Faculteit der Natuurwetenschappen, Wiskunde en Informatica
Bachelor Scheikunde
Functional Mimicry of Discontinuous
Protein Binding Sites by CLIPS- Chemistry
and Thiol-ene Reaction
Nathalie Bianca Vos
March 2011
MSc. Linde Smeenk
Dr. J.H. van Maarseveen
Prof.dr. P. Timmerman
Prof.dr. H. Hiemstra
Dr. S. Ingemann Jorgensen
Summary
To mimic discontinuous active sites, small peptide chains will be attached to scaffolds.
By coupling these scaffolds, the peptide chains are brought together in a way that they
can mimic the active site.
The peptide chains were attached to the scaffolds by CLIPS-chemistry1. To couple the
two scaffolds, the use of the thiol-ene reaction was examined. Both the thermal and the
photochemical thiol-ene reaction were tested.
The synthesis of the two required scaffolds was successful. It was possible to attach a
peptide loop to both scaffolds by CLIPS-chemistry.
The thermal thiol-ene reaction seemed not a good ligation technique because the
reaction did not reach good conversions after five hours. The reaction was oxygen
sensitive and under the current tested conditions the thermal thiol-ene reaction is not
recommended for further use in the coupling of the two scaffolds.
The UV-initiated thiol-ene reaction proved to be promising as a ligation method. After
a half hour, a good conversion was achieved. The reactions worked at a concentration of
1mM and room temperature.
Samenvatting
Om onderbroken active sites te kunnen namaken, worden kleine peptideketens op
scaffolds gezet. Door deze scaffolds aan elkaar te koppelen, worden deze peptideketens
naar elkaar gebracht zodat zij de active site kunnen nabootsen.
De peptideketens worden op de scaffolds gezet door middel van CLIPS-chemie1. Voor
het aan elkaar koppelen van de twee scaffolds wordt het gebruik van de thiol-enereactie
onderzocht. Zowel de thermische als de fotochemische thiol-enereactie worden getest.
De synthese van de twee benodigde scaffolds was succesvol. Het was mogelijk om
een peptideketen op beide scaffolds te zetten door middel van CLIPS-chemie.
De thermische thiol-enereactie bleek geen goede ligatietechniek aangezien er na vijf
uur nog geen goede omzettingen waren. De reactie was zuurstofgevoelig en onder de
huidig geteste condities niet aan te raden voor verder gebruik in het koppelen van de twee
scaffolds.
De UV-geïnitieerde thiol-enereactie bleek een veelbelovende ligatiemethode te zijn.
Na een half uur bleek er een goede omzetting te zijn. De reactie bleek bij een concentratie
van 1mM en kamertemperatuur te werken.
2
Populaire samenvatting
Eiwitten zijn heel belangrijk voor levende mensen, dieren en planten. Heel veel
processen die in levende wezens plaatsvinden, hebben te maken met eiwitten. Wanneer
deze eiwitten afgebroken moeten worden, maakt het lichaam antilichamen aan. Deze
antilichamen binden aan het eiwit op een plek die de ‘active site’ wordt genoemd.
Wanneer het antilichaam aan het eiwit gebonden is, wordt het eiwit afgebroken. Op deze
manier verweert het lichaam zich onder andere tegen eiwitten van ziekteverwekkers.
Het is gebleken dat wanneer de active site wordt nagemaakt, een antilichaam nog
steeds zich eraan kan hechten. Dit is heel handig, want wanneer er een nagemaakte active
site van een eiwit wordt gevaccineerd, zal het lichaam antilichamen maken om deze stof
af te breken.
Het probleem met het namaken van de active sites is dat het vaak heel moeilijk is. Er
zijn vaak meerdere verschillende stukjes van het eiwit nodig om de active site te kunnen
namaken. Deze stukjes, die uit peptiden bestaan, moeten bij elkaar gebracht worden zodat
ze heel erg lijken op de echte active site. Om dit te doen worden de stukjes peptiden op
stofjes gezet die we scaffolds noemen. De reactie die hiervoor zorgt wordt een CLIPSreactie genoemd1. Nadat de stukjes peptiden op een scaffold zijn gezet, worden de twee
scaffolds aan elkaar gezet.
In dit onderzoek is onderzocht of deze twee scaffold aan elkaar kunnen worden gezet
door een reactie die de ‘thiol-enereactie’ wordt genoemd. Deze reactie kan op twee
manieren werkend gemaakt worden, namelijk door te verwarmen of door UV-licht te
gebruiken.
Het bleek dat het mogelijk was om de scaffolds te maken en de peptiden eraan vast te
maken. De thiol-enereactie waarbij de reactie verwarmd werd bleek niet zo goed te
werken, aangezien de reactie na vijf uur nog niet zo ver verlopen was. Ook bleek de
reactie heel gevoelig voor zuurstof te zijn.
De UV-reactie bleek heel goed te werken. In een half uur was de reactie bijna
helemaal afgelopen. De reactie hoefde niet verwarmd te worden en kon bij hele lage
concentraties worden gedaan.
3
4
Contents:
Summary
Samenvatting
Populaire samenvatting
1. Introduction
1.1 Proteins and their active sites
1.2 Mimicking proteins and active sites
1.3 Antibodies
1.4 Mimics in vaccinations
1.5 Mimicking discontinuous active sites
1.6 CLIPS-chemistry
1.7 Scaffolds
1.8 Requirements coupling techniques
1.9 Previous studies
1.9.1 Click reaction
1.9.2 Oxim formation
1.10 Thiol-ene reaction
1.11 Aim of the project
2. The thiol-ene reaction
2.1 Oxygen free environment
2.2 Thiol-ene test reaction
2.3 Initiators
2.3.1 Thermal initiator
2.3.2 Photochemical initiator
3. Results
3.1 Synthesis of alkene scaffold
3.2 Synthesis of trityl protected thiol scaffold
3.3 CLIPS-reaction
3.4 Thiol-ene reaction
3.4.1 Products found
3.4.2 Thermal thiol-ene reaction
3.4.3. Photochemical thiol-ene reaction
4 Conclusions
5. Future prospects
6. Acknowledgements
7. Experimental
8. References
p. 2
p. 2
p. 3
p. 7
p. 7
p. 8
p. 8
p. 8
p. 8
p. 9
p. 9
p. 10
p. 10
p. 10
p. 11
p. 11
p. 11
p. 12
p. 12
p. 12
p. 13
p. 13
p. 13
p. 14
p. 14
p. 15
p. 16
p. 16
p. 16
p. 17
p. 19
p. 21
p. 22
p. 23
p. 24
p. 28
5
6
1. Introduction
Where would our life be without peptides? Presumably, we would not even have lived.
