o (54) Title: CLEAVAGE OF SYNTHETIC PEPTIDES WO 2015/ Al 5 March 2015 ( ) P O P C T

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1 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/ Al 5 March 2015 ( ) P O P C T (51) International Patent Classification: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C07K 1/04 ( ) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/EP2014/ OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 29 August 2014 ( ) TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, August 2013 ( ) SE TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (71) Applicant: BIOTAGE AB [SE/SE]; Box 8, S DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Uppsala (SE). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventor: REES, Anthony; Uppsala Nas, Focksta 19, S- GW, KM, ML, MR, NE, SN, TD, TG). Uppsala (SE). (74) Agent: BRANN AB; Box 12246, S Stockholm Declarations under Rule 4.17 : (SE). of inventorship (Rule 4.17(iv)) (81) Designated States (unless otherwise indicated, for every Published: kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, with international search report (Art. 21(3)) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, o (54) Title: CLEAVAGE OF SYNTHETIC PEPTIDES (57) Abstract: The invention relates to a method of cleaving a linker molecule attaching a peptide, polypeptide or a small protein to a solid phase comprising contacting the attached peptide, polypeptide or small protein with water under subcritical conditions. An acid may be added to the water, and the method is advantageously microwave supported.

2 CLEAVAGE OF SYNTHETIC PEPTIDES Technical Field The present invention relates to a method of peptide synthesis, and more specifically to the step of cleaving a linker molecule which attaches a synthetic peptide or a protein to a solid phase. Background Peptides are polymers of amino acids connected by amide peptide bonds, which are sometimes referred to as peptide bonds. These bonds are formed between the a-amino group of one amino acid and the a-carboxyl group of another. The possibility to produce peptides synthetically has revolutionized the research and knowledge in numerous fields, such as the chemistry of peptides and proteins, enzymology, immunology and drug design. A major breakthrough in the area of peptide synthesis was the possibility to synthesize peptides onto an insoluble or solid support, introduced by Bruce Merrifield in Until his discovery, peptides were synthesized in solution which often required tedious purification steps. The findings of Merrifield initiated further development of peptide synthesis methods using solid supports. During the last decades Solid-Phase Peptide Synthesis (SPPS) has been the primary way to synthesise peptides and small proteins on a small to medium scale. SPPS is a stepwise synthesis method, as illustrated in Figure 1. In the first step the primary amino acid of the target peptide is attached to a cleavable linker molecule connected to the solid support. Peptides are normally synthesized from the C- to the N-terminus and the amino acid is therefore attached via its carboxyl group. To make sure that the correct coupling is performed, a temporary protecting group blocks the N-terminus (N-a) and permanent protecting groups block the side chains of the amino acid residues. Thus, the temporary protecting group must be removed before the next N-a-protected amino acid can be coupled. The amino acids can either be added directly, in the

3 presence of an activator or as a pre-activated species, such as a symmetrical anhydride or active ester. After each N-a deprotection, the amino acid- or peptide resin complex is thoroughly washed. The deprotection, washing and coupling steps are repeated until the full target peptide is obtained. In the final step of SPPS, the peptide is cleaved off from the linker and the side chain protecting groups removed, preferably with the same reagent. In its early days, the SPPS methodology suffered from problems with the acid sensitivity of peptide bonds and acid catalysed side reactions. Consequently, it was of great interest to find alternative ways to carry out the synthesis. In 1978, an "orthogonal" protection strategy was introduced, where the base labile Fmoc group replaced the former acid labile N-a-protecting group in SPPS. The strategy is called orthogonal as certain protecting groups can be removed in the presence of the others. For Fmoc SPPS, this means that the removal of the Fmoc group under basic conditions does not affect the acid labile linker or the acid labile side chain protecting groups. In this orthogonal approach, the reaction time and acid cleavage cocktail for linker cleavage and removal of the side chain protecting groups will depend on the type of linker attached to the resin, the type of side chain protecting groups and the type and number of amino acids in the peptide. For Fmoc SPPS, a solution of TFA and scavengers (nucleophilic reagents to avoid side reactions under acidic conditions) is normally used for this final step. Scavengers are added to trap the highly reactive cationic species that are formed from the linker and the protecting groups thereby avoiding reaction with residues containing nucleophilic functional groups, such as Trp and Cys. For Fmocprotected amino acids, protecting groups belonging to the t-bu family are generally recommended, as shown in Table 1 below (ISIDRO-LLOBET, A., ALVAREZ, M. and ALBERICIO, F., Amino Acid-Protecting Groups. Chemical Reviews, 109(6), pp ). In this procedure, a cleavage cocktail comprised of TFA/TIS/water (95:2.5:2.5) has proved to be

