Sidechain Screening - RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

3.1.3. Sidechain Screening

Having established an efficient method for sidechain functionalization, the goal was to study the effect on cellular uptake of different groups. In particular, we were interested in introducing three types of modifications: ring tension,

3.1.3.1. Introducing Ring Tension

As mentioned before, excellent results obtained with monomeric strained disulfides made it intriguing to investigate the effect of combining them with the more active cell-penetrating poly(disulfide)s. The possibility to add an extra contribution to the thiol mediated uptake pathway led to the design of reloaded CPDs with lipoic acid units and more strained asparagusic acid units, as shown in Figure 61.

Figure 61. Comparison between monomeric strained disulfides (left) and CPDs loaded with strained disulfides (right).

A CPD loaded with lipoic acid, polymer 168, had already been obtained during the development of sidechain engineering discussed earlier. Therefore, we decided to keep the same design to introduce the more strained asparagusic acid unit. To do so, alkyne 172 was first synthesized by performing a peptidic coupling using activated asparagusic acid 173 previously synthesized in the

S S HN

O NH

S S

S n

+ N N N

S S

S n

+ N N N

NH O ⁴ S S

O SS S

35 °

S 27 ° 30

Scheme 17. Reagents and conditions: a) propargylamine, DIPEA, DMF, rt, 1 h, 50%.

The model CuAAC reaction between azide 160 and alkyne 172 was carried out next to obtain triazole 174, which was then reduced by DTT to obtain calibration standard 175 (Scheme 18).

Scheme 18. Reagents and conditions: a) CuSO4×H2O, sodium ascorbate, TBTA, THF/H2O 1:1, 0.5 h, rt, 10%; b) DTT, TEOA buffer (1.0 M, pH 7.5), 25 °C, 1000

CuAAC reaction was then performed on the azide CPD 165 using the previously optimized conditions, to obtain the asparagusic acid-containing 176 in quantitative yield (Scheme 19), as calculated after reductive depolymerization with DTT using standards 169 and 175.

Scheme 19. Reagents and conditions: a) CuSO4×H2O, sodium ascorbate, TBTA, 25 °C, H2O/THF 9:1, 1000 rpm, 17 h, >99%.

While HPLC-MS analysis after reductive depolymerization demonstrated that polymer 165 could undergo cycloaddition to give modified CPDs 168 and 176, it did not prove that the introduced rings were still intact and had not been reduced during the reaction.

In order to prove this, we turned to fluorescence measurements. Indeed, when studying the cellular uptake of strained disulfides, it was observed that the presence of ring tension quenched the emission of the fluorophore attached to the small monomers.^[60] By measuring the emission spectra of polymers 165, 168 and 176 we could indeed observe significant fluorescence quenching for the CPDs reloaded with ring tension, as shown in Figure 62. In particular, 168 containing lipoic acid was quenched by a factor of 40, while 176 loaded with even more strained asparagusic acid was quenched by a higher factor of 52,

cause of fluorescence quenching, DTT was added to the polymer solution and the emission spectra recorded one more time. A full recovery of intensity for the quenched polymers could be observed, suggesting that the closed disulfide was indeed the cause of the considerable decrease in emission intensity of the fluorophore.

Figure 62. Normalized emission spectra of CPDs 165 (red), 168 (blue) and 176 (green), before (solid) and after (dashed) reductive depolymerization with DTT.

Even though the fluorescence quenching strongly indicated that the rings introduced on the CPDs were intact, a major consequence of this was the inability to quantify the cellular uptake with precision, and thus to observe the differences between the modified polymers. Although depolymerization inside the cytosol due to the high concentration of glutathione was expected to be complete, it would still be impossible to assess whether a full recovery of fluorescence had indeed occurred. Taking this into account we decided to assess the effects of ring tension on the CPD cellular uptake by using the more qualitative, but also more informative, confocal laser scanning microscopy (CLSM).

580 600 700

λ (nm)

620 640 660 680

In order to test the effect of ring tension on the CPD uptake in HeLa cells, the images obtained after incubation with modified polymers 168 and 176 were compared to unmodified azide 165 and reference 177 containing the best performing monomer 24 reported so far. CPD 177 was obtained following reported procedures^[52] and with a size comparable to the polymers studied (Table 5).

