Bachelor Thesis Chemistry
Binding studies on 14-3-3ζ
by
Thomas Maas
10779388
30 June 2017
Research institute
Responsible teacher
AIMMS
Prof. Dr. Tom N. Grossmann
Research group
Supervisor
Gratitude
I would like to thank Prof. Dr. Tom N. Grossmann, who has made this thesis possible. I would like to thank you for offering a place in your lab and always be able to ask for help. I would like to thank the entire group, Carolin Müller, Kerstin Wallraven, Dr. Marta Pelay Gimeno, Mathias Wendt, Niall McLoughlin, for support when needed and for the great scientific and fun atmosphere. Special thanks go to Kerstin Wallraven who put great effort in supervising me and reading my manuscripts. I would like to thank Eelco Ruijter for correcting my final work.
Table of content
Summary 4
1.0 Introduction 6
2.0 Objectives 11
3.0 Results and discussions 14
3.1 Peptide synthesis 14
3.2 Determination of dissociation constants 15
3.3 Determination of inhibitory concentration 17
4.0 Conclusion and outlook 21
5.0 Experimental part 22
5.1 Chemicals, solutions and devices 22
5.2 Peptide synthesis 23
5.2.1 Solid phase peptide synthesis 23
5.2.2 Ring-closing metathesis and reduction 24
5.2.3 PEG and FITC coupling 25
5.2.4 Test cleavage 25
5.2.5 Final cleavage 25
5.2.6 Purification 25
5.2.7 Peptides 26
5.3 Analysis 29
5.3.1 Fmoc-monitoring 29
5.3.2 Biophysical assays 29
6.0 Abbreviations 31
7.0 References 32
8.0 Appendices 34
Appendix I: UV- and mass spectra of synthesized compounds 34
Summary
Protein-protein interactions (PPIs) are involved in many disease processes, but they are difficult to address due to their large interaction surfaces and absence of well defined binding pockets. A suitable method for inhibiting PPIs uses peptide-based interaction motifs as starting point for the design of inhibitors. However, a crucial limitation of unmodified peptides is their flexibility when free in solution which produces a whole spectrum of conformations while only one of them binds to the target protein. Therefore, modifications of peptides such as macrocyclization are necessary. According to this approach, the stabilized peptide βRS8 (H2N-QGR5LDS5LDLAS-CONH2) was developed in the research group
Grossmann to inhibit the exoenzyme S (ExoS) / 14-3-3ζ interaction. The macrocyclized βRS8
imitates the binding motif of ExoS to 14-3-3ζ which is known as ESp (ExoS peptide, H2
N-QGLLDALDLAS-CONH2). ExoS is a virulent factor from the opportunistic bacterium Pseudomonas aeruginosa, which is a common cause of hospital-acquired-infections, that
occur frequently and can be associated with high morbidity and mortality rates. However, 14-3-3ζ interacts with more than 500 protein interaction partners for executing its regulatory functions in cell cycle control, MAP kinase activation and apoptosis. Therefore, inhibition of the ExoS / 14-3-3ζ interaction may also cause inhibition of desired interactions. A solution can possibly be found in the different types of interactions. Most desired interactions with 14-3-3ζ depend on hydrophilic phosphorylated serine motifs, while ExoS binds through hydrophobic interactions. It is also known that peptides with hydrophilic and hydrophobic properties bind at slightly different location in the binding groove of 14-3-3ζ. For this purpose, shorter peptides based on βRS8 have been designed in the Grossmann group,
βRS8(ΔQ/AS)(Me/Me) (H2N-GR5MeLDS5MeLDL-CONH2) and βRS8(ΔQ/AS)(Et/Et) (H2
N-GR5EtLDS5EtLDL-CONH2), aiming to only inhibit the interaction between ExoS and 14-3-3ζ by
preventing an overlap with the binding site for phospho dependent binding partners. In this thesis, it has been investigated whether the peptides derived from ESp can bind to 14-3-3ζ simultaneously with a phosphorylated peptide. As phosphorylated peptide pS-Raf-259 (H2
N-LSQRQRSTpSTPNVHM-CONH2) was chosen which is derived from the protein cRaf. First, the
peptides ESp, βRS8, βRS8(ΔQ/AS)(Me/Me) and pS-Raf-259 have been synthesized by solid
present in stock, were labeled with the fluorophore FITC. The affinity (Kd) towards 14-3-3ζ was
determined by fluorescence polarization (FP) assays. From this, βRS8 (Kd = 12.3 μM) appeared
to be the best binder while the measured Kd values were higher than reported in the
literature. Also, an FP competition assay was performed to determine the half maximal inhibitory concentrations (IC50) of selected peptides. The FITC labeled βRS8(ΔQ/AS)(Et/Et) and
ESp were used as tracers while acetylated ESp and pS-Raf-259 served as competitors. It emerged that both the wild type peptide ESp and the shortened βRS8(ΔQ/AS)(Et/Et) could
not bind simultaneously with pS-Raf-259 as for both competition was visible. Thus, the phosphorylated peptide binds to the same site in the groove as βRS8(ΔQ/AS)(Et/Et) in such
1.0 Introduction
14-3-3 proteins are a family of well conserved molecules, produced in plants, vertebrates and higher eukaryotes. In the human genome seven genes encode for seven different isoforms of 14-3-3 proteins indicated with the Greek letters β, γ, ϵ, ζ, η, σ and τ. All of them occur as helical-rich cup shaped homo- or hetero dimers, giving the dimeric complex two binding grooves running in opposite direction of each other.1 The binding pocket of 14-3-3 has an
amphipathic nature, existing of a cluster of basic and polar residues (from α3 and α5) on one side and a cluster of hydrophobic residues (from α7 and α9) on the other (Figure 1).2 This
binding groove is conserved best within and across species.1
Figure 1. Crystal structure of a monomeric 14-3-3ζ in which the amino acids crucial for peptide binding are shown. Hydrophobic amino acids are coloured orange (F117,
P165, I166, G169, L172, V176, L216, I217, L220, L227, W228)3,4, acidic red (E180, D212)4, basic blue
(K48, R56, K120, R127)4 and hydrophilic pink (N42, Y127, N173, Y179, N224)4. (PDB: 4N7G)3
The family of 14-3-3 proteins performs crucial roles in the cell as regulators of intracellular signal pathways that control cell cycle checkpoints, MAP kinase activation, apoptosis and gene expression.5 14-3-3 executes these functions by interacting with more
than 500 protein interaction partners in eukaryotic cells.6 The interaction with many different
proteins is mediated by a specific phosphoserine/-threonine binding site.7 To bind 14-3-3
two phosphopeptide motifs have been found: RSXpSXP and RXXXpSXP, in which pS stands for a phosphorylated serine and can be replaced by pT (phosphorylated threonine) and X can be any amino acid.8,9
In general, the tasks of 14-3-3 proteins can be divided into two main categories, first is the cell cycle progression and apoptosis and second is intracellular protein trafficking.5
These roles are executed via three different mechanisms. Firstly 14-3-3 can introduce conformational changes of the interaction partner upon binding. Here, 14-3-3 acts as a stable support on which target proteins can be reshaped.1 The 14-3-3 dimer is a highly rigid
structure leading to conformational changes of the target protein only.
