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Leukocyte adhesion in the microvasculature influences blood rheology and plays a key role in vaso-occlusive manifestations of sickle cell disease. Notably, polymorphonuclear neutrophils (PMNs) can capture circulating sickle red blood cells (sRBCs) in inflamed venules, leading to critical reduction in blood flow and vaso-occlusion. Recent studies have suggested that E-selectin expression by endothelial cells plays a key role by sending activating signals that lead to the activation of Mac-1 at the leading edge of PMNs, thereby allowing RBC capture. Thus, the inhibition of E-selectin may represent a valuable target in this disease. Here, we have tested the biologic properties of a novel synthetic pan-selectin inhibitor, GMI-1070, with in vitro assays and in a humanized model of sickle cell vaso-occlusion analyzed by intravital microscopy. We have found that GMI-1070 predominantly inhibited E-selectin–mediated adhesion and dramatically inhibited sRBC-leukocyte interactions, leading to improved microcirculatory blood flow and improved survival. These results suggest that GMI-1070 may represent a valuable novel therapeutic intervention for acute sickle cell crises that should be further evaluated in a clinical trial.

We showed previously that HL-60 and F9 mouse embryonal carcinoma cells will take up and deblock peracetylated Galbeta1-4GlcNAcbeta-O-naphthalenemethanol (Galbeta1-4GlcNAc-NM) and use the disaccharide as a primer of oligosaccharide chains (Sarkar, A. K., Fritz, T. A., Taylor, W. H., and Esko, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3323-3327). We now report that another disaccharide, acetylated GlcNAcbeta1-3Gal-naphthalenemethanol (GlcNAcbeta1-3Gal-NM), has even greater potency and that both compounds will inhibit sialyl LewisX (sLex)-dependent cell adhesion. When fed to U937 cells, acetylated forms of Galbeta1-4GlcNAc-NM and GlcNAcbeta1-3Gal-NM primed oligosaccharides in a dose-dependent manner. Analysis of compounds assembled on Galbeta1-4GlcNAc-NM showed only one product, namely Galbeta1-4(Fucalpha1-3)GlcNAc-NM. In contrast, GlcNAcbeta1-3Gal-NM generated Galbeta1-4GlcNAcbeta1-3Gal-NM, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Gal-NM, NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Gal-NM, and NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1- 3Gal-NM. Both compounds decreased the incorporation of [3H]fucose into cellular glycoconjugates, without affecting the incorporation of [3H]mannosamine, a precursor of sialic acid residues. Moreover, the overall extent of sialylation was not affected based on the reactivity of cells to fluorescein isothiocyanate-conjugated Maackia amurensis lectin. Priming inhibited expression of sLex on cell surface glycoconjugates, which reduced E-selectin-dependent cell adhesion to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells. GlcNAcbeta1-3Gal-NM and Galbeta1-4GlcNAc-NM represent starting points for making enzyme-specific, site-directed inhibitors of glycosyltransferases that could act in living cells.

We have reported that 3-estradiol-beta-D-xyloside primes heparan sulfate synthesis in Chinese hamster ovary cells and that the proportion of heparan sulfate made rises with increasing concentration of xyloside (Lugemwa, F.N. and Esko, J.D. (1991) J. Biol. Chem. 266, 6674-6677). Using estradiol as a guide, we varied the structure of the aglycone and showed that beta-D-xylosides containing two fused aromatic rings efficiently prime heparan sulfate. Thus, 2-naphthol-beta-D-xyloside primed heparan sulfate at low dose (

Synthesis of three tetrasaccharides, namely, 0-α-L-fucopyranosyl-(1→3)-0-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-0-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose (7), 0-α-L-fucopyranosyl-(1→4)-0-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-0-(β-D-galactopyranosyl)-(1→4)-D-glucopyranose (9), and 0-α-L-fucopyransoyl-(1→3)-0-(2-acetamido-2-deoxy-β-D-glucopyransoyl)-(1→6)-0-(β-D-galactopyranosyl)-(1→4)-D-glucopyranose (15) has been described. Their structures have been established by 13C NMR spectroscopy.

