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The effect of peptide length on the cleavage kinetics of 2-chlorotrityl resin-bound ethers
Abstract
Different characteristics of cleavage kinetics of resin-bound amino alcohols and their peptide derivatives were observed in acid containing protic and aprotic solvent mixtures. The hydrolysis reactions are hindered by steric crowding around the cleaving C--O bond and accelerated by the special solvation effect of CF(3)CH(2)OH on the peptide chain as well as the increase of the strength and concentration of the acid. In trifluoroacetic acid containing mixtures, trifluoroacetylation of the peptide alcohols was detected. The appearance of O-trifluoroacetyl serine and threonine derivatives is detected in cleavage mixtures containing trifluoroacetic acid in anhydrous solvent.
... This approach would effectively counteract the significant hydrophobic nature of palmitoylated products and address the tendency toward cleavage-specific secondary reactions similar to the likely O-trifluoroacetylation of serine/threonine that we observed in peptides with an unmodified N-terminal amine. 44 Solubility. Visual assessment of the solubility at 1 mg/mL in some common solvents for peptides was carried out. ...
Peptide-based cancer vaccines have shown promising results in preclinical trials focusing on tumor immunotherapy. However, the presence of hydrophobic amino acid segments within these peptide sequences poses challenges in their synthesis, purification, and solubility, thereby hindering their potential use as cancer vaccines. In this study, we successfully synthesized peptide sequences derived from mesothelin (MSLN), a tumor-associated antigen overexpressed in pancreatic ductal adenocarcinoma (PDAC) by conjugating them with monodisperse polyethylene glycol (PEG). By PEGylating mesothelin epitopes of varying lengths (ranging from 9 to 38 amino acids) and hydrophobicity (60–90%), we achieved an effective method to improve the peptide yield and facilitate the processes of synthesis and purification. PEGylation significantly enhanced the solubility, facilitating the single-step purification of lengthy hydrophobic peptides. Most importantly, PEGylation did not compromise cell viability and had little to no effect on the immunogenicity of the peptides. In contrast, the addition of a palmitoyl group to increase immunogenicity led to reduced yield and solubility. Overall, PEGylation proves to be an effective technique for enhancing the solubility and broadening the range of utility of diverse long hydrophobic peptides.
... All washings and coupling steps were performed in dimethylsulfoxide/ethyl acetate 1:9 [40]. Peptides were released from the resin by acidic treatment [52]. The procedure must be repeated at least three times to obtain complete peptide release, and then additional treatment with HCl 3M in methanol is needed to remove the tertButyloxycarbonyl (Boc) side-chain protection (either on Lys or Api). ...
Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure–activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity.
... The syntheses of precursors N 3 -RibAFU(ip)-OH (1b) and N 3 -XylAFU(ip)-OH (2b) was completed using partly the well-known general pathways starting from d-glucose (Gruner et al. 2002b;Chandrasekhar et al. 2004). The 2-chlorotrityl chloride (2-Cl-Trt) resin was used as it offers the advantage of easy ligation of the first amino acid with ester bond and then after the final coupling the easy cleavage of the polypeptide under mild acidic conditions (Barlos et al. 1991;Kocsis et al. 2006;García-Martín et al. 2007). Determination of theoretical capacity and coupling efficiency of the resin was completed by elementary analysis (N %) and measurement of Fmoc capacity (Table 1). ...
We report the solid phase synthesis of -GG-X-GG- type α/β-carbopeptoids incorporating RibAFU(ip) (1a, tX) or XylAFU(ip) (2a, cX) sugar amino acids. Though coupling efficacy is moderate, both the lengthier synthetic route using Fmoc derivative (e.g., Fmoc-RibAFU(ip)-OH) and the azido derivative (e.g., N3-RibAFU(ip)-OH) via Staudinger reaction with nBu3P can be successfully applied. Both X-ray diffraction, (1)H- and (31)P-NMR, and theoretical (QM) data support and explain why the application of Ph3P as Staudinger reagent is "ineffective" in the case of a cis stereoisomer, if cX is attached to the preceding residue with a peptide (-CONH-) bond. The failure of the polypeptide chain elongation with N3-cX originates from the "coincidence" of a steric crowdedness and an electronic effect disabling the mandatory nucleophilic attack during the hydrolysis of a quasi penta-coordinated triphenylphosphinimine. Nevertheless, the synthesis of the above α/β-chimera peptides as completed now by a new pathway via 1,2-O-isopropylidene-3-azido-3-deoxy-ribo- and -xylo-furanuronic acid (H-RibAFU(ip)-OH 1a and H-XylAFU(ip)-OH 2a) coupled with N-protected α-amino acids on solid phase could serve as useful examples and starting points of further synthetic efforts.
