Chiral ferrocene amines derived from amino acids and peptides: synthesis, solution and X-ray crystal structures and electrochemical investigations

For the recognition of all but the simplest naturally occurring molecules, electrochemical sensors based on ferrocene will certainly require chiral centers. To advance the necessary chemistry, this work describes the synthesis and properties of ferrocene derivatives of enantiomerically pure amino acids, peptides, and other chiral amines. Ferrocene aldehyde is condensed with amino acid esters to yield the corresponding Schiff bases 2, which are reduced by NaBH4 in methanol to the ferrocene methyl amino acids 3. An X-ray single-crystal analysis was carried out on the phenylalanine derivative 3a (monoclinic space group P2(1), a = 10.301(1) A, b = 9.647(1) A, c = 18.479(2) A, beta = 102.98(2) degrees, Z = 4). Further peptide chemistry at the C terminus proceeds smoothly as demonstrated by the synthesis of the ferrocene labeled dipeptide Fc-CH2-Phe-Gly-OCH3 5 (Fc = ferrocenyl ((eta-C5H4)Fe(eta-C5H5))). We also report the synthesis of the C,N-bis-ferrocene labeled tripeptide Phe-Ala-Leu and its electrochemical characterization. Starting from the enantiomerically pure ferrocene derivative 9, which was synthesized from ferrocene aldehyde and L-1-amino-ethylbenzene, two diastereomers 10 were obtained by peptide coupling with N-Boc protected D- and L-alanine. There was, however, only very little diastereomeric induction if 0.5 equiv of a racemic mixture of alanine were used. This suggests that amino acid activation rather than coupling is the rate-determining step. A combination of NOESY (nuclear Overhauser effect spectroscopy) spectra and molecular modeling furnished detailed insights into the solution structures of 3, 9, and 10 and was used to rationalize their different reactivity.     

TLC of Phenylthiohydantoins of Amino Acids: A Review

Abstract

Proteins are among the most important components of all living systems. Their function range from catalysts 9enzymes) to regulators to structural components. The building blocks and language of proteins are about 20 amino acids (H₂N CHR COOH), linked together By peptide bonds ([sbnd]CO[sbnd]NH[sbnd]) in chains that may consist of a few dozen to more than 1000 amino acides. The determination of primary structure of proteins, namely the sequence (arrangement) of the various amino acids along the chain is still a challenging tast. Edman reaction lies virtually at the core of all modern sequencing strategies [1,2]. The N-terminal polypeptide is first coupled to phenyl isothiocyanate to from the phenylthio carbamyl peptide; this derivative is then cleaved with anhydrous acid to expose a new N-terminus and to release the original N-terminal amino acid as a 5′thiazolinone (Scheme-1). The excess reagents and by products are extracted by an organic solvent wash. The extract of thiazolinone amino acid (obtained either from liquid-phase or solid-phase degration) is evaporated and converted to the phenylhtiohydantion derivative by 5 n HCL/CH₃COOH (1:2 v/v) at 52°C for 50 min. The sample is extracted with ethyl acetate, dried and redissolved in a suitable volume of ethanol for TLC identification. Repetition of this process with identification of the released PTH-amino acidsfro the N-terminal end. For smaller peptides PITC may be used to remove the amino terminal amino acid while a chromphore or fluorophore such as dansyl chloride or DABITC, which react with the newly exposed amino terminus, is used to identify the new amino terminus. Both manual and automated methodologies are currently used for small and large polypeptides which rely upon identification of amino terminal amino acid as PTH derivative. A large number of papers have been and continue to be publihsed on the analysis of the PTH derivatives of amino acides.     

Synthesis and single cell pharmacology of potential heterocyclic bioisosteres of the excitatory amino acid antagonist glutamic acid diethyl ester

A series of heterocyclic analogues of glutamic acid diethyl ester (GDEE), an antagonist at central excitatory amino acid receptors, have been synthesized and tested biologically. (RS)-Ethyl alpha-amino-alpha-(3-ethoxyisoxazol-5-yl)acetate (7), (RS)-ethyl 2-amino-3-(3-ethoxy-5-methylisoxazol-4-yl)propionate (16) and closely related analogues were synthesized. Compound 7, a diethyl derivative of the naturally occurring excitatory amino acid ibotenic acid (IBO), was synthesized from 3-hydroxy-5-methylisoxazole (1) via 3-ethoxyisoxazol-5-ylacetic acid (5) and its ethyl ester. Nitrosation of this ester followed by catalytic reduction gave 7. The ethyl ester of IBO, 9, was synthesized in a similar manner from 3-benzyloxyisoxazol-5-ylacetic acid (8). Ethyl derivatives of the synthetic excitatory amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) were synthesized from 3-hydroxy-4,5-dimethylisoxazole (10) through a diethyl acetylaminomalonate derivative, which upon deprotection gave the 3-ethoxy derivative of AMPA (15). Esterification of 15 gave the diethyl derivative 16 and the ethyl ester of AMPA (18) as well as N-ethylated derivatives of AMPA, 21 and 22 were synthesized. The final products were tested microelectrophoretically. The derivatives 7, 9, 15, 16 and 18 were weak and non-selective excitatory amino acid antagonists, whereas 21 and 22 were found to be inactive.     