Peptides play an important role in all living organisms. In all of our body cells, peptides
are present as proteins, enzymes or for instance a class of hormones. These peptides are
the driving force behind many important processes for living organisms, like the
respiration process and the digestion process.
Knowing the importance of peptides, it is not hard to imagine that the presence or
absence of some peptide can influence the health of an organism. Reactive peptides or
lacking a specific kind of peptide can lead to illnesses. In the same way, peptides can
enhance the health of an organism. For this reason, peptides are often used in medicines
and vaccinations.
The world of chemistry is also concerned with these peptides. Many research is done
to develop or improve these medicines, and the results of these studies are of great value2.
Many of the peptides involved in this field of research are synthesized by chemists, and
therefore are called synthetic peptides.
1.1 Proteins and their active sites
The peptides involved in medicines are often proteins, a long folded peptide. A protein
is a large molecule, but only a small part of the protein is involved in the reactions it
undergoes. This part of the molecule is called the ‘active site’. At this side, a specific
reaction takes place. The molecule that reacts with the protein, the antibody, fits in the
active site and binds to it (see Scheme 1). This is often explained as the lock and key
model; if a key does not fit exactly in the lock, the door will not open. If the antibody
does not fit in a good way in the active site, it will not bind to it. When an antibody is
bound to a protein, the protein cannot fulfill its function anymore. The antibody can
degradate the protein in
smaller peptides and amino
acids. This is can be
necessary
in
biological
processes. In this way,
proteins play an important
role in biological processes.
Scheme 1: Antibody binds to active site of protein
The part of the peptide chain that forms the active site, does not necessarily have to be
a linear part of the peptide. It is possible, and even quite common, that the active site is
built up from different parts of the peptide chain, that do not lie next to each other. It is
possible that one region of the peptide chain forms a part of the active site, and that
further on the chain another region is also part of the active site. In Scheme 2 for
instance, the red and blue part of the peptide form the active site. When the peptide is
unfolded and linear, it is easy to see that these two parts on
the peptide chain are not next to each other, but separated by
a piece of chain that is not part of the active site. In this
linear chain, the protein is not active, so the two active
pieces of this chain should be brought together to get
biological activity.
Scheme 2: Discontinuous active site
7
1.2 Mimicking proteins and active sites
By mimicking a protein and making some small variations in it, the activity of the
protein can be studied. If the activity drops it can be concluded that the varied part was an
important part for the activity. This is a good way for exploring the ‘properties’ of the
active site. When the active site is known, a mimic can be made. Only the active site is
mimicked, leaving a smaller and easier to synthesize molecule with eventually the same
activities as the natural protein. In this way, the antibody can undergo the same reaction
with the mimicked active site as it would
have done with the natural active site
(compare Scheme 1 to Scheme 3). This
can for instance be quite useful for
studies on the generation of antibodies.
Scheme 3: Antibody bound to mimicked active site
1.3 Antibodies
When a pathogen enters the body, the immune system tries to break the pathogen
down. Therefore the body has to find a compound that will break the pathogen down,
called an antibody. Antibodies recognize a specific kind of pathogen and will only break
down this specific pathogen and some derivatives of it.
The antibodies are not present in the body all the time. When the body needs them,
they are generated. The first time the body meets a specific pathogen, this takes longer
because the body ‘does not know’ yet which compound will destroy the pathogen. When
the body found the right antibody, the immune system reminds which antibody is needed
against which pathogen. When the pathogen comes back a second time, the immune
system recognizes the pathogen and knows immediately which antibody should be
formed to destroy the pathogen.
1.4 Mimics in vaccinations
By mimicking the active site of a pathogen and vaccinating it into the blood, the
immune system will make antibodies which recognize this active site. When the real
pathogen will come into the blood afterwards, the body recognizes the active site in the
same way and immediately makes antibodies to protect itself to the pathogen.
Research is done also at active site mimics which can attack harmful proteins of the
body itself. Some diseases are made from our own proteins, for instance cancer. If it is
possible to mimic the active sites of these proteins, there might be a possibility that
antibodies are synthesized which will break down the pathogen proteins.
1.5 Mimicking discontinuous active sites
As explained earlier, a lot of the active sites are discontinuous. The active site is build
up from two or more pieces of the peptide chain. These discontinuous active sites make it
far more complex to mimic an active site. The mimic cannot be made of one single
peptide. When looked at an active site which exists of two peptide chain parts, the two
regions of interest need first to be synthesized apart from each other. Then they should be
coupled in such a way that they can arrange in the right form, the form of the active site.
Arranging these two small peptide chains in the right conformation is done by using
scaffolds. A scaffold is a molecule on which a peptide can be coupled.
8
When coupled to peptides, the two scaffolds can be coupled to each other, afterwards
making a connection between the two peptides. The peptides will be attached to the
scaffolds by using the in 2005 developed CLIPS-chemistry 1.
1.6 CLIPS-chemistry
CLIPS is an abbreviation voor Chemically LInkage of Peptides onto Scaffolds. The
reaction is developed by Timmerman et al.1 and will be used in this project.
A chain with a specific amino acid sequence is coupled to the scaffolds. Via the
nucleophilic attack of a sulfur atom from a cysteine amino acid at a carbon with a
bromine atom, the peptide is attached to the scaffold. The bromomethyl group is placed
on a phenyl ring. The nucleophilic attack leads to a bromine elimination and the
formation of a carbon sulfur bond. To do this type of reaction, a cysteine amino acid at
the beginning and the end of the sequence is required. In this way, a peptide loop is
formed (see Scheme 4).
Scheme 4: Mechanism of CLIPS-reaction; the orange ball is a schematic peptide
To use the CLIPS-reaction, the use of two cysteine amino acids at both ends of the
peptide is necessary. Thus, when a specific sequence is wanted to set onto a scaffold, the
sequence starts and ends with a cysteine, also when this would not be the place of a
cysteine in the natural peptide. The cysteine is only used for the linkage to the scaffold.
1.7 Scaffolds
To mimic discontinuous active sites, the peptides are coupled to scaffold. Binding
these two scaffolds should result in an active site mimic. These scaffolds should meet a
few requirements.