Treatment of benzyl O-beta-D-galactopyranosyl-(1----3)-2-acetamido-2- deoxy-4,6-O-isopropylidene-beta-D-glucopyranoside with tert-butylchlorodiphenylsilane afforded the 6'-O-tert-butyldiphenylsilyl ether, which was converted into the 3',4'-O-isopropylidene derivative. Methylation and subsequent removal of protecting groups afforded benzyl O-(2-O-methyl-beta-D-galactopyranosyl)- (1----3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (7). The trisaccharide methyl O-(2-O-methyl-beta-D-galactopyranosyl)-(1----3)-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----3)-beta-D-galactopyranosi de (17) and the tetrasaccharide O-(2-O-methyl-beta-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-b eta-D- glucopyranosyl)-(1----3)-O-beta-D-galactopyranosyl-(1----4)-D-glucopyran ose (32), both containing the 2'-O-methyllacto-N-biose I unit at the nonreducing end, were synthesized, and the structures of 7, 17, and 32 were confirmed by 13C-n.m.r. spectroscopy.

Several sialosaccharides containing the Neu5Acα2→6 glycosyl linkage have been synthesized using Ag-zeolite as an effective catalyst for exclusive α-glycosylation.

Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra- O -acetyl-β- d -galactopyranosyl-(1→4)-2-acetamido-3,6-di- O -acetyl-2-deoxy-β- d -glucopyranoside ( 6 ), 2-naphthylmethyl 2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- d -glucopyranosyl-(1→3)-2,4,6-tri- O -acetyl-β- d -galactopyranoside ( 14 ), 2-naphthylmethyl 2,3,4,6-tetra- O -acetyl-β- d -galactopyranosyl-(1→3)-2-acetamido-4,6-di- O -acetyl-2-deoxy-α- d -galactopyranoside ( 20 ), 2-naphthylmethyl 2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- d -glucopyranosyl-(1→3)-2-acetamido-4,6-di- O -acetyl-2-deoxy-α- d -galactopyranoside ( 23 ) and 2-naphthylmethyl 2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- d -glucopyranosyl-(1→6)-2-acetamido-3,4-di- O -acetyl-2-deoxy-α- d -galactopyranoside ( 27 ). These per-O-acetylated compounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6 , 14 , and 20 served as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin.

While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein beta1, 3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO3GlcUAbeta1,3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in beta-linkage to Galbeta1,3/4GlcNAcbeta-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Galbeta1, 3Galbeta-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Galbeta1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H., Tone, Y., Tamura, J., Neumann, K. W., Ogawa, T., Oka, S., Kawasaki, T., and Sugahara, K. (1998) J. Biol. Chem. 273, 6615-6618). The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production.

The biosynthesis of glycosaminoglycans (GAG) takes place while the polysaccharide chains are usually attached to a proteoglycan core protein. Cells also will assemble GAG chains on beta-D-xylosides containing hydrophobic aglycones. In order to evaluate the relationship of the structure of the sugar to priming activity of the glycoside, we synthesized beta-D-xyloside analogs in which the hydroxyls were substituted with hydrogen, fluorine, -O-methyl, amino, -O-isopropyl, and -O-benzyl groups. Epimers at the 2-, 3-, and 4-position of xylose also were made. Their ability to prime GAGs was tested in Chinese hamster ovary cells by measuring 35SO4 incorporation into polysaccharide chains and by assaying the transfer of galactose to the xylosides by galactosyltransferase I (UDP-D-galactose:xylose beta1-4-galactosyltransferase) in vitro. All of the analogs failed to act as primers of GAGs in vivo and as substrates in vitro with the following exceptions. Substitution of 2-OH and 3-OH with -OCH3 were active at high concentration (1 mM), but the deoxygenated derivatives were inactive. Efficient priming also occurred on a derivative with fluorine instead of the 3-OH group, suggesting that the oxygen atoms at C-2 and C-3 were involved as hydrogen bond acceptors. Methylated and deoxy analogs at C-4 were inactive, due to the loss of the acceptor hydroxyl group. Interestingly, benzyl-beta-D-threo-pentopyranos-4-uloside (4-keto derivative) and benzyl-4-methyl-beta-D-xyloside, with a methyl group in place of an axial hydrogen at C-4, primed GAG chains. Priming by these unusual xylosides suggests the possibility of designing inhibitors of GAG synthesis based on xyloside analogs with reactive groups in key positions.

