title: Radioiodinated O(6)-Benzylguanine derivatives containing an azido function.
abstract: BACKGROUND
  Drug resistance to alkylator chemotherapy has been primarily attributed to the DNA repair protein alkylguanine-DNA alkyltransferase (AGT); thus, personalizing chemotherapy could be facilitated if tumor AGT content could be quantified prior to administering chemotherapy. We have been investigating the use of radiolabeled O(6)-benzylguanine (BG) analogues to label and quantify AGT in vivo. BG derivatives containing an azido function were sought to potentially enhance the targeting of these analogues to AGT, which is primarily present in the cell nucleus, either by conjugating them to nuclear localization sequence (NLS) peptides or by pretargeting via bio-orthogonal approaches.
  
  
  METHODS
  Two O(6)-(3-iodobenzyl)guanine (IBG) derivatives containing an azido moiety-O(6)-(4-azidohexyloxymethyl-3-iodobenzyl)guanine (AHOMIBG) and O(6)-(4-azido-3-iodobenzyl)guanine (AIBG)--and their tin precursors were synthesized in multiple steps and the tin precursors were converted to radioiodinated AHOMIBG and AIBG, respectively. Both unlabeled and radioiodinated AHOMIBG analogues were conjugated to alkyne-derivatized NLS peptide heptynoyl-PK(3)RKV. The ability of these radioiodinated compounds to bind to AGT was determined by a trichloroacetic acid precipitation assay and gel electrophoresis/phosphor imaging. Labeling of an AGT-AIBG conjugate via Staudinger ligation using the (131)I-labeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[(131)I]iodobenzoate, also was investigated.
  
  
  RESULTS
  [(131)I]AHOMIBG was synthesized in two steps from its tin precursor in 52.2 ± 7.5% (n = 5) radiochemical yield and conjugated to the NLS peptide via click reaction in 50.7 ± 4.9% (n = 6) yield. The protected tin precursor of AIBG was radioiodinated in an average radiochemical yield of 69.6 ± 4.5% (n = 7); deprotection of the intermediate gave [(131)I]AIBG in 17.8 ± 4.2% (n = 9) yield. While both [(131)I]AHOMIBG and its NLS conjugate bound to AGT pure protein, their potency as a substrate for AGT was substantially lower than that of [(125)I]IBG. Uptake of [(131)I]AHOMIBG-NLS conjugate in DAOY medulloblastoma cells was up to eightfold higher than that of [(125)I]IBG; however, the uptake was not changed when the cellular AGT content was first depleted with BG treatment. [(131)I]AIBG was almost equipotent as [(125)I]IBG with respect to binding to pure AGT; however, attempts to radiolabel AGT by treatment with unlabeled AIBG followed by Staudinger ligation using the radiolabeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[(131)I]iodobenzoate were not successful.
  
  
  CONCLUSIONS
  Although AHOMIBG, and AIBG were synthesized successfully in both unlabeled and radioiodinated forms, the radioiodinated compounds failed to label AGT either after NLS peptide conjugation or via Staundiger ligation. Currently, other bio-orthogonal approaches are being evaluated for labeling AGT by pretargeting.
authors.affiliation: Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710, USA. ganesan.v@duke.edu
authors.forename: Ganesan
authors.initials: G
authors.lastname: Vaidyanathan
authors.forename: Benjamin
authors.initials: B
authors.lastname: White
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Affleck
authors.forename: Darryl
authors.initials: D
authors.lastname: McDougald
authors.forename: Michael R
authors.initials: MR
authors.lastname: Zalutsky
mesh_terms: D019797:3-Iodobenzylguanidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000737:chemistry*; D045744:Cell Line, Tumor; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry; D019913:Nuclear Localization Signals / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D011854:Radiochemistry
chemical_list: D001386:Azides; D007457:Iodine Radioisotopes; D019913:Nuclear Localization Signals; C064976:O(6)-benzylguanine; C049322:4-amino-3-iodobenzylguanidine; D019797:3-Iodobenzylguanidine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase