S1 SUPPORTING INFORMATION Solid-Phase Synthesis of Difficult Peptide Sequences at Elevated Temperatures A Critical Comparison of Microwave and Conventional Heating Technologies Bernadett Bacsa, Kata Horváti, Szilvia Bősze, Fritz Andreae, and C. Oliver Kappe* Christian Doppler Laboratory for Microwave Chemistry (CDLMC) and Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, A-81 Graz, Austria, Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, 1117 Budapest, and pichem Forschungs und EntwicklungsgmbH, Kahngasse 2, A-845 Graz, Austria Table of Contents General Experimental Section Description of Solid-Phase Peptide Synthesis Heating Curves, HPLC Chromatograms and MS Spectra Racemization Studies S2 S2-S4 S5-S19 S2
S2 General Experimental Details. All commercially available solvents and reagents were used without further purification. N,N -Dimethylformamide (puriss. p.a., >99.8%) (DMF), 1-methyl-2- pyrrolidone (for peptide synthesis, >99.8%) (NMP), dichloromethane (reagent grade, >99.5%) (DCM), methanol (reagent grade, >99.5%), trifluoroacetic acid (reagent grade, >98%) (TFA), piperidine (reagent grade, >98%), triisopropylsilane (99%) (TIS) and N,N -diisopropylcarbodiimide (99%) (DIC) were purchased from standard sources. Rink Amide MBHA resin (substitution.64 mmol/g, Lot No. A35327) was obtained from Novabiochem (Novabiochem, Switzerland), RAM Tentagel resin S AC (substitution.24 mmol/g, Lot No. S 3 11) was purcased from Rapp Polymere GmbH (Germany) and RAM ChemMatrix (substitution.5 mmol/g, Lot No. 7I6-13-5-29) was obtained from Martix Innovation (Canada). The Fmoc-amino acids and 1-hydroxybenzotriazole hydrate (HOBt) were purchased from Orpegen (Germany). For both microwave and conventionally heated experiments solid-phase reaction vessels (1 ml polypropylene syringe with frit and PTFE valve) were used. The comparison experiments at elevated temperature were performed in a PLS 4 6 manual solid-phase synthesizer. HPLC analyses were performed using Nucleosil-1 RP-18 column (15 mm 4.6 mm, 5 µm) was used by applying a linear gradient of 1 9% B in 15 min. The mobile phases were.1% TFA in H 2 O (eluent A), and.1% TFA in MeCN (eluent B). The flow rate was 1 ml/min and the detection was at 215 nm. The peptide masses were confirmed by MALDI-TOF MS. Synthesis of Model Peptide (H-Gly-Ile-Leu-Thr-Val-Ser-Val-Ala-Val-CONH 2 ) Using Standard SPPS Conditions at Room Temperature. In a 1 ml solid-phase reaction vessel, 15 mg of resin (RAM MBHA PS, RAM Tentagel, RAM ChemMatrix, see Table S) was swollen in 4 ml of DMF/DCM (1:1) for 3 min. After this time 2 ml of 3% piperidine in DMF was added to the resin. The reaction vessel was placed in the PLS 4 x 6 synthesizer and shaken for 2 min at room temperature. The resin was then washed with 4 ml of DMF, and 2 ml of 3 % piperidine in DMF were subsequently added to the sample which was gently agitated for 2 min in the PLS 4 6 synthesizer. The resin was then washed with DMF and DCM (5 4 ml each) and NMP (2 4 ml). In a separate vial, the corresponding Fmoc-amino acid, DIC, and HOBt were combined in NMP. The used solid supports, reagent volumes and concentrations delivered to the reaction vessel are given in Table S. The preactivated coupling cocktail after 2 min was added to the resin and the reaction mixture was agitated for 1 h in the PLS 4 6 synthesizer. After the last deprotection step, the peptidyl resin was dried under reduced pressure.
