HOT TOPICS 8 doi:10.2533/chimia.2008.8 CHIMIA 2008, 62, No. 1/2

Chimia 62 (2008) 8–12 © Schweizerische Chemische Gesellschaft ISSN 0009–4293

Automated Synthesis of Provides Access to Chemical Tools and Vaccine Candidates

Lenz Kröck and Peter H. Seeberger*

Abstract: Defined, pure oligosaccharides are important tools for biologists to study the structure and function of in biological systems and to create novel diagnostic and therapeutic approaches. Access to reasonable quantities of sufficiently pure oligosaccharides by isolation is difficult and time consuming. Synthetic chemists are therefore called upon to develop straightforward and reliable methods to produce carbohydrates. These molecules are key to the creation of molecular tools and vaccine candidate molecules. A brief overview of the state-of-the-art of automated synthesis is followed by some illustrative examples to highlight applications. Keywords: Automation ⋅ synthesis ⋅ Oligosaccharides ⋅ Solid-phase

1. Why Carbohydrate Synthesis? are hard to access. When using isolated peptide 206 (= 64 million) and for a hexa- carbohydrates, only very small quantities saccharide 192 billion. The importance of carbohydrates as carri- of usually impure molecules are available Theoretically, 224 building blocks ers of information and as key mediators of such that structure− activity relationships would be required to assemble any given cellular interactions is now well appreci- are difficult to determine since impurities mammalian when consid- ated.[ 1 ] Consequently, interest in studying may be responsible for the effects that are ering the ten mammalian monosaccharides biological processes that involve different observed. Pure and well-characterized syn- and all possible connections and . classes of carbohydrates such as glyco- thetic molecules are of course ideal tools, A recent bioinformatics study revealed that proteins, glycolipids, glycosaminoglycans particularly when incorporated into tech- nature only uses a fraction of the possible and GPI anchors has drastically increased. nology platforms such as carbohydrate mi- glycospace: just 36 building blocks suffice Biochemists and biologists interested in croarrays. to allow for the synthesis of 75% of mam- specific structural or functional questions malian carbohydrate structures.[ 3 ] often find themselves in a difficult position as the molecules they are trying to study 2. What Renders Oligosaccharide Synthesis so Challenging? 3. What Challenges Had to Be Overcome in the Automation of The challenge that lies in the synthe- Oligosaccharide Synthesis? sis of oligosaccharides becomes most ap- parent when comparing sugars to the two The automation of oligosaccharide other classes of biopolymers that are rou- synthesis required us to combine synthet- tinely synthesized by automation. Oligo- ic methodology with a synthesizer robot. nucleotides and peptides are strictly linear Both had to be developed simultaneously biopolymers while oligosaccharides exhibit and fine tuned to work hand in hand. highly branched structures. Four nucleic acid and 20 amino acid building blocks are 3.1. Synthetic Methodology required for synthesis. Mammalian carbo- 3.1.1. Synthetic Strategy hydrates consist of ten monosaccharides. First, a synthetic strategy had to be These monosaccharides each can be linked selected. Automated oligonucleotide and via multiple positions. Furthermore, a new peptide synthesis rely on the solid-phase * Correspondence: Prof. Dr. P. H. Seeberger stereogenic center is created in every cou- approach. The main advantage of the solid- Laboratorium für Organische Chemie der pling and results in two possible isomers. phase approach lies in the use of excess Eidgenössischen Technischen Hochschule (ETH) Multiple linking positions and stereoiso- reagents to drive coupling reactions to Zürich HCI F315 mers result in higher diversity of possible completion. Solid-phase peptide and oligo- Wolfgang-Pauli-Strasse 10 molecular structures as illustrated by the nucleotide synthesis is carried out as fol- CH-8093 Zürich following example:[ 2,3] The number of lows: A growing chain of the biopolymer is Tel.: + 41 44 633 2103 Fax: + 41 44 633 11235 theoretically possible structures in case of synthesized in a strictly linear fashion on a E-Mail: [email protected] a hexanucleotide is 4 6 (= 4096), for a hexa- polymeric support. A nucleophile exposed HOT TOPICS 9 CHIMIA 2008, 62, No. 1/2 on the support is reacted with excess build- ing block (coupling) that carries permanent Donor-Bound Strategy: PO and temporary protecting groups. Removal PO of the temporary protecting group (depro- O PO B PO PO tection) reveals a nucleophile that in turn HO X PO PO PO can undergo a coupling step and thereby O OP O O ActivateX Elongation