Azide/Alkyne Reactive Terminators

Having established a strategy for the engineering of new CPDs at the sidechain level, we were eager to explore post-functionalization at other sites of the polymer structure. In particular, we chose to work at the terminator level since studies on initiator modification had already been conducted in the group.^{[52, 56]}

We decided therefore to proceed by first evaluating the efficiency of the simple and commercially available iodoacetamide 23 that had so far been used as terminator for the quenching of the disulfide exchange polymerization. We were also eager to address the main disadvantages of 23, namely the moderate yields of termination even by using a large excess (1.0 M) and the inability to further functionalize the polymer at this site. For this reason, we decided to screen new terminators that could result in better termination at lower concentrations. Moreover, we decided to work with derivatives equipped with either an azide or an alkyne group in order to be able to exploit functionalization through cycloaddition reactions. In particular, we decided to introduce a model fluorescent substrate at the terminator level of the CPD that would enable the use

of techniques such as UV-Vis and fluorescence to evaluate the efficiency of incorporation, as shown previously.

Therefore, we grew two CPDs starting from a green fluorescent initiator and quenched the reaction mixture with two different terminators bearing an azide group. After purification of the two CPDs, CuAAC reaction would be performed using a red fluorescent alkyne, as shown in Figure 108. The yield of the cycloaddition would reflect the efficiency of incorporation of the terminators.

Figure 108. Schematic representation of disulfide exchange polymerization with azide reactive terminators followed by CuAAC reaction with a red-fluorescent alkyne.

The structure of the terminators we decided to screen is shown in Figure 109.

Both 240 and 241 were equipped with an azide group, essential for the cycloaddition reaction, and in particular 241 was chosen as an analog of iodoacetamide 23, while terminator 240 was chosen as a new intriguing reagent known to efficiently quench thiols in different types of reactions both on small organic molecules and on larger substrates such as cells and proteins.^[190]

S ⁿS S +

N₃

polymerization

S S

S n

+

N₃

CuAAC

S S

S n

+

N N N

Figure 109. Structure of the two terminators used in the screening study.

Terminator 240, an ethynyl benziodoxolone (EBX) derivative, was developed in the Waser group in 2013.^[232] Its historical precursor, EBX 242 (Figure 110), was first synthesized by Simonsen and coworkers in 1996 as an example of a more stable heterocyclic iodinane.^[233] The hypervalency nature of the iodine of 242, valency of III, has been however exploited by the Waser group since 2009 in mainly electrophilic alkynylation reactions, either gold or palladium catalysed, on a variety of substrates (indoles, thiophenes, olefins, keto esters, etc).^[234-236] The high reactivity of this class of reagents stems from the breaking of the octet rule by the iodine atom (hypervalency) which renders it strongly electrophilic and susceptible to nucleophilic attack.^[232] In the case of reagents 240 and 242, the reactivity is further enhanced by the combination of an excellent leaving group that is 2-iodobenzoate 243, where the valency of the iodine is restored to normality.

In 2013, the Waser group reported the use of EBX reagent 242 in the reaction of generic thiols 244 to form thio-alkynes 245, as shown in Figure 110.^[232] The thio-alkynylation proceeds under mild conditions without requiring the presence of any type of catalyst and in a highly chemoselective way. The reaction occurs on aromatic, aliphatic and peptidic thiols and a wide variety of functional groups are tolerated.^[232] In 2015, the mechanism was investigated and is shown in Figure 110.^[237] The first step involves an initial sulfur-iodine interaction followed by β-addition of the thiolate to the more electrophilic alkyne to give intermediate 246 which then undergoes α-elimination of iodobenzoate 243 and a 1,2-shift to afford final thio-alkyne 245.

I O

O N₃

I N

H O

N₃

241 240

Figure 110. Thio-alkynylation reaction and proposed mechanism. Figure adapted from reference.^[237]

Among the several variations of the original EBX 242 that were investigated,^[238] one involved the introduction of an azide group leading to derivative 240. In 2015, Adibekian and coworkers successfully integrated 240 in a proteomic study which targeted cysteine residues of biologically relevant molecules, like curcumin.^[190] In particular, 240 was efficient in alkynylating cysteines in living cells, under physiological conditions and, moreover, the azide group could be further engaged in a cycloaddition reaction.^[190]

Intrigued by the possibility of introducing a more efficient terminator that would allow for CPD post-functionalization, we started a collaboration with the Waser group that provided us with derivative 240. On the other hand, to compare with an iodoacetamide derivative, 241 was synthesized using iodoacetic anhydride 247 and 3-azidopropylamine following reported procedures^[239]

(Scheme 36).

