Bachelor Thesis Scheikunde
Towards dipeptide cyclization
in supramolecular assemblies
door
Martin Spruijt
05-07-2016
Studentnummer
10262946
Onderzoeksinstituut Verantwoordelijk docent
Van 't Hoff Institute for Molecular Sciences Prof. dr. J.N.H. Reek
Onderzoeksgroep Tweede corrector
Homogeneous, Supramolecular and Bio-Inspired Catalysis
Prof. dr. J.H. van Maarseveen
Begeleider
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1. Abstract
Cyclic peptides are polypeptides in a cyclic ring structure and there are a lot of structures known for their biological activities2. In general it is difficult to facilitate a cyclization of small peptides, especially 7-membered cyclic dipeptides and tripeptides. Using supramolecular cages is a method to control the selectivity of chemical reactions. The goal of this project is to facilitate a cyclization of the dipeptide Phenylalanine-Glycine (Phe-Gly) in a M2L4 supramolecular cage. The synthesis of M2L4 and their building
blocks is discussed. This is followed by the synthesis of the dipeptide with protection group. After that, the solubility and ligand exchange of these cages is discussed. Eventually, after synthesis of the functionalized cage, the Boc protection group has to be deprotected in order to make a cyclization happen. The consequences of the reaction conditions of the deprotection reaction towards the stability of the cage are examined. Finally, a cyclization reaction is attempted in an experiment with functionalized cages. The synthesis of M2L4 cages succeeded and can be done in non-deuterated solvents.
M12L24 cages cannot be analyzed when synthesizing in non-deuterated solvents: after
evaporation of the solvent, the cages will not dissolve again in any solvent. However, M2L4 in solid phase will re-dissolve in acetonitrile. For Pd2L4 cages, ligands will begin to
exchange after 30 minutes according to mass spectrometry measurements. According to
1
H NMR measurements, M2L4 cages decompose less rapidly than M12L24 cages in acidic
environment. Although cyclization of a dipeptide did not succeed, the results of the experiments on the properties towards cyclization with Boc deprotection in M2L4 cages
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2. Populair wetenschappelijke samenvatting
Eiwitten zijn opgebouwd uit aminozuren en vervullen allerlei functies in de natuur. Ze worden onder andere gebruikt voor het verteren van voedsel, opbouw van cellen, transport van moleculen in cellen en signaaloverdracht. Peptides zijn moleculen die zijn opgebouwd uit een kleiner aantal aminozuren dan eiwitten. Een eigenschap van veel ringvormige peptides is dat ze biologische activiteit vertonen. Zo werd Gramicidin S, een ringvormig peptide bestaande uit 10 aminozuren, ten tijde van de Tweede Wereldoorlog veelvuldig gebruikt als medicijn om geïnfecteerde wonden te genezen.
In de natuur komen veel ringvormige peptides voor. Echter, het blijkt niet gemakkelijk de reactie van een ringvorming op het lab uit te voeren. De natuur gebruikt enzymen om reacties die normaal moeizaam verlopen uit te voeren: ze zorgen ervoor dat een reactie plaatsvindt door de moleculen bij elkaar te brengen in de zogenoemde ‘active site’ van het enzym. Hiernaast is een voorbeeld van een enzym afgebeeld: Hexokinase.
Omdat enzymen het mogelijk maken moeilijke chemische reacties uit te voeren, proberen chemici deze werking na te bootsen op het lab. Een voorbeeld daarvan zijn supramoleculaire kooistructuren (zie figuur linksonder). Hierbij is er net als bij een enzym sprake van een groot molecuul met daarin een ‘active site’: een plek waar de reactie daadwerkelijk plaatsvindt. In dit onderzoek is geprobeerd een ringvorming van een dipeptide te laten plaatsvinden. Er wordt gebruik gemaakt van een beschermgroep op het amine-uiteinde van phenylalanine. In dit onderzoek zijn eerst de kooistructuren gemaakt en de eigenschappen daarvan zijn onderzocht die belangrijk zijn voor de ringvormingsreactie. Hoewel een ringvorming niet is gelukt in dit onderzoek, zijn de resultaten van de experimenten naar de eigenschappen van de kooistructuren positief en zal er dan ook nog meer onderzoek naar gedaan moeten worden om uiteindelijk een ringvorming uit te kunnen voeren.
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3. Introduction
3.1 BackgroundAlthough catalysis already plays a big role in the chemical industry and academia as well, there is a lot more to discover in chemical reactions concerning activity, stability and selectivity. These variables can be controlled by catalysts. A way to control a chemical reaction is to construct catalysts and make changes in the structure of the ligands. Another way of controlling the selectivity of a chemical reaction is to use a confined space as second coordination sphere.
