Fragment Condensation Approach Research Articles

Determining the Hydrophobicity Index of Protected Amino Acids and Common Protecting Groups

Peptides are in great demand in the pharmaceutical arena and a majority of these peptides contain 20 or more amino acids. They are infrequently synthesised using the fragment condensation approach. A key limitation in adopting this approach more commonly is that protected peptide fragments with high purity are often required prior to the final condensation steps. It is hypothesized that understanding the hydrophobic nature of the protected amino acids will assist with designing optimal fragment purification processes when needed. Whilst a myriad of hydrophobicity indices are reported in the literature for unprotected amino acids, the literature lacks any data regarding the protected amino acids which form the key precursor for the fragment condensation task. In this current study, hydrophobicity indices for protected amino acids with common α-amino and sidechain protecting groups were experimentally determined. Different positions for each amino acid within the peptide chain were considered, namely at the C-terminal and N-terminal as well as internal positions. These data give deep insights on the hydrophobicity of each amino acid with respect to its position in the peptide chain. The data acquired in this research facilitated the prediction of the retention time of protected peptide fragments with an uncertainty of less than ±1.5%.

Successful synthesis of a glial-specific blood-brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin.

Successful manual synthesis of the TD2.2 peptide acting as a blood-brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.

Synthesis of New Galanthamine-Peptide Derivatives Designed for Prevention and Treatment of Alzheimer's Disease.

Although no effective treatment for the Alzheimer's disease currently exist, some drugs acting as Acetylcholinesterase inhibitors, like galanthamine have positively affected such patients. β- and/or γ-secretase inhibitors are another type of potential drugs. Here we report synthesis of new peptide-galanthamine derivatives, with expected inhibitory activity against both Acetylcholinesterase and β-secretase. The aim of this work is obtaining new peptide derivatives of galanthamine with decreased toxicity compared to galanthamine. Syntheses were conducted in solution using fragment condensation approach. The new derivatives were characterized by melting points, angle of optical rotation, NMR and Mass spectra. Acute toxicity was determined on mice, according to a Standard protocol. All new compounds were tested in vitro for cytotoxic activity in a panel of human (HEP-G2, BV-173) and murine (Neuro-2a) tumor cell lines via a standard MTT-based colorimetric method. New derivatives of galanthamine containing shortened analogues of β-secretase inhibitor (Boc- Asn-Leu-Ala-Val-OH) and either nicotinic or isonicotinic residue, both connected with a linker (L-Asp) to position 11 of galanthamine were obtained. In vivo toxicity of some new compounds was found up to 1000 mg/kg. Cell toxicity screening against the tumor cell lines showed negligible growth-inhibiting properties of the galanthamine derivatives. Synthesis of new galanthamine derivatives comprising peptide moiety and nicotinic acid or isonicotinic acid is reported. Acute toxicity studies reveal they are about 100 times less toxic than galanthamine. This effect is due to the peptide fragment. Cytotoxicity studies show good correlation with low toxicity results. These results are encouraging for the application of this class compounds as medicines.

Click to Join Peptides/Proteins Together

Copper(I) is able to catalyze Huisgen 1,3-dipolar cycloaddition in a "click" fashion. This copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction presents excellent chemoselectivity and occurs over a wide-range of reaction conditions. It shows tolerance to variation in both pH and solvent polarity, thereby facilitating the ligation of peptides and proteins to produce peptidomimetics and synthetic proteins. In addition, the only product formed is a 1,4-disubstituted-1,2,3-triazole moiety, in many aspects resembling the natural peptide bond, including hydrogen-bonding capability, planarity, distance between the 1 and 4 substituents, and conformational restriction of the peptide backbone; thus the triazole-backbone-modified peptide, in which a triazole replaces the amide bond, may be anticipated to present a secondary structure similar to that of its natural counterpart. This Focus Review describes the scope and applications of copper(I)-catalyzed alkyne–azide cycloaddition in synthetic peptide/protein chemistry.

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach.

Characterization of the 310-helix in model peptides by HRMAS NMR spectroscopy.

A tetra- and a hepta-homopeptide from the C(alpha)-tetrasubstituted Aib (alpha-aminoisobutyric acid) residue were covalently linked to the POEPOP resin by the fragment-condensation approach. The conformational preferences of the two model peptides were determined for the first time on a solid support by means of high-resolution magic angle spinning NMR spectroscopy. The results obtained indicate that the Aib homopeptides adopt a regular 3(10)-helical structure even when they are covalently bound to a polymeric matrix, and thus confirm the remarkable conformational stability of the peptides rich in this amino acid. An ATR-FTIR spectroscopic investigation, performed in parallel, also confirmed that these polymer-bound peptides do indeed adopt a helical conformation. The results of this study open the possibility to exploit the peptide-resin conjugates based on C(alpha)-tetrasubstituted alpha-amino acids as helpful, structurally organized templates in molecular recognition studies or as catalysts in asymmetric synthesis.

