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.     

Cyclization of all-L-Pentapeptides by Means of 1-Hydroxy-7-azabenzotriazole-Derived Uronium and Phosphonium Reagents

Due to their restricted conformational flexibility, cyclic peptides are of great interest in connection with structure-activity relationships, especially the elucidation of bioactive conformations. For linear peptides that do not contain turn structure-inducing amino acid residues, the cyclization reaction may be an inherently improbable or slow process, and side reactions, such as cyclodimerization and epimerization at the C-terminal residue, may dominate. A number of 1-hydroxy-7-azabenzotriazole-based onium salts were examined for cyclization of thymopentin-derived pentapeptides and the results compared with data from more conventional coupling reagents. The azabenzotriazol-derived coupling reagents stood out as being the most effective by far. The cyclizations proceed extremely rapidly, and in contrast to other coupling reagents, C-terminal epimerization was generally less than 10%. C-terminal D-amino acid residues favor the formation of monocyclic pentapeptide rings. A similar effect was observed for cyclization of linear N-methylamino acid-containing peptides, suggesting that reversible amide bond alkylation such as Hmb-modification should be useful in promoting the cyclization of pepitdes devoid of turn-inducing amino acid residues.     

Further studies on the fragmentation of protonated ions of peptides containing aspartic acid, glutamic acid, cysteine sulfinic acid, and cysteine sulfonic acid

Here we examined the fragmentation, on a quadrupole ion-trap mass spectrometer, of the protonated ions of a group of peptides containing one arginine and two different acidic amino acids, one being aspartic acid (Asp) or glutamic acid (Glu) and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)]. Our results showed that, upon collisional activation, the cleavage of the peptide bond C-terminal to C(SO2H) is much more facile than that of the peptide bond C-terminal to Asp, Glu, or C(SO3H). There is no significant difference, however, in susceptibility to cleavage of peptide bonds that are C-terminal to Asp, Glu, and C(SO3H). To understand these experimental observations, we carried out B3LYP/6-31G* density functional theory calculations for a model cleavage reaction of GXG --> b2 + Gly, in which X is Asp, Glu, C(SO2H), or C(SO3H). Our calculation results showed that the cleavage reaction is thermodynamically more favorable when X = C(SO2H) than when X = Asp or C(SO3H). We attributed the less facile cleavage of the amide bond after Glu to that the formation of a six-membered ring b ion for Glu-bearing peptides is kinetically not as favorable as the formation of a five-membered ring b ion for peptides containing the other three acidic amino acids. The results from this study may provide useful tools for peptide sequencing.     

炔酰胺类缩合剂研究进展

缩合剂是指用于促成羧酸与胺或者醇直接缩合构建酰胺键或酯键的一类试剂的总称.由于酰胺和酯的重要性,缩合剂的开发成为了学术界与工业界广泛关注的一个重要研究方向.多肽合成就是α-氨基酸在缩合剂的作用下反复形成酰胺键的过程,因此,缩合剂在多肽合成中发挥着至关重要的作用.当前多肽合成所使用的试剂和技术大多是20世纪50~80年代发展起来的,这些试剂和技术的天生弊端逐渐显现出来.比如传统多肽缩合剂过度活化α-氨基酸而诱发的外消旋化和其它副反应导致的副产物成为药物多肽生产过程中一个极为关切的问题.另外固相多肽合成的低原子经济性给可持续发展带来了极大的挑战.这些问题只能依靠原始创新的颠覆性技术和全新的缩合方法来解决.我们课题组致力于通过发展新试剂和新反应来解决多肽与蛋白质化学合成领域的难题.本文系统介绍了我们发展的一种结构全新的炔酰胺类缩合试剂及其在酰胺、酯、大环内酯、多肽、硫代多肽合成中的应用研究进展.     

Combined quantum chemical and RRKM modeling of the main fragmentation pathways of protonated GGG. II. Formation of b(2), y(1), and y(2) ions

