A fast and convenient synthesis of new water-soluble, polyanionic dendrimers

Article abstract of DOI:10.3390/molecules26164754

Reasonably simple, efficient, and possessing aspects of generality, the methodology for the synthesis of new, water-soluble, dendrimeric polyesters with great potential applications as antiviral drugs in their own right is described. The essential aspect of the presented approach is a quite unique, immediate access to the polyanionic material at each generation during divergent synthesis. Six target polyanionic dendrimers (generations 1, 2, and 3) have been synthesized. The key monomers applied in this project were 1,3,5-benzenetricarboxylic acid derivatives, which also worked as direct precursors of the charged dendrimer surface.

Full text of DOI:10.3390/molecules26164754

molecules
Article
A Fast and Convenient Synthesis of New Water-Soluble,
Polyanionic Dendrimers
Grzegorz M. Salamon´czyk
Centre of Molecular and Macromolecular Studies, The Polish Academy of Sciences, Sienkiewicza 112,
90-363 Łódz´, Poland; gmsalamo@cbmm.lodz.pl; Tel.: +48-42-684-7113; Fax: +48-42-684-7126
Abstract: Reasonably simple, efficient, and possessing aspects of generality, the methodology for the
synthesis of new, water-soluble, dendrimeric polyesters with great potential applications as antiviral
drugs in their own right is described. The essential aspect of the presented approach is a quite unique,
immediate access to the polyanionic material at each generation during divergent synthesis. Six
target polyanionic dendrimers (generations 1, 2, and 3) have been synthesized. The key monomers
applied in this project were 1,3,5-benzenetricarboxylic acid derivatives, which also worked as direct
precursors of the charged dendrimer surface.
Keywords: dendrimer; polyanion; 1,3,5-benzenetricarboxylic acid; polyester; phosphorus; total
synthesis; chemoselectivity
1. Introduction
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Dendrimers are three-dimensional, highly branched, synthetic polymers that possess a
well-defined structure. Their graphical representation resembles tree-like units emanating
from a common spot. Naturally, dendrimers have been extensively studied for a variety
of biomedical applications involving both therapy and diagnosis. However, this research
seems to be still at an early stage [1–3]. Normally, antiviral and other drug research con-
centrates on relatively low molecular weight structures. On the contrary, dendrimers with
their structural precision and with a large number of functional groups on the surface
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Salamon´czyk, G.M. A Fast
and Convenient Synthesis of New
Water-Soluble, Polyanionic
Dendrimers. Molecules 2021, 26, 4754.
https://doi.org/10.3390/
molecules26164754
may serve as antiviral drugs in their own right [4–6]. Different types and generations of
dendrimers substituted with different charged groups have been explored in the preven-
tion and treatment of HIV. These macromolecular drug candidates can be administered as
Academic Editor: György Keglevich
topical microbicides [7–9]. Topical microbicides interfere with the virus at the early stage
Received: 22 July 2021
Accepted: 3 August 2021
Published: 6 August 2021
of virus infection sequence, which is adsorption and fusion of the virus to the cell. The
common feature of all such microbicide compounds is the presence of polyanions, which
have the capability to bind to the (e.g., the so-called gp120 protein or spike protein (in
the case of SARS-CoV-2)) outer protein located on the viral surface, thus inhibiting the
entire viral infection process [10]. There are numerous examples of this type of compounds
that can be found in the literature [11–14]. Thanks to their mode of action, polyanionic
dendrimers can be expected to be effective against a broad spectrum of pathogenic viruses.
In contrast to polycationic compounds, polyanionic dendrimers usually possess much
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
lower cytotoxicity [15
,
16]. Definitely, the most successful polyanionic dendrimer used
for antiviral purposes is SPL7013 (also known as VivaGel® or VIRALEZE
™—Starpharma
Ltd. A company developing dendrimer products for pharmaceutical applications, Aus-
tralia), a sulfonated (32 terminal sulfonate groups) polylysine dendrimer (molec. weight
16.6 kD) [17]. VivaGel® was originally developed as a topical microbicide for the avoid-
ance of sexually transmitted infections (STIs) such as HIV, HSV-2 (genital herpes), and
human papillomavirus (HPV). There is an enormous global need for a vaginal microbi-
cide offering protection of this nature. It has also been licensed as an antiviral condom
coating. This compound went effectively through all the stages of clinical trials [18]. Ex-
cept STIs causing viruses, the SPL7013 dendrimer possesses significant activity against
Copyright:
© 2021 by the author.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 4754. https://doi.org/10.3390/molecules26164754
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Molecules 2021, 26, 4754
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other pathogenic viruses such as adenovirus, HBV (hepatitis B), RSV (respiratory syncytial
virus) or Zika virus. Moreover, in April 2020, it was declared that the same dendrimer
SPL 7013 showed significant antiviral activity against the coronavirus (SARS-CoV-2) that
causes COVID-19 (coronavirus disease) [19]. Later on, in September 2020, Starpharma
announced that SPL 7013 when applied at clinically relevant concentrations as a nasal
spray (VIRALEZE™), had potent virucidal activity, inactivating more than 99.9% of highly
infectious SARS-CoV-2.9 [20]. Therefore, VIRALEZE™ can be complementary to vaccines
and other preventive measures.
Consequently, the chemical synthesis of potential antiviral compounds, including
macromolecular material, such as water-soluble, charged dendrimers, possessing various
types of tunable scaffolds and surfaces, is extremely important and very much justified.
2. Results and Discussion
Several years ago, we developed a method for the synthesis of dendrimeric polyphos-
phates and their analogs [21
of new polyester dendrimers based on a trimesic acid framework derivative have been
disclosed [26 27]. In this research paper, a general and undemanding approach to the
–25]. Later on, also from our laboratory, an effective syntheses
,
synthesis of carboxylate-terminated (polyanionic) dendrimers is reported. The key feature
of this approach is the unprecedented formation and straightforward access to a polyan-
ionic dendrimer at each generation during the divergent synthesis. Therefore, there is no
need for often complicated, post-synthetic surface modification, and at least one difficult
synthetic step is saved. Recently, we have found that medium-size (4.2–4.7 kD) carboxylate-
terminated polyanionic dendrimers [28] displayed exceedingly strong antiviral activity
against both strains of HIV, way below their cytotoxicity versus MT4 cells [29]. Therefore,
for a powerful antiviral activity, the high generation structures are not necessarily essential.
2.1. Synthesis of Monomers
The presented synthetic work commences from the preparation of both interior and
surface unit monomers. The careful reaction of commercially available 1,3,5-
benzenetricarbonyl trichloride with 2.0 equiv. of lithium tert-butoxide, followed by a
mild basic hydrolysis of the remaining acid chloride, produced chemoselectively the cor-
responding 1,3,5-benzenetricarboxylic acid di-tert-butyl ester or di-tert-butyl trimesoate
(1) (48%).
(Accompanied with the corresponding triester (11%) and monoester (17%), acc. to
NMR) in 48% isolated yield. (Scheme 1) The synthesis (very low yield—22% in a two-
step procedure) of diester
elective reaction was a reduction (rt, 24 h) of one carboxyl group in diester
the borane–dimethyl sulfide complex, which provided the key reagent—di-tert-butyl
5-hydroxymethylbenzene-1,3-dicarboxylate ( ) in a high isolated yield (86% after crystal-
lization from CH2Cl2–cyclohexane). This compound ( ) represents an AB2-type monomer.
1
has been recently reported [30]. Another highly chemos-
1
using
2
2
The A group (hydroxyl) is active and the B groups (carboxyl) 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 de-
protection may not be quantitative and may also include undesired reactions, causing
imperfections in the dendrimer skeleton. Therefore, the protective groups applied in this
work were selected very cautiously. It will be demonstrated that deprotection reactions
(cleavage of tert-butyl esters in the presence of benzoate-type esters) were complete and
did not cause any defects in the expected structures.

