Symmetrical and unsymmetrical incorporation of active biological monomers on the surface of phosphorus dendrimers

Article abstract of DOI:10.1016/j.tet.2017.01.044

Phosphorus-containing dendrimers are attractive carriers for the delivery of bioactive molecules due to their very particular architecture, globular shape and chemical and physical properties. Herein we report a simple synthetic approach of ethacrynic aci

Full text of DOI:10.1016/j.tet.2017.01.044

Accepted Manuscript
Symmetrical and unsymmetrical incorporation of active biological monomers on the
surface of phosphorus dendrimers
Nabil El Brahmi, Saïd El Kazzouli, Serge Mignani, Régis Laurent, Sonia Ladeira,
Anne-Marie Caminade, Mosto Bousmina, Jean-Pierre Majoral
PII:
S0040-4020(17)30067-4
10.1016/j.tet.2017.01.044
TET 28414
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 November 2016
Revised Date: 9 January 2017
Accepted Date: 18 January 2017
Please cite this article as: El Brahmi N, El Kazzouli S, Mignani S, Laurent R, Ladeira S, Caminade A-M,
Bousmina M, Majoral J-P, Symmetrical and unsymmetrical incorporation of active biological monomers
on the surface of phosphorus dendrimers, Tetrahedron (2017), doi: 10.1016/j.tet.2017.01.044.
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ACCEPTED MANUSCRIPT
Graphical Abstract

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Symmetrical and unsymmetrical incorporation of active biological monomers
on the surface of phosphorus dendrimers
Nabil El Brahmia, Saïd El Kazzoulia*, Serge Mignanib*, Régis Laurentc,d, Sonia Ladeirac,d, Anne-Marie Caminadec,d,
Mosto Bousminaa, Jean-Pierre Majoralc,d*
aEuromed Research Institute, Euro-Mediterranean University of Fes (UEMF), Route de Meknes,
30000, Fès, Morocco
bUniversité Paris Descartes, PRES Sorbonne Paris Cité, CNRS UMR 860,
cLaboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse Cedex 4.
France.
dUniversité de Toulouse, UPS, INPT, F-31077 Toulouse Cedex 4, France
Phosphorus-containing dendrimers are attractive carriers for the delivery of bioactive molecules due to their
very particular architecture, globular shape and chemical and physical properties. Herein we report a simple
synthetic approach of ethacrynic acid derivatives loaded-phosphorus dendrimers by symmetrical grafting of
ethacrynic acid analogues by nucleophilic substitution of each chlorine of P(S)Cl2 end groups. We report also for
the first time an unsymmetrical grafting of two different phenol derivatives by a selective substitution of one
chlorine of P(S)Cl2 end group followed by the grafting of the second phenol.
Keywords: dendrimers, phosphorus, biological entities
Introduction
Dendrimers are a class of highly branched three dimensional macromolecules characterized by a well-
defined nanostructure.1 Dendrimers possess a large number of unique properties and constitute an
increasingly important field of research in chemistry and in nanomedicine.2 After pioneering works concerning
dendrimers synthesis, the interest now is mainly focused on their applications.3 Considerable effort has been
devoted to the functionalization of dendrimers either on the periphery, between the branching units or at the
core4 to explore new properties and applications. Interestingly, the properties of these macromolecules are
mainly depending on several factors as the nature of their functional end groups, the nature of their internal
backbone and strongly, of their size.
The easiest way to achieve such multifunctional devices is the grafting in stochastic manner of several
groups on the dendrimer surface.5 This way was largely used for grafting a fluorophore6 then a drug (e.g., 5-
Fluorouracil (FU)7, Doxorubicin (DOX)8 or Methotrexate (MTX)9). For example, Baker et al.10 have synthesized
PAMAM dendrimer based multifunctional therapeutic devices containing MTX as an anticancer drug,
fluorescein isothiocyanate (FITC) as an imaging agent and folic acid (FA) as a specific targeting agent.
Unfortunately, the stochastic approach has a lot of limitations because the inconsistencies in the number of
chemotherapeutic units loaded to dendrimers led often to unreproducible biological results.11
This concept has also been applied to phosphorus dendrimers (see their synthesis in Scheme 1) with the
incorporation of diverse functional groups on their surface. For diverse applications, especially in biology, there
is a need of dendrimers having several simultaneous functions (e.g. therapeutic, targeting, imaging ) For
example, ca. one FITC was grafted per first generation of phosphorus dendrimer covered by azabisphosphonate
derivatives,12 and a phosphorus dendrimer of generation 3 was prepared via a 10% stochastic labeling of the
P(S)Cl2 dendrimer terminal groups with a conjugate phenol julolidine followed by oligomannose substitution on