Table S. Volumes of Fmoc-AA and Coupling Reagents to be Added in the Coupling Step. S3 Resin Equiv Fmoc-AA (mmol) DIC (µl) HOBt (mg) Conc (M) d RAM MBHA PS a 1.96 149 156 1. 5.48 75 78.5 3.288 45 47.3 RAM Tentagel b 1.36 56 6.4 5.18 28 3.2 3.18 17 18.1 RAM ChemMatrix c 1.75 116 122.8 5.375 58 61.4 3.225 35 36.2 a loading.64 mmol/g; b loading.24 mmol/g; c loading.5 mmol/g; d coupling solvent NMP Synthesis of Model Peptide (H-Gly-Ile-Leu-Thr-Val-Ser-Val-Ala-Val-CONH 2 ) Using Microwave Irradiation at 67 C or 86 C. A sample of 15 mg of resin (RAM MBHA PS, RAM Tentagel, RAM ChemMatrix, see Table S) was transferred into a 1 ml bottom-filtration reaction vessel and was swollen in 4 ml of DCM/DMF (1:1) for 3 min. After this time 2 ml of 3% piperidine in DMF was added to the resin. The reaction vessel was placed into the microwave cavity and irradiated for 3 s at 6 C (or 75 C) nominal temperature (SPS mode, maximum power 2 W, T = 3 C). Then the resin was subsequently washed with 4 ml of DMF, and 2 ml of 3% piperidine in DMF were added to the sample and irradiated for an additional 2.5 min at 6 C (or 75 C) (SPS mode, maximum power 2 W, T = 3 C). The suspension was then washed with DMF and DCM (5 4 ml each) and NMP (2 4 ml). In a separate vial the corresponding Fmoc-amino acid, DIC and HOBt were combined in NMP. The used solid supports, reagent volumes and concentrations delivered to the reaction vessel are given in Table S. The preactivated coupling cocktail was added to the resin after 2 min and the reaction mixture was irradiated for 5, 1 or 2 min at 6 C (SPS mode, maximum power 5 W, T = 3 C) or 75 C (SPS mode, maximum power 5W or 1 W, T = 3 C). After the last deprotection step, the peptidyl resin was dried under reduced pressure. Synthesis of Model Peptide (H-Gly-Ile-Leu-Thr-Val-Ser-Val-Ala-Val-CONH 2 ) Using Conventional Heating at 67 C or 86 C. A sample of 15 mg of resin (RAM Tentagel, RAM ChemMatrix, see Table S) was transferred into a 1 ml bottom-filtration reaction vessel and was swollen in 4 ml of DCM/DMF (1:1) for 3 min. After this time, 2 ml of 3% piperidine in DMF was added to the resin. The reaction vessel was placed in the PLS 4 6 synthesizer (preheated to 67 C or 86 C) and heated with agitation at 67 C (or 86 C) for 1.5 min. The resin was then washed with 4 ml of DMF, and 2 ml of 3 % piperidine in DMF were subsequently added to the sample which was
S4 agitated in the PLS 4 6 synthesizer for an additional 3.5 min at 86 C. The suspension was then washed with DMF and DCM (5 4 ml each) and NMP (2 4 ml). In a separate vial the corresponding Fmoc-amino acid, DIC and HOBt were combined in NMP. The used solid supports, reagent volumes and concentrations delivered to the reaction vessel are given in Table S. The preactivated coupling cocktail was added to the resin after 2 min and the reaction mixture was heated at 67 C for 21 min (or 86 C for 11 min) in the PLS 4 6 synthesizer. After the last deprotection step, the peptidyl resin was dried under reduced pressure. Scale-up of Peptide Synthesis (H-Gly-Ile-Leu-Thr-Val-Ser-Val-Ala-Val-CONH 2 ) Using Optimized Conditions. To a 25 ml bottom-filtration reaction vessel.225 mmol (45 mg, loading.5 mmol/g) ChemMatrix resin was transferred and was swollen in 12 ml of DCM/DMF (1:1) for 3 min. After this time 4 ml of 3% piperidine in DMF was added to the resin. A N 2 line was inserted for agitation and the reaction vessel was placed into the microwave cavity for 3 s at 86 C (SPS mode, maximum power 4 W, T =3 C). Then the resin was washed with 12 ml of DMF, and 4 ml of 3% piperidine in DMF were added to the sample and irradiated for an aditional 2.5 min at 86 C (SPS mode, maximum power 4 W, T =3 C). The suspension was then washed with DMF and DCM (5 12 ml each) and NMP (2 12 ml). In a separate vial the corresponding Fmoc-amino acid (.675 mmol), DIC (.675 mmol, 15 µl) and HOBt (.675 mmol, 19 mg) were combined in 3 ml of NMP. The preactivated coupling cocktail was added to the resin after 2 min, a N 2 line was inserted and the reaction mixture was irradiated at 86 C for 1 min using the SPS program (maximum power 3 W, T = 3 C). After the last deprotection step, the peptidyl resin was dried under reduced pressure. The peptide was cleaved from the solid support with a cleavage cocktail (15 ml) of TFA/triisopropylsilane/water (95: 2.5: 2.5) at ambient temperature for 2 h. The resin was filtered and washed with a small amount of cleavage cocktail. The residual product was precipitated with ice-cold diethyl ether and the peptide was collected by filtration, dissolved in deionized water and lyophilized. Racemization analysis. The determination of enantiomeric peptide purity was performed by C.A.T. GmbH & Co. (Tübingen, Germany). The samples were hydrolyzed using deuterated solvents (6 N D 2 O/DCl) and after derivatization the enantiomeric purity of the amino acids were determined by GC- MS (see Tables S2 and S3). Cecropin A(1-7) Mellitin(2-9) hybride peptide and Mangainin-II-amide were synthesized on ChemMatrix resin according to the optimized microwave-assisted solid-phase peptide synthesis protocol given in the main experimental part.