... The syntheses were performed on the L-Lol substituted 2-chlorotrityl resin 27-29 as described in detail in the Methods section. Our procedure involves the use of the strong activating method via HATU or EDC/HOAt in the coupling reactions with Aib and TOAC residues, and cleavage from the 2-chlorotrityl resin 29 in three cycles of 30% HFIP in distilled CH 2 Cl 2 (the last cycle performed overnight). When Lys or Arg are in the sequence, a removal protocol with strong acid (TFA or HCl) was required. ...
Peptaibols are peculiar peptides produced by fungi as weapons against other microorganisms. Previous studies showed that peptaibols are promising peptide-based drugs because they act against cell membranes rather than a specific target, thus lowering the possibility of the onset of multi-drug resistance, and they possess non-coded α-amino acid residues that confer proteolytic resistance. Trichogin GA IV (TG) is a short peptaibol displaying antimicrobial and cytotoxic activity. In the present work, we studied thirteen TG analogues, adopting a multidisciplinary approach. We showed that the cytotoxicity is tuneable by single amino-acids substitutions. Many analogues maintain the same level of non-selective cytotoxicity of TG and three analogues are completely non-toxic. Two promising lead compounds, characterized by the introduction of a positively charged unnatural amino-acid in the hydrophobic face of the helix, selectively kill T67 cancer cells without affecting healthy cells. To explain the determinants of the cytotoxicity, we investigated the structural parameters of the peptides, their cell-binding properties, cell localization, and dynamics in the membrane, as well as the cell membrane composition. We show that, while cytotoxicity is governed by the fine balance between the amphipathicity and hydrophobicity, the selectivity depends also on the expression of negatively charged phospholipids on the cell surface.
... Da) in several peptide fragments. It is reported to be a prominent side product of Fmoc synthesis, formed during cleavage and global side chain deprotection in TFA. [31] The levels of this side product were highly variable, even amongst almost identical syntheses, and we were unable to eliminate it by incubating the peptide at pH 10 overnight. The impurity elutes very late during RP-HPLC and is easily removed during purification. ...
We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.
Amino group represents the most versatile nucleophile in peptide synthesis. Its acylation reaction is the underlying principle of the amino acid coupling and peptide chain elongation. Due to the high reactivity amino group could undertake a plethora of side reactions in the process of peptide synthesis. The most common side reactions affecting amino group include acetylation, trifluoroacetylation, formylation, and alkylation. These side reactions could take place at the steps of Nα-deprotection, amino acid coupling, side chain global deprotection, Nα-protection, and even chromatographic purification.
We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.
We prepared by solid-phase methods, chromatographically purified, and characterized three analogs of the ten-amino acid-residue, membrane-active, lipopeptaibiotic trichogin GA IV, each containing a single (4-fluorophenyl)alanine in position 3, 7, or 10, where it replaces the hydrophobic residue Leu(3) , Leu(7) , or Ile(10) , respectively. We incorporated the fluorine probe based on the observation that the (19) F-NMR technique has been extensively utilized to analyze peptidemembrane interactions in biological systems. A detailed conformational investigation in solution, including a membrane-mimetic environment, was performed on these compounds using FT-IR absorption, CD, and 2D-NMR, combined with molecular-dynamics calculations. The experimentally observed, mixed 310 /α-helical structures unequivocally show that the principal conformational features of trichogin GA IV are preserved in all three analogs. Analogies and differences between the behavior of the natural lipopeptaibiotic and those of the peptides characterized by the side-chain monofluorinated aromatic amino acid were found in membrane-permeabilization experiments and antimicrobial assays. The results of a preliminary solution (19) F-NMR study support the view that the (19) F label is an excellent reporter for changes in the helical environment of the peptide.
