Synthesis of the sphingolipid activator protein, saposin C, using an azido-protected O-acyl isopeptide as an aggregation-disrupting element

In order to achieve an efficient synthesis of highly hydrophobic proteins by the native chemical ligation (NCL) reaction, we examined to incorporate the O-acyl isopeptide method, which is known to improve the solubility of the segment, to the NCL reaction: a peptide thioester having O-acyl isopeptide structures is prepared by the Boc mode solid-phase method using an azido group as a protecting group for the isopeptide site, and then ligated with C-terminal segment with an in situ reduction of the azido group followed by an O- to N-acyl shift. This method was successfully applied to the synthesis of the sphingolipid activator protein, saposin C.     

Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction

In one of the peptide condensation methods termed thioester method, an amino protecting group is required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an amino protecting group, we synthesized N^α-fluorenylmethoxycarbonyl (Fmoc)-N^ε-pyruvoyl-lysine and introduced it into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis. The pyruvoyl peptide could be condensed with a peptide thioester by the thioester method, and this protecting group was easily removed by o-phenylenediamine treatment without significant side reactions.     

N-Alkyl cysteine-assisted thioesterification of peptides

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous 3-mercaptopropionic acid (MPA) without significant side reactions.     

N-methyl-phenacyloxycarbamidomethyl (Pocam) group: a novel thiol protecting group for solid-phase peptide synthesis and peptide condensation reactions

In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.     

Enhancement in the rate of conversion of peptide Cys-Pro esters to peptide thioesters by structural modification

We previously reported that the peptide containing a Cys-Pro ester (CPE) moiety is spontaneously transformed into a peptide thioester via an N to S acyl shift followed by diketopiperazine formation. In an attempt to identify more reactive structures for the formation of a peptide thioester, we modified the CPE structure, in which the Pro residue in the CPE moiety was replaced with N-substituted glycine derivatives. These peptides were transformed into a peptide thioester more rapidly. Alternatively, the addition of an amino acid residue at the C-terminus of the CPE moiety also accelerated thioester formation.     

Synthesis of Heavily Glycosylated Peptide αThioester

Erythropoietin (EPO) needs to be heavily glycosylated to exhibit its bioactivity in vivo. In order to synthesize heavily glycosylated EPO analogues, corresponding glycosylated peptide ^αthioesters are essential to prepare glycosylated whole EPO peptide backbones through native chemical ligation. After construction of the peptide ^αthioester corresponding to the 1–32 amino acid sequence in EPO, we aimed to incorporate three complex-type biantennary sialyloligosaccharides to this peptide ^αthioester by the haloacetamide method. The reaction afforded the desired heavily glycosylated peptide ^αthioester.     

Structure of C-1 substituted glycopyranosyl azides: new insights based on CD measurements

CD spectra have been recorded for a series of peracetylated d-glycopyranosyl azides (d-gluco, d-galacto, d-xylo, d-arabino configuration) substituted at the anomeric position by various groups: amido, azido, cyano, ethoxy, methoxy. Application of the azide octant rule for the interpretation of the sign for the long-wavelength azide band allowed conformation of the azido group in each mono azido derivative investigated to be established. In each 1-cyano derivative, the azido group was in a gauche-like arrangement with respect to the C-1–O^ring bond, which is considered as a manifestation of the exo-anomeric effect of the azido group. For the 1-alkoxy derivatives, an antiparallel orientation of the azido group with respect to the C-1–O^ring bond was found in solution by CD measurement analysis, as already observed for methoxyazide 5 in the solid state. For azidoamide derivatives, intramolecularly (N–H–N^x_azide) H-bonded conformers are believed to prevail in methanol, in contrast to the situation in DMSO.     

The use of a cysteinyl prolyl ester (CPE) autoactivating unit in peptide ligation reactions

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) is spontaneously transformed into a diketopiperazine thioester via an intramolecular N-S acyl shift reaction, followed by diketopiperazine formation. The CPE peptide can be ligated with a Cys-peptide in a one-pot procedure. The peptide diketopiperazine thioester can also be transformed into a peptide thioester by intermolecular thiol-thioester exchange with external thiol compounds such as sodium mercaptoethanesulfonate. Since CPE peptides can be prepared by standard Fmoc solid-phase synthesis, it is a versatile alternative to the peptide thioester, providing a flexible ligation strategy that promises to be useful in polypeptide synthesis.     

