Solution synthesis of human neuropeptide Y (hNPY)

Human neuropeptide Y (hNPY) was synthesized in a conventional manner by assembling seven peptide fragments followed by reduction of the Met(O) residue with phenylthiotrimethylsilane and subsequent deprotection with 1 M trimethylsilyl trifluoromethanesulfonate (TMSOTf)-thioanisole in trifluoroacetic acid (TFA). Alternatively, deprotection was performed in a two-step manner; first, treatment with 1 M trimethylsilyl bromide-thioanisole in TFA, and then with 1 M TMSOTf-thioanisole in TFA. After purification by gel-filtration on Sephadex G-25, followed by reversed-phase high-performance liquid chromatography, a highly purified sample of synthetic hNPY was obtained in both cases. When administered in dogs, synthetic hNPY was as active as porcine NPY in terms of the effects on systemic arterial blood pressure, pancreatic blood flow, and superior mesentric artery (SMA) blood flow. Met(O)17-hNPY was found to be as active as the parent sample in these bioassays.     

Molecular modeling and dynamics of neuropeptide Y

Summary A combination of molecular modeling and molecular dynamics (MD) is used to determine a theoretical structure for neuropeptide Y (NPY). Starting with the X-ray structure for avian pancreatic polypeptide (APP), the substituted amino acids were mutated, the side chains oriented to local potential energy minima, and the entire structure minimized and subjected to an MD simulation. Comparison of the resulting NPY structure with APP X-ray and MD results showed secondary structural elements to be maintained and RMS fluctuations to be similar, although differences in both were observed. The approach presented offers a means to study the structure-function relationships of NPY and other similar polypeptides when combined with pharmacological measurements.Abbreviations NPY Neuropeptide Y - APP Avian pancreatic polypeptide - ABNR Adopted-basis Newton Raphson - MD Molecular dynamics     

Hormonal control of the neuropeptide Y system

Neuropeptide Y (NPY) and the related receptors represent a widely diffused system that is involved in the regulation of multiple biological functions. NPY, a 36-aminoacid peptide expressed in several areas of the nervous system, is a pleiotropic factor participating to the control of some physiological processes, such as cognitive functions, eating behavior, circadian rhythms, neuroendocrine mechanisms, reproductive and cardiovascular functions. NPY acts through a series of G-protein-associated membrane receptors (NPY-Rs), characterized by different tissue distribution and affinity for the ligand. The expression and secretion of NPY and the expression of NPY-R isoforms are controlled by a very wide range of agents, acting in an endocrine and/or paracrine fashion. NPY and NPY-Rs appear to be strongly involved in the control of eating behavior; their expression is modulated by changes of food intake and energy balance and is disrupted in several animal models of obesity and diabetes. Moreover, the hypothalamic NPY system appears to integrate signals of energy balance in the modulation of the reproductive axis. Agents that stimulate their expression include activators of intracellular signalling pathways (protein kinase A and C), classical neurotransmitters, steroid and peptide hormones and growth factors, while other agents (leptin, insulin and retinoic acid) have been shown to be inhibitory. Interestingly, some agents, like retinoic acid, have been shown to modulate the expression of both NPY and NPY-Rs in the same direction, thus providing a fine mechanism for the tuning of the system. The regulation of NPY/NPY-R expression and function appears to be part of a complex system controlling multiple physiological functions, and its disruption might be relevant in the pathophysiology of disease states such as obesity.     

Structure-affinity relationships of C-terminal cyclic analogue of neuropeptide Y for the Y1-receptor

We previously reported that a cyclic octapeptide amide, c[D-Cys29, Cys-34]NPY Ac-29-36 (YM-42454) showed a high affinity for Y1-receptors in SK-N-MC cells (Ki=0.047,microM) but not for Y2-receptors in the porcine hippocampus membranes (Ki>10microM). To explore the critical residues of this unique cyclic peptide for Y1-binding activity, the structure-affinity relationships were investigated by means of amino acid replacement. The results indicated that the hydrophobic side-chains of Leu30 and Ile31, the guanidinium groups of Arg33 and Arg33, and the C-terminal amide are critical for the binding affinity of YM-42454 to the Y1-receptor. On the other hand, Thr32 in YM-42454 might not be critical for the Y1-binding affinity. 1H-NMR studies for YM-42454 and its derivatives have suggested that the critical residues are involved in the direct interaction with a Y1-receptor rather than in maintaining the bioactive conformation.     

