Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Crucial role of position 40 for interactions of CCK-58 revealed by sequence of cat CCK-58

Evidence suggests that amino terminal extensions of CCK-8 affect the carboxyl terminal bioactive region of CCK. Cat CCK-58 was purified by low pressure reverse phase and ion-exchange chromatography steps and several reverse phase HPLC steps. The purified peptide and its tryptic fragments were characterized by mass spectral analysis and microsequence analysis. The structure of cat CCK-58 is: AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQSLDPSHRISDRDY(SO3) MGWMDF-amide. Cat and dog CCK-58 are identical except for position 40 which is serine in cat and asparagine in dog. Radioimmunoassay detected cat CCK-58 about 1/10th as well as dog CCK-58, indicating a marked effect on C-terminal immunoreactivity. Cat CCK-58 with a serine at position 40, the same residue found in pig, mouse, cow and rabbit CCK-58, can be used as a unique bioprobe for defining how amino terminal amino acids influence the structure and bioactivity of the carboxyl terminal region of CCK.     

Synthesis of biologically active biotinylated muramyl dipeptides

Muramyl dipeptide (MDP) is believed to interact with an innate immune receptor, Nod2. To identify the cellular receptor for MDP, we have synthesized biotinylated MDP isomers and tested the ability of these compounds to activate Nod2 in a cell-based assay. We found that the modification of MDP does not perturb its ability to activate Nod2. These tagged versions of MDP will be useful to identify the cellular receptor of the immunostimulatory molecules.     

Synthesis of biologically active transforming growth factor alpha

A 50-amino acid residue transforming growth factor, type alpha (TGF alpha), secreted in culture by feline-sarcoma-virus-transformed rat embryo fibroblasts, was synthesized by an improved stepwise solid-phase method with an overall yield of 31%. A deprotection strategy based on the SN2 mechanism using either a low concentration of HF or CF3SO3H-CF3CO2H in dimethylsulfide was employed to remove most of the benzyl-derived protecting groups. The more acid resistant protecting groups of Cys and Arg were removed by the SN2 condition using a high concentration of HF. Synthetic TGF alpha was purified to homogeneity in three steps. Synthetic and natural TGF alpha were indistinguishable from each other in HPLC and in different assays, including the assay for anchorage-independent growth of normal rat kidney fibroblasts in soft agar, binding, and stimulating to epidermal growth factor (EGF)-receptor protein kinase. Furthermore, synthetic TGF alpha showed similar biological activities when compared with EGF in these assays. Thus, the chemical synthesis of TGF alpha provided convincing evidence that TGF alpha is functionally related to EGF and is one of the active principles required for cellular transformation.     

Synthesis of two biologically active fluorescent probes of thymopentin

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.     

Production and secretion of biologically active recombinant canine growth hormone by Pichia pastoris

Production of recombinant canine (Canis familiaris) growth hormone (rCFGH) by two expression systems, methanol utilization slow (Mut^s) and methanol utilization plus (Mut⁺) based on Pichia pastoris. Led by the Saccharomyces cerevisiae -mating type signal sequence (SS), the hormone was secreted into the culture medium in its mature and active form. The level of total proteins secreted into the medium achieved at 25 ml working volume using Erlenmeyer flasks was approximately 40 and 15 μg/ml for Mut^s and Mut⁺ constructs, respectively. As judged by densitometry of proteins resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the hormone produced by the fermented Mut^s strain upon induction with methanol reached 24 μg/ml, representing around 60% of the total secreted proteins and being eight times more abundant than in its Mut⁺ counterpart. Finally, the recombinant hormone showed activity when tested in the Nb2 cell proliferation assay.     

NMR evidence for different conformations of the bioactive region of rat CCK-8 and CCK-58

Sulfated CCK-58 and CCK-8 have identical bioactive C-terminal primary sequences but distinct C-terminal solution structures and different bioactivities. To examine structural differences in greater detail, rat CCK-58 and -8 were synthesized with isotopic enrichment of C-terminal residues with (15)N at alpha-amino nitrogens. Proton and nitrogen chemical shift assignments of peptide solutions were obtained by homo- and heteronuclear NMR methods. These data show that the tertiary structure ensembles of C-terminal CCK-8 and CCK-58 differ significantly. Thus, distinct solution conformations may explain differences in CCK(A) and CCK(B) receptor interactions of large and small molecular forms of CCK.     

Synthesis of biologically active tritiated amylin and salmon calcitonin analogues

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family.     

Synthesis and characterization of biologically active recombinant elk and horse FSH

The objective of this investigation was to clone and express the elk and horse common α-subunit and FSH β-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1^® and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well.     

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.     

Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal

Interleukin 1beta (IL-1beta) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.     

