Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins
The construction of amide bonds plays a very important role in the production of small molecule drugs, preparation and modification of polymer materials, conjugation of proteins and drugs, and the synthesis of vaccines, especially in the field of covalent coupling of tumor-associated antigens and ca...
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Editorial Board of Journal of Chinese Mass Spectrometry Society
2025-05-01
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| Series: | Zhipu Xuebao |
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| Online Access: | https://zpxb.xml-journal.net/article/doi/10.7538/zpxb.2024.0135 |
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| author | Xue JIANG Huang-li YANG Xiao-yan LIU Wei XIAO Meng QIU Zhi-gang CHENG Zhen ZHOU Qi-dong TU Xiao-fei GAO |
| author_facet | Xue JIANG Huang-li YANG Xiao-yan LIU Wei XIAO Meng QIU Zhi-gang CHENG Zhen ZHOU Qi-dong TU Xiao-fei GAO |
| author_sort | Xue JIANG |
| collection | DOAJ |
| description | The construction of amide bonds plays a very important role in the production of small molecule drugs, preparation and modification of polymer materials, conjugation of proteins and drugs, and the synthesis of vaccines, especially in the field of covalent coupling of tumor-associated antigens and carrier proteins. In this study, the advantages of electrospray microdroplet technology in promoting the aminolysis reaction between activated phenolic ester and the free amino groups in nature proteins for amide bond formation, and its application in the conjugation of small organic molecules with nature proteins were discussed. The effects of sample flow rate, sheath pressure, electric field strength and solvent polarity on the coupling efficiency of amide bonds in electrospray microdroplet technique were studied by using N-carbobenzoxyglycine 4-nitrophenyl ester (Z-Gly-ONP) and nature protein lysozyme as model substrates and employing an home-made device with two independent liquid channels for microdroplet generation. When the two kinds of microdroplets coming from the two liquid channels were fused, followed by the aminolysis reaction, and the mixtures of the microdroplet reaction were collected, dissolved, desalted, diluted and detected. The results after the optimization showed that, in the microdroplet system, although a certain amount of positive and high voltage will slightly improve the reaction conversion, the sample flow rate and sheath pressure play a major role in the amide bond formation reaction. Below a certain pressure of sheath gas, high pressure of the sheath gas and low flow rate of the sample will benefit for the desolvation efficiency of the sample solution at the nozzle, generating small particle size of the microdroplets and increasing of the local concentration of reactive substates and the frequency of collision between sample molecules, leading to the fast mass transfer between liquid containing active esters and liquid containing nature proteins. As a consequence, the conversion rate of protein covalent modification reaction in microdroplets is significantly higher than that in the bulk solution system under the same reactant conditions. Furthermore, the microdroplet chemistry has been successfully applied to the covalent coupling of tumor antigen peptides to nature protein lysozyme, with a conversion rate of 89%, which is much higher than that in the bulk solution system. The results of this study indicate that microdroplet chemistry has a potential in the efficient conjugation of tumor antigen polypeptides and T-epitope-containing carrier proteins. |
| format | Article |
| id | doaj-art-91beef1aef684dc698aff68145efb172 |
| institution | Kabale University |
| issn | 1004-2997 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Editorial Board of Journal of Chinese Mass Spectrometry Society |
| record_format | Article |
| series | Zhipu Xuebao |
| spelling | doaj-art-91beef1aef684dc698aff68145efb1722025-08-20T03:48:10ZengEditorial Board of Journal of Chinese Mass Spectrometry SocietyZhipu Xuebao1004-29972025-05-0146327728510.7538/zpxb.2024.01352024-135-c14Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and ProteinsXue JIANG0Huang-li YANG1Xiao-yan LIU2Wei XIAO3Meng QIU4Zhi-gang CHENG5Zhen ZHOU6Qi-dong TU7Xiao-fei GAO8Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013 ChinaJiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, ChinaJiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013 ChinaJiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, ChinaJiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, ChinaJiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, ChinaInstitute of Mass Spectrometry and Atmospheric Environment, Jinan University, Guangzhou 510630, ChinaJiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013 ChinaJiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, ChinaThe construction of amide bonds plays a very important role in the production of small molecule drugs, preparation and modification of polymer materials, conjugation of proteins and drugs, and the synthesis of vaccines, especially in the field of covalent coupling of tumor-associated antigens and carrier proteins. In this study, the advantages of electrospray microdroplet technology in promoting the aminolysis reaction between activated phenolic ester and the free amino groups in nature proteins for amide bond formation, and its application in the conjugation of small organic molecules with nature proteins were discussed. The effects of sample flow rate, sheath pressure, electric field strength and solvent polarity on the coupling efficiency of amide bonds in electrospray microdroplet technique were studied by using N-carbobenzoxyglycine 4-nitrophenyl ester (Z-Gly-ONP) and nature protein lysozyme as model substrates and employing an home-made device with two independent liquid channels for microdroplet generation. When the two kinds of microdroplets coming from the two liquid channels were fused, followed by the aminolysis reaction, and the mixtures of the microdroplet reaction were collected, dissolved, desalted, diluted and detected. The results after the optimization showed that, in the microdroplet system, although a certain amount of positive and high voltage will slightly improve the reaction conversion, the sample flow rate and sheath pressure play a major role in the amide bond formation reaction. Below a certain pressure of sheath gas, high pressure of the sheath gas and low flow rate of the sample will benefit for the desolvation efficiency of the sample solution at the nozzle, generating small particle size of the microdroplets and increasing of the local concentration of reactive substates and the frequency of collision between sample molecules, leading to the fast mass transfer between liquid containing active esters and liquid containing nature proteins. As a consequence, the conversion rate of protein covalent modification reaction in microdroplets is significantly higher than that in the bulk solution system under the same reactant conditions. Furthermore, the microdroplet chemistry has been successfully applied to the covalent coupling of tumor antigen peptides to nature protein lysozyme, with a conversion rate of 89%, which is much higher than that in the bulk solution system. The results of this study indicate that microdroplet chemistry has a potential in the efficient conjugation of tumor antigen polypeptides and T-epitope-containing carrier proteins.https://zpxb.xml-journal.net/article/doi/10.7538/zpxb.2024.0135electrospraymicrodropletsamide bond formationaminolysismass spectrometry |
| spellingShingle | Xue JIANG Huang-li YANG Xiao-yan LIU Wei XIAO Meng QIU Zhi-gang CHENG Zhen ZHOU Qi-dong TU Xiao-fei GAO Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins Zhipu Xuebao electrospray microdroplets amide bond formation aminolysis mass spectrometry |
| title | Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins |
| title_full | Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins |
| title_fullStr | Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins |
| title_full_unstemmed | Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins |
| title_short | Facilitated by Microdroplet Mass Spectrometry on Amide Bond Construction and its Application in Conjugation of Small Molecules and Proteins |
| title_sort | facilitated by microdroplet mass spectrometry on amide bond construction and its application in conjugation of small molecules and proteins |
| topic | electrospray microdroplets amide bond formation aminolysis mass spectrometry |
| url | https://zpxb.xml-journal.net/article/doi/10.7538/zpxb.2024.0135 |
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