A Small-Molecule Modulator of Poly-2,8-Sialic Acid Expression on Cultured Neurons and Tumor Cells Lara K. Mahal, Neil W. Charter, Kiyohiko Angata, Minoru Fukuda, Daniel E. Koshland Jr., and Carolyn R. Bertozzi

Supplementary Material

Experimental details for immunofluorescence microscopy analysis of PSA expression on NT2 neurons (Figure 1C). NT2 neurons were seeded onto primary mouse astrocytes grown on Permanox chamber slides (Nunc) and treated with varying concentrations of ManProp or ManBut for 5 days. The cultures were fixed and permeablized with methanol for 20 min at -20 °C. The fixed cells were then rehydrated with PBS, blocked with 2.5% normal rabbit serum, and washed with PBS. The samples were incubated with 1:200 dilution of mouse mAb to PSA 12F8 for one hour and washed with PBS. The cells were stained with a 1:100 dilution of rhodamine-conjugated rabbit anti-mouse IgM, washed with PBS, and visualized by fluorescence microscopy at 400 x magnification.

Experimental details for protein immunoblot experiments with NT2 cell lysates (Figure 2). NT2 neurons were seeded onto Matrigel and treated with various concentrations of ManProp or ManBut for 5 days. The cells were then lysed in MPer (Mammalian Protein Extraction Reagent, Pierce) for 10 min at 4 °C, boiled for 10 min and centrifuged at 12,000 x g for 10 min. Samples were then separated on a 5% SDS-PAGE gel and transferred onto a nitrocellulose filter (Biorad). PSA and NCAM were detected with 1:10,000 mAb 735 and 1:5000 OB11 (Sigma), respectively. Filters were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:50,000, Sigma) and visualized by chemiluminescence.

Experimental details for analysis of the effects of ManBut and ManProp on PSA biosynthesis in SH-SY5Y cells. Cells were cultured in the presence of varying concentrations of ManProp or ManBut for 3 days. Control cells were rinsed 1 x with Dulbecco's modified Eagle's media (DMEM) and treated with 56 ng Endo NE (1) in 1 mL DMEM for one hour at 37 °C. Cells were then rinsed 2 x with PBS, lysed in IP-Lysis buffer (150 mM NaCl, 50 mM Tris•HCl, 1 mM EDTA, 1 % NP-40, pH 8.0) for 15 min at 4 °C and centrifuged at 10,000 x g for 10 min. For HeLa-NCAM-STX cells, NCAM was first immunoprecipitated with mAb ERIC-1 (Ancell). Samples were separated by 7% SDS-PAGE and transferred onto a nitrocellulose filter (Biorad). PSA was detected using 12F8 (1:1,000, Pharmigen) followed by horseradish peroxidase-conjugated anti-rat IgM (1:50,000, Zymed) and visualized by chemiluminescence. NCAM was detected with OB11 (1:1,000) similar to the above protocol. The data are shown for SH-SY5Y cells in Fig. S1. Similar results were obtained using H345 and HeLa-NCAM-STX cells although the concentrations required for inhibition varied (Table S1).

Notably, NCAM from control SH-SY5Y cells treated with endoneuraminidase (Endo) NE, which digests PSA chains down to five residues (1, 2), had an apparent molecular size slightly larger than NCAM from ManBut-treated cells, supporting the conclusion that PSA disruption by ManBut is complete.

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Supplemental Table 1.
Cell Type	2.5 mM ManBut	5 mM ManBut	10 mM ManBut	20 mM ManBut
SH-SY5Y (neuroblastoma)	+/-	-	-	-
H345 (small cell lung carcinoma)	+	+	+/-	+/-
HeLa-NCAM-STX (stable HeLa transfectant)	+/-	-	-	-

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Inhibition of PSA expression by ManBut in various cell lines. Cells were treated with varying amounts of ManBut for 3 days. Inhibition of PSA was determined by protein immunblot analysis of whole cell lysates from SH-SY5Y and H345 cells, and by analysis of immunoprecipitated NCAM from HeLa-NCAM-STX cells. PSA inhibition levels were defined as unihibited (+), somewhat inhibited (+/-) or > 90% inhibited (-).

ManBut is not a general inhibitor of sialylation
The specificity of the inhibitory effect of ManBut across other sialylated epitopes is a significant issue for use of this compound in biological studies. To confirm that ManBut is not a general inhibitor of CMP-sialic acid biosynthesis, we analyzed its effects on total glycoside-bound sialic acids using the periodate-resorcinol assay (3, 4). This assay has been demonstrated to ignore internal sialic acids in the polysialic chain and give a signal for only the one terminal sialic acid residue on PSA (5). HeLa-NCAM-STX cells treated with ManBut (5 mM) showed no detectable reduction in total glycoside-bound sialic acid residues compared to untreated controls (Fig. S2). In another control experiment, cells were treated with Endo NE to remove cell surface PSA and glycoside-bound sialic acids were quantified. No significant difference in glycoconjugate-bound sialic acid was observed compared to untreated controls, demonstrating that PSA contributes only a small fraction of total cellular sialic acids. Thus, inhibition of PSA biosynthesis by ManBut is not expected to affect the overall level of cell surface sialosides.