4 efficient for most peptide sequences. However, TFA is a corrosive chemical, is difficult to handle and safety procedures for its disposal are required. Since SPPS is used more and more on an industrial scale, a less corrosive, safer and more environmentally friendly alternative would be of great interest. Table 1: Fmoc amino acid derivatives Bodansky et al (PALLADINO, P. and STETSENKO, D.A., New TFA- Free Cleavage and Final Deprotection in Fmoc Solid-Phase Peptide Synthesis: Dilute HC1 in Fluoro Alcohol. Organic Letters, 14(24), pp ) have listed side reactions in SPPS as the main reason for variations in the peptide yields and purity. Side reactions that have been seen during Fmoc synthesis are cyclisation reactions such as aspartamide formation, deamination and diketopiperazine formation; epimerisation; oxidation; stability of tryptophan to oxidation and alkylation; amide bond hydrolysis of labile sequences; dehydration of alcohols; β elimination; and other radical-induced side

5 reactions. Thus, to obtain homogeneous peptides it is of great importance to use reaction conditions that minimize such side reactions. Since Merrifield's findings, the main focus for the development of the SPPS method has therefore been to minimize side reactions and to increase the yield by improving both coupling and deprotection steps. Nevertheless, there are still limitations with the method when it comes to the length of the peptides and syntheses of sequences recognized as difficult. The difficulties often arise because of intraand intermolecular aggregations or local conformational folding, the occurrence of which is related to the types of amino acid in the sequence. This aggregation or folding may lead to steric hindrance, affecting both the coupling and deprotection reaction rates. The solid support used in SPPS is usually a resin comprised of a polymer with a cleavable linker molecule and spacers to avoid steric hindrance. There are many different resins available today, such as the hydrophobic polystyrene (PS) resin, which is the most commonly used and the more hydrophilic PEGbased ChemMatrix resin (Sigma Aldrich). The latter is marketed as having a low risk of intra- and intermolecular aggregations which makes it a suitable choice for difficult sequences. A number of different linker molecules have been described, and many are commercially available, see e.g. FIELDS, G.B. and NOBLE,.L., Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids. International Journal of Peptide and Protein Research, 35, pp , and WHITE, P.D. and CHAN, W.C., Basic Principles. In: P.D. WHITE and W.C. CHAN, Fmoc Solid Phase Peptide Synthesis, A Practical Approach. USA: Oxford University Press, pp The type of linker will determine the C-terminal chemistry of the final peptide product : either an acid or an amide can be formed upon cleavage. Depending on the concentration of TFA, both cleavage of the linker and full deprotection of the acid-labile side chain protecting groups in the peptide may occur at the same time. Commonly

6 used examples of linkers are e.g. /?-alkoxybenzyl alcohol, which when attached to a solid support is often referred to as the 'Wang resin' from its discoverer S.S. Wang (WANG, S., p-alkoxybenzyl Alcohol Resin and p- Alkoxybenzyloxycarbonylhydrazide Resin for Solid Phase Synthesis of Protected Peptide Fragments. Journal of the American Chemical Society, 95(4), pp ) and the more acid labile linker Rink Amide (RAM) linker, 2',4'-di-CH 3 O-Ph-CH(NH-Fmoc)-Ph-4-OCH 2, sometimes simply denoted 'Rink resin' when attached to a solid support. Both Wang and Rink resins are commercially available, e.g. from Sigma-Aldrich. The Wang linker is suitable for peptides requiring a C-terminal acid while the Rink amide is suitable for peptide amides. Conditions suitably used for cleavage of these resins are listed below (see Table 2). This list clearly shows the difference in acid stability of the two linkers. Table 2 : Resin linkers and standard deprotection conditions All steps in SPPS have traditionally been performed at room temperature. Nevertheless, it has occasionally been shown that applying temperatures above ambient during the coupling, deprotection and washing steps may reduce

7 aggregation problems related to the difficult sequences. In conventional heating methods such as oil baths, the reaction vessel or container is heated first and then the heat is transferred to the sample, creating a temperature gradient in the sample. This may be avoided using microwave heating, introduced for organic synthesis as early as the 1990's. In microwave heating, the response of the molecules in the sample to electromagnetic radiation in the microwave region generates heat throughout the sample, enabling a more precise and even heating of the solution. Microwave instruments available today provide controllable, reproducible and fast heating under conditions where rapid cooling down of the reaction can take place. Another general advantage of microwave heating is that since no time is required for recalibration of the heat source, reaction parameters may rapidly be changed. Microwave heating, or indeed any heating, in SPPS increases the rates of the coupling and N-a-deprotection at the expense of side reactions and consequently increases the peptide purity. Microwave irradiation has also been employed for the final cleavage of the peptide from the resin and the concomitant deprotection of the side chains. This decreased the time needed for the cleavage from 2-5 hours down to minutes (PEDERSEN, S.L., TOFTENG, A.P., MALIK, L. and JENSEN, K.J., Microwave heating in solid-phase peptide synthesis. Chemical Society Reviews, 41(5), pp ). Thus, the high efficiency, in terms of purity and speed, of microwave-assisted SPPS makes it an attractive technology. However, problems due to high temperatures during deprotection are known to occur, such as racemization at the a-carbon atom or other undesired side reactions. Accordingly, there is still a need for alternative technologies in SPPS that increase the efficiency and/or reduce or avoid the problems associated with currently used protocols.