An N-protection free ligation of the peptide thioester and the peptide with an N-alkoxy- or N-aryloxyamino group at its N-terminus

Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and successfully ligated with a peptide thioester by silver ion activation under a slightly acidic condition without requiring protection of the side chain amino groups. The N-methoxy group was easily cleaved by the SmI₂ reduction in CH₃OH aq. to obtain the desired peptide with a native peptide bond. This method was successfully applied to the synthesis of the human atrial natriuretic peptide showing the efficiency of the novel ligation.     

Theoretical Analysis of the Detailed Mechanism of Native Chemical Ligation Reactions

The method of native chemical ligation between an unprotected peptide α‐thioester and an N‐terminal cysteine–peptide to give a native peptide in aqueous solution is one of the most effective peptide ligation methods. In this work, a systematic theoretical study was carried out to fully understand the detailed mechanism of ligation. It was found that for the conventional native chemical ligation reaction between a peptide thioalkyl ester and a cysteine in combination with an added aryl thiol as catalyst, both the thiol‐thioester exchange step and the transthioesterification step proceed by an anionic concerted S_N2 displacement mechanism, whereas the intramolecular rearrangement proceeds by an addition–elimination mechanism, and the rate‐limiting step is the thiol‐thioester exchange step. The theoretical method was then extended to study the detailed mechanism of the auxiliary‐mediated peptide ligation between a peptide thiophenyl ester and an N‐2‐mercaptobenzyl peptide in which both the thiol‐thioester exchange step and intramolecular acyl‐transfer step proceed by a concerted S_N2‐type displacement mechanism. The energy barrier of the thiol‐thioester exchange step depends on the side‐chain steric hindrance of the C‐terminal amino acid, whereas that of the acyl‐transfer step depends on the side‐chain steric hindrance of the N‐terminal amino acid.     

Sequential peptide chemical ligation by the thioester method and extended chemical ligation

The sequential chemical ligation of peptide thioesters by a combination of the thioester method and extended chemical ligation using a photoremovable auxiliary, 2-mercapto-1-(2-nitrophenyl)ethyl group, is described. The thiazolidine ring was used as a protecting group for the N-terminal 1,2-aminoethanethiol moiety of the auxiliary in the middle peptide thioester. After the first thioester coupling, the thiazolidine ring was opened by treatment with O-methylhydroxylamine. Second coupling by extended chemical ligation followed by UV irradiation gave the target polypeptide.     

Synthesis of Star-like Polybutadienes by a Combination of Living Anionic Polymerization and “Click” Coupling Method

Three-arm and four-arm star-like polybutadienes(PBds) were synthesized via the combination of living anionic polymerization and the click coupling method. Kinetic study showed that the click reaction between the azido group terminated PBd-t-N3 and the alkyne-containing multifunctional linking reagent was fast and highly efficient. All coupling reactions were fully accomplished within 40 min at 50 °C in toluene in the presence of the reducing agent Cu(0), proven by 1H-NMR, FTIR and GPC measurements. For the coupling reactions between the PBd-t-N3 polymer and dialkyne-containing compound, the final conversion of the coupled PBd-PBd polymer was ca. 97.0%. When a PBd-t-N3 polymer was reacted with trialkyne-containing or tetraalkyne-containing compound, the conversion of three-arm or four-arm PBd was around 95.5% or 87.0%, respectively. Several factors influencing the coupling efficiency were studied, including the molecular weight of the initial PBd-t-N3, arm numbers and the molar ratio of the azido group to the alkynyl group. The results indicated that the conversion of the target products would be promoted when the molecular weight of the PBd-t-N3 was low and the molar ratio of the azido to alkynyl groups was close to 1.     

