Sequence specificity of hydrogen-bonded molecular duplexes

Article abstract of DOI:10.1021/jo010250d

Hydrogen-bonded molecular duplexes, 1·3 and 1·4, each of which contains a mismatched binding site (acceptor-to-acceptor in 1·3, and donor-to-donor in 1·4), were designed and synthesized based on duplex 1·2. One- and two-dimensional NMR studies demonstrated that, despite their single mismatched binding sites, the backbones of duplexes 1·3 and 1·4 still stayed in register through the formation of the remaining five H-bonds. The backbones of 1·3 and 1·4 adjusted to the presence of the mismatched binding sites by slightly twisting around these sites, which alleviate any headon repulsive interactions between two H-bond donors (amide O) or between two acceptors (amide H). After 1 equiv of single strand 2, which forms a perfectly matched duplex 1·2 with single strand 1, was added into the solution of either 1·3 or 1·4, only 1·2 and single strand 3 or 4, were detected. Isothermal titration calorimetry (ITC, in chloroform containing 5% DMSO) indicated that duplexes 1·3 and 1·4 were significantly (>40 times) less stable than the corresponding perfectly hydrogenbonded duplex 1·2. These NMR and ITC results indicate that the pairing of two complementary single strands is not affected by another very similar single strand that contains only one wrong H-bond donor or acceptor, which demonstrates that the self-assembly of this class of H-bonded duplexes is a highly sequence-specific process. The role of these H-bonded duplexes as predictable and programmable molecular recognition units for directing intermolecular interactions has thus been established.

Full text of DOI:10.1021/jo010250d

3574
J . Org. Chem. 2001, 66, 3574-3583
Sequ en ce Sp ecificity of Hyd r ogen -Bon d ed Molecu la r Du p lexes
Huaqiang Zeng,† Harold Ickes,‡ Robert A. Flowers, II,‡ and Bing Gong*,†
Department of Chemistry, Natural Sciences Complex, State University of New York,
Buffalo, New York 14260, and Department of Chemistry, The University of Toledo, Toledo, Ohio 43606
bgong@chem.buffalo.edu
Received March 7, 2001
Hydrogen-bonded molecular duplexes, 1‚3 and 1‚4, each of which contains a mismatched binding
site (acceptor-to-acceptor in 1‚3, and donor-to-donor in 1‚4), were designed and synthesized based
on duplex 1‚2. One- and two-dimensional NMR studies demonstrated that, despite their single
mismatched binding sites, the backbones of duplexes 1‚3 and 1‚4 still stayed in register through
the formation of the remaining five H-bonds. The backbones of 1‚3 and 1‚4 adjusted to the presence
of the mismatched binding sites by slightly twisting around these sites, which alleviate any head-
on repulsive interactions between two H-bond donors (amide O) or between two acceptors (amide
H). After 1 equiv of single strand 2, which forms a perfectly matched duplex 1‚2 with single strand
1, was added into the solution of either 1‚3 or 1‚4, only 1‚2 and single strand 3 or 4, were detected.
Isothermal titration calorimetry (ITC, in chloroform containing 5% DMSO) indicated that duplexes
1‚3 and 1‚4 were significantly (>40 times) less stable than the corresponding perfectly hydrogen-
bonded duplex 1‚2. These NMR and ITC results indicate that the pairing of two complementary
single strands is not affected by another very similar single strand that contains only one wrong
H-bond donor or acceptor, which demonstrates that the self-assembly of this class of H-bonded
duplexes is a highly sequence-specific process. The role of these H-bonded duplexes as predictable
and programmable molecular recognition units for directing intermolecular interactions has thus
been established.
In tr od u ction
donor (D) and acceptor (A) sites have recently intensi-
fied.5 The laboratories of Meijer,6 Nowick,7 Sessler,8
Zimmerman,9 and others10 reported high affinity H-
bonded systems based on heterocycles, nucleobases, and
peptide strands containing unnatural amino acids.
We have also developed highly stable H-bonded du-
plexes based on linear oligoamide strands bearing arrays
of hydrogen bond donors and acceptors on one edge.11
These molecular duplexes are formed by pairing two
strands of complementary H-bonding sequences. Due to
the absence of secondary electrostatic interactions12 in
this system, the stability of a duplex is sequence-
independent and is proportional to the number of H-
bonds found in that duplex. By incorporating mismatched
High specificity of intermolecular interaction, which
leads to the precise assembly of multicomponent archi-
tecture, is one of the most prominent features of natural
supramolecular systems. Such high specificity results
from the cooperative action of numerous noncovalent
interactions. The formation of DNA or RNA double helix
represents one of the most well-known and intensively
studied examples of molecular recognition.1 The associa-
tion of nucleic acid strands is realized by nucleobase
complementarity, which results in highly specific inter-
molecular interactions. Sequence-specific pairing of DNA
and RNA strands is essential for the storage, transmis-
sion, and expression of genetic information, and forms
the basis for techniques such as PCR,2 hybridization
techniques,3 and DNA chip arrays.4 To develop DNA-like,
information-rich molecules that may lead to the specifi-
cation of intermolecular interactions, efforts in designing
complexes based on arrays (sequences) of hydrogen bond
(5) Zimmerman, S. C.; Corbin, P. S. Struct. Bond. 2000, 96, 63.
(6) (a) Folmer, B. J . B.; Sijbesma, R. P.; Kooijman, H.; Spek, A. L.;
Meijer, E. W. J . Am. Chem. Soc. 1999, 121, 9001. (b) Sijbesma, R. P.;
Meijer, E. W. Curr. Opin. Colloid. Interface Sci. 1999, 4, 24.
(7) Nowick, J . S.; Chung, D. M.; Maitra, K.; Maitra, S.; Stigers, K.
D.; Sun, Y. J . Am. Chem. Soc. 2000, 122, 7654.
(8) (a) Sessler, J . L.; Wang, R. Z. J . Am. Chem. Soc. 1996, 118, 9808.
(b) Sessler, J . L.; Wang, R. Angew. Chem., Int. Ed. 1998, 37, 1726.
(9) (a) Corbin, P. S.; Zimmerman, S. C. J . Am. Chem. Soc. 1998,
120, 9710. (b) Corbin, P. S.; Zimmerman, S. C. J . Am. Chem. Soc. 2000,
122, 3779.
(10) For other duplex systems, see (a) Bisson, A. P.; Carver, F. J .;
Eggleston, D. S.; Haltiwanger, R. C.; Hunter, C. A.; Livingstone, D.
