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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK594038_"><span class="title" itemprop="name">Large scale preparation of oligosaccharides from chicken eggs</span></h1><div class="contrib half_rhythm"><span itemprop="author">Yasuhiro Kajihara</span>, Ph.D.<div class="affiliation small">Department of chemistry, Osaka University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-akaso.ics.mehc@arahijak" class="oemail">pj.ca.u-akaso.ics.mehc@arahijak</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">November 11, 2021</span>; Last Revision: <span itemprop="dateModified">March 22, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g17-largeoligo.Introduction"><h2 id="_g17-largeoligo_Introduction_">Introduction</h2><p>Oligosaccharides linked to glycoproteins and glycopeptides affect several biological events. Therefore, it is essential to synthesize glycoproteins and glycopeptides for use as probes to study their biological roles. To synthesize these molecules, the protected asparaginyl-sialyloligosaccharide is essential for solid phase peptide synthesis.</p></div><div id="g17-largeoligo.Protocol"><h2 id="_g17-largeoligo_Protocol_">Protocol</h2><p>This protocol describes an efficient preparation method of Fmoc-asparaginyl-sialyloligosaccharide (<a class="figpopup" href="/books/NBK594038/figure/g17-largeoligo.F1/?report=objectonly" target="object" rid-figpopup="figg17largeoligoF1" rid-ob="figobg17largeoligoF1">Figure 1</a>) (<a class="bk_pop" href="#g17-largeoligo.REF.1">1</a>,<a class="bk_pop" href="#g17-largeoligo.REF.2">2</a>).</p><div id="g17-largeoligo.Materials"><h3>Materials</h3><p>Almost all reagents were purchased from chemical companies (Merck Millipore, Billerica, MA or Sigma-Aldrich, St. Louis, MO). Chicken eggs used were not fresh. The eggs passed at least a few days after ovulation. Actinase-E was purchased from Kaken Pharmaceutical Co. Ltd., Tokyo, Japan.</p><p>1. Tris-HCl buffer (50 mM, CaCl<sub>2</sub> 10 mM, pH 7.5)</p><p>2. 9-Fluorenylmethyl <i>N</i>-succinimidyl carbonate (Fmoc-OSu)</p><p>3. NaHCO<sub>3</sub></p><p>4. Acetone</p><p>5. H<sub>2</sub>O</p><p>6. Cs<sub>2</sub>CO<sub>3</sub></p><p>7. Benzyl bromide (BnBr)</p><p>8. <i>N, N</i>-Dimethylformamide (DMF)</p><p>9. Phenol</p><p>10. Acetone</p><p>11. Dowex-50W X8(H<sup>+</sup>)</p><p>12. Diethylaminoethyl (DEAE)-650M</p><p>13. Hen egg</p><p>14. Actinase-E</p></div><div id="g17-largeoligo.Instruments"><h3>Instruments</h3><p>1. Nuclear magnetic resonance (400 MHz)</p><p>2. Liquid chromatography-mass spectrometry (LC-MS) with an electron-transfer dissociation ion trap mass system</p><p>3. Evaporator system</p><p>4. Monitor the reaction by thin-layer chromatography (Merck). After development with appropriate solvent (2-propanol:1 M NH<sub>4</sub>OAc = 1:1), Stain the target material by H<sub>2</sub>SO<sub>4</sub> (5%)/MeOH at 200°C.</p><p>5. Gel permeation column (Sephadex G50 and G25, fine)</p><p>6. High-performance liquid chromatography (HPLC: 2 solvents gradient system)</p><p>7. Lyophilizer</p></div><div id="g17-largeoligo.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Glycopeptide 1</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare egg yolks (12 pieces) after breaking hen eggs (<b>Note 1</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add distilled water (380 mL).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add a phenol solution (34.2 g of phenol/3.8 g of water).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Stir for 2 h at 4°C.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add water (480 mL).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Centrifuge (6000 rpm) for 30 min at 4°C.