1. The peptides are set onto scaffolds via CLIPS-chemistry. To be able to do a
CLIPS-reaction, a part of the molecule should exist of a phenyl ring with two
bromomethyl groups, at which the peptide can bind.
2. Many peptide reactions are done in aqueous solutions. The scaffolds should
therefore also be soluble in aqueous solvents. To achieve this, for instance an
ionic charge can be brought in the molecule. Ions are often good soluble in
aqueous solutions.
3. The scaffold should have a part of the molecule that can easily be synthesized
with another functional group. When different functional groups can be easily
attached, the same general scaffold can be used in different reactions, using
different reactive side chains.
4. If it is possible, it would be preferable to use a non-chiral scaffold. Chiral
scaffolds should be isolated from each other, because they give different
enantiomers as a product after the coupling of the scaffolds to chiral peptides.
Assumingly, enantiomers do orientate the two peptide loops differently to each
other, leading to a difference in biological activity. It is hard to separate
9
enantiomers from each other and after the separation, it should be determined
which enantiomer leads to which biological activity. A lot of work would be
avoided if a non-chiral scaffold is used, because then no enantiomers will be
formed.
The scaffold used in this project is a combination of all previous mentioned
requirements (see Scheme 5). At the left side of the molecule, a phenyl group with two
bromomethyl groups is placed at which the peptides can couple via a CLIPS-reaction. By
bringing a tertiary amine in the molecule, a positive charge is created. This charge makes
the molecule soluble in aqueous solutions. Piperazine is the part of the backbone at which
different functional groups are easily placed. By reacting an one-side protected piperazine
with an acid which has the desired reactive group, the
functional groups are easily attached. In the end, if no
chiral side chains are added, the scaffold is non-chiral and
Scheme 5: General structure
therefore no enantiomers are formed.
used scaffold
1.8 Requirements coupling techniques
Several reaction techniques can be used to couple the two scaffolds together. A few
requirements are set to these techniques.
1. The technique should work at conditions in which peptide chemistry is performed.
This means for instance that the concentration should be low, approximately
1mM. At this concentration, peptides are soluble, but at higher concentrations
they are not soluble anymore.
2. The solution should be an aqueous solution.
3. All side chains of the amino acids are unprotected. These peptides may not react.
The coupling reaction may thus be not so reactive that it reacts with the
unprotected side chains. Also other conditions that can harm the peptides should
be avoided. A too high temperature will lead also to degradation of the peptide.
When reaction conditions are found which content all these requirements, the
conditions are called ‘peptide friendly’
1.9 Previous studies
In literature, some ligation methods are discussed as being promising for the
ligation of biomolecules3. In the past, already some of these techniques are examined for
attempting to couple above mentioned scaffolds to each other.
1.9.1 Click reaction
In a first attempt, click chemistry was
used as a ligation technique (see Scheme 6).
The two scaffolds were synthesized with an
azide and an alkyn respectively. It was
found already that when the click reaction Scheme 6: Click reaction
was catalyzed by a copper(I) catalyst, the reaction got significantly better reaction rates.
Unfortunately the click reaction did not work well for the peptide chemistry. The
copper from the catalyst seemed to form complexes with the amino acids and harmed the
peptides. Another problem was the rate at the wanted concentration. Peptide chemistry in
10
general is done at a concentration of approximately 1 mM. It was hard to get good
reaction rates for the click reaction at this concentration. To improve the rates of the
reaction at this concentration, the temperature was raised and microwave irradiation was
used. It was found that these conditions also harmed the peptides. The Cu(I) click
chemistry was thus not a promising field for coupling the two scaffolds until now.
1.9.2 Oxim formation
Another attempt to
couple
the
two
scaffolds was done by Scheme 7: Oxim formation
the oxim formation as
a ligation technique (see Scheme 7). This seemed to be a successful technique and
promising for the future. The oxim formation could occur under the low concentration of
1mM, in aqueous solutions and at room temperature. When catalyzed by aniline, the
reaction leaded in 30 minutes to almost completion (according to LC-MS).
A disadvantage of the oxim formation is that both scaffolds need to be deprotected
before the reaction can be done.
To couple the peptide bounded scaffolds, also several other ligation techniques can
possibly be used. One of these ligation techniques is the thiol-ene reaction.
1.10 Thiol-ene reaction
The thiol-ene reaction was first mentioned by
Posner in 19054. In this reaction, a linkage is
formed between a thiol and a double bond (see Scheme 8: Thiol-ene reaction
Scheme 8). This process is a radical reaction,
which starts with initiation of a radical initiator. Radical reactions are reactive reactions.
Because of this quite high reactivity, it is necessary to investigate if this coupling method
is not to reactive to be done in the presence of unprotected pepetides. There is a good
possibility that the radicals have a reactivity which can interfere with the unprotected side
chains of amino acids.
1.11 Aim of the project
The general aim of the project is the examination and the use of the thiol-ene reaction
as a ligation method for scaffolds, which are already coupled to peptides via a CLIPSreaction. The first goal is to synthesize both the scaffolds for the thiol-ene reaction and
the coupling to the peptide loops. This coupling will be done via a CLIPS-reaction. The
second goal will be finding reaction
conditions under which the thiol-ene reaction
takes place at mild, peptide friendly
conditions. Both the thermal thiol-ene
reaction and the photochemical thiol-ene
reaction will be examined.
Scheme 9: Schematical reproduction aim project
11
2. The thiol-ene reaction
As stated already in the introduction, Posner was the first person to mention the thiolene reaction in one of his articles4. In the thiol-ene reaction, a linkage is formed between
a thiol and a double bond. The mechanism for this reaction is given in Scheme 10.
Since the thiol-ene reaction is a radical reaction, the first step is the initiation of the
reaction by the initiator. This initiator becomes a radical under a specific wavelength
irradiation or at a temperature in which the initiator is unstable. This initiator radical
breaks the bond between the sulfur and hydrogen atom homolytically, leaving a radical at
the sulfur atom. This radical can react with the double bond of the alkene, forming a
sulfur – carbon bond and a radical
at the next carbon atom of the
alkene. In the last step, the radical
will uptake a proton from another
thiol group. In this way, the
reaction keeps itself going by
initiating another thiol every time.