L.; McCabe, J . F.; Rotger, C.; Rowan, A. E. J . Am. Chem. Soc. 2000,
122, 8856. (b) Archer, E. A.; Goldberg, N. T.; Lynch, V.; Krische, M. J .
J . Am. Chem. Soc. 2000, 122, 5006. (c) Berl, V.; Huc, I.; Khoury, R. G.;
Krische, M. J .; Lehn, J . M. Nature 2000, 407, 720.
(11) (a) Gong, B.; Yan, Y.; Zeng, H.; Skrzypczak-J ankunn, E.; Kim,
Y. W.; Zhu, J .; Ickes, H. J . Am. Chem. Soc. 1999, 121, 5607. (b) Zeng,
H.; Miller, R.; Flowers, II, B.; Gong, B. J . Am. Chem. Soc. 2000, 122,
2635. (c) Gong, B. Synlett 2001, (5), in press.
* To whom correspondence should be addressed. Fax: (716) 645
6963.
† State University of New York, Buffalo, NY.
‡ The University of Toledo.
(1) (a) Saenger, W. Principles of Nucleic Acid Structure; Springer:
New York, 1984. (b) Blackburn, G. M. In Nucleic Acids in Chemistry
and Biology; Blackburn, G. M.; Gait, M. J ., Ed.; IRL Press: Oxford,
1990; p 17.
(2) Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J .; Higuchi,
R.; Horn, G. T.; Mullis K. B.; Erlich, H. A. Science 1988, 239, 487.
(3) (a) Fodor, S. P. A.; Rava, R. P.; Huang, X. H. C.; Pease A. C.;
Holmes, C. P.; Adams, C. L. Nature 1993, 364, 555. (b) Mirzabekov,
A. D. Trends Biotechnol. 1994, 12, 17.
(4) (a) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.;
Stern, D.; Winkler, J .; Lockhart, D. J .; Morris, M. S.; Fodor, S. P. A.
Science 1996, 274, 610. (b) Wallace, R. W. Mol. Med. Today 1997, 3,
384.
(12) (a) Pranata, J .; Wierschke, S. G.; J orgensen, W. L. J . Am. Chem.
Soc. 1991, 113, 2810-2819. (b) J orgensen, W. L.; Pranata, J . J . Am.
Chem. Soc. 1990, 112, 2008-2010. (c) Murray, T. J .; Zimmermann, S.
C. J . Am. Chem. Soc. 1992, 114, 4010-4011.
10.1021/jo010250d CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/19/2001

Hydrogen-Bonded Molecular Duplexes
J . Org. Chem., Vol. 66, No. 10, 2001 3575
Ch a r t 1
binding sites, such a molecular duplex system should
provide an ideal platform for systematically probing the
effect of sequence-specificity on unnatural self-assembling
processes, which have so far been difficult to investigate
due to the lack of appropriate model systems. Addressing
the problem of sequence-specificity is critical to achieving
the final objective of this research, which involves
developing these molecular duplexes into a whole set of
information-rich molecular recognition units that can
lead to predictable and programmable intermolecular
interactions. Such molecular recognition units will serve
as powerful and versatile “sticky ends” for assembling
multicomponent architecture.
Single strands 1 and 2 were found to pair into a highly
stable, six-hydrogen-bonded duplex 1‚2 (Chart 1).11b
Single strands 3 and 4, with the hydrogen-bonding
sequences of DADDAA and DADDDD, were designed and
synthesized to pair with 1. Each of the resultant du-
plexes, 1‚3 or 1‚4, should contain a mismatched binding
site. Can duplexes 1‚3 and 1‚4 still form? Will the single
strands in duplexes 1‚3 and 1‚4 be partially aligned to
avoid the mismatched binding sites? What are the

3576 J . Org. Chem., Vol. 66, No. 10, 2001
Zeng et al.
Sch em e 1
stabilities of 1‚3 and 1‚4 compared to that of 1‚2? How
does the placement of the mismatch within each strand
influence the thermodynamics of duplex formation?
H-bonding sequences paired into the corresponding du-
plexes.
Resu lts a n d Discu ssion
We report here that, similar to duplex DNA, the self-
assembly of these unnatural H-bonded duplexes is a
highly sequence-specific process. Comparing duplexes 1‚
3 and 1‚4 with 1‚2, which contains a perfectly matched
H-bonding sequence, revealed the much higher stability
of the latter. When three different single strands coex-
isted in solution, only the two with perfectly matched
Syn th esis. Single oligoamide strands 3 and 4 were
synthesized by iterative coupling steps using either acid
chloride or EDC (Scheme 1). Compound 3a 11b was acy-
lated into 3b which was then hydrolyzed to give acid 3c.
Diamine 3d 11b was then monoacylated by 3c using EDC
coupling, leading to 3e.

Hydrogen-Bonded Molecular Duplexes
J . Org. Chem., Vol. 66, No. 10, 2001 3577
Dimethyl 4,6-dioctyloxy-1,3-benzoate 3f11b was par-
tially hydrolyzed by using 1 equiv of NaOH in DMSO
and water to give the monoacid 3g. The coupling between
3g and hexylamine using EDC in DMF gave 3h . Hy-
drolysis of 3h gave 3i, which was first coupled with
glycine ethyl ester to give 3j. Subsequent hydrolysis of
3j lead to 3k . Coupling between 3e and 3k using EDC
in DMF lead to 3.
Amide 4a was obtained by acylating 3-nitroaniline
using hexanoyl chloride. Hydrogenation of 4a and sub-
sequent acylation lead to ester 4c which was hydrolyzed
to give acid 4d . Coupling between 3e and 4d using EDC
in DMF lead to 4.
1
1H NMR Sp ectr oscop y. The H NMR (400 MHz, 298
K in CDCl3) spectra of single strand 3 or 4 showed broad,
poorly defined resonances, indicating a slowly equilibrat-
ing mixture of many conformations. Upon mixing 1 equiv
of 3 or 4 with 1 equiv of 1, both 3 and 4 showed sharp
sets of signals that can only be assigned to a single
conformer, suggesting the association of 3 or 4 with 1.
