Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

Reproductive performance of lactating dairy cattle after intrauterine administration of a prostaglandin F2α receptor antagonist 4 days after insemination.

Previous research has determined that PGF2α detrimentally affects pregnancy via direct effects on early embryonic development. Because early embryonic loss is relatively prevalent in lactating dairy cows, we hypothesized that pregnancy retention (and resulting conception rates) would be improved by administering a PGF2α receptor antagonist (AL-8810) shortly after insemination. Multiparous, lactating Holstein dairy cows were randomly assigned to receive one of four intrauterine treatments: (1) control group-untreated cohort (CON; n = 93); (2) control group-vehicle infusion (CON-V; n = 90); (3) 2000 nM AL-8810 infusion (AL-2000; n = 96); or (4) 10,000 nM AL-8810 infusion (AL-10,000; n = 93). Treatments were administered transcervically 4 days after insemination in the horn ipsilateral to the CL. There was no effect of treatment on conception rate (36.6%, 38.9%, 25.0%, and 35.5% for CON, CON-V, AL-2000, and AL-10,000, respectively) or calving rate (24.7%, 24.4%, 16.7%, and 28.0% for CON, CON-V, AL-2000, and AL-10,000, respectively). There was a significant effect of treatment on return to estrus with CON-V (23.6 ± 0.6) and AL-10,000 (23.3 ± 0.6) groups having a longer interval to next estrus over the CON group (21.5 ± 0.6; P < 0.05). Prior treatment did not affect conception to the subsequent insemination. It is important to note that although the addition of AL-8810 into the uterus on Day 4 after insemination did not increase conception rates in the present experiment, it also did not have a negative impact. Furthermore, the treatment procedure itself did not impair the establishment of pregnancy (CON vs. CON-V, AL-2000, and AL-10,000). These results demonstrate that a therapeutic agent can be administered directly into the uterus on Day 4 after insemination without detrimentally affecting conception rates.

Synthesis of bis- and tris-branched COOH-terminal pegylating reagents: conjugation to NH-terminal peptides.

In previous studies we reported an orthogonal protection scheme that was developed for the solution-phase synthesis of a family of bis- and tris-pegylating reagents which contain a free NH(2)-terminus. These pegylating reagents were coupled to the COOH-terminus of a model peptide. In the present study we report on the solution synthesis of a novel family of bis- and tris-pegylating reagents which contain a free COOH-terminus. To illustrate their general utility, conditions were developed for the coupling of these novel pegylating reagents to the NH(2)-function of a model pentapeptide. Taken together, our studies demonstrate that these pegylating reagents are well suited for conjugation to peptides and proteins that contain either free COOH- or NH(2)-functions. These reagents may have general utility in therapeutic development as branched pegylation has been shown to provide more effective protection of proteins from proteolysis by shielding the protein surface from approaching macromolecules.

Synthesis of symmetrically and asymmetrically branched pegylating reagents.

A solution-phase procedure using an orthogonal protection scheme was developed for the synthesis of a novel family of multi-pegylating reagents. The procedure was exemplified by the synthesis of bis- and tris-pegylating reagents prepared by stepwise insertion of the poly(ethylene glycol) units thereby enabling the preparation of both symmetrical and asymmetrical pegylating reagents. Asymmetrical pegylation and tris-pegylation of peptides and proteins introduces new variables for use in the optimization of pegylated peptides and proteins. These reagents are ideally suited for conjugation to peptides and proteins as they possess a required functional group and will be useful intermediates for the synthesis of a new generation of pegylated products. Tris-pegylation can also provide more effective protection from proteolysis by shielding the protein surface from approaching macromolecules. To illustrate this potential, conditions were developed for the successful coupling of the tris-pegylating reagent to a model pentapeptide.

The Alzheimer's peptide a beta adopts a collapsed coil structure in water.

The self-assembly of the soluble peptide Abeta into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Abeta is of significant interest. In contrast to studies in other solvents, in water Abeta is collapsed into a compact series of loops, strands, and turns and has no alpha-helical or beta-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers approximately 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular beta-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.

Activation barriers to structural transition determine deposition rates of Alzheimer's disease a beta amyloid.

Brain amyloid composed of the approximately 40-amino-acid human beta-amyloid peptide A beta is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Abeta during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of A beta congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Abeta congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type A beta. The hyperactive peptide, which is linked to a heritable A beta amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type A beta. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.

Combined solid-phase/solution synthesis of a 31-residue vasoactive intestinal peptide analog: general method for repetitive coupling of fragments without isolation and purification of intermediates.

