Cyclotriphosphazene, an old compound applied to the synthesis of smart dendrimers with tailored properties

Introduction

Hexachlorocyclotriphosphazene (N₃P₃Cl₆) is an old compound, first synthesized in 1832 by Liebig [1], and correctly analyzed by Gladstone and Holmes in 1864 [2]. The first X-ray diffraction structure of N₃P₃Cl₆ was determined in 1960 [3], showing that the N₃P₃ cycle is very nearly planar. N₃P₃Cl₆ is a very interesting precursor in various fields. Indeed, its thermal ring opening leads to polyphosphazenes [4], [5], whereas thousands of publications describe its functionalization by nucleophilic substitutions of the chlorides [6], [7], [8]. The most fascinating property of N₃P₃Cl₆ is certainly the possibility to regio- and stereo-chemically control the nucleophilic substitutions, to have one (or several) function(s) different from the others [9]. We have used such property for the synthesis of smart dendrimers.

Dendrimers are hyperbranched macromolecules constituted of repetitive monomeric units, associated in layers around the central core [10], [11], [12], [13]. Each layer is called a “generation”. The central core can be a cyclotriphosphazene, a compound that we have largely exploited for this purpose. This review will display what we have done in the field of dendrimers, using the specific functionalization of N₃P₃Cl₆, with at least one function different from the others. Two reviews have recently gathered some examples of this type of dendrimers [14], [15].

Specific functionalizations of N₃P₃ for the synthesis of dendrimers

Having one function different from the five others can be accomplished in two ways, either by grafting first the single function, then the five others, or by grafting first the five functions, and then the single function. Depending on the type of both functions, the purification is easier by one or the other way. Scheme 1 displays two ways for the grafting of one thioctic acid and five aldehydes on N₃P₃. In the way A (dotted lines), it was difficult to purify the compound having one thioctic acid and five Cl, and thus the targeted compound having one thioctic acid and five aldehydes could not be isolated pure from way A. On the contrary, following the way B, the compound with five aldehydes and one Cl was easily isolated, and thus the grafting of one thioctic acid in the last step was easily carried out to afford pure the targeted compound [16].

Scheme 1:

Two possible ways to synthesize the targeted compound functionalized by one thioctic acid and five aldehydes. Only way B afforded it as a pure compound.

Both way A and way B have been used to afford cyclotriphosphazenes functionalized with 5+1 functions. As illustrated in Scheme 2, no general rule can be inferred. Furthermore, if the nucleophilic substitution with functionalized phenols is preferred, methylhydrazine and azide have been grafted also to the cyclotriphosphazene [17].

Scheme 2:

Two ways of functionalization of N₃P₃ with two substituents in a 1/5 (or 5/1) ratio.

Besides the 1/5 (or 5/1) ratio of functional groups on N₃P₃, 2/4 (or 4/2) and 3/3 (in fact 1/2/3) ratios have been also obtained. In these special cases, in order to avoid the stochastic functionalization, a diphenol was used instead of two phenols, permitting the specific reaction of two Cl connected to the same phosphorus, to form a seven-membered ring, as illustrated in Scheme 3. One or two diphenols can be used, affording 2/4 and 4/2 ratios of functions. The 3/3 ratio can be obtained by reacting first one phenol ester, then the diphenol [18].

Scheme 3:

Synthesis of N₃P₃ derivatives having 2/4 (or 4/2) or 1/2/3 ratio of functional groups.

In almost all cases, the aldehydes are used for growing the dendritic branches, whereas the other functions are generally kept intact, especially if they are fluorophores, as illustrated in Scheme 4 (a linear representation is used in this Scheme, as in all the forthcoming Scheme and Figures) [19]. The synthesis necessitates two steps for each generation: the condensation of the phosphorhydrazide H₂NNMeP(S)Cl₂ with the aldehydes, and the nucleophilic substitution on the PCl₂ function with hydroxybenzaldehyde in the presence of a base [20], [21]. Both P(S)Cl₂ and aldehyde terminal function can display a versatile reactivity. An example is given in Scheme 4, the reaction of H₂NCH₂CH₂NEt₂ with the P(S)Cl₂ functions, instead of continuing the growing of the dendrimer. The HCl generated in this reaction is trapped by the NEt₂ groups, affording directly positively charged and water-soluble dendrimers.

Scheme 4:

Typical example of the synthesis of a dendrimer having a single fluorophore linked to the cyclotriphosphazene core. The linear representation is used for the dendrimers, with parentheses at the level of all branching points. Multiplication of the numbers after the parentheses affords the number of terminal functions, for instance 20 for the largest dendrimer.

