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Boramino acid as a marker for amino acid transporters

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Science Advances  11 Sep 2015:
Vol. 1, no. 8, e1500694
DOI: 10.1126/sciadv.1500694
  • Fig. 1 BAA is an AA mimic by substituting carboxylate group with its isosteric trifluoroborate.
  • Fig. 2 Computation studies show similarities between Phe-BF3 and Phe in interaction with LAT-1 transporter.

    (A) Molecular electrostatic potential (MEP) prediction of Phe and its mimics. As shown, Phe-BF3 has a more nearly identical charge distribution pattern to natural Phe than the other AA mimics, such as Phe-B(OH)2 (blue indicates the distribution of positive charge, and red indicates the distribution of negative charge). (B) Predicted structure of the LAT-1/Phe-BF3 complex. LAT-1 (gray) is in solid ribbon representation. Phe-BF3 and the LAT-1 residues in the binding site are in stick representation. Hydrogen bonds between Phe-BF3 and LAT-1 (involving residues Leu87, Val97, Ala98, and Leu99) are shown as dotted green lines, which are conserved with the interaction between Phe and LAT-1 (fig. S3). (C) Summary of the predicted binding free energy (ΔGbinding), inhibition constant (Ki, T = 298.15 K), and the root mean square deviation (RMSD). These values are calculated on the basis of the best docking conformation of LAT-1 in complex with Phe and Phe-BF3.

  • Fig. 3 Cell uptake of BAAs is time-dependent with high channel specificity.

    (A) Schematic depiction of system A, system L, and system P transporters. (B) U87MG tumor cell uptake of 18F-BAAs. %AD, percentage of added dose. (C) Competitive inhibition of U87MG cell uptake of 18F-labeled Leu-BF3, Phe-BF3, Ala-BF3, and Pro-BF3. Cells are incubated in sodium-free phosphate-buffered saline (PBS) buffer or co-incubated with other AAs at 25 mM for 60 min. As shown, the entry of 18F-BAAs is channel-specific and can be inhibited efficiently by the corresponding natural AAs.

  • Fig. 4 Uptake of 18F-BAA is mediated by AATs and kinetically indistinguishable from natural AAs.

    (A) Brief illustration of transporter-mediated cell uptake of BAAs. (B) The uptake-concentration correlation of 18F-BAAs fits the Michaelis-Menten equation. (C) Summary of experimentally measured Km of 18F-BAAs as compared to those of L-AA counterparts.

  • Fig. 5 18F-Phe-BF3 shows specific accumulation in U87MG xenografts and low uptake in normal brain and an inflammatory region (2 hours after injection).

    (A) Illustration of a mouse with tumor xenograft implanted on the right shoulder and inflammation introduced in the left hindlimb. (B) Representative coronal PET images of the normal skull and brain depicting 18F-BF3-Phe and 18F-FDG uptakes. (C) Representative transverse PET images of 18F-BF3-Phe and 18F-FDG showing prominent uptake in U87MG tumor (indicated by white arrows). (D) Representative transverse PET images demonstrate that 18F-BF3-Phe does not accumulate, but 18F-FDG does accumulate, in the inflammation region (indicated by cyan arrows; the inflammation was introduced by intramuscular injection of turpentine 72 hours before PET scan). (E) Whole-body maximum intensity projection image of a U87MG tumor–bearing mouse showing 18F-Phe-BF3 uptake. The tracer specifically accumulated in the tumor (t), whereas the remainder cleared to the bladder (b). Some gallbladder (gb) accumulation occurred for 18F-Phe-BF3, indicating rapid hepatobiliary excretion. (F) Representative coronal PET image of 18F-Phe-BF3. Color bar is calibrated in % ID/g, with no background subtracted.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/8/e1500694/DC1

    Scheme S1. General synthetic routes of α-amino boronic acid/ester derivatives with high enantiopurity.

    Scheme S2. BAAs are converted from BAA/ester in quantitative yield.

    Scheme S3. 18F-BAAs are radiosynthesized via one-step 18F-19F isotope exchange reaction.

    Fig. S1. Radioactive HPLC chromatogram for the purification of Leu-BF3.

    Fig. S2. The LC-HRMS spectrum of HPLC-purified Leu-BF3.

    Fig. S3. 19F NMR spectrum of HPLC-purified Leu-BF3 (δ = −148.95 ppm).

    Fig. S4. 1H NMR spectrum of HPLC-purified Leu-BF3.