Interestingly, protons 7 and 7′, which are equivalent in
single strand 1 or in duplex 1‚2 as proton d, became
nonequivalent upon mixing 1 with 3 or 4 (Figure 1). At
25 mM in CDCl3, proton 7 of 1‚3 appeared at 10.31 ppm,
and that of 1‚4 at 9.98 ppm; proton 7′ of 1‚3 appeared at
10.04 ppm, and that of 1‚4 at 9.73 ppm.13 These data
suggest the formation of duplexes 1‚3 and 1‚4 whose
unsymmetrical structures placed protons 7 and 7′ in
different chemical environment. Obviously, the exchange
of single strands 1, 3, and 4 with the corresponding
duplex 1‚3 or 1‚4 was slow on the NMR time scale.
To provide more detailed evidence for the formation
of duplexes 1‚3 or 1‚4 in solution, two-dimensional NMR
(NOESY) spectra were recorded (400 MHz, 293 K, 25 mM
in CDCl3). In each case, numerous interstrand contacts
were observed, which confirms the dimeric structures of
1‚3 and 1‚4. Figure 2 shows the regions of the NOESY
spectrum of duplex 1‚3, which indicate strong NOE
contacts between protons 13 and 1, and 20 and 8, as well
as 27 and 8′ (Figure 2a). In addition, Figure 2b shows
contacts between protons 14 and 4, 21 and 11, 23 and
11, and 26 and 1. Weak contact between protons 31 and
4′, and 31 and 7′, also exist. No NOE contact between
protons 34 and 1′ is found. These results suggest that
one end of 1‚3 is locked by H-bonds while the other is
open due to the presence of the mismatched binding site
consisting of the two amide carbonyl groups. NOESY
spectrum of 1‚4, as shown in Figure 3a, indicates strong
NOE contacts between protons 13′ and 1, and between
34′ and 1′, along with contacts between protons 20′ and
8, and 27′ and 8′, suggesting that the two strands align
in an end-to-end fashion. The NOESY results indicate
that both ends of 1‚4 are locked despite the presence of
mismatched binding site consisting of the two amide NH
groups. Figure 2b shows additional contacts between
protons 21′ and 11, 23′ and 11, 26′ and 11, and between
33′ and 4′. The fact that no cross-strand contacts are
observed between protons 28′ and 7′, and only extremely
weak contacts between protons 31′ and 7′, and between
28′ and 4′ are detected, indicates that the backbones of
1‚4 must be twisted to alleviate any head-on repulsive
interaction between the two NH groups. Consistent with
F igu r e 1. Amide NH resonances of (a) proton d of single
strand 1, (b) protons 7 and 7′ of 1‚4, and (c) protons 7 and 7′
of 1‚3 at 25 mM.
this explanation, in 1‚4, proton 28′ exhibits very strong
NOE contact with 29′, and strong NOE with 30′; while
proton 14′ exhibits no NOE contacts at all with 15′ and
16′. However, this type of twist seems not to disrupt the
formation of the other five attractive H-bonds, which is
supported by the presence of cross-strand NOE contacts.
Such a twist in backbone should also be true for 1‚3,
which is supported by the lack of NOE contact at one
end of this duplex as shown by NOESY studies. In 1‚3
the five attractive H-bonds also formed as evidenced by
cross-strand NOE contacts.
1H NMR competition titration experiments were car-
ried out to examine the sequence specificity in forming
1‚2. The 1H NMR signals within the 9.30-9.85 ppm
region are good indicators for deriving the information
1
of the sequence-specific pairing of 1‚2 (Figure 4). The H
NMR spectra of 1‚3 (Figure 4a) and 1‚4 (Figure 4e) (at 2
mM in 5%DMSO-d6 in CDCl3) show two and three sharp
peaks in this region, respectively. With the addition of 1
equiv of 2 into either 1‚3 (Figure 4b) or 1‚4 (Figure 4d),
the only signal left in this region is the one corresponding
to proton i of 1‚2 (Figure 4d). Signal of proton 14 in 1‚3
(13) The assignment for 7 and 7′ was made based on the assumption
that 7 is “locked” in the fully H-bonded side of 1‚3 or 1‚4 and should
appear at a more downfield position than that of 7′.

3578 J . Org. Chem., Vol. 66, No. 10, 2001
Zeng et al.
F igu r e 2. NOESY spectra of 1‚3 (25 mM in CDCl3, mixing time 0.5 s) showing cross-strand contacts: (a) between protons 1 and
13, 8 and 20, and 8′ and 27, (b) between protons 14 and 4, 21 and 11, 23 and 11, and 26 and 11. Weak contacts (dashed arrows)
exist between protons 31 and 4′, and 31 and 7′. No contacts can be detected between protons 34 and 1′.
shifts upfield (δ < 9.00 ppm), and the two signals of the
amide protons 26′ and 33′ in 1‚4 also show significant
upfield shifts (δ < 9.10 and 8.50 ppm, respectively). The
signals of protons 19 and 19′ remain in this range by
merging into the same position as the corresponding
proton i (9.56 ppm) in 1‚2. It needs to be pointed out that
signals in this region (9.30-9.85 ppm) are not from free
single strands 1 and 2 which, if existed in solution, should
show only one signal corresponding to protons d (9.67
ppm) and h (9.79 ppm), respectively. Free single strands
3 and 4 do not have any corresponding peaks within this
region. These results have confirmed that single strand
2 specifically pair with 1 by displacing 3 or 4 when added
into the solution of 1‚3 or 1‚4.
Isoth er m a l Titr a tion Ca lor im etr y. To quantita-
tively evaluate the effect of a single mismatched binding
site on the stabilities of the resultant duplexes, a solution
of 8 mM 1 was titrated into 1 mM of 3 or 4 in 5% DMSO/
CHCl3 by isothermal titration calorimetry (ITC). Analysis
of the resulting binding isotherm (Figure 5) gave the
dimerization constants and the thermodynamic data as
shown in Table 1. With just one mismatched hydrogen
bonding site, the two duplexes were found to be at least
40 times less stable (Ka ) (7.5 ( 0.4) × 104 M-1 for 1‚3,
and Ka ) (8.2 ( 0.6) × 104 M-1 for 1‚4) than duplex 1‚2
(Ka ) (3.5 ( 1.3) × 106 M-1) whose two single strands
are perfectly matched. Like the binding of 2 to 1, the
binding of both 3 or 4 to 1 is also largely enthalpically
driven. Unlike the formation of 1‚2 whose entropy
contributed negatively to the duplex stability (T∆S )
-0.8 kcal/mol),11b the mismatched duplex 1‚4 has an
almost negligible entropy contribution (T∆S ) -0.2 kcal/
mol), while in 1‚3, it becomes even positive (T∆S ) +0.9
kcal/mol). In combination with 2D NOESY data, these
results indicate that, due to its one open end, duplex 1‚3
has the most flexible backbones; the backbones of 1‚4 are
less flexible; in 1‚2, the backbones are relatively rigid.
