New water-soluble polyanionic dendrimers - Phosphoric and 1,3,5-benzenetricarboxylic acid derivatives

Article abstract of DOI:10.1016/j.tet.2012.09.094

Simple, very efficient, and having some aspects of generality, synthesis of water-soluble, polyanionic dendrimeric polyesters with different size, polarity, and flexibility is described. These macromolecular compounds consisting of phosphate or thiophosph

Full text of DOI:10.1016/j.tet.2012.09.094

Tetrahedron 68 (2012) 10209e10217
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
New water-soluble polyanionic dendrimersdphosphoric
and 1,3,5-benzenetricarboxylic acid derivatives
*
ꢀ
Grzegorz M. Salamonczyk
ꢀ ꢀ
Centre of Molecular and Macromolecular Studies, The Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2012
Received in revised form 21 August 2012
Accepted 21 September 2012
Available online 27 September 2012
Simple, very efficient, and having some aspects of generality, synthesis of water-soluble, polyanionic
dendrimeric polyesters with different size, polarity, and flexibility is described. These macromolecular
compounds consisting of phosphate or thiophosphate ester units and 1,3,5-benzenetricarboxylic acid
building blocks may find potential applications as pharmaceutical agents. Synthesized the title poly-
anionic dendrimers possess charged carboxyl functional groups on the surface and were obtained, in
high yields, from previously prepared series of new phosphorus-based dendrimeric polyols. The key
monomers applied in this project were 1,3,5-benzenetricarboxylic acid dibenzyl ester and 1,3,5-
benzenetricarboxylic acid bis(4-methoxybenzyl) ester. Both worked as the essential precursors of the
dendrimer polyanionic surface.
Keywords:
Dendrimer
Polyanion
1,3,5-Benzenetricarboxylic acid
Phosphorus
Ó 2012 Elsevier Ltd. All rights reserved.
Condensation
Methoxyacetate
Deprotection
1. Introduction
dendrimers, which are heteroorganic dendrimers possessing
phosphorus atoms inside the key structural elements, mainly at
Dendrimers constitute a class of highly branched, perfectly
symmetrical, three-dimensional, monodisperse polymers with an
architecture possessing three major components: the core, the in-
ternal skeleton, and the surface.1 All three main structural elements
can be adjusted to have an influence on both physical and chemical
properties of the macromolecule. The structure of the core is re-
sponsible for the 3D shape of the dendrimer (i.e., spherical, ellip-
soidal, or cylindrical framework). The interior affects the
dendrimer’s size and capability to form noncovalent complexes.
But the physical and biological properties of dendrimers are dom-
inated by the functional groups located on their molecular surface.
Additionally, monodispersity (i.e., well-defined molecular struc-
ture) of dendrimers seems to be another crucial factor, when the
growing interest in biomedical applications of dendrimers is taken
into account.2
branching points.3 Biological properties of organophosphorus
dendrimers (like, e.g., multiplication of human natural killer cells or
antiviral activity against HIV-1) have been recently comprehen-
sively summarized.4
Normally, antiviral and other drug research, concentrates on
relatively low molecular weight structures. On the contrary, den-
drimers with their structural precision, a large number of func-
tional groups on the surface and also possessing the dimensions
(high generation structures), which are only smaller over an order
of magnitude than the size of most viruses, may serve as antiviral
drugs in their own right.5 The macromolecular drug can be
designed to interfere with the virus-to-host cell binding process
thereby inhibiting infection at the stage of viral entry. Antiviral
dendrimers acting as non-natural imitations of the target cell sur-
face are commonly designed with anionic surface groups. The
polyanionic dendritic drug then competes with the cellular surface
for binding of virus, resulting in a lower cell-virus infection rate.
However, antiviral dendrimers may also be designed having cat-
ionic or even neutral surfaces, depending on their mode of action.
Study with herpes simplex virus (HSV) showed that both poly-
cationic macromolecular compounds (polylysine)6 and polyanionic
dendrimers7,8 could inhibit the attachment of the virus to cell
surfaces. This fact can be explained as an effect of either competi-
tion with the virus for a cell anionic receptor structure or by
On the other hand, phosphorus is a widespread element, which
plays various vital roles in nature. Biophosphates are present in all
known forms of life. Therefore, it is obvious that synthetic
phosphorus-containing compounds may interact with biological
systems. This turned out to be true also for phosphorus-based
* Tel.: þ48 42 680 3217; fax: þ48 42 680 3261; e-mail addresses: gmsalamo@
cbmm.lodz.pl. gmsalamo@bilbo.cbmm.lodz.pl.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.094

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G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10210
competing with the cell for a cationic part of the virus. A serious
negative aspect of polycationic dendrimers, however, is their tox-
icity.9 On the other hand, it has been established that polyanionic
dendrimers usually exhibit acceptable biotolerance.9,10 The usage of
dendrimers as antiviral agents in their own right is well-
documented. One of the most successful polyanionic dendrimers
used for antiviral purposes is VivaGelÒ (Starpharma Ltd.)
a sulfonated polylysine dendrimer. This compound is currently
undergoing phase III clinical trials.11 VivaGelÒ is being developed as
topical microbicide for the prevention of sexually transmitted in-
fections such as HIV, genital herpes, and human papillomavirus
(HPV). It has also been licensed as antiviral condom coating.
Although the chemical synthesis of dendrimers is more than
two decades old, the most important reason hampering the
broader use of dendrimers in biomedicine is difficult and time-
consuming multistep preparation required, especially for the high
generation structures. Consequently, striving for the general syn-
thesis of particularly water-soluble dendrimers possessing various
types of tunable scaffolds and surfaces is very much warranted.
causing imperfections in the dendrimer skeleton. That is why
protective groups applied in this work were selected very carefully.
It will be demonstrated that deprotection reactions were complete
and they did not cause any defects in the expected structures.
Scheme 2. Synthesis of the AB2-type monomer 5.
Phosphorus-based, mostly new dendrimers, used in this project
were synthesized, from readily available chemicals, via amido-
phosphite method and divergent manner as described pre-
viously.12,14 Corresponding intermediates were transformed into
phosphates12b,c,f or thiophosphates12a,e via chemoselective12c tert-
butylperoxytrimethyl silane16 oxidations or elemental sulfur addi-
tions to the resultant phosphite functions. The detailed synthesis of
phosphate and thiophosphate dendrimers 6, 9, 12, 15, 18 used in
this work, is presented in Supplementary data. It is worthy to point
out that thiophosphate dendrimers turned out to be well tolerated
by the tested biological system.17 In the beginning, hydroxy-
terminated, first generation, phosphate dendrimer 6 was allowed
to react with an excess of acid 1, in the presence of the water-sol-
uble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide18 (EDC), and 4-dimethylaminopyridine (DMAP) to furnish the
second generation dendrimer 7 in 91% isolated yield (Scheme 3).
The cleavage of the terminal benzyl esters in 7 using catalytic
hydrogenolysis proceeded smoothly and quantitatively as evi-
denced by the 1H NMR (no trace of protons corresponding to benzyl
groups in 8 was detected, see Fig. 1) to provide pure acid 8, which
was then transformed into dodecaanion using sodium bicarbonate.
Unfortunately, the 1,3,5-benzenetricarboxylic acid dibenzyl ester
(1) was not a universal precursor in this project. In the case of
thiophosphate-based dendrimer skeleton, the removal (various
known methods were tried) of benzyl protecting groups from its
surface was not successful, probably due to the sulfur poisoning
effect on the palladium-based catalysts. Therefore, an alternative
protection of carboxyl groups had to be found. Just for that reason,
but not exclusively (also to avoid degradation of benzyl esters
present within the dendrimer, vide compound 21), 1,3,5-
benzenetricarboxylic acid bis(4-methoxybenzyl) ester (2) was
proposed.
2. Results and discussion
A few years ago, we developed a method for the synthesis of
dendrimeric polyphosphates and their analogs.12 Also recently,
from our laboratory, an effective synthesis of new polyester den-
drimers based on a trimesic acid framework derivative has been
reported.13 In continuation of our efforts, a straightforward and
efficient synthesis of water-soluble, polyanionic dendrimers com-
posed of both phosphate and 1,3,5-benzenetricarboxylate units is
reported herein.
Simple two-step, one-pot synthesis of the two surface units
precursors, commences from commercially available 1,3,5-
benzenetricarbonyl trichloride. Its careful reactions with 2.0 equiv
of benzyl and anisyl alcohols in the presence of triethylamine,
followed by a mild basic hydrolysis produced chemoselectively the
corresponding 1,3,5-benzenetricarboxylic acid dibenzyl ester (1)
[accompanied with corresponding triester (18%) and monoester
(10%), acc. NMR] and 1,3,5-benzenetricarboxylic acid bis(4-
methoxybenzyl) ester (2) [accompanied with corresponding
triester (17%) and monoester (11%), acc. NMR] in 62% and 60%
isolated yields, respectively (Scheme 1).