Figure 2. Structural effects of dimer 14-3-3 binding. 14-3-3 binding can cause three effects: (A) A conformational change in the target protein; (B) masking of a specific region on the target or (C) colocalization of two proteins. The phosphopeptide motif illustrated as P.
Secondly 14-3-3 can physically block sequence-specific or structural features.1 As an
example Cdc25 cell cycle dual-specificity phosphatases contain a nuclear export sequence (NES) and a nuclear localization sequence (NLS). However, the NLS of Cdc2 is located very close to a 14-3-3-bindig site. Binding to 14-3-3 now increases the export rate of Cdc25 to the nucleus to be higher than the import rate, eventually causing mitotic activation of the cell.10
As a third role 14-3-3 proteins act as phosphorylation-dependent support to anchor proteins1
as for the interaction between 14-3-3 and Raf kinase proteins. One of the Raf kinases, cRaf, consists of two domains: the amino-terminal regulatory domain and the carboxy-terminal catalytic domain.11 The catalytic domain acts a kinase while the regulatory domain supresses
the activity of the catalytic domain. Several growth factors activate cRaf leading to triple phosphorylation of the protein itself and thereby making it capable of binding to 14-3-3 via one of the three binding sites at Ser233, Ser259 and Ser621.12 Engagement of the
phosphorylated bRaf and cRaf by 14-3-3 activates the catalytic domains of these two. Hereafter, the Raf kinases initiate the MAP Kinase Kinase Kinase proteins (MEKK) and extracellular signal-regulated kinase (ERK) cascade, which promotes proliferation and survival signaling.7,13
Although a majority of the binding partners of 14-3-3 need to be phosphorylated, some interactions are known to be based on hydrophobic interactions. One is the protein-protein interaction (PPI) between 14-3-3ζ and exoenzyme S (ExoS).14 ExoS is a virulent factor
from the opportunistic bacterium Pseudomonas aeruginosa, which is a common cause of hospital-acquired-infections, that occur frequently and can be associated with high morbidity and mortality rates when compared to other pathogenes.15 During an infection, the excreted
ExoS interacts with 14-3-3ζ of the host cell to activate its ADP-ribosyle transerfase domain.16
Eventually the DNA synthesis is decreased and causes apoptosis of the eukaryotic host cells.17
Therefore, inhibition of this PPI is a promising therapeutic target to deal with P. aeruginosa based infections.14
However, designing inhibitors for PPIs still remains a problem in medical chemistry. Small molecules that are typically used as drugs cannot bind properly to the target protein because of large interaction surfaces and the absence of clear binding pockets. A way to overcome this problem is by mimicking binding epitopes of PPIs with peptides.18 However,
in solution turns out to be a crucial drawback. Peptides exist in an ensemble of different conformational states when free in solution and only adopt their bioactive secondary structure upon target engagement. This results in an entropic penalty upon binding and a decrease in affinity for short peptides relative to their natural precursor in which the peptide is part of an entire protein with a given secondary structure. This challenge can be faced by forcing the peptide into its bioactive conformation using side chain to side chain cross links. Multiple techniques have been shown possible as disulphide bridges, amid bond bridges between Lys and Asp/Glu and hydrocarbon peptide.3,18 The last approach can be performed
on solid support, where natural amino acids are replaced by unnatural amino acids bearing an olefin side chain with a terminal double bond and a methyl group at the α-carbon (Figure 3). The length of these sidechains and the crosslink respectively can be varied as well as the location of the unnatural amino acid in the peptide. The unnatural amino acids can be linked by ring-closing metathesis (RCM) using Grubbs catalyst 1st generation. Usually this method
stabilizes α-helical structures and unnatural amino acids are therefore incorporated at positions i, i+3, or i i+4 for one turn of a helix and i i+7 for two turns.18
For the PPI between 14-3-3ζ and ExoS this approach has been applied based on an eleven amino acid long sequence (H2N-420QGLLDALDLAS430-COOH, ESp) resulting in
multiple macrocyclic PPI inhibitors.3 This sequence originating from the protein ExoS in which
it contributes to binding to 14-3-3. In the end, the most successful modulators were βRS8 and
βSS12. In both peptides Leu423 and Ala426 are replaced by α-methyl, α-alkenyl amino acids.
These amino acids differ in the absolute configuration of the α-carbon, indicated by the R and S supplement in the nomenclature of these peptides and the length of the crosslink is stated as number. βRS8 and βSS12 showed dissociation constants (Kd) of 0.25 μM and 41 nM
respectively, whereas the Kd for ESp is reported as 1.14 μM measured using fluorescence
polarization (FP).3
Considering all of the above, one crucial problem arises. In the case of inhibiting the PPI between 14-3-3ζ and ExoS, favourable interactions with 14-3-3ζ may also be prevented. As mentioned earlier, 14-3-3 carries out both hydrophobic and hydrophilic interactions with different proteins. Since the two types of interaction are situated on slightly opposite sides of the amphipathic groove it may be possible to inhibit the hydrophobic interaction-based PPI between 14-3-3ζ and ExoS while necessary phosphorylation-dependent PPI with 14-3-3ζ can still take place. However, it is required to limit the size of an 14-3-3/ExoS inhibitor to a relatively small structure also allowing simultaneous binding of phosphorylation-dependent binders. .
