Design and Synthesis of Macrocyclic Peptides as Potential Inhibitors of Lysine-Specific Demethylase 1
Victor Sonesten Bachelor thesis
Supervisor: Jan Kihlberg, Johan Viljanen
Uppsala University, Department of Chemistry - BMC Degree Project C in Chemistry, 15.0 c
2019-10-20
Abstract
The work done in this BSc thesis is part of a project with an overall objective to study macrocyclic peptides as inhibitors for Lysine-specific demethylase 1( LSD1). For this work, four new peptides, cyclised using lactam bridges, were designed and synthesised along with a positive and a negative control. This work solely covers the design and synthesis.
Using Fmoc solid phase peptide synthesis, the peptides were synthesised on Rink Amide resin using an automatic peptide synthesiser. For the cyclisation, the side-chains to be linked were protected using orthogonal allyl and alloc protecting groups. The protecting groups were removed under mild condition with Pd(0) and the lactam bridge formed with PyBOP, HOBt and DIPEA. The peptides were then cleaved from the resin with simultaneous side-chain deprotection. Purification was done by reverse-phase HPLC followed by
lyophilisation for storage. The purity of the peptides was analysed using analytical HPLC, but the results were inconclusive due to errors with the HPLC instrument. All manual steps were monitored by MALDI-TOF-MS following test cleavage from resin.
The results of the MALDI-TOF-MS show that the correct peptides had been
synthesised. Some impurities and side-products were detected from the synthesis process,
albeit the most common side-product was removed during purification. The final yields
ranged from 9 % to 23 % after purification, based on the capacity of the resin.
Table of Contents
1 Abbreviations ... 4
2 Introduction ... 5
2.1 Objective of overall project ... 5
2.2 Synthesis and cyclisation of macrocyclic peptides ... 6
3 Methods ... 9
3.1 Synthesis process of lactamised macrocyclic peptides ... 9
3.2 Purification ... 10
3.3 Analysis ... 11
3.3.1 Identity ... 11
3.3.2 Purity ... 11
4 Results and comparative discussion ... 11
5 Conclusion ... 15
6 Experimental ... 15
6.1 General procedure for the synthesis of all peptides ... 16
7 Acknowledgements ... 18
8 References ... 18
9 Appendix ... 20
1 Abbreviations
LDS1 – Lysine-specific demethylase 1 Ala - Alanine
Arg – Arginine Lys – Lysine (D)-Lys – D-Lysine Met – Methionine Gln – Glutamine Glu – Glutamic acid (D)-Glu – D-Glutamic acid Ser – Serine
Thr – Threonine [Cit] – Citrulline
Boc – tert. Butoxycarbonyl
Fmoc – 9-Fluorenylmethoxycarbonyl Alloc - Allyloxycarbonyl
tBu – t-Butyl ether
Pbf - 2,2,4,6,7-pentamethyl- dihydrobenzofuran-5-sulfonyl Trt - triphenylmethyl
TFA – Trifluoroacetic acid HOAc – Acetic acid
NMM – N-Methylmorpholine DMF - Dimethylformamide NMP – N-Methylpyrrolidinone DCM – Dichloromethane
PyBOP – Benzotriazole-1-yl-oxy-tris- pyrrolidino-phosphonium
hexafluorophosphate
HOBt – N-Hydroxybenzotriazole DIPEA – Diisopropylethylamine
HCTU - 2-(6-Chloro-1H-Benzotriazole-1- yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate
DEDT – Sodium diethyldithiocarbamate trihydrate
EDT - Ethanedithiol TIS - Triisopropylsilane
SPPS – solid phase peptide synthesis MALDI-TOF-MS – matrix-assisted laser desorption/ionisation time of flight mass spectrometry
HPLC – High performance liquid
chromatograph
2 Introduction
2.1 Objective of overall project
Macrocyclic peptides are a group of peptides which have shown to be useful in multiple field due to the restricted conformation which can arise from the cyclisation. Macrocyclic peptides can loosely be described as peptides with a ring connecting multiple amino acid residues. This cyclisation generally improves many pharmacological characteristics of the peptide. In the last few decades, the attention place into macrocyclic peptides have started the progress of macrocyclic peptides into their own drug modality
1.
