Solid-phase Fmoc Chemistry Research Articles

Incorporation of a lauric acid‐conjugated GRGDS peptide directly into the matrix of a poly(carbonate‐urea)urethane polymer for use in cardiovascular bypass graft applications

Gly-Arg-Gly-Asp-Ser (GRGDS) was modified by conjugation to lauric acid (LA) to facilitate incorporation into the matrix of a poly(carbonate-urea)urethane (PCU) used in vascular bypass grafts. GRGDS and LA-GRGDS were synthesized using solid phase Fmoc chemistry and characterized by high performance liquid chromatography and Fourier transform infrared spectroscopy. LA-GRGDS was passively coated and incorporated as nanoparticle dispersion on the PCU films. Biocompatibility of the modified surfaces was investigated. Endothelial cells seeded on LA-GRGDS coated and incorporated PCU showed after 48 h and 72 h a significant (p < 0.05) increase in metabolism compared with unmodified PCU. The platelet adhesion and hemolysis studies showed that the modification of PCU had no adverse effect. In conclusion, LA-conjugated RGD derivatives, such as LA-GRGDS, that permit solubility into solvents used in solvent casting methodologies should have wide applicability in polymer development for use in coronary, vascular, and dialysis bypass grafts, and furthermore scaffolds utilized for tissue regeneration and tissue engineering.

Angiotensin I-Converting Enzyme Inhibitory Peptide Derived from Glycinin, the 11S Globulin of Soybean (Glycine max)

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, catalyzes the conversion of Angiotensin I to the potent vasoconstrictor Angiotensin II and plays an important physiological role in regulating blood pressure. Inhibitors of angiotensin 1-converting enzyme derived from food proteins are utilized for pharmaceuticals and physiologically functional foods. ACE inhibitory properties of different enzymatic hydrolysates of glycinin, the major storage protein of soybean, have been demonstrated. The IC50 value for the different enzyme digests ranges from 4.5 to 35 microg of N2. The Protease P hydrolysate contained the most potent suite of ACE inhibitory peptides. The ACE inhibitory activity of the Protease P hydrolysate after fractionation by RP-HPLC and ion-pair chromatography was ascribed to a single peptide. The peptide was homogeneous as evidenced by MALDI-TOF and identified to be a pentapeptide. The sequence was Val-Leu-Ile-Val-Pro. This peptide was synthesized using solid-phase FMOC chemistry. The IC50 for ACE inhibition was 1.69 +/- 0.17 microM. The synthetic peptide was a potent competitive inhibitor of ACE with a Ki of 4.5 +/- 0.25 x 10(-6) M. This peptide was resistant to digestion by proteases of the gastrointestinal tract. The antihypertensive property of this peptide derived from glycinin might find importance in the development of therapeutic functional foods.

Synthetic Approaches to Disulfide-free Circular Bovine Pancreatic Trypsin Inhibitor (c-BPTI) Analogues

Several approaches were investigated with the goal to obtain disulfide-free circularized analogues of the 58-residue small protein bovine pancreatic trypsin inhibitor (BPTI). These approaches include (1) a semisynthesis that uses as a starting point naturally occurring BPTI and takes advantage of the native proximity of the C- and N-termini; (2) a synthesis in which a peptide thioester prepared by stepwise Fmoc solid-phase chemistry is cyclized by a solution native chemical ligation step; (3) a stepwise Fmoc solid-phase synthesis of a protected circularly permuted linear sequence, followed by an attempted selective activation and head-to-tail cyclization; and (4) a stepwise Fmoc solid-phase synthesis of the same analogue, but using a different disconnection point, that features backbone amide linker (BAL) anchoring and attempted on-resin cyclization. The first two of these approaches were indeed successful in providing the desired target molecules in excellent purities and respectable yields, and could well be amenable to generalization. It is not yet clear whether or not the latter two approaches could be salvaged by modifications in the details of the chemical procedures applied.

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

An Efficient, Convenient Solid-Phase Synthesis of Amino Acid-Modified Peptide Nucleic Acid Monomers and Oligomers

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.

Discovery of a sulfated tetrapeptide that binds to vascular endothelial growth factor

Molecules that mimic the sulfated glycosaminoglycan heparin and bind to heparin-binding growth factors would serve as important building blocks for synthetic biomaterials, e.g. to create a growth factor reservoir within a matrix. Peptide-based heparin mimetics would be particularly attractive, given the ease of peptide synthesis and modification. A sulfated tetrapeptide that fits this description and binds to vascular endothelial growth factor (VEGF) was discovered using a rationally-designed combinatorial approach. A ∼6600 member library of tetrapeptides, designed to include heparin functionality, was synthesized by solid-phase Fmoc chemistry. The library was analyzed on-resin for VEGF binding using a fluorescence assay that employed a 7-amino-4-methylcoumarin-modified VEGF 165. The beads were ranked according to fluorescent signal and SY(SO 3)DY(SO 3) was identified as the top binder. The binding affinity of the peptide for VEGF 165 was ascertained by surface plasmon resonance and compared with the heparin mimic suramin; the peptide binds to VEGF 165 100-fold stronger than the sulfonated compound. These results suggest that the identified peptide may be useful in biomaterial applications where binding of VEGF is desired.

Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides

The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists.

Synthesis of photoactive p-azidotetrafluorophenylalanine containing peptide by solid-phase Fmoc methodology.

[structure: see text] N-Fmoc-L-p-azidotetrafluorophenylalanine was prepared from achiral starting materials using an acetamidomalonate synthesis and enzymatic resolution. A photoactive peptide containing this fluorinated residue could be assembled using solid-phase Fmoc chemistry.

Radiometal-labeled peptide-PNA conjugates for targeting bcl-2 expression: preparation, characterization, and in vitro mRNA binding.

A new antisense peptide-peptide nucleic acid (peptide-PNA) conjugate, designed for targeting bcl-2 expression, has been radiolabeled, characterized, and evaluated for bcl-2 mRNA binding in a cell-free system. A PNA complementary to the first six codons of the bcl-2 gene was synthesized by standard solid-phase Fmoc chemistry and conjugated to a new derivative of 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) that allows macrocyclic radiometal chelates to be incorporated into any sequence position of a peptide-PNA conjugate. The DOTA-PNA conjugate was then coupled to a membrane-permeating transduction peptide, PTD-4, designed for intracellular delivery of the radiolabeled PNA. The conjugate was characterized by HPLC and ESI-MS and labeled with (111)In and (90)Y to high specific activities (>1000 Ci/mmol) with high radiochemical purity. Northern blot analysis showed that (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA bound specifically to as little as 50 fmol of bcl-2 mRNA, a result equivalent to that obtained with the analogous (32)P-labeled DNA antisense oligonucleotide. Thus, the mRNA targeting properties of (111)In- and (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA demonstrate potential for diagnostic imaging and targeted radiotherapy applications in bcl-2-positive cancers.

New Fmoc pseudoisocytosine monomer for the synthesis of a bis-PNA molecule by automated solid-phase Fmoc chemistry.

The synthesis of N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-(2-N-(benzyloxycarbonyl)isocytosin-5-ylacetyl)glycine monomer and its incorporation into a PNA molecule via automated Fmoc solid-phase chemistry is described.

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide-bridged beta-D-Galp-(1-->3)-alpha-D-GalpNAc O-glycopeptide.

The glycopeptide hormone catfish somatostatin (somatostatin-22) has the amino acid sequence H-Asp-Asn-Thr-Val-Thr-Ser-Lys-Pro-Leu-Asn-Cys-Met-Asn-Tyr-Phe-Trp-Lys-Se r-Arg-Thr-Ala-Cys-OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains D-GalNAc and D-Gal O-glycosidically linked to Thr5. The linear sequence was assembled smoothly starting with an Fmoc-Cys(Trt)-PAC-PEG-PS support, using stepwise Fmoc solid-phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin-22 were accessed by incorporating as building blocks, respectively, Nalpha-Fmoc-Thr(Ac3-alpha-D-GalNAc)-OH and Nalpha-Fmoc-Thr(Ac4-beta-D-Gal-(1-->3)-Ac2-alpha-D-GalNAc)-O H. Acidolytic deprotection/cleavage of these peptidyl-resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl-protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by Nalpha-dithiasuccinoyl (Dts)-glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2-fold tighter binding.

Candidacidal activity prompted by N-terminus histatin-like domain of human salivary mucin (MUC7)

Histidine-rich peptides (histatins, Hsn) in saliva are thought to provide a non-immune defense against Candida albicans. Sequence homology search of the human salivary mucin, MUC7, against histatins revealed a domain at the N-terminus (R 3–Q 17) having 53% identity to Hsn-5. To determine its candidacidal activity, this 15 residue basic histidine-rich domain of MUC7 ( I) was prepared by solid-phase Fmoc chemistry. Various N- and C-terminal protected derivatives of I were also synthesized to correlate the effect of peptide overall charge in exhibiting cidal potency. Candidacidal activity measurement of I and its variants showed considerable ED 50 values (effective dosage required to kill 50% of candida cells), albeit greater than Hsn-5 (ED 50 ∼4–6 μM). Of the various analogs tested, N-terminal free acid ( I, ED 50 ∼40 μM) and amide ( V, ED 50∼16 μM) exhibited appreciable candidacidal activities suggesting the possible role of peptide net charge in cidal action. Blocking of N-terminus with a bulky octanoyl group showed only marginal effect on the cidal activity of I or V, indicating that hydrophobicity of these synthetic constructs may not be important for exerting such activities. Membrane-induced conformational transition from random coil to helical structures of all the test peptides implied their tendency to adapt order structures at the lipid-membrane interface similar to that of Hsn-5. However, comparison of propensity for helical structure formation vs. ED 50 indicated that cidal potency of MUC7 Hsn-like peptides depends largely on electrostatic interactions irrespective of secondary structural elements. Delineation of solution structure of the most active peptide ( V) by 2D-NMR revealed essentially a non-structured conformation in aqueous medium, which further supported the fact that the peptide helical structure may not be a prerequisite for posing candidacidal activity. The formation of smaller truncated peptides and/or Hsn-like fragments on proteolytic degradation of intact MUC7 in the presence of oral flora provided indirect evidence that mucin could serve as a backup candidacidal agent to salivary Hsn.

