Structural Characterization of a Serendipitously Discovered Bioactive Macromolecule, Lignin Sulfate Arjun Raghuraman, † Vaibhav Tiwari, | Jay N. Thakkar, †,§ Gunnar T. Gunnarsson, †,§ Deepak Shukla, | Michael Hindle, ‡ and Umesh R. Desai* ,†,§ Departments of Medicinal Chemistry and Pharmaceutics, and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23298-0540, and Departments of Opthalmology, Visual Sciences, Microbiology, and Immunology, University of Illinois at Chicago, Chicago, Illinois 60612 Received May 4, 2005; Revised Manuscript Received June 10, 2005 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 -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 anionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits. Introduction Herpes simplex viruses (HSV) are human enveloped viruses that cause mucocutaneous lesions of the mouth, face, eyes, or genitalia. 1,2 These infections are highly prevalent, affecting at least 1 in 3 individuals in the U.S. Occasionally, the virus spreads to the central nervous system causing meningitis or encephalitis. Of the eight herpes viruses known to infect humans, HSV-1 is the most common, causing cold sores in the mouth, and is readily transmitted through routine intimate contact. 3 HSV infection of cells involves several molecules, especially glycoproteins gB, gC, and gD (viral glycoprotein D), which are known to be present on the viral envelope. 3-6 These viral glycoproteins interact with heparan sulfate (HS) chains present on cell surface to enhance the efficiency of infection. Removal of HS chains from the cell surface through enzymatic treatment or presence of soluble forms of HS severely retards HSV entry into cells. Heparan sulfate, a glycosaminoglycan covalently attached to the protein core of proteoglycans, is widely expressed in human tissues and has important roles in development, differentiation, and homeostasis. 7,8 Structurally, HS is a linear copolymer of glucosamine (GlcNp) and glucuronic acid (GlcAp) residues linked in a 1f4 manner, of which the GlcNp residue are typically acetylated at 2-position. 9,10 Despite this apparently simple monomeric disaccharide structure, heparan sulfate perhaps represents the most complex molecule nature biosynthesizes because of ad- ditional apparently indiscriminate epimerization of some GlcAp residues to iduronic acid (IdoAp) and sulfation of only some available -OH groups. This primary structural diversity is further complicated by another level of complex- ity wherein sulfate groups may cluster in small regions and form differentially charged mini-domains. A simple calcula- tion of a number of structural sequences possible with these variations, especially of the size recognized by proteins and receptors, shows millions of possibilities. The structural richness of HS is arguably the origin for its involvement in numerous biological processes. Yet, specific recognition sequences may be critical. A good example of specific recognition is demonstrated by the HS-gD interaction, wherein a 3-O-sulfated GlcNp residue is essential for HSV-1 to penetrate cells. 11,12 * To whom correspondence should be addressed. Phone: (804) 828- 7328. Fax: (804) 827-3664. E-mail: urdesai@vcu.edu. † Department of Medicinal Chemistry, Virginia Commonwealth Univer- sity. ‡ Department of Pharmaceutics, Virginia Commonwealth University. § Institute for Structural Biology and Drug Discovery, Virginia Com- monwealth University. | University of Illinois at Chicago. BATCH: bm5a26 USER: jld69 DIV: @xyv04/data1/CLS_pj/GRP_bm/JOB_i05/DIV_bm0503064 DATE: July 5, 2005 10.1021/bm0503064 CCC: $30.25 © xxxx American Chemical Society PAGE EST: 10.5 Published on Web 00/00/0000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67