3.4.New Biotinylated photoprobes via copper(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition

100 | P a g e  An excess of HBTU and Et3N along with 1.1 equivalents of D-(+)-Biotin were dissolved in DMF at room temperature (RT). One equivalent of propargyl amine was added dropwise at 0 °C to form the new peptide bond and the resulting solution was stirred for 25 minutes at RT (Scheme IV-11, vide supra).

Regardless of the cost, this new set of conditions was very fast and provided us with the desired molecule as identified by a peak of 282 m/z in ESI⁺-MS. The purification, however, was another matter altogether. The previously reported syntheses of biotin propargylic amide were done either on solid-phase or purified on preparative HPLC.²⁴ We, on the contrary, did not use such techniques but undertook first the purification on silica gel chromatography.²⁵ Due to the high polarity of the compound and the presence of the residues from the coupling agent, this technique was onerous even with 20 % of MeOH in CH2Cl2 as eluent. The coupling reagent residues still migrated with the compound and enormeous material losses were observed. Finally, we removed DMF as in the other cases and carried out several precipitations, first by dissolving the brown highly viscuous crude in CH2Cl2 and adding 10 % of MeOH. Unfortunately, this did not remove completely the residues of the coupling reagent. We then tried to dissolve the material in THF then add an excess of hexanes. This last method proved successful for the isolation of a clean compound 31 in 55-60 % yield.

IV-3.4.New Biotinylated photoprobes via copper(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition

IV-3.4.a.Monobiotinylated photoprobes

Having obtained the two series of desired fragments, what remained to be done was to assemble these two parts by the chosen methodology, i.e. the copper(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition reaction. A new family of labeled photoprobes would result and would be useful to undergo investigations of the structure and dynamics of the biotin-Strept(avidin) complex.

For such a method to proceed, a careful selection of the most suitable conditions was essential.

First, we started by using CuBr as the metal source with one equivalent of each of the biotin       

24 Zhao, X. Z.; Semenova, E. A.; Liao, C.; Nicklaus, M.; Pommier, Y.; Burke Jr, T. R. Bioorg. Med. Chem. 2006, 14, 7816-7825.

25 Aqueous work up was unfortunately not an option since the impurities and the final product were both water soluble.

motif and the reacting DMQA in DMF at room temperature.¹⁴ Although very simple to put into practice, less than a stoechiometric amount of copper afforded sluggish reactions which needed more than seven days to attain a decent amount of product. The reaction progress was monitored by TLC and especially ESI⁺-MS. In the positive mode, peaks 454-456 m/z corresponding respectively to the “free” DMQA 1u and 1w on one hand and 482-484 m/z, corresponding to 1v and 1x on the other hand, disappeared to generate 736.5-738.5 and 764.5-766.5 as major peaks relative to the expected products (17u-w and 17v-x). However, the increase of the amount of CuBr to 1.5 equivalents afforded acceptable results in 24 hours. These conditions were tested with the racemic DMQA derivatives (R = Me, ⁿPr, X = O, CH2; 1u-1x) and only with biotin propargylic ester 29, since it was easily formed in larger quantities than the parent amide 31.

Scheme IV-12: First selected conditions for the copper(I)-catalyzed azide alkyne 1,3-dipolar cycloaddition using stoechiometric CuBr as metal source. Yields are presented in table IV-4.

After removal of DMF by distillation and copper by filtration over a celite pad, the crude compounds were purified first by precipitation in a CH2Cl2/Et2O mixture then by silica gel column chromatography using CH2Cl2/MeOH 95:5 as eluent.

Although, very easy to perform, the reactions afforded low yields for the isolated products (23-38%). This could be explained by the unreacting starting material seen in the mass spectra that was removed each time as the first eluting fraction during the chromatographic purification. The

102 | P a g e  second reason could be the inability of the reaction to evolve due to rapid oxidation of the copper source, despite working in oxygen free conditions and the addition of an excess of biotin ester.

The numerous crystallizations performed to obtain a clean compound could also account for such low values. Finally, traces of “DAOTA-clicked” derivatives were also found due to previous contamination of the racemic DMQA salts engaged in the reaction.²⁶

The outcome being unsatisfactory, we chose another set of conditions using copper (II) salt that ought to be easier to manipulate. A catalytic amount of CuSO4.5H2O (0.15 equiv.) was thus used along with twice its amount of sodium ascorbate needed to reduce copper in situ.