Scheme 1. Synthesis of the surface monomers 1 and 2.
Another key dendrimer building block was also obtained via
one-pot,
two-step
synthetic
procedure,
from
3,5-
bis(hydroxylmethyl)benzoic acid methyl ester13,14 (3) (Scheme 2).
Its basic hydrolysis provided acid 4.15 Crude compound 4 was then
acylated with methoxyacetyl chloride, in the presence of pyridine,
to give the suitably protected acid 5 in 80% isolated yield. This
compound represents an AB2-type monomer. The A group (car-
boxyl) is active and the B groups (hydroxy) are protected such that
the A group reacts solely with the B (active) groups in the prior
generation of the dendrimer. Deprotection is necessary to activate
the B groups for the consequent reaction. This deprotection may
not be quantitative and may also include undesired reactions,
Scheme 3. Synthesis of dendrimer 8 with 12 carboxyl groups.
Thus, in the next experiment (Scheme 4), the condensation of
larger, second generation, thiophosphate dendrimer 912,14 with an
excess of acid 2, also in the presence of the water-soluble carbo-
diimide (EDC), and 4-dimethylaminopyridine afforded the third
generation, fully protected dendrimer 10 in 77% isolated yield.

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G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10211
was preserved. The desired third generation polyanionic, thio-
phosphate dendrimer 11 was obtained in 91% isolated yield.
To demonstrate the usefulness of the presented synthetic ap-
proach, another set of polyester dendrimers, starting from den-
drimeric substrates 1212,14 and 1512,14 was synthesized. On the way,
two fully protected intermediates 13 and 16 were isolated. As
a result, two additional, novel polyanionic compounds 14 and 17
(Fig. 3) were prepared. The first one (third generation) 14 (overall
yield 65%, from 12) is based on phosphate branching points and
possesses nine 5-carbon chains and twelve 6-carbon chains skel-
eton. Despite this rather flexible and carbon-rich interior, the effect
of polar phosphate groups seems to be predominant and the
macromolecule is soluble well in water, even at the stage of free
acid. The other one (fourth generation, the largest dendrimer de-
scribed here), 17 (overall yield 58%, from 15) is composed of much
more lipophilic, thiophosphate-based interior and for water-
solubility requires total ionization (48 COOꢁNaþ functions).
This methodology also enables divergent synthesis of ‘mixed’ or
layered dendrimers, which have branching points both at phos-
phorus and at carbon. Moreover, the introduction of an additional
generation consisted of carboxylate polyester residues to the den-
drimer, apparently facilitates its biodegradability,20 which is very
important, when biomedical applications of dendrimers are con-
sidered. The synthesis of the layered polyanionic dendrimer com-
mences from the first generation, hydroxy-terminated,
thiophosphate dendrimer 18. Therefore, 18 was condensed with an
excess of acid 5, in the presence EDC and DMAP to provide the
second generation dendrimer 19 in 93% isolated yield (Scheme 5).
Terminal hydroxyl groups in 19 are protected as methoxyacetate
esters. It is known that methoxyacetate is cleaved 20 times faster
than an acetate.21 It can be safely cleaved in the presence of
a benzoate.21 In this work, swift and clean removal of all methox-
yacetate groups, without affecting the benzoate functions present
in dendrimer 19, was achieved using a catalytic amount of mag-
nesium methoxide22 in methanol. Hydroxy-terminated dendrimer
20 was formed quantitatively and it was used for further reactions
without purification. Then, polyol 20 reacted readily with acid 2
using EDC and DMAP as condensation reagents to afford the third
generation dendrimer 21 in 88% (from 19) isolated yield. Next,
cleavage of terminal p-methoxybenzyl esters in 21 provided poly-
acid 22, which was finally converted into its dodecasodium salt. It is
also worthy to emphasize that benzyl esters present in 21 were
absolutely stable under applied conditions for the complete
cleavage of p-methoxybenzyl esters. Unlike more classical den-
drimers, compound 22 is not built only of repeating units. In con-
sequence, its 1H (Fig. 4B) and 13C NMR spectra are remarkably
conclusive, when compared with spectra of typical dendrimers.
This is due to the diversity of the signals corresponding to nuclei
absorbing in distinct areas.
Fig. 1. Partial 1H NMR spectra of dendrimers 7 (A) and 8 (B).
Scheme 4. Synthesis of dendrimer 11 with 24 carboxyl groups.
Practically quantitative deprotection of all 24 peripheral carboxyl
groups was achieved using a slight modification of the classical
procedure disclosed by Stewart.19 Hence, polyester 10 was stirred in
trifluoroacetic acid/anisole mixture at 45 ꢀC. The reaction was
monitored by 1H and 31P NMR; after 50 h, only a trace of protons
corresponding to p-methoxybenzyl residues was detected (for ex-
ample, see Fig. 4B). Phosphorus-containing dendrimers, particu-
larly those having phosphorus at the branching points display an
important feature. They can be easily characterized by 31P NMR,
which is an additional, invaluable, analytical tool. As confirmed by
31P NMR (Fig. 2), the cleavage of p-methoxybenzyl esters in 10
proceeded cleanly. The integrity of the whole dendritic structure
The polyanionic dendrimers were stable white solids, whose
solubility in water was strongly dependent on the type of the in-
terior and number of charged groups on the surface. As mentioned
before, compounds with P]O groups at branching points were
soluble in water even as uncharged polyacids. In the case of third
generation dendrimers with P]S groups inside the structure (11
and 22), for a decent water-solubility (50 mg/1 mL), it was neces-
sary to convert at least half of the COOH groups into corresponding
sodium salts.
Obviously, all the synthesized title polyanionic dendrimers will
be tested for their antiviral activity.
As expected, the layered dendrimer 22 degraded slowly at room
temperature under mild basic conditions (1 M aqueous ammonia;
see Supplementary data), but only at the ‘carboxylate part’ of the
macromolecule. Thiophosphate ester groups remained intact. The
detected products (by the 1H and 31P NMR, TLC) of such hydrolysis
(after acidification) were hexol 18, acid 4, and trimesic acid.
Fig. 2. 31P{1H} NMR (202.5 MHz) spectrum of the crude dendrimer 11 including ex-
pansion of signals absorbing at 69.3 ppm. Superscripts refer to the branching number.

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G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
Fig. 3. Structures of dendrimeric substrates 12, 15; fully protected intermediate dendrimers 13, 16 and polyanionic dendrimers 14, 17.
Fig. 4. (A) MALDI TOF MS spectrum of 10; (B) 1H NMR spectrum of polyanionic dendrimer 22.
The structures and high purity of all the dendrimeric products
were confirmed by NMR and MALDI TOF mass spectrometry. For
instance, Fig. 4A shows the MALDI TOF mass spectrum of third
generation dendrimer 10. Interestingly, the spectrum displays very
similar fork-like pattern observed for the dendrimer terminated
with allyl ethers.12 The signal at 7657.3 is attributed to the molec-
ular ion (MþNa). All four peaks differ in mass by exactly 121 amu,
most probably due to minor fragmentation via loss of one, two or
three stabilized p-methoxybenzyl cations.
approach seems to be somehow a general methodology, which
enables transformation of practically any polyfunctional compound
terminated with nucleophilic functions (e.g., hydroxy, thiol or
amino), into its polyanionic derivative. Moreover, it offers the
possibility to make discrete modifications layer by layer (i.e., P]O,
P]S, and/or carbon branching) within the same dendrimer skele-
ton, a key for a structureeactivity relationship study. Finally, newly
introduced residues have the potential to be hydrolyzable, which is
critical for in vivo applications.
3. Conclusion
4. Experimental section
4.1. General
In conclusion, using simple and readily available monomers,
a highly efficient method for the synthesis of polyanionic den-
drimers as potential antiviral drugs in their own right was pre-
sented. The mild conditions of both the coupling and virtually
quantitative deprotection reactions provided highly pure and
water-soluble macromolecular material in good overall yields. This
The melting points reported are uncorrected and were de-
termined using PHMK Boetius (VEB Analytik Dresden) apparatus.
