Synthesis and conformational analysis of novel trimeric maleimide cross-linking reagents

Article abstract of DOI:10.1021/jo0709293

(Figure Presented) Nine homotrifunctional cross-linking reagents are presented. Their synthesis and chemical properties as well as their characterization by classical mechanical conformational searching techniques is reported. Mixed Low Mode and Monte Car

Full text of DOI:10.1021/jo0709293

Synthesis and Conformational Analysis of Novel Trimeric
Maleimide Cross-Linking Reagents
Agnieszka Szczepanska,† Jose´ Luis Espartero,‡ Antonio J. Moreno-Vargas,†
Ana T. Carmona,† and Inmaculada Robina*,†
Department of Organic Chemistry, Faculty of Chemistry, UniVersity of SeVille, P.O. Box 1203, E-41071
SeVille, Spain, and Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy,
UniVersity of SeVille, Spain
Sarah Remmert and Carol Parish*
Department of Chemistry, UniVersity of Richmond, Gottwald Science Center, Richmond, Virginia 23173
robina@us.es; cparish@richmond.edu
ReceiVed May 10, 2007
Nine homotrifunctional cross-linking reagents are presented. Their synthesis and chemical properties as
well as their characterization by classical mechanical conformational searching techniques is reported.
Mixed Low Mode and Monte Carlo searching techniques were used to exhaustively sample the OPLS2005/
GBSA(water) potential energy surface of trisubstituted cyclohexane and benzene derivatives of C3
symmetry. Geometric structure, molecular length, and hydrogen-bonding patterns were analyzed.
Nonaromatic compounds exhibited exclusively chair conformations at low energies, with a preference
for axial or equatorial arms depending upon the presence of additional ring substituent Me groups.
Increasing chain length often resulted in overall shorter molecular length due to additional chain flexibility.
These results were consistent with one- and two-dimensional temperature-dependent NMR studies.
Introduction
interaction between the viral envelope gp120 and the CD4
cellular receptors.1 Subsequent interaction with chemokine co-
receptors CXCR4 or CCR5 triggers a cascade of conformational
changes in gp120 that induces activation of the gp41 fusion
subunit and formation of the pre-hairpin structure that may
bridge both viral and cellular membranes.2
The HIV-1 envelope glycoproteins gp120 and gp41 remain
noncovalently associated and oligomerize most likely as trimers
on the surface of the virion. This trimeric complex mediates
viral entry into target cells, which is initiated by the high affinity
The crystal structure of a complex between gp120, CD4, and
a neutralizing antibody that blocks the chemokine receptor
binding site has been solved showing that this complex has a
ternary structure.3 Furthermore, modeling studies of the orienta-
tion of gp120 in the ternary complex reveal that gp120 is
† Department of Organic Chemistry.
‡ Department of Organic and Pharmaceutical Chemistry.
(1) (a) Wyatt, R.; Sodroski, J. Science 1998, 280, 1884-1888. (b)
Weissenhorn, W.; Dessen, A.; Calder, L. J.; Harrison, S. C.; Skehel, J. J.;
Wiley, D. C. Mol. Membr. Biol. 1999, 16, 3-9. (c) Eckert, D. M.; Kim, P.
S. Annu. ReV. Biochem. 2001, 70, 777-810.
10.1021/jo0709293 CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/04/2007
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NoVel Trimeric Maleimide Cross-Linking Reagents
assemble peptide-peptoid structures.8 Trimesic acid was used
in the construction of aromatic tripodal systems as model
receptors for monosaccharides and other compounds.9 Also,
triamines derived from cyclohexane tricarboxylic acids 1 and
2 are used as chelating agents of trivalent cations.10
FIGURE 1. Structure of C3-symmetric templates.