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Filter the supernatant and then concentrate at 35°C using an evaporator system.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Prepare a solution of the residue in water (8 mL).</p></dd><dt>i.</dt><dd><p class="no_top_margin">Filter the solution by filter paper.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Purify by a gel permeation column (Sephadex G50, 0.1 M NaCl, id 2.5 cm × 100 cm).</p></dd><dt>k.</dt><dd><p class="no_top_margin">Collect the fractions containing sialyloligosaccharyl peptide <b>1</b> and then concentrate at 35°C using an evaporator system.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Desalt by Sephadex G50 column (water, id 2.5 cm × 100 cm).</p></dd><dt>m.</dt><dd><p class="no_top_margin">Collect the fractions containing sialyloligosaccharyl peptide <b>1</b> and then concentrate at 35°C by an evaporator system.</p></dd><dt>n.</dt><dd><p class="no_top_margin">Purify by DEAE-650M column (water, id 2.5 cm × 15 cm) (<b>Note 2</b>).</p></dd><li class="half_rhythm"><p class="no_top_margin">Collect the fractions containing sialyloligosaccharyl peptide <b>1</b> and then lyophilization.</p></li><dt>p.</dt><dd><p class="no_top_margin">Isolate sialyloligosaccharyl peptide <b>1</b> (113 mg) (<a class="bk_pop" href="#g17-largeoligo.REF.1">1</a>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Asparaginyl sialyloligosaccharide 2</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare a solution containing crude sialylglycopeptide <b>1</b> (809 mg) and NaN<sub>3</sub> (1%) in a Tris-HCl buffer (50 mM, CaCl<sub>2</sub> 10 mM, pH 7.5, 32 mL).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add Actinase-E (263 mg) to a solution of sialylglycopeptide <b>1</b>.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Stir this solution for 60 h at 37°C (the pH was maintained at 7.5) (<b>Note 3</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">After 60 h, add Actinase-E (25 mg) again and then incubate for 55 h.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Lyophilize the reaction solution.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Purify by gel permeation (Sephadex-G-25, 2.5 × 100 cm, H<sub>2</sub>O).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Collect the fractions containing asparaginyl sialyloligosaccharide <b>2</b> and then lyophilization.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Isolate asparaginyl sialyloligosaccharide <b>2</b> (301 mg).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Synthesis of 3</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare a solution containing asparaginyl sialyloligosaccharide <b>2</b> (80 mg, 0.034 mmol) and NaHCO<sub>3</sub> (11.5 mg, 0.137 mmol) in H<sub>2</sub>O-acetone (2.7 mL–4.1 mL).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add a solution (acetone 4.1 mL) containing 9-fluorenylmethyl-<i>N</i>-succimidylcarbonate (34.7 mg, 0.103 mmol).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Stir this mixture at room temperature for 2 h.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Evaporate this mixture to remove acetone.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Purify by reverse phase HPLC system (ODS-column C-18, 1.6 × 14 cm, H<sub>2</sub>O to 20% MeOH).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Isolate Fmoc-Asn-disialyloligosaccharide <b>3</b> (60 mg).</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Synthesis of 4</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare a solution (cold H<sub>2</sub>O, 2 mL, 4°C) containing Fmoc-Asn-(disialyloligosaccharide)-OH <b>3</b> (20 mg).