Self-association of protected Newkome-type second-generation dendrimers at nanomolar level concentrations in aqueous solution

Article abstract of DOI:10.1021/ma0498215

Protected Newkome-type second-generation dendrimers (based on Lin's amine) were synthesized with a pyrene moiety attached to the core. The photophysical property in aqueous solution of the protected dendrimers shows self-association behavior in water. Pyr

Full text of DOI:10.1021/ma0498215

5364
Macromolecules 2004, 37, 5364-5369
Self-Association of Protected Newkome-Type Second-Generation
Dendrimers at Nanomolar Level Concentrations in Aqueous Solution
Su bh en d u K. Moh a n ty, Sh ya m a la Th ir u n a vu k a r a su , Su n d a r a ba bu Ba sk a r a n ,* a n d
Ash ok K. Mish r a *
Department of Chemistry, Indian Institute of Technology Madras, Chennai-600036, India
Received J anuary 27, 2004; Revised Manuscript Received May 4, 2004
ABSTRACT: Protected Newkome-type second-generation dendrimers (based on Lin’s amine) were
synthesized with a pyrene moiety attached to the core. The photophysical property in aqueous solution
of the protected dendrimers shows self-association behavior in water. Pyrene excimer emission at 475
nm is observed in water even at very low concentrations of protected dendrimer (ca. 5 × 10-8 M). This
emission band is absent in other solvents even up to a concentration of 10-5 M. The corresponding
unprotected dendrimer does not show the pyrene excimer fluorescence. The amide of pyrene butyric acid
with tert-butylamine shows the formation of excimer, albeit with very low intensity. Quenching studies
on the dendrimer with hydrophilic quencher iodide anion (I-) reveal that there is significant quenching
of fluorescence intensity in the case of N-tert-butyl-4-pyren-1-ylbutyramide as compared to that of the
pyrene-attached second-generation protected dendrimer. This shows that the pyrene moiety in the case
of the protected dendrimer is significantly shielded from the surrounding.
formation of the corresponding triester 6.7 Protection of the
In tr od u ction
nitrogen with benzyl chloroformate produced 7, and hydrolysis
of the tert-butyl groups using formic acid resulted in the
formation of the triacid 8 quantitatively. Triacid 8 when
refluxed with catalytic amount of pTSA in ethanol and benzene
resulted in the triethyl ester 9 in quantitative yield. Triethyl
ester 9 under catalytic hydrogenation yielded the amine
derivative 10. The monomers 6 and 10 were then used for the
synthesis of dendrimers, tetraamide 2 and 3, respectively
(Scheme 2).
The triacid 8 was coupled with monomers 6 and 10 under
peptide coupling conditions, resulting in the second-generation
dendrimers 11 and 12, respectively. Hydrogenation of the
carbamates 11 and 12 gave the corresponding free amines 13
and 14, respectively, which were then coupled to pyrenebutyric
acid under similar peptide coupling conditions as was used
for the preparation of the second-generation dendrimers 11
and 12 (Scheme 2). Using similar reaction conditions, monoa-
mide 1 was synthesized from pyrenebutyric acid and tert-
butylamine.
Rea gen ts for Syn th esis. All chemicals were reagent grade.
Tetrahydrofuran (THF) used was freshly distilled from sodium
benzophenone. Column chromatography was performed on
silica gel (100-200 mesh) while TLC was performed on
aluminum-backed plates coated with 0.25 mm silica gel.
N -T r is[(2-{[(t r is{[2-(t er t -b u t ox yc a r b on y l)e t h o x y ]-
m eth yl}m eth yl)a m in o]ca r bon yl}eth oxy)m eth yl]m eth yl}-
4-p yr en -1-ylbu tyr a m id e (Tetr a a m id e 2). To a solution of
4-(1-pyrene)butyric acid (58 mg, 0.2 mmol) in dry THF (1 mL)
was added EDCl (46 mg, 0.2 mmol), HOBt (27 mg, 0.2 mmol),
and Et3N (34 µL, 0.2 mmol). To the stirred solution at room
temperature was added N-tris[(2-{[(tris{[2-(tert-butoxycarbonyl)-
et h oxy]m et h yl}m et h yl)a m in o]ca r bon yl}et h oxy)m et h yl]-
methylamine8 (360 mg, 0.2 mmol) dissolved in dry THF (3 mL).