HN

Scheme IV-13: Modified conditions for the copper(I)-catalyzed azide alkyne 1,3-dipolar cycloaddition of biotin propargylic ester 29 and 1v-x using catalytic CuSO₄.5 H₂O as metal source

Cation Copper source (1.3 equiv.) R R1 Isolated yield

Table IV-4: Yields of the new monobiotinylated derivatives 17u-x with two different stoechiometric copper sources (CuBr vs CuSO₄.5H₂O)

26 A control reaction showed no in situ generation of DAOTA from DMQA in the used conditions (CuBr, DMF, RT, 24 h) except from previous contamination during the nucleophilic aromatic substitution SNAr at 90 °C.

To be on the safe side, we chose to employ 1.2 equivalents of biotin ester instead of 1.0, and kept the cation as the deficient reagent. Solubility was important in this new system and we chose MeOH because it afforded the best solubility for the cations. Water was necessary for the dissolution of the copper source, the biotin ester and sodium ascorbate. It was added in a 1:4 ratio compared to methanol. The reaction proceeded cleanly at 23 °C in four days. The purification of the crude mixture was carried out by silica gel chromatography with the same eluent as before (CH2Cl2/MeOH 95:5) after in vacuo removal of the solvents. Better yields and purity were obtained compared to the previous system (increase from 23 to 70% for rac-17v and from 27 to 40% for rac-17x). Increasing the amount of CuSO4.5H2O to 1.3 equivalents afforded the “completion” of the reaction in 24 h. This system was deemed more satisfactory and we carried out the synthesis of the first series of monobiotinylated dyes with biotin ester; the cations being racemic and carrying only propyl side chains (Table IV-4).²⁷

In addition to monitoring the reaction progress by TLC, we chose to make use of ESI-MS. Mass spectroscopy has emerged as a very useful detecting technique due to its low sample consumption, fast analysis time and straightforwardness, making it adaptable to high throughput screening. This was the case in the attempts of purification of the diverse triazole-containing new dyes 17. The ESI source was operated in the positive ion mode and each spectrum was acquired and analyzed. A large importance was put on ESI-MS since the samples were very close in Rf on TLC and the impurities presented several similarities (for instance in the NMR spectra and their color) with the desired compounds.

These same conditions were employed for the preparation of the amide derived products 18.

However, due to the very polar character of these compounds, chromatographic purifications were difficult and larger proportions of MeOH (20 to 30 % MeOH in CH2Cl2) were required for elution. Loss of material was nevertheless unavoidable and lower yields were obtained (25-60 %;

Scheme IV-14).

27 This is due to the fact that the methyl substituents were now known to have a dramatic effect on the chromatographic behavior of the sulfoxide adducts in the resolution protocol.

104 | P a g e    Scheme IV-14: Synthetic conditions for the copper(I)-catalyzed azide alkyne 1,3-dipolar cycloaddition of

biotin propargylic amide 31 and 1v-x

Product^a Biotin R R1 Isolated yield^c

Rac-17v Ester 29 (CH2)5N3 Pr 70%

(-)-(M)-17v Ester 29 (CH2)5N3 Pr 44%

(+)-(P)-17v Ester 29 (CH2)5N3 Pr 46%

Rac-18v Amide 31 (CH2)5N3 Pr 50%

(-)-(M)-18v Amide 31 (CH2)5N3 Pr 52%

(+)-(P)-18v Amide 31 (CH2)5N3 Pr 59%

Rac-17x Ester 29 (CH2)2O(CH2)2N3 Pr 50%

(-)-(M)-17x Ester 29 (CH2)2O(CH2)2N3 Pr 42%

(+)-(P)-17x Ester 29 (CH2)2O(CH2)2N3 Pr 44%

Rac-18x Amide 31 (CH2)2O(CH2)2N3 Pr 60%

(-)-(M)-18x Amide 31 (CH2)2O(CH2)2N3 Pr --^b (+)-(P)-18x Amide 31 (CH2)2O(CH2)2N3 Pr --^b

afor the designation only the change of helicity is considered. ^blast purification step was problematic. ^cCuSO4.5H2O (1.3 equiv. /N3), Na Ascorbate (2.6 equiv./N3), DMF/MeOH/ H2O 1:2:1, biotin ester 29 or amide 31(1.2 equiv.).