The preparation of the scaffolds according to our model
requires the attachment of the spacers to the templates and the
incorporation of a maleimide moiety. Maleimide-containing
compounds have been widely employed as intermolecular cross-
linking agents in chemical and biochemical applications for
chemoselective ligation to cysteine-containing peptides and
proteins.11 It was reported that homobifunctional cross-linking
reagents can be used as molecular rulers to evaluate the distance
between two cross-linked amino acid residues; the quantitative
measure of the lengths has been estimated by stochastic dynamic
calculations.12
constrained to be 3-fold symmetric. The binding of CD4/gp120
takes place predominantly via electrostatic interactions within
a large and irregular interface. A protuberant Phe43 of CD4
inserts into a receptive hole of gp120, the “Phe-43 cavity”, which
is the conserved CD4 binding site on gp120.4 Compounds that
interfere with the binding of CD4-gp120 are therefore inhibitors
of HIV entry. Additionally, a trimeric structure bearing C-
peptide sequences of the gp41 ectodomain could also mimic
the prehairpin intermediate and therefore provide a tool to block
the fusion event. It has also been reported that a multivalent
assembly of gp41 peptides enhances the R-helix content of the
peptide and provides a model to mimic the HIV membrane
fusion state.5
In this paper we present the synthesis, conformational
behavior, and flexibility of trimeric maleimide clusters, com-
pounds 4a-6c,13 as homotrifunctional cross-linking reagents
(Figure 2). Since these derivatives are large molecules that can
adopt different conformations in solution, the study could
provide useful information regarding the molecular shape of
CD4 cellular receptors as well as the prehairpin intermediate
involved in the HIV-cell fusion event.
We have recently reported a series of trivalent CD4-mimetic
miniproteins designed to match the distance between any two
of the CD4 binding cavities by having three CD4M9 moieties
tethered through a spacer to a 3-fold symmetric template.6 They
have shown significantly enhanced activity over the monovalent
CD4M9 but activites similar to those of the bivalent system
first reported by Wang and co-workers.7 In this work, we report
the synthesis of several new C3 symmetric templates that could
serve as scaffolds to which CD4 mimetic peptides or minipro-
teins can be attached. Conformational analysis was used to
characterize the different rigidities, flexibilities, and spatial
orientations of each system to better understand their molecular
behavior.
Results and Discussions
Synthesis and NMR Study. The preparation of N-substituted
maleimide derivatives is not an easy task. Several procedures
have been described that involve the formation of the maleimide
moiety by ring closure of the intermediate amide, obtained by
reaction of amines with maleic anhydride14 or through a retro-
Diels-Alder reaction of the adduct generated between furan
and maleimide.15 These methods require refluxing at high
temperatures, and in the first case, low yields are obtained
because of the low solubility of the maleic acid derivatives.
Direct N-alkylation of maleimide under Mitsunobu conditions
has also been used to introduce this functionality. However,
For the preparation of our scaffolds we have chosen C3-
symmetric templates of well-defined architectures such as
cis,cis-1,3,5-trimethyl cyclohexane tricarboxylic acid (Kemp’s
triacid, 1), cis,cis-cyclohexane-1,3,5-tricarboxylic acid (2), and
trimesic acid (3) (Figure 1) that are joined to synthetic or
commercial spacers of different lengths. Kemp’s triacid is known
in supramolecular chemistry as a useful building block to
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Tetrahedron Lett. 2001, 42, 1325-1328. (c) Peters, K.; Richards, F. M.
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A.; Traut, R. R. J. Biol. Chem. 1992, 267, 5889-5894. (e) Kwaw, J. S.;
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Gebhard, W.; Kabsch, W. Nature 1990, 347, 44-49. (h) Lorenz, M.; Popp,
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(13) The synthesis and chemical characterization of compounds 4a-c
and 5c is reported in ref 6.
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Wang, L.-X. Bioorg. Med. Chem. 2004, 12, 3141-3148. (b) Tam, J. P.;
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J. Org. Chem, Vol. 72, No. 18, 2007 6777

Szczepanska et al.
FIGURE 2. Structure of trimeric maleimide derivatives 4-6.
this reaction is not always successful as it is extremely dependent
on the order of addition of reactants.16
functionality under Mitsunobu conditions were unsuccessful;
complex mixtures and brown polymeric material were obtained.
