Vesicle and stable monolayer formation from simple "click" chemistry adducts in water

Article abstract of DOI:10.1021/la104851g

Click chemistry has been successfully extended into the field of molecular design of novel amphiphatic adducts. After their syntheses and characterizations, we have studied their aggregation properties in aqueous medium. Each of these adducts forms stable suspensions in water. These suspensions have been characterized by dynamic light scattering (DLS) studies and transmission electron microscopy (TEM). The presence of inner aqueous compartments in such aggregates has been demonstrated using dye (methylene blue) entrapment studies. These aggregates have been further characterized using X-ray diffraction (XRD), which indicates the existence of bilayer structures in them. Therefore, the resulting aggregates could be described as vesicles. The temperature-induced order-to-disorder transitions of the vesicular aggregates and the accompanying changes in their packing and hydration have been examined using high-sensitivity differential scanning calorimetry, fluorescence anisotropy, and generalized polarization measurements using appropriate membrane-soluble probe, 1,6-diphenylhexatriene, and Paldan, respectively. The findings of these studies are consistent with each other in terms of the apparent phase transition temperatures. Langmuir monolayer studies confirmed that these click adducts also form stable monolayers on buffered aqueous subphase at the air-water interface.

Full text of DOI:10.1021/la104851g

pubs.acs.org/Langmuir
©
2011 American Chemical Society
Vesicle and Stable Monolayer Formation from Simple “Click” Chemistry
Adducts in Water
,
†,‡,§
†
Santanu Bhattacharya*
and Joydeep Biswas
†
‡
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India, and Chemical
§
Biology Unit of JNCASR, Bangalore 560 064, India J.C. Bose Fellow, DST, New Delhi, India
Received May 28, 2010
Click chemistry has been successfully extended into the field of molecular design of novel amphiphatic adducts. After
their syntheses and characterizations, we have studied their aggregation properties in aqueous medium. Each of these
adducts forms stable suspensions in water. These suspensions have been characterized by dynamic light scattering (DLS)
studies and transmission electron microscopy (TEM). The presence of inner aqueous compartments in such aggregates
has been demonstrated using dye (methylene blue) entrapment studies. These aggregates have been further characterized
using X-ray diffraction (XRD), which indicates the existence of bilayer structures in them. Therefore, the resulting
aggregates could be described as vesicles. The temperature-induced order-to-disorder transitions of the vesicular
aggregates and the accompanying changes in their packing and hydration have been examined using high-sensitivity
differential scanning calorimetry, fluorescence anisotropy, and generalized polarization measurements using appro-
priate membrane-soluble probe, 1,6-diphenylhexatriene, and Paldan, respectively. The findings of these studies are
consistent with each other in terms of the apparent phase transition temperatures. Langmuir monolayer studies
confirmed that these click adducts also form stable monolayers on buffered aqueous subphase at the air-water
interface.
1
. Introduction
coupling between each of the corresponding propargylated ester
precursors and appropriate either mono-, or di-, or tetra-azides,
respectively (Schemes 1 and 2). Since these compounds were syn-
thesized by click chemistry, they are exclusively made up of 1,4-
substituted 1,2,3-triazoles. In the present set of adducts, appro-
priate alkyl groups replace the tautomeric H-atom of the triazole
backbone. It occurred to us that since the triazoles are significantly
Copper(I)-catalyzed cycloaddition between an alkyne and an
1-3
azide is popularly known as “click chemistry”. Click chemistry
has been introduced by Sharpless, and it describes a step that is
tailored to generate adducts quickly and reliably by joining sui-
table molecular units together. This is inspired by the fact that
nature also generates larger molecules by joining small mo-
lecular units. Click chemistry has already found widespread use
1
0
polar, the same part of the adduct molecules would get hydrated
preferentially than their hydrophobic segments if brought into
4
in diverse fields such as bioconjugation of oligonucleotides,
1
1
5
6
contact with water. On the basis of the published reports on the
basicity constant for N-methyl-1,2,3-triazole, each adduct de-
scribed herein should remain mainly protonated in water at pH e
3.0, as shown in Chart 1.
glycoclusters syntheses, design of enzyme inhibitors, and even
7
in polymer and materials science. Further this reaction has been
adapted to join lipids with proteins and used to generate syn-
thetic analogues of a type of lipids that occur in archaebacteria.
8
9
Upon brief sonication each of these adducts formed stable
suspensions in aqueous medium, which were characterized by
dynamic light scattering (DLS) studies and transmission electron
microscopy (TEM) to determine the average sizes of the aggre-
gates. Such aggregates were found to possess inner aqueous com-
partments as evidenced from dye entrapment studies, indicating
that these adducts on dispersal in aqueous media afforded vesicles.
Cast films formed from each of these aggregates were also char-
acterized by X-ray diffraction (XRD) studies, which indicate the
formation of a tilted bilayer type arrangements from the aggre-
gates of M1 whereas regular bilayer structures were predominant
from the aggregates derived from each of M2, D1, and T1. The
temperature-induced order-to-disorder transitions of the vesicles
and the accompanying changes in packing and hydration were
examined using high-sensitivity differential scanning calorimetry
However, so far there has been no attempt to exploit this useful
chemistry for the design of amphiphilic systems that would aggre-
gate in aqueous media to produce nanoscopic ensembles.
Here we present four novel adducts (Figure 1) synthesized us-
ing click chemistry: an appropriately chosen monomer (M1) along
with a dimer (D1) and a tetramer (T1). A long-chained variant,
M2 of the monomer, M1, was also prepared for comparison. The
syntheses of M1, M2, D1, and T1 were accomplished by click
*
Corresponding author: e-mail sb@orgchem.iisc.ernet.in; Ph (91)-80-2293-
664; Fax (91)-80-2360-0529.
1) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40,
004.
2
(
2
(
(
(
2) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
3) Hassane, F. S.; Frisch, B.; Schuber, F. Bioconjugate Chem. 2006, 17, 849.
4) Morvan, F.; Meyer, A.; Pourceau, G.; Vidal, S.; Chevolot, Y.; Souteyrand,
E.; Vasseur, J.-J. Nucleic Acids Symp. Ser. 2008, 52, 47.
5) Chevolot, Y.; Bouillon, C.; Vidal, S.; Morvan, F.; Meyer, A.; Cloarec, J.-P.;
Jochum, A.; Praly, J.-P.; Vasseur, J.-J.; Souteyrand, E. Angew. Chem., Int. Ed.
(DSC), fluorescence anisotropy, and generalized polarization mea-
(
surements using a membrane-soluble probe, DPH, and a polarity
2
007, 46, 2398.
6) Srinivasan, R.; Li, J.; Ng, S. L.; Kalesh, K. A.; Yao, S. Q. Nature Protoc.
007, 2, 2655.
(
2
(10) Nulwala, H.; Takizawa, K.; Odukale, A.; Khan, A.; Thibault, R. J.; Taft,
B. R.; Lipshutz, B. H.; Hawker, C. J. Macromolecules 2009, 42, 6068.
(11) (a) Elguero, J.; Katritzky, A. R.; Denisko, O. V. Adv. Heterocycl. Chem. 2000,
76, 1. (b) Abboud, J.-L. M.; F-Foces, C.; Notario, R.; Trifonov, R. E.; Volovodenko, A. P.;
Ostrovskii, V. A.; Alkorta, I.; Elguero, J. Eur. J. Org. Chem. 2001, 3013.
(
7) Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun. 2007, 28, 15.
(
8) Musiol, H.-J.; Dong, S.; Kaiser, M.; Bausinger, R.; Zumbusch, A.; Bertsch,
U.; Moroder, L. ChemBioChem 2005, 6, 62.
9) O’Neil, E. J.; DiVittorio, K. M.; Smith, B. D. Org. Lett. 2007, 9, 199.
