Parent Analogue Research Articles

Parent visual analogue scale ratings of children’s pain do not reliably reflect pain reported by child

To determine whether parent and child visual analogue scale (VAS) scores for the pain associated with acute conditions in the child agree sufficiently for these methods of measurement to be considered interchangeable in pain and analgesia research. This was a prospective, two-group, repeated measures, blinded study in an urban pediatric emergency department. Children aged 8 to 15 years seeking treatment for painful conditions and the parents of these children were asked to rate the child's pain independently using a VAS on as many as four occasions at 20-minute intervals. Both participants were blinded to their previous rating and the rating of the other participant. The main outcome measure was the correlation of child and parent VAS pain scores by Pearson correlation and bias plot (Bland-Altman) analysis of agreement between tests. Seventy-eight child-parent sets participated, yielding 289 VAS pain score comparison pairs for evaluation. The correlation between child and parent VAS pain scores was 0.63 (95% CI, 0.56-0.70). Bias plot analysis revealed a bias of 5% and 95% limits of agreement from -38 to +47 mm. The degree of difference between child and parent scores was variable, but there was an increasing tendency for parents to underestimate the child's pain when the child recorded VAS pain scores at the higher end of the scale. Parents' VAS score ratings of their children's pain correlate only moderately with the children's VAS pain scores and show poor levels of agreement. The difference between the measures is variable and appears to be more marked when the child reports a higher VAS score. This research raises doubt about whether parental rating of a child's pain is an appropriate surrogate marker in pediatric pain and analgesia research.

O-Galloyl- C-glycosylflavones from Pelargonium reniforme

The unique series of C-2″-acylated C-glycosylflavones is extended by the discovery of the C-8-glucosyl derivatives 2″- O-galloylvitexin and 2″- O-galloylorientin and their C-6 analogues 2″- O-galloylisovitexin and 2″- O-galloylisoorientin, representing the first described O-galloyl-C-glycosylflavones. They are accompanied in the aerial parts of Pelargonium reniforme by the known non-galloylated parent analogues vitexin, orientin, isovitexin and isoorientin, as well as several known flavonoid- O-glycosides. The structures of these compounds were established from spectroscopic studies. Differentiation between C-glycosylation at C-6 and C-8 is discussed on the basis of the effects of dynamic rotational isomerism.

Highly active analogs of 1α,25-dihydroxyvitamin D 3 that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1α,25(OH) 2-3-epi-D 3. A rational design for the synthesis of potent analogs of 1α,25(OH) 2D 3 is developed based on the knowledge of the various metabolic pathways of 1α,25(OH) 2D 3. Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D 3 analogs 1α,25(OH) 2-16,23-E-diene-26,27-hexafluoro-19-nor-D 3 (Ro 25–9022), 1α,25(OH) 2-16,23-Z-diene-26,27-hexafluoro-19-nor-D 3 (Ro 26–2198), and 1α,25(OH) 2-16-ene-23-yne-26,27-hexafluoro-19-nor-D 3 (Ro 25–6760), as indicated by their antiproliferative activities.

Synthesis and enantioselectivity of the antiviral effects of (R,Z)-,(S,Z)-methylenecyclopropane analogues of purine nucleosides and phosphoralaninate prodrugs: influence of heterocyclic base, type of virus and host cells.