Chemists have a tendency to learn from nature. After all, a lot of chemical reactions in nature are enantioselective, have a high reaction rate and take place despite energetically unfavorable transition states.1 Nature’s solution to unfavorable chemical reaction in terms of energy and selectivity is the use of enzymes. These proteins have an active site that has a strong binding with the transition states of molecules of the reaction in order to stabilize them. Furthermore, enzymes show a proximity effect: the reaction products are preorganized in a way the reactive groups are brought close together which lowers the entropy of the reactants. Enzymes can also steer the selectivity of the reaction. The second coordination sphere of the enzyme plays here an important role.
3.2 Cyclic peptides
Cyclic peptides are polypeptides in a cyclic ring structure and there are a lot of structures known for their biological activities. Some cyclic peptides are antibacterial active or show immunosuppressive and anti-tumor activity.
The advantages of peptide-drugs are that peptides are less toxic than other drugs because the degradation products are amino acids and these do not accumulate in organs.2 Because of their biological activity and advantages compared to small synthetic molecules, there are many peptide drugs available on the market. An example of an antibiotic cyclic peptide is Gramicidin S (see figure 1), widely used during the Second World War to treat infected wounds2. Another cyclic peptide used as a drug is Cyclo RGD (Arg-Gly-Asp-D-Phe-Val), used in the treatment of cancer.
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Figure 1: Gramicidin S Figure 2: Cyclo RGD
The length of the peptide chain is an important factor in cyclization reactions. The longer the peptide chain, the easier the cyclic product is formed. For 6-membered cyclic dipeptide rings, the products are easily formed3, but in general it is difficult to facilitate a cyclization of small peptides. Especially 7-membered cyclic dipeptides and tripeptides are difficult to form.4 The geometry of the amine bonds favors a reaction where the linear product is formed instead of the cyclic form. Using direct peptide coupling reagents would result in low yields of cyclic products.5
Dendrimers (see figure 3) are well-defined macromolecules with a regular and highly branched three-dimensional structure that can be used to form the cyclic product of small peptide chains (see figure 3).6
Figure 3: Peptide cyclization inside dendrimeric carbodiimides.6 The site-isolation diminishes oligomerization.
The two bulky macromolecular parts are linked with a carbodiimide, which acts as an active center where the cyclization reaction takes place. The macromolecular parts contain cavities that steer the selectivity of the reaction by site-isolation. With these dendrimers, 7-membered rings have successfully been synthesized (figure 4).
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Figure 4: Synthesis of 7-membered rings using dendrimers6
3.3 Self-assembled metallocages
In order to synthesize cyclic peptides, it is important to construct a catalyst for the purpose of controlling the selectivity of the reaction. In transition metal catalysis, traditionally the activity, selectivity and stability of the reaction are controlled by the ligands directly bound to the catalytically active metal.7 In chapter 3.2, it was described that reaction selectivity can be controlled using dendrimers. Using supramolecular cages is another method to control the selectivity of chemical reactions. The second coordination sphere (the chemical environment of the cage itself) plays an important role. The bis-pyridyl functionalized building blocks are held together by non-covalent interactions with the metal ions.
An example of a supramolecular assembly is the M12L24 cage. The group of Fujita
reported on the synthesis of different M12L24 cages by varying in endo- and/or
exo-functionalization (see figure 5).8 The cage structure is the same in each case, but the groups attached to it are varying. It is possible to functionalize the cage with ether groups, porphyrins, polysaccharides, peptides and many more chemical structures.
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Figure 5: Functionalized M12L24 cages synthesized by Fujita et al.8
M2L4 cages are another example of self-assembled metallocages (see figure 6).9 These
cages could contribute to a new drug delivery method with cisplatin drugs, used in anticancer therapeutics. The cavities of the cage enable the encapsulation of two cisplatin molecules.9
Another use of these cages is the functionalization of it, comparable with M12L24.
However, since these structures are smaller, the possibilities of functionalization are more size-limited than with the M12L24 cages.
Figure 6: Pd2L4cage9
Free amines can coordinate to the metal-ions in supramolecular metallocages (see figure 7 on the next page). This causes decomposition of the cage. When functionalizing of the cage takes place with peptides, this can cause decomposition of the cage. This problem can be prevented by adding a protection group to the free amine.
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Figure 7: Left: Coordination of free amine to metal-ion in Pd2L4 cage.
Right: Using a Boc-protection group (red square) to prevent this.
3.4 Goal
The goal of this project is to facilitate a cyclization of the dipeptide Phenylalanine Glycine (PheGly) in a M2L4 supramolecular cage (see figure 8). Furthermore, a cyclization
to a 6-membered ring is attempted because these cyclic products are easily formed. The focus in this research is not to synthesize complex cyclic products, but to proof the principle of facilitating a cyclization in a supramolecular cage.
Figure 8: Schematic drawing of the cyclization-in-a-cage concept.