2.0 Objectives
The objective of this thesis was to perform initial experiments approaching the question whether phosphorylation-dependent interactions of 14-3-3ζ are blocked by ESp derived inhibitors. This is done by synthesizing peptides derived from ExoS and cRaf using SPPS and determining their affinity towards 14-3-3ζ in a fluorescence polarization (FP) assay. Also IC50
values of competing peptides were determined using a FP competition assay.
To state a hypothesis on the outcome of the competition assay between ESp, ESp derivatives and pS-Raf-259 two crystal structures, PDB: 4N7G (ESp bound to 14-3-3ζ) and PDB: 3NKX (a phosphorylated peptide cRafpSer259 [H
2N-255QRSTpSTPNVH264-COOH] bound
to 14-3-3ζ), are superimposed using Pymol (Figure 4).
Figure 4. Superimposed crystal structures of the peptides ESp (blue) and cRafpSer259 (orange) bound to the amphipathic groove of 14-3-3ζ. The
The amphipathic nature of the binding groove becomes clear as the two peptides bind on a slightly different location in the pocket, but they also show a partial overlap making it unlikely that ESp or βRS8 and pS-Raf-259 can bind 14-3-3ζ simultaneously. Based on these
assumption, also two truncated versions of βRS8 were tested as inhibitor for the PPI of
14-3-3ζ and ExoS, reducing the area of overlap with the phosphorylated peptide: βRS8(∆Q/AS)(Me/Me) (H2N-421GR5MeLDS5MeLDL428-COOH) and βRS8(∆Q/AS)(Et/Et) (H2N-420
GR5EtLDS5EtLDL430-COOH). In both peptides three amino acids have been removed compared
to βRS8, Gln from the N-terminus as well as Ala and Ser at the C-terminus.
hese shorter versions are based on not published work of Kerstin Wallraven. From an alanine scan it was concluded that Ser and Ala at the C-terminus did not show high contributions to binding, so these were cut of first. Based on this, peptides have been tested without Gln and without Gln and Gly at the N-terminus. When Gly was cut of a significant decrease in affinity was observed, but with Gln this was not. Furthermore, it could be estimated from a crystal structure of βRS8(∆Q/AS)(Me/Me) bound to 14-3-3ζ that space
remained unoccupied between 14-3-3ζ and the methyl groups on the unnatural amino acids. In order to increase hydrophobic interactions and hence affinity, it was proposed to use ethyl instead of methyl groups. This proved to be a 5.9 fold increase in Kd for βRS8(∆Q/AS)(Et/Et)
(Kd = 0.14 μM) compared to βRS8(∆Q/AS)(Me/Me) (Kd = 0.82 µM).
Table 1. Nomenclature and the sequences of the peptides.
Nomenclature Sequence N-terminal modification pS-Raf-259 H2N-LSQRQRSTpSTPNVHM-COOH Ac
ESp H2N-QGLLDALDLAS-COOH FITC-PEG, Ac
βRS8 H2N-QGR5MeLDS5MeLDLAS-COOH FITC-PEG, Ac
βRS8(∆Q/AS)(Me/Me) H2N-GR5MeLDS5MeLDL-COOH FITC-PEG, Ac
βRS8(∆Q/AS)(Et/Et) H2N-GR5EtLDS5EtLDL-COOH FITC-PEG, Ac
The binding motif of cRaf including phosphor-serine at position 259 was chosen as the competing peptide representing phosphorylation-dependent binder. In the literature this peptide is known as pS-Raf-259 (H2N-251LSQRQRSTpSTPNVHM265-COOH).2 pS-Raf-259 and
the ESp derived peptides were synthesized by solid-phase peptide synthesis (SPPS), except for the already provided f-βRS8(∆Q/AS)(Et/Et) and Ac-βRS8(∆Q/AS)(Et/Et). Peptides that were
modifications. The constrained peptides are formed by RCM using Grubbs catalysts 1st generation with subsequential reduction of the double bond with TPSH in order to avoid formation of E/Z diastereoisomer mixtures. Hereafter, the Kd values of the FITC labelled
peptides have been determined by use of FP assays and a FP competition assay was performed in order to determine the half maximum inhibitory concentrations (IC50) using
3.0 Results and discussions
3.1 Peptide synthesis
All peptides in Table 1 have been synthesized successfully according to HPLC-MS analysis (see paragraph 5.2.7 and Appendix I). For FITC labelled peptides the final yields were determined by measuring the absorbance at a wavelength of 495 nm which is the absorbance peak of FITC at pH 8.5. Using a molar extinction coefficient of 77000 M-1cm-1 and a path
length of d = 1 cm the concentration of the FITC labelled peptides could be calculated using Lambert Beer’s law (Equation (1)).
𝐴 = 𝜀 ∙ 𝑐 ∙ 𝑑 (1)
For acetylated peptides the concentrations were determined using HPLC analysis at 210 nm and a reference peptide of known concentration, by comparing the integral of the acetylated peptides peak to the integral of the internal standards peak. The yields (Table 2) are calculated based on 25 μmol as 100 % since this was the amount of loading estimated to have on the resin.
Table 2. Synthesized peptides and their yields.
Peptide yield [%] Ac-pS-Raf-259 2.1 f-ESp 36 Ac-ESp 17 f-βRS8 0.23 Ac-βRS8 1.1 f-βRS8(∆Q/AS)(Me/Me) 0.076 Ac-βRS8(∆Q/AS)(Me/Me) 0.42
During the final cleavage of pS-Raf-259 from the resin Met265 was oxidized. This problem could be overcome by using EDT instead of ODT in the cleavage solution and performing work up and purification directly after the final cleavage in order to retain methionine in its reduced state (see Scheme 1). Using these conditions, the Ac-pS-Raf-259 was synthesized.
Scheme 1. Oxidation of methionine.
At first, it was planned to synthesize a FITC labelled pS-Raf-259, but in the end coupling of the fluorophore was not successful. From a test cleavage after PEG coupling it was seen that this step worked well (Appendix III), after which the synthesis was continued with coupling FITC. This is a different kind of coupling than for amino acids or PEG spacer since no coupling reagents are used. The fluorophore itself is activated by a base, forming a thiourea unit. But despite the difference of coupling all other peptides have been labelled successfully, thus maybe the pS-Raf-259 sequence itself caused the inconveniency.
