Bachelor Thesis Chemistry

Using TIRF Microscopy to Understand theMechanism of the Biginelli Reaction

by

Teun IJntema

4th of July 2017

Student Number10717242

Research Institute SupervisorVan ’t Hoff Institute for Molecular Sciences Prof. Dr. A.M. Brouwer

Research Group Daily SupervisorMolecular Photonics MSc D. Zheng

Abstract

Mechanistic studies have been performed on the Biginelli reaction, the condensation of ethylacetoacetate, urea and benzaldehyde forming dihydropyrimidones. Previous studies involved trac-ing the reaction with 1H NMR spectroscopy and mass spectrometry, but they have not been suc-cessful in proving the mechanism unambiguously. By using total internal reflection fluorescencemicroscopy, the reaction can be followed on a single molecular level, which can give new insightinto the mechanism. With the use of fluorophoric analogues for benzaldehyde and ethyl acetoac-etate, the association and dissociation to immobilised urea can be displayed. In this project, wefound the reaction proceeds via a condensation of urea and benzaldehyde, followed by the reactionwith ethyl acetoacetate, according to the proposal made by Kappe.

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Samenvatting

De Biginelli reactie is uitgevonden in 1893 en is sindsdien steeds belangrijker geworden. Dereactie producten, dihydropyrimidonen, vertonen een hoge biologische activiteit en worden daar-door veel gebruikt in de farmaceutische industrie. Onder de toepassingen vallen middelen die degroei van tumorcellen hinderen, maar ook helpen tegen bijvoorbeeld epilepsie en malaria. Hetnadeel van deze reactie is dat de opbrengst laag is en dat de reactietijd erg lang is.

Om dit probleem op te lossen worden katalysatoren gebruikt, maar in deze reactie is dat lastig.Doordat er drie reactanten (ureum, benzaldehyde en ethyl acetoacetaat) met elkaar reageren ishet erg lastig om er achter te komen welke reagentia de eerste reactie vormen. Hier achter komenis van uiterst belang, omdat je daarna je katalysatoren op een rationele manier kan ontwerpen.Dit kan veel tijd besparen in het onderzoek, waardoor de reactie eerder efficient gemaakt worden.

Er zijn drie mogelijke eerste reacties: ureum kan eerst reageren met benzaldehyde of eerstmet ethyl acetoacetaat of de twee laatstgenoemde moleculen kunnen eerst met elkaar reageren.Volgens de literatuur is de kans het grootst dat ureum als eerste zal reageren met de anderemoleculen, dus zal het onderzoek zich richten op deze reacties.

Met behulp van een nieuwe techniek binnen de scheikunde, totale interne reflectie fluorescentiemicroscopie, kan er op het niveau van individuele moleculen naar deze reactie gekeken worden.Als een fluorescerend molecuul in de buurt van een glazen plaatje komt, wordt er een signaalontvangen in de detector. In dit onderzoek is ureum vastgezet aan een glazen plaatje. Vervolgenskunnen benzaldehyde en ethyl acetoacetaat daar dan wel of niet aan binden. Deze moleculenvertonen uit zichzelf geen emissie, daarom zijn er groepen aan gezet die dat wel doen. Op dezemanier kan er dus gekeken worden naar welk van de twee moleculen liever met ureum kan reageren.Dit zal dan ook de meest voorkomende volgorde zijn in de Biginelli reactie.

In dit onderzoek zijn we er achter gekomen dat ethyl acetoacetaat uit zichzelf niet zal bindenaan ureum, maar dat dit pas gebeurd als ook benzaldehyde in het reactiemengsel aanwezig is. Ditbetekent dus dat benzaldehyde en ureum eerst met elkaar reageren, voordat de reactie met ethylacetoacetaat ook mogelijk is.

Schematische weergave van een TIRF microscoop. Fluorofore moleculen in de buurt van het oppervlakvertonen fluorescentie, verder weg niet.

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Contents

1 Introduction 6

2 Experimental 92.1 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3 Results & Discussion 113.1 General properties of the Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2 Characterisation of the cover slip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.3 Tracking of the Biginelli Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.4 TIRF microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4 Conclusion 17

5 Outlook 17

Acknowledgements 18

References 19

Supporting Information 20

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1 Introduction

Since the discovery of the Biginelli reaction in 1893 by Pietro Biginelli, a lot of research has beencarried out; both on the mechanism and the products of the reaction. The one-pot condensation ofurea, benzaldehyde and ethyl acetoacetate yields dihydropyrimidones, as shown in Figure 1. Thesereaction products are of huge interest in the pharmaceutical world. Modification of the substratesresults in molecules with altered activity and applications, including anti-tumour activity, potassiumchannel antagonists, anti-HIV agents, anti-malarial drugs, antibiotics and several more.1

Figure 1. The Biginelli reaction.2

The efficiency of this reaction, however, is low. Brønsted acids catalyse the reaction substantially,but the reaction still takes several days.3 Besides the enhancement of the reaction speed, catalysts areneeded to control the enantioselectivity as well. The pharmaceutical applications of the dihydropy-rimidines require specific enantiomers instead of racemic mixtures. Organocatalysts have proven to besuccessful in providing this.3–5 In order to enhance the reaction speed or the enantioselectivity, knowl-edge of the mechanism has to be gained. Several mechanisms have been proposed already, which interms of reaction sequence comes down to three different mechanisms, which are displayed in Figures2, 4 and 6. All mechanisms will be discussed in the following paragraphs.