8 Subcritical water has been used e.g. for the destruction of organic wastes by hydrolysis of the organic compounds. It can also be useful in the recycling of plastics for recovery of valuable chemical resources and hydrolysis of biomass, providing chemical feedstocks. In 2002, AKIYA et al (see AKIYA, N. and SAVAGE, P.E., Roles of Water for Chemical Reactions in High- Temperature Water. Chem. Rev., 102, pp ) showed that depending on the reactivity of the organic compounds, they hydro lyse to different extent under sub-critical conditions. Summary of the Present Invention The present invention relates to a method of solid phase peptide synthesis (SPPS), and more specifically to efficient cleavage of a linker molecule and optionally the concomitant removal of the protecting groups with minimal peptide breakdown and side reactions. One object of the invention is to provide a method which avoids the use of harsh chemicals, such as trifluoroacetic acid (TFA), in one or more steps of the process of synthesizing of peptides, polypeptides or proteins. According to the invention, this may be achieved by a method of cleaving a linker molecule attaching a peptide, polypeptide or protein to a solid phase by contacting said attached peptide, polypeptide or protein with water or aqueous acid under subcritical conditions. In one embodiment, microwave energy is applied while carrying out the linker cleavage according to the invention. In one embodiment of the method according to the invention, the linker molecule is selected from the group of linkers consisting of p-alkoxybenzyl alcohol (Wang linker), N-alpha-(9-fluorenylmethoxycarbonyl)-2,4-dimethoxy- 4'-(carboxymethyloxy)-benzhydrylamine (Rink amide linker) or trityl linker.

9 In an advantageous embodiment, the method according to the invention is performed in a vessel wherein the temperature and/or pressure is controlled, and optionally monitored and/or moderated. In one embodiment, the subcritical conditions applied to the cleavage reaction involve a temperature of at least about 205 C, such as C. In one embodiment, the subcritical conditions are applied to the cleavage reaction for less than about 10 minutes, such as less than about 6 minutes. In one embodiment of the method according to the invention, the subcritical conditions involve using an aqueous acid of at least 0.36M HCl, such as at least about 0.60 M HCl. In one embodiment, the subcritical conditions used herein involve using a pressure of at least about 2 1 bar, such as at least about 25 bar. In a specific embodiment, the method according to the invention is performed for 6 min at a temperature in the range of C; at a pressure of about 18 and in an aqueous acid of about 0.06M HCl. A second aspect of the present invention is a process of solid phase synthesis of a peptide, polypeptide or a protein comprising one or more of the steps of deprotecting, activating, coupling and cleaving, wherein at least the cleavage step is performed according to the invention as described in this application. In one embodiment, the deprotection step is also performed in water or aqueous acid under subcritical conditions, as described in the present application.

10 Definitions and Abbreviations AA Amino acid (specific amino acids are also referred to using their respective conventional three letter abbreviations) Boc DCM DMF FHT Fmoc H 2 CO 3 HFIP HPLC MeOH Mpe Mtt PS SPPS t-bu Trt TIS TFA t rt-butyloxycarbonyl Dichloromethane Dimethylformamide Fixed hold time 9-Fluorenylmethoxycarbonyl Carbonic acid Hexafluoroisopropanol High-performance liquid chromatography Methanol /3-3-methylpent-3-yl 4-methyltrityl Polystyrene Solid-Phase Peptide Synthesis t rt-butyl Trityl Triisopropylsilane Trifluoracetic acid The term "subcritical region" is used herein for above 100 C and 0.1 MPa and below the supercritical point at 374 C, 22 MPa ( 1 Bar = 0.1 MPa)). Subcritical water is sometimes also denoted "superheated water" or "pressurized hot water." Brief description of the drawings Figure 1 illustrates schematically the repeated steps of conventional SPPS.

11 Figure 2 shows Matrix I according to Example 3 below, illustrating cleavage of Phe from Wang resin with a variation of HC1 concentration, pressure and time according to Table 6. Figure 3 shows Matrix II according to Example 3, illustrating cleavage of Phe from Wang resin with a variation of pressure and reaction time at a lower HC1 concentration than Matrix I. Figure 4 shows Matrix III according to Example 3 below, illustrating cleavage of Phe from Wang resin at a supplemented range of pressures and reaction times as compared to Matrix I and II. Figure 5 shows Matrix IV according to Example 3 below, illustrating cleavage of Phe from Wang resin with a further decrease of the reaction time. Figure 6 shows is a summary of the results of Matrix I, II, III and IV when used for the cleavage of Phe from Wang resin as described in Example 3 below. Figure 7(a), (b) and (c) shows Matrix 6, Matrix 7 and Matrix 8 as described in Example 4 below. Detailed Description of the Invention As discussed above, the present invention shows that subcritical water under acidic conditions as described herein can replace TFA in the final cleavage and deprotection step of SPPS. Subcritical water is cheap, non-toxic, non-explosive and environmentally friendly and is therefore an excellent alternative to other solvents. As mentioned above, a first aspect of the invention is a method of cleaving a linker molecule attaching a peptide, polypeptide or protein to a solid phase by contacting said attached peptide, polypeptide or protein with water or aqueous acid under subcritical conditions. As is well known in this field, when liquid water is heated under pressure to temperatures above 100 C and below 374 C, it is in a superheated or subcritical state, where the properties are very different from water under