    Fig. S5. Radioactive HPLC chromatography of preparing Phe-BF3.

    Fig. S6. The LC-HRMS spectrum of HPLC-purified Phe-BF3.

    Fig. S7. 19F NMR spectrum of HPLC-purified Phe-BF3 (δ = −151.96 ppm).

    Fig. S8. 1H NMR spectrum of HPLC-purified Phe-BF3.

    Fig. S9. Radioactive HPLC chromatography of preparing Ala-BF3.

    Fig. S10. The LC-HRMS spectrum of HPLC-purified Ala-BF3.

    Fig. S11. 19F NMR spectrum of HPLC-purified Ala-BF3 (δ = −149.11 ppm).

    Fig. S12. 1H NMR spectrum of HPLC-purified Ala-BF3.

    Fig. S13. Radioactive HPLC chromatography of preparing Pro-BF3.

    Fig. S14. The LC-HRMS spectrum of HPLC-purified Pro-BF3.

    Fig. S15. 19F NMR spectrum of HPLC-purified Pro-BF3 (δ = −149.02 ppm).

    Fig. S16. 1H NMR spectrum of HPLC-purified Pro-BF3.

    Fig. S17. Radioactive HPLC chromatography of Sep-Pak purified Leu-BF3, Phe-BF3, Ala-BF3, and Pro-BF3, respectively (from top to bottom).

    Fig. S18. In vitro stability assay of 18F-Leu-BF3.

    Fig. S19. In vitro stability assay of 18F-Phe-BF3.

    Fig. S20. In vitro stability assay of 18F-Ala-BF3.

    Fig. S21. In vitro stability assay of 18F-Pro-BF3.

    Fig. S22. LAT-1–AdiC alignment.

    Fig. S23. Schematic representation of the binding site of LAT-1.

    Fig. S24. Predicted structure of the LAT-1–Phe complex.

    Fig. S25. Predicted structure of the LAT-1–Phe-BF3 complex.

    Fig. S26. Predicted binding modes for LAT-1 ligands: Phe and Phe-BF3.

    Fig. S27. Predicted structure of the LAT-1–Leu complex.

    Fig. S28. Predicted structure of the LAT-1–Leu-BF3 complex.

    Fig. S29. Predicted binding modes for LAT-1 ligands.

    Fig. S30. The 18F-Leu-BF3 uptake in the presence of different concentrations of nonradioactive Leu-BF3.

    Fig. S31. The 18F-Phe-BF3 uptake was measured at 0.3, 1, 3, 10, 30, 100, and 300 μM in PBS and plotted against the concentration of Phe-BF3.

    Fig. S32. The 18F-Ala-BF3 uptake was measured at 1, 3, 10, 30, 100, 300, and 1000 μM in PBS and plotted against the concentration of Ala-BF3.

    Fig. S33. The 18F-Pro-BF3 uptake was measured at 10, 30, 100, 400, 800, 1500, 2000, 3000, 6000, and 10,000 μM in PBS and plotted against the concentration of Pro-BF3.

    Fig. S34. The biodistribution of 18F-Phe-BF3 with U87MG-bearing mice.

    Fig. S35. 3D projection PET images of 18F-FDG (left) and 18F-Phe-BF3 (right) in mice bearing U87MG xenografts and inflammation (h, heart; t, tumor; i, inflammation; b, bladder; gb, gallbladder).

    Fig. S36. Representative coronal 18F-FDG (left) and 18F-Phe-BF3 (right) PET images in mice bearing U87MG xenografts and inflammation (h, heart; t, tumor; i, inflammation; b, bladder; gb, gallbladder).

    Fig. S37. 18F-Phe-BF3 is stable at in vivo conditions.

    Table S1. A brief summary of 18F-AAs and proposed 18F-BAAs.

    Table S2. Summary of the half-lives of BAAs.

    Table S3. Structure of BAAs studied in this work and their counterpart AAs.

    Table S4. Structure of AAs and their predicted ΔGbinding and Ki with LAT-1.

