₁ with a = 12.882(7) Å, b = 15.430(5) Å, c = 18.330(5) Å and Z = 4. The structure was determined by direct methods and refined by a least-squares procedure to an R-value of 0.073. The peptide adopts a right-handed 3₁₀-helical conformation with backbone torsion angles: φ₁ = 56.0(6)°, ψ₁ = –38.0(6)°, φ₂ = –53.8(6)°, ψ₂ = 23.6(6)°, φ₃ = –82.9(6)°, ψ₃ = –10.6(7)°, φ₄ = 124.9(5)°. All the peptide bonds are trans. The conformation is stabilized by intramolecular 4→1 hydrogen bonds involving Boc carbonyl oxygen and NH of ΔPhe³ and CO of Val¹ and NH of Ile⁴. It is noteworthy that the two other chemically very similar peptides: Boc-Val-ΔPhe-ΔPhe-Ala-OCH₃ (i) and Boc-Val-ΔPhe-ΔPhe-Val-OCH₃ (ii) with differences only at the fourth position have been found to adopt folded conformations with two overlapping β-turns of types II and III′, respectively, whereas the present peptide adopts two overlapping β-turns of type III. Thus the introduction of Ile at fourth position in a sequence Val-ΔPhe-ΔPhe-X results in the formation of a 3₁₀-helix. The crystal structure is stabilized by intermolecular hydrogen bonds involving NH of Val¹ and carbonyl oxygen of a symmetry related (–x, y – 1/2, 1/2 + z) ΔPhe² and NH of ΔPhe² with carbonyl oxygen of a symmetry related (x, y1/2, 1/2 + z) Ile⁴. This gives rise to long columns of helical molecules linked head to tail running along [010] direction.     

Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several α-melanotropin (α-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ and Ac-[Tyr⁴]-α-MSH_4–11-NH₂ were compared with α-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ was found to be less active than Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr⁴]-α-MSH_4–11-NH₂ was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ over Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on frog melanocytes may be related to the fact that the shorter 4–10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of α-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle⁴]-α-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity. Similarly, incorporation of D-Phe-7 into Tyr-4 containing melanotropin fragments produced analogues Ac-[Tyr⁴, D-Phe⁷]-αMSH_4–10-NH₂ and Ac-[Tyr⁴, D-Phe⁷]-α-MSH_4–11-NH₂, which also exhibited greatly increased biological activity in all three assay systems. Both of these analogues were also found to have prolonged activity in the frog skin bioassay but little or no prolonged activity in the lizard skin bioassay. These two analogues turned out to be full agonists in the mouse melanoma adenylate cyclase bioassay and were equipotent to α-MSH. These results demonstrate that substitution of tyrosine for methionine at position-4 dramatically affects the potency and prolonged activity of these melanotropin analogues and the melanotropic activities observed as a result of such substitutions are themselves affected by concomitant substitutions at the 7(Phe) and 11 (Lys) positions of the analogues.     

Crystal structures of a nonapeptide helix containing α, α-di-n-butylglycine (Dbg), Boc-G1y-Dbg-Ala-Val-Ala-Leu-Aib-Val-Leu-OMe

Two crystal structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue α,α-di-n-butylglycyl, reveal a mixed 3₁₀-α-helical conformation. Residues 1-7 adopt φ, ψ values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has φ, ψ values of -40, -37° and -46, -407° in the two crystals with the two butyl side chains mostly extended in each. Peptide molecules in the crystals pack into helical columns. The crystal parameters are: C₅₀ H₉₁ N₉ O₁₂, space group P2₁, with a= 9.789(1)Å;, b= 20.240(2) Å. c= 15.998(3) Å. β= 103.97(1): Z= 2, R=10.3% for 1945 data observed < 3σ(F) and C₅₀H₉₁N₉O₁₂· 3H₂O, space group P2₁ with a= 9.747(3)Å, b= 21.002(8) Å, c= 15.885(6) Å, β= 102.22(3). Z= 2. R=13.6% for 2535 data observed < 3σ(F) The observation of a helical conformation at Dbg suggests that the higher homologs in the α,α-dialkylated glycine series also have a tendency to stabilize peptide helices. © Munksgaard 1996.     