The reaction was stirred at room temperature, and after the
completion of reaction, as indicated by TLC, THF was removed
under vacuum, and the reaction mixture was diluted with
EtOAc (10 mL) and washed with water (5 mL), 0.5 M HCl
solution (5 mL), and then with saturated solution of brine
(5 mL). The organic layer was separated, dried over anhydrous
Na2SO4, and concentrated under reduced pressure to yield
the crude compound, which was purified over silica gel using
50-80% gradient elution of EtOAc in hexane to yield the
pure pyrene attached dendrimer, tetraamide 2 (200 mg), in
48% yield. IR (neat): 3440, 2912, 1728, 1667, 1523, 1369,
Higher generation dendrimers assume a globular
shape and as such act like micelles1 with the core of
the dendrimer completely shielded from the environ-
ment. On the other hand, lower generation dendrimers
due to the partial shielding of the core from the
environment reveal greater detail about the structural
features of the dendrimer. Hyperbranch dendrimers2
have been the focus of recent studies pertaining to
structural morphology of dendrimers. Cardona and co-
workers3 have reported the synthesis and the photo-
physical properties of Newkome-type4 dendrimers with
different fluorescent chromophores (fluorophores). A
fluorophore when attached to the core provides insight
into the physical properties of the molecule as a whole.
Pyrene,5 due to its long fluorescence lifetime and its
characteristic excimer emission, is an ideal fluorophore
for such studies. Sluch and co-workers6 reported the
excitation dynamics of poly(allylcarbosilane) dendrimers
with pyrene at the core at a concentration of ca. 10-3
M. Recently Chou and co-workers7 reported the self-
association of hyperbranched poly(sulfone-amine) den-
drimers in water. This was studied by the addition of
pyrene-labeled dendrimer to an aqueous solution con-
taining free pyrene and monitoring the resultant exci-
mer emission.
A protected Newkome-type second-generation den-
drimer has a hemiellipsoid structure (Figure 1) with the
core being accessible to the environment. In this article,
we report the self-association of such a dendrimer in
water even at nanomolar concentration (5 × 10-8 M).
Exp er im en ta l Section
Syn th esis of Den d r im er s. Scheme 1 depicts the synthesis
of Lin’s amine, monomers 6 and 10, starting from TRIS. Tris-
(hydroxymethyl)aminomethane was treated with tert-butyl
acrylate in the presence of DMSO and NaOH, resulting in the
* Corresponding authors. E-mail: sbhaskar@iitm.ac.in (S.B.);
mishra@iitm.ac.in (A.K.M.).
10.1021/ma0498215 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/18/2004

Macromolecules, Vol. 37, No. 14, 2004
Newkome-Type Second-Generation Dendrimers 5365
F igu r e 1. Second-generation dendrimer having tert-butyl and ethyl groups at the periphery (tetraamides 2 and 3, respectively)
and the N-tert-butyl-4-pyren-1-ylbutyramide {monoamide 1}.
Sch em e 1
1257 cm-1. 1H NMR (CDCl3, 400 MHz): δ 1.42 (s, 81H); 2.20-
2.44 (m, 28H); 3.57-3.75 (m, 52H); 6.35 (bs, 3H); 7.72-8.30
(m, 9H). 13C NMR (CDCl3, 100 MHz): δ 27.9, 28.0, 36.0, 37.0,
59.8, 60.0, 60.3, 67.0, 67.4, 69.0, 80.4, 123.4, 124.6, 124.7, 124.8,
124.9, 125.0, 125.6, 125.7, 126.4, 127.2, 127.3, 127.4, 128.6,
129.7, 136.1, 170.8, 171.0, 173.1. MALDI-TOF MS (R-cyano-
4-hydroxycinnamic acid matrix): 2069.11 (M+, calcd 2069.35),
2092.89 (M + Na)+, 2108.79 (M + K)+.