These results also suggest that, although the positions
and the types of the mismatched bonding sites have a
considerable effect on the mismatched duplexes in terms
of enthalpy and entropy, the overall stability of these
mismatched duplexes are similar within experimental
error. There seems to be a mechanism for duplexes 1‚3
and 1‚4 to compensate their loss in binding energy caused
by mismatched sites to achieve similar stabilities that
are consistent with the number of H-bonds in each
complex.

Hydrogen-Bonded Molecular Duplexes
J . Org. Chem., Vol. 66, No. 10, 2001 3579
F igu r e 3. NOESY spectra of 1‚4 (25 mM in CDCl3, mixing time 0.5 s) showing cross-strand contact between (a) protons 1 and
13′, 8 and 20′, 8′ and 27′, and 1′ and 34′, (b) between protons 21′ and 11, 23′ and 11, 26′ and 11, and between 33′ and 4′. Very
weak contacts (dashed arrows) exist between protons 31′ and 7′, and between 28′ and 4′. No contacts can be detected between
protons 28′ and 7′.
Con clu sion s
duplexes can be applied to direct the assembly of a wide
variety of structural fragments. The design and construc-
tion of self-assembling, multicomponent architectures can
be envisioned.
The above data have demonstrated that replacement
of a hydrogen bond by single mismatched binding sites
in a six-hydrogen-bonded duplex still lead to complexes
with registered backbones. However, the mismatched
sites resulted in duplexes with significantly reduced
stabilities. Such a reduction in stabilities indicates that
a single strand will indeed only pair with another single
strand with a complementary sequence, forming a duplex
with perfectly matched single strands. Other strands
with imperfect sequences are not likely to interfere with
the formation of the “correct” duplex, which is confirmed
by NMR competition studies. The high precision as
demonstrated by the complementary molecular strands
in their pairing has thus confirmed this class of com-
pounds as unnatural information-rich molecular strands.
Combined with results previously reported by us,11 the
role of this class of H-bonded duplexes as versatile
unnatural molecular recognition units have now been
established. With their adjustable stabilities and pro-
grammable sequence specificities that parallel the mo-
lecular recognition characteristics of duplex DNA, these
Exp er im en ta l Section
Gen er a l Meth od s. All chemicals were purchased from
Aldrich, Fluka, and Sigma and used as received unless
otherwise noted. The organic phase from all liquid extractions
was dried over Na2SO4 unless specified otherwise. All products
were detected as single spots by thin-layer chromatography
(precoated 0.25 mm silica plates from Aldrich). All samples
were purified either by recrystallization or by flash column
chromatography and dried completely under high vacuum
before characterized by 1H NMR (400 MHz), 13C NMR (100
MHz), and elemental analysis. NMR chemical shifts were
reported in ppm relative to TMS. For the 1H NMR dilution
experiments, CDCl3 (99.8% D) and DMSO-d6 (99.8% D) were
purchased from Cambridge Isotope Laboratory and used
without further purification. T1 measurements were carried
out with degassed samples (freeze-thaw cycle with nitrogen).
NOE measurements were performed with the steady-state
NOEDIF protocol on degassed samples.
N-Eth oxyca r bon ylm eth yl-5-h exa n oa m id o-2-octyloxy-
ben za m id e (3b). To a solution of amine 3a (2.00 g, 5.70 mmol)

3580 J . Org. Chem., Vol. 66, No. 10, 2001
Zeng et al.
tion of the solvent gave the crude product which was recrystal-
lized from MeOH, giving the pure product as a white solid (2.28
g, 89%). 1H NMR (CDCl3) δ 8.714 (s, 1H), 8.192 (d, 1H; J )
8.8 Hz), 7.849 (s, 1H), 7.607 (br, 1H), 6.941 (d, 1H; J ) 8.8
Hz), 4.273-4.220 (m, 4H), 4.124 (t, 2H; J ) 6.4 Hz), 2.353 (t,
2H; J ) 7.2 Hz), 1.932 (m, 2H), 1.716 (m, 4H), 1.477-1.285
(m, 15H), 0.898-0.863 (m, 6H). 13C NMR (CHCl3)δ 171.91,
169.80, 164.87, 153.85, 131.76, 125.40, 123.08, 120.72, 113.11,
69.78, 61.37, 42.16, 37.58, 31.77, 31.41, 29.26, 29.18, 20.07,
26.14, 25.31, 22.61, 22.41, 14.18, 14.04, 13.89. Anal. Calcd for
C
25H40N2O5: C, 66.93; H, 8.99; N, 6.25. Found: C, 67.07; H,
9.06; N, 6.27.
N-Ca r b oxym et h yl-5-h exa n oa m id o-2-oct yloxyb en za -
m id e (3c). The ester 3b (2.24 g, 5.00 mmol) was dissolved in
hot MeOH (30 mL), to which 1 N NaOH (5.50 mL, 5.50 mmol)
and 10 mL of H2O was added. The mixture was heated under
reflux for 30 min, to which more water (100 mL) was added.
The aqueous layer was neutralized by addition of concentrated
HCl to pH 3.0. The precipitated crude product was collected.
Recrystallization of the crude product from MeOH gave a white
1
solid (2.01 g, 96%). H NMR (CDCl3) δ 8.799 (s, 1H), 8.425 (s,
1H), 8.143 (q, 1H; J ) 2.3 Hz, 9.2 Hz), 7.829 (d, 1H, J ) 2.4
Hz), 7.462 (br, 1H), 6.856 (d, 1H, J ) 9.2 Hz), 4.243 (d, 2H, J
) 4.0 Hz), 4.045 (t, 2H; J ) 6.4 Hz), 2.348 (t, 2H; J ) 7.2 Hz),
1. 698 (m, 2H), 1.668 (m, 2H), 1.410-1.235 (m, 14H), 0.845 (t,
6H; J ) 6.4 Hz). 13C NMR (CHCl3) δ 172.51, 172.12, 165.64,
153.82, 131.87, 125.82, 123.24, 119.79, 112.85, 69.64, 42.37,
37.31, 31.72, 31.37, 29.193, 28.94, 25.99, 25.34, 22.60, 22.38,
14.05, 13.88. Anal. Calcd for C23H36N2O5: C, 65.69; H, 8.63;
N, 6.66. Found: C, 65.49; H, 8.67; N, 6.59.