Aptamer-functionalized graphene-gold nanocomposites for label-free detection of dielectrophoretic-enriched neuropeptide Y

We present an electrokinetically enhanced aptamer sensing platform on a disposable plastic chip for label-free detection of neuropeptide Y (NPY), which is a key neurological biomarker. The sensor consists of aptamer-functionalized graphene-gold nanocomposites (Gr-AuNs) patterned inside a nanoslit that is embossed on cyclic olefin copolymer via nanoimprint lithography. Analyte molecules are dielectrophoretically focused through the nanoslit onto aptamer-immobilized Gr-AuNs for rapid and selective electrochemical detection of NPY at picomolar levels.     

Protease-catalyzed peptide synthesis on solid support

The direct enzymatic synthesis of peptides from amino acids is widely used as a useful alternative to chemical synthesis. However, good yields of such enzyme-catalyzed reactions require altered reaction conditions to overcome the bias for hydrolysis in aqueous medium. We argue that the synthesis/hydrolysis equilibrium can be shifted toward synthesis in aqueous medium by immobilizing the amine on solid support. In this report, we show the first examples of solid-phase peptide synthesis catalyzed by a protease in bulk aqueous buffer.     

Protease-catalyzed Synthesis of Bz-Arg-Gly-Asp-OMe in Full Aqueous Medium

Synthesis of N-benzoyl-argininylglycylasparagine methyl ester(Bz-Arg-Gly-Asp-OMe),a precursor tripeptide of Arg-Gly-Asp)was catalyzed by papain under kinetic control,at alkaline pH,in a full aqueous medium.The substrates were N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester.An aqueous solution of 0.1 mol/L KCl/NaOH containing 8 mmol/L EDTA and 2 mmol/L DTT was selected as the reaction medium.The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process,however,the secondary hydro-lysis of the tripeptide product was not considerable.The effects of different factors,including water content,temperature,reaction time,and molar ratio of the substrates,on the yield of Bz-Arg-Gly-Asp-OMe were examined.The optimal reaction conditions were 0.05 mol/L Bz-Arg-Gly-OEt and 0.15 mol/L Asp(-OMe)2·HCl in 0.1 mol/L KCl/NaOH solution(pH 8.5),at 40 ℃,and a reaction time of 60 min,with a maximum conversion yield of 62.4%.     

Protease-catalyzed monoacylation of 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid in pyridine

2-O-alpha-D-Glucopyranosyl-6-O-octanoyl-L-ascorbic acid was enzymatically synthesized from 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and vinyl octanoate with a protease from Bacillus subtilis in pyridine. Furthermore, with various linear saturated fatty acid vinylesters as acyl donors, AA-2G was also converted to their corresponding 6-O-acyl AA-2G in the same manner. The reactivities of transacylation decreased with increasing length of the acyl groups. Thus, short chain acyl groups were transferred to AA-2G by this protease more efficiently than were long chain acyl groups. This enzymatic method is recommended for the synthesis of 6-Acyl-AA-2G with short or medium length chain acyl groups.     

Chemical and semisynthesis of posttranslationally modified proteins

Posttranslational modifications of proteins play crucial roles in health and disease by affecting numerous aspects of protein structure, function, stability and sub cellular localization. Yet understanding the effects of these modifications on several of these processes at the molecular level has been hindered by the lack of homogeneously modified proteins obtained via traditional biochemical and molecular biology approaches. Moreover, the preparation of such bioconjugates at a workable level is highly demanding. Recent advances in protein chemistry applying chemical and semisynthetic approaches are becoming increasingly beneficial to overcome these challenges. These methods allow site-specific modifications of a desired protein and afford the product in large quantities for biochemical and structural analyses. In this review, we survey these efforts and their importance in dissecting the role of several posttranslational modifications in various proteins. Several examples are presented where glycosylated, phosphorylated, ubiquitinated, lipidated, acetylated and methylated proteins were prepared.     

A semisynthesis of isepamicin by fragmentation method

Garamine derivative, key intermediate, was obtained from acid cleavage of sisomicin derivative. Its subsequent product was glycosylated with 6-azido-2,3,4-tri-O-benzyl-6-deoxy-α-d-glucopyranosyl chloride using silver triflate as a promoter to give isepamicin.     