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the remaining P(S)Cl2 end groups.13 In another interesting work, the specific unsymmetrical functionalization of
phosphorus dendrimers bearing P(X)Cl2 (X = O or S) end groups was carried out up to the generation 7 using
different amine moieties to yield P(X)ClNRR’ terminal functions. In a second step, either a second amine was
reacted to afford P(X)NRR’NHR1 terminal functions (up to generation 4) or a phenol to afford P(O)NRR’OAr
terminal functions (up to generation 3).14 To summarize, the symmetrical substitution of the chlorine group of
P(X)Cl2 units (X = O or S) could be attempted by amines,15 phenolic entities,16 organometallics,16(b,e) fluorophoric
moieties12,17 or sugar entities18 to name as a few. Nevertheless, the sequential unsymmetrical substitution of
the chlorines of the P(S)Cl2 groups remains an exception.
In this paper, we report an approach for the simple control of the reactivity at the periphery of phosphorus-
containing dendrimers allowing the sequential introduction of one or two different active molecules bearing a
phenol group. For this purpose three phenol derivatives were selected: an azabisphosphonate 119 the 4-
hydroxybenzaldehyde, EA-220 (Figure 1) and modified ethacrynic acids (EA-3 and EA-4 see syntheses in Scheme
2).
Fig. 1: Structure of the monomers 1 and EA-2
As far as we know, two different phenols have never been grafted selectively on the surface of dendrimers.
The choice of these three different phenols was dictated by the following considerations.
i)
Ethacrynic acid or [2,3-dichloro-4-(2-methylene-1-oxobutyl)phenoxy] acetic acid belongs to the
diuretic drugs,21 has an acid function and a carbonyl unit α, β-unsaturated which is often used as
inhibitor of glutathione-S-transferase P1-1 (GSTP1-1) overexpressed in a variety of cancer cells,
especially, in chemotherapy-resistant cancer cells.22 Very recently, we reported the grafting of
ethacrynic acid analogues onto phosphorus dendrimer surface which afforded complexes with
strong anti-proliferative activities against liquid and solid tumors.20
ii) Beside the advances made in chemotherapy with the use of specific anticancer drugs, the increase of
the immune system efficiency to fight against cancers is also another interesting challenge.23 The
artificial increase of the number of blood immune cells is very difficult recurring complex and
poorly available biological entities. Interestingly, a phosphorus dendrimer of generation 1 prepared
by nucleophilic substitution of its 6 terminal P(S)Cl2 by aminobisphosphonate functionalized by a
phenol displays a very interesting property, highly and selectively promoting the multiplication of
human natural killer cells (NK).19 Therefore it was of great interest to check if such an active phenol
can be sequentially and unsymmetrically grafted to phosphorus dendrimers,
iii) Grafting of a bifunctional reagent as 4-hydroxybenzaldehyde on residual P-Cl bonds offers the
advantage to incorporate an additional function at the surface of the dendrimers (in this case
aldehyde units) and therefore the possibility to diversify the properties of the resulting
dendrimers.
Results and Discussion
Phosphorus starting dendrimers were prepared as described in Scheme 1 using the reiteration of two steps,
e.g., nucleophilic substitution and Schiff base reactions.24 So, after the nucleophilic substitution of the six
chlorine of the hexachlorocyclotriphosphazene P3N3Cl6 25with 4-hydroxybenzaldehyde under basic conditions,
the condensation reaction between the terminal aldehyde and H2NNMeP(S)Cl2 was carried out leading to
generation 1 dendrimer (G1). This two-step route was repeated once or twice to achieve the formation of

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dendrimers G2 and G3, respectively. The synthesized dendrimers (G1, G2 and G3) have P(S)Cl2 as reactive end
groups.
Scheme 1: Dendrimers generation G1-G3 with P(S)Cl2 end groups
The grafting of several types of phenolic groups at each terminal P(S)Cl2 unit could be attempted by simple
substitution reaction. In continuation of our research concerning ethacrynic acid derivatives,20 other phenolic
ethacrynic acid derivatives were prepared (Scheme 2) and were later grafted on the surface of different
generations of dendrimers Gn (G1, G2 and G3).
Scheme 2: Synthesis of EA derivatives EA-3 and EA-4 from EA
First, we started with the synthesis of EA derivatives EA-3 and EA-4 as shown in Scheme 2. Phenol EA-3 was
prepared in one step using the classical conditions of the esterification reaction, starting from the commercially
available EA and 4-(2-hydroxyethyl)phenol in the presence of paratoluenesulfonic acid (APTS). The reaction
mixture was heated at 50°C for 12h to provide EA-3 in almost a quantitative yield (92%). For the preparation of
EA amide (EA-4) a strategy based on two steps starting from 4-hydroxybenzaldehyde was used. The
condensation of the 4-hydroxybenzaldehyde with methylhydrazine, led to (E)-4-((methylimino)methyl)phenol
A, then
a
peptidic type reaction using EA in the presence of EDCI (1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide) and a catalytic amount of DMAP (4-Dimethylaminopyridine) in DCM/DMF,
afforded EA-4 in 40 % yield.
Then, we attempted the direct coupling of EA-3 with Gn (n = 1, 2, 3) dendrimers. Using Cs2CO3 as base in
THF, EA bearing a phenol group was successfully coupled to dendrimers bearing P(S)Cl2 end groups. The
reaction, carried out using different dendrimer’s generations, led to EA decorated dendrimers 1-Gn (n = 1-3,
Scheme 3). The new dendrimers 1-G1, 1-G2 and 1-G3 bear 12, 24 and 48 peripheral EA derivatives on their
surface, respectively. These products were isolated in 90, 88 and 85% yield, respectively. Reactions were
monitored by 31P NMR spectroscopy which shows the disappearance of the signal due to the external P(S)Cl2
units at 63.1 ppm on behalf of a new signal at 62.9 ppm whatever the generation. It is noteworthy also that, in
all Gn, the chemical shift corresponding to 31P of P(S)Cl2 end groups changes when the substitution of one
chlorine occurred and changes again when two chlorine are substituted. In contrast, the chemical shift