Octyl 3-Am in o-5-[(5′-h exa n oa m id o-2′-octyloxy)p h en yl-
ca r bon yla m in om eth yl-ca r bon yla m in o]ben zoa te (3e). To
a solution of acid 3c (1.96 g, 4.66 mmol) in 30 mL of methylene
chloride were added EDC [1-ethyl-3-(3-dimethyl aminopropyl)-
carbodiimide hydrochloride] (0.90 g, 4.67 mmol) and 1-hydoxy-
benzotriazole (0.71 g, 4.67 mmol). After stirring for 30 min,
the solution was added dropwise within 10 min to octyl 3,5-
diaminobenzoate 3d (3.38 g, 12.8 mmol) in 40 mL of methylene
chloride. The reaction was allowed to proceed for 6 h. The
precipitated solid was filtered and dried in the air to give the
1
pure product as a white solid (2.37 g, 76%). H NMR (DMSO-
d6) δ 10.000 (s, 1H), 9.868 (s, 1H), 8.646 (s, 1H), 8.051 (d, 2H;
J ) 2.8 Hz), 7.783 (q, 1H; J ) 2.8 Hz, J ) 8.8 Hz), 7.405 (s,
1H), 7.102 (d, 1H; J ) 8.8 Hz), 7.066 (s, 1H), 6.896 (s, 1H),
5.413 (s, 2H), 4.187 (t, 2H; J ) 6.0 Hz), 4.135 (d, 2H; J ) 4.8
Hz), 4.110 (t, 2H; J ) 6.4 Hz), 2.251 (t, 2H; J ) 6.0 Hz), 1.818
(m, 2H), 1.648 (m, 2H), 1.564 (m, 2H), 1.380-1.157 (m, 22H),
0.859-0.829 (m, 6H), 0.767 (t, 3H; J ) 6.8 Hz). 13C NMR
(DMSO) δ 171.04, 167.09, 166.17, 164.23, 152.57, 149.35,
139.54, 132.67, 130.78, 123.69, 121.87, 121.22, 113.55, 109.83,
108.56, 107.77, 69.28, 64.35, 43.60, 36.26, 31.26, 30.92, 28.79,
28.76, 28,68, 28.65, 28.58, 28.28, 25.80, 25.50, 24.87, 22.10,
21.92, 13.95, 13.93, 13.90. Anal. Calcd for C38H58N4O6: C,
68.43; H, 8.77; N, 8.40. Found: C, 68.13; H, 8.62; N, 8.35.
Dim eth yl 4,6-Dioctyloxy-1,3-ben zen edicar boxylate (3f).
A mixture of 3l (6.79 g, 30 mmol), K2CO3 (24.86 g, 180 mmol),
and 1-bromooctane (23.2 g, 120 mmol) in 150 mL of DMF
containing 30 mL of methanol was heated at 100 °C for 2 days.
The solid was filtered off and the solvent was removed in vacuo
at 130 °C. The residue was dissolved in ethyl acetate and
washed with diluted HCl and NaOH solutions alternatively.
Evaporation of the solvent gave pure 3f (11.9 g, 88%). 1H NMR
(CDCl3) δ 8.458 (s, 1H), 6.423 (s, H), 4.057 (t, 4H; J ) 6.4 Hz),
3.853 (s, 6H), 1.867 (m, 2H), 1.507 (m, 4H), 1.348-1.284 (m,
16H), 0.884 (t, 3H; J ) 6.8 Hz). 13C NMR (CDCl3) δ 165.43,
163.63, 137.01, 112.05, 97.70, 69.28, 51.62, 31.80, 29.28, 29.21,
29.06, 25.53, 22.64, 14.02. Anal. Calcd for C26H42O6: C, 69.30;
H, 9.40. Found: C, 69.46; H, 9.68.
F igu r e 4. Amide NH resonances of (a) 1‚3, (b) 1‚3 + 2, (c)
1‚2, (d) 1‚4 + 2, and (e) 1‚4 (2 mM) in the region of 9.30-9.85
ppm in CDCl3 containing 5% DMSO-d6 at 400 MHz. There are
no peaks corresponding to single strands 3 and 4 exist within
this region.
2,4-Dioctyloxy-5-m eth oxyca bon ylben zoic Acid (3g). To
a solution of Dimethyl 4,6-dioctyloxy-1,3-benzenedicarboxylate
3f (13.52 g, 30 mmol) in 50 mL of DMSO at 130 °C was added
KOH (1.68 g, 30 mmol) in 15 mL of MeOH drop by drop. The
reaction was kept at 130 °C for 3 h. After cooling the solution,
5 mL of concentrated HCl and 150 mL of distilled water was
added. The aqueous layer was extracted with 2 × 200 mL ethyl
and triethylamine (0.57 g, 5.70 mmol) in 60 mL of methylene
chloride was added dropwise hexanoyl chloride (0.79 g, 5.70
mmol) over 5 min. at 0 °C. The ice-water bath was removed.
After 6 h the solvent was evaporated, and the residue was
dissolved in ethyl acetate and washed with diluted aqueous
HCl and NaOH solutions alternatively. Drying and evapora-

Hydrogen-Bonded Molecular Duplexes
J . Org. Chem., Vol. 66, No. 10, 2001 3581
F igu r e 5. Calorimetry binding isotherms for the titration of (a) 3 with 1, and (b) 4 with 1 in 5% DMSO/chloroform.
Ta ble 1. Associa tion Con sta n ts (KA) a n d
Th er m od yn a m ic Da ta for th e Bin d in g of 2-4 to 1 in
CHCl3 Con ta in in g 5% DMSO.
28.89, 28.97, 29.05, 29.15, 29.18, 29.23, 31.69, 31.79, 51.80,
69.64, 70.65, 97.07, 109.86, 114.70, 138.40, 161.49, 164.23,
164.78. Anal. Calcd for C25H40O6: C, 68.78; H, 9.24; Found:
C, 68.53; H, 9.33.