The mechanism displayed in Figure 2 shows the condensation of ethyl acetoacetate and benzalde-hyde as the primary step, followed by the reaction with urea.6 The main argument presented wasthat the enone intermediate, as displayed in Figure 2, was prepared by the condensation of the twoprecursors with piperidine. This was followed by the addition of urea and catalytic hydrochloric acidin a separate step. As the Biginelli reaction traditionally is a one pot reaction, separating both stepswill not represent the reactivity for the standard reaction. This might alter the reactivity of themolecules thus also the mechanism.

Figure 2. Reaction mechanism of the Biginelli reaction proposed by Sweet and Fissekis.6

Using thiourea instead of normal urea has shown that this sequence of the reaction is even moreunlikely. Under standard Biginelli conditions, the substitution of sulphur for an oxygen results in aproduct shown in Figure 3a.7 When the enolic intermediate (Figure 2) reacts with thiourea, a differentproduct is formed. In this case, the sulphur takes the place of a nitrogen in the ring and forms theproduct displayed in Figure 3b. This is compelling evidence against the enolic intermediate in theBiginelli reaction, as this results in a different product than the original reaction.

(a) (b)

Figure 3. Reaction products of the Biginelli reaction with thiourea (a) and of the enolic intermediatewith thiourea (b).

The second mechanism is displayed in Figure 4, where the aldehyde and urea initially condensateto an iminium intermediate.7 This is followed by the addition of the ethyl acetoacetate, resulting inthe dihydropyrimidone. This mechanism has been proven experimentally using 1H NMR spectroscopyand mass spectrometry.7,8 Even though no intermediates have been isolated or observed by 1H NMRspectroscopy, a bisureide has been detected. This is a byproduct when an excess of urea was present.

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The excess urea reacts with the iminium intermediate, as shown in Figure 5. This is compellingevidence for the iminium pathway, as this molecule can only occur when the benzaldehyde and ureareact.

Figure 4. Reaction mechanism for the Biginelli reaction proposed by Kappe.7

Figure 5. Bisureide that is formed when an excess of urea is present in the Biginelli reaction.7

Intermediates have been detected using mass spectrometry in different studies.8 To prevent anyfragmentation in the reaction mixture, the soft ionisation method electrospray ionisation (ESI) wasused. Continuous snapshots of the reaction mixture have been made using on-line MS. The tan-dem version ESI/MS-MS can characterise the intermediates more profoundly. Little evidence for thepathway described in Figure 2 was found.

Besides the experimental evidence, multiple computational efforts confirm that the iminium routeis most likely in terms of energy.8–10 The energies of the transition states of the iminium route aresubstantially lower than those of the enone route from Figure 2.

In the computational studies described above, a third mechanism was considered as well. Thisstarts with the condensation of ethyl acetoacetate and urea, followed by the reaction with the aldehydegroup, as displayed in Figure 6. The energies of the transition states are supposed to be in between theother two.8 Besides the calculations, there is also experimental evidence that this mechanism couldexist. The 1H NMR- and ESI-MS spectra previously discussed didn’t reveal any intermediates, butlater experiments performed by Cepanec et al. did.7,11

Figure 6. Reaction mechanism for the reaction proposed by Cepanec.11

Instead of Brønsted acids, the inorganic Lewis acid SbCl3 was used to perform this reaction. Theenamine intermediate displayed in Figure 6 was isolated in this study, while no proof of any otherintermediates was found. Even though this thesis is focused on organocatalysis with Brønsted acids,this sequence of the reaction has to be considered.

The problem of finding the right sequence of the reaction could be solved by using total internalreflection fluorescence microscopy (TIRF microscopy). This novel method has shown its worth in bio-physical applications by e.g. clarifying the walking pattern of myosin V and extending the knowledgeof RNA polymerase transcription.12,13 It is relatively new in organic synthesis and catalytic reactions,but there is a lot of promise in this field.14

Total internal reflection is the most important physical basis for this experimental method.15 Thismeans that all the light shun on a surface is reflected and none of the photons penetrates the reflectivesurface. There are two physical requirements for this, the first being that the refractive index of themedium where the laser comes from, n3 in Figure 7, needs to be higher than that of the other medium,n1. This is the case for our experiments, as the 3rd medium is glass, which has a refractive indexhigher than the solvents used.16 The second requirement is for the angle of the incident light needsto exceed the critical angle θ(c) of the two surfaces. The critical angle of two media can be calculatedby Equation 1, where the n’s are the refractive indices as displayed in Figure 7.

θ(c) = sin-1(n1)

(n3)(1)

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If both requirements are met, all the incident light is refracted.15 Even though no light penetratesthe glass cover slip, a small electromagnetic field is created at the interface. This electromagneticfield, called the evanescent field, propagates perpendicular to the interface and covers several hundrednanometers into the solution. This evanescent field is able to excite molecules that are close tothe surface. If those molecules are fluorophoric, they can emit light upon excitation, meaning thatmolecules in the vicinity of the surface light up, while fluorophores further away in the solution remaindark (Figure 7). The background in TIRF experiments is relatively low due to the limited number ofexcited molecules.

