Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.     

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.     

Efficient Chemical Protein Synthesis using Fmoc-Masked N-Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

Peptide dithiodiethanol esters for in situ generation of thioesters for use in native ligation

A new approach is described for the general Fmoc-based solid-phase synthesis of C-terminal peptide (thio)esters. One hydroxy group of 2,2-dithiodiethanol (used in large excess) was anchored on trityl resin, and the remaining hydroxy group was loaded with the first amino acid. Standard chain elongation and TFA-based peptide release yielded peptide C-terminal dithiodiethanol esters in good purities. Under standard conditions of native chemical ligation (excess thiol, neutral pH), the dithiodiethanol function is presumably reduced and rearranged (or equilibrated) to the thioester via a 5-membered intermediate. The resulting thioesters are shown to undergo native chemical ligation with N-terminal cysteine peptides. Notably, hydrolysis of the reduced ester is a major competing reaction, especially in the presence of 6 M guanidinium chloride, which is often required for solubilization of large peptide fragments.     

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.     

Synthesis of a selenocysteine-containing peptide by native chemical ligation

[reaction in text] A new method for the synthesis of selenocysteine derivatives and selenocysteine-containing peptides is described. Fmoc-Se-p-methoxybenzylselenocysteine (1) was prepared and used for solid-phase synthesis of peptides with an N-terminal unprotected selenocysteine. Subsequent native chemical ligation with a peptide thioester provided a 17-mer that corresponds to the C-terminus of ribonucleotide reductase with selenocysteine in place of cysteine.     

On-resin native chemical ligation for cyclic peptide synthesis

A novel cysteine derivative, N(alpha)-trityl-S-(9H-xanthen-9-yl)-l-cysteine [Trt-Cys(Xan)-OH] has been introduced for peptide synthesis, specifically for application to a new strategy for the preparation of cyclic peptides. The following steps were carried out to synthesize the cyclic model peptide cyclo(Cys-Thr-Abu-Gly-Gly-Ala-Arg-Pro-Asp-Phe): (i). side-chain anchoring of Fmoc-Asp-OAl via its free beta-carboxyl as a p-alkoxybenzyl ester to a solid support; (ii). stepwise chain elongation of the peptide by standard Fmoc/tBu solid-phase chemistry; (iii). removal of the N-terminal Fmoc group; (iv). coupling of Trt-Cys(Xan)-OH; (v). selective Pd(0)-promoted cleavage of the C-terminal allyl ester; (vi). coupling of the C-terminal residue, i.e., H-Phe-SBzl, preactivated as a thioester; (vii). selective removal of the N(alpha)-Trt and S-Xan protecting groups under very mild acid conditions; (viii). on-resin cyclization by native chemical ligation in an aqueous milieu; and (ix). final acidolytic cleavage of the cyclic peptide from the resin. The strategy was evaluated for three supports: poly[N,N-dimethacrylamide-co-poly(ethylene glycol)] (PEGA), cross-linked ethoxylate acrylate resin (CLEAR), and poly(ethylene glycol)-polystyrene (PEG-PS) graft resin supports. For PEGA and CLEAR, the desired cyclic product was obtained in 76-86% overall yield with initial purities of approximately 70%, whereas for PEG-PS (which does not swell nearly as well in water), results were inferior. Solid-phase native chemical ligation/cyclization methodology appears to have advantages of convenience and specificity, which make it promising for further generalization.     

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.     

Aziridine-2-carboxylic acid-containing peptides: application to solution- and solid-phase convergent site-selective peptide modification

The development of a method for site- and stereoselective peptide modification using aziridine-2-carboxylic acid-containing peptides is described. A solid-phase peptide synthesis methodology that allows for the rapid generation of peptides incorporating the aziridine residue has been developed. The unique electrophilic nature of this nonproteinogenic amino acid allows for site-selective conjugation with various thiol nucleophiles, such as anomeric carbohydrate thiols, farnesyl thiol, and biochemical tags, both in solution and on solid support. This strategy, combined with native chemical ligation, provides convergent and rapid access to complex thioglycoconjugates.     

