Synthesis of phosphorothioate oligonucleotide-peptide conjugates by solid phase fragment condensation

Phosphorothioate oligonucleotide-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). Arginine rich peptides could be successfully conjugated in 2.8-13.4% isolated yields. All the products were fully characterized by reversed phase HPLC and MALDI-TOF-MS to give satisfactory results.     

Synthesis and purification of self‐assembling peptide‐oligonucleotide conjugates by solid‐phase peptide fragment condensation

Peptide‐oligonucleotide conjugates (POCs) are interesting molecules as they covalently combine 2 of the most important biomacromolecules. Sometimes, the synthesis of POCs involves unexpected difficulties; however, POCs with self‐assembling propensity are even harder to synthesize and purify. Here, we show that solid‐phase peptide fragment condensation combined with thiol‐maleimide or copper‐catalyzed azide‐alkyne cycloaddition click chemistries is useful for the syntheses of self‐assembling POCs. We describe guidelines for the selection of reactive functional groups and their placement during the conjugation reaction and consider the cost‐effectiveness of the reaction. Purification is another important challenge during the preparation of POCs. Our results show that polyacrylamide gel electrophoresis under denaturing conditions is most suitable to recover a high yield of self‐assembling POCs. This report provides the first comprehensive study of the preparation of self‐assembling POCs, which will lay a foundation for the development of elegant and sophisticated molecular assemblies.     

An alternative solid phase peptide fragment condensation protocol with improved efficiency.

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.     

Synthesis of a fully active snake venom cardiotoxin by fragment condensation on solid polymer.

A polypeptide cardiotoxin containing 60 amino acids with 4 disulfide bonds has been synthesized by the "fragment solid-phase" method. The identity of the synthetic product with native cardiotoxin was established by chromatography on Sephadex G-50, carboxymethyl-cellulose column chromatography, thin layer chromatography, disc gel electrophoresis, amino acid analysis, end group analysis, peptide mapping, circular dichroism spectra, and four biological tests.     

Synthesis of an open-chain asymmmetrical cystine peptide corresponding to the sequence A18-21--B19-26 of bovine insulin by solid phase fragment condensation

An insulin fragment containing residues A 18-21 and B 19-26 linked by the disulfide bond between residues A 20 and B 19 was synthesized. The sequence B 21-26 was assembled on a solid support by the Merrifield technique. The protected fragments A 18-21 and B 19-20 were prepared by conventional methods. After forming the disulfide bridge through cleavage of the S-thiocarbonate derivative of A 18-21 by the thiol peptide B 19-20, the resulting assymmetrical cystine peptide A 18-21--B 19-20 was coupled via the carboxyl group of residue B 20 to the free NH 2-terminal amino group of the protected B 21-26 resin. The product was deprotected, cleaved from the resin, and purified to give the homogenous dodecapeptide A 18-21--B 19-26.     

Synthesis of metallointercalator-DNA conjugates on a solid support

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.     

A novel approach for the solid phase synthesis of DNA-peptide conjugates

The strategy of this study involves automated synthesis of oligonucleotides on a CPG support using standard cyanoethyl phosphoramidite chemistry followed by covalent linkage to peptide fragments bearing a free terminal alpha-amino group and residues with protected side chains. Conjugation was formed through an alkyldiisocyanate linker. Conjugates were isolated by cleavage from the solid support and deprotection in one step.     

Direct Fmoc/tert-Bu solid phase synthesis of octamannosyl polylysine dendrimer-peptide conjugates

The mannose binding proteins on the surface of the dendritic cells are responsible for capture of pathogens in the early stages of immune response. Conjugation to mannose dendrimers is a rarely explored but potentially powerful strategy for enhancing immunogenicity of synthetic peptides relying on direct delivery to dendritic cells. We describe a general protocol for preparation of pure, monodisperse third-generation mannosylated poly-L-lysine dendrimer-peptide conjugates using direct, machine-assisted Fmoc/t-Bu solid phase peptide synthesis. The glycodendrons were elaborated onto the N- or C-terminus of sequences derived from HIV-1 gp41, SARS-CoV S2 protein, and Influenza Hemagglutinin (consisting of 15-44 residues). The products were obtained in a homogeneous state after cleavage from the resin, deprotection, and a single purification on semipreparative RP-HPLC.     

