Heating and Microwave Assisted SPPS of C-Terminal Acid Peptides on Trityl Resin: the Truth

Heating and Microwave assisted SPPS of C-terminal acid peptides on trityl resin: The truth behind the yield

Cécile Echalier a†•Soultan Al-Halifaa • Aude Kreiter†• Christine Enjalbal• Pierre Sanchez • Luisa Ronga†• Karine Puget †• Pascal Verdié•Muriel Amblard • Jean Martinez • Gilles Subra

Table of Contents

Experimental Conditions

Analytical HPLC analysesS2

LC/MS analysesS2

RP-Preparative HPLCS2

First amino acid anchoring on hydroxymethylphenoxy PS resin (Wang)S3

First amino acid anchoring on Fmoc-Rink amide aminomethyl PS resinS3

Synthesis of compound 8c[Phe-Ala] DKPS3

Titration of Fmoc-Ala-OH 4’ (HPLC =214 nm) S5

• Calibration curve of Fmoc-Ala-OH 4’S5
• Titration of released Fmoc-Ala-OH from the resin at room temperature in DMFS5
• Titration of released Fmoc-Ala-OH from the resin at 70°C in DMFS6

Titration of Fmoc-Phe-Ala-OH 5’ (HPLC =214 nm) S6

• Calibration curve of peptide 5’S6
• Titration of released peptide 5’ from the resin at room temperature in DMFS7
• Titration of released peptide 5’ from the resin at 70°C in DMFS7

Titration of Fmoc-Leu-Phe-Ala-OH 7’ (HPLC =214 nm) S8

• Calibration curve of peptide 7’S8
• Titration of released peptide 7’ from the resin at room temperature in DMFS8
• Titration of released peptide 7’ from the resin at 70°C in DMFS9

Calibration curve of DKP 8(LC/MS, SIR MRM m/z 219.2)S9

Calibration curve of protected peptide 1’ (HPLC =214 nm)S10

LC/MS analyses of crude peptide 1’S10

LC chromatogram of purified peptide 1’S11

Titration of released protected peptide 1’S12

LC/MS analyses of Crude Peptide 1H-Glu-Lys-Arg-Tyr-Cys-Ser-OHS13

Determination of stereochemistry of released peptide from resin 7S15

Synthesis scheme of R2 with resin weighting and calculationsS17

Synthesis scheme of R3 with resin weighting and calculationsS17

LC/MS Analysis of peptide 2S18

LC/MS Analysis of peptides 3S19

Analytical HPLC analyses

Conditions A

Analytical HPLC were run on a Beckman instrument, equipped with a photodiode array and ChromolithSpeedROD (50 – 4.6 mm) reversed-phase column (RP-18e). Standard conditions were eluent system A (water/0.1% TFA), system B (acetonitrile/0.1% TFA). A flow rate of 5 mLmL/min and a gradient of (0–100)% B over 3 min were used, detection at 214 nm, unless otherwise stated. Retention times (Rt(A)) are given in minutes.

Conditions B

Analytical HPLC were run on anAgilent 1100 instrument, equipped with a photodiode array and XBridge (150 – 3 mm) reversed-phase column (C18). Standard conditions were eluent system A (H2O/ACN/MSA - 98/2/0.1%), system B (ACN/H2O/MSA - 98/2/0.1%). A flow rate of 5mLmL/min and a gradient of (0–50)% B for2 min, (50–100)% B for 5 minwere used, detection at 214 nm, unless otherwise stated. Retention times (Rt(B)) are given in minutes.

Conditions C

LC analyses were run on a Waters Alliance 2695 HPLC, equipped with monochromatic detector array and ChromolithHigh resolution (25 – 4.6 mm) reversed-phase column (RP-18e). A flow rate of 3 mLmL/min and a gradient of (0–100) % B over 2.5 min (or over 15 min) were used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. Retention times (Rt(C)) are given in minutes

Conditions D (for separation of diastereoisomers of 7’)

LC analyses were run on a Beckman instrument, equipped with a monochromatic detector and Onyx monolithic HD (50 – 4.6 mm) reversed-phase column (C18). Standard conditions were eluent system A (water/0.1% TFA), system B (acetonitrile/0.1% TFA). A flow rate of 5 mLmL/min and a gradient of (32–34)% B over 30 min were used, detection at 214 nm. Retention times (Rt(D)) are given in minutes.