(Scheme 1)
Coupling reactions between templates 1-3 and monopro-
tected N-Boc diamino spacers 7a, 7b, and 7c using EDCI and
HOBt gave compounds 8-10 in moderate-to-good yield. Boc
deprotection with HCl/MeOH and subsequent neutralization with
Na/MeOH gave the corresponding triamines in quantitative
yield, which were made to react with N-methoxycarbonylma-
leimide affording the maleimide scaffolds 4a-6c in moderate-
to-good yield. Other attempts to introduce the maleimide
The structures of the new compounds were based on
spectroscopic and analytical data (see experimental details in
Supporting Information and ref 6). In order to assess the
existence of hydrogen bonded amide protons, NMR experiments
at different temperatures, 218, 248, 273, and 303 K, in CDCl3
were carried out.17 These experiments provided information
about the presence of rotational isomers around the CO-NH
bond.18 At 303 K, only one set of signals in each NMR spectrum
was observed, indicating that no rotational isomers along the
NH-CO bond are present. Compounds 4a, 5c, and 6c were
chosen for this study. The choice was based on their solubility
in CDCl3 at low temperatures. Compound 4a derived from
Kemp’s triacid and with the shortest spacer was totally soluble
in chloroform at low temperatures. For compounds derived from
cis,cis-cyclohexane-1,3,5-tricarboxylic acid and trimesic acid,
only derivatives 5c and 6c with the longest spacers were soluble
(16) (a) Walker, M. A. J. Org. Chem. 1995, 60, 5352-5355. (b) Boeckler,
C.; Frish, B.; Scuber, F. Bioorg. Med. Chem. Lett. 1998, 8, 2055-2058.
(c) Antczak, C.; Bauvois, B.; Monneret, C.; Florent, J. C. Bioorg. Med.
Chem. Lett. 2001, 9, 2843-2848.
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2001, 14, 463-467. (b) Gung, B. W.; Zhu, Z.; Everingham, B. J. Org.
Chem. 1997, 62, 3436-3437.
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61-65. (b) Iriepa, I.; Madrid, A. I.; Ga´lvez, E.; Bellanato, J. J. Mol. Struct.
2006, 787, 8-13.
6778 J. Org. Chem., Vol. 72, No. 18, 2007

NoVel Trimeric Maleimide Cross-Linking Reagents
SCHEME 1
in chloroform at low temperatures. For both cyclohexane
derivatives 4a and 5c, the NH chemical shift [δ(NH)] at 303 K
is 7.42 and 6.28 ppm, respectively. This downfield shift of δ(NH)
observed for 4a is an indication of an intramolecular hydrogen-
bonded state. On decreasing temperature, there were very little
differences in the chemical shifts (δ) of all of the signals
(variations between 0.01 and 0.07 ppm), except for the NH in
compounds 5c and 6c. This may be an indication of low
flexibility in the respective moieties. The cyclohexane ring in
both 4a and 5c is a rigid moiety as well. The variation of δ(NH)
with temperature is indicated in Figures 3-5, and a graph of
the amide proton chemical shift as a function of the temperature
is also displayed in Figure 6.
Kemp’s triacid derivative 4a presented a slight variation of
δ(NH) with temperature from 303 to 218 K, indicating the
existence of intramolecular hydrogen bonding at these temper-
atures. For compounds 5c and 6c, a variation of δ(NH) with
FIGURE 3. Variation of δ(NH) with temperature in compound 4a.
FIGURE 5. Variation of δ(NH) with temperature in compound 6c.
FIGURE 6. Plot of amide proton chemical shift as a function of
temperature.
FIGURE 4. Variation of δ(NH) with temperature in compound 5c.
J. Org. Chem, Vol. 72, No. 18, 2007 6779

Szczepanska et al.
TABLE 2. Summary of Conformational Search Results for 4-6
no. of structures found
within 4 kcal/mol of the
lowest E structure
no. of blocks of
5000 steps
completed
compound
4a
4b
4c
5a
5b
5c
6a
6b
6c
12
240
2626
271
3173
1955
2563
8430
1388
16
35
35
35
35
35
35
35
40
FIGURE 7. Rotamers along the CO-NH bond.
TABLE 1. Temperature Coefficients
compound
NH (∆δ/∆T) (ppb/K)
4a
5c
6c
-1
-6.6a
-6.3a
a Considering variation from 303 to 248 K.
These results are in accordance with the OPLS05 based
computational data (see below).