12 ambient conditions. To keep the water in the liquid state during the heating, the pressure should be at least the saturated vapour pressure for the specific temperature. For hydrolysis reactions, the cleavage of a bond is caused by water or by an acid or base produced by water reacting with a salt. In subcritical water, it has been shown that hydrolysis occurs mainly due to acid catalysis by an increased concentration of H + ions (and OH ) from water under subcritical conditions. Because of this high ionic product, acid- and base catalysed reactions occur readily. The hydrolysis rate in subcritical water is dependent on the temperature, density and salt concentration of the water, since these parameters affect the ability of subcritical water to solvate polar and ionic species. As is also well known, in SPPS the side chain protecting groups and resin linkers are normally based on esters, ethers, carbamates or activated amides. The hydrolysis kinetics available in the literature for these reactants in water at room temperature and neutral ph show that amide bonds are more stable than ester and carbamate bonds. Rogalinski et al (ROGALINSKI, T., LIU, K., ALBRECHT, T. and BRUNNER, G., Hydrolysis kinetics of biopolymers in subcritical water. The Journal of Supercritical Fluids, 46(3), pp ) teaches that in acid catalysed hydrolysis of proteins, the addition of a proton to the peptide bond is the rate-determining step and an addition of acid to the water subsequently accelerates the hydrolysis reaction. Therefore, in SPPS an addition of acid to the water might be expected to increase the risk of breakdown of the desired peptide. However, as appears in more detail from the experimental part below, the present invention shows that it is possible to optimise the conditions to use acidic water under subcritical conditions for selective cleavage of the peptide from a solid support without cleavage of the peptide amide bonds.

13 Equipment and reagents of standard type may be used in the present method. In one embodiment, the method according to the invention is performed in a vessel wherein the temperature and/or pressure may be monitored and optionally moderated. Subcritical conditions of the water or acidic water may be achieved herein using any well-known equipment, as is commercially available and familiar to the skilled person in this field. One example is the Biotage Initiator + Microwave Synthesizer, which was used in the experimental part below. The temperature may be adjusted to a suitable value by the skilled person based on the teachings of this application and optionally by further routine experimentation as appropriate. In one embodiment of the invention, the subcritical conditions are applied at a temperature of at least about 205 C, such as C. Cleavage according to the invention may be performed at any acceptable reaction times, such as about 20 minutes or below. In one embodiment, the subcritical conditions involve a reaction time below about 10 minutes, such as below about 6 minutes. The skilled person will realise and learn from the present application that the reaction time may be shortened by optimization of other parameters such as acidity and/or pressure and/or temperature to obtain a desired yield. The acidity of the subcritical water may be provided by any conventionally used acid such as hydrochloric acid or acetic acid. In one embodiment, the acidity is obtained by using at least about 0.12M HC1. In a specific embodiment, the subcritical conditions involve using an aqueous solution of at least about 0.36M HC1, such as at least about 0.60 M HC1. As discussed above, temperature and acidity may compensate for a lower pressure in order to obtain a desired yield. In one embodiment, the subcritical

14 conditions involve using a pressure of at least about 2 1 bar, such as about 27 bar or at least about 25 bar. In addition to the subcritical conditions discussed above, further energy may be added to the cleavage reaction performed according to the invention. In one embodiment, the present method involves applying microwave energy while carrying out the linker cleavage. The present method may be applied to any acid labile linker commonly used in SPPS, provided the appropriate conditions are identified. In a specific embodiment, the linker molecule is selected from the group of linkers consisting of p-alkoxybenzyl alcohol (Wang linker), N-alpha-(9- fluorenylmethoxycarbonyl)-2,4-dimethoxy-4'-(carboxymethyloxy)- benzhydrylamine (Rink amide linker) or trityl linker. In an advantageous embodiment, the method according to the invention is performed for about 6 min at a temperature in the range of C; at a pressure of about 18 bar and in aqeuous acid of about 0.06M HC1. A second aspect of the present invention is a process of solid phase synthesis of a peptide, polypeptide or a protein comprising deprotecting, activating, coupling and cleaving steps wherein at least the cleavage step is performed according to the present invention. In one embodiment, the deprotection step is also performed in water; acidified water; or aqueous acid under subcritical conditions as discussed above. Detailed Description of the Drawings Figure 1 illustrates schematically the repeated steps of conventional SPPS. Figure 2 illustrates the results related to Matrix I, as described in example 3 below and presented in Table 5. More specifically, a result of about 68% was