Experimental details for in vitro assays of PSA biosynthesis using unnatural CMP-sialic acids (Figure 4A). Soluble NCAM-Fc and chimeric protein A-PST and -STX immobilized on IgG-Sepharose 6FF (Pharmacia) as a suspension in MEM (enzyme suspension) were prepared as previously reported (6). CMP-sialic acid derivatives (CMP-SiaProp and CMP-SiaBut) were synthesized by the methods of Halcomb and coworkers (7). Upon isolation, 50% of CMP-SiaBut and 36% of CMP-SiaProp had hydrolyzed to produce CMP and the free sialic acid. Additional CMP (Sigma) was added to CMP-sialic acid (Sigma) and CMP-SiaProp samples to normalize any inhibitory effects by CMP on the polysialyltransferases. The substrate solution contained 1 L of 0.2 g/L NCAM-Fc in buffer (20 mM Tris•HCl pH 7.4, 0.05% Tween-20), 5 L 2x sodium cacodylate buffer (0.2 M sodium cacodylate pH 6.0, 10 mM MnCl₂, 4 mM CaCl₂, 2% Triton CF-54)), various concentrations of CMP-sialic acid, CMP-SiaProp or CMP-SiaBut, CMP and H₂O to a final volume of 10 L. To this substrate solution, 10 L of enzyme suspension was added and the reaction mixture was incubated for various times at 37 °C, centrifuged and the supernatant recovered. SDS-PAGE samples were prepared by adding 1 L of 1 M DTT and 5 L of sample buffer (10 % glycerol, 60 mM Tris•HCl pH 8.0, 2% SDS) to 4 L of the supernatant. Samples separated by electrophoresis and transferred to nitrocellulose. NCAM-Fc was detected with a horseradish peroxidase-conjugated anti-human IgG antibody (1:120,000, Fc-specific, Sigma) and visualized by chemiluminescence.

Experimental details for in vitro assays of PSA biosynthesis using NCAM primed with unnatural sialic acids (Figure 4B). NCAM-Fc, 2 g in 100 L reaction buffer (0.1 M sodium cacodylate pH 6.0, 5 mM MnCl₂, 2 mM CaCl₂, 1% Triton CF-54) containing 100 nmol CMP-SiaProp or CMP-SiaBut was treated with 100 L STX enzyme suspension for 4 hours at 37 °C. The reaction was centrifuged, and the supernatant containing the NCAM-Fc was combined with buffer (Tris•HCl pH 7.4, 0.05 % Tween-20) washes of the enzyme beads. The unnatural sialic acid primed-NCAM-Fc was desalted by repeated concentration and addition of buffer using Microcon 100 (Amicon) and resuspended in 30 L of buffer. The SiaProp- and SiaBut-primed NCAM-Fcs were assayed as follows. The substrate solution contained 3 L of NCAM-Fc in buffer (20 mM Tris•HCl pH 7.4, 0.05 % Tween-20), 5 L 2x sodium cacodylate buffer (0.2 M sodium cacodylate pH 6.0, 10 mM MnCl₂, 4 mM CaCl₂, 2 % Triton CF-54), 10 nmol of CMP-Sia and H₂O to a final volume of 10 L. To this substrate solution 10 L of enzyme suspension was added and the reaction mixture was incubated for 4 hours at 37 °C, centrifuged and the supernatant recovered. Samples were analyzed as described above.

In vitro PSA biosynthesis by PST and STX using unnatural CMP-sialic acid analogs and NCAM-Fc primed with unnatural sialic acid. In vitro PSA biosynthesis using recombinant PST, NCAM-Fc and CMP-sialic acid gave qualitatively similar results to assays using STX. CMP-SiaBut was the least efficient substrate; a significant amount of NCAM-Fc lacking any apparent PSA remained after an 8-hour incubation (Fig. S3A). CMP-SiaProp was also used less efficiently than CMP-sialic acid, but a larger fraction of NCAM-Fc was converted to a high-molecular size than observed using CMP-SiaBut. Similar assays in which the concentrations of the CMP-sialic acid analogs were varied confirmed the rank order of substrate efficiencies with both PST (Fig. S3B) and STX (Fig. S3C). For both enzymes, CMP-SiaBut was used least efficiently and CMP-sialic acid was used most efficiently.

Synthesis of CMP-SiaProp and CMP-SiaBut
CMP-SiaBut (5.5) and CMP-SiaProp (5.6) were synthesized using the procedures Halcomb and coworkers (7). The synthesis involved the coupling of a selectively-protected sialic acid derivative (5.7 or 5.8, Fig. S4) with a cytidine phosphoramidite (5.9) to form the crucial glycosidic linkage. Compounds 5.7 and 5.8 can be derived from the respective free sialic acids, which in turn come from the N-acylmannosamine analogs 5.2 and 5.3.

The selectively-protected sialic acid derivatives 5.7 and 5.8 were synthesized in four steps as shown in Fig. S5. An efficient method for the synthesis of unnatural sialic acid analogs is through the action of N-acetylneuraminic acid aldolase (NANA aldolase), previously used by Whitesides and coworkers for the enzymatic synthesis of N-Cbz sialic acid (54). The NANA aldolase-catalyzed condensation of pyruvate with unnatural N-acylmannosamine analogs 5.2 and 5.3 afforded their respective sialic acids (5.10 and 5.11) in good yield (54,55). Treatment of compounds 5.10 and 5.11 with trifluoroacetic acid in dry methanol yielded their corresponding methyl esters 5.12 and 5.13. The selective acetylation of sialic acid methyl esters to directly yield the tetra-O-acetyl derivatives 5.7 and 5.8 has been reported (56,57). However, the major products that we observed from this reaction were the peracetylated sialic acid derivatives 5.14 and 5.15. Thus, we treated compounds 5.14 and 5.15 with hydrochloric acid in acetyl chloride and the resulting anomeric chlorides were immediately hydrolyzed by treatment with silver carbonate in acetone/H₂O to yield the desired tetra-O-acetyl sialic acid derivatives 5.7 and 5.8 in good yields.