Synthesis of the15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Synthesis of zervamicin IIB, specifically labeled at the α-position of glutamine-11 with15N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF3-PyBOP. Using the strategy developed, zervamicin IIB specifically15N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the15N-label was clearly detected. The isotope enrichment (98 ±2%) was determined by FAB-mass spectrometry.

Total synthesis of zervamicin IIB and its deuterium-labelled analogues.

For the first time the total synthesis of the peptaibol zervamicin IIB is described. Synthesis of this peptaibol was achieved by the Fmoc/tert-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered alpha-aminoisobutyric acid residues BOP/DMAP activation was applied. The fmoc group was removed by reaction with 0.1 M NaOH in dioxane/methanol/water (30/9/1, v/v/v). Peptide fragments were coupled by means of a new coupling reagent, CF3-PyBOP. Using the strategy developed, zervamicin IIB and two analogues specifically deuterium-labelled at different positions of the glutamine-11 residue have been synthesized in 40% overall yield based on the isotopically labelled amino acid and with 98 +/- 2% of isotope enrichment. FAB mass spectroscopy, 600 MHz 1H-NMR spectroscopy and high-performance liquid chromatography provided convincing evidence that the synthetic products, zervamicin IIB and its deuterium-labelled analogues, fully correspond to the naturally occurring zervamicin IIB.

The Use of Crown Ethers in Peptide Chemistry – V. Solid‐phase Synthesis of Peptides by the Fragment Condensation Approach using Crown Ethers as Non‐covalent Protecting Groups

The Use of Crown Ethers in Peptide Chemistry – V. Solid‐phase Synthesis of Peptides by the Fragment Condensation Approach using Crown Ethers as Non‐covalent Protecting Groups

The use of crown ethers in peptide chemistry-V. Solid-phase synthesis of peptides by the fragment condensation approach using crown ethers as non-covalent protecting groups.

We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N alpha-amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N alpha-complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantitative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly52 to Leu72 (21-mer) and Pro33 to Leu72 (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product has been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.

The use of crown ethers in peptide chemistry IV. Solid phase synthesis of peptides using peptide fragments Nα protected with 18-crown-6

The conditions under which peptide synthesis by the fragment condensation approach in the solid phase can be carried out using crown ethers as non-covalent protecting groups for the Nα amino group of peptides were determined. The dipeptide Gly-Gly was complexed with 18-crown-6 to establish the feasibilty of this new protection scheme and to optimise the reaction conditions. Nearly quantitative incorporation of the complex onto resin-bound amino acids possessing either proline or an Nα-alkylated amino acids was achieved using DCM as the activation and coupling reactions solvent. The use of DMF as the solvent and resin-bound primary amino acids were found detrimental to the reaction yields due to the removal of the crown ether protection. Extending the length of the peptide fragment to model pentapeptide complexes bearing no reactive functionalities on the side-chain gave essentially quantitative incorporation yields in the coupling reactions to both resin-bound amino acids and short peptides. The non-covalent nature of the protection afforded by the crown molecule allowed its mild removal from resin-bound complexes by rapid treatments with 1% DIEA solutions. Thus the continuation of chain assembly was possible. The results obtained served as the basis for extending the concept of non-covalent protection to the side chains of Lys and Arg containing peptides.

Conformational and Nucleic Acid Binding Studies on the Synthetic Nucleocapsid Protein of HIV-1

A 55 residue peptide corresponding to the nucleocapsid protein of HIV-1 (NCp7) containing two zinc binding domains as well as three truncated peptides were synthesized by Fmoc-based solid phase synthesis using the fragment condensation approach. Circular dichroism (CD) data support a conformational model in trifluoroethanol/buffer solution consisting of two helical segments at the chain ends with two Zn-modules in the center of the molecule. CD titration experiments show that the synthetic protein binds two equivalents of Zn 2+ stoichiometrically, and Zn 2+ induced conformational changes are completely reversible by addition of EDTA. NCp7 and its S-acetamidomethylated analog (NCp7-Acm), devoid of the zinc co-ordination centers, exhibit preferential binding to RNA with a K d =∼10 -9M irrespective of the cysteine modification as determined by filter binding assays. The binding affinity of the NCp7 protein to single-stranded DNA is lower than to RNA. Binding to double-stranded DNA is lower than to ssDNA. The NCp7-Acm protein exhibits reduced single-stranded DNA binding affinity compared to the unmodified protein. Nucleic acid binding analyses with the fragments of NCp7 protein suggest that two basic amino acid stretches are involved in RNA binding of the NCp7.