Table 5. GPC Data for Polymer 177.

Polymer Mwa Mnb PDI

177 9.3 kDa 11.4 kDa 1.22

aMass average molecular weight. ^bNumber average molecular weight.

The collection of CPDs used for cellular uptake studies is represented in Figure 63.

Figure 63. Schematic representation of unmodified CPDs 177, 165 and CPDs reloaded with ring tension 168 and 176 tested for cellular uptake in HeLa cells.

177 O

The cellular uptake experiments in HeLa cells were conducted at different concentrations of polymer. We decided to start by incubating the CPDs for 1 h at 37 °C at a concentration of 500 nM, as reported in previous work from the group.^[53] With this molecular weight, reference CPD 177 showed an uneven distribution, reaching the cytosol, the nucleus and the nucleoli in some cells and showing punctuate emission in others (Figure 64a). On the other hand, azide 165 showed an even punctuate emission for all cells, indicating more likely localization in the endosomes (Figure 64b). With much disappointment, CPDs 168 and 176 that were expected to outperform reference polymer 177, showed weaker uptake. 168 bearing lipoic acid units mainly aggregated outside the cells, even though it could also reach the cytosol (Figure 64c), while 176 with higher ring tension showed overall weaker emission coming from inside the cell and aggregated mainly on the cell membrane (Figure 64d).

Figure 64. CLSM images of HeLa cells after 1 h incubation with 500 nM of CPDs a) 177, b) 165, c) 168 and d) 176, at 37 °C in Leibovitz’s medium. Laser power (LP) = 6%, scale bar = 10 µm.

By lowering the concentration to 250 nM and increasing the incubation time to 4 h, all CPDs became more evenly localized in the cytosol of the cell, as shown in Figure 65. Aggregates for sidechain-modified polymers started to disappear, especially for 168 (Figure 65c).

Figure 65. CLSM images of HeLa cells after 4 h incubation with 250 nM of CPDs a) 177, b) 165, c) 168 and d) 176, at 37 °C in Leibovitz’s medium. LP = 8%, scale bar = 10 µm.

Finally, the best conditions for cellular uptake were met when the concentration was lowered to 100 nM, while keeping an incubation time of 4 h.

All CPDs showed equal activity, localizing in the cytosol, partly in endosomes as demonstrated later by colocalization studies with FITC-dextran 40 kDa, an

endosomal marker. No aggregation outside the cell or on the cell membrane could be observed, as shown in Figure 66.

Figure 66. CLSM images of HeLa cells after 4 h incubation with 100 nM of CPDs a) 177, b) 165, c) 168 and d) 176, at 37 °C in Leibovitz’s medium. Laser power (LP) = 10%; scale bar = 10 µm.

Having seen that all polymers, with and without ring tension, showed no difference in activity in HeLa cells in these conditions, we decided to decrease the concentration even more. Indeed, at a concentration of 50 nM, reference CPDs 177 and 165 became inactive. On the contrary, modified CPDs 168 and 176 could be visualized in the cytosol with punctuate emission, as shown in Figure 67. Cells incubated with 176, equipped with asparagusic acid, showed less intensity suggesting either less uptake efficiency of this CPD compared to lipoic acid loaded 168, or an incomplete recovery of emission intensity due to a

stronger quenching effect of the more strained asparagusic unit. However, these are only speculative remarks and the technical difficulties in distinguishing the punctuate emission of the red fluorophore of the CPDs from the background called for a more qualitative analysis.

Figure 67. CLSM images of HeLa cells after 4 h incubation with 50 nM of CPDs a) 177 (LP = 8%), b) 165 (LP = 10%), c) 168 and d) 176 (LP = 15%), at 37 °C in Leibovitz’s medium. Scale bar = 10 µm.

The cellular uptake of 168, which showed the highest intensity inside the cells, was also tested at concentrations as low as 25 nM and 10 nM (Figure 68).

Even though it became much harder to distinguish the red fluorescence coming from inside the cell from the background, very weak red punctuate emission

Figure 68. CLSM images of HeLa cells after 4 h incubation with a) 25 nM, b) 10 nM, c) 5 nM concentration with CPD 168 and d) 5 nM with CPD 177 at 37 °C in Leibovitz’s medium. LP = 15%, scale bar = 10 µm.

