Synthesis and physicochemical characterization of the lanthionine analog of somatostatin[1–14]

Summary The synthesis of the lanthionine analog of somatostatin[1–14] on a Kaiser-oxime resin is described. The 12-residue peptide segment [3–14] was assembled and cyclized on the resin by using the method of peptide cyclization on an oxime resin (PCOR); the product was obtained with good yield (41%) and purity (94%). The Fmoc protecting group on the N-terminus was cleaved with DBU, followed by a 2+12 segment condensation in solution. The chromatographic (HPLC, CZE) and spectral (UV, NMR) properties of the lanthionine and the natural somatostatins have been studied and compared. Preliminary biological tests show that the lanthionine and the natural somatostatins exhibit similar binding affinities to somatostatin receptor SSTR2.Abbreviations Ala_L one end of a lanthionine unit - Boc tert-butyloxycarbonyl - BOP benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - Cbz benzyloxycarbonyl - DQF-COSY double-quantum-filtered correlated NMR spectroscopy - CZE capillary zone electrophoresis - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DMSO-d₆ hexadeuterated dimethylsulfoxide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - Fmoc 9-florenylmethoxycarbonyl - For formyl - HMPA hexamethylphosphoramide - HOBt N-hydroxybenzotriazole - HOHAHA homonuclear Hartmann-Hahn experiment - HPLC high-performance liquid chromatography - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - TFA trifluoroacetic acid - PCOR peptide cyclization on an oxime resin - Tmac₂O trimethylacetic or pivalic anhydride - Tos p-toluenesulfonyl     

Isolation and characterization of the siderophore N-deoxyschizokinen from Bacillus megaterium ATCC 19213

N-Deoxyschizokinen, a novel siderophore, was isolated from stationary phase cultures of Bacillus megaterium ATCC 19213 and identified as 4-[(3(acetylhydroxyamino)propyl)amino]-2-[2-[(3-(acetylamino)propyl)amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid. The siderophore was purified by HPLC and its structure determined using ¹H and ¹³C NMR, ¹H-¹H COSY and electrospray mass spectroscopy. The monohydroxamate siderophore has the same carbon skeleton as schizokinen but the hydroxyl group on one hydroxamate is replaced by a hydrogen. A detailed ¹H NMR study of schizokinen, N-deoxyschizokinen and their imides, schizokinen A and N-deoxyschizokinen A is presented.     

SYNTHESIS AND STRUCTURE DETERMINATION OF SOME OXADIAZOLE-2-THIONE AND TRIAZOLE-3-THIONE GALACTOSIDES

The syntheses of 5-pyridyl-3((β-D-galactopyranosyl)-1,3,4-oxadiazole-2-thiones 3a–3c and 5-pyridyl-2((β-D-galactopyranosyl)-4-benzyl-1,2,4-triazole-3-thiones 6a–6c are reported. The existence of N-galactosides – not S-galactosides – was proven by IR and ¹⁵N NMR spectroscopy. The structures of the final products and the intermediates were elucidated by IR, ¹H, ¹³C and ¹⁵N NMR spectroscopy and mass spectrometry.

     

Efficient solid-phase synthesis of a large peptide by a single coupling protocol with a single HPLC purification step

Summary Peptide chain assembly is now routinely performed by the use of automated synthesizers, although purification and characterization of large peptides still requires knowledge and experience. Structural biology has recently become closely involved in molecular recognition studies that often require the analysis of relatively large peptides using high-resolution NMR spectroscopy, for which synthesis of high-quality peptides in 5–10 mg amounts is of prime importance. The present study describes a solid-phase synthesis of a 7 kDa peptide related to the recently characterized ethylene-responsive element binding protein of tobacco, which is the conserved sequence among these proteins. The rapid and efficient preparation was carried out through a single coupling in combination with a single HPLC separation step. Assembly was performed in 63 h. Different coupling chemistries were employed and compared, involving benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and/or the recently introduced reagent,N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphateN-oxide. After each synthesis, purified material was characterized by mass spectrometry, sequencing and enzymatic mapping and shown to contain a high proportion of the desired peptide.     

On the Manner of Cyclization of <Emphasis Type="Italic">N</Emphasis>-Acylated Aspartic and Glutamic Acid Derivatives

Abstract  

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of N-acylated glutamic acid derivatives. During concomitant cleavage and cyclization two alternative routes were possible—either formation of six-member imide (glutarimide) or five-member lactam. Application of MS/MS and ¹H NMR method allowed us to establish that cyclization of N-acylated glutamic acid derivatives preceded to lactams—N-acylated pyroglutamic acid derivatives.     

Synthesis of cyclic analogues of loop 4 of nerve growth factor

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of theβ-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52–83% yields by the solid-phase method with the use of the Fmoc/Bu ^t strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxytris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphorylazide (DPPA) at a dilution of 10^?3 M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five-and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization, and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.     