Quantum chemical and RRKM calculations were performed on protonated GGG in order to determine the atomic details of the main fragmentation pathways leading to formation of b(2),y(1), and y(2) ions. Formation of y(1) ions on the "diketopiperazine" pathway is initiated from relatively high-energy C-terminal amide nitrogen protonated species for which the N-terminal amide bond is in the cis isomerization state. The reaction goes through a transition structure which is only slightly less favored than the reactive configuration itself. RRKM calculations indicate that this reaction is extremely fast as soon as the fragmenting species have more internal energy than the reaction threshold. The calculated energetics suggests that y(1) ions are formed on the "diketopiperazine" pathway with a non-negligible (6-10 kcal/mol) reverse activation barrier. Investigation of species occurring during the formation of b(2) ions having an oxazolone structure indicates that y(1) ions can be formed also from intermediates previously thought to result in only b(2) ions. As the first step of the "b(x)-y(z)" pathway proposed here the extra proton must reach the nitrogen of the C-terminal amide bond. Attack of the N-terminal amide oxygen on the carbon center of the C-terminal amide bond results in formation of the oxazolone ring while the detaching G leaves the precursor ion. Under low-energy collision conditions the complex of protonated 2-aminomethyl-5-oxazolone and G can rearrange to form a proton-bonded dimer of these species. In such circumstances the extra proton is shared by the two monomers and dissociation of the dimer will be determined by the thermochemistry involved. Based on the "b(x)-y(z)" pathway one can easily explain the linear relationship between the logarithm of the y(1)/b(2) ion abundance ratio and the proton affinity of the C-terminal amino acid substituent for the series of H-Gly-Gly-Xxx-OH tripeptides where Xxx was varied (Morgan DG, Bursey MM. Org. Mass. Spectrom. 1994; 29: 354). The calculated energetics indicates that both y(1) and b(2) ions are formed with no reverse activation barrier on the "b(x)-y(z)" pathway.     

Synthetic Methods of Phosphonopeptides

Phosphonopeptides are phosphorus analogues of peptides and have been widely applied as enzyme inhibitors and antigens to induce catalytic antibodies. Phosphonopeptides generally contain one aminoalkylphosphonic acid residue and include phosphonopeptides with C-terminal aminoalkylphosphonic acids and phosphonopeptides with a phosphonamidate bond. The phosphonamidate bond in the phosphonopeptides is generally formed via phosphonylation with phosphonochloridates, condensation with coupling reagents and enzymes, and phosphinylation followed by oxidation. Pseudo four-component condensation reaction of amides, aldehydes, alkyl dichlorophosphites, and amino/peptide esters is an alternative, convergent, and efficient strategy for synthesis of phosphonopeptides through simultaneous construction of aminoalkylphosphonic acids and formation of the phosphonamidate bond. This review focuses on the synthetic methods of phosphonopeptides containing a phosphonamidate bond.     

Protein assembly by orthogonal chemical ligation methods

Chemical synthesis harbors the potential to provide ready access to natural proteins as well as to create nonnatural ones. The Staudinger ligation of a peptide containing a C-terminal phosphinothioester with a peptide containing an N-terminal azide gives an amide with no residual atoms. This method for amide bond formation is orthogonal and complementary to other ligation methods. Herein, we describe the first use of the Staudinger ligation to couple peptides on a solid support. The fragment thus produced is used to assemble functional ribonuclease A via native chemical ligation. The synthesis of a protein by this route expands the versatility of chemical approaches to protein production.     

Specific cleavage at peptide backbone Cα-C and CO-N bonds during matrix-assisted laser desorption/ionization in-source decay mass spectrometry with 5-nitrosalicylic acid as the matrix

The use of 5-nitrosalicylic acid (5-NSA) as a matrix for in-source decay (ISD) of peptides during matrix-assisted laser desorption/ionization (MALDI) is described herein. Mechanistically, the decay process is initiated by a hydrogen abstraction from a peptide backbone amide nitrogen by 5-NSA. Hydrogen abstraction results in formation of an oxidized peptide containing a radical amide nitrogen. Subsequently, the C(α)-C bond N-terminal to the peptide bond is cleaved to form an a·/x fragment pair. The C(α)-C bonds C-terminal to Gly residues were less susceptible to cleavage than were those of other residues. C(α)-C bonds N-terminal to Pro and Sar residues were not cleaved by the aforementioned mechanism; instead, after hydrogen abstraction from a Pro or Sar C(α)-H bond, the peptide bond N-terminal to the Pro was cleaved yielding b- and y-series ions. We also show that fragments produced by MALDI 5-NSA-induced ISD were formed independently of the ionization process.     

Efficient synthesis of complex glycopeptides based on unprotected oligosaccharides

N-Glycopeptides containing 1 to 4 trisaccharide chains, with the carbohydrates vicinal to each other in the multivalent glycopeptides, were efficiently synthesized by using the glycosylated Fmoc-asparagine as a key building block. While the couplings of amino acids with glycopeptides could be achieved in the homogeneous solutions in N-methylpyrrolidinone (NMP) to give excellent yields, all products were conveniently isolated from the reaction mixtures through a precipitation method by using the free carbohydrate chains as phase tags. Commercially available pentafluorophenyl (Pfp) esters of amino acids were employed for the glycopeptide elongation. Longer glycopeptides were constructed by means of a highly convergent synthetic design that is based on the coupling of glycopeptide/peptide fragments. Hydrogen bond interactions between free oligosaccharides were proposed to explain the exceptionally high efficiency of the couplings between two glycosylated building blocks.     

