HPLC and PDMS analysis of cleavage products aid in the design of small,stable ANP analogues

The degradation of a prototypical small analogue of atrial natriuretic peptide (ANP) has been studied using HPLC and mass spectrometric techniques. These studies revealed that removal of the N-terminal amino acid as the primary catabolic event in vitro. Based on this information the N-terminus was remanufactured to provide a family of more stable analogues. Additional stabilization was provided through modification of the C-terminal tripeptide. Though dramatically more stable in vitro, these new analogues do not appear to have longer in vivo half-lives.     

Opioid peptides Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) were replaced by N^α-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of μ, Δ, and κ opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the κ binding site. The introduction of N^α-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed μ/δ specificity.     

Cholecystokinic activity of Nα-hydroxysulfonyl-[Nle28,31]CCK26-33 analogues modified at the C-terminal residue

Three new analogues of Nα-hydroxysulfonyl-[Nle^28,31]CCK_26-33 are reported in which the C-terminal l -Phe³³ residue has been replaced by l -Leu, d -Phe or N-methyl-l -Phe. Biological evaluation in a series of binding and bioassays demonstrates that both l -stereochemistry and an aromatic side chain at position-33 are essential for full agonist activity. While the l -Leu³³ and d -Phe³³ analogues had reduced potencies in stimulating contraction of the guinea pig ileum or gall bladder, the d -Phe³³ analogue was fourfold selective for the ileum. This latter analogue also exhibited apparent partial agonism in the rat pancreatic amylase release assay. The N-methyl-l -Phe³³ analogue was almost equipotent to the parent analogue in all bioassays, suggesting that this modification might be useful for introducing enzymatic stability in CCK analogues.     

New fMLF-OMe Analogues Containing Constrained Mimics of Phenylalanine Residue

In the context of a research program aimed at elucidating the HCO-Met-Leu-Phe-OMe (fMLF-OMe) structural features which control interactions with the receptor in correspondence with the C-terminal residue, four different analogues of the native ligand have been synthesized and evaluated. Compounds 1-3 possess the general formula HCO-Met-Leu-Xaa-OMe with Xaa = N-benzylglycine, N-benzylphenylalanine, and α,α-dibenzylglycine, respectively. In the analogue 4 the constraint at the C-terminus has been obtained by incorporating a 2-oxopiperazine ring, made up of two phenylalanine residues, to replace the native C-terminal Phe residue. The consequences of the chemical modifications on the activity of the new analogues are discussed.     

Synthesis of potent agonists of Substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of Substance P

The analogues [Glp⁶,Glu(OBzl)¹¹]SP_(6-11) and [Glp⁵,Glu(OBzl)¹¹]SP_(5-11)) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH₂C₆H₅ ester group has replaced the SCH₃ group of the Met¹¹ side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively. © Munksgaard 1995.     

Synthesis,Antifolate and Anticancer Activities of N5‐Substituted 8,10‐Dideazatetrahydrofolate Analogues

Based on our previous work, seven N⁵‐substituted 8,10‐dideazatetrahydrofolate analogues and one 8‐deazatetrahydrofolate analogue were designed and synthesized as human dihydrofolate reductase (hDHFR) inhibitors. All compounds were assayed versus DHFR and five different cancer cell lines. The biological assay indicated that replacing N¹⁰ with carbon would significantly increase inhibitory activities against DHFR and cytotoxicities against cancer cell lines. Compound 19a with 4‐amino and N⁵‐formyl showed great antitumour activities against HL‐60, Bel‐7402 and BGC823 which were much better than MTX.     

ISOLATION AND CHARACTERIZATION OF HIGHLY PURIFIED ALPHA-1-ANTITRYPSIN

Highly purified human alpha-1-antitrypsin (phenotype MM) was obtained by an original method of preparative electrophoresis. The criteria of homogeneity were assured by one arc in crossed immunoelectrophoresis and one band on polyacrylamide gel. A unique N-terminal amino acid (pyroglutamic acid) and a unique C-terminal residue (lysine) were identified. Determined by gel electrophoresis, its molecular weight was 47,000 daltons.     