Structure-activity studies of analogs of β, γ-methylene-ATP at P2X-purinoceptors in the rabbit ear central artery

Structure-activity studies using naphthylmethyl analogs of β, γ-methylene-ATP were conducted at the P_2X-purinoceptor that mediates contraction of the rabbit ear central artery by ATP, α, β-m-ATP. On the adenine base, substitution at the C²-position (WRC-0440) increased the agonist potency 2-fold and substitution at the C⁸-position (WRC-0431) did not change agonist potency, and both compounds had the same maximal response as β, γ-m-ATP, whereas substitution at the N⁶-position (WRC-0416) abolished activity. On the D-ribose sugar, substitution on the 2′-hydroxyl generated a partial agonist (WRC-0479), which had a maximal effect of only 39% of that of β-γ-m-ATP. Attempts to substitute the 3′-hydroxyls by naphthylmethyl failed, but substitution by p-methoxybenzyl (WRC-0617) did not change potency or the maximal response. Cyclic substitution of both the 2′- and 3′-hydroxyls by naphthylmethylidine (WRC-0498) had no effect on the agonist potency or the maximal response relative to β-γ-m-ATP. On the β, γ-methylenetriphosphonate chain, substitution on the methylene linkage by naphthylmethyl (WRC-0433) had no effect on agonist potency, but the maximal response increased to 122% that of β-γ-ATP. However, the contractile response to WRC-0433 was not desensitized by α, β-γ-ATP (contractile responses to all other agonists were abolished by α-β-γ-ATP pretreatment), but was blocked by the α₁ antagonist prazosin (10^?6 M). WRC-0433 appears to act at a prejunctional site that mediates ATP-induced release of norepinephrine. Purine nucleotides with substituents at the 2′-position of the ribose sugar could provide a lead to the generation of P_2X-purinoceptor antagonists. © 1995 Wiley-Liss, Inc.     

Conformational versatility of the Nα-acylated tripeptide amide tail of oxytocin

The synthesis, physical and analytical characterization, and crystal-state structural analysis by X-ray diffraction of three analogues of the N^α-acylated tripeptide amide tail of oxytocin, each containing a cyclic C^{α, α-} disubstituted glycine at position 2, have been performed. The peptides arc Boc-L-Pro-Ac₃c-Gly-NH₂, Z-L-Pro-Ac₅c-Gly-NH₂ and Z-L-Pro-Ac₅c-Gly-NH₂. While the former is folded in a type-II β-turn conformation at the -L-Pro-Ac₃c- sequence, the two latter tripeptides form two consecutive (type-II, type-I′) β-turns. The Ac₅c- and Ac₆c-tripeptides are the first examples of such a highly folded structural combination in a position-2 analogue of the N^α-acylated -L-Pro-L-Leu-GIy-NH² sequence.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

Synthesis,crystal structure and molecular conformation of the tBuCO-D,L-Ala-Δz-Phe-NhiPr α,β-unsaturated dipeptide

The crystal structure of the tBuCO-d,l -Ala-Δ^z-Phe-NHiPr dipeptide has been solved by X-ray diffraction. The peptide crystallizes in monoclinic space group P2JC with a = 13.445 (3) Å, b = 35.088 (4) Å, c = 14.755(3) Å, β= 116.73(1)°, Z = 12 and d_c= 1.151 g.cm^?3. The three independent molecules per asymmetric unit accommodate a βII-folded conformation, but only one of them contains the typical i + 3 → i interaction characterizing a β-turn. In the other two molecules, the N…O distance exceeds 3.2 Å, a value generally considered the upper limit for hydrogen bonds in peptides. In solution, the βII-turn conformation is largely predominant.     

A convenient synthesis of 2′,3′‐dideoxyinosine‐13C5 and related 2′,3′‐dideoxyadenosine‐13C5

2′,3′‐Dideoxyinosine‐¹³C₅ (ddI‐¹³C₅) and the related 2′,3′‐dideoxyadenosine‐¹³C₅ (ddA‐¹³C₅) were prepared from (S)‐5‐[¹³C₅]2,3‐dideoxyribonolactone 1 . From a batch of this starting material ddI‐¹³C₅ was made in 27% overall yield in seven steps and ddA‐¹³C₅ in five steps and 14% overall yield. The known synthesis of ddI‐¹³C₅ from glucose‐¹³C₆ took 18‐steps_; therefore the present work is a substantial improvement. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative biological activities of potent analogues of α-melanotropin

Several α-melanotropin (α-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO₂), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle⁴] -α-MSH_4–11 - NH₂ fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle⁴, Ala⁷]-α-MSH_4–11-NH₂ was more potent than Ac-[Nle⁴, D-Ala⁷]-α-MSH_4–11-NH₂ in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of α-MSH, to biological potency of the hormone.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.