Figure 7. An illustration of the principle of TIRF microscopy. The critical angle (θc is exceeded,causing the total internal reflection, illustrated with the white arrow. The evanescent field is encircled,exciting the fluorophores near the surface (white), but not far away (black).15

The TIRF microscopy is thus appropriate for observing molecules that are close to a surface.15

In this project, the goal is to exploit that characteristic by attaching a reactive group to the surface.If a fluorophoric molecule attaches to this group, this group emits light from a specific position fora longer time. You can spot this on the microscope by a fluorescent spot on one position. Whenthe fluorophore is released from the surface, the luminescent spot disappears. The result of this isconstant blinking of all molecules that attach to and are released from the surface. It is important tonote that after disengaging of the fluorophoric groups from the surface, they are still emitting light.However, due to the fast motion of the molecules, the results is an average background emission. Thisis distributed over many locations near the surface and thus less strong than the emission from onefixed position.

The focus of this project is to understand and clarify the reaction mechanism of the Bignellireaction. Much earlier research using for example 1H NMR- and mass-spectrometry has not beenconclusive, so other methods have to be used. The newer, less well known experimental method oftotal internal reflection fluorescence microscopy will be used to understand this mechanism. Therefore,the research question is as follows: Can the reaction mechanism of the Biginelli reaction be clarifiedusing TIRF microscopy?

As explained earlier, the two most likely mechanisms for the Biginelli reaction are the ones proposedby Kappe and by Cepanec (Figures 4 and 6 respectively). This project focuses on distinguishingbetween those two and looking for a preferred mechanism using TIRF microscopy. Both mechanismsstart by a condensation on urea, either with benzaldehyde or ethyl acetoacetate. To exploit thestrengths of TIRF, we are immobilising urea on the surface, as this is present in the first step of bothmechanisms.

When fluorophoric groups are present on ethyl acetoacetate and benzaldehyde, insight in thisreaction can be provided. When looking at the on-time of the blinking, it can be determined whichof the two molecules stays associated to the urea for a longer time. This longer on-time provides alarger possibility for a reaction to occur and. When similar concentrations of fluorophores are used,the amount of blinking can be compared, as this is related to the association constants. The absoluteassociation constants cannot be determined, as there is no way of figuring out the total amount of

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immobilised urea molecules on the surface. Both values, association constant as kbind and dissociationconstant as kunbind are displayed in Figure 8

Figure 8. Association and dissociation of a fluorophoric aldehyde on an immobilised urea. The Rrepresents a fluorophoric group, for example one in Figure 9. The kbind and kunbind are the associationand dissociation constants for this reaction.

As an indication that the reaction is successful, we can look at whether any product is formed.The product will be attached to the surface, which results in permanently illuminated spots.

There are, however, several requirements for the use of the fluorophores. To make a comparisonbetween both experiments, the same settings have to be used on the TIRF microscope. This meansthat the absorption spectra of the two fluorophores have to overlap, as well as the emission spectra. Ifthe absorption spectra do not overlap, a different wavelength has to be used to excite the molecules,which makes a comparison harder. The same holds for the emission spectra, but then different filtershave to be used.

Another requirements is a high quantum yield for the fluorophores. This is the fraction of photonsthat are emitted in respect to the number that are absorbed.17 If the quantum yield is too low, thesignal of the blinking will be too low, causing an insufficient signal to give an accurate representation.

2 Experimental

2.1 Methods

First of all, several basic properties of the fluorophores were measured. These include the absorptionand emission spectra, the molar absorption coefficient, the fluorescence quantum yield and the lifetimeof the excited state.

For the TIRF experiment, fluorophoric analogues of molecules 1 and 3 have been used. Thechemical properties of these molecules differ from the ones in the standard Biginelli reaction, whichis why the reactivity in this reaction has to be tested. The progress of the reaction is monitored bymeasuring the emission spectra, which are expected to change upon the formation of the product.

Several reactions were tested, the reactants and solvents for each individual reaction can be foundin Table 1. All were performed under the same conditions and concentrations, as described in theSupporting Information.3 The reactants that have been used can be found in Figures 1, 9, 10 and 11.

Table 1. The reactions that have been carried out to check the reactivity of the fluorophores. Thereactants can be found in Figures 1, 9, 10 and 11. The concentrations and reaction conditions aredescribed in the Supporting Information.

Reaction Ethyl Acetoacetate Urea Benzaldehyde Catalyst Acid Solvent1 1 2 4 7 Present DMSO2 1 2 4 7 Present THF/EtOH3 1 2 4 No catalyst Not present THF/EtOH4 1 11 4 7 Present THF5 1 11 4 No catalyst Not present THF6 1 2 4 8 Present THF/EtOH7 1 2 4 9 Present THF/EtOH8 1 2 4 10 Present THF/EtOH9 1 2 4 No catalyst Not present THF/EtOH10 1 2 5 7 Present DMSO11 6 2 3 7 Present DMSO12 6 2 5 7 Present DMSO13 Not present 2 5 7 Present DMSO14 6 2 5 7 Present Toluene

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(a) 3-perylenecarboxaldehyde (b) BODIPY aldehyde (c) PMI acetoacetate

Figure 9. The Fluorophores used during this project. Figure (a) shows molecule 4, 3-perylenecarboxaldehyde. Figure (b) shows molecule 5, a BODIPY with a benzaldehyde side group.Figure (c) shows molecule 6, a perylene monoimide with an ethyl acetoacetate side group.