The enzyme Lysine-specific demethylase 1 (LSD1) catalyses the demethylation of methyl groups from both histone and non-histone lysine residues. It has been found to be overexpressed in certain types of cancer, such as ovarian cancer. Therefore, chemical
inhibitors of LSD1 have been proposed as candidates in cancer therapy
2. In a study at Uppsala University, it was found that macrocyclic peptides can be used as possible inhibitors for LSD1. The study compared several different peptides and methods for cyclisation
3.
The aim of this BSc thesis was to design and synthesise macrocyclic peptides as inhibitor of LSD1. The peptides designed and synthesised are found in Table 1.
Table 1: The six peptides designed and synthesised in the project. The sequences use the three letter coding for the amino acids. The underlined amino acids in the sequences indicate which amino acids form the macrocyclic bridge.
Code Sequence
I Ala-Arg-(D)-Lys-Met-Gln-Glu-Ala-Arg-Lys-Ser-Thr-NH
2II Ala-Arg-(D)-Glu-Met-Gln-Lys-Ala-Arg-Lys-Ser-Thr-NH
2III Ala-Arg-(D)-Lys-Met-Gln-Glu-Ala- NH
2IV Arg-(D)-Lys-Met-Gln-Glu-Ala-Arg-Lys-Ser-Thr-NH
2V Ala-[Cit]-(D)-Lys-Met-Gln-Glu-Ala-Arg-Lys-Ser-Thr-NH
2VI Ala-Arg-(D)-Lys-Ala-Gln-Glu-Ala-Arg-Lys-Ser-Thr-NH
2Peptide I was used as a basis for the design of the other peptides, as it has been found to be a
promising inhibitor for LSD1
3. Peptide II, which had also been studied previously alongside
I, was included as a negative control
3. The other peptides were chosen to study how certain
amino acids affect the binding to LSD1. Two figures representing peptide I can be found in
figure 1. As only the first seven amino acids are shown in figure 1, the last four appear not to be directly involved in the binding. Peptide III was chosen to have the same sequence as peptide I, but without the last four C-terminal amino acids, to study their effect on LSD1 binding. In figure 1 the terminal alanine (Ala 1) appear to be directly involved in the binding, thus its removal in peptide IV was to confirm its involvement. In peptide V, the positively charged arginine was changed to the similarly structured neutral citrulline. Peptide VI, where the methionine was substituted for alanine, was designed to evaluate the involvement of methionine in the LSD1 binding.
Figure 1: The crystal structure of peptide I and the active site on LSD1 in two different models. Red colour indicates negative charge and blue colour indicates positive charge (from Talibov in Yang et al.
4).
2.2 Synthesis and cyclisation of macrocyclic peptides
There are different methods to synthesise and cyclise peptides. This study focuses on Fmoc- based solid phase peptide synthesis (SPPS) and lactamisation to form the macrocyclic peptides.
In Fmoc SPPS, an initial amino acid is coupled to a solid support via a linker, leaving
its a-amino group to be coupled to other amino acids. The a-amino group is temporarily
protected, using an Fmoc protecting group. To prevent undesired reactions, reactive side-
chains located on the amino acids are semi-permanently protected using different protecting
groups. To build the peptide, the temporary protecting group is removed, and the next amino acid is introduced in excess. This amino acid forms an amide bond with the initial amino acid using either a coupling reagent or an already activated amino acid in basic conditions (figure 2). The peptidyl resin is then washed to remove excess or unreacted coupling reagent and amino acids. The deprotection of the N-terminal a-amino group and subsequent binding to new amino acids are repeated until the whole sequence has been built
5,6.
Figure 2: A SPPS scheme, where the initial compound is an amino acid coupled to the solid support (dark circle) via a linker. Reaction a) is the deprotection of Fmoc, leaving the a-amino group ready to react with the carboxyl group of another amino acid. Reaction b) is the amide bond formation resulting in the elongation of the peptide.
After the whole peptide sequence has been synthesised and if no further work is to be done on the peptide, it is cleaved from the solid support with simultaneous side-chain deprotection.
The temporary protecting used in this study is the base-labile Fmoc protecting group
7. The deprotection of the Fmoc moiety from the a-amino group is done with a mild base, such as diluted piperidine
5,6. As the temporary protecting group is base-labile, the semi-permanent protecting groups are chosen to be acid-labile.
The cleaving cocktail used at this step is often TFA-based for Fmoc synthesis. Most of the removed protecting groups can generate reactive species during the cleaving process.