We next performed colocalization experiments with FITC-dextran 40 kDa, a marker for endosomes. HeLa cells were first incubated with CPDs 177 and 168 for 4 h at 37 °C and the last 15 minutes, FITC-dextran was added. Several images were taken and the correlation coefficient between the two dyes taken as an average from at least 4 pictures. We used Pearson’s correlation coefficient (PCC) to analyze in an objective way if the green-emitting endosomal marker overlapped with the red-emitting fluorophore of the polymer. With both CPDs 177 and 168, a value of PCC = 0.28 was obtained. Generally, a value of PCC >

0.5 is accepted as evidence for correlation, thus, only a partial colocalization with endosomes was achieved (Figure 69).

Figure 69. CLSM images of HeLa cells after 4 h incubation with 100 nM of a) 177 and b) 168 at 37 °C in Leibovitz’s medium, followed by 15 min post-incubation with 100 µM of FITC-dextran 40 kDa. Left: FITC-dextran 40 kDa, LP = 10%; centre: CPDs, LP = 4%; right: merged. Scale bar = 10 µm.

One of the major points of loading CPDs with strained disulfides was to promote thiol-mediated uptake over counterion mediated uptake. Having demonstrated that, at lower concentration, ring tension enabled the CPDs to remain active in HeLa cells compared to unmodified ones, we decided to prove that thiol-mediated uptake accounted for the efficiency of CPDs 168 and 176. In order to assess this, we performed inhibition experiments using DTNB, so-called Ellman’s reagent, which represents a hallmark of thiol-mediated uptake since it oxidizes exofacial thiols, and thus, destroys their ability to interact with disulfides like the ones contained in CPDs. As expected, both reference CPDs 177, 165 and modified 168 showed a complete shutdown of the uptake in cells that were pre-incubated with DTNB, as shown in Figure 70.

FITC-dextran

merged

Figure 70. CLSM images of HeLa cells after 4 h incubation with 100 nM of a) 177, b) 165 and c) 168 at 37 °C in Leibovitz’s medium with (top) and without (bottom) pre-incubation with 1.2 mM DTNB for 30 min before addition of CPDs.

LP = 10%, scale bar = 10 µm.

In conclusion, the sidechain engineering method developed in section 3.1.2.

was applied to obtain two new CPDs from the same batch of azide polymer.

Alkyne partners were easily synthesized and CuAAC reaction was performed affording modified CPDs with excellent yield and reproducibility.

Unfortunately, the introduced strained disulfides, while still being intact after reacting with the CPD, induced quenching of the fluorophore used to track the cellular uptake of the polymers. This aspect complicated uptake studies and led to the solely qualitative analysis by confocal microscopy. Working at high dilution, which is desirable when dealing with precious cargos, we were able to prove that ring tension does indeed contribute to maintaining the CPDs active compared to unmodified ones. Activity of the new CPDs was detected at concentrations that are below the detection limit of the fluorophore, proving how powerful thiol-mediated uptake can be.

a) b)

+ DTNB

+ DTNB + DTNB

Finally, introducing ring tension enabled the polymers to still enter through thiol-mediated uptake, as shown by the inhibition with Ellman’s reagent. The next direction we decided to take was to promote, on the other hand, counterion-mediated uptake by introducing cumulative positive charges on the CPD sidechain.

3.1.3.2. Introducing Extra Charges

CPDs with the sidechain modified with strained disulfides showed higher activity than unmodified ones at high dilution. The promotion of thiol-mediated uptake, due to the presence of ring tension, over counterion-mediated uptake, accounted for this efficiency. We were therefore intrigued by the possibility to boost, complementary to the previous strategy, counterion-mediated uptake over thiol-mediated uptake and to evaluate if differently engineered CPDs could discriminate the two mechanisms. In order to achieve this, we decided to use the comparable CPD backbone provided by the sidechain modification strategy used before, to introduce extra charges as shown in Figure 71.

Figure 71. Schematic representation of CPDs loaded with strained disulfides entering mainly through thiol-mediated uptake and CPDs with extra charges entering mainly through counterion-mediated uptake.

S S

S n

+ SS

S S

S n

+ +

thiol-mediated uptake

counterion-mediated uptake

- - -

-S S

S S

bidentate hydrogen bonding with exofacial polyanions; 2) the more hydrophobic phosphonium cation with an extra targeting function. The obtained extra cationic CPDs are represented in Figure 72.