N -Tr is[(2-{[(t r is{[2ca r b oxye t h oxy]m e t h yl}m e t h yl)-
a m in o]ca r bon yl}eth oxy)m eth yl]m eth yl}-4-p yr en -1-ylbu -
tyr a m id e (Un p r otected Den d r im er ). Tetraamide 2 (140
mg, 0.07 mmol) was stirred in 10 mL of 96% formic acid for

5366 Mohanty et al.
Macromolecules, Vol. 37, No. 14, 2004
Sch em e 2
24 h. Then the formic acid was removed at reduced pressure
to produce the title compound in quantitative yield. H NMR
(15 mL) was added 10% Pd/C (170 mg), and the mixture was
stirred at room temperature under an atmosphere of hydrogen
(balloon). After completion of the reaction catalyst was filtered
using a pad of Celite, and the filterate was concentrated under
reduced pressure to yield the pure amine 10 (1.1 g, 87% yield).
1
(CD3COCD3, 400 MHz): δ 2.20-2.44 (m, 28H); 3.57-3.75 (m,
52H); 6.35 (bs, 3H); 7.72-8.30 (m, 9H). 13C NMR (CDCl3, 100
MHz): 28.0, 36.0, 37.0, 60.0, 60.3, 67.0, 67.4, 69.0, 80.4, 123.4,
124.6, 124.7, 124.8, 124.9, 125.0, 125.6, 125.7, 126.4, 127.2,
127.3, 127.4, 128.6, 129.7, 136.1, 170.8, 171.0 MS (ESI): 1565
(M + 1).
IR (neat): 3440, 2992, 1731 cm-1 1H NMR (CDCl3, 400
.
MHz): δ 1.26 (t, J ) 7.0 Hz, 9H), 1.68 (bs, 2H), 2.54 (t, J )
6.3 Hz, 6H), 3.31 (s, 6H), 3.69 (t, J ) 6.3 Hz, 6H), 4.14 (q, J )
7.0 Hz, 6H). 13C NMR (CDCl3, 100 MHz): δ 14.2, 35.1, 60.4,
66.8, 66.98, 72.8, 171.5. MS (ESI): 422 (M + 1).
Synthesis ofN-tert-Butyl-4-pyren-1-ylbutyramide (Monoa-
m id e 1). To a stirred solution of 4-(1-pyrene)butyric acid (50
mg, 0.17 mmol) in THF (1 mL) was added HOBt (24 mg, 0.17
mmol) at room temperature. The reaction mixture was stirred
for 5 min, after which Et3N (29 µL, 0.2 mmol) was added
followed by EDCl (40 mg, 0.2 mol). Finally, tert-butylamine
(22 µL, 0.2 mol) was added dropwise to the reaction mixture.
The reaction mixture was then allowed to stir at room
temperature. After completion of the reaction THF was
removed under vacuum, and the crude mass was diluted with
ethyl acetate (10 mL). The organic layer was then washed
successively with 0.5 M HCl (5 mL) and saturated brine
solution (5 mL). The organic layer was separated, dried over
anhydrous Na2SO4, and concentrated under reduced pressure
to yield the crude compound, which was purified over silica
gel using gradient elution of 20-30% EtOAc in hexane to
afford the pure compound (28 mg) in 47% yield. IR (neat):
Ben zyl N-Tr is[(2-{[(tr is{[2-(eth oxyca r bon yl)eth oxy]-
m eth yl}m eth yl)a m in o]ca r bon yl}eth oxy)m eth yl] Meth -
ylca r ba m a te (12). To a solution of triacid 8 in dry THF (6.0
mL) was added HOBt (115 mg, 0.85 mmol), Et3N (0.42 mL,
3.5 mmol), and EDCl (575 mg, 3.5 mmol). The amine 10 in
THF was then added, and the reaction mixture was stirred at
room temperature for 24 h. After completion of the reaction,
the THF was removed under vacuum and the reaction mixture
was diluted with ethyl acetate. The organic layer was then
washed with 0.5 M HCl and brine, separated, dried over
anhydrous Na2SO4, and concentrated to yield the crude
compound, which was then purified by column chromatogra-
phy using 70-80% EtOAc in hexane as the eluent to yield the
pure second-generation dendrimer, 12 (900 mg, 63% yield). IR
(neat): 3360, 2944, 1731, 1664 cm-1 1H NMR (CDCl3, 400
.
3392, 2998, 1721, 1657, 1257 cm-1
.
