Design, Synthesis and Characterization of Small Peptide Conjugates as Protein Actors

Design, Synthesis and Characterization of Small Peptide Conjugates as Protein Actors
Linköping Studies in Science and Technology
Dissertation No. 960
Design, Synthesis and Characterization of Small
Molecule Inhibitors and Small Molecule –
Peptide Conjugates as Protein Actors
Jonas W. Nilsson
Division of Chemistry
Department of Physics and Measurement Technology, Biology and Chemistry
Linköpings Universitet, SE-581 83 Linköping, Sweden
Linköping 2005
© 2005 Jonas W. Nilsson
ISBN 91-85457-00-0
ISSN 0345-7524
Printed in Sweden by UniTryck
Linköping 2005
Abstract
This thesis describes different aspects of protein interactions. Initially the function of
peptides and their conjugates with small molecule inhibitors on the surface of Human
Carbonic Anhydrase isoenzyme II (HCAII) is evaluated.
The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated
with a series of spacered inhibitors were measured by fluorescence spectroscopy and
found in the best cases to be in the low nM range. Dissociation constants show
considerable dependence on linker length and vary from 3000 nM for the shortest
spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an
intermediate length. A rationale for binding differences based on cooperativity is
presented and supported by affinities as determined by fluorescence spectroscopy.
Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC)
spectroscopic experiments with 15N-labeled HCAII were used for the determination of
the site of interaction.
The influence of peptide charge and hydrophobicity was evaluated by surface plasmon
resonance experiments. Hydrophobic sidechain branching and, more pronounced,
peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a
cooperative manner, with affinities spanning almost two orders of magnitude.
Detailed synthesis of small molecule inhibitors in a general lead discovery library as
well as a targeted library for inhibition of α-thrombin is described. For the lead
discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid
scaffolds derivatized in two dimensions employing a combinatorial approach on solid
support.
The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the
scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine
mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics
resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three
orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly,
the best inhibitors showed reversed stereochemistry in comparison with a previously
reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.
Publications
This thesis is based on four scientific papers and one summary, discussed in the
introductory text and enclosed as appendices:
Paper I
Andersson, T.; Lundquist, M.; Dolphin, G. T.; Enander, K.; Jonsson, B.-H.; Nilsson, J.
W. and Baltzer, L. “The binding of human Carbonic Anhydrase II by functionalized
folded polypeptide receptors” Chem. & Biol. Submitted.
Paper II
Nilsson, J. W. and Baltzer, L. “Electrostatic and hydrophobic contributions to protein –
peptide surface interactions.” In manuscript.
Paper III
Nilsson, J. W.; Thorstensson, F.; Kvarnström, I.; Oprea, T.; Samuelsson, B. and
Nilsson, I. “Solid-phase synthesis of libraries generated from a 4-Phenyl-2-carboxypiperazine scaffold.” J. Comb. Chem. 2001, 3, 546-553.
Paper IV
Nilsson, J. W.; Kvarnström, I.; Musil, D.; Nilsson, I. and Samulesson, B. ”Synthesis and
SAR of Thrombin Inhibitors Incorporating a Novel 4-Amino-Morpholinone Scaffold:
Analysis of X-ray Crystal Structure of Enzyme Inhibitor Complex.“ J. Med. Chem.
2003, 46, 3985-4001.
Unpublished results
Nilsson, J. W.; Kvarnström, I.; Nilsson, I. and Samuelsson, B. “Synthesis of Thrombin
Inhibitors Based on a (1R,2R)-cyclopropane-1,2-dicarboxylic acid scaffold.”
Unpublished results summarized by Jonas Nilsson, December 2001.
Contents
1 Introduction
11
2 Small molecule – peptide conjugates based on enzyme inhibitors
15
2.1 Polypeptide design
16
2.2 The peptide conjugate – HCAII system
16
2.3 Evaluation of linker length
19
2.3.1 Design and synthesis of a helix-loop-helix motif linked to a
benzenesulfonamide group
19
2.3.2 Conjugate – HCAII interactions
20
2.3.3 Conclusions
24
2.4 Evaluation of scaffold properties
24
2.4.1 Affinity evaluation by SPR
25
2.4.2 Secondary structure
28
2.5 Conclusions
29
3 Drug candidate identification and generation
31
3.1 Generating a lead
3.1.1 Synthetic strategy of the piperazine scaffolds
3.1.2 Derivatization of the piperazine scaffolds
3.2 Rational design of enzyme inhibitor
3.2.1 α-Thrombin inhibitors
3.2.2 The leads
3.2.3 Mimetics of D-Phe-Pro-Arg
3.3 Initial attempts – the cyclopropane scaffold
3.3.1 Synthesis of the cyclopropane scaffold
3.3.2 Derivatization of cyclopropane scaffolds
3.3.3 Inhibitor evaluations
3.4 The story continues – the N4-amino-morpholinone scaffolds
3.4.1 Synthesis of N4-amino-morpholinone scaffolds
3.4.2 Derivatization of N4-amino-morpholinone scaffolds
3.4.3 Inhibition data
3.4.4 Structure activity relationships
3.5 Conclusions
4 Future prospects and unpublished results
4.1 A possible route to optimizing a peptide conjugate
4.2 Selective peptide cleavage N-terminal of cysteine
5 Notations and abbreviations
5.1 Peptide notations
Quick reference to α-thrombin substrates
Quick reference to peptide notations
5.2 Reagents and substructures
31
32
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Fold-out on page 57
Fold-out on page 57
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6 References
61
Acknowledgements
65
1
Introduction
Protein actors have a profound role in bioorganic chemistry. The comprehension of
protein interactions are crucial for the understanding of cellular transport and
communication, biosensing (diagnostics and screening) and drug design. The diverse set
of tools available for the evaluation of protein interactions include techniques for
biophysical characterization such as X-ray crystallography, NMR spectroscopy and
surface plasmon resonance. Structure-activity relationships may be utilized as well as
biomolecules capable of binding recognition and reporting. Biological or bio-mimicking
constructs such as antibodies, aptamers and synthetic peptides may be used for the
identification, quantification and inhibition of biomolecular interactions involving
proteins.
Peptide conjugates come in many flavors and range from side chain labeled, small
molecule derivatized and branched peptides to more complex macromolecular
constructs such as conjugations of PNA/RNA/DNA to peptide oligomers. Peptides have
the advantage of being accessible via routs that are completely synthetic, making
directed modifications easy, selective and flexible.
Since more than 25 years instruments for automated peptide synthesis have been
commercially available. Even though the automation initially might have been seen as
an expensive luxury, instruments can now be afforded even by small research groups.
The speed and ease with which even longer peptides can be synthesized by this tool is
remarkable, and with an ever expanding set of available building blocks, protection
groups and unnatural amino acids, peptide conjugates have been made available leading
to a plethora of new molecules and applications.
In general most peptides with as many as 50 or even 60 amino acids can be synthesized.
By employing orthogonal protection strategies and self catalyzed selective
condensations, post-synthesis modifications in three, four or even five dimensions are
achievable. The variability is immense. The synthesis of one molecule each of every
peptide with 58 amino acids that can be constructed from the 20 natural amino acids
would require 5 times the mass of the visible universe!
11
In sensing applications, modifications of any residue by the incorporation of
fluorescense-, spin- and radio-labels can be used to probe biomolecular surfaces as well
as other targets. Peptides can also be used as vehicles for drug delivery. Cytotoxins
conjugated with specific peptides can target cancer cells to induce cell death1. PNApeptide conjugates can be used to site specifically cleave or modify DNA2,3 or as
antisense-based imaging agents when chelated with radioactive nuclei for the detection
of pathological overexpression of certain mRNA4.
In this thesis I demonstrate affinity modulation of small molecule inhibitors conjugated
to peptides. Not only can peptides be synthesized in any sequence, but linkage length,
character as well as position of conjugation can easily be varied, to generate affinity
probes suitable for almost any target. When peptides are combined with sensing labels,
the affinities can be determined with high accuracy by direct measurement of single
samples or in microtiter plate format for large-scale drug screening and drug target
evaluation.
The prerequisite for drug screening, however, is a potential drug. In chapter 3 the
rational design and synthesis of α-thrombin inhibitors are described. α-Thrombin is a
key enzyme in the coagulation cascade, and modulation of its activity is crucial for
patients suffering from thromboembolic disorders, the major cause of morbidity and
mortality in the developed world.
The starting point for the design of a new enzyme inhibitor or other drug candidate is to
gain as much information as possible about the dedicated target. This includes the
deduction of key structural elements used in previously known inhibitors or substrates,
lead library screening, structure activity relationships in combination with X-ray
crystallography analysis of enzyme with and without inhibitor or natural substrate.
Lead libraries exist in two forms. General lead discovery libraries (Chapter 3.1) are drug
like or lead like substances without any designated target. High throughput screening of
thousands or millions of compounds can lead to the discovery of new leads unrelated to
previously known inhibitors5. Targeted libraries (Chapter 3.2) are created as an
expansion of a known lead where some of the key structural elements are predefined.
Further optimization includes in vitro activity screening and X-ray crystallography
analyses of inhibitors bound to the enzyme leading to the design of new targeted
libraries. Thus, the circle is closed and this iterative structure based drug design process
will hopefully lead to a preclinical drug.
This is, however, only the first step towards a new drug. Before reaching the market a
series of evaluations must be performed. The first step being evaluation of bioavailability and toxic side effects most commonly performed on cell culture or animal.
If no obstacles are found, tests on humans are performed in three phases. First on a
small number of healthy humans to confirm that no side effects undetected in animals
12
occur (Phase I), secondly on a small number of patients (Phase II) and finally on a large
number of patients (Phase III). Only a small number of the preclinical drugs actually
reach the market.
13
14
2
Small molecule – peptide conjugates based on enzyme inhibitors
Interactions between biomacromolecules are required for the function of all biological
systems. Protein assemblies working together as a single unit perform functions such as
transcription and ATP synthesis. In vivo transport, signal transduction and pathogen
recognition all depend on surface interactions with proteins or antibodies. Advances in
proteomics demand a deeper understanding of the nature of the complicated framework
of hydrogen bonds and electrostatic/hydrophobic interactions that are needed to match
the intricate polymorph surface contours of proteins.
The large pool of available biomacromolecules such as antibodies, aptamers and
proteins optimized by phage display is a valuable source of components for
biomolecular recognition. The biological approach is a well-established and useful tool
for the production of high affinity macromolecules. The structural control, however, is
mostly left to the biological system and directed optimizations as well as the
incorporation of non-natural building blocks might be difficult to assess.
Pharmaceutical Industry, working with small molecules as inhibitors can achieve high
affinities, not by flexibility or complexity, but by cumbersome optimization of small,
rigid lead substances. Systematic trial and error, together with computational aids such
as molecular modeling and in silico docking are used to find the “perfect fit”.
This chapter describes a conjugate approach, which combines the affinity and
specificity of known small molecule inhibitors with the variability of pre-organized
polypeptide constructs. Since they are produced in vitro, the available modifications are
almost unlimited, thereby giving us tools such as unnatural amino acid constructs and
fluorescent reporter functionality. The conjugate consists of a polypeptide connected via
a linker to a small molecule inhibitor that can be anchored into the active site of the
target enzyme. By systematic variation of linker length or position as well as of peptide
charge distribution or hydrophobic character it can be used to probe peptide – protein
interactions and to optimize affinity.
Even though synthetic strategies employed in lead optimizations or in peptide conjugate
syntheses limit the accessible size of libraries of screened species to several orders of
15
magnitude below those of aptamers and antibodies (1012-1014) the directed approach is
expected to compensate for this. Intelligent selections replace random screening.
2.1
Polypeptide design
To be able to present a pre-organized surface, the peptide needs to adopt a defined
secondary and supersecondary structure. The scaffold in this investigation is a 42 amino
acid residue helix-loop-helix motif that dimerizes to form a four-helix bundle6. This was
achieved partly by choosing amino acids with high helix propensities and by
introducing a helix breaking proline at position 21 to generate the loop motif.