A novel analog of vasoactive intestinal peptide (VIP) has been reported which exhibits high potency and enhanced duration of in vivo biological activity. This VIP analog, cyclo-(Lys21-Asp25)Ac[Glu8 Lys12 Nle17 Ala19, Asp25 Leu26,Lys27,28,Gly29,30,Thr31]-VIP, which also has a lactam bridge, has been reported to have relaxant effects that are significantly more potent than other beta-agonists such as salbutamol and salmeterol. Because it has potential use for the treatment of bronchial asthma in humans, various convergent syntheses were evaluated to enable the economic preparation of large quantities of this medium-sized hentriacontapeptide. From these studies we developed a combined solid-phase/solution synthesis which uses four protected fragments (each prepared by solid-phase synthesis with highly acid-labile resins) possessing Nalpha-9-fluorenylmethyloxycarbonyl and side-chain tert-butyl protection. Only equivalent amounts of each fragment were required to achieve near-quantitative coupling reactions using N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmeth anaminium hexafluorophosphate N-oxide/N-hydroxybenzotriazole. All reagents and side products were removed at each stage by simple extraction procedures. Final deprotection was carried out with 90% trifluoroacetic acid. Under these conditions only low levels of epimerization were observed (<2%). These diastereoisomers and other trace impurities were removed from the product in a single purification by preparative high-performance liquid chromatography. The procedure has been scaled up (10-g scale) and the final product obtained in an overall (nonoptimized) yield of 24%. This procedure for the repetitive coupling of fragments, without isolation of intermediates, may be generally applicable for the economic synthesis of other medium-sized and longer peptides.

Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo.

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity.

A beta deposition inhibitor screen using synthetic amyloid.

The formation, growth, and maturation of brain amyloid "senile" plaques are essential pathological processes in Alzheimer's disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of A beta at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-through put screen for inhibitors of A beta deposition onto a synthetic template (synthaloid) of fibrillar A beta immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both A beta analogs and inhibitors. Synthaloid, in contrast to current A beta aggregation screens, accurately predicted inhibitor potency for A beta deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.

Point substitution in the central hydrophobic cluster of a human beta-amyloid congener disrupts peptide folding and abolishes plaque competence.

Alzheimer's disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.

Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs.

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.

Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.

1H NMR of A beta amyloid peptide congeners in water solution. Conformational changes correlate with plaque competence.

To begin to examine the structural basis for the deposition of soluble A beta amyloid peptide onto senile plaques in Alzheimer's disease, we have prepared A beta congeners and measured their activity in an in vitro plaque growth assay. The N-terminal fragment, A beta (1-28)-OH, was inactive at all pH values tested. While the central fragment, A beta (10-35)-NH2, and the full length peptide, A beta (1-40)-OH, were inactive below pH 4, both were active (plaque competent) between pH 5 and 9. The active and inactive fragments were studied by nuclear magnetic resonance spectroscopy in water at submillimolar concentrations at pH 2.1 and 5.6. Changes in chemical shifts, coupling constants, and nuclear Overhauser enhancements indicate a pH dependent folding transition in A beta (10-35)-NH2 as it becomes active. The conformation of the active fragment is not helical, and preliminary data indicate the presence of several turns and at least two short strands. In contrast, the inactive fragment A beta (1-28)-OH did not undergo a similar folding transition. Earlier nuclear magnetic resonance studies of amyloid peptides in fluorinated alcohols or detergent micelles at low pH described a helical conformation and proposed a helix to sheet transition in plaque formation; the present study demonstrates that no such conformations are present in water under conditions where the peptides can adhere to authentic amyloid plaques.

Rational design, synthesis, and biological evaluation of novel growth hormone releasing factor analogues.

Since its initial discovery in 1982, growth hormone-releasing factor (GRF) has been the subject of intense investigation. This interest was prompted by the potential application of GRF for stimulating growth in dwarf humans and for performance enhancement in livestock. Substantial research has been focused upon the development of potent, long-acting analogs as therapeutics. Herein is described a summary of the cumulative efforts of various laboratories endeavoring in this quest. The rationale utilized in GRF analog development is discussed: 1) determination of bioactive core, 2) evaluation of secondary structure, and 3) elucidation of degradation pathways (chemical and enzymatic). Using this information, several series of linear (unnatural and natural sequence) and cyclic GRF analogs were designed, synthesized, and evaluated. Stimulated by the constraints of commercial production, innovative, alternative methods of synthesis were explored: solid-phase, solution-phase, enzymatic, and recombinant. To date, the most promising candidate for drug development is [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH. This natural sequence analog, consisting of rodent and human sequences, incorporates the bioactive core, preferred secondary structure, resistance to chemical and enzymatic degradation; with the added benefit of amenability to large-scale recombinant synthesis.

Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency.

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), from the precursor, [Ala15,29]-GRF(4-29)-OH (1). C-Terminal amidation of 1 to form [Ala15]-GRF(4-29)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala29-OH for Arg-NH2. The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3. GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 degrees C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp3-Ala4 and Asp25-Ile26. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH2Tyr-D-Ala-Asp(OH)-OR (3) [R = CH3CH2- (3a), CH3- (3b), ClCH2CH2- (3c), C6H5CH2- (3d), 4-NO2C6H4CH2- (3e)] revealed that rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4, increase with increasing specificity (Vmax/Km) of GSE for the tripeptide ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Solid-phase synthesis of extended lactam ring systems: preparation of amino acid alpha-fluorenylmethyl esters for the synthesis of reverse-extended lactams.

