The first example of linear peptides containing a N-trifluoroethylated backbone amide linkage and the surprising solution dynamics observed by F NMR

The α-amino group of (l)phenylalanine methyl ester was trifluoroethylated using (2,2,2-trifluoroethyl)phenyliodonium N,N-bis(trifluoromethylsulfonyl)imide. A dipeptide Gly(l)Phe containing a trifluoroethylated peptide bond was synthesized by removing the α-amino proton of N^α-trifluoroethyl (l)phenylalanine methyl ester followed by coupling with N^α-phthaloyl glycine acid fluoride. The dipeptide was further coupled with (l)leucine methyl ester under conventional carboxyl activation conditions to provide two diastereomers of the tripeptide Gly(d,l)Phe(l)Leu. The solution dynamic behavior of the tripeptide was investigated as a function of solvents, by NOESY and variable temperature (VT) ¹⁹F NMR experiments.     

Halbsandwich‐Komplexe von Ruthenium(II), Rhodium(III) und Iridium(III) mit Tripeptidestern aus α‐, β‐ und γ‐Aminosäuren als Liganden. — Peptidsynthese und Cyclisierung zu Cyclotripeptiden am Metallzentrum

Metal Complexes with Biological Important Ligands. CXLII. Half Sandwich Complexes of Ruthenium(II), Rhodium(III), and Iridium(III) with Tripeptide Esters from α‐, β‐, and γ‐Amino Acids as Ligands. — Peptide Synthesis and Cyclization to Cyclotripeptides at Metal Centers Halfsandwich complexes of ruthenium, rhodium and iridium with deprotonated N, N', N"‐tripeptide ester ligands were obtained from chloro bridged compounds and tripeptide methyl esters ( 1—6 ) or by peptide synthesis at a metal centre ( 9—15 ). For the peptide synthesis at the complex (C₆Me₆)Ru coordinated dipeptide methyl esters from glycine and β‐alanine or γ‐amino butyric acid were elongated by an a‐amino acid methylester. The tripeptide ester Ru(η⁶‐C₆Me₆) complexes with chiral amino acid components and an “asymmetric” metal atom are formed with high diastereoselectivity. The tripeptide esters Gly‐Gly‐β‐AlaOMe, Val‐Gly‐β‐AlaOMe and Phe‐Gly‐β‐AlaOMe can be condensated at the (C₆Me₆)Ru complex with sodium methanolate to give triple deprotonated cyclic tripeptides.     

Synthesis and screening of libraries of synthetic tripodal receptor molecules with three different amino acid or peptide arms: identification of iron binders

A novel selectively deprotectable triazacyclophane scaffold was used for the design and split-mix synthesis of two libraries of solid-phase bound tripodal synthetic receptors possessing three different amino acid or peptidic arms. In the synthesis of the first library, the two outer arms consisted of amino acid Ala, Arg, Asp, Gln, Gly, Lys, Phe, Ser, Tyr, or Val and the middle arm consisted of amino acid Asn, Glu, His, Leu, or Pro. The second library contained amino acid and/or (di)peptide arms. The arms were different in all library members. The first outer arm consisted of amino acid(s) Ala, Arg, Gln, Phe, or Ser, the second outer arm consisted of amino acid(s) Asp, Gly, Lys, Tyr, or Val, and the middle arm consisted of amino acid(s) Asn, Glu, His, Leu, or Pro, leading to a 27 000 member library of synthetic tripodal receptor molecules. In on-bead screening experiments, a remarkable selectivity of some library members for Fe(3+) was observed and decoding of their structures by Edman degradation revealed consensus sequences with structural resemblance to non-heme iron proteins.     

Stabilizing and destabilizing effects of phenylalanine --> F5-phenylalanine mutations on the folding of a small protein

We report a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe --> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F5-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than aryl-aryl interactions and because perfluoroaryl units are more hydrophobic than are analogous aryl units. One mutant, Phe-10 --> F5-Phe, provides enhanced tertiary structural stability relative to the native sequence. The other six mutants analyzed caused a decrease in stability.     

