This project elucidates the fundamental molecular design of the heparin/heparan sulfate class of biological regulatory polysaccharides (H/HS) and studies how the high degree of diversity of its sulfated structures relates to its parallel, multi-functional capacity. This capacity is exerted through specific binding to, and thus modulating the functional activity of, many different protein partners in normal and disease processes (e.g., cell growth, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and in viral and other infections). Established and/or newly devised biological, biochemical, and physical methods are utilzed. Previously: To study putative unique structures required for specific H/HS functions, and focussing on its anti-viral capacities against HIV-1, we started with a pharmaceutical heparin-mimetic sulfated xylan to devise a simpler, model system composed of a family of heparin-mimetic sulfated xylan oligosaccharides (S-oligoS). A macro combinatorial strategy with five in vitro target bioassays (anti-thrombin, anti-Xa, anti-cytotoxicity and -syncytium-forming infectivity of HIV-1, and clinical aPTT) was used and demonstrated that distinct functional Components were separable and isolated by low pressure liquid chromatography based on structural differences. These differential functional capacities among the S-oligoS in HIV-1 inhibition and other heparin functions indicated that, like those in the H/HS family, each was governed by a degree of structural specificity. This established the usefulness of our model system and we thereby isolated and characterized CpF-PkII, a potent anti-HIV-1 agent which lacks anti-thrombin activity. We developed an upscaled method to produce sufficient CpF-PkII for Phase I testing, and preparation continued this year. Details of results were given in previous reports. [HD 01315-01-06]. Structural features of S-oligoS are studied by analytical, chemical, sugar and specroscopic-FTIR, NMR, dye-coupling analysis, and by titration. Early findings showed that S-oligoS must contain a tetrasaccharide motif (beta-1,4-linked trixyloside with one alpha 1,2-linked branch) [HD 01315-01-05] In addition, FTIR spectra unexpectedly revealed the presence of axial sulfate moieties as well as the expected equatorial sulfates; heparin also contains both types of sulfate substituents. This finding indicated a structural basis for the heparin-mimetic capacity of S-oligoS for the first time. [HD 01315-03-04] Moreover, the relative proportions of these multiple forms differred among Components with different functional specificities. [HD 01313-05]. We reasoned that axial and equatorial sulfates must originate from different ring conformations of the glycopyranose moieties, i.e., an alternate and normal chair, resp., and that the corresponding equatorial and axial anomeric hydrogens would be seen in proton NMR spectra of the S-oligoS. Proton NMR spectra of CpF-PkII and several other S-oligoS last year were consistent with the interpretation that both beta-D Xyl and alpha-D Glc had tendency towards alternate conformation upon the sulfation of xylan, but the data were not conclusive because unambiguous assignment of the proton peaks of heavily sulfated oligoS was difficult. Further studies were required. [HD 01313-06] Because of the complex diversity of sulfated oligosaccharide structures within H/HS the structure-function relations of all but a few well- characterized HS-protein partners remains to be clarified. Findings from the S-oligoS family may contribute to this end. This year, continued production of CpF-PkII as a potential anti-AIDS agent, and the structure function studies of various other functionally distinct S-oligoS were pursued, using the same and more sophisticated methods. Two significant advances were made. In one, the macro combinatorial strategy above was extended to elucudate the anti-malaria capacity of S-oligoS and H/HS [See HD 008733-01]. In the second, compelling NMR evidence confirmed our results from FTIR spectroscopy. CpF-PkII was analyzed by two-dimensional NMR proton-13C -correlation spectroscopy using an inverted probe in a 600 MHz spectrometer at 60 degrees and the heteronuclear multi-dimensional quantum coherences (HMQC) method. In that study, If a given hydrogen were bonded to a sugar carbon that existed in two distinct conformations, the hydrogen would exhibit two complex interactions with that carbon. The correlation spectrum displayed two pairs of 13C-satellite signals about 71 ppm on the carbon spectrum, demonstrating that at least one sugar moiety existed in two conformations in CpF-PkII. The mid-points between these satellites were at 3.75 ppm and 4.65 ppm on the proton spectrum. This provides a basis for the peak assignments for the hydrogen and hence the position of the carbon the ring. Experimental data for the proton-NMR peaks of the sugars in similar S-oligoS indicates that the peaks of the 13C-satellite pairs originated from splitting by the proton bound to the 13C3 of a glycopyranose, that the sugar existed in normal chair and in an alternate form. and that the CpF-PkII structure contained axial sulfates on the C3 position of a portion of one of its sugars. This finding is important because it raises the idea that specific sugar conformation of individual sugars might be an additional governing parameter in structural specificity of the protein-binding sugar sequences of H/HS. This work will be reported at the November meeting of the Society for Glycobiology and the abstract published: MO Longas and AL Stone "Structure-function relations of heparin-mimetic sulfated oligosaccharides: Study of alternate chair conformations by NMR spectroscopy" Glycobiology vol. 11, 2001. Similar and/or different satellite pairs might be displayed by S-oligoS having different functional capacities. These studies will be continued next year.