A series of R and S enantiomers of 2-aminopurine methylenecyclopropane analogues of nucleosides was synthesized. Two diastereoisomeric lipophilic phosphate prodrugs derived from R and S enantiomers of 2,6-diaminopurine analogue were also prepared. Enantioselectivity (diastereoselectivity in case of prodrugs) of in vitro antiviral effects was investigated with human and murine cytomegalovirus (HCMV and MCMV, respectively), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV). Strong differences in enantioselectivity were found between the R and S enantiomers of adenine analogue and enantiomeric 2-aminopurine analogues. Thus, the enantiomers of adenine analogue were equipotent against HCMV but not MCMV, where the S enantiomer is strongly preferred. The same S preference was found throughout the 2-aminopurine series for both HCMV and MCMV. In contrast, R-synadenol in HIV-1 assays was the best agent, whereas the S enantiomers of moderately effective 2-amino-6-cyclopropylamino and 2-amino-6-methoxypurine analogues were preferred. Little enantiomeric preference was found for R and S enantiomers of synadenol and the corresponding enantiomers of 2,6-diaminopurine analogue against HBV. A mixed pattern of enantioselectivity was observed for EBV depending on the type of host cells and assay. Against VZV, the R and S enantiomers of adenine analogue were equipotent or almost equipotent, but throughout the series of 2-aminopurine analogues a distinct preference for the S enantiomers was found. The stereoselectivity pattern of both diastereoisomeric prodrugs mostly followed enantioselectivity of the parent analogues. The varying enantioselectivities in the series of purine methylenecyclopropane analogues are probably a consequence of differences in the mechanisms of action in different virus/host cell systems.

Impact of α,β-dehydroamino acid residues on the binding abilities of di-, tri- and tetra-peptides

Insertion of a dehydroamino acid residue into a sequence of di-, tri- or tetra-peptide changed considerably the binding abilities of peptide ligands towards copper(II) ions. Potentiometric and spectroscopic (EPR, UV-VIS and CD) data have shown that the amide nitrogen of the dehydroamino acid residue is more effective in co-ordination than its parent analogue. In the case of the bulky ΔPhe residue also the (Z–E) isomerisation has a critical impact on the co-ordination equilibria in the system studied.

Enhanced biological activity of 1α,25-dihydroxy-20-epi-vitamin D 3, the C-20 epimer of 1α,25-dihydroxyvitamin D 3, is in part due to its metabolism into stable intermediary metabolites with significant biological activity

1α,25-Dihydroxy-20-epi-vitamin D 3 (1α,25(OH) 2-20-epi-D 3), the C-20 epimer of the natural hormone 1α,25(OH) 2D 3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D 3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1α,25(OH) 2D 3 with that of 1α,25(OH) 2-20-epi-D 3 using the technique of isolated perfused rat kidney. The results indicated that the C-24 oxidation pathway plays a major role in the metabolism of both compounds in the rat kidney. However, it was noted that the concentrations of two of the intermediary metabolites of 1α,25(OH) 2-20-epi-D 3, namely, 1α,24( R),25(OH) 3-20-epi-D 3 and 1α,25(OH) 2-24-oxo-20-epi-D 3 in the kidney perfusate, exceeded the concentrations of the corresponding intermediary metabolites of 1α,25(OH) 2D 3. Furthermore, 1α,25(OH) 2-24-oxo-20-epi-D 3 induces the conformation of the vitamin D receptor similar to that induced by its parent analog and is nearly as potent as its parent in inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element. We conclude that 1α,25(OH) 2-20-epi-D 3 by itself is not metabolically stable when compared to 1α,25(OH) 2D 3, but it acquires its metabolic stability because of the reduced rate of catabolism of its intermediary metabolites. Furthermore, 1α,25(OH) 2-24-oxo-20-epi-D 3, the stable bioactive intermediary metabolite plays a significant role in generating the enhanced biological activities ascribed to 1α,25(OH) 2-20-epi-D 3.

High level of aspartic acid-bond isomerization during the synthesis of an N-linked tau glycopeptide.

An increased degree of utilization of the potential N-glycosylation site in the fourth repeat unit of the human tau protein may be involved in the inability of tau to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a beta-peptide. The anomalous chromatographic behavior of the acetylated beta-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated tau into the paired helical filaments in the affected brains.

