Team:Freiburg/Labbook/chemical synthesis
This subgroup was responsable for the synthesis of all peptides we needed for the project. This was accomplished via solid phase peptide synthesis (SPPS). For two reasons we couldn't use normal expression for our peptides: We needed to synthesize random dodecamer peptides without any knowledge about it's interactions with living cells or microbes. Secondly we synthesized D-peptides which is not possible in vivo at all. We managed to synthesize over 20 different peptides with this method includig some modification.
Click on an item for a detailed description
Today we had an appointment at 2 pm with Prof. Dr. Maja Köhn. In her Lab she's got a synthesizer for SPPS, which we want to use for the synthesis of PSMα3 and potential ligands.
She was very cooperatively and permitted us to use the synthesizer under the advice of Nico, her TCA.
To test how good PSMα3 can be synthesized with SPPS and to learn about the process we first synthesized the Modulin in fragments, 100 µmol each:
Sequence PSMα3:
MEFVA KL | FKF FKDLL | GKFLG NN
- Frag S1: GKFLGNN
- Frag S2: FKFFKDLL
- Frag S3: MEFVAKL
Materials
- Syringes, 5 mL
- Preloaded Wang resin (Asn, Leu)
- Fmoc-XXX(yyy)-OH AA:
- Fmoc-Ala-OH
- Fmoc-Asn(Trt)-OH
- Fmoc-Asp(OtBu)-OH
- Fmoc-Glu(OtBu)-OH
- Fmoc-Gly-OH
- Fmoc-Leu-OH
- Fmoc-Lys(Boc)-OH
- Fmoc-Met-OH
- Fmoc-Phe-OH
- Fmoc-Val-OH
- HBTU
- DMF
- NMM
- TFA
- TIPS
- Piperidine
- Formic acid
- DCC
- Diethyl ether
Procedure
- Weight in the preloaded resin:
- Leucine Wang resin (loading: 76.8 mmol/g): 2x 130 mg
- Asparagine Wang resin (loading: 65.3 mmol/g): 1x 150 mg
- Add 1 mL DMF to each syringe an let the resins swell for 20 min.
- Prepare the Activated AA solution (2 eq)
- For each AA weight in 75.8 mg (0.5 eq, 200 µmol) HBTU
- Add 1.5 mL DMF
- Add 11 µL (0.1 eq, 0.1 nmol) NMM
- Add 2 eq of the next AA (reverse direction!): N (119,4 mg), L (70.6 mg), K (93.7 mg)
- Press the syringes carefully out and refill them with the activated AA solution.
- Shake the syringes for 60 min.
- Wash the peptides 3 times with DMF.
- Repeat steps 3-6 with to other AA.
- Formylation of Met:
- Prepare formic acid (1.88 mL, 50 mmol, 25 eq) with DCC (5.14 g, 25 mmol, 12.5 eq) in diethyl ether (29 mL)
- Stir for 4 h at 0°C
- Remove the solvent by rotary evaporatoration to a volume of 1-2 mL.
- Fill it into the respective syringe and shake it over night.
- Cleavage:
- Prepare solution: 95% TFA + 2.5% H2O, 2.5% TIPS, 1 mL.
- Fill the syringes with the cleavage solution and shake it for 4 h.
- Prpare 50 mL falcon tubes with -20°C cold diethylether.
- Press out the syringes in the falcon tubes and centrifuge them down (5 min at 13,200 rpm).
- Decant the tubes and let them open to dry.
- Analysis (performed by Nico, TCA)
All fragments have been analized via HPLC-MS. A C-18 column was used and a gradient of 10% to 90% acetonitrile in water was driven over 14 min.
Protocol analysis
Fig. 1: Gradient of the HPLC-runs in % acetonitrile in water, each containing 0.05% TFA.
Analysis fragment S1
(a)
(b)
m = 748.93 g/mol
Fig. 2: UV spectrum (a) at 220 nm of the sample. As you can see in (b) the repective mass was found at a retention time of 7.25 min.
Analysis fragment S2
(a)
(b)
m = 1057.41 g/mol
Fig. 3: UV spectrum (a) at 220 nm of the sample. As you can see in (b) the repective mass was found at a retention time of 9.35 min.
Analysis fragment S3
(a)
(b)
No MS spectrum could be obtained due to a low yield.
m = 837.14 g/mol
Fig. 4: UV spectrum (a) at 220 nm of the sample. The respective mass was not found.