When the proton is taken, the
thiol and the alkene form a
saturated bond. (see Scheme 10)
Scheme 10: Mechanism thiol-ene reaction
2.1 Oxygen free environment
The thiol-ene reaction has to be carried out in an oxygen free environment. When any
oxygen is present, the thiol can be oxidized by the oxygen, forming a disulfide with
another thiol. A disulfide cannot react in a thiol-ene reaction anymore. Another reaction
that can take place in the presence of oxygen is when the radical in the last step does not
take up a proton but a molecule of oxygen5. When a lot of oxygen is dissolved in the
solvent, this is a likely side reaction. To avoid the forming of these side products, the
reaction has to be carried out under nitrogen or argon gas.
2.2 Thiol-ene test reaction
The reaction conditions for the thiol-ene reaction were examined with compounds that
are more-or-less comparable to the scaffolds. Fmoc protected trityl cysteine (14) will be
used as thiol and 1-Cbz-piperazine-4-allyl formate (13) will be used as alkene. The
desired product after a thiol-ene reaction is 15.
Scheme 11: Test reaction thiol-ene
To obtain the Fmoc-cystein (14), trityl protected Fmoc-cystein was first deprotected
by 5% Trifluoro acetic acid and 3 % triethyl silane in dichloromethane (according to
Triola et al.6). The thiol scaffold that will be used in the thiol-ene reaction, is also trityl
protected, and will be deprotected in the same way as 14.
12
Certain conditions of the test reaction follow the procedure of Dondoni et al7. The
specific initiators and the use of a phosphate buffer are described in this article.
For the photochemical reaction, a mercury lamp of 366 nm was used. The reaction
tube was left approximately three centimeters under the mercury lamp. To avoid heating
up due to the lamp, the reaction tube was cooled during the reaction by compressed air.
The temperature was measured to stay at room temperature using this cooling method.
The thiol-ene test reaction was followed by LC-MS. Using the peak ratio of the LCMS data, an estimation was made of the progress of the reaction.
2.3 Initiators
A radical reaction cannot take place without initiation. A radical is formed of a
specific molecule, called an initiator, which starts with the radical reactions. The thiolene reaction can be initiated by a thermal and a photochemical method. For both
methods, different initiators are needed.
2.3.1 Thermal initiator
For the thermal thiol-ene reaction, azobisisobutyronitrile
(AIBN) is used as an initiator (see Scheme 13Scheme 13). When
heated up to temperatures above 40°C, the bond between the
carbon atom and nitrogen atom in the middle of the molecule Scheme 13: AIBN
breaks homolytically, leaving two
radicals. Nitrogen gas escapes from
the solution and two similar radicals
are formed (see Scheme 13: Scheme 12: Mechanism initiation of AIBN
Mechanism initiation of AIBN).
2.3.2 Photochemical initiator
For the fotochemical reaction, 2,2-dimethoxy-2-phenylacetophenone (DPAP) is used as an initiator (see Error!
Reference source not found.). When irradiated at 365 nm, the
bond between the carbon of the carbonyl and the carbon Scheme 14: DPAP
substituted with two metoxy
groups
breaks
homolytically. In this way,
two radicals are formed
which both can react as the
initiator for the thiol-ene reaction (see Scheme 15: Mechanism initiation of DPAP).
Scheme 15: Mechanism initiation of DPAP
13
3. Results
3.1 Synthesis of alkene scaffold
The synthesis of the alkene scaffold (5) was done via the route which is drawn
retrosynthetically in Scheme 16. First, an amide bond is formed by the coupling of the
Boc-protected piperazine (1) with an acid (2). The product (3) is deprotected. In the last
step, 4 is coupled to T4 via a SN2-reaction.
Scheme 16: Retrosynthesis of alkene scaffold
In the first step, Boc-protected piperazine (1) is coupled to Allyl chloroformate (2)
under basic conditions in DCM. The product (3), is deprotected by a mixture of
TFA:DCM (1:1), to form 4. The final alkene scaffold is formed by adding T4 to 4 under
basic conditions in ACN. A total of 3 equivalents of T4 is added to avoid the formation
of two piperazines that react on one molecule of T4. In the NMR-spectrum, no dimer is
seen in this step.
Scheme 17: Total synthesis of alkene scaffold (5)
The total yield of this three step synthesis was 49 %. There is an opportunity that the
total rate of the synthesis can be optimized by optimizing the work up step in the last
reaction step. During the decantation process, there is no obvious precipitation, so it is
hard to make a good separation by decantation. If the solution would first be
centrifugated, it is possible that solvent removal goes easier.
14
3.2 Synthesis of trityl protected thiol scaffold
The synthesis of the (trityl protected) thiol scaffold was done in a comparable way as
the alkene scaffold was synthesized. The retrosynthesis can be seen in Scheme 18.
Fmoc-piperazine (9) instead of Boc-piperazine (1) was used because of expected
problems with the Boc-deprotection. The Boc-group is deprotected by acid, as is the trityl
group. So when the piperazine would be deprotected, the thiol would deprotect as well.
To avoid this, the acid stable Fmoc-protecting group is used.
Scheme 18: Retrosynthesis of trityl protected thiol scaffold
First of all, starting material 8 had to be synthesized. This is done by following the
procedure of Röhrig et al.8 by adding 1.24eq DIPEA in a solution of 6 and 7. From this
step, the total yield of the trityl protected thiol scaffold is 16 %. Under basic conditions in
THF and using HBTU as a coupling reagens, 8 is coupled to Fmoc-piperazine (9). The
Fmoc-group is removed by using a DEA:THF (1:1) mixture. In the final step, 11 was
added to 3 eq T4 under basic conditions, to give the trityl protected scaffold 12.
Scheme 19: Total synthesis of trityl protected thiol scaffold (12)
Also in the synthesis of this scaffold, the last step has a low yield. In this last coupling,
the yield could possibly be optimized by improving the work up method by for instance
centrifugation. According to NMR-data, a 1:8 ratio dimer:scaffold is formed. This is not
problematic because the dimer cannot compete in the CLIPS-reaction.
15
3.3 CLIPS-reaction
To both scaffolds, the sequence of a small part of a FSH-hormone (follicle stimulating
hormone) was coupled. The sequence of this peptide is Cys-Glu-Lys-Glu-Glu-Cys(Acm)Arg-Phe-Ala-Cys. To couple the peptide to the scaffold, 1.25 eq of the scaffold is added
to a 0.5 mM peptide solution in 1:3 ACN:H2O. 200 mM of Sodium carbonate is used as a
buffer to maintain the pH at 8. The reaction is followed by LC-MS.