Nα-Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride: A convenient building block for the solid-phase synthesis of phosphotyrosyl peptides

Summary Fmoc-O,O-(dimethylphospho)-l-tyrosine was converted into stable Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride by means of (diethylamino) sulfur trifluoride or cyanuric fluoride. This building block was used for efficient coupling of phosphotyrosine to the adjacent sterically hindered amino acid Aib or Ac₆c in, model peptide sequences as well as for the synthesis of the ‘difficult’ phosphotyrosine peptide Stat91^695–708. The phosphate methyl groups were cleaved on solid phase after peptide assembly by means of trimethylsilyl iodide in MeCN. Aib, α-aminoisobutyric acid Ac₆c, 1-amino-cyclohexyl-l-carboxylic acid; BOP, benzotriazol-l-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate, CIP, 2-chloro-l, 3-dimethylimidazolidium hexafluorophosphate, DAST, (diethylamino)sulfur trifluoride; DBU 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DIEA, drisopropylethylamine; DMA dimethylacetamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU,O-(7-azabenzotriazol-l-yl)-1.1,3,3-tetramethyluronium hexafluorophosphate; HOAt, I-hydroxy-7-azabenzotriazole; HOBt,N-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; MBHA, 4-methylbenzhydrylamine; MeCN, acetonitrile; NMP,N-methyl-2-pyrrolidinone; NMR, nuerear magnetic resonance; PS, polystyrene; PyBroP, bromotris(pyrrolidino)phosphonium hexafluorophosphate; Rink amide MBHA-PS, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminophenyl)-phenoxyacetamido-norleucyl-MBHA-PS; TFA, trifluoroacetic acid; TMSI, trimethylsilyl iodide; TPTU, 2-(2-pyridon-l-yl)-1,1,3,3-tetramethyluroniumfluoroborate; t_R, retention time; UNCA, arethane-protected amino acidN-carboxy anhydride Abbreviations for amino acids and nomenclature of, peptide structures follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9].     

Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

Summary The cyclization of a linear dynorphin A (Dyn A) analogue to give the lactam derivative cyclo[d-Asp², Dap⁵]Dyn A(1–13)NH₂ (where Dap=,-diaminopropionic acid) was studied to evaluate the usefulness of different coupling reagents for side chain to side chain lactam formation. This cyclization proved to be difficult and yielded substantial byproducts that varied depending upon the activating reagent used. On-line HPLC-ion spray mass spectrometry was more practical and useful than conventional HPLC alone for characterizing the products of these cyclization reactions. Peptide byproducts could be identified from the series of multiply charged ions observed, even when some of these peptides eluted from the HPLC with similar retention times. In addition to the desired cyclic peptide, the peptide byproducts observed following the cyclization using BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate) were the linear peptide, the cyclic dimeric peptide and the linear peptide resulting from aspartimide rearrangement. The peptide byproducts obtained following cyclization using HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HAPyU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate) were predominantly linear tetramethylguanidinium (Tmg) and dipyrrolidinylguanidinium (Dpg) derivatives resulting from alkylation of the side chain of Dap by HATU and HAPyU, respectively; in addition to monomeric guanidinium derivatives, dimeric and aspartimide-containing peptides were also produced. Peptide sequencing by ion spray tandem mass spectrometry was performed to confirm the structure of both pure peptides and peptide byproducts in the crude samples. A unique fragmentation for the ,-bond of the Dap side chain was demonstrated and could be used to identify linear peptide byproducts. The distinctive fragment ions from this cleavage were also observed for the peptides containing the Tmg and Dpg functionalities on the Dap side chain.     

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999     

NMR Studies of N 1- and N 3-(D-2′-Deoxyribofuranosyl) Nucleosides from Ethyl 5-Aminoimidazole-4-Carboxylate

Abstract

The conformation of deoxyribosides derived from N¹ and N³ substitution of the aglycone ethyl 5-aminoimidazole-4-carboxylate (AICE) has been determined by ¹H and ¹³C NMR spectroscopy. The deoxyribose ring shows considerable variation in the form and population of the contributing N and S conformers but the syn/anti ratio is similar to that in the purine analogues.     

Chemical Modification of Insulin by <Emphasis Type="Italic">N</Emphasis>-phosphorylation

In this paper, the reactions of bovine insulin and small peptides, such as actin binding domain of thymosin β₄ and Growth Hormone Releasing Factor (GRF 1–29 amino acids) with diisopropyloxyphosphite (DIPPH) and dimethyloxyphosphite (DMPH) were studied by modified Todd reaction. The MALDI-TOF or ESI-MS results showed that lysine, histidine and arginine residues in insulin could be phosphorylated under the water/ethanol system. The N,N,N-diisopropyloxyphosphorylated insulin analogues were characterized using MALDI-TOF and ³¹P NMR. These insulin analogues with different phosphorylation degree were separated and identified through LC-ESI-MS. In addition, circular dichroism (CD) spectra showed that the conformation of N,N,N-dimethyloxyphosphorylated insulin were only changed a little, whereas, that of N,N,N-diisopropyloxyphosphorylated insulin was changed completely.     