This synthesis was performed with the SPPS machine from Prof. Köhn. We synthesized L-PSMα3 at a 100 µmol scale. Therefore we typed in the sequence and weighted in the amino acids and all the other reagents according to the following list.
Sequence: MEFVA KLFKF FKDLL GKFLG NN
solutions to prepare
(a)
(b)
(c)
Fig. 5: (a) List of amino acids and (b) (c) reagents which have been prepared for the synthesis.
Layout synthesizer
Fig. 6: Scheme of the synthesizer and a corrensponding picture.
Due to a low yield in the previous synthesis of the fragments, half of the resin was transferred into another syringe to formylate it, the other half was kept as a backup without formylation. Formylation, cleavage and analysis were executed like described above (May 13th - 15th, steps 8, 9 and 10). The purification was also performed by Nico with a protocol similar to the analysis protocol.
L-PSM alpha 3 without formylation
(a)
(b)
m = 2605.4 g/mol
Fig. 7: UV spectrum (a) at 220 nm of the sample. As you can see in (b) the repective mass was found at a retention time of 12.69 min. Further the dimer was found in the mass spectrum at (m + Z) / Z = 1737.7 for Z = 3.
L-PSM alpha 3 with formylation
(a)
(b)
m(carboxyl) = 2653.1 g/mol
m(formyl) = 2637.1 g/mol
Fig. 8: UV spectrum (a) at 220 nm of the sample. As you can see in (b) the repective mass was found at a retention time of 12.69 min. Further the dimer was found in the mass spectrum at (m + Z) / Z = 1737.7 for Z = 3.
Synthesized sequences
- Fragment A: MEFVA KLFKF FK
- Fragment B: SLLGK FLGNN
- L-PSMα3 D13S: MEFVA KLFKF FKSLL GKFLG NN
- D-PSMα3: mevfa klfkf fkdll GkflG nn
- Biotin-D-PSMα3: mevfa klfkf fkdll GkflG nn
All sequences were synthesized with the Synthesizer of Prof. Köhn at a 100 µmol scale. For the L-peptides the normal preloaded wang resins have been used. The D-peptides have been coupled to a 2-chlorotrityl chloride resin or to a biotin resin.
Solutions to prepare
(a)
(b)
(c)
Fig. 9: (a) List of amino acids and (b) (c) reagents which have been prepared for the synthesis.
A subsequent formylation was porformed for the half of each full length PSMα3 (L-PSMα3 D13S, D-PSMα3, Biotin-D-PSMα3) as describt above (May 13th - 15th, step 8). Cleavage and purification did neither change to the above description, but the analysis was done with a MALDI-TOF MS.
MS diagrams of the samples
(a) + (b)
(c) + (d)
(e)
Fig. 10:
(a) MS spectrum of fragment A. m(fragment A) = 1534.9 g/mol
(b) MS spectrum of fragment B. m(fragment B) = 1062.2 g/mol
(c) MS spectrum of L-PSMα3 D13S, not formylated. m(L-PSMα3 D13S) = 2579.1 g/mol
(d) MS spectrum of L-PSMα3 D13S, formylated = 2607.1 g/mol
(e) MS spectrum of D-PSMα3 C-(biotinylethylendiamin). m(D-PSMα3 C-NEtN-biotin) = 2877.5 g/mol
As the spectra show, the two fragments and PSMα3 with the mutation and without formylation could be isolated very pure and with a high yield. The formylation did work but not well and the synthesis with the biotin resin worked but neither well. The other syntheses unfortunately didn't work.
Synthesized sequences
- surf 11: KHVQI TSTFG VI
- surf 8: KHLHY HSSVR YG
- surf 27: MPMFK HRMFH TH
- s 1: TLEDL QLLAD LF
- us 1: FTREL FYKLF KE
- us 2: SKMLN TCYRI LG
- us 3: QFQFH EALCK EA
All sequences were synthesized with the Synthesizer of Prof. Köhn at a 50 µmol scale. For the L-peptides the normal preloaded wang resins have been used.
Solutions to prepare
(a)
(b)
(c)
Fig. 9: (a) List of amino acids and (b) (c) reagents which have been prepared for the synthesis.
Cleavage and purification did neither change to the above description, the analysis was done via HPLC-MS.