The NMR spectra (1H, 13C, 31P) were recorded on Bruker Avance AV-
200, AV-500 or AV-600 spectrometer (200, 500 or 600 MHz,

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G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10213
Scheme 5. Synthesis of polyanionic dendrimer 22 with phosphorus and carbon-based branching points.
respectively). Superscripts in the NMR spectra description refer to
the dendrimer generation number. 13C NMR spectra were assisted
with DEPT 90 and DEPT 135 experiments. High resolution mass
spectra were recorded on Finnigan MAT 95 spectrometer and ma-
trix assisted laser desorption ionization time of flight mass spectra
(MALDI TOF MS) were run on Voyager Elite (PerSeptive Biosystems
Inc.) mass spectrometer using 2,5-dihydroxybenzoic acid as the
matrix. FTIR spectra were measured on ATI Mattson Infinity 60 AR
spectrophotometer. Microanalyses were performed on EuroVector
3018 and/or on Vario (Elementar Analysensysteme GmbH) ana-
lyzers. Thin layer chromatography was carried out on Merck silica
gel 60F254 aluminum sheets using UV light (254 nm) or phospho-
molybdic acid (5% solution in ethanol) for the spots visualization.
Preparative flash chromatography was performed on silica gel
columns (Merck, Kieselgel 230e400 mesh). The terms: ‘short col-
umn’ or ‘short pad (plug) of silica gel’ used throughout this section
refer to the column of silica gel with a length 25 mm and diameter
30 mm. Solvents were obtained from commercial sources and
distilled or dried according to standard methods.
MeOH mixture (1:1, 100 mL) was added to the residue and its pH
was adjusted to w10 (with 1 M NaOH). The resulting mixture was
extracted with hexane (2ꢂ20 mL) and the hexane solutions were
discarded. After that, the organic volatiles were removed in vacuo
once again and water (50 mL) was added to the residue and its pH
was set to w8.5 (with 0.1 M HCl). The mixture was extracted with
EtOAc (4ꢂ30 mL). Combined EtOAc solution was concentrated. The
residual material was purified by flash chromatography using
CH2Cl2/acetone (30:1) as an eluent. Yield of 1 (white solid) 2.4 g
(62%). Rf 0.32 (CH2Cl2/acetone/AcOH 20:1:0.1). Crystallization from
CH2Cl2/hexane mixture afforded the crystalline compound; mp
151e152 ꢀC; nmax 3035, 1725, 1692, 1264, 1247, 957, 741 cmꢁ1
(200 MHz, CDCl3) 5.37 (s, 4H), 7.35e7.45 (m, 10H), 8.89 (s, 3H) ppm;
dC (50 MHz, CDCl3) 67.41 [2C, (PhCH2O)], 128.4 [4C, (PhC )], 128.5
[2C, (PhC )], 128.6 [4C, (PhC )], 130.6 (ipso Ar), 131.3 [2C, (ipso Ar)],
; dH
a
g
b
135.2 [2C, (Ar)], 135.3 [2C, (ipso Ph)], 135.5 (Ar), 164.6 [2C, (C]O)],
170.2 (C]O) ppm; m/z (FAB) 183 (90), 299 (100), 389 [MꢁH (75%)];
HRMS (FAB): MꢁHþ, found 389.1032. C23H17O6 requires 389.1025.
4.2.2. Bis-4-methoxybenzyl 1,3,5-benzenetricarboxylate (2). This
compound was prepared from 1,3,5-benzenetricarbonyl trichloride
(2.65 g, 10 mmol), 4-methoxybenzyl alcohol (2.5 mL, 20 mmol,
2 equiv), and triethylamine (4.2 mL, 30 mmol, 3 equiv) in dry
CH2Cl2/THF (100 mL, 8:1) mixture following the procedure de-
scribed for 1. The reaction product was then purified by flash
chromatography using CH2Cl2/acetone (30:1) as an eluent. Yield of
2 (white solid) 2.7 g (60%). Rf 0.36 (CH2Cl2/AcMe/AcOH 20:1:0.1).
Crystallization from CH2Cl2/hexane mixture gave the crystalline
material; mp 176e178 ꢀC. Found: C, 66.85; H, 5.01, O, 28.43.
C25H22O8 requires C, 66.66; H, 4.92, O, 28.42%; nmax 2838, 1729,
4.2. Synthesis of monomers
4.2.1. Dibenzyl 1,3,5-benzenetricarboxylate (1). To a solution of
1,3,5-benzenetricarbonyl trichloride (2.65 g, 10 mmol) in dry
dichloromethane/THF (90 mL, 8:1) mixture at ꢁ20 ꢀC were slowly
[for about 1 h (syringe pump recommended)] added benzyl alcohol
(2.1 mL, 20 mmol, 2 equiv) and triethylamine (4.2 mL, 30 mmol,
3 equiv) in dry dichloromethane/THF (9 mL, 8:1) solution, and the
resulting mixture was stirred for an additional 1 h, at ꢁ20 ꢀC, then
for the next 1 h at rt. Afterward, 2 M aqueous solution (15 mL) of
sodium bicarbonate was added and the mixture was stirred for
30 min at rt. The organic solvents were removed in vacuo and H2O/
1695, 1613, 1526, 1280, 1250, 1170, 1035, 814, 738 cmꢁ1
;
dH
3
(200 MHz, CDCl3) 3.83 (s, 6H), 5.35 (s, 4H), 7.16 (AB, JAB¼8.6 Hz,

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G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10214
8H), 8.91 (s, 3H) ppm; dC (50 MHz, CDCl3) 54.95 [2C, (CH3O)], 67.00
[2C, (p-MeOC6H4CH2O)], 113.7 [4C, (p-MeOC6H4C )], 127.3 [2C, ipso
(p-MeOC6H4)], 130.0 [4C, (p-MeOC6H4C )], 130.9 [2C, (ipso Ar)],
131.5 (ipso Ar), 134.3 (Ar), 134.6 [2C, (Ar)], 159.5 [2C, (ipso p-
(P0OCH2CH2CH2OP1)], 64.72 [d, 2J(C,P)¼5.7 Hz, 3C, (P0OCH2CH2
-
b
CH2OP1)], 65.88 [6C, (O]COCH2CH2CH2CH2O)], 66.98 [d, 2J(C,P)¼
a
5.7 Hz, 6C, (OCH2CH2CH2CH2OP)], 67.21 [12C, (PhCH2)], 128.2 [24C,
(PhCa)], 128.3 [12C, (PhCg)], 128.4 [24C, (PhCb)], 131.1 [18C, (ipso
MeOC6H4C
g
)], 164.9 [2C, (C]O)], 167.3 (C]O) ppm; m/z (FAB) 183
Ar)], 134.5 [18C, (Ar)], 135.3 [12C, (ipso Ph)], 164.5 [12C, (C]O)],
164.6 [6C, (C]O)] ppm; dP {1H} (81 MHz, CDCl3) ꢁ0.66 (P0), ꢁ0.62
(3P1) ppm; MALDI TOF MS calcd for C171H168O52P4, M¼3179.0.
Found m/z¼3203.3 (100) (MþNa), 3112.5 (50) [Mꢁ(benzylþ)þNa],
3021.4 (15%) [Mꢁ2ꢂ(benzylþ)þNa].
