Protected Tetrapeptide Research Articles

Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2

For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1H and 15N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B1 and B2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1H and 15N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For Hα and HN correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.

Pressure dependence of side chain 13C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of 13C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200MPa. The size of the polynomial pressure coefficients B 1 and B 2 is dependent on the type of atom and amino acid studied. For HN, N and Cα the first order pressure coefficient B 1 is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically 13C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.

Pressure dependence of backbone chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

For a better understanding of nuclear magnetic resonance (NMR) detected pressure responses of folded as well as unstructured proteins the availability of data from well-defined model systems are indispensable. In this work we report the pressure dependence of chemical shifts of the backbone atoms (1)H(α), (13)C(α) and (13)C' in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of these nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200MPa. The polynomial pressure coefficients B 1 and B 2 are dependent on the type of amino acid studied. The coefficients of a given nucleus show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure are also weakly correlated.

Pressure response of amide one-bond J-couplings in model peptides and proteins.

The pressure dependence of the one-bond indirect spin-spin coupling constants (1)J(N-H) was studied in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (with Xxx being one of the 20 proteinogenic amino acids). The response of the (1)J(N-H) coupling constants is amino acid type specific, with an average increase of its magnitude by 0.6 Hz at 200 MPa. The variance of the pressure response is rather large, the largest pressure effect is observed for asparagine where the coupling constant becomes more negative by -2.9 Hz at 200 MPa. The size of the J-coupling constant at high pressure is positively correlated with its low pressure value and the β-propensity, and negatively correlated with the amide proton shift and the first order nitrogen pressure coefficient and the electrostatic solvation free energy.

Synthesis of enzymatically crosslinkable peptide-poly(L-lysine) conjugate and creation of bio-inspired hybrid fibers.

Poly(L-lysine)s having an Nepsilon-substituted tetrapeptide, Lys-Gly-Tyr-Gly, were synthesized by the coupling of the protected tetrapeptide active ester, Boc-Lys(Z)-Gly-Tyr(Bzl)-Gly (4-hydroxyphenyl)dimethylsulfonium methylsulfate and Nepsilon-group of the poly(L-lysine) side chain. The Nepsilon-substituted tetrapeptide functions as the substrate of tyrosinase and is responsible for the enzyme-mediated interpolymer cross-linking. The degree of Nepsilon-substitution (DS) was mostly controlled by changing the stoichiometry between the Nepsilon-amino groups of the parent poly(L-lysine) and the protected tetrapeptide active ester. Two kinds of samples having DS values of 8.6 and 18 mol-% were prepared. The resulting cationic Nepsilon-(Lys-Gly-Tyr-Gly)-poly(L-lysine) (abbreviated as PLL(GYGK)) was spun into hybrid fibers with the anionic polysaccharide gellan via a polyionic complexation reaction at the interface between aqueous solutions of the two polymers. The mechanical strengths of the PLL(GYGK)-gellan hybrid fibers were superior to those of the original poly(L-lysine)-gellan fibers. The mechanical strength of the hybrid fibers further increased upon the tyrosinase-mediated cross-linking reaction of the PLL(GYGK). This result indicates that the covalent cross-bridge formation between the Nepsilon-substituted peptides significantly contributed to reinforcement of the hybrid fibers. The present study affords a new methodology for reinforcement inspired by a biological process.

Synthesis and Evaluation of Enantiomeric Purity of Protectedα-Amino and Peptide Aldehydes

The synthesis of enantiomerically pure Ac-Tyr-Val-Ala-Asp(OtBu)-H dimethyl acetal ((S)-1) is reported, a protected tetrapeptide C-terminal aldehyde belonging to a class of potent, reversible inhibitors of cysteine proteases (e.g., interleukin-1β-converting enzyme (ICE), also called caspase-1). The coupling of the precursors Ac-Tyr-Val-Ala-OH ((S)-8) and H-Asp(OtBu)-H dimethyl acetal ((S)-6) gave (S)-1 in a yield of 85%, with epimerization of <2% at the alanine and aspartic-acid residue. (S)-6 itself was synthesized in four steps in an overall yield of 83% with an ee >98%.

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin.

A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by 1H, 1H-1H, 13C and 1H-13C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.

Solid-Phase Synthesis of Tyrosine Peptide Aldehydes. Analogues of (S)-MAPI.