Biomimetic semisynthesis of arglabin from parthenolide

The semisynthesis of arglabin, an anticancer drug in clinical application, is developed from abundant natural product parthenolide via three steps. Each step in this sequence is highly stereoselective, and the substrate-dependent stereoselectivity in the epoxidation step can be explained by computational calculations. The success of chemical semisynthesis of arglabin suggests that the biosynthesis of arglabin might proceed in a similar pathway.     

In vitro selection of aptamers with affinity for neuropeptide Y using capillary electrophoresis

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) was used to select aptamers for neuropeptide Y (NPY). This is the first example of a CE-SELEX selection for aptamers that bind a target molecule smaller than itself. One of the limitations of CE-SELEX is that the aptamer must exhibit a significant mobility shift when it binds the target to facilitate fraction collection. Before this study, it was not clear if smaller targets would be capable of inducing a large enough shift in mobility for CE-SELEX to be successful. NPY is a 36-amino acid peptide (MW = 4272 g/mol), much smaller than the 80-base ssDNA used in the selection ( approximately 25 kDa). NPY binding aptamers with 300-1000 nM dissociation constants were obtained after only four rounds of selection. The specificity of the aptamers was tested using human pancreatic polypeptide (hPP). hPP is a 36-amino acid peptide with approximately 50% homology with NPY. Aptamers with up to 42-fold selectivity for NPY over hPP were observed.     

Practical synthesis of a neuropeptide Y antagonist via stereoselective addition to a ketene

[Reaction: see text]. The synthesis of neuropeptide Y antagonist 1, currently under clinical investigation for the treatment of obesity, is described. The convergent synthesis from trans-spirolactone carboxylic acid intermediate 2a and aminopyrazole 3 is predicated on a stereoselective route to the former. The coupling reaction of ethyl 4-oxocyclohexanecarboxylate (10a) with lithiated isonicotinamide 11 was investigated in detail, but even optimized conditions only provided a 45:55 ratio of trans:cis isomers (12a:12b). While selective crystallization schemes were developed to isolate the thermodynamically less stable trans isomer 2a, improved stereocontrol was subsequentially achieved by the application of ketene chemistry. The ketene formation and quench was investigated under a variety of conditions aimed at maximizing the trans:cis ratio. Reacting a mixture of carboxylic acids 2a and 2b with POCl3 in THF, followed by concomitant addition of tert-butyl alcohol in the presence of TMEDA at 35 degrees C provided a 4:1 ratio of trans:cis tert-butyl esters (18a:18b) via in situ ketene formation. Ester hydrolysis, followed by selective crystallization of undesired 2b as the HCl salt, led to isolation of 2a in 47% overall yield. Aminopyrazole intermediate 3 was synthesized via the condensation reaction of 2-fluorophenylhydrazine hydrochloride (4a) with acrylonitrile derivative 5 in 65-70% yield. Coupling of advanced intermediates 2a and 3b via activation with thionyl chloride gave a 92% yield of 1.     

Spinosyn g: proof of structure by semisynthesis

[reaction: see text] Spinosyn G was isolated in the late 1980s as a minor component from the broth of our potent, fermentation-derived insecticide spinosad. Its structure was then tentatively identified as 5' '-epispinosyn A (3) on the basis of (1)H and (13)C NMR data, but the 4' '-epi compound 4 could not be conclusively ruled out with the data available. Described herein are unambiguous syntheses of both 3 and 4, whereby 3 was proved identical to the natural product. Compound 4 was prepared from intact spinosyn A by a novel F-TEDA-promoted oxidative deamination to the 4' '-ketone 5, stereoselective reduction to the equatorial alcohol 6, and nitrogen incorporation via the axial azide 7. Compound 3 was obtained by coupling spinosyn A 17-pseudoaglycone (9) with the N-protected dihydropyran 16 derived from methyl l-ossaminide (14). This gave an approximately 2:1 mixture of anomeric products 17 with the desired equatorial glycoside predominating, which was then converted to 3 by N-deprotection and methylation.     

HPLC-MS analysis of the riboflavin crude product of semisynthesis

An accurate and simple analysis method by high-performance liquid chromatography-mass spectrometry for the riboflavin crude product of semisynthesis is established in this study. In this method, a C(18) column is used, and the mobile phase is methanol-water. Under the optimal analysis conditions established in this study, all components in the riboflavin crude product are well separated and identified, respectively. The external standard method is used for the quantitative analysis, and the results are proven to be accurate.