(
Langmuir 2011, 27(5), 1581–1591
Published on Web 02/03/2011
DOI: 10.1021/la104851g 1581

Article
Bhattacharya and Biswas
Figure 1. Molecular structures of the triazole-based adducts that have been synthesized and used in the present investigation.
a
1
Scheme 1
for H) spectrometer. The chemical shifts (δ) are reported in ppm
downfield from the internal standard, TMS, for H NMR. Mass
1
spectra were recorded on a MicroMass ESI-TOF spectrometer.
Infrared (IR) spectra were recorded using neat samples on a Jasco
FT-IR 410 spectrometer.
Caution! Reactions of azide salts with organic compounds and
transformations involving organic azides are potentially explosive.
Only small amounts of materials should be used and cautiously
handled. Although we did not experience any problem, proper
caution must be exercised while handling with all new azide com-
pounds.
2.2. Synthesis. Prop-2-ynyl Dodecanoate (1). Propargyl
alcohol (616 mg, 11.00 mmol) was added to a stirred solution of lauric
acid (2.0 g, 10.00 mmol), DMAP (1.59 g, 13.00 mmol), and DCC
(
2.68 g, 13.00 mmol) in dry THF (12 mL), and the resultant mixture
was stirred at room temperature for 6 h. The reaction mixture was
then diluted with acetone and filtered. The filtrate was concentrated,
diluted with ethyl acetate (50 mL), and washed with saturated
a
Reagents and conditions: (i) DCC, DMAP, THF, rt, 6 h; (ii), (iii),
iv), and (v) NaN
, DMF, 80 °C, 10 h.
(
3
aqueous KHSO
and finally using saturated brine (20 mL). The organic phase was
separated, dried over anhydrous Na SO , and evaporated, leaving a
4
solution (20 mL ꢀ 3) followed by water (20 mL)
sensor, Paldan, respectively. Interestingly, each of these adducts
also formed a stable film at the air-water interface as characterized
by the Langmuir isotherm studies. Click chemistry thus allows
a simple preparation of novel amphiphiles with assorted varia-
tion in their head/tail groups and adds to the growing list of new
2
4
residue. The product residue, prop-2-ynyl dodecanate (1), was found
to be of sufficient purity for direct use in the next step without any
need for further purification. Yield: light yellow oil, 2.0 g (8.4 mmol,
-
1
1
8
4.0%). IR (neat) (cm ): 3313, 2927, 2856, 2131, 1747, and 1158. H
NMR (300 MHz, CDCl ): δ 0.86 (3H, m), 1.34-1.18 (16H, m), 1.62
2H, m), 2.36 (2H, t), 2.44 (1H, t), 4.65 (2H, d). ESI-MS calcd. for
1
2a-f
3
amphiphiles.
(
[
þ
C H O þ Na ]: 261.3555; found: 261.183.
15
26 2
2
. Experimental Section
Azidoethane (2). NaN (598 mg, 9.20 mmol) was added to a
3
solution of bromoethane (200 mg, 1.84 mmol) in DMF (5 mL)
kept in a screw-top pressure tube, and the resulting mixture was
heated to 80 °C for 10 h. Then the solvent was evaporated under
vacuum to leave a residue, to which EtOAc was added. This af-
fordedanorganic layer, whichwaswashedwith water(20 mL ꢀ 2)
and followed by saturated brine (20 mL). Because of the potential
2
.1. Materials and Methods. All reagents, solvents, and
chemicals usedinthese studieswere obtainedfromthe bestknown
commercial sources. The solvents were dried prior to use. Column
chromatography was performed using a 60-120 mesh silica gel.
NMR spectra were recorded using a Jeol JNM λ-300 (300 MHz
13
explosiveness of azides, the organic layer was dried over anhy-
drous Na SO and concentrated gently below 40 °C under vacuum.
(
12) (a) Bhattacharya, S.; Biswas, J. Langmuir 2010, 26, 4642. (b) Menger, F. M.;
2
4
Shi, L.; Rizvi, S. A. A. J. Colloid Interface Sci. 2010, 344, 241. (c) Ahmad, R. K.;
Faure, D.; Goddard, P.; Oda, R.; Bassani, D. M. Org. Biomol. Chem. 2009, 7, 3173. (d)
Aim ꢀe , C.; Manet, S.; Satoh, S.; Ihara, H.; Park, K.-Y.; Godde, F.; Oda, R. Langmuir
007, 23, 12875. (e) Jiao, T.; Liu, M. Langmuir 2006, 22, 5005. (f) Grinberg, S.; Linder,
C.; Kolot, V.; Waner, T.; Wiesman, Z.; Shaubi, E.; Heldman, E. Langmuir 2005, 21,
The crude residue thus obtained was purified by column chroma-
tography using EtOAc/hexane (2:98), which afforded azidoethane
-1
(
2) as a brown oil, 120 mg (1.69 mmol, 92.0%). IR (neat) (cm ):
2930, 2867, 1598, 1495, 1467, 1366, 1188, and 1174. H NMR
2
1
7
2
7
638. (g) Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Angew. Chem., Int. Ed.
001, 40, 1228. (h) Bhattacharya, S.; De, S.; Subramanian, M. J. Org. Chem. 1998, 63,
640. (i) Bhattacharya, S.; Kumar, V. P. Langmuir 2005, 21, 71. (j) Bhattacharya, S.;
(
CDCl
3
, 300 MHz): δ 1.74 (3H, t), 3.39 (2H, q). ESI-MS calcd for
þ
[C
2 5
H N
3
þ Na ]: 94.0711; found: 94.0381.
Snehalatha, K.; Kumar, V. P. J. Org. Chem. 2003, 68, 2741. (k) Bhattacharya, S.;
Haldar, J. Langmuir 2004, 20, 7940. (l) Haldar, J.; Kondaiah, P.; Bhattacharya, S. J.
Med. Chem. 2005, 48, 3823.
(13) Klapotke, T. M.; Rienacker, C. M. Propellants, Explos., Pyrotech. 2001, 26, 43.
1
582 DOI: 10.1021/la104851g
Langmuir 2011, 27(5), 1581–1591

Bhattacharya and Biswas
Article
a
Scheme 2
a
Reagents and conditions: (i), (ii), (iii), and (iv) CuI, DIEA, MeOH, rt, 6-8 h.
Chart 1
1
-Azidotetradecane (3). NaN
3
(234 mg, 3.60 mmol) was
purified by column chromatography (eluent: EtOAc/hexanes,
4:96) to furnish 1,3-diazidopropane (4) as a brown oil, 110 mg
added to a solution of 1-bromotetradecane (200 mg, 0.72 mmol)
in DMF (20 mL) in a round-bottom flask, and the resulting
mixture was warmed to 80 °C for 10 h. Then the solvent was
evaporated under vacuum to leave a residue, to which EtOAc was
added. This afforded an organic layer, which was washed with
water (20 mL ꢀ 2) and followed by saturated brine (20 mL). Be-
-
1
1
(0.87 mmol, 88.0%). IR (neat) (cm ): 2924, 2852, and 2100. H
NMR (CDCl
ESI-MS calcd for [C
3
, 300 MHz): δ 2.26-2.29 (2H, m), 3.30-3.34 (4H, t).
þ
3
H
6
N
6
þ Na ]: 149.11; found: 149.0552.
