Ac10c: a medium‐ring,cycloaliphatic Cα,α‐disubstituted glycine. Incorporation into model peptides and preferred conformation

Abstract: Two complete series of N‐protected oligopeptide esters to the pentamer level from 1‐amino‐cyclodecane‐1‐carboxylic acid (Ac₁₀c), an α‐amino acid conformationally constrained through a medium‐ring C^α_i ? C^α_i cyclization, and either the l ‐Ala or Aib residue, along with the N‐protected Ac₁₀c monomer and homo‐dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT‐IR absorption and ¹H NMR techniques. Furthermore, the molecular structures of two derivatives (Z‐Ac₁₀c‐OH and Fmoc‐Ac₁₀c‐OH) and two peptides (the dipeptide ester Z‐Ac₁₀c‐l ‐Phe‐OMe and the tripeptide ester Z‐Aib‐Ac₁₀c‐Aib‐OtBu) were determined in the crystal state using X‐ray diffraction. The experimental results support the view that β‐bends and 3₁₀‐helices are preferentially adopted by peptides rich in Ac₁₀c, the third largest cycloaliphatic C^α,α‐disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1‐amino‐cycloalkane‐1‐carboxylic acid (Ac_nc, with n = 3–12) series, which represents the prerequisite for our recent proposal of the ‘Ac_nc scan’ concept.     

Bioactive N‐terminal undecapeptides derived from parathyroid hormone: the role of α‐helicity*

Abstract: The N‐terminal 1–34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N‐terminal fragments presenting the principal activating domain such as PTH(1–11) and PTH(1–14) with helicity‐enhancing substitutions yield potent analogues with PTH(1–34)‐like activity. To further investigate the role of α‐helicity on biological potency, we designed and synthesized by solid‐phase methodology the following hPTH(1–11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix‐promoting C^α‐tetrasubstituted α‐amino acids α‐amino isobutyric acid (Aib), 1‐aminocyclopentane‐1‐carboxylic acid (Ac₅c) and 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c): Ac₅c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( I ); Aib‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( II ); Ac₆c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( III ); Aib‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IV ); Aib‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( V ); S‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VI ), S‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VII ); Ac₅c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VIII ); Ac₆c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IX ); Ac₅c‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( X ); Ac₆c‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( XI ). All analogues were biologically evaluated and conformationally characterized in 2,2,2‐trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I – V , which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand‐stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III , VI and VII are very weakly active and analogues IV , V , VIII–XI are inactive. The most potent analogue, I exhibits biological activity 3500‐fold higher than that of the native PTH(1–11) and only 15‐fold weaker than that of the native sequence hPTH(1–34). Remarkably, the two most potent analogues, I and II , and the very weakly active analogues, VI and VII , exhibit similar helix contents. These results indicate that the presence of a stable N‐terminal helical sequence is an important but not sufficient condition for biological activity.     

Amphipathic control of the 310‐/α‐helix equilibrium in synthetic peptides

Abstract: A series of short, amphipathic peptides incorporating 80% C^α,C^α‐disubstituted glycines has been prepared to investigate amphipathicity as a helix‐stabilizing effect. The peptides were designed to adopt 3₁₀‐ or α‐helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3₁₀‐helix in peptides containing amino acids with large side‐chains, as with 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3₁₀‐helix.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

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.     

Crystallographic characterization of conformation of 1-aminocyclopropane-1-carboxylic acid residue (Ac3c) in simple derivatives and peptides*