Computational Studies
Force Field Analysis. Seven different force fields are
available in the MacroModel program. It is important to choose
a force field that is well parametrized for the molecular system
under study. Accurate torsional parameters are particularly
important in flexible molecular systems since they control
conformational interconversions. Force field analysis proceeded
as follows. Conformational searches of the surfaces generated
by each force field (MM2*, MM3*, AMBER*, MMFF, OPLS,
OPLS01, and OPLS05), in conjunction with the GBSA (water)
model, for 4a, were performed for 125,000 steps. Low-energy
structures found on each surface were selected and subjected
to unrestrained quantum mechanical minimization using B3LYP/
6-311G**-SCRF(water). The quantum calculations found chair
and boat conformations of 4a to be surprisingly similar in
enthalpy (within 2 kcal/mol); however, these results were not
in agreement with the observed spectra or previous reports.20
We are investigating further these aspects of the conformational
preferences of cis,cis derivatives of Kemp’s triacid. The
computational outcome will be reported elsewhere.21 Force field
analysis was repeated for 5a and 6a, but in these cases only a
subset of force fields (AMBER*, OPLS05) was examined as
preliminary data showed the results to be invariant. From these
data, along with a comparison with the experimental NMR
results, it was determined that the OPLS05 force field was, on
average, the best suited for describing 4-6 and was used in
the remainder of the report.
FIGURE 8. NOE contacts for compounds 4a, 5c, and 6c.
temperature is observed, but on cooling to 218 K, no appreciable
variation is detected. A steeper slope is observed from 303 to
248 K with an inflection point around 250 K. This behavior
indicates that there is no intramolecular hydrogen bonding at
higher temperatures and that, at temperatures lower than 250
K, a more ordered structure is observed. Temperature coef-
ficients (Table 1), which are reasonably good indicators of
hydrogen bonding,19 confirm this behavior.
To elucidate which rotational isomer (E or Z) along the CO-
NH bond is present (Figure 7), NOE contacts were determined
and are shown in Figure 8.
The conformational flexibility of each of these systems was
investigated using the LM:MC conformational search method.
Details of each search, including convergence, can be found in
Supporting Information; a summary of the results appears in
Table 2. The results indicate that each search is convergent with
respect to the number of structures found, the energy of the
lowest conformer, and the number of times this structure is
visited. Very few new structures are found toward the end of
each search, and searches on the smaller molecular systems
(4a-6a, 4b, and 5b) find the lowest energy structure within
the first block of 5000 steps. This suggests that the method is
For 4a, whose structure is well defined by the intramolecular
hydrogen bonds, NOE is observed between pairs of protons NH/
H2′ and NH/H1′ of the ethylene chain (not shown). A strong
NOE between pairs NH/H3e and, to a lesser extent, for NH/
CH3 is also observed. The absence of an NOE between H1′ or
H2′ with either Me or H3a and H3e is an indication of the presence
of the E rotational isomer. For 5c, NOE signals are observed
for H2′ and H1′ (not shown) of the ethylene chain interacting
with H3a/H3e and between the pair of protons NH/H3a, NH/H3e,
and NH/H2. This can be an indication of free rotation along the
C(H2)-CO bond. For the aromatic derivative 6c a strong NOE
between the pair of protons NH/HAr is observed, indicating the
presence of the E rotational isomer (Figure 8).
(20) (a) Bencini, A.; Bianchi, A.; Burguete, M. I.; Dapporto, P.;
Domenech, A.; Garcia-Espana, E.; Luis, S. V.; Paoli, P.; Ramirez, J. A. J.
Chem. Soc., Perkin Trans. 1994, 2, 569-577. (b) Kemp, D. S.; Petrakis,
K. S. J. Org. Chem. 1981, 46, 5140-5143. (c) Chan, T.-L.; Cui, Y.-X.;
Mak, T. C. W.; Wang, R.-J.; Wong, H. N. N. J. Crystallogr. Spectrosc.
Res. 1991, 21, 297-308. (d) Rebek, J.; Marshall, L.; Wolak, R.; Parris, K.;
Killoran, M.; Askew, B.; Nemeth, D.; Islam, N. J. Am. Chem. Soc. 1985,
107, 7476-7481.