1,3-Diazido-2,2-bis(azidomethyl)propane (5). NaN (169 mg,
3
2.60 mmol) was added to a solution of 1,3-dibromo-2,2-bis-
1
3
cause of the potential explosiveness of azides, the organic layer
was dried over anhydrous Na SO and concentrated gently below
(bromomethyl)propane (200 g, 0.52 mmol) in DMF (5 mL) in a
round-bottom flask, and the resulting mixture was warmed slowly
to 80°C. After 10h ofreaction atthistemperature, the solvent was
evaporated under vacuum. The reaction mixture was diluted with
EtOAc. The organic layer was washed with water (20 mLꢀ 2) and
followed by saturated brine (20 mL). Because of the potential ex-
2
4
40 °C under vacuum. The crude product thus obtained was puri-
fiedbycolumnchromatographyusingEtOAc/hexane(2:98)which
afforded 1-azidotetradecane (3) as a brown oil, 164 mg (0.69
-1
1
mmol, 95.0%). IR (neat) (cm ): 2930, 2867, and 2104. H NMR
CDCl , 300MHz):δ0.78-0.84(3H,t),1.18-1.32 (22H, m), 1.64-
.82 (2H, m), 3.30-3.36 (2H, t). ESI-MS calcd for [C14
1
3
(
1
3
plosiveness of azides, the organic layer was dried over anhydrous
H N
29 3
þ
Na SO and concentrated gently under vacuum <20 °C. The re-
2
4
þ
Na ]: 262.3901; found: 262.2259.
sulting oil was purified by column chromatography (eluent: EtOAc/
hexanes, 6:94), which afforded 1,3-diazido-2,2-bis(azidomethyl)-
propane (5) as a colorless solid, 97 mg (0.41 mmol, 80.0%). IR
1
,3-Diazidopropane (4). NaN (322 mg, 4.95 mmol) was ad-
3
ded to a solution of 1,3-dibromopropane (200 mg, 0.99 mmol) in
DMF (5 mL) in a round-bottom flask, and the resulting mixture
was warmed gently to 80°C. After10h,thesolventwasevaporated
under vacuum. The reaction mixture was diluted with EtOAc. The
organic layer was washed with water (20 mL ꢀ 2) and followed by
saturated brine (20 mL). Because of the potential explosiveness of
-
1
1
(neat) (cm ): 2923, 2850, and 2099. H NMR (CDCl , 300 MHz):
3
þ
δ 3.27 (8H, s). ESI-MS calcd for [C
found: 259.0893.
5
H
8
N12 þ Na ]: 259.1877;
1-Ethyl-1H-[1,2,3]triazol-4-yl-methyl Dodecanoate (M1).
Compound 2 (50 mg, 0.70 mmol) was added to a solution of com-
pound 1 (336 mg, 0.70 mmol) in MeOH (6 mL) in a round-bottom
1
3
2 4
azides, the organic layer was dried over anhydrous Na SO and
0
0
concentrated under vacuum gently <40 °C. The residue was
Langmuir 2011, 27(5), 1581–1591
flask. After that, N ,N -diisopropyl-N-ethylamine (DIEA) (452 mg,
DOI: 10.1021/la104851g 1583

Article
Bhattacharya and Biswas
þ
3
.5 mmol) and CuI (667 mg, 3.5 mmol) were added, and the
reaction mixture was stirred for 8 h at room temperature. After
h, MeOH was evaporated and the reaction mixture was diluted
with CHCl . The organic layer was washed with saturated aqu-
eous KHSO
7.19 (4H, s). ESI-MS calcd for [C65
found: 1211.6624.
H N O
112 12 8
þ Na ]: 1212.6506;
8
2.3. Computational Studies. An optimization of the energy-
minimized structure of each click chemistry based triazole adduct
was performed using Gaussian 98 program and RHF/6-31G*
level of theory. The ball-stick structure of each molecule was
drawn using Gauss View software, and then the coordinates of
these structures were taken and run in Gaussian 98 to get the opti-
mized geometry. The distance between the “head” to the “tail”
3
4
solution (3 ꢀ 20 mL), water (20 mL), and saturated
1
4
brine solution (20 mL). Finally, the organic layer was separated
and dried over anhydrous Na SO . The solvent was removed us-
2
4
ing a rotary evaporator, and the product was purified by column
chromatography over silica gel using a mixture of CHCl and MeOH
v/v = 96:4] to afford a white solid, 158 mg (0.51 mmol, 73.0%). IR
3
1
5
[
carbon atom was determined using ChemCraft software.
-1
1
(
neat) (cm ): 2924, 2852, 1739, and 1625. H NMR (CDCl , 300
3
2.4. Aggregate Preparation. Each adduct was dissolved in
chloroform in separate autoclaved glass vial. Thin film was made
by evaporation of the organic solvent under a steady stream of
dry nitrogen. The lasttraces of chloroformwereremoved bykeep-
ing the film under vacuum overnight. Freshly autoclaved water
(Milli-Q) or particular buffer was added to the individual film
such that the final concentration of the adduct was maintained
at ∼1 mM. The mixtures werekept for hydration at 4 °C for 6-8 h
and were repeatedly freeze-thawed (ice-cold water to 70 °C) with
intermittent vortexing to ensure optimal hydration. Sonication of
these suspensions for 15 min in a bath sonicator at 70 °C afforded
approximately spherical aggregates as evidenced from transmis-
sion electron microscopy. The aggregates were prepared and kept
under sterile conditions. The aggregates were found to be stable
even after keeping them for more than 1 month.
MHz): δ 0.81-0.86 (3H, t), 1.23 (16H, m), 1.81-1.85 (5H, m),
2.23-2.8 (2H, t), 4.24-4.29 (2H, q), 5.14 (2H, s), 7.52 (1H, s). ESI-
þ
MS calcd for [C H N O þ Na ]: 332.4368; found: 332.2314.
17
31
3 2
1
-Tetradecyl-1H-[1,2,3]triazol-4-yl-methyl Dodecanoate (M2).
Compound 3 (50 mg, 0.21 mmol) was added to a solution of com-
pound 1 (50 mg, 0.21 mmol) in MeOH (6 mL) in a round-bottom
flask. After that, DIEA (136 mg, 1.05 mmol) and CuI (200 mg,
1
8
.05 mmol) were added, and the reaction mixture was stirred for
h at room temperature. After 8 h, MeOH was evaporated and
3
the reaction mixture was diluted with CHCl . The organic layer
was washed with saturated KHSO (3 ꢀ 20 mL), water (20 mL),
4
and saturated brine solution (20 mL). Finally, the organic layer
2 4
was separated and dried over anhydrous Na SO . The solvent was
removed using a rotary evaporator, and the product was purified
by column chromatography over silica gel using a mixture of
CHCl and MeOH [v/v = 96:4] to afford a white solid, 78 mg
2.5. pK Measurements of Click Adduct Aggregates.
a
UV-vis spectra of the aggregate of each click adduct in aqueous
3
-1
media or media of different acidities (by adding solutions of H SO )
were recorded using Shimadzu UV-2100 spectrophotometer at
2
4
(
0.16 mmol, 78.0%). IR (neat) (cm ): 2929, 2868, 1743, and 1632.
1
H NMR (CDCl , 300 MHz): δ 0.81-0.86 (6H, t), 1.23 (38H, m),
3
2
5 °C. Then the ratios of absorbance maxima before and after
1
.85-1.90 (4H, m), 2.27-2.32 (2H, t), 4.29-4.34 (2H, q), 5.19
þ
protonation of triazole groups in the aggregates of each click ad-
duct of each spectrum were plotted against the pH of the media.
The pointsobtained werefitted (Boltzmann) using Origin8.0 soft-
(
5
2H, s), 7.56 (1H, s). ESI-MS calcd for [C H N O þ Na ]:
29
55
3 2
00.7558; found: 500.4192.
-[3-(4-Dodecanoyloxymethyl-[1,2,3]triazol-1-yl)-propyl]-
H-[1,2,3]triazol-4-yl-methyl Dodecanoate (D1). Compound 4
1
a
ware, and from the break we obtained the pK value of the aggre-
gates of each click adduct.
1
(
0
50 mg, 0.40 mmol) was added to a solution of compound 1(191mg,
.80 mmol) in MeOH (6 mL) in a round-bottom flask. After that,
2.6. Dye Entrapment Studies. Films of a given click adduct
were prepared as described above and an appropriate amount
of 0.1 mM methylene blue (MB) (λmax = 665 nm) was added such
that eachclick adduct concentration was 5 mM. A 2 mL aliquot of
the resulting click adduct suspension was loaded on to a column
packed with pre-equilibrated Sephadex G-50. Gel filtration was
performed using freshly autoclaved water (Milli-Q) as the eluent
until elution of free dye was complete. Neat Triton-X-100 was
added to aliquots of fractions to give a 1 wt % concentration of
Triton X 100, and the vesicles were lysed by bath sonication of the
resulting solution for 2 min at room temperature. The absor-
bances at 665 nm for all the fractions containing lysed solutions
was determined and plotted against the elution volume.