Thus, different scavengers are included in the cocktail to prevent possible side-reactions
8.
The peptides are intended to undergo cyclisation, before removal of the peptide from
the solid support. Thus, the protecting groups for the side-chains of the amino acids, to be
linked, are deprotected separately from the other semi-permanent protecting groups. Thus, an
orthogonal protecting group strategy is chosen for the cyclisation on solid support
5. All the
peptides form a lactam bridge between the side-chains of a lysine and a glutamic acid. The
chosen protecting groups in this study are the Allyl group for the glutamic acid and Alloc for the chosen lysine. The allyl and alloc protecting groups are both stable in TFA and piperidine conditions, but can be selectively removed in mild conditions with a Pd(0) catalyst
3. One of the most useful is tetrakis(triphenylphosphine)Pd(0) (Pd(PPh
3)
4)dissolved in
CHCl
3/HOAc/NMM
5.
A lactam is a cyclic amide with varying ring size
7. After the allyl/alloc deprotection of the lysine and glutamic acid side-chains, the lactam bridge formation can be done using coupling reagents under basic conditions. This step is preferably done using a large volume of solvent. A large volume lowers the risk of lactam bridges formed between different peptides and instead favour the intramolecular bridge formation. The allyl/alloc deprotection and sequent lactamisation can be seen in figure 3.
Figure 3: A general allyl/alloc deprotection (a) and lactamisation of (D)-Lys and Glu (b). The dark circle represents the solid support, whilst X, Y and Z in the sequence represents any number and any order of amino acids.
A common technique to determine the peptides obtained is matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). In MALDI-TOF- MS, the sample is placed on a MALDI plate together with a matrix that co-crystallises with the sample molecules. The sample is ionised by a bombardment from a laser. A high voltage is applied, and the ionised molecules enter a high vacuum and eventually hit a detector.
Peptides travel at different velocities depending on their masses. The result is a spectrum showing a high intensity peak close to the m/z ratio of the peptide. Some other peaks may occur from the matrix or different salt adducts formed on the MALDI plate
8.
As the MALDI cannot be used to determine the purity of a sample, analytical HPLC is a common method to check purity
8. A small amount of peptide is dissolved and injected into a reverse phase HPLC. The fraction obtained are gathered and analysed with MALDI to
determine their masses. If the peptide analysed is pure, the resulting chromatogram will only
show one peak for the peptide.
3 Methods
3.1 Synthesis process of lactamised macrocyclic peptides
The synthesis of the un-cyclised peptides was done using an automatic peptide synthesiser.
The amino acids, solid phase, solvent, base, activation reagent and deprotection reagent were prepared and added to the synthesiser. The synthesiser then performed the SPPS according to a pre-set method.
The amino acids used were all Fmoc protected on the a-amino group, except for the last amino acid in the sequence, which was always protected with a Boc protecting group.
This was done so that the Boc protecting group could be removed during the cleaving process.
All amino acids that contained reactive side-chains were protected using common protecting groups, except for the amino acids intended for lactamisation, which instead were protected with allyl and alloc protecting groups.
For this work, Rink Amide resin was used as the solid phase and NMP was the most common solvent. The Fmoc deprotection was done using piperidine diluted in NMP, followed by activation done by HCTU alongside the base DIPEA.
After the peptide synthesis was performed, the resin was transferred to another vessel and washed with NMP, followed by washing with DCM. The DCM was evaporated using an Ar gas flow and the vessel was left in a desiccator overnight.
Pd(PPh
3)
4was dissolved in CHCl
3/HOAc/NMM. Pd(PPh
3)
4is sensitive to both oxygen and light. Hence, both the resin and the solution were protected using Ar to remove oxygen. The Pd solution was added to the reaction vessel, which was sealed and left to react for approximately 2 hours in a dark environment. The reaction vessel was stirred using a vortex machine every 10-15 min.
After 2 hours the supernatant was removed and the resin was washed alternately with 0.5 % DEDT in DMF and 0.5 % DIPEA in DMF to get rid of excess Pd
5. After 4 washes of each solution, the resin was washed thrice with only DMF and thrice with DCM, followed by evaporation of the DCM using Ar. After evaporation, the reaction vessel was left in the desiccator.
