Maleimido peptides

Maleimido-peptide and thiol-oligonucleotides are deblocked just before use. 

A. Synthesis of Oligonucleotide-Peptide Conjugates Using Strategy A Preparation and Conjugation of Maleimido-Peptides with Thiol-Oiigonucleotides... 

HOBt in a small amount of DMF were added. The maleimido-peptide was deprotected and released from the resin by treatment with trifluoroacetic acid (TFA)-H20 (19 1) for 4 h at room temperature. The resin was filtered and washed several times with H2O. The combined solutions were evaporated under reduced pressure. The residue was treated three times in diethyl ether and concentrated to dryness to eliminate the excess of TFA. The peptide was analyzed by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry. 

Synthesis of Thiol-Containing Oligonucleotides and Conjugation with Maleimido-Peptides... 

Protection of the 7-hydroxyl group in Paditaxd (taxol ) 10 as the carbonate 13 of the maleimido-peptide-benzyl alcohol 12 was carried out through the corresponding chloroformate using diphosgene in the presence of pyridine/DIE [19]. 

For the synthesis of the peptide-protein conjugates, peptides with a readive linker group, ie a maleimido group at the N-terminal amino function, were required. The synthesis of these peptides... 

The Ras proteins were then allowed to react with the MIC-modified peptides in stoichiometric amounts. The maleimido group1261 reacts specifically with mercapto groups of proteins by conjugate addition of the thiol to the a, 3-unsaturated carbonyl compound. The Ras mutants reacted smoothly with the MIC modified peptides and in... 

Maleimido activated core syntheses are performed on a Symphony synthesizer by Fmoc solid-phase peptide synthesis. These methods can easily be adapted for other solid phase peptide synthesizers. 

Dissolve 8pmol of monomeric peptide containing a unique cysteine and 1 pmol of maleimido activated core in Ar-purged lx PBS/0.01M EDTA. Vortex it at room temperature for 2h (see Notes 5 and 29). 

The maleoyl-) -alanine N-hydroxysuccinimide ester was conveniently prepared following the procedure of scheme 3 (101,102), and was then used to convert [Nle15]-gastrin-[2-17] and [Thr,Nlel-CCK-9 into the reactive maleimido-derivatives for the final coupling of the 1,2-diacyl-3-mercaptoglycerols to produce the lipo-peptide derivatives DM-gastrin and DM-CCK shown in fig. 11 (51,104). 

Abbreviations IgGl. immunoglobulin Gl KLH, keyhole limpet hemocyanin BSA, bovine serum albumin RNase A. ribonuclease A LDH. lactate dehydrogenase MHC, major histocompatibility complex LHRH, luteneizing hormone releasing hormone CCK. cholecystokinin VIP. vasoactive intestinal peptide TASP, template-assembled synthetic proteins MAPS, multiple antigen presenting system SUV, small unilamellar vesicle DCC. dicyclohexylcarbodiimide HOSu. N-hydroxysuccinimide Mal>. maleimido TFA, trifluoroacelic acid TFE. triiluoroethanol DMF, dimethylformamide DMSO. 

The maleimido-protein approach which today represents the most frequently used selective conjugation technique is based on maleimido-benzoylated proteins and synthetic peptides containing at their N- or C-termini an additional cysteine residue (61). This method, however, bears two main difficulties ... 

Most commonly used. Maleimido-oligonucleotide is prepared from 5 -amino-oligonucleotide. Introduction of a thiol group in peptides is performed by adding a Cys residue. 

Solid-Phase Synthesis of Peptides Carrying Maleimido Groups... 

We have observed that the maleimido-containing peptides are not completely stable in aqueous solutions. After 3-4 days in neutral aqueous solutions, the purified peptides were transformed to a complex mixture of products formed by reaction of the maleimido groups with the side-chain amino groups. To avoid this problem, the peptidyl-resin is stored in the freezer and small aliquots of the resin (5-10 mg per 20-30 OD units at 260 nm of crude oligonucleotide) are deprotected just before the coupling with the thiol-containing oligonucleotide. 

Figure 3 Reversed-phase HPLC profiles at 260 nm of (a) thiol-hexanucleotide, (b) maleimido-Ala-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-Asn-CONHa, and (c) oligonucleotide-peptide conjugate (see conditions in the text).

An aliquot of the thiol-peptide ( 1 fjumol, 10 equiv.) was dissolved in 0.2 mL of 0.1 M triethylammonium acetate buffer, pH 6.5, mixed, and the solution was added to a solution of 5 -maleimido-oligonucleotide (0.1 tmol) in the same solution (0.2 mL), The reaction was allowed to occur for 2-3 h at room temperature and the product was purified as described in Section... 

The chemoselective Michael addition of sulfhydryl group to the maleimido group is a well-known conjugation reaction tmder neutral pH, which has been commonly used for the coupling of fiuorophores to proteins with surface-exposed cysteine residues. The reaction was used to conjugate a maleimidocaproyl (MIC) peptide to a C-terminally truncated Ras protein bearing a C-terminal cysteine (Scheme 12). 

Keyhole limpet hemocyanin (Sigma, USA) using maleimido-benzoyl-N-hydroxysuccin-imide ester essentially as described by Harlow and Lane (1988). Antipeptide antiserum were raised in rabbits using a schedule of boosting at three weeks intervals followed by bleeding ten days after each boost. Antipeptide antibodies were affinity purified with immobilised synthetic peptide on epoxy-activated Sepharose 6B (Pharmacia). 

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