Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

Synthesis and characterization of branched phosphopeptides: Prototype consolidated ligands for SH(32) domains

Summary This paper details the solid-phase synthesis by N -9-fluorenylmethyloxycarbonyl (Fmoc) chemistry of a series of bivalent consolidated ligands, branched peptides with lengths of 22 to 25 residues. The target peptides were designed to, and in fact do, interact with greater specificity and higher affinity with the SH2 and SH3 domains of Abelson kinase in an SH(32) dual domain construct. Fmoc-O-phospho-l-tyrosine[Fmoc-Tyr(PO₃H₂)-OH] was used to introduce the required phosphotyrosine residues, and Fmoc-N -1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-l-lysine [Fmoc-Lys(Dde)-OH] was used to introduce a branch point that allowed proper orientation of individual ligands. The resultant product peptides were characterized by amino acid analyses and electrospray mass spectra.This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Synthesis of Nα-(fluoren-9-ylmethoxycarbonyl)-Nε-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine for use in solid-phase synthesis of fluorogenic substrates

N -(fluoren-9-ylmethoxycarbonyl)-N -[(7-methoxycoumarin-4-yl)acetyl]-L-lysine[Fmoc-Lys(Mca)-OH] has been conveniently prepared. The copper complex of L-lysine hydrochloride was initially prepared, followed by the addition of the (7-methoxycoumarin-4-yl)acetyl (Mca) group to the -amino group. The copper complex was decomposed, and the Fmoc group was introduced to the -amino group. Fmoc-Lys(Mca)-OH was recrystallized from hexane and could be used directly for the solid-phase synthesis of fluorogenic substrates.     

Opioid activity of synthetic deltorphin II analogues and their spin labeled derivatives

Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH₂, Del II), an endogenous linear heptapeptide, is a highly selective agonist of the -opioid receptor. To study the effect of the position 4 residue (Glu) on the opioid activity of Del II, we designed and synthesized three analogues of Del II by solid-phase peptide synthesis. They were [Val⁴,Glu⁵]Del II, [Val⁴,Glu⁶]Del II and [Gly⁴,Glu⁷]Del II. To study the effect of spin labeling on peptide bioactivities, all the peptides were labeled using a free radical. The labeling material was a stable nitrogen–oxygen free radical which was linked to the N-terminal via an amide bond. We investigated the opioid bioactivities of these analogues both in vivo and in vitro, and concluded that the differences in opioid activity of Del II and its analogues were due to structural differences. When the Glu residue is at position 5 or 6, the internal hydrogen bonds in Del II are affected and there is a change in three-dimensional structure and opioid activity. The antinociceptive activity of all the peptides decreased after spin labeling. This indicates that the stable nitrogen–oxygen free radical is a dual-function spin-labeling molecule.     

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One area of the search for hormonal signaling systems regulators is development of peptides that correspond to the cytoplasmic regions of G protein-coupled receptors (GPCR). Modification of such peptides with hydrophobic radicals increases their efficiency and selectivity. However, at present it has not been studied how the activity of the peptide depends on the localization of hydrophobic radicals, their number, and chemical nature. The aim of this work consisted in synthesis of peptide 562–572 derivatives modified by fatty-acid radicals and corresponding to the C-terminal region of the luteinizing hormone receptor (LHR) and in the study of regulatory effects of the acylated LHR peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in rat tissues. To elucidate the effects of localization of hydrophobic radicals and of their number, modifications of peptide 562–572 were carried out only at the N-or at the C-terminus or at both ends. To study the effect of hydrophobicity, residues of palmitic (Pal) and decanoic (Dec) acids were chosen. Using a solid-phase strategy synthesis was performed of the unmodified peptide NKDTKIAKK-Nle-A^562-572-KA (1) and five of its acylated analogues, N[K(Dec)]DTKIAKK-Nle-A^562-572-KA (2), NKDTKIAKK-Nle-A^562-572-[K(Dec)]A (3), N[K(Dec)]DTKIAKK-Nle-A^562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A^562-572-KA (5), and NKDTKIAKK-Nle-A^562-572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the basal AC activity and reduced the AC stimulating effect of human chorionic gonadotropin (hCG) in testes of rats; peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the unmodified peptide 1; and peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by tissue and the receptor specificity. Thus, modification of the LHR peptide 562–572 with fatty-acid radicals at the C-terminus enhances its regulatory effect on the functional activity of the adenylyl cyclase system in rat testes, which indicates a promising modification of GPCR peptides with hydrophobic radicals. These data confirm the hypothesis that the hydrophobic radical is to be localized in the locus of GPCR peptide, where a transmembrane domain is located in the receptor.     