However, a sequential reactivity can be also obtained by growing first the dendrimer, then by modifying in the last step the single function, as illustrated in Scheme 5 [22]. Indeed, after grafting Boc-protected tyramine as terminal functions, the methylester function linked to the core is reduced with LiAlH₄ then hydrolyzed, to afford a benzylalcohol, which is then added to an isocyanate bearing the desired triethoxysilyl group.

Scheme 5:

Sequential reactivity on a dendrimer, first on the terminal groups, then at the single function linked to the core.

All these syntheses have been generally carried out for precise purposes, as will be illustrated in the forthcoming paragraphs.

Specific functionalization of N₃P₃ for accelerated syntheses of dendrimers

The synthesis of dendrimers is generally a lengthy process. Two steps are generally needed to obtain one generation, and to multiply most often by two the number of terminal functions. Thus, any improvement to multiply more rapidly the number of terminal functions is desirable. Using the cyclotriphosphazene not only as core but also as branching points induces a multiplication by five of the number of terminal functions. Furthermore, using the cyclotriphosphazene at each step induces a multiplication by 25 every two steps, instead of by two in the classical methods. A dendrimer with 750 phosphine terminal groups was obtained after only three steps. Such method affords highly dense dendrimers, and the synthesis could not be carried out up to higher generations (Scheme 6) [17]. This method can be also applied in alternation with the classical method, every two generations [23].

Scheme 6:

Three steps for the synthesis of a dendrimer bearing 750 phosphine end groups.

A previous report claimed that other dendrimers having the cyclotriphosphazene as branching points were synthesized up to the eighth generation [24], but only the first generation was unambiguously characterized [25]. Very recently, cyclotriphosphazenes bearing one function different from the five others have been used as terminal branching points for the synthesis of “onion peel” dendrimers [26], [27], i.e. dendrimers constituted of layers of different chemical composition. A compound with 180 alcohol terminal groups was synthesized in this way [28]. Two cyclotriphosphazenes linked by an arylether linkage have been grafted to the N₃P₃ core, to afford a kind of star dendritic structure. The relative position of the two functions of the cyclotriphosphazenes that are both linked to the core and to the external N₃P₃ are not controlled, thus a mixture of diastereoisomers was obtained [29].

Association of two cyclotriphosphazenes at the core: towards Janus dendrimers

Many fluorophores are sensitive to the presence of water, which induces a quenching of the fluorescence; thus, including a fluorophore at the core of a rather lipophilic dendrimer might screen water and preserve the fluorescence. In case of large fluorophores, a better protection should be obtained if using two cyclotriphosphazenes instead of one. This idea has been applied in particular for the protection of fluorophores having two-photon absorption (TPA) properties, i.e. fluorophores able to absorb simultaneously two photons of identical (or different) frequencies, most generally issued from a laser. TPA offers several advantages in biology, such as a deeper imaging with reduced photo-damages, and reduced background fluorescence [30]. Scheme 7 displays two phenols linked to a TPA fluorophore, used for the reaction with two equivalents of N₃P₃Cl₆. The 10 remaining Cl are all substituted by hydroxybenzaldehyde, then the growing of the dendrimer is pursued, using the method already shown in Scheme 4. When having P(S)Cl₂ terminal functions, two equivalents of H₂NCH₂CH₂NEt₂ can be reacted, instead of continuing the growing of the dendrimer, affording positively charged and water-soluble dendrimers (Scheme 7). These compounds remain brightly fluorescent, and were used for imaging in vivo the vascular network of a rat olfactory bulb [31]. With the same fluorophore as core, the 10 remaining Cl were used also for the grafting of 10 fluorophores having also TPA properties, but at a different wavelength. A partial quenching due to the interaction between chromophores was observed [32]. With a longer diphenol chromophore (38 bonds instead of 22), and ammonium terminal groups, the photoluminescence was low in water, showing that the dendritic branches are not large enough to isolate the chromophore from water [33], [34].

Scheme 7:

Example of a TPA fluorophore protected against water by dendritic branches emanating from two N₃P₃ rings. Water soluble dendrimers of generations 1, 2, and 3 were obtained.

The use of an expanded core obtained by association of two N₃P₃ rings has been proposed also by a few other authors. For instance, the reaction of two equivalents of N₃P₃Cl₆ with bisphenol A, followed by the reaction of the remaining 10 Cl with iodophenol, and finally by Sonogashira couplings with propargyl α-D-mannopyranoside afforded a small dendrimer with 10 mannose terminal functions [35]. The bisphenol A has also been used for grafting a new layer of cyclotriphosphazenes, linked to the core by aryl ether linkers, and having 50 derivatives of 2-naphthol as terminal functions [36].