Molecules 2021, 26, 4754
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Scheme 1. Synthesis of the monomers 1 and 2.
2.2. Synthesis of Dendrimers
To express the usefulness of the presented synthetic methodology, the author split this
project into three subsections.
Firstly, the core compound, tris(4-hydroxybutyl) phosphate [28] (3) was allowed to
react with an excess of dibenzyl 1,3,5-benzenetricarboxylate [28], (1a) in the presence of
the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
and 4-dimethylaminopyridine (DMAP) in anhydrous dichloromethane to furnish the first
generation (G1) dendrimer
benzyl esters in using catalytic hydrogenolysis proceeded smoothly and quantitatively
as evidenced by the 1H NMR to provide pure acid
(no trace of protons corresponding
to benzyl groups in was detected) (96%), which can be effortlessly transformed into the
corresponding hexaanion using, e.g., aqueous sodium bicarbonate. Then, hexacarboxylic
acid reacted readily with an excess of alcohol using EDC and DMAP as condensation
4 in 92% isolated yield. (Scheme 2) The cleavage of the terminal
4
5
5
6
5
2
reagents (in CH2Cl2-THF 1:1 solution) to afford the second generation (G2) dendrimer
88% isolated yield.
7 in
Scheme 2. Reagents and conditions: (a) for
acid , EDC, DMAP, DCM, rt; (c) H2, Pd/C, MeOH; (d) NaHCO3 aq.; (e) CF3COOH, DCM, rt; (f) alcohol
DCM, THF, rt; (g) for dodecaacid 8 as the substrate: alcohol 2, EDC, DMAP, DCM, DMF, rt.
3
as the substrate: acid 1a, EDC, DMAP, DCM, rt; (b) for 13 as the substrate:
1
2, EDC, DMAP,

Molecules 2021, 26, 4754
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Next, polyester 7 was stirred in a trifluoroacetic acid-dichloromethane 3:2 mixture at
room temperature. The reaction was closely monitored by the 1H NMR. After 4 h, neither
the trace of protons related to tert-butyl residues nor any damage (cleavage of an internal
ester bond) to the dendrimer skeleton was detected. The expected polyacid
in 90% isolated yield (all of the polyacids ( 11 15 18 22) reported here were purified
by their precipitation from a THF solution with acetone). At this stage, another polyanionic
(dodecaanion) dendrimer (sodium salt of ) was in hand. Repetition of the condensation
of acid , this time, with alcohol provided the third generation polyester 10 with a 76%
isolated yield. It should be noted that the condensation of polyacid with alcohol requires
the DCM-DMF mixture as the solvent due to its (acid ) solubility issues. The structures,
8 was obtained
5
,
8,
,
,
,
9
8
8
2
8
2
8
monodispersity, and high purity of all the dendrimeric products were confirmed by the
NMR and MALDI TOF mass spectrometry. For instance, Figure 1 shows the MALDI TOF
mass spectrum of third generation dendrimer 10. The signals at 5464.7 and 5477.8 are
attributed to the molecular ion (M + Na) and (M + K), respectively.
Figure 1. The MALDI TOF MS spectrum of dendrimer 10.
The minor peak (5420.4), which is 57 atom mass units in mass lower than the major
one (M + K), presumably arose due to the insignificant fragmentation of one (out of 24)
tert-butyl cation. The subsequent cleavage (TFA—DCM) of all tert-butyl esters (91%) led
to the formation of third generation dendrimer 11 with 24 free carboxyl groups, which
was further converted into the corresponding polyanion 12 by the reaction with aqueous
sodium bicarbonate. It is worth noting that the terminal bulky tert-butyl groups do not
cause any adverse congestion effects at the surface, and the third generation dendrimer
was formed smoothly.
Secondly, when catalytic hydrogenolysis could not be used during the synthesis
(such as the presence of nucleophilic sulfur in the substrate), an obvious solution was
the replacement of dibenzyl ester 1a with di-tert-butyl ester
reaction of another core triol, tris(4-hydroxybutyl) thiophosphate 13
of di-tert-butyl trimesoate ( ), again in the presence of the EDC and DMAP provided the
1
(Scheme 2). Therefore, the
[
21 28] with an excess
,
1
first generation dendrimer 14 in 90% isolated yield. Similarly, the removal of the terminal
tert-butyl ester groups from 14 occurred clearly in the TFA-DCM 3:2 mixture at room
temperature. No trace of any desulfuration product was detected by means of the 31P

Molecules 2021, 26, 4754
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NMR. The hexacarboxylic acid 15 as well as its sodium salt 16 were obtained in high yields.
The reaction of the acid 15 with an excess of the benzyl alcohol
2 (EDC, DMAP, DCM-THF
2:1) furnished another second generation polyester 17 in 84% isolated yield. Dendrimer
17 was an evident precursor of the polyanionic dendrimer 19, which was obtained by the
use of tert-butyl ester cleavage in 17 (via polyacid 18) using the reagents and conditions
mentioned above.
At last, di-tert-butyl trimesoate (1) can also be applied as a monomer for the modifica-
tion of the surface of practically any small and medium-size dendrimer, terminated with
nucleophilic functions such as: Hydroxyl, amino, thio, etc. Obviously, this conversion will
eventually lead also to the polyanionic compound. A first generation hydroxyl-terminated,
thiophosphate dendrimer 20 was synthesized using the amidophosphite approach [31
–33],
as described previously [21 22 28]. Therefore, the hexa-hydroxyl compound 20 possess-
,
,
ing both four phosphorus and sulfur atoms was swiftly converted (via fully protected
polyester 21) into a second generation polyanionic dendrimer precursor–dodecaacid 22
using the two-step condensation and deprotection procedure, as described above. In the
end, polyacid 22 was naturally transformed into the corresponding dodecaanion 23 using
diluted aqueous sodium bicarbonate (Scheme 3).
Scheme 3. Synthesis of the second generation polyanionic dendrimer 23 and its precursors—
dodecacarboxylic acid 22 and polyester 21.
Dendrimers 21
–
23 are the examples of “mixed” or layered dendrimers, which have
1
branching points both at phosphorus and at the carbon. In consequence, their H 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. For instance, Figure 2 shows the 13C NMR (CD3OD-CDCl3 3:1) spectrum of
the polyacid 22. Signals at 26.4 and 28.1 ppm are attributed to 12 “inner” carbon atoms from
four-carbon chains, whereas the signal (multiplet, coupling with two phosphorus atoms) at
30.6–30.7 (very minor) is attributed to the three “middle” carbons of the three-carbon chains.
Carbon atoms, which are in alpha position to the ester moieties, resonate at 63.4 (m, 6C,
three-carbon chain), 66.5 (6C, four-carbon chain), and 69.1 (doublet, 6C four-carbon chain)
ppm, respectively. The aromatic range of signals looks quite clear. There are 36 carbons
distributed as follows: 6 CH, 12 CH, 12 C ipso, 6 C ipso. Finally, there are six ester carbonyls
(166.6 ppm) and twelve carboxylic acid carbonyls (167.9 ppm).