Unwanted trifluoroacetylation occurred at the N-terminus of prolinyl peptides during detachment from the solid phase. This was observed when the N-alpha-Fmoc protecting group had been removed prior to the final TFA treatment. Subtly changing the SPPS protocol and incorporating Boc-in place of the Fmoc-protected proline as the N-terminal building block efficiently suppressed this side reaction. (c) 2012 Elsevier Ltd. All rights reserved.
A set of three analogs of the 10-residue, membrane-active lipopeptaibiotic trichogin GA IV, labeled with the promising 4-nitrophenylalanine IR absorption probe for local polarity, was synthesized by the solid-phase methodology, chromatographically purified, and extensively characterized. A single residue modification was inserted near the N-terminus, in the central region, or at the C-terminus. A solution conformational analysis, carried out by FT-IR absorption, CD, and 2D-NMR combined with molecular dynamics calculations, indicates that the mono-labeled analogs maintain the overall helical properties of the parent compound. Membrane permeabilization measurements and antimicrobial tests revealed that they possess membrane-modifying properties and in vitro antibacterial activities analogous to those of the natural lipopeptaibiotic. Our IR absorption and attenuated total reflectance investigations in various environments, from which water was excluded for solubility reasons, showed that the 1350 cm(-1) 4-nitrobenzyl band is a reporter group of rather limited sensitivity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.
The Wang resin is one of the most commonly used solid polymers in SPPS. An Fmoc strategy for the lengthening of the peptide chain is compatible with the final acid cleavage of the peptide-resin bond. To increase tactic possibilities, it would be interesting to be able to use the Boc group on this resin type. Classical acid conditions (TFA 30 to 90% in DCM) for Boc removal cleave the peptide from the Wang resin simultaneously. Therefore, we investigated new conditions for deprotecting the Boc group which would preserve the peptide-resin bond.
Isocyanates 7 were formed from monoprotected diamines 3 or 6, which in turn can be easily prepared from commercially available N-BOC- or N-FMOC-protected amino acid derivatives. Isocyanates 7, formed in situ, could be coupled directly to a solid support functionalized with amine groups or to amino acids anchored on resins using CH2Cl2 as solvent and an 11 h coupling time at 25 °C. Such couplings afforded peptidomimetics with an N-phthaloyl group at the N-terminus. The optimal conditions identified for removal of the N-phthaloyl group were to use 60% hydrazine in DMF for 1−3 h. Several sequences of amino acids coupled to ureas (“peptidic ureas”) and of sequential urea units (“oligoureas”) were prepared via solid phase syntheses and isolated by HPLC. Partition coefficients were measured for two of these peptidomimetics, and their water solubilities were found to be similar to the corresponding peptides. A small library of 160 analogues of the YGGFL-amide sequence was prepared via Houghten's tea bag methodology. This library was tested for binding to the anti-β-endorphin monoclonal antibody. Overall, this paper describes methodology for solid phase syntheses of oligourea derivatives with side chains corresponding to some of the protein amino acids. The chemistry involved is ideal for high-throughput syntheses and screening operations. The products can be expected to have an interesting range of pharmacological properties and enhanced proteolytic stabilities relative to the corresponding peptides.
The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr.
The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the mu-opioid receptor selective agonist synthetic peptide (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at mu-opioid receptors in both organs. The -ol derivatives were slightly (2.3-4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the mu-opioid receptor pool in mouse vas deferens by 5x10(-7) M beta-funaltrexamine. The calculated receptor constants indicated a "high-affinity, low intrinsic efficacy" profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.