In a perfect helix there are 3.6 residues per turn. In the folded state residues with
approximately this interresidue distance, i.e. residues in positions three and seven of the
heptad repeat pattern will form one side of the helix. Introduction of hydrophobic
residues in these positions (e.g. pos 16, 12, 9, 5 upstream from loop and pos 27, 31, 35,
38 downstream) leads to a hydrophobic face that can interact with other helical
hydrophobic faces and form helical bundle structures. If the other residues in the helix
are polar and charged the helix is referred to as amphiphilic. When helical bundles are
formed the hydrophobic faces from several amphiphilic helices generate a hydrophobic
core and the polar residues ensure solubility. Water is excluded from the core and the
free energy in minimized.
The dimerization not only stabilizes the construct, but in the system described in this
thesis it also enhances the effect of reporter functions such as fluorescent probes. Upon
binding to a host protein, hydrophobic and charged interactions between the monomer
subunits are replaced by interactions between each monomer and the target protein,
making it possible for the peptide to bind as a monomer. A fluorescent probe attached to
the peptide may have a self-quenching effect due to the spatial proximity in the dimer.
As this quenching is removed upon binding, a “light-up” effect will occur, making
binding interactions directly measurable by fluorescence.
2.2
The peptide conjugate – HCAII system
Human Carbonic Anhydrase isoenzyme II (HCAII) is a well-characterized enzyme that
has been used for studies of protein folding7,8, ligand design9,10 and enzyme-template
based inhibitor generation employing “click chemistry”.11 In human erythrocytes it
catalyzes the equilibrium between carbon dioxide and water, forming bicarbonate and
oxonium ion. It is a monomeric protein constituted of 259 amino acids. In its native
form, a Zinc ion is coordinated by three histidine residues in the active site located in a
15 Å deep funnel. Several high resolution X-ray crystal structures of HCAII cocrystallized with high-affinity inhibitors have been reported.
One class of inhibitors is based on substituted benzenesulfonamides as their common
motif. Benzenesulfonamide itself has been shown to have a Kd of 1.5 µM12 and the alkyl
16
substituted 4-sulfamoyl-benzamides show chain length dependent Kd values ranging
from approximately 80-150 nM (methyl) to 1.2-2.5 nM (heptyl or octyl)9,12 (Figure 2.1).
O
H2N
O
O
O
O
HN
H2N
Figure 2.1. Benzenesulfonamide (top) and the 4-sulfamoyl-benzamide group (bottom) used as an
anchoring unit into the active site of HCAII.
X-ray crystallography derived structures13 strongly imply that the directional vector of
the Zn – NH2SO2 bond as well as the position of the aromatic ring are well defined for
all para substituted benzenesulfonamides as well as for the ortho,ortho and meta,meta
difluoro substituted ones (Figure 2.2). Deviations are found for 3-mercuri-4-aminobenzenesulfonamide (3CA2) and to some extent for 3-nitro-4-(2-oxo-pyrrolidin-1-yl)benzenesulfonamide10,14 (1KWQ) which displays an aromatic ring dislocated from the
plane defined by the other inhibitors.
Employing the 4-sulfamoyl-benzamide as a basal connector into the active site ensures a
well-defined positioning of the anchoring group. The conjugates were constructed by
connecting the benzamide to the helix-loop-helix scaffold using an aliphatic linker.
Interactions can be probed and tuned employing the conjugate principle. The linker
extends the conjugate to the surface of the protein, placing the peptide in position for
multiple hydrophobic/electrostatic interactions with HCAII (Figure 2.3). Subtle changes
of linker length or position as well as changes in peptide charge or hydrophobic
character as well as in the distribution of charges and hydrophobic groups within the
peptide scaffold can affect both binding strength and protein distortion.
17
Figure 2.2. Aligned overlay showing a stick representation of nine inhibitors from the Protein Databank
together with the Zinc coordinating histidines of HCAII at position 94, 96 and 119. The rest of HCAII is
shown shaded in an overlay ribbon representation The aromatic rings of the benzenesulfonamides are
well aligned for the para substituted (1CNW; 1CNX; 1CNY; 1IF4; 1OQ5) and the symmetrically
difluoro substituted (1IF5; 1IF6) inhibitors. The ortho mercury substituted (3CA2, green) and the meta
nitro substituted (1KWQ10,14, purple) show different positions of the aromatic ring.
Helix-loop-helix
ε-N-Lys34
C8-linker
Zn-Benzenesulfonamide linkage
HCAII
Figure 2.3. Principle schematic of possible interactions between HCAII and a peptide – small molecule
conjugate. The nature of the true interactions is not known in detail. HCAII structure and ligand position
is based on X-ray crystallographic structure of HCAII co-crystallized with N-(2-{2-[2-(2-aminoacetylamino)-ethoxy]-ethoxy}-ethyl)-4-sulfamoyl-benzamide, pdb entry 1CNW.
18
2.3
Evaluation of linker length
A systematic variation of linker length was performed to evaluate the dependence of the
distance between the active site Zinc ion of HCAII and the peptide helix-loop-helix
motif [Paper I]. The linker needs a specific length to be able to reach down into the cleft
of the active site without suffering the penalty of severely distorting the peptide or
protein. However, if the linker is too long there may be an entropic loss due to residual
degrees of freedom in the bound state leading to independent binding of the
benzenesulfonamide and the peptide. Optimally, binding contributions from ligand,
linker and peptide should work simultaneously and in synergy. The chelate effect can
then generate cooperativity between the different binding events leading to a strong
binder. Cooperativity arises when the pre-organization of the peptide resulting from the
binding of the benzenesulfonamide into the active site is such that it is superior to that
of a freely moving peptide.
2.3.1
Design and synthesis of a helix-loop-helix motif linked to a benzenesulfonamide
group
Two 42 amino acid residue polypeptide templates were utilized (KE2, TA4). In
solution these helix-loop-helix templates were found to be dimers at low µM
concentration but it has been previously shown that the peptides are bound as monomers
to HCAII15. A fluorophore was introduced to enable direct measurement of the strength
of the interaction by standard fluorometry titrations.
The peptides were synthesized according to standard Fmoc chemistry protocols using
acid labile protection groups. The side chain of the lysine in position 15 was protected
with an allyloxycarbonyl protection group which after solid-phase synthesis of the
peptide could be selectively removed by Pd[PPh3]4 and then sulfonylated with a dansyl
probe for fluorometric assay. Cleavage of the peptide from the resin by trifluoroacetic
acid was followed by purification on reversed phase HPLC.
The ligands were prepared from 4-sulfamoyl-benzoic acid and unbranched primary
amino acids using carbodiimide chemistry (Scheme 2.1).
O
O
H2N S
O
HO N
O
O
DCC
DMF/Dioxane
OH
O
H2N S
O
O
O
O N
O
O
H2N (CH2)n
O
H2N S
O
OH
Aceton
Aqueous borate (pH=8-9)
O
O
O
HN (CH2)n
O
HO N
O
O
N
DCC
DMF/Dioxane
O
H2N S
O
O
O
HN (CH2)n
OH
O
Scheme 2.1. Synthesis of the activated benzensulfonamide ligands.
19
The helix-loop-helix motif (KE2-DNS(15)) was selectively modified at position 34
using the site selective self-modification reaction, a valuable tool for easy access to
modified peptides16. Previous studies have shown that the histidine in position 11
preferrably directs acylation to a lysine in position 15 in similar peptides. When this
position does not contain an amine, a lysine in position 34 can be selectively targeted.
This would in principle be applicable to this system and both the dansyl probe and the
benzenesulfonamide group could be introduced in the correct positions in aqueous
solution by sequential addition of the corresponding reagents to a solution of the peptide
scaffold. However, due to solubility issues, the dansyl probe was introduced on solid
support employing a selective protection group strategy.
Selective modification of lysine 34 was achieved in hepes buffer, pH 8 using the Nhydroxy succinimide activated ligand to yield the fluorophore labeled conjugates KE2DNS(15)-Cn(34), n=6; 7; 8; 11 (Scheme 2.2). The alternative conjugate TA4-DNS(15)C8(34) was produced in an analogous manner.
N
H
OO
S
N
O
NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG OH
KE2-DNS(15)
Hepes buffer
pH=8
O
H2N S
O
O
O
HN (CH2)(n-1)
O
O
N
O
N
H
OO
S
N
O
NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG OH
O
H2N S
O
O
HN (CH2)(n-1)
KE2-DNS(15)-Cn(34)
n=6; 7; 8; 11
NH
O
Scheme 2.2. Selective side chain modification with linked benzenesulfonamide.
2.3.2
Conjugate – HCAII interactions
All peptide conjugates were screened for affinity to HCAII. This was done in three
modes. Initially, direct titration of a 0.2 µM solution of conjugate with HCAII was used
to determine the dissociation constant of the conjugate – HCAII complex. However for
direct titration to be a reliable method, the conjugate concentration should be in the
same order of magnitude as the dissociation constant. Due to limitations in signal
intensities and surface adhesion problems at low concentrations, additional
measurements utilizing indirect determination of dissociation constants with a
competitive inhibitor (acetazolamide) were carried out. Solutions containing 2 µM
20
peptide conjugate and 1 µM HCAII was titrated with acetazolamide. The determined
affinities was dependent on whether the wavelength of maximum fluorescence intensity
or the absolute value of the intensity was used, and thus both will be used in the
following discussion.
It is evident that for short linkers (0-4 methylene groups) the affinities are all low and
surprisingly indifferent to linker length (Table 2.1). This might illustrate a state in which
severe distortions to the peptide or protein structure is needed to accommodate binding.
By introducing a fifth methylene group however (KE2-DNS(15)-C6(34)) the affinity
increased markedly showing Kd values of 20; 25 and 16 nM when determined by the
three modes of measurement. The consistent values and good empirical fit indicates that
the 1:1 binding model is valid. For the longest linkers however (KE2-DNS(15)-C8(34)
and KE2-DNS(15)-C11(34)) there are some discrepancies between the different modes
of measurement. Also in the case of C11 the fluorescence intensity increases with
increasing acetazolamide concentration, which is opposite to the expected behavior.
This might indicate a more complex way of binding to the protein.
Table 2.1. Affinities of conjugates for HCAII recognition and binding.
No. CH2
Kd / nM
Conjugate
in linker
[a]
[b]
[c]
*
KE2-DNS(15)-C0(34)
-
3000
KE2-DNS(15)-C2(34)
1
1000
KE2-DNS(15)-C4(34)
3
700
KE2-DNS(15)-C5(34)
4
800
KE2-DNS(15)-C6(34)
5
20
KE2-DNS(15)-C7(34)
6
10
KE2-DNS(15)-C8(34)
7
4
TA4-DNS(15)-C8(34)
7
70
23±2
28±4
19±2
13±2
29±8
22±4
6±2
6±2
24±4
60±16
22±4
53±13
[a] As determined from direct titration of conjugate with HCAII. [b] and [c] As determined from titration
of HCAII – conjugate complex with a competitive inhibitor using fluorescence intensities [b] or
wavelength maxima [c] to extract data. Data is presented as duplicates ± one standard deviation as
determined by the Levenberg-Marquardt curve-fitting algorithm. Dissociation constants for entries 1-4
are from ref. 15.
KE2-DNS(15)-C11(34)
10
Measures were taken to determine the nature of these complexes. A quencher peptide
(KE2-DAB(15)) in which the dansyl probe was replaced by 4-(4-dimethylaminophenylazo)-benzoate (dabcyl) was utilized. By titrating the conjugate – HCAII
complexes with KE2-DAB(15) the dissociation constants of the ternary complex
HCAII – KE2-DNS(15)-Cn(34) ↔ KE2-DAB(15) was determined to be 290; 110 and
90 µM for C6; C8 and C11 respectively. Even though longer linkers show greater
*
KE2-DNS(15)-C0(34) has no linker. The 4-sulfamoyl-benzoic acid is directly connected to Lys34.
21
tendencies for aggregation the dissociation constants are much higher than the
concentration used for fluorometric assay (2 µM). Arguing that the ternary complex
studied should have similar dissociation constants as that of HCAII – KE2-DNS(15)Cn(34) ↔ KE2-DNS(15)-Cn(34) the population of ternary complexes from HCAII and
two polypeptide conjugates is low under the experimental conditions and thus no
conclusive model could be presented.
In general, affinities based on measurements of wavelengths of maximum fluorescence
should be considered more reliable than those based on fluorescence intensities due to
self-quenching phenomena that are not linearly related to the dissociation equilibrium.