A A B completes the synthetic cycle. The solid ActivateLG O LG O O X support serves as a handle for washing steps OP OP that remove byproducts generated during OP coupling and deprotection. Upon comple- tion of the synthesis, the material is discon- Acceptor-Bound Strategy: PO nected from the solid support (cleavage) PO O and purified by HPLC. Permanent protect- PO PO B PO ing groups are generally removed (global P 2 O LG PO PO PO deprotection) in the cleavage step. Since Elongation Deprotection O O OP O B A A the coupling, deprotection and washing Activate LG P 2 O O O HO O steps are repeated, the solid-phase approach OP OP OP lends itself to automation. Solid-phase oligosaccharide synthesis was first explored in the early 1970s.[ 4 ] In Scheme 1. Donor- and acceptor-bound approach solid-supported oligosaccharide synthesis either the sugar carrying the anomeric leav- ing group () or the nucleo- phile (glycosyl acceptor) can be attached O SR to the solid support (Scheme 1). Both ap- [ 5 ] proaches have been explored. The accep- O tor-bound approach was found to be supe- O R O O HO O n R O O rior since the glycosyl donor is the more n reactive species and is more prone to de- or or CM O O O R O O R O OH composition. The acceptor-bound approach O n n allows the glycosylating agent to be used in O HO O excess. Side products deriving from donor O decomposition remain in solution and can R O OR' n be simply washed away. When the donor O is bound to the solid phase, decomposi- R O O tion of the limiting reagent would result in n an unproductive reaction and diminish the overall yield. Scheme 2. Octenediol and ester linkers To establish a suitable synthetic meth- odology based on the acceptor-bound solid- phase approach the following issues had to be addressed: The linker connects the first unit of therefore limits the methods i) development of a linker that is compat- the growing oligosaccharide chain to the that can be used. ible with all synthetic operations (Sec- solid support. It has to remain intact dur- tion 3.1.2) and can be cleaved efficiently ing the synthesis but has to be cleaved ef- 3.1.3. Building Blocks (Section 3.1.5) ficiently, selectively and under relatively The identification and synthesis of the ii) identification of building blocks re- mild conditions at the end of the synthesis. monosaccharide building blocks required quired to access the relevant structures Furthermore, the cleaved linker should al- for the automated synthesis is currently the (Section 3.1.3) equipped with readily low attachment to a protein carrier or a chip most laborious and time consuming task. removable protecting groups (Section surface and ideally enable release of a free Nevertheless it is a very important aspect 3.1.6) reducing end (Scheme 2). in the development of a commercial syn- iii) rapid and selective coupling and depro- A linker containing a double bond ful- thesizer. Once the most suitable building tection conditions (Section 3.1.4) fills these prerequisites. The double bond blocks have been identified and tested, their iv) purification of the final product (Section was incorporated in form of an octenediol large scale synthesis will be commercial- 3.1.6) linker that is connected to the support ei- ized as is the case for peptide and oligo- ther by an ether[ 7 ] or an ester[ 8 ] linkage. The nucleotide building blocks. 3.1.2. Solid Support and Linker product is released by cross metathesis us- A building block for oligosaccharide Merrifield’sresin[ 6 ] wasfoundtobemost ing Grubbs catalyst in presence of ethylene synthesis generally carries permanent and suitable among the commercially available without affecting the protecting groups. temporary protecting groups. The anomeric solid supports. This chloromethylated poly- Cleavage results in the oligosaccharide re- position is equipped with a leaving group. styrene resin also used in peptide synthesis lease in form of a n -pentenyl . The In our approach benzyl ethers (Bn), pivaloyl shows the best performance with respect terminal olefin can be chemically modified esters (Piv), azides and N-trichloroacetyl to loading, swelling, solvent and reagent to connect the sugar to a protein carrier or (TCA) groups serve as permanent protect- compatibility. The stability towards acids chip surface.[ 9 ] Removal of the pentenyl ing groups – i.e. those groups will only be and bases, used in the glycosylation and glycoside furnishes the free reducing sugar. removed during global deprotection at the deprotection steps of the synthetic cycle, is The octenediol linker works well although end of the synthesis (Scheme 3). 9-Fluo- an important advantage. it precludes the use of electrophiles and renylcarbyloxymethyl (Fmoc), levulinoyl HOT TOPICS 10 CHIMIA 2008, 62, No. 1/2

