The 2,4-dinitrophenyl group for protection of hydroxyl function of tyrosine during solid-phase peptide synthesis

The facile thiolytic cleavage of the O-2,4-dinitrophenyl (Dnp) tyrosine bond was applied to the solid-phase synthesis of the 22-amino acid residue peptide H-Asp-Ala-Val-Tyr -Thr-Gly-Leu-Asn-Thr-Arg-Asn-Gln-Glu-Thr-Tyr -Glu-Thr-Leu-Lys-His-Glu-Lys-OH, corresponding to positions 62-83 in the chain of the type 1 receptor for Fc_ε, domains expressed on the rat mucosal-type mast cells (line RBL-2H3). A method for the spectrophotometric determination of insoluble O-Dnp as well as of unprotected phenolic moieties of tyrosine was developed. It is based on monitoring S-Dnp-2-mercaptoethanol, produced upon O-Dnp thiolysis by 2-mercaptoethanol. © Munksgaard 1995. Dedicated to the memory of Dr. Susumu Funakoshi, a dear friend and a leader in peptide chemistry.     

Peptides related to the calcium binding domains II and III of calmodulin

Three hexadecapeptides which correspond to the putative Ca²⁺ binding domains II and III of calmodulin were synthesized employing solid phase methodology. One of the peptides contained an internal cystine bridge which was formed while the corresponding linear peptide was still attached to the polymeric carrier. The interaction of the synthetic peptides with calcium ions was investigated using Tb³⁺mediated fluorescence. Binding was of the order Cal2 > Cal3 > Cal3C (Fig. 1) with binding constants K_Tb= 0.68 times 10^-5, 0.54 times 10^-5, and 0.21 times 10^-5 M^-1 respectively. Biological activity of the compounds was assessed by measuring their stimulatory effect on erythrocyte membrane (Ca²⁺⁺ Mg²⁺)-ATPase activity For 50% activity as compared with CaM, the concentration of peptides required was for Cal2, Cal3 and Cal3C, 50, 100 and 167 times higher than CaM, respectively. The results suggest that the three synthetic peptides possess certain calmodulin-like features.     

Binding of human serum amyloid A (hSAA) and its high-density Iipoprotein3 complex (hSAA-HDL3) to human neutrophils. Possible implication to the function of a protein of an unknown physiological role

Serum amyloid A (SAA) is an acute-phase serum protein which exists in the body in a complex with high-density lipoprotein (HDL₃). It is involved in chronic inflammation and neoplastic diseases in an as yet unknown manner. Toward an understanding of the possible physiological role of SAA we initiated a study of its association with blood proinflammatory cells with which it may interact functionally in vivo. In the following we describe the binding characteristics of recombinant human SAA to human neutrophils (polymorphonuclear leukocytes; PMNLs) and their plasma membranes. Scatchard analysis of rSAA binding and displacement curves revealed K_d in the nanomolar range. The C-terminal domain of the protein, i.e. amino acid residues 77-104, which might reside in serum following SAA degradation and amyloid A formation, was found to inhibit efficiently the binding of the whole protein to neutrophils. The interaction of SAA, and of its related peptides while complexed in HDL₃, with human PMNs was also studied. The results suggest that SAA may be involved, in an as yet unknown manner, in the neutrophil-associated inflammatory mechanism.     

Peptides derived from human C-reaetive protein inhibit the enzymatic activities of human leukocyte elastase and cathepsin G: use of overlapping peptide sequences to identify a unique inhibitor

Ten overlapping 15-mer peptides, spanning the entire inner disulfide loop of human C-reactive protein (residues 36-97), were used to isolate a potent inhibitor of the enzymes human leukocyte elastase and human leukocyte cathepsin G, which are associated with chronic inflammatory tissue damage. In contrast to the inability of intact C-reactive protein to inhibit both enzymes, the synthetic peptide E₆₂ILIFWSKDIGYSFT₇₆ inhibited leukocyte elastase (K_i= 0.18 μm ) and cathepsin G (K_i= 0.25 μm ) at concentrations far lower than the acute-phase concentration of C-reactive protein. Several peptide-enzyme binding motifs were elucidated by structure-function studies, with the Glu₆₂ residue being crucial in establishing long-range subsite interactions. Peptides derived from C-reactive protein, which may be generated in vivo by neutrophil-mediated proteolysis as part of a complex regulatory homeostatic mechanism, may play an important role in regulating the activity of matrix-degrading enzymes, specifically at sites of inflammation. The present results thus may shed additional insight on the physiological functions of the major acute-phase reactant C-reactive protein, and perhaps be used as a basis for the design of novel therapeutic substances.     

POLYMERIC 2-MERCAPTOPYRIDINE AND 2-MERCAPTO-NITROBENZENE DERIVATIVES: New Reagents for Peptide Synthesis

Insoluble 2-mercaptopyridine and 2-mercapto-nitrobenzene derivatives were prepared by modification of commercially available polystyrene. Applicability of these polymers as reagents for the thiolytic removal of the 2-nitrophenyl-sulphenyl amino-protecting group and as supports for preparation of polymeric active esters was evaluated. Polymeric 2-mercaptopyridine (PMP) was found efficient for both purposes. It was used in the stepwise synthesis of Leu-enkephalin via the polymeric reagent approach, serving as an Nps-cleaver. Polymeric esters derived from PMP and Boc-amino acids proved to be excellent acylators. Their usefulness is exemplified in the preparation of two dipeptides, which were produced rapidly and in high yields and purity.     

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production

Six Thr¹ (O-glyco)-derivatives of the “phagocytosis stimulating peptide” tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(β-d -GlcNAc)-Ser-Thr-OH, which correspond to the “tuftsin-region” at the Fc-domain of immunoglobulin G (amino acid residues 289–299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.     

Activity of two synthetic amphiphilic peptides and magainin-2 against herpes simplex virus types 1 and 2

The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin-1 (mod-1) and modelin-5 (mod-5), and of the natural antibacterial peptide magainin-2 (mag-2) against herpes simplex viruses type 1 (HSV-1) and 2 (HSV-2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod- displayed a strong antiviral effect against HSV-1 and HSV-2, with 50% effective dose (ED₅₀) values of 4.6 and 4.1 μg/mL, respectively. Mag-2, mod-5 and a mixture of both had no significant inhibitory effect. Addition of mod-1 up to a concentration of 100μg/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blue-exclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod-1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod-1 may be an effective topical antiviral agent against herpes viruses.     

Synthetic peptides derived from the sequence of human C-reactive protein inhibit the enzymatic activities of human leukocyte elastase and human leukocyte cathepsin G

Peptides derived from the primary sequence of the acute phase reactant C-reactive protein (CRP) are shown to inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with tissue damage occurring in the course of several chronic inflammatory conditions. CRP-derived peptides were synthesized based on their sequence similarity to domains within the natural inhibitors of hLE and hCG. The octapeptide Val₈₉-Thr-Val-Ala-Pro-Val-His-Ile₉₆, (CRP 89-96) is shown to inhibit hLE and hCG to a larger extent than peptides of similar chain lengths corresponding to the active sites of their natural inhibitors, α₁,-protease inhibitor and α-antichymotrypsin, respectively. Several additional peptides containing this core sequence were synthesized and shown to be inhibitors, in contrast to peptides derived from other regions of CRP as well as the intact protein, which are totally inactive. The inhibitory capability of CRP-derived peptides, which may be generated in vivo by neutrophil-mediated proteolysis as part of a complex regulatory homeostatic mechanism, may now be used as a basis for the design of novel therapeutic substances. The present finding may shed some light on the enigmatic physiological functions of CRP. © Munksgaard 1996.