In both cases, the right masses calculated beforehand for a multiple positive charge,
were found in the LC-MS data. The reaction mixtures were lyophilized, and a small
amount of white powder is obtained. Further analysis has not been done, because the
reaction was done at a small scale just to examine if it was possible to couple a peptide
chain to both scaffolds without any side reactions.
Scheme 20: CLIPS-reaction with both scaffolds and a FSH-peptide
3.4 Thiol-ene reaction
The thiol-ene reaction as described in chapter 2.2 (Scheme 11) was followed by LCMS. By analyzing the mass data of the LC-MS, the desired product as well as some side
products were found. These products can be seen in scheme (Scheme 21).
3.4.1 Products found
Scheme 21: Products found on LC-MS
Product A is the desired product. When oxygen was present, it was possible that the
thiol got oxidized and formed a disulfide, B. The other product which involved oxygen,
C, had uptaken an oxygen molecule instead of a proton first to terminate the reaction.
The last compound, D, has an oxidized sulfur atom.
16
3.4.2 Thermal thiol-ene reaction
Nr.
Solvent Temp Conc.
(°C)
(mM)
[1]
DCM
50
10
[2]
ACN
80
10
[3]
DMSO 80
100
[4]
ACN
80
10
[5]
DMF
80
10
[6]
ACN
80
10
[7]
ACN
80
10
[8]
ACN
50
10
[9]
ACN
80
10
[10] ACN
80
1
Time
(h)
40
1
2.5 d
24
24
3
3
5
4.5
4.5
A
B
C
D
13.
2
1
0.5
1
4
3
1
1
3
6
1
1
1
1
0.5
0.5
1
1
1
1
5
-
1
1
1
2
0.5
2
4
1
3
2
6
2
20
3
2
Tcep
added
yes
yes
yes
yes
-
Scheme 22: Results thermal thiol-ene reaction, based on LC-MS data
The first test reaction [1] was performed at 50°C, using DCM as a solvent. At this
temperature, DCM is refluxing. After 40 hours, product (A) was seen on LC-MS, as well
as disulfide (B) and the product which had reacted with oxygen (C). Still a lot of starting
material is left, showing that the reaction is not completed.
To get a quicker conversion, the temperature was raised to 80°C, and the solvent was
changed to ACN [2]. After one hour, product was seen, but the reaction was still not
completed.
When the temperature is kept at 80°C and the solvent is changed to DSMO [3], only
disulfide and starting material are seen. Tcep was added in an attempt to reduce the
disulfide back to the thiols so that the thiol-ene reaction could still occur. Also after the
adding of Tcep, only disulfide was seen. DMSO as a solvent has oxidizing properties,
and therefore it was assumed that DMSO would not be a useful solvent for the reaction.
The reaction was done again in ACN [4] and DMF [5], both at 80°C and in the
presence of Tcep. In both reactions, no product was seen. Because the reaction in ACN
did form product without Tcep [2], it was assumed that Tcep could interfere with the
radicals of the initiator. If Tcep reacts with the radicals from the initiator, the thiol-ene
reaction can not be initiated and will therefore not occur.
To test whether Tcep really influences the reaction, reaction [6] and [7] are performed
parallel. In reaction [6] Tcep was added and in reaction [7] Tcep was absent. The
temperature was kept at 80°C and the solvent remained ACN. After three hours, a small
amount of product was formed in the reaction with Tcep. In the reaction without Tcep no
product seemed to have formed at all. This was a strange result, because the reaction
conditions in [7] were exactly the same as the conditions in reaction [2], but the results
were quite different. In [7], no product was seen at all, where in [2] the forming of
product was observed.
17
This strange outcome gave rise to the idea that it was not the presence of Tcep which
influenced the forming of product, but the presence of oxygen. In the same time, a leak in
the gas system was observerd. This leak is probably the reason why in the reactions [3][7] hardly any product was formed.
When the leak was solved, the thiol-ene reaction was tested in ACN at 50°C [8]. After
five hours, a little bit of product was seen, but there was only a small conversion.
According to this finding the temperature was raised again to 80°C [9]. After 4,5
hours, there seemed to be a good conversion to the product. The reaction was still not
fully completed, but according to de LC-MS data there was more product than side
product or starting material. This finding supports the theory that the reactions [3]-[7]
where influenced by the oxygen, because in reaction [9] the problem of the leak was
already solved.
In reaction [10] the reaction was performed at a concentration of 1 mM. At this
concentration it seemed that is was still possible to obtain product from the thiol-ene
reaction. Unfortunately, the reaction after five hours was still far from complete.
Noteworthy, the ratios are based on LC-MS Mass detection, which can vary for every
compound.
The thiol-ene reaction was performed more times than represented in Scheme 22. In
many of these reactions, product was seen, but the solvent was evaporated. The
concentration would therefore be unreliable. These result would also not be useful for the
peptides, while reactions involving peptides cannot be done under solventless or highly
concentrated conditions.
Other reactions showed that adding the cystein solution slowly (in 30 – 60 minutes) to
the alkene solution leaded to slightly more product comparable to disulfide. When the
cystein is added at once, more disulfide is formed. When the alkene is added to the
cystein, also more disulfide is formed.
Dondoni et al.7 tested also the thermal thiol-ene reaction and found only 25 %
conversion. The fact that none of the test reactions in Scheme 22 reached full conversion
thus seems not to be strange.
18
3.4.3. Photochemical thiol-ene reaction
Nr. Solvent
Conc.
(mM)
[11] ACN/phospate buffer
10
(aq) (1:4)
[12] ACN/phospate buffer
1
(aq) (1:4)
[13] ACN/phospate buffer
10
(aq) (1:4)
[14] ACN/phospate buffer
1
(aq) (1:4)
[15] ACN
1
[16] ACN/H2O (1:4)
1
Time
(min)
30
A
B
C
D
E
20
1
-
-
2
Tcep
added
-
60
1
10
-
0.5
1
-
60
20
-
-
1
1
yes
60
4
1
-
-
1
yes
60
60
2
2
-
1
-
3
4
1
yes
yes
Scheme 23: Results photochemical thiol-ene reaction, based on LC-MS data
The UV initiated thiol-ene reactions are all done in a total volume of 10 ml. The
phosphate buffer (pH 7.4) is made by adding 1.98 ml 1M KH2PO4 to 8.02 ml 1M
K2HPO4 and diluting it with 90 ml H2O.
In the first reaction [11], it seemed that after 30 minutes the thiol-ene reaction was
almost completed, with only a small amount of disulfide.
