A new "native ligation" procedure for peptide-oligonucleotide conjugation

"Native ligation", a powerful method of joining peptide fragments, has been applied successfully to peptide-oligonucleotide conjugation. Novel reagents are described for the solid-phase synthesis of peptide N-terminal thioesters and 5'-cysteinyl oligonucleotides suitable for ligation reactions.     

Synthesis and use of a pseudo-cysteine for native chemical ligation.

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.     

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.     

'100 years of peptide synthesis': ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies.

A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, 'Synthesis of Proteins by Chemical Ligation' a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo-prolines, 'unrestricted' amino-acid residues (using the Staudinger reaction), as well as solid-phase segment coupling by thioligation of unprotected peptides. In another section, 'Synthesis of beta-peptides by Thioligation', couplings involving beta2- and beta3-peptides are described (with experimental details).     

A NEW “NATIVE LIGATION” PROCEDURE FOR PEPTIDE-OLIGONUCLEOTIDE CONJUGATION

“Native ligation”, a powerful method of joining peptide fragments, has been applied successfully to peptide-oligonucleotide conjugation. Novel reagents are described for the solid-phase synthesis of peptide N-terminal thioesters and 5′-cysteinyl oligonucleotides suitable for ligation reactions.     

The synthesis of beta-peptides containing guanidino groups

The synthesis of the beta-peptide 1 by the postsynthetic modification of the corresponding amino-containing peptide 3 is described. The potential of 1 to act as a template for the ligation of complementary negatively-charged peptides is discussed.     

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.     

Total synthesis by modern chemical ligation methods and high resolution (1.1 A) X-ray structure of ribonuclease A

The total chemical synthesis of RNase A using modern chemical ligation methods is described, illustrating the significant advances that have been made in chemical protein synthesis since Gutte and Merrifield's pioneering preparation of RNase A in 1969. The identity of the synthetic product was confirmed through rigorous characterization, including the determination of the X-ray crystal structure to 1.1 Angstrom resolution.     

The Chemical Synthesis of the Collagenous Domain of the Hormone Adiponectin

As a first step towards the preparation of a functional biomolecule, a chemical synthesis of the collagenous domain of adiponectin is described. The 76 residue polypeptide (without post-translational modifications) could be assembled efficiently from smaller unprotected peptides using two native chemical ligation reactions followed by a reductive desulfurisation step. In turn, the polypeptides were synthesised in high purity by microwave enhanced solid phase synthesis.     

A one-pot multistep approach to alpha-azido-phosphonate and phosphonothioate diesters: key intermediates in the synthesis of haptens for the generation of antibody ligases

A four-step, one-pot synthesis of mixed alpha-azido-phosphonates and phosphonothioates 12a-d is described. This chemistry has provided a facile route to haptens 6a b and 7 that have been employed for the elicitation of antibody ligases. Five hapten-specific antibodies have been identified as modest catalysts of a model peptide ligation reaction between thioester 1b and thiol 2 to give the amide product 5.     

New phosphoramidite reagents for the synthesis of oligonucleotides containing a cysteine residue useful in peptide conjugation

The preparation is described of four 2-cyanoethyl-N,N-diisopropyl phosphoramidites of N-alpha-Fmoc-S-protected cysteine hydroxyalkyl amides. The phosphoramidites were used in solid-phase synthesis of 5'-cysteinyl oligonucleotides, useful intermediates in the preparation of peptide-oligonucleotide conjugates through reaction with a maleimide peptide or with a peptide thioester via "native ligation".     

Chemico-enzymatic method of synthesis of mixed oligonucleotides

Oligo (ribodeoxyribo)nucleotide d(TGTGTAT)r(GCCAU) was synthesized from oligonucleotide blocks by chemical ligation, the oligoribonucleotide and oligodeoxynucleotide blocks being prepared by enzymatic and chemical synthesis, resp. A general procedure of the hybride duplex strand separation is described.     