15 obtained at a concentration of 0,60M HC1, a pressure of 25 bar and a reaction time of 20 minutes. Figure 3 illustrates the results related to Matrix II, as described in example 3 below and presented in Table 6. More specifically, a result of about 48% was obtained at a concentration of 0,1 2M HC1, a pressure of 2 1 bar and a reaction time of 20 minutes. Figure 4 illustrates the results related to Matrix III, as described in example 3 below and presented in Table 7. More specifically, a result of about 76% was obtained at a concentration of 0,36M HC1, a pressure of 27 bar and a reaction time of 20 minutes. Figure 5 illustrates the results related to Matrix IV, as described in example 3 below and presented in Table 8. More specifically, a result of about 58% was obtained at a concentration of 0,36M HC1, a pressure of 25 bar and a reaction time of 2 minutes. Figure 6 is a summary of the results related to Matrix I-IV, as described in example 3 below and presented in Table 9. More specifically, Figure 6 shows how cleavage of a Wang linker is obtained under subcritical conditions, and that high yields can be obtained at low additions of acid and short reaction times compared to conventional cleavage and deprotection. Figure 7(a), (b) and (c) shows matrix 6, 7 and 8 as described in example 4. In all of these matrices, the maximum amount of released hippuric acid was observed at the longest time (6 min) and the highest values could be seen for 0.06 M HC1. EXPERIMENTAL The experiments presented below are provided for illustrative purposes only, and should not be construed as limiting the invention in any way. All references provided below and elsewhere in the present application are hereby included by reference.

16 In this study, the Biotage Initiator Microwave Synthesizer is used, which is limited to a maximum pressure of 30 bar. Therefore a temperature range of C and the corresponding saturated vapour pressure range , where the dielectric constant of the subcritical water is equal to that of DMF or MeOH at 25 C 5, was suitable to investigate. MATERIALS AND METHODS Sample preparation The Fmoc group on the Fmoc-Phe-Wang (PS) complex (Iris GmbH, loading 58 mmol/g) is removed with piperidine in DMF ( 1:4), using Biotage Initiator + Alstra Automated Microwave Peptide Synthesizer. Thereafter 30 mg of the deprotected Phe-Wang complex is dried with DCM and put in a 2-5 ml Biotage Microwave Vial containing a magnet followed by addition of 3 ml of deionized water acidified by 37% v/v HC1 (aq) to concentrations of 0.06,0.12, 0.36 and 0.60 M. The vial is capped and put in the Biotage Initiator + Microwave Synthesizer Robot Sixty instrument. The reaction conditions are programmed in advanced mode: Temperature set to off, pressure ramped from 9 bar and 30s up to the set pressure and an FHT of 2, 5, 10 or 20 minutes, cooling down set to off, stir rate set to 300 rpm, pre-stirring set to off, initial power set to off, absorption level set to very high. After microwave heating, the vial is decapped and the sample mixture filtered using a plastic syringe into a glass container. The container should preferably be able to tolerate heat if used for the ninhydrin test. Sample analysis For the ninhydrin test, the sample is evaporated or put in the freezer followed by freeze-drying. The sample is diluted in 1 ml 60% ethanol (EtOH) in deionized H 2 O. The Kaiser test kit from Sigma-Aldrich is used for the ninhydrin test: addition of 75 µΐ of the PhOH (-80% in EtOH), 100 µΐ KCN (in H O/pyridine), 75 µΐ ninhydrin (6% in EtOH). The sample is mixed and put in

17 a sand bath of 100 C for 10 minutes. Thereafter the container is put in cold water. For the UV/VIS-measurements, 50 µΐ of the sample is dissolved in 8 ml of 60% EtOH in deionized H 2 O. The UV/VIS- spectrometer is set to 570 nm. The absorption is compared to the standard curve of Phe in solution. For HPLC-MS, Agilent Technologies 6120 Quadrupole LC/MS is used with Biotage Resolux 200 CI8 (4.5 µιη, 150x2.1 mm), P/N R , S/N Example 1: Identification of operating conditions for subcritical conditions In the first part of the study, the optimal operating conditions for subcritical conditions controllable with the Biotage Initiator + Microwave Synthesizer were established. Biotage Microwave Vials were suitable for reactions under subcritical water conditions. These glass vials are available in four different sizes: ml, ml, 2-5 ml and ml ( The glass vials do not absorb microwaves and are designed for safe and efficient heating within their specific volume and beyond pressures of 30 bar. The vials are sealed with a cap and a septum and magnetic stirring creates an even temperature distribution in the reaction mixture. The largest vial (10-20 ml) cannot handle the high pressures used in this project and were excluded from the experiments. In addition, the smallest vial ( ml) was excluded because the total sample volume would have been too small for HPLC analysis. Therefore the two middle-sized vials, ml and 2-5 ml, were used for the scans of deionized water. There are a number of parameters that can be controlled and specified with the Biotage Initiator + Microwave Synthesizer. These are time, temperature, pressure, cooling, power, stirring rate, pre-stirring, initial power, vial type and