Synthesis,metabolic stability and chemotactic activity of peptide T and its analogues

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD₄ present on T₄ helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4–8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10^-11-10^-10M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4–8)-OH abolished the biological action. In the octapeptide series, whereas the α-amino butyric acid (Abu) substitution for Thr⁴ was well tolerated, the same “slight” structural change at Thr⁵ or Thr⁸ was very detrimental. Finally, [d -Asn⁶]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr⁴ does not interfere with biological characteristics of peptides. With regard to the enzymic degradation, the in vitro experiments showed the susceptibility of peptide T to rapid metabolism by human or rat plasma (T1/2 = 5.2 min), rat brain (T1/2 = 2.1 min) and kidney (T1/2 = 0.4 min) enzymes. The main metabolic product appears to be the C-terminal heptapeptide, which retains, in turn, a low enzymatic stability.     

4-Sulfobenzyl ester – an anionic protecting group for peptide synthesis

The preparation of the 4-sulfobenzyl esters of 18 amino acid derivatives is described. This carboxyl protecting group was introduced according to Hubbuch et al. (1980). The caesium or dicyclohexylammonium salts of N-terminal protected amino acids were reacted with 4-(bromomethyl)benzenesulfonate (1). After N-terminal deblocking, the amino acid-4-sulfobenzyl esters were isolated as zwitterions. The protecting group was removable by catalytic hydrogenation and by saponification. The 4-sulfobenzyl esters could be easily converted to amides and hydrazides. They were stable to 2 M hydrogen bromide in acetic acid as well as to a 10-fold excess of trifluoromethane sulfonic acid in trifluoro-acetic acid. The behaviours of ⁺H₂-Gly-Phe-Leu-OBzl-SO^-₃ and the corresponding methyl, benzyl and 4-nitrobenzyl esters were compared under various conditions.     

异硫氰酸荧光素标记人血清白蛋白

目的：用异硫氰酸荧光素（FTTC）对人血清白（HSA）进行荧光标记。方法：采用直接标记法,并考查不同料比时的荧光素分子与蛋白分子的结合情况,荧光标记物（FTTC－HSA）的荧光光谱及荧光寿命情况,结果：FTTC－HSA的激发波长为500nm,荧光波长为530nm;其固态和液态低温保存时荧光寿命分别为一年和37天。     

A new enkephalin analogue: trans-4-hydroxycinnamoyl-glycyl-glycyl-phenylalanyl-leucine. Synthesis and biological properties

A new analogue of the leucine-enkephalin in which the N -terminal tyrosine has been replaced by trans -4-hydroxycinnamic acid, has been synthetized by liquid-phase coupling methods. The central cardiovascular effects of this analogue were investigated and the results discussed.     

Isolation and sequence determination of trichorzianines A antifungal peptides from Trichoderma harzianum

Trichorzianines A, membrane active peptides of the peptaibol class, were isolated from cultures of the mould Trichoderma harzianum. Trichorzianines A were separated into pure components by HPLC on octadecyl bonded and SiO₂ phases successively. Nine trichorzianines A (IIa, IIIa, IIIb, IIIc, IVb, Vb, VIa, VIb and VII) were isolated from the complex microheterogeneous mixture. Their N-terminal amino acid is acetylated, the C-terminal amino alcohol is either tryptophanol or phenylalaninol, 7 to 8 of the 19 residues are α-aminoisobutyric acid. Gas chromatography on a chiral phase showed isovaline to have the d -configuration and all the other optically active amino acids and amino alcohols to have the l -configuration. The amino acid sequences were determined from their positive ion FAB mass spectra which exhibited the preferential cleavage of the Aib 12-Pro 13 amide bond as a main fragmentation. The resulting fragments subsequently underwent amide bond ruptures that generated two series of abundant acylium ions which enabled direct determination of the 1–19 sequence. The relative position of the isomeric amino acids in the sequence of trichorzianine AVII was assigned from analysis of the N- and C-terminal oligopeptides yielded by its selective acidic hydrolysis. The microheterogeneity of trichorzianines A results mainly from single or multiple substitution of amino acids at the specific positions 5, 14, 16 and 19.     