For the lactamisation, PyBOP and HOBt were dissolved in DMF
9. The solution was added
to the resin in the reaction vessel, which was then filled with DMF to a volume roughly five
times the resin volume. DIPEA was added to the reaction vessel before placing it on a rocking
table. The reaction vessel was left on the rocking table for a minimum of 2 hour, up to overnight. The resin was then washed with DMF and DCM, and the DCM was evaporated.
The peptide was cleaved from the resin with simultaneous side-chain deprotection using a TFA-based cleaving cocktail. The cocktail contained TFA, H
2O, EDT, TIS and thioanisole
3,5. As both the thioanisole and EDT are malodorous, all material in contact with the cocktail was put into a quenching bath containing sodium carbonate and hydrogen peroxide
8.
The reaction vessel was filled with about 10 ml of the cleaving cocktail and left on a rocking table for 2 hours. Every 10-15 min the vessel was stirred with a vortex machine.
After 2 hours the cleavage solution was filtered into a Falcon tube. The resin was washed with neat TFA and the filtrates were combined. The volume was reduced using Ar gas flow, before cold ether was added to the solution to initiate precipitation. The tube was then centrifuged, and the ether decanted leaving only the precipitate. This was repeated twice before the precipitate was flushed with Ar for several minutes to evaporate as much solvent as possible. The crude peptide was then dissolved in 2-3 drops of 0.1 % TFA in 50 %
acetonitrile in water. Then 2 ml of deionised water was added once the precipitate was dissolved and the sample was freeze-dried
8.
3.2 Purification
The lyophilised sample was dissolved in 0.1 % TFA in 50 % acetonitrile, after which 2 ml of deionised water was added. A portion of the solution was injected into a reverse phase HPLC equipped with a C-8 column, using gradient elution with 0.1 % TFA in H
2O and 0.1 % TFA in acetonitrile as the mobile phases. The fraction showing a clear peak was collected and analysed using MALDI. Once the fraction containing the peptide was found, the rest of the sample was injected, and the fractions collected.
The fractions were combined in a round bottom flask, and the acetonitrile was
evaporated using a rotavapor, followed by lyophilisation. Once lyophilised, the peptide was
dissolved in 0.1 % TFA in 30 % acetonitrile and transferred to a clean falcon tube. The round
bottom flask was washed with H
2O, which was added to the falcon tubes. The falcon tubes
were then freeze-dried. After the second lyophilisation the peptide was weighed and stored in
freezer until further use.
3.3 Analysis 3.3.1 Identity
After each step in the synthesis process, a test-cleavage was done and analysed with MALDI- TOF-MS. A small amount of resin from each step prior to the cleaving step were cleaved in a small amount of the cleaving cocktail. 1 µl of the cleaving solution was then added to 30 µl 0.1 % TFA in 50 % acetonitrile and stirred using a vortex machine. 1 µl of the new solution or alternatively 1 µl of eluate from the HPLC was added to a MALDI plate. On top of that 1 µl of the matrix was placed. The matrix used in this case was a solution of a-cyano-4-
hydroxycinnamic acid
8. The sample was then analysed using the MALDI-TOF-Ms with ionisation in the positive mode. The matrix without any sample was also analysed to determine the peaks originating from the matrix.
3.3.2 Purity
The purity of a sample was checked using an analytical HPLC. Using a C-18 column and the same mobile phases as the purification step, but with a steeper gradient elution. The peptide was dissolved in 0.1 % TFA in 30 % acetonitrile, vortexed, and injected into the HPLC.
Whenever the chromatogram showed a peak, a small amount of that fraction was collected to be analysed with MALDI-TOF-MS.
4 Results and comparative discussion
After the peptidyl resins were removed from the synthesiser, a small amount of each resin was
taken out for a test cleavage, and was subsequently analysed using MALDI-TOF-MS. The
m/z of the highest non-matrix peaks were noted in table 2 and compared to the calculated m/z
for each peptide. The spectra for all samples at this stage showed a clear peak with a m/z ratio
less than 1 unit away from the calculated m/z. Other noticeable peaks were matrix peaks, or a
peak with a m/z +163 units higher than each peptide. As this peak is present in each peptide,
it is believed to be a side-product formed during the synthesis. As all the spectra showed a
clear peak for their respective peptide, the synthesis was seen as successful at this stage.
Table 2: The calculated m/z of each peptide, as well as the m/z of the highest peak observed in the spectra obtained using MALDI-TOF-MS. The data is based on the spectra found in the appendix.
