Synthesis and characterization of phosphorus-containing dendrimers bearing rhodamine derivatives as terminal groups

The synthesis of two new derivatives of Rhodamine B functionalized by phenols is reported. For one rhodamine derivative, the equilibrium between the open form and the ring-closed (spirolactam) form is completely shifted toward the latter, whereas the constitution of the other rhodamine derivative precludes ring closure. The spirolactam derivative was grafted as terminal group to a first generation phosphorus-containing dendrimer. Attempts to open the spirolactam form by adding HCl failed and resulted only in protonation of the NEt 2 substituent of rhodamine.


Introduction
Rhodamine derivatives pertain to one of the most largely used family of chromophores, owing to their photostability, high fluorescence intensity, and low cost compared to many other types of fluorophores.Beside the application as textile dyes, rhodamines are often used as sensitive labels or tracers for biological experiments, and as chemo-sensors for sensing ions. 1 This property is due to a pH-driven ring-opening / ring-closure (Scheme 1); the open form 1A is colored and highly fluorescent, whereas the ring-closed form 1B is colorless and non-fluorescent. 2 Scheme 1. pH-Driven equilibrium between open and closed (spirolactam) forms of rhodamines.
Frequently, grafting of a functionality as terminal groups of dendrimers enhances its properties.In the case of fluorescent groups, the presence of multiple fluorescent labels linked to dendrimers generally induces a brilliant fluorescence provided no quenching is caused by the close proximity of the fluorophores. 3We have shown that phosphorus-containing dendrimers are suitable for such purpose, 4 also for obtaining brilliant two-photon absorption organic "nanodots", as efficient as inorganic quantum dots. 5Grafting rhodamine derivatives as terminal groups of phosphorus-containing dendrimers may allow an enhancement of their sensing properties in biology (fluorescence in acidic conditions).Here we report the synthesis of new derivatives of Rhodamine B and their grafting as terminal groups of phosphorus-containing dendrimers.

Results and Discussion
The phosphorus-containing dendrimers we synthesized contain P(S)Cl2 or aldehyde groups as terminal groups.Since the P(S)Cl2 group readily reacts with phenols Rhodamine B should be first functionalized to provide a phenol group, and the carboxylic acid group could serve for this purpose.As a linker for grafting various functionalities on P(S)Cl2 groups we have used tyramine, 6 which appears to be suitable here too.The reaction of the tyramine NH2 function with the carboxylic acid group of Rhodamine B was attempted by two different ways.First, we tried the direct coupling using a classical peptidic coupling strategy based on the activation with hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC); however, this attempt failed to afford the expected product.Then, we tried an indirect method (two steps) derived from the protocol for grafting of Rhodamine B to glycine. 7The first step was the esterification of the carboxylic acid group in Rhodamine B 1 with N-hydroxysuccinimide (NHS) in the presence of DCC.The resulting ester 2 was characterized by 13 C NMR, displaying the CO signal at δ 160.74, while Rhodamine B 1 displays the CO signal at δ 167.7.The reaction of ester 2 with tyramine in acetonitrile and borate buffer formed compound 3, which was isolated as a white powder upon column chromatography (Scheme 2).Unexpectedly, compound 3 is colorless and non-fluorescent.Thus, the structure of 3 cannot be the open form 3A but the spirolactam form 3B; this was confirmed by the 13 C NMR signal at δ 65.13 assigned to the spiro-carbon.
This result was unexpected but may be interesting for practical synthesis.THF and chloroform are good solvents for dendrimers and for spirolactam 3B.An open form is anticipated to be insoluble in such solvents and permanently fluorescent.Therefore, the synthesis of a phenol derivative of Rhodamine B was devised that is prevented from ring closure: 4-hydroxyphenylpiperazine was chosen for this purpose.The peptide coupling strategy used previously was successfully applied to afford compound 4, which is dark purple in color and fluorescent as anticipated (Scheme 3).The open form 4A was confirmed by the absence of a 13 C NMR signal at about δ 65 (spiro carbon) and by the signal at δ 167.87 (N-C=O).

