Site-specific Folate Conjugation to a Cytotoxic Protein

Abstract

Conjugation to folic acid is known to enhance the uptake of molecules by human cells that over-produce folate receptors. Variants of bovine pancreatic ribonuclease (RNase A) that have attenuated affinity for the endogenous ribonuclease inhibitor protein (RI) are toxic to mammalian cells. Here, the random acylation of amino groups in wild-type RNase A with folic acid is shown to decrease its catalytic activity dramatically, presumably because of the alteration to a key active-site residue, Lys41. To effect site-specific coupling, N(δ)-bromoacetyl-N(α)-pteroyl-l-ornithine, which is a folate analogue with an electrophilic bromoacetamido group, was synthesized and used to S-alkylate Cys88 of the G88C variant of RNase A. The pendant folate moiety does not decrease enzymatic activity, enables RI-evasion, and endows toxicity for cancer cells that over-produce the folate receptor. These data reveal a propitious means for targeting proteins and other molecules to cancer cells.

Figure 1

Scheme for the synthesis of N^δ-bromoacetyl-N^α-pteroyl-l-ornithine (1). Pteroyl azide was synthesized from folic acid in 4 steps as described previously.

Figure 2

Mass spectra of random and site-specific ribonuclease–folate conjugates. (A) Mass spectrum of K41R/G88R–folate, which was prepared by the random conjugation of folic acid. (n = 1, m/z 14250; expected: 14232). (B) Mass spectrum of G88C–folate, which was prepared by site-specific conjugation of folate analogue 1 (m/z 14205; expected: 14195). Major peaks are labeled with the number of folate moieties, n. Minor peaks are photochemically generated adducts of the matrix (3,5-dimethoxy-4-hydroxycinnamic acid) with the proteins.

Figure 3

Three-dimensional structure of the complex between RNase A (blue) and RI (red). The location of the three residues substituted herein are shown explicitly. Images were made with PyMol (Delano Scientific) and Protein Data Bank entry 1dfj.

Figure 4

Agarose gel-based assay of the inhibition of the enzymatic activity of RNase A variants and folate conjugates by RI. Inhibition was assessed by visualizing the ribonuclease-catalyzed degradation of 16S and 23S rRNA in the absence or presence of excess RI.^,