In this work, the synthesis of M2L4 and their building blocks is discussed. This is followed
by the synthesis of the dipeptide with protection group. After that, the solubility and ligand exchange of these cages are discussed. Eventually, after synthesis of the functionalized cage, the Boc protection group has to be removed in order to facilitate a cyclization. The consequences of the reaction conditions of the deprotection reaction towards the stability of the cage are examined. Finally, a cyclization reaction is performed in an experiment with functionalized cages.
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4. Synthesis & general properties cages
To prepare functionalized and non-functionalized Pd2L4 and Pt2L4 cages and examine the
chemical properties, several building blocks were synthesized. Next to that, the dipeptide of Phenylalanine and Glycine with a protection group Boc-Phe-Gly-OMe was prepared in order to functionalize the cages. The goal of that functionalization is to facilitate a cyclization of the dipeptide.
4.1 Synthesis
I. Synthesis of blank building block 1
Building Block H was synthesized for use in experiments where the properties of Pd2L4 and Pt2L4 cages were studied.
Figure 9: Synthesis of “blank” building block
This synthetic route yielded “blank” building block via a Sonogashira coupling between 1,3-dibromobenzene and 3-ethynylpyridine. PdCl2(PhCN)2 was added
to effectuate the complexation with 1,3 dibromobenzene via oxidative oxidation. Compound 1 was obtained as a brown powder after column chromatography.
II.Synthesis of functionalized Building Block OMe 2
1
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Figure 10: Synthesis functionalized building block
Sonogashira coupling between 1,Diiodo-2-methoxybenzene and 3-ethynylpyridine yielded desired compound 2 in 74%. Next, the reaction of 2 with BBr3 did not succeed, nor with 2 or 1 equivalents BBr3. A possible explanation for
the undesired product being formed in the reaction, is that the HBr that is formed in the reaction reacts with the alkyne in an addition reaction.
A modified synthesis route has been pursued:
+
Figure 11: Modified synthesis route towards functionalized building block 6
First, a synthesis of benzyl acetate 4 has been performed with acetic anhydride and 1,3-dibromophenol which yielded the product in 88% yield as a white/yellow powder. Next, via a Sonogashira coupling between 5 and 3-ethynylpyridine, compound 6 was obtained. It was found that in the reaction of
3 2 4 2 5 2 6 2 7 2
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5 with KOH, the main product that was formed was 7. This product is confirmed
with 1H NMR.
III.Synthesis of dipeptide with Boc protection group
The amino acids phenylalanine and glycine were chosen to form the dipeptide because phenylalanine contains a phenyl group, so the reaction can easily be tracked with TLC under UV light (254 nm). Glycine is chosen because it is the smallest amino acid and would not cause steric hindrance in the cyclization reaction.
Figure 12: Synthesis of BOC protected dipeptide
The dipeptide Phe-Gly-OMe 7 has been synthesized with use of EDC as a coupling reagent in a yield of 52%. Next, 8 was obtained in 88% yield by hydrolysis of the methylester.
IV.Synthesis of functionalized building block 9
Unfortunately, due to time constraints, the last step in the functionalization of the building block for M2L4 cages could not be carried out. However, with para
positioned building blocks made in house, it was possible to do a functionalization of building blocks for M12L24 cages.
Figure 13: Synthesis of dipeptide-functionalized building block 9
7 2 8 2 9 2
- 13 - V.Assembly of cages
The M2L4 cages were synthesized by mixing 5.2 equivalents of metal precursors with 1.0
equivalents of building blocks. The metal precursors that were used in the assembly are Pt(BF4)2(MeCN)4 and Pd(BF4)2(MeCN)4. Both the building blocks and metal precursors
were first dissolved separately in CD3CN and then combined in a vial and heated to 60 ºC
with stirring for a minimum of 3 hours (palladium precursor) and 24 hours (platinum precursor).
First, M2L4 cages were synthesized with blank building blocks in order to look at general
properties of these cages like solubility, ligand exchange and stability under higher temperatures and in acidic environment. Next, functionalized cages were prepared to explore if the facilitation of a cyclization is successful. The following cages, depicted in figure 14, were synthesized:
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4.2 Cage-characterization
1H NMR
All synthesized cages were characterized with 1H NMR. Certain peaks show a clear downfield shift when coordination of the building block to the metal occurs (see figure 15). Furthermore, free building blocks can easily be observed in these spectra.
Figure 15: 1H NMR spectra of the blank building block and the cage. The red squares show the downfield shifting of the peaks in the aromatic region when coordination of
the building blocks to the metal occur. The blue square indicates the presence of unreacted building block that is not coordinated to the metal.