Molecules 2021, 26, 4754
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Figure 2. 13C NMR spectrum of polyanionic dendrimer 22.
The synthesized polyanionic dendrimers (
6
-G1,
9-G2, 12-G3, 16-G1, 19-G2, 23-G2)
were water-soluble non-hygroscopic white solids (powders). All of the synthesized polyan-
ionic dendrimers possessed a decent water-solubility (at least 50 mg/1 mL). They are
chemically stable in the aqueous solutions, at least, at the pH range from 5 to 9. How-
ever, as expected, for the macromolecular compounds, they were not stable enough for
the melting point measurements. The synthesized dendrimers, as polyesters, have the
biodegradability potential, which is critical for biomedical applications. These compounds
will be tested against commonly known pathogenic viruses. The author also hopes that
they will be tested against the SARS-2 coronavirus.
3. Materials and Methods
3.1. General Procedures
The melting points reported here are uncorrected and were determined using the Mel-
Temp® Digital Melting Point Apparatus. The NMR spectra (1H, 13C, 31P) were recorded on
Bruker Avance AV-200, AV-500 or AV-600 spectrometers (Bruker Corporation, Billerica, MA,
USA) (200, 500 or 600 MHz, 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 the SYNAPT
G2-Si (Waters Corporation, Milford, MA, USA) spectrometer and the matrix assisted
laser desorption ionization time of flight mass spectra (MALDI TOF MS) were run on the
MALDI AXIMA Performance ToF/ToF (Shimadzu Corporation, Kyoto, Kyoto, Japan) mass
spectrometer using 2,5-dihydroxybenzoic acid as the matrix. FT-IR spectra were measured
on the ATI (American Thermal Instruments, Dayton, OH, USA) Mattson Infinity 60 AR
spectrophotometer. Microanalyses were performed on EuroVector 3018 and/or on Vario
(Elementar Analysensysteme GmbH, Hanau, Germany) analyzers. Chemical reagents
were purchased from Sigma-Aldrich and were used without any further purification.
Thin layer chromatography was carried out on Merck silica gel 60F254 aluminum sheets
using UV light (254 nm) or phosphomolybdic acid (5% solution in ethanol) for the spots
visualization. Preparative flash chromatography was performed on silica gel columns
(Merck, Kieselgel 230-400 mesh). The terms: “Short column” or “short pad (plug) of silica
gel” used throughout this section refer to the column of silica gel with a length of 25 mm

Molecules 2021, 26, 4754
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and diameter of 30 mm. Solvents were obtained from commercial sources (usually from
POCh S.A., Poland) and distilled or dried according to the standard methods. The NMR
and mass spectra for the synthesized compounds are shown in Supplementary Materials.
Please note, the NMR spectra of sodium salts:
with the spectra of their corresponding precursors—acids:
they are not reported.
6
,
9
,
12
,
16
,
19
,
23 were virtually identical
11 15 18 22. Therefore,
5,
8
,
,
,
,
3.2. Synthetic Procedures and Analyses for the Monomers 1 and 2
Di-tert-butyl 1,3,5-benzenetricarboxylate (
ride (2.65 g, 10 mmol) in dry THF (60 mL) at
1
). To a solution of 1,3,5-benzenetricarbonyl trichlo-
◦
−
20 C, was slowly (during about 1 h (syringe
pump useful) added a 1.0 M solution of lithium tert-butoxide (20 mL, 20 mmol, 2 equiv.) in
dry THF, and the resulting mixture was stirred for an additional 1 h, at −20 ◦C, then for
the next 1 h at room temperature. Afterwards, a 2 M aqueous solution (15 mL) of sodium
bicarbonate was added and the mixture was stirred for 30 min at room temperature. Then,
the THF was removed in a vacuo and the H2O–MeOH mixture (2:1, 30 mL) was added to
the residue and its pH was adjusted to ~10 (with 1 M NaOH). The resulting mixture was
extracted with hexane (2
×
20 mL) and the hexane solutions were concentrated and the
residue (tri-tert-butyl 1,3,5-benzenetricarboxylate 415 mg, 11%) was analyzed by the 1H
NMR (200 MHz, CDCl3) δ = 1.52 (s, 27H), 8.54 (s, 3H) ppm.
Next, the organic volatiles were removed in vacuo once again and water (50 mL) was
added to the residue and its pH was set to ~8.5 (with 0.1 M HCl). The mixture was extracted
with EtOAc (4
material was then purified by flash chromatography using CH2Cl2-acetone (gradually
increasing polarity from 30:1 to 20:1) as an eluent. Yield of (white solid) 1.55 g (48%). Rf
0.15 (CH2Cl2-acetone 20:1). Crystallization from the CH2Cl2-cyclohexane mixture afforded
×
30 mL). The combined EtOAc solution was concentrated. The residual
1
the crystalline title compound 1 ν max 2966, 1737, 1482,
; mp 202–204 ◦C (decomposed); IR
1273, 1112, 761 cm−1; NMR: δH (600 MHz, CDCl3) 1.66 (s, 18H), 8.83 [t, 3J(H,H) = 1.7 Hz
,
3
1H], 8.86 [d, J(H,H) = 1.7 Hz, 2H] ppm; δC (50 MHz, CDCl3) 28.11 {6C, [(CH3)3C)]},
82.32 {2C, [(CH3)3C)]}, 129.9 (Ar), 132.7 [2C, (Ar)], 134.6 [2C (ipso Ar)], 135.2 (ipso Ar),
164.1 [2C, (C=O)], 170.7 (C=O) ppm; HRMS (APCI): M
requires 321.1338.
−
H+, found 321.1338. C17H28O6
The remaining aqueous solution was acidified to the pH ~ 3.0 with 6 M HCl, yielding
a white dispersion. The organic material was then extracted with EtOAc (2 15 mL). The
×
solvent was finally removed to give tert-butyl 1.3.5-benzenetricarboxylate as a white solid
1
(450 mg, 17%). H NMR (500 MHz, CD3OD:CDCl3 1:1)
δ
= 1.56 (s, 9H), 8.80 [d, 4J(H,H) =
1.6 Hz, 2H], 8.82 [t, 4J(H,H) = 1.6 Hz, 1H] ppm.
Di-tert-butyl
5-hydroxymethylbenzene-1,3-dicarboxylate
(
2
).
Di-tert-butyl
1,3,5-
benzenetricarboxylate (1) (1.29 g, 4.0 mmol) was dissolved in dry THF (8 mL). The borane-
methyl sulfide complex (4.0 mL of 2 M solution in toluene, 8.0 mmol) was added slowly,
accompanied by effervescence. After 20 h of stirring at room temperature, methanol (15 mL
)
was added. The solution was stirred at room temperature for 30 min and concentrated
under reduced pressure. The resulting white solid was dissolved in ethyl acetate (30 mL)
and washed with water (25 mL) and saturated NaHCO3 (20 mL). The solvent was removed
under reduced pressure, and the crude product was purified by a short column silica
chromatography (CH2Cl2-acetone 50:1) to give
(CH2Cl2-acetone 35:1). Crystallization from the CH2Cl2-cyclohexane mixture afforded the
crystalline compound: Mp 144–145 ◦C; 1H NMR (200 MHz, CDCl3)
= 1.56 (s, 9H), 4.74
(s, 2H), 8.08 [d, J(H,H) = 1.6 Hz, 2H], 8.42 [t, J(H,H) = 1.6 Hz, 1H] ppm; δC (50 MHz,
CDCl3) 28.63 {6C, [( H3)3C)]}, 64.59 ( H2OH), 82.20 {2C, [(CH3)3 )]}, 129.9 (Ar), 132.0 [2C,
=O)]; HRMS (APCI): M
H+, found
2 as a white solid (1.09 g, 88%). Rf 0.29
δ
4
4
C
C
C
(Ar)], 132.9 [2C (ipso Ar)], 142.3 (ipso Ar), 165.7 [2C, (
307.1541. C17H28O6 requires 307.1545.
C
−

