Sequence-specific association in aqueous media by integrating hydrogen bonding and dynamic covalent interactions

Article abstract of DOI:10.1021/ja064515n

Oligoamide strands that associate in a sequence-specific fashion into hydrogen-bonded duplexes in nonpolar solvents were converted into disulfide cross-linked duplexes in aqueous media. Thus, by incorporating trityl-protected thiol groups, which allows the reversible formation of disulfide bonds, into the oligoamide strands, only duplexes consisting of complementary hydrogen-bonding sequences were formed in aqueous solution as well as in methanol. The sequence-specific cross-linking of oligoamide strands was confirmed by MALDI-TOF, reverse-phase HPLC, and by isolating a cross-linked duplex. This study demonstrates that the sequence-specificity characteristic of multiply hydrogen-bonded systems can be extended into competitive media through the interplay of H-bonding and reversible covalent interactions, based on which a new class of molecular associating and ligating units that are compatible with both polar and nonpolar environments can be conveniently obtained. Copyright

Full text of DOI:10.1021/ja064515n

Published on Web 09/13/2006
Sequence-Specific Association in Aqueous Media by Integrating Hydrogen
Bonding and Dynamic Covalent Interactions
Minfeng Li, Kazuhiro Yamato, Joseph S. Ferguson, and Bing Gong*
Department of Chemistry, UniVersity at Buffalo, The State UniVersity of New York, Buffalo, New York 14260
Received June 26, 2006; E-mail: bgong@chem.buffalo.edu
Strategies for controlling intermolecular association will lead to
the creation of a variety of novel self-assembled architectures, most
of which would otherwise be difficult to obtain based on covalent
synthesis.1 Due to its strength and directionality, hydrogen bonding
has attracted intense interest in the design of self-assembling
structures.2 H-bonded complexes consisting of components carrying
arrays (sequences) of H-bond donors and acceptors have been used
as associating units for directing intermolecular interactions.3 Highly
specific H-bonded units have been applied to the creation of
supramolecular constructs, such as supramolecular polymers,4
dendrimers,5 and liquid crystal materials.6 In recent years, we
reported H-bonded duplexes based on the zippering of oligoamide
strands bearing complementary H-bonding sequences.7 The du-
plexes we developed are featured by programmable sequence
specificity and tunable stability. These duplexes have been used as
sequence-specific associating units for the nucleation of â-sheet
structures when attached to natural oligopeptides,8 for the templation
and direction of chemical reactions,9 and for the noncovalent ligation
of strongly phase-separating polymer blocks.10 Like most other
H-bonded complexes, our H-bonded duplexes are stable only in
nonpolar solvents. Their dissociation in aqueous media hampers
applications involving biological conditions. Although several
systems with microenvironments that promote H-bonding in aque-
ous media have been reported,11 in competitive environments, the
association of relatively small molecular components based on
H-bonding is still a largely unsolved fundamental problem. In
contrast, in nature, nearly all water-soluble self-assembling systems
are formed based on the cooperative interaction of multiple
noncovalent forces. For example, the extensively studied DNA
duplexes are stabilized by both H-bonding and aromatic stacking
interactions.
A possible strategy for achieving specific intermolecular as-
sociation in polar media is to combine H-bonding with other forces.
In this paper, we would like to report the sequence-specific
association of molecular strands into covalently cross-linked
duplexes in aqueous media based on the interplay of H-bonding
and dynamic covalent12 interactions.
Oligoamides 1, 2, 3, and 4 were designed. In nonpolar media,
strands 1 and 2, and similarly, 3 and 4, can sequence-specifically
associate into highly stable, six-H-bonded duplexes 1‚2 and 3‚4.7b,d
It is known that, under redox conditions, S-trityl groups can lead
to the reversible formation of disulfide bonds.13 The formation of
disulfide bonds between the oligoamide strands should result in
their covalent cross-linking. In this system, the reversibility in the
formation of disulfide bonds is crucial to the retention of the
H-bonding sequence specificity. The fully matched H-bonding
sequences between 1 and 2 and between 3 and 4, along with the
presence of two disulfide linkages in each pair, should lead to
thermodynamically stable, disulfide cross-linked duplex products
1-2 and 3-4.14 Other combinations with mismatched sites, such
as the self-dimers of the individual oligoamide strands, should be
corrected due to the reversible nature of both the noncovalent and
covalent interactions.
Amides 1, 2, 3, and 4 were synthesized based on the same
procedures we reported previously7-10 and were fully characterized
by NMR and mass spectrometry.14 The 1:1 mixture of 1 and 2 (0.5
mM each) was treated with iodine (6 mM) in methylene chloride,
methanol, or water containing 10% THF. The reaction mixture was
then examined using MALDI-TOF. In CH2Cl2, the disulfide cross-
linked 1-2 was the overwhelmingly major product.14 This is not a
surprising result since in CH2Cl2 1 and 2 can form a highly stable,
six-H-bonded duplex in which the S-trityl groups are placed in close
proximity. In methanol, the effect of H-bonding on the association
