Proton,calcium, and magnesium binding by peptides containing γ-carboxyglutamic acid

γ-Carboxyglutamic acid (Gla) is believed to bind Ca [II] ions and Mg [II] ions in prothrombin and other coagulation proteins. Binding constants for H ⁺, Ca [II] ions, and Mg [II] ions to Gla-containing peptides are determined using pH and ion selective electrode titrations. The binding constants for peptides containing a single Gla residue are similar to the constants for malonic acid. Peptides containing two Gla residues in sequence (di-Gla peptides) bind Ca [II] ions and Mg [II] ions more strongly. K_MgL for the di-Gla peptides is similar to the site-binding constant for Ca [II] ions in denatured BF1. These di-Gla peptides may be useful analogs for metal binding by the disordered Gla domain in BF1.     

SYNTHESIS OF γ-CARBOXYGLUTAMIC ACID AND ITS DERIVATIVES VIA MANNICH-BASE CONDENSATION

This report describes the synthesis of N^α-formyl and N^α-benzyloxycarbonyl-γγ-di-t-butyl-γ-carboxyglutamic acid via Mannich-base condensation starting from di-t-butyl malonate and diethyl formamido or benzyloxycarbonylamidomalonate.     

Binding of calcium to synthetic peptides containing γ-carboxyglutamic acid

The Ca²⁺ binding properties of various γ-carboxyglutamic acid (gla)-containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca²⁺-selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca²⁺, with some peptides probably involved in intermolecular bridging of the Ca²⁺. The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g-atom of Ca²⁺ interacts with 2 mol of peptide (n= 0.5) with a K_d value of 1.6 mM. A second g-atom of Ca²⁺ interacts with 2 mol of this same peptide (n= 0.5) and is characterized by a K_d of 8.8 mM. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (n= 2.0)for Ca²⁺ of K_d=16.1 mM, as well as a tighter site (n= 1), Of K_d= 0.4 mM. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38-residue peptide that possesses all nine of the gla-residues of protein C in their proper sequence positions. The strongest Ca²⁺ binding sites (n= 3–4) possess an average K_d of 0.4 mM, followed by another class of sites (n= 5–10, average K_d= 1.5–3.0 mM). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca²⁺ to the gla region of prothrombin fragment 1. By selective [¹³C]-labeling of the essential gla 16 residue of the 38-mer peptide, we demonstrate that this particular gla residue participates as a donor for a high-affinity Ca²⁺ site. These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca²⁺ to intact gla-containing proteins.     

β-Endorphin

A double-headed analog of human β-endorphin (β_h-EP), N, N'-bis (β-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with β_h-EP-Cys(CH₂CONH₂)-OH (I) and (Tyr³¹]-β_h-EP (III). Their relative potencies in a radioreceptor-binding assay were: B_h-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: β_hEP, 100; II, 86; I, 93; and III, 116.     

Synthesis of N-tert.-butoxycarbonyl(α-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide α-carboxamides

N-tert.-butoxycarbonyl-aminomethyl(α-phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid-phase synthesis of peptide α-carboxamides. This handle was prepared with an 82% yield when N-tert.-butoxycarbonyl-(p-hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C-terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH₂Cl₂ (< 1% loss after 10h). Upon treatment for 30min with HF:anisole(9:1) at 0°, 92% cleavage of glycinamide from Glyhandle-resin was obtained. In a test synthesis of a peptide α-carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.-butoxycarbonyl-aminomethylphenoxyacetic acid and N-tert.-butoxycarbonyl-aminornethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid-phase synthesis.     

β-Endorphin

Ostrich β-endorphin has been synthesized by the solid-phase method. Opiate activity in a radioreceptor binding assay is about seven times that of human β-endorphin. Structural differences within positions 6–15 account for the increased binding potency.     

Homologation of α-amino acids to β-amino acids using Fmoc-amino acid pentafluorophenyl esters

The homologation of α-amino acids to β-amino acids by the two-step Arndt–Eister method is achieved by using Fmoc-α-amino acid pentafluorophenyl esters for the acylation of diazomethane, synthesizing the key intermediates Fmoc-aminoacyldiazomethanes as crystalline solids in good yields and purity.     

Synthesis and properties of human β-endorphin-(1–17) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–17) (I), [D-Ala²]-β_h-EP-(1–17) (II), [Gln⁸]-β_h-EP-(1–17) (III) and [D-Ala², Gln⁸]-β_h-EP-(1–17) (IV). Measurement in a radio-receptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 33; II, 47; III, 889; IV, 123; β_h-endorphin, 2253.     