Metabolically stable isotope labeling prior to electrophoretic protein separation reveals differences in fractional synthesis rates between mitochondrial aldehyde dehydrogenase isoforms

Living mice were subjected to whole body labeling by intravenous infusion of [(13)C]glucose as the sole carbon source. After 10 h infusion the mice were sacrificed, and liver proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Five spots were found to contain mitochondrial aldehyde dehydrogenase (ALDH2) by matrix assisted time of flight mass spectrometry protein identification. By measuring the isotopologue mass distributions of peptide ions, and modeling the (13)C content of the precursor amino acid pool, the fractional synthesis rate of ALDH2 molecules synthesized during the labeling period was determined. One of the five spots was observed to have a five-fold higher fraction of (13)C-containing newly synthesized ALDH2 than the spot with the highest ALDH2 content, and contained more than 60% of newly synthesized ALDH2 although it accounted for less than 20% of the total ALDH2 detected. The total range in the fraction of (13)C-containing proteins between different ALDH2 spots approached 50-fold. The ability to quantitatively characterize different protein isoforms of biological origin for ALDH2 and other proteins from living animals provides new avenues for the exploration of protein function.     

Chiral analysis of biogenic DL-amino acids derivatized by urethane - protected alpha-amino acid N-carboxyanhydride using capillary zone electrophoresis and micellar electrokinetic chromatography

A new analytical method for enantioselective separation of DL-amino acids derivatized by N-fluorenylmethoxycarbonyl-L-alanyl N-carboxyanhydride (FMOC-L-Ala-NCA) using capillary electrophoresis was developed. Separation parameters, such as composition and pH of the background electrolyte, and concentration of gamma-cyclodextrin (in capillary zone electrophoresis) and sodium dodecyl sulfate (in micellar electrokinetic chromatography) were optimized. The separation method was validated and it suits well for purity analysis. Detection limit of the method was 0.2% of the minor enantiomer in the major one. The level of racemization in coupling during solid-phase peptide synthesis was studied using capillary electrophoresis with gamma-cyclodextrin as a chiral selector. The anchorage of the first (C-terminal) amino acid derivative to the solid supports bearing the hydroxylic groups is the key step of the synthesis affecting the extent of its racemization. FMOC-L-phenylalanine was chosen as the suitable model amino acid derivative making it possible to study the degree of racemization of N-fluorenylmethoxycarbonyl-L-alanine-L-phenylalanine synthesized on different polymer resins, using the different condensation agents.     

Solution-phase synthesis of porcine brain natriuretic peptide (pBNP) using S-trimethylacetamidomethylcysteine

The hexadodecapeptide corresponding to the entire amino acid sequence of porcine brain natriuretic peptide (pBNP) was synthesized by assembling four segments in solution, followed by HF deprotection and subsequent oxidation to establish an intramolecular disulfide bridge. The synthesis using the newly developed S-trimethylacetamidomethylcysteine [Cys(Tacm)] derivative gave a better yield than that using the S-2,4,6-trimethylbenzylcysteine [Cys(Tmb)] derivative. The chick rectum relaxant activity of the synthetic pBNP was 2.9 times more potent than that of alpha-rat atrial natriuretic peptide (alpha-rANP).     

酸敏性PEG载体的合成及其在多肽合成中的应用

本文通过聚苯乙烯固载聚乙二醇氨基树脂与羟甲基苯氧乙酸缩合制备了具有酸敏手臂结构的对羟甲基苯氧乙酰胺ＰＥＧ树脂。在合成中改进了ＰＳ－ＰＥＧＮＨ２树脂的制备方法,提高了树脂上功能基的转化率,不必进行封闭剩余活性基的操作,就得到具有单一功能基的树脂。     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Fullerene-derivatized amino acids: synthesis, characterization, antioxidant properties, and solid-phase peptide synthesis