(Lev), acetyl (Ac) and silyl-protecting amine protecting groups the corresponding HO Monosaccharide groups were chosen for temporary protec- Building Blocks naturally occurring N-acetyl moieties are tion. These groups are cleaved upon cou- O procured by reduction with zinc in the pres- pling to allow for elongation of the growing ence of acetic acid. The commonly used biopolymer. benzyl ethers and any esters are removed The development of anomeric leaving Coupling by Birch reduction using sodium in liquid groups has been the focus of carbohydrate ammonia. Alternatively, base-assisted ester chemistry for decades.[ 10] For the automat- cleavage followed by palladium-catalyzed O O ed approach we found glycosyl trichloro- HO RO hydrogenolysis has been applied. This pro- acetimidates[ 11] and phosphates[ 12] the most O O tocol results in reduction of the n -pentenyl suitable choice. Both classes of compounds O O residue that has to be modified prior to hy- are reasonably stable − they can be stored drogenolysis in order to allow attachment longterm in the freezer without decomposi- of the oligosaccharide to surfaces.[ 9 ] tion and both compounds can be activated UV Quantitation Deprotection The unprotected sugar finally has to be by TMSOTf addition – a caustic, but non- of Cleaved Fmoc prepared for use in biological studies. It toxic reagent. Most importantly, both gly- is passed through size exclusion columns Cleavageand cosylating agents generally result in high Purification and dialyzed to remove any salts. Char- yielding and selective . acterization relies on full analysis by 1 H-, 13 Products C-NMR and mass spectrometry. Once the synthesis process has been streamlined and OBn BnO OAc O has proven to be reliable a less thorough NH Scheme 4. Coupling cycle of automated FmocO O OP(OBu) BnO O analysis e.g. by mass spectrometry only BnO 2 BnO oligosaccharide synthesis OPiv O CCl3 may become sufficient.