1‚2
1‚3
1‚4
N -H e x y l-2,4-d io c t y lo x y -5-m e t h o x y c a b o n y lb e n za -
m id e (3h ). To a solution of acid 3g (5.72 g,13.1 mmol), EDC
(2.52 g, 13.1 mmol), and 1-hydoxybenzotriazole (1.77 g, 13.1
mmol) in 40 mL of DMF was added hexylamine (1.33 g, 13.1
mmol) in 10 mL of DMF. The reaction mixture was kept
heating for 1 h to dissolve any precipitate and was allowed to
proceed for additional 5 h at room temperature. Distilled water
(150 mL) and ethyl acetate (150 mL) were added. The ethyl
acetate layer was washed with distilled water (100 mL) at least
four times to ensure complete removal of DMSO. Drying and
evaporation of ethyl acetate gave pure 3h as a white solid (5.58
g, 82%).1H NMR (CDCl3) δ: 0.885 (9H, d, J ) 4.4 Hz), 1.300-
1.327 (22H, m), 1.501 (4H, m), 1.580 (2H, m), 1.882 (4H, m),
3.429 (2H, q, J ) 6.8 Hz, J ) 12.4 Hz), 3.838 (3H, s), 4.043
Ka (M-1
∆G (kcal/ -8.9 ( 0.2
mol)
∆H (kcal/ -9.7 ( 0.2
mol)
T∆S (kcal/ -0.8 ( 0.4
mol)
)
(3.5 ( 1.3) × 106 (8.2 ( 0.4) × 104 (7.5 ( 0.6) × 104
-6.7 ( 0.4
-5.8 ( 0.2
0.9 ( 0.5
-6.6 ( 0.4
-6.8 ( 0.2
-0.2 ( 0.4
acetate. After drying and evaporation of ethyl acetate the
residue was recrystallized twice from 100 mL hexane to give
pure 3g as a white solid (7.99 g, 61%). 1H NMR (CDCl3) δ:
0.870-0.904 (6H, m), 1.293-1.361 (16H, m), 1.462-1.538 (4H,
m), 1.847-1.972 (4H, m), 3.859 (3H, s), 4.081 (2H, t, J ) 6.4
Hz), 4.260 (2H, t, J ) 6.8 Hz), 6.488 (1H, s), 8.694 (1H, s).
13CNMR (CDCl3) δ: 13.99, 14.02, 22.58, 22.63, 25.86, 25.88,

3582 J . Org. Chem., Vol. 66, No. 10, 2001
Zeng et al.
(2H, t, J ) 6.4 Hz), 4.135 (2H, t, J ) 6.0 Hz), 6.425 (1H, s),
7.709 (1H, s), 8.741 (1H, s). 13C NMR (CDCl3) δ: 13.99, 14.02,
22.59, 22.63, 25.91, 26.26, 26.88, 29.09, 29.19, 29.20, 29.23,
29.28, 29.33, 29.66, 31.59, 31.77, 31.80, 39.80, 51.55, 69.43,
97.13, 113.44, 114.21, 136.94, 160.85, 162.60, 164.22, 165.40.
Anal. Calcd for C31H53NO5: C, 71.64; H, 10.28; N, 2.69.
Found: C, 71.50; H, 10.48; N, 2.98.
N-H exyl-2,4-d ioct yloxy-5-h yd r oxyca b on ylb en za m id e
(3i). Hydrolysis of 3h based on the similar procedures as
described for 3c gave the crude product, which was crystallized
from MeOH to give the pure product as a white solid (94%).
1H NMR (CDCl3) δ: 0.868-0.893 (9H, m), 1.301-1.596 (26H,
m), 1.913 (4H, m), 3.428 (2H, q, J ) 6.8 Hz, J ) 12.8 Hz),
4.155 (2H, t, J ) 6.4 Hz), 4.228 (2H, t, J ) 6.4 Hz), 6.466 (1H,
s), 7.561 (1H, t, J ) 3.2 Hz), 8.957 (1H, s). 13C NMR (CDCl3)
δ: 13.99, 22.59, 25.86, 26.23, 26.87, 28.93, 29.06, 29.17, 29.20,
29.32, 29.63, 31.59, 31.70, 31.78, 39.91, 69.85, 70.66, 96.60,
111.21, 138.70, 160.59, 161.53, 163.58, 164.06, 194.62. Anal.
Calcd for C30H51NO5: C, 71.25; H, 10.16; N, 2.77. Found: C,
71.10; H, 10.39; N, 3.00.
167.41, 166.10, 165.15, 164.45, 164.10, 160.47, 160.32, 153.29,
139.11, 136.45, 132.64, 131.36, 124.88, 123.02, 120.81, 116.08,
114.86, 114.65, 114.12, 112.82, 96.40, 69.77, 69.56, 69.46,
65.05, 44.33, 33.87, 31.64, 31.60, 31.45, 31.34, 29.44, 29.21,
29.11, 29.05, 28.93, 28.80, 28.60, 26.14, 26.08, 25.78, 25.30,
24.88, 22.48, 22.35, 14.02, 13.95. Anal. Calcd for C70H110
-
N6O11: C, 69.39; H, 9.15; N, 6.94. Found: C, 69.54; H,9.27; N,
7.02.
N-(3-Nitr op h en yl)h exa n oa m id e (4a ). Hexanoyl chloride
(1.34 g) in CH2Cl2 was added to a solution of 3-nitroaniline
(1.38 g) and triethylamine (1.01 g) in CH2Cl2 (50 mL) at 0-5
°C. After stirring for overnight, the solvent was removed and
the residue was dissolved in ether and washed with acidic and
basic solutions. Evaporation of ether gave the pure product
as a yellow oil, which solidified upon standing (1.98 g, 84%).
1H NMR (CDCl3) δ 9.351 (s, 1H), 9.932 (m, 2H), 7.649 (s, 1H),
7.458 (t, 1H, J ) 8.4 Hz), 2.388 (t, 2H, J ) 7.6 Hz), 1.715 (m,
2H), 1.324-1.350 (m, 4H), 0.905 (t, 3H, J ) 7.2 Hz). 13C NMR
(CDCl3) δ 171.85, 148.48, 139.02, 129.83, 125.40, 118.73,
114.39, 37.66, 31.34, 25.08, 22.39, 13.90. Anal. Calcd for
N-Eth oxyca r bon ylm eth yl-N′-h exyl-4,6-d ioctyloxy-1,3-
ben zen ed ica r boxa m id e (3j). To a solution of acid 3i (0.384
g, 0.76 mmol) and EDC (0.146 g, 0.76 mmol) and HOBt (1-
hydoxybenzotriazole) (0.103 g, 0.76 mmol) in 30 mL of DMF
were added glycine ethyl ester hydrochloride (0.420 g, 3.0
mmol) and triethylamine (0.203 g, 2 mmol) in 10 mL DMSO.