Because of this good result of the first reaction, the concentration was lowered to 1
mM [12]. After one hour, a lot of disulfide was formed. To be certain that this results did
not come from any oxygen presence, the reaction was repeated twice. It appeared that the
disulfide was formed during the reaction. Apparently, at this low concentration, the
forming of disulfide proceeds quicker than the thiol-ene reaction.
To find a way for avoiding the forming of disulfide, the influence of Tcep was also
studied in the UV initiated thiol-ene reaction. An excess of Tcep was added in the next
reaction [13], which had the same conditions as reaction [11]. Tough after one hour,
again an almost full completion of the thiol-ene reaction was seen, and no disulfide was
formed. It seemed that Tcep did not react with the the radicals, but did prevent the
formation of disulfide.
The concentration was lowered again in reaction [14] to 1 mM, but now in the
presence of Tcep. After one hour, it seemed there had been already a good conversion
and the ratio between the product and the disulfide improved very well in comparison
with reaction [12].
To see if the phosphate buffer was really necessary for the reaction, reaction [15] was
performed with only ACN as a solvent. After one hour it seemed that no product was
formed. Despite the fact that Tcep was added, the disulfide seemed to be formed the
most. It can be concluded from this reaction that only ACN is actually not a good solvent
for a thiol-ene reaction.
19
In the last reaction, the influence of the buffer was examined by taking H2O as solvent
instead of the buffer [16]. It seemed that after one hour product was formed but that there
was even more side product formed with the oxidized sulfur atom. It seems like the
function of the buffer is the avoidance of the sulfur oxidation.
It can be concluded that the photochemical thiol-ene reaction is more promising for
the peptide chemistry than the thermal one. The reaction can be done at low
concentrations and reaches almost completion in a short time, according to the LC-MS
data.
20
4 Conclusions
Synthesis of both the alkene scaffold and the trityl protected thiol scaffold was
succesful. The alkene scaffold was synthesized in an overall yield of 49 % in a three step
synthesis. The thiol scaffold was synthesized in an overall yield of 16 % in a three step
synthesis.
Both scaffolds are successfully CLIPSed with a peptide chain from a FSH peptide.
The reaction was followed on LC-MS and the corresponding masses were found.
Both the thermal and photochemical initiation of the thiol-ene reaction were tested
under several conditions. These reactions were followed on LC-MS.
The thermal thiol-ene reaction never showed full conversion. This is in agreement
with the results of Dondoni et al.7 which found only 25 % conversion. Raising the
temperature did also not lead to a full conversion. For this test reaction, the influence of
oxygen was high. The Fmoc-cystein formed disulfides quickly. It is possible, and even
likely, that the forming of disulfides will be less if the peptide scaffolds will be used.
Possibly the thermal reaction will then proceed in a better way than it does now with the
test reaction. According to the results of the test reaction, the thermal thiol-ene reaction
will not be recommended for the ligation of the two scaffolds. To make it useful, more
research will have to be done at the thermal thiol-ene reaction.
The photochemical thiol-ene reaction is more promising for the ligation of the
scaffolds. In one hour, the reaction leads to a good conversion. There is also less forming
of side product, compared to the thermal version. It seemed in this reaction that using a
mixture of ACN:Phosphate buffer (aq) (1:4) as a solvent worked well and that purely
ACN was not a good solvent. This can also be the reason why the thermal thiol-ene
reaction did not proceed as well as one might hope, since almost all the thermal reactions
were carried out in purely ACN.
The question that still remains is if the thiol-ene reaction can be used in the peptide
chemistry. The thiol-ene reaction has not been done with the CLIPSed peptides, so it is
not know yet if the radicals in the thiol-ene reaction will harm the peptides. If the
peptides will remain unharmed, the UV-initiated thiol-ene reaction can be a good ligation
method. This is likely the case when the reaction times are short, because it is known that
peptides will not be harmed if they are irradiated by UV for one hour.
21
5. Future prospects
Regarding to the synthesis of the scaffolds, the yields of the coupling with T4 can
possibly be optimized. A possibility to do this is to centrifugate the solutions before the
decantation work up. In this way, it is easier to remove solvents.
In the thermal thiol-ene reaction, a lot of improvement can still be done. In the
photochemical thiol-ene reaction, it seemed that a mixture of ACN:Phosphate buffer (aq)
(1:4) as a solvent works well. The thermal thiol-ene reaction can also be tested using this
solvent, to see if this will improve the reaction rate. The experiments using DMSO or
DMF as a solvent can also be repeated. This could be useful because the test reactions
done with these solvents were probably influenced by the presence of oxygen, due to a
leak in the gas system.
The role of Tcep can also be studied again for the thermal thiol-ene reaction. It seemed
at first sight that Tcep reacted with the initiators, but in the photochemical thiol-ene
reaction Tcep only prevented the forming of disulfide. Tcep can thus be used to avoid the
forming of disulfide.
In the photochemical thiol-ene reaction, more investigation can be done to solvent
mixtures of ACN and the phosphate buffer. Different ratios can be examined and even a
reaction can be done with only the phosphate buffer, to see which will give the best
results.
An interesting examination is to test the UV-initiated thiol-ene reaction with both
peptide scaffolds. The results will show if it is possible to get a good conversion with the
scaffolds and there can be concluded if the radical chemistry involved in the thiol-ene
reacton is too reactive for unprotected peptides or not.
22
6. Acknowledgements
I would like to thank Linde Smeenk for the help, guidance and patience during my
project. I appreciated it very much that I could work independently and I have really
learned a lot in the months that I did the project. I think it was not easy all the time to
attempt to translate my chaotic personality into structured chemistry.
I would like Jan van Maarseveen for his advices and the support in all the things I had
to do. I was a great pleasure to me to work with you as my supervisor. I am also thankful
to Peter Timmerman for his advices. I would like to thank Henk Hiemstra for the
possibility to do my project in his group, and for the good questions he asked and the
advices he gave after these good questions.
In the end, I would like to thank all the other people of the Synthetic Organic
Chemistry Group, for all the help they gave me during my project and the nice ambience
they gave to the group, which made me feeling very well in this group.
23
7. Experimental
1-Boc-piperazine-4-allyl formate (3)
1 (0.82 mmol, 153 mg, 1eq) was dissolved in 20 ml DCM. 2 (1.23
mmol, 0.13 ml, 1.5eq) was added. triethylamine (1.23 mmol, 0.17
ml, 1.5eq) was added. The solution was stirred overnight. The
DCM and triethylamine were evaporated in vacuo. The solution is
dissolved in 20 ml ethylacetate and is washed with a 1 M KHSO4 solution to remove 1.