1H NMR (CDCl3, 400
MHz): δ 1.26 (t, J ) 7.1 Hz, 27H), 2.40 (t, J ) 6.3 Hz, 6H),
2.52 (t, J ) 6.2 Hz, 18H), 3.64-3.69 (m, 48H), 4.13 (q, J ) 7.0
Hz, 18H), 5.03 (s, 2H), 6.22 (s, 4H), 7.30-7.34 (m, 5H). 13C
NMR (CDCl3, 100 MHz): δ 14.2, 21.0, 34.9, 37.2, 58.8, 59.7,
60.3, 60.6, 67.5, 69.0, 127.9, 128.0, 128.3, 128.4, 136.7, 151.1,
171.0, 171.7. MS (ESI): 1680 (M+).
MHz): δ 1.2 (s, 9H), 2.10 (bs, 4H), 3.27 (t, J ) 6.9 Hz, 2H),
5.15 (s, 1H), 7.16-8.20 (m, 9H). 13C NMR (CDCl3, 100 MHz):
δ 27.5, 28.3, 32.6, 36.8, 51.2, 123.4, 124.7, 124.9, 125.0, 125.1,
125.9, 126.7, 127.3, 127.4, 127.5, 128.8, 129.9, 130.9, 136.0,
172.0. MS (ESI): 344 (M + 1), 343 (M+).
Ben zyl
N-Tr is[2-(et h oxyca r bon yl)et h oxy]m et h yl}-
N-Tr is[(2-{[(tr is{[2-(eth oxyca r bon yl)eth oxy]m eth yl}-
m eth yl)a m in o]ca r bon yl}eth oxy)m eth yl] Meth ylca r ba m -
a te (14). To a solution of ester 12 (900 mg, 0.53 mmol) in
ethanol (7 mL) was added 10% Pd/C (90 mg). The reaction
mixture was stirred at room temperature for 24 h under a
hydrogen atmosphere (balloon). After completion of the reac-
tion the catalyst was filtered using a pad of Celite, and the
filterate was concentrated to yield the pure amine 14 (700 mg,
m eth ylca r ba m a te (9). A mixture of triacid 87 (1.5 g, 3.18
mmol) and a catalytic amount of pTSA in ethanol (3 mL) and
benzene (7 mL) was refluxed using a Dean-Stark apparatus.
After completion of the reaction, the reaction mixture was
diluted with ethyl acetate and washed with a saturated
solution of NaHCO3. The organic layer was separated, dried
over anhydrous Na2SO4, and concentrated to give the crude
compound. The crude compound was purified by column
chromatography using 20% ethyl acetate-hexane as the eluent
to give the pure benzyl N-tris[2-(ethoxycarbonyl)ethoxy]methyl}-
methylcarbamate in quantitative yield (1.7 g). IR (neat): 3392,
1
85% yield). IR (neat): 3360, 2944, 1731, 1664 cm-1. H NMR
(CDCl3, 400 MHz): δ 1.26 (t, J ) 7.1 Hz, 27H), 2.43 (t, J ) 6.0
Hz, 6H), 2.53 (t, J ) 6.2 Hz, 18H), 3.67-3.73 (m, 48H), 4.14
(q, J ) 7.0 Hz, 18H), 6.35 (s, 3H). 13C NMR (CDCl3, 100
MHz): δ 14.2, 18.3, 34.9, 37.2, 58.2, 59.8, 60.4, 60.5, 66.7, 66.9,
171.7, 1714.6. MS (ESI): 1547 (M + 1).
N-Tr is[(2-{[(tr is{[2-(eth oxyca r bon yl)eth oxy]m eth yl}-
m eth yl)am in o]car bon yl}eth oxy)m eth yl]m eth yl}-4-pyr en -
1-ylbu tyr a m id e (Tetr a a m id e 3). To a stirred solution of 4-(1-
pyrene)butyric acid (141 mg, 0.49 mmol) in dry THF (2 mL)
at room temperature was added EDCl (94 mg, 0.49 mmol),
HOBt (66 mg, 0.49 mmol), and Et3N (68 µL, 0.49 mmol)
1
2928, 1734, 1507 cm-1. H NMR (CDCl3, 400 MHz): δ 1.73 (t,
J ) 6.8 Hz, 9H), 3.02 (t, J ) 6.2 Hz, 6H), 4.15 (s, 6H), 4.18 (t,
J ) 6.4 Hz, 6H), 4.62 (q, J ) 7.0 Hz, 6H), 5.53 (s, 2H), 5.75 (s,
1H), 7.77-8.03 (m, 5H). 13C NMR (CDCl3, 100 MHz): δ 14.2,
35.0, 58.7, 60.4, 66.1, 66.8, 69.4, 127.9, 128.0, 128.3, 136.7,
155.0, 171.4. MS (ESI): 557 (M + 1).