Induction of avidin in the chick oviduct by tissue damage and prostaglandins

In immature, diethylstilboestrol-treated chicks, ligation of the oviduct caused local avidin synthesis in the immediate vicinity of the ligature. PGF-2alpha injected directly into the oviduct also induced avidin synthesis, whereas saline or PGE-2 had no effect. PGE and PGF-2alpha concentrations increased in the oviduct within 24 h of ligation: the PGE increase could be partly inhibited by indomethacin, whereas that of PGF-2alpha was less inhibited. An LD50 dose of indomethacin alone and with ligation had a clear stimulatory effect on avidin synthesis, whereas aspirin alone, or with ligation, was not effective. Ligation alone and with indomethacin appeared to alter the PGF-2alpha/PGE ratio. These results suggest that PGF-2alpha may be involved in the regulation of avidin synthesis in the chick oviduct.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Carboproteins: a 4-alpha-helix bundle protein model assembled on a D-galactopyranoside template

We have recently introduced the concept of monosaccharides as templates for de novo design of protein models and described the synthesis of a model 'carbopeptide'. Here, we report the synthesis of a 64 amino acid (AA) 'carboprotein' by chemoselective ligation of a C-terminal hexadecapeptide aldehyde to a tetra-aminooxy functionalized methyl alpha-D-galactopyranoside (D-Galp) template. Biophysical characterizations by CD spectroscopy and NMR amide H-D exchange experiments indicated that the four-stranded carboprotein forms a 4-alpha-helix bundle structure.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

New Phosphoramidite Reagents for the Synthesis of Oligonucleotides Containing a Cysteine Residue Useful in Peptide Conjugation

Abstract

The preparation is described of four 2-cyanoethyl-N,N-diisopropyl phosphoramidites of N-α-Fmoc-S-protected cysteine hydroxyalkyl amides. The phosphoramidites were used in solid-phase synthesis of 5′-cysteinyl oligonucleotides, useful intermediates in the preparation of peptide-oligonucleotide conjugates through reaction with a maleimide peptide or with a peptide thioester via “native ligation”.     

The use of 5''-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA.

Methodology is described for the synthesis and chemical aminoacylation of the hybrid dinucleotide 5'-phospho-2'-deoxyribocytidylylriboadenosine (pdCpA). Ligation of aminoacylated pdCpA to a truncated amber suppressor tRNACUA (-CA) using T4 RNA ligase generates an aminoacylated suppressor tRNA which can be used for site-specific incorporation of unnatural amino acids into proteins. Both the ligation and in vitro suppression efficiencies are the same when either pCpA or pdCpA is used. The use of deoxycytidine simplifies the chemistry involved in the synthesis of the dinucleotide pCpA. In addition, these results demonstrate that ribocytidine is not required for recognition of the aminoacylated tRNA during protein synthesis.     

Synthesis of 4-mercapto-l-lysine derivatives: Potential building blocks for sequential native chemical ligation

A general and diastereoselective synthesis of (2S, 4S)-4-mercapto-l-lysine derivative was described. The key features of this synthesis include Zn-mediated diastereoselective Reformatsky reaction and selective reduction of methyl ester with sodium borohydride. Introduction of thiol functional group on lysine side chain proved to be appropriate for dual native chemical ligation. This methodology allows to develop various 4-substituted l-lysine derivatives.     

Total synthesis of artificial zinc-finger proteins: Problems and perspectives

The total synthesis of a peptide segment corresponding to the DNA-binding segment of Sp1 (positions 532-623) using a native chemical ligation approach is described. The folding of the synthetic segment in the presence of Zn(II) gave a zinc-coordinated protein. The dissociation constant (K(D)) for the DNA binding of the resulting protein, determined by a gel mobility shift assay, was 130 nM, almost nine times higher than that of the genetically prepared protein. However, methylation interference assay showed an identical sequence specificity of both proteins in DNA recognition. The chemical ligation method to connect the respective zinc-finger units was also accomplished. Successive ligation between a cysteine-containing peptide segment and a chloroacetylated peptide segment gave an artificial three-finger protein, which corresponds to the above DNA-binding segment of Sp1. However, this protein failed to bind DNA, even at 1.25 mM. Assessment of their folding structure based on the absorption spectra of their Co(II) complexes showed that the linker design to connect the respective finger units is critical for the proper folding of the proteins as well as the occurrence of the DNA-binding function.