Molecules 2021, 26, 4754
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3.3. Synthetic Procedures and Analyses for Dendrimers: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19
First generation polyester dendrimer 4. Tris(4-hydroxybutyl) phosphate 3 (FW 314.3, 190 mg,
0.6 mmol), was dispersed in dry dichloromethane (6 mL). Dibenzyl trimesoate (1a) (800 mg
,
2.05 mmol, 3.4 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (FW
191.7, 403 mg, 2.1 mmol, 3.5 equiv.) were added under argon atmosphere. The suspension
was stirred vigorously and quickly dissolved. After 5 min, 4-dimethylaminopyridine
(DMAP) (FW 122.2, 36 mg, 0.3 mmol, 0.4 equiv.) was added and the mixture was stirred
at room temperature overnight. The reaction mixture was then diluted with ethyl acetate
(60 mL), washed with 0.1 M citric acid (15 mL), dried (MgSO4), and evaporated under
reduced pressure. The residue was placed on a short plug of silica gel. Elution with the
CH2Cl2–MeOH 100:1 mixture and gradually increasing the polarity to CH2Cl2–MeOH 20:1,
gave the title ester
CDCl3) 1.86-1.87 [m, 3J(H,H) = 6.4 Hz, 6H, (POCH2C
= 6.4 Hz, 6H, (POCH2CH2C
2CH2OC)], 4.14 [dt, 3J(H,H) = 5.8 Hz, 3J(P,H) = 11.2 Hz, 6H,
(POC
4
(790 mg). Yield 92%. Rf 0.3 (CH2Cl2-MeOH 20:1); NMR: δH (500 MHz,
H
2CH2CH2OC)], 1.91–1.92 [m, 3J(H,H)
H
H
2CH2CH2CH2OC)], 4.42 [t, 3J(H,H) = 5.8 Hz, 6H, (POCH2CH2CH2C
H2OC)], 5.39 [s,
12H, (PhCH2)], 7.32–7.47 (m, 30H, Ph), 8.85 [d, 4J(H,H) = 1.5 Hz, 6H, Ar], 8.88 [t, 4J(H,H) =
1.5 Hz, 3H, Ar] ppm; δC (125 MHz, CDCl3) 25.05 [3C, O=COCH2CH2CH2CH2O)], 26.94 [d,
3J(C,P) = 6.3 Hz, 3C, (OCH2CH2
C
H2CH2OP)], 65.05 [3C, (O
[d, 2J(C,P) = 5.0 Hz, 3C, (OCH2CH2CH2
H2OP)], 67.43 [6C, (Ph
128.5 [6C, (PhC )], 128.7 [12C, (PhC )], 131.3 [6C, (ipso Ph)], 134.4 [3C, (Ar)], 134.7 [6C,
(Ar)], 135.5 [9C, (ipso Ar)], 164.8 [6C, ( =O)], 164.9 [3C, (
=O)] ppm; δP {1H} (202 MHz,
CDCl3) 0.51 ppm; MALDI TOF MS calcd. for C81H75O22P, M = 1430.4. Found m/z =
1453.9 M + Na, 1469.9 M + K.
Polyanionic dendrimer . Dendrimer
C
H2CH2CH2CH2OP)], 67.20
C
C
H2)], 128.4 [12C, (PhC )],
α
γ
β
C
C
−
6
4 (FW 1431.4, 300 mg, 0.21 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 10 h. The catalyst
was filtered off, the filtrate concentrated to dryness in vacuo to furnish analytically
pure acid
5 (181 mg, 97%) as a white solid. NMR: δH (500 MHz, CDCl3:CD3OD 1:1)
3
3
1.73-1.74 [m, J(H,H) = 6.4 Hz, 6H, (POCH2CH2CH2CH2OC)], 1.83–1.84 [m, J(H,H)
3
3
= 6.4 Hz, 6H, (POCH2CH2CH2CH2OC)], 4.09 [dt, J(H,H) = 5.8 Hz, J(P,H) = 11.2 Hz,
6H, (POCH2CH2CH2CH2OC)], 4.31 [t, 3J(H,H) = 5.8 Hz, 6H, (POCH2CH2CH2CH2OC)],
4
4
8.71 [d, J(H,H) = 1.6 Hz, 6H, Ar], 8.74 [t, J(H,H) = 1.6 Hz, 3H, Ar] ppm; δC {1H,
31P} (125 MHz, CD3OD-CDCl3 2:1) 24.35 [3C, O=COCH2
(OCH2CH2 H2CH2OP)], 64.45 [3C, (O H2CH2CH2CH2OP)], 67.07, 130.4 [3C, (ipso Ar)],
131.2 [6C, (ipso Ar)], 133.5 [6C, (Ar)], 134.0 [3C, (Ar)], 164.5 [3C, ( =O)], 166.0 [6C, ( =O)]
ppm; δP {1H} (202 MHz, CD3OD-CDCl3 2:1)
0.42 ppm; MALDI TOF MS calcd. for
C39H39O22P, M = 890.2. Found m/z = 890.2. Next, acid (60 mg, 0.066 mmol) was dis-
CH2CH2CH2O)], 26.00 [3C,
C
C
C
C
−
5
persed in water (2 mL) and solid sodium bicarbonate (34 mg, 0.4 mmol, 6 equiv.) was
added. After 20 min, the resulting solution was frozen and lyophilized under high vacuum
(0.1 mmHg) to give
Second generation polyester dendrimer
drimer (FW 890.7, 100 mg, 0.112 mmol), was dissolved in dry THF (2 mL). Then, the reac-
tion mixture was diluted with dry dichloromethane (2 mL). Di-tert-butyl 5-
hydroxymethylbenzene-1,3-dicarboxylate ( ) (FW 308.4, 230 mg, 0.74 mmol, 6.6 equiv.) and
6
as a hexasodium salt of
5 (68 mg), as a nonhygroscopic white powder.
7. Carboxy-terminated, first generation phosphate den-
5
2
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (172 mg, 0.896 mmol, 8.0 equiv.)
were added under argon atmosphere. The suspension was stirred vigorously and quickly
dissolved. After 5 min, 4-dimethylaminopyridine (DMAP) (15 mg, 0.12 mmol, 1.0 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 the cyclohexane–acetone 20:1 mixture and gradually
increasing the polarity to cyclohexane–acetone 8:1, gave the title ester
5 (260 mg). Yield
88%. NMR: δH (500 MHz, CDCl3-C6D6 2:1) 1.58 [s, 108H, C(CH3)3], 1.81–1.86 [m, 6H,