In the key coupling reaction, tertiary alcohols 5.7 and 5.8 were condensed with cytidine phosphoramidite 5.9 (52) to yield phosphites 5.16 and 5.17 (Fig. S6). Oxidation of phosphites 5.16 and 5.17 with dimethyldioxirane (DMDO) and removal of the allyl protecting groups on the resultant phosphates afforded the protected CMP-sialic acid derivatives 5.18 and 5.19 (Fig. S7). Deprotection of 5.18 and 5.19 with sodium methoxide in methanol followed by sodium hydroxide treatment yielded the desired final products 5.5 and 5.6. Upon isolation of the final products it was discovered that 36% of CMP-SiaProp (5.6) and 50% of CMP-SiaBut (5.5) had hydrolyzed to produce CMP (5.20) and the free sialic acids (Fig. S8). Due to the precious nature of the material and the difficulty in obtaining pure product, the impure substrates were used in enzyme assays with normalization across control reactions achieved by adding equivalent amounts of CMP.

Synthetic details and spectral data for all synthetic compounds

N-Butanoylmannosamine (ManBut, 5.2, mixture of anomers). To a solution of mannosamine hydrochloride (2.0 g, 9.3 mmol) in anhydrous MeOH (31 mL) was added 9 mL of 1 M NaOMe/MeOH. The solution was stirred at rt under N₂. After 15 min, butyric acid anhydride (1.6 mL, 9.7 mmol) was added dropwise by syringe. After 16 h, the solvent was removed in vacuo. Silica gel chromatography eluting with 7:1 CH₃Cl₃:MeOH yielded 1.9 g (80%) of product as an amorphous white foam. The product was characterized as a mixture of anomers: ¹H NMR (300 MHz, D₂O): 0.86-0.91 (m, 6), 1.50-1.63 (m, 4), 2.20-2.31 (m, 4), 3.37 (ddd, 1, J = 2.3, 4.8, 7.1), 3.47 (app t, 1, J = 9.8), 3.58 (app t, 1, J = 9.6), 3.72-3.87 (m, 6), 4.01 (dd, 1, J = 4.7, 9.8), 4.29 (dd, 1, J = 1.5, 4.7), 4.41 (dd, 1, J = 1.5, 4.4), 4.98 (d, 1, J = 1.6), 5.07 (d, 1, J = 1.5); ¹³C NMR (125 MHz): 13.0, 13.0, 21.5, 21.6, 27.5, 35.3, 35.5, 53.0, 53.9, 60.3, 66.4, 68.7, 71.9, 76.3, 92.9, 93.1, 178.0, 178.9; HRMS (FAB⁺) calcd for C₁₀H₂₀NO₆ (M+H)⁺ 250.1291, found 250.1290.

N-Propanoylmannosamine (ManProp, 5.3, mixture of anomers). To a solution of mannosamine hydrochloride (2.0 g, 9.3 mmol) in 31 mL of anhydrous MeOH was added 9.4 mL 1 M sodium methoxide in MeOH. The solution was stirred 20 min under N₂ and propanoic acid anhydride (1.2 mL, 9.7 mmol) was added dropwise by syringe. The reaction was allowed to stir overnight at rt and the solvent was removed in vacuo. The crude product was purified by silica gel chromatography eluting with 7:1 CH₃Cl₃:MeOH to afford 1.9 g (88%) of a white foam. The product was characterized as a mixture of anomers: ¹H NMR (500 MHz, D₂O): 1.08-1.18 (m, 6), 2.28-2.38 (m, 4), 3.40 (ddd, 1, J = 2.3, 5.0, 7.0), 3.50 (app t, 1, J = 10), 3.61 (app t, 1, J = 9.5), 3.77-3.88 (m, 6), 4.04 (dd, 1, J = 4.5, 10), 4.31 (dd, 1, J = 1.5, 4.5), 4.44 (dd, 1, J = 1.5, 4.5), 5.01 (d, 1, J = 1.5), 5.09 (app s, 1); ¹³C-NMR (125 MHz): 9.4, 9.4, 28.9, 29.0, 53.0, 53.8, 60.4, 66.4, 66.7, 68.7, 71.9, 72.0, 76.3, 92.9, 93.1, 178.7, 179.5; HRMS (FAB⁺) calcd for C₉H₁₈NO₆ (M+H)⁺ 236.1134, found 236.1133.

Peracetylated N-butanoylmannosamine (AcManBut, 5.4, mixture of anomers). Compound 5.2 (0.20 g, 0.80 mmol) was dissolved in 10 mL of acetic anhydride and 5 mL of anhydrous pyridine. The reaction mixture was stirred for 18 h at rt and diluted with EtOAc. The solution was washed with copious amounts of sat. NaHCO₃, H₂O and sat. NaCl. The organic extract was dried over Na₂SO₄ and the solvent was removed in vacuo. Purification of the crude product by silica gel chromatography eluting with 1:1 hexanes:EtOAc followed by HPLC on a C18 reversed-phase column eluting with a gradient of acetonitrile (20%-60% over 35 min) in H₂O yielded 0.075 g (22%) of a clear syrup. The compound was characterized as a mixture of anomers: ¹H NMR (500 MHz, CDCl₃): 0.96-1.01 (m, 6), 1.64-1.72 (m, 4), 1.98-2.00 (m, 6), 2.05-2.06 (m, 6), 2.09-2.10 (m, 9), 2.17 (m, 3), 2.22-2.82 (m, 4), 3.80 (ddd, 1, J = 2.4, 5.1, 7.5), 4.02-4.06 (m, 2), 4.79 (ddd, 1, J = 1.7, 3.9, 5.6), 5.04 (dd, 1, J = 4.0, 10.0), 5.10-5.19 (m, 2), 5.32 (dd, 1, J = 4.4, 10.2), 5.75 (d, 1, J = 9.2), 5.79 (d, 1, J = 9.0), 5.85 (d, 1, J = 1.7), 6.01 (d, 1, J = 1.7); ¹³C NMR (125 MHz): 13.7, 13.8, 19.3, 19.4, 20.8, 20.8, 20.9, 20.9, 20.9, 21.0, 38.6, 38.8, 49.2, 49.5, 62.0, 62.2, 65.4, 65.6, 69.1, 70.3, 71.6, 73.6, 90.8, 91.9, 168.4, 169.9, 170.2, 170.3, 170.7, 173.2, 173.8; HRMS (FAB⁺) calcd for C₁₈H₂₈NO₁₀ (M+H)⁺ 418.1713, found 418.1723.