Thus based on the entries in columns [a] and [c] in table 2.1 the dissociation constants
are approximately 20; 10; 5 and 57 nM for conjugates having linkers with 5, 6, 7 and 10
methylene groups respectively. This indicates cooperativity with an optimum at 7
methylene groups. Shorter linkers exert excessive strain on the system and longer
linkers indicate an entropic loss due to discrete behavior of peptide and
benzenesulfonamide, both phenomena leading to an affinity decrease.
Two different peptide scaffolds with the C8 linkers were used. Previous studies showed
that the KE2 scaffold is essentially a non-binder of HCAII by itself. In contrast, TA4
was shown to bind in the µM range. Surprisingly, this is not at all reflected in the
conjugates where KE2-DNS(15)-C8(34) shows more than an order of magnitude
stronger binding to HCAII than TA4-DNS(15)-C8(34) does. To understand the nature
of the peptide – HCAII interactions in more detail a NMR spectroscopy investigation
was initiated.
HSQC spectroscopic experiments with 15N-labeled HCAII in the presence and absence
of conjugate were undertaken. The chemical shifts of HCAII are very conserved under
the experimental conditions. Seven different samples of HCAII were analyzed for
perturbations of the amide proton – nitrogen shifts and all cross peaks were reproduced
within +/- 1.5 Hz in the 15N dimension and +/- 2.5 Hz in the 1H dimension. When the
environment of an amide is moderated, either by close proximity to the conjugate or by
structural perturbations due to binding, the chemical shifts are affected. To distinguish
natural variations from real interactions, shifts exceeding 10 Hz in either the 1H or 15N
dimension were considered significant.
The NMR spectrum has been assigned for HCAII17, which enables visualization of the
polypeptide conjugate binding interactions with HCAII. Figure 2.4 reveals a plausible
explanation for why TA4 and KE2 show reversed behavior when binding
independently or as conjugates. Unmodified KE2 did not affect the chemical shifts of
HCAII due to its low affinity, an observation confirmed by SPR measurements. Even
though residues around the active site of HCAII were affected by TA4 as well as by the
conjugate TA4-DNS(15)-C8(34), binding regions were significantly different.
22
Figure 2.4. Residues of HCAII affected (dark) by TA4 (left) and TA4-DNS(15)-C8(34) (right).
Apparently, simultaneous binding of the scaffold to its medium-affinity binding site and
sulfonamide to the Zinc ion was not possible. However, the area affected by TA4 seems
to be located within reach of a linker originating in the active site. Redesigning the
conjugate to adapt to the preferred location of the peptide would be an interesting
challenge that might be achieved by systematic variations of linker length and
hydrophobicity in combination with its attachment point to the peptide (see chapter 4.1).
For the KE2 – conjugate series differential perturbations of the HCAII surface were
observed. No shifts were observed for KE2-DNS(15)-C5(34) possibly due to the
inability of the bound peptide to retain its helical structure when the linkers are too
short. It is unlikely that HCAII can accommodate a folded helical peptide within its
binding cleft, but possibly an unordered peptide chain. This would lead to a large
number of randomized conformations incapable of distinct interactions with HCAII. For
longer linkers however, several perturbed amide protons were identified (Figure 2.5).
This is also reflected by KE2-DNS(15)-C5(34) having a 40-fold lower affinity than
KE2-DNS(15)-C6(34). The fact that the affinities for even shorter linkers are largely
constant supports a model where the dominating binding event is the interaction
between the aryl-sulfonamide and HCAII active site.
Figure 2.5. Residues of HCAII affected (dark) by KE2-DNS(15)-C6(34) (left), KE2-DNS(15)-C8(34)
(middle) and KE2-DNS(15)-C11(34) (right).
For KE2-DNS(15)-C8(34) the interactions are significantly fewer than for KE2DNS(15)-C6(34). In this complex 30% of the NMR intensity is lost suggesting some
23
degree of aggregation and the observation of fewer interactions between scaffold and
HCAII remain unexplained.
For the longest linker, KE2-DNS(15)-C11(34), the observed interactions are very
strong which might be due to a more complex binding model. Even though the nature of
this complex is uncertain, a simple 1:1 interaction can be ruled out. Attempts to
crystallize conjugate – HCAII complexes have so far failed, but NMR indicates a high
molecular weight aggregate that might contain two or more HCAII. This would result in
a molecular weight of >65kD for which the NMR signals would be broadened beyond
detection and when looking at the total intensity of the NMR signal only 30% remains
after addition of the conjugate. Thus, the majority of the complex ensembles are
undetectable by NMR.
2.3.3
Conclusions
For optimum binding to HCAII no less than 5 methylene groups should be present in a
polypeptide – benzenesulfonamide conjugate such as KE2-DNS(15)-Cn(34). Furthermore, 5 methylene groups are optimal if minimum translational freedom of the peptide
is imperative. This however severely restricts the accessible binding area of HCAII, and
puts rigorous constraints on the electrostatic contours of the peptide. In fact, a slightly
longer linker might be preferred as this flexibility relaxes the demands on the peptide.
This is also supported by the fact that maximum affinity was observed for a conjugate
with 7 methylene groups.
If very long hydrophobic linkers are used, unwanted aggregation occurs. If increased
hydrophobicity alone is the reason for the aggregation, hydrophilic elements might be
introduced in linkers of similar length to enhance the probability that the scaffold binds
in an optimal fashion to the target protein. It has been shown, however that polar
polypeptide linkers provide poor binding, and thus the hydrophilic elements should be
sparse keeping the linker character mainly hydrophobic.
2.4
Evaluation of scaffold properties
To deepen the understanding of the peptide Table 2.2. Properties of scaffold peptides
properties that influence HCAII binding, a set
Peptide
Charge γ-branched
pH=7.4a leucines
of new peptides was synthesized [Paper II].
KE2
-0.9
Yes
Starting from a non-binder (KE2) and a µM
JWN-H8
+1.1
Yes
binder (TA4) as initial templates, two new
JWN-H9
-0.9
No
peptides were designed to have intermediate
TA4
+1.1
No
TA4+
+4.1
No
properties between the two (Figure 2.6). The
a
Based on typical pKa values of amino
differences between the peptides were reduced
acid residues in short peptides.
to two parameters: peptide charge (-0.9 and
+1.1 for KE2 and TA4 respectively) and hydrophobic branching (3 leucines and 3
norleucines for KE2 and TA4 respectively). The peptide JWN-H8 was designed to
24
have the charge of TA4 and the hydrophobic branching of KE2 and vice versa for
JWN-H9 (Table 2.2). Initial analysis indicated charge as the more important parameter
and thus TA4+ was designed by extrapolation of this attribute to probe the validity of
this conclusion.
KE2
Ac-NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-OJWN-H8 Ac-NAADLEAKIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-NH2
JWN-H9 Ac-NAADJEAAIRHLAEKJAARGPVDAAQJAEQLAKKFEAFARAG-OTA4
Ac-NAADJEAKIRHLAEKJAARGPVDAAQJAEQLARKFEAFARAG-NH2
TA4+ Ac-NAADJEAKIRHLREKJAARGPRDAAQJAEQLARKFERFARAG-NH2
Figure 2.6. Representation of differences in charge and side chain branching between peptide scaffolds.
Residue charges at experimental conditions are shown in white with border and black for negative and
positive respectively. Hydrophobic branching are shown in light gray (leucines (L); γ-branched) and
darker gray (norleucines (J); linear). Peptide net charge at pH=7.4 changes from –0.9 (KE2; JWN-H9) to
+1.1 (JWN-H8; TA4). Hydrophobic branching is reduced going from leucines (KE2; JWN-H8) to
norleucines (JWN-H9; TA4). TA4+ is based on TA4 but has 3 additional positive charges.
2.4.1
Affinity evaluation by SPR
Surface plasmon resonance spectrometry (SPR) is a powerful technique for observing
binding phenomena at surfaces. It is a highly sensitive method for detecting refractive
index changes in a small space above a gold-surface. On this surface a thin layer of
carboxymethylated dextran matrix is immobilized.* The matrix functions as a
convenient handle for attachment of proteins, peptides or other species. By activating
the carboxyl groups by EDC / NHS chemistry and then flushing the surface with HCAII
some of the carboxyl groups from the matrix react with free amines from the protein
forming amide bonds and thus irreversible covalent links to the protein.
The sensing range is approximately 150 nm from the surface and the sensitivity can be
high enough to detect changes as small as 0.0001º in angle of incidence, corresponding
to approximately 10-6 refractive index units. This is called one response or resonance
unit (RU) and is proportional to the adsorbed mass. For typical biomolecules one RU
corresponds to approximately 1 pg/mm2.
For the experiments discussed in this thesis the typical amount of immobilized HCAII
was 2300 RU. This would correspond to approximately 47600 HCAII molecules within
the sensor range (150 nm) per µm2 or typically 2.5 % volume coverage by protein
(Figure 2.7).
*
This is one type of commercial surface sold by Biacore Sweden; Rapsgatan 7; SE-754 50 Uppsala
and is referred to as CM5-type sensor chip.
25
Figure 2.7. Typical surface element sensed by SPR measuring 150 by 150 by 150 nm. The volume is
filled to an extent of approximately 2.5 % by 1070 randomly positioned HCAII molecules which
corresponds to a typical immobilization level of 2300 RU. HCAII is represented as spheres with Ø = 45
Å.
By flowing peptide solutions at different concentrations over the surface, interactions
between HCAII and peptide could be quantified. The response parameter (r) is the
relative amount of HCAII complexed with peptide and can be calculated from equation
2.1 using the experimentally determined number of response units (RUresponse). Due to
the covalent nature of immobilization some of the HCAII will be nonnative. The
fraction of native protein (n) and the dissociation constant were used as fitting
parameters, employing the Leverberg-Marquadt algorithm on equation 2.2 to search for
minimum χ2.
Using the strongest binder (TA4+) as a surface quality descriptor and searching for the
best fit of equation 2.2 to the experimental results led to the values of Kd=227 µM and
n=0.9 indicating that only 90 % of the immobilized HCAII was available for binding
(Figure 2.8). Under the assumption that this is a valid global surface quality descriptor,
the peptide – HCAII dissociation constants for all peptides were calculated (Table 2.3).
26
Equation 2.1a.
1
RU response
0.9
M peptide
r=
RU HCAIIimmobilized
0.8
0.7
M HCAII
a
0.6
Response parameter (r) calculated from
refractive index responses and molecular weights.
0.4
Equation 2.2b.
[E ][P ]
[EP]
0.3
⎫
⎪
⎪
E act = n × Etot ⎪
n[P ]
⇒r=
[E ] + [EP] = E act ⎬⎪
K d + [P ]
⎪
[EP]
r=
⎪
Etot
⎭
Kd =
r 0.5
0.2
0.1
0
1.0E-01
1.0E-02
1.0E-03
1.0E-04
1.0E-05
1.0E-06
[TA4+] (M)
b
Dissociation constant (Kd) and fraction of native
HCAII (n) were used as parameters in fitting
function. Enzyme (E) is HCAII and peptide (P) is
KE2, JWN-H8, JWN-H9, TA4 or TA4+.
Figure 2.8. Response parameter (r) as function of
TA4+ concentration (+). The solid line represents
the best fit of equation 2.2 with Kd=227µM and
n=0.9.
Variable descriptions: r (the unitless response parameter); RUresponse (response from peptide in refractive
units); RUHCAIIimmobilized (amount of immobilized HCAII in refractive units); Mpeptide (peptide molecular
weight); MHCAII (HCAII molecular weight); Kd (dissociation constant); [E] (concentration of free
enzyme); [P] (concentration of free peptide); [EP] (concentration of peptide – enzyme complex); Eact
(total concentration of active enzyme); Etot (total concentration of enzyme); n (native fraction of enzyme).
The results show that neither charge nor hydrophobic branching alone explains the
affinity differences. The weak binder KE2 has a Kd of 12 mM. Replacing the branched
L by the linear J leads to JWN-H9 and an observed change in affinity by less than a
factor of two, while the addition of two charges to KE2 leads to JWN-H8 and an
almost four-fold increase in affinity. The combined effect of changing both charge and
hydrophobic branching, leads to TA4 for which the effect is even more pronounced and
an affinity increase of a factor of 26 was observed.
Table 2.3. HCAII Dissociation constants.
Peptide
Kd (mM)
Charge
KE2
JWN-H8
JWN-H9
TA4
TA4+
12
3.2
7.6
0.46
0.23
-0.9
+1.1
-0.9
+1.1
+4.1
γ-branched
leucines
Yes
Yes
No
No
No
Expressing the values in ΔΔG˚ clearly indicates that the parameters work cooperatively.