Molecules 2021, 26, 4754
9 of 14
(POCH2CH2CH2CH2OC)], 1.87–1.96 [m, 6H, (POCH2CH2CH2CH2OC)], 4.13 [dt, 3J(H,H)
= 6.3 Hz, 3J(P,H) = 12.3 Hz, 6H, (POCH2CH2CH2CH2OC)], 4.40 [t, 3J(H,H) = 6.5 Hz, 6H,
4
(POCH2CH2CH2CH2OC)], 5.29 [s, 12H, (ArCH2)], 8.20 [d, J(H,H) = 1.3 Hz, 12H, Ar2],
4
3
3
8.53 [t, J(H,H) = 1.3 Hz, 6H, Ar2], 8.85 [d, J(H,H) = 1.5 Hz, 6H, Ar1], 8.87 [t, J(H,H)
= 1.5 Hz, 3H, Ar1] ppm; δC (125 MHz, CDCl3) 25.04 [3C, O=COCH2
26.96 [d, 3J(C,P) = 7.1 Hz, 3C, (OCH2CH2
H2CH2OP)], 28.21 [36C, C(
(O H2CH2CH2CH2OP)], 67.43 [6C, (Ar
H2)], 67.20 [d, 2J(C,P) = 5.7 Hz, 3C,
(OCH2CH2CH2 H2OP)], 81.92 [12C,
(CH3)3], 130.6 [6C, (Ar2)], 131.1 [6C, (ipso Ar1)],
131.5 [3C, (ipso Ar1)], 132.9 [12C, (ipso Ar2)], 133.2 [12C, (Ar2)], 134.9 [3C, (Ar1)], 135.0 [6C,
(Ar1)], 136.1 [6C, (ipso Ar2)], 164.7 [18C, ( =O)] ppm; δP {1H} (202 MHz,
=O)], 164.8 [3C, (
CH2CH2CH2O)],
C
CH3)3], 65.16 [3C,
C
C
C
C
C
C
CDCl3) 0.53 ppm; MALDI TOF MS calcd. for C141H171O46P, M = 2631.1. Found m/z =
2670.3 M + K.
Polyanionic dendrimer 9. Dendrimer 7 (FW 2632.8, 134 mg, 0.05 mmol) was dissolved in dry
dichloromethane (2 mL), and trifluoroacetic acid was added (2 mL). The reaction mixture
was stirred at room temperature under argon atmosphere for 5 h. All the volatiles were
removed in vacuo, and the residue was dissolved in THF (1 mL). Next, acetone (10 mL) was
added to that solution and the resulting mixture was kept in the refrigerator for about an
hour. The precipitate was filtered off to provide acid
8 (88 mg, 90%) as a white solid. NMR:
δH (500 MHz, CDCl3-CD3OD 2:1) 2.03–2.08 [m, 6H, (POCH2CH2CH2CH2OC)], 2.25–2.28
[m, 6H, (POCH2CH2CH2CH2OC)], 3.90-3.92 [m, 6H, (POCH2CH2CH2CH2OC)], 4.14–4.16
[m, 6H, (POCH2CH2CH2CH2OC)], 5.22 [s, 12H, (ArCH2)], 8.36 [s, 6H, Ar2], 8.48 [s, 12H,
Ar2], 8.56 [s, 6H, Ar1], 8.61 [s, 3H, Ar1] ppm; δP {1H} (202 MHz, CD3OD-CDCl3 2:1)
−
2.80
ppm; MALDI TOF MS calcd. for C93H75O46P, M = 1959.3. Found m/z = 1982.6 M + Na.
Later on, acid (35 mg, 0.0179 mmol) was dispersed in water (2 mL) and solid sodium
8
bicarbonate (18 mg, 0.2 mmol, 12 equiv.) was added. After 20 min, the resulting solution
was frozen and lyophilized under high vacuum (0.1 mmHg) to give 9 as a dodecasodium
salt of 8 (40 mg), as a nonhygroscopic white powder.
Third generation polyester dendrimer 10. Carboxy-terminated, second generation phosphate
dendrimer
the resulting solution was diluted with dry dichloromethane (2 mL). Di-tert-butyl 5-
hydroxymethylbenzene-1,3-dicarboxylate ( ) (FW 308.4, 82 mg, 0.264 mmol, 13.2 equiv.)
8 (FW 1959.5, 40 mg, 0.02 mmol), was dissolved in dry DMF (1.5 mL). Then,
2
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (172 mg, 0.3 mmol, 15 equiv.)
were added under argon atmosphere. The suspension was stirred vigorously and quickly
dissolved. After 5 min, 4-dimethylaminopyridine (DMAP) (5 mg, 0.04 mmol, 1.8 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 the cyclohexane–acetone 20:1 mixture and
gradually increasing the polarity to cyclohexane–acetone 6:1, provided the title ester
10
(83 mg). Yield 76%. NMR: δH (500 MHz, CDCl3) 1.56 [s, 216H, C(CH3)3] 1.82–1.87
[m, 6H, (POCH2C
H
2CH2CH2OC)], 1.88–1.95 [m, 6H, (POCH2CH2C
H
2CH2OC)], 4.13 [dt,
3
3
3J(H,H) = 5.9 Hz, J(P,H) = 12.2 Hz, 6H, (POCH2CH2CH2CH2OC)], 4.39 [t, J(H,H) =
6.4 Hz, 6H, (POCH2CH2CH2CH2OC)], 5.44 [s, 24H, (Ar3CH2)], 5.46 [s, 12H, (Ar2CH2)],
8.19 [s, 24H, Ar3], 8.35 [s, 12H, Ar3], 8.51 [s, 12H, Ar2], 8.69 [s, 6H, Ar2], 8.83 [s, 6H,
Ar1], 8.85 [s, 3H, Ar1] ppm; δC (125 MHz, CDCl3) 24.94 [3C, (O=COCH2CH2CH2CH2O)],
26.91 [d, 3J(C,P) = 7.4 Hz, 3C, (OCH2CH2
(O
H2CH2CH2CH2OP)], 66.27 [6C, (Ar2
= 6.7 Hz, 3C, (OCH2CH2CH2 H2OP)], 81.81 [24C,
C
C
H2CH2OP)], 28.14 [72C, C(
C
H3)3], 65.12 [3C,
2
C
H2)], 66.33 [12C, (Ar3
C
H2)], 67.09 [d, J(C,P)
C
C
(CH3)3], 130.5 [12C, (Ar3)], 131.0
[6C, (Ar2)], 133.0 [24C, (Ar3)], 130.5 [12C, (Ar3)], 130.8 [6C, (ipso Ar1)], 131.0 [12C, (ipso
Ar2)], 131.5 [3C, (ipso Ar1)], 132.8 [24C, (ipso Ar3)], 134.2 [12C, (Ar2)], 135.0 [9C, (Ar1)],
136.2 [12C, (ipso Ar3)], 136.7 [6C, (ipso Ar2)], 164.5 [9C, (C1=O)], 164.6 [24C, (C3=O)], 165.0
[12C, (C2=O)] ppm; δP {1H} (202 MHz, CDCl3) -0.46 ppm. MALDI TOF MS calcd. for
C297H339O94P, M = 5440.1. Found m/z = 5477.8 M + K.