N-Butanoyl sialic acid (SiaBut, 5.10). A solution of N-butanoylmannosamine (5.2, 1.8 g, 7.3 mmol), sodium pyruvate (9.0 g, 82 mmol), N-acetylneuraminic acid aldolase (E.C. 4.1.3.3, 136 units) and sodium azide (365 L of a 1% solution) in potassium phosphate buffer (41 mL, 0.05 M, pH 7.2) was incubated at 37 °C. After 21 h, the product was isolated via anion exchange chromatography on Dowex AG1X-8 (formate form) eluting with a formic acid gradient (1-2.5 M). Fractions containing the product were pooled and the solvent was removed in vacuo to yield the product in quantitative yield as a white solid: ¹H NMR (500 MHz, D₂O): 0.85 (app t, 3, J = 7.2), 1.56 (app sxt, 2, J = 7.3), 1.81 (app t, 1, J = 12.4), 2.21 (app t, 2, J = 7.3), 2.24 (dd, 1, J = 4.6, 12.9), 3.48 (d, 1, J = 9.3), 3.54 (dd, 1, J = 6.3, 11.8), 3.67-3.70 (m, 1), 3.76 (dd, 1, J = 2.5), 3.87 (app t, 1, J = 10.2), 3.97-4.02 (m, 2); ¹³C NMR (125 MHz): 12.8, 18.9, 37.8, 38.9, 51.8, 63.1, 66.5, 68.2, 70.0, 70.3, 95.2, 173.3, 177.8; HRMS (FAB⁺) calcd for C₁₃H₂₄NO₉ (M+H)⁺ 338.1451, found 338.1444.

N-Propanoyl sialic acid (SiaProp, 5.11). A solution of N-propanoylmannosamine (5.3, 1.9 g, 8.2 mmol), sodium pyruvate (9.0 g, 82 mmol), ), N-acetylneuraminic acid aldolase (E.C. 4.1.3.3, 158 units) and sodium azide (410 L of a 1% solution) in potassium phosphate buffer (41 mL, 0.05 M, pH 7.2) was incubated at 37 °C. After 19 h, the product was isolated via anion exchange chromatography on Dowex AG1X-8 (formate form) eluting with a formic acid gradient (1-2.5 M). Fractions containing the product were pooled and the solvent was removed in vacuo to yield the product in 99% yield as a white solid: ¹H NMR (500 MHz, D₂O): 1.06 (app t, 3, J = 7.7), 1.81 (dd, 1, J = 11.6, 13.0), 2.22-2.27 (m, 3), 3.46 (dd, 1, J = 0.9, 9.2), 3.54 (dd, 1, J = 6.4, 11.9), 3.68 (ddd, 1, J = 2.7, 6.4, 9.1), 3.77 (dd, 1, J = 2.7, 11.9), 3.86 (app t, 1, J = 10.3), 3.98-4.03 (m, 2); ¹³C NMR (125 MHz): 9.4, 29.2, 38.8, 51.8, 63.0, 66.5, 68.1, 70.0, 70.3, 95.2, 173.2, 178.7; HRMS (FAB⁺) calcd for C₁₂H₂₂NO₉ (M+H)⁺ 324.1294, found 324.1288.

Compound 5.12. To a solution containing compound 5.10 ( 0.57 g, 1.8 mmol) in MeOH (17.5 mL) was added 100 L trifluoroacetic acid (TFA). The reaction mixture was stirred under Ar for 10 h at rt. The solvent was removed in vacuo to yield the product as a white solid in a quantitative yield: ¹H NMR (500 MHz, D₂O): 0.86 (app t, 3, J = 7.4), 1.56 (app sxt, 2, J = 7.4), 1.85 (dd, 1, J = 11.6, 13.1), 2.21 (app t, 2, J = 7.3), 2.25 (dd, 1, J = 4.9, 13.0), 3.48 (dd, 1, J = 1.1, 9.3), 3.54 (dd, 1, J = 6.3 11.9), 3.67 (ddd, 1, J = 2.6, 6.3, 9.1), 3.75-3.78 (m, 4), 3.87 (app t, 1, J = 10.3), 4.00 (m, 2); ¹³C NMR (125 MHz, D₂O): 12.7, 18.9, 37.8, 38.7, 51.8, 53.3, 63.0, 66.4, 68.2, 70.0, 70.2, 95.2, 171.3, 177.9; HRMS (FAB⁺) calcd for C₁₄H₂₆NO₉ (M+H)⁺ 352.1608, found 352.1610.