DIEA (517 mg, 4.00 mmol) and CuI (762 mg, 4.00 mmol) were
added, and the reaction mixture was stirred for 8 h at room tem-
perature. After 8 h, solvent was evaporated from the reactionmix-
ture and the residue was dissolved in CHCl
was washed with saturated KHSO
(3 ꢀ 20 mL), water (20 mL),
and saturated brine (20 mL). Finally, the organic layer was sepa-
rated and dried over anhydrous Na SO . The solvent was removed
using a rotary evaporator, and theproduct was purifiedby column
chromatography over silica gel using a mixture of CHCl and
MeOH [v/v = 94:6] to furnish a white solid, 167 mg (0.28 mmol,
3
. The organic layer
4
2
4
3
-1
1
7
0.0%). IR (neat) (cm ): 2934, 2854, 1726, and 1630. H NMR
(
(
CDCl , 300 MHz): δ 0.81-0.86 (6H, t), 1.23 (32H, m), 1.68-1.76
3
2.7. Transmission Electron Microscopy. The aqueous ag-
4H, m), 2.25-2.30 (6H, m), 3.72-3.83 (4H, d), 5.34 (4H, s), 7.26
þ
gregatesoftheadducts(1 mM) were examinedundertransmission
electron microscopy by negative staining using 0.5% uranyl ace-
tate. A 10 μL sample of the suspension was loaded onto Formvar-
coated, 400 mesh copper grids and allowed to remain for 1 min.
Excess fluid was removed from the grids by touching their edges
with filter paper, and 10 μL of 0.1% uranyl acetate was applied on
the same grid after which the excess stain was similarly removed
from the grids. First, the grids were air-dried, and then the last
traces of water were removed under vacuum. The samples were
observed under TEM (TECNAI T20) operating at an accelera-
tion voltage (dc voltage) of 100 keV. Micrographs were recorded
at a magnification of 10000-80000ꢀ.
(
6
2H, s). ESI-MS calcd for [C H N O þ Na ]: 625.8414; found:
3
3
58
6
4
25.4417.
1
-[3-(4-Dodecanoyloxymethyl-[1,2,3]triazol-1-yl)-2,2-bis(4-
dodecanoyloxymethyl-[1,2,3]triazol-1-yl-methyl)propyl]-1H-[1,
,3]triazol-4-yl-methyl Dodecanoate (T1). Compound 5 (50 mg,
.21 mmol) was added to a solution of compound 1 (200 mg, 0.84
2
0
mmol) in MeOH (6 mL) in a round-bottomed flask. After that,
DIEA (543 mg, 4.2 mmol) and CuI (800 mg, 4.2 mmol) were ad-
ded, and the reaction mixture was stirred for 8 h at room tem-
perature. After 8 h, solvent was evaporated from the reactionmix-
ture and the residue was dissolved in CHCl . The organic layer
3
was washed with saturated KHSO
and saturated brine (20 mL). Finally, the organic layer was sepa-
rated and dried over anhydrous Na SO . The solvent was removed
4
(3 ꢀ 20 mL), water (20 mL),
2.8. Dynamic Light Scattering. Unilamellar vesicles (1 mM)
prepared in buffered aqueous media (as mentioned under the
aggregate preparation) werediluted to 0.33 mM and were usedfor
dynamic light scattering (DLS) measurements. DLS measurements
were performed at room temperature using a Malvern Zetasizer
2
4
using a rotary evaporator, and theproduct was purifiedby column
chromatography over silica gel using a mixture of CHCl and
MeOH [v/v = 92:8] to furnish a white solid, 166 mg (0.14 mmol,
3
-1
1
6
(
1
6.0%). IR (neat) (cm ): 2936, 2876, 1749, and 1634. H NMR
CDCl , 300 MHz): δ 0.83-0.88 (12H, t), 1.22 (64H, m),
.52-1.56 (8H, m), 2.23-2.28 (8H, t), 3.67 (8H, s), 5.24 (8H, s),
(
14) (a) Kumar, V. P.; Ganguly, B.; Bhattacharya, S. J. Org. Chem. 2004, 69, 8634.
b) Bhattacharya, S.; Vemula, P. K. Org. Chem. 2005, 70, 9677.
(15) Kress, J. W. J. Phys. Chem. A 2005, 109, 7757.
3
(
1
584 DOI: 10.1021/la104851g
Langmuir 2011, 27(5), 1581–1591

Bhattacharya and Biswas
Article
Nano ZS particle sizer (Malvern Instruments Inc., Westborough,
MA). Samples were prepared and examined under dust-free con-
ditions. Mean hydrodynamic diameters reported were obtained
from the Gaussian analysis of the intensity-weighted particle size
distributions.
ratio of 1000:4. Steady-state fluorescence anisotropies sensed by
DPH-doped suspensions were measured in a HORIBA Jobin Yvon
Fluorolog spectrofluorimeter equipped with polarizers upon ex-
citation of DPH at 360 nm. The emission intensity was followed at
430 nm. The slit widths were kept at 5 nm for both the excitation
and the emission. The fluorescence intensities of the emitted light
2
.9. Differential Scanning Calorimetry. Thermotropic pro-
perties of the adduct aggregate samples in aqueous media were
investigated by high-sensitivity DSC using a CSC-4100 model
multicell differential scanning calorimeter (Calorimetric Sciences
Corp., Lindon, UT). Click adduct aggregate samples were pre-
pared as mentioned under the aggregate preparation. Each click
adduct aggregate sample (0.5 mL) in buffered aqueous media (pH
polarized parallel (IVV) and perpendicular to the excited light (IVH)
were measured at different temperatures. These fluorescence inten-
sities were corrected for scattered light intensity. The fluorescence
anisotropy values (r) at each temperature were calculated employ-
ing the equation r = (IVV - GIVH)/(IVV þ 2GIVH), where G is the
instrumental grating factor. The grating factor, G, was calculated
using the equation G = IHV/IHH, where IHV and IHH represent the
fluorescence intensities of vertically polarized and horizontally
polarized light, respectively, when a horizontally polarized light
was used for the probe excitation. The systemic gel-to-liquid crys-
2.0 or 7.4) was carefully transferred into three of the four DSC
ampules, and the fourth one was filled with 0.5 mL of buffered
aqueous media. Then the ampules were sealed. The measurement
was carried out in the temperature range of 20-70 °C at a scan rate
of 20 °C/h. At least two consecutive heating and cooling
were performed. Baseline thermograms were obtained using same
amount of the degassed buffer in the respective DSC cells. The
thermograms for the adduct aggregate samples were obtained by
subtracting the respective baseline thermogram from the sample
thermogram using “CpCalc” software. From the plot of excess heat
capacity vs temperature, solid-to-fluid transition temperature (Tsff),
fluid-to-solid transition temperature (Tffs), and calorimetric enthal-
m
talline phase transition temperatures (T ) of each click adduct were
calculated from the midpoints of the breaks related to the tempera-
ture-dependent anisotropy values.
20a,b
2.12. Cast-Film XRD Measurements. An aqueous aggre-
gates of each adduct (1 mM) was placed on a precleaned glass plate
which, upon air-drying, afforded a thin film of the adduct aggregate
on the glass plate. X-ray diffraction (XRD) of an indi-
vidual cast film was performed using the reflection method with
2
1a-c
c
pies (ΔH ) of the transitions were obtained. The size of the coopera-
a Phillips X-ray diffractometer.
ated with a Cu anode, and the Cu KR
The X-ray beam was gener-
1
beam of wavelength 1.5418
tivity unit (CU) for the melting transition was determined using the
formula CU = ΔHvH/ΔH , where the ΔH is the van’t Hoff
˚
A was used for the experiments. Scans were performed for 2θ
values up to 18°.
cal
vH
enthalpy and the ΔHcal is the calorimetric enthalpy. The van’t Hoff
2
enthalpy was calculated from the equation ΔHvH = 6.9T
where T is the phase transition temperature and ΔT1/2 is the full
width at half-maximum of the thermogram.
