Biomedical subjects

Umesh R Desai

Publications and source records attributed to Umesh R Desai.

18 recordsLinked to original sources

PubMed2006

Novel chemo-enzymatic oligomers of cinnamic acids as direct and indirect inhibitors of coagulation proteinases.

Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers.

Anticoagulants↗

PubMed2006

Finding a needle in a haystack: development of a combinatorial virtual screening approach for identifying high specificity heparin/heparan sulfate sequence(s).

We describe a combinatorial virtual screening approach for predicting high specificity heparin/heparan sulfate sequences using the well-studied antithrombin-heparin interaction as a test case. Heparan sulfate hexasaccharides were simulated in the 'average backbone' conformation, wherein the inter-glycosidic bond angles were held constant at the mean of the known solution values, irrespective of their sequence. Molecular docking utilized GOLD with restrained inter-glycosidic torsions and intra-ring conformations, but flexible substituents at the 2-, 3-, and 6-positions and explicit incorporation of conformational variability of the iduronate residues. The approach reproduces the binding geometry of the sequence-specific heparin pentasaccharide to within 2.5 A. Screening of a combinatorial virtual library of 6,859 heparin hexasaccharides using a dual filter strategy, in which predicted antithrombin affinity was the first filter and self-consistency of docking was the second, resulted in only 10 sequences. Of these, nine were found to bind antithrombin in a manner identical to the natural pentasaccharide, while a novel hexasaccharide bound the inhibitor in a unique but dramatically different geometry and orientation. This work presents the first approach on combinatorial library screening for heparin/heparan sulfate GAGs to determine high specificity sequences and opens up huge opportunities to investigate numerous other physiologically relevant GAG-protein interactions.

Antithrombins↗

PubMed2005

Importance of tryptophan 49 of antithrombin in heparin binding and conformational activation.

Tryptophan 49 of antithrombin, the primary inhibitor of blood clotting proteinases, has previously been implicated in binding the allosteric activator, heparin, by chemical modification and mutagenesis studies. However, the X-ray cocrystal structure of the antithrombin-pentasaccharide complex shows that Trp49 does not contact the bound saccharide. Here, we provide a detailed thermodynamic and kinetic characterization of heparin binding to a Trp49 to Lys variant of antithrombin and suggest a model for how Trp49 participates in heparin binding and activation. Mutation of Trp49 to Lys resulted in substantial losses of 16-24% in heparin-binding energy at pH 7.4, I 0.15, and 25 degrees C. These losses were due to both the loss of one ionic interaction ( approximately 30%) and the loss of nonionic interactions ( approximately 70%). Rapid kinetics analyses showed that the mutation minimally affected the initial weak binding of heparin to antithrombin or the rate constant for the subsequent conformational activation of the serpin. Rather, the principal effect of the mutation was to increase the rate constant for reversal of the conformational activation step by 70-100-fold, thereby destabilizing the activated conformation. This destabilization could be accounted for by the disruption of a network of interactions involving Trp49, Glu50, and Lys53 of helix A and Ser112 of helix P, which stabilizes the activated conformation.

Amino Acid Sequence↗

PubMed2005

Mechanism of poly(acrylic acid) acceleration of antithrombin inhibition of thrombin: implications for the design of novel heparin mimics.

The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive approximately 2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA-dependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K(D) of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the "bridging" mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics.

Acrylic Resins↗

PubMed2005

Synthesis of per-sulfated flavonoids using 2,2,2-trichloro ethyl protecting group and their factor Xa inhibition potential.

The synthesis of per-sulfated flavonoids, organic compounds with multiple sulfate groups, is challenging. We present here a two-step synthesis of fully sulfated flavonoids in high overall yields using the 2,2,2-trichloroethyl moiety as a protecting group. The two-step synthesis results in exclusive formation of the per-sulfated product in contrast to common sulfating agents that yield differentially sulfated mixture of compounds. Most per-sulfated flavonoids studied are activators of antithrombin for accelerated inhibition of factor Xa, a key enzyme of the blood coagulation cascade. As a group the per-sulfated flavonoids possess a range of factor Xa inhibition potential.

Chromatography, High Pressure Liquid↗

PubMed2005

Antithrombin activation by nonsulfated, non-polysaccharide organic polymer.