18 absorption. To find the most suitable parameters for the reaction mixtures, different volumes of deionized water were irradiated in the microwave vials. The volumes chosen were the minimum and maximum volumes possible in the vial chosen and one intermediate volume. The irradiation of the water was regulated by either temperature or pressure. The time for the irradiation may be chosen as a certain time period for the whole irradiation process, including the time it takes until the specified temperature or pressure is reached. Another possibility is to choose a fixed hold time (FHT) that starts as soon as the specified temperature or pressure is reached. At the end of the irradiation, diagrams of the pressure, temperature and power are generated and used to determine which vial, volume, time, pressure and temperature conditions provided the most stable process. As discussed above, the dielectric constant of subcritical water is close to that of DMF or MeOH in a temperature interval of C and saturated vapour pressures of bar. The temperature and pressure can only be programmed to integer values and therefore the intervals used throughout the present experiments were C and bar. At the beginning of the microwave irradiation of deionized water it was established that the irradiation should be controlled by pressure, since regulation by temperature failed to give high enough pressures. Also, this is a better choice as the regulation by pressure is more exact (±1 bar from the desired pressure) for the Biotage Initiator + Microwave Synthesizer than for temperature regulation (± 5 C from the desired temperature). Four different pressures in the pressure range were tested: 16, 19, 24 and 27 bar. The times for the irradiations were chosen to a FHT of 1 and 5 minutes. For an FHT of 1 minute the curves did not have time to stabilise and therefore the longer FHT was better to determine the stability of the operating conditions.

19 The volumes tested for deionized water were 1 and 2 ml for the ml vial. The smallest volume of 0.5 ml is still not large enough to give sufficient amounts of sample. From the temperature and pressure curves it was established that the minimum pressure (16 bar) rendered unstable temperature and/or pressure curves for both volumes. 19 bar showed stable temperature and pressure curves for both volumes, while 24 bar showed some instability. The highest pressure (27 bar) gave stable temperature and pressure curves for both volumes. The volumes tested for the 2-5 ml vial were 2, 3 and 5 ml. From the temperature and pressure curves, it was realized that the minimum pressure (16 bar) rendered unstable temperature and/or pressure curves for all volumes. 19 bar gave unstable temperature and pressure curves for the smallest volume, but stable curves for the two highest volumes. For the smallest volume (2 ml) it took time until the set pressure was reached for both these pressure values. Therefore this volume was excluded and not tested for higher pressures. As for the smaller vial, 24 bar showed some instabilities, but the highest pressure (27 bar) generated stable temperature and pressure curves for both 3 and 5 ml. The scans of water showed that stable temperatures and pressures were obtained for 27 bar and 19 bar for all volumes except one for the 19 bar pressure. The higher pressure was chosen for the first cleavage experiments, since it is a threshold value that makes it more straightforward to interpret the results. For the smaller vial the resulting temperature at this pressure was above 230 C, and therefore the larger vial was a better choice. For the larger vial, both a volume of 3 and of 5 ml showed stable results. Using the 3ml vial the operating conditions for subcritical conditions of pure water that showed a stable trend and could be used for the first experiments were 3 ml water in a 2-5 ml vial at a set pressure of 27 bar, giving a maximum temperature of 226 C, and a FHT of 5 minutes.

20 Table 3 : Sample volumes Example 2 : Cleavage of acid labile linker in subcritical water with no additives Phenylalanine-Rink Amide resin conjugate was used for the first cleavage experiments. The Rink-Amide linker is more acid labile than e.g. the Wang linker, see Table mg of the conjugate was put in a 2-5 ml Biotage Microwave vial with 3 ml deionized water. The mixture was irradiated in the Biotage Initiator + Microwave Synthesizer at a set pressure of 27 bar and an FHT of 5 minutes (total irradiation time around 7 minutes). After the irradiation, the sample was filtered so that the resin could be removed.

21 As earlier tests on the analysis of resulting cleavage product with HPLC/ESI- MS had indicated that this method may be difficult the ninhydrin test was chosen since it is a sensitive method for the detection of free amines. When free amines exist, the solution turns blue/purple, called the Ruhemann's purple, and this can be analysed with a UV-VIS spectrometer at 547 nm. It was therefore a suitable method to see whether any cleavage had occurred. The ninhydrin test is normally used to monitor the completion of each coupling step during SPPS, while here it was used to detect free amine present in any cleaved amino acid. To be able to quantify the amount of cleavage, a standard curve of ninhydrin colour values using standard solutions of different concentrations of Phe was performed. For this first trial, the solution did not turn blue during the ninhydrin test and no absorption was seen. Therefore it appeared that no cleavage had occurred. Example 3 : Cleavage of acid labile linker in subcritical water with addition of acid As discussed above, the addition of acid to subcritical water would be expected to enhance the hydrolysis rate. Only very small amounts of acid can increase the reaction rate significantly and scans were therefore performed on low concentrations of acid. HC1 was the acid of choice, since it has a low pk a of -8 (i.e. complete dissociation in water). In comparison, TFA has a pk a of To test the concentrations of HC1 the Biotage Initiator + Microwave Synthesizer could tolerate, 1 ml of 37% HC1 (aq) was added to 100 ml of H 2 O, giving a concentration of 0.12 M HC1 (ph at ambient conditions = 2.9), and microwave irradiation was performed with the conditions determined above. During the heating, the pressure suddenly increased uncontrollably and the instrument stopped the irradiation process. Therefore a new trial was performed, this time with 0.1 ml 37% HC1 (aq) per 100 ml of H O (concentration = M HC1). The same uncontrollable pressure rise occurred. A third trial with an even lower concentration of only 0.01 ml 37%