LC/MS analyses

LC/MS analyses – samples were prepared in acetonitrile/water (50/50 v/v) mixture, containing 0.1% TFA. The LC/MS system consisted of a Waters Alliance 2695 HPLC, coupled to a Micromass (Manchester, UK) ZQ spectrometer (electrospray ionization mode, ESI+). All the analyses were carried out using a Merck Chromolith Speed rod C18, 25 × 4.6 mm reversed-phase column. A flow rate of 3 mLmL/min and a gradient of (0–100) % B over 2.5 min (or over 15 min) were used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. Retention times (RT) are given in minutes. Positive ion electrospray mass spectra were acquired at a solvent flow rate of 200 mLmL/min. Nitrogen was used for both the nebulizing and drying gas. The data were obtained in a scan mode ranging from 100 to 1000 m/z in 0.1 s intervals; 10 scans were summed up to get the final spectrum.

MRM analysis of compound 8 c[Phe-Ala]

Diketopiperazinec[Phe-Ala] analysis was perform using MRM acquisition mode (multiply reaction monitory) on a Waters Alliance 2695 coupled to a MicromassQuatro Micro. A flow rate of 3 mLmL/min and a gradient of (0–100) % B over 2.5 min were used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. Retention times (RT) are given in minutes. Detection of DKP was done at 214 nm using the same acquisition (ES+) and the following parameters: Cone voltage: 20 V; Collision energy: 12 eV.

RP-Preparative HPLC

RP-preparative HPLC purification was performed on a Waters HPLC 4000 instrument, equipped with a UV detector 486 and Waters Delta-Pack 40×100 mm, 15 Å, 100 µm, reversed-phase column. Eluent system A (water/0.1% TFA), system B (acetonitrile/0.1% TFA). A flow rate of 50 mLmL/min and a gradient of (10–70)% B over 30 min were used, detection at =214 nm.

First amino acid anchoring on hydroxymethylphenoxyPS resin (Wang), example of Fmoc-Ala-OH.

The hydroxymethylphenoxy PS resin (1g, 0.7 mmol, initial theoretical loading = 0.7 mmol/g) was conditioned for 15 min in DCM. Then, 10mmLl of DMF solution of Fmoc-Ala-OH (3 eq., 2.1 mmol, 654mg) containing DIC (1.5 eq, 1.05 mmol, 165 µl), DMAP (0.15 eq, 105 µmole, 13 mg) was poured unto the resin and stirred overnight. The resin was filtered and then washed with DMF (2×), DCM/MeOH 1/1 v/v(2×), DMF (2×), DCM (2×) and dried in a desiccator overnight.

First amino acid anchoring on Fmoc-Rink amide aminomethyl PS, example of Fmoc-Ala-OH.

The Fmoc rink amide aminomethyl PS resin (1g, 0.96 mmol, initialtheoritical loading 0.96 mmol/g) was conditioned for 15 min in DCM. The Fmoc protecting group was removed with a standard deprotection cycle using 80/20 v/v DMF/pip solution (see main article Material and Methods). After washing step, the first aminoacid was loaded on the resin through a standard coupling cycle (see main article Material and Methods) using HBTU and NMM.

Synthesis of compound 8 c[Phe-Ala] DKP. (3R,6S)-3-benzyl-6-methylpiperazine-2,5-dione

Boc-Ala-OH (1.1 eq) was dissolved in a solution of HBTU (1.1 eq), N-Methylmorpholine (NMM; 2 eq) in N,N-dimethylformamide (DMF), and the resulting solution was added to the H-Phe-OMe dissolved in DMF. The reaction mixture was stirred at room temperature for 2 h and then DMF was evaporated under vacuum. The residue was dissolved in AcOEt and washed with KHSO4 (3 times), NaHCO3 (3 times), NaCl (3 times) and then dried over anhydrous sodium sulfate and evaporated to give an oily residue (compound 1). The compound 1 was dissolved in trifluoroacetic acid (TFA) and stirred for 1 hour, followed by removal of the solvent by evaporation under vacuum to give compound 2. This compound 2 was refluxed in DCM for 6 hours and DKP (compound 8) precipitates directly into the flask. The precipitate was washed with DCM, Et2O, frozen and lyophilized in water/acetonitrile (50/50).

MS (ESI) calcd for C12H14N2O2, 218.11; [M + H]+m/z found 219.20 , Rt(C): 0.82 min.

TIC/LC and LC/MS analyses are presented in the following figures

Titration of Fmoc-Ala-OH 4’ (HPLC =214 nm)

• Calibration curve of Fmoc-Ala-OH 4’

A solution of Fmoc-Ala-OH (stock solution, 10-3 M) was prepared in 4mlmL of DMF. Stock solution was used to make a dilution range of 10-3M to 10-6 M and 10 µl of each solution was injected on ananalytical HPLC (conditions A). Data are summarized in the following table.