A series of [60]fullerene-substituted phenylalanine (Baa) and lysine derivatives have been prepared by the condensation of 1,2-(4'-oxocyclohexano)fullerene with the appropriately protected (4-amino)phenylalanine and lysine, respectively. Conversion of the imine to the corresponding amine is achieved by di-acid catalyzed hydroboration. The reduction of the imine is not accompanied by hydroboration of the fullerene cage. The [70]fullerene phenylalanine derivative has also been prepared as have the di-amino acid derivatives. The compounds were characterized by MALDI-TOF mass spectrometry, UV/Vis spectroscopy, and cyclic voltammetry. 1H and 13C NMR spectroscopy allowed the observation of diastereomers. Fullerene-substituted peptides may be synthesized on relatively large scale by solid-phase peptide synthesis. The presence of the C60-substituted amino acid in a peptide has a significant effect on the secondary structures and self-assembly properties of peptides as compared to the native peptide. The antioxidant assay of Baa and a Baa-derived anionic peptide was determined to be significantly more potent than Trolox.     

Accurate evaluation method of the polymer content in Monomethoxy(polyethylene glycol) modified proteins based on Amino acid analysis

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.     

Amino acid conjugates of 1,1'-diaminoferrocene. Synthesis and chiral organization

1,1'-bis(tert-butoxycarbonylamino)ferrocene (6), a protected derivative of 1,1'-diaminoferrocene, has been synthesized by a very convenient method and serves as a synthon for 1,1'-diaminoferrocene. Its structure in solid state and in solution has been studied by NMR and X-ray crystallography. 1,1'-bis(tert-butoxycarbonylamino)ferrocene serves as starting material for the synthesis of amino acid conjugates of L- and D-alanine. The structures of these bioconjugates have been studied by NMR and CD spectroscopy and X-ray crystallography and reveal that the chiral organization of the podant amino acid chains is controlled by the chirality of the attached amino acid. The substituents engage in strong intramolecular H-bonding generating 14-membered H-bonded rings, a motif previously unrealized in ferrocene-amino acid and peptide conjugates.     

Preparation, isolation, and characterization of Nalpha-Fmoc-peptide isocyanates: solution synthesis of oligo-alpha-peptidyl ureas

The N(alpha)-Fmoc-peptide isocyanates 3a-q, 4a-c, and 5a-c were prepared by the Curtius rearrangement of N(alpha)-Fmoc-peptide acid azides in toluene under thermal, microwave, and ultrasonic conditions. All the N(alpha)-Fmoc-oligo-peptide isocyanates made were isolated as stable crystalline solids with 71 to 94% yield and were fully characterized by 1H NMR, 13C NMR, and mass spectroscopy. Their utility for the synthesis of oligo-alpha-peptidyl ureas 7a-f and 8a-c by the divergent coupling approach was demonstrated. The coupling of N(alpha)-Fmoc-dipeptide isocyanates with amino acid ester or with N,O-bis(trimethylsilyl)amino acids resulted in N(alpha)-Fmoc-tripeptidyl urea ester and acids containing one each of peptide bond and urea bond. The divergent approach is extended to the synthesis of tetrapeptidyl ureas by the 2 + 2 strategy using bis-TMS-peptide acid as an amino component. To incorporate urea bonds in adjacent positions, N(alpha)-Fmoc-peptidyl urea isocyanates 9a-d were prepared and employed in the synthesis of three tetrapeptidyl ureas 10a-b and 11 containing one peptide bond and two urea bonds in series from the N-terminal end. The protocol was then employed for the synthesis of five urea analogues 13-15, 18, and 21 of [Leu5]enkephalin containing urea bonds at the 2, 3, 4 positions as well as at the 2, 4 and 2, 3, 4 positions. The analogue 2l was made by the convergent synthesis by the N --> C terminal chain extension. Finally, two urea analogues 22 and 23 of repeat units of bioelasto polymers, namely Val-Pro-Gly-Val-Gly-OH and Pro-Gly-Val-Gly-Val-OH, were synthesized incorporating the urea bond by the concomitant isocyanate generation and urea bond formation under thermal conditions.     

Peptide affinity chromatography media that bind N fusion proteins under chaotropic conditions

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N^pro fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N^pro from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7–15 mg protein mL⁻¹ of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.     

爱滋病病毒中肽段的酶促合成

为了进一步研究酶促合成在多肽合成中的实际应用,选择合成了爱滋病病毒(人类免疫缺损病毒,HIV-I)的gp41中氨基酸序列598-609的三个肽段,该部分是HIV-I中的2个抗原决定簇部分,H-Leu-Glg-Leu-Trp-Glg-cgs-Ser-Glg-Lgs-Leu-Ile-Cgs-OH可以作为抗原来检测HIV抗体.     

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: a novel protected spin labeled beta-amino acid for peptide and protein chemistry

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta-amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxycarbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (All, DRVYIHPF) was synthesized by replacing Pro7 with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC7-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta-amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.     