Results show 8.0 kJ/mol for the combined effect of charge and branching, markedly
27
more than the sum of the individual effects being 1.1 and 3.2 kJ/mol for hydrophobic
branching and charge respectively.
TA4+ was designed by extrapolation based on the conclusion drawn from the
measurements described above that charge was the dominant reason for the higher
affinity of TA4 in comparison with that of KE2. The design was based on the sequence
of TA4 in which A13, V22 and A37 were replaced by arginines to give a charge of
+4.1, three more than in the parent peptide. Even though this peptide was an even better
binder of HCAII than all other peptides, the effect was not as pronounced as expected.
For the series JWN-H9, TA4 and TA4+ displaying the charges –0.9, +1.1 and +4.1
respectively the corresponding dissociation constants were 7.6, 0.46 and 0.23 mM. Thus
changing the charge by two units from –0.9 to +1.1 gave more than a 16-fold increase in
affinity while the change of three units from +1.1 to +4.1 gave only an additional twofold increase. We concluded that with regards to charge magnitude, the peptide TA4+ is
nearly optimized and that other modification such as change of charge distribution or
hydrophobic character are needed to further increase its affinity.
An alternative model for interpretation of the data reproduced the differences as
described in paper II. And thus, we are confident that the applied interpretation model
gives us accurate relative affinities of the peptides for HCAII as well as significant
differences between the binding abilities of the peptides. The accuracies of the absolute
values of dissociation constants are, however, limited and the values should be
considered approximate. This has however no implications regarding the ΔΔG˚ values
and cooperativity reasoning.
2.4.2 Secondary structure
The helical contents of all peptides were evaluated by circular dichroism spectroscopy
(CD) (Table 2.4). The mean residue ellipticity at 222 nm ([Θ]222) is considered a
quantifier of helical content where a fully developed helix has [Θ]222 = -35 700 deg cm2
dmol-1 based on helical poly-L-lysine at pH 11.118.
Table 2.4. Mean residue ellipticities of peptide scaffolds.
Peptide
KE2
JWN-H8
JWN-H9
TA4
TA4+
[Θ]222
(deg cm2 dmol-1)
-18 600
-20 700
-24 400
-20 000
-15 000
Conc. (µM)
100a
100b
100b
250c
100b
Charge
-0.9
+1.1
-0.9
+1.1
+4.1
γ-branched
leucines
Yes
Yes
No
No
No
a
In 10 mM phosphate buffer at pH 7.4 from reference 19. bIn 20 mM Trizma® buffer at pH 7.4 cIn 50 mM
phosphate buffer at pH 7.4 from reference 20.
For the peptides in which the total charge is small the mean residue ellipticities are close
to –20 000 deg cm2 dmol-1 with JWN-H9 as an exception, possibly due to higher helical
28
propensity of the unbranched norleucines. This effect however is cancelled in TA4 for
no obvious reason. Not surprisingly, TA4+ shows considerably lower helical content
than all the other peptides. Since peptides in this series are believed be helical only
when dimerized, a low helical content suggests that TA4+ is dissociated to a high
degree. An overall large positive charge might destabilize the structure due to
electrostatic repulsion. Furthermore, two alanines, usually considered highly helix
inducing, were replaced by arginines.
2.5
Conclusions
To design high affinity conjugates many parameters must be taken into consideration.
Only a few of all possible variations have been evaluated so far. Chapter 2.3 concluded
that for each peptide scaffold there should exist an optimal length of the linker that
might also differ between different templates. A set of linkers should therefore be
evaluated for each peptide template. Linker character might also have an impact on both
complex stability and morphology.
Peptide charge and hydrophobic branching were evaluated in chapter 2.4 and charge
was seen to have a great impact on affinity. To further optimize binding, charge
distribution would be a good candidate. There are also many hydrophobic amino acids
of different length, branching and type (aliphatic, aromatic) that should be introduced in
various positions and configurations to match the complex contour of the host protein.
29
30
3
3.1
Drug candidate identification and generation
Generating a lead
In the process of producing drug candidates there are generally a set of lead substances
that show potential for evolving into active substances. When starting with a new target,
or when the target is unknown it can be advantageous to have access to general lead
discovery screening libraries of lead-like or drug-like compounds. To be able to
distinguish between drug-like as opposed to nondrug-like21 substances we have tried to
find general structural motifs that seem to lead to biological activity for a broad range of
possible targets.
Drug-like properties as defined by Lipinski’s “rule of 5”22 states that the molecular
weight should be less than 500 g/mol, the calculated logarithm of the octanol-water
partition coefficient (logP) should be less than 5, the number of hydrogen-bond donor
atoms should be less than five and the hydrogen bond acceptors should be less than 10.
It is anticipated that both molecular weight and lipophilicity increases during the drug
optimization. In order to make “room” for these modifications a lead-like substance
could be defined as having low molecular weight (100-350 g/mol) and relatively low
lipophilicity (logP=1-3)23.
By searching in the MDDR database24 for templates showing structural similarities
attributed to biological activity, we have come up with three similar templates that was
found in 3084 different substances (Figure 3.1) [Paper III].
N
O
O
N
N
O
N
N
N
N
H
N
H
N
673 Hits
2271 Hits
140 Hits
Y
N
N
O
A1
N
N
O
A2
Figure 3.1. An overview of hits found in the MDDR database.
31
These three templates were reduced to an initial set of two scaffolds, A1 and A2, both
having 3 points of diversity.
3.1.1 Synthetic strategy of the piperazine scaffolds
The scaffolds A1 and A2 were both synthesized in solution from racemic piperazine-2carboxylic acid dihydrochloride. Since piperazine-2-carboxylic acid has some adverse
properties related to solubility in organic solvents and several points of reaction,
orthogonal protection of the carboxylic acid and the two amines was achieved in 78 %
in a three-step procedure as described by Bigge25. The protected amino acid 1 thus
formed can easily be mono deprotected at any point by using different deprotection
strategies i.e. alkali, acid or catalytic hydrogenation. By treatment with 4.5 M
hydrochloric acid in dioxane / water the BOC protection group was easily removed in
quantitative yield to form 2 (Scheme 3.1).
Scheme 3.1a. Synthesis of piperazine scaffolds.
H
N
N
H
O
OH
x 2HCl
i-iii
Z
N
O
O
N
BOC
iv
Z
N
Z
N
O
O
N
H
v or vi
O
H
N
O
vii
N
NO2
N
O
O
CF3SO3H
NO2
3a (o-NO2)
4a (o-NO2, 87 %)
3b (m-NO2)
4b (m-NO2, 66 %)
a
Reagents and conditions: (i) BOC-ON, pH 11, dioxan/H2O; (ii) benzyl chloroformate, pH 9.5; (iii)
methyliodide, aqueous sodium bicarbonate, DCM, Adogen 464; (iv) HCl, dioxane; (v) 2fluoronitrobenzene, TEA, 60°C (for 3a); (vi) Pd(OAc)2, BINAP, CsCO3, 3-bromonitrobenzene, toluene,
100°C (for 3b); (vii) TFMSA, anisole, DCM.
1
2 (quant.)
The o-nitro-phenyl group was introduced by nucleophilic aromatic substitution
employing 2-fluoronitrobenzene. Several methods were tried including refluxing in
aqueous ethanol with sodium hydrogen carbonate and heating in DMF (150°C).
However the most successful procedure was stirring a mixture of 1 eq. amine, 2 eq. of
3-fluoronitrobenzene and 2 eq. of triethylamine for 5 days at 60°C or 20 days at rt. The
desired aniline scaffold 4a was achieved after acidic deprotection of the Z protection
group employing TFMSA in 87 % total yield.
The m-nitro-phenyl group could not be introduced in the same manner since 3fluoronitrobenzene is much less prone to nucleophilic attack than 2-fluoronitrobenzene.
However over the last decade, advances in palladium catalyzed aryl aminations mainly
achieved by the Buchwald26 and Hartwig27 groups have given us valuable tools to attain
these transformations.
Numerous factors, such as choice of phosphine ligand and base excessively affects the
outcome. In figure 3.2 a typical catalytic cycle employing BINAP as a bidentate
phosphine ligand and cesium carbonate as base is shown. The cycle is initiated by an
active monodentate palladium(0) complex (4) that can be formed in several ways from
32
both palladium(0) and palladium(II) sources. Starting from palladium(II) acetate (1) an
initial addition of the phosphine ligand occurs (2) after which the species is reduced (3).
An equilibrium exists between the resting bidentate species (3) and the active
monodentate palladium(0) species (4). The arylbromide is then oxidatively added to the
active species to form complex 5. When a primary or secondary amine is present the
complex reorganizes (6) and a base is used to remove a proton from the amine and the
bromine ligand from palladium (7). The fate of complex 7 can be either the usually
unwanted β-hydride elimination pathway (8) or reductive elimination forming the
product. The active complex (4) is thus recycled and ready for another catalytic round.
OAc Ligand addition
(BINAP)
P
Pd(II)
OAc Reduction
P
BINAP Pd(II)
OAc
1
P
BINAP Pd
OAc
P
(Resting state)
3
2
P
BINAP Pd
P
ArNR2
(Product)
(Active complex)
4
ArBr
L
Pd
8
N
R'
ArH
H
ß-hydride
elimination
Pd
Pd
Ar
Br
L
7
L
R
Ar
NR2
Pd
Ar
Cs2CO3
Br
5
L
Br
L
CsHCO3 +
CsBr
Ar
Pd
NHR2
HNR2
6
Figure 3.2. The catalytic cycle of palladium catalyzed aryl aminations. For detailed explanations see text.
The palladium catalyzed aryl amination scheme above was used to achieve aniline
scaffold 4b in 66% total yield after deprotection.
3.1.2 Derivatization of the piperazine scaffolds
The combination of a combinatorial approach with chemistry on solid support is a
valuable tool that can produce a diverse set of hundreds or thousands of spatially
separated compounds28,29 as well as mixtures of millions of compounds30,31. Hence, 4a
and 4b were connected to a Merrifield resin via an activated Wang linker (Figure 3.3)
and then reduced to have the first handle ready for derivatization (6a/6b) (Scheme 3.2).
33
O
O
NO2
O
O
Figure 3.3. Merrifield PS/DVB copolymer resin bead with p-nitrophenyl activated Wang-linker.
Scheme 3.2a. Connection to solid support and activation of the scaffold.
H
N
N
O
O
N
O
CF3SO3H
viii
O
N
O
ix
N
NO2
O
N
NO2
HCl
NH2
5a(0.57 mmol/g, 81 % load.eff.b)
6a/6b
5b(0.43 mmol/g, 61 % load.eff.b)
a
Reagents and conditions: (viii) DIPEA, DMF, activated PS/DVB-Wang resin; (ix) SnCl2·H2O, DMF.
b
Loading efficiency corresponds to PS-DVB-Wang resin with 0.89 mmol/g loading.
4a/4b
The two different solid phase-supported scaffolds (6a, 6b) were each put in 80 spatially
separated wells in an 8 rows by 10 columns configuration. To each of the rows a unique
electrophile was added, together with pyridine / DCM (Scheme 3.3). When the reaction
was complete, the fluid was rinsed off and the next point of diversity was exposed by
ester hydrolysis employing potassium trimethylsilanoate in THF. Each column was
treated with a unique amine, together with the coupling reagent pyBOP and the base
NMM in DMF. The library members were cleaved from the solid support by treatment
with trifluoroacetic acid in DCM to yield the final products 10a and 10b.
Scheme 3.3a. Reactions involved in derivatization.
O
N
N
O
O
N
O
HCl
NH2
x
N
O
xi
N
NH
X
O
N
O K
xii
N
NH
X
H
N
Y
xiii
N
NH
X
O
Y
N
NH
X
10a{1-8;1-10}
8a{1-8}
9a{1-8;1-10}
7a{1-8}
10b{1-8;1-10}
8b{1-8}
9b{1-8;1-10}
7b{1-8}
a
Reagents and conditions: (x) XCl, Pyridine, DCM; (xi) TMSOK, THF; (xii) HY, PyBOP, NMM,
DMF;(xiii) TFA, 30 % in DCM. Typical yields for the total reaction 5a or 5b to 10a or 10b was 50-70 %.