Peptide Calculated m/z
5,8Highest non-matrix peak m/z
I 1429 1428
II 1429 1428
III 956 956
IV 1357 1357
V 1430 1429
VI 1368 1368
After the allyl/alloc deprotection a small amount of each sample was cleaved to observe if the deprotection had been completed. The samples were analysed using MALDI-TOF-MS and the m/z of the highest non-matrix peak was noted down in table 3, along with the calculated mass of each peptide at this stage. For all spectra, except for peptide III, the highest peak was found to be close to the calculated m/z. Thus, those peptides were deemed to have undergone allyl/alloc deprotection. For peptide III, the highest peak was 1689, with is more than double the calculated value of 832. Instead, in the spectrum, shown in figure 4, the second highest peak has a m/z ratio close to the calculated value. As the second highest peak has an intensity similar to the highest peak, and the MALDI-TOF-MS is not a quantitative analysis, the peptide was deemed to have gone through deprotection. The matrix peaks were more noticeable in the spectra, as was the side-product with a m/z +163 units higher than each peptide. The side-product appears to have undergone the same deprotection as the peptides.
Table 3: The m/z ratio obtained from the MALDI-TOF-MS spectra and calculated m/z for each peptide after the removal of the allyl and alloc protecting groups. In the case of peptide III, both the highest and second highest peaks were recorded in the table. This was done as both shared a similar level of intensity, but the highest peak showed a m/z ratio with a large difference to the expected value.
Peptide Calculated m/z
5,8Highest non-matrix peak m/z
I 1305 1304
II 1305 1304
III 832 1689 / 832
IV 1233 1233
V 1306 1305
VI 1244 1244
Figure 4: The MALDI-TOF-MS spectrum for the sample containing III, post allyl/alloc deprotection. The most noticeable peaks are in 1689 and 832. The second highest peak belongs to the peptide, whilst the highest peak is from an unknown substance in the sample. The calculated m/z of the peptide is 832.
After the cyclisation the samples were analysed with MALDI-TOF-MS to determine if the
correct masses had been obtained, the obtained and calculated m/z can be seen in table 4. For
all spectra, the highest non-matrix peak had a m/z ratio close to the calculated m/z. There was
still a presence of the side-product with a m/z ratio of +163 that of each peptide. There was
also a noticeable peak with a m/z ratio double that of the peptides. This could be caused by
two intermolecular lactam bridge formed instead of an intramolecular side-chain to side-chain
lactam bridge. The MALDI-TOF-MS does not give the amount of each peak, so no data on
how much of each peptide have been lost to this undesired reaction. The use of a large volume
during lactamisation should prevent this from occurring, albeit it appears that it has not been
completely successful in blocking it. Some spectra also showed a few peaks just above the
peptide peaks, this could be salt adduct or some uncyclized peptide.
Table 4: The calculated m/z of each peptide, as well as the m/z of the highest peak. The data is based on the spectra from the MALDI-TOF-MS analysis on all samples.
Peptide Calculated m/z
5,8Highest non-matrix peak m/z
I 1287 1286
II 1287 1286
III 814 814
IV 1215 1215
V 1288 1287
VI 1226 1226
Each sample was purified using reverse-phase HPLC. All chromatograms of the initial injections showed a few peaks. Of these peaks, the peaks with the highest area were found to contain the peptides after a MALDI-TOF-MS analysis. The obtained spectra from the peptide fraction showed no clear peak for the side-product with double m/z nor with a m/z +163 units higher than the peptides. This indicates that these side-product were removed during the purification process. Thus, the rest of the samples were injected. The fractions containing the peptides was collected.
After the purified peptides had been lyophilised their masses was measured and the yield calculated. The masses and yields for each peptide can be found in table 5. The yield ranged from 9 % to 23 %. This is a low yield, but for a person who has not performed peptide chemistry previously, it is an acceptable result. Due to the many steps from the SPPS to the final cleavage of the peptide from the resins, the overall yield is not expected to be high. A higher yield could be achieved from a more experienced peptide chemist or by tweaking the process.
Table 5: The masses obtained for each purified peptide and the yield for each peptide.
Peptide Mass [mg] Yield %
I 13 21
II 6 9
III 5 13
IV 10 17
V 15 23
VI 12 19
A purity analysis using an analytical HPLC was performed, but the results obtained were inconclusive. All chromatograms showed a phenomenon consisting of high peaks, broad peaks and a general increase in absorbance in the second half of the chromatograms. As no blanks were run during the analysis, no conclusion on the origin of the phenomenon can be drawn.