Titration (M) / 0 / 6.2x10-5 / 7.75x10-5 / 1.03x10-4 / 1.55x10-4
Areas / 0 / 261479 / 453235 / 543635 / 730797

• Titration of released Fmoc-Ala-OH from the resin at room temperature in DMF

100 mg of resin 4were weighted and placed in a test tube. 4 mlmL of DMF were added and kineticswas carried outat room temperature on an analytic HPLC (Conditions A) for1200 min. Data are summarized in the following table.

Time (min) / 0 / 60 / 150 / 240 / 360 / 1200
Area / 73117 / 90434 / 298272 / 271477 / 199759 / 203332
Titration (M) / 1.25 x10-5 / 1.61 x10-5 / 5.89 x10-5 / 5.23 x10-5 / 3.86 x10-5 / 3.93 x10-5

• Titration of released Fmoc-Ala-OH from the resin at 70°C in DMF

In a closed vessel containing 100 mg of resin 4, 4 mlmL of DMF were addedand the mixture washeat till 70°C. Kineticswas carried outon an analytic HPLC (conditions A) for400 min. Data are summarized in the following table.

Time (min) / 0 / 60 / 150 / 240 / 400
Area / 11407110 / 32217440 / 49795400 / 11987300 / 0
Titration (M) / 2.349 x10-3 / 6.640 x10-3 / 1.026 x10-3 / 2.469 x10-3 / 0

Titration of Fmoc-Phe-Ala-OH 5’ (HPLC =214 nm)

• Calibration curve of peptide 5’

A solution of Fmoc-Phe-Ala-OH (stock solution, 10-3 M) was prepared in 4mlmL of DMF. Stock solution was used to make a dilution range of 10-3M to 10-5M and 10 µl of each solution was injected on ananalytical HPLC (Conditions A). Data are summarized in the following table.

Titration (M) / 2.10-5 / 1.10-4 / 2.10-4 / 3.10-4 / 4.10-4
Areas / 43035 / 237928 / 469037 / 797828 / 963521

• Titration of released peptide 5’ from the resin at room temperature in DMF

100 mg of resin 5’were weighted and placed in a test tube. 4 mlmL of DMF were added and kineticswas carried outat room temperature on an analytic HPLC (Conditions A) for400 min. Data are summarized in the following table.

Time (min) / 0 / 67 / 132 / 206 / 315 / 388
Area / 34564 / 184223 / 243800 / 215285 / 229580 / 258306
Titration (M) / 1.41 x10-5 / 7.49 x10-5 / 9.91 x10-5 / 8.75 x10-5 / 9.33 x10-5 / 1.05 x10-4

• Titration of released peptide 5’ from the resin at 70°C in DMF

In a closed vessel containing 100 mg of resin 5’, 4 mlmL of DMF were addedand the mixture was heat till 70°C. Kineticswas carried outon an analytic HPLC (Conditions A) for400 min. Data are summarized in the following table.

Time (min) / 5 / 75 / 137 / 215 / 302 / 390
Area / 691751 / 3676338 / 1353920 / 2037955 / 1669751 / 1303901
Titration (M) / 2.81 x10-4 / 1.49 x10-3 / 5.50 x10-3 / 8.28 x10-3 / 13.6 x10-3 / 15.9 x10-3

Titration of Fmoc-Leu-Phe-Ala-OH 7’ (HPLC =214 nm)

• Calibration curve of peptide 7’

A solution of Fmoc-Phe-Ala-OH (stock solution, 10-2 M) was prepared in 4 mlmL of DMF. Stock solution was used to make a dilution range of 10-2M to 10-5M and 10 µl of each solution was injected on an analytical HPLC (Conditions A). Data are summarized in the following table.

Titration (M) / 1.67 x10-4 / 3.33 x10-4 / 6.66 x10-4 / 1 x10-3 / 1.5 x10-3 / 2.25x10-3 / 3.38 x10-3 / 5.07 x10-3
Areas / 76041 / 174998 / 352007 / 626643 / 829560 / 1180207 / 1730425 / 2417291

• Titration of released peptide 7’ from the resin at room temperature in DMF

100 mg of resin 7’were weighted and placed in a test tube. 4 mlmL of DMF were added and kineticswas carried outat room temperature on an analytic HPLC (Conditions A) for400 min. Data are summarized in the following table.

Time (min) / 0 / 65 / 129 / 209 / 313 / 391
Area / 34564 / 184223 / 243800 / 215285 / 229580 / 258306
Titration (M) / 1.20 x10-5 / 1.14 x10-4 / 1.35 x10-4 / 1.42 x10-4 / 1.75 x10-4 / 1.64 x10-4

• Titration of released Peptide 7’ from the resin at 70°C in DMF

In a closed vessel containing 100 mg of resin 5’, 4 mlmL of DMF were addedand the mixture was heat till 70°C. Kineticswas carried outon an analytic HPLC (Conditions A) for400 min. Data are summarized in the following table.