Oligonucleotide conjugation to a cell-penetrating (TAT) peptide by Diels-Alder cycloaddition

Modifed oligonucleotides are routinely employed as analytical probes for use in diagnostics, e.g. in the examination of specific RNA sequences for infectious diseases, however, a major limiting factor in oligonucleotide-based diagnostics is poor cellular uptake of naked oligonucleotides. This problem can be overcome by covalent attachment of a so-called 'cell-penetrating peptide' to form an oligonucleotide peptide conjugate. Stepwise solid phase synthesis of such a conjugate is difficult and expensive due to the conflicting chemistries of oligonucleotides and peptides. A simple approach to overcome this is post-synthetic conjugation. Diels-Alder cycloaddition is an attractive methodology for oligonucleotide peptide conjugation; the reaction is fast, chemoselective and the reaction rate is greatly enhanced in aqueous media - ideal conditions for biological moieties. An oligodeoxyribonucleotide sequence has been derivatised with a series of dienes at the 5'-terminus, using a series of unique dienyl-modified phosphoramidites, and investigation into the effect of diene type on the efficiency of conjugation, using Diels-Alder cycloaddition with a maleimido-derivatised cell-penetrating (TAT) peptide, has been performed. This led to the observation that the optimal diene for conjugation was cyclohexadiene, allowing conjugation of oligodeoxyribonucleotides to a cell-penetrating peptide by Diels-Alder cycloaddition for the first time.     

Assessment of the Full Compatibility of Copper(I)-Catalyzed Alkyne-Azide Cycloaddition and Oxime Click Reactions for bis-Labelling of Oligonucleotides

The conjugation of oligonucleotides with reporters is of great interest for improving their intrinsic properties or endowing new ones. In this context, we report herein a new procedure for the bis-labelling of oligonucleotides through oxime ligation (Click-O) and copper(I)-catalyzed alkyne–azide cycloaddition (Click-H). 5′-Azido and 3′-aldehyde precursors were incorporated into oligonucleotides, and subsequent coupling reactions through Click-O and Click-H (or vice versa) were successfully achieved. In particular, we exhaustively investigated the full compatibility of each required step for both tethering strategies. The results demonstrate that click Huisgen and click oxime reactions are fully compatible. However, whilst both approaches can deliver the targeted doubly conjugated oligonucleotide, the route involving click oxime ligation prior to click Huisgen is significantly more successful. Thus the reactions investigated here can be considered to be key elements of the chemical toolbox for the synthesis of highly sophisticated bioconjugates.     

Synthesis of N alpha-(1-phenyl-2-mercaptoethyl) amino acids, new building blocks for ligation and cyclization at non-cysteine sites: scope and limitations in peptide synthesis

A new and convenient method for the synthesis and incorporation of N(alpha)-(1-phenyl-2-mercaptoethyl)-derivatized amino acids applicable to chemical ligation at non-cysteine sites is presented. N(alpha)-Auxiliary derivatives of glycine and alanine were easily prepared using reductive amination approaches. Several strategies for the incorporation of these derivatives into peptide chains were investigated: coupling without protection, with acid-labile protection, with base-labile protection, and via a novel protection strategy using the thiazolidine derivative. All amino acid derivatives were successfully coupled to various peptide resins, and with the exception of those incorporating Boc-protected derivatives, all resins yielded the desired peptide fragments. However, the coupling of the two alanine derivative diastereomers generated some epimerization. Finally, N-terminal auxiliary glycine and alanine peptides were cyclized, and the corresponding native circular peptides were obtained upon successful removal of the auxiliary.     

"Volatilizable" supports for high-throughput organic synthesis

The concepts and use of "volatilizable" solid supports are presented. Such supports, which are completely removed by volatilization following decomposition, improve the efficiency of the solid-phase synthesis of both individual and mixtures of low-molecular weight acyclic and heterocyclic compounds as well as peptides, peptidomimetics, and protected peptide fragments. The "volatilizable" silica-based solid supports and linkers used to illustrate this concept were found to be completely removed by their decomposition and ultimate "volatilization" during the final cleavage step of synthetic process to yield solely the desired synthetic product(s) in the final reaction vessel.     

