Sidechain Engineering with Hydrazone Exchange

There are three possibilities to intrinsically change the properties of cell-penetrating poly(disulfide)s: modify the nature of the initiator, the terminator or the monomer. In order to evaluate different modifications of the scaffold or compare small differences on the same backbone, a new method had to be developed. Different points had to be taken into account, such as the strategy to use and the most convenient and efficient site of the CPD to host the new functionality.

As far as the strategy is concerned, once again several points should be considered. Firstly, the dynamic poly(disulfide) scaffold of the CPDs has to remain intact, that is, the use of basic conditions that could destroy the polymer should be avoided. Secondly, a practical approach requiring reduced synthetic effort and a limited number of purification steps would be highly desirable.

Therefore, we decided to exploit orthogonal dynamic chemistry encouraged also by the experience acquired over the years in the group.^{[51, 71]} Considering that the CPD scaffold is constituted of disulfides, an orthogonal bond to disulfides had to be chosen to ensure the stability of the polymer during and after functionalization. For this reason, and taking inspiration from the SOSIP-TSE methodology discussed in the introduction,^[71] hydrazones were chosen. Their orthogonality to disulfides, that is, stable in basic conditions, but labile in acidic conditions where the disulfides are stable, made them the perfect choice for modifying CPDs while keeping the scaffold intact. Moreover, we decided to construct a fully dynamic system that involved not the formation of hydrazones, for example by having hydrazines on the polymer scaffold, but hydrazone exchange. In order to achieve this, hydrazone units would have to be integrated in the polymer backbone and the modifying units to achieve post-functionalization would have to contain aldehydes, as shown in Figure 50.

Figure 50. Schematic representation of a fluorescent CPD containing a hydrazone functional group and its exchange with substrates containing aldehydes.

Having decided on the type of strategy to use, the site of the modification had to be chosen next. Since we wanted to introduce a hydrazone as the functional moiety, three positions on the polymer were available: the initiator, the monomer and the terminator. Initiator and terminator were discarded since they could offer only one modification site per polymer while, on the other hand, the monomer could enable to study the effect of modification to its fullest extent.

Therefore, the first idea was to prepare a monomer containing the hydrazone moiety which, after polymerization, would represent at least 10 sites of modification per polymer for the shortest CPDs. This new propagator would have to contain three functionalities: 1) the strained disulfide, that is, the lipoic acid moiety required for polymerization and for disulfide exchange with exofacial thiols; 2) the guanidinium group which represents the CPP-mimic necessary for the bidentate hydrogen bonding with the exofacial anions for direct translocation; 3) a hydrazone group essential for the sidechain modification.

Putting these characteristics together led to the design of the monomer shown in Figure 51 where lipoic acid is coupled to an arginine residue and to a most labile aromatic hydrazone.

O N N O

H N N

H +

Figure 51. Structure of hydrazone monomer and hydrazone polymer.

It is worth mentioning that previous studies in the grouphad shown how small modifications of the monomers could lead to total loss of activity during cellular uptake of the resulting polymers.^[53-54] Therefore, there was the possibility that after polymerization, the hydrazone CPD would be either more or less efficient in the delivery of a model substrate to HeLa cells than the best performing available polymer, or, on the contrary, be completely inactive. We reasoned, however, that the possible loss of activity could turn to our advantage, enabling us to introduce a modification that could promote or increase the cellular uptake of a cell-impermeable system.

The synthesis of the hydrazone monomer is shown in Scheme 1. The two building blocks needed for the synthesis of the hydrazone monomer 130 were the aromatic aldehyde 131 and the protected hydrazide 132. Aldehyde 131 was synthesized following reported procedures.^[208] Namely, 4-carboxybenzaldehyde 133 was protected as the acetal derivative 134 which was then engaged in a peptidic coupling with protected arginine 135. Having obtained derivative 136, deprotection using a mixture of TFA/CHCl₃ 1:1 gave the free aldehyde 131. The protected hydrazide 132 was synthesized from lipoic acid 137 using tert-butyl

S S S I S S S S

n

polymerization

n

HN O N

O HN O

O HN

H₂N NH₂

HN O N

O HN O

O HN H₂N NH₂

carbazate. To obtain the final hydrazone 130, the hydrazide 132 was first deprotected using 20% TFA in EtOAc, followed by the addition of the aldehyde 131 to the reaction mixture. Any form of work up of the deprotected intermediate needed to be avoided since it led to decomposition of the hydrazide and possible polymerization products.

Scheme 1. Reagents and conditions: a) CH₃OH, NH₄Cl, reflux, 20 h, >99%; b)

Having obtained the hydrazone monomer, polymerization was attempted using a fluorescent initiator and iodoacetamide as a terminator. Unfortunately, due to solubility problems, hydrazone monomer 130 could not be polymerized and the obtained mixtures did not contain any polymer.

In order to solve the issue of polymerization, we decided to reconsider the design of the polymer. As discussed in the introduction (section 1.1.2.), the Matile group previously reported how to prepare copolymers with the purpose of studying the effect of different monomers on the cellular uptake, overcoming at the same time solubility issues.^[54] Indeed, it was observed that small variations of the sidechain of the arginine moiety in the monomer led to solubility problems that, in turn, precluded polymerization.^[53] Among the arginine derivatives that were tested, the methyl ester proved to not only give perfectly soluble copolymers, but also homopolymers with the best activity in cells reported so far.^[52-54] A point worth mentioning is that in this study all the copolymers that were made used the methyl ester derivative as the major monomer with ratios of major/minor monomer of 16:1 and 8:1.^[54]

In order to vary the solubility of the hydrazone monomer 130 during polymerization, we decided to remove the positive charge coming from the guanidinium group and therefore construct a new monomer bearing only the strained disulfide, necessary for polymerization, and the aromatic hydrazone, necessary for the exchange. This strategy led to the design of monomer 138 which was copolymerized together with monomer 24, as depicted in Figure 52.

Figure 52. Structure of the building blocks necessary for the preparation of the hydrazone copolymer.

The synthesis of the new hydrazone monomer 138 was very straightforward and is shown in Scheme 2. The same lipoic acid hydrazide 132 used before was engaged in hydrazone formation with benzaldehyde 139 to yield monomer 138.

Monomer 24 was synthesized according to reported procedures,^[52] by coupling lipoic acid 137 with arginine derivative 140.

S S S I

polymerization S S m

n

S S

S n S S S

m

HN O

O O HN H₂N NH₂

HN O N

HN O

O O HN H₂N NH₂

HN O N

24 138

Scheme 2. Reagents and conditions: a) 1. CHCl₃/TFA/thioanisole 66:33:1, rt, 1 h, 2. rt, 2 h, 60%; b) 1. CDI, DMF, rt, 0.5 h, 2. DIPEA, DMF, 4 h, 84%.

Monomer 138 showed excellent solubility in DMF, which was the solvent used for polymerization, and was copolymerized together with monomer 24 (Scheme 3).

O

a) 139

132 138

S S

N HN O

b) 140

137 24

S S NH₂

O O

HN NH₂

NH

O NH NH H₂N

NH₂

O O SS

HN O NHBoc

SS

OH O