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corresponding to the dendrimer core N3P3 did not change if a mono or a disubstitution occurred on the surface
of the dendrimer.
Scheme 3: Grafting of the EA-3 on the surface of Gn dendrimers
The same reaction was applied for the grafting of EA-4 on the surface of different dendrimer generations
Gn (n= 1-3). The resulting EA-decorated dendrimers 2-Gn (n = 1-3, Scheme 4), bearing 12, 24 and 48 peripheral
EA moieties, were obtained in 88, 85 and 85 % yield, respectively. Analogous modifications of the 31P spectra
than those observed for the 1-Gn family were also observed.
Scheme 4: Grafting of the EA-4 on the surface of Gn dendrimers
Then, we tried the direct coupling of EA-2 onto Gn. We started by grafting EA-2 on G1 under the same
reaction conditions as indicated above. The reaction, monitored by 31P NMR spectroscopy, showed at the
beginning two signals at 8.3 ppm (N3P3) and at 62.8 ppm (P=S groups) corresponding to the starting material
G1. Then, the appearance of a signal at 68.3 ppm characteristic of the formation of the mono-substitution at
the P(S)Cl2 groups was detected. Subsequently, precipitation was observed in the reaction medium with the
disappearance of all the NMR peaks, probably due to the formation of the di-substitution derivative. All the

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attempts to solve this problem by varying the reaction solvent or changing the reaction temperature (from
room temperature to 50°C) failed.
To explain this phenomenon, we thought about the possible formation of intra or inter hydrogen bonds,
between the oxygen of the acrylate and the hydrogen of the amide function O-H…O=C, which can lead to the
formation of aggregates due to di-substitution of P(S)Cl2. This hypothesis seems to be supported by a X-ray
study realized on phenol AE-2 (Figure 2) which showed the formation of a hydrogen bond between the oxygen
of the acrylate moiety and the hydrogen of the amide (distance between H(2)-O(8) = 2.811(10) Å).
Fig. 2: X-ray structure of the monomer AE-2
Bond lengths N(2)-H(2)= 0.864(10) Å, C(13)-O(8)=1.230(04) Å and H(2)-O(8)=2.26(2) Å
It is very important to note that during this reaction, we observed the formation of the mono-substitution
intermediate first then solubility problems occurred when the di-substitution started. Based on this interesting
observation we decided to exploit this selectivity to introduce two different phenol moieties on dendrimer
surface by an unsymmetrical di-substitution of the phosphorus end groups P(S)Cl2.
The mono-substitution of P(S)Cl2 end groups of G1 was achieved using 6 equivalents of EA-2 and 12
equivalents of Cs2CO3 in THF. The reaction was monitored particularly by 31P NMR, which displays, in addition to
the signal at 8.4 ppm corresponding to the chemical shift of the N3P3 core, a deshielding of the signal
corresponding to the terminal groups on going from 63.1 ppm (P(S)Cl2) to 68.3 ppm typical to the mono-
substitution and a signal at 62.8 ppm corresponding to 5% of the di-substitution product (Figure 3).
Fig. 3: 31P NMR spectrum of mono-substitution dendrimer G1mono
This compound was obtained in situ and engaged in a second coupling reaction with other phenol
derivatives. In the first example, the mono-substituted intermediate (G1 mono) was treated with 6 equivalents
of 4-hydroxybenzaldehyde and 12 equivalents of Cs2CO3 in THF. The reaction mixture was stirred for 12h at
room temperature and monitored by 31P NMR; when the substitution of the second chlorine occurred, the
chemical shift of N3P3 core was not modified while the P1 (phosphorus of the first generation) shifted to 61.7
ppm (Figure 3). The desired product 3-G1 was obtained as a white powder in 72 % yield (Scheme 5).