Figure 10. Organocatalysts used for the enantioselective Biginelli reaction.

The TIRF experiment has been performed according to a general procedure, which can be foundin the Supporting Information. The reactants that have been used during the TIRF experiment aredisplayed in Table 2. The concentrations for the reactants are described in the Supporting Informationas well.

Table 2. Reactants for the TIRF experiments. The concentrations and the conditions are given inthe Supporting Information.

Experiment Ethyl Acetoacetate Urea Benzaldehyde Catalyst Acid Solvent1 Not Present Immobilised 5 7 Present Toluene2 1 Immobilised 5 7 Present Toluene3 6 Immobilised Not Present 7 Present Toluene4 6 Immobilised 3 7 Present Toluene5 1 Not Present 5 7 Present Toluene

2.2 Equipment

Figure 11. The substitutedurea that will be immobilisedon a cover slip.

For the TIRF experiment, a stabilite 2017 laser was used at 476 nmwith an average power of 51 mW. An emission filter blocking all lightup to 496 nm was used, together with an extra notch blocking the lightfrom 470 nm to 490 nm. For the recording of the microscope image, aHamamatsu C11440 digital camera was used, recording movies of 1000frames. For the absorption measurements, a Shimadzu UV 2700 UV-vis spectrophotometer has been used. For the emission measurements,several different spectrofluorometers have been used. For the generalproperties of the molecules, the Spex fluorolog 3-22 by Horiba JobinYvon has been used. Due to technical issues with this machine, aFluorolog 3 by Horiba Jobin Yvon has been used to measure the emission spectra for all the reactionsin Table 1. This machine was equipped with a synapse CCD dectector. A Shimadzu LC 10-AT liquidchromatograph was used for the HPLC separation.

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3 Results & Discussion

In this section, the results of the project will be displayed together with a discussion of these results.First of all, the basic properties of the fluorophores 4, 5 and 6 will be looked at. Those include theabsorption and emission spectra, fluorescence quantum yields and lifetime of the excited state. Afterthat, functionalisation of the cover slips will be examined, together with the emission spectra of thereactions from Table 1. The last part includes the analysis of the TIRF experiment.

3.1 General properties of the Fluorophores

Figure 12 shows the normalised absorption and emission spectra of molecules 4, 5 and 6 in toluene.The lines show the absorption spectra, where the dotted lines depict the emission spectra. The spectrawere measured in DCM, DMSO and THF as well, which are included in the Supporting Information.The main difference is that the peaks are less well defined in polar solvents than for the apolar solvents.

Figure 12. Absorption- and emission spectra of molecules 4, 5 and 6 in toluene. The full linesrepresent the absorption spectra, where the dotted lines depict the emission spectra.

In Table 3 the absorption coefficients for molecules 5 and 6 are given. Those have been measuredin duplo, due to the sensitivity of the analysis. As can be seen in the table, a difference betweenboth measurements is observed. The cause of this is the low amount of fluorophores used in theanalysis (below 2 mg) and the weighting scale is not very accurate at those values. Taking that intoconsideration, all duplo measurements for molecule 5 show reasonable similarities, but the ones formolecule 6 are further off.

Table 3. Molar absorption coefficients (ε) · 104 at λ max for molecules 5 and 6. The λmax aredisplayed in brackets following the absorption coefficients. The measurements have been performedin duplo.

Molecule Toluene DCM THF DMSO5 (1st measurement) 12.8 (506) 11.5 (504) 12.3 (503) 11.6 (504)5 (2nd measurement) 12.0 (506) 11.0 (504) 11.4 (503) 10.4 (504)6 (1st measurement) 4.59 (505) 4.50 (512) 3.81 (504) 4.51 (514)6 (2nd measurement) 5.94 (505) 6.48 (512) 6.07 (504) 5.66 (514)

The fluorescence quantum yields of molecule 6 are displayed in Table 4, together with the lifetimeof the excited state. The quantum yield has been calculated according to the method described inliterature, with Rhodamine 6 G as a reference.16,18 As stated in literature, the quantum yield lowerswith increasing polarity of the solvent and the same happens for the primary lifetime.17 There couldbe two explanations for there being two lifetimes. The first one is that there is an impurity in thesample, but HPLC has shown that this was not the case. The second explanation could be that this isa property of the molecule. The similarities in amplitude across all solvents for the τ1 and τ2 supportthis explanation.

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Table 4. Quantum yields and lifetimes (τ) of molecule 6, measured in several solvents. The excitationwavelength used was 494 nm for the quantum yield measurements. For the lifetime experiments, theexcitation wavelength was 488 nm and the emission wavelength was 610 nm.

