Radiometal-Containing Aryl Diazonium Salts for Chemoselective Bioconjugation of Tyrosine Residues

Abstract

Tyrosine is an attractive target for chemo- and site-selective protein modification. The particular chemical nature of tyrosine residues allows bioconjugation chemistry with reactive aryl diazonium salts via electrophilic aromatic substitution to produce diazo compounds. In this work, we describe the preparation of 64Cu- and 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-diazonium salts as building blocks for azo coupling chemistry with tyrosine and tyrosine-containing peptides and proteins under mild conditions. 2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA) was used to form the corresponding 64Cu- and 68Ga-labeled complexes, followed by diazotization with NaNO2 in the presence of HCl. 64Cu- and 68Ga-labeled NOTA complexes were prepared in high radiochemical yields >80% starting from 20 μg of p-NH2-Bn-NOTA. Conversion of p-NH2-Bn-NOTA complexes into diazonium salts followed by azo coupling with l-tyrosine afforded 64Cu- and 68Ga-labeled tyrosine in radiochemical yields of 80 and 56%, respectively. Azo coupling with tyrosine-containing hexapeptide neurotensin NT(8–13) afforded 64Cu- and 68Ga-labeled NT(8–13) in radiochemical yields of 45 and 11%, respectively. Azo coupling of 64Cu-labeled NOTA-diazonium salt with human serum albumin (HSA) gave 64Cu-labeled HSA in radiochemical yields of 20%. The described azo coupling chemistry represents an innovative and versatile bioconjugation strategy for selective targeting of tyrosine residues in peptides and proteins.

Tyrosine Conjugation Methods for Protein Labelling

“…Wuest and co‐workers also studied the chemoselectivity of the diazo‐coupling reaction by reacting radiometal‐diazo‐Phe species with an equimolar mixture of l ‐tyrosine and l ‐histidine. Radio‐HPLC analysis confirmed a better diazo‐coupling with Tyr at pH 9, whereas a preferential coupling to His was observed at neutral pH (pH 7) [70] . The genetic incorporation of more reactive handles than Tyr or His towards diazo‐coupling has been shown to efficiently circumvent these selectivity issues.…”

“…Radio-HPLC analysis confirmed a better diazo-coupling with Tyr at pH 9, whereas a preferential coupling to His was observed at neutral pH (pH 7). [70] The genetic incorporation of more reactive handles than Tyr or His towards diazo-coupling has been shown to efficiently circumvent these selectivity issues. The electron-rich 2-naphtol analogue of Tyr (NpOH) was genetically incorporated into proteins in E. coli by Tsao and co-workers ( Figure 18).…”

“…These successful Tyr modifications were achieved with arydiazonium reagents bearing a formyl group for oxime ligation, which also proved essential for the stability of the diazonium ion. Although direct peptide and protein ligation with aryldiazonium salts bearing PEGs, radionuclide complexes or grafted on polymethacrylate polymers has been reported, [69, 70] examples remain scarce and one‐step Tyr coupling by the diazo procedure seems to be limited to a restricted number of conjugates. Indeed, a major limitation of diazo coupling is the general instability of diazonium reagents, which are prepared in situ just prior to use and in strongly acidic conditions whereas the protein coupling is performed at mildly basic pH.…”

Catalyst-free site-selective cross-aldol bioconjugations

“…The field of protein bioconjugation has developed rapidly over the past three decades and a plethora of techniques now exist for modifying the side-chains of canonical amino acid residues such as lysine 1, 2 , tyrosine, 3,4 tryptophan, 5 methionine, [6][7][8] histidine, 9 and cysteine. 1,[10][11][12] However, the targeting of canonical amino acids in proteins is seldom site-specific, as multiple copies of the same amino acid may be solventexposed.…”

Bio‐Orthogonal Conjugation of a Cationic Metalloporphyrin to BSA and HSA via “Click” Chemistry

“…Under basic conditions, the phenolate group of tyrosine residues becomes active towards electrophilic substitution with moieties such as diazonium heterobifunctional linkers [14] . This nature has been exploited to yield radiolabelled proteins and polypeptides to high effect from pH 7–9 [15] . Historically, diazonium salts have enjoyed some promiscuity due to poor residue‐selectivity as the reaction between aryl diazo compounds and histidine amino acids are also possible [11] .…”