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Summary The backbone ¹H and ¹⁵N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the ¹⁵N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.Abbreviations 2D, 3D two-, three-dimensional - drkN SH3 N-terminal SH3 domain of Drosophila drk - HSQC heteronuclear single-quantum spectroscopy - NOE nuclear Overhauser enhancement - SH3 Src homology domain 3 - TOCSY total correlation spectroscopy     

Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the α-tetrasubstituted amino acid residue α-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with ¹⁵N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled ¹⁵N and ³¹P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional ¹⁵N chemical shift - ¹H-¹⁵N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed α-/3₁₀-helical structures which can be explained by the restraints imposed by the membranes and the bulky α-aminoisobutyric acid residues. The ¹⁵N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Total Synthesis of the Peptaibols Hypomurocin A3 and Hypomurocin A5, and Their Conformation Analysis

The total syntheses of hypomurocin A3 and hypomuricin A5 (HM A3 and HM A5, resp.) in solution phase are described. These syntheses have been successfully achieved by applying the ‘azirine/oxazolone method’ to introduce the two Aib‐Pro units into the backbone of these undecapeptaibols in one step with methyl 2,2‐dimethyl‐2H‐azirine‐3‐prolinate as the ‘Aib‐Pro synthon’. The coupling of Z‐protected (Z=(benzyloxy)carbonyl) amino acids or peptide acids with amino acid tert‐butyl esters and of peptide segments was carried out according to the TBTU (=O‐(benzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium tetrafluoroborate) and HOBt (=1‐hydroxybenzotriazole) protocol. Purification by reversed‐phase HPLC gave the peptides in pure form. The products were characterized by optical rotation, NMR and IR spectroscopy, mass spectrometry, and elemental analysis. The crystal structures of HM A3 and of an octapeptide fragment of HM A5 could be obtained. An NMR analysis was also carried out with HM A3 and HM A5 to determine their conformations in solution. A global structural comparison between the three sequences of HM A1, HM A3, and HM A5 was performed, as well as the HPLC correlation of the natural HM A family and the synthetic samples.     

Methylated N-aryl chitosan derivative/DNA complex nanoparticles for gene delivery: Synthesis and structure–activity relationships

In this study, three kinds of methylated chitosan containing different aromatic moieties were synthesized by two steps, reductive amination and methylation, respectively. The chemical structures of all methylated derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMChC), methylated N-(4-N,N-dimethylaminobenzyl) chitosan chloride (MDMBzChC), and methylated N-(4-pyridinylmethyl) chitosan chloride (MPyMeChC) were characterized by ATR–FTIR and ¹H NMR spectroscopy. The complexes between the chitosan derivatives and plasmid DNA at different N/P ratios were characterized by gel electrophoresis, dynamic light scattering, and atomic force microscopic techniques. The smallest particle sizes of these complexes were obtained at N/P ratio of 5 and ranged from 95 to 124 nm while the zeta-potentials were in the range of 18–27 mV. Transfection efficiencies of these complexes were investigated by expression of the plasmid DNA encoding green fluorescence protein (pEGFP-C2) on human hepatoma cells (Huh 7 cells) compared to N,N,N-trimethyl chitosan chloride (TMChC). The rank of transfection efficiency was MPyMeChC > MDMBzChC > TMChC > MDMCMChC, respectively. The cytotoxicity of these complexes was also studied by MTT assay where the MPyMeChC complex exhibited less toxicity than other derivatives even at high N/P ratios. Therefore, MPyMeChC demonstrated potential as its safe and efficient gene carrier.     

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐N^α [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A proteomics approach to study in vivo protein Nα-modifications

In this article we present a simple method to enrich peptides containing in vivo Nα-modified protein N-termini. We demonstrate that CNBr-activated Sepharose, a commercial amine reactive matrix, can selectively couple peptides via the α-NH₂ group under mild conditions. Following digestion by trypsin, a simple incubation step with the CNBr-activated Sepharose by which the free α-NH₂ containing peptides are coupled with matrix through a covalent bond, allows the separation of N^α-modified peptides from massive free α-NH₂ containing peptides. The removal of contaminant peptides with artificial N^α-modifications, like cyclization of N-terminal S-carbamoylmethylcysteine and glutamine, are also discussed. Application of this method to tryptic digests of HeLa cell proteins resulted by a single LC-MS/MS analysis in the identification of 588 in vivo N^α-modified peptides, of which 507 contain IPI (International Protein Index) annotated protein N-termini and 81 contain IPI unannotated protein N-termini. Most of the identified modifications are acetylations with only a few formylations and propionylations present. Furthermore, Lys-N digestion was also applied and resulted in the identification of 394 in vivo N^α-modified peptides, of which 371 contain IPI annotated protein N-termini and 23 contain IPI unannotated protein N-termini. Combination of the two datasets leads to the identification of 675 N^α-modified IPI annotated protein N-termini and 88 N^α-modified IPI unannotated protein N-termini. Our results suggest that N-terminal acetyltransferases (NATs) may function as N-terminal formyltransferases (NFTs) and N-terminal propionyltransferases (NPTs) in vivo.