Solvent Quantum Yield (Φ) τ1 in ns/amplitude τ2 in ns/amplitudeToluene 75.5 % 4.69 (0.81) 0.28 (0.19)DCM 69.4 % 4.62 (0.79) 0.19 (0.21)THF 70.8 % 4.67 (0.79) 0.23 (0.21)

DMSO 62.4 % 4.33 (0.79) 0.68 (0.21)

3.2 Characterisation of the cover slip

Table 5 shows the properties of the functionalised and the clean cover slips. Both cover slips have beencleaned according to the method described in the Supporting Information, the functionalised one hasbeen silinised as well. The goal of performing the three described experiments was to determine if theimmobilisation had been successful. The significant difference in contact angle shows that the surfaceof the cover slips has definitely changed. This is caused by the difference in hydrophilicity betweenthe two surfaces. The TIRF experiment itself shows the most compelling evidence that the surfacehas been functionalised. Bright spots are visible for the functionalised cover slips, which means thefluorophores are attached to the surface. This is not the case for the clean cover slips, therefore thefunctionalisation has been successful.

Table 5. Characterisation of the functionalised and clean cover slips. The contact angle images canbe found in the Supporting Information. The TIRF image for a functionalised cover slip can be foundin Figure 18, the clean cover slip can be found in the Supporting Information.

Clean cover slip Functionalised cover slipContact angle (o) 11 ± 1 32 ± 2

TIRF measurement No signal Blinking

3.3 Tracking of the Biginelli Reaction

For this part, all the result of the reactions described in Table 1 will be discussed in chronologicalorder. Reaction 1 was performed in DMSO, since this is described as the best solvent in terms ofyield and enantioselectivity.3 Molecule 4 turns out to be insoluble in DMSO, hence this reaction wasunsuccessful. THF was described as the second best solvent, so this was chosen for reactions 2 to 9.Urea, however, is poorly soluble in this solvent, so 10 vol% ethanol was added to the THF to solve thisproblem. Reaction 3 was carried out as a control experiment, with the same reactants but withoutacid and catalyst.

Figure 13. Normalised emission spectrum for reactions 2 (full lines) and 3 (dotted lines). Theexcitation wavelength was 490 nm.

The spectra for these two reactions can be found in Figure 13. These spectra give the impressionthat this reaction is not successful. No shift in the emission spectra is seen and the spectra with and

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without acid and catalyst overlap. The reaction does not proceed without the acid, so the similarspectra show that this reaction is unsuccessful without it as well.3 The only shift that can be seen isfrom reaction 2 after 4 days, but the change is insignificantly small, so this will not be discussed.

When the reaction is successful, a change in emission for molecule 4 is expected. The conjugatedsubstituent changes drastically upon forming the reaction product, which influences the wavelengthof the emission. Not seeing this change is a good indication that this reaction is not possible, but noevidence. This evidence can be obtained by performing HPLC, 1H NMR or mass spectrometry withsoft ionisation methods. This was not carried out due to time limitations, which also applies for theother reactions.

Reactions 4 and 5 were performed to check whether the reactivity of the urea molecules withsilicon tails (molecule 11) is different from normal urea. The reactants in those reacions were ethylacetoacetate 1, urea 11 and benzaldehyde 4. Reaction 4 did contain catalyst 7 and trifluoroaceticacid (TFA), where reaction 5 did not. After several minutes a precipitate formed is both samples,most likely due to an aggregation product of the silicon molecules due to a condensation reaction. Forthis reason, the reaction was not tracked.

Other catalysts (8, 9 and 10) have been tested for the reaction of benzaldehyde 4 with moleculesethyl acetoacetate 1 and urea 2 in reactions 6, 7 and 8. Reaction 9 is a control experiment for thosereactions. The emission spectra show no significant change for all cases, which suggests that theBiginelli reaction with molecule 4 is not possible. As this is no evidence that those reactions do notwork, it was tried to measure an 1H NMR spectrum of the reaction mixtures. The concentrations inwhich these reactions have been performed turned out to be too low to obtain a good spectrum.

(a) Reaction 6 (b) Reaction 7

(c) Reaction 8 (d) Reaction 9

Figure 14. Normalised emission spectra for reactions 6 to 9 for the excitation wavelength of 450 nm.All spectra are zoomed in on the peaks, the full spectra can be found in the Supporting Information.

The normalised emission spectra for reactions 10 and 13 can be found in Figure 15. In bothreactions urea 2, benzaldehyde 5, catalyst 7 and trifluoroacetic acid were present. The difference wasthe presence of ethyl acetoacetate 1 in reaction 10, where it was absent in reaction 13. As shown inFigure 15a, there is a shift of the emission spectrum, as described in literature.3 This indicates thatthe reaction is possible with molecule 5 as a replacement for molecule 3. The control reaction 13,which is the same as reaction 13 but without ethyl acetoacetate, shows that this does not happen

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when molecule 1 is absent. This indicates that the Biginelli reaction is successful with benzaldehyde5 as a fluorophore. The small shift that is visible in Figure 15b might be caused by the condensationof molecules 2 and 5.

(a) Reaction 10 (b) Reaction 13

Figure 15. Normalised emission spectra of the Biginelli reaction with fluorophores for the excitationwavelength of 490 nm. Both pictures are zoomed in on the peaks, the full spectra can be found in theSupporting Information.

In reaction 11 and 12, ethyl acetoacetate 6, urea 2, catalyst 7 and TFA were used. In reaction 11benzaldehyde 3 was present, whereas benzaldehyde 5 was present in reaction 12. As shown in Figure16a, no shift of the emission spectrum was observed for reaction 11. As explained earlier, this doesn’tmean that the reaction is not possible.