Identification of degradation products of aspartyl tripeptides by capillary electrophoresis-tandem mass spectrometry

Capillary electrophoresis-electrospray tandem mass spectrometry (CE-MS/MS) has been used to identify degradation products of the aspartyl tripeptides Phe-Asp-GlyNH(2) and Gly-Asp-PheNH(2) following incubation of the peptides in acidic and alkaline solution. At pH 2, the dominant decomposition products resulted from cleavage of the peptide backbone amide bonds to yield the respective dipeptides and amino acids. In addition, the cyclic aspartyl succinimide intermediate was identified by its [M+H](+) at m/z = 319 and the MS/MS spectrum exhibiting a simple fragmentation pattern with the [C(8)H(10)N](+)-ion as the principal daughter ion (a(1) of Phe-Asp-GlyNH(2)). Deamidation of the C-terminal amide as well as isomerization and enantiomerization of the Asp residue occurred upon incubation at pH 10. alpha-Asp and the isomeric beta-Asp and most of the diastereomeric forms (corresponding to D/L-Asp) could be separated by CE. All isomers could be identified based on their MS/MS spectra. Peptides with the amino acid sequence Phe-Asp-Gly containing the regular alpha-Asp bond displayed a highly intense b(2) fragment ion and a low abundant y(2) ion. In contrast, the y(2) and a(1) fragment were high abundant daughter ions in the mass spectra of beta-Asp peptides while the b(2) ion exhibited a lower abundance. Differences in the MS/MS spectra of the isomers of the peptides with the sequence Gly-Asp-Phe were obvious but less pronounced. In conclusion, CE-MS/MS proved to be a useful tool to study the decomposition and enantiomerization of peptides including the isomerization of Asp residues, due to the combination of efficient separation of isomers by CE and their identification by MS/MS.     

Total Enzymatic Synthesis of the Cholecystokinin Octapeptide (CCK‐8)

The enzymatic synthesis of the cholecystokinin octapeptide (CCK‐8) is reported. The target octapeptide CCK‐8 is the minimum active sequence with the same biological activity as naturally occurring cholecystokinin and is a potential therapeutic agent in the control of gastrointestinal function as well as a drug candidate for the treatment of epilepsy and obesity. The protected CCK‐8 was obtained by incubation of Bz‐Arg‐Asp(OEt)‐Tyr‐Met‐OAl and Gly‐Trp‐Met‐Asp(OMe)‐Phe‐NH₂ with immobilized α‐chymotrypsin. The Bz‐Arg group was used as an N‐terminal protecting group in the synthesis of the tripeptide fragment. The protected CCK‐8 was treated with trypsin to remove the Bz‐Arg group successfully. Free or immobilized enzymes were used as catalysts. The effect of the acyl donor ester structure, the C(α) protecting group of the nucleophile, reaction media, enzyme, and the carrier of the enzymes on the outcome of the coupling reaction was studied.     

Cyclic RGD Peptides Containing Azabicycloalkane Reverse‐Turn Mimics

The Fmoc‐protected lactams 3 and 4 were used to prepare cyclo(Arg‐Gly‐Asp‐lactam) 1 and cyclo(Arg‐Gly‐Asp‐Phe‐lactam) 2 , which contain the Arg‐Gly‐Asp (RGD) recognition motif. Their solid‐phase synthesis, conformational analysis, and binding to purified α_Vβ₃ and α_Vβ₅ integrins are reported. Compound 1 was found to act as an active and selective inhibitor of the α_Vβ₅ integrin.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Designed Beta‐Turn Mimic Based on the Allylic‐Strain Concept: Evaluation of Structural and Biological Features by Incorporation into a Cyclic RGD Peptide (Cyclo(‐L‐arginylglycyl‐L‐α‐aspartyl‐))

The (3R,5S,6E,8S,10R)‐11‐amino‐3,5,8,10‐tetramethylundec‐6‐enoic acid (ATUA; 1 ), which was designed as a βII′‐turn mimic according to the concepts of allylic strain and 2,4‐dimethylpentane units, was incorporated into a cyclic RGD peptide. The three‐dimensional structure of cyclo(‐RGD‐ATUA‐) (=cyclo(‐Arg‐Gly‐Asp‐ATUA‐)) 4 in H₂O was determined by NMR techniques, distance geometry calculations and molecular‐dynamics simulations. The RGD sequence of 4 shows high conformational flexibility but some preference for an extended conformation. The structural features of the RGD sequence of 4 were compared with the RGD moiety of cyclo(‐RGDfV‐) (=cyclo(‐Arg‐Gly‐Asp‐D ‐Phe‐Val‐)). In contrast to cyclo(‐RGDfV‐), which is a highly active αvβ3 antagonist and selective against αIIbβ3, cyclo(‐RGD‐ATUA‐) shows a lower activity and selectivity. The structure of the ATUA residue in the cyclic peptide resembles a βII′‐turn‐like conformation. Its middle part, adjacent to the C?C bond, strongly prefers the designed and desired structure.     

