DEVELOPMENT OF MICROWAVE-ASSISTED SYNTHESIS METHODS FOR PREPARATION OF PEPTIDES. Ph.D. thesis BERNADETT BACSA

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1 DEVELOPMENT OF MICROWAVE-ASSISTED SYNTHESIS METHODS FOR PREPARATION OF PEPTIDES Ph.D. thesis BERNADETT BACSA Supervisors: Dr. Gábor Dibó associate professor ELTE Department of Organic Chemistry Dr. Gábor Mező professor MTA-ELTE Research Group for Peptide Chemistry Ph.D. School of Chemistry, Eötvös Loránd University School leader: Dr. György Inzelt professor Synthetic Chemistry, Materials Science and Biomolecular Chemistry Ph.D. Program Head of the Program: Dr. András Perczel professor Budapest 2010

2 1. Introduction Recently, heating chemical reactions by microwave energy continues to be a popular theme in the organic and medicinal chemistry community. Since the first published reports on the use of microwave irradiation to carry out organic chemical transformations by the groups of Gedye and Giguere in 1986 [1,2], more than 4,000 articles have been published in this fastmoving and exciting field. In many of the published examples, microwave heating has been shown to dramatically reduce reaction times, increase product yields, and enhance product purities by reducing unwanted side reactions compared with conventional heating methods [3,4]. The advantages of this enabling technology are exploited not only in organic and medicinal chemistry/drug discovery, but have also penetrated related fields such as polymer synthesis, materials science, nanotechnology, and biochemical processes. At least in the field of organic synthesis, the use of microwave irradiation has become such a popular technique that it might be assumed that, in a few years, microwave reactors will have the Bunsen burners of the 21 st century and will be standard equipment of every chemical laboratory [3]. According to market research studies the potential of peptide therapeutics has recently intensified. Their role as mediators of key biological functions make them particularly attractive therapeutic agents: peptides show high biological activity associated with low toxicity and high specificity. Additionally, compared to small molecules, peptides offer valuable chemical and biological diversity in which intellectual property is widely available. By 2005, there were more than 40 peptides on the market worldwide and around 700 peptide based drugs were under different stages of preclinical and clinical testing [4]. In the past few years the use of microwave irradiation to enhance solid-phase peptide synthesis has been growing at a rapid rate. By using microwave-assisted SPPS, several research groups [5,6] reported impressive improvements both in the speed of coupling/deprotection steps and in the purity/yield of the final products obtained. Successful applications of this enabling technology have additionally been published for the generation of notoriously difficult peptide motives such as β-peptides, glycopeptides, phosphopeptides, pseudopeptides, peptoids, cyclic peptides, biopolymers, peptide conjugates and certain types of peptidomimetics. 2

3 2. Aims The advent of solid-phase peptide synthesis (SPPS) has led to dramatic developments in peptide chemistry and related fields. Since Merrifield s pioneering work on SPPS in the 1960 s, peptide preparation on small to medium scale has almost exclusively been performed on solid supports. A common phenomenon in SPPS, however, is the occurrence of so-called difficult sequences which are problematic if not impossible to synthesize using standard coupling and deprotection protocols. The desired peptide products are often contaminated with a series of structurally and chemically very similar peptides such as incomplete, mismatch or deletion sequences. The separation of these undesired byproducts from the target peptide can sometimes be very tedious, and elusive on a preparative scale. In the past few years several successful attempts have been published for the application of microwave irradiation for the preparation of peptides and their derivatives. The first part of my PhD work are focusing on developing a general method for the preparation of peptides and their derivatives by using microwave technology. Therefore my goal was to achieve the following plans: Development of a general microwave-assisted synthesis methods for the preparation of peptides by using Fmoc/ t Bu orthogonal protection strategy. Initially, it was found that the standard Pyrex glass microwave reaction vessel was not optimal for solid-phase synthesis in the CEM Discover reactor. To overcome this problem, I was planning to introduce the MicroKan reactor for encapsulating the resin beads, which was placed inside the Pyrex microwave process vials. Optimization of reaction parameters of microwave-assisted peptide synthesis (reaction time, temperature, magnetron output power, ramping time, and temperature monitoring). Application of the developed method for the fast and effective synthesis of model peptides and their derivatives. Fluorescent labeling N-terminal and side chain of model peptides on solid phase under microwave irradiation. Development of a general method for the synthesis of difficult peptide sequences in a new generation of manual microwave peptide synthesizer (CEM Discover SPS). It was planned to test different solid supports (polystyrene, Tentagel, ChemMatrix ), various combinations of coupling solvents, excess of coupling reagents, different coupling and cleavage temperatures under microwave conditions. 3