  • Supplementary Materials

    This PDF file includes:

    • Scheme S1. General synthetic routes of α-amino boronic acid/ester derivatives with high enantiopurity.
    • Scheme S2. BAAs are converted from BAA/ester in quantitative yield.
    • Scheme S3. 18F-BAAs are radiosynthesized via one-step 18F-19F isotope exchange reaction.
    • Fig. S1. Radioactive HPLC chromatogram for the purification of Leu-BF3.
    • Fig. S2. The LC-HRMS spectrum of HPLC-purified Leu-BF3.
    • Fig. S3. 19F NMR spectrum of HPLC-purified Leu-BF3 (δ = −148.95 ppm).
    • Fig. S4. 1H NMR spectrum of HPLC-purified Leu-BF3.
    • Fig. S5. Radioactive HPLC chromatography of preparing Phe-BF3.
    • Fig. S6. The LC-HRMS spectrum of HPLC-purified Phe-BF3.
    • Fig. S7. 19F NMR spectrum of HPLC-purified Phe-BF3 (δ = −151.96 ppm).
    • Fig. S8. 1H NMR spectrum of HPLC-purified Phe-BF3.
    • Fig. S9. Radioactive HPLC chromatography of preparing Ala-BF3.
    • Fig. S10. The LC-HRMS spectrum of HPLC-purified Ala-BF3.
    • Fig. S11. 19F NMR spectrum of HPLC-purified Ala-BF3 (δ = −149.11 ppm).
    • Fig. S12. 1H NMR spectrum of HPLC-purified Ala-BF3.
    • Fig. S13. Radioactive HPLC chromatography of preparing Pro-BF3.
    • Fig. S14. The LC-HRMS spectrum of HPLC-purified Pro-BF3.
    • Fig. S15. 19F NMR spectrum of HPLC-purified Pro-BF3 (δ = −149.02 ppm).
    • Fig. S16. 1H NMR spectrum of HPLC-purified Pro-BF3.
    • Fig. S17. Radioactive HPLC chromatography of Sep-Pak purified Leu-BF3, Phe-BF3, Ala-BF3, and Pro-BF3, respectively (from top to bottom).
    • Fig. S18. In vitro stability assay of 18F-Leu-BF3.
    • Fig. S19. In vitro stability assay of 18F-Phe-BF3.
    • Fig. S20. In vitro stability assay of 18F-Ala-BF3.
    • Fig. S21. In vitro stability assay of 18F-Pro-BF3.
    • Fig. S22. LAT-1–AdiC alignment.
    • Fig. S23. Schematic representation of the binding site of LAT-1.
    • Fig. S24. Predicted structure of the LAT-1–Phe complex.
    • Fig. S25. Predicted structure of the LAT-1–Phe-BF3 complex.
    • Fig. S26. Predicted binding modes for LAT-1 ligands: Phe and Phe-BF3.
    • Fig. S27. Predicted structure of the LAT-1–Leu complex.
    • Fig. S28. Predicted structure of the LAT-1–Leu-BF3 complex.
    • Fig. S29. Predicted binding modes for LAT-1 ligands.
    • Fig. S30. The 18F-Leu-BF3 uptake in the presence of different concentrations of nonradioactive Leu-BF3.
    • Fig. S31. The 18F-Phe-BF3 uptake was measured at 0.3, 1, 3, 10, 30, 100, and 300 μM in PBS and plotted against the concentration of Phe-BF3.
    • Fig. S32. The 18F-Ala-BF3 uptake was measured at 1, 3, 10, 30, 100, 300, and 1000 μM in PBS and plotted against the concentration of Ala-BF3.
    • Fig. S33. The 18F-Pro-BF3 uptake was measured at 10, 30, 100, 400, 800, 1500, 2000, 3000, 6000, and 10,000 μM in PBS and plotted against the concentration of Pro-BF3.
    • Fig. S34. The biodistribution of 18F-Phe-BF3 with U87MG-bearing mice.
    • Fig. S35. 3D projection PET images of 18F-FDG (left) and 18F-Phe-BF3 (right) in mice bearing U87MG xenografts and inflammation (h, heart; t, tumor; i, inflammation; b, bladder; gb, gallbladder).
    • Fig. S36. Representative coronal 18F-FDG (left) and 18F-Phe-BF3 (right) PET images in mice bearing U87MG xenografts and inflammation (h, heart; t, tumor; i, inflammation; b, bladder; gb, gallbladder).
    • Fig. S37. 18F-Phe-BF3 is stable at in vivo conditions.
    • Table S1. A brief summary of 18F-AAs and proposed 18F-BAAs.
    • Table S2. Summary of the half-lives of BAAs.
    • Table S3. Structure of BAAs studied in this work and their counterpart AAs.
    • Table S4. Structure of AAs and their predicted ΔGbinding and Ki with LAT-1.

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