A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1H NMR assignments and their application in solid-phase glycopeptide synthesis.

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp and Nalpha-Fmoc-Thr-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. Nalpha-Fmoc-Ser-OPfp or Nalpha-Fmoc-Thr-OPfp) with protected beta-D-Gal(1-3)-D-GalN3 synthon mediated by silver salts resulted in only alpha- and/or beta-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO4 at -40 degrees C which afforded a-isomer in a quantitative yield (alpha:beta = 5:1). On the other hand, the beta-isomer was formed exclusively when the reaction was performed in the presence of Ag2CO3/AgClO4 at room temperature. A complete assignment of 1H resonances to individual sugar ring protons and the characteristic anomeric alpha-1 H and beta-1 H in Ac4Galbeta(1-3)Ac2GalN3 alpha and/or beta linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned alpha- and beta-isomers of the O-glycosylated amino acid building blocks carrying protected beta-D-Gal(1-3)-D-GalN3, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. The transformation of the 2-azido group into the acetamido derivative was carried out with thioacetic acid on the polymer-bound glycopeptides before the cleavage step. After cleaving the glycopeptide from the resin, the acetyl groups used for sugar OH-protection were removed with sodium methoxide in methanol. Finally, the glycopeptides were purified by reversed-phase high-performance liquid chromatography and their integrity was confirmed by proton NMR as well as by mass spectral analysis. Secondary structure analysis by circular dichroism of both the glycosylated and nonglycosylated peptides revealed that carbohydrates did not exert any profound structural effect on the peptide backbone conformation.

Synthesis of a cyclic-thioether peptide which binds anti-cardiolipin antibodies

Thioether cyclized peptide analogue, 1, was synthesized using solid phase FMOC chemistry on HMPB-MBHA resin. α-Methylproline was introduced as an FMOC-alanyl-α-methylproline dipeptide, and the thioether was incorporated as FMOC-protected thioether tetrapeptide intermediate, 8. Compound 1 has been shown to bind anti-cardiolipin antibodies.

Design of a novel class of bifunctional thrombin inhibitors, synthesised by the first application of peptide boronates in solid phase chemistry

Borologs containing peptide boronates are new bifunctional biologically active molecules which bind to and inhibit thrombin. These compounds are designed based on the C-terminal sequence of hirudin. The inhibitors consists of four parts, i) an active site inhibitor, D-Phe Pro-Boro(aa)-OPin. ii) an anion binding exosite association moiety, Hirudin, iii) a spacer to link these components and iv) a novel ‘flexor’ non-peptide unit to allow correct orientation. The bivalent nature of the inhibitor [-D-PheProBoroBpgOPin]CO(CH 2) 3COGly 2Hir enhanced binding up to 10 fold greater than the corresponding native peptide Z-D-PheProBoroBpgOPin or the mixture of non covalently linked units, and resulted in a potent and selective inhibitor of thrombin having a Ki of 0.6nM. For the synthesis of these compounds suitably protected aminoboronate derivatives were shown to be compatible with FMOC solid phase chemistry.

Synthesis and pharmacology of novel analogues of oxytocin and deaminooxytocin: directed methods for the construction of disulfide and trisulfide bridges in peptides.