Depsipeptide analogues of elastin repeating sequences: Synthesis

Depsipeptide analogues of peptide sequences can help in elucidating the role of specific hydrogen bonds in determining the conformation in peptides. The repeating pentapeptide and hexapeptide sequences of elastin have been suggested to contain a type II beta-turn with a 4----1 hydrogen bond. Depsipeptide analogues of the repeating sequences of elastin in which this 4----1 hydrogen bond cannot exist were synthesized. A fragment condensation approach was employed in which the depsipeptide ester bond was introduced early in the synthesis. This approach proved to be effective, although the increased lability of the depsipeptide ester bond resulted in side products and low yields in some reactions.

ChemInform Abstract: SYNTHESIS OF FRAGMENTS OF ARGININE VASOPRESSIN AND OXYTOCIN CONTAINING A CYSTINE RESIDUE IN POSITION 6

AbstractDie Synthese der Titelverbindungen (VI), (VII), (XIa), (Xlc) (Arginin‐Vasopressin‐Fragmente) bzw. (IX), (XIb), (Xld) (Oxytocin‐Frag‐ mente) wird beschrieben.

Synthesis of fragments of arginine vasopressin and oxytocin containing a cystine residue in position 6

AbstractThe synthesis is described of several fragments of the neurohypophyseal nonapeptide hormones arginine vasopressin and oxytocin. One group of fragments is characterized by the presence of an asymmetrical disulfide (a cystine residue in position 6), a second group consists of dimeric (symmetrical disulfides) fragments.As common intermediates, peptides containing an S‐tritylcysteine residue were synthesized by the fragment condensation approach. Treatment with methoxycarbonylsulfenyl chloride followed by reaction with the free thiol function of cysteine gave the asymmetrical disulfides, while treatment of the S‐tritylcysteine containing peptide with iodine resulted in the corresponding dimers. Peptides with an N‐terminal glutamine residue (position 4) were found to convert spontaneously into the corresponding pyroglutamic acid products.

The synthesis of precursors of labelled neuropeptides

AbstractModified amino acids which can serve as precursors in the synthesis of tritium‐labelled peptides were prepared and incorporated into biologically active peptides. Thus, p‐iodophenylalanine was incorporated at position 4 of H‐Met(O2)‐Glu‐His‐Phe‐D‐Lys‐Phe‐OH (Org 2766) and in des‐Tyr1‐γ‐endorphin. As a second labelling site, the precursor of L‐leucine, L‐methallylglycine, was incorporated in both des‐Tyr1‐γ‐endorphin and the shorter des‐enkephalin‐γ‐endorphin. Finally, the lysine residue in Org 2766 (D‐Lys) and des‐enkephalin‐γ‐endorphin (L‐Lys) was replaced by its analogue 2,6‐diamino‐4‐hexynoic acid. The precursor peptides were synthesized via the fragment condensation approach. With the exception of the methallylglycine‐containing des‐Tyr1‐γ‐endorphin, in which partial desulfurization of the methionine residue could not be prevented, these peptides could be converted by means of catalytic reduction into their respective parent compounds.

Synthesis of fragments of human β‐lipotropin, βh‐LPH. Part VI. The synthesis of des‐1‐tyrosine‐α‐endorphin and shortened peptides

AbstractThe synthesis of des‐1‐tyrosine‐α‐endorphin and fourteen fragments of this psychostimulant type of neuropeptide is described. In these syntheses use has been made of the classical fragment condensation approach.

ChemInform Abstract: SYNTHESIS AND MELANOTROPIC ACTIVITY OF Γ‐MELANOTROPIN

AbstractDas Titelpeptid (IIIb), eine weitere (dritte) melanotrope Sequenz im N‐terminalen Teil der ACTH‐ bzw. β‐LPH‐ Vorstufe Pro‐opiocortin, wird durch Kondensation der nach konventionellen Methoden aufgebauten Fragmente (I) und (II) [Azidmethode; ‐ (IIIa)] und nachfolgende Schutzgruppenabspaltung synthetisiert.

Synthesis and melanotropic activity of γ‐melanotropin

Abstractγ‐MSH, a third melanotropic sequence in the N‐terminal cryptic portion of the ACTH/β‐LPH precursor molecule pro‐opiocortin, was synthesized by the fragment condensation approach. The dodecapeptide H‐Tyr‐Val‐Met‐Gly‐His‐Phe‐Arg‐Trp‐Asp‐Arg‐Phe‐Gly‐OH was tested for melanocyte‐stimulating activity on the skin of the lizard Anolis Carolinensis and was found to possess only 2.5 × 10−4 of the activity of α‐MSH.

Peptides—XXXXII: Synthesis of the 76–93 fragment of a lysozyme analogue

The synthesis of the (76–93) fragment of a lysozyme analogue was achieved using a fragment condensation approach employing the protected subfragments (76–79), (80–86), and (87–93). The utility of Bates' reagent in conjunction with N-hydroxysuccinimidc was examined for the linking of fragments. The resulting protected peptide (76–93) was found to be one of the most insoluble encountered in this whole programme directed towards the synthesis of a lysozyme analogue.