Acid-base properties and microspeciation of six angiotensin-type octapeptides

The acid-base chemistry of six angiotensin II analogues (A 11) was analyzed by determining two types of terms. Macroconstants were used to characterize the molecular proton-binding and dissociating ability of the octapeptides, and group constants to quantitate the submolecular basicity of each individual protona-tion site of the derivatives. The group constant values indicated that some sites (Arg-guanidino, Tyrphenolate, C-terminal carboxylate) were of similar basicity in the different analogues, while others (Hisimidazole, N-terminal amino) were significantly different. The group constant values are interpreted by taking into consideration the intramolecular effects of the adjoining moieties and are used for microspeciation.     

Binding affinities to rat brain synaptosomes – synthesis of biotinylated analogues of Substance P

The synthesis of four biotinylated analogues of Substance P is described. The affinities of these analogues and of their complexes with avidin for the ¹²⁵I-Bolton Hunter Substance P binding sites on rat brain synaptosomes were determined. While these biotinylated peptides complexed to avidin retain a good biological activity on the guinea-pig ileum bioassay, we observe a net decrease in their binding affinities in the central nervous system. The present study confirms that in the central nervous system the higher affinity is related to the N-terminal tetrapeptide and establishes that the free amino group (N-α-Arg and N-α-Lys) are not essential in the binding.     

GAMMA-MELANOTROPIN IS NOT PRESENT IN AN N-TERMINAL PEPTIDE OF SALMON PROOPIOCORTIN

The N-terminal peptide of salmon proopiocortin has been isolated and the primary structure including two disulfide bonds elucidated. The peptide consisted of 76 amino acid residues, which is 27 residues shorter than the bovine and human peptides. The N-terminal 44 residues of the teleost peptide exhibited significant sequence homology to those of the mammalian peptides. The salmon peptide, however, is lacking in the counterpart of gamma-MSH which is located between residues 51 and 64 in the mammalian peptides.     

Orthogonal solid-phase synthesis of human gastrin-I under mild conditions*

An approach to the solid-phase segment condensation synthesis of the 17-peptide amide human gastrin-I has been developed. N^α-amino and side-chain protection were provided by 9-fluorenylmethyloxycarbonyl (Fmoc) and tert.-butyl groups, and a series of anchors cleavable under mild conditions were used. The N-terminal pentapeptide pGlu-Gly-Pro-Trp-Leu-OH was prepared using a p-alkoxybenzyl ester linkage made by a preformed handle strategy. Cleavage, in 65% yield, was with the new Reagent M: CF₃ COOH—CH₂ Cl₂—β-mercaptoethanol-anisole (70:30:2:1), which was optimized to preserve the labile tryptophan residue. A new preformed handle procedure expedited solid-phase synthesis of the protected “middle” hexapeptide, Fmoc-(Glu(OtBu))₅-Ala-OH, anchored as an o-nitrobenzyl ester. Chains were not lost during this assembly, and final photolytic cleavage (350nm) in toluene—CF₃ CH₂ OH (4:1) occurred in 59% yield. Both protected intermediates were purified by simple gel filtration, whereupon they were shown to be pure by analytical HPLC, and gave satisfactory NMR and FABMS spectra. Last, the C-terminal hexapeptide, Tyr(tBu)-Gly-Trp-Met-Asp(OtBu)-Phe, was assembled on a tris(alkoxy)benzylamide “PAL” support. For the polymer-supported segment condensation, the middle and N-terminal pieces were added respectively in > 98% and 89% yields (judged by amino acid analysis and solid-phase sequencing), by overnight couplings in N,N-dimethylformamide (DMF) mediated by benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). Racemization was 4% and 11% respectively at Ala and Leu. Cleavage with Reagent M followed by reversed-phase chromatography gave pure gastrin-I in an overall 30% isolated yield. These results compare favorably with those from a stepwise assembly.     