Conventional side-chain to side-chain cyclization between Lys/Orn and Asp/Glu side chains has the inherent shortcoming of limited variability in the ring sizes; limited by the amino acids involved in the lactamization. A novel approach to modulate the ring size has been introduced by using "spacers" (e.g., Gly, beta-Ala, gamma-Aba). General solid-phase procedures were developed that enabled the insertion of these spacers for the preparation of extended and reverse-extended lactam ring systems. Extended lactam ring systems were prepared using the Boc/Bzl strategy with Fmoc protection on the side chain of the basic residue, Lys/Orn. The spacer was introduced as the Fmoc amino acid, and chain elongation proceeded by the Boc/Bzl strategy. The acidic residue, Asp/Glu, involved in the lactam bridge was introduced with side-chain -OFm protection. Selective deprotection of the Fmoc and -OFm functions was followed by BOP cyclization, further chain elongation and HF cleavage. Reverse-extended lactam ring systems were prepared analogously using the Boc/Bzl strategy with the initial introduction of -OFm protection on the side chain of the acidic residue. In this case, amino acid alpha-fluorenylmethyl esters were used as the spacers. Amino acid alpha-fluorenylmethyl ester hydrochlorides were prepared in two steps from N-t-butyloxycarbonyl amino acids in excellent yield. These intermediates have general utility in peptide synthesis, including their specific application as "spacers" for the solid-phase synthesis of extended cyclic peptides (lactams). Using these intermediates, we prepared a model dicyclo-peptide that contains both an extended and reverse-extended lactam ring system.

Pegylated peptides. II. Solid-phase synthesis of amino-, carboxy- and side-chain pegylated peptides.

General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)2-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry.

Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences.

Native human GRF(1-44)-NH2(hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala2 and Asp3. The hGRF44 is also subject to chemical rearrangement (Asn8-->Asp8, beta-Asp8 via aminosuccinimide formation) and oxidation [Met27-->Met(O)27] in aqueous environments, greatly reducing its bioactivity. It is therefore advantageous to develop long-acting GRF analogues using specific amino acid replacements at the amino-terminus (to prevent enzymatic degradation): residue 8 (to reduce isomerization) and residue 27 (to prevent oxidation). Inclusion of Ala15 substitution (for Gly15), previously demonstrated to enhance receptor binding affinity, would be predicted to improve GRF analogue potency. Substitution of [His1,Val2]-(from the mouse GRF sequence) for [Tyr1,Ala2]-(human sequence) in [Ala15,Leu27]hGRF(1-32)-OH analogues completely inhibited (24-h incubation) DPP-IV cleavage and greatly increased plasma stability in vitro. Additional substitution of Thr8 (mouse GRF sequence), Ser8 (rat GRF sequence), or Gln8 (not naturally occurring) for Asn8 (human GRF sequence) resulted in analogues with enhanced aqueous stability in vitro (i.e., decreased rate of isomerization). These three highly stable and enzymatically resistant hGRF(1-32)-OH analogues, containing His1, Val2, Thr/Gln8, Ala15, and Leu27 replacements, were then bioassayed for growth hormone (GH)-releasing activity in vitro (rat pituitary cell culture) and in vivo (SC injection into pigs). Enhanced bioactivity was observed with all three hGRF(1-32)-OH analogues. In vitro, these analogues were approximately threefold more potent than hGRF44, whereas in vivo they were eleven- to thirteenfold more potent.(ABSTRACT TRUNCATED AT 250 WORDS)

Pegylated peptides I: Solid-phase synthesis of N alpha-pegylated peptides using Fmoc strategy.

The feasibility of pegylating peptides by the solid-phase procedure was examined. Although polyethyleneglycol (PEG) was shown to be partially degraded by HF, the use of TFA was fully compatible with the PEG system. Therefore, the Fmoc/tBu solid-phase strategy was utilized for the synthesis of a series of model tetra-, octa- and dodecapeptides, and the corresponding N alpha-pegylated peptides, which were prepared from common peptide-resin intermediates. PEG-OCH2-CO-Nle-OH, 3, proved to be an ideal reagent for N-terminal pegylation. This intermediate served as a diagnostic for the determination of the number of PEG units/mole of peptide. Solid-phase coupling reactions proceeded by standard procedures using BOP-activation. The authentic pegylated peptides (readily purified by conventional methods of preparative HPLC) were fully characterized by amino acid analysis, 1H-NMR spectroscopy, analytical HPLC and laser desorption ionization mass spectrometry, leading to the values that are identical with the expected structures.