S5 Figure S1. Aggregation potential of GILTVSVAV analyzed using Peptide Companion Software. The prediction program characterizes the coupling difficulty of the amino acids from the fifth residue in the sequence using an aggregation potential value. An aggregation potential value below.9 indicates that the coupling of the current amino acid is easy, a value between.9-1.1 means the difficulty of the coupling is intermediate. The sequence starts being potentially difficult after two or three consecutive potentials have exceeded the value 1.1. The observed aggregation potential values of > 1.2 indicate a very difficult peptide sequence. TABLE S1. Synthesis of GILTVSVAV on Rink Amide MBHA PS resin. a entry Fmoc-amino acid coupling deprotection purity d equiv conc (M) temp b ( C) power c (W) time (min) temp b a Peptide synthesis was performed on a.1 mmol scale using Rink Amide MBHA PS resin (loading.64 mmol/g) in a 1 ml solid-phase reaction vessel (ca. 1.1 ml solvent for the coupling step; 2 ml 3% piperidine in DMF for the deprotection step), CEM Discover SPS. b Set temperature monitored by internal fiber-optic probe. c Maximum magnetron microwave output power for pulsing. d Purity of crude peptides (analytical RP- HPLC peak area %, UV absorbance at 215 nm). Peaks between 5 and 8 min retention time were used for integration. The identidy of the target peptide was established by MALDI-TOF MS. e Isolated yield 61% (crude product). f Using.8 M LiCl in NMP as coupling solvent. g Using 2% DMSO in NMP as coupling. h Standard room temperature SPPS, Advanced Chemtech PLS 4 6. ( C) power c (W) time (min) 1 1.96 6 5 2 6 2.5 + 2.5 85 e 2 2 5.48 6 5 2 1 6 2.5 + 2.5 88 3 f 2 5.48 6 5 2 1 6 2.5 + 2.5 66 4 g 2 5.48 6 5 2 1 6 2.5 + 2.5 86 5 1.96 75 1 1 75 2.5 + 2.5 81 6 3.23 75 5 5 75 2.5 + 2.5 57 7 h 1.96 25-6 25-2 + 2 44 (%)
S6 entry 1 entry 7 [M+H]+ Purity: 85% Purity: 44% Figure S2. Comparison of HPLC chromatograms (UV absorbance at 215 nm) of GILTVSVAV peptide prepared under reaction conditions described in Table S1: (a) microwave conditions (Table S1, entry 1); (b) room temperature conditions (Table S1, entry 7). The identity of the target peptide and the deletion sequence was established by MALDI-TOF MS. entry 1 ( 3 equiv of Fmoc-AA) entry 2 (5 equiv of Fmoc-AA) entry 3 (1 equiv of Fmoc-AA) Purity: <1% Purity: 32% Purity: 37% Figure S3. Comparison of HPLC chromatograms (UV absorbance at 215 nm) of GILTVSVAV peptide prepared on Tentagel resin demonstrating the influence of excess amino acid on coupling efficiency under reaction conditions described in Table 1, entries 1-3. The identity of the target peptide and the deletion sequence was established by MALDI-TOF MS.