22 HC1 (aq) per 100 ml of H 2 O (concentration = 1.2 mm) was tested, and this time the complete irradiation process was carried out. For this concentration and irradiation conditions, the maximum temperature reached was C. After establishing that the maximum practical HC1 concentration was 1.2 mm, the first experiment on cleavage of the acid-labile Wang linker was performed. For efficient and reproducible experiments, preloaded Fmoc-Phe-Wang resin (loading 0.59 mmol/g) was purchased from Iris GmbH. The Fmoc group was removed with piperidine in DMF ( 1:4) in the Biotage Initiator + Alstra Automated Microwave Peptide Synthesizer, followed by air-drying. Thereafter, 30 mg of the resin complex was added to a microwave vial containing a magnet, and 3 ml of acidic water solution of 1.2 mm was added. The vial was closed with a cap and a septum and put in the rack of the Biotage Initiator + Microwave Synthesizer, which was programmed with the desired reaction conditions, i.e. FHT 5 and 30 minutes and set pressure 27 bar. After the irradiation, the vial was decapped and the sample mixture filtered with a plastic syringe into a glass container tolerant to high temperatures. The same experiment was performed, but with a solution of 99% TFA (aq), in the same amounts as for HC1. This gave a concentration of 3.4 mm TFA (ph at ambient conditions = 2.5). For the analysis of any cleavage of Wang linker in subcritical water under mild acidic conditions it was necessary first to prepare a standard curve in order to enable quantification of the yield. Solutions of different concentrations of Phe were prepared. The maximum concentration of Phe available in the cleaved samples is calculated below, and this was used as a benchmark for the concentration of the stock solution. ^-Resin complex 0 C Loading Wang Phe = 0.59 mmol/g

23 Solvent = 3 ml Phe = u -degree Wang m Phe = 0.59 mmol/g 30 mg = mmol Phe 3 ml of each standard solution was put in containers and evaporated or freezedried. Thereafter 1 ml of 60% EtOH in water was added to each sample to dissolve the dried sample, followed by a ninhydrin test performed with a Kaiser test kit. 75 µΐ of the PhOH (-80% in EtOH), 100 µΐ KCN (in H 2 O/pyridine) and 75 µΐ ninhydrin (6% in EtOH) were added to each mixture. The glass containers were then heated in a sand bath of 100 C for 10 minutes together with a reference sample. During this time, mixtures containing free amines should turn blue. After the heating, the containers were put in cold water. 100 µΐ of each sample was put in 2 ml of 60% EtOH in water. These solutions were then analysed in a UV-spectrometer at 547 nm and a standard curve for UV absorbance of free Phe was obtained. The equation for the standard curve obtained here is y = x The sample filtrates were also evaporated or put in the freezer, followed by freeze-drying before the ninhydrin test. This showed that the cleavage product for FHT's of 5 and 30 minutes were 4 and 17% respectively. For the TFA, two tests were made, which gave a cleavage product of 3 and 6%, respectively. The experiments were not repeated and hence no exact conclusions were drawn, but since the cleavage product with both TFA and HC1 showed very low yields, a concentration of 1.2 mm of acid does not seem sufficient despite the high pressure (and high temperature) used. Thus, a direct inject through Isolera Dalton Nanolink to Isolera Dalton Mass Detector was made. This mass spectrum indicated a possible degree of decarboxylation of the Phe.

24 Since such a small amount of cleavage product was obtained in the first experiments higher concentrations of HCl were investigated. To avoid the accelerated pressure problems previously seen with the higher acid concentrations, a stepwise pressure increased was used with the Biotage Initiator + Microwave Synthesizer. In this protocol, specific time intervals can be programmed for each pressure step up to the desired pressure, after which a final time interval is chosen. Trials of different concentrations of HCl, varying starting points and time periods were tested, followed by tests with addition of the resin complex. With time intervals of 30 seconds and a starting pressure of 9 bar, the pressure could be increased up to 27 bar for mixtures containing resin complex and concentrations of HCl up to 1.12 M. It was observed that the lowest pressure to obtain temperatures above 200 C for the mixtures tested was 2 1 bar. This was therefore chosen as the minimum pressure for the cleavage experiments described in the next section. For the analysis of cleavage of Wang linker in subcritical water under mild to strong acidic conditions, a number of test matrices were prepared. Matrix I : Variation of HCl concentration, pressure and time The possibility to acidify the water to a higher extent opened up a range of possible conditions for cleavage of the Wang-linker. Since barely any cleavage was obtained with a low concentration of acid, even at high pressures and temperatures and a long reaction time of 30 minutes, the focus here was to see if a higher concentration could increase the cleavage. To significantly increase the harshness of the conditions, solutions of 5 and 10 ml 37% HCl (aq) per 100 ml of H 2 O were prepared. This gave concentrations of 0.60 M HCl and 1.21 M, and ph values at ambient conditions of 0.22 and Since the pressure is increased stepwise, the time period until the set pressure is around 10 minutes. The short FHT of 5 minutes in the former experiment showed a lower cleavage product than the longer FHT of 30 minutes and therefore FHT ' s of 10 and 20 minutes were chosen, giving total irradiation