3.2. Synthesizer Scheme 3. Representative examples of building in DMF). The main advantage of Fmoc in The synthesizer has to execute the syn- blocks comparison to acetate or levanulinyl es- thetic cycle including coupling, deprotec- ters lies in the fast and mild deprotection tion and washing steps. Initially, a modified conditions and in the UV-activity of the ABI peptide synthesizer was used (Fig.).[ 13] Alternative protecting and leaving dibenzofulvene byproduct generated dur- The modification enabled temperature con- groups can be envisioned. The examples ing deprotection. Coupling efficiency can trol of the reaction vessel as the coupling described above can be seen as a starting be assessed by the UV absorbance of the steps generally take place below 0 °C. The point and certainly will be further devel- deprotection solution. machine uses argon pressure to drive re- oped in the future. agent solutions and solvents through Teflon 3.1.5. Cleavage from the Resin lines and solenoid valves that are opened 3.1.4. Synthetic Cycle After the coupling cycles have been and closed to control the amount of reagent The assembly of oligosaccharides in- completed the protected oligosaccharide dispensed. The building blocks are stored volves a two-step synthetic cycle consist- has to be removed from the solid support. in plastic vessels. Delivery of the building ing of glycosylation and deprotection. Be- Currently this task is achieved by olefin block solutions takes place via a needle that tween those two steps the resin is washed cross metathesis. The resin is transferred to punches a hole into the container. The solu- thoroughly to remove any byproducts. In a round-bottom flask and is treated over- tion then is delivered by argon pressure to the glycosylation step a solid support bound night with Grubbs catalyst under an atmo- the reaction vessel. This vessel is a double- nucleophile is reacted with the anomeric sphere of ethylene. The liberated products jacketed glass tube with a frit in the bot- position of the incoming building block to are obtained in the form of n -pentenyl furnish a glycosidic linkage. In the initial that can be separated from the glycosylation, the incoming nucleophile catalyst by filtration. At this point the re- Fig. The fi rst is the hydroxyl of the linker to anchor the sult of the synthesis can be assessed by automated growing biopolymer to the solid support. HPLC− mass spectrometry (LC-MS) using oligosaccharide Selective removal of a temporary protect- a small aliquot of the product. Unwanted synthesizer ing group furnishes a hydroxyl group as side-products are removed from the fully nucleophile for the next coupling (Scheme protected oligosaccharide by HPLC, since 4). separation is significantly easier than at the In the glycosylation step the building fully deprotected stage. Nevertheless, about blocks are commonly used twice in five- 40− 50% of the desired product is lost dur- fold excess with respect to the solid phase ing the HPLC purification. Although recov- bound nucleophile. These so-called ‘double ery rates are similar to those for peptides couplings’ have been shown to result in purification further improvements of the coupling yields that exceed 98%. In case purification protocols have to be achieved. of glycosyl phosphates a five-fold excess of TMSOTf as activating reagent is used 3.1.6. Global Deprotection and while glycosyl trichloroacetimidates only Purification of the Final Product require 0.75 equiv. of this activating agent. All protecting groups have to be re- The temperature for coupling reactions var- moved from the oligosaccharide to afford ies from − 15 °C to room temperature. the final product. Depending on the nature Fmoc is currently used as the standard of the protecting groups one or more trans- temporary protecting group.[ 8 ] This car- formations are required. When N-trichlo- bamate is cleaved with piperidine (20% roacetyl groups were used as permanent HOT TOPICS 11 CHIMIA 2008, 62, No. 1/2 tom. The double jacket is used to circulate a cryogenic fluid that allows for cooling of OH O BnO O BnO the reaction vessel. The frit in the vessel PivO HO retains the solid phase in the vessel while O O washing solutions can be filtered away. Cleavage and Purification LevO OBn H CCH OLev The first prototype required manual ad- O 2 2 O BnO O BnO O PCy Deprotection Coupling O O 3 BnO O P OBu justment of the temperature as the peptide BnO PivO Cl Ph BnO BnO Ru H NNH TMSOTf PivO O Cl 2 2 OBu synthesizer did not communicate with the BnO 15°C -15°C BnO O PCy3 cryostat. A syringe pump-driven oligosac- OBn PivO O Alternate Donors BnO O BnO O charide synthesizer has been developed to BnO O OLev BnO PivO BnO O BnO O O OLev overcome this limitation and to have more O BnO OBn O BnO PivO O PivO BnO O BnO freedom with regards to the chemistry that OBn O O O P OBu O BnO PivO BnO O BnO O BnO OBu can be used. BnO O BnO PivO BnO O BnO O OBn PivO O BnO O BnO O 4. Automated Oligosaccharide BnO O BnO PivO O BnO O Synthesis: Beyond the Proof of BnO Principle?! PivO

Three examples of automated syntheses Scheme 5. Automated synthesis of the β -glucan dodecamer are discussed in order to illustrate the cur- rent state of the art of our system.