S7 entry 2 (reaction time: 6 min) entry 5 (reaction time: 2 min) entry 4 (reaction time: 1 min) Purity: 37% Purity: 5% Purity: <1% Figure S4. Comparison of HPLC chromatograms (UV absorbance at 215 nm) of GILTVSVAV peptide demonstrating the influence of reaction time on coupling efficiency under reaction conditions described in Table 1, entries 2, 4 and 5. The identity of the target peptide and the deletion sequence was established by MALDI-TOF MS.
S8 a) b) c) for Figure legend, see following page
S9 d) e) Figure S5. Identification of of GILTVSVAV peptide and deletion sequences via MALDI-TOF MS. (a), Table 1, entry 4: MS spectra corresponding to the main peak (t R = 7.51 min) [M+Na] + = 88.6, [M+K] + = 896.5 (M cal = 857.1). (b): MS spectra corresponding to the impurity peak (t R = 6.58 min), [M- Ile+H] + = 745., [M-Ile+Na] + = 767.2, [M-Ile+K] + = 783.2. (c), Table 2, entry 9: MS spectra corresponding to the main peak (t R = 7.4 min) = 858.1, [M+Na] + = 88.2, [M+K] + = 896.2 (M cal = 857.). (d), Table 4 entry 7: MS spectra corresponding to the main peak (t R = 7.44 min) [M+Na] + = 88.6, [M+K] + = 896.6. (e) Table 4, entry 8: MS spectra corresponding to the main peak (t R = 7.42 min) = 858.6, [M+Na] + = 88.6, [M+K] + = 896.6; M cal =857..
S1 Table 1, entry 1 Table 2, entry 14 Table 2, entry 4 25 C [M-Gly-Ile+H] + 4 C [M-Ala+H] + [M-Ala+H] + 6 C Purity: <1% Purity:9% Purity: 42% Table 2, entry 11 Table 2, entry 13 75 C 85 C Purity: 61% Purity: 78% Figure S6. Comparison of HPLC chromatograms (UV absorbance at 215 nm) of GILTVSVAV peptide synthesized on Tentagel resin using 3 equiv of Fmoc-amino acid demonstrating the influence of reaction temperature on the efficiency of the coupling step. For exact reaction conditions, see Tables 1 and 2. The identidy of the target peptide and the deletion sequence was established by MALDI-TOF MS.
S11 entry 2 entry 4 room temperature [M+H] Purity: 91% Purity: 95% Purity: 47% Figure S7. Comparison of HPLC chromatograms (UV absorbance at 215 nm) of GILTVSVAV peptide prepared on ChemMatrix resin under reaction conditions described in Table 3 (entries 2 and 4) and at room temperature (6 min coupling time and 2+2 min deprotection time). The identity of the target peptide and the deletion sequences was established by MALDI-TOF MS. Temperature [ C] 14 12 1 8 6 4 NMP.18M Leu.18M Leu.36M Leu 2 1 2 3 4 5 Time [s] Figure S8. Heating profiles (OpSens fiber-optic probe) for different concentration of coupling cocktails (3, 5, 1-fold excess of Fmoc-Leu-OH, DIC and HOBt calculated to.36 mmol RAM Tentagel resin) in 1 ml of NMP using constant 1 W microwave power.
S12 9 8 7 Opsens 1 Temperature [ C] 6 5 4 3 P Discover FO 5 Power [W] 2 1 5 1 15 2 Time [min] Figure S9. Temperature (Discover FO, OpSens FO) and microwave power (P) profiles for the microwave-assisted peptide coupling step (5-fold excess of Fmoc-amino acids, DIC and HOBt on.36 mmol RAM-Tentagel resin) in 1 ml NMP at 6 C (set temperature) for 2 min irradiation time using the Discover SPS program (maximum power 5 W, T = 3 C). The average temperature measured by the OpSens probe is 67 C whereas only 59 C was montiored by the Discover FO. Temperature [ C] 9 8 7 6 5 4 3 2 1 OpSens FO DIscover P 2 4 6 8 1 Time [min] 5 4 3 2 1 Power [W ] Figure S1. Temperature (Discover FO, OpSens FO) and microwave power (P) profiles for a typical microwave-assisted peptide coupling step (3-fold excess of Fmoc-amino acid, DIC and HOBt on.18 mmol RAM-Tentagel resin) in 3 ml NMP at 75 C (set temperature) for 1 min irradiation time using the Discover SPS program (maximum power 3 W, T = 3 C). The average temperature measured by the OpSens FO probe is 75 C and is in agreement with the temperature measured by the Discover FO.