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Desalt sodium salt from Fmoc-Asn-(disialyloligosaccharide)-OH <b>3</b> by passing through a Pasteur pipette column (0.5 cm × 5 cm) containing resin of Dowex-50W X8(H<sup>+</sup>) and then wash the resin with water.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Collect all eluents and then lyophilize them.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Prepare a solution (water, 2 mL) containing Fmoc-Asn-(disialyloligosaccharide)-OH <b>3</b>.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add a solution of aq. Cs<sub>2</sub>CO<sub>3</sub> (2.5 mg/1 mL) and adjust the pH to 6.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Lyophilize this solution.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Prepare a solution (dry DMF, 1.3 mL) containing Fmoc-Asn-(disialyloligosaccharide)-OH <b>3</b>.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Add BnBr (5.1 μL) to the above DMF solution and then stir for 45 h at room temperature.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Add diethyl ether (10 mL) and then collect precipitate generated.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Purify ODS-column (1.6 cm × 14 cm, H<sub>2</sub>O to 40% MeOH).</p></dd><dt>k.</dt><dd><p class="no_top_margin">Isolate Fmoc-Asn-(dibenzyl-sialyloligosaccharide)-OH <b>4</b> (18 mg).</p></dd></dl></dd></dl></div><div id="g17-largeoligo.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Because sialylglycopeptide <b>1</b> is a metabolized material, very fresh eggs do not include sialylglycopeptide <b>1</b>.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Protocol 14 of glycopeptide <b>1</b> can be skipped. The DEAE-650M column purification can remove a byproduct, such as monosialyloligosaccharide. The final purification step of Fmoc-Asn-(dibenzyl-sialyloligosaccharide)-OH <b>4</b> can remove byproduct glycan.</p></dd><dt>3.</dt><dd><p class="no_top_margin">In terms of Actinase-E reaction, a key point in this protocol is to keep pH 7.5 during the enzymatic reaction. This reaction was monitored by TLC (1 M NH<sub>4</sub>OAc:isopropanol = 1:1).</p></dd><dt>4.</dt><dd><p class="no_top_margin">NMR data</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">
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<b>Asparaginyl sialyloligosaccharide 2</b>
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</p><p><sup>1</sup>H-NMR (400 MHz, 30°C in D<sub>2</sub>O, HOD = δ 4.81) δ 5.21 (s, 1H, Man4-H-1), 5.15 (d, 1H, <i>J</i> = 9.5 Hz, GlcNAc1-H-1), 4.03 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-1), 4.70 (m, 3H, GlcNAc2,5,5’-H-1), 4.53 (d, 2H, <i>J</i> = 8.0 Hz, Gal6,6’-H-1), 4.34 (bs, 1H, Man3-H-2), 4.28 (bd, 1H, Man4-H-2), 4.20 (bd, 1H, Man4’-H-2), 3.03 (dd, 1H, <i>J</i> = 4.4 Hz, 17.2 Hz, Asn-bCH<sub>2</sub>), 2.95 (dd, 1H, <i>J</i> = 7.0 Hz, 17.2 Hz, Asn-bCH<sub>2</sub>), 2.76 (bdd, 2H, <i>J</i> = 4.6 Hz, 12.4 Hz, NeuAc7,7’-H-3<sub>eq</sub>), 2.16 (s, 3H, Ac), 2.15 (s, 6H, Ac × 2), 2.28 (s, 6H, Ac × 2), 2.10 (s, 3H, Ac), 1.80 (dd, 2H, <i>J</i> = 12.4 Hz, 12.4 Hz, NeuAc7,7’-H-3<sub>ax</sub>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">
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<b>Fmoc-Asn-disialyloligosaccharide 3</b>
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</p><p><sup>1</sup>H-NMR (400 MHz, 30°C in D<sub>2</sub>O, HOD = δ 4.81) δ 8.01 (d, 2H, <i>J</i> =7.5 Hz, Fmoc), 7.80(d, 2H, <i>J</i> = 7.5 Hz, Fmoc), 7.60 (dd, 2H, <i>J</i> = 7.5 Hz,　Fmoc), 7.53 (dd, 2H, <i>J</i> = 7.5 Hz, Fmoc),　5.22 (s, 1H, Man4-H-1), 5.09 (d, 1H, <i>J</i> = 9.4 Hz, GlcNAc1-H-1), 5.03 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-1), 4.69 (m, GlcNAc2,5,5’-H-1), 4.53 (d, 2H, <i>J</i> = 7.8 Hz, Gal6, 6’-H-1), 4.44 (1H, Fmoc), 4.34 (bd, 1H, Man3-H-2), 4.29 (bd, 1H, Man4-H-2), 4.20 (bd, 1H, Man4’-H-2), 2.83-2.72 (m, 3H, Asn-bCH<sub>2</sub>, NeuAc7,7’-H-3<sub>eq</sub>), 2.61 (bdd, 1H, Asn-bCH<sub>2</sub>), 2.15 (s, 9H, Ac × 3), 2.12 (s, 6H, Ac × 2), 1.98 (s, 3H, Ac), 1.80 (dd, 2H, <i>J</i> = 12.1 Hz, 12.1 Hz, NeuAc7,7’-H-3<sub>ax</sub>); HRMS Calcd for C<sub>103</sub>H<sub>154</sub>N<sub>8</sub>NaO<sub>66</sub>[M+Na<sup>+</sup>] 2581.8838, found 2581.8821.</p></dd><dt>c.</dt><dd><p class="no_top_margin">
