A novel ring opening reaction of peptide N-terminal thiazolidine with 2,2′-dipyridyl disulfide (DPDS) efficient for protein chemical synthesis

In the protein chemical synthesis via native chemical ligation (NCL) method with three peptide segments, the N-terminal cysteine residue of middle segment is generally protected by thiazolidine ring. In this paper, we show the novel method for thiazolidine ring opening using 2,2′-dipyridyl disulfide (DPDS). The N-terminal thiazolidine was converted into S-pyridylsulfenylated cysteine residue with DPDS under acidic conditions, and this N-terminally Cys peptide protected with disulfide was applicable to NCL reaction without purification and deprotection steps. DPDS treatment did not remove other Cys protecting groups generally used for regioselective disulfide bond formation reactions. These results indicate that this thiazolidine ring opening reaction is quite useful for the protein chemical synthesis with three-segment NCL strategy.     

Efficient conjugation of peptides to oligonucleotides by "native ligation"

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.     

Preparation of a multitopic glycopeptide-oligonucleotide conjugate

[structure: see text] A novel strategy to prepare glycopeptide-oligonucleotide conjugates bearing a glycocluster is reported. The strategy utilizes a cyclodecapeptide scaffold as a key intermediate to anchor the carbohydrate cluster and the oligonucleotide through sequential oxime bond formation. The oligonucleotide glycocluster retains the binding affinity and recognition specificity for the target sequence. Furthermore, the conjugate shows enhanced binding to the specific lectins due to the cooperative effect produced by the carbohydrate cluster.     

Generation of oligonucleotide conjugates via one-pot diselenide-selenoester ligation–deselenization/alkylation

A breadth of strategies are needed to efficiently modify oligonucleotides with peptides or lipids to capitalize on their therapeutic and diagnostic potential, including the modulation of in vivo chemical stability and for applications in cell-targeting and cell-permeability. The chemical linkages typically used in peptide oligonucleotide conjugates (POCs) have limitations in terms of stability and/or ease of synthesis. Herein, we report an efficient method for POC synthesis using a diselenide-selenoester ligation (DSL)-deselenization strategy that rapidly generates a stable amide linkage between the two biomolecules. This conjugation strategy is underpinned by a novel selenide phosphoramidite building block that can be incorporated into an oligonucleotide by solid-phase synthesis to generate diselenide dimer molecules. These can be rapidly ligated with peptide selenoesters and, following in situ deselenization, lead to the efficient generation of POCs. The diselenide within the oligonucleotide also serves as a flexible functionalisation handle that can be leveraged for fluorescent labelling, as well as for alkylation to generate micelles.

An efficient and versatile approach for the late-stage generation of oligonucleotide conjugates by diselenide-selenoester ligation (DSL)–deselenization/alkylation was developed.     

New method to prepare peptide-oligonucleotide conjugates through glyoxylic oxime formation

A new method to prepare peptide-oligonucleotide conjugates through chemoselective glyoxylic oxime linkage is reported. A novel phosphoramidite reagent, readily accessible from serine, was prepared and used in automated DNA synthesis to prepare oligonucleotides carrying a glyoxylic aldehyde functionality at the 5' terminus. This was efficiently coupled to a peptide functionalized with an aminooxy group. The method could be of general use to prepare a broad range of oligonucleotide conjugates.     

New strategy for the synthesis of 3',5'-bifunctionalized oligonucleotide conjugates through sequential formation of chemoselective oxime bonds

A convenient strategy for the synthesis of bifunctionalized oligonucleotide conjugates bearing two different reporters at the 3' and 5' ends of the oligonucleotide is presented. The method involves the preparation of oligonucleotides bearing an aldehyde and/or aminooxy functionality at each end, followed by reaction to form oxime bonds with appropriately functionalized reporters. The conjugation reactions are carried out under mild aqueous conditions with good reaction yield.     

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (aminooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with liquid chromatography and tandem mass spectrometry has been used. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.     