S13 18 25 16 Opsens FO 1 14 Opsens FO 2 2 Temperature [ C] 12 1 8 6 Opsens FO 3 Discover FO 15 1 Power [W] 4 2 P 5 2 4 6 8 1 Time [min] Figure S11. Temperature (Discover FO, OpSens FO1, OpSens FO2, OpSens FO3) and microwave power (P) profiles for 1 ml NMP in a 1 ml SPS vessel equipped with four internal fiber-optic sensors placed at different positions. The sample was heated to 8 C (set temperature) for 1 min irradiation time using the standard non-pulsed irradiation method (maximum power 2 W, no stirring). Shown are the profiles for the three OpSens fiber-optic probes and the CEM FO probe. The good agreement between the OpSens FO probes confirms the absence of temperature gradients in the microwave-heated experiments and lends further confidence to the reliability of the faster OpSens FO probes. 9 OpSens FO 3 8 Temperature [ C] 7 6 5 4 3 P Discover FO 2 1 Power [W] 2 1 5 Time [s] 1 15 Figure S12. Temperature (Discover FO, OpSens FO) and microwave power (P) profiles for a typical microwave-assisted peptide deprotection step (2 ml 3% piperidine in DMF) at 75 C (set temperature) for 15 sec irradiation time using the Discover SPS program (maximum power 2 W, T = 3 C).
S14 entry 1 entry 2 Purity: 77% Purity: 82% entry 3 entry 4 Purity: 89% Purity: 92% entry 5 entry 6 Purity: 88% Purity: 91% Figure S13. Comparison of peptide purities (HPLC chromatograms, UV absorbance at 215 nm) of GILTVSVAV peptide synthesized on Tentagel and ChemMatrix resins under conventional and microwave conditions at the same temperature. For exact conditions, see Table 4. See also Figure 4.
15 125 mau 215nm4nm (1.) S15 1 75 5 25 Purity: 88% 2.5 12.5 min Figure S14. Peptide purity (HPLC chromatogram, UV absorbance at 215 nm) of GILTVSVAV peptide synthesized on ChemMatrix resins on.225 mmol scale. Reactions were performed on a.225 mmol scale applying RAM-ChemMatrix resin (loading.5 mmol/g) in a 25 ml solid-phase reaction vessel mixing via nitrogen bubbling (ca. 3.2 ml solvent for the coupling step; 4 ml 3% piperidine in DMF for deprotection step), CEM Discover SPS. Set temperature monitored by internal fiber-optic probe (see Figure S1). Figure S15. Aggregation potential of KWKLFKKIGAVLKVL analyzed using Peptide Companion Software. The prediction program characterizes the coupling difficulty of the amino acids from the fifth residue in the sequence using an aggregation potential value. An aggregation potential value below.9 indicates that the coupling of the current amino acid is easy, a value between.9-1.1 means the difficulty of the coupling is intermediate. The sequence starts being potentially difficult after two or three consecutive potentials have exceeded the value 1.1. The observed aggregation potential values of > 1.2 indicate a very difficult peptide sequence.
S16 a) 4 mau 215nm,4nm (1.) + [M-Lys+H] + b) 5 mau 215nm4nm (1.) c) 5 mau 215nm4nm (1.) 3 4 3 4 3 2 2 2 1 1 1 Purity:34%. 2.5 12.5 min Purity:87%. 2.5 12.5 min Purity:91% -1. 2.5 12.5 min [M-Lys+H] + =1771.6 [M-Lys+H] + =1643.9 a) b) =1771.4 [M+H] c) + =1771.2 Figure S16. Comparison of peptide purities (HPLC chromatograms, UV absorbance at 215 nm) of KWKLFKKIGAVLKVL peptide synthesized on ChemMatrix resin using 3 equiv of Fmoc-amino acids under: (a) room temperature conditions, (b) at 86 C coupling and deprotection temperature under conventional conditions, and (c) at 86 C coupling and deprotection temperature under microwave conditions. Purity of crude peptides (analytical RP-HPLC peak area %, UV absorbance at 215 nm). Peaks between 5 and 8 min retention time were used for integration. Peptide synthesis was performed on a.75 mmol scale applying RAM ChemMatrix resin (loading.5 mmol/g) in a 1 ml solid-phase reaction vessel (ca. 1.1 ml solvent for the coupling step; 2 ml 3% piperidine in DMF for the deprotection step. For further details of the reaction conditions, see Table 4. Also shown are the MALDI-TOF mass spectra of the major peak (M cal = 177.3).