Characterization of specific calcitonin gene related peptide receptors present in hamster pancreatic β cells

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of¹²⁵I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound¹²⁵I-[His]hCGRP I could be induced in the presence of 1 M chicken CGRP (cCGRP). The specific¹²⁵I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC₅₀) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0–2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of¹²⁵I-[His]hCGRP I and sCT was only effective at a high concentration (1 M). Binding of¹²⁵I-[His]hCGRP I was dose dependently inhibited by guanosine-5-O-(3-thiotriphosphate) or (GTPS) and a 70% reduction of binding was obtained with 0.1 mM GTPS. The IC₅₀ value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTPS. Human CGRP I and cCGRP at 2.5 M did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 M) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.Abbreviations CGRP calcitonin gene-related peptide - IAPP islet amyloid polypeptide - IC₅₀ half-maximal inhibitory concentration - GTPS guanosine-5-O-(3-thiotriphosphate) - ¹²⁵I [His]hCGRP I, (2[¹²⁵I]iodohistidyl¹⁰) human CGRP I     

The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs.

In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.     

The coordination of copper(II) with β-casomorphin and its fragments

The synthesis of β-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly, H₂L) and a number of its peptide fragments is described. Complexes formed between these peptides and Cu(II) have been investigated spectrophotometrically, using CD and EPR spectroscopy, and potentiometrically. Results show that, with tyrosine as the N-terminal residue, the major complex formed at physiological pH is the dimeric species, [Cu₂L₂], bonded through the phenolic O^? of the Tyr residue of one ligand and the N-terminal amine nitrogen of the second ligand molecule. There is no evidence for coordination through the peptide nitrogens unless the terminal Tyr group is removed.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.     

Rearrangement of Glu(OtBu)- and Asp(OtBu)-containing peptides upon fluoride treatment in solid-phase synthesis

Summary Although fluoride-labile protecting groups and linkers have been developed in solid-phase peptide synthesis to add an extra level of orthogonality, fluoride ions were found to convert -peptides to their corresponding - or -peptides in Glu(OtBu)- and Asp(OtBu)-containing peptidyl resins. Different peptide sequences and fluoride reagents were examined to determine the scope of this side reaction. CZE analysis was found to be a useful analytical technique to characterize peptides with respect to this phenomenon.     

Synthesis and spectroscopic properties of 4-ethoxymethylene-2-(1)-naphthyl-5(4H)-oxazolone-labeled fluorescent peptides

Strategies for the preparation of new fluorescent oligopeptide conjugates labeled with 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) at the N-terminal on solid support or in solution have been devised. These procedures are simple and easy to carry out by reacting naOx-OEt or N(alpha)-naOx-amino acid with side chain protected peptide chains attached to resins. The integrity of the N-alkyl bond was maintained even after the trifluoracetic acid or HF based cleavages procedures. Our data show that the naOx fluorophore is compatible with both Fmoc/tBu and Boc/Bzl methods and also suggest that naOx-amino acid could be utilized as building blocks for solid phase peptide synthesis. Comparative analysis of fluorescence properties of naOx-conjugates indicated that the spectral properties of the fluorophore do not change after incorporating into peptides. The compact size, the definite chemical reaction for its introduction in combination with the appropriate spectral features (e.g., intense emission, pH independent fluorescent characteristics, and beneficial photobleaching dose constant and rates) and with chemical and spectral stability, naOx-based labeling could be attractive for novel cellular fluorescent techniques (e.g., in laser scanning confocal FRET) to study peptide-protein and protein-protein interactions even in biological matrices.     

