György Kóczán

Conjugation of HS-Oligopeptides with Polymeric Branched Chain Polypeptides Containing Multiple Amino Groups:

For the preparation of bioconjugates containing polymeric polypeptides with well-defined structure and composition, we systematically studied 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester (SPDP). SPDP as amino- and thiol-reactive heterobifunctional coupling agent is mainly used for protein-based conjugates, and very little data are available on its application for the modification of polymers. In this communication, we describe the effect of polymer/oligopeptide structure and of the reaction condition on the incorporation of oligopeptides with free thiol group (CAVKDEL, CTGRGDSP) into polymeric polypeptides possessing multiple amino groups. For these studies, linear poly[L-lysine] with free e-amino groups and its XAK-type branched polypeptide derivatives {poly[Lys(X i -DL-Ala m )] (i < 1, m ∼ 3, XAK)} either with polycationic character {X =O (AK), X = Ser (SAK)} or with amphoteric nature {X = Glu (EAK)} were utilized. First, the polymers were modified with SPDP under various conditions, and the degree of substitution was determined. We found that the efficacy of the nucleophilic substitution of NH 2 groups with SPDP depended not only on the pH and the concentration of the coupling reagent but also on the polymer composition, mainly on the pK a of the branch-terminal amino group of the polymers. SPDP-modified polymeric polypeptides were reacted with the HS-oligopeptides, and the effects of polymer/oligopeptide structure as well as the reaction conditions (pH, peptide/(SSP)polymer molar ratio) on the composition of the product were evaluated. The results suggest that acidic pH is more favorable for the thiol-disulphide exchange, and the side chain composition of the polymers had a pronounced effect while the chemical structure of oligopeptides had only moderate influence on the average degree of substitution.

Cellular Uptake Mechanism of Cationic Branched Polypeptides with Poly[l-Lys] Backbone

Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes, or nucleic acids. Polylysine-based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study, we report our findings on the cellular uptake characteristics of nine structurally related polylysine-based polypeptides with cationic side chains composed of (i) single amino acid (poly[Lys(Xi)], XiK) or (ii) oligo[dl-alanine] (poly[Lys(dl-Alam)], AK) or (iii) oligo[dl-alanine] with an additional amino acid (X) at the terminal position (poly[Lys(Xi-dl-Alam)] (XAK)) or (iv) at the position next to the polylysine backbone (poly[Lys(dl-Alam-Xi)] (AXK)). In vitro cytotoxicity and cellular uptake were characterized on HT-29 human colon carcinoma and HepG2 human hepatocarcinoma cell lines. Data indicate that the polycationic polypeptides studied are essentially nontoxic in the concentration range studied, and their uptake is very much dependent on the side chain structure (length, identity of amino acid X, and distance between the terminal positive charges) and also on the cell lines. Our findings in uptake inhibition studies suggest that predominantly macropinocytosis and caveole/lipid raft mediated endocytosis are involved. The efficacy of their internalization is markedly influenced by the hydrophobicity and charge properties of the amino acid X. Interestingly, the uptake properties of the these polypeptides show certain similarities to the entry pathways of several cell penetrating peptides.

Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates

Methotrexate (MTX) conjugates with poly[Lys(DL-Alam)] based polymeric polypeptides are efficient against Leishmania donovani parasite infection, but the mechanism of the effect is not known yet. We prepared therefore the 5(6)-carboxyfluorescein (Cf) labeled oligopeptide [Cf-K(AaAa)] (a: D-alanine, A: L-alanine) and the corresponding MTX conjugates [Cf-K(MTX-AaAa)] as model compounds for structure–activity experiments. The conjugate aimed to be synthesized with solid phase methodology on MBHA resin with Boc strategy, using Fmoc-Lys(Boc)-OH. However, various side reactions were identified. Here we report three problems observed during the synthesis as well as solutions developed by us: (1) unexpected cyclopeptide-formation with the lactone-carboxylic group of the Cf was detected, when Cf was attached to the α-amino group of the Lys residue on solid phase. This was avoided by changing the order of Cf incorporation with using Fmoc/Dde strategy. Alternatively, we have built the peptide with Fmoc strategy on solid phase first and performed the labeling with Cf-OSu subsequently in solution. (2) During HF cleavage of the protected conjugates, MTX was demonstrated to form adducts with anisole and p-cresol scavengers, and the TMSOTf cleavage methodology was also found to be inadequate due to the large number of side products formed. We report here that using Fmoc/Dde strategy is an appropriate method to circumvent the cleavage with HF or TMSOTf. (3) During the coupling of MTX with oligopeptide, structural and stereo isomers are formed. We have described here the suitable conditions of HPLC separation of these products.