S17 c) Figure S17. Aggregation potential of CGIGKFLHGAKKFGKAFVGEIMNS analyzed using Peptide Companion Software. The prediction program characterizes the coupling difficulty of the amino acids from the fifth residue in the sequence using an aggregation potential value. An aggregation potential value below.9 indicates that the coupling of the current amino acid is easy, a value between.9-1.1 means the difficulty of the coupling is intermediate. The sequence starts being potentially difficult after two or three consecutive potentials have exceeded the value 1.1. The observed aggregation potential values of > 1.1 indicate a difficult peptide sequence.
S18 a) mau 215nm4nm (1.) 15 b) 5 mau 215nm4nm (1.) 4 c) 6 mau 215nm4nm (1.) 5 1 5 3 2 4 3 2 1 1-5 Purity:18% Purity:48% Purity:54% 2.5 12.5 min 2.5 12.5 min 2.5 12.5 min a) b) =2541.5 =254.7 c) =254.3 Figure S18. Comparison of peptide purities (HPLC chromatograms, UV absorbance at 215 nm) of CGIGKFLHGAKKFGKAFVGEIMNS peptide synthesized on ChemMatrix resin using 3 equiv of Fmoc-amino acids under: (a) room temperature conditions, (b) at 86 C coupling and deprotection temperature using conventional heating, and (c) at 86 C coupling and deprotection temperature using microwave heating. Purity of crude peptides (analytical RP-HPLC peak area %, UV absorbance at 215 nm). Peaks between 5 and 8 min retention time were used for integration. Peptide synthesis was performed on a.75 mmol scale applying RAM ChemMatrix resin (loading.5 mmol/g) in a 1 ml solid-phase reaction vessel (ca. 1.1 ml solvent for the coupling step; 2 ml 3% piperidine in DMF for the deprotection step. For further details of the reaction conditions, see Table 4. Also shown are the MALDI-TOF mass spectra of the major peak. (M ca l = 2539.)
S19 1 9 8 Temperature [ C] 7 6 5 4 3 2 1 2 4 6 8 1 Time [min] Figure S19. Temperature profile (internal OpSens FO) for a conventionally heated coupling step (3- fold excess of Fmoc-amino acids, DIC and HOBt) in 1 ml NMP using the PLS 4 x 6 syntheszer. Set temperature 86 C. The temperature was recorded starting at the time the solid-phase vessel was inserted into the preheated aluminum block of the PLS synthesizer (see Figure 3 for the same experiment carried out using microwave heating). The heating rate for the deprotection cocktail is very similar (data not shown). mau 215nm4nm (1.) 125 1 75 5 25 Purity: 82% 2.5 12.5 min Figure S2. Peptide purity (HPLC chromatogram, UV absorbance at 215 nm) of GILTVSVAV peptide synthesized on Tentagel resin under conventional conditions using 7 C for the coupling and deprotection steps. The other conditions are described as in Table 4 for entry 1.
S2 Table S2. Racemization Analysis of Nonapeptide GILTVSVAV Synthesized on Tentagel Resin Using Room Temperature (25 C) or Microwave (86 C) Conditions. Amino acid % D Enantiomer SPPS 25 C MW-SPPS 86 C Isoleucine a <.1 <.1 Leucine.26.26 Threonine b <.1 <.1 Valine <.1 <.1 Serine.16.19 Alanine.13.24 a L-allo Isoleucine <.1, D-allo Isoleucine <.1; b L-allo Threonine <.1, D-allo Threonine Table S3. Racemization Analysis of Modified Mangainin-II-amide Synthesized on ChemMatrix Resin Using Conventionally Heated (86 C) or Microwave (86 C) Conditions. % D Enantiomer Amino acid CONV-SPPS 86 C MW-SPPS 86 C Serine.23.24 Cysteine 2.8 1.88 Methionine.36.47 Histidine 6.29 7.59