Compound 5.13. To a solution containing compound 5.11 (1.3 g, 4.0 mmol) in freshly distilled MeOH (40 mL) was added 300 L of TFA. The reaction mixture was stirred under Ar at rt. After 2 d, the solvent was removed in vacuo to yield the product as a white solid in a 75% yield: ¹H NMR (500 MHz, D₂O): 1.08 (app t, 3, J = 7.6), 1.86 (app t, 1, J = 12.3), 2.24-2.28 (m, 3), 3.47 (app d, 1, J = 9.2), 3.56 (dd, 1, J = 6.3, 11.8), 3.68 (ddd, 1, J = 1.9, 6.2, 8.5), 3.77-3.78 (m, 4), 3.87 (app t, 1 J = 10.2), 3.99-4.05 (m, 2); ¹³C NMR (125 MHz): 9.5, 29.2, 38.6, 51.8, 53.4, 63.0, 66.5, 68.1, 70.0, 70.3, 95.3, 171.3, 178.7; HRMS (FAB⁺) calcd for C₁₄H₂₄NO₉ (M+H)⁺ 338.1451, found 338.1444.

Compound 5.14. Compound 5.12 was dissolved in 15 mL of acetic anhydride to which 100 L of perchloric acid was added. The reaction mixture was stirred under N₂. After 1 h, the reaction was diluted with EtOAc and washed with H₂O, sat. NaHCO₃, H₂O and sat. NaCl. The organic extracts were combined and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with 1:1 hexanes:EtOAc to yield 1.1 g (43%) of 5.14 as a white foam: ¹H NMR (400 MHz, CDCl₃): 0.88 (app t, 3, J = 7.4), 1.48-1.60 (m, 2), 1.95-2.12 (m, 18), 2.52 (dd, 1, J = 5.0, 13.4), 3.76 (s, 3), 4.05-4.15 (m, 3), 4.46 (dd, 1, J = 2.6, 12.4), 5.00-5.04 (m, 1), 5.18-5.28 (m, 1), 5.33 (dd, 1, J = 1.8, 5.1), 5.42 (d, 1, J = 9.3); ¹³C NMR (100 MHz): 18.9, 20.9, 20.9, 21.0, 36.1, 38.7, 49.3, 53.3, 62.3, 68.0, 68.3, 71.5, 73.0, 97.7, 166.5, 168.4, 170.3, 170.4, 170.7, 171.0, 173.2; HRMS (FAB⁺) calcd for C₂₄H₃₅NO₁₄Li (M+Li)⁺ 568.2218, found 568.2227.

Compound 5.15. Compound 5.13 was dissolved in 10 mL acetic anhydride to which 67 L of perchloric acid was added. The reaction mixture was stirred under N₂. After 1.5 h, the reaction was diluted with EtOAc and washed with H₂O, sat. NaHCO₃ and sat. NaCl. The organic extracts were combined and the solvent removed in vacuo. The crude product was purified by silica gel chromatography eluting with a gradient of 1:1-2:1 hexanes:EtOAc to yield 0.70 g (44%) of a white foam: ¹H NMR (500 MHz, CDCl₃): 1.07 (app t, 3, J = 7.6), 2.00-2.13 (m, 18), 2.53 (dd, 1, J = 5.0, 13.7), 3.78 (s, 3), 4.08-4.14 (m, 3), 4.48 (dd, 1, J = 2.5, 12.4), 5.03-5.06 (m, 1), 5.23-5.30 (m, 1), 5.35-5.37 (m, 2); ¹³C NMR (125 MHz): 9.8, 21.0, 21.0, 21.0, 21.0, 21.1, 29.9, 36.1, 49.3, 53.4, 62.3, 67.9, 68.4, 71.5, 73.0, 97.7, 166.5, 168.4, 170.4, 170.4, 170.7, 171.1, 174.1; HRMS (FAB⁺) calcd for C₂₃H₃₃NO₁₄Li (M+Li)⁺ 554.2061, found 554.2049.

Compound 5.7. To a solution of compound 5.14 (1.0 g, 1.8 mmol) in 4 mL of 3:1 CH₂Cl₂/acetyl chloride was added 2 drops of conc. HCl. The reaction was allowed to stir overnight under N₂ at rt followed by the addition of 2 additional drops of conc. HCl. The reaction was again allowed to stir overnight. The mixture was concentrated by coevaporation with toluene and then redissolved in 18 mL of 4:1 acetone/H₂O. To this solution Ag₂CO₃ (1.5 g, 5.4 mmol) was added. The reaction mixture was covered with foil and allowed to stir in the dark at rt overnight. The mixture was filtered through Celite and the solvent was removed in vacuo to yield 0.79 g (85%) of a white solid. The product was 95% pure by ¹H-NMR and was taken on without further purification: ¹H NMR (500 MHz, CDCl₃): 0.89 (app t, 3, J = 7.4), 1.51-1.62 (m, 2), 1.97-2.20 (m, 16), 3.83 (s, 3), 4.00 (dd, 1, J = 8.0, 12.3), 4.14-4.23 (m, 2), 4.56 (dd, 1, J = 2.5, 12.3), 5.16-5.24 (m, 2), 5.34 (dd, 1, J = 2.1, 4.6), 5.94 (d, 1, J = 9.6); ¹³C NMR (125 MHz): 13.9, 19.1, 21.0, 21.1, 21.2, 36.4, 38.8, 49.2, 53.5, 62.8, 68.4, 69.3, 71.6, 72.0, 95.0, 169.2, 170.3, 171.0, 171.3, 171.5, 173.3; HRMS (FAB⁺) calcd for C₂₂H₃₄NO₁₃ (M+H)⁺ 520.2030, found 520.2036.