We report an efficient solid-phase synthesis of C-terminal tyrosine peptide aldehydes based on the HIV protease inhibitors (S)-MAPI and GE 20372 A. Our strategy consisted of anchoring the side chain of Dde-Tyrosinol (5) onto the brominated Wang linker derivative ((4-bromomethyl)-phenoxy-allyl acetate) (6) to give after ester hydrolysis the N(alpha)-(Dde)-O-(4-methylphenoxyacetic acid)-L-Tyrosinol template (8). This was attached to aminomethyl resin and elongated using standard Fmoc protocols. Importantly there was no evidence of esterification side reactions. The unsymmetrically substituted urea linkage of the (S)-MAPI family was incorporated using the N(alpha)-(4-nitrophenyloxycarbonyl)amino acid tert-butyl esters following which the protected tetrapeptide alcohol immobilized on the solid support was oxidized to its corresponding aldehyde using sulfur trioxide-pyridine. The efficiency and reliability of the oxidation step was dramatically improved by the incorporation of a small PEG-spacer between the linker and the solid support. The tetrapeptides 12a and 12b were cleaved by acidolysis, purified by RP HPLC, and isolated in high yield and purity, demonstrating the success of the whole synthetic process.

Unusual conformations adopted by standard amino acids in Aib-containing oligopeptides

Abstract The structures of the protected tetrapeptides Z-Aib-Aib-Aib-Val-OtBu (I) and Z-Val-Aib-Aib-Gln-OtBu (II), which contain the conformationally constrained residue α-aminoisobutyric acid (Aib), have been studied by X-ray crystallography. Both molecules in the asymmetric unit of I adopt left-handed 310-helical conformation, stabilized both by two 4 → 1 intramolecular hydrogen bonds. The structure of II consists of a β-turn of type II and a consecutive β-turn of type III′ and is extended at the C-terminus. This structure is stabilized by three intramolecular hydrogen bonds. All non-Aib residues (Val, Gln) in I and II adopt rather unusual backbone conformations compared with other helical structures in proteins.

Three protected tetrapeptides.

The structures of three protected tetrapeptides, containing the Boc-Gly-Gly-Phe-X-OMe chain, tert-butoxycarbonyl-glycy-glycl-phenylalanine-leucine methyl ester dihydrate, Boc-Gly-Gly-L-Phe-D-Leu-OMe, C25H38N4O7.2H2O, tert-butoxycarbonyl-glycy-glycl-phenylalanine-methionine methyl ester dihydrate, Boc-Gly-Gly-L-Phe-D-Met-OMe, C24H36N4O7S.2H2O and tert-butoxycarbonyl-glycy-glycl-phenylalanine-norleucine methyl ester dihydrate, Boc-Gly-Gly-D-Phe-L-Nle-OMe, C25H38N4O7.2H2O, are described. The three molecules have the same conformation on the tetrapeptide chain and display the same packing, consisting of couples of molecules linked head-to-tail by two hydrogen (N--H...O) bonds; other hydrogen bonds, also involving two water molecules of crystallization, link these couples together and give rise to a planar structure.

Effect of a hydrophobic amino acid at position (i −1) on the stability of β-turns in hydrophilic pentapeptides as studied by NMR and molecular mechanics

A detailed NMR study of the peptide NAc-FDEKA-NH2 in aqueous and in CD3OH/H2O solutions as well as the N-acetylpentapeptide amides YDEKA, VDEKA, GDEKA, and the protected tetrapeptide NAc-DEKA-NH2 in methanolic solutions indicates the importance of the first amino acid (at i −1) on stabilizing the type I β-turn. The data illustrate the hydrophobic stabilization of this turn, which is present in FDEKA, YDEKA, and VDEKA. For GDEKA and DEKA, the NMR data indicate that this turn is not present. Molecular mechanics calculations support this conclusion and indicate that for FDEKA and GDEKA the type I β-turn is distorted in both the vacuum and the solvated structures. For the solvated structures, the Cα(i) − Cα(i + 3) distance is 4.87 Å for FDEKA and 6.00 Å for GDEKA, which are to be compared with the value of 4.64 Å for an ideal type I β-turn, i.e., the distortion is far greater in GDEKA than in FDEKA. The calculations can be interpreted to indicate the presence of two major conformations in solution. Keywords: β-turns, FDEKA, pentapeptide.