of 1 and 2 is less clear, given that 1 and 2 cannot stably associate
in polar solvents.3b It is interesting that, despite the high polarity
of methanol, the sequence-specifically paired 1-2 still appeared
as the dominant product.14 To probe whether the same sequence-
specific cross-linking can be achieved in the highly competitive
aqueous solution, strands 1 and 2 were mixed in water (with 10%
THF) in the presence of iodine.14 In this case, MALDI indicated
that 1-2 again appeared as the overwhelmingly major product
(Figure 1a). That the formation of 1-2 was indeed sequence-
dependent was also supported by the results obtained when 1, 2,
or 3 alone, or the 1:1 mixture of 1 and 3, or 2 and 3, was treated
under the same redox conditions. Without its complementary strand,
1 or 2 self-cyclized into the monomeric 1′ or 2′ via the formation
of a disulfide bond. Neither 1 nor 2 paired with 3 to form the
corresponding cross-linked duplex.14
To further verify the sequence dependence of the cross-linked
products in aqueous media, a mixture containing 1 equiv (0.5 mM)
of each of 1, 2, and 3 was treated with iodine and then examined
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C O M M U N I C A T I O N S
nonpolar solvents. (2) The programmable specificity of our H-
bonded duplexes allows the generation of a large number of
duplexes that are cross-linked sequence-specifically by disulfide
bonds, which greatly expands the diversity of this important, but
symmetrical, dynamic covalent interaction. Using these supra-
molecular-dynamic covalent linking units, the directed, specific
formation of covalently linked structures and materials based on a
self-assembling fashion under mild, biocompatible conditions can
be envisioned. (3) Fundamentally, this system offers a platform
for studying the role of H-bonding interaction in water and other
competitive media. Obviously, H-bonding interactions play a critical
role in this case. Such a role can be either kinetic, based on the
sequence-specific association of the complementary stands, which
increases the effective molarity of the disulfide formation reaction,
or thermodynamic, based on the stabilization of the cross-linked
products through fully matched H-bonding sequences. Elucidating
the corresponding mechanism should lead to a deeper understanding
and better control of H-bonding in aqueous media, a challenge
worthy of addressing.
Figure 1. MALDI-TOF MS spectra of (a) the 1:1 mixture of 1 and 2 and
(b) the 1:1:1 mixture of 1, 2, and 3, in H2O/THF (9/1, v/v) in the presence
of iodine (6 mM).
Figure 2. HPLC traces of aliquots of the solutions of (a) the 1:1 mixture
of 1 and 2 and (b) the 1:1:1 mixture of 1, 2, and 3, in H2O/THF (9/1, v/v)
in the presence of iodine (6 mM). Retention time: 1-2 (21 min); 3-3 (35
min); 3′ (42 min).
Acknowledgment. This work was supported by the Petroleum
Research Fund, administered by the ACS (37200-AC4). ONR, NSF,
and NIH are acknowledged for partial funding.
using MALDI-TOF. It was found that, although having four and
three matched sites with 1 and 2, respectively, strand 3 did not
interfere with the formation of 1-2. The cross-linked 1-2 was
detected as the major product (Figure 1b). Compared to 1-2, other
possible products, such as the cross-linked 3-1 or 3-2, did not
appear in significant portion. In contrast, when mixed in water in
the presence of iodine, amide 3 did pair with 4 and form the cross-
linked 3-4 as the dominant product,14 suggesting that the observed
specific pairing and cross-linking were not a special case associated
with 1 and 2 only.
While MALDI-TOF experiments provide clear, yet qualitative,
evidence for the sequence-specific formation of the cross-linked
duplexes in aqueous solution, the cross-linking of 1 and 2 in water
was also examined by reverse phase HPLC. As shown in Figure
2a, the 1:1 mixture of 1 and 2, after being treated with iodine in
water, formed the cross-linked 1-2 exclusively. In contrast, strands
1 or 2 alone formed mostly the disulfide cyclized monomeric 1′
and 2′.14 The presence of strand 3 did not interfere with the
exclusive formation of 1-2 (Figure 2b). The identity of each HPLC
fraction was confirmed by comparing with standards or by ESI
and MALDI. These results are fully consistent with those from the
MALDI experiments, which provide conclusive evidence for the
sequence-dependent nature of the formation of the disulfide cross-
linked duplexes in aqueous media.
The efficiency of the sequence-dependent disulfide cross-linking
was also demonstrated by isolating 1-2 from a reaction on a larger
scale.14 The cross-linked crude product 1-2 formed nearly quan-
titatively and was still obtained in 80% yield even after exhaustive
purification using flash column chromatography followed by reverse
phase HPLC.
The above results clearly indicate that, in highly competitive
media, such as methanol and water, by introducing reversible
covalent forces into a H-bonded system, the high sequence
specificity offered by the H-bond arrays can be completely
preserved. This discovery is significant in several aspects: (1) The
combination of the sequence specificity of H-bond arrays and the
strength of disulfide bonds offers a novel class of molecular
associating and ligating units that are applicable in both polar and
Supporting Information Available: Experimental details, NMR
data, MS spectra, HPLC traces, and structures of cross-linked products.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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