Accelerated antithrombin inhibition of procoagulant enzymes has been exclusively achieved with polysulfated polysaccharides. We reasoned that antithrombin activation should be possible with nonsulfated activators based only on carboxylic acid groups. As a proof of the principle, linear poly(acrylic acid)s were found to bind to antithrombin and accelerate inhibition of factor Xa and thrombin. Our work demonstrates that molecules completely devoid of sulfate groups can activate antithrombin effectively and, more importantly, suggests that it may be possible to develop orally bioavailable, carboxylate-based antithrombin activators.

Acrylic Resins↗

PubMed2005

Capillary electrophoresis of highly sulfated flavanoids and flavonoids.

Flavanoids and flavonoids are natural products present in our diet and known to possess multiple biological activities. Sulfated species of these natural products represent highly charged water-soluble organic molecules that possess unique biochemical properties. We describe here the first studies on capillary electrophoresis of these highly charged molecules. Fully sulfated flavanoids and flavonoids can be electrophoresed and resolved under reverse polarity at pH 3.5 using 5-10 kV in less than 20 min. In contrast, at high pH under normal polarity these species can be electrophoresed only if a pressurized capillary is employed. (+/-)-Catechin sulfate, a racemic sulfated flavanoid, was resolved into its enantiomers using 15% beta-cyclodextrin, a chiral selector, but not with alpha- or gamma-cyclodextrins. Yet, the high charge density of these molecules challenges the resolving capability of capillary electrophoresis as diastereomers (-)-epicatechin sulfate and (+)-catechin sulfate do not resolve, even in the presence of cyclodextrins or chiral positively charged amino acids. Overall, capillary electrophoresis of highly sulfated flavanoids and flavonoids is expected to be useful in rapid structure analysis of sulfated flavonoids, either synthetic or natural.

Buffers↗

PubMed2004

Hydropathic interaction analyses of small organic activators binding to antithrombin.

Recently we designed the first small organic ligands, sulfated flavanoids and flavonoids, that act as activators of antithrombin for accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade [Gunnarsson and Desai, Bioorg. Med. Chem. Lett. (2003) 13:579]. To better understand the binding properties of these activators at a molecular level, we have utilized computerized hydropathic interaction (HINT) analyses of the sulfated molecules interacting in two plausible electropositive regions, the pentasaccharide- and extended heparin-binding sites, of antithrombin in its native and activated forms. HINT analyses indicate favorable multi-point interactions of the activators in both binding sites of the two forms of antithrombin. Yet, HINT predicts better interaction of most activators, except for (-)-catechin sulfate, with the activated form of antithrombin than with the native form supporting the observation in solution that these molecules function as activators of the inhibitor. Further, whereas (+)-catechin sulfate recognized the activated form of antithrombin better in both the pentasaccharide- and extended heparin- binding sites, the native form was better recognized by (-)-catechin sulfate, thus explaining its weaker binding and activation potential in solution. A reasonable linear correlation between the overall HINT score and the solution free energy of binding of the sulfated activators was evident. This investigation indicates that HINT is a useful tool in understanding interactions of antithrombin with small sulfated organic ligands at a molecular level, has some good predictive properties, and is likely to be useful for rational design purposes.

Amino Acids↗

PubMed2004

Roles of N-terminal region residues Lys11, Arg13, and Arg24 of antithrombin in heparin recognition and in promotion and stabilization of the heparin-induced conformational change.

The N-terminal region residues, Lys11, Arg13, and Arg24, of the plasma coagulation inhibitor, antithrombin, have been implicated in binding of the anticoagulant polysaccharide, heparin, from the identification of natural mutants with impaired heparin binding or by the X-ray structure of a complex of the inhibitor with a high-affinity heparin pentasaccharide. Mutations of Lys11 or Arg24 to Ala in this work each reduced the affinity for the pentasaccharide approximately 40-fold, whereas mutation of Arg13 to Ala led to a decrease of only approximately 7-fold. All three substitutions resulted in the loss of one ionic interaction with the pentasaccharide and those of Lys11 or Arg24 also in 3-5-fold losses in affinity of nonionic interactions. Only the mutation of Lys11 affected the initial, weak interaction step of pentasaccharide binding, decreasing the affinity of this step approximately 2-fold. The mutations of Lys11 and Arg13 moderately, 2-7-fold, altered both rate constants of the second, conformational change step, whereas the substitution of Arg24 appreciably, approximately 25-fold, reduced the reverse rate constant of this step. The N-terminal region of antithrombin is thus critical for high-affinity heparin binding, Lys11 and Arg24 being responsible for maintaining appreciable and comparable binding energy, whereas Arg13 is less important. Lys11 is the only one of the three residues that is involved in the initial recognition step, whereas all three residues participate in the conformational change step. Lys11 and Arg13 presumably bind directly to the heparin pentasaccharide by ionic, and in the case of Lys11, also nonionic interactions. However, the role of Arg24 most likely is indirect, to stabilize the heparin-induced P-helix by interacting intramolecularly with Glu113 and Asp117, thereby positioning the crucial Lys114 residue for optimal ionic and nonionic interactions with the pentasaccharide. Together, these findings show that N-terminal residues of antithrombin make markedly different contributions to the energetics and dynamics of binding of the pentasaccharide ligand to the native and activated conformational states of the inhibitor that could not have been predicted from the X-ray structure.