Synthesis and antiviral activity of phosphoralaninate derivatives of methylenecyclopropane analogues of nucleosides

Phenylmethylphosphoro-L-alaninate prodrugs of antiviral Z-methylenecyclopropane nucleoside analogues and their inactive E-isomers were synthesized and evaluated for their antiviral activity against HCMV, HSV-1, HSV-2, HHV-6, EBV, VZV, HIV-1 and HBV. The adenine Z-analogue was a potent inhibitor of all these viruses but it displayed cellular toxicity. The guanine Z-derivative was active against HCMV, HBV, EBV and VZV and it was not cytotoxic. The 2,6-diaminopurine analogue was the most potent against HIV-1 and HBV and somewhat less against HHV-6, HCMV, EBV and VZV in a non-cytotoxic concentration range. The 2-amino-6-cyclopropylamino and 2-amino-6-methoxypurine prodrugs were also more active than parent analogues against several viruses but with a less favorable cytotoxicity profile. In the E-series of analogues, adenine derivative was active against HIV-1, HBV and EBV, and it was non-cytotoxic. The guanine analogue exhibited a significant effect only against HBV. The 2,6-diaminopurine E-analogue was inactive with the exception of a single EBV assay. The 2-amino-6-methoxypurine Z-methylenecyclopropane nucleoside analogue was an effective inhibitor of HCMV, MCMV and EBV. The 2,6-diaminopurine Z-prodrug seems to be the best candidate for further development.

The gas phase acidities of substituted silanoic acids

Ab initio molecular orbital methods at the CBS-Q level of theory have been used to study the structure, gas phase acidities and vibrational spectra of silanoic acid, HSiO2h, and several of its derivatives RSiO2H (R = F, Cl, NH2, OH and CH3). Geometry optimizations were performed at the MP2(FC)/6-31G(†) level of theory. The syn forms always are more stable than the anti. The silanoic acid derivatives are more acidic than their parent oxygen acid analogues. The fundamental vibrational frequencies have been computed using the MP2(Full)/6-31G(D) method. The ν(Si=O) = 1261 cm−1 value agrees with the experimentally determined normal mode (1249 cm−1) in silanoic acid.

L-687,414, a low efficacy NMDA receptor glycine site partial agonist in vitro, does not prevent hippocampal LTP in vivo at plasma levels known to be neuroprotective.

N-methyl-D-aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long-term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK-801 and glycine-site antagonism by 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414). In vitro experiments using rat cortical slices revealed L-687,414 to be approximately 3.6 fold more potent than its parent analogue, R(+)HA-966 at antagonizing NMDA-evoked population depolarizations (apparent Kbs: 15 microM and 55 microM, respectively). Whole-cell voltage-clamp experiments using rat cultured cortical neurones revealed L-687,414 to shift to the right the concentration-response relationship for NMDA-evoked inward current responses (pKb=6.2+/-0.12). L-687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration-inhibition curves, pKi=6.1+/-0.09. In the latter experiments, L-687,414 and R(+)HA-966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity = approximately 10 and 20% of glycine, respectively). L-687,414 and MK-801 were compared for their effects on NMDA receptor-dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg(-1) followed by 0.0298 ml min(-1)), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK-801 dosing regimen (0.12 mg kg(-1) i.v. followed by 1.8 microg kg(-1) h(-1)) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L-687,414 treatment (28 mg kg(-1) i.v. followed by 28 mg kg(-1) h(-1)). These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor-dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L-687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of 'normal' synaptic transmission whilst curtailing excessive receptor activation.

Oral midazolam for conscious sedation of children during minor procedures.

OBJECTIVE: To compare the safety and efficacy of two doses of oral midazolam, and to assess the drug induced amnesia obtained, when used for conscious sedation of children undergoing minor...

How acidic are thiocarboxylic S-acids and thiosilanoic S-acids (XYOSH, X = H, F, Cl, CH3, NH2; Y = C, Si)?

