Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates

Abstract

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin-I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies.

Monofunctional Derivatives of Coproporphyrins for Phosphorescent Labeling of Proteins and Binding Assays

“…The platinum-and palladium-coproporphyrin I pisothiocyanatophenyl derivatives (PtCP-NCS, PdCP-NCS, shown in Scheme 1) were synthesized according to the described method (27). Mouse IgG (I-8765), rabbit anti-mouse IgG (M-7023), goat anti-rabbit-IgG-peroxidase conjugate (A-1949), avidin (A-9390), bovine serum albumin, poly(L-lysine) (MW 52.3 kDa), Triton X-100 (TX-100), Tween 20, cetyltrimethylammonium bromide (CTAB), PD-10 desalting columns, Sigma Fast o-phenylenediamine dihydrochloride tablets, dimethyl sulfoxide (DMSO), dimethyl formamide, biotin-NHS, and fluorecein isothiocyanate (FITC) were purchased from Sigma-Aldrich.…”

Peptide-Mediated Cell Transport of Water Soluble Porphyrin Conjugates

“…The biological efficacy of porphyrin-based sensitizers for the photodynamic therapy (PDT) , and the boron neutron capture therapy (BNCT) , of tumors depends on their efficient translocation across cellular membranes and delivery into specific organelles within cancer cells. For example, the amount of boron-10 needed for effective BNCT treatment has been estimated to be between 15 and 30 μg/g tumor, depending on the exact location of the boron atoms; 2−5 times less boron is required when it localizes preferentially near the cell nucleus rather than on the plasma membrane. , Several strategies have been developed to improve the selective delivery of porphyrin sensitizers to tumor tissues, including their conjugation to carrier proteins, − oligonucleotides, , monoclonal antibodies, − epidermal growth factors, , carbohydrates, , and hydrophilic polymers (such as HPMA, PVA, PEG, dextran, and polylysine). , The efficient delivery of such conjugates to cancer cells and their subcellular distribution does not only depend on cellular processes, such as transport across the plasma membrane, movement through the cytoplasm, and transport across organelle membranes, but also on the physicochemical properties and structural characteristics that these compounds exhibit. − Although enhanced cellular uptake and selectivity for tumor tissues have been achieved with some porphyrin sensitizers, most of these are usually found in cytoplasmic membranes rather than in sensitive intracellular sites, such as the mitochondria, the endoplasmic reticulum (ER), and the nuclei. One strategy currently used for drug delivery to these intracellular sites is based on the natural ability of certain proteins and peptide sequences for crossing cell membranes and specifically targeting these organelles.…”

Synthetic approaches for the conjugation of porphyrins and related macrocycles to peptides and proteins

“…57, 58 The sequences chosen were HIV-derived Tat (48-56), R9, and bactenectin 7-derived peptide (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). For the synthesis of the labelled peptides, two different ligation strategies were examined: one relied on the reaction of the amino-reactive isothiocyanate-bearing PtCP 52 59 with the N-terminus of the peptide, and the other on the reaction of maleimido Pt-CP 53 with the thiol group of a Cys appended at the N-termini of the sequences. The latter strategy was adopted when more than one Scheme 12 PpIX conjugation to GnRH agonist and antagonist described by Rahimipour et al 55 amino group was present on the peptide, as it is in the case of the Tat sequence (Fig.…”

“…In a later work, the synthesis of four porphyrin-peptide conjugates linked to bi-functional cell-penetrating/nuclear-localising sequences was described. 71 Amino-reactive porphyrin 78 was coupled to four hybrid sequences containing a cell penetrating string, Tat (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) or Antp (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), and a NLS, either the minimal sequence of nucleoplasmin or the SV40-derived GPKKKRKV. TBTU/HOBt/DIEA-mediated coupling on solid support, and TFA-mediated cleavage and deprotection afforded the desired species in 18 to 72% yields.…”