The organic layer is also washed with a saturated solution of NaHCO3, to remove 2. The
organic layer was washed with brine and dried over Na2SO4. The solvent of the organic
layer was evaporated in vacuo. Yield is 206 mg (0.76 mmol, 93 %). Product is found as
light yellow cristals.
H-NMR (400 MHz, CDCl3): δ 1.43 (s, 9H), 3.49 – 3.31 (m, 8H), 4.57 (dt, J = 5.6, 1.4
Hz, 2H), 5.18 (ddd, J = 10.5, 2.6, 1.2 Hz, 1H), 5.26 (ddd, J = 17.2, 3.1, 1.5 Hz, 1H), 5.97
– 5.84 (m, 1H) 13C-NMR (100 MHz, CDCl3): 28.3 (C1), 43.5 (C2), 66.1 (C2), 80.0 (C2),
117.5 (C2), 132,8 (C3), 154.5 (C4), 154.9 (C4)
IR : 1698, 1416, 1231 cm-1
1
Piperazinyl allyl formate (4)
3 (0.76 mmol, 206 mg, 1eq) was dissolved in a 20 ml
dichloromethane / trifluoro acetic acid mixture (1:1). The mixture
was stirred for one hour. The solvent was evaporated in vacuo. Yield
is 254 mg (0.954 mmol, >99 %). Product is found as a brown oil.
H-NMR (400 MHz, MeOD): δ 3.30 – 3.18 (m, 4H), 3.76 (s, 4H) , 4.60 (d, J = 5.6 Hz, 2
H), 5.22 (dd, J = 10.5, 1.1 Hz, 1H), 5.31 (dd, J = 17.2, 1.4 Hz, 1H), 5.94 (ddd, J = 22.7,
10.8, 5.6 Hz, 1H) 13C-NMR (400/100 MHz, MeOD): 40.4 (C2), 42.9 (C2), 66.4 (C2),
116.9 (C2), 132.4 (C3), 154.8 (C4)
IR: 1672, 1438, 1257, 1171 cm-1
1
Alkene scaffold (5)
T4 (0.56 mol, 250 mg, 3eq) was dissolved in 27.8 ml
dry ACN. 0.0645 ml dipea (0.37 mmol, 64.5 µl, 2eq)
was added. 4 (31 mg, 0.185 mmol, 1eq) was
dissolved in 1.00 ml dry ACN and added slowly to
the solution with dipea. The reaction was stirred and followed by LC-MS for 1.5h at
room temperature. ACN was evaporated in vacuo until approximately 5 ml ACN was
left. 30 ml diethyl ether is added. After 10 minutes of stirring, the solvent was removed
by decantation. 30 ml diethyl ether was added again, stirred, decantated (this decantation
was done for 4 times). The remaining solvent was evaporated in vacuo. Yield is 45 mg
(0.098 mmol, 53 %). Product is found as a light brown power.
24
H-NMR (400 MHz, ACN:D2O 1:9): δ 3.71 (d, J = 4.7 Hz, 4H), 3.94 (s, 4H), 4.80 (s,
2H), 5.00 (s, 4H), 5.30 (dd, J = 10.5, 1.2 Hz, 1H)* 13C NMR (100 MHz, ACN:D2O) δ
29.7 (C2), 39.0 (C2), 42.9 (C2), 59.4 (C2), 67.1 (C2), 67.3 (C2), 118.0 (C2), 126.3 (C3),
132.3 (C3), 133.3 (C4), 138.5 (C4), 155.9 (C4)
IR: 1698, 1441, 1249 cm-1
* The missing 2H are probably under solvent peak.
1
Tritylthio acetic acid (8)8
6 (7.48 mmol, 1.039 g, 1eq) and 7 (8.22 mmol, 2.273 g, 1.1eq)
were dissolved in 8 ml DMF. Dipea (9.27 mmol, 1.61 ml, 1.24eq)
was added. The mixture was stirred at room temperature for 4h.
The solvent was evaporated in vacuo. The product was dissolved
in DCM and purified on a silica 60 A column (PE:EA 9:1  1:1).
The product containing fractions were evaporated in vacuo. Yield
is 631 mg (1.89 mmol, 50%*). Product is found as a light yellow
solid compound.
H-NMR (400 MHz, MeOD): δ 2.93 (s, 2H), 7.27 – 7.20 (m, 3H), 7.31 (ddd, J = 7.8, 4.6,
1.3 Hz, 6H) 7.41 (dt, J = 3.4, 2.2 Hz, 6H) 13C-NMR (100 MHz, MeOD): 34.3 (C2), 66.7
(C4), 126.1 (C3), 127.6 (C3), 129.3 (C3), 144.2 (C4), 171.7 (C4)
IR: 1708, 1490, 1444, 743, 700 cm-1
*Due to a procedural mistake, 50% of total reaction volume was lost during work up. The
remaining half gave a 50% yield.
1
Fmoc-4-((tritylthio)acetate)piperazine (10)
9 (0.40 mmol, 156 mg*, 1eq) was dissolved in 19 ml THF.
8 (0.48 mmol, 161 mg, 1.2eq) was dissolved in 1.00 ml
THF and added to the solution of 9. HBTU (0.6 mmol, 228
mg, 1.5eq) and dipea (1.00 mmol, 0.174ml, 2.5eq) was
added. The mixture was stirred overnight at room
temperature. The solvent was evaporated in vacuo. The
product was dissolved in ethyl acetate and purified in a
separation funnel by washing with 1M KHSO4, saturated
NaHCO3 and brine. The organic layer was dried with
Na2SO4. The solvent was evaporated in vacuo. Yield is 270 mg (0.43 mmol, >99 %)
Product is found as a white crystal foam.