Tr is[2-(eth oxyca r bon yl)eth oxy]m eth yl}m eth yla m in e
(10). To a solution of compound 9 (1.7 g, 3 mmol) in ethanol

Macromolecules, Vol. 37, No. 14, 2004
Newkome-Type Second-Generation Dendrimers 5367
F igu r e 2. UV spectra of (A) tetraamide 2 and (B) monoamide
1 (both at 10-5 M concentration) in water.
F igu r e 3. (A) Emission spectra of tetraamide 2 in different
solvents. (B) Normalized spectra of monoamide (10-5 M) and
tetraamide 2 (10-5 M) in water [λex at 342 nm].
followed by a solution of N-tris-[(2-{[(tris{[2-(ethoxycarbonyl)-
et h oxy]m et h yl}m et h yl)a m in o]ca r bon yl}et h oxy)m et h yl]-
methylamine (700 mg, 0.45 mmol) in dry THF (5 mL). After
the completion of reaction, as indicated by TLC, THF was
removed under vacuum, and the reaction mixture was diluted
with EtOAc (15 mL) and washed with water (10 mL), 0.5 M
HCl solution (10 mL), and then with saturated solution of brine
(10 mL). The organic layer was separated, dried over anhy-
drous Na2SO4, and concentrated under reduced pressure to
yield the crude compound, which was purified over silica gel
using 50-80% gradient elution of EtOAc in hexane to yield
the pure pyrene attached dendrimer 3 (600 mg) in 73% yield.
IR (neat): 3360, 2928, 1734, 1667, 1545, 1523, 1472, 1372,
to the chromophore offered by the comparatively larger
size and hydrophobicity of the dendrimer could be a
reason for the observation.
The emission spectra of the tetraamide 2 in different
solvents are shown in Figure 3 A.
The structural features of pyrene monomer emission
are very similar in all solvents. Only in water medium
an intense, structureless, broad, and highly red shifted
emission at 475 nm is observed, which is readily
ascribable to excimer emission. This and the observation
that there is a total absence of this new emission in all
other solvents suggests that the excimer emission could
be arising out of a water-induced hydrophobic aggrega-
tion of the molecules. Tetraamide 2 is moderately large
and fairly hydrophobic. In a nonpolar solvent like
acetonitrile, both monoamide 1 and tetraamide 2 showed
similar fluorescence spectra, corresponding to monomer
emission only. It was further noticed that, in acetoni-
trile, the fluorescence intensity of tetraamide is not
substantially enhanced as compared to that of monoa-
mide. Thus, in this nonaggregating solvent, pyrene
moieties for both tetraamide and monoamide experience
similar environments.
To ascertain that the aggregation is hydrophobic in
nature, the same studies were carried out with the
monoamide 1. The monoamide 1 did not give the
excimer peak with the same intensity as the tetraamide
2 (Figure 3B).
On decreasing the hydrophobicity of the dendrimer
end group, from a bulky tert-butyl to a relatively less
bulky ethyl group, tetraamide 3, the same trend was
observed as in the case of tetraamide 2. This, coupled
with the fact that the unprotected dendrimer, where
there are nine acid groups instead of the esters, did not
show any excimer formation (Figure 4), confirms that
the excimer formation is a consequence of hydrophobic
aggregation.
1
1187 cm-1. H NMR (CDCl3, 400 MHz): δ 1.23 (t, J ) 7.2 Hz,
27H), 2.04-2.18 (m, 4H), 3.36-3.40 (m, 2H), 2.42 (t, J ) 6.3
Hz, 6H), 2.48 (t, J ) 6.2 Hz, 18H), 3.61-3.75 (m, 48H), 4.10
(qt, J ) 7.2 Hz, 18H), 6.20 (s, 3H), 6.66 (s, 1H), 7.22-8.35 (m,
9H). 13C NMR (CDCl3, 100 MHz): δ 14.7, 21.0, 27.6, 34.9, 36.5,
37.1, 59.7, 59.9, 60.3, 60.4, 66.7, 67.5, 69.0, 123.5, 124.6, 124.7,
124.8, 124.9, 125.0, 125.7, 126.5, 127.4, 127.5, 128.7, 129.7,
130.9, 131.3, 136.3, 171.2, 171.5, 171.6. MS (ESI): 1840 (M +
Na)+.