(a) Reaction 11

(b) Reaction 12

Figure 16. Normalised emission spectra of reactions 11 and 12 for excitation wavelength 490 nm.

The reaction with molecule 6 needed to work for the TIRF experiment to be successful, so extraeffort was put in to proving the reaction works. This is why reaction 12 has been carried out, where

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fluorescent analogues for both 1 and 3 were used. Since the absorption of molecule 6 overlaps withthe emission of molecule 5, Forster resonance energy transfer (FRET) should be observed when theproduct is formed. This would result in a significant increase in the emission peak around 575 nm,while the peak at 515 nm should decrease in intensity. As shown in Figure 16b, this phenomenon isnot observed. The intensities of the peaks relative to each other stays the same, but there is still asmall shift for the peak around 515 nm. So FRET is not observed, but there is another indicationthat the reaction happens.

To make a definitive conclusion, HPLC was performed on the reaction sample. First of all, theHPLC graphs of molecules 5 and 6 were measured separately, they can be found in the SupportingInformation. As shown in Figure 17, these two molecules were still present after the reaction, but anew product showed up at 14 minutes. This could very well be the reaction product, but if it was,the BODIPY peak is expected to be more prominent due to its higher absorption coefficient. It stillhas to be investigated what the cause is of this peak.

Figure 17. HPLC graph of reaction mixture 14. A normal phase column was used with a 2% ethylacetate in DCM as eluent and a UV-Vis detector. The absorption spectrum of the peak at 14 min isdisplayed on the left.

3.4 TIRF microscopy

(a) (b)

Figure 18. TIRF figures of the experiments with BODIPY aldehyde 5. (a) Is the image of theexperiment 1, with urea 11 on the surface, BODIPY aldehyde 5, catalyst 7 and TFA. (b) is theimage of experiment 2, where ethyl acetoacetate was added to the reactants of experiment 1. The Ris BODIPY. A time trace of those experiments is included in the Supporting Information

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For the final part of the discussion, the results of the TIRF microscopy experiment will be discussed.The first experiment was performed to look at the association of BODIPY aldehyde to the immobilisedurea, in presence of catalyst 7 and TFA. As shown in Figure 18a, a signal was found for these tworeactants. Some of the spots were on for a time period of up to multiple seconds, as shown in theSupporting Information. This might indicate the formation of the condensation product of urea andmolecule 5, as shown in Figure 18a. However, most of the dots were merely blinking in the time frameof hundreds of milliseconds.

For experiment 2, a new cuvette with ethyl acetoacetate 1, urea 11 on the cover slip, benzaldehyde5, catalyst 7 and TFA was observed under the TIRF microscope. When comparing the videos of thisexperiment to the ones of experiment one, it was clear that the amount of blinking molecules did notincrease. What did happen was that the luminescence of the spots was more intense. The productof this experiment, which is shown in Figure 18b, has a higher fluorescence quantum yield than thereaction product of experiment 1.3 Permanently illuminated spots appeared as well. The lifetime ofthis luminescence was up to 50 seconds, after which bleaching killed the fluorescence.

More telling were the TIRF experiments with PMI acetoacetate 6. This fluorophore was presentin reactions 3 and 4, together with urea 11 immobilised on the surface, catalyst 7 and TFA. Thedifference can be found in the presence of benzaldehyde 3 in reaction 4, where it was absent inreaction 3. As shown in Figure 19a, no signal is observed in this reaction. This means that the isno association between those molecules and that condensation does not work and there is little to noattraction between them.

As shown in Figure 19b, permanent fluorescent sport were observed after the addition of ben-zaldehyde 3 in reaction 4. This observation suggests that for the product to be formed, urea andbenzaldehyde need to react first. Ethyl acetoacetate reacts with the condensate and thus does notparticipate in the first step.

(a) (b)

Figure 19. TIRF figures of the experiments with PMI acetoacetate 6. (a) is experiment 3 with urea11 on the surface, PMI acetoacetate 6, catalyst 7 and TFA. (b) is experiment 4, where benzaldehydewas added to the reactants of experiment 3. The R is perylene monoimide. A time trace of thisexperiment is included in the Supporting Information

In the introduction, more objective ways of identifying a preferred reaction path were discussed,such as the association and dissociation constant. Those values were not obtained during this exper-iments, as not enough molecules were blinking. This means not enough data could be gathered onthe values. Besides that, a vast amount of the blinking molecules were not bright enough to analyse,especially for experiments 3 and 4.

The reason for both limitations could be found in the that the silinisation was not optimal. Themethod used has been optimised for another molecule instead of molecule 11. The silinisation hasto be optimised for this molecule itself. Another reason is that the fluorescence quantum yield formolecule 5 is lower than optimal. This means that not all excited molecules actually fluoresce, so theamount of of illumination is limited.

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4 Conclusion

From the TIRF microscopy experiment the conclusion can be drawn that the association betweenmolecule benzaldehyde and urea is the first step of the Biginelli reaction. The fluorophoric analogueof benzaldehyde shows blinking on surface on which urea has been immobilised. This happens im-mediately after the addition, which implies that there is an attractive force between these molecules.After one hour, a condensation product in the form of a permanently illuminated spot was found aswell. Both these phenomenon were not the case for the fluorophoric analogue for ethyl acetoacetatewith the immobilised urea.

