TrifluoroacetylHYNIC-peptides have recently been shown to label directly with 99mTc as efficiently as their non-trifluoroacetylated analogs. In this work, the trifluoroacetyl (Tfa) moiety has been evaluated as a protecting group for HYNIC against reaction with strong electrophiles. Fmoc-(trifluoroacetylHYNIC)-lysine, the chosen model starting material, was found to be resistant against acetaldehyde and benzylchloroformate challenges, at 1molequiv and a 1000M excess, respectively. In contrast, the Fmoc-(HYNIC)-lysine derivative, with a free hydrazine group, was quantitatively converted to the corresponding hydrazone after a 1h incubation with acetaldehyde. Fmoc-(trifluoroacetylHYNIC)-lysine was also found to be stable over a wide pH range (3.6–10) to the acetaldehyde challenge. High efficiency 99mTc-radiolabelling (99%) was achieved in the presence of acetaldehyde using Fmoc-(trifluoroacetylHYNIC)-lysine, as compared to a poor radiolabelling yield (34%) obtained with the non-trifluoroacetylated analog. These findings firmly establish the trifluoroacetyl group as a convenient and effective protecting group for HYNIC, and as a promising alternative to currently available labelling strategies.

Electrospray mass spectrometry (ESMS) of certain peptides labelled with 99mTc via hydrazinonicotinamide (HYNIC) with tricine as co-ligand shows one Tc-bound tricine, whereas typically two are observed. We speculated that this was due to coordination of a neighbouring histidine (His) or glutamate (Glu). To investigate this possibility, several short peptides incorporating lysine (HYNIC), with and without His and Glu at different positions in the sequence, were radiolabelled with 99mTc, using tricine, ethylenediaminediacetic acid (EDDA) and nicotinic acid as co-ligands. The products were examined by HPLC-ESMS, cysteine challenge and bovine serum albumin (BSA) challenge. Peptides with His nearby on either side of lysine (HYNIC) contained only one tricine and showed markedly enhanced structural homogeneity and stability to cysteine challenge and BSA binding, except those with His located at the N-terminus. Peptides without His, or with neighbouring N-terminal His, contained two tricines and were less stable to cysteine challenge and BSA binding. Glu participated in Tc-binding but did not enhance stability. We conclude that neighbouring His or Glu side chains coordinate to Tc and this could alter peptide or protein conformation. Inclusion of His in a neighbouring position to lysine (HYNIC) enhances stability, improves homogeneity and reduces the demand of the metal center for binding to additional co-ligands.

The aim of this study was to determine the effects of assisted coordination by amino acids such as histidine and glutamic acid on the function of Tc-99m-labeled gastrin peptide-hydrazinonicotinamide (HYNIC) conjugates and their ability to target cholecystokinin-R in small-animal models. Methods: Three peptide-HYNIC conjugates containing the -AYGWMDF-NH2 C-terminal sequence and combinations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with Tc-99m/Tc-99 using either tricine or ethylenediaminediacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography-mass spectrometric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed. Results: When radiolabeling was performed using tricine as a coligand, the insertion of a histidine residue near the HYNIC residue resulted in the displacement of one molecule of tricine from the coordination sphere, a reduction in the number of radiolabeled species formed, an improvement in the in vitro stability, an increase in the rate of radiopeptide internalization, and a significant improvement in tumor uptake in vivo. When radiolabeling was performed using ethylenediaminediacetic acid as a coligand, no effect on coligand binding, homogeneity, or in vitro stability was observed but a significant improvement in the internalization in vitro and tumor uptake in vivo was again found. All of the complexes formed showed similar receptor affinity in competitive radioligand binding assays. Conclusion: The insertion of histidine into the sequence of peptide-HYNIC conjugates can result in more stable, more homogeneous complexes that show improvements in tumor-targeting performance both in vitro and in vivo.