Ma ter ia ls for P h otop h ysica l Stu d ies. The stock solution
(10-3 M) for all the studies was prepared using acetonitrile.
The stock solution was diluted to an appropriate concentration
using water. Aqueous solutions of dendrimer were made up
by using triply distilled water. KI for quenching studies was
bought locally and used as such.
In str u m en ta tion . 1H and 13C were recorded on a 400 MHz
Bruker NMR spectrometer. MALDI-TOF mass spectra of the
pyrene attached protected dendrimers were recorded on an
Applied Biosystems Voyager System 6316 using R-cyano-4-
hydroxycinnamic acid as the matrix. Ultraviolet (UV) spectra
were measured on a Perkin-Elmer Lamda 25 spectrophotom-
eter. Fluorescence measurements were recorded on a Hitachi
F-4500 spectrofluorimeter. The excitation wavelength was
fixed at 342 nm, and emission spectra were recorded in the
wavelength range 352-600 nm with the excitation and emis-
sion slit width 2.5 nm. For the excitation spectra, the emission
wavelength was fixed at 396 nm. The value of IE/IM was the
ratio of the emission intensity at 475 nm (excimer) to the
intensity at 376 nm (monomer).
Resu lts a n d Discu ssion
The protected second-generation dendrimers (tetraa-
mides 2 and 3) with pyrene in the core and the amide
N-tert-butyl-4-pyren-1-ylbutyramide (monoamide 1) are
shown in Figure 1.
The UV spectra of the tetraamide 2 and monoamide
1 show the typical absorption spectral features of pyrene
(Figure 2). The vibronic bands of the longest wavelength
transition (300-350 nm) in monoamide 1 are somewhat
less sharp than those of tetraamide 2. Some protection
Figure 5 gives the normalized emission spectra at
different concentrations of tetraamide 2. It is seen that
even at a very low concentration of 5 × 10-8 M there is
an appreciable emission due to the excimer. With
increasing concentration there is an initial increase in
excimer intensity followed by a leveling effect at higher
concentrations. The plot of excimer-to-monomer ratio
(IE/IM) of the tetraamide 2 against concentration shows
that there is an initial increase in the ratio of IE/IM.

5368 Mohanty et al.
Macromolecules, Vol. 37, No. 14, 2004
F igu r e 4. Comparison of the emission spectrum of the
F igu r e 7. Change in emission spectrum with increasing vol
% of acetonitrile in a water-acetonitrile mixture of tetraamide
2 at 10-6 M, normalized at the 0-0 vibronic band.
deprotected dendrimer and the tetraamide
3 in water
(10-5 M).
Since at 10-5 M concentration of tetraamide 2 the
excimer emission is completely absent in acetonitrile,
progressive addition of acetonitrile to water should
decrease the relative excimer emission intensity. If the
excimer formation is related to aggregation, then this
emission should disappear when there is complete
disaggregation.
Figure 7 shows the effect of change in the excimer
intensity with increasing amount of the acetonitrile in
water. It was observed that on increasing the concen-
tration of acetonitrile there is gradual decrease in the
emission intensity of the excimer, and at 50% v/v the
excimer peak completely disappears (Figure 7).
A similar disggregation was also observed in the case
of tetraamide 3; the disaggregation was complete at 25%
v/v of acetonitrile in water. The relatively lesser hydro-
phobicity of ethyl groups of tetraamide 3, as compared
to the tertiary butyl groups of tetraamide 2, appears to
be a likely reason for this observation.
F igu r e 5. Emission spectra of tetraamide 2 in water at
different concentrations, normalized at 0-0 vibronic band [λex
at 342 nm].
Quenching studies on the tetraamide 2 in water by
using hydrophilic fluorescence quencher can provide
information regarding the accessibility of the pyrene
core to the aqueous environment. We carried out the
quenching studies with hydrophilic I- as the quencher
which essentially quenches by the heavy atom effect.9
The quenching studies with amide revealed a significant
quenching of monomer fluorescence band with 0.02 M
solution of I-. However, there was only a weak quench-
ing in the case of tetraamide 2 even with 0.5 M solution
of I-. The variation of very weak excimer emission of
monoamide 1 as a function of quencher concentration
could not be monitored.