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Scheme 5: Grafting of the EA-2 and 4-hydroxybenzaldehyde on the surface of G1 dendrimers
In the second example, azabisphosphonate phenol 1 was grafted as a second active phenol onto the
remaining Cl of the mono-substituted dendrimer (intermediate G1 mono) using 6 equivalents of 1 and 12
equivalents of Cs2CO3 in THF. The signal corresponding to the terminal groups moves from 68.3 ppm to 63.1
ppm with appearance of another signal at 26.5 ppm corresponding to the phosphonate groups of phenol 1
(Figure 4). The dendrimer 4-G1 was obtained as a white powder (65%) (Scheme 6). To our knowledge, two
different phenols have never been grafted on such phosphorus dendrimers thus allowing to have on their
surface two different bioactive molecules (NK multiplication, target 1 and caspase 3/7 activation (apoptotic
enzymes),26 target 2).

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Scheme 6: Structure of 4-G1
P1
P0
Pdi
G1 mono
P1
P0
Pdi
3-G1
Cl
O
Pp
Cl
O
O
NH
N
S
P0
O
O
N3P0
O
P1
P1
3
O
6
N
N
O
O
P
O
P
O
O
4-G1
Fig. 4: 31P NMR spectrum of G1mono, 3-G1 and 4-G1
Conclusions
In this study, we have developed a simple synthetic method allowing to graft phenol derivatives of drugs on
the periphery of phosphorus dendrimers. We have also described the first phosphorus dendrimers containing
two different phenol moieties in their periphery in a controlled fashion. This current approach will offer several
opportunities for phosphorus containing dendrimers to diversify their surface by unsymmetrical substitution of
the P(S)Cl2 end groups. Taking into account the huge number of functionalized phenols that exist; this method
opens thus an original and versatile way for the functionalization of dendrimer’s surface.

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Experimental Part
The syntheses were carried out using standard high vacuum and dry-argon techniques. All chemicals were
purchased from Acros, Aldrich, Fluka, and used without further purification. The solvents were freshly dried
and distilled according to standard procedures prior to use.
1H, 13C, and 31P NMR spectra were recorded with Bruker AV300, DPX300, AV400, spectrometers. All 13C NMR
and 31P NMR spectra were generally recorded decoupled {1H}. The signal of the non-deuterated solvent served
as internal standard, relative to SiMe4 for 1H and 13C NMR, to H3PO4 for 31P NMR. Fourier transformed infrared
(FTIR) spectra were obtained with a Perkin–Elmer Spectrum 100 FT-IR spectrometer on neat samples (ATR FT-
IR) or in solutions. Mass spectrometry was carried out with a Thermo Fisher DS QII (DCI/NH3 or DCI/CH4). The
purity of phosphorhydrazone dendrimers cannot be assessed by mass spectrometry (ESI or MALDI-Tof) as both
techniques induce dramatic cleavages of the structure.27
X-ray data were collected at low temperature (193(2) K) on a Bruker Kappa Apex II diffractometer equipped
with a 30 W air-cooled microfocus, using MoKα radiation (λ = 0.71073 Å), and an Oxford Cryosystems
Cryostream cooler device. Phi- and omega- scans were used for data collection. The structure was solved by
direct methods with SHELXS-97.28 All non-hydrogen atoms were refined anisotropically by means of least-
squares procedures on F² with SHELXL-97.Error! Bookmark not defined. The hydrogen atoms were refined isotopically at
calculated positions using a riding model except the N-bound H atoms which have been located in a difference
Fourier map and refined with the N-H bond-length fixed at 0.88(1) Å. In the crystal structure, disorders were
modeled successfully with the atom ellipsoids restrain by SIMU and DELU commands.The data of the crystal
structure were deposited at the Cambridge crystallographic data centre and have been assigned to the
following deposition numbers CCDC 1473464.
Compounds 119 and EA-220 were prepared following the previously reported procedures.
Synthesis of EA-3:
To a mixture of paratoluenesulfonic acid (APTS) (342 mg, 1.8 mmol) and ethacrynic acid (500 mg, 1.8 mmol) in
DCM (10 mL), 4-(2-hydroxyethyl)phenol (250 mg, 1.8 mmol) was added at 0°C. The reaction mixture was
heated to 50°C for 12h and then evaporated to dryness. The crude residue was purified by flash
chromatography (hexane/EtOAc (6/4)) to furnish EA-3.
1
EA-3. White solid, 92% yield (0.642 g). H NMR (400 MHz, CD2Cl2) δ 1.18 (t, J =7.4 Hz, 3H), 2.49 (q, J = 7.4 Hz,
2H), 2.91 (t, J = 6.5 Hz, 2H), 4.40 (t, J = 6.5 Hz, 2H), 4.76 (s, 2H), 5.54 (s, 1H), 5.63 (s, 1H), 6.02 (s, 1H), 6.62 (d, J =
8.6 Hz, 1H), 6.69 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.6 Hz, 1H); 13C {1H} NMR (101 MHz,
CD2Cl2) δ 12.2, 23.4, 33.9, 66.1, 66.2, 110.4, 115.3, 126.8, 129.2, 129.8, 122.7, 129.3, 131.1, 133.4, 150.2, 154.5,
155.4, 167.7, 196.3. HRMS (+ESI) m/z: [M+H]+ calcd for C21H20Cl2O5: 423.0766, found, 423.0773. Anal. Calcd for
C21H20Cl2O5: C, 59.59; H, 4.76; found: C, 59.49; H, 4.70. IR (neat): ν = 3316 (OH), 1661 (C=O) cm-1
Synthesis of EA-4:

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To a solution of para-hydroxybenzaldehyde (1.65 g, 13.5 mmol) in dry THF (40 mL), monomethylhydrazine (0.71
mL, 621 mg, 13.5 mmol) was added dropwise. The reaction mixture was stirred overnight at room temperature
and monitored by TLC. The reaction mixture was concentrated under reduced pressure to give the expected 4-
((2-methylhydrazono)methyl)phenol as a yellow oil.
To a solution of crude 4-((2-methylhydrazono)methyl)phenol (1.0 g, 6.66 mmol) in a DCM/DMF mixture (60 mL,
2:1), ethacrynic acid (2.10 g, 6.93 mmol), EDCI (1.50 g, 7.82 mmol) and a catalytic amount of DMAP were
added. The reaction mixture was stirred for 40 h at room temperature. Ethyl acetate (300 mL) was added and
the organic layer was washed with water (2 × 100 mL) and brine (4 × 100 mL), dried over anhydrous MgSO4 and
then concentrated under reduced pressure. The crude product was purified by flash chromatography
(DCM/EtOAc 98:2 to 95:5) to give the EA-4 as a yellow powder.
EA-4. Yellow solid, 40% yield (1.2 g). 1H NMR (400 MHz, d8-THF) δ 1.16 (t, J =7.4 Hz, 3H), 2.47 (q, J = 7.4 Hz, 2H),
3.38 (s, 3H), 5.48 (s, 2H), 5.61 (s, 1H), 5.95 (s, 2H), 6.82 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.6
Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 8.68 (s, 1H); 13C {1H} NMR (101 MHz, d8-THF) δ 11.9, 23.4, 26.7,
67.2, 111.1, 115.4, 122.2, 126.2, 126.6, 126.9, 128.5, 130.3, 132.8, 140.1, 150.4, 156.7, 159.5, 166.9, 194.5.
HRMS (+ESI) m/z: [M+H]+ = 435.0870. Anal. Calcd for C21H20Cl2N2O4: C, 57.94; H, 4.64; N,6.44; found C, 57.89; H,
4.58; N, 6.40. IR (neat): ν = 3475 (OH), 1688 (C=O), 1649 (C=N) cm-1
Synthesis of 1-Gn:
General procedure
A dendrimer Gn (100 mg, 0.055 mmol, n = 1), (100 mg, 0.021 mmol, n = 2) or (100 mg, 0.01 mmol, n = 3), was
dissolved in THF (20 mL), and then the appropriate masses of phenol EA-3 (291 mg, 0.687 mmol, n = 1), (222
mg, 0.525 mmol, n = 2) or (201 mg, 0.48 mmol, n = 3), and cesium carbonate (430 mg, 1.32 mmol, n = 1), (328
mg, 1.01mmol, n = 2), (312 mg, 0.96 mmol, n = 3), were added. The reaction mixture was stirred overnight at
room temperature, and then centrifuged to remove salts. The solution was concentrated and precipitated two
times in pentane/Et2O (9/1). The product was filtered and dried under vacuum to give 1-G1 (generation 1), 1-
G2 (generation 2) or 1-G3 (generation 3) as white powders.
Synthesis of 1-G1:
White solid, 90% yield (0.393 g). 31P {1H} NMR (121 MHz, CD2Cl2) δ 8.32 (s, P0), 62.90 (s, P1). 1H NMR (300 MHz,
CD2Cl2) δ 1.14 (t, J = 7.4 Hz, 36H), 2.45 (q, J = 7.4 Hz, 24 H), 2.91 (t, J = 6.8 Hz, 24H), 3.28 (d, J = 10.2 Hz, 18H),
4.37 (t, J = 6.8 Hz, 24H), 4.71 (s, 24H), 5.58 (s, 12H), 5.95 (s, 12H), 6.74 (d, J = 8.6 Hz, 12H), 7.03 (d, J = 8.6 Hz,
12H ), 7.08-7.22 (m, 60H), 7.60-7.68 (m, 18H); 13C {1H} NMR (75 MHz, CD2Cl2) δ 12.2, 23.4, 32.9 (d, J = 12.0 Hz),
34.1, 65.6, 66.1, 110.7, 121.2, 121.3 (d, J = 4.7 Hz), 122.8, 126.9, 128.2, 128.6, 130.0, 131.1, 132.3, 133.7, 134.8
(d, J = 1.7 Hz), 138.5 (d, J = 13.5 Hz), 149.4 (d, J = 7.1 Hz), 150.1, 151.2, 155.3, 167.5, 195.4. Anal. Calcd for
C300H276N15Cl24O66P9S6: C, 55.70; H, 4.30; N, 3.25; found: C, 55.59; H, 4.19; N, 3.14.
Synthesis of 1-G2:

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1
White solid, 88% yield (0.334 g). 31P {1H} NMR (121 MHz, CD2Cl2) δ 8.3 (s, P0), 62.6 (s, P1), 62.9 (s, P2); H NMR
(300 MHz, CD2Cl2) δ 1.12 (t, J = 7.4 Hz, 72H), 2.43 (q, J = 7.4 Hz, 48H), 2.90 (t, J = 6.8 Hz, 48H), 3.29 (m, 56H),
4.36 (t, J = 6.8 Hz, 48H), 4.70 (s, 48H), 5.56 (s, 24H), 5.94 (s, 24H), 6.72 (d, J = 8.5 Hz, 24H), 7.00 (d, J = 7.8 Hz,
12H), 7.08-7.28 (m, 144H), 7.58 -7.74 (m, 54H); 13C {1H} NMR (75 MHz, CD2Cl2) δ 12.2, 23.4, 32.9 (d, J = 12.3 Hz,
2C), 34.1, 65.6, 66.1, 110.7, 121.2, 121.4 (d, J = 4.0 Hz), 121.71 (d, J = 4.0 Hz), 122.8, 126.9, 128.2, 128.3, 128.6,
130.0, 131.1, 132.2, 132.5, 133.7, 134.8, 138.9 (d, J =13.5 Hz), 139.3 (d, J = 13.5 Hz), 149.4 (d, J = 7.1 Hz), 150.1,
151.2 (d, J = 7.1 Hz, 2C), 155.3, 167.5, 195.4. Anal. Calcd for C648H600Cl48N39O138P21S18: C, 55.31; H, 4.30; N, 3.88;
found: C, 55.04; H, 4.28; N, 3.32.
Synthesis of 1-G3:
White solid, 85% yield (0.321 g). 31P {1H} NMR (121 MHz, CD2Cl2) δ 7.9 (s, P0), 62.6 (s, P1), 62.8 (s, P2 and P3). 1H
NMR (300 MHz, CD2Cl2) δ 1.11 (t, J = 7.4 Hz, 144H), 2.42 (q, J = 7.4 Hz, 96H), 2.80-2.97 (m, 96H), 3.29 (d, J = 9.8
Hz, 126H), 4.26-4.45 (m, 96H), 4.69 (s, 96H), 5.55 (s, 48H), 5.92 (s, 48H), 6.70 (d, J = 8.5 Hz, 48H), 7.02-7.29 (m,
324H), 7.69 (d large, 126H). 13C {1H} NMR (75 MHz, CD2Cl2) δ 12.2, 23.4, 32.9 (d, J = 12.6 Hz, 3C), 34.1, 65.6,
66.1, 110.7, 121.2, 121.4 (d, J = 4.5 Hz), 121.8 (s, 2C), 122.8, 126.9, 128.2 (s, 2C), 128.3, 128.5, 130.0, 131.1,
132.4 (s, 2C), 133.6, 134.8 (s, 2C), 138.9 (d, J = 13.5 Hz), 139.3 (d, J = 13.5 Hz, 2C), 149.4 (d, J = 7.1 Hz, 2C), 150.1,
151.3 (d, J = 6.9 Hz, 2C), 155.3, 167.5, 195.4.M.W.29274.63. Anal. Calcd for C1344H1248Cl96N87O282P45S42: C, 55.14;
H, 4.30; N, 4.16; found: C, 54.81; H, 4.12; N, 3.89.
Synthesis of 2-Gn:
General procedure
A dendrimer Gn (100 mg, 0.055 mmol, n = 1), (100 mg, 0.021 mmol, n = 2) or (100 mg, 0.01 mmol, n = 3), was
dissolved in THF (20 mL), and then the appropriate masses of phenol EA-4 (300 mg, 0.687 mmol, n = 1), (228
mg, 0.525 mmol, n = 2) or (210 mg, 0.48 mmol, n = 3), and cesium carbonate (430 mg, 1.32 mmol, n = 1), (328
mg, 1.01 mmol, n = 2), (312 mg, 0.96 mmol, n = 3), were added. The reaction mixture was stirred overnight at
room temperature, and then centrifuged to remove salts. The solution was concentrated and precipitated two
times in pentane/Et2O (9/1). The product was filtered and dried under vacuum to give 2-G1 (generation 1), 2-
G2 (generation 2) or 2-G3 (generation 3) as white powders.
Synthesis of 2-G1:
White solid, 88% yield (0.411 g). 31P {1H} NMR (162 MHz, CD2Cl2) δ 8.81 (s, P0), 62.24 (s, P1). 1H NMR (300 MHz,
CD2Cl2) δ 1.12 (t, J = 7.4 Hz, 36H), 2.43 (q, J = 7.4 Hz, 24H), 3.27 (d, J = 10.1 Hz, 18H), 3.34 (s, 36H), 5.31 (s, 24H),
5.60 (s, 12H), 5.91 (s, 12H), 6.75 (d, J = 8.7 Hz, 12H), 6.88 (d, J = 8.5 Hz, 12H ), 7.05 (d, J = 8.6 Hz, 12H), 7.22 (d, J