The aim of this study was to determine the effects of assisted coor- dination by amino acids such as histidine and glutamic acid on the function of 99m Tc-labeled gastrin peptide-hydrazinonicotinamide (HYNIC) conjugates and their ability to target cholecystokinin-R in small-animal models. Methods: Three peptide-HYNIC conjugates containing the -AYGWMDF-NH2 C-terminal sequence and com- binations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with 99mTc/99Tc using either tricine or ethylenediamine- diacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography-mass spectro- metric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed. Re- sults: When radiolabeling was performed using tricine as a coli- gand, the insertion of a histidine residue near the HYNIC residue resulted in the displacement of one molecule of tricine from the co- ordination sphere, a reduction in the number of radiolabeled species formed, an improvement in the in vitro stability, an increase in the rate of radiopeptide internalization, and a significant improvement in tumor uptake in vivo. When radiolabeling was performed using ethylenediaminediacetic acid as a coligand, no effect on coligand binding, homogeneity, or in vitro stability was observed but a signif- icant improvement in the internalization in vitro and tumor uptake in vivo was again found. All of the complexes formed showed similar receptor affinity in competitive radioligand binding assays. Conclu- sion: The insertion of histidine into the sequence of peptide-HYNIC conjugates can result in more stable, more homogeneous com- plexes that show improvements in tumor-targeting performance both in vitro and in vivo.

The trifluoroacetyl group protects the nucleophilic HYNIC from unwanted side reactions yet allows radiolabelling with technetium-99m under mild conditions, due to efficient and specific promotion of deprotection by a technetium complex intermediate.

Fmoc-lys(HYNIC-Boc)-OH, a precursor for solid-phase synthesis of 99mTc-labeled peptides, was synthesized efficiently without HPLC purification. HPLC-ESMS showed that deprotection and decoupling of peptide from the resin with trifluoroacetic acid gave initially HYNIC-peptide, which was trifluoroacetylated upon prolonged incubation. The trifluoroacetyl-HYNIC group was hydrolyzed during 99mTc labeling, rendering deprotection unnecessary. Trifluoroacetyl-HYNIC peptide was 99mTc-labeled as efficiently, producing the same product, as HYNIC-peptide. These modifications enhance the versatility of HYNIC for 99mTc peptide labeling.

Hydrazinonicotinamide (HYNIC) is an established bifunctional complexing agent for technetium-99m ((99m)Tc) but the structure of the technetium coordination sphere remains uncertain. To gain further insight into this, we have prepared conjugates of HYNIC and hydrazinobenzoic acid (HYBA) with a model peptide, and radiolabelled them with (99m)Tc using three well-established co-ligand systems: EDDA, tricine and tricine-nicotinic acid. The labelled peptides were studied by LC-MS and by subjecting them to serum stability and protein binding assays. For each co-ligand system, HYNIC conjugates formed fewer and more stable labelled species than the corresponding HYBA conjugates. LC-MS analysis showed that all conjugates contained one hydrazine moiety bound to Tc, that binding of Tc to HYNIC-peptide and co-ligand occurs with displacement of 5H(+) indicating a Tc formal oxidation state of +5, and that the Tc has no oxo- or halide ligands. LC-MS also shows that complexes formed with the HYNIC conjugate contain fewer coordinating co-ligand molecules than the HYBA conjugate indicating that HYNIC is able to more effectively satisfy the coordination requirement of technetium, perhaps by binding in chelating mode.

Inositol monophosphatase is widely held to be the therapeutic target for inhibition by lithium ion in the treatment of bipolar disorder. In a continued effort to improve the bioavailability of alternative inhibitors, we have designed and tested two new series of compounds; phosphonates and product-like mimics. Phosphonate substrate mimics were competitive inhibitors of reduced potency as compared to phosphate based inhibitors. Product mimics however, showed various inhibitory modes of action. The 6-butylamino derivative 6p was an uncompetitive inhibitor when acting alone (Ki = 0.3 mM) but displayed non-competitive inhibition in the presence of inorganic phosphate. This compound represents a new lead in the search for a viable replacement for lithium ion therapy.

A series of product-like inhibitors of inositol monophosphatase have been prepared and tested for activity in vitro as possible leads for treatment of bipolar disorders. Compounds possessing a 6-alkyloxy side chain were inhibitors but less efficacious than those possessing a 6-aminoalkyl side chain. These new structures show promise as inhibitors possessing the bioavailability and characteristics necessary for drug development. (C) 2003 Elsevier Ltd. All rights reserved.

Pd/C and Pd(OH)(2)/C catalysts are both poisoned by bis-benzyl protected 6-aminocyclitols thereby inhibiting the hydrogenolysis of benzyl ethers but removing N-Cbz groups chemoselectively. This outcome was unaltered by the use of different hydrogen donors. Replacement of the C-6 nitrogen atom by oxygen resulted in the starting materials being fully deprotected under similar conditions. These findings add cyclitolamines to the list of amines/bases that are poisonous to Pd/C catalysts during hydrogenolysis. (C) 2003 Elsevier Ltd. All rights reserved.