3.2 Determination of dissociation constants
The dissociation constants (Kd) of the peptides were determined based on a fluorescence
polarization (FP) assay. In FP assays a fluorescence labelled molecule is excited using linear polarized light. Afterwards the degree of polarization of the emitted light can be measured. The duration between excitation and emission of light is referred to as the fluorescence lifetime. The principle of FP arises from the fact that the degree of polarization of a fluorophore is inversely related to its molecular rotation within this fluorescence lifetime. For a low molecular mass, the rotational speed of the molecule is high, resulting in a high depolarization of the emitted light. Hence, when a fluorescence labelled molecule is bound to an interaction partner, the total mass and thus the polarization of the emitted light are increased.19–21 Consequently, a labelled peptide binding to its target protein will emit higher
polarized light than a peptide free in solution. From a FP assay the Kd values of a labelled
peptide in complex with its target protein can be determined since it corresponds to the inflectionpoint of the curve that arises from titrating a peptide solution with constant concentration to a dilution row of protein solution.
dissociation constants can vary between different FP measurements. The relation to the references and between the references, on the other hand, is supposed to remain constant. The FP data are presented in Figure 5 and Figure 6.
Figure 5. Graphical plot of the fluorescence polarization of f-ESp f-βRS8, f-βRS8(∆Q/AS)(Me/Me) and
f-βRS8(∆Q/AS)(Et/Et) after 1 h incubation against the logarithm of the 14-3-3ζ concentration for the determination
of dissociation constants.
From the first FP assay it can be concluded that the best binder is f-βRS8 (Kd = 12.3
μM), having the lowest Kd of the four peptides. It is 1.3 fold better binder than f-ESp (Kd =
16.3 μM), 1.77 fold better than f-βRS8(∆Q/AS)(Me/Me) (Kd = 21.8 μM) and 1.6 fold better than
f-βRS8(∆Q/AS)(Et/Et) (Kd = 19.8 μM). In the literature a 4.6 fold increase of affinity is mentioned
of f-βRS8 relative to f-ESp, which is a bigger improvement than is found here.3 The dissimilarity
may be due to a difference in purity of the peptide stocks or because the measurement was performed as single instead of triple for more robust data. Furthermore, both truncated peptides show a slightly lower affinity towards 14-3-3ζ than the wild type; f-βRS8(∆Q/AS)(Me/Me) is a 1.3 fold worse binder than f-ESp and f-βRS8(∆Q/AS)(Et/Et) 1.2 fold.
Because these peptides have fewer contact points with 14-3-3ζ than ESp and βRS8 this is as
expected. Nonetheless, the difference is so small that it could also be within the error of the single FP measurement. In Kerstin Wallraven’s research a 1.5 fold decrease of affinity was found for f- βRS8(∆Q/AS)(Me/Me) (Kd = 0.82 μM) relative to f-ESp (Kd = 0.55 μM), which is
consistent with what is seen in this FP assay. Nonetheless, f-βRS8(∆Q/AS)(Et/Et) (Kd = 0.14 μM)
although the difference is relatively small. Nevertheless, it could be that the ethyl groups do contribute to hydrophobic interactions with 14-3-3ζ and thereby increase the target affinity.
Figure 6. Graphical plot of the fluorescence polarization of ESp and f-βRS8(∆Q/AS)(Et/Et) after 1 h incubation against the logarithm of the 14-3-3ζ
concentration for the determination of dissociation constants.
The second FP assay that was performed shows different Kd values, which are in
general lower than the Kd values of the first measurement. f-ESp (Kd = 22.4 nM) is 727 fold
better than in the first measurement. This difference is most likely due to an error in some pipetting steps in one of the two measurements. In the second measurement f-βRS8(∆Q/AS)(Et/Et) (Kd = 5.56 nM) shows a 4 fold increase of affinity relative to f-ESp (Kd =
22.4 nM). This is different from the first FP assay but consistent with Kerstin Wallraven’s study in which a 3.9 fold increase was observed. Nonetheless, the absolute Kd values differ from
the literature, but again this measurement was only performed as single measurement; a triple measurement would be more robust.
3.3 Determination of inhibitory concentration
A FP competition assay was performed to investigate if phosphorylation-dependent interactions with 14-3-3ζ are blocked by ESp derived inhibitors. FP competition assays rely on the same physics as the FP assay, but takes another approach. It works by measuring the decrease of fluorescence polarization caused by a non-labeled ligand that replaces a
the protein and the tracer solution need to be incubated in order to form a protein/peptide complex. The concentration of the protein should be chosen at ~ 50% to 80% of the increase in FP between the free and the fully bound state of the ligand, based on binding curves from the previous measured FP assay. Hereafter, the competition can be executed with different, non-labelled competitors by titrating these to the protein/tracer complex. 20 The inflection
point of the resulting curve indicates the IC50.
Although it was planned to use pS-Raf-259 as tracer FITC-pS-Raf-259 could not be synthesized. It would have been scientifically valuable to use the phosphoserine peptide as tracer and the ESp derivatives as competitors to make all competitions comparable. However, coupling of FITC to pS-Raf-259 did not work out as stated earlier. Therefore, it has been chosen to use two tracers, f-ESp and f-βRS8(∆Q/AS)(Et/Et), while Ac-ESp and Ac-pS-Raf-259
were applied as competitors. f-βRS8(∆Q/AS)(Et/Et) was chosen instead of
f-βRS8(∆Q/AS)(Me/Me) or f-βRS8 because of its relatively good affinity towards 14-3-3ζ and a
because it is a shorter sequence compared to ESp and βRS8, which makes it more like to bind
14-3-3ζ simultaneously with pS-Raf-259. While the competitor Ac-ESp is a suitable reference to compare the inhibitory capability of the ESp derivatives to its unmodified precursor. The resulting curves are shown in Figure 7 and the IC50 values in Table 3.
Table 3. IC50 values determined in a FP competition assay using ESp and
f-βRS8 (∆Q/AS)(Et/Et) as tracers and Ac-ESp and Ac-pS-Raf-259 as competitors.
Tracer - competitor IC50
ESp – ESp 0.652 μM ESp – pS-Raf-259 2.34 μM βRS8(∆Q/AS)(Et/Et) – ESp 7.63 μM
Figure 7. Competitive displacement of f- ESp and f-βRS8(∆Q/AS)(Et/Et) by Ac-ESp and Ac-pS-Raf-259.