Compound 5.8. To a solution of compound 5.15 (0.70 g, 1.3 mmol) in 4 mL of 3:1 CH₂Cl₂/acetyl chloride was added 2 drops of conc. HCl. The reaction was allowed to stir overnight under N₂ at rt followed by the addition of 3 additional drops of conc. HCl. The reaction was again allowed to stir overnight. The mixture was concentrated by coevaporation with toluene and then redissolved in 13 mL of 4:1 acetone/H₂O. To this solution Ag₂CO₃ (1.1 g, 3.8 mmol) was added. The reaction mixture was covered with foil and allowed to stir in the dark at rt. After 4 d, the mixture was filtered through Celite and the solvent was removed in vacuo to yield 0.60 g (93%) of a white solid: ¹H NMR (500 MHz, CDCl₃): 1.08 (app t, 1, J = 7.6), 1.99 (s, 3), 2.02-2.56 (m, 13), 3.84 (s, 3), 4.00 (dd, 1, J = 7.9, 12.3), 4.12-4.23 (m, 2), 4.55 (dd, 1, J = 2.4, 12.3), 5.18-5.26 (m, 2), 5.34 (dd, 1, J = 2.0, 4.9) 5.83 (d, 1, J = 9.6); ¹³C NMR (125 MHz): 9.9, 21.0, 21.0, 21.0, 21.2, 36.3, 49.4, 53.6, 62.8, 68.3, 69.4, 71.4, 71.8, 95.1, 169.3, 170.4, 171.1, 171.2, 171.3, 174.3; HRMS (FAB⁺) calcd for C₂₁H₃₂NO₁₃ (M+H)⁺ 506.1874, found 506.1882.

Compound 5.16. An anhydrous solution of compound 5.7 (0.79 g, 1.5 mmol) and tetrazole (0.64 g, 9.1 mmol) in 4 mL of acetonitrile was cooled to -40 °C and a solution of cytidine phosphoramidite (5.9, 2.5 g, 4.6 mmol (52)) in 17 mL of acetonitrile was added by syringe. The solution was stirred under Ar. After 20 min, 2.1 mL of triethylamine (TEA) was added and the solvent was removed in vacuo. The crude product was dissolved in EtOAc, washed with H₂O followed by sat. NaCl and the organic layer was dried over Na₂SO₄. The crude product was purified by silica gel chromatography eluting with 5:1:0.3 EtOAc:hexanes:TEA to yield 1.1 g (74%) of the labile phosphite 5.16. Due to the instability of the phosphite, the product of the following step was fully characterized: mass spectrum (ES⁺) C₄₀H₅₅N₄O₂₂PNa (M+Na)⁺ calcd. 997.8, found 997.2.

Compound 5.17. An anhydrous solution of compound 5.8 (0.56 g, 1.1 mmol) and tetrazole (0.47 g, 6.6 mmol) in 6 mL of acetonitrile was cooled to -40° C and a solution of cytidine phosphoramidite (5.9, 2.2 g, 4.0 mmol (52)) in 12 mL of acetonitrile was added by syringe. The solution was stirred under Ar. After 15 min, 1.5 mL of TEA was added and the solvent was removed in vacuo. The crude product was dissolved in EtOAc, washed with H₂O followed by sat. NaCl and the organic layer was dried over Na₂SO₄. The crude product was purified by silica gel chromatography eluting with 6:1:0.3 EtOAc:hexanes:TEA to yield 0.66 g (62%) of labile phosphite 5.17. Due to the instability of the phosphite, the product of the following step was characterized spectroscopically.

Dimethyldioxirane (DMDO) (60). To a solution of H₂O (160 mL), spectroscopic grade acetone (103 mL), and NaHCO₃ (96 g) was added Oxone (200 g) in 2 portions over 20 min. The solution was distilled under partial vacuum into a cooled receiving flask (-78 °C). After 15 min, the resulting distillate was dried over CaSO₄ and filtered to yield the desired DMDO solution (~30 mL). The concentration of the DMDO solution was assumed to be ~0.1-0.08 M.

Compound 5.18. A solution of compound 5.16 (0.69 g, 0.71 mmol) in CH₂Cl₂ (7.1 mL) was treated with 9.7 mL of dimethyldioxirane in acetone. The reaction was stirred under Ar. After 15 min, the solvent was removed in vacuo and the crude product was redissolved in 7.1 mL of acetonitrile. To this solution was added diisopropyl amine (14 L, 0.12 mmol) and Pd(PPh₃)₄ (42 mg, 0.035 mmol). The reaction was stirred for 1 h and the solvent was removed in vacuo. The crude product was purified by HPLC on a silica gel column eluting with a step gradient of MeOH (0% for 5 min, 0-20% over 5 min, 20% for 15 min) in EtOAc (all solvents contained 0.1% TEA) to yield 0.50 g (68%) of compound 5.18. The product was characterized as the triethylammonium salt: ¹H NMR (400 MHz, CD₃OD): 0.91 (app t, 3, J = 7.4), 1.29 (t, 9, J = 7.4), 1.56 (app sxt, 1, J = 7.4), 1.93 (s, 3), 1.96-2.10 (m, 18), 2.18 (s, 3), 2.71 (dd, 1, J = 4.8, 13.2), 3.17 (q, 6, J = 7.3), 3.79 (s, 3), 4.00-4.08 (m, 2), 4.17-4.25 (m, 2), 4.28 (ddd, 1, J = 2.4, 5.2, 7.8), 4.40-4.41 (m, 1), 4.45 (dd, 1, J = 2.0, 10.4), 4.56 (dd, 1, J = 2.8, 12.4), 5.21-5.24 (m, 1), 5.27-5.33 (m, 1), 5.41 (dd, 1, J = 2.4, 4.8), 5.47-5.53 (m, 1), 5.27-5.33 (m, 1), 5.41 (dd, 1, J = 2.4, 4.8), 5.47-5.53 (m, 1), 6.19 (d, 1, J = 4.8), 7.53 (d, 1, J = 7.6), 7.81 (d, 1, J = 10), 8.47 (d, 1, J = 7.6); ¹³C NMR (100 MHz): 9.2, 10.2, 20.2, 20.3, 20.5, 20.8, 20.8, 20.8, 21.1, 24.6, 30.5, 38.3, 38.3, 47.7, 50.2, 53.3, 63.6, 65.5, 65.5, 69.6, 70.6, 71.9, 72.6, 72.9, 75.5, 83.4, 83.4, 89.5, 98.9, 99.0, 99.0, 146.7, 158.1, 164.6, 169.2, 171.3, 171.5, 171.7, 171.7, 171.9, 172.5, 172.9, 177.0; ³¹P (162 MHz, H₃PO₄ external standard set to 0.0 ppm): -6.9; HRMS (FAB⁺) calcd for C₃₇H₅₂N₄O₂₃P(M+H)⁺ 951.2760, found 951.2752.