(100), 329 (80), 449 [MꢁH (70%)]; HRMS (FAB): MꢁHþ, found
449.1244. C25H21O8 requires 449.1236.
4.2.3. 3,5-Bis[(2-methoxyacetoxy)methyl]benzoic acid (5). Methyl
3,5-bis(hydroxymethyl)benzoate (3) (4.0 g, 20 mmol) was dissolved
in MeOH (40 mL). Aqueous NaOH (40 mL of 1.0 M, 40 mmol,
2.0 equiv) was added. The dispersion was stirred vigorously and
slowly dissolved for 1 h. After 18 h, organic solvent (MeOH) was
removed under reduced pressure. The remaining aqueous solution
was acidified (pH 2.0) with 6 M HCl and saturated with NaCl. The
resulting mixture was extracted with ethyl acetate (6ꢂ15 mL). The
combined organic phases were dried (MgSO4), the solvent re-
moved, and the remaining colorless oil was kept under high vac-
uum (0.5 mmHg) at 40 ꢀC for 2 h to afford 3,5-bis(hydroxymethyl)
benzoic acid (4)14 as a white powder (3.8 g, 98%). dH (200 MHz,
CDCl3) 4.42 (s, 4H), 7.32 (s, 1H), 7.67 (s, 2H) ppm. Next, compound 4
(1.8 g, 10 mmol) was dissolved in dry dichloromethane/pyridine
(30 mL, 2:1). Methoxyacetyl chloride (2.3 mL, 25 mmol, 2.5 equiv)
was slowly added at ꢁ5 ꢀC, and the resulting mixture was stirred at
ꢁ5 ꢀC for 1 h, then at rt overnight. Solvents were removed under
reduced pressure, and the residue was dissolved in water (50 mL)
and acidified with 6 M HCl to pH 3 to give a milky suspension. The
suspension was extracted with ethyl acetate (4ꢂ25 mL), and the
combined organic washes were concentrated. The residue was
purified by flash chromatography using CH2Cl2/acetone (15:1) as an
eluent. Yield of 5 (white powder) 2.7 g (82%). Rf 0.35 (CH2Cl2/ace-
tone/AcOH 10:1:0.1). Crystallization (CH2Cl2/hexane) provided
crystalline material; mp 88e89 ꢀC; nmax 2994, 2958, 2937, 2897,
4.3.2. Polyanionic dendrimer 8. Dendrimer 7 (318 mg, 0.1 mmol)
was dissolved in methanol (4 mL) and palladium on activated
charcoal (10%, 100 mg) was then added. The reaction mixture was
stirred under a hydrogen atmosphere for 16 h. The catalyst was
filtered off, the filtrate concentrated to dryness in vacuo to furnish
analytically pure acid 8 (205 mg, 98%) as a colorless oil. Next, acid 8
(105 mg, 0.05 mmol) was dissolved in water (2 mL) and solid so-
dium bicarbonate (51 mg, 0.6 mmol, 12 equiv) was added. After
10 min, the resulting solution was frozen and lyophilized under
high vacuum (0.1 mmHg) to give dodecasodium salt of 8 (118 mg)
as nonhygroscopic white powder. dH [acid (200 MHz, CD3OD)] 1.89
[m, 24H, (OCH2CH2CH2CH2O)], 2.07 [quintet, 3J(H,H)¼5.8 Hz, 6H,
(OCH2CH2CH2O)], 4.16 [dt, 3J(H,H)¼5.6 Hz, 3J(P,H)¼6.4 Hz, 24H,
(POCH2CH2)], 4.37 [t, 3J(H,H)¼5.6 Hz, 12H, (O]COCH2CH2CH2
-
CH2O)], 8.70 [d, 4J(H,H)¼1.5 Hz, 12H, Ar], 8.75 [d, 4J(H,H)¼1.5 Hz,
6H, Ar] ppm; dC [sodium salt (125 MHz, D2O)] 24.32 [6C, (O]
COCH2CH2CH2CH2O)], 26.32 [d, 3J(C,P)¼6.3 Hz, 6C, (OCH2CH2CH2
-
CH2OP)], 30.07 [t, 3J(C,P)¼5.0 Hz, 3C, (POCH2CH2CH2OP)], 64.50 [dd,
2J(C,P)¼5.7 Hz, 3J(C,P)¼18.8 Hz, 6C, (P0OCH2CH2CH2OP1)], 65.35
[6C, (O]COCH2CH2CH2CH2O)], 68.44 [d, 2J(C,P)¼6.2 Hz, 6C,
(OCH2CH2CH2CH2OP)], 129.5 [6C, (Ar)], 132.5 [12C, (Ar)], 134.0 [12C,
(ipso Ar)], 134.6 [6C, (ipso Ar)], 166.6 [12C, (C]O)], 170.8 [6C, (C]
O)] ppm; dP {1H} [acid (81 MHz, CD3OD)] ꢁ1.11 (4P) ppm; MALDI
TOF MS calcd for C87H96O52P4 (acid), M¼2096.9. Found m/z¼2121.3
(MþNa).
1765, 1731, 1695, 1284, 1211, 1132, 928 cmꢁ1
; dH (200 MHz, CDCl3)
3.45 (s, 6H), 5.24 (s, 4H), 7.60 (s, 1H), 8.05 (s, 2H), 10.7 (br s,
1H) ppm; dC (50 MHz, CDCl3) 59.28 [2C, (CH3O)], 65.38 [2C,
(ArCH2O)], 69.77 [2C, (O]CCH2O)], 129.1 [2C, (Ar)],130.5 (Ar),132.5
(ipso Ar), 136.6 [2C, (ipso Ar)], 169.8 [2C, (OC]OCH2)], 170.0 (C]
O) ppm; m/z (FAB) 149 (95), 237 (95), 327 [MþH (100%)]; HRMS
(FAB): MþHþ, found 327.1081. C15H19O8 requires 327.1079.
4.3.3. Third generation polyester dendrimer 10. This compound was
prepared from hydroxy-terminated, second generation thio-
phosphate dendrimer 9 (FW 2450.6, 74 mg, 0.03 mmol), 1,3,5-
benzenetricarboxylic acid bis(4-methoxybenzyl) ester (2)
(216 mg, 0.48 mmol, 16 equiv), EDC (92 mg, 0.48 mmol, 16 equiv),
and DMAP (6 mg, 0.05 mmol, 1.6 equiv) in dry dichloromethane
(5 mL) following the procedure described for 7. The product was
then purified through a short pad of silica gel. Eluting with CH2Cl2/
acetone 80:1 mixture and gradually increasing the polarity to
CH2Cl2/acetone 30:1, furnished the title ester 10 (163 mg) as an oil.
Yield 71%. Rf 0.26 (CH2Cl2/acetone 15:1); dH (200 MHz, CDCl3) 1.78
[m, 48H, (POCH2CH2CH2CH2OP)1 and (POCH2CH2CH2CH2OC)2], 1.85
[m, 24H, (POCH2CH2CH2CH2OC)2], 2.09 [quintet, 3J(H,H)¼6.1 Hz,
6H, (OCH2CH2CH2O)], 3.78 [s, 72H, (OCH3)], 4.09 [m, 60H,
(POCH2CH2e)0,1,2], 4.37 [t, 3J(H,H)¼5.4 Hz, 24H, (O]
COCH2CH2CH2CH2O)], 5.30 [s, 48H, (OCH2Ar)], 7.09 [AB, 3J(HA,HB)¼
8.6 Hz, 96H, (Ar)], 8.79 [s, 24H, (Ar)], 8.80 [s, 12H, (Ar)] ppm; dC
(50 MHz, CDCl3) 24.94 [12C, (O]COCH2CH2CH2CH2OP)2], 26.22 [d,
3J(C,P)¼7.6 Hz, 12C, (POCH2CH2CH2CH2OP)1], 26.57 [d, 3J(C,P)¼
4.3. Synthesis of dendrimers
4.3.1. Second generation polyester dendrimer 7. Hydroxy-termi-
nated, first generation phosphate dendrimer 6 (FW 944.8, 190 mg,
0.2 mmol) was dispersed in dry dichloromethane (5 mL). Dibenzyl
trimesoate (1) (600 mg, 1.54 mmol, 7.7 equiv) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) (296 mg, 1.54 mmol,
7.7 equiv) were added under argon atmosphere. The suspension
was stirred vigorously and quickly dissolved. After 5 min, 4-
dimethylaminopyridine (DMAP) (20 mg, 0.16 mmol, 0.8 equiv)
was added and the mixture was stirred at room temperature
overnight. The reaction mixture was then diluted with ethyl acetate
(50 mL), washed with 0.1 M citric acid (10 mL), dried (MgSO4), and
evaporated under reduced pressure. The residue was placed on
a short plug of silica gel. Elution with CH2Cl2/MeOH 50:1 mixture
and gradually increasing the polarity to CH2Cl2/MeOH 10:1, gave
the title ester 7 (578 mg). Yield 91%. Rf 0.29 (CH2Cl2/MeOH 10:1); dH
(200 MHz, CDCl3) 1.87 [m, 24H, (OCH2CH2CH2CH2O)], 2.09 [quintet,
3J(H,H)¼5.6 Hz, 6H, (OCH2CH2CH2O)], 4.16 [dt, 3J(H,H)¼5.6 Hz,
3J(P,H)¼6.4 Hz, 24H, (POCH2CH2)], 4.39 [t, 3J(H,H)¼5.6 Hz,12H, (O]
COCH2CH2CH2CH2O)], 5.38 [s, 24H, (PhCH2)], 7.32e7.51 (m, 60H,
Ph), 8.83 [d, 4J(H,H)¼1.5 Hz, 12H, Ar], 8.87 [d, 4J(H,H)¼1.5 Hz, 6H,
Ar] ppm; dC (50 MHz, CDCl3) 24.83 [6C, (O]COCH2CH2CH2CH2O)],
26.73 [d, 3J(C,P)¼6.9 Hz, 6C, (OCH2CH2CH2CH2OP)], 31.04 [t,
3J(C,P)¼6.4 Hz, 3C, (POCH2CH2CH2OP)], 63.75 [d, 2J(C,P)¼5.7 Hz, 3C,
7.7 Hz, 12C, (COCH2CH2CH2CH2OP)2], 30.74 [m, 3C, (POCH2CH2
-
CH2OP)], 55.18 [24C, (OCH3)], 64.43 [d, 2J(C,P)¼5.7 Hz, 6C,
(P0OCH2CH2CH2OP1)], 64.91 [12C, (O]COCH2CH2CH2CH2OP)2],
67.10 {s, 24C [OCH2Ar(PMB)]}, 67.48 [d, 2J(C,P)¼5.7 Hz, 24C,
(POCH2CH2CH2CH2OP)1 and (COCH2CH2CH2CH2OP)2], 113.9 {48C,
[Cb Ar(PMB)]}, 127.5 {24C, [ipso Ar(PMB)]}, 130.2 {48C [Ca
Ar(PMB)]}, 131.2 [36C, (ipso Ar)], 134.4 [36C, (Ar)], 159.7 {24C,
[ipso(g
) Ar(PMB)]}, 164.7 [36C, (C]O)] ppm; dP {1H} (81 MHz,
CDCl3) 68.50 (6P2), 68.58 (4P1þP0) ppm; MALDI TOF MS calcd for
C381H414O126P10S10, M¼7634.0 [exact (monoisotopic) mass]. Found
m/z¼7653.3 (100) (MþNa), 7536.0 (77) [Mꢁ(p-methoxybenzylþ)þ

ꢀ
G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10215
Na], 7415.0 (38) [Mꢁ2ꢂ(p-methoxybenzylþ)þNa], 7294.2 (18%)
[Mꢁ3ꢂ(p-methoxybenzylþ)þNa].