The scope and limitations of reagents for the cleavage of ethers are discussed. The reagents are conveniently classified in the five main headings. Selectivity patterns of some of the reagents are discussed in cases where sufficient data has been given in the literature. 1. Introduction 2. Acidic Reagents 2.1. Bronsted Acids 2.2. Lewis Acids 3. Basic Reagents 3.1. Alkali Hydroxides 3.2. Alkali Alkoxides 3.3. Alkali Amides 3.4. Alkali Metals 3.5. Organo-Alkali Metal Compounds 3.6. Sodium Cyanide/Dimethyl Sulfoxide 3.7. Sodium Ethanethiolate 3.8. Sodium Thiocresolate 3.9. Lithium Iodide 3.10. Sodium Benzeneselenolate 4. Miscellaneous Reagents 4.1. Iodotrimethylsilane 4.2. Iodotrichlorosilane 4.3. Dichloroiodomethylsilane 4.4. Bromotrimethylsilane 4.5. Alkylthiotrimethylsilanes 4.6. Ethanethiol or Ethanedithiol/Boron Trifluoride Etherate 4.7. Aluminium Halide/Thiol Systems 4.8. Acetyl Iodide and Pivaloyl Iodide 4.9. Diiodomethyl Ether/Hydrogen Iodide 5. Reductive Cleavage of Ethers 5.1. Lithium Tris[t-butoxy]aluminium Hydride/Triethylborane Complex 5.2. Hydrogenolysis 6. Oxidative Cleavage of Ethers 6.1. Ceric Ammonium Nitrate 6.2. Silver Oxide 6.3. Dichlorodicyanoquinone 6.4. Tris[p-bromophenyl]ammonium Hexachloroantimonate 7. Photochemical Cleavage of Ethers 8. Selectivity in Ether Cleavage 8.1. Stereoelectronic Characteristics of the Ether-Cleaving Agent 8.2. Structural Features of the Groups Cleaved 8.3. Molecular Environment of the C-O Bond not Undergoing Cleavage 9. Addendum
The synthesis of a new polymeric support for the preparation of fully protected peptide fragments by the Fmoc/t-butyl method is described. Data for coupling yields and racemization data are given. Cleavage from the resin is performed by very mild acidolysis.1
Total synthesis of novel DMT-phosphoramidites of thymidine (11 and 15) and 2′-deoxyguanosine (8 and 20) have been accomplished. The utility of these modified building blocks in the preparation of triple helix forming oligodeoxyribonucleotides with a stretched phosphodiester backbone has been evaluated. It was found that the oligonucleotides with extended backbones were unable to enhance the binding to duplex targets containing CG or TA base pairs.
N-Protected α-amino alcohols were prepared by reduction of N-protected α-amino acid esters by sodium acetoxyborohydride in dioxane at elevated temperature. The reductions proceed with excellent yields and without racemisation. Reduction of the carbamate protecting groups was not observed
In this new convergent peptide synthetic approach the advantages of the Fmoc and Boc techniques are combined. Boc-protected peptide fragments prepared on 2-chlorotrityl resin by Fmoc-technique were coupled sequentially in solution. As an example, the C-terminal (91–131) part of the agouti protein comprising all the 5 disulfide bridges in the original molecule has been synthesized by this method.
Application of 4-Polystyryltriphenylmethyl Chloride to the Syntheses of Peptides and Amino Acid Derivatives
Use of polymer-bound trityl chloride as an α-amino protecting group of amino acids allows simple and high-yield syntheses of amino acid derivatives and peptides.
A repetitive solid-phase method for the synthesis of polyamines is described. Primary amino groups attached to a crosslinked polystyrene resin are monoalkylated by acid labile, benzhydryl-based alkyl chlorides. Reductive alkylation of the resulting secondary amino group by Fmoc-protected aminoaldehydes gives a N-benzhydryl polyamine backbone. Treatment of the resin with trifluoroacetic acid cleaves both the benzhydryl protective group and the polyamine derivative from the resin. By using benzhydryl protective groups with different acid stability, unbranched, branched and partly branched polyamines are synthesized.