.10. Measurements of Aggregate Hydration. Paldan,
m
/ΔT1/2
,
2.13. Langmuir Monolayer Studies. Isotherms of the in-
dividualclick adduct monolayeratthe air-bufferedaqueousmedia
interface were recorded on a Nima Langmuir trough (Nima Tech-
nology, Coventry, England) equipped with computer control. A
paper Wilhelmy plate was used as the surface pressure sensor and
situated in the middle of the trough. Two barriers compressed or
expanded symmetrically at the same velocity from two sides of the
trough. Monolayers were obtained by spreading the chloroform
solutions of desired volumes on buffer containing subphases. The
subphase temperature was maintained at 25 °C. After spreading,
30 min was allowed for solvent evaporation, and the monolayers
were then compressed at a rate of 5 mm/min to record isotherms.
m
2
which is a palmitoyl analogue of Prodan, was synthesized by
the procedures of Weber, Davis, and Balter et al. The synthesis of
1
6,17
Paldan was performed by following the literature procedure.
First, 2-methoxynaphthalene was treated by n-C15 31COCl in
the presence of AlCl to get 6-palmitoyl-2-methoxynaphthalene.
H
3
6
corresponding dimethylamino derivative (Paldan) by following
-Palmitoyl-2-methoxynaphthalene was then converted to the
1
6,17
the appropriate literature procedure.
After purification and
spectral characterizations, the synthetic Paldan was used for spe-
ctroscopic studies. All the fluorescence experiments were carried
out onsonicated click adductaggregate samples inaqueous media
using adduct-to-probe ratio of 1000:3. The width of the excitation
and emission slit was 10 nm. Generalized polarization of emission
3
. Results and Discussion
Upon hydration followed by bath sonication for 15 min at 70 °C,
each adduct having a 1,2,3-triazole unit was found to get dispersed
in aqueous media easily. The suspensions formed from the adducts
were found to be stable and optically clear suspensions in aqueous
buffer solutions. No precipitation or noticeable increase in turbi-
dity was observedevenafter1 month when these suspensions were
stored at 4 °C under sterile conditions.
(
GP) was calculated using the equation
I
440 - I490
440 þ I490
GPem
¼
I
where I440 and I represent the fluorescence intensities at 440
490
3.1. pK Measurements of Click Adduct Aggregates.
a
1
8,19
and 490 nm.
For the GP values reported herein, an excitation
UV-vis spectra of the aggregates of each click adduct prepared
in aqueous media of various pH values were recorded at 25 °C. A
representative spectral profile for the aggregates of M1 recorded
at various pH values is shown in Figure 2A. At pH 7.6, the aggre-
gates M1 showed a peak at 252 nm due to the presence of the
wavelength of 350 nm was used. For excitation, the spectra
wavelength was kept at 440 nm.
2
.11. Fluorescence Anisotropy Measurements. For the
preparation of click adduct suspensions doped with fluorescent
probe, 1,6-diphenylhexatriene (DPH), the probe in dry CHCl3
was cosolubilized with a given click adduct in chloroform. A thin
film was generated upon evaporation of solvent in the dark, and
then DPH-doped vesicles (adduct concentration = 1.0 mM) were
prepared by the freeze-thaw method followed by sonication as
mentioned above. All the fluorescence anisotropy experiments were
carried out on sonicated suspensions using an adduct-to-probe
1
1b
triazole moiety. Progressive decreases in the pH values of the
same aggregates resulted in the evolution of a new peak at 284 nm
at the expense of the 252 nm peak. This occurred at pH e 3 due to
the formation of protonated triazolium moiety. Then the ratios of
the absorbance maxima before and after protonation of triazole
(
(
16) Weber, G.; Davis, F. J. Biochemistry 1979, 18, 3075.
17) Lakowicz, J. R.; Bevan, D. R.; Maliwal, B. P.; Cherek, H.; Balter, A.
(20) (a) Lakowicz, J. Principles of Fluorescence Spectroscopy, 1st ed.; Plenum
Press: New York, 1983. (b) Shinitzky, M.; Barenholtz, Y. Biochim. Biophys. Acta 1978,
525, 367.
(21) (a) Bhattacharya, S.; Dileep, P. V. J. Phys. Chem. B 2003, 107, 3719. (b)
Kimizuka, N.; Kawasaki, T.; Kunitake, T. J. Am. Chem. Soc. 1993, 115, 4387. (c)
Kimizuka, N.; Kawasaki, T.; Hirata, K.; Kunitake, T. J. Am. Chem. Soc. 1998, 120,
4094.
Biochemistry 1983, 22, 5714.
18) Parasassi, T.; Di Stefano, M.; Loiero, M.; Ravagnan, G.; Gratton, E.
Biophys. J. 1994, 66, 763.
19) Parasassi, T.; De Stasio, G.; Ravagnan, G.; Rusch, R. M.; Gratton, E.
(
(
Biophys. J. 1991, 60, 179.
Langmuir 2011, 27(5), 1581–1591
DOI: 10.1021/la104851g 1585

Article
Bhattacharya and Biswas
Figure 2. (A) UV-vis spectra of the aggregates of click adduct M1 (conc.=1 mM) in aqueous buffered media of different pH values. (B) Plot of
the ratio of absorbance maxima before and after protonation of triazole groups in the aggregates of click adduct M1 of each spectrum vs the pH
of the media.
Table 1. Apparent pK
a
Values of the Aggregates of Each Click Adduct
a
Formed in Aqueous Media
click adduct
M1
pK
a
2.9
2.9
3.1
3.1
M2
D1
T1
a
The pK values were within (0.1 units in duplicate experiments.
a
groups in the aggregates of each click adduct of each spectrum
were plotted against the pH of the media. The points obtained
were fitted using Origin 8.0 software, and from the break we ob-
tained the systemic pK value of the adduct aggregates of M1
a
(
Figure 2B). The pK values of the aggregates of each click adduct
a
including that of M1 are given in Table 1. The apparent pK values
a
of the aggregates of monomeric click adducts M1 and M2 were
found to be 2.9 (same for the two adducts), whereas that of the ag-
gregates of click adducts D1 and T1 was found to be ∼3.1.
3
.2. Aggregate Characterizations. Upon hydration fol-
lowed by dispersal by bath sonication for 15 min at 70 °C in freshly
autoclaved water (Milli-Q) or a particular buffer, each of the four
compounds (1 mM) formed optically clear, stable suspensions.
No precipitation or noticeable increase in turbidity was observed
even after 1 month when these suspensions were stored at 4 °C
under sterile conditions. Examination of freshly prepared aqueous
suspensions of each compound by TEM revealed the existence of
vesicle-like nanoscopic structures (Figure 3). The dispersions of
the dimeric adduct D1 showed a few elongated nonspherical struc-
tures as well, whereas that of the other adducts formed spherical
morphologies as evident from the TEM study. Interestingly, the
vesicular aggregates were observed from both M1 (one fatty acid
ester-linked chain with a delocalized protonated triazole) and M2
Figure 3. Negative-stain transmission electron micrographs of
aqueous suspensions of (A) M1, (B) M2, (C) D1, and (D) T1.
adduct probably caused a slight increase in the interamphiphilic
headgroup repulsion, resulting in an increase in the hydrodynamic
diameter. Aggregates of T1 showed the largest hydrodynamic
diameter, whereas that of D1 showed the smallest hydrodynamic
size in both pH values of the buffered aqueous media. Among the
monomeric adducts, aggregates of M1 showed smaller size, which
was also evidenced from their TEM images. The sizes of the aggr-
egates obtained from DLS and TEM experiments were not the
same but followed an approximately similar trend. The shrinkages
in aggregate sizes observed in TEM could be due to drying of the
2
3
samples;a step required before imaging the micrographs.