Figure 8 gives the Stern-Volmer plots for the quench-
ing of monomer emissions of monoamide 1, tetraamide
2, and tetraamide 3, as per the Stern-Volmer equation
I0/I ) 1 + KSV[Q] ) 1 + kqτ[Q]
F igu r e 6. Plot of IE/IM (λ475/λ376) with concentration of tet-
raamide 2 and monoamide 1. Inset depicts the variation of
IE/IM between 5 × 10-7 and 5 × 10-6 M of tetraamide 2 [λex at
342 nm].
In this expression, I0 and I are the fluorescence intensi-
ties in the absence and presence of quencher, respec-
tively, KSV is the Stern-Volmer quenching constant, kq
is the bimolecular quenching constant, τ is the fluores-
cence lifetime in the absence of quencher, and [Q] is the
analytical concentration of the quencher. We find that
the Stern-Volmer plot is linear over the concentration
range of the quencher.
Further increase of concentration results in saturation
of the IE/IM (Figure 6). A similar plot was obtained for
the tetraamide 3. On the other hand, the ratio is not
significantly affected by the change in concentration in
the case of the monoamide.

Macromolecules, Vol. 37, No. 14, 2004
Newkome-Type Second-Generation Dendrimers 5369
tion, even at nanomolar concentrations. The hydropho-
bic self-association of this dendrimer in aqueous me-
dium, as reflected in the appearance of the pyrene
excimer emission even at nanomolar concentration of 5
× 10-8 M, is a novel observation. Pyrene appears to be
reasonably protected from the aqueous environment in
the aggregated form.
Ack n ow led gm en t. We thank DRDO New Delhi for
financial support. S.K.M. thanks CSIR New Delhi for
fellowship, and S.T. thanks IIT Madras for fellowship.
We are grateful to R.S.I.C. IIT Madras for UV and
LRMS mass spectra. We thank DST, New Delhi, for
MALDI mass data and NMR facilities.
Refer en ces a n d Notes
(1) Van Hest, J . C. M.; Delnoye, A. P.; Baars, M. W. P. L.; Van
Genderen, M. H. P.; Meijer, E. W. Science 1995, 268, 1592.
(2) (a) Gao, C.; Yan, D. Y.; Zhang, B.; Chen, W. Langmuir 2002,
18, 3708. (b) Winnik, M. A.; Bystryak, S. M.; Liu, Z.
Macromolecules 1998, 31, 6855.
F igu r e 8. S-V plot of monoamide 1, tetraamide 2, and
tetraamide 3 in water.
(3) (a) Cardona, C. M.; J annach, S. H.; Huang, H.; Itojima, Y.;
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The KSV value for monoamide 1 is 80 M-1, for
tetraamide 2 it is 1.3 M-1, and for tetraamide 3 it is 1.8
M-1. Thus, there is a very substantial reduction of
quenching efficiencies in the case of tetraamide 2 and
tetraamide 3. We could not measure the fluorescence
lifetime of these molecules. Taking a rough value of 200
ns for pyrene in water,10 the corresponding bimolecular
quenching constant kq is 4.0 × 108 M-1 s-1 for monoa-
mide 1, 6.5 × 106 M-1 s-1 for tetraamide 2, and 9.0 ×
106 M-1 s-1 for tetraamide 3. Since the bimolecular
diffusion constant, kdiff, for water is 8 × 109 M-1 s-1 11
,
it is seen that kq of monoamide 1 is close to the diffusion-
controlled limit. The marked reduction of kq for tetraa-
mide 2 and tetraamide 3 indicates that the accessibility
of pyrene to the hydrophilic I- is significantly restricted
because of the aggregation of the protected dendrimers.
Thus, the pyrene excimer moiety is reasonably protected
from the aqueous environment in these aggregates. The
slightly higher quenching efficiency in the case of
tetraamide 3 as compared to the tetraamide 2 could be
due to the relatively greater accessibility of the quencher
to the pyrene core in the case of tetraamide 3.
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Con clu sion s
In this dendrimer architecture, the hydrophobicity
provided by ester functional groups results in aggrega-
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