Aminothiols were attached through their thiol group onto the 4,4%-dimethoxytrityl (Dmt)-, 4-methoxytrityl (Mmt)-, 4-methyltrityl (Mtt)-, trityl (Trt)-and 2-chlorotrityl (Clt)-resins. The new resins were used in the solid-phase synthesis of aminothiol containing peptides utilizing N-Fmoc amino acids. The synthesized peptides were cleaved from the resins by treatment with trifluoroacetic acid (TFA) solutions using triethylsilane (TES) or ethanedithiol (EDT) as scavengers. © 2002 Published by Elsevier Science Ltd. Aminothiols are constituents of important biologically active compounds such as coenzyme A. In addition, they are contained in the antihypertensives 1, 1 the wide range cytoprotectant amifostine (ethyol, 2) 2–4 and the antitumor activity revealing Ras protein farnesyltrans-ferase inhibitors, such as 3. 5,6 For the development of new lead structures using solid-phase combinatorial methods, suitable resin-bound aminothiol derivatives are required. For their synthesis we applied commer-cially available resins of the trityl-type. 7–9 In fact, the reaction of the trityl resins 4 (polystyrene based polymers, 100–200 and 200–400 mesh, crosslinked with 1% divinylbenzene and loaded as fol-lows: 4a=1.63; 4b=1.92; 4c=1.48; 4d=1.81; 4e=1.74 mmol Cl − /g resin) with a two-fold molar excess of the linear aminothiols 5 or aminothiols derived from natu-rally occurring amino acids 7 10 (Scheme 1) and
N-Benzoyl-2′-deoxyformycin A (5) was prepared from formycin A and incorporated into the triple helix forming oligonucleotide PRE2ap at CG inversion sites. The modified oligonucleotide containing three substitutions of 2′-deoxyformycin A displayed a 10-fold increase in binding affinity as compared to its unmodified counterpart. This provided a method to accommodate CG inversion sites within target sites for antiparallel triple helix formation.
The standard methods of stepwise solid phase synthesis according to Merrifield could not previously be applied to the synthesis of the important naturally occurring peptaibols because of difficulties arising from the pronounced steric hindrance caused by alpha,alpha-dialkylated amino acids (incomplete coupling, especially to adjacent similarly constituted units, racemization due to slow coupling to hindered amino acids, etc.), chain degradation due to the presence of acid-labile Aib-Pro linkages, and the lack of any general method for the loading of C-terminal amino alcohols to resin supports. Following recent work on model systems, it is now shown that the adoption of Fmoc amino acid fluorides as coupling reagents makes possible the facile, general assembly of such peptides. The method was demonstrated for alamethicin F30 and F50, saturnisporin SA III, and trichotoxin A50-J. The crude products were of remarkable purity. Amino acid analysis, mass spectral data, and comparison of the synthetic alamethicins with samples of naturally occurring material confirmed the success of the syntheses. No significant amount of racemization (<0.8%) was found for any of the chiral amino acids present. The first step of the synthesis involved a new general method for assembly of C-terminal peptide alcohols via the use of 0-chlorotrityl resin. In addition, model studies on the question of racemization during the coupling of Fmoc amino acid fluorides are reported.
The success of solid-phase peptide synthesis is highly dependent on the accessibility of the growing resin-bound peptide chain to reagents. To maximize this accessibility, the relationship between solvent properties and peptide-resin solvation has been explored. The tridecapeptide [Lys9]-alpha-conotoxin G I (from Conus geographus), which contains nine side-chain-protected residues, was used to study solvation effects of protected peptide-resins. Efficient solvation of (aminomethyl)copoly(styrene-1% DVB) and peptide-copoly(styrene-1% DVB) could be directly correlated to solvent Hildebrand and hydrogen-bonding solubility parameters (delta and delta-h, respectively). Solvation was also highly dependent on the side-chain protecting group strategy (benzyl (Bzl), tert-butyl (tBu), or p-methoxybenzyl (Mob)) utilized. The most efficient solvation by a single solvent occurred with NMP, regardless of side-chain protection, although the relative solvation is greater for the Bzl-based versus tBu-based side-chain-protected conotoxin-resin. Mixed-solvent systems with optimized delta and delta-h, values, such as 45% THF/NMP and 20% TFE/DCM, offered greater solvation than single solvents for the Bzl and tBu side-chain-protected conotoxin-resins. Solvation results for Mob side-chain-protected conotoxin-resin suggested that replacement of the tBu side-chain protecting group by the Mob group improves solvation by single solvents, such as NMP, while still providing the weak acid lability desired for side-chain deprotection following solid-phase peptide synthesis utilizing Fmoc chemistry.
The synthesis of fully protected peptide fragments by the Fmoc/ t-butyl-method on a new polymeric support is described. The fragments are obtained in good yields and high purity upon cleavage from the resin with 0.5 - 1 % trifluoroacetic acid in methylene chloride.