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= 7.9 Hz, 24H), 7.55 (d, J = 8.5 Hz, 12H), 7.57 (d, J = 7.9 Hz, 24H), 7.63 (s, 6H), 7.67 (s, 1H). 13C {1H} NMR (75
MHz, CD2Cl2) δ 12.4, 23.4, 28.1, 32.9 (d, J = 11.8 Hz), 67.5, 111.1, 121.4, 121.7 (d, J = 4.5 Hz), 123.1, 126.7,
128.3, 128.5, 128.7, 131.1, 131.5, 132.1, 133.2, 138.9 (d, J = 14.8 Hz), 139.2, 150.1, 150.8-151.4 (m, C), 151.7 (d,
J = 7.1 Hz), 156.1, 167.9, 195.9. Anal. Calcd for C300H276Cl24N39O54P9S6: C, 54.48; H, 4.21; N, 8.26; S, 2.91; found:
C, 54.20; H, 4.06; N, 7.98.
Synthesis of 2-G2:
White solid, 85% yield (0.355 g). 31P {1H} NMR (162 MHz, CDCl3) δ 8.55 (s, P0), 62.28 (s, P2), 62.59 (s, P1). 1H NMR
(400 MHz, CDCl3) δ 1.10 (t, J = 7.4 Hz, 72H), 2.41 (q, J = 7.1 Hz, 48H), 3.19 (d, J = 9.8 Hz, 18H), 3.23-3.40 (m,
108H), 5.31 (s, 48H), 5.57 (s, 24H), 5.89 (s, 24H), 6.74 (d, J = 8.6 Hz, 24H), 6.88 (d, J = 8.1 Hz, 12H), 7.04 (d, J = 8.6
Hz, 24H), 7.13 (d, J = 7.9 Hz, 24H), 7.21 (d, J = 8.0 Hz, 48H), 7.53 (d, J = 8.0 Hz, 12H) 7.53-7.65 (m, 90H), 7.66 (s,
24H). 13C {1H} NMR (101 MHz, CDCl3) δ 12.4, 23.4, 28.1, 32.1-33.7 (m, 2C), 67.5, 110.7, 121.2, 121.6, 121.8 (d, J
= 3.6 Hz), 122.9, 126.8, 128.3 (s, 2C), 128.5, 128.7, 131.1, 131.4, 132.1, 132.3, 133.2, 139.0, 139.1, 139.3, 150.0,
151.3 (d, J = 7.2 Hz, 2C), 151.7 (d, J = 7.2 Hz), 156.1, 167.4, 195.9. Anal. Calcd for C648H600Cl48N87O114P21S18: C,
54.20; H, 4.21; N, 8.49; found: C, 54.04; H, 3.97; N, 8.15.
Synthesis of 2-G3:
White solid, 85% yield (0.351 g). 31P {1H} NMR (162 MHz, CDCl3) δ 7.88 (s, P0), 62.56 (s, P1), 62.27 (s, P2), 61.76
1
(s, P2). H NMR (400 MHz, CDCl3) δ 1.05 (t, J = 7.1 Hz, 144H), 2.27-2.47 (m, 48H), 3.11-3.42 (m, 270H), 5.28 (s,
96H), 5.52 (s, 24H), 5.84 (s, 24H), 6.71 (d, J = 8.4 Hz, 48H), 6.99 (d, J = 8.2 Hz, 60H), 7.11 (d, J = 6.4 Hz, 84H), 7.18
(d, J = 7.2 Hz, 96H), 7.44-7.71 (m, 262H). 13C {1H} NMR (101 MHz, CDCl3) δ 12.4, 23.4, 28.1, 32.7-33.2 (m, 3C),
67.5, 111.7, 121.8 (s, 5C), 122.9, 126.8, 128.3 (s, 3C), 128.5, 128.7, 131.0, 131.4, 132.2, 132.3 (s, 2C), 133.1,
138.9-139.3 (m, 3C), 139.4, 150.0, 150.9-151.4 (m, 3C), 151.7 (d, J = 7.2 Hz), 156.1, 167.8, 195.9. Anal. Calcd for
C1344H1248Cl96N183O234P45S42: C, 54.08; H, 4.21; N, 8.59; found: C, 53.28; H, 3.86; N, 8.19.
Synthesis of 3-G1:
To a mixture of dendrimer G1 (100 mg, 0.055 mmol) and cesium carbonate (215 mg, 0.66 mmol) in THF (20
mL), six equivalents of phenol EA-2 (140 mg, 0.33 mmol) were added. The reaction mixture was stirred at room
temperature overnight and monitored with 31P NMR. When the monosubstitution was achieved, six
equivalents of 4-hydroxybenzaldehyde (40 mg, 0.33 mmol) and cesium carbonate (215 mg, 0.66 mmol) were
added. Again the reaction mixture was stirred at room temperature overnight. Salts were then removed by
centrifugation and the clear solution was concentrated under reduced pressure. The residue was then
dissolved in a minimum amount of THF (4 mL) and precipitated with a mixture of pentane/Et2O (9:1) to afford
3-G1 as a white powder in 75 % yield.