Molecules 2021, 26, 4754
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Polyanionic dendrimer 12
.
Dendrimer 10 (80 mg, 0.015 mmol) was dissolved in dry
dichloromethane (1.5 mL), and trifluoroacetic acid was added (2 mL). The reaction mixture
was stirred at room temperature under argon atmosphere for 5 h. All the volatiles were
removed in vacuo, and the residue was dissolved in THF (1 mL). Next, acetone (10 mL) was
added to that solution and the resulting mixture was kept in the refrigerator for about an
hour. The precipitate was filtered off to provide acid 11 (54 mg, 88%) as a white solid. NMR:
δH (500 MHz, THF-d8) 1.75–1.80 [m, 6H, (POCH2CH2CH2CH2OC)], 1.82–1.88 [m, 3J(H,H)
= 6.4 Hz, 6H, (POCH2C
H
2CH2CH2OC)], 4.07 [dt, 3J(P,H) = 12.3 Hz, 3J(H,H) = 6.2 Hz, 6H,
(POCH
2CH2CH2CH2OC)], 4.33 [t, 3J(H,H) = 6.2 Hz, 6H, (POCH2CH2CH2C
H2OC)], 5.48 [s,
24H, (Ar3CH2)], 5.49 [s, 12H, (Ar2CH2)], 8.30 [d, 4J(H,H) = 1.3 Hz, 33H, 9HAr1, 24HAr3],
8.58 [s, 12H, Ar3], 8.76 [d, 4J(H,H) = 1.6 Hz, 12H, Ar2], 8.81 [t, 4J(H,H) = 1.5 Hz, 6H, Ar2]
ppm; δC (125 MHz, THF-d8) 26.60 [d, 3J(C,P) = 7.2 Hz, 3C, (OCH2CH2
C
H2CH2OP)], 29.24
H2)], 66.08 [12C, (Ar3
H2)], 67.09 [3C,
H2CH2CH2CH2OP)], 67.34 [d, 2J(C,P) = 6.7 Hz, 3C, (OCH2CH2CH2
CH2OP)], 130.6
[3C, (O=COCH2
C
H2CH2CH2O)], 64.84 [6C, (Ar2
C
C
(OC
[12C, (Ar3)], 131.1 [12C, (Ar2)], 131.4 [6C, (Ar2)], 131.7 [24C, (ipso Ar3)], 133.3 [12C, (ipso
Ar2)], 133.3 [6C, (Ar1)], 133.4 [24C, (Ar3)], 133.6 [3C, (Ar1)], 134.1 [6C, (ipso Ar2)], 134.2 [12C,
(ipso Ar3)], 136.8 [9C, (Ar1)], 164.0 [12C, (C2=O)], 164.1 [9C, (C1=O)], 165.9 [24C, (C3=O)]
ppm; δP {1H} (202 MHz, THF-d8)
−
0.95 ppm; MALDI TOF MS calcd. for C201H147O94P,
M = 4096.7.3. Found m/z, fragmentation: 4129.6, 3965.6, 3949.9, 3844.2, 3731.4, 3700.0,
3601.9 (major peaks). Elemental analysis: Found (acid): C, 58.87; H, 3.66; C201H147O94P
requires C, 58.92; H, 3.62; P, 0.76%. Afterwards, acid 11 (FW 4097.2, 40 mg, 0.0098 mmol)
was dispersed in water (2 mL) and solid sodium bicarbonate (20 mg, 0.235 mmol, 24 equiv.)
was added. After 20 min, the resulting solution was frozen and lyophilized under high
vacuum (0.1 mmHg) to give 12 as a tetracosasodium salt of 11 (45 mg), as a nonhygroscopic
white powder.
First generation polyester thiophosphate dendrimer 14. Tris(4-hydroxybutyl) thiophosphate 13
(FW 330.4, 166 mg, 0.5 mmol), was dissolved in dry dichloromethane (5 mL). Di-tert-butyl
trimesoate (1) (322.4, 560 mg, 1.7 mmol, 3.4 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) (FW 191.7, 336 mg, 1.75 mmol, 3.5 equiv.) were added under argon
atmosphere. The suspension was stirred vigorously and quickly dissolved. After 5 min,
4-dimethylaminopyridine (DMAP) (FW 122.2, 25 mg, 0.2 mmol, 0.4 equiv.) was added and
the mixture was stirred at room temperature overnight. The reaction mixture was then
diluted with ethyl acetate (60 mL), washed with 0.1 M citric acid (15 mL), dried (MgSO4),
and evaporated under reduced pressure. The residue was placed on a short plug of silica gel.
Elution with the cyclohexane–acetone 100:1 mixture and gradually increasing the polarity to
cyclohexane–acetone 20:1, gave the title ester 14 (560 mg). Yield 90%. Rf 0.25 (cyclohexane–
acetone 25:1). NMR: δH (500 MHz, CDCl3) 1.59 [s, 54H, C(CH3)3], 1.79-1.84 [m, 3J(H,H) =
6.4 Hz, 12H, (POCH2CH2CH2CH2OC) and (POCH2CH2CH2CH2OC)], 4.12 [dt, 3J(H,H)
= 5.8 Hz, 3J(P,H) = 12.6 Hz, 6H, (POCH2CH2CH2CH2OC)], 4.42 [t, 3J(H,H) = 5.7 Hz, 6H,
(POCH2CH2CH2C
[3C, O=COCH2
H2CH2CH2O)], 26.77 [d, 3J(C,P) = 7.8 Hz, 3C, (OCH2CH2
28.19 [18C, C( H3)3], 64.93 [3C, (O
H2CH2CH2CH2OP)], 67.70 [d, 2J(C,P) = 5.4 Hz, 3C,
(OCH2CH2CH2 H2OP)], 82.24 [6C, (CH3)3], 130.9 [3C, (ipso Ar)], 132.8 [6C, (ipso Ar)],
134.0 [6C, (Ar)], 134.4 [3C, (Ar)], 164.3 [6C, ( =O)], 165.3 [3C, (
=O)] ppm; δP {1H} (202
H
2OC)], 8.70 [s, 3H, Ar], 8.71 [s, 6H, Ar] ppm; δC (125 MHz, CDCl3) 25.13
C
C
H2CH2OP)],
C
C
C
C
C
C
MHz, CDCl3) 68.52 ppm; MALDI TOF MS calcd. for C63H87O21PS, M = 1242.5. Found m/z
= 1282.5 M + K.
Polyanionic dendrimer 16. Dendrimer 14 (FW 1243.4, 125 mg, 0.1 mmol) was dissolved in dry
dichloromethane (1.5 mL), and trifluoroacetic acid was added (2 mL). The reaction mixture
was stirred at room temperature under argon atmosphere for 5 h. All the volatiles were
removed in vacuo, and the residue was dissolved in THF (1 mL). Next, acetone (10 mL)
was added to that solution and the resulting mixture was kept in the refrigerator for about
an hour. The precipitate was filtered off to provide acid 15 (84 mg, 93%) as a white solid.
NMR: δH (500 MHz, CDCl3-THF-d8 1:5) 1.60–1.69 [m, 12H, (POCH2CH2CH2CH2OC) and
(POCH2CH2CH2CH2OC)], 3.86 [dt, 3J(H,H) = 6.0 Hz, 3J(P,H) = 12.6 Hz, 6H,