109 mg, 0.015 mmol) using gaseous hydrogen and Pd/C (10%,100 mg) in
methanol (4 mL) following the procedure described for 8. The catalyst
was filtered off, the filtrate concentrated to dryness under reduced
pressure to give analytically pure acid 14 (72 mg, 94%) as a colorless oil.
Polyacid 14 was then converted into its sodium salt (24ꢂNaþ) by the
reaction with NaHCO3 (29 mg, 0.34 mmol, 24 equiv) in water (2 mL)
4.3.4. Polyanionic dendrimer 11. Dendrimer 10 (230 mg, 0.03 mmol)
was dissolved in anisole (1 mL) and trifluoroacetic acid was added
(4 mL). The reaction mixture was stirred at 45 ꢀC under argon at-
mosphere for 50 h. All the volatiles were removed in vacuo, and the
residue was washed with hexane (3ꢂ2 mL) to provide analytically
pure acid 11 (130 mg, 91%) as a colorless oil. Then, acid 11 (95 mg,
0.02 mmol) was dispersed in water (2 mL) and solid sodium bi-
carbonate (40 mg, 0.48 mmol, 24 equiv) was added. After 10 min, the
resulting solution was frozen and lyophilized under high vacuum
(0.1 mmHg) to give sodium salt (24ꢂNaþ) of 11 (105 mg) as non-
hygroscopic white powder. dH [acid (500 MHz, CDCl3/CD3OD 2:1)]
1.80 [m, 48H, (POCH2CH2CH2CH2OP)1 and (POCH2CH2CH2CH2OC)2],
1.85 [quintet, 3J(H,H)¼6.9 Hz, 24H, (POCH2CH2CH2CH2OC)2],
1.98 [quintet, 3J(H,H)¼6.0 Hz, 6H, (OCH2CH2CH2O)], 4.03e4.09
[m, 60H, (POCH2CH2e)0,1,2], 4.34 [t, 3J(H,H)¼6.1 Hz, 24H, (O]
COCH2CH2CH2CH2O)], 8.73 [d, 4J(H,H)¼1.0 Hz, 24H, (Ar)], 8.78 [d,
4J(H,H)¼1.0 Hz, 12H, (Ar)] ppm; dC [acid (125.7 MHz, CDCl3/CD3OD
2:1)] 24.94 [12C, (O]COCH2CH2CH2CH2OP)2], 26.81 [d, 3J(C,P)¼
7.4 Hz, 12C, (POCH2CH2CH2CH2OP)1], 26.57 [d, 3J(C,P)¼7.6 Hz, 12C,
(COCH2CH2CH2CH2OP)2], 31.27 [brs, 3C, (POCH2CH2CH2OP)],
65.08 [d, 2J(C,P)¼5.7 Hz, 6C, (P0OCH2CH2CH2OP1)], 65.65 [12C, (O]
COCH2CH2CH2CH2OP)2], 68.22 [d, 2J(C,P)¼5.7 Hz, 24C,
(POCH2CH2CH2CH2OP)1 and (COCH2CH2CH2CH2OP)2], 131.6 [12C,
(Ar)], 132.2 [24C, (Ar)], 135.2 [24C, (ipso Ar)], 135.7 [12C, (ipso Ar)],
165.8 [12C, (C]O)], 167.6 [24C, (C]O)] ppm; dP {1H} [acid
(202.5 MHz, CDCl3/CD3OD 2:1)] 69.32 (6P2), 69.36 (3P1), 69.43
(P0) ppm; MALDI TOF MS calcd for C189H222O102P10S10 (acid),
M¼4753.7. Found m/z¼4775.69 (MþNa).
following the procedure described for sodium salt of
8 to
afford 79 mg of nonhygroscopic white powder. dH [acid (500 MHz,
CDCl3/CD3OD 10:1)] 1.39 [m, 66H, (POCH2CH2CH2CH2CH2OP)0,1
and (POCH2CH2CH2CH2CH2CH2OC)2], 1.62 [m, 48H, (POCH2CH2
CH2CH2CH2CH2OC)2], 1.72 [m, 36H, (POCH2CH2CH2CH2CH2OP)0,1], 4.01
[t, 3J(H,H)¼3J(P,H)¼7.9 Hz, 60H, (POCH2CH2e)], 4.27 [t, 3J(H,H)¼6.4 Hz,
24H, (POCH2CH2CH2CH2CH2CH2OC)2], 8.73 [s, 24H, Ar], 8.78
[s, 12H, Ar] ppm; dC [acid (125.7 MHz, CDCl3/CD3OD 10:1)] 21.12 [t,
4J(C,P)¼6.5 Hz, 9C (POCH2CH2CH2CH2CH2OP)0,1], 24.89 [12C,
(POCH2CH2CH2CH2CH2CH2OC)2], 25.31 [12C, (POCH2CH2CH2CH2CH2
-
CH2OC)2], 28.21 [12C, (POCH2CH2CH2CH2CH2CH2OC)2], 29.47 [d,
3J(H,H)¼6.3 Hz, 18C, (POCH2CH2CH2CH2CH2OP)0,1], 29.88 [d, J(H,H)¼
3
5.3 Hz, 12C, (POCH2CH2CH2CH2CH2CH2OC)2], 65.35 [12C,
(POCH2CH2CH2CH2CH2CH2OC)2], 67.41 [d, 2J(C,P)¼5.1 Hz, 18C
(POCH2CH2CH2CH2CH2OP)0,1], 67.65 [d, 2J(H,H)¼5.3 Hz, 12C,
(POCH2CH2CH2CH2CH2CH2OC)2],130.9 [12C, (Ar)],131.5 [24C, (ipso Ar)],
134.4 [24C, (Ar)], 134.9 [12C, (ipso Ar)], 165.1 [12C, (C]O)], 166.6 [24C,
(C]O)] ppm; dP {1H} [acid (81 MHz, CDCl3-CD3OD 10:1)] 2.90
(10P) ppm; MALDI TOF MS calcd for C225H294O112P10 (acid), M¼5098
(80), 5099 (100), 5100 (83%). Found m/z¼5120.3 (MþNa) (80), 5123.1
(MþNa) (100), 5137.9 (MþK) (75%).
