Sizing of amino acid based dendrimers in Langmuir monolayers

Article abstract of DOI:10.1039/a801541d

The size and monolayer morphology of synthesised amphiphilic amino acid based dendrimers is studied in a Langmuir trough using Brewster Angle microscopy.

Full text of DOI:10.1039/a801541d

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Sizing of amino acid based dendrimers in Langmuir monolayers
Suzanne J. E. Mulders,a Arwin J. Brouwer,a Peter Kimkes,†,b
Ernst J. R. Sudhölterb and Rob M. J. Liskamp*,a
a Dept. of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences,
Utrecht University, PO Box 80082, NL-3508 TB Utrecht, The Netherlands
b Dept. of Biomolecular Sciences, Laboratory of Organic Chemistry,
Wageningen Agricultural University, NL-6703 HB Wageningen, The Netherlands
The size and monolayer morphology of synthesised
amphiphilic amino acid based dendrimers is studied in a
Langmuir trough using Brewster Angle microscopy.
4–7 were obtained in a convenient manner. To each generation
a fatty acid moiety was attached affording dendrimers 9–13
(Scheme 2). In addition, the fourth generation dendrimer 6 was
saponified and 14 without a fatty acid chain was obtained for
spreading experiments (Scheme 1).¶
Dendrimers 9–13 were spread on pure water in a Langmuir
trough. In Fig. 1 the surface pressure–area isotherms of 9–13 at
20 ЊC are depicted. The isotherms correspond to a gaseous,
liquid-expanded (LE) and liquid-condensed (LC) state.7 In the
isotherms of dendrimers 9 (1st generation), 10 (2nd gener-
ation) and 11 (3rd generation) a plateau was observed.
The lift-off area A1, obtained by extrapolation of the first
slope of the isotherm to the end of the two-dimensional gas-
phase situation, afforded the molecular area occupied by one
dendrimer molecule. Naturally, this area increased with the
dendrimer generation and is the direct experimentally observed
size of the dendrimer molecule on the water surface when the
molecules start to interact with each other (Table 1).
The synthesis of dendrimers remains an exciting challenge in
view of their size, number of end-groups, properties and pos-
sible applications. Among the presently available large diversity
of dendrimers,1 we are especially interested in the synthesis,
properties and applications of dendrimers which are obtained
by repeated formation of amide bonds.2 Recently we have con-
tributed to this field by describing a novel class of amino acid
based dendrimers and by expanding this to the synthesis of a
diversity of dendrimers.3
Concomitant with increasing possibilities for the synthesis of
dendrimers, there is an increasing desire to be able to study
some of their fundamental properties. Examples of these prop-
erties include size, orientation of end-groups and presence of
cavities.
As a promising approach towards realising part of these aims
we turned to spreading of dendrimers at the water–air interface
using a Langmuir trough.‡ We thought that this convenient
technique would offer one of the few possibilities to directly
determine their size and might also offer some perspective
for the ordering of dendrimer end-groups.4 Furthermore, by
employing Brewster Angle microscopy (BAM) in combination
with the Langmuir technique the monolayer morphology can
be studied by direct imaging.5
Our prototype dendrimers were used for formation of
Langmuir monolayers.3a The dendrimer itself is hydro-
phobic and insoluble in an aqueous system. The carboxylic acid
moiety, which is obtained after saponification of the methyl
ester, can then insert into the water surface. However, in order
to provide sufficient space for a possible undistorted alignment
of the dendrimers and their end-groups a fatty acid chain
was connected first to the carboxylic acid moiety, which pos-
sibly could also lift the dendrimers from the water surface. For
comparison purposes we have included the fourth generation
dendrimer without a fatty acid chain.
From the relatively steep part of the isotherm, the condensed
monolayer, the second intercept A2 was obtained by extra-
polation which gave the area of one dendrimer molecule in the
condensed monolayer. The latter area was roughly one third of
the area taken up by the dendrimer at the phase transition from
the ‘gas’ to the liquid-expanded state (A1) for the first to the
third generation dendrimer, going up to about half for the fifth
generation dendrimer. This might indicate that less change of
structural organisation takes place in dendrimers of higher
generations going from the first to the second phase transition.
The area taken up per Boc end-group varied between 13 and
18 Å2 in the monolayer and is significantly larger in the two-
dimensional ‘gas’ phase where it varied between 38 and 60 Å2.
An estimation of the ‘theoretical’ area of the Boc group by
looking mainly at the tert butyl end amounts to 25 Å2, at least
indicating that the Boc end-groups were not arranged in some
kind of densely packed orderly aligned fashion but were partly
intertwined with the dendrimer skeleton.