Analysis of amide bond formation with an alpha-hydroxy-beta-amino acid derivative, 3-amino-2-hydroxy-4-phenylbutanoic acid, as an acyl component: byproduction of homobislactone

In the synthesis of peptidomimetics containing alpha-hydroxy-beta-amino acid, the coupling of this N(beta)-protected beta-amino acid with amine components was generally performed without the protection of its alpha-hydroxyl group. However, the formation of dipeptides in low yield was often observed when sterically hindered amine components were used. Boc-Apns-OH [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid, allophenylnorstatine] (6), which is one of such beta-amino acid derivatives, is intensively employed as a core structure in the development of HIV-1 protease inhibitors. There have been no precise studies, to date, that have examined amide bond formation with alpha-hydroxy-beta-amino acid derivatives as an acyl component. To determine the cause of this low-yield reaction, we studied the amide bond formation focusing on the activation step of N(beta)-protected alpha-hydroxy-beta-amino acid by using a model coupling reaction between 6 and H-Dmt-OR [Dmt: (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid] (7). A significant amount of homobislactone 9 was formed through the activation of the carboxyl group of 6 to the benzotriazole-type active esters such as OBt and OAt. In addition, this homobislactone formation was markedly increased in the presence of a catalytic amount of a base, which exhibited good correlation with the low yield of the amide bond formation, suggesting that homobislactone formation is one major reason for the low yield of the amide bond formation. Moreover, homobislactones were also formed in other derivatives of the N(beta)-protected alpha-hydroxy-beta-amino acid, suggesting a common feature of this type of amino acids. The use of a strong activation method like EDC--HOAt without base addition enhanced amide bond formation, although a small amount of homobislactone may be formed during the coupling reaction.     

New selectivity in peptide hydrolysis by metal complexes. Platinum(II) complexes promote cleavage of peptides next to the tryptophan residue

Tryptophan-containing N-acetylated peptides AcTrp-Gly, AcTrp-Ala, AcTrp-Val, and AcTrp-ValOMe bind to platinum(II) and undergo selective hydrolytic cleavage of the C-terminal amide bond; the N-terminal amide bond remains intact. In acetone solution, bidentate coordination of the tryptophanyl residue via the C(3) atom of indole and the amide oxygen atom produces complexes of spiro stereochemistry, which are characterized by (1)H, (13)C, and (195)Pt NMR spectroscopy, and also by UV-vis, IR, and mass spectroscopy. Upon addition of 1 molar equiv of water, these complexes undergo hydrolytic cleavage. This reaction is as much as 10(4)-10(5) times faster in the presence of platinum(II) complexes than in their absence. The hydrolysis is conveniently monitored by (1)H NMR spectroscopy. We report the kinetics and mechanism for this reaction between cis-[Pt(en)(sol)(2)](2+), in which the solvent ligand is water or acetone, and AcTrp-Ala. The platinum(II) ion as a Lewis acid activates the oxygen-bound amide group toward nucleophilic attack of solvent water. The reaction is unimolecular with respect to the metal-peptide complex. Because the tryptophanyl fragment AcTrp remains coordinated to platinum(II) after cleavage of the amide bond, the cleavage is not catalytic. Added ligand, such as DMSO and pyridine, displaces AcTrp from the platinum(II) complex and regenerates the promoter. This is the first report of cleavage of peptide bonds next to tryptophanyl residues by metal complexes and one of the very few reports of organometallic complexes involving metal ions and peptide ligands. Because these complexes form in nonaqueous solvents, a prospect for cleavage of membrane-bound and other hydrophobic proteins with new regioselectivity has emerged.     

Fragmentation reactions of deprotonated peptides containing proline. The proline effect

The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the 'b(2) ion; further evidence is presented that the 'b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline.     

Mechanism of thio acid/azide amidation

A combined experimental and computational mechanistic study of amide formation from thio acids and azides is described. The data support two distinct mechanistic pathways dependent on the electronic character of the azide component. Relatively electron-rich azides undergo bimolecular coupling with thiocarboxylates via an anion-accelerated [3+2] cycloaddition to give a thiatriazoline. Highly electron-poor azides couple via bimolecular union of the terminal nitrogen of the azide with sulfur of the thiocarboxylate to give a linear adduct. Cyclization of this intermediate gives a thiatriazoline. Decomposition to amide is found to proceed via retro-[3+2] cycloaddition of the neutral thiatriazoline intermediates. Computational analysis (DFT, 6-31+G(d)) identified pathways by which both classes of azide undergo [3+2] cycloaddition with thio acid to give thiatriazoline intermediates, although these paths are higher in energy than the thiocarboxylate amidations. These studies also establish that the reaction profile of electron-poor azides is attributable to a prior capture mechanism followed by intramolecular acylation.     