‘Tethered ligand’derived pentapeptide agonists of thrombin receptor: a study of side chain requirements for human platelet activation and GTPase stimulation

Proteolytic action of α-thrombin on human thrombin receptor results in cleavage of a portion of the N-terminus, thereby generating a‘tethered ligand’at the newly exposed N-terminus, which then activates the receptor in an intramolecular fashion. Agonist peptides incorporating the amino acid sequence of the newly exposed N-terminal portion of the cleaved receptor cause receptor activation without requiring prior cleavage of the receptor by thrombin. The pentapeptide amide Ser-Phe-Leu-Leu-Arg-NH₂, which retains the N-terminal sequence of the‘tethered ligand’of the receptor, has been shown to be the minimum sequence to cause receptor activation. To understand the importance of the side chains of various residues within the pentapeptide amide, we carried out an extensive structure-activity study of the ability of peptides to stimulate gel-filtered platelet aggregation. In this study 106 pentapeptide amides were synthesized, utilizing naturally occurring l -amino acids, unnatural amino acids, D-amino acids and N-methyl amino acids for replacements. At position-1, charged residues (acidic or basic) were not tolerated, and the size and shape of the residue were important. Position-2 tolerated only aromatic residues. Position-3 accommodated various residues. A significant finding of this study was that two very different residues, [3-(2-naphthyl)]-l -alanine and l -arginine, when substituted for leucine residue at position-3, resulted in more active agonists. At position-4 aromatic and aliphatic residues were well tolerated, whereas basic and acidic residues were less tolerated. Position-5 mimicked position-3 in its ability to tolerate a wide range of residues. In general, an acidic residue was not preferred in any position. In all positions a -Phe-residue was tolerated. Positions 1 and 3 tolerated -Pro-residue, whereas positions 2, 4 and 5 did not. d -Amino acid substitution was not tolerated in any position. While N-methylation of residues at positions-3 and -4 led to poor agonists, at position-2 it resulted in an inactive analog. These studies define the side chain requirements for the‘tethered ligand’derived pentapeptide agonists of thrombin receptor for human platelet activation. Selected peptides were tested for ability to stimulate platelet membrane GTPase. A good correlation was observed between peptides that stimulate GTPase and those that stimulate platelet aggregation, with the general finding that 3-10-fold higher concentrations were required to half maximally activate GTPase when compared with the concentrations needed for activation of platelet aggregation. © Munksgaard 1995.     

Synthesis and properties of equine β-melanotropin analogs with substitution in residue position 1

Five analogs of equine β-melanotropin have been synthesized by the solid phase method. The NH₂-terminal aspartic acid was substituted with amino acids (Gly, Trp, Ile, Lys and Nα-acetyl-Asp) differing widely in physicochemical properties. On the basis of their lipolytic potencies it was concluded that this position plays a negligible role in this activity.     

Synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by a β-alanine

Syntheses of analogues of the C-terminal heptapeptide of cholecystokinin are described. These analogues were obtained by replacing glycine 29 by a β-alanine. The C-terminal phenylalanine amide was in some cases substituted by 2-phenylethyl alcohol and/or residues of the C-terminal tetrapeptide by their d -enantiomers. These compounds were tested for their action on stimulation of amylase release from rat pancreatic acini and for their ability to inhibit binding of labeled CCK to rat pancreatic acini and guinea pig brain membranes. Some of these derivatives behaved as CCK receptor antagonists.     

Synthesis of a cyclic analogue of the C-loop region of epidermal growth factor,containing 1-aminocyclopropane-1-carboxylic acid

We report the synthesis of a cyclic analogue of epidermal growth factor sequence 33–42 with substitution of 1-aminocyclopropane-1-carboxylic acid for glycine at position 39 (N-acetyl-Cys-Val-Ile-Gly-Tyr-Ser-ACPCA-Asp-Arg-Cys-NH₂). The analogue was synthesised by solid-phase methods, using t-Boc chemistry and acid-labile side-chain protecting groups. The use of the 4-methoxybenzyl protecting group for C- and N-terminal cysteine residues resulted in the spontaneous formation of the desired intramolecular disulfide bond after HF deprotection.