6a/6b
The 2 different regioisomers combined with 8 electrophiles and 10 nucleophiles
produced a total of 160 compounds, still having one point of diversity left for further
substitution possibly for use in lead to drug optimization. (Figure 3.4)
34
regioisomers
H
N
10a
N
H
N
10b
-Y =
-X =
O
Y
H
N
N
S
OO
O
NH
N
S
X
O
S
OO
O
NH
N
O
O
O
O
Y
O
O
N
O
N
NH
X
NH
O
N
O
O
N
N
H
N
N
S
OO
O
NH
O
N
O
Figure 3.4. Library diversity showing 2 x 8 x 10 = 160 compounds.
3.2
Rational design of enzyme inhibitor
By studying existing inhibitors of a given enzyme, and by using existing knowledge of
enzyme-inhibitor interactions, a rationale for the design of new targeted libraries of
potential inhibitors can be generated. Synthesis of these libraries and evaluation of
theoretical models in comparison to empirical results further adds to the total
knowledge and can be used for further optimization. This completes the cycle that
eventually leads to better understanding of general and specific interactions and
hopefully more efficient inhibitors.
3.2.1 α-Thrombin inhibitors
α-Thrombin, also known as Factor IIa is a serine protease having a central role in the
coagulation cascade where the proteolytic capability is used to cleave soluble fibrinogen
into fibrin. Furthermore it activates Factor XIII resulting in fibrin cross-linking and it
induces platelet activation and aggregation that enables a blood clot to form. In a
healthy organism the purpose is to protect against excessive blood loss upon injury.
However in several pathological conditions such as hypertension and artery plaque
formation unwanted clot formation can lead to lethal conditions such as deep venous
thrombosis, stroke, pulmonary embolism and myocardial infarction. For these patients
regulation of blood coagulation is imperative, and administration of α-thrombin
inhibitors is a common treatment.
Inhibition/activity is often shown by short peptide sequences that display similar motifs
as natural substrates i.e. have a similar peptide sequence as the natural substrate at some
key points. These key points include active sites and cofactor binding sites. Bioavailability and metabolic rates of short peptides often limits their suitability as drugs.
Oral availability, which is considered crucial, is poor, and metabolic rates are often too
high, to be of therapeutic interest. The tri-peptide D-Phe-Pro-Arg (Figure 3.5) that
mimics the α-thrombin sensitive region (-Phe-(AA)5-Gly-Val-Arg-↓-Gly-Pro-Arg-)32
in a loop of the pro-protein fibrinogen has shown inhibitory activity against α-thrombin.
35
H3N
N
O
NH
O
NH2
O
O
HN
D-Phe-Pro-Arg
NH2
Figure 3.5. Tripeptide fragment capable of α-thrombin inhibition.
Knowledge of short peptides showing activity or knowledge of natural substrate
configuration in enzyme binding site is a valuable starting point for inhibitor
development. Peptidomimetics are small organic molecules that mimic the important
structural motifs of these peptide sequences. The advantages over peptides are many, as
synthetic constructs introduce many possibilities for structural diversity as well as
enhanced bioavailability and metabolic rates.
H3N
N
NH
NH2
O
O
O
N
H
NH2
Cl
OH N
O
O
NH
O
PPACK
IC50=1.5 nM
NH3
L-372.460
IC50=4 nM
Ki=1.5 nM
O
NH
H
N
N
O
O
NH2
NH2
Melagatran
IC50=2 nM
Ki=2 nM
Figure 3.6. Known synthetic α-thrombin inhibitors.
Known α-thrombin inhibitors include PPACK33,34, L-372.46029 and Melagatran35
(Figure 3.6) for which the similarities with the tri-peptide D-Phe-Pro-Arg are apparent.
Two amide bonds connect three fragments named P3, P2 and P1 from left to right
having a proline or proline-like scaffold in the P2 position. The P1-position all have
positively charged guanidine, amine or amidine in coherence with the tri-peptide and in
the P3-position hydrophobic elements are present.
Amide bonds are a common motif seen in many pharmaceutical substances. They work
as both hydrogen bond acceptors and donors, which is an important factor in inhibitor
binding36,37. The chemistry around amide bonds is also well known, and there are
36
numerous commercially available chiral and achiral amines, carboxylic acids and amino
acids to use for structural variation*.
Analysis of a crystal structure of PPACK bound to α-thrombin active site from the
protein databank38 shows three important binding pockets in the active site of αthrombin (Figure 3.7). Key amino acids in α-thrombin active site are shown in gray
together with PPACK inhibitor (white). The interacting fragments are presented in
black. The S2 and D pockets both bind the P2 and P3 fragment of the inhibitor
respectively. The interactions are mainly hydrophobic in character and include the side
chains of Tyr60A and Trp60D in the S2 pocket as well as Ile174, Trp215 and Leu99 in
the D pocket. The S1 / P1 interaction is a very strong ionic bond between the side chain
amidine moiety of the inhibitor arginine-chloromethylketone and the Asp189 carboxylic
function. Hydrogen bonds are also established between inhibitor and α-thrombin, e.g.
Phe-NH2 ··· O=C-Gly216 (HB1), Arg-NH ··· O=C-Ser214 (HB2) and His57-N ···
CH2C(Arg)(OH) ··· O-Ser195 (HB3, a hemiacetal formed by two covalent bonds with
the arginine chloromethylketone).
S2
HB3
P2
P3
HB2
D
HB1
P1/S1
Figure 3.7. Schematic of PPACK bound to α-thrombin active site. For details see text.
*
A search in the MDL® Available Chemicals Directory (http://www.chemweb.com/databases) reveals
that there are approximately 4500 primary amines, 2500 secondary amines, 9500 carboxylic acids
and 500 acid chlorides with a molecular weight below 250 g/mol that are commercially available.
Using only 10 % of these will make a possible library of above half a million or half a billion
compounds for 2 or 3 points of diversity respectively.
37
3.2.2 The leads
By reducing structural information from known inhibitors into pseudo-structures that
display the important key features, new leads can be generated (Figure 3.8). From these
pseudo-structures new targeted libraries can emanate whose potential will be evaluated
and added to the total knowledge base for a specific target. This iterative process, if
successful, eventually generates inhibitors with drug potential. Out of the eight proline
mimicking scaffolds presented, detailed analysis of the cyclopropane scaffold (B) and
the N4-amino-morpholinone scaffolds (C1 and C2) with numerous substituents are
included in this thesis. Design, synthesis and activity for the templates D1 and D2 has
been discussed by Dahlgren at al.39 and templates E, F and G has been discussed by
Thorstensson et al.40
O
R
O
X
O
N
O
N
O
N
R
N
O
O
D1
N
O
O
X=C, N
D2
O
N
N
O
N
N
O
C2
N
N
O
N
N
O
N
O
E
O
=single or double bond
n=0, 2, 3
N
O
C1
n
O
N
O
N
O
N
O
F
N
N
O
O
N
N
O O
B
G
Figure 3.8. Deduction of six scaffolds from two general pseudo-structures. The boxes show which
scaffolds that are discussed in this thesis.
3.2.3
Mimetics of D-Phe-Pro-Arg
The choice of arginine mimetic is restricted by the
very strong S1 / P1 bidentate electrostatic
interaction, that is crucial for the function of this
class of inhibitors. Indeed P1 in known αthrombin inhibitors often include an amidine type
structure41. In this design 4-aminomethylbenzamidine (Pab)42 (Figure 3.9) was used.
H2N
NH2
NH
Figure 3.9. 4-Aminomethyl-benzamidine
(Pab), the positively charged P1 fragment
common for all presented α-thrombin
inhibitors.
The proline isoster should enable interaction with the hydrophobic S2 pocket in αthrombin. Three distinct structural motifs as discussed in chapter 3.2.2 were chosen. For
each scaffold, numerous hydrophobic P3 substituents were selected to interact with the
distal pocket (D) and for their ability to obtain hydrogen bonds in analogy with Dphenylalanine in the tripeptide.
38
3.3
Initial attempts – the cyclopropane scaffold
1,2-Disubstituted cyclopropanes have a very rigid framework lacking the flexibility
displayed by larger ring systems. Preliminary studies showed potential for this scaffold
with α-thrombin activities in the low µM range and therefore a more thorough
investigation was onset with the production of a targeted library.
3.3.1 Synthesis of the cyclopropane scaffold
Enantiomerically pure cyclopropane-1,2-dicarboxylic acid can be generated by
asymmetric alkylation of succinic acid and demonstrates the usefulness of a chiral
auxiliary [Unpublished results]. α-Alkylation of a succinic acid template would give a
racemic mixture of preferably trans diacid (Scheme 3.4). However, a chiral auxiliary
predominately produces one of the diastereomers43 that can be recrystallized to optical
purity of above 99 %.
Scheme 3.4.
The use of chiral auxilarya to produce enatiomerically enriched material is shown.
O
O
X
X
O
No chiral auxillary, X
O
O
X
O
Xc
Xc
Alkylation
Xc
Xc
X
X
X
X
O
O
O
1:1 mixture of trans enatiomers plus
some meso-cis form.
O
O
O
O
X
Alkylation
Xc
Xc
Xc
Xc
O
O
O
96:4 mixture of trans diastereomers plus
some cis form.
a
Chiral auxillary, Xc=(+)-Menthyl
Succinic acid anhydride was condensed with (+)-menthol in high yield (95 %) using a
Dean Stark apparatus (Scheme 3.5). The dienolate was formed by treatment with 2
equivalents of lithium 2,2,6,6-tetramethylpiperidide.
Scheme 3.5a. Synthesis of the cyclopropane scaffold.
O
O
O
O
O
i
O
ii
O
O
O
O
O
12 (95 %)
13 (26 %)
11
Reagents and conditions: (i) (+)-Menthol, (±)-10-camphorsulfonic acid, reflux. toluene; (ii) LTMP,
CH2BrCl
a
39
Upon treatment with the electrophile bromochloromethane, addition was achieved on
the side with least steric shielding which in the case of the dienolate of (+)-menthylester
predominately results in the cyclopropane with the R,R configuration.
3.3.2 Derivatization of cyclopropane scaffolds
Ideally derivatization is achieved with minimum effort starting from a common
platform. When a library of similar compounds is needed, the total effort will be less if
divergence can be introduced late in the reaction scheme. Since reaction steps might
differ in yield, ease and purity there isn’t an absolute correlation between minimum
effort and number of steps from the common platform. In this case two different
approaches was implemented (Scheme 3.6).
Method A. First the di-(+)-menthylester 13 was monohydrolysed using sodium
hydroxide in hot isopropanol. The carboxylate was used after purification as the
common starting point for the diverse set of substances. Creation of the first amide bond
using HATU / DIPEA / DMF and a diverse set of amines was followed by the second
ester hydrolysis. After purification the PabZ was coupled using HATU and the synthesis
converged with 17 of method B.
Method B. The di-(+)-menthyester (13) was first transesterfied to the dimethylester
followed by mild mono hydrolysis using 0.9 eq. of lithium hydroxide in dioxane / water
which after extraction resulted in a mixture of the diacid and the monomethylester (18).
PabZ was coupled using HATU after which a gentle hydrolysis employing lithium
hydroxide achieved 20 as the common platform for diversity. The second amide
coupling with a diverse set of amines formed the converging products 17.
The main difference between method A and B is the ester used. Method A uses the (+)menthyl ester, which makes it easy to visualize on tlc and easy to purify by column
chromatography. The ester hydrolysis conditions however, are too harsh to be
performed in the presence of the Z-protected amidine. Therefore the first amide
coupling must be the diverse, and the total steps from the common platform to final
products are three.
Method B however employs the methyl ester that can be hydrolyzed under mild
conditions. Therefore the second amide coupling can be the diverse, and the number of
steps from the common platform is reduced to one. Drawbacks to this scheme are that
no effective purification strategy was found for the steps 13-19, and thus the yield of
this path is somewhat erratic. The physical properties of 18, 19 and 20 are also adverse
being very polar and hard to purify using the straight phase column chromatography,
which for many organic chemists can be seen as the purification method of choice.
The conclusion is that neither of method A or B could be seen as more advantageous
than the other. Thus both were used as appropriate.
40
Scheme 3.6a. Derivatization of cyclopropane scaffolds.