Figure 72. Structure of new CPDs loaded with extra guanidinium groups (178-180) and with a triphenyl phosphonium cation 181.

Regarding polymer design, new CPDs 178-180 presented cumulative guanidinium groups coming from arginine (178), diarginine (179) and triarginine (180) residues. Meanwhile, CPD 180 contained a triphenyl phosphonium cation that, other than being bulky and hydrophobic unlike the small hydrophilic guanidinium, has been shown to be attracted by the strong negatively charged surface of the mitochondrial membrane, thus making it interesting for potential targeting.^[215-218]

In order to synthesize the new polymers, the adequate alkynes were designed, as shown in Figure 73.

178 : m = 1

Figure 73. Structure of alkynes for the synthesis of new extra charge-containing CPDs.

Alkyne derivative 157, bearing only one arginine, had already been synthesized when making alkyne monomer 154. The synthesis of alkynes 182 and 183, bearing two and three arginine residues, respectively, was achieved through a series of coupling-deprotection-coupling steps, as shown in Scheme 20.

Derivative 185 was used as a common precursor for the synthesis of alkynes 182 and 183. The alkyne group was introduced via peptidic coupling using Fmoc-protected arginine 186 and propargylamine to obtain derivative 187 in good yield. The Fmoc group of 187 was removed using piperidine to obtain derivative 188 which was then engaged in a second peptidic coupling with a second Fmoc protected arginine 186 to obtain the first diarginine peptide 189 in excellent yield. A second Fmoc deprotection afforded common precursor 185.

At this point, deprotection in acidic conditions using TFA yielded final diarginine alkyne 182, while triarginine derivative 190 was obtained by a coupling reaction of 185 to Boc-protected arginine 155. Final Boc and Pbf removal of 190 afforded final triarginine alkyne 183.

H₂N NH

O H

N NH₂ NH₂ 157 : m = 1 182 : m = 2 183 : m = 3

184 P

O NH

Scheme 20. Reagents and conditions: a) propargylamine, HATU, DIPEA, DMF,

The synthesis of triphenyl phosphonium alkyne 184 was more straightforward, as shown in Scheme 21. Starting from free carboxylic acid 191, propargylamine was used to obtain alkyne 184 in moderate yield.

Scheme 21. Reagents and conditions: a) propargylamine, HATU, DIPEA, DMF, rt, 0.2 h, 47%.

As for the CPDs functionalized with strained disulfides, the next step was to obtain the calibration standards necessary to then determine the yield of sidechain engineering. For this purpose, CuAAC was carried out using azide monomer 160 and alkynes 157, 182, 183 and 184 to obtain triazoles 192-195, respectively, as shown in Scheme 22.

184 P a)

OH O Br

P NH O Cl

191

Scheme 22. Reagents and conditions: a) CuSO4×H2O, sodium ascorbate, TBTA,

Derivatives 192-195 were then treated with DTT and the reduced monomers 196-199 analysed by HPLC-MS to obtain calibration standards used in the next step to determine the yield of CuAAC on azide CPD 165. The structures of the calibration standards are shown in Figure 74.

Figure 74. Structure of standards 196-199 used to determine the yield of sidechain engineering.

Having synthesized the required alkynes and obtained the corresponding standards, CuAAC was performed next using the same comparable scaffold as for the previous CPDs, that is, azide polymer 165 (Scheme 23). The copper-catalyzed cycloaddition reaction was carried out using the same conditions developed for the ring tension containing CPDs, proving the robustness of the strategy for sidechain functionalization with substrates of different nature. All the stock solutions of arginine alkynes, 157, 182 and 183, were made in water,

199

A decrease in the yield of CuAAC was observed going from one to three arginine-functionalized CPDs. However, this decrease was consistent with an increase in the bulkiness of the substrate and a similar trend was also reported by Fréchet and coworkers.^[92] Unfortunately, due to an overlap between the peaks of standard 195 and free azide monomer 169, the yield of sidechain modification for 181 could not be determined.