Exploring water-soluble Pt(II) complexes of diethylenetriamine derivatives functionalized at the central nitrogen. Synthesis, characterization, and reaction with 5′-GMP

The aims of our program are to develop coordination complexes that can be used as selective probes, fluorescent agents and inorganic medicinal agents. In order to accomplish this, the design, synthesis, characterization and X-ray structure of new water-soluble monofunctional Pt(II) complexes with useful spectroscopic properties for assessing metal binding to biomolecules were investigated. Two diethylenetriamine (dien) derivatives, 2-(bis(2-aminoethyl)amino)acetic acid (acdien) and N′-[7-(acetamido)-4-(trifluoromethyl)coumarin]diethylenetriamine (atfcdien), were used. The latter was designed to allow the fluorophore group, 7-amino-4-(trifluoromethyl)coumarin (atfc), to be attached to metal centers through the dien moiety. ¹H NMR spectroscopy and X-ray crystallography were employed to characterize the [Pt(atfcdien)Br][Pt(Me₂SO)Br₃] (8a) and [Pt(acdien)Br]Br (9a) complexes. ¹H NMR and fluorescence spectroscopic methods were used to characterize the [Pt(atfcdien)Br]Br (8b) and [Pt(acdien)Br]Br (9a) complexes. ¹H NMR studies of the monofunctional [Pt(acdien)Br]Br (9a) complex conducted to examine its interaction with guanosine 5′-monophosphate (5′-GMP) in D₂O solutions revealed one downfield-shifted H8 and one downfield-shifted H1′ signal, consistent with 5′-GMP binding via N7 and fast rotation about the Pt-N7 bond.     

DNA-binding studies of XSPTSPSZ,derivatives of the intercalating heptad repeat of RNA polymerase II

The synthesis, solution conformation, and interaction with DNA of three 8-residue peptides structurally related to the heptad repeat unit found at the C-terminus of RNA polymerase II are reported. Peptides QQ, XQ, and PQ are derived from the parent sequence YSPTSPSY (peptide YY), which was reported to bind to DNA by bisintercalation [M. Suzuki (1990) Nature, Vol. 344, pp. 562–565], and contain either a 2-quinolyl (Q), 2-quinoxolyl (X), or 5-phenanthrolyl (P) group in place of the aromatic side chains of the N- and C-terminal tyrosine residues present in the parent sequence. The combined results of linear dichroism and induced CD measurements of peptides QQ, XQ, and PQ with calf thymus DNA are consistent with weak binding of the peptides to DNA in a preferred orientation in which the chromophores are intercalated. Small increases in the melting temperatures of poly[d(A-T)₂] are also consistent with the peptides interacting with DNA. While enzymatic footprinting with DNase I showed no protection from cleavage by the enzyme, chemical footprinting with fotemustine showed that the peptides modify the reactivity of the major groove, presumably via minor groove binding. Peptide QQ inhibited fotemustine alkylation significantly more than either XQ or PQ, and slightly more than YY. In aqueous solution, nmr experiments on QQ, XQ, and PQ show a significant population of a conformation in which Ser2-Pro3-Thr4-Ser5 form both type I and type II β-turn conformations in equilibrium with open chain conformations. Nuclear magnetic resonance titration experiments of PQ with (GCGTACGC)₂ showed small changes in chemical shifts, consistent with the formation of a weak nonspecific complex. Analogous experiments, using peptides QQ and XQ with (GCGTACGC)₂, and peptide YY with (CGTACG)₂, showed no evidence for the interaction of the peptides with these oligonucleotides. These results show that peptides of general structure XSPTSPSZ are weak nonspecific DNA binders that differ significantly from previously characterized S(T)PXX DNA-binding motifs that are generally AT-selective minor groove binders. © 1997 John Wiley & Sons, Inc. Biopoly 42: 387–398, 1997     

Peptide synthesis on a phenolic resin support

Protected peptides assembled on a phenolic resin support were cleared by peroxide-catalysed hydrolysis. In genenal peptide phenyl ester resins were more labile to nucleophiles than were corresponding Merrifield resin derivatives; transesterification with dimethylaminoethanol providing on alternative cleavage method for peroxide-sensitive peptides. Losses of radiolabelled peptide from both Merrifield and phenolic resins were determined during acid deprotection, base wash and coupling steps in the synthesis of a tetrapeptide. Using 40% (v/v) trifluoroacetic acid in dichloromethane for Boc-deprotection the phenolic resin gave improved results compared to the Merrifield resin. The merits of the procedure for the preparation of protected peptide acids suitable for subsequent condensation reactions were exemplified by the synthesis of an octapeptide sequence of a modified lysozyme.     