Thermolytic properties of 3-(2-pyridyl)-1-propyl and 2-[N-methyl-N-(2-pyridyl)]aminoethyl phosphate/thiophosphate protecting groups in solid-phase synthesis of oligodeoxyribonucleotides

Thermolytic groups may serve as alternatives to the conventional 2-cyanoethyl group for phosphate/thiophosphate protection in solid-phase oligonucleotide synthesis to prevent DNA alkylation by acrylonitrile generated under the basic conditions used for oligonucleotide deprotection. Additionally, thermolytic groups are attractive in the context of engineering a "heat-driven" process for the synthesis of oligonucleotides on diagnostic microarrays. In these regards, the potential application of pyridine derivatives as thermolytic phosphate/thiophosphate protecting groups has been investigated. Specifically, 2-pyridinepropanol and 2-[N-methyl-N-(2-pyridyl)]aminoethanol were incorporated into deoxyribonucleoside phosphoramidites 7a-d and 9, which were found as efficient as 2-cyanoethyl deoxyribonucleoside phosphoramidites in solid-phase oligonucleotide synthesis. Whereas the removal of 3-(2-pyridyl)-1-propyl phosphate/thiophosphate protecting groups from oligonucleotides is effected within 30 min upon heating at 55 degrees C in concentrated NH4OH or in an aqueous buffer at pH 7.0, cleavage of 2-[N-methyl-N-(2-pyridyl)]aminoethyl groups occurs spontaneously when their phosphate or phosphorothioate esters are formed during oligonucleotide synthesis. The deprotection of these groups follows a cyclodeesterification process generating the bicyclic salts 13 and 14 as side products. These salts do not alkylate or otherwise modify any DNA nucleobases and do not desulfurize a phosphorothioate diester model under conditions mimicking large-scale oligonucleotide deprotection.     

An efficient reagent for 5′-azido oligonucleotide synthesis

A new bromohexyl phosphoramidite was synthesized and used for the introduction of an azide function at the 5′-end of oligonucleotides after a treatment on solid support with sodium azide and sodium iodide. The corresponding 5′-azido oligonucleotide could be further used for ‘Click’ conjugation with alkyne derivatives or by Staudinger ligation.     

Synthetic procedure for N-fmoc amino acyl-N-sulfanylethylaniline linker as crypto-Peptide thioester precursor with application to native chemical ligation

N-Sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Site-selective conjugation of thiols with aziridine-2-carboxylic acid-containing peptides

The synthesis and convergent site-selective conjugation of aziridine-2-carboxylic acid-containing peptides with thiols, both in solution and on solid support, are described. The synthesis and use of FmocAzyOH in this capacity demonstrate both the efficient incorporation and tolerance of the Azy moiety in multistep Fmoc solid-phase peptide synthesis (SPPS), as well as the competence of solution and on-bead ligation through a highly regioselective base-promoted aziridine ring-opening process.     

Template-free segmental synthesis of oligonucleotides containing nonnative linkages

Protected oligonucleotides containing 3'-alkyl carboxylic acids or 3'-alkylamines were obtained from photolabile solid-phase synthesis supports (1 and 4). Protected oligonucleotides containing 5'-alkylamines and 3'-hydroxyl groups were obtained using a photolabile solid-phase synthesis support (2) and a commercially available phosphoramidite reagent (3). Depending upon the source of alkylamine-containing oligonucleotide, the segments were coupled under mild conditions to form products containing either 5'-3' or 3'-3' linkages in good yield and high purity. Oligonucleotides as long as 40 nucleotides were prepared, and coupling yields of protected biopolymer segments were independent of length over the range examined. This method is particularly well suited for the convergent synthesis of oligonucleotides containing nonnative linkages and should be useful for the rapid assembly of modified biopolymers that are useful in biochemical studies.     

(R)-2,4-Dihydroxybutyramide seco-pseudonucleosides: new versatile homochiral synthons for synthesis of modified oligonucleotides

[reaction: see text] Two series of seco-pseudonucleoside synthons were synthesized from (R)-(+)-alpha-hydroxy-gamma-butyrolactone and (R)-(-)-pantolactone by aminolysis, side-chain protection, dimethoxytritylation, and phosphitylation or solid-phase attachment. The phosphoramidites and solid supports were used in automated DNA synthesis to prepare oligonucleotides modified with one or more 2,4-dihydroxybutyramide units bearing side-chain reporter groups. These new oligonucleotide modification reagents allow the introduction of a label into any desired position within an oligonucleotide chain during solid-phase assembly.