The molecular and crystal structures of the C^α,α-dialkylated α-amino acid residue 1-aminocyclopropane-1-carboxylic acid hemihydrate (H₂^⊕-Ac₃c-O^?·½ H₂O) and nine derivatives and dipeptides have been determined by X-ray diffraction. The derivatives are pBrBz-Ac₃c-OH, Piv-Ac₃c-OH, Z-Ac₃c-OH, the α- and β-forms of t-Boc-Ac₃c-OH, Z-Ac₃c-OMe, and the 5(4H)-oxazolone from pBrBz-Ac₃c-OH; the dipeptides are H-(Ac₃c)₂-OMe and c(Ac₃c)₂. The values determined for the torsion angles about the N-C^α (φ) and C^α-C′ (φ) bonds for the single Ac₃c residue of Piv-Ac₃c-OH, the α- and β-forms of t-Boc-Ac₃-OH and Z-Ac₃c-OMe, and the C-terminal Ac₃c residue of H-(Ac₃c)₂-OMe correspond to folded conformations in the “bridge” region of the Ramachandran map. The structures of pBrBz-Ac₃c-OH and Z-Ac₃c-OH, however, are unusual in having a semi-extended conformation for the φ,ψ angles. The N-terminal Ac₃c residue of H-(Ac₃c)₂-OMe adopts a novel type of C₅ conformation, characterized inter alia by an (amino) N ? H-N (peptide) intramolecular hydrogen bond. While the acyl N^α-blocking groups form trans amides (pBrBz-Ac₃c-OH and Piv-Ac₃c-OH), the urethane groups may adopt either the trans [Z-Ac₃c-OH and t-Boc-Ac₃c-OH(α-form)] or the cis amide conformations [t-Boc-Ac₃c-OH(β-form) and Z-Ac₃c-OMe]. The five- and six-membered rings of the 5(4H)-oxazolone and the 2,5-dioxopiperazine, respectively, are planar. The four independent molecules in the asymmetric unit of the free α-amino acid are zwitterionic.     

Conformational studies on host-guest peptides containing chiral α-methyl-α-amino acids

The conformational behaviour of host-guest peptides of the type Ac-Ala-Xxx-Ala-Ala-Xxx-Ala-Ala-Xxx-Ala-Ala-NH-PEGM (Xxx =α-aminoisobutyric acid (Aib), (S)-2-ethylalanine ((S)-Iva). (S)-2-methyiserine ((S)-α-MeSer)) has been studied by CD spectroscopy in CF₃CH₂OH. CH₃OH. and water and by i.r. spectroscopy in CHCl₃ and in the solid state. In this way the relative helix-inducing potential of the two chiral α-methyl-α-amino acids (S)-Iva and (S)-α-MeSer could be established in comparison to the strong helix-former Aib. The results show that (S)-Iva exerts a comparable helix-inducing effect as Aib, making this amino acid a valuable complementary tool for the stabilization or induction of helices. No significant helix-promoting effect was observed for (S)-α-MeSer in polar solvents; however, the i.r.-spectroscopic data in CHCl₃ and in the solid state point to a helical conformation under these conditions. Possible reasons for the different behaviour of (S)-Iva and (S)-α-MeSer are briefly discussed.     

(αMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides

Abstract: Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, C^α‐methylated α‐amino acid l ‐(αMe)Nva with a short, linear side‐chain. A set of terminally protected model peptides to the pentamer level containing either (αMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (αMe)Nva peptides were also synthesized using side‐chain hydrogenation of the corresponding C^α‐methyl, C^α‐allylglycine (Mag) peptides. A detailed solution and crystal‐state conformational analysis based on FT‐IR absorption, ¹H NMR and X‐ray diffraction techniques allowed us to define that: (i) (αMe)Nva is an effective β‐turn and 3₁₀‐helix former; and (ii) the relationship between (αMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. l ‐(αMe)Nva induces the preferential formation of right‐handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on α‐amino acids with a C^α‐methyl substituent and a C^α‐linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Chiral recognition in dipeptides containing 1-aminocyclopropane carboxylic acid or α-aminoisobutyric acid: NMR studies in solution