One‐Pot Four‐Segment Ligation Using Seleno‐ and Thioesters: Synthesis of Superoxide Dismutase

The synthesis of a peptide selenoester was efficiently carried out by the 9‐fluorenylmethoxycarbonyl (Fmoc) method using N‐alkylcysteine, at the C‐terminus of the peptide, as the N‐to‐S acyl shift device. The selenoester selectively reacted with the terminal amino group of the peptide aryl thioester in the presence of N ,N ‐diisopropylethylamine and dipyridyldisulfide, thus leaving the aryl thioester intact. Combined with silver‐ion‐promoted and silver‐ion‐free thioester activation methods, a one‐pot four‐segment ligation was realized. The method was successfully used to assemble the entire sequence of superoxide dismutase (SOD), which is composed of 153 amino‐acid residues, in one pot. After the folding reaction, the fully active SOD was obtained.     

Nitrido-Azido-Komplexe des Molybdän(VI): Synthese und Kristallstruktur von [MoN(N3)2(terpy)]+ [MoN(N3)4]−·MoN(N3)3 (terpy)

Nitrido-Azido-Complexes of Molybdenum(VI). Synthesis and Crystal Structure of [MoN(N₃)₂(terpy)]⁺[MoN(N₃)₄] ^? · MoN(N₃)₃(terpy) (CH₃)₃SiN₃ reacts with Mo(CO)₃(terpy) in CH₃CN yielding red crystals of [MoN(N₃)₂(terpy)]⁺[MoN(N₃)₄] ^?· MoN(N₃)₃(terpy) (space group P1 , a = 1039.3 pm, b = 1384.6 pm, c = 1685.4 pm, α = 112.4°, β = 108.1°, γ = 88.3°, Z = 2, R = 0.035 for 4376 independent reflections). The structure consists of three different mononuclear complexes. In the neutral complex MoN(N₃)₃(terpy) Mo exhibits the coordination number 7 in form of a distorted pentagonal bipyramid, with the terpyridine ligand and two azido groups in the equatorial plane. The axial positions are occupied by the nitrido ligand and another azido group. The triply bonded nitrido nitrogen atom (Mo1? N1 = 165.6 pm) causes a strong trans effect resulting in a long distance of 245.7 pm to N_α of the trans bonded azido group. The cationic complex [MoN(N₃)₂(terpy)]⁺ derives from MoN(N₃)₃-(terpy) by abstraction of the trans bonded azido group. For the molybdenum atom remains the coordination number 6 in form of the rarely found pentagonal pyramid. In the anion [MoN(N₃)₄]^? the molybdenum atom exhibits the coordination number 5 in form of a tetragonal pyramid with the nitrido ligand in the apex. The square basic plane is formed by the N_α atoms of the azido groups.     

Peptide thioester formation using standard Fmoc-chemistry

A highly efficient and simple Fmoc-based preparation of peptide ^αthioesters is presented. After Fmoc/t-butyl solid-phase synthesis on 2-chlorotrityl resin the C-terminal carboxylic group of the protected peptide is directly converted to the corresponding thioester. The method leads to very high yields, shows a low level of epimerization and can be easily applied also for the preparation of long peptide ^αthioesters as demonstrated for the 41 amino acid N-terminal fragment of pro-neuropeptide Y (proNPY 1-40).     

15N NMR spectra and reactivity of 2,4,6‐triazidopyridines, 2,4,6‐triazidopyrimidine and 2,4,6‐triazido‐s‐triazine

2,4,6‐Triazido‐s‐triazine, 2,4,6‐triazidopyrimidine and six different 2,4,6‐triazidopyridines were studied by ¹⁵N NMR spectroscopy. The assignment of signals in the spectra was performed using the gauge‐independent atomic orbital (GIAO)–Tao‐Perdew‐Staroverov‐Scuseria exchange‐correlation functional (TPSS)h/6‐311+G(d,p) calculations on the M06‐2X/6‐311+G(d,p) optimized molecular geometries. The Truhlar and coworkers' continuum solvation model called SMD was applied to treat solvent effects. With this approach, the root mean square error in estimations of the ¹⁵N chemical shifts for the azido groups was just 1.9 ppm. It was shown that the different reactivity of the α‐ and γ‐azido groups in pyridines correlates well with the chemical shifts of the N_α signals of these groups. Of two nonequivalent azido groups of azines, the azido group with the most shielded N_α signal is the most electron‐deficient and reactive toward electron‐rich reagents. By contrast, the azido group of azines with the most deshielded N_α signal is the most reactive toward electron‐poor reagents. Copyright © 2013 John Wiley & Sons, Ltd.     