Using as models the neurohypophyseal nonapeptide hormone oxytocin and its analogue deaminooxytocin, several directed routes to formation of sulfur-sulfur bridges have been developed and evaluated. The linear sequences (through common octapeptide-resin intermediates) were assembled smoothly on tris(alkoxy)benzylamide (PAL) poly(ethylene glycol)-polystyrene (PEG-PS) graft supports, using stepwise Fmoc solid-phase chemistry. Side-chain protection of beta-mercaptopropionic acid (Mpa) and/or cysteine (Cys) was provided by S-2,4,6-trimethoxybenzyl (Tmob), S-acetamidomethyl (Acm), and/or a series of sulfenyl thiocarbonate and carbamoylsulfenyl protecting/activating groups: S-(methoxycarbonyl)sulfenyl (Scm), S-(methoxycarbonyl)disulfanyl (Sscm), S-(N-methyl-N-phenylcarbamoyl)sulfenyl (Snm), and S-(N-methyl-N-phenylcarbamoyl)disulfanyl (Ssnm). Thiolytic displacement of S-Snm (preferred) or S-Scm provided intramolecular cyclized peptide disulfides, and homologation of the chemistry with S-Ssnm (again preferred) and S-Sscm provided the corresponding trisulfides along with smaller amounts of disulfides and tetrasulfides. These chemistries could be implemented both in solution and in solid-phase modes. Various parameters were studied systematically and optimized, and the novel trisulfides of oxytocin and deaminooxytocin were synthesized and purified to homogeneity. The trisulfide compounds were evaluated in three assays: uterotonic in vitro, uterotonic in vivo, and pressor tests, and they showed substantial potencies, ranging from 5% to 40% of the parent (disulfide) activities, as well as protracted actions. The affinities of the peptide trisulfides to uterine membrane receptors were only 3.3-3.6-fold lower than those of the parent disulfides. Possible explanations of the biological results are discussed.

Alpha-fetoprotein derived synthetic peptides: assay of an estrogen-modifying regulatory segment

This study describes the estrogen bioassay of a synthetic peptide fashioned after an amino acid sequence from human alpha-fetoprotein (HAFP). The synthetic peptide (P149), modeled after a portion of the estrogen binding pocket of rat/human AFP chimeras, was produced via F-MOC solid phase chemistry. Bioassay of P149 in the estrogen-sensitive immature rodent uterus demonstrated an anti-estrogenic (40–50% inhibitory) activity in the 23 h but not the 3–4 h uterine response. In contrast to purified HAFP, incubation of the peptide with estrogen was not a prerequisite for inhibitory activity. The estrogen-dependent increase in uterine thrombin and tissue factor, as determined by an enzymatic esterase assay, was inhibited by 30% in rat uterine cytosols. In an in vitro bioassay of estrogen-induced focus formation in MCF-7 human breast cancer cultures, focus development was inhibited by 70% following peptide exposure. The mechanism of the AFP-derived peptide inhibition of estrogen-dependent growth remains to be determined.

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Trypsin‐mediated semisynthesis of salmon calcitonin

Salmon calcitonin, CT(1-32).NH2, was synthesised by the trypsin-mediated coupling of the peptide fragments CT(1-24) and CT(25-32).NH2, prepared by conventional Fmoc solid-phase chemistry. Optimal conditions regarding reaction time course, pH, proportion of catalyst, substrate concentration and composition of the reaction medium were determined from initial studies on the coupling of CT(1-11) to CT(12-24) and of CT(12-24) to CT(25-32).NH2. For the final successful semisynthesis, we found that it was unnecessary to protect lysine residues not involved in the coupling, and that secondary hydrolysis at these sites could be prevented by increasing the pH of the reaction medium. The reaction achieved equilibrium after 30-45 min, with overall conversion of around 30% of the initial amount of CT(1-24) substrate into product. Yields were depressed due to cyclisation of the CT(1-24) substrate via air-oxidation of the Cys1 and Cys7 residues.

Design and synthesis of novel inhibitors of prohormone convertases

Prohormone convertase-1 (PC1) and furin are subtilisin-like endopeptidases involved in the biosynthesis of peptide hormones. Five decapeptides representing the junction between the pro-region and the catalytic region of PC1 were prepared. The core sequence corresponded to D-Tyr-Arg-Ser-Lys-Arg- Xaa-Val-Gln-Lys-Asp where D-Tyr replaces the native Glu residue and Xaa, representing the P1' position, corresponds to L-Ser, L-Leu or the unnatural amino acids, D-Ser, beta-Ala, gamma-Abu, beta-Cha or gamma-Hyp. Another analog incorporating an Orn residue in place of the Arg at the P1 site was also prepared. These peptides, synthesized by solid-phase Fmoc chemistry, were fully characterized by FAB-MS, 1H-NMR and amino acid composition. Except for Orn, gamma-Hyp, L/D-Ser and L-Leu containing analogs, the others were found to be moderate to potent competitive inhibitors of hPC1 activity in the following order: gamma-Abu > beta-Cha > beta-Ala, with Ki values ranging from 1 to 8.6 microM. Both L-Ser and L-Leu analogs were correctly cleaved at the acyl carbon COOH-terminal to the Lys-Arg pair by human PC1, whereas beta-Cha, gamma-Abu, beta-Ala and D-Ser analogs proved to be very poor substrates. The Orn and gamma-Hyp derivatives were not cleaved by the enzyme at all. The three analogs containing beta-Cha, gamma-Abu and beta-Ala also proved to be potent inhibitors of the human furin activity in the following order: beta-Ala > beta-Cha > gamma-Abu, with Ki ranging from 0.8 to 2.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)