Synthesis and biological activity of polygalloyl-dendrimers as stable tannic acid mimics

Article abstract of DOI:10.1016/S0960-894X(02)00245-7

Inspired by the structure of tannic acid, first- to third-generation dendrimers containing two, four, and eight galloyl moieties were synthesized. Stability, antioxidant activity and collagen cross-linking activity of the natural product and its dendrimer analogues were compared. The experimental results indicate that polygalloyl dendrimers might be used as new lead compounds to improve the long-term healing characteristics of burn wounds.

Full text of DOI:10.1016/S0960-894X(02)00245-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1567–1570
Synthesis and Biological Activity of Polygalloyl-Dendrimers as
Stable Tannic Acid Mimics
S. Bart A. Halkes, Ioannis Vrasidas, Gilbert R. Rooijer, Albert J. J. van den Berg,
Rob M. J. Liskamp and Roland J. Pieters*
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082,
3508 TB Utrecht, The Netherlands
Received 15 February 2002; revised 9 April 2002; accepted 12 April 2002
Abstract—Inspired by the structure of tannic acid, first- to third-generation dendrimers containing two, four, and eight galloyl
moieties were synthesized. Stability, antioxidant activity and collagen cross-linking activity of the natural product and its dendrimer
analogues were compared. The experimental results indicate that polygalloyl dendrimers might be used as new lead compounds to
improve the long-term healing characteristics of burn wounds. # 2002 Elsevier Science Ltd. All rights reserved.
Advances in surgical techniques and the general care of
burn patients over the past century have resulted in a
strong decrease in the mortality due to thermal injury.
Attention now has to be focused on the development
of therapeutic regimens that can improve the cosmetic
outcome of the treatment of extensively burned
patients. One of the most promising candidates in this
respect is tannic acid (Fig. 1), a mixture of poly-
galloylglucose esters or gallotannins, which is found in
high concentrations in the galls of Rhus and Quercus
species.1 It has been topically applied with success in the
past, in particular in the period between 1920 and 1940
just before the introduction of penicillin.2 In the early
1990s, the beneficial effects on wound healing were fur-
ther substantiated as it was shown that tannic acid
positively influenced inflammation and processes involved
in granulation tissue formation, re-epithelialization, and
scar-tissue formation.3,4
structurally related analogues that combine a higher
stability with a similar biological activity.
To achieve this objective we have chosen to synthesize
first-, second-, and third-generation dendrimers con-
taining two, four, and eight galloyl moieties, respec-
tively. Dendrimers were used with
a
3,5-di(2-
aminoethoxy)benzoic acid repeating unit.7 They were
previously used as multivalent scaffolds8 for carbohy-
drate ligands which resulted in enhanced binding to
cholera toxin and galectins.9 A convergent synthesis was
used that started with the benzyl protection of the three
hydroxyl groups of the starting material methyl gallate
1 (Scheme 1). After saponification of the methyl ester
However, a major shortcoming of tannic acid is its
relative instability in pharmaceutical formulations.
Facile hydrolysis of its meta-depsidic galloyl ester bonds
results in the release of gallic acid, besides other low
molecular weight constituents.5 Since elevated gallic
acid levels have recently been related to the hepatotoxic
effects ascribed to the tannic acid treatment of burns in
the past,2,6 we considered it worthwhile to synthesize
Figure 1. General structural representation of tannic acid. On average,
five to eight gallic acid residues are attached to the glucose-nucleus at the
positions shown, depending on the plant source. The larger structures
contain the encircled labile meta-depsidic galloyl ester bond.
*Corresponding author. Tel.: +31-30-253-6944; fax: +31-30-253-
6655; e-mail: r.j.pieters@pharm.uu.nl
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00245-7

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the resulting carboxylic acid 2 was coupled to the first
generation dendrimer, diamine 3,7 using the peptide
coupling reagent BOP10 in the presence of base in di-
chloromethane. The coupling product was subjected to
basic conditions to hydrolyze the methyl ester to pro-
duce compound 4. Removal of the benzyl groups by
hydrogenation yielded the di-galloyl compound 5. In
order to create the larger structures, 4 was coupled to
dendrimer monomer 3, which after removal of methyl
ester functionality and benzyl groups gave the tetra-
galloyl dendrimer 7. Repeating the sequence from the
benzyl protected 6 resulted ultimately in octa-galloyl
dendrimer 9 and completed the series.11
The higher molecular weight polygalloylglucose esters
in tannic acid contain a pentagalloylglucose nucleus to
which additional galloyl groups are attached by depsidic
linkages.12 Due to the lability of these linkages, tannic
acid in solution is easily degraded. Stability-testing was
performed with ethanolic solutions of a highly purified
tannic acid product (BrewtanTM, Omnichem B.V.,
Wetteren, Belgium; 3.6 mM) and 9 (0.6 mM) which were
kept for 2 and 4 weeks. Degradation was monitored
using a modified version of the HPLC-system described
by Beasley et al.13 In agreement with literature data,5
degradation of tannic acid was characterized by the
appearance in the HPLC-chromatogram of peaks cor-
responding with mono-, di-, and trigallic acid deriva-
tives (data not shown). In comparison, the stability of
the synthetic analogue 9 was found to be far superior.
Even after 4 weeks in solution, only the single peak of 9
was detectable. No additional peaks of degradation
products were present (data not shown).
Two biological assays were used in this study. One assay
determines antioxidant activity and another provides
information about the collagen cross-linking abilities.
The assays were selected because they can provide
important information with which it seems possible to
predict the in vivo efficacy of new drugs for the treat-
ment of burns. Thus, the tannic acid product which
improved wound healing in an animal-experimental as
well as clinical setting,3,4 is also found to be highly
active in these assays.
The antioxidant activity of Brewtan, gallic acid (Carl
Roth GmbH, Karlsruhe, Germany), and the synthetic
analogues 5, 7, and 9, was determined as the decolor-
ization of the stable free radical 2,2-diphenyl-1-picryl-
hydrazyl (DPPH; Sigma Chemical Co., St. Louis, MO,
USA).14,15 Antioxidant activity varied considerably
among the compounds tested (Table 1). Tannic acid was
most active in this respect. The antioxidant activity of 9
was slightly but significantly lower and further declined
Table 1. Antioxidant and collagen cross-linking activity of tannic
acid, gallic acid, and the synthetic analogues 5, 7, and 9
Compd
Antioxidant activity
ED50 (mM)a,b
Collagen cross-linking
Ts (ꢀC)a,c
Tannic acid
Gallic acid
5
7
9
2.9 (ꢁ0.2)
14.3 (ꢁ3.6)d
13.7 (ꢁ1.0)d
7.4 (ꢁ0.6)d
4.7 (ꢁ0.4)d
59.5 (ꢁ0.3)e
57.5 (ꢁ0.2)d,e
58.4 (ꢁ0.3)e
57.9 (ꢁ0.1)d,e
57.0 (ꢁ0.1)d
aValues are means of five experiments, standard error of the mean is
given in parentheses.
Scheme 1. Synthesis of the galloyl dendrimers. Reaction conditions:
(a) (i) BnCl, K2CO3, DMF, 100 ꢀC, 14 h, 81%; (ii) NaOH, H2O/
MeOH/dioxane, 95%; (b) (i) 2, BOP, iPr2NEt, CH2Cl2, 82%; (ii)
NaOH, H2O/MeOH/dioxane 92%; (c) H2, Pd/C, DMF, quant for 5,
51% for 7, 91% for 9; (d) (i) 3, BOP; iPr2NEt, CH3CN/DMF; (ii)
NaOH, H2O/MeOH/dioxane, 78% for 6, 36% for 8.
bEffective dose giving 50% decolorization of DPPH.
cTemperature at which collagen shrinkage is initiated. Values depicted
were obtained with sample concentrations of 4 mM in 85% glycerol.
Ts for untreated collagen matrix is 56.4 (ꢁ0.2)ꢀC.
dSignificantly different from tannic acid, p <0.05.
eSignificantly different from control, p <0.05.

S. B. A. Halkes et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1567–1570
1569
in the series of 7, 5, and gallic acid. However, these dif-
ferences can be totally accounted for by the number of
galloyl moieties per molecule and the relative size of the
backbone to which these galloyls are attached.16
Netherlands Organization for International Coopera-
tion in Higher Education (NUFFIC) for a Huygens
fellowship. The Royal Netherlands Academy of Arts
and Sciences (KNAW) is gratefully acknowledged for a
fellowship to R.J.P.
Effects on cross-linking of collagen were assessed
according to the method of Heijmen et al.17 (Table 1).
Compounds were dissolved in 85% glycerol and incu-
bated for 48 h at room temperature with purified skin
collagen matrices. The degree of collagen cross-linking
was determined by the hydrothermal shrinkage tem-
perature (Ts). The Ts denotes the change in molecular
conformation of collagen from triple helix to random
coil and will be increased when cross-linking has taken
place.18 The strongest increase in Ts was observed for
the collagen matrices pretreated with tannic acid. For 5,
elevation of the Ts was less pronounced but not sig-
nificantly different from tannic acid. In comparison, the
cross-linking capacity of 7 and 9 lagged behind, the Ts
of collagen matrices pretreated with 9 not even being
distinct from the controls without tannins/dendrimers.