O
O
O
v
OLi
OLi
+
O
Method B
vi, vii
O
O
O
O
O
O
vii
NH
NH
NH2
19
(57 %)
20
(100 %)
Xc
v
iii, iv
NR1R2
NR1R2
O
O
ZN
NH2
O
OH
NH
O
ZN
O
NR1R2
ii
O
O
14 (79 %)
O
OLi
ii
OH
13
(14 %)
Xc
Xc
OLi
18 (60 %)
O
Method A
i
Xc
O
16a-e
(92-95 %)
15a-e
(59-82 %)
ZN
NH2
(+)-methyl
Xc =
17a-h
(70-90 %)
O
a
Reagents and conditions: (i) NaOH, reflux. iPrOH; (ii) HATU, DIPEA, NHR1R2, DMF; (iii) NaOH, hot
PrOH / water; (iv) HCl(aq); (v) HATU, DIPEA, PabZ×2HCl, DMF; (vi) reflux. MeOH, H2SO4. (vii)
LiOH in dioxane / water.
i
The final targets were synthesized from 17a-h by hydrogenation (Scheme 3.7). After
purification using preparative HPLC eight compounds (21a-h) were isolated as acetic
acid complexes. Method A was used for 21a-e and method B for 21f-h.
Scheme 3.7a. Release of final products.
O
O
NR1R2
NR1R2
viii
NH
O
NZ
O
NH
O
NH2
17a-h
a
O
NH2
Index
R1
a; d
Et; H
b; c
Et; H
R2
O
NH2
21a-h (50-100 %)
Reagents and conditions: (viii) Pd-C (10 %), EtOH, H2, preparative
HPLC MeOH / H2O / AcOH.
e
H
f
g
h
H
H
H
Et
Bn
i
Bu
O
3.3.3 Inhibitor evaluations
All compounds were screened for α-thrombin activity. It was disappointing to see that
none of the inhibitors reproduced the low µM activity. The best substrate was 21b
having an IC50 value of 9µM. Reinvestigation by in silico docking revealed a plausible
explanation for the lack of activity (Figure 3.10). In comparison with proline the
directional vectors of the two bonds protruding from the ring are quite different in
cyclopropane. This renders it almost impossible for the P3 hydrophobic fragment to
make favorable interactions with the distal pocket. Furthermore it limits the possibility
for a hydrogen bond between the P2-P3 linkage and Gly216 to be established.
41
O
NH
NH
NH2
O
NH2
21g
Figure 3.10. Computer generated picture of 10 best fits calculated for inhibitor 21g in α-thrombin active
site. The surface structure of α-thrombin binding site is a “true” model based on X-ray crystallographic
data, while the inhibitor conformations are computer generated.
This is in agreement with other published α-thrombin inhibitors that utilizes rigid P2motifs and can be summarized by the P2-directional vector axiom that states that for a
potent α-thrombin inhibitor the directional vector of the P2-framework should obey the
following rules:
An sp3 atom or occasionally in certain series an sp2 atom for the group moving into the
S1 pocket.
An sp2 atom for the group moving into the D pocket.
In some cases an sp2/sp2 combination can be viable but only when substituents are in a
meta-type correlation and never for an ortho-type correlation.
Also reevaluation of the initial low µM activity showed that results were misinterpreted
due to solubility issues. The actual activity should be interpreted as >1.3µM and thus, as
the potential for the cyclopropane scaffold in α-thrombin inhibitors was considered
limited, evaluation of other scaffolds were prioritized.
3.4
The story continues – the N4-amino-morpholinone scaffolds
By expanding the set of scaffolds with N4-amino-morpholinones we were hoping to
reestablish the lost hydrogen bond with Gly216 by introduction of a lone pair located on
the hydrazone N4 nitrogen [Paper IV]. The directional vectors are also more in
alignment with what is expected to fit nicely into the binding pockets of α-thrombin.
42
3.4.1 Synthesis of N4-amino-morpholinone scaffolds
Natural L-malic acid (S-form), and the unnatural D-malic acid (R-form) are
commercially available chemicals. Starting with the very cheap (S)-malic acid the (S)form of the scaffold (C2, p.38) will result. Initially esterfication was achieved using
thionylchloride in methanol, followed by O-allylation employing allylbromide in Ag2O
/ toluene to afford 24S in excellent yields (Scheme 3.8). Diol formation of the olefin
was achieved with osmiumtetraoxide. The diol was then oxidatively cleaved using
periodate to afford the aldehyde 25S in 82 % yield. Reductive alkylation of the aldehyde
with BOC-protected hydrazine in two steps was followed by lactamization to afford
28S. Worth mentioning is that only extremely slow (days) lactamization was seen using
refluxing toluene with or without triethylamine. When hot DMF was used as solvent
some unexplored side reaction was dominating. Surprisingly however is that when hot
or refluxing water was used for lactamization, not only did the reaction take place in
high yield and in a short period of time (hours), it was also accompanied by the
desirable BOC-group deprotection.
Scheme 3.8a. Synthesis of N4-amino-morpholinone scaffold.
O
OH O
i
OH O
HO
O
OH
O
t-BOC
N
H
N
H
O
O
vi
O
O
26S (78 %)
N
H
H
N
O
O
O
24S (84%)
t-BOC
O
O
O
O
O
23S (91%)
22S
iii, iv
O
O
O
O
v
ii
O
H
25S (82 %)
H
O
O
O
O
vii
O
O
27S
H2N
N
O
O
O
28S (89 %, two steps)
Analogous for 22R to 28R
a
Reagents and conditions: (i) SOCl2, MeOH; (ii) allyl bromide, silver(I) oxide, toluene; (iii)
osmium(VIII) oxide, N-methyl morpholine-N-oxide, THF / H2O 3:1; (iv) sodium periodate, THF / H2O
3:1; (v) hydrazine carboxylic acid tert-butyl ester, toluene, 65 ºC; (vi) H2 / Pd-C, THF; (vii) reflux H2O.
The R-form of the scaffold (28R, C1) was synthesized using the same procedure, but
starting from the unnatural (R)-malic acid.
3.4.2 Derivatization of N4-amino-morpholinone scaffolds
The N4-amino-morpholinone nucleophilic amine of scaffolds 28S and 28R were reacted
with different electrophiles (Scheme 3.9). Reaction with bromobenzene and 3bromonitrobenzene was achieved employing palladium-catalyzed aryl aminations as
described in chapter 3.1.1 (Table 3.1). Reactions with acylchlorides and
sulfonylchlorides were performed in pyridine to achieve the amides and sulfonamides
respectively, and reactions with phenylisocyanate and aldehydes were performed in
43
toluene, forming the urea and imines correspondingly. The imines were further reduced
by catalytic hydrogenation to form the alkylated products.
Scheme 3.9. Derivatization, general strategy.
R, S or racemic
R or S
O
H2N
N
electrophile
(R-X)
O
O
O
28S or 28R
O
R
N
R or H
N
O
O
O
29{a-d,f-r,t-u}
Table 3.1a. Derivatization of the N4-amino-morpholinone scaffolds.
Cond.a Reagent
R1
viii 3-Bromonitrobenzene
3-Nitrophenyl
R2 Productb
H
29a
H
29b
viii Bromobenzene
Phenyl
=R1 29c
ix Benzoylchloride
Benzoyl
H (R)-29d
(R)-29f
ix Benzenesulfonyl chloride
Benzenesulfonyl
H
(S)-29f
ix Phenylacetyl chloride
Phenylacetyl
H (R)-29g
(R)-29h
ix Phenylmethanesulfonyl chloride
Phenylmethanesulfonyl
H
(S)-29h
x Phenylisocyanate
N-Anilinocarbonyl
H (R)-29i
ix Isopropylchloroformate
Isopropoxycarbonyl
H (R)-29j
(R)-29k
x, xi Phenyl-acetaldehyde
Phenethyl
H
(S)-29k
H (R)-29l
x, xi 3-Phenyl-propionaldehyde
3-Phenyl-propyl
=R1 (R)-29m
H (S)-29l
(R)-29n
x, iv Benzaldehyde + Benzaldehyde dimethylacetal Benzyl
H
(S)-29n
xii benzylbromide
Benzyl
=R1 (S)-29o
ix 2,5-Dimethoxybenzenesulfonyl chloride
2,5-Dimethoxybenzenesulfonyl
H (S)-29p
ix 2,4-Difluorobenzenesulfonyl chloride
2,4-Difluorobenzenesulfonyl
H (S)-29q
ix 4-Chloro-2,5-dimethylbenzenesulfonyl chloride 4-Chloro-2,5-dimethylbenzenesulfonyl H (S)-29r
ix 2,3-Dihydro-benzofuran-5-sulfonyl chloride 2,3-Dihydro-benzofuran-5-sulfonyl H (S)-29t
x, xi p-Tolyl acetaldehyde
2-p-Tolyl-ethylamino
H (S)-29u
a
Reagents and conditions: (viii) Pd(II) acetate, XANTPHOS, toluene; (ix) pyridine; (x) toluene; (xi) H2 /
b
Pd-C, THF; (xii) DIPEA, NaHCO3, LiI, DMF. Scaffold 28S was used for racemic and S configured
products. Scaffold 28R was used for R configured products.
Ester hydrolysis employing lithium hydroxide in methanol / water was followed by
coupling with PabZ using HATU and DIPEA in DMF (Scheme 3.10). Deprotection by
hydrogenolysis was followed by purification using preparative HPLC to isolate the
acetate salt.
44
Scheme 3.10a. Introduction of P1-moety in N4-amino-morpholinone derivatives.
O
X
N
Y
O
O
xiii
N
X
O
O
29{a-r,t-u}b
O
N
N
Y
OLi
O
30{a-r,t-u}
xiv
O
X
N
Y
O
O
N
O
N
H
O
O
xv
c,d
NH2
X
N
Y
N
O
O
N
H
Z
N
NH2
NH2
32{a-u}
31{a-r,t-u}
a
Reagents and conditions (xiii) 3 eq. LiOH in MeOH / H2O; (xiv) PabZ, HATU, DIPEA, DMF; (xv) H2 /
Pd-C, preparative HPLC in MeOH / H2O / AcOH. b(R)-29e was synthesized by other strategy, described
in paper IV. cThe aromatic nitro-group of 31a is reduced to amine in this step. dChlorine in (S)-31r is
partially cleaved by hydrogenation leading to (S)-32r and (S)-32s.
The resulting library contained 26 different compounds as potential α-thrombin
inhibitors.
3.4.3 Inhibition data
In parallel with synthesis of new derivatives, enzyme-binding data was acquired. This is
crucial to be able to direct synthetic efforts in an efficient manner and to make the best
use of available resources. Therefore inhibitory properties of all compounds directed to
α-thrombin inhibition were continuously analyzed. A summary of α-thrombin IC50values for N4-morpholinone substances are shown in table 3.2.
Table 3.2. IC50 for inhibition of α-thrombin.
Structurea
Comp.
identifier
O
32a
H2N
N
H
N
H
Pab
32c
Pab
N
N
O O
S
N
N
H
Pab
54.7
O
O
Pab
O
O
O
N
H
Pab
9.15
Pab
>133
O
O
O
Pab
80.6
O
O
(R)-32k
36.8
O
N
N
37.7
Pab
O
O
O
N
H
>133
O
O
(R)-32j
O
O
O
N
H
Pab
O
O O
S
N
N
H
N
H
2.89
O
N
O
(R)-32i
O
O
Pab
O O
S
N
N
H
(R)-32h
IC50
(μM)
O
O
N
H
105.7
O
O
O
(R)-32g
(S)-32h
Pab
O
N
H
4.9
O
N
O
(R)-32f
34.4
O
Pab
O
N
H
O
O O
S
N
N
H
O
O
(R)-32e
(S)-32f
Structurea
O
N
O
(R)-32d
>32
O
O
N
Comp.
identifier
O
N
O
32b
IC50
(μM)
N
O
Pab
35.6
O
45
Structurea
Comp.
identifier
O
(S)-32k
N
H
IC50
(μM)
O
N
1.79
O
N
H
N
Pab
N
H
N
F
(S)-32q
O
Pab
4.74
Pab
36.2
O
O
(R)-32n
N
H
(S)-32n
N
H
Pab
(S)-32o
NH
a
Pab =
13.6
O
Pab
10.6
O
O
Pab
O
(S)-32t
O
N
N
0.130
O
O
Pab
8.14
O
O
O
Pab
O
O
0.164
O
Pab
O
0.247
O
O
O
N
O O
S
N
N
H
(S)-32s
N
O
O O
S
N
N
H
Cl
O
N
Pab
O O
S
N
N
H
O
O
N
O
O
F
(S)-32r
(R)-32m
O
IC50
(μM)
O
45.4
O
O O
S
N
N
H
(S)-32p
O
O
(S)-32l
O
Pab
O
(R)-32l
Structurea
Comp.
identifier
O O
S
N
N
H
O
N
H
Pab
0.486
Pab
0.961
O
O
(S)-32u
O
N
O
O
19.8
NH2
NH2
.