The crystal structure of Z-Leu-Aib-Pro-Leuol, the synthetic, protected C-terminal tetrapeptide of trichovirin

Abstract The structure of the synthetic, protected tetrapeptide Z-Leu-Aib-Pro-Leuol(C29H46O6N4, Mw = 546.71) which contains the conformationally constrained residue α-aminoisobutyric acid (Aib), has been determined by X-ray crystallography. The title compound comprises the C-terminal tetrapeptide of trichovirin and has been chosen as a first step in a systematic analysis of the trichovirin structure-sequence relationships. Furthermore, the tetrapeptide has an interesting amino acid sequence, containing a strong helix former (Aib) and a helix breaker (Pro). The title compound crystallizes in P21 with a = 10.523(1) Å, b = 23.033(5) Å, c = 13.462(4) Å, β = 91.81(1)°, V = 3261(2) Å3, Z = 4, Dcalc = 1.135 g cm−3 (R = 0.054 for 3572 observed reflections). Both independent molecules in the asymmetric unit form a β-turn of type I with a 4 → 1 intramolecular hydrogen bond; the overall folding of each molecule fits approximately the pattern of a right-handed 310-helix, although the conformational angles φ, ψ of the Leu residues deviate significantly from the standard helical values. In the crystal the molecules are hydrogen-bonded head-to-tail forming infinitely long, helical columns along the a-axis; these columns are associated by hydrogen bonds forming layers parallel to the ac-plane.

Nα-Benzyloxycarbonyl-α-aminoisobutyrylglycyl-L-isoleucyl-L-leucine methyl ester monohydrate

(Z)-Aib-Gly-L-Ile-L-Leu-OMe.H2O, C27H42N4O7.H2O, is a protected analogue of the C-terminal sequence of the membrane-active peptaibol antibiotic trichogin A IV. The peptide backbone is folded. The urethane carbonyl O atom acts as the acceptor of two intramolecular hydrogen bonds which give rise to a beta bend and to an alpha bend. The geometry of the latter is significantly distorted from that observed in alpha helices. This structure represents the first observation of an alpha bend in a protected tetrapeptide sequence.

CCK-B agonist or antagonist activities of structurally hindered and peptidase-resistant Boc-CCK4 derivatives.

Replacement of Met31 by (N-Me)Nle in CCK8 or CCK4 has been shown to improve the affinity and selectivity for CCK-B receptors. In order to obtain molecules with enhanced bioavailability, two novel series of protected tetrapeptides of the general formula Boc-Trp30-X-Asp-Y33 have been developed. Introduction of (N-Me)Nle and the bulky, aromatic naphthylalaninamide (Nal-NH2) in positions X and Y, respectively, does not greatly modify the affinity for guinea pig brain CCK-B receptors. In contrast, incorporation of hindering N-methyl amino acids such as (N-Me)Phe, (N-Me)Phg, or (N-Me)Chg, but not their non-methylated counterparts, in position X induced a large decrease in affinity for the CCK-B binding sites. Among the various peptides synthesized, Boc-[(N-Me)Nle31,1Nal-NH2(33)]CCK4 (2) (KI = 2.8 nM), Boc-[Phg31,1Nal-NH2(33)]CCK4 (15) (KI = 14 nM), and Boc-[Phg31,1Nal-N(CH3)2(33)]CCK4 (17) (KI = 39 nM) displayed good affinities for brain CCK-B receptors and had good selectivity ratios. These pseudopeptides, in which the presence of unnatural and hydrophobic residues is expected to improve their penetration of the central nervous system, were shown to be very resistant to brain peptidases. Interestingly, whereas compounds 2 and 15 proved to be full agonists for rat hippocampal CCK-B receptors when measured in an electrophysiological assay, compound 17 behaved as a potent antagonist in the same test and displayed a good affinity in rat brain KI(CCK-B) = 51 nM as compared to the Merck antagonist L365,260,KI(CCK-B) = 12 nM. This illustrates a simple means to obtain CCK-B antagonists and suggests that the free, CONH2 group plays a critical role in the recognition of the agonist state of brain CCK-B receptors.

ChemInform Abstract: Synthesis of a Glycopeptide Containing the Amino Acid Sequence 4 to 7 of the NH2‐Terminus of the Protein Core‐Structure of the Beef‐Skin Proteodermatan Sulfate.

AbstractCoupling of the protected tetrapeptide (VIII) synthesized as shown in the reaction scheme with the xylose derivative (IX) and subsequent deprotection yield the title compound (Xb).

NMR studies of a free and protected tetrapeptide glycyl-L-prolylglycylglycine in beta-turn-supporting environment.

AbstractNmr studies of the protected and free tetrapeptide Gly‐Pro‐Gly‐Gly were carried out in β‐turn‐supporting solvents, that is, in CDCl3 for Z‐Gly‐Pro‐Gly‐Gly‐OMe and in Me2SO‐d6 for H‐Gly‐Pro‐Gly‐Gly‐OH. Comparisons with specifically α‐deuterated analogs gave complete assignments of the NH and methylene regions. Analysis of chemical shifts, coupling constants, and the temperature dependence of chemical shifts show that the peptide adopts a type II β‐turn conformation. This turn is stabilized for the protected tetrapeptide by two hydrogen bonds between (i) CO (Gly1) and NH(Gly4), and (ii) urethane function NH and methyl ester CO.