The reaction mixture was allowed to proceed for 6 h at room
temperature. Distilled water and ethyl acetate were added.
Cooling the solution gave pure 3j as a white solid (0.40 g, 89%).
1H NMR (CDCl3) δ: 0.875-0.900 (9H, m), 1.299-1.612 (31H,
m), 1.894 (2H, m), 1.974 (2H, m), 3.440 (2H, q, J ) 6.8 Hz, J
) 12.4 Hz), 4.138 (4H, q, J ) 6.4 Hz, J ) 12.4 Hz), 4.245 (4H,
m), 6.442 (1H, s), 7.614 (1H, t, J ) 4.2 Hz), 8.272 (1H, t, J )
4.8 Hz), 9.016 (1H, s). 13C NMR (CDCl3) δ: 14.03, 14.06, 14.20,
22.62, 26.16, 26.26, 26.89, 28.97, 29.18, 29.23, 29.26, 29.34,
29.63, 31.60, 31.78, 39.78, 42.08, 61.27, 69.42, 69.74, 96.20,
114.42, 115.37, 137.19, 160.23, 160.28, 164.21, 164.25, 170.23.
Anal. Calcd for C34H58N2O6: C, 69.12; H, 9.89; N, 4.74.
Found: C, 68.89; H, 9.75; N, 4.77.
N-H yd r oxyca r b on ylm et h yl-N′-h exyl-4,6-d ioct yloxy-
1,3-ben zen ed ica r boxa m id e (3k ). To a solution of ester 5h
(0.591 g, 1 mmol) in 10 mL of DMSO under reflux was added
NaOH (0.4401 g, 1.1 mmol) in 3 mL of H2O. The reaction was
kept refluxing for 20 min. After cooling the solution, 0.3 mL
of concentrated HCl and 150 mL of distilled water were added.
The precipitate was filtered off and recrystallized from MeOH
to give pure 5i as a white solid (0.512 g, 91%). 1H NMR (CDCl3)
δ: 0.858-0.907 (9H, m), 1.301-1.596 (28H, m), 1.921 (4H, m),
3.432 (2H, q, J ) 6.8 Hz, J ) 12.4 Hz), 4.131 (4H, t, J ) 6.0
Hz), 4.273 (2H, d, J ) 4.4 Hz), 6.430 (1H, s), 7.681 (1H, t, J )
4.8 Hz), 8.310 (1H, t, J ) 4.4 Hz), 8.947 (1H, s). 13C NMR
(CDCl3) δ 13.99, 14.02, 22.60, 25.74, 26.28, 26.96, 28.52, 29.12,
29.26, 29.32, 29.50, 31.66, 31.73, 31.80, 39.79, 43.86, 69.38,
69.59, 96.06, 113.77, 114.17, 136.28, 160.00, 160.46, 164.24,
164.46, 174.67. Anal. Calcd for C32H54N2O6: C, 68.29; H, 9.67;
N, 4.98; Found: C, 68.46; H, 9.95; N, 5.17.
Octyl 3-[(5′-Hexa n oa m id o-2′-octyloxy)p h en ylca r bon y-
la m in om eth yl ca r bon yl-a m in o]-5-[(5′′-h exyla m in oca r bo-
n yl-2′′,4′′-d ioct yloxy)p h en ylca r b on yla m in om et h yl-ca r -
bon yla m in o]ben zoa te (3). To a solution of 3k (0.348 g, 0.618
mmol), EDC (0.119 g, 0.618 mmol) and HOBt (0.095 g, 0.618
mmol) in 40 mL of CH2Cl2 was added amine 3e (0.412 g, 0.618
mmol) in 5 mL of DMF. The reaction mixture was stirred for
6 h. The solvent was removed in vacuo. The residue was
trituated with hot MeOH, which was filtered off and washed
with acetone to give pure white solid 3 (0.370 g, 50%). 1H NMR
(DMSO) δ 10.399 (1H, s), 10.382 (1H, s), 9.879 (1H, s), 8.676
(2H, t, J ) 4.0 Hz), 8.470 (1H, s), 8.086 (1H, s), 8.061 (1H, d,
J ) 2.8 Hz), 8.040 (1H, s), 8.012 (1H, s), 7.854 (2H, t, J ) 4.4
Hz), 7.788 (1H, q; J ) 2.4 Hz, J ) 8.8 Hz), 7.087 (1H, d, J )
9.2 Hz), 6.752 (1H, s), 4.234-4.194 (8H, m), 4.093 (2H, t, J )
6.0 Hz), 3.252 (2H, q, J ) 6.4 Hz, J ) 12.4 Hz), 2.245 (2H, t,
J ) 7.2 Hz), 1.865-1.784 (5H, m), 1.671 (2H, m), 1.563 (2H,
m), 1.477-1.126 (50H, m), 0.850-0.799 (9H, m), 0.736-0.707
(6H, m). 13C NMR (10% DMSO in CDCl3) δ 172.05, 167.50,
C12H16N2O3: C, 61.00; H, 6.83; N, 11.86. Found: C, 61.27; H,
6.95; N, 12.01.
N-(3-Am in op h en yl)h exa n oa m id e (4b). This compound
was prepared from 4a based on the same procedure for
1
preparing 3a . Yield: 89%. H NMR (DMSO) δ 7.581 (s, 1H),
7.137 (s, 1H), 7.033 (t, 1H, J ) 8.0 Hz), 6.702 (d, 1H, J ) 7.6
Hz), 6.393 (d, 1H, J ) 7.2 Hz), 3.190 (s, 2H), 2.293 (t, 2H, J )
7.2 Hz), 1.689 (m, 2H), 1.295 (m, 4H), 0.881 (t, 3H, J ) 6.4
Hz). 13C NMR (CDCl3) δ 171.68, 147.13, 139.03, 129.55, 110.90,
109.76, 106.67, 37.72, 31.35, 25.28, 22.35, 13.84.