Alanine↗

PubMed2004

New antithrombin-based anticoagulants.

Clinically used anticoagulants are inhibitors of enzymes involved in the coagulation pathway, primarily thrombin and factor Xa. These agents can be either direct or indirect inhibitors of clotting enzymes. Heparin-based anticoagulants are indirect inhibitors that enhance the proteinase inhibitory activity of a natural anticoagulant, antithrombin. Despite its phenomenal success, current anticoagulation therapy suffers from the risk of serious bleeding. The need for safer and more effective antithrombotic agents clearly exists. The past decade has seen enormous effort directed toward discovering and/or designing new molecules with anticoagulant activity. These new molecules can be classified into (a). antithrombin and its mutants, (b). natural polysaccharides, (c). synthetic modified heparins and heparin-mimics, (d). synthetic oligosaccharides, and (e). synthetic non-sugar antithrombin activators. This review focuses on these efforts in designing or discovering new molecules that act through the antithrombin pathway of anticoagulation.

Animals↗

PubMed2004

1,2-dithiole-3-ones as potent inhibitors of the bacterial 3-ketoacyl acyl carrier protein synthase III (FabH).

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 microM (ecFabH) and 0.16 microM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 microM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 microM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered.

Anti-Bacterial Agents↗

PubMed2003

Exploring new non-sugar sulfated molecules as activators of antithrombin.

New non-sugar, small, sulfated molecules, based on our de novo rationally designed activator (-)-epicatechin sulfate (ECS), were investigated to bind and activate antithrombin, an inhibitor of plasma coagulation enzyme factor Xa. For the activators studied, the equilibrium dissociation constant (K(D)) of the interaction with plasma antithrombin varies nearly 53-fold, with the highest affinity of 1.8 microM observed for morin sulfate, while the acceleration in factor Xa inhibition varies 2.6-fold. The results demonstrate that antithrombin binding and activation is a common property of these small sulfated molecules and suggests plausible directions for designing more potent activators.

Antithrombin III↗

PubMed2002

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin.

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.

Apoptosis↗

PubMed2002

Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators.

Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity ( approximately 10.7 microM) comparable to the reference trisaccharide DEF ( approximately 4.5 microM), it accelerated the inhibition of factor Xa only 10-fold as compared to the approximately 300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K(D) = 3.5 microM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M(-1) s(-1)). On the contrary, the K(D) and k(ACT) were found to be lower approximately 7.4- and approximately 2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion ( approximately 55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected approximately 4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.

Antithrombins↗

PubMed2002

Importance of lysine 125 for heparin binding and activation of antithrombin.

The anticoagulant sulfated polysaccharide, heparin, binds to the plasma coagulation proteinase inhibitor, antithrombin, and activates it by a conformational change that results in a greatly increased rate of inhibition of target proteinases. Lys125 of antithrombin has previously been implicated in this binding by chemical modification and site-directed mutagenesis and by the crystal structure of a complex between antithrombin and a pentasaccharide constituting the antithrombin-binding region of heparin. Replacement of Lys125 with Met or Gln in this work reduced the affinity of antithrombin for full-length heparin or the pentasaccharide by 150-600-fold at I = 0.15, corresponding to a loss of 25-33% of the total binding energy. The affinity decrease was due both to disruption of approximately three ionic interactions, indicating that Lys125 and two other basic residues of antithrombin act cooperatively in binding to heparin, and to weakened nonionic interactions. The mutations caused a 10-17-fold decrease in the affinity of the initial, weak binding step of the two-step mechanism of heparin binding to antithrombin. They also increased the reverse rate constant of the second, conformational change step by 10-50-fold. Lys125 is thus a major heparin-binding residue of antithrombin, contributing an amount of binding energy comparable to that of Arg129, but less energy than Lys114. It is the first residue identified so far that has a critical role in the initial recognition of heparin by antithrombin, but also appreciably stabilizes the heparin-induced activated state of the inhibitor. These effects are exerted by interactions of Lys125 with the nonreducing end of the heparin pentasaccharide.