4 While the reported improvements using microwave-assisted SPPS compared to conventional SPPS have been impressive, little effort has so far been devoted to provide a definitive scientific rationalization for the observed effects. The question is the enhancements achieved are of purely thermal origin (the result of efficient dielectric heating during the irradiation processes), or so-called nonthermal microwave effects are also involved in the direct interaction of the electromagnetic field (not related to a macroscopic temperature effect) with, for example, the peptide backbone or other substrates/intermediates in the reaction mixture. It has recently been suggested that due to the very high dipole moment of the amide bond, irradiation of peptides with microwave energy may lead to the aggregation of the peptide backbone via direct interaction of the peptide chain with the electric field [7,8]. Microwave effects of this type would not be reproducible by conventional heating at the same reaction temperature. Therefore I planned a detailed evaluation of microwave-assisted Fmoc/ t Bu solid-phase peptide synthesis involving several difficult sequences under strictly controlled conditions: carefully monitor and optimize the reaction temperatures under microwave-assisted coupling and deprotection steps by using recently developed fast responding internal fiber-optic temperature probes; perform adequate control experiments for microwave and conventional heating at the same reaction temperature; compare the purity of the difficult peptides synthesized under microwave and conventional heating in order to distinguish between thermal and nonthermal microwave effects during solid-phase peptide synthesis; investigate the effect of high temperature on the racemization of the individual amino acids during SPPS; develop a racemization-free synthesis method for the preparation of β-amyloid (1 42) peptide using microwave and conventional heating protocols; evaluate and compare the in vitro neurotoxicitiy of Aβ (1 42) peptide synthesized under different SPPS methods on SH-SY5Y neuroblastoma cell lines. 4

5 . Results The primary goal of my Ph.D. thesis was to develop a general method for the preparation of peptides and their derivatives using microwave technology. Additionally, second part of my work was dedicated to the examination of a recently suggested hypothesis. Accordingly, the observed enhancements during microwave-assisted peptide synthesis are related to the microwave field (nonthermal microwave effect) and are not only purely thermic. 1. Firstly, the synthesis of the selected calmodulin binding nonapeptide (H-Trp-Asp-Thr- Val-Arg-Ile-Ser-Phe-Lys-OH) was optimized in a general CEM Discover reactor. The synthesis protocol was based on the use of cycles of pulsed microwave irradiation with intermittent cooling of the reaction mixture to subambient temperatures. When the resin beads were encapsulated in Microkan ciontainer, the preparation of the model peptide required in a significantly shorter reaction time (12 h vs 2.5 h), and resulted in nearly identical purity and yield compared to conventional peptide synthesis carried out at room temperature. 2. Applying the developed microwave-assisted method, the streptavidin binding heptapeptide (H-Phe-Ser-His-Pro-Gln-Asn-Thr-OH) and it s N-terminal labelled derivative with 5(6)-carboxyfluorescein was prepared. By using the new method the reaction time was shortened and the purity of the peptide was nearly identical in comparison with conventional synthesis at room temperature. 3. The synthesis of the calmodulin binding peptide (H-Trp-Asp-Thr-Val-Arg-Ile-Ser-Phe- Lys-OH) was optimized for the newly available microwave peptide synthesizer (CEM Discover SPS). The reaction vessel of this instrument was specially designed for solidphase synthesis, allowing for bottom filtration and therefore mimicking the workflow of a conventional peptide synthesizer. Applying very short (5 min) coupling time at 60ºC the model peptide was obtained in 95% purity and 90% yield. 4. Employing the developed method for this microwave peptide synthesizer (CEM Discover SPS), a model peptide {tetratuftsin. OT20; H-(Thr-Lys-Pro-Lys-Gly) 4 -NH 2 } and its derivatives modified at the side-chain with 5(6)-carboxyfluorescein was successfully synthesized under less than15 min in a coupling step. 5. For further optimization studies, a model peptide (H-Gly-Ile-Leu-Thr-Val-Ser-Val-Ala- Val-CONH 2 ) was selected which suffers from poor synthetic efficiency under standard 5