Chemical synthesis of a designed β-protein through the flow-polyamide method

A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a β-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70%β structure, Accordingly, it was anticipated, and actually found, to be a “difficult sequence” for all its length. Comparison of the standard, “minimum redundancy” protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected IonSpray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.     

Preparation and properties of Nα-Bpoc-amino acid pentafluorophenyl esters

The preparation and properties are reported of several N^x-Bpoc -amino acid pentafluorophenyl esters, including those bearing tert-butyl-, allyl- and trityl-based protecting groups. These derivatives have been used in the solid-phase peptide synthesis of sevral short peptides.     

Convenient and efficient synthesis of Boc‐/Z‐/Fmoc‐β‐amino acids employingN‐protected α‐amino acid fluorides

Abstract: A new and efficient method for the synthesis ofN^α‐Fmoc‐/Boc‐/Z‐β‐amino acids using the two‐step Arndt‐Eistert approach is described. Fmoc‐/Boc‐/Z‐α‐Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc‐/Boc‐/Z‐α‐aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding β‐amino acids using PhCOOAg/dioxane/H₂O.     

β-ENDORPHIN: SYNTHESIS AND RADIOLIGAND BINDING ACTIVITY OF ANALOGS CONTAINING CYSTINE BRIDGES

Two analogs of human β-endorphin containing cystine bridges between positions 21 and 26 or 14 and 26 have been synthesized and their radioligand binding activity using rat brain membranes has been determined. Both peptides showed three to four times the binding activity of human β-endorphin.     

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.     

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.     

β-ENDORPHIN: SYNTHESIS AND RADIORECEPTOR BINDING ACTIVITY OF βh-ENDORPHIN-(1–27) AND ITS ANALOGS

β_h-Endorphin-(1–27) (I), [Ac-Tyr¹]-β_h-endorphin-(1–27) (II), [Gln⁸]-β_h-endor-phin-(1–27) (III), and [Ac-Tyr¹, Gln⁸]-β_h-endorphin-(1–27) (IV) were synthe sized by the solid-phase method. The binding potency of I-IV to rat brain membrane preparations was measured by radioreceptor binding assay. The relative potencies were: β_h-endorphin, 100; I, 30; II, 0.04; III, 90; IV, 0.07.     

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.     

Susceptibility of glycans to β-elimination in Fmoc-based O-glycopeptide synthesis

order to investigate the possible extent of β-elimination occuring in Fmoc-based continuous-flow solid-phase glycopeptide synthesis, the influence of the pK_b of the base used for N^α-deprotection has been studied. A glycosylated pentapeptide as synthesized using 50%morpholine, 10%, piperidine or 2% DBU, respectively, in DMF for deprotection. The dehydropentapeptide N^α-.Ac-Thr-Thr-δAba-Val-Thr-NH₂, which would be formed in the case of β-elimination, was prepared independently and used as a control in HPLC analysis; however, this product was not formed under any of the deprotection conditions applied. Furthermore, a 23 amino acid long glycopeptide from human intestinal mucin was prepared using 2% DBU as a base for Fmoc cleavage, and similarly no β-elimination was observed. The glycopeptide products were subjected to a prolonged treatment with sodium hydroxide in methanol/water without significant formation of byproducts, and the pure glycopeptides were isolated and characterized by ¹H-NMR spectroscopy.     

Solid phase synthesis of human growth hormone-releasing factor analogs containing a bicyclic β-turn dipeptide

Three analogs derived from the N-terminal 29-residue fragment of human growth hormone-releasing factor (hGRF) which contained a bicyclic β-turn dipeptide (BTD) at 7-8,8-9, and 9-10 positions were synthesized by solid phase methodology to ascertain if the β-turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-response curves to that of hGRF(1-29)NH₂, except their relative potencies were very low.     

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.     

Variation of amino acid properties in protein secondary structures, α-helices and β-strands

A study was made on the physical, chemical, energetic, conformational, geometric, and dynamic property potentials of amino acid residues in protein secondary structures: α-helix and β-strand. Property patterns were obtained by computing the average property values for specified residue units partitioned longitudinally and transversely about the chain. It was found that in r-helices with not more than 15 residues, there exist longitudinally opposing portions, one characteristically higher in average property potentials than the other. The helical chain, in general, acquires either an increasing or decreasing average potential in the N-terminal to C-terminal direction. The sequence-wise and surface-wise variations of property potentials in the elements of β-structure also revealed such general patterns. Possible wrong predictions in statistical methods of one secondary structural class over the other are pointed out.