Fluorescence polarization is plotted as a function of unlabelled competitor peptide concentration using a logarithmic scale. The concentration of the tracers is 10 nM and the concentration of 14-3-3ζ is 2 µM.
For the competition it was expected that Ac-pS-Raf-259 (f-pS-Raf-259, Kd = 412 nM)22
would replace both f-ESp (Kd = 1.14 μM)3 and f-βRS8(∆Q/AS)(Et/Et) (Kd = 19.8 μM) better than
Ac-ESp, due to the lower affinity of f-ESp to 14-3-3ζ and based on the overlay of crystal structures showing that the phosphorylated and the non-phosphorylate peptide partially address the same binding site. The affinity of Ac-pS-Raf-259 that is used for this hypothesis was obtained in a FP assay using a the isoform 14-3-3γ instead of ζ.22
For the competition with f-ESp as tracer this is not the case. The ESp – ESp tracer – competitor combination shows an IC50 of 0.652 μM, which is lower than the 2.34 μM for the
ESp – pS-Raf-259 combination. This IC50 value is lower than should be possible to measure,
since the protein concentration in the assay was 2 μM, resulting in a lowest possible IC50 of 1
μM. Yet, if the found value is within the error of this measurement there is competition between Ac-pS-Raf-259 and f-ESp for binding, but this is not as strong as the competition between Ac-ESp and f-ESp. The reason for this could be that f-ESp and Ac-Esp share the same entire binding sequences to 14-3-3ζ and therefore the same binding place on 14-3-3ζ, whilst pS-Raf-259 does only partly bind the same spot in the amphipathic groove.
In the competitions using f-βRS8(∆Q/AS)(Et/Et) as tracer the opposite is observed.
When Ac-ESp is used as competitor the IC50 is higher than when using Ac-pS-Raf-259 (see
Table 3). This would suggest that pS-Ra259 is a better competitor towards f-βRS8(∆Q/AS)(Et/Et) than Ac-ESp.
Also, the competition assay insinuates that Ac-pS-Ra259 outcompetes f-βRS8(∆Q/AS)(Et/Et) (IC50 = 1.50 μM) more easily than f-ESp (IC50 = 2.34 μM). This is contrary
to the expectations since βRS8(∆Q/AS)(Et/Et) leaves more space within the binding groove for
pS-Raf-259 to bind than ESp, but conform with the lower Kd of f-βRS8(∆Q/AS)(Et/Et).
Nonetheless, conclusions should be drawn carefully because no plateau is reached at the higher concentrations of Ac-ESp in the tracer – competitor combination βRS8(∆Q/AS)(Et/Et) – ESp. Therefore, it is not possible to calculate a precise fit of the IC50
curve and thus the position of the inflection point can’t be determined that accurate. Reason for this could be shortage of measure points at the higher concentrations of competitor. Additionally, the FP competition assay was only performed as a single measurement.
4.0 Conclusion and outlook
The goal of this thesis was to take a first step in giving the answer to whether phosphorylation-dependent interactions with 14-3-3ζ are blocked by ESp derived PPI inhibitors. This was done by synthesising ESp, βRS8, βRS8(∆Q/AS)(Me/Me) and pS-Raf-259 by SPPS and measuring a FP
assay and a FP competition assay using the FITC labelled peptides. The found Kd values were
higher than those found in literature. It has been reported that the interaction between 14-3-3ζ and f-ESp reveals a Kd of 1.14 μM and βRS8 showed an 0.25 μM affinity.3 These numbers
differ 14 fold for ESp and 49 fold for βRS8. Furthermore, the Kd values differ between the two
measurements. According to the first measurement ESp is a better binder than βRS8(∆Q/AS)(Et/Et), the second FP assay gives an inverse result. These unexpected behaviors
might be cause of the single measurements that were performed, so triple measurements are recommended to be done in further research. Furthermore, the FP competition assay showed that ESp as well as βRS8(∆Q/AS)(Et/Et) were outcompeted by pS-Raf-259, showing the overlap
in binding between the phosphorylation-dependent and phosphorylation-independent interactions. Concluding, ESp and βRS8(∆Q/AS)(Et/Et), cannot bind simultaneously with
pS-Raf-259 to 14-3-3. For ESp this result is conform with the expectations, but for the truncated peptide the competition was stronger than expected. For a more detailed answer to the question whether phosphorylation-dependent interactions of 14-3-3ζ are blocked by ESp derived inhibitors it is suggested to use a fluorescence labelled pS-Raf-259 as tracer in a competition assay and to do triple measurements. Furthermore, overlap of ESp derivatives and phosphorylation-dependent interaction partners of 14-3-3ζ might be impossible to avoid. So although further truncation seems tempting, it also is likely to lose affinity doing so, resulting in being outcompeted by phosphorylation-dependent binders even easier. Therefore, further truncation of the ESp derived peptides remain a challenge but might be a solution for inhibiting the ExoS/14-3-3ζ PPI.