To establish a framework for extrapolating the helix-forming properties of peptides from TFE/H2O mixtures (TFE = 2,2, 2-trifluoroethanol) back to water, the thermal unfolding curves have been measured by circular dichroism for four repeating-sequence peptides, with chain lengths from 7 to 22 residues. The unfolding curves were measured between 0 and 50 volume percent TFE and were fitted to the modified Lifson-Roig theory. A single set of helix-coil parameters fits the results for the four peptides at each TFE concentration; only two of the basic helix-coil parameters, , the mean helix propagation parameter of residues in the sequence repeat, and DeltaH, the enthalpy change per residue on unfolding the helix, are allowed to vary with TFE molarity. The success in fitting these curves over a wide range of experimental variables shows that helix formation is basically the same in TFE/H2O mixtures as in water. Moreover, a simple model based on a linear dependence of ln and DeltaH on TFE molarity can be used to extrapolate the results from 25% TFE (approximately 4 M) back to water. The results also give curves of helix formation induced by TFE at constant temperature, and the properties of these helix induction curves explain some of the puzzling results shown by other peptides in the literature. The average helix propensity increases regularly from 0 to 25% TFE but levels off at higher TFE concentrations, which explains why the extent of helix formation levels off in this range. The change in the apparent cooperativity of thermal unfolding curves in concentrated TFE solutions results from the decrease of the enthalpy change for helix unfolding at higher TFE concentrations. The rapid decrease in the plateau values of apparent helix content with increasing temperature results mainly from the strong temperature dependence of the ellipticity of the complete helix. To determine whether the helix-stabilizing effect of TFE arises from strengthening the hydrogen bonds in the helix backbone, the strength of the hydrogen bond in a model compound, salicylic acid, has been measured in TFE/H2O mixtures from the pKa difference between salicylic acid and a similar compound which cannot form the hydrogen bond. The curve of hydrogen bond strength versus increasing TFE concentration matches both in shape and magnitude the increase in average helix propensity in TFE/H2O mixtures.
The kinetics of cleavage reactions of 16 resin-bound carbamates, ureas, secondary amides, and sulfonamides from four different acid labile linkers including benzyl, benzhydryl, and indole linkers has been investigated. The optimized cleavage conditions are generally milder than those commonly used and reported (e.g., 0.5% TFA as opposed to 5%). Among various linkers studied in this work, the indole linker has been found to be the most acid labile followed by the Rink linker. The rate of cleavage of compounds linked to the resin via various functional groups can be summarized as follows: sulfonamide >carbamate approximately urea > amide. This study shows that cleavages of 16 compounds from four different acid labile linkers have been optimized to much milder conditions in terms of TFA concentration and the reaction time. It also demonstrates that single bead FTIR is an effective tool for optimizing cleavage conditions.
In order to achieve an EPR sensitive probe for DNA, 3-carboxy-Proxyl free radical was linked to O-6 of dG through a five-atoms-tether. The modified base was incorporated into a 30-mer ODN, then annealed to its complementary DNA strand. Hydrodynamic parameters show only a slight destabilization with respect to the equivalent unlabeled hybrid. EPR could monitor the hybrid formation showing a progressive enlargement of the upfield signal in passing from the labeled ss- to the ds-30-mer.
A solid-phase-Fmoc-based-synthesis strategy is described for oligourea peptidomimetics as well as a convenient general synthesis approach for the preparation of the required building blocks 5a-j and 5k. These are suitable for use in peptide or robot synthesizers, which is illustrated by the synthesis of oligourea peptidomimetics of part of Leu-enkephalin (10) and a neurotensin derivative (17).
In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra- and pentapeptides selective for the kappa-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [(35)S]GTPgammaS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K(e)) of 2.44 nM, and no agonist effect at 10 microM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the delta-opioid receptor agonist [D-Ala(2),D-Leu(5)]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [(3)H]nociceptin-NH(2) binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [(35)S]GTPgammaS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N- and the C-terminal region of the molecule.
Structure and Mechanism in Organic Chemistry
- Ck Ingold
Ingold CK. Structure and Mechanism in Organic Chemistry. Cornel University Press: New York, 1953.