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<b>Fmoc-Asn-(dibenzyl-sialyloligosaccharide)-OH 4</b>
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</p><p><sup>1</sup>H-NMR (400 MHz, 30°C in D<sub>2</sub>O, HOD = δ 4.81) δ 8.00 (d, 2H, Fmoc), 7.80 (d, 2H, Fmoc), 7.65-7.50 (m, 12H, Ph, Fmoc), 5.46 (d, 2H, <i>J</i> = 11.6 Hz, PhC<i>H</i><sub>2</sub>), 5.40 (d, 2H, <i>J</i> = 11.6 Hz, PhC<i>H</i><sub>2</sub>), 5.21 (s, 1H, Man4-H-1), 5.08 (d, 1H, <i>J</i> = 9.3 Hz, GlcNAc1-H-1), 5.02 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-1), 4.67 (m, 3H, GlcNAc2,5,5’-H-1), 4.41 (bd, 3H, Gal6, 6’- H-1, Fmoc), 4.33 (bd, 1H, Man3- H-2), 4.27 (bd, 1H, Man4’-H-2), 4.20 (d, 1H, Man4- H-2), 2.79 (bd, 3H, Asn-bCH<sub>2</sub>, NeuAc7, 7’- H<sub>3eq</sub>), 2.61 (bdd, 1H, Asn-bCH<sub>2</sub>), 2.15 (s, 3H, Ac), 2.12 (s, 6H, Ac × 2), 2.10 (s, 6H, Ac × 2), 1.98 (s, 3H, Ac), 1.93 (2H, dd, <i>J</i> = 12.2, 12.2 Hz, NeuAc7,7’-H-3<sub>ax</sub>);HRMS Calcd for C<sub>117</sub>H<sub>165</sub>N<sub>8</sub>Na<sub>2</sub>O<sub>66</sub> [M+Na<sup>+</sup>] 2783.9597, found 2783.9501.</p></dd></dl></dd></dl></div></div><div id="g17-largeoligo.References"><h2 id="_g17-largeoligo_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g17-largeoligo.REF.1">Seko A, Kotetsu M, Nishizono M, Enoki Y, Ibrahim H R, Juneja L R, Kim M, Yamamoto T. Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg york. <span><span class="ref-journal">Biochim Biophys Acta. </span>1997 Apr 17;<span class="ref-vol">1335</span>:23–32.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9133639" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9133639</span></a>] [<a href="http://dx.crossref.org/10.1016/s0304-4165(96)00118-3" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g17-largeoligo.REF.2">Yamamoto N, Ohmori Y, Sakakibara T, Sasaki K, Juneja L R, Kajihara Y. Solid-phase synthesis of sialylglycopeptides through selective esterification of the sialic acid residues of an Asn-linked complex-type sialyloligosaccharide. <span><span class="ref-journal">Angew Chem Int Ed Engl. </span>2003 Jun 6;<span class="ref-vol">42</span>(22):2537–2540.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12800181" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12800181</span></a>] [<a href="http://dx.crossref.org/10.1002/anie.200250572" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594038_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g17-largeoligo.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g17-largeoligo.F1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201%3A%20.%20Synthesis%20of%20Fmoc-Asn-(Benzyl-disialyloligosaccharide)-OH%204%20from%20glycopeptide%201%20isolated%20from%20egg%20yolk.&p=BOOKS&id=594038_g17-largeoligo-Image001.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK594038/bin/g17-largeoligo-Image001.jpg" alt="Figure 1: . Synthesis of Fmoc-Asn-(Benzyl-disialyloligosaccharide)-OH 4 from glycopeptide 1 isolated from egg yolk." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Synthesis of Fmoc-Asn-(Benzyl-disialyloligosaccharide)-OH <b>4</b> from glycopeptide <b>1</b> isolated from egg yolk.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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