Titanium dioxide photocatalyzed oxidation of proteins in biocontaminated waters

The TiO2 photocatalyzed oxidation of the proteins serum albumin, ovalbumin and gamma globulin, is reported. All the amino acids were susceptible to photocatalytic oxidation. However, some were especially vulnerable. Tyrosine was particularly sensitive, as was the semiaromatic histidine, although to a lesser extent. The lack of an activating group on the aromatic ring in Phe, renders the system less amenable to degradation. The photocatalytic degradation of the aliphatic amino acids Gly and Asp, was particularly slow, like in the Fenton oxidation where production of glycine was observed during the cleavage of collagen induced by hydroxyl radicals. Intermediate degradation rate was noticed in Ser, Arg, Val, Cys and Phe.     

天花粉蛋白的化学 III:溴化氰降解片段CBa的氨基酸顺序

The results from the study on the separation, purification, amino acid composition and amino acid sequence of CBa, one of the four CNBr degradation fragments of crystalline trichosanthin, are presented. Its amino acid composition is: Asp3, Thr2, Ser2, Hse1, Glu2, Gly2, Ala6, Val1, Tyr3, Phe3, Lys2, Arg1. The sequence of the CBa is Gly-Tyr-Arg-Ala-Gly-Asp-Thr-Ser- Tyr-Phe-Phe-Asn-Glu-Ala-Ser-Ala-Thr-Glu-Ala-Ala-Lys-Tyr-Val- Phe-Lys-Asp-Ala-Hso.     

Determination of sequence-specific intrinsic size parameters from cross sections for 162 tripeptides

Ion mobility and mass spectrometry techniques have been used to measure cross sections for 162 tripeptide sequences (27 different sets of six sequence isomers). The isomers have the general forms ABC, ACB, BAC, BCA, CAB, and CBA, where A corresponds to the amino acids Asp, Glu, or Gly, B corresponds to Lys, Arg, or Leu, and C corresponds to Phe, Tyr, or Ser. From these data, we derive a set of size parameters for individual amino acids that reflect the position of the amino acid in the sequence. These sequence-specific intrinsic size parameters (SSISPs) are used to retrodict cross-section values for the 162 measured sequences and to predict cross sections for all remaining tripeptide sequences (567 different sequences) that are comprised of these residues. In several types of peptide compositions, the position of the amino acid in the sequence has a significant impact on the parameter that is derived. For example, the sequence-specific intrinsic size parameter for leucine in the third position of a peptide (SSISP(Leu3)) is approximately 10% larger than SSISP(Leu1). On average, cross sections that are derived using SSISPs provide a better representation of the experimental value than those derived from composition only intrinsic size parameters, derived as described previously (Valentine et al. J. Phys. Chem. 1999, 103, 1203). Finally, molecular modeling techniques are used to derive some insight into the origin of cross-section differences that arise from sequence variation.     

Liquid chromatographic study of the enzymatic degradation of endomorphins, with identification by electrospray ionization mass spectrometry.

The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp3-Phe4 or Phe3-Phe4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe3-Phe4 bond. Aminopeptidase M cleaves the peptides at the Pro2-Trp3 or Pro2-Phe3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.     

Longicalycinin A, a new cytotoxic cyclic peptide from Dianthus superbus var. longicalycinus (MAXIM.) WILL

A new cyclic peptide, longicalycinin A (1), and six known compounds, vaccaroside A, dianoside A, dianoside G, 3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde were isolated from the MeOH extract of Dianthus superbus var. longicalycinus. The amino acid sequences of 1 was elucidated as cyclo(Gly(1)-Phe(2)-Tyr(3)-Pro(4)-Phe(5)-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. Furthermore, compound 1 showed cytotoxicity to Hep G2 cancer cell line.     

Conformational analysis of an active chemotactic peptide analog containing z-dehydrophbnylalanine at position 3

Formyl-Met-Leu-Δ^Z-Phe-OMe, an analog of the chemotactic tripeptide Formyl-Met-Leu-Phe has been synthesized to evaluate the effect of substitution of α, β -dehydrophenylalanine on activity and conformation. The analog peptide shows high biological activity in stimulating superoxide production by rabbit neutrophils. An NMR analysis of the solution conformation of the Δ^Z-Phe analog, using nuclear Overhauser effects and comparisons with the corresponding saturated peptides, favours a significant population of extended backbone conformations.     

