title: Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications.
abstract: High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California at San Francisco, San Francisco, California 94143-0446, USA.
authors.forename: Zhaohua
authors.initials: Z
authors.lastname: Huang
authors.forename: Joshua I
authors.initials: JI
authors.lastname: Park
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Watson
authors.forename: Peter
authors.initials: P
authors.lastname: Hwang
authors.forename: Francis C
authors.initials: FC
authors.lastname: Szoka
mesh_terms: D001710:Biotin / Q000737:chemistry; D043525:Cytosine Deaminase / Q000737:chemistry / Q000378:metabolism; D016503:Drug Delivery Systems; D019793:Fluorescein / Q000737:chemistry; D008081:Liposomes; D015394:Molecular Structure; D009571:Nitrilotriacetic Acid / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D008081:Liposomes; D001710:Biotin; D043525:Cytosine Deaminase; D009571:Nitrilotriacetic Acid; D019793:Fluorescein