5 Conclusion
All in all, six peptides were designed and synthesised. Four of these were new peptides and the two others were the peptide upon which the new were based and a negative control.
The MALDI-MS show that the synthesis and cyclisation steps used, resulted in the correct peptides with some repeating side-products. The yield of the purified peptides was low, but acceptable.
The analysis of the purified samples via analytical HPLC was fruitless as the results show errors with the machine and not conclusive regarding the purity of each sample. To verify the purity the sample would have to be re-analysed via analytical HPLC. Otherwise a different method to analyse the purity would have to be used.
The objective of this study was to design and synthesise a few macrocyclic peptides as possible inhibitor for LSD1. The design was based on a peptide shown by a previous study to be an inhibitor for LSD1. No analysis to determine if they could be used as inhibitor for LSD1 has been conducted in this thesis, instead the focus lied on design and synthesis of the
peptides.
For use in future works, these peptides need to be re-analysed using an analytical HPLC, with blanks. If the peptides are confirmed to be of a high enough purity, they can be used to study their interaction with LSD1. This would then be done with techniques such as Surface plasmon resonance (SPR)
10.
6 Experimental
The peptides were synthesised on a Prelude peptide synthesiser (Protem Technologies). The
MALDI-TOF-MS was done on an Autoflex II (Bruker). The preparative column was a Grace
Vydac C8-column (size: 22 mm x 150 mm, particle size: 10 µm, pore size: 300 Å). The
mobile phases used was: A, 0.1 % TFA in H
2O; B, 0.1 % TFA in acetonitrile. Most solvents,
acids and bases were bought from Sigma-Aldrich or VWR. Standard amino acids were bought
from ChemPep. More special amino acids were bought from sources such as Novabiochem.
The resin used was a NovaPEG Rink Amide resin LL (0.17 mmol/g) bought from Novabiochem.
6.1 General procedure for the synthesis of all peptides
The synthesis was done on a 50 µmol scale, with 5 molar equivalents of amino acids and coupling reagent, and 10 molar equivalents of base. The amino acid concentration was 125 mM. The deprotection reagent used was 20 % piperidine in NMP and the base was 2 M DIPEA in NMP. The coupling reagent was 450 mM HCTU in DMF. The sequence was put into the software. The resin was left in NMP for 10 minutes prior to use to swell. The resin was drained of solvent and the machine was started.
The synthesis method consisted of an initial Fmoc deprotection step. After this step, the resin was washed with NMP and drained. An amino acid was added, followed by the coupling reagent and base. After occasional N
2bubbling the vessel was drained and the resin washed with NMP. The amino acid was added again with the coupling reagent and base. The liquid was drained, and the resin washed with NMP. This was then repeated starting with the Fmoc deprotection and using the next amino acid in the sequence for the sequent steps.
Once the program was done, the resin was transferred to a new reaction vessel and washed thrice with NMP and then thrice with DCM. The remaining DCM was evaporated using Ar flushing and the vessel was left in a desiccator overnight. A small amount of resin was used in a test cleavage. 1 µl of the cleavage solution was added to 30 µl 0.1 % TFA in 50
% acetonitrile. 1 µl of the new solution was added to a MALDI plate and then 1 µl of the matrix was added on top. The sample was then analysed with MALDI-TOF-MS.
3-5 equivalents of Pd(PPh
3)
4was dissolved in CHCl
3/HOAc/NMM in a ratio of 37:2:1. Both the solution and the resin were flushed with Ar to create an oxygen free environment. The solution was added to the reaction vessel and left sealed in a dark
environment for circa 2 hours, with 20 seconds of stirring using a vortex machine every 10-15 minutes.
Once the 2 hours had past, the solution was drained from the vessel. The resin was washed
alternately using 0.5 % DEDT in DMF and 0.5% DIPEA in DMF, starting with the DEDT
and ending with the DIPEA. After 4 washes of each solution the resin was washed thrice with
DMF and thrice with DCM. Any leftover DCM was evaporated using Ar and the vessel was
placed into a desiccator. A small amount of the resin was used in a test cleavage.