The analysis of the platinum cages has a difference compared with palladium cages, because the peaks are broad (see figure 16). This observation is supported by literature.10 The broad peaks are caused by the slow tumbling motion with respect to the NMR timescale. An experiment has been carried out were 1H NMR spectra were taken at elevated temperatures (20 – 70 ºC). However, there was no difference notable
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Figure 15: 1H NMR spectra of the Pt2L4 cage with blank building blocks
DOSY NMR
The synthesized cages were also analyzed with DOSY NMR (Diffusion-Ordered 2D NMR spectroscopy), a technique that determine diffusion coefficients of compounds. The diffusion coefficient is related to the radius of the species in the sample.11 With 1H NMR, the difference between the spectra of the building blocks and the cages is the downfield shifting when the building block coordinates to the metal. With DOSY NMR, the difference in size of the molecules gave other diffusion coefficients. Furthermore, when the compound is pure, the diffusion coefficient does not vary in the sample. A DOSY NMR spectrum shows the 1H NMR spectrum on the x-axis and the diffusion trace on the
y-axis. The diffusion coefficient could be read from the y-axis. The lower the diffusion coefficient, the larger the molecule. In figure 16, a DOSY NMR spectrum is shown for a
palladium cage (red) and the blank building block (blue).
Figure 16: DOSY spectrum of Pd2L4 and the blank building block. The dots at 2 ppm (log D ~ -8.2)
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Although the 1H NMR spectrum of platinum cages gives broad peaks, DOSY NMR gives more information about the reaction product of the self-assembly. In figure 17, the DOSY NMR spectrum for the Pt2L4 cage with blank building blocks (blue) and the DOSY
NMR spectrum for the blank building blocks (red) are known. This spectrum shows the Pt2L4 caged is obtained in the self-assembly.
Figure 17: DOSY spectrum of Pt2L4 and the blank building block.
4.3 General properties M2L4 cages
Solubility
A disadvantage in the synthesis of M12L24 cages is that when the cages are in solid phase
(for example, when the solvent is evaporated) the solid compound will not dissolve again in any solvent. For analyticaly reasons, that self-assembly always has to be performed in deuterated solvents, usually CD3CN. The use of deuterated solvents is
more expensive and can give limitations in synthesis routes.
However, with M2L4 cages it is possible to re-dissolve the cages in acetonitrile. When the
solvent is evaporated, the cages in solid phase (stored for 24 hours) will dissolve again and no decomposition is observed in the 1H NMR spectrum. When evaporating the solvent and re-dissolving the cages, the yield is 95% .
- 17 - Ligand exchange
Since supramolecular cages are dynamic systems, it is important to know what the ligand exchange is. The concept of using supramolecular cages in this research is that the second coordination sphere plays a role in the selectivity of the reaction. But, when the ligand exchange rate is higher than the rate of the cyclization reaction, then the ligand with the dipeptide attached to it will exchange before the cyclic product would have been formed inside the cage.
First, two different palladium cages were synthesized (see figure 18). Then, two samples of these cages were injected in a Mass Spectrometer (Ionization Mode: ColdSpray+). When ligand exchange takes place, new peaks would be appearing (see figure 19 for an example when ligands exchange).
Figure 18: Cages used in the ligand exchange experiment
Figure 19: Example of what can happen if ligand exchange takes place. The right cage contains after ligand exchange, one methoxy building block. The left cage lost one methoxy building block which has been replaced by a “blank” building block. These new cages have a different
mass, and therefore new m/z peaks should appear on the mass spectrum if ligand exchange takes place.
Ligand exchange ?
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In Figure 19 the outcome of the ligand exchange experiment is shown. Directly after injection, a mass spectrum is taken. The peak a 1714.2 m/z belongs to Pd2OMe4 (Ligand=
methoxy building block) minus BF4- anion. The peak at 1593.2 m/z belongs to Pd2L4
(Ligand = “blank” building block) minus BF4- anion. The other peaks could not be
assigned. When time passes, new peaks appear (at 1624.2 m/z and 1683.2 m/z). That means that some exchange of species takes place. However, since the peaks could not be assigned properly, it is not possible to draw profound conclusions from this experiment.
Figure 19: Ligand exchange experiment. The orange rectangle denotes the appearing of a new peak. There is exchange of species between the two cages.
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5. Stability M
2L
4cages towards deprotection
There are two important things to consider concerning the functionalization of supramolecular cages with peptides. First, free amines can coordinate to the metal ions of the supramolecular cage. The coordination of the free amine can be prevented by adding protection of the free amine. In this research the Boc protection group is used. The second important thing is the deprotection of the Boc group. It is a key step in the overall process because cyclization can only happen when the amine attacks on the ester binding of the peptide attached to the cage. For the deprotection of the Boc group, an acid is needed. Figure 20 shows the mechanism for the deprotection.