Time (min) / 10 / 89 / 149 / 225 / 318 / 400
Area / 1109903 / 3631953 / 2184003 / 1135121 / 1441678 / 852544
Titration (M) / 3.4 x10-4 / 1.12 x10-3 / 3.38 x10-3 / 7.03 x10-3 / 8.93 x10-3 / 7.92 x10-3

Calibration curve of DKP8 (LC/MS, SIR MRM m/z 219.2)

A solution of DKP (stock solution, 10-2 M) was prepared using 2.18 mg of DKP in 1 mlmL of water/acetonitrile (50/50). Stock solution was used to make a dilution range of 10-2 M to 5.10-6 M and 1 µl of each solution was injected on a mass spectrometer (Mass lynx power). Data are summarized in the following table.

Titration (M) / 1 x10-3 / 5 x10-4 / 1 x10-4 / 5 x10-5 / 1 x10-5 / 5 x10-6
areas / 7859 / 3700 / 791 / 374.21 / 59 / 51.54

Calibration curve of protected peptide 1’ (HPLC =214 nm):Boc-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-Tyr(tBu)-Cys(trt)-Ser(tBu)-OH MW = 1646.8 g/mol.

Protected peptide 3was synthesized on a LibertyTM Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC) providing MW irradiation at 2450 MHz usingFmoc/tBu SPPS strategy in the conditions described in Material and methods section of the article. The crude peptide 3 wasreleased from the resin using a solution of HFIP for 45 min in order to keep the side chain protecting groups.LC/MS (conditions C) analyses are presented below.

The crude peptide was purified on an autopurification system Waters Micromass equipped with an injector/collector sample manager (Waters 2767), a pump (Waters 2767), a photodiode array detector (Waters 2996) and a mass spectrometer ZQ (Waters). LC chromatogram (conditions C) of purified 1’is presented below.

A calibration curve of the pure peptide (stock solution, 10-3 M) was prepared in 4 mlmL of DMF. Stock solution was used to make a dilution range of 10-3M to 10-6M and 10 µl of each solution was injected on an analytical HPLC (Conditions B). Data are summarized in the following table.

Titration (M) / 7.80x10-6 / 1.56x10-5 / 3.12x10-5 / 6.25x10-5 / 1.25x10-4 / 2.50x10-4 / 5.00x10-4 / 7.60x10-4
Area / 514.5 / 562.1 / 746.9 / 1046 / 1697 / 2625 / 4725 / 6543

Titration of released Peptide 1’ from the resin at 70°C in DMF

In a closed vessel containing 100 mg of resin R1’, 4 mlmL of DMF were addedand the mixture was heat till 70°C. Aliquots were analyzed (conditions A) for400 min. Data are summarized in the following table.

Time (hour) / Peak area / Titration (M)
1 / 1678 / 2.80x10-4
2 / 2727 / 4.60x10-4
3 / 3929 / 6.60x10-4
4 / 6791 / 1.10x10-3
5 / 4001 / 1.35x10-3
6 / 4313 / 1.45x10-3
7 / 4853 / 1.60x10-3
8 / 3161 / 1.60x10-3
9 / 4424 / 2.20x10-3
10 / 3055 / 2.10 x10-3
11 / 3257 / 2.20 x10-3
12 / 3896 / 2.60 x10-3
13 / 4173 / 2.80 x10-3
14 / 4517 / 3.00 x10-3
15 / 4628 / 3.10 x10-3
16 / 4086 / 3.40 x10-3
17 / 4050 / 3.40 x10-3
18 / 4752 / 4.00 x10-3
19 / 5293 / 4.50 x10-3

LC/MS analysis of Crude Peptide 1H-Glu-Lys-Arg-Tyr-Cys-Ser-OH,MW = 784.8 g/mol

Determination of stereochemistry of released peptide from resin 7

Chromatogram of 7’ Fmoc-Leu-Phe-Ala-OH (conditions D)

Chromatogram of 7’(D) Fmoc-Leu-Phe-(D)Ala-OH (conditions D)

Coinjection of 7’(D) and 7’ (conditions D)

Released peptide from resin 7 after 6 hours heating (70°C)

Coinjection of released peptide after 6 hours heating (70°C) and 7’(D) (Fmoc-Leu-Ala-(D)Phe-OH)

Synthesis scheme of R2with resin weighting and calculations

Synthesis scheme of R3 with resin weighting and calculations

LC/MS analysis of peptide 2b

LC/MS analysis of peptide 3b

S1