4.3.7. Fourth generation polyester dendrimer 16. This compound
was prepared from hydroxy-terminated, third generation thio-
phosphate dendrimer 15 (FW 5081.2, 102 mg, 0.02 mmol), 1,3,5-
benzenetricarboxylic acid bis(4-methoxybenzyl) ester (2) (315 mg,
0.7 mmol, 35 equiv), EDC (134 mg, 0.7 mmol, 35 equiv), and DMAP
(9 mg, 0.07 mmol, 3.5 equiv) in dry dichloromethane (5 mL) fol-
lowing the procedure described for 7. Dendrimer 16 (colorless oil)
was purified on a short pad of silica gel using the gradient of the
eluent from CH2Cl2/acetone 40:1 to CH2Cl2/acetone 5:1. Yield
205 mg, 65%. Found: C, 57.51; H, 5.20; S, 4.44. C759H822O282P22S22
requires C, 57.54; H, 5.23, S, 4.45%; Rf 0.22 (CH2Cl2/acetone 5:1); nmax
4.3.5. Third generation polyester dendrimer 13. This compound
was prepared from hydroxy-terminated, second generation
phosphate dendrimer 12 (FW 2794.9, 84 mg, 0.03 mmol), 1,3,5-
benzenetricarboxylic acid dibenzyl ester (1) (187 mg, 0.48 mmol,
16 equiv), EDC (92 mg, 0.48 mmol, 16 equiv), and DMAP (6 mg,
0.05 mmol, 1.6 equiv) in dry dichloromethane (5 mL) following the
procedure described for 7. The product was then purified through
a short pad of silica gel. Elution with CH2Cl2/MeOH 30:1 mixture and
gradually increasing the polarity to CH2Cl2/MeOH 20:1, afforded the
title ester 13 (150 mg) as an oil. Yield 69%. Rf 0.20 (CH2Cl2/MeOH 20:1);
(liquid film) 2958, 1726, 1516, 1238, 1031, 988 cmꢁ1
; dH (500 MHz,
CDCl3) 1.76e1.81 [m, 96H, (POCH2CH2CH2CH2OC]O)],1.87 [m, 36H,
(OCH2CH2CH2O)1,2], 2.04 [quintet, 3J(H,H)¼5.7 Hz, 6H, (OCH2CH2
-
nmax (liquid film) 2954, 1728, 1516, 1239, 997 cmꢁ1
;
dH
CH2O)0], 3.76 [s, 144H, (OCH3)], 4.08 [m, 132H, (POCH2CH2CH2
-
(500 MHz, CDCl3) 1.45 [m, 66H, (POCH2CH2CH2CH2CH2OP)0,1
and (POCH2CH2CH2CH2CH2CH2OC)2], 1.68e1.80 [br m, 84H,
(POCH2CH2CH2CH2CH2OP)0,1 and (POCH2CH2CH2CH2CH2CH2OC)2],
4.01 [dd, 3J(H,H)¼6.4 Hz, 3J(P,H)¼12.9 Hz, 60H, (POCH2CH2e)], 4.34 [t,
3J(H,H)¼6.6 Hz, 24H, (POCH2CH2CH2CH2CH2CH2OC)2], 5.39 [s, 48H,
CH2OC]O) and (OCH2CH2CH2O)], 4.35 [t, 3J(H,H)¼6.9 Hz, 48H,
(POCH2CH2CH2CH2OC]O)], 5.28 [s, 96H, (OCH2Ar)], 7.11 [AB,
3J(HA,HB)¼10.0 Hz, 192H, (Ar)], 8.76 [s, 48H, (Ar)], 8.78 [s, 24 H,
(Ar)] ppm; dC (125.7 MHz, CDCl3) 25.77 [24C, (O]COCH2CH2CH2
-
CH2OP)], 26.99 [d, 3J(C,P)¼7.5 Hz, 24C, (COCH2CH2CH2CH2OP)],
27.35 [d, 3J(C,P)¼7.5 Hz, 21C, (POCH2CH2CH2OP)], 55.99 [48C,
(OCH3)], 65.70 [24C, (O]COCH2CH2CH2CH2OP)], 67.91 {48C
[OCH2Ar(PMB)]}, 68.31 [m, 66C, (POCH2CH2CH2OP) and (O]
4
PhCH2e], 7.35e7.42 (m, 120H, Ph), 8.84 [d, J(H,H)¼1.0 Hz, 24H, Ar],
8.87 [d, 4J(H,H)¼1.0 Hz, 12H, Ar] ppm; dC (125.7 MHz, CDCl3) 21.35 [t,
4J(C,P)¼6.3 Hz, 9C (POCH2CH2CH2CH2CH2OP)0,1], 25.06 [12C,
(POCH2CH2CH2CH2CH2CH2OC)2], 25.41 [12C, (POCH2CH2CH2CH2CH2
COCH2CH2CH2CH2OP)], 114.7 {96C, [Cb Ar(PMB)]}, 128.2 {48C, [ipso
-
CH2OC)2], 28.42 [12C, (POCH2CH2CH2CH2CH2CH2OC)2], 29.73 [d,
3J(C,P)¼5.0 Hz, 18C, (POCH2CH2CH2CH2CH2OP)0,1], 30.06 [d, 3J(C,P)¼
6.3 Hz, 12C, (POCH2CH2CH2CH2CH2CH2OC)2], 65.47 [12C,
(POCH2CH2CH2CH2CH2CH2OC)2], 67.14 [d, 2J(C,P)¼6.3 Hz, 12C,
(POCH2CH2CH2CH2CH2CH2OC)2], 67.23 [24C, (PhCH2e)], 67.42 [d,
Ar(PMB)]}, 131.0 {96C [C Ar(PMB)]}, 131.9 [24C, (ipso Ar)], 132.0
a
[48C, (ipso Ar)], 135.3 [72C, (Ar)], 160.5 {48C, [ipso(g) Ar(PMB)]},
165.5 [72C, (C]O)] ppm; dP {1H} (202.5 MHz, CDCl3) 69.47 (18P2,3),
69.52 (3P1), 69.59 (P0) ppm; MALDI TOF MS calcd for
C759H822O282P22S22
,
M¼15,831.8 [exact (monoisotopic) mass].
2J(C,P)¼6.3 Hz, 18C (POCH2CH2CH2CH2CH2OP)0,1], 128.2 [48C, (PhC
a
)],
Found m/z, fragmentation: 15,850.3, 15,651.0, 15,507.9, 15,328.4,
14,266.6, 14,152.6, 13,100.7, 12,916.9, 11,600.5, 11,430.9, 11,226.0,
10,201.9, 9330.3.
128.3 [24C, (PhCg)], 128.5 [48C, (PhCb)], 131.1 [24C, (Ar)], 131.3 [12C,
(Ar)], 134.5 [36C, (ipso Ar)], 135.3 [24C, (ipso Ph)], 164.6 [24C, (C]O)],
164.7 [12C, (C]O)] ppm; dP {1H} (202 MHz, CDCl3) 0.56 (4P0,1), 0.61
(6P2) ppm; MALDI TOF MS calcd for C393H438O112P10, M¼7262.6 (100),
7263.6 (92.5%). Found m/z¼7266.2 (100%).
4.3.8. Polyanionic dendrimer 17. This compound was prepared
from fully protected, fourth generation dendrimer 16 (FW 15,843.4,
158 mg, 0.01 mmol) using anisole (1 mL) and TFA (5 mL) and fol-
lowing the procedure described for 11. All the volatiles were re-
moved under high vacuum (0.1 mmHg), and the residue was
4.3.6. Polyanionic dendrimer 14. This compound was prepared from
fully protected, third generation phosphate dendrimer 13 (FW 7263.4,

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G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10216
washed with hexane (3ꢂ2 mL) to provide analytically pure acid 17
(87 mg, 90%) as colorless glass. Then, acid 17 (80 mg,
(CH2Cl2/MeOH 7:1); dH (200 MHz, CDCl3) 1.89 [quintet, 3J(H,H)¼
6.4 Hz, 6H, (OCH2CH2CH2O)0], 1.99 [quintet, 3J(H,H)¼6.1 Hz, 12H,
a
0.0082 mmol) was dispersed in water (2 mL) and solid sodium
bicarbonate (34 mg, 0.4 mmol, 48 equiv) was added. After 10 min,
the resulting solution was frozen and then lyophilized under high
vacuum (0.1 mmHg) to give sodium salt (48ꢂNaþ) of 17 (114 mg) as
nonhygroscopic white powder. Found (acid): C, 46.41; H, 4.50; S,
7.22. C375H438O210P22S22 requires C, 46.47; H, 4.55, S, 7.28%; nmax
(OCH2CH2CH2O)1], 4.00 [s, 12H, (OH)], 4.08 [m, 24H, (OCH2CH2
-
CH2O)0 and (POCH2CH2CH2O)1], 4.25 [t, 3J(H,H)¼6.1 Hz, 12H,
(POCH2CH2CH2O)1], 4.50 [s, 24H, (ArCH2O)], 7.40 (s, 6H, Ar), 7.71 (s,
12H, Ar) ppm; dC (125.7 MHz, CD3OD/CDCl3 1:4) 29.65 [d, 3J(C,P)¼
7.3 Hz, 6C, (OCH2CH2CH2O)1], 30.93 [t, 3J(C,P)¼7.3 Hz, 3C,
(OCH2CH2CH2O)0], 61.60 [6C, (POCH2CH2CH2O)1], 64.01 [12C,
(ArCH2O)], 64.77 [d, 2J(C,P)¼4.7 Hz, 6C, (POCH2CH2CH2O)1], 65.32
[d, 2J(C,P)¼5.3 Hz, 6C, (OCH2CH2CH2O)0], 127.1 (12C, Ar), 130.2 (6C,
Ar), 130.3 [6C, (ipso Ar)], 142.3 [12C, (ipso Ar)], 167.1 [6C, (C]
O)] ppm; dP {1H} (202.5 MHz, CD3OD/CDCl3 1:4) 69.34 (P0), 69.50
(3P1) ppm.