Scheme 36. Reagents and conditions: a) 3-azidoproprylamine, NEt₃, CH₂Cl₂, 0 °C, 0.5 h, 47%.

We then introduced an alkyne group on red-emitting fluorophore 150 by performing a peptidic coupling with propargylamine which afforded derivative 248 in moderate yield (Scheme 37). 248 would serve the dual purpose of enabling the evaluation of 240 and 241 in the polymerization step as well as serving as a model substrate for post-functionalization at the terminator level.

Scheme 37. Reagents and conditions: a) propargylamine, HATU, NEt₃, DMF, rt, 1 h, 40%.

The next step was to use the two terminators to quench the disulfide exchange polymerization to evaluate a possible difference in efficiency. Two CPDs were therefore prepared using green-emitting initiator 21 and monomer 24, as shown in Scheme 38. The same stock solutions were used to synthesize both polymers in order to be able to attribute the differences in reactivity to the nature of the terminator used. After 30 min of polymerization in aqueous TEOA buffer, the terminator solution, either 240 or 241, was added in a 1:1 ratio with the initiator that is at 100-fold higher dilution compared to the previous reported conditions (4.0 mM versus 1.0 M).^[52] Stoichiometric amounts were chosen as a means to eliminate the large excess needed with standard iodoacetamide 23 to

I N

achieve efficient thiol quenching. The GPC data obtained for CPDs 249, terminated using EBX reagent 240, and 250, terminated with 241, is shown in Table 7. It is noteworthy to highlight that the values of Mw, Mn and PDI were almost the same for both polymers enabling a more precise comparison of the terminators used in the study.

Scheme 38. Reagents and conditions: a) DMF, TEOA buffer (1.0 M, pH 7.0), 25 °C, 1000 rpm, 0.5 h.

Table 7. GPC Data for Polymers 249- 250.

Polymer M_w^a M_n^b PDI

249 10.3 kDa 9.9 kDa 1.04

250 10.4 kDa 9.7 kDa 1.07

aMass average molecular weight. ^bNumber average molecular weight.

As mentioned before, a method we envisioned to evaluate possible differences in terminator incorporation made use of the azide group introduced on the CPD. A cycloaddition reaction was therefore performed using fluorescent alkyne 248 on CPDs 249-250. The same conditions used for sidechain modification were applied for the terminator functionalization to obtain 251-252, as shown in Scheme 39, namely using copper sulfate, sodium ascorbate as a reducing agent, TBTA as a ligand, in THF/H2O 9:1. The alkyne 248 was used in large excess and the reaction mixture then purified using once again dialysis centrifugal filters with a molecular weight cut-off of 3.0 kDa.

Scheme 39. Reagents and conditions: a) CuSO₄×H₂O, sodium ascorbate, TBTA, THF/H₂O 9:1, 25 °C, 1000 rpm, 17 h.

The functionalized CPDs 251-252 contained two fluorophores: green-emitting at the initiator level and red-green-emitting at the terminator level. We decided to exploit the presence of the second fluorophore to determine the yield of CuAAC.

In order to check that the reaction had indeed occurred and that no left over fluorescence coming from alkyne 248 was still present, we analysed CPD 251 by GPC. As shown in Figure 111, by exciting the red-fluorophore and by monitoring its emission, we could observe a single peak with the same retention

S S

No extra peaks coming from alkyne 248 could be observed showing that the conditions used in the cycloaddition reaction did not affect the stability of the CPD.

Figure 111. GPC profile of CPDs 249 (green) and 251 (red) detected at λ_em = 517 nm (λ_ex = 492 nm) for CPD 249 and λ_em = 582 nm (λ_ex = 552 nm) for CPD 251.

The yield of terminator functionalization for 251-252 was then determined using UV-Vis. As shown in Figure 112, both green and red fluorophores could be detected in the absorption spectra of the CPDs. However, the ratio of the two fluorophores was much lower in the case of 252 which was terminated with iodoacetamide derivative 241 compared to CPD 251 terminated with hypervalent iodine reagent 240.

30 45

t (min)

35 40

Figure 112. Absorption spectrum of CPDs 251 (blue) and 252 (pink) in TEOA buffer (1.0 M pH 7.0).