Scheme 23. Reagents and conditions: a) CuSO4×H2O, sodium ascorbate, TBTA,

Before testing the new CPDs in cellular uptake experiments, we decided to record their fluorescence emission spectra, since an important quenching of emission intensity had occurred for the CPDs containing strained disulfides in their sidechain (namely, a factor of 52 for more strained polymer 176). However, the emission intensities of CPDs 178-180 were only quenched by a negligible factor of 1.1-2.1 and not quenched at all for 181 compared to azide 165, as shown in Figure 75. The lesser degree of quenching for the polymers loaded with extra charges compared to the polymers loaded with ring tension supported once again that the presence of many strained disulfide rings on the CPD scaffold was the cause of such an important decrease in fluorescence intensity.

Figure 75. Normalized emission spectra of polymers 165 (red), 168 (blue), 176 (green), 178 (orange), 179 (pink), 180 (light blue), 181 (grey), before (solid) and after (dashed) reductive depolymerization with DTT.

Cellular uptake of the newly sidechain modified CPDs was investigated next. In order to compare the effect of counterion mediated uptake on the cell entry of the new CPDs, we used the same conditions applied to the CPDs bearing ring tension on the sidechain, namely CPD 168, which had shown the highest

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λ (nm)

620 640 660 680

activity by confocal laser scanning microscopy. The structure of the CPDs used for cellular uptake is shown in Figure 76.

Figure 76. Structure of CPDs tested for cellular uptake in HeLa cells: reference 177, 168 loaded with ring tension and 178-181 loaded with extra charge.

All new CPDs were then incubated in HeLa cells for 4 h at 100 nM and the uptake followed by confocal microscopy. Unfortunately, no differences could be observed neither with reference 177 nor with strained disulfide-containing CPD 168 which, on the contrary, gave slightly brighter pictures. All images showed punctuate emission and, moreover, the presence of increasing amounts of charges on the backbone (CPD 178-180) or the presence of a more hydrophobic charge (CPD 181) did not have any effect (Figure 77).

177 O

Figure 77. CLSM images of HeLa cells after 4 h incubation with 100 nM of polymers a) 178 (LP = 12%), b) 179 (LP = 12%), c) 180 (LP = 12%), d) 181 (LP

= 6%), d) 177 (LP = 10%) and e) 168 (LP = 10%), at 37 °C in Leibovitz’s medium. Scale bar = 10 µm.

Since the CPDs bearing ring tension had become more active at lower concentrations compared to the reference polymers, we decided to measure the activity of CPD 178 at high dilution. Indeed, we could still observe activity for extra cationic 178, comparably to 168 bearing strained disulfides at 50 nM concentration where the reference CPDs became inactive. However, when further diluting to 25 nM and 10 nM, a higher emission intensity of 178 could be detected compared to 168, as shown in Figure 78. Again, it is important to highlight that the conditions in which the images were taken, that is at very high dilution and with much interference from the background, led to speculative and only qualitative comparisons between the polymers.

a) b)

f) e)

Figure 78. CLSM images of HeLa cells after 4 h incubation with CPDs a) 178 and b) 168 at, from left to right, 50 nM, 25 nM and 10 nM concentration, at 37

°C in Leibovitz’s medium. LP = 15%, scale bar = 10 µm.

Since CPD 178 seemed to be more active than 168 at lower concentration, we decided to evaluate the uptake of CPDs 179-180 bearing even more charges at an ultrahigh dilution of 5 nM (Figure 79). Once again, all extra cationic polymers 178-180 gave brighter images compared to CPD 168, but, unfortunately, no increase in emission intensity could be detected with increasing charges on the CPD scaffold. One possible explanation could rely on the concentration at which the uptake was measured. Being the concentration so low, below the detection limit of the fluorophore attached to the polymers, it is probable that differences could not be appreciated entirely. Moreover, the yield of sidechain engineering decreased going from mono- to di- to tri-arginine-containing CPDs, meaning that the number of guanidinium groups was similar for all extra cationic CPDs even if the branching of the charges changed.

b) 50 nM

50 nM

25 nM

10 nM

Figure 79. CLSM images of HeLa cells after 4 h incubation with 5 nM CPDs a) 178, b) 179, c) 180 and d) 168, at 37 °C in Leibovitz’s medium. LP = 15%, scale bar 10 = µm.

Overall, the results obtained from the cellular uptake experiments revealed that the extra charge on the CPD scaffold played a bigger role than the presence

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