5.0 Experimental part
5.1 Chemicals, solutions and devices
Table 4. Solutions used in solid phase peptide synthesis
Supplier Chemicals
Acros, Geel, Belgium Diethyl ether
Iris Biotech, Marktredwitz, Germany Amino acids, PEG (Fmoc-O2Oc-OH),
HMBA-resin, NMP, Fmoc-Ser(HPO3Bzl)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH*H2O, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pdf)-OH
Okeanos, Beijing, China (S)-N-Fmoc-2-(4'-pentenyl)alanine,
(R)-N-Fmoc-2-(4'-pentenyl)alanine
Pan Biotech GmbH, Aidenbach, Germany DMSO
Roth, Karlsruhe, Germany COMU, DIPEA, Na3PO4, Piperidine, PyBOP, TFA
Serva, Heidelberg, Germany Tween-20
ACN, DCM, HEPES, NaCl
Sigma-Aldrich, Steinheim, Germany DCE, EDT, FITC, Grubbs 1st Generation-
Katalysator, ODT, TPSH, TIS (Triisopropylsilan),
Table 5. Solutions used in solid phase peptide synthesis
Solution Composition
Deprotecting solution 25% piperidine in NMP
Capping solution 1 part piperidine, 1 part Ac2O and 8 parts of
NMP
Cleavage solution 1% TIPS, 2.5% H2O and 2.5% ODT in TFA
Table 6. Buffer solutions
Solution Composition FP Buffer HEPES NaCl Tween-20 10 mM 150 mM 0.1% pH 7.4 FITC-Buffer Na3PO4 100 mM pH 8.5
Table 7. Devices used in research
Device Type
Centrifuge Hettich universal 320 R
Centrifuge Nippon Genetics Europe model: N6002B HPLC semipreparative Shimadzu SCL-10A (controller)
HPLC-MS Agilent Technologies 1290 Infinity (column: 125/10 Nucleodur C18 gravity (5 µm, 110 Å))
Lyophilizer Labconco Freezon 4.5 Microplate reader Tecan infinite® M1000 PRO
Nanodrop Thermo scientific isogen lifescience Nanodrop one
Orbital shaker GFL 3005 Thermomixer HTA Biotec
Ultrasonic bath VWR ultrasonic cleaner Vortex Star lab vortex
5.2 Peptide synthesis
5.2.1 Solid phase peptide synthesis
Peptides were synthesised manually on Fmoc Rink amide MBHA resin by standard Fmoc-based solid-phase peptide synthesis (SPPS) using the following protocol. After the resin was weighed into a syringe reactor it was swollen for 1-2 h in NMP (1 mL / 100 mg). The amino acids used as well as the coupling reagents were dissolved in NMP and the solvent volume was chosen to obtain a final concentration of 0.2 M in relation to the resin loading. All reactions were carried out at RT in closed syringe reactors, which were placed on a shaking plate. All washing and reaction solutions were discarded, unless otherwise stated. The washing steps comprise rinsing the resin three times with 2 mL of NMP, three times with 2 mL of DCM and three times with 2 mL of NMP.
1. Deprotection of the resin:The resin was treated with deprotecting solution (1 mL / 100mg) for 15 min.
2. Coupling for natural amino acids and PEG: Every amino acid was coupled twice using different coupling reagents. For the first coupling 4 equivalents of the amino acids were mixed with 4 equivalents of PyBOP and 8 equivalents of NMM and added to the resin for 45 min. Hereafter, the reaction mixture was washed. For the second coupling, also called re-coupling, 4 equivalents of amino acids were mixed with 4 equivalents of COMU, 4 equivalents of Oxyma and 8 equivalents of DIPEA and added to the resin for 20 min.
Coupling of unnatural amino acids: For the unnatural olefin-containing amino acids, (S)-N-Fmoc-2-(4'-pentenyl)alanine and (R)-N-Fmoc-2-(4'-pentenyl)alanine, coupling
equivalents of NMM and adding the mixture to the resin for 4 h. For the phosphorylated serine in pS-Raf-259 three couplings were performed, once PyBOP/NMM and twice COMU/Oxyma/DIPEA, using the same conditions as for natural amino acids.
Coupling of natural amino acids onto an unnatural amino acid: 6 equivalents of amino acid was mixed together with 6 equivalents of COMU, 6 equivalents of Oxyma and 12 equivalents of DIPEA and added to the resin for 20 min. Hereafter, the reaction mixture was washed and the another coupling under the same conditions was performed. The reaction mixture was washed again and the third coupling was done using 6 equivalents of amino acid, 6 equivalents of PyBOP and 12 equivalents of NMM for 45 min
3. Capping:The resin was treated with 2 mL capping solution once for 5 min.
4. Fmoc-removal: To deprotect the amino acid, the resin was treated with 2 ml deprotecting solution and shaken for 5 min twice. The first reaction solution was collected and used in Fmoc-monitoring.
After step 1,2 3, and 4, the resin was washed. Step 2 until 4 were repeated for every further amino acid or PEG coupling. When the peptide was finished the resin was washed three times with DCM and dried on vacuum.
5.2.2 Ring-closing metathesis and reduction
Crosslinking the unnatural olefin amino acids for βRS8 and βRS8(∆Q/AS)(Me/Me) was done
before PEG and FITC coupling by ring closing metathesis (RCM), using 4 mg/mL of Grubbs catalysts 1st generation in dry DCE. The reaction was performed four times for 1-2 h each
under a constant nitrogen stream. Between different RCM reactions the resin was washed with DCM three times. After all RCM reactions, the resin was washed with a 1:1 mixture of DMSO/DCM. The double bond was reduced using a solution of TPSH (0.6 M) and piperidine (1.2 M) in NMP for four times 100 min at 60 °C and 600 rpm orbital shaking. Afterwards the resin was washed three times with NMP, three times with DCM and again three times with NMP.
5.2.3 PEG and FITC coupling
As PEG linker 8-(9-Fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid was coupled to the N-terminus of the peptides. Thereafter, the Fmoc protection group was removed and the fluorophore FITC was attached. FITC coupling was done by adding a solution of 4 equivalents FITC and 8 equivalent DIPEA in NMP to the resin twice for 1 h each.
5.2.4 Test cleavage
During peptide synthesis the success of certain reactions could be verified by test cleavages. For this, a few resin beads were removed from the reaction syringe and transferred to another syringe reactor. The beads were then exposed to 200 μL of cleavage solution (Table 5) under constant shaking for 1 h. The cleaved peptide was then precipitated with cold diethyl ether (-20 °C), centrifuged (4 °C, 10 min, 13200 rpm) and the supernatant was removed. The remaining pellet was dissolved in 50-100 μL of a 30% ACN solution in water for a subsequent HPLC-MS analysis
5.2.5 Final cleavage
After completed synthesis the cleavage of the peptides without methionine was performed with 500 μL of a cleavage solution per 10 μmole of resin loading. The resin was treated with the cleavage mixture twice for 1 h and then washed twice with TFA (200 μL / 10 μmole resin). Both the cleavage and washing solutions were collected. For peptides containing methionine EDT was used instead of ODT in the cleavage solution to prevent oxidation of methionine. From the collected solution the TFA was evaporated until a volume of 200 μL remained. Then the peptide was precipitated with cold diethyl ether (-20 °C) and centrifuged (4°C, 10 min 4000 rpm). The supernatant was removed and the crude product was dried under a nitrogen stream before dissolving in a 30% ACN solution in water.
5.2.6 Purification
Purification of the peptides was carried out by preparative HPLC using a SP 125/10 Nucleodur C18 gravity (5 µm, 110 Å) column. Depending on the gradient the mobile phase consisted of the two components A (MilliQ + 0.1%TFA) and B (ACN + 0.1% TFA). After purification, the
purity of the collected fractions was tested with HPLC-MS analysis. Fractions containing the pure peptide were combined and the solvent was removed by lyophilization.