Molecules 2021, 26, 4754
11 of 14
(POCH2CH2CH2CH2OC)], 4.11 [t, 3J(H,H) = 6.0 Hz, 6H, (POCH2CH2CH2CH2OC)], 8.52 [s,
6H, Ar], 8.55 [s, 3H, Ar] ppm; δC (125 MHz, CDCl3-THF-d8 1:5) 25.06, 26.22 [d, 3J(C,P) = 7.5
Hz, 3C, (OCH2CH2CH2CH2OP)], 67.04 [d, 2J(C,P) = 5.2 Hz, 3C, (OCH2CH2CH2CH2OP)],
67.25 [3C, (O
C
H2CH2CH2CH2OP)], 130.6 [9C, (ipso Ar)], 131.3 [9C, (Ar)], 164.3 [3C, (
=O)] ppm; δP {1H} (202 MHz, CDCl3-THF-d8 1:5) 68.52 ppm; HRMS (TOF
H+: found 905.1371. C39H39O21PS requires 905.1364. Soon after, acid 15
C=O)],
165.6 [6C, (
MS ES) M
C
−
(35 mg, 0.0179 mmol) was dispersed in water (2 mL) and solid sodium bicarbonate (10 mg,
0.11 mmol, 6 equiv.) was added. After 20 min, the resulting solution was frozen and
lyophilized under high vacuum (0.1 mmHg) to give 16 as a hexasodium salt of 15 (40 mg),
as a nonhygroscopic white powder.
Second generation polyester thiophosphate dendrimer 17. Carboxy-terminated, first genera-
tion thiophosphate dendrimer 15 (FW 906.8, 40 mg, 0.044 mmol) was dissolved in dry
THF (1.0 mL). Then, the reaction mixture was diluted with dry dichloromethane (2 mL).
Di-tert-butyl 5-hydroxymethylbenzene-1,3-dicarboxylate (2) (FW 308.4, 67 mg, 0.3 mmol,
6.6 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (70 mg, 0.36 mmol,
8.0 equiv.) were added under argon atmosphere. The suspension was stirred vigorously
and quickly dissolved. After 5 min, 4-dimethylaminopyridine (DMAP) (FW 122.2, 5.0 mg
,
0.04 mmol, 0.9 equiv.) was added and the mixture was stirred at room temperature
overnight. The reaction mixture was then diluted with ethyl acetate (20 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 the cyclohexane–acetone
100:1 mixture and gradually increasing the polarity to cyclohexane–acetone 15:1, afforded
the title ester 17 (95 mg). Yield 84%. δH (500 MHz, CDCl3) 1.58 [s, 108H, C(CH3)3], 1.80–
1.87 [m, 6H, (POCH2CH2CH2CH2OC)], 1.88–1.96 [m, 6H, (POCH2CH2CH2CH2OC)], 4.12
3
3
3
[dt, J(H,H) = 6.3 Hz, J(P,H) =14.3 Hz, 6H, (POCH2CH2CH2CH2OC)], 4.40 [t, J(H,H)
= 6.4 Hz, 6H, (POCH2CH2CH2CH2OC)], 5.46 [s, 12H, (ArCH2)], 8.20 (s, 12H, Ar2), 8.53
(s, 6H, Ar2), 8.85 (s, 3H, Ar1), 8.87 (s, 6H, Ar1) ppm; δC (125 MHz, CDCl3) 25.01 [3C,
O=COCH2
[36C, C( H3)3], 65.13 [3C, (O
= 5.7 Hz, 3C, (OCH2CH2CH2
(ipso Ar1)], 131.6 [3C, (ipso Ar1)], 132.8 [12C, (ipso Ar2)], 133.1 [12C, (Ar2)], 134.9 [3C, (Ar1)],
135.0 [6C, (Ar1)], 136.0 [6C, (ipso Ar2)], 164.6 [21C, ( =O)] ppm; δP {1H} (202 MHz, CD3OD-
C
H2CH2CH2O)], 26.66 [d, 3J(C,P) = 7.6 Hz, 3C, (OCH2CH2
C
H2CH2OP)], 28.16
H2)], 67.61 [d, 2J(C,P)
C
(CH3)3], 130.6 [6C, (Ar2)], 130.9 [6C,
C
C
H2CH2CH2CH2OP)], 66.51 [6C, (Ar
C
CH2OP)], 81.87 [12C,
C
CDCl3 2:1) 68.60 ppm; MALDI TOF MS calcd. for C141H171O45PS, M = 2647.0. Found m/z
= 2670.4 M + Na, 2686.7 M + K.
Polyanionic dendrimer 19. Dendrimer 17 (FW 2648.9, 70 mg, 0.0264 mmol) was dissolved in
dry dichloromethane (1.5 mL), and trifluoroacetic acid was added (1.5 mL). The reaction
mixture was stirred at room temperature under argon atmosphere for 5 h. All the volatiles
were removed in vacuo, and the residue was dissolved in THF (1 mL). Next, acetone (10 mL)
was added to that solution and the resulting mixture was kept in the refrigerator for about
an hour. The precipitate was filtered off to provide acid 18 (48 mg, 92%) as a white solid.
NMR: δH (500 MHz, THF-d8) 1.82–1.88 [m, 6H, (POCH2C
(POCH2CH2CH2CH2OC)], 3.59-3.64 [m, 6H, (POCH2CH2CH2CH2OC)], 3.80 [t, 3J(H,H) =
6.8 Hz, 6H, (POCH2CH2CH2C 2OC)], 5.47 [s, 12H, (ArC
2)], 8.30 (s, 12H, Ar2), 8.36 (s, 6H,
Ar2), 8.54 (s, 6H, Ar1), 8.61 (s, 3H, Ar1), 10.87 (brs, 12H, COOH) ppm; δC (125 MHz, THF-
d8) 25.40 [3C, O=COCH2 H2CH2CH2O)], 28.96 [3C, (OCH2CH2 H2CH2OP)], 65.92 [3C,
(OCH2CH2CH2 H2OP)], 66.96, [3C, (O H2CH2CH2CH2OP)], 67,24 [6C, (Ar H2)], 128.6
[9C, (Ar1)], 130.5 [12C, (ipso Ar2)], 130.9 [6C, (ipso Ar2)], 131.6 [6C, (Ar2)], 133.5 [12C, (Ar2)],
137.1 [9C, (ipso Ar1)], 163.9 [3C, (
=O)], 164.5 [6C, ( =O)], 165.8 [12C, (HO =O)] ppm δP
{1H} (202 MHz, THF-d8) 68.71 ppm; MALDI TOF MS calcd. for C93H75O45PS, M = 1974.3
H2CH2CH2OC)], 1.90-1.98 [m, 6H,
H
H
C
C
C
C
C
C
C
C
.
Found m/z, fragmentation: 1149.8, 1121.7, 1093.5, 1065.5 (major peaks). Elemental analysis:
Found (acid): C, 56.51; H, 3.85; S, 1.62%. C93H75O45PS requires C, 56.54; H, 3.83; S, 1.62%.
Afterwards, acid 18 (35 mg, 0.0179 mmol) was dispersed in water (2 mL) and solid sodium
bicarbonate (18 mg, 0.2 mmol, 12 equiv.) was added. After 20 min, the resulting solution