A representative optimized molecular structure of M1 using the
(
one fatty acid ester-linked chain and one n-C H chain with a
14 29
protonated triazole). Observations of vesicle formation from a
single-chain systems are not unprecedented. Indeed, vesicle for-
mation from a single-chain compound having a large polar head-
2
5
RHF/6-31G* level of theory is shown in Figure 4. Other molec-
ular structures obtained using the same computational method
are shown in the Supporting Information (Figure S1). From the
optimized structure, we determined the theoretical length of the
adduct M1 by measuring the distance from the “head” carbon
atom to the “tail” carbon atom with the help of ChemCraft
software. Then the theoretical bilayer width was calculated by
2
2
group (not related to the present design) is already reported.
All the adduct suspensions in two buffer media (pH 2 and 7.4)
were further characterized using DLS, as shown in Table 2. The
hydrodynamic diameter of the aggregates of each click adduct
increased modestly with the decrease in the pH of the media. Thus,
the protonation of the triazole groups in the aggregates of each click
(
23) Bhattacharya, S.; Bajaj, A. Langmuir 2007, 23, 8988.
(
24) Feng, Z. V.; Spurlin, T. A.; Gewirth, A. A. Biophys. J. 2005, 88, 2154.
(
22) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S.; Takarabe, K.
(25) Spelios, M.; Nedd, S.; Matsunaga, N.; Savva, M. Biophys. Chem. 2007, 129,
137.
J. Am. Chem. Soc. 1981, 103, 5401.
1
586 DOI: 10.1021/la104851g
Langmuir 2011, 27(5), 1581–1591

Bhattacharya and Biswas
Article
Table 2. DLS Hydrodynamic Diameters, Sizes from TEM, Thermal Phase Transition Temperatures, and Unit Bilayer Thicknesses of the Click
Adduct Aggregates
a
b
˚
unit bilayer thickness (A)
DLS size ((5 nm)
c
TEM diameter (nm)
d
dye entrapment capacity (%)
e
f
adduct
pH 2
pH 7.4
obsd
calcd
M1
M2
D1
T1
a
170
220
145
280
165
210
130
275
80-100
160-180
60-80
0.97
1.24
0.76
1.52
37.3
41.1
42.0
42.1
40.3
46.6, 37.5 (w)
45.8, 37.4 (w)
45.8, 37.4 (w)
140-150
b
Each aggregate (1 mM) was prepared by sonication (Elma, Transsonic bath T460/H, Germany) for 15 min at 70 °C and at 35 kHz. Dynamic light
scattering (DLS) was examined with aqueous suspensions of each adduct prepared under the same conditions. Average sizes of the aggregates obtained
from negative stain TEM. [Click adduct] = 5 mM; [methylene blue] = 0.1 mM. As obtained from reflection X-ray diffraction of cast films. Sum of the
lengths of two molecular layers in their energy-minimized, fully extended conformation as obtained from theoretical models.
c
d
e
f
Figure 4. Energy-minimized structure of the adducts M1, M2, D1, and T1 (yellow: C; light blue: H; purple: N; red: O).
multiplying the monolayer width obtained from the optimized
structure by a factor of 2. The bilayer widths of each adduct
aggregates were experimentally determined from the cast films
obtained by a reported procedure followed by XRD experiments,
and the XRD patterns obtained from the cast films of all the four
2
6
click chemistry based adducts are shown in Figure 5. The bilayer
widths of each of the adduct aggregates are given in Table 2.
From the XRD experiments, the 2θvalues for each of the sample
were obtained; the bilayer width of each adduct aggregate was
obtained from Bragg’s equation (nλ = 2d sin θ). Each adduct ag-
gregate showed two peaks under XRD, one strong (in the region
7
.55-7.75 of 2θ values) and the other one weak, except for the
monomer M1, which showed only one peak at 9.48 of 2θ value
corresponding to the bilayer width of 37.32 A. One can interpret
˚
these data on the basis of formation of slightly tilted bilayer struc-
tures in case of M1 in aqueous media as its bilayer width deter-
mined from XRD is less than the theoretical bilayer width
determined from its optimized structure (Scheme 3). On the other
hand, the adduct aggregates of M2, D1, and T1 manifested both
Figure 5. XRD patterns of the cast films obtained from the sus-
pensions of each of the four click chemistry based adducts.
Scheme 3. Schematic Representation of Possible Bilayer-Forming Motifs with Various Triazole-Based Adducts
Langmuir 2011, 27(5), 1581–1591
DOI: 10.1021/la104851g 1587

Article
Bhattacharya and Biswas
Figure 6. Dye entrapment profile of aqueous suspension of (A) M1 and (B) T1 in methylene blue (MB). [M1] or [T1] = 5.0 mM; [MB] =
.1 mM. The absorbance at 665 nm was checked for all fractions.
0
Figure 7. (A) Thermotropic phase transitions of various adduct suspensions (1 mM) in buffered aqueous media (pH 7.4) as evidenced by
DSC heating scans and (B) cooling scans. Both the thermograms have been successively raised from the baseline by 1 kcal/(K mol) steps for
clarity.
regular bilayer structures. The untilted bilayer structures are more
prominent for the adduct aggregates of M2, D1, and T1 as all of
them show strong peaks in the region 9.47-9.49 of 2θ values
small initial portion containing vesicles entrapping approximately
0.76-1.52% of total dye followed by a large peak which was due
to the free, unentrapped dye. It is clear that the MB molecules as-
sociated with vesicles could be distinctly separated from the free
dye. Thus, each click chemistry based adduct formed closed vesi-
cular aggregates with inner aqueous compartments. The percen-
tage of dye entrapment capacity of each vesicular aggregate of a
given click adduct is given in Table 2.
˚
corresponding to the bilayer width in the region 45.80-46.65 A.
3
.2.1. Inner Aqueous Compartments. To confirm vesicular
aggregate formation from these newly synthesized click adducts,
we employed dye entrapment studies to examine whether these
aggregates contained closed inner aqueous compartments. For
instance, the amphiphiles which form micelles in aqueous media do
not have the ability to entrap hydrophilic water-soluble molec-
ules (dye). For this purpose, we chose a dye, methylene blue (MB).
3.3. ThermalPropertiesoftheAdduct Aggregates. 3.3.1.
Differential Scanning Calorimetry (DSC). To determine the
temperature (T ) at which the phase transition of the individual
m
2
7
28
Gokel et al. showed using this dye that the aggregates of steroidal
azacrown derivatives in aqueous media possessed entrapment
capacities. To find the entrapment capacities (if any) of the
aggregates generated from each click adduct (M1, M2, D1, and
T1), we made vesicles from each adduct by vortexing followed by
sonication for 10 min at 70 °C of the hydrated films to afford
a final concentration of 5 mM in water containing 0.1 mM MB.
Then freshly autoclaved water (Milli-Q) was used for elution of the
gel filtration column (Sephadex G-50), and separate fractions of
each were collected. The profile of the gel filtration was obtained by
plotting the absorbance at 665 nm of all the fractions obtained
from the gel filtration column vs the elution volume. Dye entrap-
ment profile of aqueous suspension of click adducts M1 and T1 are
shown in Figure 6. In all the cases, it was observed that there was a
aggregate from solidlike phase to fluidlike phase takes place, we
examined their aqueous suspensions by DSC at pH 7.4 (Figure 7).
Each adduct suspension demonstrated sharp chain-melting tran-
sitions both during heating and cooling scans. The dependence of
the thermotropic phase transition (T ) of various click adduct
m
aggregates at this pH is shown in Figure S3 (Supporting In-
formation). The phase transition temperatures (T ) and other
m
thermotropic parameters as obtained from the DSC studies with
each adduct aggregates are given in Table 3.
As the number of long chains in the adducts increased from one
to four, the phase transition temperatures also increased from 42.1
to 50.3 °C. The adduct M1, which contains only one C chain,
1
2
showed a T at ∼42.1 °C, whereas the incorporation of four C
m
12
chains in the adduct T1 showed the highest T at ∼50.3 °C. The
m
adducts M2 and D1 which contain two C12 chains showed T at
m
(
26) Bhattacharya, S.; Bajaj, A. J. Phys. Chem. B 2007, 111, 13511.
