{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T03:44:14Z","timestamp":1790307854146,"version":"4.1.0"},"reference-count":51,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2017,3,23]],"date-time":"2017-03-23T00:00:00Z","timestamp":1490227200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["chemistry-europe.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["ChemBioChem"],"published-print":{"date-parts":[[2017,4,18]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>By using O\u2010SP\u2010core (O\u2010SPcNH<jats:sub>2<\/jats:sub>) polysaccharide, isolated from <jats:italic>Vibrio cholera<\/jats:italic> O1 lipopolysaccharide (LPS) and related synthetic substances, a detailed study of factors that affect conjugation of bacterial polysaccharides to protein carriers through squaric acid chemistry to form conjugate vaccines has been carried out. Several previously unrecognized processes that take place during the squarate labeling of the O\u2010SPcNH<jats:sub>2<\/jats:sub> and subsequent conjugation of the formed squarate (O\u2010SPcNH\u2010SqOMe) have been identified. The efficiency of conjugation at pH\u20058.5, 9.0, and 9.5 to bovine serum albumin (BSA) and to the recombinant tetanus toxin fragment C (rTT\u2010Hc) has been determined. The study led to a protocol for more efficient labeling of O\u2010SPcNH<jats:sub>2<\/jats:sub> antigen with the methyl squarate group, to yield a higher\u2010quality, more potent squarate conjugation reagent. Its use resulted in about twofold increases in conjugation efficiency (from 23\u201326\u2009% on BSA to 51\u2009% on BSA and 55\u2009% on rTT\u2010Hc). The spent conjugation reagent could be recovered and regenerated by treatment with MeI in the absence of additional base. The immunological properties of the experimental vaccine made from the regenerated conjugation reagent were comparable with those of the immunogen made from the parent O\u2010SPcNH\u2010SqOMe.<\/jats:p>","DOI":"10.1002\/cbic.201600699","type":"journal-article","created":{"date-parts":[[2017,2,9]],"date-time":"2017-02-09T16:36:34Z","timestamp":1486658194000},"page":"799-815","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["Conjugate Vaccines from Bacterial Antigens by Squaric Acid Chemistry: A Closer Look"],"prefix":"10.1002","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1867-0059","authenticated-orcid":false,"given":"Peng","family":"Xu","sequence":"first","affiliation":[{"name":"NIDDK LBC Section on Carbohydrates National Institutes of Health (NIH)  Bethesda MD 20892-0815 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meagan","family":"Kelly","sequence":"additional","affiliation":[{"name":"Department of Immunology and Infectious Diseases Massachusetts General Hospital  55 Fruit Street Boston MA 02114 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Willie F.","family":"Vann","sequence":"additional","affiliation":[{"name":"Laboratory of Bacterial Toxins CBER FDA  Silver Spring MD 20993 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Firdausi","family":"Qadri","sequence":"additional","affiliation":[{"name":"International Centre for Diarrhoeal Disease Research Bangladesh icddr b)  Dhaka Bangladesh"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edward T.","family":"Ryan","sequence":"additional","affiliation":[{"name":"Department of Immunology and Infectious Diseases Massachusetts General Hospital  55 Fruit Street Boston MA 02114 USA"},{"name":"Department of Medicine Harvard Medical School  Shattuck Street Boston MA 02115 USA"},{"name":"Department of Immunology and Infectious Diseases Harvard School of Public Health  677 Huntington Avenue Boston MA 02115 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5044-3449","authenticated-orcid":false,"given":"Pavol","family":"Kov\u00e1\u010d","sequence":"additional","affiliation":[{"name":"NIDDK LBC Section on Carbohydrates National Institutes of Health (NIH)  Bethesda MD 20892-0815 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2017,3,23]]},"reference":[{"key":"e_1_2_7_1_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00731506"},{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1021\/bc00009a003"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.carres.2007.10.015"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1039\/c3cs60153f"},{"key":"e_1_2_7_5_1","first-page":"69","volume-title":"Vaccine Design: Innovative Approaches and Novel Strategies","author":"Bundle D. 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T. C.","year":"2011"},{"key":"e_1_2_7_38_1","doi-asserted-by":"publisher","DOI":"10.1021\/j100719a013"},{"key":"e_1_2_7_39_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules20022892"},{"key":"e_1_2_7_40_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0008-6215(00)83938-5"},{"key":"e_1_2_7_41_1","unstructured":"To minimize hydrolysis ofBunder the conditions of its preparation the conversion O-SPcNH2\u2192O-SPcNH-SqOMe is conducted with an appropriate excess of dimethyl squarate (determined experimentally with each antigen; the protocol described in the Experimental Section for O-SPcNH2fromV.\u2005choleraO1 should serve as a guideline) and without extending the reaction time unnecessarily (see text above and Figure\u20056). The capacity of the buffer has to be sufficient to maintain the pH near neutral. Using a too large excess of dimethyl squarate can cause a cascade of unwanted events: hydrolysis of4[to give monoester acid Sq(OMe)OH 5] might generate enough acid to break the buffer resulting in protonation of O-SPcNH2and in turn termination of the squarate formation; more extensive hydrolysis of the squarateBmight occur which would result in free acid O-SPcNH-SqOH instead of salt\u2005Cbeing collected with squarate\u2005B and the use of conjugation reagent containing a large amount of O-SPcNH-SqOH might in turn result in inefficient conjugation."},{"key":"e_1_2_7_42_1","unstructured":"When recovery and reactivation of the conjugation reagent is anticipated excess of the O-SPcNH-SqOMe reagent and the reaction time of the conjugation should be such (based on preliminary experiments) that preferentially noBremains unchanged when the desired carbohydrate loading onto protein is reached. This is because if a considerable amount of the unchangedBis present at the end of the conjugation some of it will be irreversibly destroyed during workup [dialysis against a solution of (NH4)2CO3; see later] to give O-SPcNH-SqNH2(F)."},{"key":"e_1_2_7_43_1","unstructured":"The use of (NH4)2CO3 rather than water is important to prevent losses of the conjugate due to its binding to microporous membranes through hydrophobic interactions."},{"key":"e_1_2_7_44_1","unstructured":"It could be present in the conjugation mixture because it had been used in excess at the onset of conjugation and might not be fully saponified at the time the conjugation was deemed complete."},{"key":"e_1_2_7_45_1","unstructured":"We had verified the formation ofFby treating model compound12with 10\u2005mm(NH4)2CO3and identified the corresponding amide14(see Scheme\u20055 and the Experimental Section for details)."},{"key":"e_1_2_7_46_1","first-page":"125","volume-title":"Methods in Enzymology, Vol.\u2005362: Conjugating Low Molecular Mass Carbohydrates to Proteins. 2: Recovery of the Excess Ligand Used in the Conjugation Reaction","author":"Saksena R.","year":"2003"},{"key":"e_1_2_7_47_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0008-6215(01)00018-0"},{"key":"e_1_2_7_48_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.200500518"},{"key":"e_1_2_7_49_1","doi-asserted-by":"publisher","DOI":"10.1021\/ol048614m"},{"key":"e_1_2_7_50_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10719-013-9490-x"},{"key":"e_1_2_7_51_1","unstructured":"The contribution to overall efficiency of recovery=% of recovered spent conjugation reagent\u00d7yield of methylation (regeneration)\u00d7conjugation efficiency of conjugation\u20052. 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