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Research in Inorganic Biochemistry:
Vanadium Analogues as Orally Active Insulin Enhancing Agents
Matthew Ward, Assistant Professor, Natural Sciences
Background
It is estimated that nearly 300 million people by the year 2025 will suffer from diabetes mellitus. The condition of diabetes mellitus is characterized by abnormal glucose tolerance with a tendency to hyperglycemia and is a result of a relative or absolute deficiency of insulin. Two types of diabetes mellitus have thus far been identified. Type 1 or insulin dependent diabetes mellitus (IDDM) has an acute onset and typically affects the younger population; patients of Type 1 do not produce the insulin hormone, requiring the administration of daily injections of the hormone to control blood glucose levels. Type II or non-insulin dependent diabetes mellitus (NIDDM) has a gradual onset and is the most common form of diabetes in human adults; patients of Type II produce the insulin hormone but the body is unable to properly use it, initially requiring the administration of oral hypoglycemic drugs but eventually requiring insulin injections to control glucose levels in the blood stream. If blood glucose levels remain uncontrolled, diabetic patients are subject to numerous pathologies associated with the disease, including neuropathy, retinopathy, nephropathy and arterial disease that ultimately leads to death. The hormone Insulin is essential in controlling fat and carbohydrate metabolism. Insulin also serves to counteract catabolic hormones and suppress glucose production in the liver. The normal uptake and metabolism of glucose is initiated by a series of intracellular reactions known as the insulin-signaling cascade [1]. The binding of insulin to the extra-cellular side of the cell membrane at the insulin receptor site triggers a series of phosphorylation/dephosphorylation steps in the insulin-signaling cascade, leading to the control of blood glucose levels. Insulin is secreted by the pancreas in response to elevated levels of glucose; promoting glucose uptake by the liver, splanchnic tissue and adipose and muscle tissues; resulting in energy production and storage [2]. The absence of secreted insulin (Type I) or cellular resistance to the hormone (Type II) leads to inadequate disposal of blood glucose and to the numerous pathologies associated with the disease, diabetes mellitus. The oral ingestion of insulin to offset the lack of production or resistance to the hormone, however, produces a biologically inactive compound unable to ameliorate blood glucose levels. Therefore, diabetic patients are required to inject insulin daily to control blood glucose levels and to avoid the harmful pathologies associated with the disease. Inorganic salts of vanadium have long been known to act as orally viable mimics or enhancing agents for increased activity of insulin in vitro and in vivo. Oral doses of sodium metavanadate to diabetic patients resulted in a decrease of glucosuria in 2 out of 3 patients was first reported over 100 years ago by Lyonnet et al. [3]. This result remained relatively unnoticed until a more recent study in 1979 by Tolman et al. [4] demonstrated that a millimolar addition of sodium metavanadate to fat cells stimulated glucose uptake and inhibited lipid breakdown in a tissue specific manner, similar to insulin. Since this report, the research in this field has focused on developing new vanadium compounds to mimic insulin activity and determine the role vanadium compounds play in the insulin-signaling cascade. The search for new vanadium-containing compounds that enhance the activity of insulin as orally administered drugs has primarily focused on three general classes of compounds: (1) inorganic vanadium salts, both anionic (vanadates [VO4]3-) and cationic (vanadyl VO2+), (2) complexes resulting from the combination of vanadium(V) and hydrogen peroxide (mono- and diperoxovanadates, [VO(O2)(H2O)2(L-L′)]n- (n = 0,1) and [VO(O2)2(L-L′)]n- (n = 1, 2, 3), and (3) chelated oxovanadium(IV) complexes. Although inorganic vanadium salts of class (1) have been successful at enhancing the activity of insulin, the poor in vivo absorption and high dose requirement, resulting in increased toxicity, have led to further investigations of the more organic-vanadium compounds of class (2) and (3). Various model compounds have been reported in the recent literature; comparing the activity, biocompatibility, and stability of these class (2) and (3) organic-vanadium compounds that have shown enhanced insulin activity [5-10]. Amavandin was discovered as the vanadium-containing compound in the mushroom Aminita muscoria [11]. The members of this genus accumulate vanadium to levels of up to 400 times those typically found in plants [12]. Amavandin was first thought to contain an oxovanadium(IV) center coordinated to the pro-ligand (S,S)-2,2′-(hydroxyimino)dipropionic acid. Subsequent studies have shown the structure of amavandin and its analogues consist of an octa-coordinate V4+ (or V5+ analogue) center with two coordinated pro-ligands [12]. Amavandin coordination occurs from both ligands via an η2 N-O unit and the two-deprotonated carboxylates [12]. Although the biological role of amavandin remains elusive, it is postulated that it can act as an electron-transfer catalyst or mediator, mediating the electrocatalytic oxidation of thiols [13,14] and phenols [14]. Amavandin-analogues and ligands derived from the pro-ligand (S,S)-2,2′-(hydroxyimino)dipropionic acid should generate very stable, “pseudo-natural” complexes that act as carriers of vanadium with exceptional cell tolerance and that enhance the activity of insulin in vitro and in vivo. The research goals are: • To understand bonding models as applied to inorganic compounds. • To use the current scientific literature to identify potential target compounds. • To design, synthesize and characterize a new series of ligands to promote greater stereochemical flexibility and enhanced stability of the vanadium complexes. • To model the stability of the complexes using modern computer techniques. • To structurally characterize the new vanadium complexes by standard techniques. • To coordinate the new ligands to oxovanadium(IV) and dioxovanadium(V). • To present the research outcomes at the Daemen College Academic Festival.
Proposed Work for Fall 2004 & Spring 2005
With the success of the class (3) organic-oxovanadium(IV) complexes and the recent discovery of the first organic vanadium(V) insulin-mimetic agent, the proposed work will focus on developing a new series of vanadium compounds, both oxo-vanadium(IV) and the bisoxovanadium(V), that could be tested in vitro and in vivo for enhancement in the activity of insulin in lowering blood glucose levels. The new class of compounds will be designed to have a more stereochemically flexible structure and should offer greater stability in a more physiologically suitable pH range. The new series of ligands are designed from amavandin and its analogues, with (S,S)- 2,2’-(hydroxyimino)dipropionic acid type ligands. The newly designed ligands are broken into three series; Series 1: hydroxyamines, Series 2: hydroxyalkylamines, and Series 3: hydroxyaryl-amines, Figure 1. Each series is designed to add stability through successive replacement of the carboxylate arm with a pyridyl arm. The solution state and solid-state properties of one hydroxyalkylamine has been previously reported for both oxovanadium(IV) and bisoxovanadium(V) [15]; however, no further tests into the in vitro or in vivo insulin-mimetic properties were conducted. It is therefore likely the desired complexes can be easily synthesized (Scheme 1), characterized, and tested for insulin enhancing activity. Computer Modeling Molecular modeling software will be used to aid in the characterization of the new complexes and to help determine future complexes to synthesize.