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1
White solid, 75% yield (0.340 g). 31P {1H} NMR (121 MHz, CD2Cl2) δ 8.3 (s, P0), 61.69 (s, P1), 62.87 (P1, dis). H
NMR (300 MHz, CD2Cl2) δ 1.15 (t, J = 7.4 Hz, 18H), 2.46 (q, J = 7.4 Hz, 12H), 2.83 (t, J = 6.6 Hz, 12H), 3.33 (d, J =
10.4 Hz, 18H), 3.58 (q, J = 6.6 Hz, 12H), 4.53 (s, 12H), 5.58 (s, 6H), 5.97 (s, 6H), 6.79 (s, 6H), 6.87 (d, J= 8.6 Hz,
6H), 7.05 (d, J = 8.5 Hz, 12H), 7.11-7.23 (m, 30H), 7.36 (d, J = 7.6 Hz, 12H), 7.62 (d, J = 8.5 Hz, 12H), 7.68 (s, 6H),
7.81 (d, J= 7.6 Hz, 12H), 9.90 (s, 6H). 13C {1H} NMR (75 MHz, CD2Cl2) δ 12.2, 23.4, 32.9 (d, J = 12.4 Hz), 34.9, 39.9,
68.2, 111.0, 121.2, 121.4 (d, J = 4.6 Hz), 121.9 (d, J = 4.9 Hz), 122.7, 127.3, 128.2, 128.7, 129.9, 130.9, 131.2,
132.1, 133.62, 133.9, 136.2, 139.2 (d, J = 14.3 Hz), 149.1 (d, J = 7.1 Hz), 150.1, 151.3, 154.5, 155.2 (d, J = 7.1 Hz),
166.4, 190.6, 195.3. Anal. Calcd for C216H198Cl12N21O42P9S6: C, 55.71; H, 4.29; N, 6.32; found: C, 55.26; H, 4.11; N,
6.05.
Synthesis of 4-G1:
To a mixture of dendrimer G1 (100 mg, 0.055 mmol) and cesium carbonate (215 mg, 0.66 mmol) in THF (20
mL), six equivalents of phenol EA-2 (140 mg, 0.33 mmol) were added. The reaction mixture was stirred at room
temperature overnight and monitored with 31P NMR. When the mono substitution was achieved, six
equivalents of 1 (126 mg, 0.33 mmol) and cesium carbonate (215 mg, 0.66 mmol) were added. Again the
reaction mixture was stirred at room temperature overnight. Salts were then removed by centrifugation, and
the clear solution was concentrated under reduced pressure. The residue was dissolved in a minimum amount
of THF (4 mL) and then precipitated with a mixture of pentane/Et2O (9:1) to afford 4-G1 as a white powder in
65% yield.
White solid, 65% yield (0.525 g). 31P {1H} NMR (121 MHz, CD2Cl2) δ 8.3 (s, P0), 26.5 (s, PO3Me2), 62.8 (P1, dis),
1
63.1 (s, P1). H NMR (300 MHz, CD2Cl2) δ 1.14 (t, J = 7.3 Hz, 18H), 2.45 (q, J = 7.3 Hz, 12H), 2.70-2.87 (m, 24H),
3.03 (t, J = 7.4 Hz, 12H), 3.16 (d, J = 9.3 Hz, 24H), 3.29 (d, J = 10.1 Hz, 18H), 3.56 (q, J = 6.5 Hz, 12H), 3.70 (d, J =
10.5 Hz, 72H), 4.53 (s, 12H), 5.58 (s, 6H), 5.97 (s, 6H), 6.79-6.93 (m, 12H), 6.99-7.25 (m, 66H), 7.60-7.72 (m,
18H). 13C {1H} NMR (75 MHz, CD2Cl2) δ 12.0, 23.2, 32.2, 32.8 (d, J = 12.4 Hz), 34.7, 40.0, 49.1 (dd, J = 157.5Hz, J =
8.2Hz), 52.0 (d, J = 6.9 Hz), 58.1 (t, J = 7.6Hz), 68.3, 111.8, 121.0 (d, J = 4.3 Hz), 121.20 (d, J = 4.4 Hz), 121.3,
122.1, 127.4, 128.3 (s, 2C), 129.9, 130.1, 130.2, 132.6, 133.5, 136.5, 137.3, 139.3 (d, J = 11.3 Hz), 148.9 (d, J =
6.8 Hz), 149.3 (d, J = 7.0 Hz), 150.1, 151.3, 155.3, 166.4, 194.9. Anal. Calcd for C258H312Cl12N27O72P21S6: C, 49.89;
H, 5.06; N, 6.09; found: C, 49.22; H, 4.99; N, 5.95.

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Acknowledgements
Thanks are due to CNRS (Toulouse France), CEFIPRA (project number5303-2) and Euro-Mediterranean
University of Fes (Morocco) for financial support.