Structural studies of C-amidated amino acids and peptides: crystal structures of Z-Gly-Phe-NH2, Tyr-Lys-NH2, and Asp-Phe-NH2

As part of the series investigating the structural features of C-terminal amidated amino acids and peptides, three crystal structures of Z-Gly-Phe-NH2, Tyr-Lys-NH2, and Asp-Phe-NH2 were analyzed by the X-ray diffraction method, and their molecular conformations and intermolecular interactions were investigated. Although the respective dipeptides exhibited an energetically allowable torsion angle concerning each backbone or side chain, the observed extended (Z-Gly-Phe-NH2, Asp-Phe-NH2) and folded (Tyr-Lys-NH2) conformations were considerably different from those of the corresponding unamidated peptides, due to the conformational flexibility of the respective dipeptides. The comparison between the crystal packings of the amidated and unamidated dipeptides indicated that the C-terminal amides tend to associate with the same neighboring group through hydrogen bonds, in which both the amide NH and O=C groups participate, while the unamidated peptides prefer a linear molecular connection, where both or either of the two carboxyl oxygens participate in the hydrogen bond formation. The difference in hydrogen bonding ability between the C-terminal amide and carboxyl groups has been considered to be based on the structural data of the related peptides analyzed so far.     

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol(-1)); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the alpha-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol(-1)); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol(-1)). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol(-1)) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.     

Efficient amidation from carboxylic acids and azides via selenocarboxylates: application to the coupling of amino acids and peptides with azides

A facile one-pot procedure for the coupling of carboxylic acid and azide via selenocarboxylate and selenatriazoline has been developed and successfully applied to the coupling of amino acids and peptides with azides. Selenocarboxylates are readily prepared by the reaction of the activated carboxylic acids with LiAlHSeH under mild conditions. The selenocarboxylates formed in situ are used to react directly with azides to form the corresponding amides via a selenatriazoline intermediate. Excellent yields were obtained for electron-deficient azides, and moderate to good yields were obtained for electron-rich azides. The selenocarboxylate/azide amidation reaction is clean and chemoselective. It provides an attractive alternative method to the conventional acylation of amines when an amide bond needs to be formed without going through an amine intermediate.     

Nickel‐Catalyzed Synthesis of Dialkyl Ketones from the Coupling of N‐Alkyl Pyridinium Salts with Activated Carboxylic Acids

While ketones are among the most versatile functional groups, their synthesis remains reliant upon reactive and low‐abundance starting materials. In contrast, amide formation is the most‐used bond‐construction method in medicinal chemistry because the chemistry is reliable and draws upon large and diverse substrate pools. A new method for the synthesis of ketones is presented here that draws from the same substrates used for amide bond synthesis: amines and carboxylic acids. A nickel terpyridine catalyst couples N‐alkyl pyridinium salts with in situ formed carboxylic acid fluorides or 2‐pyridyl esters under reducing conditions (Mn metal). The reaction has a broad scope, as demonstrated by the synthesis of 35 different ketones bearing a wide variety of functional groups with an average yield of 60±16 %. This approach is capable of coupling diverse substrates, including pharmaceutical intermediates, to rapidly form complex ketones.     

The reaction of thio acids with azides: a new mechanism and new synthetic applications

A new amide synthesis strategy based on a fundamental mechanistic revision of the reaction of thio acids and organic azides is presented. The data demonstrate that amines are not formed as intermediates in this reaction. Alternative mechanisms proceeding through a thiatriazoline intermediate are suggested. The reaction has been applied to the preparation of simple and architecturally complex amides that are difficult to access using conventional methods. The reaction is chemoselective, effective for unprotected substrates, and compatible with aprotic and protic solvents, including water.     

Sugar-assisted ligation for the synthesis of glycopeptides

Chemical synthesis of glycoproteins from readily available materials is a powerful method for obtaining a pure product with full control of its atomic structure. Sugar-assisted ligation (SAL) is an emerging approach that allows the synthesis of a large glycopeptide from two unprotected fragments. Contrary to other ligation methods that are limited to the use of a cysteine residue or depend on external auxiliary, SAL takes advantage of the existing sugars in glycopeptides to promote proximity between the two peptides to facilitate an amide bond formation.