(19) (a) A temperature coefficient more positive than -4.5 ppb/K
indicates a hydrogen bond, whereas if it exchanges rapidly and has a
temperature coefficient more negative than -4.5 ppb/K, it is not hydrogen
bonded. See: Baxter, N. J.; Williamson, M. P. J. Biomol. NMR 1997, 9,
359-369. (b) Sosnicki, J. G.; Hansen, P. E. J. Mol. Struct. 2004, 700, 91-
103.
(21) Remmert S.; Parish, C. Unpublished work.
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NoVel Trimeric Maleimide Cross-Linking Reagents
FIGURE 9. Lowest-energy structures and hydrogen-bonding patterns found on the OPLS2005/GBSA(water) surface of 4-6.
indeed exhaustively sampling the available conformational space
and that the generated ensembles are representative of the low-
energy structures of 4-6 on the OPLS05/GBSA(water) surface.
of 4a-4c. Each C6 ring adopts a chair conformation with the
maleimide (M) arms in the equatorial positions. Structures 5a
and 5b do not contain any hydrogen bonding interactions
between different M arms; however, in 5b the heteroaromatic
ring on one arm wraps back around to hydrogen bond with the
amide hydrogen of the same arm. Structure 5c possesses C3
symmetry, no hydrogen bonding between arms, and three
hydrogen bonds between heteroaromatic rings and the amide
hydrogen on the same arm.
Analysis of the Global Minima. The lowest energy structures
for 4-6 are shown in Figure 9. A comparison of the global
minima for compounds based on the Kemp’s triacid 1,3,5-
trimethyl cyclohexane (TMC6) scaffold (4a, 4b, and 4c)
indicates that on the OPLS05/GBSA(water) surface these
molecules preferentially adopt chair conformations with the
methyl substituents arranged equatorially, and the maleimide
(M) arms arranged axially for optimal hydrogen bonding
between the amide proton on one arm and the amide carbonyl
on another arm. Three such hydrogen-bonding interactions occur
in each of the lowest energy structures for 4a-4c. The lowest
energy structure for 4a is a very ordered C3-symmetric structure;
in addition to the structural features described above, a carbonyl
moiety on each maleimide points toward the nitrogen on a
neighboring heteroaromatic ring (distances ∼3.4 Å). The NMR
data are in accordance with these results (see above). The lowest
energy structure for 4b contains maleimide-ethoxy-ethyl car-
boxamide (MEEC) arms that are long enough to allow the chains
to fold back toward the side of the central TMC6 ring, allowing
relatively close (3.6 Å) interactions between the amide carbonyl
on one chain and the maleimide ring on another chain. This
results in a more compressed and less ordered structure relative
to the lowest energy conformer for 4a. The maleimide-diethoxy-
ethyl carboxamide (MDEC) arms in 4c are quite flexible, and
although three hydrogen bonds still exist, the molecule appears
more disordered than either 4a or 4b.
Systems 6a, 6b, and 6c contain central aromatic rings in place
of the C6 or TMC6 rings. This causes the lowest energy
structure for 6a to be roughly planar with the maleimide arms
radiating from the aromatic core, preventing any hydrogen
bonding interactions. The larger arm size of 6b and 6c allow
these molecules to adopt nonplanar lowest energy structures
wherein the MEEC and MDEC arms wrap back toward the
central aromatic ring allowing hydrogen bonding between the
arms and π-stacking interactions between the central ring and
heteroaromatic maleimide rings. In 6b and 6c, one arm wraps
back to form a bifurcated hydrogen bond between the carbonyl
of the heteroaromatic ring and the amide of the same arm and
an adjacent arm.
Analysis of the Conformational Ensembles. All of the C6
and TMC6 global minimum energy structures (4a-4c, 5a-5c)
shown in Figure 9 adopt the chair conformation on the OPLS05/
GBSA(water) surface. Global minimum energy structures are
often not representative of the overall ensemble of structures22
but in this case they are, at least with respect to ring orientations;
100% of the structures found within 4 kcal/mol for 4a-4c and
A comparison of the global minima for 5a, 5b, and 5c,
molecules with a simple cyclohexane (C6) core, reveals much
“squatter,” more disordered molecular shapes relative to those
(22) Parish, C.; Lombardi, R.; Sinclair, K.; Smith, E.; Goldberg, A.;
Rappleye, M.; Dure, M. J. Mol. Graphics Modell. 2002, 21, 129-150.