(
27) DeWall, S. L.; Wang, K.; Berger, D. R.; Watanabe, S.; Hernandez, J. C.;
(28) De, S.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. J. Phys. Chem. 1996,
100, 11664.
Gokel, G. W. J. Org. Chem. 1997, 62, 6784.
1
588 DOI: 10.1021/la104851g
Langmuir 2011, 27(5), 1581–1591

Bhattacharya and Biswas
Article
∼
46.0 and 45.8 °C, respectively. The other thermal transition
The adduct aggregates containing 1,2,3-triazole moieties
showed λem in the range of 435-443 nm in their gel states, but in
their fluid-melted states, the range of λem was between 436 and
450 nm (Figure 8A). This change in the emission λmax of the
adduct aggregates from the solidlike states to the fluid-melted
states is caused due to the increase in the temperature, when a
large number of water molecules penetrate inside the adduct ag-
gregates (loosening of the aggregates at higher temperature), and
hence there is a bathochromic shift in the emission λmax of the
adduct aggregates.
The excitation λmax’s of Paldan in these adduct aggregates in
their solid and in the fluid phase are given in Table 4. This further
confirms the fact that the emission characteristics of Paldan in
these novel triazole-based adduct aggregates are purely due to
the excess solvent (water)-mediated stabilization of the excited
state. In the solid state (20 °C), the monomer M1 has the lowest
GP value and the tetramer T1 has highest GP value, whereas the
monomer M2 has larger GP value than that of the dimer D1
parameters, e.g., transition enthalpy (ΔH ) and entropy rise as one
c
moves from M1 to D1 to T1, which is consistent with the increase
in the number of their hydrocarbon chains. Between M1 and M2,
the same trend is seen as M2 is significantly more hydrophobic
(
two hydrocarbon chains and one triazole headgroup) than the
other monomeric adduct, M1. It appears the dimer D1 is slightly
less hydrophobic (two long chains and two triazole head groups)
compared to M2, and consistently its thermal parameters were
lower than that of M2. The corresponding cooperativity units of
the thermal transition of the adducts followed similar trends. The
transition of aggregates of the tetrameric T1, which contains four
long chains and four triazole units, was found to be the most co-
operative among the aggregates of each type of adduct.
Further we performed the DSC experiments with the click ad-
duct aggregates prepared at pH 2.0. We did not observe any signi-
ficant changes in the thermal phase transition temperatures using
DSC of the aggregates of each click adduct. This confirms again
that the protonation of the triazole groups in the aggregates of the
click adduct did not influence the packing and the Tm values in
any significant way. The DSC thermogram plots at pH 2.0 are in-
cluded in the Supporting Information (Figure S2).
(
Supporting Information, Figure S4). So, in the solid state, the
tetramer T1 is the least hydrated among all the four adducts,
whereas the aggregate of monomer M1 is the most hydrated. But
in the fluid-melted states, the dimer D1 is the most hydrated one
(
lowest GP value), whereas the tetramer T1 is the least hydrated.
3
.3.2. Polarity of the Click Adduct Aggregates. The appar-
From this experiment, we also determined the apparent phase
transition temperature from the break in the generalized polar-
ization (GP) vs temperature plot (Figure 8B). The aggregate of
monomer M1 showed the least Tm value as it contains only one
long chain (C12 chain) attached to the triazole moiety ,whereas
ent hydration of the adduct aggregates was determined by study-
ing a steady-state fluorescence emission of a polarity sensitive
1
5,16
fluorophore, Paldan,
which is the palmitoyl derivative of the
much studied polarity sensitive fluorophore, Prodan. As the chain
length of the adducts match approximately with the chain length
of Paldan, the fluorophore is expected to report the hydration of
the present set of adduct aggregates correctly. Paldan was found
to be highly sensitive to the polarity of the surrounding medium.
that of the tetramer T1 showed the highest T value as it contains
m
four C12 chains.
3
.3.3. Fluorescence Anisotropy. To further understand the
thermally induced order-to-disorder transitions in the aggregates
formed by each of these four adducts in buffered aqueous media
(pH 7.4), the hydrocarbon chain melting were also measured by
Table 3. Thermotropic Parameters As Obtained from DSC Studies
with Various Click Adduct Aggregates (1 mM) in Buffered Aqueous
Media (pH 7.4)
a
b
c
c
T
m
(°C)
ΔH
(kcal/mol)
ΔS (cal/(K mol))
Table 4. Summary of Fluorescence Characteristics of Paldan in Click
Adduct Aggregates (1 mM)
up down
adduct scan scan
up
scan
down
scan
up
scan
down
scan
d
C.U.
a
b
λ
ex (nm)
λ
em (nm)
GP
c
M1
M2
D1
42.1 40.2
46.0 43.7
45.8 43.5
50.3 48.8
5.6
8.3
6.4
14.8
5.7
8.8
6.9
14.1
19
34
23
44
22
37
26
40
37
56
46
81
adduct 20 °C 70 °C 20 °C 70 °C 20 °C 70 °C
T
m
(°C)
M1
M2
D1
355.4 349.6 435.8 436.2 0.205 0.183
373.6 373.2 440.0 445.2 0.215 0.183
376.6 375.0 442.8 449.2 0.212 0.180
41.6
46.9
46.1
49.3
T1
a
m
Accuracy of T was (0.2 °C between successive runs of the same
sample; two different sample preparations gave a difference of (1.0 °C.
T1
a
378.4 378.2 437.4 446.0 0.219 0.187
b
b
d
c
Values were within (0.2 kcal/mol. Values varied within (2 cal/(K mol).
Size of the cooperativity unit.
Probe was excited at 350 nm. Emission was monitored at 440 nm.
These values were within (0.5 °C.
c
Figure 8. (A) Fluorescence emission of Paldan doped in adduct suspension of T1 in solid (20 °C) and in fluid-melted states (70 °C). (B)
Changes in the generalized polarization (GP) of Paldan-doped aggregates (1 mM) of all the four adducts as a function of temperature.
Langmuir 2011, 27(5), 1581–1591
DOI: 10.1021/la104851g 1589

Article
Bhattacharya and Biswas
determining the fluorescence anisotropy (r) of DPH-doped ad-
ducts as a function of temperature. The r vs T profiles in Figure 9
show systemic breaks related to the main-chain thermotropic
phase transition processes for the individual adduct aggregates.
T1 is relatively loosely packed in the aggregates under analogous
conditions. This could be due to the fact that M1 has only one
long chain, whereas T1 has four long chains and four triazoles in
the same structure, so to fit into aggregate an element of
“disorderness” in T1 is manifested more compared to the other
adducts in aqueous media. Again the adducts M2 and D1
have two long chains, so the disorder in their packing is more
compared to that of adduct M1 as the r values of M2 and D1
(0.239 and 0.232, respectively) are less than that of adduct M1. In
fluid-melted state (70 °C), again M1 has the highest r value
(0.176), whereas T1 has the lowest r value (0.163), which explains
that in the fluid-melted state also the disorder in packing is more
in the aggregates of adduct T1 compared to that of the other
adducts.
We also performed the fluorescence anisotropy experiments
at pH 2.0 with the click adduct aggregates. We did not, however,
observe any noticeable changes in the anisotropy values and in the
breaks related to Tm values in the thermotropic phase transition
temperature measurements using the DPH-doped aggregates of
each click adduct. This confirms that protonation of triazole groups
in the aggregates of each click adduct did not affect the packing
and thermotropic phase transition temperature in any major way.
Fluorescence anisotropy vs T plots at pH 2.0 are included in the
Supporting Information (Figure S5).
The phase transition temperatures (T ) of all adduct aggregates
m
observed in this study are consistent with the results obtained us-
ing DSC. A summary of the thermal order-to-disorder transition
parameters as obtained from the steady-state anisotropy experi-
ment due to DPH doped in various click adduct aggregates is
given in Table 5.
