Efficient preparation of azodye-labeled aminoxy acids and peptides

1-(4-Arylazobenzoyl)benzotriazoles 2a – c react in aqueous acetonitrile at 20 o C with aminoxy acids 3a – c to give azodye-labeled aminoxy acids 4a – i in 65-80% yields. Similarly, reaction of Fmoc-protected aminoxy-dipeptides 6a – c with 2a gave azodye-labeled aminoxy peptides 7a – c in 55–65% yield.


Introduction
][3] Azodye-labeled peptides are important for pharmaceutical and biological investigations (Figure 2).Peptide based azodye-labeled molecules show substrate specificity for prostate membrane antigens 4 and in some cases are potent inhibitors of m-calpain. 3Azodye-labeled peptides may also serve as markers in biological applications. 5 Attaching azodye carboxylic acids to host molecules is the key to the synthesis of azophotoresponsive systems.Amino acids/peptides or amines acting as links between azo-dye acyl groups and host molecules are common in many photobiological switches and bioprobes.Azophotoresponsive systems frequently incorporate azodye-labeled ()-amino acids/peptides. 6-Aminoxy acids are analogs of β-amino acids in which the β-carbon atom is replaced by an oxygen atom.The incorporation of -aminoxy acids into peptidomimetics has attracted interest, since -aminoxy acid units are more rigid than the corresponding β-amino acid units, 7 and aminoxy amide bonds RCONHOR' resist enzymatic degradation. 8-Aminoxy peptides have also attracted interest as novel foldamers 9 with unusual conformations and diverse bioactivity.10a-c Aminoxy peptides also feature strong intramolecular hydrogen bonds between adjacent residues in peptidomimetic foldamers 11 and may provide useful labels.
N-Acylbenzotriazoles are easily prepared, non-hygroscopic, chirally stable analogs of acid halides that are relatively insensitive to water, 12,13 and are therefore advantageous for N-, O-, C-, or S-acylation; [12][13][14][15][16][17][18] especially where the corresponding acid chlorides are unstable or difficult to prepare. 19,20We previously acylated amino acids and amines with N-(4-arylazobenzoyl)-1Hbenzotriazoles 21 and we recently synthesized azodye-labeled peptides in good yields by a milder procedure than published methods. 22e have not located any previous syntheses of azodye-labeled aminoxy acids or peptides.Because of the interesting properties of aminoxy peptides, we now report the synthesis of azodye labeled aminoxy acids and peptides by reaction of N-(4-arylazobenzoyl)-1H-benzotriazole with aminoxy acids and aminoxy peptides under mild conditions.

Conclusions
In conclusion, we have synthesized novel azodye labeled aminoxy acids and aminoxy hybrid peptides in a convenient and efficient manner by reacting N-(4-arylazobenzoyl)-1H-benzotriazoles with aminoxy acids and aminoxy hybrid peptides.All the azodye labeled aminoxy acids and aminoxy hybrid peptides were obtained under mild reaction conditions in moderate to good yields.These novel azodye labeled aminoxy acids and aminoxy hybrid peptides may be useful in the preparation of peptidomimetic foldamers and in biological applications.

Experimental Section
General.Aminooxy acid 3a was purchased commercially and used without purification.Aminooxy acids 3b, 3c were prepared according to our previously reported method. 23Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected.NMR spectra were recorded in acetone-d6, CDCl3 or DMSO-d6 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference.Elemental analyses were performed on a Carlo Erba-1106 instrument.Mass spectrometry was done on Agilent 6210 TOF-MS with electro spray ionization (ESI).CH2Cl2 was dried and distilled over CaH2, whereas THF was used after distillation over Nabenzophenone.

Synthesis of azo-dye carboxylic acid labeled aminooxy acids (4a-i)
N-(4-Arylazobenzoyl)-1H-benzotriazoles 2a-c (1.0 mmol) were added to a solution of the appropriate α-aminooxy acids 3a-c (1.2 mmol) in THF-H2O (3:1) in the presence of Et3N (2.0 mmol).The reaction mixture was stirred at 20 °C for about 3 h until TLC showed the absence of 2ac, then the solvent was removed under reduced pressure.The residue was dissolved in EtOAc (50 mL) and the solution was washed with 4 N HCl (3 × 50 mL), saturated NaCl solution (50 mL), and dried over anhydrous Na2SO4.After evaporation of the solvent, the residue was crystallized from appropriate solvents to give 4a-i.

Synthesis of Fmoc-protected aminooxy peptides (6a-e)
Benzotriazole derivatives of Fmoc-amino acids 5a-c (1 mmol) were added to a solution of the appropriate α-aminooxy acids 3a-b (1 mmol) in MeCN-H2O (3:1) in the presence of Et3N (2.0 mmol).The reaction mixtures were stirred at 20 °C for about 1 h until TLC showed the absence of 5a-c when the solvent was removed under reduced pressure.The residue was dissolved in EtOAc (150 mL) and the solution was washed with 4 N HCl (3 × 50 mL), saturated NaCl solution (50 mL), and dried over anhydrous Na2SO4.After evaporation of the solvent, the residue was crystallized from appropriate solvents to give 6a-e.Compounds 6a-c were used for reaction without further purifications.Fmoc-L-Phe-AO-Gly-OH (6a

Synthesis of azodye labeled aminooxy peptides (7a-c)
Fmoc protected aminooxy dipeptide 6a-c (1 mmol) were added to a solution of DBU (1 mmol) in dry THF (6 mL) and after stirring for 2 h at 0-5 o C, 4-Phenylazobenzoyl-1H-benzotriazole 2a (1 mmol) was added and the reaction mixtures were stirred at 20 o C for another 10 h.Solvent was evaporated and residue was dissolved in CH2Cl2 (20 mL), washed with 3N HCl (3x10 mL) and dried over anhydrous Na2SO4.After evaporation of solvent, the residue was recrystalized from CH2Cl2/hexanes to give 7a-c.