5.2.7 Peptides
The following peptides were synthesized in a 250 μmole batch as previously described, before labeling. The batch sizes of the subsequent syntheses were calculated based on the last Fmoc monitoring of the LDL sequence.
5.2.7.1 Ac-pS-Raf-259
The peptide Ac-pS-Raf-259 was synthesized in a batch of 25 μmole. A solvent gradient of 5 to 50% B in 40 min was used for purification on a preparative HPLC.
Yield: 0.54 μmole, 2.1% HPLC-MS: Calculated: 1862.9 u [M+H]+ 932.5 u [M+2H]2+ 620.6 u [M+3H]3+ Gradient: 5% to 95% of B in 10 min, tr = 7.7 min Found (m/z): 932.2 621.8 5.2.7.2 f-ESp
The peptide f-ESp was synthesized in a batch of 25 μmole. The FITC fluorophore coupled once for 1 h and a second time overnight as a departure from the protocol. A solvent gradient of 30 to 80% B in 40 min was used for purification on a preparative HPLC.
Yield 9.03 μmole, 36%
HPLC-MS: Calculated: 1649.8 [M+H]+
825.4 [M+2H]2+
825.0
5.2.7.3 Ac-ESp
The peptide Ac-ESp was synthesized in a batch of 25 μmole. A solvent gradient of 30 to 80% B in 40 min was used for purification on a preparative HPLC.
Yield 4.12 μmole, 17% HPLC-MS: Calculated: 1157.3 [M+H]+ 579.2 [M+2H]2+ Gradient: 30% to 95% of B in 10 min, tr = 7.6 min Found (m/z): 1156.6 579.0 5.2.7.4 f-βRS8
The peptide f-βRS8 was synthesized in a batch of 25 μmole. Reduction of the double bond
after RCM was performed four times, chronologically having a duration of 2 h, 1.5 h, 1,5 h and 1 h. The coupling of the FITC fluorophore took place 1 h and a second time overnight as a departure from the protocol. A solvent gradient of 30 to 70% B in 40 min was used for purification on a preparative HPLC. Yield 0.06 μmole, 0.2% HPLC-MS: Calculated: 1717.9 [M+H]+ 859.5 [M+2H]2+ Gradient: 30% to 95% of B in 10 min, tr = 9.5 min Found (m/z): 1717.8 859.0 5.2.7.5 Ac-βRS8
The peptide Ac-βRS8 was synthesized in a batch of 25 μmole. Reduction of the double bond
after RCM was performed four times, chronologically having a duration of 2 h, 1.5 h, 1,5 h and 1 h. The coupling of the FITC fluorophore coupled once for 1 h and a second time overnight as a departure from the protocol. A solvent gradient of 30 to 70% B in 40 min was used for purification on a preparative HPLC.
Yield 0.28 μmole, 1.1% HPLC-MS: Calculated: 1225.4 [M+H]+ 613.2 [M+2H]2+ Gradient: 30% to 95% of B in 10 min, tr = 9.1 min Found (m/z): 1224.9 612.9 5.2.7.6 f-βRS8(∆Q/AS)(Me/Me)
The peptide f-βRS8(∆Q/AS)(Me/Me) was synthesized in a batch of 25 μmole. Reduction of
the double bond after RCM was performed four times, chronologically having a duration of 2 h, 1.5 h, 1,5 h and 1 h. The coupling of the FITC fluorophore coupled once for 1 h and a second time overnight as a departure from the protocol. A solvent gradient of 30 to 60% B in 40 min was used for purification on a preparative HPLC.
Yield 0.02 μmole, 0,076% HPLC-MS: Calculated: 1431.6 [M+H]+ 716.3 [M+2H]2+ Gradient: 30% to 95% of B in 10 min, tr = 9.7 min Found (m/z): 1430.7 716.0 5.2.7.7 Ac-βRS8(∆Q/AS)(Me/Me)
The peptide Ac-βRS8(∆Q/AS)(Me/Me) was synthesized in a batch of 25 μmole. Reduction of
of 2 h, 1.5 h, 1,5 h and 1 h. A solvent gradient of 30 to 60% B in 40 min was used for purification on a preparative HPLC. Yield 0.11 μmole, 0.4% HPLC-MS: Calculated: 939.1 [M+H]+ 470.0 [M+2H]2+ Gradient: 30% to 95% of B in 10 min, tr = 10. 3 min
Found (m/z): No spectrum available.
5.3 Analysis
5.3.1 Fmoc-monitoring
The SPPS was followed by Fmoc monitoring. After the removal of Fmoc the deprotecting solution was collected and the absorbance of the cleavage product 9-methylidenefluorene was measured at a wavelength of 295 nm. Using a molar extinction coefficient of 7800 M-1cm -1 and a path length of d = 1 cm the concentration could be calculated using Lambert Beer’s
law (Equation (1)), which corresponds stoichiometricely to the concentration of peptide that was synthesized.
5.3.2 Biophysical assays
Two kinds of FP measurements were performed: FP assays to determine Kd values of the
different peptides to 14-3-3ζ and FP competition assays to determine IC50 values.
5.3.2.1 FP assay
For FP assays 0.1 mM stocks of the FITC labelled peptides in DMSO were prepared and diluted in FP buffer to obtain 40 nM solutions. For the protein 14-3-3ζ a 200 μM solution was obtained by diluting a stock solution with FP buffer. Hereafter, a dilution series of the protein was made in a 384 well plate, decreasing the protein concentration by a factor 2.5 in each dilution step. To these prepared wells the FITC-labelled peptides were added, resulting in 10 nM as final concentration of the peptides whilst the protein concentrations ranged from 13.4 nM to 150 μM. After incubation at room temperature for one hour the fluorescence
reader. For this purpose an excitation wavelength of 485 nm was selected and the emission at a wavelength of 525 nm was measured. Using the software Prism, the results were analysed. In Prism the datapoints at the highest 14-3-3ζ concentrations were excluded as well as outliers. The 14-3-3ζ concentrations were transformed to a logarithmic scale in order to create a sigmoidal FP curve. Hereafter, a nonlinear regression was applied of which the inflection point indicates the Kd values for the 14-3-3/peptide complexes.