3.4.4 Structure activity relationships
The N4-amino-morpholinone-scafford based inhibitors first synthesized (32a through
(R)-32m) were all based on the assumption that the preferred stereochemistry would be
(R) as this configuration showed the highest affinities for a previously reported
morpholinone scaffold39(See D1 in figure 3.8 p.38). Since the additional nitrogen in the
new series was believed to introduce possible hydrogen bond interactions with the C=O
of Gly216 in α-thrombin it was disappointing to see that the new compounds with
comparable length of the P3 side chain showed only modest affinity and in no case was
the affinity significantly improved over those of the previous series.
However, docking studies using the Glide44,45 and MacroModel46 modeling software
indicated that structures with the (S) stereochemistry would fit in the active site at least
as well as the (R) form. Thus some (S) form inhibitors ((S)-32{f,h,k,l}) were
synthesized and the affinities were significantly improved showing 4-20 times higher
affinity than the corresponding (R) form.
Based on this a new set of 141 potential inhibitors were enumerated using commercially
available aldehydes and benzenesulfonyl chlorides. Based on virtual docking employing
the Glide software a set of six compounds ((S)-32{p-u}) were selected for synthesis
46
together with two compounds ((R)-32n and (S)-32n) that were selected for comparison
with the previous scaffold39.
Out of the resulting analogs, (S)-32p having a 2,5-dimethoxybenzenesulfonyl in the P3
position, showed an affinity improvement by more than an order of magnitude giving an
IC50 of 0.130 µM. As one can see from the IC50 values, the lipophilic substitution of
benzenesulfonamide derivatives is well accommodated in the distal pocket of αthrombin giving more than an order of magnitude higher IC50 values for ((S)-32{p,r,s};
IC50=0.130; 0.164; 0.247µM) compared to that of the unsubstituted ((S)-32f;
IC50=2.89µM). The more polar 2,4-difluoro benzenesulfonyl seems to make less
favorable interactions with the hydrophobic pocket ((S)-32q; IC50=8.14µM).
To gain more insight on the binding of this series of compounds to the active site of αthrombin, the most potent inhibitor ((S)-32p) was cocrystallized in complex with αthrombin and subjected to X-ray analysis. The Connolly surface map of the α-thrombin
– (S)-32p complex and an outline of the important interactions between the two are
shown in figure 3.11. Analysis of X-ray data show the expected strong salt-bridge
between Asp189 and the strongly basic P1-p-amidinobenzyl with N-O distances of 2.70
and 2.74Å. There is also a weak hydrogen bond between the N-H of the P1-P2 amide
linkage and Ser214 with an N-O distance of 3.15Å. Moreover the morpholinone ring
moves deep into the S2 pocket leading to a significant movement of Tyr60A and
Trp60D compared to that of similar inhibitors47,48,49,50. This makes it impossible for the
NH of the P2-P3 linkage to reach the C=O of Gly216 and as a result no hydrogen bond
could be established. This may also be caused by a potential deprotonation of the NH
leading to unfavorable electrostatic repulsion between the negatively charged nitrogen
and the electronegative C=O of Gly216. Structural elements as these do show depressed
pKa values e.g. N'-acetyl-N'-methyl-benzenesulfonohydrazide have a pKa of 7.85. The
2,5-dimethoxy benzenesulfonyl group fits nicely into the hydrophobic distal pocket with
the 5-methoxy group in a small cavity between Leu99 and Tyr60A.
47
Trp215 Leu99
O
D
Ile174
O
O
Tyr60A
S
O
N
H
O
S2
Trp60D
Asp102
His57
O
Ser214
N
O
O
N
H
H
N
NH2 S1
O
Gly216
NH2
Gly219
O
Asp189
Figure 3.11. An outline of the important interactions between (S)-32p and α-thrombin (top) and the
Connolly surface map of the X-ray structure of the α-thrombin – (S)-32p complex (bottom) at 1.85 Å
resolution.
3.5
Conclusions
The initial library containing cyclopropane scaffolds showed low potency as expected
from the lack of previously reported ortho-sp3-sp3 scaffolds as α-thrombin inhibitors.
The N4-amino-morpholinone scaffold however resulted in several sub-µM binders and
further optimizations might result in compounds with preclinical drug potential.
Even though an additional nitrogen was introduced compared to that of the previously
reported morpholinone scaffold no hydrogen bond with Gly216 could be established for
(S)-32p. If this is due to the partially negatively charged nitrogen, successful
reestablishment of the hydrogen bond might be possible with substrates showing higher
pKa. Also substrates with a distinctly different P3-conformation might not move the
Tyr60A and Trp60D as much, making the Gly216 accessible for hydrogen bonding. An
48
interesting substrate is (S)-32u showing an IC50 of 0.961µM and probably obeying the
two above-mentioned features. If in fact the hydrogen bond is present for this substrate,
further optimizations might result in high affinity inhibitors.
49
50
4
Future prospects and unpublished results
This is a chapter dedicated to some speculations about what could be done and why.
This is my personal thoughts and its scientific plausibility and completeness might be
argued. To reflect this, the language is chosen in many cases to be presented in first
person.
4.1
A possible route to optimizing a peptide conjugate
In one of our less successful projects I was aiming at self-catalyzed selective
modifications of peptides using peptide active esters as substrates. Simple substrates
such as p-nitrophenyl fumarate can be introduced in this way16, but the success of
peptide active esters was limited. Thinking that this might be a sequence dependent
problem, an idea arose to generate a library of peptides, and then to find the most
appropriate by some “fishing out” strategy. Even though the concept failed due to the
fact that the problem wasn’t sequence dependent the method of synthesis is still valid
and could easily be adapted to conjugate optimization as described in chapter 2.3.2. The
idea was that by a slight reprogramming of the peptide synthesis machine to be able to
produce a large number of similar peptides in a combinatorial way.
The original sequence was RYESYGQ with Q being attached to the resin by its side
chain rather than the C-terminus. This allowed post synthetic activation forming a pnitrophenyl ester on the C-terminus.
The synthesis protocol was modified so that instead of supplying the peptide synthesis
machine with Y, S, E, Y for the 3rd, 4th, 5th and 6th synthesis step I supplied it with
equimolar mixtures of the amino acid and alanine. In this way there was not only one
peptide, but actually 24=16 peptides in equimolar amount with amino acid 2, 3, 4, 5
being either alanine or Y, S or E. Usually, to perform an amino acid coupling cycle, a
four-fold excess of Fmoc protected amino acid is used. This was seen as a potential
problem due to the fact that different amino acids couple at different rates. Thus, instead
of four equivalents, initially only one equivalent was used (0.5 equivalent of each amino
acid). After a prolonged coupling time the resin was rinsed and to complete coupling
51
another four equivalents (two of each amino acid) was introduced without prior Fmoc
deprotection.
The protocol was very successful and all expected fragments could be identified on
MALDI. Surprisingly, a simple reverse phase HPLC purification allowed spatial
separation of almost every individual fragment (Figure 4.1).
no yes
15
16
17
18
19
Tyr 973.4(0.1)
TyrTyr 881.4(0.2)
TyrTyrGlu 823.4(0.2)
Absorbance
14
20
21
22
TyrGlu 915.4(0.1)
no yes
yes
no
yes
no yes
23 24 25 26
Time (minutes)
no
27
28
yes
no yes no yes
29
30
31
32
Ser 1049.4(0.1)
SerGlu 991.4(0.4)
no
no
none 1065.4(0.0)
Glu 886.4(0.1)
yes
Tyr 973.4(0.1)
TyrGlu 915.4(0.2) TyrSer 957.4(0.1)
no
one
yes
yes
no
13
one
no
yes
no
Glu->Ala
two
TyrTyrSer 865.4(0.3)
TyrTyrSerGlu 807.4(0.4)
Ser->Ala
TyrSerGlu 899.4(0.2)
TyrSer 957.4(0.3)
TyrSerGlu 899.4(0.3)
Tyr->Ala
33
34
35
Figure 4.1. HPLC chromatogram using a standard reversed phase C8-column. All peaks were identified
on MALDI. For each peak a label indicating which amino acids that are substituted for alanine is shown.
Numbers are theoretical masses (u) and within parenthesis are experimental deviations (u). It is clear that
the substitution Tyr→Ala has greatest impact on reversed phase HPLC mobility. Next is Ser→Ala, and
Glu→Ala has the smallest impact. Also isomers of singly Tyr→Ala substituted peptides are separated.
Optimization of the peptide conjugates could be conceived by an adaptation of the
protocol mentioned above by starting from a TA4 variant, having the sequence of TA4
with the exception of lysine at position 34 being exchanged for alanine. Consider
selecting 6 key points, including position 34 as attachment points for the linker.
Introduction of Mtt protected lysines at 1/6th equivalents in these positions leaving 5/6th
as the original sequence amino acid could be done by the method described above. This
52
would in theory create (1-1/6)6=33%* of the unmodified mutant (TA4-K34A), (11/6)5=40% of mono modified species (6.7% of each peptide) and the rest (26%), would
be poly modified. After selective deprotection of the Mtt groups, benzenesulfonamides
attached to linkers could be introduced on solid phase. Adding 6 different linkers in
equimolar amount first in deficit, then after prolonged reaction time, in excess, would in
principle generate 6 x 6 = 36 different mono modified conjugates available in equimolar
amounts (approx. 1 µmol each for 100 µmol peptide synthesis scale). As the ligands are
mainly hydrophobic it would be plausible to separate the mono modified species from
the rest by reversed phase HPLC.
To what use could this conjugate mix be? There are several fishing-out strategies for
finding the strongest binders that spring to mind. Two of them are:
Mix excess conjugate with HCAII. Separate by centrifugation with a 10000 MW cutoff
filter or by gel filtration chromatography.
Use polymer bound HCAII to enrich the most potent inhibitor.
And to identify the HCAII – conjugate complex one could use:
MALDI in combination with proteolytic digestion.
MS/MS fragmentation studies.
This strategy could also be used to further expand the search for the “perfect fit”.
Choosing the best candidate from the initial ligand length/position optimization one can
start working on peptide structural variations by the same means.
4.2
Selective peptide cleavage N-terminal of cysteine
It would be a great benefit to expand the range of proteases and chemical agents that
selectively cleave peptides or proteins. Well, I did just that – mostly by accident.
In an effort to produce peptides with an ability to transfer acyl groups to a designated
target (such as a protein), we designed a peptide named JWN-CYS1. The peptide is a 42
amino acid residue helix-loop-helix motif containing one lysine residue with an ability
to carry an anchoring group for a specific protein and one cysteine residue with a
potentially volatile thioester that can transfer acylgroups to the target by
transesterfication/transamidation. This doubly acylated species, JWN-CYS1[diBSAC6],
is depicted in figure 4.2. When absence of function was observed, I tried to hydrolyze
*
A general formula of the fraction of product with x modifications when n points are modified to the
n!
n− x x
fraction of p is (1 − p) p x!(n − x)! . If one modification is desired (x=1) the formula is reduced to
(1 − p ) n −1 p ⋅ n and maximal yield is given at the function maximum which is p=1/n. For the example
given where n=6, the optimal fraction of introduced Mtt protected lysine should thus be 1/6th.
53
the formed thioester. Remarkably it withstand both hydroxide (pH=10) and even hot
nuclephiles such as hydrazine at pH=9 without being cleaved.
O
NAADLEAAIRHLAEKLAARGPVDAAQLAEQLACRFEAFARAG NH2
O
O
NH
S
O
O
H2N
H2N
S
S
O
O
N
H
N
H
Figure 4.2. The original peptide JWN-CYS1[diBSAC6].
However when the pH was raised to 12.2, things started to happen after 30 minutes.
