Palaniappa Nanjappan

Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

An efficient synthesis of some 6-substituted 4,8-diaza-3,3,9,9- tetramethylundeca-2,10-dione dioximes (propylene amine oximes, PnAOs): Ligands for 99mTc complexes used in structure distribution relationship (SDR) studies

Abstract Technetium complexes of the ligand PnAO [4,8-diaza-3,3,9,9-tetramethylundeca-2,10-dione dioximes (3)] are of interest as commercial radiopharmaceuticals. In general, PnAOs are synthesized by alkylation of a propylenediamine derivative with 3-chloro-3-methyl-2-nitrosobutane (2). This alkylation reaction proved to be low yielding. With modestly bulky substituents at the 2-position of 1,3-diaminopropane, little or none of the required PnAO was obtained. As a result, an alternative approach of the synthesis of PnAO was developed. This method involved the alkylation of the propylenediamine with 3-bromo-3-methylbutan-2-one (18) followed by oximation of the resulting diamine-diketone (19). By this method, PnAOs were prepared in good yield, even with bulky C-2 substituents. Fourteen PnAO derivatives were prepared by this method. We also describe the syntheses of several new propylenediamine derivatives.

SYNTHESIS OF FUNCTIONALIZED 3,3,9,9-TETRAMETHYL-4,8-DIAZAUNDECANE-2,10-DIONE DIOXIMES (PROPYLENE AMINE OXIMES, PNAOS)

Abstract The syntheses of several new derivatives of PnAO and their chloronitroso precursors are described. The PnAOs were prepared by an improvement of the reported general procedure. An unexpected product, an isooxalone, was formed while attempting to prepare 1-carbomethoxyl-PnAO.

Separation of the stereoisomers of hexamethyl-propyleneamine oxime (HM-PAO) by high-performance liquid chromatography

Abstract The Resolvosil BSA-7, Chiracel OD, and Chiralpak AD columns were examined to determine whether one might be suitable for the separation of enatiomers of the ligand hexamethyl-propyleneamine oxime (HM-PAO) and/or its technetium-99m complex. Using hexane/IPA as eluent, partial separation (Rs = 0.96) of the d- and l-ligand enantiomers was achieved on the OD column, but as the meso-form of the ligand was eluted midway between the d- and l-enantiomers. There was excellent separation of the technetium-99m complexes of d- and 1-HM-PAO on the OD column, and partial separation (Rs = 0.90) of the meso- and d-complexes. Only partial resolution of the Tc-99m complexes of d,l- and meso-diastereoisomers was achieved on the AD column, but this column provided baseline resolution of all three stereoisomeric forms of the ligand. Thus, the OD column could be used for HPLC analysis of Tc-99m complexes of HM-PAO stereoisomers, while the AD column is suitable for analysis of the ligand stereoisomers.

Synthesis of the four stereoisomers of 1-azabicyclo[2.2.2]oct-3-yl-α-hydroxy-α-(4-phenylboronic acid)-α-phenylacetate (QNB-boronic acid), including a preparative HPLC method to separate diastereoisomeric mixtures with high optical purity

Abstract The four stereoisomers of 1-azabicyclo[2.2.2]oct-3-yl-α-hydroxy-α-(4-phenylboronic acid)-α-phenylacetale (QNB boronic acids, 1a-1d ) were synthesized initially via 1-azabicyclo[2.2.2]oct-3-yl-α-hydroxy-α-(4-iodophenyl)-α-phenylacetate (iodo-QNBs, 6a-6d) using an adaptation of a published procedure. The number of steps involved were reduced substantially using a distinctly different approach. This involved the synthesis of diastereoisomers of QNB-boronic acid, followed by separation and isolation of enantiomers by preparative reversed-phase HPLC. An improved method of synthesis of α-hydroxy-α-(4-aminophenyl)-α-phenyl-acetic acid ( 3a and 3b ) from α-hydroxy-α-(4-nitrophenyl)-α-phenylacetic acid ( 2a and 2b ) is also described.