In all the previous examples, the two N₃P₃ ring are associated at the beginning of the synthesis process, and thus fully symmetrical structures are obtained. On the contrary, if the association of two N₃P₃ rings is carried out at the end of the synthesis process, non-symmetrical structures, also called Janus dendrimers in case of two faces [37], will be obtained. The nature of the single function linked to the core is crucial in this case, as it should not react during the synthesis of the branches [38], [39], but it should be activated in the last step, i.e. the association of the two molecules to afford the Janus dendrimer. The Boc-protected tyramine, and the methylester function shown in Scheme 2 are particularly suitable for such purpose. Indeed, the amine can be deprotected with trifluoroacetic acid (TFA), and the methyl ester can be deprotected by reaction with N₂H₄, to afford a hydrazide, or by reaction with KOH/H₂O, to afford the carboxylic acid. Peptide couplings and condensation reactions have been used to afford diverse types of Janus dendrimers. The Boc-protected tyramine can be used also as terminal functions, and the deprotection affords water-soluble Janus dendrimers. The main examples are shown in Scheme 8 [40]. The FTIR and FT Raman spectra of some of these Janus-type dendrimers were also studied [41].

Scheme 8:

Examples of Janus dendrimers, obtained by peptide coupling (DIPEA, diisopropylethylamine; TBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) or by condensation reactions. Deprotection with trifluoroacetic acid (TFA) affords water-soluble Janus dendrimers.

A single different function on N₃P₃ for grafting dendrimers to materials

Depending on the nature of the single different function, the type of grafting, and the type of materials, the expected uses are different. Four typical examples will be given below.

It is well known that pyrene is able to interact by π-stacking with many multiaromatic compounds, including with itself [42]. A single pyrene was chosen to interact by π-stacking with graphene, and was surrounded by 5 or 10 phosphines suitable for the complexation of palladium (Fig. 1a), to be used as catalyst in Suzuki couplings. At room temperature, the dendrimer is stacked on cobalt nanoparticles covered by graphene layers. Heating decreases the strength of the π-stacking, and the dendrimers go inside the solution, to perform the catalysis. The solution is cooled at the end of the catalysis, and the dendrimers go back onto the graphene layers on the cobalt nanoparticles, which are magnetic. These nanoparticles can be easily recovered using a magnet, with the dendrimer attached, and they can be re-used in another catalytic run. Felbinac (biphenylylacetic acid) was obtained in quantitative yields, even after 11 re-uses [43].

Fig. 1:

Examples of dendrimers grafted to materials. (a) Pyrene and Pd complexes for catalysis. (b) Phosphonates and maleimides as chemical sensor. (c) and (d) Thioctic acid and ammoniums (c) or carboxylate salts (d) for the culture of cells.

A dendrimer having a single triethoxysilyl group linked to the core and Boc-protected amines as terminal functions (Scheme 5) was covalently grafted to nanoporous silica. After deprotection of the amines, the silica functionalized by the dendrimers was used for trapping CO₂. Among various types of phosphorus-containing dendritic structures used for such purpose, this dendrimer was the most efficient, albeit less efficient than the simple aminopropyltriethoxysilane [44].

A dendrimer comprising two azabisphosphonates and five maleimide fluorophores (Fig. 1b) was grafted through the phosphonates to a titanium oxide film. Such grafting afforded a highly fluorescent film, usable a chemical sensor. Indeed, the fluorescence of the maleimide group is sensitive to the presence of phenol, as the H-bonding with the C=O groups of the maleimide induces a non-radiative decay of the absorbed energy. A low sensitivity to water was also observed. Among phenols, nitrophenols in particular induced the quenching of the fluorescence of the film. The detection of the presence of nitrophenols was by far more efficient with the dendrimers linked to the TiO₂ film than with the dendrimer in solution [45].