These results fit within the concept for tannic acid–col-
lagen interactions as proposed by Haslam.19 In this
model, pentagalloylglucose as the prototype molecule
representative for tannic acid, has in its most favored
conformation a disc-like shape with a thickness of
approximately 0.7 nm and a diameter of approximately
2.1 nm. These dimensions are ideally suited to infiltrate
in the gaps between the tropocollagen molecules that
compose the collagen fibrils. Hydrophobic effects are
thought to initiate the association with hydrogen bond-
ing acting to reinforce the structure. The decline in col-
lagen cross-linking capacity going from the first- to
third-generation dendrimers is consistent with the disc
model. Bis-galloyl compound 5 is more likely to adopt a
flat conformation than the more three-dimensional 7
and 9. Alternatively, spatial contraints may simply not
allow the accommodation of the significantly larger
structures 7 and 9, as compared to 5 or tannic acid.
Furthermore the difference in hydrophobicity between
the galloyl dendrimers cannot be ruled out as a deter-
mining factor either considering the importance of
hydrophobicity in the initiation of the association.
References and Notes
1. (a) Haslam, E. In Plant Polyphenols—Vegetable Tannins
Revisited; Cambridge University Press: Cambridge, 1989. (b)
Nishizawa, M.; Yamagishi, T.; Nonaka, G.-I.; Nishioka, I.
J. Chem. Soc., Perkin Trans. 1 1982, 2963.
2. Halkes, S. B. A.; Van den Berg, A. J. J.; Hoekstra, M. J.;
Du Pont, J. S.; Kreis, R. W. Wounds 2001, 13, 144.
3. Hoekstra, M. J.; Dutrieux, R. P.; Kreis, R. W. Tannin
Ointment Alternated with Silversulfadiazine 1% Cream Versus
Tannin Ointment Alternated with Silversulfadiazine 1% Liquid.
Pre-clinical investigation report, no: 92871008. Burns Research
Institute: Beverwijk, The Netherlands, 1992.
4. Kreis, R. W.; Vloemans, A. F. P. M. Tannin Ointment.
Clinical Pilot Trial Report; Burns Research Institute: Bev-
erwijk, The Netherlands, 1992.
5. Delahaye, P.; Verzele, M. J. Chromatogr. 1983, 265, 363.
6. Halkes, S. B. A.; Van den Berg, A. J. J.; Hoekstra, M. J.;
Du Pont, J. S.; Kreis, R. W. Burns 2001, 27, 299.
7. (a) Mulders, S. J. E.; Brouwer, A. J.; Liskamp, R. M. J.
Tetrahedron Lett. 1997, 38, 3085. (b) Mulders, S. J. E.;
Brouwer, A. J.; van der Meer, P. G. J.; Liskamp, R. M. J.
Tetrahedron Lett. 1997, 38, 631. (c) Brouwer, A. J.; Mulders,
S. J. E.; Liskamp, R. M. J. Eur. J. Org. Chem. 2001, 1903.
8. For a review on the potential benefits of multivalency, see:
Mammen, M.; Chio, S.-K.; Whitesides, G. M. Angew. Chem.,
Int. Ed. 1998, 37, 2755.
9. (a) Vrasidas, I.; de Mol, N. J.; Liskamp, R. M. J.; Pieters,
R. J. Eur. J. Org. Chem. 2001, 4685. (b) Andre, S.; Pieters,
R. J.; Vrasidas, I.; Kaltner, H.; Kuwabara, I.; Liu, F.-T.; Lis-
kamp, R. M. J.; Gabius, H.-J. ChemBioChem. 2001, 2, 822.
10. BOP=1H-benzotriazol-1-yloxy)tris(dimethylamino) phos-
phonium hexafluorophosphate).
11. 13C NMR data, see ref 9a for numbering of the dendrimer
backbone, Gal=galloyl moiety. 5: 13C NMR (CD3OD,
75.4 MHz) d 171.7, 170.8 (C¼OI, II), 161.1 (C3/5), 146.6 (Gal-
C3/5), 138.1, 136.8 (Gal-C4, C1), 126.0 (Gal-C1), 109.2 (C1),
107.9 (Gal-C2/6), 106.3 (C4), 67.7 (CH2a), 40.5 (CHb2); ESI-MS:
m/z=545.1 [M + H]+ (100%), 567.0 [M + Na]+; 7: 13C
NMR (CD3OD, 75.4 MHz) d 170.8, 170.2 (C¼OI, II, III), 161.3,
0
0
161.1 (C3/5, C3 /5 ), 146.7 (Gal-C3/5), 138.1, 137.5 (Gal-C40, C1,
As shown in this paper, the synthetic analogues 5, 7,
and 9, retain a satisfactory, albeit lower, biological
activity in comparison to tannic acid, but have the
advantage of a much enhanced stability. Therefore,
polygalloyl-dendrimers may serve as potential leads for
the development of new topical drugs to be used in burn
wound treatment. Considering the importance of both
the number of galloyl moieties and possibly molecular
size for the biological activity, it might finally be of
interest to search for other synthetic analogues of tannic
acid in which the backbone is reduced in dimension but
still can accomodate a maximal load of gallic acid residues.