6 SPPS conditions. Synthesis of the nonapeptide was performed using various combinations of solid supports (polystyrene, Tentagel, ChemMatrix ), solvents (DMF, NMP, NMP/DMSO, LiCl/NMP), various excess of the coupling reagents (10, 5, 3 molar) and different coupling and cleavage temperatures (60 90 C) employing Fmoc/tBu orthogonal protection strategy. Microwave-assisted SPPS was performed using the manual microwave peptide synthesizer (CEM Discover SPS). The best support for the preparation of the nonapeptide (H-Gly-Ile-Leu-Thr-Val-Ser-Val-Ala-Val-CONH 2 ) proved to be the fully PEG-based ChemMatrix material. Using RAM-ChemMatrix resin, the desired peptide could be synthesized in very high purity (ca. 95%). In the coupling steps (at 86 C, 10 min), only 3-fold excess of the Fmoc-amino acid was sufficient to allow the preparation of a highly pure peptides. A control experiment at room temperature using 10 equiv of amino acid and 60 min coupling time furnished the desired peptide in moderate 47% purity. The results presented clearly demonstrate the effectiveness of microwaveassisted solid-phase synthesis for the generation of difficult peptide sequences, thus confirm previous reports on the general usefulness of microwave-assisted methods over conventional SPPS carried out a room temperature 6. It has recently been suggested that due to the very high dipole moment of the amide bond, irradiation of peptides with microwave energy may lead to the segregation of the peptide backbone via direct interaction of the peptide chain with the microwave field. My goal was to examine this hypothesis with strictly controlled comparative experiments performed under microwave and conventional heating. Using recently developed fast responding OpSens internal fiber-optic temperature probes, the reaction temperature during microwave-assisted peptide couplings has been carefully monitored and optimized. Applying the optimized reaction conditions for the preparation of the model peptide (H- Gly-Ile-Leu-Thr-Val-Ser-Val-Ala-Val-NH 2 ), coupling and deprotection reactions under microwave-heated and conventionally-heated conditions were compared at the exactly same temperature. For this purpose the same solid-phase reaction vessel used in the Discover SPS for microwave-assisted couplings and deprotections was applied in a conventional manual solid-phase synthesizer keeping all other reaction parameters the same. There is a surprisingly close match in terms of peptide purity between the results obtained by using conventional and microwave heating at the same coupling and deprotection temperatures, indicating that nonthermal microwave effects are probably not involved. 6

7 The general effectiveness of elevated temperature SPPS was tested on other difficult and longer peptide sequences. The generation of the 15-mer Cecropin A(1 7) Mellitin(2 9) hybride peptide (H-Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu- NH 2 ) and the 24-mer Magainin-II-amide peptide derivative (H-Cys-Gly-Ile-Gly-Lys-Phe- Leu-His-Gly-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-NH 2 ) was attempted. The synthesis of both model peptides was performed on ChemMatrix resin applying the microwave-assisted DIC/HOBt coupling and piperidine/dmf deprotection conditions optimized for the nonapeptide (H-Gly-Ile-Leu-Thr-Val-Ser-Val- Ala-Val-NH 2 ) utilizing 3-fold excess of activated Fmoc-amino acids at 86 C coupling temperature (10 min reaction time) and 3 min deprotection cycles at 86 C provided the Cecropin A(1 7) Mellitin(2 9) hybride peptide in remarkable high (91%) purity. The identical experiment using conventional heating at 86 C for coupling and deprotection led to similar peptide purity (87 %). Applying the optimized coupling/deprotection SPPS conditions described above, Magainin-II-amide peptide derivative was obtained on ChemMatrix resin in 54% purity using microwave conditions, compared to 48% purity applying conventional heating at the same temperature. This demonstrates the absence of any significant nonthermal microwave effect, even for longer peptide sequences. Standard room temperature SPPS was not successful in providing this peptide in a reasonable purity. 7. In order to investigate the effect of temperature on amino acid racemization during solidphase pepide synthesis at elevated temperature, model peptides synthesized by conventional and microwave heating were compared. While for most amino acids no significant racemization was observed, the high coupling temperature led to considerable levels of racemization for the sensitive amino acids, histidine and cysteine. The racemization levels were very similar comparing peptide samples obtained from microwave and conventionally heating experiments at 86 C. Subsequently, coupling of these two sensivitve amino acids was performed at room temperature to eliminate the effect of racemization. 8. An improved synthetic protocol was developed for the direct microwave-assisted synthesis of the 42-mer β-amyloid peptide. While standard solid-phase protocols typically result in peptides of poor quality, the application of controlled microwave heating provides the Aβ (1 42) peptide in high purity in only 15 h of total processing time. Our best conditions utilized 5 equiv of activated Fmoc-amino acid at 86 C for 10 min 7