Figure 19: Reaction mechanism for deprotection of the BOC group
The reaction mechanism shows that an acid is needed to remove the Boc group. It is important to know is the cages are stable in acidic environment. An experiment has been set up to determine what the stability of M2L4 cages are in acidic environment. A
titration with HCl in dioxane has been carried out and followed with 1H NMR. The ratio of the integrals of free building blocks and cage is calculated in order to determine what the percentage is of the intact cage. Figure 20 shows the result of the titration with Pd2L4
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cages. A titration with Pt2L4 has been carried out as well, but the results were
inconclusive. The peaks could not properly be integrated and a lot of peaks not assigned.
Figure 20: Titration experiment of Pd2L4 cage (L=blank building block). When HCl is added
(equiv. with respect to cage), peaks are appearing that belong to free building blocks. Integrating the peaks and calculating the ratio between cage : building block result in an
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6. Cyclization experiment
In the last experiment, a cyclization reaction was attempted in a M12L24 cage (Pd12L24 and
Pt2L24 L=functionalized building block). In this experiment, 1 equivalent HCl in dioxane
with respect to the functionalized cage was added and the mixture analyzed with 1H NMR. Figure 21 shows the 1H NMR spectrum of the starting product and the reaction mixture after adding HCl. In comparison with M2L4 cages, the cage decomposes very
rapidly, (an upfield shifting of the peak that belongs to the cage is observed after 1 equiv. HCl added) so it is not possible to determine if a cyclic product is formed. For the Pt12L24 cages the peaks could not properly be integrated and a lot of peaks not assigned.
Figure 21: Attempt of facilitating a cyclization in Pd12L24 cage. The upfield shifting of the peaks
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7. Conclusion
The blank building block 1 and building block OMe (2) for M2L4 cages were successfully
synthesized. For M12L24 cages, the functionalized building block with Boc-Phe-Gly-OH (9)
is prepared. The building blocks were successfully applied in the self-assembly of M2L4
and M12L24 cages.
A disadvantage of M12L24 cages is that when the cages are in solid phase (for example,
when the solvent is evaporated) the solid compound will not dissolve again in any solvent. However, with M2L4 cages it is possible to re-dissolve the cages in acetonitrile,
so it is possible to synthesize the cages in non-deuterated solvents and analyze it afterwards in deuterated acetonitrile. When the solvent is evaporated, the cages in solid phase (stored for 24 hours) will dissolve again and no decomposition is observed in the
1
H NMR spectrum. When evaporating the solvent and re-dissolving the cages, the yield is 95% .
For Pd2L4 cages, ligands will begin to exchange after 30 minutes according to mass
spectrometry measurements.
According to 1H NMR measurements, M2L4 cages decompose less rapidly than M12L24 in
acidic environment.
Although cyclization of a dipeptide did not succeed, the results of the experiments on the properties towards cyclization with Boc deprotection in M2L4 cages are promising
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8. Outlook
It is difficult to synthesize the functionalized building block for M2L4 cages. The main
product in the reaction is depicted below in figure 22.
Figure 22: Problems with functionalizing building block for M2L4 cage
To prevent this intramolecular cyclization, it may be a possibility to first attach the dipeptide to 1,3-dibromobenzene and then carry out the Sonogashira coupling (see figure 23).
Figure 23: Modified synthesis route towards dipeptide-funtionalized building block. First functionalization of 1,3-dibromobenzene,followed by subsequent Sonogashira coupling
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Another modification that could be done in the synthesis route is to use phenyl groups (see figure 24) in the structure of the building block instead of alkyne-moieties, since there were troubles with the reaction of the methoxy building block with BBr3. The more
BBr3 was added, the more precipitation was observed.
Figure 24: An example of a building block with an extra phenyl group instead of alkyne binding
For the ligand exchange experiment, it would be useful to do a MS MS experiment with the compound that belongs to the peaks that could not be assigned.
Furthermore, the cyclization experiment could be further explored. The stability of the M2L4 cages are higher in acidic environment than the M12L24 cages. It would be useful to
carry on the synthesis of the functionalized building blocks for the M2L4 cages and do
cyclization experiments with the functionalized cages. Next to 1H NMR, Mass Spectroscopy can be used as a method to determine whether a cyclic product is formed or not.
Concerning the Boc protection group, it could be further explored how much equivalents HCl are needed exactly to deprotect the peptide. Then it will be also known how much HCl is needed to deprotect the Boc group inside the cage. In addition, other acids than HCl could be tried out. It is important that the cage does not decompose while deprotection happens. Maybe with the use of HBF4 the cage decomposes less
rapidly.
There are many other protection groups that are available for the amine group. Teoc is an example and has been tested by Tobias van Enk.12 However, fluoride ions are needed for deprotection and they cause immediately decomposition of the M12L24 cage.
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Furthermore, the deprotection takes a very long time (80 hours), so the Boc protection group is more promising with shorter deprotection time.