25 times of 20 and 30 minutes. The former cleavage experiments also showed that the lowest pressure to obtain a temperature above 200 C was 2 1 bar and this pressure was chosen as a minimum for the experiment in this matrix. The maximum pressure was maintained at 27 bar. A third pressure of 25 bar was also used to expand the set of conditions in the matrix. This resulted in experiments at two different concentrations of HCl, at three different pressures and for two different time periods. The cleavage products were analysed with the ninhydrin test. The colour developed was much more concentrated than previously and therefore a new standard curve was made where 50 µΐ of each solution was diluted in 8 ml of 60% EtOH in water before the UV analysis. The results are presented in Table 4 below: Table 4 : Ninhydrin test of cleavage of Phe from Wang resin From these results it appeared that it is possible to obtain a much higher cleavage of the Wang linker than observed previously. A significant amount of cleavage was obtained for the lower concentration of HCl, with yields of

26 around 70%. The cleavage product for the higher concentration of HC1 was significantly lower. Because of the many steps involved, the ninhydrin test is time consuming and also not always reliable or reproducible. HPLC/ESI-MS was therefore reinvestigated, this time successfully. It was now possible to quantify the cleavage obtained more efficiently. Since almost all samples for the first matrix had been used in the ninhydrin test, only single samples could be analysed in the HPLC for these first samples. As expected, the HPLC results deviated somewhat, giving an average yield of 60% for the lower concentration and around 40% for the higher concentration, see results in Table 5 below: Table 5 : Matrix I The higher concentration of HCI still appeared to give a lower yield, which may be due to secondary acid-catalysed reactions. This explanation was strengthened by the HPLC, where a broad peak was observed for Phe. The

27 yield results obtained from the HPLC indicated that the difference in time did not seem to have a substantial effect on the amount of cleavage, while the yield increased somewhat with pressure. Matrix II: Variation of pressure and reaction time at a lower HCl concentration The second set of experiments was planned using the results of the first matrix. Since the concentration of 1.21 M provided lower yields than 0.60 M, it was decided to decrease the acid concentration further. A solution with 1 ml HCl per 100 ml water was prepared, giving a concentration of 0.12 M and a ph of 0.92 at ambient conditions. Since no major difference was noted between the times tested, it was important to check if a shorter time had any impact on the yield. The time periods tested were therefore expanded to FHT's of 5, 10 and 20 minutes. Since the difference in yield between 25 and 27 bar was small, only 2 1 and 25 bar were employed. For this HCl concentration, all pressures and times tested, gave yields in the same range, see Table 6 below: Table 6 : Matrix II

28 Overall, the results presented slightly lower yields than the results of the samples performed with 0.60 M HCl. Matrix III: Supplemented range of pressures and reaction times Additional samples for the Matrix I and Matrix II were made to obtain a more complete data collection including results for pressures between bar and times of 5-20 minutes at all concentrations. Two additional concentrations of acid were also tested. Solutions of 0.05 and 3 ml per 100 ml water were prepared, giving concentrations of 0.06 and 0.36 M respectively and ph of 0.44 and 1.22 at ambient conditions. The total yields showed that the lowest concentration (0.06M) of HCl gave the lowest yields, see Table 7 below: Table 7 : Matrix III

29 nd nd nd nd nd nd nd nd nd

30 nd nd (nd = not determined) nd The reaction time continued to have a minimal impact on the yield for each concentration. The difference in yield was only significant when comparing the 2 1 bar with either 25 or 27 bar for each acid concentration. Overall the samples with 0.36 M HC1 showed higher yields than the other concentrations. The irradiation of these samples was carried out on an equivalent Biotage Initiator + Microwave Synthesizer to avoid some temperature related operational problems and to ensure the method was not instrument-specific. To confirm the high yields obtained with the 0.36 M HC1 solution, the same experiments were performed on the more stable microwave instrument that was able to generate higher temperatures. The difference in temperature could be as much as 20 C. Nevertheless, the yields were in the same range for both instruments. The yields appeared to be lower for higher temperatures suggesting that at too high a temperature breakdown of product may occur or that there is a decreased effect of acid with increasing temperature. Matrix IV: Further decrease of the reaction time The results above indicated that the cleavage yield may not be substantially improved by increasing time. Consequently, it was possible that the cleavage of the linker may occur relatively early in the irradiation process. To investigate this, the reaction times were decreased further.

31 In this matrix (IV), an FHT of 2 minutes was tested for 2 1 and 25 bar, with the concentrations of HC1 used that gave the highest yields previously (0.12, 0.36 and 0.60 M). No significant difference in yield was seen for this short FHT compared to the longer times used for the other matrices, except for the 0.12 M samples that showed higher yields than previously, see Table 8 below: Table 8 : Matrix IV (Numbers in bold results from the experiments carried out on the more stable microwave instrument) A summary of Matrix I, II, III and IV is provided in Table 9 below: Table 9 : Matrix I-IV

32

33 nd nd nd nd nd nd nd nd nd