A simple and versatile route to ketomethylene dipeptide analogs

A facile and versatile procedure for the synthesis of ketomethylene dipeptides by using N^α-Z-amino acid halomethyl ketones and dimethyl malonate as starting materials is reported. By application of this method, alanyl and phenylalanyl derivatives containing C-terminal Gly, Ala, Asp, Phe and Trp residues have been prepared.     

The SAAP force field: Development of the single amino acid potentials for 20 proteinogenic amino acids and Monte Carlo molecular simulation for short peptides

Molecular simulation by using force field parameters has been widely applied in the fields of peptide and protein research for various purposes. We recently proposed a new all‐atom protein force field, called the SAAP force field, which utilizes single amino acid potentials (SAAPs) as the fundamental elements. In this article, whole sets of the SAAP force field parameters in vacuo, in ether, and in water have been developed by ab initio calculation for all 20 proteinogenic amino acids and applied to Monte Carlo molecular simulation for two short peptides. The side‐chain separation approximation method was employed to obtain the SAAP parameters for the amino acids with a long side chain. Monte Carlo simulation for Met‐enkephalin (CHO‐Tyr‐Gly‐Gly‐Phe‐Met‐NH₂) by using the SAAP force field revealed that the conformation in vacuo is mainly controlled by strong electrostatic interactions between the amino acid residues, while the SAAPs and the interamino acid Lennard‐Jones potentials are predominant in water. In ether, the conformation would be determined by the combination of the three components. On the other hand, the SAAP simulation for chignolin (H‐Gly‐Tyr‐Asp‐Pro‐Glu‐Thr‐Gly‐Thr‐Trp‐Gly‐OH) reasonably reproduced a native‐like β‐hairpin structure in water although the C‐terminal and side‐chain conformations were different from the native ones. It was suggested that the SAAP force field is a useful tool for analyzing conformations of polypeptides in terms of intrinsic conformational propensities of the single amino acid units. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Synthesis of Aib‐ and Phe(2Me)‐Containing Cyclopentapeptides

Some recently described pentapeptides containing the α,α‐disubstituted α‐amino acids Aib and Phe(2Me) have been cyclized in DMF solution using diphenyl phosphorazidate (DPPA), O‐(1H‐benzotriazol‐1‐yl)‐N,N,N′,N′‐tetamethyluronium tetrafluoroborate/1‐hydroxybenzotriazole (TBTU/HOBt), and diethyl phosphorocyanidate (DEPC), respectively, to give the corresponding cyclopentapeptides in fair‐to‐good yields. In the case of peptides with L ‐amino acids, and (R)‐ and (S)‐Phe(2Me), the yields differed significantly in favor of the L /(R) combination. The conformations in the crystals of cyclo(Gly‐Aib‐(R,S)‐Phe(2Me)‐Aib‐Gly) and cyclo(Gly‐(R)‐Phe(2Me)‐Pro‐Aib‐Gly) have been determined by X‐ray crystallography, leading to quite different results. In the latter case, the conformation in solution has been elucidated by NMR studies.     

Synthesis and Biological Evaluation of RGD Peptidomimetic–Paclitaxel Conjugates Bearing Lysosomally Cleavable Linkers

Two small‐molecule–drug conjugates (SMDCs, 6 and 7 ) featuring lysosomally cleavable linkers (namely the Val–Ala and Phe–Lys peptide sequences) were synthesized by conjugation of the α_vβ₃‐integrin ligand cyclo[DKP–RGD]‐CH₂NH₂ ( 2 ) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP–RGD]–PTX conjugate with a nonpeptide “uncleavable” linker ( 8 ) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified α_Vβ₃‐integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8 , which possesses a nonpeptide “uncleavable” linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF‐CEM (α_Vβ₃?) and its subclone CCRF‐CEM α_Vβ₃ (α_Vβ₃+). Fairly effective integrin targeting was displayed by the cyclo[DKP–RGD]–Val–Ala–PTX conjugate ( 6 ), which was found to differentially inhibit proliferation in antigen‐positive CCRF‐CEM α_Vβ₃ versus antigen‐negative isogenic CCRF‐CEM cells. The total lack of activity displayed by the “uncleavable” cyclo[DKP–RGD]–PTX conjugate ( 8 ) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.     

Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7 %, whereas CVs for the area ratio under the curve were 2.1 and 2.9 % for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8 %, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.