Spreading of the fourth generation dendrimer 14, i.e. the
dendrimer without the fatty acid chain, gave a lift-off area of
613 Å2, which is virtually identical to that found for dendrimer
12 having the fatty acid chain. The condensed monolayer area
(A2: 300 Å2) of 14 is somewhat larger than that of 12 (253 Å2),
possibly indicating that the former underwent less further
organisation than the latter upon increasing compression.
Indeed, for dendrimer 14 close to the water surface, there
are probably less alternative arrangements possible than for
dendrimer 12, containing the long, flexible, fatty acid chain.
The dendrimers were synthesised by the convergent method
using 3,5-bis(2-tert-butoxycarbonylaminoethoxy)benzoic acid
methyl ester 1.6 The synthesis of the dendrimers up to the fifth
generation is depicted in Scheme 1.3a Briefly, amino acid ester 1
was used to prepare the ‘surface’ monomer 2 as well as the
‘branching’ monomer 3. After successive BOP§ coupling and
deprotection (by saponification) steps, subsequent generations
† Present address: Graphics Division, Avery Dennison, NL-2394 ZG
Hazerswoude, The Netherlands.
‡ A thermostatted (0.1 ЊC), computer controlled Lauda Langmuir
balance FW2 installed on a vibration-free marble table.
§ BOP reagent = benzotriazol-1-yloxytris(dimethylamino)phosphon-
ium hexafluorophosphate.
¶ The convergent method allows the preparation of pure single molecu-
lar weight monodisperse dendrimers, which were fully characterised by
1H and 13C NMR, FAB and electron-spray mass spectra, HPLC and
GPC.
J. Chem. Soc., Perkin Trans. 2, 1998
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Table 1 Experimental and theoretical areas (Å2) of 9–14 in the monolayer at T = 20 ЊC
Condensed
Dendrimer,
generation
Number of
end-groups
Theoretical
areaa
‘gas’ phase, lift-
off area: A1
monolayer area
b,c
b,c
A1(Boc)
A2
A2(Boc)
A1/A2
9, 1
10, 2
11, 3
12, 4
13, 5
14, 4
2
4
8
16
32
16
50
100
200
400
800
400
104
240
366
620 15
1210
613
1
2
6
52
60
46
39
38
38
29
74
102
253
585
300
0
0
6
3
5
0
15
18
13
16
18
9
3.6
3.2
3.6
2.5
2.1
2.0
0
3
a The theoretical area per end-group, i.e. Boc group, is ca. 25 Å2. b For comparison: A1 and A2 for stearic acid are 24 and 20 Å2, respectively. c Two
independent experiments.
N(H)
N(H)
Boc Boc
Repeating unit:
N
N
Boc Boc
NaOH
N
N
O
N
N
O
O
100%
100%
HCl
2
HO
O
MeO
O
HCl•H
N
H•HCl
N
O
MeO2C
(Boc)2
1:
G1
3
MeO
O
Boc Boc
Boc Boc
N
N
N
N
Boc Boc
Boc Boc
Boc Boc Boc Boc
N
N
N
N
N
N
N
N
O
O
N
N
1. NaOH
1. NaOH
BOP
100%
2 + 3
O O
O
O
O
O
N
N
N
N
2. 3, BOP
2. 3, BOP
84%
65%
N
N
MeO
O
MeO
O
5: MeO2C
(Boc)8
MeO2C
(Boc)4
4:
G3
G2
Boc
Boc
Boc
Boc
Boc
Boc
N
N
N
N
N
N
Boc
N
Boc
N
O
O
N
Boc
Boc
N
O
O
N
N
N
N
O
N
O
Boc
Boc
Boc
NaOH
N
1. NaOH
O
O
HO2C
(Boc)16
G4
14
Boc
MeO2C
(Boc)32
G5
7
N
N
N
N
N
N
2. 3, BOP
O
O
Boc
Boc
60%
N
N
N
N
N
N
N
N
O
O
O
O
MeO
MeO2C
O
(Boc)16
6:
G4
Scheme 1 Convergent synthesis of the first to fifth generation amino acid based dendrimer
The monolayer behaviour of dendrimers 9 to 13 was also
studied by Brewster Angle microscopy (BAM). After spread-
ing, none of the compounds showed formation of domains and
the molecules do not aggregate after spreading of the chloro-
form solution of the dendrimer,|| or crystallise before compres-
sion. The first and second generation dendrimers 9 and 10,
respectively, showed the presence of a plateau in the isotherm
that was not observed with the higher generation dendrimers 12
and 13 (Figs. 1 and 2). Although a plateau is present in the
isotherm of 11, it is less distinct.
At the onset of the plateau (18 mN mϪ1) the first generation
dendrimer 9 started to crystallise and small regular nuclei
appeared, which were observed by BAM [Fig. 3(a)]. As the
compression was continued, the surface pressure remained con-
stant and the formation of nuclei progressed towards the form-
ation of domains until a complete probably polycrystalline
layer was obtained, which finally collapsed at a surface pressure
of 26 mN mϪ1.
|| The dendrimer solutions contained 1 mg mlϪ1 of dendrimer in
chloroform.