Rhamnogalacturonan-II--a biologically active fragment

Rhamnogalacturonan-II inhibited the uptake of [¹⁴C]leucine and,consequently, the incorporation of [¹⁴C]leucine into acid-precipitableproteins by suspension-cultured tomato cells. Fractionationof rhamnogalacturonan-II showed that the lower molecular componentswere the most effective. KDO and apiose, both constituents ofrhamnogalacturonan-II, also inhibited [¹⁴C]leucine incorporationweakly, suggesting that these sugar residues may be an integralrequirement for the biological activity of rhamnogalacturonan-II.The incorporation of [¹⁴C]glutamate and [¹⁴C]histidine, andto a lesser extent [¹⁴C]proline and [¹⁴C]arginine, was alsoinhibited by rhamnogalacturonan-II; the incorporation of [¹⁴C]tyrosineand [¹⁴C]phenylalanine was little affected. This suggests thatrhamnogalacturonan-II exerts its effect by acting on certainmembrane transport systems. Key words: Rhamnogalacturonan-II, inhibition, protein synthesis, amino acid incorporation     

Synthesis of biologically active steroid derivatives by the utility of Lawesson's reagent

Steroid derivatives V, VI, VII and VIII reacted with Lawesson's reagent (LR) to produce spiro-oxazaphosphole-4',17-androstene derivative XI, diazaphospholoandrostane XIV and the thionated derivatives XVI and XVII, respectively. The structures of the new compounds were confirmed by analytical and spectroscopic evidence. A mechanism accounting for the formation of the new compounds was given. The in vitro antimicrobial activity of the new compounds were tested.     

Synthesis and application of methyleneoxy pseudodipeptide building blocks in biologically active peptides

Summary. Pseudodipeptides H-Phe[CH₂O]Phe-OH, H-Tyr[CH₂O] Asp-OH and H-Pro[CH₂O]-D-Thr-OH were synthesized using the intramolecular Williamson reaction via substituted morpholin-3-one ring with the nitrogen atom protected with bulky Boc group. This protection and the substituent at C₅ position induced the stereospecific alkylation at the C₂ position introducing the side chain of the C-terminal amino acid mimetic. In the first pseudodipeptide a quenching of the enolate with benzaldehyde was followed by dehydration and corresponding double bond was hydrogenated with high stereospecific purity. In the other pseudodipeptides, this alkylation was carried out directly by tert-butyl 2-bromoacetate or acetaldehyde. However, in the latter reaction an R configuration of C₃ substituent in conjugated lactame ring was determined using a NOE NMR. Consequently, after opening this ring by acidic hydrolysis, the C-terminal part of corresponding pseudodipeptide possessed the side-chain of D-Thr mimetic, contrary to former one. Synthesized pseudodipeptides were introduced into HIV protease inhibitors and into peptides with oostatic activity.     

Synthesis of Pro-XylaneTM: A new biologically active C-glycoside in aqueous media

The scope and limitation of Lubineau’s reaction were evaluated for the synthesis of C-glycosides (compounds 1–13). Further transformation of side chain carbonyl was also achieved (compounds 16–23). Optimization of these two steps was investigated in xylose case. Some of the compounds were shown to stimulate sulfated glycosaminoglycans (GAGs) synthesis. Compound 20 (called Pro-Xylane^TM) was identified as the best activator of GAGs biosynthesis. Pro-Xylane^TM was developed using environmentally friendly conditions relevant to ‘Green-Chemistry’ principles and launched on the market in September 2006. This compound is the first example of ‘Green’ chemical used in cosmetic.     

CCK-58 is the only detectable endocrine form of cholecystokinin in rat

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 +/- 0.5 and 17 +/- 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.     

Formation of biologically active peptides

Many small biologicaly active peptides are derived from larger precursor forms which fulfil a variety of roles in the synthesis, segregation and intracellular migration of secretory products. Limited proteolysis may occur at several stages during this process, giving rise to products that are either degraded (e.g. the prepeptides) or discharged coordinately from their cells of origin during exocytosis (e.g. insulin and C-peptide). Molecular defects have recently been found to occur at cleavage sites in proinsulin as well as in other proproteins, and these point mutations may, in some instances, be responsible for familial metabolic disorders. The nature and cell specificity of the proteolytic enzymes involved in the conversion of the various precursor forms remains unresolved. Recent studies in our laboratory have led to the identification of precursors of glucagon and somatostatin in rat islets of Langerhans. Analysis of tryptic maps of these precursors has shown that a trypsin-like enzyme would be sufficient to cleave the C-terminally located somatostatin sequence from its precursor (relative molecular mass 12,500), but that both trypsin-like and carboxypeptidase B-like enzymes would be necessary to cleave the internal glucagon sequence from its prohormone (relative molecular mass 18,000). Molecular cloning techniques have provided valuable new approaches to analysing the structures of a variety of precursor forms, including those for insulin, gastrin, growth hormone, adrenocorticotropic hormone and the endorphins, and in the future will undoubtedly shed more light on the structures of their chromosomal genes, the mechanisms regulating their expression, and their evolutionary origins.     

Synthesis and properties of the first all-aza analogue of a biologically active peptide

The synthesis of the first all-aza-amino acid analogue ( 2 ) of a peptidic renin inhibitor is described. The X-ray structural analysis and molecular modelling investigations of this novel compound reveal interesting conformational features which have a significant impact on its biological activity. In addition, insight into conformational features of azapeptides in general in comparison with the corresponding purely peptidic compounds is given.     

Synthesis and anti-inflammation evaluation of new C60 fulleropyrrolidines bearing biologically active xanthine

We designed and prepared the new C60 fullerene hybrids bearing a xanthine moiety as potential double-action anti-inflammatory agents, capable of simultaneous inhibition of LPS-induced NO and TNF-alpha production. The 10 microM of fulleropyrrolidine-xanthine dyad 2a and b were effective in suppressing LPS-induced NO production by 55.1+/-2.1% and 58.6+/-2.6%, respectively, but only 2b was also effectively in suppressing LPS-induced TNF-alpha production by 34.0+/-2.7%. We believed that the agents synthesized herein would hold promise for future development of a new generation of potent anti-inflammatory agents.