Compound 5.19. A solution of compound 5.17 (0.66 g, 0.69 mmol) in CH₂Cl₂ (14 mL) was treated with 8.4 mL of a ~0.08 M solution of dimethyldioxirane in acetone. The reaction was stirred under Ar. After 10 min, the solvent was removed in vacuo and the crude product was redissolved in 7.1 mL of acetonitrile. To this solution was added diisopropyl amine (14 L, 0.12 mmol) and Pd(PPh₃)₄ (42 mg, 35 mol). The reaction was stirred for 3 h and the solvent was removed in vacuo. The crude product was purified by silica gel chromatography eluting with 6:0.5:0.1 EtOAc:MeOH:TEA. Further purification by HPLC on a silica gel column eluting with a step gradient of MeOH (0% for 5 min, 0-20% over 5 min, 20% for 15 min) in EtOAc (all solvents contained 0.1% TEA) yielded 0.31 g (43%) of compound 5.19. The product was characterized as the triethylammonium salt: ¹H NMR (400 MHz, CD₃OD): 1.07 (app t, 3, J = 7.6), 1.28 (t, 9, J = 7.3), 1.92-2.11 (m, 21), 2.17 (s, 3), 2.72 (dd, 1, J = 4.9, 13.3), 3.19 (q, 6, J = 7.3), 3.80 (s, 3), 4.03 (app t, 1, J = 10.5), 4.18-4.26 (m, 2), 4.30 (ddd, 1, J = 2.4, 5.2, 7.6), 4.39-4.43 (m, 1), 4.47 (dd, 1, J = 2.2, 10.7), 4.58 (dd, 1, J = 2.7, 12.2), 5.22-5.27 (m, 1), 5.29-5.35 (m, 1), 5.43 (dd, 1, J = 2.2, 4.8), 5.49-5.54 (m, 2), 6.20 (d, 1, J = 4.6), 7.54 (d, 1, J = 7.5), 8.48 (d, 1, J = 7.6); ¹³C NMR (100 MHz): 9.2, 10.2, 20.4, 20.6, 20.8, 20.8, 20.8, 21.1, 24.6, 30.5, 38.3, 47.7, 50.2, 53.3, 63.6, 65.5, 65.5, 69.6, 70.6, 71.9, 72.6, 72.9, 75.5, 83.4, 83.4, 89.5, 98.9, 99.0, 99.0, 146.7, 158.1, 164.6, 169.2, 171.3, 171.5, 171.7, 171.7, 171.9, 172.5, 172.9, 177.0; ³¹P (162 MHz, H₃PO₄ external standard set to 0.0 ppm): -6.9; HRMS (FAB⁺) calcd for C₃₆H₅₀N₄O₂₃P(M+H)⁺ 937.2603, found 937.2632.

CMP-N-butanoyl sialic acid (CMP-SiaBut, 5.5). To a solution of compound 5.18 (0.20 g, 0.19 mmol) in MeOH (15 mL) was added 3.8 mL of a 1 M solution of NaOMe/MeOH (1.9 mmol). The reaction was allowed to stir for 30 min and then diluted with 15 mL of NH₄HCO₃ solution (25 mM). The product was purified by size exclusion chromatography (Biogel P-2). Fractions containing product were combined and repeatedly lyophilized. The resultant solid was redissolved in 2 mL of H₂O and 1 mL of 1 M NaOH solution was added. After 10 min, the reaction was diluted with 12 mL of NH₄HCO₃ solution (25 mM) and purified by size exclusion chromatography (Biogel P-2). Fractions containing product were lyophilized, diluted with H₂O and passed down cation exchange resin (Na⁺ form, Biorad AGX8). The product obtained was impure. Purification by anion-exchange chromatography (DEAE resin, eluting with 0-1 M pyridinium acetate pH 5.6) followed by cation exchange (Na⁺ form, Biorad AGX8) yielded 0.05 g (40%) of a fluffy white solid. The ¹H NMR spectrum of the product revealed that 50% of the desired CMP-SiaBut (5.5) had hydrolyzed. The ¹H NMR spectrum was assigned by comparison to the known spectra for CMP-sialic acid and 5.10: ¹H NMR (400 MHz, D₂O): 0.86 (app t, 3, J = 7.4), 1.48-1.62 (m, 3), 2.21 (app t, 2, J = 7.4), 2.43 (dd, 1, J = 4.6, 13.2), 3.38 (d, 1, J= 9.7), 3.54 (dd, 1, J = 6.6, 11.7), 3.80-3.92 (m, 3), 3.97-4.01 (m, 1), 4.08 (d, 1, J = 10.6), 4.14-4.29 (m, 5), 5.91 (d, 1, J = 4.1), 6.12 (d, 1, J = 7.7), 7.99 (d, 1, J = 7.8); ¹³C NMR (HMQC, 125 MHz): 12.8, 19.0, 37.9, 41.1, 51.6, 62.9, 64.7, 66.7, 68.9, 69.2, 69.6, 71.7, 74.2, 83.0, 89.0, 96.1, 142.5; HRMS (FAB⁺) calcd for C₂₂H₃₆N₄O₁₆P(M+H)⁺ 643.1864, found 643.1860.