To test if disaccharides might serve as primers of oligosaccharide synthesis in animal cells, we synthesized 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 4)-beta-D-xylopyranoside, 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 3)-beta-D-galactopyranoside, and 2-naphthyl O-(beta-D-glucopyranosyluronic acid)-(1 --> 3)-beta-D-galactopyranoside. These three disaccharides are related to subunits of the linkage tetrasaccharide of heparan sulfate and chondroitin sulfate chains in animal cell proteoglycans. The disaccharides were synthesized with coupling efficiencies of 40-70% using thioglycosides or by activating the monosaccharides with trichloroacetimidate. The structures of these compounds were confirmed by 1H NMR, 13C NMR and elemental analysis. The ability of these disaccharides to prime glycosaminoglycan chains was examined in a Chinese hamster ovary cell mutant, p gsA 745, which lacks xylosyltransferase. The missing enzyme renders the cells dependent on exogenous primers for making glycosaminoglycan chains. 2-Naphthyl O-(beta-D-galactopyranosyl)-(1 --> 3)-beta-D-galactopyranoside and 2-naphthyl O-(beta-D-glucopyranosyluronic acid)-(1 --> 3)-beta-D-galactopyranoside did not stimulate glycosaminoglycan synthesis, but 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 4)-beta-D-xylopyranoside at high concentration primed chains. The peracetylated derivative (2-naphthyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1 --> 4)- 2,3-di-O-acetyl-beta-D-xylopyranoside) primed chains at lower concentration (100 microM), suggesting that cells took up the compound and removed the acetyl groups apparently in the compartment where glycosaminoglycan synthesis occurs.

Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

11103 Background: Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells (MMCs) in the peripheral blood and homing back to the BM. Selectins are adhesion molecules involved in the primary interaction of lymphocytes with the endothelial cells (ECs) of blood vessels. In this study we studied the role selectins in the pathogenesis of MM. Methods: We have characterized the expression of E, L and P-selectins and their ligands on MM cell lines, patient sample and plasma cells from normal subjects (NPCs). We have tested the effect of blockade of each of the selectins and selectin-ligands on the interaction of MMCs with ECs. Moreover, we tested the effect of a pan selectin inhibitor on MMCs adhesion to ECs, and trans-well (through filter) and trans-endothelial SDF1-induced migration in vitro, and characterized its effect on cytoskeletal signaling induced by the interaction of MMCs and ECs. Moreover, we have tested the effect of the inhibitor on homing of MMCs to the BM in mice using in vivo flow cytometry to detect the number of circulating cells, and in vivo confocal microscopy to directly visualize the homing. Results: All MM cell lines and patient samples had low expression of all selectins and high expression of L and P, but not E, selectin ligands. While NPCs showed low expression of all selectins and ligands. Blockade of L and P-selectin ligands reduced the interaction of MMCs with ECs in vitro, while blockade of E-selectin ligand or any of selectins did not show any effect. The pan-selectin inhibitor reduced the interaction of MMCs with ECs in vitro, did not alter their SDF1-induced migration through filter, but reduced significantly the migration through ECs. The inhibitor inhibited the activation of FAK and ERK induced by interaction of MMCs and ECs. Moreover, the selectin inhibitor extending the circulation time of MM cells in mice, and reduced the homing of MMCs. Conclusions: We found that L and P selectin ligands are highly expressed in MMCs compared to NPCs, and that those play a major role in homing of MMCs to the BM. Moreover, the pan-selectin inhibitor prevented the homing of MMCs to the BM. This provides a basis for testing the effect of the inhibitor on MM tumor progression and initiation. No significant financial relationships to disclose.

Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model (Berkeley) suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. In addition, mice deficient in both P-and E-selectins are protected from vaso-occlusion (VOC) induced by surgical trauma and TNF-α stimulation, suggesting that targeting selectins or their ligands represents a potentially useful strategy. Selectins bind to specific sialylated and fucosylated carbohydrate structures presented by glycoprotein or glycolipid ligands. Here, we tested the effect of novel small glycomimetic selectin inhibitors, GMI-1070 and GMI-1077, on leukocyte behavior and sickle cell VOC. Berkeley SCD mouse bone marrow was transplantated into lethally irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle 90 min later. GMI-1070, GMI-1077 (both 20 mg/kg) or vehicle (PBS) were administered immediately prior to cytokine stimulation (t=0 min), and an additional dose was given at t=70min. Another group of mice was injected with antibodies against P-and E-selectins (PES, 1 mg/kg) as positive control. Several post-capillary and collecting venules were examined between t= 90min and t= 150min. Antibody blockade of endothelial selectins completely ablated leukocyte rolling, whereas GMI-1070 and GMI-1077 significantly increased the rolling flux fractions (PBS: 5.0±1.2 GMI-1070: 10.6±1.3%%; GMI-1077: 9.9±1.0%; p< 0.001). Furthermore GMI-1070 and GMI-1077 significantly reduced the recruitment of adherent leukocytes (914±172 and 1433±119 cells/mm2, respectively) compared to sickle mice injected with PBS control (2400±392 cells/mm2, p< 0.001). Although the reduction in leukocyte adhesion was not as marked as with anti-P and E-selectins (61±25 cells/mm2, p< 0.001), GMI-1070, in particular, dramatically inhibited the capture of sickle RBCs by adherent leukocytes (PBS: 0.9±0.4, GMI-1077: 0.6±0.2, GMI-1070: 0.07±0.05 and PES: 0.01±0.01 RBC interactions/WBC/min, p< 0.05) and markedly improved the blood flow in venules (PBS: 312±24, GMI-1077: 398±41, GMI-1070: 710±68 and PES: 683±75 nL/s, p< 0.001), to levels observed in non-sickle mice. The increased leukocyte rolling fluxes by these glycomimetics suggest that they inhibit E-selectin > P-selectin. Since the hallmark of E-selectin-mediated adhesion is the slow leukocyte rolling, we analyzed leukocyte rolling velocities in the various group and indeed found a 2-fold increase in rolling velocities in sickle mice treated with GMI-1070 compared to PBS control (PBS: 21±1 μm/s, GMI-1070: 38±1 μm/s, p