H NMR (400 MHz, CDCl3) δ 2.96 (s, 2H), 3.59 – 3.06 (m, 8H), 4.24 (t, J = 6.3 Hz, 1H),
4.52 (s, 2H), 7.39 – 7.23 (m, 11H), 7.43 (t, J = 7.4 Hz, 2H), 7.49 (t, J = 8.5 Hz, 6H ), 7.57
(t, J = 6.7 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H) 13C NMR (100 MHz, CDCl3) δ 34.4 (C2),
41.4 (C2), 43.6 (C2), 45.4 (C2), 47.2 (C3), 67.2 (C4), 119.9 (C3), 124.7 (C3), 126.9 (C3),
126.9 (C3), 127.6 (C3), 128.0 (C3), 129.3 (C3), 141.2 (C4), 143.7 (C4), 143.7 (C4), 154.8
(C4), 167.0 (C4)
IR: 1736, 1372, 1233, 1043 cm-1
*Calculated for the HBr salt
1
25
(Tritylthio)acetyl piperazine (11)
10 (0.40 mmol, 250 mg, 1eq) was dissolved in a 28.58 ml THF
/ DEA mixture (1:1). The solution was stirred for one hour. The
solvent was evaporated in vacuo. The product was purified on a
silica 60 A column (DCM/MeOH 100:0  95.5). The solvent
was evaporated in vacuo. Yield is 124 mg (0.308 mmol, 77 %).
Product is found as a brown oil.
H NMR (400 MHz, MeOD) δ 2.81 – 2.75 (m, 1H), 2.89 – 2.81
(m, 2H), 3.03 (s, 2H), 3.19 – 3.09 (m, 2H), 3.58 – 3.47 (m, 2H), 7.27 (t, J = 7.2 Hz, 3H),
7.34 (t, J = 7.5 Hz, 6H), 7.45 (d, J = 7.8 Hz, 6H) 13C NMR (100 MHz, MeOD) δ 33.64
(C2), 41.06 (C4), 44.49 (C2), 45.41 (C2), 126.59 (C3), 127.59 (C3), 129.09 (C3), 143.80
(C4), 167.72 (C4).
IR: 1637, 1444, 841, 701 cm-1
1
Triyl protected thiol scaffold (12)
11 (0.08 mmol, 32 mg, 1eq) was dissolved in 0.43
ml dry ACN. T4 (0.24 mmol, 107 mg, 3eq) was
dissolved in 11.90 ml dry ACN. The solution of 11
was added slowly to the solution of T4. The reaction
was stirred and followed by LC-MS for 1.5h at room temperature. ACN was evaporated
in vacuo until approximately 5 ml ACN was left. 30 ml diethyl ether was added. After 10
minutes of stirring, the solvent was removed by decantation. 30 ml diethyl ether was
added again, stirred, decantated (this decantation was done for 4 times). The remaining
solvent was evaporated in vacuo. Yield is 12 mg (0.017 mmol, 21 %). Product is found as
light brown powder.
H NMR (400 MHz, CD3CN) δ 7.49 (s, 2H), 7.47 – 7.42 (m, 6H), 7.39 – 7.33 (m, 6H),
7.32 – 7.26 (m, 3H), 4.85 (s, 4H), 4.75 (s, 4H), 3.85 – 3.74 (m, 2H), 3.56 – 3.48 (m, 2H),
3.47 – 3.39 (m, 4H), 3.04 (s, 2H).
IR: 1190, 910, 798, 696 cm-1
1
1-Cbz-piperazine-4-allyl-formate (13)
Cbz-piperazine (1.64 mmol, 0.315 ml, 1eq) was dissolved in
40 ml DCM. 2 (2.46 mmol, 0.263 ml, 1.5eq) was added.
Triethylamine (2.46 mmol, 0.343 ml, 1.5eq) was added. The
reaction was stirred overnight at room temperature. The
solvent was evaporated in vacuo. The product was dissolved in ethyl acetate and purified
in a separation funnel by washing with 1M KHSO4, saturated NaHCO3 and brine. The
organic layer was dried with Na2SO4. The solvent was evaporated in vacuo. Yield is 494
mg (1.62 mmol, 99%) Product is found as a dark yellow liquid.
26
H NMR (400 MHz, CDCl3) δ 3.27 (s, 8H), 4.42 (dd, J = 5.5, 1.2 Hz, 2H), 4.96 (s, 2H),
5.02 (dd, J = 10.4, 1.3 Hz, 1H), 5.11 (dd, J = 17.2, 1.5 Hz, 1H), 5.75 (ddd, J = 22.7, 10.7,
5.5 Hz, 1H), 7.23 – 7.04 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 43.2 (C2), 65.7 (C2),
66.9 (C2), 117.1 (C2), 127.6 (C3), 127.7 (C3), 128.2 (C3), 132.7 (C3), 136.3 (C4), 154.4
(C4), 154.6 (C4)
IR: 1700, 1426, 1228 cm-1
1
CLIPS-reaction with alkene scaffold1
The peptide CEKEEC(Acm)RFAC (0.75 µmol, 1mg, 1eq) was dissolved in a 1.505 ml
mixture ACN:H2O (1:3). 5 (0.94 µmol, 0.43 mg, 1.25eq) was dissolved in 94 µl ACN
and added to the peptide solution. 0.188 ml of a 200mM Na2CO3 solution in H2O was
added as buffer. The reaction was stirred and followed by LC-MS for one hour at room
temperature. The solvent was lyophilized.
CLIPS-reaction with trityl protected scaffold1
The peptide CEKEEC(Acm)RFAC (0.75 µmol, 1mg, 1eq) was dissolved in a 1.505 ml
mixture ACN:H2O (1:3). 12 (0.94 µmol, 0.65 mg, 1.25eq) was dissolved in 94 µl ACN
and added to the peptide solution. 0.188 ml of a 200mM Na2CO3 solution was added as
buffer. The reaction was stirred and followed by LC-MS for one hour at room
temperature. The solvent was lyophilized.
27
8. References
1. Timmerman, P.; Beld, J.; Puijk, W.C.; Meloen, R. H.; Chembiochem, 2005, 6, 821-824
2. www.peptideguide.com - Last view 16 march 2011
3. Tiefenbrunn, T. K.; Dawson, P. E.; Peptide Science, 2010, 94, 95–106
4. Posner, T.; Ber. Dtsch. Chem. Ges. 1905, 38, 646 – 657.
5. Kasprzak, S.E.; Martin, B.; Raj, T.; Gall, K.; Polymer, 2009, 50, 5549-5558
6. Triola, G.; Brunsveld, L.; Waldmann, H.; J. Org. Chem., 2008, 73, 3646-3649
7. Dondoni, A.; Massi, A.; Nanni, P.; Roda, A; Chemistry - A European Journal, 2009,
15, 11444–11449
8. Röhrig, C.; Loch, C.; Guan, J.-Y.; Siegal, G.; Overhand, M.; ChemMedChem, 2007,
2, 1054–1070
28