CMP-N-propanoyl sialic acid (CMP-SiaProp, 5.6). To a solution of compound 5.19 (0.12 g, 0.12 mmol) in MeOH (9.2 mL) was added 2.3 mL of a 1 M solution of NaOMe/MeOH (1.2 mmol). The reaction was allowed to stir for 30 min and then diluted with 15 mL of NH₄HCO₃ solution (25 mM). The product was purified by size exclusion chromatography (Biogel P-2). Fractions containing product were combined and repeatedly lyophilized. The resultant solid was redissolved in 2 mL H₂O and 1 mL 1 M NaOH solution was added. After 10 min, the reaction was diluted with 12 mL of NH₄HCO₃ solution (25 mM) and purified by size exclusion chromatography (Biogel P-2). Fractions containing product were lyophilized. Purification by anion-exchange chromatography (DEAE resin, eluting with 0-1 M pyridinium acetate pH 5.6) followed by cation exchange (Na⁺ form, Biorad AGX8) yielded 0.04 g (50%) of a fluffy white solid. The ¹H NMR spectrum of the product revealed that 36% of the desired CMP-SiaProp (5.6) had hydrolyzed. The ¹H NMR spectrum was assigned by comparison to the known spectra for CMP-sialic acid and 5.11: ¹H NMR (500 MHz, D₂O): 1.03 (app t, 3, J = 7.7), 1.54 (m, 1), 2.17-2.25 (m, 2), 2.39 (dd, 1, J = 4.7, 13.2), 3.32 (app d, 1, J = 10.6), 3.46-3.55 (m, 1), 3.72-4.25 (m, 9), 4.37 (dd, 1, J = 2.3, 8.9), 5.88 (d, 1, J = 4.0), 6.01 (d, 1, J = 7.6). 7.91 (d, 1, J = 7.6); HRMS (FAB⁺) calcd for C₂₁H₃₃N₄O₁₆PNa (M+Na)⁺ 651.1527, found 651.1530.

References for Supplementary Material

1. R. Gerardy-Schahn, et al., Mol. Microbiol. 16, 441 (1995).
2. S. Pelkonen, J. Pelkonen, J. Finne, J. Virol. 63, 4409 (1989).
3. G. W. Jourdian, L. Dean, S. Roseman, J. Biol. Chem. 246, 430 (1971).
4. C. F. Culling, P. E. Reid, C. W. Ramey, W. L. Dunn, M. G. Clay Can. J. Biochem. 55, 778 (1977).
5. M. A. Maliakal, M. H. Ravindranath, R. F Irie, D. L. Morton Glycoconjugate J. 11, 97 (1994).
6. K. Angata, M. Suzuki, M. Fukuda, J. Biol. Chem. 273, 28524 (1998).
7. M. D. Chappell, R. L. Halcomb, Tetrahedron 53, 11109 (1997).

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Supplemental Figure 1. Western analysis of NCAM from SH-SY5Y cells. Cells were grown in the presence of varying concentrations of ManBut (Lanes 2-6) or ManProp (Lanes 8-12) for 3 days, and whole cell lysates were analyzed. The blot was stained with mAb OB11 as before. Control cells were untreated (Lane 1, C) or treated with Endo NE for 1 hour at 37° C prior to lysis (Lane 7, E).

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Supplemental Figure 2. ManBut does not generally inhibit sialoside biosynthesis. HeLa-NCAM-STX cells were treated with 5 mM ManBut for 3 days. Glycoside-bound sialic acid in cell lysates was quantified by the periodate/resorcinol assay. A similar amount of sialic acid was observed with ManBut-treated and untreated cells, and control cells pretreated with endo NE.

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Supplemental Figure 3. In vitro PSA biosynthesis by PST and STX. (A) NCAM-Fc was treated with PST and 10 nmol of either CMP-Sia (CMP-SA, Lanes 1-4), CMP-SiaProp (CMP-SP, Lanes 5-8) or CMP-SiaBut (CMP-SB, Lanes 9-12) at 37° C for the indicated times. Samples were analyzed by Western blot, stained with anti-Human IgG (Fc-specific)-HRP conjugate and visualized by chemiluminescence. (B) NCAM-Fc was treated with PST and various amounts of either CMP-SA (Lanes 2-5), CMP-SP (Lanes 6-9) or CMP-SB, (Lanes 10-13) for 4 hours at 37° C. Samples were analyzed by Western blot as before. (C) Experiment was performed with STX in an identical manner to the experiment shown in (B).

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Supplemental Figure 4. Retrosynthetic analysis of CMP-sialic acid derivatives 5.5 and 5.6.

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Supplemental Figure 5. Synthesis of selectively-protected sialic acid derivatives 5.7 and 5.8.

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Supplemental Figure 6. Key coupling reaction between selectively-protected sialic acid derivatives 5.7 and 5.8 and the cytidine phosphoramidite 5.9.

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Supplemental Figure 7. Synthesis of unnatural CMP-sialic acids, CMP-SiaBut (5.5) and CMP-SiaProp (5.6).

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Supplemental Figure 8. CMP-sialic acid derivatives 5.5 and 5.6 are hydrolyzed to give the corresponding sialic acids 5.10 and 5.11 and CMP (5.20). Hydrolysis of 50% of CMP-SiaBut and 36% of CMP-SiaProp was observed upon isolation.

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