A novel class of chemically modified iodo-containing resins: design, synthesis and application to mass spectrometry-based proteome analysis

A novel class of chemically modified iodo-containing resins with isotope-labeled tagging for mass spectrometry-based proteome analysis is described. This iodo-containing resin contains a thiol-reactive group that is used to capture the cysteine (Cys)-containing peptides from peptide mixtures, one 'tag' amino acid, and an aminomethyl polystyrene resin with Rink Amide Linker. The 'tag' amino acid is synthesized in both heavy and light isotope-coded forms and therefore permits the direct relative quantification of peptides/proteins through mass spectrometric analysis. In the iodo-containing resin strategy, the Cys-containing peptides of two samples covalently captured by either light or heavy iodo-containing resin were mixed and washed extensively under stringent conditions. Then the Cys-containing peptides were retrieved by acid-catalyzed elution. Finally, the eluted peptides were directly analyzed by micro liquid chromatography/mass spectrometry for identification and relative quantification. The iodo-containing resins were synthesized by a simple but effective method. Their abilities to identify and quantify the Cys-containing part in two samples were proved by the analysis of mixtures of amino acids, peptides and proteins.     

One‐pot three‐component synthesis of novel 2‐(3‐nitro‐phenyl)‐quinazoline‐4‐carboxylic acid derivatives

A simple and easy synthesis of 2‐(3‐nitro‐phenyl)‐quinazoline‐4‐carboxylic acid ( 3 ) has been successfully developed through a one‐pot three‐component condensation reaction of (2‐amino‐phenyl)‐oxo‐acetic acid sodium salt ( 1 ) obtained from the hydrolysis of isatin with ammonium acetate and 3‐nitrobenzaldehyde. Some novel quinazoline‐ester derivatives 4‐7 were then obtained by the reaction between the new compound 3 and various alcohols. Then, quinazoline‐amide derivatives 10‐14 were synthesized from the reaction of various amines and 2‐(3‐nitro‐phenyl)‐quinazoline‐4‐carbonyl chloride ( 8 ), obtained by the reaction of compound 3 with SOCl₂. Finally, some novel quinazoline‐azo derivatives 17‐19 were synthesized by the coupling reaction between β‐dicarbonyl compounds and the novel amino‐quinazoline derivative compound 15 , obtained by reduction of nitro‐quinazoline derivative compound 11 . Thus, a new series of quinazoline‐4‐carboxylic acid, ester, amide, and azo derivatives was synthesized and fully characterized by ¹H NMR, ¹³C NMR, IR, and mass spectrometry analysis.     

Nojirimycin Based 2,5-Diketopiperazines

2,5-Diketopiperazines were prepared and characterized where one of the amino acids is (2S,3R,4R,5S)-3,4,5-trihydroxypipecolic acid. The protected pipecolic acid was synthesized from a selectively protected deoxynojirimycin derivative. The ring closure to give the diketopiperazines, from the dipeptides, was performed by nucleophilic attack of the amino function of the pipecolic acid moiety onto the carbonyl group of the methyl esters.     

Tandem mass spectrometric analysis of (13)C-containing ions from a mixture of homologous peptides differing by one mass unit at a residue

Tandem mass spectrometry of a mixture of two peptides that differ from each other by a single mass unit due to mutation is presented. The mutant beta-globin of hemoglobin Hoshida is present along with the normal counterpart, and the amino acid substitution of glutamine for glutamic acid is located within tryptic peptide T5 of M(r) 2057. 9. The mass of the mutated peptide is 1 u lower. In the isotopic cluster for the doubly charged ion of the peptide T5, the resolved ion with mass of 1030.0 represents the normal peptide with 93 (12)C atoms and the mutated one with 92 (12)C and one (13)C atoms. Collision-induced dissociation (CID) of this composite ion identified the mutation by presenting a key fragment derived from the (12)C-only mutant peptide, as reported in a previous study. Similarly, when an ion containing multiple (13)C atoms was selected as a precursor for CID, the mutation could be identified, even in large fragments, by a marked change in the shape of the isotopic cluster for the consecutive product ions. This study demonstrates the merit of selecting a resolved ion rather than the whole isotopic cluster as a precursor in the CID measurements of large peptides or proteins for characterizing heterozygous mutations.     

N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging application in biochemistry. It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restricted. Indeed, in acidic conditions required for linker cleavage, N-ter FITC-labeled peptides undergo a cyclization leading to the formation of a fluorescein with subsequent removal of the last amino acid. This can be avoided when a spacer such as amino hexanoic acid is used or if non-acidic cleavage is operated to release targeted peptide from the resin.