5.3.2.2 FP competition assay
From a stock solution of 14-3-3ζ a 2.67 μM solution was obtained by diluting with FP buffer. f-ESp and f-βRS8(∆Q/AS)(Et/Et) solutions of 13 nM were prepared from their 0.1 mM DMSO
stocks using the 2.67 µM protein solution. The mixture was incubated for 30 min in order to form protein/peptide complexes. Meanwhile, a dilution series of the competitors was made in a 384 well plate, decreasing the concentration by a factor 1.5 in each dilution step. Afterwards the peptide/protein complex was added to each well, resulting in 2 μM as final concentration of the protein and a final concentration of 10 nM for the tracer peptides. The competitor concentrations ranged from 11.1 nM to 37.0 μM The fluorescence polarization of the peptides was measured in the same manner as for FP assays. In Prism the highest competitor concentrations as well as outliers were excluded for the analysis. The competitor concentrations were transformed to a logarithmic scale in order to create a sigmoidal FP curve. Hereafter, a nonlinear regression was applied.
6.0 Abbreviations
Ac Acetyl ACN Acetonitrile COMU 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy-dimethylamino-morpholino-carbenium hexafluorophosphate DCE 1,2-Dichloroethane DCM Dichloromethane ddH2O Double distilled waterDIPEA N,N-diisopropylethylamine
EDT 1,2-Ethanedithiol
ERK Extracellular signal-regulated kinase ExoS Exoenzyme S
FITC Fluorescein isothiocyanate FP fluorescence polarization
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IC50 Half maximum inhibitory concentrations
Kd Dissociation constant
MEKK MAP Kinase Kinase Kinase NES Nuclear export sequence NLS Nuclear localization sequence NMM N-Methylmorpholine
NMP N-Methyl-2-pyrrolidone
ODT 1,8-Octanedithiol
Oxyma Ethyl 2-cyano-2-(hydroxyimino)acetate PEG Polyethylene Glycol
PPI Protein-protein interaction
PyBOP Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro-phosphate
RCM Ring-closing metathesis TFA Trifluoroacetic acid
7.0 References
[1] Bridges, D.; Moorhead, G. B. G. Sci. Signal. 2005, 296.
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[8] Muslin, A. J.; Tanner, J. W.; Allen, P. M.; Shaw, A. S. Cell 1996, 84 (6), 889–897. [9] Yaffe, M. B.; Rittinger, K.; Volinia, S.; Caron, P. R.; Aitken, A.; Leffers, H.; Gamblin, S.
J.; Smerdon, S. J.; Cantley, L. C.; Street, W. Cell 1997, 91, 961–971. [10] Margolis, S. S.; Kornbluth, S. Cell Cycle 2004, 34, 425–428.
[11] Michaud, N. R.; Fabian, J. R.; Mathes, K. D.; Morrison, D. K. Mol. Cell. Biol. 1995, 15 (6), 3390–3397.
[12] Molzan, M.; Schumacher, B.; Ottmann, C.; Baljuls, A.; Polzien, L.; Weyand, M.; Thiel, P.; Rose, R.; Rose, M.; Kuhenne, P.; Kaiser, M.; Rapp, U. R.; Kuhlmann, J.; Ottmann, C.
Mol. Cell. Biol. 2010, 30 (19), 4698–4711.
[13] Aghazadeh, Y.; Papadopoulos, V. Drug Discov. Today 2016, 21 (2).
[14] Ottmann, C.; Yasmin, L.; Weyand, M.; Veesenmeyer, J. L.; Diaz, M. H.; Palmer, R. H.; Francis, M. S.; Hauser, A. R.; Wittinghofer, A.; Hallberg, B. EMBO J. 2007, 26, 902– 913.
[15] Driscoll, J. A.; Brody, S. L.; Kollef, M. H. Drugs 2007, 67 (3), 351–368.
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8.0 Appendices
Appendix I: UV- and mass spectra of synthesized compounds
Figure 8. UV- (left) and mass (right) spectra of Ac-pS-Raf-259. tr = 7.7 min, [M+2H]2+ = 932.2,
[M+3H]3+ = 621.8
Figure 9. UV- (left) and mass (right) spectra of f-ESp. tr = 9.0 min, [M+H]+ = 1648.9, [M+2H]2+ = 825.0
Figure 10. UV- (left) and mass (right) spectra of Ac-ESp. tr = 7.6 min, [M+H]+ = 1156.6, [M+2H]2+ =
Figure 11. UV- (left) and mass (right) spectra of f-βRS8. tr = 9.5 min, [M+H]+ = 1717.8, [M+2H]2+ =
859.0
Figure 12. UV- (left) and mass (right) spectra of Ac-βRS8. tr = 9.1 min, [M+H]+ = 1224.9, [M+2H]2+ =
612.9
Figure 13. UV- (left) and mass (right) spectra of f-βRS8(∆Q/AS)(Me/Me). tr = 9.7 min, [M+H]+ = 1430.9,
Appendix II: UV- and mass spectra of test cleavages before and after RCM
Figure 15. UV- (left) and mass (right) spectra of f-βRS8 before RCM expecting the sequence
Fmoc-QGR5MeLDS5MeLDLAS. Calculated m/z: [M+H]+ = 1431.67.
Figure 16. UV-spectrum of f-βRS8 after RCM and reduction.
Figure 17. Mass-spectra of f-βRS8 after RCM and reduction. The left spectrum depicts masses found
at a retention time of 6.2 min (not reduced; calculated m/z: [M+H]+ = 1181.37) and the right shows
Figure 18. UV- (left) and mass (right) spectra of f-βRS8(∆Q/AS)(Me/Me) before RCM expecting the
sequence Fmoc-GR5MeLDS5MeLDL. Calculated m/z: [M+H]+ = 1145.4.
Figure 19. UV-spectrum of f-βRS8(∆Q/AS)(Me/Me) after RCM and reduction.
Figure 20. Mass-spectra of f-βRS8(∆Q/AS)(Me/Me) after RCM and reduction. The left spectrum
depicts masses found at a retention time of 6.3 min (not reduced; calculated m/z: [M+H]+ = 894.1)
Appendix III: UV-spectrum of test cleavage after PEG coupling to pS-Raf-259
Figure 21. UV-spectrum of Fmoc-PEG-pS-Raf-259 after PEG coupling. tr = 4.3 min.