Molecules 2021, 26, 4754
12 of 14
was frozen and lyophilized under high vacuum (0.1 mmHg) to give 19 as a dodecasodium
salt of 18 (40 mg), as a nonhygroscopic white powder.
Second generation fully protected dendrimer 21. Hydroxy-terminated, first generation thiophos-
phate dendrimer12 20 (FW 1009.1, 51 mg, 0.050 mmol) was suspended in dry dichloromethane
(3.0 mL). Di-tert-butyl 1.3.5-benzenetricarboxylate (1) (FW 322.4, 105 mg, 0.325 mmol,
6.5 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (77 mg, 0.4 mmol,
8.0 equiv.) were added under argon atmosphere. The suspension was stirred vigor-
ously and quickly dissolved. After 5 min, 4-dimethylaminopyridine (DMAP) (FW 122.2,
5.0 mg, 0.04 mmol, 0.9 equiv.) was added and the mixture was stirred at room tem-
perature overnight. The reaction mixture was then diluted with ethyl acetate (20 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 the neat
dichloromethane provided the title dendrimer 21 (127 mg). Yield 90%. δH (500 MHz,
CDCl3) 1.58 [s, 108H, C(CH3)3], 1.78–1.87 [m, 12H, (POCH2CH2CH2CH2OC)], 1.88–1.95
[m, 12H, (POCH2CH2CH2CH2OC)], 2.12–2.21 [m, 6H, (POCH2CH2CH2OP)], 4.12 [dt,
3
3
3J(H,H) = 6.3 Hz, J(P,H) = 13.6 Hz, 12H, (POCH2CH2CH2CH23OC)], 4.24 [dt, J(H,H)
3
= 6.3 Hz, J(P,H) = 14.3 Hz, 12H, (POCH2CH2CH2OP)], 4.45 [t, J(H,H) = 6.1 Hz, 12H,
(POCH2CH2CH2CH2OC)], 8.70 (s, 6H, Ar), 8.71 (s, 12H, Ar) ppm; δC (125 MHz, CDCl3),
25.07 [6C, O=COCH2
28.13 [36C, C(
61.73 [m, 6C, (PO
= 5.4 Hz, 6C, (OCH2CH2CH2
C
H2CH2CH2O)], 26.71 [d, 3J(C,P) = 7.8 Hz, 6C, (OCH2CH2
C
H2CH2OP)],
3
C
H3)3], 29.41–29.49 [m, J(C,P) = 7.8 Hz, 3C, (POCH2
C
H2CH2OP)], 61.62–
H2CH2CH2CH2OP)], 67.64 [d, 2J(C,P)
H2OP)], 82.17 [12C, (CH3)3], 130.9 [6C, (ipso Ar)], 132.8
=O)], 165.2 [6C, ( =O)]
C
H2CH2CH2OP)], 64.87 [6C, (OC
C
C
[12C, (ipso Ar)], 134.0 [12C, (Ar)], 134.4 [6C, (Ar)], 164.2 [12C, (
C
C
ppm; δP {1H} (202 MHz, CDCl3), 68.29 (P0), 68.58 (3P1) ppm; MALDI TOF MS calcd. for
C135H192O48P4S4, M = 2833.0. Found m/z = 2845.8 M + Na.
Polyanionic dendrimer 23
. Dendrimer 21 (80 mg, 0.028 mmol) was dissolved in dry
dichloromethane (1.5 mL), and trifluoroacetic acid was added (1.5 mL). The reaction mix-
ture was stirred at room temperature under argon atmosphere for 5 h. All the volatiles were
removed in vacuo, and the residue was dissolved in THF (1 mL). Next, acetone (11 mL) was
added to that solution and the resulting mixture was kept in the refrigerator for about an
hour. The precipitate was filtered off to provide acid 22 (56 mg, 93%) as a white solid. NMR:
δH (200 MHz, CD3OD) 1.75–1.96 [m, 24H, (POCH2CH2
(POCH2C
2CH2OP)], 4.09–4.21 [m, 3J(H,H) = 6.3 Hz, 12H, (POC
4.30 [m, 12H, (POC 2CH2C
2OP)], 4.37 [t, 3J(H,H) = 6.0 Hz, 12H, (POCH2CH2CH2C
8.70 (s, 12H, Ar), 8.74 (s, 6H, Ar) ppm; δC (125 MHz, CD3OD-CDCl3 3:1), 26.44 [6C,
O=COCH2 H2CH2OP)], 30.60–
H2CH2CH2O)], 28.09 [d, 3J(C,P) = 7.4 Hz, 6C, (OCH2CH2
30.70 [m, 3C, (POCH2 H2CH2OP)], 66.27–66.40 [m, 6C, (PO H2CH2 H2OP)], 66.53 [6C,
(O H2OP)], 132.5
H2CH2CH2CH2OP)], 69.15 [d, 2J(C,P) = 5.6 Hz, 6C, (OCH2CH2CH2
[6C, (ipso Ar)], 133.3 [6C, (Ar)], 135.6 [12C, (Ar)], 136.1 [12C, (ipso Ar)], 166.6 [6C, ( =O)],
167.9 [12C, (
=O)] ppm; δP {1H} (202 MHz, CD3OD-CDCl3 3:1), 68.61 (P0), 68.80 (3P1)
C
H2CH2OC)], 2.12–2.21 [m, 6H,
2CH2CH2CH2OC)], 4.24–
2OC)],
H
H
H
H
H
C
C
C
C
C
C
C
C
C
ppm; MALDI TOF MS calcd. for C87H96O48P4S4, M = 2160.3. Found m/z, fragmentation:
1470.0, 1450.8, 1005.7, 947.5, 930.7 (major peaks). Elemental analysis: Found (acid): C,
48.39; H, 4.41; S, 6.00%. C87H96O48P4S4 requires C, 48.34; H, 4.48; S, 5.93%. Finally, acid
22
(40 mg, 0.0185 mmol) was dispersed in water (2 mL) and solid sodium bicarbonate
(19 mg, 0.22 mmol, 12 equiv.) was added. After 20 min, the resulting solution was frozen
and lyophilized under high vacuum (0.1 mm Hg) to give 23 as a dodecasodium salt of 22
(47 mg), as a nonhygroscopic white powder.
4. Conclusions
In conclusion, using simple and readily available monomers, an easy and highly
efficient method for the synthesis of new polyanionic dendrimers as potential broad-
spectrum antiviral drugs in their own right was presented. The mild conditions of both the
coupling and virtually quantitative deprotection reactions, provided highly pure and water-
soluble macromolecular materials in good overall yields. For example, the total yields

Molecules 2021, 26, 4754
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of the key compounds were as follows: Second generation acid
8
—64%, third generation
polyacid 11—42%, and second generation acid 18—60%. The roughly estimated time of
the preparation of each generation (two steps—condensation, purification, cleavage of the
terminal esters, and purification) should fit easily within the 2-day-period. The important
advantage of the presented strategy is direct access to the polyanionic material at each
generation of the prepared dendrimer. Therefore, the synthesis is at least one difficult
synthetic step shorter. This approach seems to be somehow a general methodology, which
enables the transformation of practically any macromolecular compound terminated with
hydroxy functions, 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 skeleton, a key for a structure-activity relationship study.
Although the chemical synthesis of dendrimers is more than two decades old, the
most significant reason hampering the broader use of dendrimers in biomedicine is usually
difficult and their multistep preparation is time-consuming, especially for the high gen-
eration structures. Consequently, developing methodologies offering faster access to the
important macromolecular material is especially warranted.
Supplementary Materials: 1H, 31P, 13C NMR, and mass spectra of the products synthesized in this
work are available online.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the Supplementary
Materials.
Acknowledgments: The author thanks The Polish Academy of Sciences for financial support.
Conflicts of Interest: The author declares no conflict of interest.
Sample Availability: Samples of the polyanionic dendrimers 6, 9, 12, 16, 19, 23 are available from
the author.
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