Internalization of a peptide into multilamellar vesicles assisted by the formation of an alpha-oxo oxime bond

As part of a drug-delivery project, we designed and synthesised a novel hydroxylamine cholesterol-based anchor to ensure the chemoselective ligation of recognition patterns onto multilamellar vesicles by oxime ligation. The entry of a glyoxylyl peptide into the vesicles was unexpectedly assisted by the formation of the alpha-oxo oxime bond. We studied extensively the kinetic and thermodynamic aspects of this phenomenon. Briefly, for a glyoxylyl peptide, the speed and ability to enter the vesicle were dependent upon 1) the presence of a hydroxylamine anchor of the type CholE3ONH2, 2) the amount of peptide engaged in the ligation and 3) the flip-flop motion permitted by the different formulations, in which the presence of cholesterol seems to play an important role.     

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

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

Multimeric cyclic RGD peptides as potential tools for tumor targeting: solid-phase peptide synthesis and chemoselective oxime ligation

The alpha v beta 3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging alpha v beta 3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.     

Chemoselectively addressable template: a valuable tool for the engineering of molecular conjugates

We herein report the modular design and the synthesis of new molecular conjugates, which can combine a cell targeting function (ligand domain) with potential cytotoxic molecules (effector domain). The present approach utilizes a cyclic peptide template, Chemoselectively Addressable Template (CAT) as a key intermediate. These CAT molecules exhibit two independent and chemically addressable domains which permits the sequential and regioselective assembly of different ligand and/or effector domains. The attachment of various units to the template was achieved by the formation of iterative oxime bonds. The chemoselective oxime bonds were produced by the reaction of glyoxylyl aldehyde groups obtained from serine precursors. The process was further developed to prevent transoximation reactions. RAFT(c[-RGDfK-])4, a synthetic vector targeting the tumor-associated a alpha(V)beta3 integrin was prepared and coupled to either a cytotoxic peptide or oligonucleotide as an illustration of present approach. The potential application of this approach has been further demonstrated by the synthesis of high molecular weight compounds such as RAFT(c[-RGDfK-])16, a alpha(V)beta3-targeting ligand of high valency index.     

Synthesis of an oxyamino-containing phenanthroline derivative for the efficient preparation of phenanthroline oligonucleotide oxime conjugates

A phenanthroline derivative bearing an oxyamino linker was efficiently prepared from commercial 5-nitro-1,10-phenanthroline. The subsequent reaction with an oligonucleotide containing an aldehyde either at the 5′ end or the 3′ end afforded, in good yield, the phenanthroline-oligonucleotide conjugates through oxime bond formation.     

A New and Efficient Method for Synthesis of 5′‐Conjugates of Oligonucleotides through Amide‐Bond Formation on Solid Phase

An efficient method for synthesis of oligonucleotide 5′‐conjugates through amide‐bond formation on solid phase is described. Protected oligonucleotides containing a 5′‐carboxylic acid function were obtained by use of a novel non‐nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2‐chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid‐phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5′‐Carboxylate‐modified oligonucleotides were efficiently conjugated on solid support under normal peptide‐coupling conditions to various amines or to the N‐termini of small peptides to yield products of high purity. The method is well‐suited in principle for the synthesis of peptide‐oligonucleotide conjugates containing an amide linkage between the 5′‐end of an oligonucleotide and the N‐terminus of a peptide.     

Double‐Clicking Peptides onto Phosphorothioate Oligonucleotides: Combining Two Proapoptotic Agents in One Molecule

Described here is a method for the conjugation of phosphorothioate oligonucleotides (PSOs) with peptides. PSOs are key to antisense technology. Peptide–PSO conjugates may improve target specificity, tissue distribution, and cellular uptake of PSOs. However, the highly nucleophilic phosphorothioate structure poses a challenge to conjugation chemistry. Herein, we introduce a new method which involves a sequence of oxime ligation and strain‐promoted [2+3] cycloaddition. The usefulness of the method was demonstrated in the synthesis of peptide–PSO conjugates that targeted two suppressors of both the intrinsic and the extrinsic pathway of apoptosis. It is shown that the activity of a PSO sequence targeted against mRNA from c‐Flip can be enhanced by conjugation with a peptide mimetic designed to inhibit the X‐linked inhibitor of apoptosis protein (XIAP).     