In the BAM image of dendrimer 10 the formation of nuclei
was also observed, however there is a noticeable difference.
1536
J. Chem. Soc., Perkin Trans. 2, 1998

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NaOH
1. NaOH
G1-5
MeO2C
(Boc)32
G1-5
MeO2C(CH2)11N(H)CO
(Boc)32
9–13
2. 8, BOP
1,4,5,6,7
8: MeO2C(CH2)11NH2
Boc
Boc
Boc
Boc
N
N
N
N
Boc
Boc
Boc
Boc
N
N
N
N
Boc
Boc
O O
O O
N
N
Boc
N
N
Boc
Boc
N
N
O
O
N
N
Boc
N
N
O
O
N
N
O
N
O
N
N
N
Boc
N
Boc
N
N
O
O
N
N
Boc
N
Boc
N
N
N
O
O
O
N
O
N
O
O
O O
O O
N
N
N
N
N
Boc
Boc
N
N
O
N
N
O
O
Boc
Boc
Boc
N
N
N
N
N
HN
O
O
N
N
N
N
O
N
O
N
N
Boc
O
O
Boc
Boc
N
N
N
N
Boc
Boc
N
N
Boc
Boc
Boc
Boc
G5
13
HO2C(CH2)11N(H)CO
(Boc)32
O
OH
Scheme 2 Synthesis of dendrimers 9–13
ation. During compression the higher generation dendrimers
11, 12 or 13 gave only homogenous BAM images indicative of
the absence of crystallisation. Clearly, the higher generation
dendrimers 11, 12 and 13 fail to crystallise.
BAM studies were also performed at 40 ЊC. Again all com-
pounds gave no domains after spreading, i.e. no aggregation or
crystallisation before compression. During compression crys-
tallisation was not only observed in the images of dendrimers 9
and 10, but also in the third and fourth generation dendrimers
11 and 12, respectively. In addition to crystallisation and/or the
occurrence of domains, meandering structures were observed
with dendrimers 10–12, but not with 9. The fifth generation
dendrimer 13 did not show any crystallisation and/or aggre-
gation. Therefore, it seemed that the increased mobility at a
higher temperature, i.e. 40 ЊC, allowed for an easier orientation
of the third and fourth generation dendrimer for crystallisation.
Interestingly, two different monolayer areas (A2) were observed
at T = 20 ЊC and at T = 40 ЊC for the third to fifth generation
dendrimers. The area at the higher temperature for the fourth
and fifth generation dendrimer was about half that observed at
T = 20 ЊC indicating that either a better or different packing
was achieved at a higher temperature (illustrated by 13 in Fig.
4), since at the higher temperature the crystallisation can better
accommodate the compression.
Fig. 1 Surface pressure–area isotherm at 20 ЊC of compounds 9–13
(from left to right)
Fig. 2 Isotherms of compounds 9 (––) and 10 (- - -) at 20 ЊC
In conclusion, we have shown that investigation of Langmuir
monolayers is a convenient technique for determining the size
of an individual dendrimer molecule at the beginning of the
liquid-expanded state, i.e. when a coherent monolayer starts to
be present as well as at the liquid-condensed phase when the
monolayer is highly ordered. The attachment of a fatty acid
chain was not required per se for successful spreading since
saponification of the methyl ester of one of our dendrimers,
i.e. 14, seemed to be sufficient for study of the Langmuir
monolayers. Finally, the observed areas are smaller than the
After the first incline of the isotherm the pressure remained
constant at π = 22 mN mϪ1. Simultaneously, nucleation cores
became visible. During the plateau in the isotherm further
nuclei formation was observed, but the pressure started to rise
before a fully covered image was observed, i.e. in the BAM
image only domains of crystallised structures appeared, which is
in contrast to observations with dendrimer 9. At π = 28 mN mϪ1
the monolayer of dendrimer 10 collapsed (Fig. 2). Apparently,
the rate of compression is higher than the rate of crystallis-
J. Chem. Soc., Perkin Trans. 2, 1998
1537

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Acknowledgements
We thank Dr E. E. Moret (Dept. Medicinal Chemistry) for
assistance with molecular modelling, C. Versluis (Bijvoet
Centre for Biomolecular Research) as well as R. Fokkens of the
Institute of Mass Spectrometry of the University of Amster-
dam for recording FAB and electron-spray mass spectra. These
investigations were supported in part (S. J. E. M. and A. J. B.)
by The Netherlands Foundation for Chemical Research (SON)
with financial aid from The Netherlands Technology found-
ation (STW).
