3.2.Resolution of the novel functionalized unsymmetrical derivatives

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Cation R yield

1q (CH2)5N3 80%

1r (CH2)2O(CH2)2N3 85%

1s (CH2)5OH 70%

1t (CH2)2O(CH2)2OH 60%^a

a unoptimised data

Table IV-2: Yields of the new symmetrical DMQA derivatives 1q-t

The purification of 1q, 1s and 1t was performed as previously described, by precipitation in 100 mL of a 0.2M solution of KPF6(aq) followed by a thorough wash with water. All the compounds were then further purified by dissolving in CH2Cl2 and adding an excess of Et2O and/or pentane to afford a dark green precipitate in good yields (60-80%). However, in the case of DMQA 1r, the purification procedure was different. The excess of amine and NMP was distilled from the crude reaction mixture followed by silica gel column chromatography to remove unreacted material with CH2Cl2/MeOH 95:5 as eluent. The isolated fraction was evaporated and dried on the vacuum line but the material appeared viscuous and did not give a solid precipitate as in the previous cases. The ¹H-NMR spectrum showed no presence of solvent responsible for such a behavior and an entirely clean compound in 85% yield. It seems that the presence of the oxygen is as expected, affecting its general aspect and increasing its water solubility. Indeed, this derivative is water soluble even in the presence of the hydrophobic PF6 counterion.

In any case, compound 1t could be transformed into 1r using the traditional sequence of mesylation (MsCl, Et3N, CH2Cl2, 25 °C, 20 h) and nucleophilic substitution (NaN3, DMF, 25°C, 24 h) in 85% overall yield.²¹ Overall, it was found that the direct addition of the amine 23b was easier to perform.

IV-3.2.Resolution of the novel functionalized unsymmetrical derivatives

Rather than perform the photophysical studies with racemic substrates only, we considered that it would be interesting to use enantiopure dyes, as single enantiomers could possibly interact differently. Consequently, this new set of compounds needed to be resolved.

21 i.e. the same purity and yield as in the direct addition of 23b.

The same protocol established in the group through the enantiopure sulfoxide addition was also most useful in this case. For the unsymmetrical structures, we will only detail the ones with the n-propyl side chains since the reactions with methyl group-containing cations led to complex mixtures of diastereoisomers that were extremely hard to separate. In this case, the process was time-consuming and poorly efficient overall.

As shown with 1n in chapter II, unsymmetrical dyes form upon addition of the enantiopure sulfoxide 9, four diastereoisomers. The usual conditions employed and detailed previously were not changed and the quenching of the reaction was performed immediately upon the final addition step of the anion on the corresponding DMQA.

To simplify matters, it was decided to group some fractions and isolate two M and two P diastereoisomers together; this being possible due to the consecutive elution of isomers M1, M2

and then P3, P4 as seen with 1n. As such, two mixtures of diastereoisomers, each containing two isomers of the same helicity were observed on TLC, in ¹H-NMR and on HPLC using a MN-Nucleosil column (Figure IV-4).²² Their chromatographic separation was afforded on silica gel using EtOAc/Et2O 30:70 (ΔR_f = 0.26-0.30). Both fractions were isolated in good yields. In the methylene series, (M) and (P)-10v were obtained in slightly higher yield (44% and 42%

respectively) than their oxo-analogues (M) and (P)-10x (38% and 37% respectively) (Scheme IV-8).

Scheme IV-8: Enantiopure sulfoxide addition on unsymmetrical azide-containing DMQA 1v-x

Let us note that since we are dealing with mixtures, we will only relate to them simply by (R, M) and (R, P) without considering the absolute configuration of the new stereocenter obtained upon addition.

22 Mentioned in chapter II.

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Fraction R R1 yield

(R, M)-10v (CH2)5N3 Pr 44%

(R, P)-10v (CH2)5N3 Pr 42%

(R, M)-10x (CH2)2O(CH2)2N3 Pr 38%

(R, P)-10x (CH2)2O(CH2)2N3 Pr 37%

Table IV-3: Yields of the separated fractions of 10v and 10x

The separated fractions were then characterized by ¹H-NMR, UV and CD spectroscopies and as seen in chapter II, the first eluted fractions afforded the derivatives of M helicity whereas the least eluted fraction was shown as having the P helicity. Moreover, the same observations were detected in terms of a large chemical shifts for the methylene protons next to the sulfoxide moiety ΔδCH2SO and a small one for the methoxy groups ΔδOMe in the (-)-(R, M) series. As expected, the opposite was noticed with the (+)-(R, P) series.

Figure IV-4: HPLC chromatogram of the crude mixture from 10v (top) and the first separated fraction corresponding to (R, M₁)-10v and (R, M₂)-10v (bottom)

Figure IV-5: ¹H-NMR spectra of the crude mixture of 10v, with each of the separated fractions of M and P helicity, containing two isomers

Figure IV-6: HPLC chromatogram of the crude mixture from 10x. The first two eluted fractions are of the same (M)-helicity, whereas the two least eluted ones are of the opposite (P)-helicity.

96 | P a g e  Figure IV-7: ¹H-NMR spectra of the crude mixture of 10x, with each of the separated fractions

of M and P helicity, containing two isomers

The following Pummerer fragmentation steps did not pose any problem. The release of the enantiopure cations was done using TFAA in excess and afforded the desired material after 20 minutes in excellent yields in both enantiomeric series (85-90%; Scheme IV-9).

Scheme IV-9: DMQA regeneration in enantiopure form by Pummerer fragmentation with trifluoroacetic anhydride

The same conditions were applied to the bis-azide derivatives 1q-1r. Their resolution was successful via the “sulfoxide” protocol that gave, to our surprise, lower yields for one diastereomer compared to the other. For instance, in the case of 10q with the fully carbonated side chain, 26% of the (-)-(R, M) was isolated whereas the (+)-(R, P) diastereoisomer was obtained in 48% yield. The opposite was however detected for the derivatives of 10r. In this case, 43% of (-)-(R, M) and 24% of (+)-(R, P) were recovered. It seems that, in each case, the minor diastereomer is less stable, undergoing degradation and regenerating the cation spontaneously (Figures IV-8 and IV-9). The same Pummerer fragmentation reaction also gave the enantiopure material in both series for each enantiomer in 80-90% yield

Figure IV-8: ¹H-NMR spectra of the crude mixture of 10q, with each of the separated fractions of M and P helicity

98 | P a g e  Figure IV-9: ¹H-NMR spectra of the crude mixture of 10r, with each of the separated fractions of

M and P helicity

With the azido dyes synthesized and resolved, we turned our attention to the making of the biotin motifs in order to get the propargylic ester and amide needed for the implementation of the mono and double “click” reactions that would lead to the new biotinylated photoprobes.

IV-3.3.Synthesis of the functionalized Biotin derivatives