3-Amino-3-(2-nitrophenyl)propionic acid

[5678-48-8]  · C9H10N2O4  · (MW 210.19)

(reagent used as a photocleavable linker for the solid-phase synthesis of C-terminal carboxamides)

Physical Data: mp 222 °C.

Form Supplied in: commercially available as the Fmoc protected b-amino acid, 3-Na Fmoc-amino-3-(2-nitrophenyl)propionic acid (3), or in the pre-functionalized form of 3-Na Fmoc-amino-3-(2-nitrophenyl)propionic acid-aminomethyl resin.

Handling, Storage, and Precautions: no special handling requirements but prevent excess exposure to light.

Preparation of Resin-Bound ANP Linker

Synthesis of b-amino acid (2) is accomplished by reaction of 2-nitrobenzaldehyde (1)1,2 with malonic acid in ammonium acetate/acetic acid. The amino function of 3-amino-3-(2-nitrophenyl)propionic acid (2) is Fmoc protected prior to coupling the carboxylic acid onto aminomethyl TentaGel resin.3 Removal of the Fmoc moiety liberates the amino unit to give 4 ready for sequential amino acid couplings (1).

Utility as a linker

3-Amino-3-(2-nitrophenyl)propionic acid (ANP) (2) is a linker that is compatible with use of both acid- and base-sensitive protecting groups used in peptide synthesis.3 The compatibility of the ANP linker with common acid- and base-labile protecting groups was assessed by the individual coupling and subsequent photolytic release of 20 different amino acid residues possessing a varied range of Na (Boc, Fmoc, Cbz) and side chain (Bzl, OBut, Ac, Alloc, Boc, Tos, Trt) protecting groups. Photolytic cleavage of 2-nitrobenzyl systems has been reviewed.4,5 In the case of ANP the cleavage is performed at 365 nm. The mechanism is presumed to be analogous to the related 4-aminomethyl-3-nitrobenzyl amide (NBA) resin (5),6 resulting in release of a C-terminal carboxamide.

The cleaved products (carboxamides) were analyzed by electrospray ionization-mass spectrometry (ESI-MS).3 In general, of the amino acids surveyed, most were well suited to the cleavage protocol. Notable exceptions include Fmoc-Asp(OBut)NH2, which revealed trace amounts of product due to side-chain ester deprotection, and 2,4-Dnp-Pro-NH2, which produced trace amounts of material derived from nitro group reduction. In addition, cleavage of thiol-containing residues, Boc-Met-NH2 and Fmoc-Cys-(4-Me-Bzl)-NH2 showed products of oxidation by ESI-MS. Photolysis is an extremely efficient method of cleavage from the ANP-resin when compared to closely related NBA-resin 5. Model studies, as verified from the mass spectroscopic analysis of carboxamide Fmoc-Arg(Tos)-NH2 as a representative example, showed that light-initiated cleavage at 365 nm for a period of 3 h yields 52% of carboxamide from the ANP resin whereas only 12% was obtained from NBA resin. A water:methanol (4:1) solvent mixture was found to be superior for the cleavage protocol than methanol alone.

Use of ESI-MS as Decoding Strategy for Resin-Bound Peptides

Tripeptide Fmoc-Asp-Arg(Tos)-Val-NH2 was synthesized and the product from a single bead photocleaved (365 nm for 1 h) in H2O.3 ESI-MS analysis revealed a molecular ion in accordance with the protonated molecular mass of the peptide. Tandem mass spectrometry (MS/MS) analysis on MH+ indicated fragments that allowed full characterization of the tripeptide. Resuspension of the same bead in H2O and further irradiation at 365 nm for 16 h once more showed the MH+ ion that allowed for complete sequencing by tandem mass spectrometry. As a result, analyte molecular weight and structure can be ascertained from a single bead, thus eliminating the need for complex tagging techniques.

Limitations

The ANP appendage is susceptible to degradation under a combination of Lewis acid-amine reaction conditions, a problem attributed to b-elimination side reactions.7 This difficulty can be overcome by use of 3-amino-3-(2-nitrophenyl)-2,2-dimethylpropionic acid (6).7 Another recent variant includes 6-hydroxy-6-(2-nitrophenyl)-hexanoic acid (7).8

Related Reagents.

5-(3-Iodopropoxy)-2-nitrobenzyl alcohol [185994-27-8];9 3-amino-3-(2-nitrophenyl)-2,2-dimethylpropionic acid;7 6-hydroxy-6-(2-nitrophenyl)-hexanoic acid [198065-04-2];8 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid [175281-76-2];10 4-[4-(1-(9H-fluoren-9-ylmethoxycarbonylamino)-ethyl)-2-methoxy-5-nitrophenoxy]butyric acid [162827-98-7].11


1. Profft, E.; Becker, F.-J., J. Prakt. Chem. 1965, 30, 18.
2. Dallemagne, P.; Tembo, O.; Rault, S.; Robba, M., Bull. Soc. Chim. Fr. 1989, 98.
3. Brown, B. B.; Wagner, D. S.; Geysen, H. M., Molecular Diversity 1995, 1, 4.
4. Pillai, V. N. R., Synthesis 1980, 1.
5. Patchornik, A.; Amit, B.; Woodward, R. B., J. Am. Chem. Soc. 1970, 92, 6333.
6. Rich, D. H.; Gurwara, S. K., Tetrahedron Lett. 1975, 16,301.
7. Sternson, S. M.; Schreiber, S. L., Tetrahedron Lett. 1998, 39, 7451.
8. Rodebaugh, R.; Fraser-Reid, B.; Geysen, H. M., Tetrahedron Lett. 1997, 38, 7653.
9. Nicolaou, K. C.; Winssinger, N.; Pastor, J.; DeRoose, F., J. Am. Chem. Soc. 1997, 119, 449.
10. Whitehouse, D. L.; Savinov, S. N.; Austin, D. J., Tetrahedron Lett. 1997, 38, 7851.
11. Holmes, C. P.; Jones, D. G., J. Org. Chem. 1995, 60, 2318.

Mark Bradley & Stifun Mittoo

University of Southampton, Southampton, UK



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