N-[3-(Eth oxyca r bon ylm eth ylca r bon yla m in o)p h en yl]-
h exa n oa m id e (4c). To a solution of 4b (0.80 g, 3.88 mmol)
and triethylamine (0.39 g, 3.88 mmol) in 40 mL of methylene
chloride was added dropwise ethyl 3-chloro-3-oxo-propionate
(1.05 g, 6.98 mmol) over 5 min. at 0 °C. The ice-water bath
was removed. After 6 h the solvent was evaporated, and the
residue was dissolved in ethyl acetate and washed with diluted
HCl and NaOH solutions alternatively. Drying and evapora-
tion of the solvent gave the crude product which was recrystal-
lized from MeOH, giving the pure product as a white solid (1.03
1
g, 83%). H NMR (CDCl3) δ 9.263 (s, 1H), 7.777 (s, 1H), 7.423
(s, 1H), 7.245 (d, 2H; J ) 5.6 Hz), 7.218 (q, 1H; J ) 8.0 Hz, J
) 10.8 Hz), 4.234 (q, 2H; J ) 6.8 Hz, J ) 14.0 Hz), 3.456 (s,
3H), 2.326 (t, 2H; J ) 7.2 Hz), 1.704 (m, 2H), 1.349-1.312 (m,
6H), 0.896 (t, 3H; J ) 7.2 Hz). 13C NMR (CDCl3) δ 171.63,
169.71, 163.20, 138.58, 137.90, 129.50, 115.85, 115.54, 111.20,
61.94, 41.64, 37.74, 31.37, 25.23, 22.41, 14.02, 13.92. Anal.
Calcd for C17H24N2O4: C, 63.73; H, 7.55; N, 8.74. Found: C,
63.63; H, 7.39; N, 8.64.
N-[3-(Hyd r oxyca r bon ylm eth ylca r bon yla m in o)p h en yl]-
h exa n oa m id e (4d ). This compound was prepared from 4c
based on the same procedure for preparing 3c. Yield: 0.82 g,
1
94%. H NMR (DMSO): δ 10.116 (s, 1H), 9.880 (s, 1H), 7.899
(s, 1H), 7.243 (d, 2H; J ) 7.2 Hz), 7.197 (t, 1H; J ) 7.2 Hz),
3.322 (s, 2H), 2.266 (t, 2H; J ) 7.2 Hz), 1.564 (m, 2H), 1.295-
1.261 (m, 4H), 0.855 (t, 3H; J ) 6.8 Hz). 13C NMR (DMSO) δ
171.48, 169.44, 164.62, 139.71, 139.22, 128.95, 114.35, 113.90,
110.05, 44.03, 36.45, 30.98, 24.94, 22.00, 13.97. Anal. Calcd
for C15H20N2O4: C, 61.63; H, 6.90; N, 9.58. Found: C, 61.69;
H, 7.94; N, 9.64.
Octyl 3-[(5′-Hexa n oa m id o-2′-octyloxy)p h en ylca r bon y-
la m in om eth ylca r bon yl-a m in o]-5-[(3′′-h exa n oa m id op h e-
n yl)a m in oca r bon ylm eth ylca r bon yla m in o]ben zoa te (4).
To a solution of acid 4d (0.61 g, 2.10 mmol) and EDC (0.41 g,
2.13 mmol) and HOBt (0.33 g, 2.16 mmol) in 40 mL of DMF
was added amine 3e (1.32 g, 5 mmol) in 10 mL of DMF. The
reaction was allowed to proceed overnight. Distilled water (100
mL) was then added to the reaction mixture. The precipitated
solid was filtered and stirred with acetone under reflux for 30
min. The solution was cooled to room temperature and filtered,
giving the pure product as a white solid (1.01 g, 51%). 1H NMR
(DMSO) δ 10.399 (s, 1H), 10.368 (s, 1H), 10.142 (s, 1H), 9.860
(s, 2H), 8.653 (t, 1H; J ) 4.8 Hz), 8.153 (s, 1H), 8.057 (t, 2H;
J ) 6.4 Hz), 7.927 (d, 2H; J ) 1.6 Hz), 7.794 (q, 1H; J ) 2.8
Hz, 8.8 Hz), 7.307-7.251 (m, 2H), 7.187 (t, 1H, J ) 8.0 Hz),

Hydrogen-Bonded Molecular Duplexes
J . Org. Chem., Vol. 66, No. 10, 2001 3583
7.102 (d, 1H; J ) 8.8 Hz), 4.246 (t, 2H; J ) 6.4 Hz), 4.183 (d,
2H; J ) 5.2 Hz), 4.110 (t, 4H; J ) 6.4 Hz), 3.474 (s, 2H), 2.252
(q, 4H; J ) 7.6 Hz, 11.2 Hz), 1.824 (m, 2H), 1.683 (m, 2H),
1.570 (m, 4H), 1.419-1.139 (m, 24H), 0.870-0.803 (m, 9H),
0.750 (t, 3H; J ) 6.4 Hz). 13C NMR (DMSO) 171.26, 170.94,
167.55, 165.75, 165.40, 165.16, 164.26, 152.50, 139.63, 139.53,
139.42, 139.09, 132.65, 130.70, 128.78, 123.60, 121.80, 121.26,
114.71, 114.63, 114.23, 113.84, 113.53, 110.04, 69.24, 64.74,
45.89, 43.54, 36.33, 36.19, 31.16, 30.85, 28.72, 28.66, 28.57,
25.54, 28.15, 25.72, 25.38, 24.79, 22.01, 21.99, 21.84, 13.86,
13.82. Anal. Calcd For C53H76N6O10: C, 66.50; H, 8.00; N, 8.78.
Found: C, 66.12; H, 8.12; N, 8.79.
Isoth er m a l Titr a tion Ca lor im etr y. The thermodynamic
binding parameters were determined by titrating 1 mM
solutions of 3 or 4 with 8 mM 1 in 5% DMSO/CHCl3 in an
Omega isothermal titration calorimeter (MicroCal, Northamp-
ton, MA). The cell was thermostated to (0.1 °C using a
circulating bath. All experiments were performed at 25 °C. The
enthalpy of binding between strands were determined from
multiple single injections. Injection volumes were 5 µL, with
3 min of equilibration time allowed between injections. The
heat of dilution of 1 into 5% DMSO/CHCl3 was determined
and the 1-3 and 1-4 heats were adjusted via subtraction of
the heat of dilution from the two data sets. The equilibrium
binding constants K were extracted from the calorimetric data
by employing the Origin data analysis software supplied with
the calorimeter. A complete description of the data analysis
has been published by Brandts and co-workers.14
Ack n ow led gm en t. This work was supported by
grants from NASA (NAG5-8785), NIH (GM63223), and
ACS-PRF (34456-AC4).
J O010250D
(14) Wiseman, T.; Williston, S.; Brandts, J . F.; Lin, L.-N. Anal.
Biochem. 1989, 179, 131.