Facile one‐pot/one‐step technique for preparation of side‐chain functionalized polymers: Combination of SET‐RAFT polymerization of azide vinyl monomer and click chemistry

An azido‐containing functional monomer, 11‐azido‐undecanoyl methacrylate, was successfully polymerized via ambient temperature single electron transfer initiation and propagation through the reversible addition–fragmentation chain transfer (SET‐RAFT) method. The polymerization behavior possessed the characteristics of “living”/controlled radical polymerization. The kinetic plot was first order, and the molecular weight of the polymer increased linearly with the monomer conversion while keeping the relatively narrow molecular weight distribution (M_w/M_n ≤ 1.22). The complete retention of azido group of the resulting polymer was confirmed by ¹H NMR and FTIR analysis. Retention of chain functionality was confirmed by chain extension with methyl methacrylate to yield a diblock copolymer. Furthermore, the side‐chain functionalized polymer could be prepared by one‐pot/one‐step technique, which is combination of SET‐RAFT and “click chemistry” methods. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Convenient high yield and stereoselective synthesis of O-glycopeptides using N-α-Fmoc-Tyr/Ser[β-d-Glc(OAc)4]OPfp generated in solution

Fmoc-AA-OPfp (AA=Tyr or Ser) (1 equiv) was reacted with β-d-Glc(OAc)₅ (6 equiv) in the presence of BF₃.Et₂O (6 equiv) in CH₂Cl₂ at room temperature for 2 h, and the glycosylation reaction mixture was used directly to couple to the amino group of the peptide resin without isolation and purification of the Fmoc-AA[β-d-Glc(OAc)₄]-OPfp. Moreover, the -OAc protecting groups of glucose was removed just prior to releasing the peptide from the resin using 6 mM NaOMe in 85% DMF-MeOH. The crude product obtained by TFA cleavage contained >90% of the target O-glycopeptide, and the 500 MHz ¹H NMR analysis revealed that the glycosylation reaction was nearly stereoselective (>97% β-anomer). This method is rapid and stereoselective, and can now be exploited for the routine synthesis of O-glycopeptides.     

Study of complexation between Cu<Superscript>2+</Superscript> ions and <Emphasis Type="Italic">ortho</Emphasis>-azidobenzoic acid

An aqua complex of copper(II) o-azidobenzoate, [Cu(OH)ABA2H₂O]₂ (ABA is o-azidobenzoic acid), was synthesized in an aqueous solution and identified by IR and electronic absorption spectroscopy. In the complex, the azido group is not coordinated by Cu²⁺. When dissolved in dry organic solvents (DMF, DMSO, dioxane, and methanol), the complex undergoes dehydration to give a chelate complex (CC) containing the Cu²⁺-coordinated azido group as a result of the electron density redistribution at its N atoms. The IR spectrum of the chelate complex contains no absorption band at 2135 cm^–1 corresponding to the stretching vibrations of the azido group. The resulting CC is unstable in solutions and spontaneously decomposes with a release of molecular nitrogen. The interaction of a Cu²⁺ ion with o-azidobenzoic acid in dry organic solvents affords a CC similar to the complex obtained on the dissolution of [Cu(OH)ABA2H₂O]₂ in dry solvents.Translated from Koordinatsionnaya Khimiya, Vol. 31, No. 3, 2005, pp. 195–198.Original Russian Text Copyright © 2005 by Budruev, Karyakina, Levina, Oleinik.This revised version was published online in April 2005 with a corrected cover date.This revised version was published online in April 2005 with a corrected cover date.