The main argument for this sequence of the reaction is that permanently illuminated spots arefound after the addition of benzaldehyde to the fluorophoric analogue of ethyl acetoacetate and theimmobilised urea. This means that benzaldehyde reacts with the immobilised urea first, followed bythe attachment of the fluorophore. Therefore, the correct mechanism for the Biginelli reaction is theone proposed by Kappe.7

5 Outlook

The main issue we encountered during this project was the low amount of blinking molecules duringthe TIRF experiment. The solution for this problem can be found in three parts. First of all, thesilinisation method has to be optimised for molecule 11. The method that was used has been optimisedfor an amine instead of the urea that was used here. Optimisation should result in a higher amountof immobilised urea on the surface, which will improve the amount of molecules that can attach tothe surface during the experiment. Another solution could be the use of different fluorophoric groupswith a higher fluorescence quantum yield. This results in more emission when a molecule is attachedthus a better signal. Simply using a higher concentration of fluorophore could also solve the problem.The problem with the last two methods is that the background signal will also increase, so the firstmethod is recommended.

Once the problem of the low signal has been solved, other, more objective measurements can becarried out. As described in the introduction, the goal was to find dissociation and reaction constants.Those values can give a better insight in the reaction mechanism than the comparative study we havedone so far.

Another interesting characteristic would be getting to know how much silanised groups are presenton the surface. If this is known, the association constants can be measured instead of comparing themusing similar concentrations of fluorophores. This, together with the measurement of the dissociationconstant, could actually prove which of the sequences of the reactions is more likely to be the rightone.

Finally, what could still be done is checking if the reactions from Table 1 have actually failed orsucceeded. Measuring the emission spectra could indicate that a change or reaction has happened,but is no unambiguous proof. This could be done by performing 1H NMR spectroscopy or massspectrometry on the reaction samples.

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Acknowledgements

First of all, I would like to thank prof. dr. Fred Brouwer for giving me the opportunity to do thisfascinating project to finish of my bachelor. The project has been very inspirational and I enjoyedmy time thoroughly. Secondly, I would like to thank my daily supervisor Dongdong Zheng for hishelp every day, the interesting discussions and finally for great company during the last three months.I would also like to thank Dina Petrova, Michiel Hilbers and Chia-Ching Huang for the help withthe TIRF-, lifetime- and emission measurements and Hans Sanders and Mina Raeisolsadati Oskoueifor the preparation of the fluorophores. I would also like to thank Nicole Oudhof for the excellentcompany, the free coffee and reviewing my thesis. For the last reason, I would like to thank Christiaanvan Campenhout and Rosa Brakkee as well. My final gratitude is for the whole Molecular Photonicsgroup for the great atmosphere during and after the working hours. I really enjoyed my stay at thisamazing research group.

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References

[1] Sandhu, J. S.; Suresh, Arkivoc 2012, Part (i), 66–133.

[2] Biginelli, P. Gazz. Chim. Ital 1893, 23, 360–416.

[3] Raeisolsadati Oskouei, M. Fluorogenic Organocatalytic Reactions. Ph.D. thesis, University of Amsterdam, 2017.[4] Chen, X.-H.; Xu, X.-Y.; Liu, H.; Cun, L.-F.; Gong, L.-Z. J. Am. Chem. Soc. 2006, 128, 14802–14803.

[5] Wang, Y.; Yang, H.; Yu, J.; Miao, Z.; Chen, R. Adv. Synth. Catal. 2009, 351, 3057–3062.

[6] Sweet, F.; Fissekis, J. D. J. Am. Chem. Soc. 1973, 95, 8741–8749.[7] Kappe, C. O. J. Org. Chem. 1997, 62, 7201–7204.

[8] De Souza, R. O.; da Penha, E. T.; Milagre, H.; Garden, S. J.; Esteves, P. M.; Eberlin, M. N.; Antunes, O. A.Chem. Eur. J. 2009, 15, 9799–9804.

[9] Ma, J. G.; Zhang, J. M.; Jiang, H. H.; Ma, W. Y.; Zhou, J. H. Chin. Chem. Lett. 2008, 19, 375–378.

[10] Lu, N.; Chen, D.; Zhang, G.; Liu, Q. Int. J. Quantum Chem. 2011, 111, 2031–2038.[11] Cepanec, I.; Litvic, M.; Filipan-Litvic, M.; Grungold, I. Tetrahedron 2007, 63, 11822–11827.

[12] Yildiz, A.; Forkey, J. N.; McKinney, S. A.; Ha, T.; Goldman, Y. E.; Selvin, P. R. Science 2003, 300, 2061–2065.

[13] Kapanidis, A. N.; Margeat, E.; Ho, S. O.; Kortkhonjia, E.; Weiss, S.; Ebright, R. H. Science 2006, 314, 1144–1147.[14] Zhang, Y.; Song, P.; Fu, Q.; Ruan, M.; Xu, W. Nat. Commun. 2014, 5, 4238.

[15] Axelrod, D. Methods Cell Biol. 2008, 89, 169–221.[16] Refractive Index. http://macro.lsu.edu/HowTo/solvents/Refractive%20Index.htm, accessed on 29th of June 2017.

[17] Lakowicz, J. R., Ed. Principles of Fluorescence Spectroscopy; Springer US: Boston, MA, 2006; pp 205–235.