Table IV-5: Yields of the monobiotinylated unsymmetrical DMQA in racemic and enantiopure form.

With the biotin ester, crude reaction mixtures were easier to purify than with the amide since the compounds were slightly less polar. The presence of unidentified compounds which were almost unseparable from the desired products demanded the recourse to two or even three consecutive column chromatographies with up to 50% of methanol in the eluent mixture.

Having established the synthesis of 17v, 17x, 18v and 18x in racemic series, the enantiopure cations were then used in the coupling reactions, their resolution having been detailed earlier (§IV-3.2.). Slightly lower yields were isolated for the enantiopure material (44-46% respectively for the M and P enantiomers of 17v) probably because of the difference in the counterion. In all the cases, the full conversion was never attained. There was always a portion of unreacted starting material as the first eluted fraction. A so far unidentified compound always appeared as the second eluted fraction, preceding the desired product. Its mass always corresponded to a value of 44 m/z over the mass of the expected product (M+44). Since biotin, is a cofactor responsible for carbon dioxide transfer in several carboxylase enzymes, it is prone to bind CO2

and this may explain the secondary product. Surprisingly, we were able to “isolate” this material which presented almost the same ¹H-NMR as the one for the expected dye with few differences.

With biotin amide, as already mentioned products were even more difficult to obtain pure. As the slightest contamination could influence the extremely delicate photophysical studies and induce false results, it was essential to perform as many columns as possible in order to isolate satisfactorily clean fractions. This however played a major part in lowering the yields since many fractions were contaminated with slight traces of impurities. At the end, we were left with as much as 2-6 mg maximum of almost every compound, albeit in pure form (Table IV-5).

IV-3.4.b.Bisbiotinylated photoprobes

To form the bis-biotinylated derivatives, we used the same conditions as for the mono and the material was afforded first in racemic form. Some of the enantiopure compounds were isolated and characterized as well (8-15 % yields).²⁸

28 By ESI⁺-MS, we have noticed the presence of “mono-click” adducts. Driving the reaction to completion was not always feasible necessitating the isolation of the mono-triazole derivatives and their resubmission to the reaction conditions.

106 | P a g e    Scheme IV-15: Synthetic conditions for the bis-copper(I)-catalyzed azide alkyne 1,3-dipolar cycloaddition

with biotin propargylic ester 29 or amide 31 and 1q-r

Entry Biotin R R1 Isolated yield^a

Rac-19q Ester 29 (CH2)5N3 (CH2)5N3 15%

(-)-(M)-19q Ester 29 (CH2)5N3 (CH2)5N3 14%

(+)-(P)-19q Ester 29 (CH2)5N3 (CH2)5N3 13%

Rac-20q Amide 31 (CH2)5N3 (CH2)5N3 --^b

Rac-19r Ester 29 (CH2)2O(CH2)2N3 (CH2)2O(CH2)2N3 8%

Rac-20r Amide 31 (CH2)2O(CH2)2N3 (CH2)2O(CH2)2N3 --^b

aCuSO4.5H2O (1.3 equiv. /N3), Na Ascorbate (2.6 equiv./N3), DMF/MeOH/ H2O 1:2:1, biotin ester 29 or amide 31(1.2 equiv.). ^b only mono amide isolated (10%).

Table IV-6: Yields of the bis-biotinylated symmetrical DMQA in racemic and enantiopure form.

The bis-biotinylated compounds are even less soluble than the mono derivatives and a few drops of deuterated methanol in d2-CH2Cl2 in the NMR tube were required to solubilize everything for NMR detection. These compounds were extremely polar and difficult to purify even after three chromatographic separations; no decent amounts were isolated and always in slightly contaminated fashion. Only scarce amount of enantiopure materials, due to the many steps

needed for their preparation and purification in particular. Overall the yields for the bis-biotinylated compounds 19 ranged from 8 to 15 %.