8 coupling and 3 min deprotection time. The coupling of His residues was performed at room temperature to eliminate the effect of racemization for this sensitive amino acid. 9. In vitro neurotoxicitiy of Aβ (1 42) peptide synthesized under different SPPS methods was evaluated. Therefore, SH-SY5Y human neuroblastoma cells were treated with the two differently prepared Aβ (1 42) peptides (synthesized under microwave and conventionally heated conditions) and the viability of the cells was determined by using the MTT-assay. The two differently synthesized Aβ (1 42) peptides show almost identical cytotoxicity effects on SH-SY5Y human neuroblastoma cells. Since irradiation of peptides with microwave energy has been claimed to result in a segregation of the peptide backbone via direct interaction of the peptide chain with the electric field, these results are important in demonstrating the bioequivalency of conventionally and microwave heated synthetic peptides, in particular as the toxicity of the Aβ (1-42) peptide is known to depend strongly on aggregation phenomena. It can therefore be concluded that the observed enhancement effects in microwave-assisted SPPS are of purely thermal nature and not related to the microwave field. No evidence for the recently proposed segregation of the peptide backbone via direct interaction of the peptide chain with the microwave field could therefore be obtained. Finally, it should be emphasized that increasing the reaction temperature from ambient conditions by 60 C for both coupling and deprotection steps represents an estimated 50-fold increase in the reaction rate for both processes based on the Arrhenius equation. This kinetic effect is probably responsible for the highly efficient coupling and deprotection in microwave-assisted solid-phase peptide synthesis, providing peptides in high purity within extremely short time. Publications related to this summary: 1. Giguere RJ, Bray TL, Duncan SM, Majetich G. Application of commercial microwave ovens to organic synthesis. Tetrahedron Lett. 1986, 27, Gedye R, Smith F, Westaway K, Ali H, Baldisera L, Laberge L, Rousell J. The Use of Microwave Ovens for Rapid Organic Synthesis. Tetrahedron Lett. 1986, 27, Kappe CO. Controlled microwave heating in modern organic synthesis. Angew. Chem., Int. Ed. Engl. 2004, 43, Kappe CO, Dallinger D. Controlled microwave heating in modern organic synthesis: highlights from the literature. Mo.l Divers. 2009, 13, Marx V. Watching peptide drugs grow up. Chem. Eng. News 2005, 83,