Synthesis and DNA binding ability of cyclam-amino acid conjugates

N-Substituted cyclam–amino acid conjugates have been synthesised both in solution and on the solid phase. The DNA binding affinity of these species has been studied: the nature of the amino acid strongly influences the change in melting temperature suggesting that simple cyclam–peptide conjugates could interact with DNA in a highly selective manner.     

Syhthesis of peptides by fragment condensation on a solid support. I. Application in preparation of bradykinin.

Two tripeptides and one dipeptide, protected except at the carboxyl ends, have been prepared in solution and used as intermediates in a new synthesis of bradykinin on a solid support. Condensation of the two tripeptides to the resin was effected in satisfactroy yield with dicyclohexylcarbodiimide plus N-hydroxysuccinimide. The partial epimerization that might occur by such an approach was explicitly verified to be without practical importance in this case. The crude product contained only traces of impurities and yielded, after purification, bradykinin of high purity. Both the crude and purified bradykinin exhibited full biological activity.     

DNA condensation by multivalent cations

In the presence of multivalent cations, high molecular weight DNA undergoes a dramatic condensation to a compact, usually highly ordered toroidal structure. This review begins with an overview of DNA condensation : condensing agents, morphology, kinetics, and reversibility, and the minimum size required to form orderly condensates. It then summarizes the statistical mechanics of the collapse of stiff polymers, which shows why DNA condensation is abrupt and why toroids are favored structures. Various ways to estimate or measure intermolecular forces in DNA condensation are discussed, all of them agreeing that the free energy change per base pair is very small, on the order of 1% of thermal energy. Experimental evidence is surveyed showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions. The various intermolecular forces whose interplay gives rise to DNA condensation are then reviewed. The entropy loss upon collapse of the expanded wormlike coil costs free energy, and stiffness sets limits on tight curvature. However, the dominant contributions seem to come from ions and water. Electrostatic repulsions must be overcome by high salt concentrations or by the correlated fluctuations of territorially bound multivalent cations. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Undulations of the DNA in its confined surroundings extend the range of the electrostatic forces. The condensing ions may also subtly modify the local structure of the double helix. © 1998 John Wiley & Sons, Inc. Biopoly 44: 269–282, 1997     

Synthesis of peptides by fragment condensation on a solid support. II. A scheme for preparation of 4,8-disubstituted vasopressins evaluated on 8-arginine-vasopressin.

A convenient scheme for the synthesis of 4,8-disubstituted vasopressins has been designed and its usefulness evaluated in the preparation of one of the parent hormones, 8-arginine-vasopressin. The main feature of the scheme involves preparation of two protected tripeptide fragments, Z-Cys(Bzl)-Tyr(Bzl)-Phe and Boc-Asn(Mbh)-Cys(Bzl)-Pro which are incorporated in a synthesis on a solid support. Both tripeptides were prepared conventionally with the carboxyl groups protected as benzyl esters. The benzyl-ester groups were removed by transesterification with 2-dimethylaminoethanol and subsequent hydrolysis. To avoid racemization in the coupling step with the fragment containing a C-terminal phenylalanine, N-hydroxysuccinimide was added. After removal of the peptide from the resin, deprotection, oxidation and desalting, final purification was effected by ion-exchange chromatography. Apart from the main product, which exhibited full pressor activity, only small amounts of impurities could be isolated.     