0
0
C1 ), 126.0 (Gal-C1), 109.4, 107.2, 106.1, 105.7 (C2/6, C2 /6 , C4,
a0
0
C4 ), 107.9 0 (Gal-C2/6), 67.8 (CH2 ), 67.5 (CH2a), 40.8 (CHb2),
+
40.5 (CHb2 ); MALDI-TOF-MS: m/z=1293 [M H]+
(100%); 9: 13C NMR (DMSO-d6, 75.4 MHz)0 d 167.0, 166.2
00 00
0
(C¼OI,
IV), 159.8 (C3 /5 ), 159.7 (C3 /5 ), 159.4 (C3/5),
II, III,
10
100
145.8 (C3/5), 136.7, 136.6, 136.5 (Gal-C4, C01, 0 C ,00C ), 124.8
00
0
(Gal-C1), 107.8, 106.2, 104.4, 104.1 (C2/6, C2 /6 , C2 /6 , C4, C4 ,
a00
a0
00
C4 ), 107.1 (Gal-C2/6), 66.6 (CH2 ), 66.5 (CH2 ), 66.3 (CHa2),
0
00
(CH2b,b ,b signals obscured by DMSO-d6 signals).
12. Armitage, R.; Bayliss, G. S.; Gramshaw, J. W.; Haslam,
E.; Haworth, R. D.; Jones, K.; Rogers, H. J.; Searle, T. J.
Chem. Soc. 1961, 1842.
13. Beasley, T. H.; Ziegler, H. W.; Bell, A. D. Anal. Chem.
1977, 49, 238. HPLC analysis was performed on a Gilson
chromatographic system (Gilson 360 pumps combined with a
Gilson 805 manometric module, a Gilson 811C dynamic
mixer, a Gilson 234 autoinjector, and a Gilson 170 diode-
array-detector) equipped with an Alltima Silica 5U column,
250 Â 4.6 mm (Alltech, Deerfield, IL, USA; Cat. No. 88127).
Solvent system: mobile phase A: hexane with 0.5%
Acknowledgements
S.B.A.H. is financially supported by ‘Stichting ACH-
MEA Slachtoffer en Samenleving’, Zeist and the Dutch
Burn Foundation, Beverwijk. I.V. is grateful to the

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CH3COOH and mobile phase B: EtOH/C4H8O (3:1) with
0.5% CH3COOH, gradient: 0–60 min: isocratic 27% B, 60–
70 min: 27!100% B, 70–75 min: isocratic 100% B, 75–80 min:
100!27% B, 80–90 min: isocratic 27% B, total flow: 1 mL/
min. Injection volume: 10l. Detection: 254 and 280 nm.
14. Blois, M. S. Nature 1958, 181, 1199.
15. Test compounds were serially diluted (0.1–180 mM) in flat-
bottom microtiter plates and incubated with a 250 mM DPPH
solution in 75% ethanol for 15 min at room temperature.
Subsequently, absorbance was read at 550 nm using an auto-
matic ELISA-reader (SLT Labinstruments, Salzburg, Aus-
tria). ED50 values were calculated by linear regression analysis
of the absorbance curves obtained for each compound.
16. Since the galloyl groups present in tannic acid and 5, 7,
and 9, are considered to be the parts in these molecules to
accept the free radical from DPPH, it is possible to predict
antioxidant activities on basis of the experimental ED50-value
of gallic acid using the formula: ED50(x)=ED50(gallic acid)/
n(x)*RMW(x), in which n(x) is the total number of galloyl
groups in compound x and RMW(x) the ratio between the
molecular weight of all galloyl groups present in compound x
and the total molecular weight of this compound. The latter
ratio is introduced in the formula to correct for the differences
in weight of the backbone structure to which the galloyl
groups are attached. This scaffold is significantly larger in the
dendrimeric analogues yielding RMW-values of 0.88, 0.56, 0.47,
and 0.44 for tannic acid and 5, 7, and 9, respectively. ED50-
values calculated according to the above formula are 2.0
(ꢁ0.5), 12.7 (ꢁ3.2), 7.5 (ꢁ1.9), and 4.1 (ꢁ1.0) mM for tannic
acid and 5, 7, and 9, respectively. These values do not differ
significantly from the experimental data.
17. Heijmen, F. H.; Du Pont, J. S.; Middelkoop, E.; Kreis,
R. W.; Hoekstra, M. J. Biomaterials 1997, 18, 749.
18. Heidemann, E. J. Soc. Leather Technol. Chem. 1982, 66,
21.
19. Haslam, E. In Practical Polyphenolics, from Structure to
Molecular Recognition and Physiological Action; Cambridge
University Press: Cambridge, 1998; p. 396.