The oxime bond formation as a useful tool for the preparation of oligonucleotide conjugates

Synthetic oligonucleotides are attracting considerable interest as potential therapeutic agents for the selective inhibition of gene expression. The attainment of effective cellular delivery however remains a problem. The conjugation of oligonucleotides to cell penetrating peptides is one of the most promising alternatives, that is being currently investigated to improve the uptake efficiency of oligonucleotides. The synthesis of peptide–oligonucleotide conjugates (POC) is however still a problem. Work from our laboratory has attempted to address the problem of POC synthesis by using the chemoselective oxime bond formation. Herein, we present an account of the work accomplished in our laboratory in the recent past, concerning the conjugation of various reporters to oligonucleotides. To cite this article: Y. Singh et al., C. R. Chimie 8 (2005).     

Head-to-tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues

A convenient strategy for the synthesis of the analogue of cyclic oligodeoxyribonucleotides is presented. The cyclization of the oligonucleotide was accomplished through intramolecular oxime bond formation between a 5'-oxyamine moiety and a 3'-aldehydic group.     

Oligonucleotides with 2'-O-carboxymethyl group: synthesis and 2'-conjugation via amide bond formation on solid phase

An efficient method for synthesis of oligonucleotide 2'-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2'-O-carboxymethyl group were obtained by use of a novel uridine 3'phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2'-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide-oligonucleotide conjugates containing an amide linkage between the 2'-position of an oligonucleotide and the N-terminus of a peptide.     

Straightforward synthesis of cholic acid stabilized loop mimetics

We here report on a new straightforward strategy for the synthesis of cyclic cholic acid–peptide conjugates. A solid-phase synthesis method is presented in which a selected anti-lysozyme CDR3 fragment, Asp-Ser-Thr-Ile-Tyr-Ala-Ser-Tyr-Tyr-Glu-Ser, is immobilized onto a steroidal cholic acid derived scaffold in order to yield a loop-like structure. Therefore, part of the desired sequence, that is, Ser-Tyr-Tyr-Glu-Ser, is introduced, at the C12 position of the scaffold. Subsequently, the remainder of the envisaged sequence is introduced at C3 via a Cu-catalyzed cyclo-addition reaction. Finally, amide bond formation delivers the desired cyclic peptidosteroid. This new synthetic strategy offers an easy and short access to cyclic peptidosteroids via convergent peptide ligation and macrocyclization.     

New solid support for the synthesis of 3'-oligonucleotide conjugates through glyoxylic oxime bond formation

A novel solid support 1 was synthesized to incorporate glyoxylic aldehyde functionality at the oligonucleotide 3'-terminus. 6-mer and 11-mer oligonucleotide sequences containing 3'-glyoxylic aldehyde functionality were prepared by using this support. These modified oligonucleotides were coupled to reporters containing an aminooxy group to prepare oligonucleotide 3'-conjugates through glyoxylic oxime bond formation. The hydrolytic stability of a glyoxylic oxime linkage was also investigated. [reaction: see text].     

A new strategy for synthesis of branched cyclic peptide by Asn side-chain hydrazide ligation

Here, we report a new strategy for rapid synthesis of branched peptide by side-chain hydrazide ligation at Asn. The hydrazide was converted to thioester at Asn side chain by NaNO₂ and thiol reagent, and sequential ligation with an N-terminus Cys-peptide efficiently afforded the branched peptide. A branched cyclic peptide was successfully synthesized by side-chain ligation with a two-Cys-peptide and formation of a disulfide bond. This approach provides a new way for expeditious synthesis of branched peptides and facilitates the design of neopeptides as functional bio-mimics.