J. Org. Chem, Vol. 72, No. 18, 2007 6781

Szczepanska et al.
FIGURE 10. Longest molecular distance: (a) compounds 4a (red), 4b (black), 4c (green); (b) compounds 5a (red), 5b (black), 5c (green); (c)
compounds 6a (red), 6b (black), 6c (green).
TABLE 3. Molecular Length Data (Å) for 4-6
If every conformation is structurally distinct from all other
conformations, then the number of unique minima provides
quantitative information about molecular flexibility and the
complexity of each potential energy surface. A larger number
of unique minima indicate a more complex potential energy
surface. Judging by the number of minima found on each
surface, the molecules with methyl groups on the cyclohexane
rings are less flexible for the small and medium length chains
than are either the aromatic or unsubstituted C6 systems. For
systems with large chains (4c, 5c, and 6c), the aromatic molecule
has the smallest number of minima. According to the number
of minima, the overall ordering of flexibility is 4a < 4b < 5a
< 6c < 5c < 6a < 4c < 5b < 6b. However, it is possible that
the minima are not structurally distinct, i.e., that multiple
structures can be grouped into conformationally similar families.
When this occurs, the number of families is a better indicator
of flexibility than the number of conformations. Therefore, the
XCluster program was used to determine if structures within
each ensemble naturally form structurally related groupings.
Clustering by atomic RMS after rigid body superposition of all
heavy atoms in all ensembles and ring atoms for cyclohexane
ensembles 4a-4c, 5b, and 5c did not lead to significant
clustering as evidenced by distance maps, mosaics, and separa-
tion ratios. These data, along with ring conformer analysis and
visual inspection of the smaller ensembles, indicates that the
low-energy structures within each ensemble adopt similar core
ring structures (chair or aromatic) but with maleimide arm
orientations that are in fact geometrically distinct and that span
compound
average
minimum
maximum
range
4a
4b
4c
5a
5b
5c
6a
6b
6c
12.7
14.4
15.7
15.6
15.5
13.9
17.9
14.0
14.6
12.5
12.0
12.7
11.1
12.9
12.6
13.4
11.8
12.4
12.9
18.9
20.8
20.3
21.1
17.1
20.6
18.4
16.2
0.4
6.9
8.1
9.2
8.2
4.5
7.2
6.6
3.8
5a-5c adopt the chair conformation on the OPLS05/GBSA-
(water) surface.23 For 4a-4c, 100% of the ensemble contains
structures with arms in the axial orientation, whereas 100% of
the structures of 5b and 5c display arms in the equatorial
position. The ensemble of 5a, the C6 system with the smallest
arms, contains a mixture of structures with 40% all axial and
60% all equatorial arms.
(23) Structures were determined to be chairs or boats by measuring the
dihedral angle of each arm relative to the central ring. All angles greater
than 125° were identified as axial substituents, whereas those less than or
equal to 125° were defined as equatorial. Those structures that contained
all axial or all equatorial arms were counted as chairs, and those with
mixtures of axial and equatorial arms were defined to be boats. We were
concerned that perhaps our conformational search was overly biased by
the chair starting structure; however, simulations purposely seeded with
boat conformations as initial structures also resulted in 100% chair
ensembles.
6782 J. Org. Chem., Vol. 72, No. 18, 2007

NoVel Trimeric Maleimide Cross-Linking Reagents
FIGURE 11. Hydrogen bonding and energetic analysis for ensembles 4a-c, 5a-c, and 6b,c. The ensemble for 6a did not contain any structures
with hydrogen bonding, most likely due to the short M arms and aromatic nature of the compound.
the available conformational space. The only system that
exhibited statistically significant clustering (separation ratios
greater than 2.0)24 was 5a. Clustering of this ensemble by ring
atom RMSD resulted in two families; analysis of the average
and representative structure indicated that both families adopted
a chair orientation and 60% of the ensemble contained structures
with equatorial maleimide arms while the other 40% of the
ensemble contained axial arms in agreement with the indepen-
dent geometric analysis above.