Solution synthesis of 14C‐labeled thymopoietin32–36 using catalytic transfer hydrogenation as the final deprotection step

AbstractA 14C‐labeled pentapeptide, [14C]Arg‐Lys‐Asp‐Val‐Tyr (6), corresponding to the active fragment 32–36 of thymopoietin II has been synthesized by a solution method with a 44 percent radiochemical yield. 14C‐labeled Z‐Arg(HCl)‐OH was coupled to the protected tetrapeptide Lys(Z)‐Asp(OBzl)‐Val‐Tyr(Bzl)‐OBzl using dicyclohexylcarbodiimide and 1‐hydroxybenzotriazole to give the protected pentapeptide 5 with a specific activity of 340 mCi/mmol. Catalytic transfer hydrogenation was used to remove the protecting groups and the free peptide was purified by gel filtration and high performance liquid chromatography.

A conformational study of the tetrapeptide CH3CO-Ala-Asp-Gly-Lys-NHCH3 corresponding to a β-bend in staphylococcal nuclease

The tetrapeptide sequence Ala-Asp-Gly-Lys occurs as a type I′ β-bend at residues 94–97 in staphylococcal nuclease. We have synthesized theN-acetyl,N′-methylamide derivative of this tetrapeptide and studied its conformation in solution, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. In the synthesis, special attention was paid to the possibility of cyclic aspartimide formation giving rise to mixtures of α- and β-Asp-Gly products. The presence of such a mixture was excluded by infrared, NMR, and other analytical procedures applied to the products and to models for α- and β-linked aspartyl residues. The CD spectra of the protected tetrapeptide in water, methanol, and trifluoroethanol show no evidence of preferred chain conformations. In dimethylsulfoxide-d 6 , however, the NMR spectra are consistent with the presence of a population of conformers in which the Lys and C-terminal NHCH3 amide protons are shielded from solvent. Taken together with the observed3JNH-CαH coupling constants for all residues, this permitted the construction and energetic evaluation of possible conformations in solution. Only one such conformation was fully compatible with the NMR data; this is a type II β-bend in which the Lys and C-terminal NHCH3 amide protons are close to the Ala C=O group and may form bifurcated hydrogen bonds with it. This conformation can be converted into the conformation existing in staphylococcal nuclease by rotating the plane of the Ala-Asp peptide group by about 120° around a line connecting the Ala and Asp Cα atoms and by making small shifts in dihedral angles elsewhere in the peptide.

Synthesis and Catalytic Activity of Poly-l-Histidyl-l-Aspartyl-l-Seryl-Glycine

Poly(His-Asp-Ser-Gly) was synthesized from the fully protected tetrapeptide active ester hydrochloride, which was prepared by stepwise coupling, using pentachlorophenyl ester and mixed anhydride methods. Complete deprotection of the protected tetrapeptide polymer was achieved by using 90% trifluoroacetic acid. The free polymer was dialyzed for 24hr using a membrane (which retains molecules with molecular weights >5000). The catalytic activity was determined by studying the hydrolysis of p-nitrophenyl acetate in 0.2M phosphate buffer (pH7.4) at 37°. The catalytic coefficient of the dialyzed polymer was found to be 138 liters/mole/min.

Preparation and properties of some citrulline p-nitroanilide derivatives for possible use as protease substrates

A number of citrulline p-nitroanilides have been synthesised as potential substrates for proteolytic enzymes. N ∝-Benzyloxycarbonyl- l-citrulline p-nitroanilide, a key starting material, was prepared by the phosphoazo method. During this reaction, depending on the conditions, lactam formation and decarbamoylation took place. It is probable that decarbamoylation took place subsequent to the lactamisation step. The derivatives prepared included some protected tripeptide nitroanilides, benzyloxycarbonylglycyl- l-prolyl- l-citrullin p-nitroanilide, bezyloxycarbonyl- d-phenylalanyl- lprolyl- l-citrulline P-nitroanilide, benzyloxycarbonylglycyl- l-phenylalanyl- l-citrulline p-nitroanilide, methyloxycarbonylglycyl- l-phenylalanyl- l-citrulline p-nitroanilide and a protected tetrapeptide, benzyloxycarbonylglycyl glycyl- l-phenylalanyl-- l-citrulline p-nitroanilide. Prelimnary results have indicated that citrulline pnitroanilides are far more susceptible to hydrolysis by plant thiol enzymes such as papain, ficin and bromelain than by mammaalian serine proteases.