The structure, gas-phase acidities and vibrational spectra of thioformic S-acid, thiosilanoic S-acid and several of their derivatives XY(O)SH ( X = H, F, Cl, NH 2, CH 3; Y = C, Si) were investigated by DFT and ab initio HF calculations. Geometry optimizations and frequency computations were performed at the Becke3LYP level of theory with 6–311+G ∗ basis set. For all acids studied the syn conformers are predicted to have the lowest energy. The syn-anti energy difference is varying between 1 and 2.7 kcal mol −1. Thioformic and thiosilanoic S-acid are, according to calculated ionization energy, about 8 kcal mol −1 and 19.5 kcal mol −1 more acidic than their parent oxygen acid analogues. The substitution of carbon by silicon in thiolcarboxylic acids studied leads to considerable increase of acidity of those species. The greater acidity of thiosilanoic acid derivatives over their thiocarboxylic acid counterparts is largely due to electrostatic interactions. The more electropositive silicon imparts more ionic character to the bonds in thiosilanoic acids compared to carboxylic acids. Computed vibrational frequencies for thioformic S-acid, thioformic O-acid and thioacetic S-acid were in average 3–5% higher than the observed values.

Diels-Alder addition of butadiene to various thiocarbonyl(R2C?SOn,n=0-2) heterodienophiles andendo-lone pair effect in heterocumulene

Diels–Alder reactions of various thiocarbonyl S-oxides and thiocarbonyl S,S-dioxides with buta-1,3-diene were studied by semiempirical AM1 and PM3 methods. Calculations show that the reactions of such unsymmetrical heterodienophiles pass through asynchronous transition states (TSs) with C=S π* involved in the reaction. Calculated activation energies indicate that the reactivity of these heterodienophiles, viz. R2C=S, R2C=SO and R2C=SO2, decrease gradually with successive addition of oxygen atoms to the thiocarbonyl sulfur. This is in good agreement with the experimental observations. The predictions based on LUMO and deformation energies show that the above reactivity trend corresponds to a gradual destabilization of LUMO of the dienophile and increase in deformation energy of both diene and dienophile with increase in the number of oxygen atoms around sulfur. Thiophosgene and their S-oxides in Diels–Alder reactions are found to be less reactive than the parent analogues. Monosubstituted (Z/E)-sulfines react with buta-1,3-diene to form cis and trans products through closely lying TSs. Calculations predict that (E)-sulfine has a higher reactivity than the Z-isomer, in reasonable agreement with experimental results. © 1997 John Wiley & Sons, Ltd.

Diels–Alder addition of butadiene to various thiocarbonyl(R2C=SOn, n=0–2) heterodienophiles and endo-lone pair effect in heterocumulene

Diels–Alder reactions of various thiocarbonyl S-oxides and thiocarbonyl S,S-dioxides with buta-1,3-diene were studied by semiempirical AM1 and PM3 methods. Calculations show that the reactions of such unsymmetrical heterodienophiles pass through asynchronous transition states (TSs) with C=S π* involved in the reaction. Calculated activation energies indicate that the reactivity of these heterodienophiles, viz. R2C=S, R2C=SO and R2C=SO2, decrease gradually with successive addition of oxygen atoms to the thiocarbonyl sulfur. This is in good agreement with the experimental observations. The predictions based on LUMO and deformation energies show that the above reactivity trend corresponds to a gradual destabilization of LUMO of the dienophile and increase in deformation energy of both diene and dienophile with increase in the number of oxygen atoms around sulfur. Thiophosgene and their S-oxides in Diels–Alder reactions are found to be less reactive than the parent analogues. Monosubstituted (Z/E)-sulfines react with buta-1,3-diene to form cis and trans products through closely lying TSs. Calculations predict that (E)-sulfine has a higher reactivity than the Z-isomer, in reasonable agreement with experimental results. © 1997 John Wiley & Sons, Ltd.