Rational design of glycomimetic inhibitors based on the bioactive conformation of functional carbohydrates provides new therapeutic opportunities for the development of small molecule drugs with improved activity, pharmacokinetics, and bioavailability relative to native ligands. In designing more active glycomimetic selectin inhibitors, the low enthalpy (ΔH0) of the reaction is compensated by pre-forming the bioactive conformation thereby improving the entropy (ΔS0), known as S/H compensation. Modifications of the molecule that also stabilize the core structure, further improve ΔS0 and binding activity. To improve ΔH0, second site interactions were explored. To address the requirements for P and L-selectins, interactions were combined for both carbohydrate and sulfate-binding domains to produce heterobifunctional molecules. By stabilizing the bioactive core and exploring second site molecular interactions, we have produced a family of pan-selectin antagonists, one of which (GMI-1070) is now in development and scale-up synthesis as a lead compound. GMI-1070 is a potent inhibitor of E, P, and L-selectins in vitro and inhibits E and P-selectin-mediated leukocyte adhesion to endothelial monolayers under flow conditions. More importantly, GMI-1070 is active in several animal models of diseases requiring leukocyte adhesion and migration such as: a delayed-type hypersensitivity (DTH) response, cardiac ischemia/reperfusion injury, and vaso-occlusive crisis in sickle cell disease. GMI-1070 significantly inhibited infarct size in an ischemia/reperfusion cardiac injury model in rats. A single dose at 10mg/kg gave maximal inhibition. The effects of GMI-1070 on T-cell migration was studied in a DTH model. Mice were sensitized with oxazolone on the abdomen and then, 7 days later challenged on the ear. Donors T-cells from a different cohort of similarly sensitized mice were fluorescently labeled and injected into the test cohorts at the time of administration of GMI-1070. Three hours following injection, migration of fluorescent T-cells to the challenged area was determined. Selectin-dependent T-cell migration in the DTH response was completely eliminated with a dose of 10mg/kg of GMI-1070, suggesting the potential clinical application in diseases involving skin homing T-cells such as graft vs. host disease (GVHD) after bone marrow transplantation, and other inflammatory skin diseases. We have also shown that acute myelogenous leukemia (AML) cells adhere to endothelial cells under flow in a selectin-dependent mechanism suggesting that graft vs. leukemia as well as graft vs. host may be affected by treatment with GMI-1070. GMI-1070 was also tested in a model of vaso-occlusive crisis in sickle cell disease using Berkeley sickle cell mice. Blood flow was restored to normal values and adhesion of sickle red blood cells to adherent leukocytes was essentially eliminated as determined intravital microscopy using a dose of 20mg/kg administered at the time of elicitation of the vaso-occlusive challenge (TNFα) and at the start of intravital microscopy (70 minutes later). Based on the encouraging results in disease models, we are advancing the clinical development of GMI-1070 into Phase 1 studies to support indications which include the treatment of sickle cell patients in vaso-occlusive crisis.

Methyl 4-O-(2,3,4,6-tetra-O-acetyl- β -D-galactopyranosyl)-2,3-O-isopropylidene- α -L-rhamnopyranoside was obtained by condensing methyl 2,3-O-isopropylidene- α -L-rhamnopyranoside with acetobromogalactose. This compound was benzylated, the iso-propylidene group was removed and the product was then partially benzylated to give methyl 2-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-β -D-galactopyranosyl)- α -L-rhamnopyranoside. Koenigs Knorr condensation of this compound with methyl (2,3,4-tri-O-acetyl-α -D-glucopyranosyl bromide) uronate gave a trisaccharide derivative which after deprotection gave the title trisaccharide.

Syntheses of methyl 3-O-alpha-D-galactopyranosyl-alpha-D-mannopyranoside (10) and methyl 3-O-alpha-D-galactopyranosyl-2-O-(beta-D-glucopyranosyluronic acid)-alpha-D-mannopyranoside (11) in good yield are described. Both 10 and 11 significantly inhibit antigen-antibody precipitation in the Klebsiella Type 10 immune system. The results provide more evidence for the structure (1) of the antigen from Klebsiella K-10 and its immunodominant grouping.

A search has been made for electroinitiators of postpolymerization. A number of hydroxycarboxylic acids and some of their sodium salts have been found to be quite efficient. Factors controlling the rate of polymerization and yield have been described. Surprisingly, citric acid, though not an electroinitiator of polymerization, is a good initiator of postpolymerization. Other active species that deserve mention are tartaric acid and its salts. A free radical mechanism involving formation of oligomeric initiator fragments, growth by attachment of imbibed monomer, and termination by chain transfer has been proposed to explain the observed facts.

The capsular polysaccharide from Klebsiella Type 10 was found to contain D-galactose, D-glucose, D-mannose, and D-glucuronic acid in the ratios 3:1:1:1. Acid hydrolysis of the polysaccharide gave one aldobiouronic acid, one aldotriouronic acid, one aldotetraouronic acid, and two neutral disaccharides the structures of which were established. The native and carboxyl-reduced polysaccharide have been subjected as appropriate to methylation analysis and Smith degradation. Degradation of the methylated polysaccharide with base established the identity of the sugar unit preceding the glucosyluronic acid residue. The anomeric configurations of the sugar residues were determined by oxidation of the acetylated native and carboxyl-reduced polysaccharides with chromium trioxide. Based on these studies, the hexasaccharide structure 1 has been assigned to the repeating unit of the K-10 polysaccharide.