Figure 25: Protecting the free amine with a Teoc group.12
A problem with the deprotection of the Teoc and Boc groups are that the compounds needed for deprotection interferes with the cages. Using photolabile protection groups could possible solve this problem, because only the protection group is light-sensitive. An example of a light-sensitive group is NPPOC.13
M2L4 cages are promising to function as a mimetic enzyme. It is possible to functionalize
them with a great variety of chemical groups and the structure of the cage could lead to a steering of selectivity in reactions. It should be further explored what the capabilities are of these cages.
9. Acknowledgements
First of all, I want to thank prof. dr. Joost Reek for giving me the opportunity to do my research project in this group and prof. dr. Jan van Maarseveen for being my second reviewer. Furthermore, I want to thank Arnout Hartendorp, MSc for being my daily supervisor. You really learned me a lot about chemistry and I want to wish you all of luck with your PhD research. Ed Zuidinga for Mass Analysis and Jan Meine Ernsting for help with the NMR measurements. The rest of the homkat group for the nice work environment.
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10. Bibliography
(1) Bartlett, G. J.; Porter, C. T.; Borkakoti, N.; Thornton, J. M. J. Mol. Biol. 2002, 324 (1), 105–121.
(2) Joo, S. H. Biomolecules and Therapeutics. 2012, pp 19–26.
(3) Müller-Hartwieg, J. C. D.; Akyel, K. G.; Zimmermann, J. J. Pept. Sci. 2003, 9 (3), 187–199.
(4) Rutters, J. P. A.; Verdonk, Y.; de Vries, R.; Ingemann, S.; Hiemstra, H.; Levacher, V.; van Maarseveen, J. H. Chem. Communm. 2012, 48 (65), 8084–8086.
(5) Davies, J. S. Journal of Peptide Science. 2003, pp 471–501.
(6) Amore, A.; Van Heerbeek, R.; Zeep, N.; Van Esch, J.; Reek, J. N. H.; Hiemstra, H.; Van Maarseveen, J. H. J. Org. Chem. 2006, 71 (5), 1851–1860.
(7) Leenders, S. H. A. M.; Gramage-Doria, R.; de Bruin, B.; Reek, J. N. H. Chem. Soc. Rev. 2015, 44 (2), 433–448.
(8) Harris, K.; Fujita, D.; Fujita, M. Chem. Commun. (Camb). 2013, 49 (60), 6703– 6712.
(9) Lewis, J. E. M.; Gavey, E. L.; Cameron, S. A.; Crowley, J. D. Chem. Sci. 2012, 3, 778–784.
(10) Fujita, D.; Takahashi, A.; Sato, S.; Fujita, M. J. Am. Chem. Soc. 2011, 133 (34), 13317–13319.
(11) Stilbs, P. Progress in Nuclear Magnetic Resonance Spectroscopy. 1987, pp 1–45. (12) Van den Enk, T. Towards Oligomerization-free Peptide Cyclization in an MnL2n
Coordination Sphere 2014. Bachelor thesis Scheikunde, Universiteit van Amsterdam
(13) Wöll, D.; Smirnova, J.; Galetskaya, M.; Prykota, T.; Böhler, J.; Stengele, K. P.; Pfleiderer, W.; Steiner, U. E. Chem. - A Eur. J. 2008, 14 (21), 6490–6497. (14) Suzuki, K.; Kawano, M.; Sato, S.; Fujita, M. J. Am. Chem. Soc. 2007, 129 (35),
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11. Experimental procedures
All reagents and solvents were purchased from commercially available sources. Solvents were distilled prior utilization by conventional methods. Deuterated solvents were not distilled or dried. 1H-NMR spectra were recorded in DMSO, CD3CN solutions using a
Bruker Bruker 300. Electrospray ionization mass spectra (positive ions) were recorded in full scan mode with varying m/z ranges. 2D 1H-DOSY were performed on a DRX 300 with the temperature and gradient calibration prior to the measurements. The temperature was controlled at 298 K during the measurements.
Synthesis of 1,3-bis(pyridine-3-ylethynyl)benzene (1)
Schlenk 1: In a flame dried 100 ml Schlenk PdCl2(PhCN)2 (243,9 mg, 0.636 mmol, 0.1
equiv.) and HP(t-Bu)3BF4 (369 mg, 1.27 mmol, 0.2 equiv) were added. This was dissolved
in 12 ml dioxane. Dry, degassed NEt3 (6 ml, 35,9 mmol, 5 equiv.) was added.
Schlenk 2: In a 100 ml flame dried Schlenk 2,6-dibromobenzene (1500 mg, 6.36 mmol, 1 equiv.), 3-ethynylpyridine (1500 mg, 15.45 mmol, 2.43 equiv.) and copper iodide (181.7 mg, 0.951 mmol, 0.15 equiv.) were added and then 20 ml dioxane.