Structure of hypothalamic coronaro-constrictory peptide factors

The following peptide structure in 3 of 5 coronaro-constrictory peptide factors isolated from bovine hypothalamus was determined by amino acid analysis and Edman degradation: 1) (P₁)-Val-Val-Tyr-Pro-Trp; 2) (P₂)-Val-Val-Tyr-Pro-Trp-Thr; 3) (P₃)-Leu-Val-Val-Tyr-Pro-Trp-Thr. A computer search for these amino acid sequences revealed that these peptides represent fragments 33-37; 33-38; 32-38 of the -chain of bovine hemoglobin. Solid phase peptide synthesis of 2 peptides (P₂ and P₃) was carried out. It was established that synthetic peptides had the properties of coronaro-constrictory peptides. The possibility of the formation of hypothalamic coronaro-constrictory peptides in vivo is discussed.     

Peptide signals for plant defense display a more universal role

Hydroxyproline-rich systemins (HypSys) are small defense signaling glycopeptides found within the Solanaceae family that until recently were thought to only induce defense genes to herbivore attack. The glycopeptides are processed from larger proproteins with up to 3 different glycopeptides being processed out of a single precursor protein. A conserved central hydroxyproline motif within each HypSys is the site of pentose sugar attachment. Recently, it was found that in Petunia hybrida, these defense signaling glycopeptides did not induce protease inhibitor but instead, increased levels of defensin, a gene that is involved in pathogen attack. More recently, a HypSys peptide was isolated from Ipomoea batatas (sweet potato) of the Convolvulaceae family and found to induce sporamin. The proprotein precursor contained six putative peptide signals and had a propeptidase processing region with homology to solanaceous proHypSys. Thus, the HypSys defense peptides are no longer confined to defense against herbivory or exclusivity to the Solanaceae family, redefining both function and dispersion.Key words: systemin, hydroxyproline-rich systemin glycopeptides, HypSys, plant defense, proteinase inhibitorsPlants have evolved an arsenal of defense mechanisms for survival against the wide array of predators and pathogens that they encounter. Each species has evolved within its unique environment and the protective defense mechanisms must evolve and refine over time to allow a plant to compete in its niche.¹ Plant peptide signals have recently been discovered that induce defense genes for protection against both herbivores and pathogens.² This raises the issue of how these peptides, their receptors, signaling pathways, and the downstream regulated defense proteins and compounds have evolved to meet the unique and specific needs of each plant. Our recent papers^3,4 reveal that these defense signaling peptides are not confined to a single family of plants and that the end products of the signaling pathway may be more diverse than expected.Systemin was the first peptide signal discovered in plants.⁵ The 18 amino acid peptide is processed from the C-terminal of a 200 amino acid precursor; prosystemin.⁶ Although lacking a signal sequence, prosystemin reaches the apoplast and the mature peptide is processed upon insect attack, signaling downstream events leading to the production of defense proteins, such as polyphenol oxidase and protease inhibitors.⁷ Systemin has only been found in the Solanaceae family and more specifically, only in the subfamily Solanoideae, which contains tomato, potato, nightshade and pepper.The hydroxyproline-rich systemin glycopeptides are similar to systemin in size (18–20 amino acids in length) and, like systemin, are processed from larger precursors.^2,8 Both systemin and HypSys induce the production of methyl jasmonate and function to amplify the defense response. Each HypSys peptide contains a hydroxyproline-rich inner core that is the site of glycosylation and both the peptide backbone and the carbohydrate moieties are important for receptor recognition (9^,10 Although there is no sequence similarity between prosystemin and hydroxyproline-rich systemins, it has been suggested that because of their size, structure and functional similarities, they should be classified together.¹¹

Table 1

Comparisons of the amino acid sequences of isolated and putative Systemin and HypSys peptides

Non-amidated and amidated members of the C-type allatostatin (AST-C) family are differentially distributed in the stomatogastric nervous system of the American lobster, <Emphasis Type="Italic">Homarus americanus</Emphasis>