Amino Acid Sequence↗

PubMed2002

Designing small, nonsugar activators of antithrombin using hydropathic interaction analyses.

Conformational activation of antithrombin is a critical mechanism for the inhibition of factor Xa, a proteinase of the blood coagulation cascade, and is typically achieved with heparin, a polyanionic polysaccharide clinically used for anticoagulation. Although numerous efforts have been directed toward the design of better activators, a fundamental tenet of these studies has been the assumed requirement of an oligo- or a polysaccharide backbone. We demonstrate here a concept that small nonsaccharidic nonpolymeric molecules may be rationally designed to interact with and activate antithrombin for enhanced inhibition of factor Xa. The rational design strategy is based on a study of complexes of natural and mutant antithrombins with heparin-based oligosaccharides using hydropathic interaction (HINT) technique, a quantitative computerized tool for analysis of molecular interactions. A linear correlation was observed between the free energy of binding for antithrombinminus signoligosaccharide complexes and the HINT score over a wide range of approximately 13 kcal/mol, indicating strong predictive capability of the HINT technique. Using this approach, a small, nonsugar, aromatic molecule, (minus sign)-epicatechin sulfate (ECS), was designed to mimic the nonreducing end trisaccharide unit DEF of the sequence specific heparin pentasaccharide DEFGH. HINT suggested a comparable antithrombin-binding geometry and interaction profile for ECS and trisaccharide DEF. Biochemical studies indicated that ECS binds antithrombin with equilibrium dissociation constants of 10.5 and 66 microM at pH 6.0, I 0.025, and pH 7.4, I 0.035, respectively, that compare favorably with 2 and 80 microM observed for the natural activator DEF. ECS accelerates the antithrombin inhibition of factor Xa nearly 8-fold demonstrating for the first time that conformational activation of antithrombin is feasible with appropriately designed small nonsugar organic molecules. The results present unique opportunities for de novo activator design based on this first-generation lead.

Antithrombins↗

PubMed2002

Probing reactive center loop insertion in serpins: a simple method for ovalbumin.

Insertion of the reactive center loop in beta-sheet A in serpins has been typically inferred from the increased stability of the cleaved form to thermal- and urea-induced denaturation. We describe a convenient and rapid fluorescence-based method that differentiates the loop-inserted form from the loop-exposed form in ovalbumin, a prototypic noninhibitory serpin. Recombinant wild-type and R345A ovalbumins in the intact form bind ANS with equilibrium dissociation constants of 116 and 125 microM and a maximal fluorescence increase of 200 and 264%, respectively, in pH 6.8 buffer. Cleavage of the two proteins with porcine pancreatic elastase results in a 1.6- and 2.6-fold increase in the ANS-binding affinity. While cleavage of the reactive center loop in rR345A ovalbumin results in a approximately 200% increase in the ANS fluorescence, the rWT protein exhibits a approximately 50% decrease. Similar experiments with alpha(1)-proteinase inhibitor and antithrombin, two inhibitory serpins that exhibit reactive center loop insertion, show a decrease in ANS fluorescence on cleavage with porcine pancreatic elastase and thrombin, respectively. Denaturation studies in guanidinium hydrochloride indicate that the reactive center loop is inserted in the main body of the serpin in the cleaved form of rR345A mutant, while it is exposed in the cleaved form of rWT ovalbumin. These results demonstrate that ANS fluorescence change is an indicator of the loop-inserted or loop-exposed form in these recombinant ovalbumins, and thus could be advantageously used for probing reactive center loop insertion in ovalbumins. The major increase in fluorescence for the rR345A mutant on cleavage primarily arises from a change in ANS binding rather than from the generation of an additional ANS-binding site.

Amino Acid Substitution↗

PubMedDate not supplied

Structural characterization of a serendipitously discovered bioactive macromolecule, lignin sulfate.

The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed beta-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.

Biocompatible Materials↗