Analysis of Molecular Lengths. To obtain an idea of the
distribution of structures in each ensemble as well as the average
molecular length of each system, we measured the longest
interatomic distance of each structure and visualized histograms
of these data (Figure 10 and Table 3). For TMC6 systems (4a-
4c), the overall molecular behavior, as measured by this metric,
is what one would expect from the 2D structures shown in
Figure 2. As the chain joined to the maleimide moeity increases,
the overall molecular length increases, from an average of 12.7
Å for 4a to 14.4 and 15.7 Å for 4b and 4c, respectively,
suggesting an increase in the corresponding 3D molecular size.
These averages are accompanied by a corresponding increase
in the ranges associated with each system: 12.5 - 12.9 Å for
4a; 12.0 - 18.9 Å for 4b; 12.7 - 20.8 Å for 4c, indicating a
positive correlation between molecular length and flexibility.
The situation for molecules with C6 and aromatic cores is
different. For 6a, 6b, and 6c (aromatic core), the ensemble data
indicates a decrease in molecular size as arm length increases,
along with a corresponding loss in flexibility likely due to
increased opportunities for hydrogen bonding and π-stacking,
as demonstrated in the low-energy structures in Figure 9. For
C6 systems, the ensembles for 5a and 5b are relatively similar
with respect to molecular lengths; however, 5a is slightly more
flexible than 5b judging by the range of distances sampled (for
5a, range ) 11.1 -20.3 Å and average ) 15.6 Å; for 5b, range
) 12.9 - 21.2 Å and average ) 15.5 Å). Ensemble 5c contains
the smallest range of intramolecular distances for C6 systems
(12.6-17.1 Å), as well as significantly shorter distances sampled
(average ) 13.9 Å), even though this molecule contains the
longest arms. According to the average longest interatomic
distances, the overall ordering of molecular length is 4a < 5c
≈ 6b < 4b ≈ 6c < 5b ≈ 5a ≈ 4c < 6a.
Hydrogen Bonding. The number of classical hydrogen bonds
(defined as having a maximum distance between donor and
acceptor of 2.5 Å, and a minimum donor and acceptor angle of
120° and 90°, respectively) in each ensemble was measured
(Figure 11 and Table 4.) The overwhelming majority of TMC6
structures (4a, 4b, and 4c) found on the OPLS2005/GBSA-
(water) surface contain three hydrogen bonds; all structures for
4a and 4b contain at least one hydrogen bond while the number
of structures without any hydrogen bonds for 4c is less than
1%. As shown in Figure 11, structures with less hydrogen
bonding interactions typically occur in the ensemble at higher
energies. Taken together with the flexibility and longest distance
data, this information provides a clear picture of the overall
(24) Shenkin, P. S.; McDonald, D. Q. J. Comput. Chem. 1994, 15, 899-
916.
J. Org. Chem, Vol. 72, No. 18, 2007 6783

Szczepanska et al.
FIGURE 12. Superimposition of 271 low-energy structures in the ensemble of 5a using cyclohexane ring atoms.
TABLE 4. Percent of Structures within the Ensembles of 4-6
Four of the compounds presented in this study (4a-4c, 6c)
were ligated to miniproteins, and their virus inhibition was
measured.6 Compounds 4a-4c behave similarly (IC50 values
are 4a ) 0.79 µM, 4b ) 1.8 µM, and 4c ) 1.0 µM) in a
biological setting. This is not surprising given the overall
similarities of the molecular ensembles and the resulting rigidity
due to the preponderance of intramolecular hydrogen bonding.
Interestingly the aromatic compound 6c behaves similarly (IC50
values ) 0.33 µM), and of all of the aromatic systems, this
ensemble is the least flexible (judging by the distance data),
contains the largest number of hydrogen-bonded structures, and
appears to behave in a very compacted fashion. The modeling
results indicate that, in most cases, the trimeric compounds with
longer arms would provide a more conformationally flexible
scaffold to which to attach CD4 mimetic miniproteins. However,
such increased mobility generally results in ensembles of shorter
molecular length.
