Alexei V. Demchenko
Professor Demchenko graduated from the Mendeleev University of Chemical Technology of Russia with a Diploma (M.S.) in Chemical Engineering (1988) before joining the laboratory of the late Professor Kochetkov at the Zelinsky Institute of Organic Chemistry in Moscow. In 1993, he was awarded a Ph.D. in organic chemistry by the Russian Academy of Sciences for his work on the development of thiocyanate methodology for glycosylation. After two post-doctoral years under Kochetkov, he joined Professor Boons’ group at the University of Birmingham (UK) as a BBSRC post-doctoral research fellow. In 1998, he moved to the Complex Carbohydrate Research Center, University of Georgia (USA) as a research associate. In 2001, he joined the faculty at the University of Missouri - St. Louis as an Assistant Professor where he was promoted to the rank of Associate Professor with tenure (2007) and Professor (2011). In 2014, Demchenko was appointed Curators’ Distinguished Professor of Chemistry and Biochemistry. In 2021, Demchenko joined the faculty at Saint Louis University as Professor and Department Chair.
1. Abstract
The rapid growth of R&D in glycosciences demands the development of rapid, efficient, and simple procedures for glycan synthesis. High Performance Liquid Chromatography equipment-based Automation (HPLC-A) of carbohydrate synthesis seeks to meet this demand by focusing on the development of an affordable and accessible automation platform that will enable both specialists and non-specialists to perform the synthesis of glycans from renewable precursors. Current methods for the synthesis of glycans are highly sophisticated and operationally complex. By contrast, HPLC-A represents a highly accessible method for synthesis because many scientists already have easy access to HPLC equipment. Automated synthesis offers operational simplicity by delivering all reagents using standard HPLC components, but also convenient real-time reaction monitoring of every step.
Keywords : Automation, Building blocks, Chemical synthesis, Glycosylation, HPLC, Oligosaccharides, Solid phase synthesis
2. Carbohydrates as molecules of life and death
Carbohydrates are known as the “essential molecules of life”1 due to their involvement in many fundamental processes: fertilization, supply of nutrients, joint lubrication, cell growth, antigenic determination, anti-inflammation, and immune response among others (Scheme 1)2,3. The explosive growth of glycobiology4,5, glycomics6,7, and glycoproteomics8,9 has increased our understanding of the roles of sugars also as “molecules of death” due to their involvement in every major disease including cancers, AIDS, pneumonia, septicemia, diabetes, and malaria among others. The discovery of new methods and accessible technologies that will offer new capabilities for obtaining glycans in high purity is at the heart of this article.
Scheme 1. Glycans as molecules of life and death
3. Chemical synthesis of glycosides and oligosaccharides
Glycans are composed of monosaccharide building blocks connected via O-glycosidic linkages10. This linkage is obtained by glycosylation, a displacement of an anomeric leaving group of the donor with a hydroxyl group of the acceptor. Current understanding is that a typical glycosylation falls at a certain position on a continuum of mechanisms spanning from ideal SN1 to ideal SN2 extreme11-13. Due to significant advances in synthesis, many glycosidic bonds can now be prepared by using classical methods or promising recent inventions. Nevertheless, glycosylation remains challenging, and reactions that proceed with high rates, complete conversion, flawless stereoselectivity, minimal side reactions, and work with a broad range of substrates are rare.
Oligosaccharide synthesis requires additional protection and/or leaving group modifications between each glycosylation step, which can be streamlined by using advanced strategies. Expeditious strategies that streamline glycan synthesis are based on (chemo)selective activation of leaving groups14,15. Fraser-Reid’s armed-disarmed16,17, Danishefsky’s18, Roy’s19 and Boons’20 active-latent, Ogawa-Ito’s orthogonal21,22, Ley’s tunable23,24, Wong’s programmable25,26, Huang’s preactivation27-29 are select examples of such strategies. We have also introduced a number of strategies for glycan synthesis including temporary deactivation30,31, inverse armed-disarmed strategy32, O-2/O-5 cooperative effect in superarming/disarming33-36, STICS: surface-tethered iterative carbohydrate synthesis37, thioimidate-only orthogonal strategies38,39, and reverse orthogonal strategy40,41. Regardless of the strategy chosen, chemical assembly of glycans is hard to implement in non-specialized labs to accelerate innovations in glycosciences.
4. Automation of solid-phase and solution synthesis
Solid-phase synthesis of glycans on polymer supports also involves reiteration of glycosylation – deprotection steps, but it eliminates the need for conventional reaction work-up and purification of intermediates. Another strength of solid-phase synthesis is its automation amenability. As the 21st century unfolds with rapid changes, new challenges in R&D emerge. A marked improvement has been achieved in 2001 by Seeberger who adapted a peptide synthesizer to glycan synthesis42. In 2012, Seeberger reported “the first fully automated solid-phase oligosaccharide synthesizer43.” This synthesizer was then commercialized as Glyconeer 2.144, and more recently as Glyconeer 3.1. Also in 2012, we reported HPLC-based automation (HPLC-A) of solid-phase synthesis45. Very recently, Hsieh-Wilson and Huang reported an automated solid-phase synthesis of a heparan sulfate library46,47. Efforts to automate synthesis of glycans in solution have been reported by Takahashi48,49, Pohl50,51, and Nokami52-54. Recently, Ye reported a very impressive automated synthesis of a 1,080-mer glycan55. Being still relatively new, these approaches may offer viable alternatives to automated enzymatic syntheses being developed by Wong56,57, Chen58-60, Wang61,62, and Boons63. The availability of dependable and transferable methods for glycan synthesis is essential for boosting innovations and practical applications in glycosciences. This Article describes the invention and key steps that have already been made towards initial refinement of the HPLC-A.