[18] Wurth, C.; Grabolle, M.; Pauli, J.; Spieles, M.; Resch-Genger, U. Nat. protoc. 2013, 8, 1535–1550.[19] Suhina, T.; Weber, B.; Carpentier, C. E.; Lorincz, K.; Schall, P.; Bonn, D.; Brouwer, A. M. Angew. Chem. 2015,

127, 3759–3762.

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Supporting Information

General Procedure 1: Tracking the Biginelli Reaction

Ethyl acetoacetate (1.3 · 10-5 mole, 1 eq), benzaldehyde, (1.3 · 10-5 mole, 1 eq) and urea (1.3 · 10-5

mole, 1 eq) were dissolved in 0.2 mL solvent. For the cases where the fluorophoric analogues of ethylacetoacetate and benzaldehyde were used, the same amount was added as for the standard molecules.Afterwards, a catalyst from Figure 10 (2.6 · 10-6 mole, 0.2 eq) and trifluoroacetic acid (2.6 · 10-6 mole,0.2 eq) were added. For the control experiments, this step was not carried out. The reaction was runat 35 oC with the use of a water bath. The emission spectrum was measured immediately after addingthe TFA or the last component in the case of the control experiments and several days of reactingonwards.

General Procedure 2: Cleaning and Silanisation of Cover Slips19

The cover slips were washed in 0,3 % Extran AP 12 solution by sonication for 30 minutes at 40 oC,followed by sonication in deionised water for 10 minutes and in ethanol for 30 minutes. The coverslips were dried in an oven for 1 hour and further cleaned in an ozone photoreactor for 2 hours.The cuvettes and stops used in the TIRF experiment were cleaned according to the same procedure.Afterwards, the cover slips were silanised with 2% (volume) 1-[3-(Trimethoxysilyl)propyl]urea in 96%ethanol in which 2% water was added. The pH of this solution was adjusted to approximately 5 byaddition of acetic acid. A teflon rack with cover slips was kept for 1 hour in this solution with stirring.The cover slips were sonicated afterwards for three times in ethanol (20 minutes in total) and washedwith acetone and DCM subsequently. Finally, the cover slips were dried in air and put in an oven for3 hours at 110 oC to keep them clean.

General Procedure 3: TIRF microscopy experiment

Cuvettes were glued to the functionalised cover slips using Devcon two component epoxy glue. Thefirst step for all experiments was checking the microscope’s image for just the solvent. 0.2 mL of thesolvent was deposited in the cuvette and observed under the microscope. The second step was theaddition of the reaction components, as displayed in Table 2. The concentrations in the cuvettes wereas follows: the concentration of the fluorophores (molecule 5 or 6) was 10-10 M, of the other reactant(molecule 1 or 3) it was 10-4 M and for the catalyst and the trifluoroacetic acid 2·10-5 M. The reactionmixture was observed using the TIRF microscope immediately and after one to two hours.

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Graphs and Images

Figure 20. Absorption- and emission spectra of molecules 4, 5 and 6 in DMSO. The full linesrepresent the absorption spectra, where the dotted lines depict the emission spectra.

Figure 21. Absorption- and emission spectra of molecules 4, 5 and 6 in DCM. The full lines representthe absorption spectra, where the dotted lines depict the emission spectra.

Figure 22. Absorption- and emission spectra of molecules 4, 5 and 6 in THF. The full lines representthe absorption spectra, where the dotted lines depict the emission spectra.

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(a) (b)

Figure 23. Contact angle of a functionalised and a clean cover slip (Figures (a) and (b) respectively).

Figure 24. TIRF image of a cover slip cleaned according to the method described in the SupportingInformation. No silinisation was performed. Benzaldehyde 5 was used as a fluorophore.

Figure 25. Histogram of the lifetime measurement of molecule 6 in Toluene, excited at 610 nm.

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Figure 26. Histogram of the lifetime measurement of molecule 6 in DCM, excited at 610 nm.

Figure 27. Histogram of the lifetime measurement of molecule 6 in THF, excited at 610 nm.

Figure 28. Histogram of the lifetime measurement of molecule 6 in DMSO, excited at 610 nm.

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(a) Reaction 6 (b) Reaction 7

(c) Reaction 8 (d) Reaction 9

Figure 29. Emission spectra for reactions 6 to 9. The excitation wavelength was 450 nm.

Figure 30. Emission spectrum for reaction 10, excited at 490 nm.

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Figure 31. Emission spectrum for reaction 13, excited at 490 nm.

Figure 32. HPLC graph of benzaldehyde 5. A normal phase column was used with a 2% ethylacetate in DCM as eluent and a UV-Vis detector. The absorption spectrum of the peak at 3 minutesis displayed in small.

Figure 33. HPLC graph of benzaldehyde 6. A normal phase column was used with a 2% ethyl acetatein DCM as eluent and a UV-Vis detector. The absorption spectrum of the peak at 6.5 minutes isdisplayed in small.

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Figure 34. Time trace of a blinking spot during TIRF experiment 1.

Figure 35. Time trace of a permanently fluorescing spot until bleaching occured during TIRFexperiment 2.

Figure 36. Time trace of a permanently fluorescing spot until bleaching occured during TIRFexperiment 4.

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