Cryptophane-Folate Biosensor for ¹²⁹Xe NMR

Abstract

Folate-conjugated cryptophane was developed for targeting cryptophane to membrane-bound folate receptors that are overexpressed in many human cancers. The cryptophane biosensor was synthesized in 20 nonlinear steps, which included functionalization with folate recognition moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized 129Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane. Cellular internalization of biosensor was monitored by confocal laser scanning microscopy and quantified by flow cytometry. Competitive blocking studies confirmed cryptophane endocytosis through a folate receptor-mediated pathway. Flow cytometry revealed 10-fold higher cellular internalization in KB cancer cells overexpressing folate receptors compared to HT-1080 cells with normal folate receptor expression. The biosensor was determined to be nontoxic in HT-1080 and KB cells by MTT assay at low micromolar concentrations typically used for hyperpolarized 129Xe NMR experiments.

NMR Hyperpolarization Techniques of Gases

“…To date, cryptophane-based sensors that use specific binding to the target have been implemented for sensing the enzyme MMP7, [142] nucleotides, [143] human carbonic anhydrase, [144] MHC class II, [145] zinc ions, [146] the glycoprotein CD14 [147] and the receptors for integrin, [148] transferrin, [149] EGF, [150] and folate. [151] An indirect binding approach was pursued through in situ click chemistry with metabolically labeled cell surface glycans. [152] Conformational changes of the sensor induced by changes in pH represent an approach for stimulus-induced binding.…”

A cryptophane-based “turn-on” 129Xe NMR biosensor for monitoring calmodulin

“…13 Cryptophanes can be made water-soluble and provide a hydrophobic cavity with internal volume roughly double that of Xe ( D = 4.3 Å, V ≈ 40 Å 3 ). 13f–h, 14 Our laboratory 11, 15 and others 16 have designed cryptophane-xenon biosensors targeted to specific biomarkers indicated in cancer, including integrin 11b and folate receptors, 15b EGFR, 16h mIgM, 16i MMP-7, 17 and carbonic anhydrase. 15a Additionally, studies have demonstrated the use of xenon biosensors to target live cells.…”

“…13f–h, 14 Our laboratory 11, 15 and others 16 have designed cryptophane-xenon biosensors targeted to specific biomarkers indicated in cancer, including integrin 11b and folate receptors, 15b EGFR, 16h mIgM, 16i MMP-7, 17 and carbonic anhydrase. 15a Additionally, studies have demonstrated the use of xenon biosensors to target live cells. 15c, 18 In these prior examples, Xe binding to cryptophane biosensors produced a specific hp 129 Xe NMR chemical shift, which shifted downfield when bound to protein analyte.…”

“…15a Additionally, studies have demonstrated the use of xenon biosensors to target live cells. 15c, 18 In these prior examples, Xe binding to cryptophane biosensors produced a specific hp 129 Xe NMR chemical shift, which shifted downfield when bound to protein analyte.…”

“…Briefly, the synthesis of tripropargyl cryptophane 1 was performed in six non-linear steps with a yield for the five linear steps of 9.9%. 15c Azido-FRRIAR peptide 2 was prepared with standard Fmoc-based synthetic methods. A single peptide was attached to the cryptophane via copper(I)-catalyzed [3+2] azide-alkyne cycloaddition (CuAAC) to form 3 after a 3 h reaction.…”