The contents of Schlenk 1 were added to schlenk 2 and placed in a preheated oil bath (45oC) and stirred overnight. The brown mixture was cooled to room temperature, filtered and the filtrate washed with 10 mL ethyl acetate. The solvents (ethyl acetate and dioxane) were evaporated under reduced pressure. The compound was purified by column chromatography DCM : methanol (100:1 100:2). Yield: 1.782 g, 76%. 1
H NMR (300 MHz, CD3CN) δ 8.77 (s, 2H), 8.58 (d, J = 4.8 Hz, 2H), 7.92 (dt, J = 7.9, 1.9 Hz, 2H), 7.78 (s, 1H), 7.66 – 7.59 (m, 2H), 7.49 (dd, J = 8.5, 7.0 Hz, 1H), 7.40 (dd, J = 7.9, 4.9 Hz, 2H)
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Synthesis of 3,3’-((2-methoxy-1,3-phenylene)bis(ethyne-2,1-diyl))dipyridine (2)
Schlenk 1: In a flame dried 100 ml Schlenk PdCl2(PhCN)2 (266 mg, 0.694 mmol, 0.1
equiv.) and HP(t-Bu)3BF4 (403 mg, 1.39 mmol, 0.2 equiv) were added. This was dissolved
in 12 ml dioxane. Dry, degassed NEt3 (9.67 ml, 69.4 mmol, 10 equiv.) was added.
Schlenk 2: In a 100 ml flame dried Schlenk 1,3-dibromo-2-methoxybenzene (2499 mg, 6.94 mmol, 1 equiv.), 3-ethynylpyridine (1862 mg, 18.05 mmol, 2.6 equiv.) and copper iodide (198 mg, 1.04 mmol, 0.15 equiv.) were added and then 20 mL dioxane.
The contents of schlenk 1 were added to schlenk 2 and placed in a preheated oil bath (45 oC) and stirred overnight. The brown mixture was cooled to room temperature, filtered and the filtrate washed with 15 mL ethyl acetate. The solvents (ethyl acetate and dioxane) were evaporated under reduced pressure. The compound was purified by column chromatography DCM : methanol (100:1 100:2). Yield: 1.648 g, 74%.
1H NMR (300 MHz, CD3CN) δ 8.78 (d, J = 1.4 Hz, 2H), 8.59 (dd, J = 4.9, 1.6 Hz, 2H), 8.05 – 7.83 (m, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.49 – 7.33 (m, 2H), 7.20 (t, J = 7.7 Hz, 1H), 4.15 (s, 3H)
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Synthesis of 2,6-dibromophenyl acetate14(4)
2,6-dibromophenol (10.5 g, 42.0 mmol) was dissolved in 500 ml acetic anhydride under nitrogen. Pyridine (35 mL, 432 mmol) was slowly added. The mixture was heated to 80oC and refluxed for 3 hours. The solvent was evaporated under reduced pressure. The mixture was purified using column chromatography eluting with hexane : CH3Cl (70:30)
to obtain a white powder. Yield: 1.476 g, 88%.
1
H NMR (300 MHz, CDCl3) δ 7.57 (d, J = 8.1 Hz, 2H), 7.02 (t, J = 8.1 Hz, 1H), 2.39 (d, J =
15.4 Hz, 3H).
Synthesis of 2,6-bis(pyridine-3-ylethynyl)phenyl acetate (5)
Schlenk 1: In a flame dried 100 ml Schlenk PdCl2(PhCN)2 (1495 mg, 3.9 mmol, 0.1 equiv.)
and HP(t-Bu)3BF4 (2260 mg, 7.8 mmol, 0.2 equiv) were added. This was dissolved in 20
mL dioxane. Dry, degassed NEt3 (27.2 ml, 390 mmol, 10 equiv.) was added.
Schlenk 2: In a 250 ml flame dried Schlenk 2,6-dibromo phenyl acetate (4) (1476 mg, 39 mmol, 1 equiv.), 3-ethynylpyridine (9652 mg, 93.6 mmol, 2.6 equiv.) and copper iodide (1100 mg, 5.85 mmol, 0.15 equiv.) were added and then 60 mL dioxane.
The contents of schlenk 1 were added to schlenk 2 and placed in a preheated oil bath (45 oC) and stirred overnight. The brown mixture was cooled to room temperature and filtered. The solvent (dioxane) was evaporated under reduced pressure. The compound was purified by column chromatography DCM : methanol (100:1 100:2). Yield: 2.80 g, 21%.
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1
H NMR (300 MHz, CD3CN) δ 8.76 (s, 2H), 8.60 (d, J = 4.5 Hz, 2H), 7.91 (d, J = 7.5 Hz, 2H),
7.69 (d, J = 7.7 Hz, 2H), 7.46 – 7.35 (m, 3H), 2.46 (s, 3H).