OBn BnO BnO OBn OBn BnO HO 4.1. The First Automated Synthesis O O O O BnO O O O O Thefirstautomatedoligosaccharidesyn- O PivO BnO O TCAHN OPiv [ 13] O thesis was reported by our group in 2001 OBn O BnO PivOOPiv using a phytoalexin elicitor β -glucan dode- PivOOPiv casaccharide as example. This elicitor gly- can triggers soybean plants to release anti- OBn BnO OBn BnO BnO OBn OBn BnO BnO HO O O O O O O biotic phytoalexins as a defense mechanism BnO O O O O O O O O PivO BnO against fungi which exhibit the β -glucan. O TCAHN PivO TCAHN OPiv O O OBn OBn O The molecule had been previously pre- BnO OPiv PivOOPiv PivO pared in solution[ 14] and on solid support[ 15] PivOOPiv and served as a benchmark for our early au- tomation endeavors. The synthesis was ac- complished using two glycosyl-phosphate BnO OBn O OBn OBn OBn O O BnO O O building blocks: a monosaccharide and O O O P (OBu)2 LevO O O FmocO O BnO OP(OBu)2 OP(OBu)2 FmocO OP(OBu)2 OP(OBu) OBn FmocO BnO 2 a that were alternately used OFmoc PivOOPiv TCAHN PivO OPiv for coupling. The levulinoyl ester served as temporary protecting group. Therefore, real-time monitoring of the coupling yields Scheme 6. Retrosynthesis of Lewisy hexasaccharide and Ley-Lex nonasaccharide was impossible. This synthesis was an im- portant milestone. Nevertheless, the syn- thetic challenge imposed by the branching of the structure was circumvented using second protecting group orthogonal to the yields comparable or better than previous disaccharide building blocks, exclusively levulinoyl group used in the previous syn- solution-phase syntheses, but in a fraction trans-glycosidic linkages were established theses was required. Fmoc was identified as of the time previously required. and the final compound was not deprotect- the optimal choice since it can be cleaved ed (Scheme 5). in presence of levalinoyl protection. Fmoc cleavage can be used to monitor the cou- 4.3. A First Application of Auto- pling yields, a feature that was of particular mated Oligosaccharide Synthesis 4.2. Synthesis of Blood Group importance at the development stage of our – An Anti-Toxin Malaria Vaccine Determinants and Tumor-Associat- synthetic system. Candidate ed Antigens The Lewisy hexasaccharide was assem- Automated oligosaccharide synthesis Lewisy hexasaccharide and Ley -Lex bled from five different building blocks in holds great potential to accelerate the as- nonasaccharide are tumor markers that are an isolated overall yield of 10%. Ley -Lex sembly of oligosaccharides to be used in currently under evaluation as anti-cancer nonasaccharide was assembled from the conjugated vaccines. An illustrative first vaccines.[ 16] Their branched structures im- same five building blocks. The synthesis example from our laboratories is the use of posed a serious challenge to our approach required nine glycosylations, eight depro- automated oligosaccharide synthesis in the and gave us the opportunity to improve the tections and one cleavage and purification construction of a molecule currently in pre- overall protocol.[ 7 ] The resin was modi- step. The isolated overall yield of the syn- clinical evaluation as an anti-toxin malaria fied to include an ester moiety between the thesis was 6.5% (Scheme 6). Given the size vaccine candidate.[ 17] linker and the resin. The modified linker and complexity of the molecule, the result Malaria infects currently 5% of the enabled faster and more reliable cleavage, underscores the power of our methodol- world’s population, resulting in 100 mil- but at the same time precluded the use of ogy. lion clinical cases and 3 million deaths an- temporary acetyl protecting groups. As Though fully protected, the target oli- nually. We demonstrated that vaccination the structures contain branching points a gosaccharides were obtained in overall with anti-toxin malaria vaccine candidate HOT TOPICS 12 CHIMIA 2008, 62, No. 1/2 can protect mice from death caused by ma- [ 18] OAc laria parasites. OBn O The oligosaccharide part of the vaccine BnO BnO candidate was synthesized in a semi-auto- O NH mated fashion as the α -linkage between OBn OBn OBn OAc CCl3 O O inositol and glucosamine was deemed too BnO BnO OH challenging for automation. A tetra-manno- OH BnO BnO O O TIPSO NH HO O O syl fragment was synthesized by automa- + automated HO P O(CH2 ) 2 NH3 O BnO CCl3 tion and joined to a disaccharide that was O - TIPSO O O BnO OAc O OBn O obtained by solution phase synthesis. The HO O BnO HO O BnO oligosaccharide was conjugated to a car- BnO OH O NH O BnO rier protein to obtain the vaccine candidate O HO O CCl 3 OBn OBn (Scheme 7). HO O OBn BnO O In this case solution-phase synthesis can O BnO OH BnO OR BnO O provide larger amounts of the oligosaccha- HO O NH ride, but automated synthesis allows for fast HO OH + O CCl3 access. This feature will be instrumental in fu- O OBn HO + O ture studies where fast access to structurally H 3 N O OH HO O OH BnO diverse oligosaccharide libraries is required. O OH N 3 O OBn O OBn P O OBn - O O