Serum stability of phosphopeptides

Synthetic phosphopeptides are increasingly used to mimic phosphoprotein fragments and in rational drug design. Both applications require a study of the stability of phosphopeptides in biological environments. Here we report a study of the stability of three sets of synthetic un-, mono-, and diphosphorylated peptides in diluted human serum. The synthetic peptides contained the phosphoserines or phosphothreonines in mid-chain position corresponding to immunodominant regions of abnormally hyperphosphorylated variants of the human τ protein present in the paired helical filaments of Alzheimer's disease. The peptide degradation in human serum was monitored, and the first metabolites formed were collected from reversed-phase high performance liquid chromatography (RP-HPLC). Seven of nine monophosphorylated peptides exhibited increased stability compared to the unphosphorylated parent analogs. All peptides phosphorylated at immunodominant sites displayed higher serum stability than phosphopeptide isomers of the same sequences giving evidence of the extended presence of the antigenic stimulus in the hosts. Analysis of the degradation pathway of the fastest degrading peptide family showed that the mono- and diphosphorylated peptides, like the unphosphorylated version, underwent aminopeptidase cleavage and the phosphate group remained attached to the serine and threonine side chains. These results indicate that phosphopeptides are suitable models of phosphoprotein fragments in biochemistry assays, and that phosphorylation can be a viable modification of peptide leads in drug design.

Separation of sets of mono- and diphosphorylated peptides by reversed-phase high performance liquid chromatography

The effect of phosphorylation on the behavior of synthetic peptides in reversed-phase high performance liquid chromatography (RP-HPLC) was re-examined using sets of peptides that included four unphosphorylated, 10 monophosphorylated and 11 diphosphorylated analogs. Trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA) were compared as anionic ion-pairing reagents in water/acetonitrile gradient runs on a standard C 18 analytical column. The separation of the unphosphorylated and monophosphorylated peptides appeared to be dependent upon multiple factors: oncolumn time, and hydrophilicity of the phosphate group when TFA was used, and general hydrophobicity and partial negative charge when HFBA was used. Among the chromatograms developed in the TFA system, increased on-column times resulted in better separation. The retention times of some phosphopeptides in which the phosphoamino acids were located proximal to basic residues were reduced compared with other phosphopeptide isomers in the TFA system, indicating that local in-peptide ion bridges retain more hydrophilic character of the phosphate side-chain group at pH 1.9. In identical gradients HFBA generally separated the monophosphorylated peptides from the unphosphorylated parent analogs better than TFA. Nevertheless, most phosphopeptide isomers of the same sequences coeluted in both systems. In identical Chromatographic conditions, TFA produced sharper peaks with a considerably smoother baseline. Remarkably, diphosphorylated peptides frequently eluted as broad or very broad peaks in both systems, and their retention times were sometimes increased compared to the unphosphorylated and monophosphorylated analogs suggesting that the predictive methods for phosphopeptide retention times are not fully applicable for multiphosphorylated sequences. Our data indicate that the RP-HPLC of multiphosphorylated peptides and protein fragments need to be further investigated or expanded with unconventional mobile phases.

Metabolism of 1α,25(OH)2-20-epi-D3 Into a Stable, Biologically Active, Intermediary Metabolite. † 418

1α,25(OH)2-20-epi-D3, a synthetic analog of 1α,25(OH)2D3, differs from the natural hormone, by a simple modification in the stereochemistry of the methyl group at carbon-20 of the side chain. Previous studies have shown that the activity of the analog in inhibiting proliferation and inducing differentiation of leukemic cells is several fold greater than that of 1α,25(OH)2D3. At present, the various mechanisms responsible for the enhanced biological activities of the analog have not been clearly elucidated. In one study it is shown that the 20-epi analog induces a conformation of the vitamin D receptor(VDR) that is distinct from that induced by 1α,25(OH)2D3. The modified conformation of the VDR enhances dimerization of VDR with the retinoid X receptor resulting in increased transactivation activity. It is now established that the enhanced biological activities of synthetic analogs of 1α,25(OH)2D3 also depend on their stability and the rate at which they are metabolized. Therefore, in our present study we compared the metabolism of 1α,25(OH)2-20-epi-D3 with that of 1α,25(OH)2D3 in the isolated perfused rat kidney. The metabolism studies reveal that there is a partial block in the further metabolism of the analog, resulting in accumulation of the intermediary metabolite, 1α,25(OH)2-20-epi-24-oxo-D3 and its precursor, 1α,24,25(OH)3-20-epi-D3. We determined that 1α,25(OH)2-20-epi-24-oxo-D3 also induces a conformation of the VDR similar to that induced by the parent analog. Furthermore, this intermediary metabolite is nearly as potent as the parent analog at inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element linked to the thymidine kinase promoter and growth hormone reporter gene. Thus, we conclude that 1α,25(OH)2-20-epi-D3 is metabolically stable and the stability is at the level of its intermediary metabolites, which in turn contribute significantly to the overall biological activity of the analog.