Synthesis of 2,6-bis(pyridine-3-ylethynyl)phenol
In a round bottom flask, 2,6-bis(pyridine-3-ylethynyl)phenyl acetate(5) (1.00 g, 2.96 mmol) was dissolved in 30 mL methanol. Then, 3M KOH was added (1.82 mL, 5.46 mmol). The solution was stirred for 1 hour and after that 50 mL water was added and the reaction mixture was neutralized with 2M HCl. Extraction was performed with three times with 50 mL DCM. The solvents were evaporated under reduced pressure and a yellow solid was obtained. Yield: 75.6 mg, 8%.
1
H NMR (300 MHz, CD3CN) δ 8.78 (s, 2H), 8.62 – 8.47 (m, 2H), 7.94 (dt, J = 7.9, 1.9 Hz,
2H), 7.53 (d, J = 7.7 Hz, 3H), 7.41 (dd, J = 7.9, 4.9 Hz, 2H), 6.99 (t, J = 7.7 Hz, 1H).
Synthesis of Boc-Phe-Gly-OMe
In a 250 mL flame dried schlenk, Boc-Phe (2.0 g, 7.54 mmol, 1 equiv.) was dissolved in 60 mL dry DCM and 60 mL dry DMF. The solution is cooled in an ice bath. After that, DIPEA (1.956 g, 15.08 mmol, 2 equiv.), HOBT (1.223 g, 9.048 mmol, 1.2 equiv.) and EDC (1.734 g, 9.048 mmol, 1.2 equiv.) were added. The mixture was stirred for 15 minutes. Then, Gly-OMe HCl (0.946 g, 7.54 mmol, 1 equiv.) was added and stirred overnight on the ice bath. Extraction was done with 3x 80 mL water. The solvents were reduced under reduced pressure. The white foam is obtained with column chromatography (PE:EtOAc = 7:3). Yield: 1.324 g, 52%.
1
H NMR (300 MHz, CDCl3) δ 7.37 – 7.28 (m, 4H), 4.95 (s, 2H), 4.39 (s, 3H), 4.17 – 3.92 (m,
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Synthesis of Boc-Phe-Gly-OH
In a round bottom flask, Boc-Phe-Gly-OMe (1.324 g, 3.94 mmol, 1 equiv.) was dissolved in 24 mL water and 6 mL MeOH. Then, LiOH is added (1.65 g, 9.4 mmol, 10 equiv.) and the solution is stirred for 1 hour. After that, 1 M KHSO4 was added until pH ~ 2. The
reaction mixture was transferred to a separation funnel and extractred three times with 100 mL EtOAc. The combined organic layers were dried with MgSO4, filtered and the
solvents were removed with under reduced pressure. Yield: 1.17 g, 88%.
1
H NMR (300 MHz, CDCl3) δ 7.32 (d, J = 6.7 Hz, 6H), 7.23 – 7.08 (m, 9H), 6.73 (s, 4H), 4.57
(s, 10H), 4.07 (dd, J = 35.3, 21.5 Hz, 11H), 3.07 (s, 11H), 1.64 – 1.19 (m, 41H).
Synthesis functionalized building block for M12L24 cage
In a 100 mL flame dried schlenk, 2,6-bis(pyridine-3-ylethynyl)phenol (213 mg, 0.718 mmol, 1 equiv.) was dissolved in 11.3 mL dry DCM and 2.26 mL dry THF. Then, Boc-Phe-Gly-OH (231 mg, 0.718 mmol, 1 equiv.) was added. The solution is cooled in an ice bath. After that, EDC HCl (172.1 mg, 0.898 mmol, 1.25 equiv.) and DMAP (2.2 mg, 0.0169 mmol, 0.025 equiv.) were added. The mixture was stirred overnight. Extraction was done with 3x 50 mL water. The solvents were evaporated under reduced pressure. The white powder is obtained with column chromatography (PE:EtOAc = 7:3). Yield: 75.6 mg, 18%. 1 H NMR (300 MHz, CD3CN) δ 7.64 (dd, J = 4.4, 1.6 Hz, 4H), 6.74 (d, J = 7.8 Hz, 2H), 6.57 (dd, J = 4.4, 1.6 Hz, 4H), 6.49 – 6.42 (m, 1H), 6.33 – 6.10 (m, 5H), 4.49 (d, J = 18.3 Hz, 1H), 3.41 (d, J = 6.1 Hz, 3H), 2.17 (dd, J = 14.0, 4.9 Hz, 1H), 1.92 – 1.69 (m, 1H), 0.41 – 0.14 (m, 9H).
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Assembly cages
The M2L4 and M12L24 cages were synthesized by mixing 5.2 equivalents of metal
precursors with 1.0 equivalents of building blocks. The metal precursors that were used in the assembly are Pt(BF4)2(MeCN)4 and Pd(BF4)2(MeCN)4. Both the building blocks and
metal precursors were first dissolved separately in CD3CN and then combined in a vial
and heated to 60 ºC with stirring for a minimum of 3 hours (palladium precursor) and 24
hours (platinum precursor).