5 molar equivalents of PyBOP and HOBt were dissolved in DMF and added to the reaction vessel. The vessel was filled with DMF to a volume five times the volume of the resin. 10 molar equivalents of DIPEA was added to the vessel. The vessel was left on a rocking table for a minimum of 2 hours or overnight. After the time had passed, the vessel was drained and the resin washed with DMF, DCM. The leftover DCM was evaporated, and the vessel placed in a desiccator. A small amount of resin was used for test cleavage.
The peptide cleavage from the resin was done simultaneously as the side-chain deprotection. The cleaving cocktailed consisted of 94 % TFA, 2 % Thioanisole, 2 %
deionised water, 1 % TIS and 1 % EDT. A quenching bath was prepared to remove smell of thioanisole and EDT. About 10 ml of the cocktail was added to the vessel. The vessel was left on a rocking table for approximately 2 hours, with vortex stirring every 10-15 minutes.
The cleaving solution was then filtered into a clean 50 ml falcon tube. The resin was washed with TFA, after which the TFA was added to the falcon tube. The tube was flushed with Ar to reduce the volume until 3-4 ml remained. Cold ether was added drop-wise to the solution under constant stirring to start precipitation. Once precipitation started, ether was added till about 40 ml. The tube was centrifuged, and the ether decanted. About 20 ml of cold ether was added again, before the tube was centrifuged and the ether decanted. This was repeated once more. The precipitate was flushed with Ar for several minutes to evaporate as much solvent as possible. The precipitate was dissolved in a 2-3 drops of 0.1 % TFA in 50 % acetonitrile, with 2 ml of deionised water being added after. The solution was then freeze- dried.
In preparation for the HPLC, the sample was dissolved in 0.1 % TFA in 50 % acetonitrile with 2 ml of deionised water added once dissolved. With a flow of 15 ml per minute, the gradient elution started at 5 % B. The same percentage was kept for the first 5 minutes, after which the percentage of B was increased till 40 % at 20 minutes. An initial injection of 0.5 ml of the solution was done and the elute at each peak was collected and analysed using MALDI-TOF-MS. Once the fraction with the peptide was identified the rest of the dissolved peptide was injected and the fractions collected.
All fractions were combined, and the acetonitrile evaporated using a rotavapor. The peptides were then freeze-dried in the round bottom flasks used in the rotavapor. Once
lyophilised, the peptides were dissolved in 2 ml 0.1 % TFA in 30 % acetonitrile. The solution was transferred to a falcon tube. The round bottom flask was washed thrice with 2 ml
deionised H
2O, which was added to the falcon tube. The tubes were freeze-dried once more.
The falcon tubes were removed from the freeze-dryer and the peptides were weighed and stored in a freezer until further use.
For the analytical HPLC, the peptides were dissolved in 0.1 % TFA in 30 %
acetonitrile. The solution was vortexed and 10 µl was injected into the analytical HPLC. The starting conditions for the gradient elution was a composition of 5 % B at a flow rate of 1.5 ml per minute. The condition was kept for 5 minutes, afterwards the percentage of mobile phase B increased until it reached 60 % at 30 minutes. After 30 minutes the column was washed.
For each peak shown in the chromatogram a small amount of the elute was collected and analysed with MALDI.
7 Acknowledgements
I would like to thank Professor Jan Kihlberg for being my supervisor, allowing me to dip my toes into peptide chemistry. I would also like to thank Johan Viljanen for helping and teaching me how peptide chemistry works in the lab. I also send my thanks to Fabio and Duy for creating a welcoming atmosphere in the lab.
8 References
1
Vinogradov, A.A.; Yin, Y.; Suga, H. J. Am. Chem. Soc. 2019, 141, 10, 4167–4181.
2
Konovalov, S.; Garcia-Bassets, I. J. Ovarian. Res., 2013, 6, 75.
3
Yang J. Development of Peptide Binders: Applied to Human CRP, Carbonic Anhydrase (II, IX) and Lysine Demethylase 1 [Internet] [PhD dissertation]. [Uppsala]; 2017. (Digital
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology). Available from: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-330489
4
Yang, J.; Talibov, V.O.; Peintner, S.; Poongavanam, V.; Geitmann, M.; Rhee, C.; et al.
Macrocyclic peptides as inhibitors of human LSD1. ACS Omega, submitted.
5
Merck Millipore, Novabiochem®. Peptide synthesis. Anniversary edition 2014/2015.
6
Chan, W.; White P.. Fmoc solid phase peptide synthesis: a practical approach. Oxford University Press. 1999.
7
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