Scheme 3
Using cesium carbonate as a base, Rhodamine derivatives 3B and 4A were grafted as terminal groups on the first generation dendrimer 5-G1. 7Monitoring the reaction of compound 3B by 31 P NMR showed the disappearance of the P(S)Cl2 signal at δ 62.5 and the appearance of a signal at δ 68.5 corresponding to mono-substitution [P(S)Cl(OAr)]; disappearance of the latter signal and the appearance of a new signal at δ 62.7 indicated full substitution (dendrimer 5-G1) (Scheme 4). 31P NMR is a very sensitive tool for the characterization of phosphorus-containing dendrimers, 8,9 mass spectrometry, including MALDI-Tof, is not useful for proving the purity of the phosphorhydrazone-containing dendrimers. 10endrimer 5-G1 is not fluorescent, indicating that the closed rhodamine form is retained upon grafting to dendrimers.In an attempt to open the spiro-form (cf.Scheme 1), an equivalent amount (1 H + per terminal group) of aqueous HCl (0.1 N) was added to the solution of dendrimer 5-G1 in THF; a pink color developed after 1.5 h. 31 P and 1 H NMR showed that the overall structure of the dendrimer was not changed by the addition of HCl.The 13 C NMR signal of the spiro carbon was still present indicating that most of the rhodamine moieties were not opened.Additional HCl did not open all spiro forms, the closed form prevailing in the equilibrium as confirmed by the very low fluorescence intensity of 5-G1HCl compared to Rhodamine B (Figure 1) even in DMSO, which should favor the open form as polar solvents do in general.Comparison of the 1 H and 13 C NMR spectra of 5-G1 before and after addition of HCl reveals several differences (Figure 2; data from compounds 2, 3B, and 4A are also included).After  Addition of HCl affected the 13 C NMR chemical shifts of the xanthene ring, as well as the NCH2CH3 signal (5-G1: δ 44.04; 5-G1HCl: δ 47.36).This is taken as evidence that the NEt2 groups were protonated, and that protonation opens only a few spirolactam rings, as indicated also by UV-Visible and fluorescence spectra (Figure 1).A gradient enhanced hydrogen-nitrogen multiple quantum coherence NMR experiment ( 1 H-15 N HMBC) served to prove protonation at NEt2.In this experiment, dendrimer 5-G1 afforded four 15 N NMR signals as expected (Figure 3).By comparison with a previous experiment, 12 the signals at δ -52 and δ -249 are attributed to the CH=N and N-Me groups of the dendrimer skeleton, respectively. 1H/ 15 N correlation allows to assign the signals at δ -226 and δ -304 to N-C=O and NEt2 groups of Rhodamine, respectively.5-G1HCl is less soluble than 5-G1, and the same type of experiment required a longer time (48 h).A single new signal was observed at δ -328, compatible with Et2NH + .However, this signal is less intense than the signal at δ -304 (neutral NEt2) (Figure 3), and the 1 H NMR spectrum recorded after the experiment displays a more intensive signal at δ 1.1 for CH3 of the NEt2 groups beside the signal at δ 1.3 (CH3 of Et2NH + ), indicating that protonation is reversible.The 1 H- 15 N HMBC experiments confirm that protonation occurs mainly at the NEt2 groups.Rhodamine derivative 4, which cannot cyclize, was reacted with dendrimer 5-G1.The solubility of both compounds is different: dendrimer 5-G1 was dissolved in THF and the rhodamine derivative 4 in acetonitrile.Upon mixing both reactant solutions the partly substituted dendrimer precipitates in the solvent mixture preventing the reaction to drive to completion.

Conclusions
We have synthesized two new rhodamine derivatives functionalized with a phenol, one for which the equilibrium is shifted toward the colorless and non-fluorescent spirolactam form 3B and one which can be only in the open colored and fluorescent form 4A. Due to problems of solubility, the salt 4 could not be grafted on a phosphorus-containing dendrimer, but the neutral rhodamine derivative 3B was successfully grafted as terminal groups.A few papers have reported the synthesis of dendrimers labeled with rhodamine (rhodamine being linked to one or a few terminal functions 13,14 ) but to the best of our knowledge, 5-G1 is the first example of a dendrimer bearing rhodamine moieties on all terminal groups.Addition of HCl did not damage the structure of the dendrimer, and does not open the spirolactam form of the rhodamine terminal groups (or only some of them).Protonation of the NEt2 substituent of rhodamine takes place; this shows that the spirolactam form of the rhodamine derivative 3B is particularly stable.

Experimental Section
General.All manipulations were carried out with standard high vacuum and dry-argon techniques.Filtrations were also carried out under argon.The solvents were dried (THF and diethyl ether over sodium/benzophenone, pentane and dichloromethane over phosphorus pentoxide, acetonitrile over calcium hydride) and distilled prior to use. 1 H, 13 C, and 31 P NMR spectra were recorded with Bruker AC 200, AC 250, DPX 300 or AMX 400 spectrometers.References for NMR chemical shifts are 85% H3PO4 in water for 31 P, SiMe4 for 1 H and 13 C, and CH3NO2 for 15 N NMR. 13C NMR signals were assigned using Jmod, two dimensional HMBC, and HMQC, broad band or CW 31 P decoupling experiments when necessary, and were found consistent with previously reported data for the rhodamine derivatives. 15The numbering used for NMR assignments for open forms 2, 4A and for spirolactam forms (3B, dendrimers 5-G1 and 5-G1HCl) is shown in Scheme 3 and Figure 2, respectively.Yields are not optimized.