The general idea for developing the HPLC-A is that a computer interface, coupled with standard HPLC components, will allow recording a successful automated sequence as a computer program. In our original (Generation A) set-up for solid phase synthesis, resin-immobilized acceptor was packed into an Omnifit column that was integrated into a HPLC system. All reactants and reagents were delivered via intake lines of a HPLC pump. Glycosylations were performed by recirculating the premixed mixture containing the imidate donor and TMSOTf. After the desired number of glycosylation/Fmoc group deprotection iterations, the glycan was cleaved off from the resin. Although this Generation A HPLC-A set-up offered operational convenience, reaction monitoring using a UV detector, faster reaction times, it remained semi-manual45.
In 2016, we introduced Generation B HPLC-A. In this set-up, we implemented a standard autosampler as a highly reproducible mode for injecting promoter (TMSOTf) and identified JandaJel resin as an optimal solid support64,65. To advance the original design, Generation B introduced in 2016 implemented for automated synthesis. Also, the incorporation of an HPLC autosampler introduced into the HPLC-A system. However, this Generation B HPLC-A set-up remained semi-manual because switching between the reaction, discharge, and collection modes required the operator.
To improve our capabilities, we introduced Generation C HPLC-A, which was supplemented with a 2-way split valve as a mode for complete automation66. The programmable two-way split valve allowed for automated switching between discharge and the product collection modes. This platform was also supplemented with a preparative autosampler, which was programmed to deliver all reagents and all glycosyl donors for all steps of the HPLC-A synthesis (Scheme 2). The sequence consisting of four glycosylation/deprotection cycles, followed by the off-resin cleavage and product collection was performed with a single press of a button. All donors and reagents were delivered by the autosampler, and the pump supplied standard ACS-spec solvents. We also developed PanzaGel resin as a dedicated support for solid-phase synthesis that can be created as beads or monoliths67.
Scheme 2. Fully automated HPLC-A synthesis on polymer support
Unexpected recent pandemic revealed unpreparedness of our society, but also gave us an opportunity to demonstrate how HPLC-A can be utilized to maintain high productivity by performing the synthesis of many products with a single press-of-a-button, even during reduced-work-hour and time-shift protocols, which were implemented to ensure social distancing68. To enable this capability, we switched to the solution phase and supplemented the system with a fraction collector (Scheme 3). To enable recirculation, along with the waste and the collection modes, we implemented a four-way split valve. Pump line A was used for delivering DCM needed for glycosylation and washing. Pump line D was dedicated to recirculation, and the autosampler was used to deliver all reagents. The column was repurposed to hold activated molecular sieves. The software was programmed to perform completely automated synthesis of six di- and tri-saccharides from randomly selected donor-acceptor pairs in 12 h68. We also showed that HPLC-A reactions can accurately be reproduced by a high-school researcher.
Scheme 3. Fully automated HPLC-A for sequential synthesis of multiple products in solution
However, we were still unable to automate all stages of glycan synthesis and purification starting from renewable commercial precursors. With the goal of developing the synthesis of building blocks, glycosylation, and purification in a fully automated manner, we combined HPLC-A with the reactor-based synthesis69. The modular character of HPLC allows for straightforward modification using the plug-in approach. Our new set-up comprised the upstream (reaction circuit) and downstream (separation circuit) operations (Scheme 4). The upstream operations were equipped with an autosampler, a quaternary pump, and a reactor. The reactor was equipped with a magnetic stirrer, a bottom outlet, and reactions could be either heated or cooled. The autosampler was programmed to deliver all necessary reagents to the reactor. The reaction mixture was then stirred for the same time as that for the manual synthesis.
Scheme 4. Fully automated HPLC-A for reactor-based synthesis and chromatographic purification in the single platform
Downstream operation circuit was equipped with a quaternary pump, a disposable flash chromatography cartridge, a UV detector, and a fraction collector (Scheme 4). We have implemented a programmable logic controller (PLC) to operate a proximity sensor, an optical sensor, and a solenoid valve needed for controlled reaction mixture transfer from the reactor to the chromatography column. Subsequent chromatographic separation and fraction collection concluded the automation sequence. The reaction and separation circuits were interfaced with a 2-way split valve, which allows to use both pumps for both circuits.
5. Conclusions and outlook
Described herein are fully automated HPLC-A applications. With the fully automated platform we were able to record and save entire synthetic sequences as a computer program. With the refined operational techniques, we are well positioned to investigating how well the developed platforms work in application to the synthesis of various carbohydrate derivatives. Automated synthesis ensures rigorous experimental design to obtain robust results and to eliminate any variables. The delivery of standard ACS-spec solvents ensures authentication of key chemical resources and reproducibility of experiments. The recorded sequences can then be accurately reproduced anywhere by anybody who has access to HPLC, even non-specialists, who may not have expertise to do conventional synthesis. The acquired knowledge will benefit scientists in academia, government, and industry who have access to standard HPLC equipment and an interest in creating glycans or other biomolecules for their own studies.
What should you do if you were inspired by our work and interested in our automation platform? If you have a recent Agilent HPLC system, you have everything you need to adapt our technology to your machine; no investment will be necessary. We have mainly been using glass Omnifit columns, but you can use any compatible chromatography column. If we have already done the reaction that interests you, we already have a basic sequence of parameters that has been recorded as a computer program. The program can be shared, you then load it to your system, and you are ready to automate. What if you change your mind? You can always repurpose your system back to chromatographic separation. What do you do if you do not have an Agilent HPLC system? Certainly, you can write your own operating programs following our general guidelines and parameters or you may decide to develop your own sequences. Alternatively, you can interface practically any recent model machine with Agilent software, but this requires an add-on unit from the manufacturer of your HPLC.
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