Protected dipeptides containing 1-aminocyclopropane carboxylic acid (Ac₃c) or α-aminoisobutyric acid (Aib) residues at the C-terminus and Phe, Val or Ala residues at the N-terminus displayed different proton NMR spectra for the pure enantiomers and the racemic mixtures in deuterochloroform (CDCl₃) solution. An unequal mixture of enantiomers showed two sets of resonances (NMR nonequivalence), one corresponding to major and the other to minor enantiomer. The NMR nonequivalence was originated by the presence of the C-terminal Ac₃c or Aib residues, which have been known for their unique spatial preferences in avoiding an extended (C₅) conformation. When a C₅ conformation favoring residue such as glycine was incorporated in place of Ac₃c or Aib, negligible NMR nonequivalence was observed. The magnitude of the NMR nonequivalence depended on the side chain as well as on the protecting groups at N-terminus α-amino acid. For the same peptide, the magnitude of nonequivalence increased with increasing solution concentration and/or with decreasing the solution temperature. The NMR nonequivalence disappeared in polar solvent-like deuterated dimethylsulfoxide (DMSO-d₆). A preference for hetero-chiral recognition leading to dimeric association under fast exchange conditions had been invoked to explain the observed phenomenon. The dipeptides thus prepared could well serve as ‘model peptides’ for the evaluation of any preparative methods.     

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.     

Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c)

The crystal structures of four peptides incorporating l-aminocycloheptane-l-carboxylic acid (Ac₇c) are described. Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe adopt β-turn conformations stabilized by an intramolecular 4 × 1 hydrogen bond, the former folding into a type-I/III β-turn and the latter into a type-II β-turn. In the dipeptide esters, Boc-Aib-Ac₇c-OMe and Boc-Pro-Ac₇c-OMe, the Ac₇c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac₇c-OMe found in the asymmetric unit. The cycloheptane ring of Ac₇c residues adopts a twist-chair conformation in all the peptides studied. ¹H-NMR studies in CDCl₃ and (CD₃)₂SO and IR studies in CDCl₃, suggest that Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe maintain the β-turn conformations in solution.     

Conformational studies on model dipeptides of Gly,L -Ala and their Cα-substituted analogs

As a part of the development of conformational guidelines for the design of metabolically altered peptidomimetics, we present conformational energy calculations on model dipeptide compounds with glycine (Gly), L-alanine (Ala), α-aminoisobutyric acid (Aib), L-tert-butylglycine (Tle), L-phenylglycine (Phg), (α,α)-diphenylglycine (Dφg), L-2-aminobutyric acid (Abu), 2-amino-2-ethylbutync acid (Deg), L-2-amino-2-vinylacetic acid (Ava) and (α,α)-divinylglycine (Dvg). The energy calculations have been made using molecular mechanics methods with a force field derived from MM2. The salient features are expressed in terms of conformational energy plots, drawn as a function of the backbone torsion angles φ(C_i-1′-N_i-C_i^α-C_i′) and ψ(N_i- C_i^α-N_{i + 1}). The low-energy structures of these compounds are qualitatively consistent with the X-ray crystal structure analyses of peptides and peptidomimetics. They are also in agreement with the results of the solution-phase studies carried out by NMR and IR techniques. The results obtained have important implications in the design of conformationally restricted peptidomimetics.     

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.     

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs

Analogs of the Saccharomyces cerevisiaeα-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys⁷ and Gln¹⁰ were replaced with Cys, Cys(CH₃), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo^7,10[Cys⁷,X⁹,Cys¹⁰,Nle¹²]α-factor, where X = D-Val, D-Ala, l -Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20–30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys⁷ and Gln¹⁰ with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5–10-fold more active than their sulfhydryl-contaihing homologs, and cyclo^7,10[Cys⁷,L-Ala⁹,Cys¹⁰,Nle¹²] α-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH₃) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the α-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form β-turns spanning residues 7–10 than their less active linear counterparts. The results provide strong evidence that this β-turn is important for optimal signal transduction by α-factor.     

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.     

(αMe)Aun: a highly lipophilic,chiral, Cα‐tetrasubstituted α‐amino acid. Incorporation into model peptides and preferred conformation

Abstract: Using a chemo‐enzymatic approach we prepared the highly lipophilic, chiral, C^α‐methylated α‐amino acid (αMe)Aun. Two series of terminally protected model peptides containing either d ‐(αMe)Aun in combination with Aib or l ‐(αMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT‐IR absorption, ¹H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its α‐carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that d ‐(αMe)Aun favors the formation of the left‐handed 3₁₀‐helical conformation.     