(liquid film) 2954, 2920, 2850, 1739, 1462, 1246, 1019 cmꢁ1
; dH [acid
(500 MHz, CD3OD/CDCl3 2:1) 1.74 [m, 96H, (POCH2CH2CH2CH2]
O)], 1.88 [m, 42H, (OCH2CH2CH2O)], 3.96e4.18 [br m, 132H,
(POCH2CH2CH2CH2OC]O) and (OCH2CH2CH2O)], 4.37 [brt,
3J(H,H)¼6.9 Hz, 48H, (POCH2CH2CH2CH2OC]O)], 8.64 [s, 24H,
(Ar)], 8.69 [s, 48H, (Ar)] ppm; dC [acid (125.7 MHz, CD3OD/CDCl3
2:1)] 25.30 [24C, (O]COCH2CH2CH2CH2OP)], 26.20 [d, 3J(C,P)¼
7.4 Hz, 24C, (POCH2CH2CH2CH2OP)], 26.55 [d, 3J(C,P)¼7.4 Hz, 21C,
(POCH2CH2CH2OP)], 64.99 [24C, (O]COCH2CH2CH2CH2OP)], 67.56
4.3.11. Third generation polyester dendrimer 21. This compound was
prepared from hydroxy-terminated, second generation dendrimer
20 (FW 1909.9, 190 mg, 0.1 mmol), 1,3,5-benzenetricarboxylic acid
bis(4-methoxybenzyl) ester (2) (720 mg, 1.6 mmol, 16 equiv), EDC
(310 mg, 1.6 mmol, 16 equiv), and DMAP (20 mg, 0.16 mmol,
1.6 equiv) in dry dichloromethane (6 mL) following the procedure
described for 7. The crude product was then purified through a short
pad of silica gel. Elution with CH2Cl2/acetone 50:1 mixture and
gradually increasing the polarity to CH2Cl2/acetone 15:1, gave den-
drimer 19 (688 mg) as glassy white solid. Yield 97%. Rf 0.18 (CH2Cl2/
acetone 15:1); nmax (liquid film) 2957, 2837, 1782, 1725, 1515, 1232,
[m, 24C, (O]COCH2CH2CH2CH2OP)], 67.74 [m, 42C (POCH2CH2
-
CH2OP)], 131.2 [24C, (ipso Ar)], 132.0 [48C, (ipso Ar)], 134.5 [48C,
(Ar)],135.1 [24C, (Ar)],165.3 [24C, (C]O)],166.9 [48C, (C]O)] ppm;
dP {1H} (202.5 MHz, CDCl3) 68.44 (12P3), 68.53 (6P2), 68.62
(4P0,1
) ppm; MALDI TOF MS calcd for C375H438O210P22S22,
M¼9685.2 [exact (monoisotopic) mass]. Found m/z, fragmentation:
9684.0, 9674.6, 9659.9, 9621.9, 9605.2, 9370.6, 9256.4, 9143.5,
8975.5, 8860.0, 8733.0, 8429.6, 8338.2, 8211.5, 8025.3, 7901.6,
7629.5.
1174, 1031 cmꢁ1
;
dH (200 MHz, CDCl3) 2.02 [quintet, 3J(H,H)¼6.1 Hz,
6H, (OCH2CH2CH2O)0], 2.12 [quintet, 3J(H,H)¼6.0 Hz, 12H,
4.3.9. Second generation polyester dendrimer 19. This compound
was prepared from hydroxy-terminated, first generation thio-
phosphate dendrimer 18 (FW 924.9, 280 mg, 0.3 mmol), 3,5-bis[(2-
methoxyacetoxy)methyl]benzoic acid (5) (750 mg, 2.3 mmol,
7.7 equiv), EDC (442 mg, 2.3 mmol, 7.7 equiv), and DMAP (30 mg,
0.24 mmol, 0.8 equiv) in dry dichloromethane (5 mL) following the
procedure described for 7. Dendrimer 19 (colorless oil) was purified
on a short pad of silica gel using the gradient of the eluent from
CH2Cl2/acetone 15:1, to CH2Cl2/acetone 5:1. Yield 790 mg, 95%. Rf
0.26 (CH2Cl2/acetone 5:1); nmax (liquid film) 2957, 2928, 2902, 1754,
(OCH2CH2CH2O)1], 3.76 [s, 72H, (OCH3)], 4.18 [m, 24H, (OCH2CH2
-
CH2O)0 and (POCH2CH2CH2O)1], 4.39 [t, 3J(H,H)¼6.0 Hz, 12H,
(POCH2CH2CH2O)1], 5.26 [s, 48H, (ArCH2O)3], 5.37 [s, 24H,
(ArCH2O)2], 7.08 [AB, 3J(HA,HB)¼8.4 Hz, 96H, (Ar)], 7.68 [s, 6H, (Ar)2],
8.05 [s, 12H, (Ar)2], 8.87 [s, 36H, (Ar)3] ppm; dC (50.3 MHz, CDCl3)
30.09 [d, 3J(C,P)¼5.7 Hz, 6C, (OCH2CH2CH2O)1], 31.41 [t, 3J(C,P)¼
6.3 Hz, 3C, (OCH2CH2CH2O)0], 55.90 [24C, (OCH3)], 62.20 [6C,
(POCH2CH2CH2O)1], 65.17 [d, 2J(C,P)¼5.0 Hz, 6C, (POCH2CH2CH2O)1],
65.67 [d, 2J(C,P)¼5.2 Hz, 6C, (OCH2CH2CH2O)0], 67.13 [12C,
(ArCH2O)2], 67.86 [24C, (ArCH2O)3],114.6 {48C, [C
b
Ar(PMB)]},128.2
1723, 1215, 1187, 1125, 1024, 987 cmꢁ1
;
dH (500 MHz, CDCl3) 2.03
{24C, [ipso Ar(PMB)]}, 130.3 [12C, (Ar)2], 131.0 {48C [C
a Ar(PMB)]},
[quintet, 3J(H,H)¼6.1 Hz, 6H, (OCH2CH2CH2O)0], 2.14 [quintet,
131.4 [12C, (ipso Ar)3], 131.7 [6C, (ipso Ar)2], 132.0 [24C, (ipso Ar)3],
133.4 [6C, (Ar)2],135.4 [24C, (Ar)3],135.5 [12C, (Ar)3],137.3 [12C, (ipso
3J(H,H)¼6.1 Hz, 12H, (OCH2CH2CH2O)1], 3.44 [s, 36H, (OCH3)], 4.15
[double quintet, 3J(H,H)¼6.0 Hz, 3J(P,H)¼5.9 Hz, 12H, (OCH2CH2
Ar)2],160.4 {24C, [ipso( ) Ar(PMB)]},165.3 [12C, (C]O)3],165.4 [24C,
g
-
CH2O)0], 4.09 [s, 24H, (O]CCH2O)], 4.22 [double quintet, 3J(H,H)¼
6.1 Hz, 3J(P,H)¼8.9 Hz, 12H, (POCH2CH2CH2O)1], 4.41 [t, 3J(H,H)¼
6.3 Hz, 12H, (POCH2CH2CH2O)1], 5.22 [s, 24H, (ArCH2O)], 7.55 (s, 6H,
Ar), 7.98 (s, 12H, Ar) ppm; dC (125.7 MHz, CDCl3) 29.65 [d, 3J(C,P)¼
7.0 Hz, 6C, (OCH2CH2CH2O)1], 30.94 [t, 3J(C,P)¼7.0 Hz, 3C,
(C]O)3], 166.1 [6C, (C]O)2] ppm; dP {1H} (81.03 MHz, CDCl3)
68.61 (P0), 68.80 (3P1) ppm; MALDI TOF MS calcd for
C381H348O120P4S4, M¼7097.9 (100%). Found m/z, 7120.8 [(MþNa)
100], 6999.2 [Mꢁ(p-methoxybenzylþ)þNa (71%)].
(OCH2CH2CH2O)0], 59.69 [12C, (OCH3)], 61.66 [6C, (POCH2CH2
4.3.12. Polyanionic dendrimer 22. This compound was prepared
from fully protected, third generation dendrimer 21 (FW 7099.0,
142 mg, 0.02 mmol) using anisole (1 mL) and TFA (4 mL) and fol-
lowing the procedure described for 11. All the volatiles were re-
moved under high vacuum (0.1 mmHg), and the residue was
washed with hexane (3ꢂ2 mL) to provide analytically pure acid 22
-
CH2O)1], 64.70 [d, 2J(C,P)¼5.0 Hz, 6C, (POCH2CH2CH2O)1], 65.19 [d,
2J(C,P)¼5.0 Hz, 6C, (OCH2CH2CH2O)0], 65.81 [12C, (ArCH2O)], 69.97
[12C, (O]CCH2O)], 129.6 (12C, Ar), 131.1 [6C, (ipso Ar)], 132.8 (6C,
Ar), 136.7 [12C, (ipso Ar)], 165.8 [6C, (C]O)], 170.2 [12C, (C]
O)] ppm; dP {1H} (81 MHz, CDCl3) 68.58 (P0), 68.77 (3P1) ppm;
MALDI TOF MS calcd for C117H156O60P4S4, M¼2773.7 (100%). Found
m/z, 2797.3 (MþNa).