In this study, we observed that in solid state (20 °C) the
adduct M1 doped with DPH has the highest fluorescence
anisotropy (r) value (0.243), whereas the adduct T1 has the lowest
r value (0.226). This suggests that the monomer M1 is tightly
packed in the aggregates in their solid state, but the tetramer
3.4. Langmuir-Blodgett Monolayer Studies. Investigat-
ing the interfacial properties of the click chemistry based four
adducts should be useful as it can provide pertinent information
on the interactions occurring between these adducts in their
aggregates. The surface properties of each adduct at the air--
water interface were studied at 25 °C using the Langmuir film
balance technique. Comparisons were also made using water
subphase made of two pH values in buffered aqueous media.
Data have been presented as a compression isotherm of sur-
face pressure as a function of the mean molecular area and reveal
details about the adduct monolayers such as states of order, two-
dimensional phase transitions, collapse areas, and collapse pres-
sures.
Figure 9. Fluorescence anisotropy of DPH-doped adduct aggre-
gates of each of the four adducts in buffered aqueous media (pH
7.4) as a function of temperature.
Table 5. Thermal Phase Transition Parameters As Obtained from the
Steady-State Anisotropy Measurements of the DPH Doped in Click
Adduct Aggregates (1 mM)
The monolayer of monomer M1 collapsed approximately at
a surface pressure and mean molecular area (collapse area) of
fluorescence
anisotropy
2
˚
2
2.43 mN/m and 15.92 A , respectively (Figure 10A). The
a
adducts
r
20
r
70
T
m
(°C)
breaks from liquid-expanded phase to liquid-condensed phase
were not sharp enough for all the four click adducts. The
monolayers of the adducts M2, D1, and T1 have relatively
sharper breaks from their liquidlike phases to the solidlike
phases. For each of the four adducts, the lift of area (i.e., a
break from gaslike phase to liquidlike phase) were not the
M1
M2
D1
0.243
0.239
0.232
0.226
0.176
0.173
0.164
0.163
42.3
47.1
47.0
51.8
T1
a
m
Accuracy of T values were within (0.5 °C.
Figure 10. Surface pressure-mean molecular area isotherms of click chemistry based adducts at 25 °C spread on buffered aqueous media
pH 7.4). Monolayers at the air-water interface were compressed at a constant rate of 5 mm/min. (A) Adducts M1, M2, and D1 and (B)
adduct T1.
(
1
590 DOI: 10.1021/la104851g
Langmuir 2011, 27(5), 1581–1591

Bhattacharya and Biswas
Article
Table 6. Isotherm Properties for Click Adducts Obtained at the
Air-Liquid Interface on Buffered Aqueous (pH 2.0 and 7.4)
Subphases at 25 °C
4. Conclusions
In conclusion, we have synthesized four simple but new 1,2,3-
triazole adducts using click chemistry, which on sonic dispersal in
aqueous media afforded vesicular aggregates as evidenced from
dye entrapment, TEM, and DLS studies. Dye entrapment studies
confirmed that each click chemistry based adduct formed closed
vesicular aggregates with inner aqueous compartments. XRD
experiments with their cast films of the aqueous suspensions
indicate the formation of a tilted bilayer type arrangement for
the aggregates of M1, whereas regular bilayer structures are
predominant for the aggregates derived from each of M2, D1,
and T1. Recording of UV-vis spectra at various pH values
2
lift-off area (A ) collapse area (A ) collapse pressure (mN/m)
2
˚
˚
adduct pH 2.0 pH 7.4 pH 2.0 pH 7.4
pH 2.0
pH 7.4
M1
M2
D1
T1
32.4
41.6
34.6
34.8
47.4
38.3
11.8
11.6
17.5
87.1
15.9
15.9
22.5
94.3
22.4
24.3
13.9
20.4
22.4
24.8
13.8
21.4
202.9 217.1
2
˚
same. The adduct T1 has the highest lift of area (217.12 A ), so
we represent the surface pressure-mean molecular area iso-
therm plot of adduct T1 separately from the others
afforded the apparent pK values of each click adduct aggregates.
a
The aggregates of monomeric click adducts (M1 and M2) possess
slightly lower pK value than that of the aggregates of dimeric
(
Figure 10B). The lift of areas of adducts M1, M2, and D1
2
˚
are respectively 34.85, 47.38, and 38.28 A . The monolayer of
a
2
˚
(D1) and tetrameric (T1) analogues, and the values lie within the
range of 2.9-3.1. The hydrodynamic diameter of the aggregates
of each click adduct increased modestly with a decrease in the pH
of the media. The temperature-induced order-to-disorder transi-
tions of the vesicles and the accompanying changes in hydration
were examined using fluorescence anisotropy and generalized
polarization measurements with the aid of membrane-soluble
probes, DPH and Paldan, respectively. In the solid state, M1
remains as the most hydrated species, whereas in the fluid-melted
phase, D1 maintains as the most hydrated aggregate. Clearly
simple changes in the adduct molecular architecture bring about
significant variation in their packing in aggregates and also the
hydration of the resulting vesicles. These results are in agreement
with the calorimetric data obtained with each of the suspensions.
Langmuir monolayer studies confirmed that these click adducts
form stable monolayers as well on buffered aqueous subphase at
the air-water interface. The mean molecular areas (collapse
areas) from the Langmuir monolayer studies were derived from
the monolayer studies, and as perhaps expected the adduct T1
possess the highest headgroup area.
adduct T1 collapsed at a mean molecular area of 94.33 A (at a
surface pressure of 21.35 mN/m), which is highest among all the
four adducts. The collapse of the monolayers of the adducts
M1, M2, and D1 occurs at a mean molecular area of 15.92,
2
˚
1
5.98, and 22.47 A , respectively. From this surface pressur-
e-mean molecular area isotherm studies, it may be concluded
that the adduct T1 has the highest headgroup area among the
four adducts. Among the monomers, M1 has higher headgroup
area. Isotherm properties of each click adduct obtained at the
air-liquid interface on buffered aqueous media (pH 2.0 and
7
.4) subphases at 25 °C are given in Table 6.
We observed almost the same collapse area for M1 and M2 as
both of them contain only one triazole moiety (as headgroup). In
M2, the second long chain most likely folds back and behaves like
a double-chain compound. As the adduct D1 contains two
triazole moieties, so its collapse area is more compared to that
of the monomeric counterparts (M1 and M2) but not quite as
large as one would expect from two “headgroups”. A possible
explanation for the lower collapse area may be due to the partial
water solubility of these adducts (M1, M2, and D1) in aqueous
media causing a diving down into the subphase during the
compression of the films. As the adduct T1 contains four long
chains and four triazole rings within the same molecule, T1 acts
differently during monolayer formation in aqueous media.
Hence, we observed a significantly higher collapse area for T1
as expected in Langmuir monolayer studies.
Thus, these click chemistry based simple triazole adducts,
which can be very easily prepared, are good candidates for further
investigations involving syntheses of novel self-assembling struc-
tures. Such adducts when suitably tailored may find wide-ranging
2
9
30
applications as novel nanocapsules or in gene delivery. Work
is now underway toward this direction in our laboratory.
We also performed Langmuir film formation with the above
click adducts at pH 2.0. Consistent with the previous observa-
tions, we did not observe major changes in the monolayer film
formation at the air-water interface due to each click adduct.
This confirms again that protonation of the triazole groups in the
aggregates of each click adduct did not remarkably alter the
isotherm properties in any significant way. Surface pressure-
mean molecular area isotherms of click chemistry based
adducts at pH 2.0 are included in the Supporting Information
Acknowledgment. J.B. thanks the CSIR, New Delhi, for the
award of senior research fellowship.
Supporting Information Available: Figures S1-S6 and
Table S1. This material is available free of charge via the
Internet at http://pubs.acs.org.
(
29) Yan, X.; He, Q.; Wang, K.; Duan, L.; Cui, Y.; Li, J. Angew. Chem., Int. Ed.
007, 46, 2431.
2
(
Figure S6).
(30) Bhattacharya, S.; Bajaj, A. Chem. Commun. 2009, 4632.
Langmuir 2011, 27(5), 1581–1591
DOI: 10.1021/la104851g 1591