9 6. Sabatino G, Papini AM. Advances in automatic, manual and microwave-assisted solid-phase peptide synthesis. Curr. Opinion Drug Discov. Dev. 2008, 11, Collins JM, Leadbeater NE. Microwave energy: a versatile tool for the biosciences. Org. Biomol. Chem. 2007, 5, Palasek SA, Cox ZJ, Collins JM. Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis. J. Peptide Sci. 2007, 13, Publications related to PhD activity: 1. Bacsa B, Bősze Sz, Kappe CO. Direct solid-phase synthesis of the β-amyloid (1 42) peptide using controlled microwave heating. J. Org. Chem. 2010, 75, [IF 3,952] 2. Bacsa B, Horváti K, Bősze Sz, Andreae F, Kappe CO. Solid-phase synthesis of difficult peptide sequences at elevated temperatures A critical comparison of microwave and conventional heating technologies. J. Org. Chem. 2008, 73, [IF 3,952], Citations: Bacsa B, Kappe CO. Rapid solid-phase synthesis of a calmodulin binding peptide using controlled microwave irradiation, Nature Protocols 2007, 2, [IF 1,671], Citations: 7 4. Bacsa B, Desai B, Dibó G, Kappe CO. Rapid solid-phase peptide synthesis using thermal and controlled microwave irradiation. J. Peptide Sci. 2006, 12, [IF 1,801], Citations: *Bacsa B, Gombosuren N, Kappe, CO, Dibó G. Microwave-assisted peptide synthesis, Peptide Science 2005, (Hidaka Y, Wakamiya T, Ed.), Protein Foundation, Osaka, pp Citations: 2 *Short communication in Conference Book Conference materials (oral presentations, posters) related to the PhD activity 1. Bacsa B, Horváti K, Bősze Sz, Andreae F, Kappe CO. Microwave-assisted solid-phase peptide synthesis, ZING Microwave and Flow Chemistry Conference, Antigua, invited speaker 2. Bacsa B, Horváti K, Bősze Sz, Andreae F, Kappe CO. MAOPS 2009 Microwave assisted organic and peptide synthesis, Montpellier, France, oral lecture 3. Bacsa B, Horváti K, Bősze Sz, Andreae F, Kappe CO. Solid-phase peptide synthesis at elevated temperatures A comparison of conventional and microwave heating technology. 30th European Peptide Symposium, Helsinki, Finnland, Kappe CO, Bacsa B. Solid-phase peptide synthesis using controlled microwave heating. 236th ACS National Meeting, Philadelphia, PA, USA, Bacsa B, Desai B, Dibó G, Kappe CO. Rapid solid-phase synthesis of a calmodulin-binding nonapeptide using thermal and controlled microwave irradiation. Advances in Microwave- Assisted Organic Synthesis, MAOS 2006, Budapest, poster award 6. Bacsa B, Kappe CO, Dibó G. Tapasztalatok a mikrohullámú peptidkémiában. HAS Hungarian Peptideand Nucleic Acid Society Annual Meeting,, Balatonszemes, Bacsa B, Gombosuren N, Kappe CO, Dibó G. Mikrohullámmal kiváltott szerves szintézisek, XI. International Chemistry Congress, Cluj, Romania,

10 8. Bacsa B, Gombosuren N, Kappe CO, Dibó G. Microwave-assisted peptide synthesis. 42nd Japanese Peptide Symposium, Osaka, Japan, Bacsa B, Kappe CO, Dibó G. A novel approach for microwave-assisted peptide synthesis. Symposium on Microwave Accelerated Synthesis (MAS-5), Düsseldorf, Germany, Bacsa B, Gombosuren N, Kappe CO, Dibó G. Application of microwave technology for the synthesis of peptides and their derivatives. 4th Bulgarian Peptide Symposium, Dolna Bania, Bulgaria, Publications, not related to the dissertation 1. *Gombosuren N, Schlosser G, Pócsfalvi G, Bacsa B, Furka Á, Dibó G. Mass spectrometric monitoring of binding assays, Peptide Science 2004, (Shimohigashi Y, Ed.), Protein Foundation, Osaka, pp poster award 2. *Bacsa B, Gombosuren N, László L, Furka Á, Dibó G. Application of fluorescent labeling for combinatorial libraries, Peptide Science 2004, (Shimohigashi Y, Ed), Protein Foundation, Osaka, pp *Bacsa B, Gombosuren N, Furka Á, Dibó G. Fluorescently labelled protein libraries, Peptides 2004, Wiley, London, UK, pp Schlosser G, Gombosuren N, Bacsa B, Dibó G, Malorni A, Hudecz F, Pócsfalvi G. Detection of noncovalent interactions by solid-phase affinity capture mass spectrometry, Bioorganic Chemistry Meeting-2, Budapest, Hungary, Bacsa B, Gombosuren N, Furka Á, Dibó G. Fehérjekönyvtárak fluoreszcens jelzése, X. International Chemistry Congress, Cluj, Romania, Gombosuren N, Schlosser G, Bacsa B, Furka Á, Dibó G. A tömegspektrometria alkalmazása kötődésvizsgálatokban, X. International Chemistry Congress, Cluj, Romania, poster award 7. Gombosuren N, Bacsa B, Furka Á, Dibó G. Fluoreszcens jelzés alkalmazása a kombinatorikus kémiában, HAS Hungarian Peptide Society Annual Meeting, Balatonszemes, Gombosuren N, Bacsa B, László L, Furka Á, Dibó G. Peptid fehérje kölcsönhatások vizsgálata fluoreszcens jelzés alkalmazásával, IX. International Chemistry Congress, Cluj, Romania, *Short communication in Conference Book 10