In order to quantify these results, we assumed quantitative CuAAC and calibrated the maxima of emission of the two fluorophores against their respective extinction coefficients. We were therefore able to calculate an incorporation yield of 46% of EBX 240 in CPD 249 and 11% of terminator 241 in CPD 250. This remarkable difference in terminator functionalization reflected a different reactivity between the two thiol-alkylating reagents. In particular, EBX 249, bearing a more reactive hypervalent iodine, proved to be four times superior in efficiently terminating the disulfide exchange polymerization in conditions of high dilution.

After having analysed the UV-Vis spectra of CPDs 251-252, we decided to also record their emission spectra. This was of interest to investigate whether FRET could occur from initiator to terminator since the green fluorophore could potentially act as a donor when excited at its maximum and the red fluorophore act as an acceptor. Indeed, when exciting the green initiator of 251, emission coming from both green and red fluorophores could be detected, as shown in

400 700

λ (nm)

500 600

A (a.u.)

0 0.04 0.08

terminator and the initiator were close enough to be able to record a strong FRET signal. The same measurement was also repeated after treatment of the polymer with DTT leading to a disappearance of the emission coming from the red fluorophore. This observation indicated that the observed FRET in the intact CPD came from an efficient functionalization and not from the presence of aggregates.

Figure 113. Emission spectrum of CPD 251 in TEOA buffer (1.0 M pH 7.0) before (solid) and after (dashed) incubation with 100 mM DTT (20 min, rt).

In sharp contrast, very weak FRET signal could be detected when exciting the green initiator of CPD 252 which was characterized by a lower yield of incorporation of terminator 241, as shown in Figure 114.

500 700

λ (nm)

550 600

I (rel)

0 0.05 1

650

Figure 114. Emission spectrum of CPD 252 in TEOA buffer (1.0 M pH 7.0) before (solid) and after (dashed) incubation with 100 mM DTT (20 min, rt).

Having succeeded in finding a more reactive terminator for the construction of the CPDs, we decided to also investigate the cellular uptake of the doubly-labelled polymer 251. Entry of the CPD into HeLa cells was analysed using confocal laser scanning microscopy. Images were taken after 15 minutes incubation using 500 nM concentration of 251 and are shown in Figure 115. In particular, we were eager the see whether it was possible to measure the strong FRET signal inside the cells as well. In order to evaluate this, emission coming from the red fluorophore (at 610 nm) was analysed by excitation of the green (at 488 nm) and the red (at 561 nm) fluorophores.

Upon excitation of the green fluorophore, the green emission could be observed mainly in the nucleoli and in the cytosol (Figure 115a). On the other hand, when the emission of the red fluorophore was detected upon excitation of the green initiator, the image was dominated by mainly punctuated spots suggesting endosomal or lysosomal localization (Figure 115b). The occurrence of FRET in these conditions could be due to intact doubly-labelled CPD that did

500 700

λ (nm)

550 600

I (rel)

0 0.05 1

650

While the absence of FRET in the cytosol suggested that the CPD that escaped the endosomes was readily degraded at least in half. Finally, the emission of the red fluorophore detected upon excitation of the red fluorophore itself appeared much stronger than in the other two cases and resulted, as expected, in a localization in nucleus, cytosol and probably endosomes. The different localization obtained by detecting red emission upon different excitation, supported that the reduction of the poly(disulfide) backbone inside the cytosol did occur most likely operated by the high concentrations of glutathione and accounting for the lack of toxicity of the CPDs.

Figure 115. CLSM images of HeLa cells after 15 min incubation with 500 nM of CPD 251 at 37 °C in Leibovitz’s medium. The emission of the green fluorophore was detected at a) 517 ± 18 nm upon excitation at 488 nm, the emission of the red fluorophore was detected at 610 ± 40 nm upon excitation at b) 488 nm and d) 610 nm. Merged image of a) and b) is shown in c). LP = 4%, scale bar = 10 µm.

In conclusion, we were able to introduce a more reactive reagent to terminate the disulfide-exchange polymerization. By using a hypervalent iodine derivative, the amount of terminator needed to quench the reaction was reduced to 100-fold and moreover, the yield of incorporation compared to iodoacetamide was far

b) a)

c) d)

ex 488 nm em 517 nm

ex 488 nm em 610 nm

merged ex 561 nm

em 610 nm

superior as detected using post-functionalization. The EBX reagent enabled to not only efficiently terminate the polymerization, but also to functionalize CPDs at the terminator level as showed by UV-Vis studies. Finally, FRET measurements conducted both in solution and in living cells confirmed both the successful double labelling of the polymer and the occurrence of intracellular reductive depolymerization which destroys the disulfide-rich CPD liberating the cargo and eliminating cytotoxicity.