A solid phase method of determining DNA sequence

A solid-phase method for DNA sequencing has been developed which involves immobilization of the terminally labeled DNA fragment on the DEAE-paper followed by chemical modification and cleavage at G, A + G, C + T, and C sites. As compared to the Maxam and Gilbert method, the new technique is more rapid and less laborious, being of the same efficiency.     

Synthesis and characterization of covalently linked single-stranded DNA oligonucleotide-dendron conjugates

A solution-phase synthesis and characterization of covalent DNA-dendron conjugates is presented. Thiol-terminated 12-base oligonucleotides were added to second- and third-generation triazine-based dendrons via thiol/disulfide exchange chemistry. Single-stranded DNA oligonucleotides were successfully attached to dendrons at the core, the periphery, and both. Proof of structure for these architectures is derived primarily from mass spectrometry and polyacrylamide gel electrophoresis and complemented by labeling analysis using Ellman's reagent and degradation analysis using a reducing agent.     

Selective isolation of in vitro phase II conjugates using a lipophilic anionic exchange solid phase extraction method

Identification, characterization and structure elucidation of human metabolites of drug candidates is crucial for the pharmaceutical industry to assess their activity against the therapeutic target of interest and potential toxicological effects. It often requires in vitro synthesis of microgram quantities of metabolites of interest with enzymatic preparations, pre-concentration of the reaction mixture by solid phase extraction (SPE), metabolite isolation using HPLC systems coupled to fraction collectors prior to nuclear magnetic resonance characterization. The method reported herein is a rapid and simple technique using solely off-line mixed phase anionic exchange lipophilic SPE cartridges to selectively isolate glucuronide and sulfate metabolites from their parent compound. This approach capitalizes on the pKa differences between the parent compound, devoided of acidic moieties, and the negatively charged glucuronide and/or sulfate metabolites. Once loaded on the SPE cartridge, the incubation mixture is washed successively with a basic aqueous solution, methanol to elute the non-anionic parent compounds, and then with an acidic methanolic solution to protonate and recover the phase II conjugates. Over 100 microg (>95% purity) of 17 alpha-ethynylestradiol-3-glucuronide and 6-gingerol-4'-glucuronide were successfully isolated using this technique, as well as glucuronide and a sulfate conjugates of 1-{4'-[(1R)-2,2-difluoro-1-hydroxyethyl]biphenyl-4-yl}cyclopropanecarboxamide (DHBC) synthesized in-house. Their structures were confirmed by Ultra Performance Liquid Chromatography coupled to Quadrupole-Time of flight (UPLC-QTof) and nuclear magnetic resonance analysis.     

The enzymatic segment condensation of peptides on a solid phase in organic medium

The segment condensation of peptides on a solid phase (Aminosilochrom) in organic medium catalyzed by a subtilisin complex with sodium dodecylsulfate was studied. The dependence of the efficiency of the enzymatic coupling of tripeptides with the basic structure X-Ala-Ala-Y-OMe [where X = Z, Boc, or Dnp and Y = Leu or Glu(OMe)] on the spacer content on the support and on the structure of the acylating component was investigated. The tripeptide segments were successively coupled to Aminosilochrom containing the Met-Ala-Gly spacer, and the peptidylaminosilochroms Dnp-Ala-Ala-Leu-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A (A is the Aminosilochrom residue) were obtained in satisfactory yields. It was shown by these examples that the second and third segments are attached in yields higher than that for the first segment and the coupling efficiency does not depend on the amino acid composition of the acylating component.     

The semisynthesis of portions of hen''s-egg lysozyme by fragment condensation.

1. We have evaluated several methods for coupling protected tryptic peptides and have selected one that is satisfactory in terms of efficiency and degree of racemization. 2. We report the use of the chosen method (which must be slightly modified for each application in the light of the result of micro-scale pilot experiments) for the preparation of five fragments of hen's-egg lysozyme ranging in length from 31 to 51 residues. 3. We report methods for the complete deprotection of these fragments. They have been characterized by end-group determination, amino acid analysis and tryptic digestion.