Conformation—activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating l-aminocycloalkane-l-carboxylic acid (Ac_nc) and α, α-dialkylglycines (Deg, diethylglycine; Dpg, N, N-dipropylglycine and Dbg, N, N-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx = Ac₇c. I: Ac₈c. II: Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded β-turn conformations in CDCl₃, and (CD³)₂SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C₅) conformations in solution. Peptides I-V exhibit high activity in inducing β-glucosaminidase release from rabbit neutrophils, with ED₅₀ values ranging from 1.4–8.0 × 10–¹¹. M. In human neutrophils the Dxg peptides III-V have ED₅₀ values ranging from 2.3 × 10^?8 to 5.9 × 10^?10 M, with the activity order being V>IV>III. While peptides I-IV are less active than the parent. For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors. © Munksgaard 1996.     

A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1HNMR assignments and their application in solid-phase glycopeptide synthesis

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, N^α-Fmoc-Ser-[Ac₄-β-d -Gal-(1-3)-Ac₂α or β-d -GalN₃]-OPfp and N^α-Fmoc-Thr-[Ac₄-β-d -Gal-(1-3)-Ac₂-α or β-d -GalN₃]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. N^α-Fmoc-Ser-OPfp or N^α-Fmoc-Thr-OPfp) with protected β-d -Gal(1-3)-d -GalN₃ synthon mediated by silver salts resulted in only α-and/or β-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO₄ at -40°C which afforded α-isomer in a quantitative yield (α:β= 5:1). On the other hand, the β-isomer was formed exclusively when the reaction was performed in the presence of Ag₂CO₃AgClO₄ at room temperature. A complete assignment of ¹H resonances to individual sugar ring protons and the characteristic anomeric α-1H and β-1H in Ac₄Galβ(1-3)Ac₂GalN₃α and/or β linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned a- and p-isomers of the O-glycosylated amino acid building blocks carrying protected β-d -Gal(1-3)-d -GalN₃, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic     

Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitors

A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural l -amino acids (except for cysteine), plus the non-natural l -amino acids ornithine and norleucine. The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN′, α-chymotrypsin, trypsin, anhydro-α-chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to α-chymotrypsin, two of which also bind to anhydro-α-chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K₁) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K₁ values. In the case of the α-chymotrypsin inhibitors, the peptides with the lowest K_i (0.1–0.05 mM) were the only peptides that bound to anhydro-α-chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that α-chymotrypsin and trypsin are inhibited by these cyclic peptides.     

AMINONITRILES AND AMINOTHIOAMIDES RELATED TO NATURAL AMINO ACIDS

N-p-Methoxybenzyloxycarbonyl and N-tert.-butyloxycarbonyl amino acid amides related to a series of natural amino acids were dehydrated to the corresponding Meoz- and Boc-α-aminonitriles. Deprotection of the latter derivatives afforded α-aminonitriles related to alanine, tyrosine, phenylalanine, dihydrophenylalanine, histidine, Dopa, ornithine, asparagine and glutamine. Thioamidation with H₂S/NH₃ or H₂S/NEt₃ in general converted the protected amino nitriles to Meoz- and Boc-α-aminothioamides. When deprotected these furnished the α-aminothioamides corresponding to alanine, tyrosine, phenylalanine, dihydrophenylalanine and histidine. For dehydration and thioamidation of histidine and Dopa, Nα-Boc-^im trityl-histidine and N-Boc-0, 0′-diacetyldihydroxyphenylalanine were useful. Dopa was obtained as the free and Boc-thiohydrazide. Also prepared were Nα,ω -diMeoz-ornithine DCHA, Meoz-2,5-dihydrophenylalanine DCHA and N,0-diMeoz-tyrosine as starting materials and N,0-dicarbobenzyloxycarbonyltyrosinamide, N,0-diZ-tyrosine nitrile and Z-β-cyano-β-alaninamide as model compounds. During deprotection of Meoz-alanine thioamide, transfer of an anisyl group from the N-Meoz protecting group to sulfur took place as a side reaction that yielded alanine p-methoxybenzyl β-imidothiolic ester. This study provides two new series of amino acid analogs with potential antimetabolite activity. Also suitable for incorporation into peptide analogs, these afford approaches to relating structure and conformation to activity in biologically active peptides.