Two series of dendrimers having one thioctic acid linked to the core, and either ammoniums or carboxylic acid salts as terminal functions (Fig. 1c and d) were tentatively grafted to glass surfaces covered by a thin layer of gold. It was possible to graft only the first generation dendrimers. Indeed, the thioctic acid is buried too profoundly inside the second generation dendrimers, and thus could not be grafted. The surfaces covered by the first generation dendrimers were used as support of cultures of human osteoblasts (HOB) cells, responsible for the building of bones. Dramatic differences on the behavior of the HOB cells were observed, depending on the charge of the dendrimers. HOB cells proliferated well on surfaces covered by the negatively charged dendrimers, leading to the confluence (full coverage of surface by the cells) after about 10 days. After the same time, all the HOB cells were died or dying by apoptosis on surfaces covered by the positively charged dendrimers [16]. It must be emphasized that this is not a general trend, as a previous experiment demonstrated on the contrary that neurons of fetal rats proliferated better on surfaces covered by positively charged dendrimers, compared to negatively charged dendrimers [46].

Useful dendritic tools in biology, obtained by the specific functionalization of N₃P₃

The previous example is in between materials and biology, but in many cases of biological uses, the single function on the N₃P₃ core is a fluorophore. Indeed, a fluorophore inside the structure, should not modify the biological properties of the dendrimers, but should allow imaging the biological events associated with the targeted properties [47]. Positively charged dendrimers are useful carriers of plasmids, DNA, or RNA, for transfection experiments [48]. The presence of a fluorophore should allow the detection of the behavior of the dendrimers when penetrating inside cells and after. A single maleimide grafted to the core of a dendrimer having ammonium terminal groups (Scheme 4) has been tentatively used for such purpose. The maleimide dendrimers are highly fluorescent in organic solvents [19], and were found poorly sensitive to water when linked to a solid surface [45], but deceptively, the fluorescence of the positively charged second generation dendrimer was too low in water for any practical purpose. It was shown only that the interaction of the dendrimer with plasmid DNA induced a possible disturbing of the helical B-type structure of DNA [49].

Surprisingly, the same fluorophore linked to the core of a first generation dendrimer having azabisphosphonic terminal functions was enough fluorescent to monitor the interaction of this dendrimer with human monocytes (Fig. 2a). It was used in particular to study fluorescence resonance energy transfer (FRET) experiments with phycoerythrin-coupled antibodies, showing that the innate Toll-like receptor (TLR)-2 was involved, but not alone, in the interaction [50]. The fully substituted dendrimer (12 azabisphosphonic terminal functions) possesses many properties towards cells of the human immune system. The multiplication of Natural Killer (NK) cells [51], the anti-inflammatory activation of monocytes [52], which are specifically recognized [53], the activation of dendritic cells [54], a therapeutic potential in vivo in the treatment of inflammatory diseases, such as rheumatoid arthritis [55], endotoxin induced uveitis [56], and neuro-inflammation [57] have already been demonstrated. Among the dendritic tools used to decipher the interaction of this dendrimer with the cells, besides the maleimide fluorophore shown in Fig. 2a, the julolidine fluorophore shown in Fig. 2b has been used for showing the inhibition of the activation, and therefore the proliferation, of human pro-inflammatory CD4+ T cells [58].

Fig. 2:

Examples of water-soluble dendrimers ended by azabisphosphonate groups. (a) and (b) fluorescent tools for imaging interactions. (c) highly dense first generation dendrimer with 30 azabisphosphonate groups.

In view of the numerous biological properties of this dendrimer, the structure/activity relationship was studied. Many different dendritic structures have been synthesized, in particular those issued from the reactivity of the functional cores shown in Scheme 3. The highly dense dendrimer shown in Fig. 2c was also synthesized for this purpose. From a large family of dendritic structures having from 2 to 30 azabisphosphonate groups, it was shown that the best biological activities were obtained with dendrimers having from 8 to 12 azabisphosphonate terminal functions [18]. Different aspects of the structure/activity relationship have been investigated, such as various modifications of the internal structure, using other families of dendrimers [polyamidoamines (PAMAM), polypropyleneimine (PPI), polylysine, polycarbosilane, etc.]. Those having the cyclotriphosphazene as core were found the most efficient [59].

Conclusion

Hexachlorocyclotriphosphazene is known since almost two centuries, but there is still plenty of work to be carried out to expand the versatility of its functionalization. A recent paper by J. Chabre, R. Roy et al. using the specific functionalization of N₃P₃ for the synthesis of onion-peel, dumbbell shapes, and a wide variety of structurally diversified dendritic platforms, has led to diverse biologically active glycoconjugates [60]. We have used N₃P₃ as core of many dendrimers, but also as branching points inside the structure in some cases. Its use for the synthesis of specifically engineered dendrimers, with properties in the fields of catalysis, materials, imaging, or biology, is a recent illustration of its ability to become a ubiquitous tool in chemistry, and beyond, in particular towards biology.