5. Conclusion and Outlook synthesizedinsolution

Automated oligosaccharide synthesis to Scheme 7. Retrosynthesis of the malaria vaccine candidate. The disaccharide is synthesized in date has provided access to a broad range solution, the tetrasaccharide by automation from four building blocks. of biologically relevant oligosaccharides – three examples have been highlighted in this review. [ 8 ] K. R. Love, P. H. Seeberger, Angew. Chem. [ 15] K. C. Nicolaou, N. Winssinger, J. Pastor, We were able to address the key chal- Int. Ed. 2004, 43, 602. F. DeRoose, J. Am. Chem. Soc. 1997, 119, lenges towards a general approach of au- [ 9 ] T. Buskas, E. Soederberg, P. Konradson, 449. tomated oligosaccharide synthesis. Still, B. Fraser-Reid, J. Org. Chem. 2000, 65, [ 16] G. Ragupathi, P. P. Deshpande, D. M. there is ample room for improvement. In 958. Coltart, H. M. Kim, L. J. Williams S. J. particular, the steps following the assembly [ 10] K. Toshima, K. Tatsua, Chem. Rev. 1993, Danishefsky, P. O. Livingston, Int. J. of the structure like cleavage, purification 93, 1503. Cancer 2002, 99, 207. and deprotection have to be streamlined. [ 11] R. R. Schmidt, W. Kinzy, Adv. Carbohydr. [ 17] M. C. Hewitt, D. A. Snyder, P. H. Chem. Biochem. 1994, 50, 21. Seeberger, J. Am. Chem. Soc. 2002, 124, Further challenges like difficult linkages 13434. and complex sequences remain to be tack- [ 12] O. J. Plante, E. R. Palmacci, R. B.Andrade, P. H. Seeberger, J. Am. Chem. Soc. 2001, [ 18] L. Schofield, M. C. Hewitt, K. Evans, M. led. Overall we are confident that our ap- 123, 9545. A. Siomos, P. H. Seeberger, Nature 2002, proach will become a powerful tool for [ 13] O. J. Plante, E. R. Palmacci, P. H. 418, 785. glycomics research. Seeberger, Science 2001, 291, 1523. [ 14] P. Fugedi, W. Birberg, P. J. Garegg, A. Acknowledgements Pilotti, Carbohydr. Res. 1987, 164, 297. We gratefully acknowledge the ETH Zürich and the Swiss National Science Foundation (SNF, grant 200020 – 109555) for fi nancial support. We thank all of the coworkers that have made the automated carbohydrate synthesizer a reality during the past nine years. Their names are listed in the references. We thank the Roche Research Foundation (Doctoral stipend for L. K.).

Received: October 24, 2007

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