Specific binding of Cu 2+ and Ni 2+ ions by thioamide analogs of tetraalanine

Thioamide derivatives of short peptides are much more efficient ligands for Cu 2+ and Ni 2+ ions than their parent analogs. The most effective is the insertion of the thiocarbonyl group into the first, second, or third peptide linkage. In all of these cases, the sulfur atom is involved directly in metal ion binding, leading to the formation of very stable complexes. In all cases discussed, sulfur binding does not prevent an amide nitrogen deprotonation and its coordination to metal ion in basic solutions, although at the region close to physiological pH, sulfur is the basic donor for both Cu 2+ and Ni 2+ ions.

Identification of high-potency neuropeptide Y analogues through systematic lactamization.

In the pursuit of potent analogues of neuropeptide Y (NPY) that are selective for the Y1 receptor subtype, two lactam bridge scans of a centrally truncated parent compound were synthesized. A single lactam bridge (gamma-carboxyl of Glu to epsilon-amino of Lys) extending from residues i to i + 3 or i to i + 4 of the proposed alpha-helical region (residues 25-31 of NPY) was introduced in des-AA7-24[Gly6]NPY. Cyclogues (contraction of cyclic analogues), which were approximately one-half the size of native NPY, were initially screened for binding affinity at two discrete NPY receptor types using human neuroblastoma cell membranes, SK-N-MC and SK-N-BE2. Exploitation of the subtle differences present on each receptor type allowed for the identification of cyclogues which bound specifically to Y1 receptors with increased affinity when compared to the corresponding linear parent analogue, while one short Y1 specific cyclogue, des-AA2,3,5,7-24cyclo-(26/29)[Gly6,Glu26,Lys2 9,Pro34]NPY, bound with Ki = 16 nM. Other cyclogues showed distinct preference for Y2 receptors and bound in the low-nanomolar range. Functionally, the compounds inhibited the norepinephrine-stimulated accumulation of cAMP indicating that all acted as agonists with varying potencies.

Solution structure and adenylyl cyclase stimulating activities of C-terminal truncated human parathyroid hormone analogues.

Analogues of human parathyroid hormone (hPTH) truncated at the C-terminal end have been studied for adenylyl cyclase (AC) activity and for solution conformation by circular dichroism (CD) spectroscopy. Analogues of hPTH-(1-34)-NH2, containing the first 28-31 residues, had only a slightly diminished ability to stimulate AC in rat osteosarcoma (ROS) cells as compared to that of the parent analogue. CD data on hPTH-(16-34)-NH2 and C-terminal deletion mutants of hPTH-(1-34)-NH2 supported the presence of a partially stable alpha-helix over residues 17-28. A carboxyl-terminal mutant, hPTH-(1-30)-OH, showed both reduced helix and greatly reduced AC-stimulating activity as compared to the corresponding amide analogue. In contrast, both of these analogues, in the presence of palmitoyloleoylphosphatidylserine (POPS) vesicles, showed an equal stabilization of alpha-helix. All other analogues showed at least some enhancement of alpha-helix in the presence of POPS. However, both in neutral, aqueous buffer and in POPS, the relative amount of alpha-helix decreased greatly as the peptide was shortened below the 1-28 sequence. These data provide additional support for an amphiphilic alpha-helix over residues 21-28 being the conformation for receptor binding of hPTH for stimulation of AC activity. Modeling human parathyroid hormone-related peptide as an alpha-helix over this same region, and comparison to hPTH, suggests that both may bind via the hydrophobic face to the receptor.