The crustacean stomatogastric nervous system (STNS) is a well-known model for investigating neuropeptidergic control of rhythmic behavior. Among the peptides known to modulate the STNS are the C-type allatostatins (AST-Cs). In the lobster, Homarus americanus, three AST-Cs are known. Two of these, pQIRYHQCYFNPISCF (AST-C I) and GNGDGRLYWRCYFNAVSCF (AST-C III), have non-amidated C-termini, while the third, SYWKQCAFNAVSCFamide (AST-C II), is C-terminally amidated. Here, antibodies were generated against one of the non-amidated peptides (AST-C I) and against the amidated isoform (AST-C II). Specificity tests show that the AST-C I antibody cross-reacts with both AST-C I and AST-C III, but not AST-C II; the AST-C II antibody does not cross-react with either non-amidated peptide. Wholemount immunohistochemistry shows that both subclasses (non-amidated and amidated) of AST-C are distributed throughout the lobster STNS. Specifically, the antibody that cross-reacts with the two non-amidated peptides labels neuropil in the CoGs and the stomatogastric ganglion (STG), axons in the superior esophageal (son) and stomatogastric (stn) nerves, and ~ 14 somata in each commissural ganglion (CoG). The AST-C II-specific antibody labels neuropil in the CoGs, STG and at the junction of the sons and stn, axons in the sons and stn, ~ 42 somata in each CoG, and two somata in the STG. Double immunolabeling shows that, except for one soma in each CoG, the non-amidated and amidated peptides are present in distinct sets of neuronal profiles. The differential distributions of the two AST-C subclasses suggest that the two peptide groups are likely to serve different modulatory roles in the lobster STNS.     

Solid (gel) phase synthesis of fully-protected acylpeptide hydrazides with hydrazine-labile sidechains

A simple, efficient strategy for the preparation of fully-protected acylpeptide hydrazides with hydrazine-labile sidechains is described. The method involves controlled hydrazinolytic cleavage of target peptides from assemblies elaborated by solid (gel) phase synthesis on phenolic peptide resins.     

Peptide derivatives of tylosin-related macrolides

Approaches to the synthesis of model compounds based on the tylosin-related macrolides desmycosin and O=mycaminosyltylonolide were developed to study the conformation and topography of the nascent peptide chain in the ribosome tunnel using specially designed peptide derivatives of macrolide antibiotics. A method for selective bromoacetylation of desmycosin at the hydroxyl group of mycinose was developed, which involves preliminary acetylation of mycaminose. The reaction of the 4″-bromoacetyl derivative of the antibiotic with cesium salts of the dipeptide Boc-Ala-Ala-OH and the hexapeptide MeOTr-Gly-Pro-Gly-Pro-Gly-Pro-OH led to the corresponding peptide derivatives of desmycosin. The protected peptides Boc-Ala-Ala-OH, Boc-Ala-Ala-Phe-OH, and Boc-Gly-Pro-Gly-Pro-Gly-Pro-OH were condensed with the C23-hydroxyl group of O-mycaminosyltylonolide.     

Specificity of cathepsin B to fluorescent substrates containing benzyl side-chain-substituted amino acids at P1 subsite

We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures NH₂-Cap-Leu-X-MCA and Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp, respectively. Abz (ortho-aminobenzoic acid) and EDDnp (2,4-dinitrophenyl-ethylenediamine) are the fluorescent donor-acceptor pair, and X was Cys(SBzl), Ser(OBzl), and Thr(OBzl) containing benzyl group (Bzl) at the functional side chain of Cys, Ser, and Thr. The peptidyl-coumarin-containing Cys(SBzl), Ser(OBzl), and Thr(OBzl) have higher affinity cathepsin B, supporting the interpretation of the crystal structure of rat cathepsin B complexed with the inhibitor Z-Arg-Ser(OBzl)-CH₂Cl that the benzyl group attached to Ser hydroxyl side chain occupies the enzyme S₁ subsite [Jia et al. (1995), J. Biol. Chem. 270, 5527]. A similar effect of benzyl group was also detected in the internally quenched peptides. Finally, the benzyl group in substrates containing Cys(SBzl) amino acid at P₁ seems to compensate the absence of adequate S₂-P₂ interaction in the hydrolysis of the peptides having Pro or Ala at P₂ position.