Expected Outcome
Vanadium compounds have shown the ability to mimic or enhance the activity of insulin in vitro and in vivo in a variety of coordination motifs. The newly design ligands provide a
stable, “pseudo-natural” environment that can act as a carrier of vanadium with exceptional cell
tolerance. The coordination of the designed ligands with the replacement of the carboxylate arms
with pyridyl arms should offer advantages in greater pH stability and cell tolerance. Model
ligands have been previously synthesized and shown to coordinate to both oxovanadium(IV) and
bis oxovanadium(V), providing the general synthetic scheme and general characterization
outcome for the work proposed. The work proposed is likely to succeed and should be
publishable in refereed journals and/or presentable to the scientific community at local, regional
or national meetings.
Budget: $1500.00
A budget of $1500 is requested to financially support the research.
Description
Daemen College Faculty Research Funding:

The research has been generously supported by the Daemen College Faculty Research Fund. The
monies provided have been used to purchase the chemicals and software package needed to
perform the research.

Budget: $2000.00
Description
Spartan ’04 for Windows (Academic Price): Molecular Modeling Software to perform the modeling studies proposed. Funds needed to purchase instrument time from the University at Buffalo or Buffalo State College (instruments not at Daemen College). Funds needed to purchase chemicals and glassware necessary to synthesize desired References
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