Some of the thioester was cleaved as indicated on MALDI, but the major products were
cleaved peptide containing two fragments whose mass was identified to be cleavage
products from cysteine N-terminal cleavage (Figure 4.3). Evidently the found fragments
both contained the original sidechain modification. However it is likely that it has
migrated to the terminal amine forming an amide for fragment carrying the N-terminal
cysteine.
O
NAADLEAAIRHLAEKLAARGPVDAAQLAEQLA OH
O
NH
O
S NH2
CRFEAFARAG NH2
Fragment
O
S NH2
O
NH
O
Formula
O
N
H
C62H91N19O16S2
O
C157H256N46O51S
MALDI
(experimental)
1422
1423
1424
1425
1426
1427
1428
1429
1430
3646
3645
3644
3643
3642
3641
3640
3639
3638
3637
3636
3635
3634
1422
1423
1424
1425
1426
1427
1428
1429
1430
3646
3645
3644
3643
3642
3641
3640
3639
3638
3637
3636
3635
3634
MALDI
(simulated)
Figure 4.3. Cleavage fragments of peptide JWN-CYS1[diBSAC6].
The applications of this might seem limited but it is an unexpected and interesting
feature. Experiments indicate that the cleavage will not occur unless the cysteine is
acylated. This might be taken advantage of to further specify cleavage sites. Concerning
the generality of this procedure, it has been shown to work for at least one other peptide.
54
At these high pH values there will probably be some amino acid racemization but when
used as an analysis tool this might not be important.
55
56
Peptide notations – Quick reference
KE2
TA4
TA4+
JWN-H8
JWN-H9
5
Notations and abbreviations
(Ac-NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-OH)
(Ac-NAADJEAKIRHLAEKJAARGPVDAAQJAEQLARKFEAFARAG-NH2)
(Ac-NAADJEAKIRHLREKJAARGPRDAAQJAEQLARKFERFARAG-NH2)
(Ac-NAADLEAKIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-NH2)
(Ac-NAADJEAAIRHLAEKJAARGPVDAAQJAEQLAKKFEAFARAG-OH)
[J=Norleucine]
Substructure
Not. Modification
C0
5.1 Peptide notations
In general peptides are named as NAME-M(pos)-M(pos)
NAME is the peptide abbreviation, which can be one of:
(Ac-NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-OH)
KE2
(Ac-NAADJEAKIRHLAEKJAARGPVDAAQJAEQLARKFEAFARAG-NH2)
TA4
(Ac-NAADJEAKIRHLREKJAARGPRDAAQJAEQLARKFERFARAG-NH2)
TA4+
JWN-H8 (Ac-NAADLEAKIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-NH2)
JWN-H9 (Ac-NAADJEAAIRHLAEKJAARGPVDAAQJAEQLAKKFEAFARAG-OH)
[J=Norleucine]
M(pos) indicates sidechain modification at position pos with modifier M, where M
can be:
DNS (Dansylation)
DAB (Dabcylation)
C0 (4-Sulfamoyl-benzoylation)
C2 (2-(4-Sulfamoyl-benzoylamino)-acetylation)
C4 (4-(4-Sulfamoyl-benzoylamino)-butyrylation)
And analogous for C6, C7, C8 and C11.
Modifications of peptides KE2 and TA4 are mentioned in this thesis. The
modifications are DNS or DAB (position 15, shaded in light gray) and C0, C2, C4,
C6, C7, C8 or C11 (position 34, shaded in dark gray).
C2
C4
C6
C7
C8
C11
O
H2N S
O
O
H2N S
O
O
H2N S
O
4-Sulfamoyl-benzoylation
2-(4-Sulfamoyl-benzoylamino)-acetylation
4-(4-Sulfamoyl-benzoylamino)-butyrylation
O
7-(4-Sulfamoyl-benzoylamino)-heptanoylation
8-(4-Sulfamoyl-benzoylamino)-caprylation
11-(4-Sulfamoyl-benzoylamino)-undecanoylation
OO
S
HN
HN
O
O
O
HN
O
N
OO
S
O
HN
O
HN
O
O
H2N S
O
O
HN
OO
S
O
NH
HN
O
NH
Ac-NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-OH
KE2-DNS(15)-C6(34)
57
DAB (Dabcylation)
O
Sample Conjugate
H2N
N
O
O
H2N S
O
O
H2N S
O
O
H2N S
O
6-(4-Sulfamoyl-benzoylamino)-hexanoylation
DNS (Dansylation)
O O
O
N
N
N
α-Thrombin substrates – Quick reference
N
H
NH2
NH
Pab
O
O
O
O
Pab
N
O
O O
N
S
N
H
21b
O
O
Pab
N
H
O
O O
N
S
N
H
21c
21d
N
O
Pab
O
O
(R)-32m
O
N
H
O
O
O
N
Pab
O
O
O
O
Pab
N
H
21e
O
O
Pab
N
H
21f
21g
O
(R)-32h
O
O
O
Pab
O
F
O
Pab
O
(R)-32i
21h
O
O
O
N
H
O
32a
N
H
32b
N
H
Cl
O
(S)-32q
O
(R)-32k
Pab
O
(S)-32k
O
Pab
O
O
32c
N
H
O
(R)-32l
Fold out again for peptide notations
(R)-(+)-2,2'-Bis(diphenylphosphino)-1,1'binaphthyl
BOC
tert-Butoxy carbonyl protection group
BOC-ON
2-(tert-Butoxycarbonyloxyimino)-2phenylacetonitrile
Dabcyl group
NAME is the peptide abbreviation, which can be one of:
(Ac-NAADLEAAIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-OH)
KE2
(Ac-NAADJEAKIRHLAEKJAARGPVDAAQJAEQLARKFEAFARAG-NH2)
TA4
(Ac-NAADJEAKIRHLREKJAARGPRDAAQJAEQLARKFERFARAG-NH2)
TA4+
JWN-H8 (Ac-NAADLEAKIRHLAEKLAARGPVDAAQLAEQLAKKFEAFARAG-NH2)
JWN-H9 (Ac-NAADJEAAIRHLAEKJAARGPVDAAQJAEQLAKKFEAFARAG-OH)
[J=Norleucine]
DCC
N,N'-Dicyclohexylcarbodiimide
DCM
DIPEA
Dichloromethane
N,N-Diisopropylethylamine
DMF
N,N-Dimethylformamide
DMPU
1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone
M(pos) indicates sidechain modification at position pos with modifier M, where M
can be:
DNS (Dansylation)
DAB (Dabcylation)
C0 (4-Sulfamoyl-benzoylation)
C2 (2-(4-Sulfamoyl-benzoylamino)-acetylation)
C4 (4-(4-Sulfamoyl-benzoylamino)-butyrylation)
And analogous for C6, C7, C8 and C11.
DNS
Dansyl group
Modifications of peptides KE2 and TA4 are mentioned in this thesis. The
modifications are DNS or DAB (position 15, shaded in light gray) and C0, C2, C4,
C6, C7, C8 or C11 (position 34, shaded in dark gray).
LDA
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'tetramethyluronium hexafluorophosphate
Lithium diisopropylamide
LTMP
Lithium 2,2,6,6-tetramethylpiperidide
NMM
N-Methyl morpholine
4-Aminomethyl-benzamidine
also known as p-amidinobenzylamine
[Amino-(4-aminomethyl-phenyl)methylene]-carbamic acid benzyl ester
HATU
Pab
PabZ
O
Pab
O
O O
N
S
N
H
O
PS/DVB
(CH2)xCH3
N
H
N
O
(S)-32t
O
Pab
O
(S)-32u
x=7-9
N (CH2)xCH3
(CH2)xCH3
Cl
P
P
O
O
O
N
O
O
CN
N
N
N
O
N
N
C
CH2Cl2
NEt(iPr)2
O
N
H
O
N
N
OO
S
N N
N
N
N
N
O
N
F
F
F
P
F
F
F
Li+ -N(iPr)2
Li
N
N
O
NH
H2N
NH2
H2N
N
O
NH2 O
Polystyrene/divinylbenzene copolymer
n
pyBOP
(Benzotriazol-1-yloxy)tripyrrolidino
phosphonium hexafluoro phosphate
TEA
TFA
TFMSA
THF
Triethylamine
Trifluoroacetic acid
Trifluoromethanesulfonic acid
Tetrahydrofuran
Pab
O
O
Pab
BINAP
DAB
O
O
N
Structure
(S)-32s
O
N
O
(S)-32r
O O
N
S
N
H
Pab
O
O
Pab
O
O
Pab
O
O O
S
N
N
H
O
N
O
Pab
N
O O
N
S
N
H
O
N
O
Pab
(R)-32j
O
Pab
O
O
O
O
O
N
O
Pab
O
(S)-32p
F
O
N
O O
N
S
N
H
O
(S)-32h
N
O
Pab
N
H
N
(S)-32o
Pab
O
N
H
N
H
O
O
O
N
H
O
O
Pab
N
H
N
H
O O
N
S
N
H
O
O
H2N
O O
N
S
N
H
5.1 Peptide notations
In general peptides are named as NAME-M(pos)-M(pos)
(S)-32n
O
N
O
Pab
Pab
(R)-32g
Notations and abbreviations
(R)-32n
N
O
N
5
Pab
O
N
H
O
N
O
(S)-32f
O
Chemical Name
Methyltrialkyl(C8-C10)ammonium
Adogen 464
chloride
O
N
Pab
O
N
H
Pab
N
H
O
(R)-32f
O
Reagents and substructures
Abbrev.
Pab
O
5.2
(S)-32l
O
(R)-32e
O
O
O
O
Pab
O
Pab
O
O
N
(R)-32d
N
21a
N
H
Pab
O
O
N
H
O
O
O
N
O
Pab
N
O
O
NH2
This is a quick reference fold-out page. Please fold out one time
for α-thrombin substrates and two times for peptide notations.
57
58
N N
N N
N O P
N
F
F
F
P
F
F
F
NEt3
CF3COOH
CF3S(O2)OH
O
Abbrev.
Chemical Name
TMSOK
Potassium trimethylsilanolate
XANTPHOS
9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene
Z
Benzyloxy carbonyl protection group
Structure
K+ -O Si
O
P
P
O
O
59
60
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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Acknowledgements
Thank you…
Lars Baltzer, my main supervisor during the last three years, for sharing your
knowledge and enthusiasm with me, and for the wonderful espresso.
Ingemar Kvarnström my main supervisor during the “thrombin era” (1998-2002), for
sharing your great knowledge in organic synthesis with me.
Ingemar Nilsson, for all help, and for giving me of your time when you had little to
spend. Thank you for your support and helpfulness when I was stationed at AstraZeneca
in Mölndahl.
Bertil Samuelsson, for novel ideas about synthetic strategies.
For participation in joint projects with me…
Fredrik Thorstensson for sharing my first PhD time and publication, for being a friend
and for your fierce persuasion into hanging out on bars instead of staying home with my
family.
Karin Enander, our group’s fluorescence pioneer, for sharing paper I with me and for all
the interesting discussions.
Martin Lundquist, NMR in paper I, and for my whisky connoisseur training.
Gunnar Dolphin, for sharing paper I with me and for always being so willing to help.
Tess Andersson for sharing paper I with me, and your positive attitude.
Nalle Jonsson for biochemistry discussions.
Djordje Musil, for doing the X-ray crystallography in paper IV.
Tudor Oprea, for your input on paper III.
65
Special thanks for helps on particular matters…
Shenghua Huang and Elisabeth Sauer-Eriksson (conjugate – HCAII complex
crystallization attempts)
Johanna Deinum, Karolina Åkesson and Olle Karlsson (Thrombin screening)
Karl-Erik ”Kirre” Karlsson (High Resolution MS)
Shalini Andersson (chiral HPLC)
Stefan Svensson and Per Hammaström (for oral discussions)
Thanks to present and former colleagues not mentioned above. For sharing discussions,
knowledge and free time activities with me…
Alina, Andreas, Cissi, Gunnar H., Helena, Janosch, Jesus, Johan R., Johan V., Kerstin,
Klas, Laila, Lotta, Maria, Patrik, Sofia and Susanne in the espresso corridors.
Anders, Daniel, David, Per-Ola, Åsa in the old organic chemistry.
I have the great opportunity to share vacations, and weekends with my wonderful
family…
My parents, Kenneth and Gunnel and my siblings Fredrik and Jenny and their families.
And finally for your love and support, and for giving my life purpose…
My beloved sons Adam and Erik and my Lotta.
Thank you all!
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