References
1 For recent reviews see, e.g. D. A. Tomalia, A. M. Naylor and W. A.
Goddard III, Angew. Chem., Int. Ed. Engl., 1990, 29, 138; G. R.
Newkome, C. N. Moorefield and G. R. Baker, Aldrichim. Acta, 1992,
25, 31; F. H.-B. Mekelburger, W. Jaworek and F. Vögtle, Angew.
Chem., Int. Ed. Engl., 1992, 31, 1571; G. R. Newkome, C. N.
Moorefield and F. Vögtle, Dendritic Molecules Concepts, Synthesis,
Perspectives, VCH, Weinheim, 1996.
2 D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin,
J. Roeck, J. Ryder and P. Smith, Macromolecules, 1986, 19, 2466;
A. M. Naylor, W. A. Goddard III, G. E. Kiefer and D. A. Tomalia,
J. Am. Chem. Soc., 1989, 111, 2339; D. A. Tomalia, Adv. Mater., 1994,
6, 529; P. R. Dvornic and D. A. Tomalia, Macromol. Symp., 1994, 88,
123; G. R. Newkome, X. Lin and C. D. Weis, Tetrahedron Asymm.,
1991, 2, 957; K. E. Uhrich and J. M. J. Fréchet, J. Chem. Soc., Perkin
Trans. 1, 1992, 1623; C. Rao and J. P. Tam, J. Am. Chem. Soc., 1994,
116, 6975; J. Shao and J. P. Tam, J. Am. Chem. Soc., 1995, 117, 3893;
L. J. Twymam, A. E. Beezer and J. C. Mitchell, Tetrahedron Lett.,
1994, 35, 4423; J. C. Spetzler and J. P. Tam, Int. J. Pept. Protein Res.,
1995, 45, 78; R. Roy, W. K. C. Park, Q. Wu and S.-N. Wang,
Tetrahedron Lett., 1995, 36, 4377; D. Zanini, W. K. C. Park and
R. Roy, Tetrahedron Lett., 1995, 36, 7383; P. J. Dandliker,
F. Diederich, M. Gross, C. B. Knobler, A. Louati and E. M. Sanford,
Angew. Chem., Int. Ed. Engl., 1994, 33, 1739; P. J. Dandliker,
F. Diederich, J.-P. Gisselbrecht, A. Louati and M. Gross, Angew.
Chem., Int. Ed. Engl., 1995, 34, 2725; S. Mattei, P. Seiler, F. Diederich
and V. Gramlich, Helv. Chim. Acta, 1995, 78, 1904.
3 (a) S. J. E. Mulders, A. J. Brouwer, P. van der Meer and R. M. J.
Liskamp, Tetrahedron Lett., 1997, 38, 631; (b) S. J. E. Mulders, A. J.
Brouwer and R. M. J. Liskamp, ibid., 1997, 38, 3085.
4 To our best knowledge there are only three other studies of
dendrimer behaviour at the air–water interface: P. M. Saville, P. A.
Reynolds, J. W. White, C. J. Hawker, J. M. J. Fréchet, K. L. Wooley,
J. Penfold and J. R. P. Webster, J. Phys. Chem., 1995, 99, 8283; Z. Bo,
X. Zhang, X. Yi, M. Yang, J. Shen, Y. Rehn and S. Xi, Polym. Bull.,
1997, 38, 257; Y. Sayed-Sweet, D. M. Hedstrand, R. Spindler and
D. Tomalia, J. Mater. Chem., 1997, 7, 1199.
Fig. 3 BAM image of compound 9 (a) in the plateau (at π = 18
mN mϪ1) and (b) in the monolayer area (18 < π < 26 mN mϪ1
)
5 M. A. Cohen Stuart, R. A. J. Wegh, J. M. Kroon and E. J. R.
Sudhölter, Langmuir, 1996, 12, 2863.
6 Convergent method: C. J. Hawker and J. M. J. Fréchet, J. Chem. Soc.,
Chem. Commun., 1990, 1010; J. M. J. Fréchet, C. J. Hawker and A. E.
Phillippides, US Pat. 5 041 516 (August 20, 1991); C. J. Hawker and
J. M. J. Fréchet, J. Am. Chem. Soc., 1990, 112, 7638.
7 A. Ulman, Ultrathin Organic Films, Academic Press, New York, 1991,
p. 113.
Fig. 4 Isotherms of 13 at T = 20 ЊC (––) and at T = 40 ЊC (- - -)
theoretical areas taken up by the Boc groups or the aromatic
residues of the dendrimer skeleton (data not shown), which
indicates that at least a simple parallel arrangement is absent.
Paper 8/01541D
Received 23rd February 1998
Accepted 6th May 1998
1538
J. Chem. Soc., Perkin Trans. 2, 1998