(78 mg, 93%) as
a colorless glass. Then, acid 22 (70 mg,
0.0166 mmol) was dispersed in water (2 mL) and solid sodium bi-
carbonate (17 mg, 0.2 mmol, 12 equiv) was added. After 10 min, the
resulting solution was frozen and lyophilized under high vacuum
(0.1 mmHg) to give sodium salt (12ꢂNaþ) of 22 (103 mg) as non-
hygroscopic white powder. nmax (liquid film) 3407 (br), 2952 (br),
4.3.10. Hydroxy-terminated second generation intermediate den-
drimer 20. Dendrimer 19 (FW 2774.6, 555 mg, 0.2 mmol) was
dissolved in CH2Cl2/MeOH 1:4 (4 mL) mixture. Magnesium meth-
oxide, 6e10 wt. % solution in MeOH (150
mL) was added. After
1640,1658,1450,1203,1012 cmꢁ1
; dH [acid (600 MHz, CD3OD/CDCl3
stirring at rt for 1 h the reaction mixture was acidified to pH 4e5
using hydrochloric acid (36e38%)/methanol 1:2 mixture. The
resulting solution was then concentrated under high vacuum and
the residue was redissolved in acetone/MeOH 10:1 mixture. In-
soluble material was filtered off, the solution was concentrated in
vacuo to give the title dendrimer 20 (colorless oil, 370 mg), which
was used as a substrate later without further purification. Rf 0.1
1:2)] 1.77 [dt, 3J(H,H)¼5.8 Hz, 3J(H,H)¼5.7 Hz, 6H, (OCH2CH2
-
CH2O)0], 1.90 [dt, 3J(H,H)¼5.8 Hz, 3J(H,H)¼5.7 Hz, 12H, (OCH2CH2
-
CH2O)1], 3.90 [ddt, 3J(H,H)¼5.5 Hz, 3J(P,H)¼5.9 Hz, 3J(H,H)¼5.4 Hz,
12H, (OCH2CH2CH2O)0], 3.99 [ddt, 3J(H,H)¼5.5 Hz, 3J(H,H)¼5.4 Hz,
3J(P,H)¼5.7 Hz, 12H, (POCH2CH2CH2O)1], 4.18 [dt, 3J(H,H)¼5.7 Hz,
2J(H,H)¼4.7 Hz, 12H, (POCH2CH2CH2O)1], 5.21 [s, 24H, (ArCH2O)2],
7.55 [s, 6H, (Ar)2], 7.86 [d, 4J(H,H)¼3.4 Hz, 12H, (Ar)2], 8.57 [s, 12H,

ꢀ
G.M. Salamonczyk / Tetrahedron 68 (2012) 10209e10217
10217
(Ar)3], 8.58 [d, 4J(H,H)¼1.6 Hz, 24H, (Ar)3] ppm; dC (150.9 MHz,
CD3OD/CDCl3 1:2) 29.59 [d, 3J(C,P)¼6.9 Hz, 6C, (OCH2CH2CH2O)1],
30.91 [t, 3J(C,P)¼7.5 Hz, 3C, (OCH2CH2CH2O)0], 61.90 [6C,
3. (a) Majoral, J. P.; Caminade, A. M. Chem. Rev. 1999, 99, 845e880; (b) Majoral, J.
P.; Caminade, A. M.; Maraval, V. Chem. Commun. 2002, 2929e2942.
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5. (a) McCarthy, T. D.; Karellas, P.; Henderson, S. A.; Giannis, M.; O’Keefe, D. F.;
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312e318; (b) Rojo, J.; Delgado, R. Anti-infect. Agents 2007, 6, 151e174; (c) Ji-
(POCH2CH2CH2O)1], 64.74 [d, 2J(C,P)¼4.6 Hz, 6C, (POCH2CH2
-
CH2O)1], 65.30 [d, 2J(C,P)¼5.0 Hz, 6C, (OCH2CH2CH2O)0], 66.77 [12C,
(ArCH2O)2], 129.9 [12C, (Ar)2], 130.9 [12C, (ipso Ar)3], 131.2 [6C, (ipso
Ar)2], 132.0 [24C, (ipso Ar)3], 133.2 [6C, (Ar)2], 135.0 [24C, (Ar)3],
135.5 [12C, (Ar)3], 137.1 [12C, (ipso Ar)2], 165.3 [12C, (C]O)3], 166.2
[6C, (C]O)2], 167.1 [24C, (C]O)3] ppm; dP {1H} (81.03 MHz, CD3OD/
CDCl3 1:2) 68.29 (P0), 68.58 (3P1) ppm; MALDI TOF MS calcd for
C189H158O95P4S4, M¼4199.5 (96), 4200.5 (100%). Found m/z, 4223.8
[(MþNa) 100], 4241.2 [(MþK) 65%].
~
~
menez, J. L.; Pion, M.; de la Mata, F. J.; Gomez, R.; Munoz, E.; Leal, M.; Munoz-
Fernandez, M. A. New J. Chem. 2012, 36, 299e309.
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Acknowledgements
11. Starpharma Holdings Limited announcement on March 22, 2012, http://www.
starpharma.com/news/113.
12. (a) Salamonczyk, G. M.; Kuznikowski, M.; Skowronska, A. Tetrahedron Lett.
This work was supported by the Ministry of Science and Higher
Education, Grant No. N N204 030836.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2000, 41, 1643e1645; (b) Salamonczyk, G. M.; Kuznikowski, M.; Poniatowska, E.
ꢀ
ꢀ
Chem. Commun. 2001, 2202e2203; (c) Salamonczyk, G. M.; Kuznikowski, M.;
Poniatowska, E. Tetrahedron Lett. 2002, 43, 1747e1749; (d) Poniatowska, E.;
Supplementary data
ꢀ
ꢀ
Salamonczyk, G. M. Tetrahedron Lett. 2003, 44, 4315e4317; (e) Salamonczyk, G.
ꢀ
M. Tetrahedron Lett. 2003, 44, 7449e7453; (f) Salamonczyk, G. M. Phosphorus,
Detailed synthetic procedures and analyses of compound 3, all
dendrimeric substrates: 6, 9, 12, 15, 18 as well as their precursors
are provided. The NMR spectra of the key dendrimers 13, 14, 15, 16,
21, 22 are also presented. Supplementary data associated with this
article can be found in the online version, at http://dx.doi.org/
10.1016/j.tet.2012.09.094. These data include MOL files and InChi-
Keys of the most important compounds described in this article.
Sulfur Silicon Relat. Elem. 2005, 180, 1051e1056.
13. Salamonczyk, G. M. Tetrahedron Lett. 2011, 52, 155e158.
ꢀ
14. For detailed synthetic procedure, see Supplementary data.
15. Nakhle, B. M.; Trammell, S. A.; Sigel, K. M.; Meyer, T. J.; Erickson, B. W. Tetra-
hedron 1999, 55, 2835e2846.
16. (a) Buncel, E.; Davies, A. G. J. Chem. Soc. 1958, 1550e1556; (b) Fan, Y. L.; Shaw, R.
G. J. Org. Chem. 1973, 38, 2410e2412.
ꢀ
ꢀ
17. Domanski, D. M.; Bryszewska, M.; Salamonczyk, G. M. Biomacromolecules 2004,
5, 2007e2012.
18. The use of more traditional dicyclohexyl carbodiimide (DCC) instead of EDC
resulted in a significantly lower (w65e70%) yield in the coupling reaction. Also,
purification of the product from dicyclohexyl urea was very tedious.
19. Stewart, F. H. C. Aust. J. Chem. 1968, 21, 2543e2550.
References and notes
1. Caminade, A. M.; Turrin, C. O.; Laurent, R.; Ouali, A.; Delavaux-Nicot, B. Den-
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Parker, S.; Valliant, J. F.; Adronov, A. J. Am. Chem. Soc. 2009, 131, 2906e2916; (b)
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Almutairi, A.; Akers, W. J.; Berezin, M. Y.; Achilefu, S.; Frechet, J. M. J. Mol.
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(b) Dendrimer-based Nanomedicine; Majoros, I. J., Baker, J. R., Jr., Eds.; Pan
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Pharmaceut. 2008, 5, 1103e1110; (c) Guillaudeu, S. J.; Fox, M. E.; Haidar, Y. M.;
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Dy, E. E.; Szoka, F. C.; Frechet, J. M. J. Bioconjugate Chem. 2008, 19, 461e469; (d)
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Gillies, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137e14146.
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