That Contain 0, 1, 2, and 3 Hydrogen-Bonding Interactions
no. of hydrogen bonds
compound
0
1
2
3
4a
4b
4c
5a
5b
5c
6a
6b
6c
0
8
1
7
41
45
30
0
59
44
17
12
7
3
4
56
0
24
41
75
87
86
3
<1
9
0
2
12
0
<1
53
51
5
0
15
3
behavior of these systems. The ensembles contain structures in
the chair conformation with equatorial methyl groups and axial
maleimide arms whose flexibility of motion sweeps out the
available conformational space in a coordinated way so as to
maintain optimal hydrogen bonding. The situation is somewhat
different for systems based on the unsubstituted cyclohexane
core (5a-5c). Ensembles with shorter arms (5a and 5b) contain
a mix of structures, with (∼50%) and without (∼50%) hydrogen
bonding interactions, whereas the ensemble with the longest
arms (5c) contains a much smaller percentage of non-hydrogen-
bonded structures (5%). The hydrogen bonding in 5c causes
this system to be much less flexible and smaller on average
than 5b. As Figure 11 indicates, structures with larger numbers
of hydrogen bonds tend to occur at higher energies in the
ensemble of 5b. As with ensembles based on the TMC6 core,
the ensembles of 5 contain structures that adopt the chair
conformation with equatorial maleimide arms that sweep out
all of the available conformational space. This behavior is
illustrated in the superposition of all 271 low-energy structures
of 5a as shown in Figure 12. There were no hydrogen-bonded
structures found for 6a because of the planar aromatic nature
of the molecule and short maleimide arms. For all other
structures with aromatic cores, the maleimide arms are long
enough to wrap around to form 1-3 hydrogen bonding
interactions, with the majority of the structures for 6b and 6c
containing at least one such interaction.
Conclusions
The synthesis and characterization of nine homotrifunctional
cross-linking reagents are presented. These systems contain
maleimide arms of varying length attached to three well-defined
molecular architectures: Kemp’s triacid, cis,cis-cyclohexane-
1,3,5-tricarboxylic acid, and trimesic acid. Temperature-de-
pendent NMR analysis of those systems soluble in chloroform
over a range of temperatures (4a, 5c, and 6c) confirmed the
existence of hydrogen-bonding interactions. Molecular behavior
and conformational flexibility was further probed using the
mixed Low Mode-Monte Carlo conformational searching
technique to exhaustively sample the corresponding OPLS2005/
GBSA(water) surface. Geometric structure, molecular length,
and hydrogen-bonding patterns were analyzed. Low-energy
ensembles based on Kemp’s triacid and cis,cis-cyclohexane-
1,3,5-tricarboxylic acid contain structures that adopt preferen-
tially chair conformations with axial and equatorial maleimide
arms, respectively. Increasing chain length often resulted in
overall shorter molecular length due to additional chain flex-
6784 J. Org. Chem., Vol. 72, No. 18, 2007

NoVel Trimeric Maleimide Cross-Linking Reagents
The Low Mode (LM) search method29 was used in a 1:1
combination22 with the Monte Carlo (MC) search method30 to
explore the potential energy surfaces of 4-6. Interconversion of
ring structures was enabled using the ring-opening method of Still.31
Searches utilized the usage-directed structure selection method30
that identifies the least-used structure from among all known
conformations and then uses this structure as the starting point for
each new search. This ensures that a variety of different starting
structures from different regions of the potential energy surface
are used to begin each new block of steps. During the conforma-
tional search all structures were subjected to 500 steps of the
Truncated Newton Conjugate Gradient (TNCG)32 minimization
method to within a derivative convergence criterion of 0.01 kJ Å-1
ibility. These results were consistent with two-dimensional
temperature-dependent NMR studies.
Experimental Section
Synthesis and NMR. General Remarks. 1H NMR and 13C
NMR spectra were obtained for solutions in CDCl3 and DMSO-
1
d6, J values are given in Hz, and δ in ppm. H NMR spectra at
different temperatures were obtained for solutions in CDCl3 at 500
MHz. All assignments were confirmed by homonuclear 2D COSY
and NOESY and heteronuclear 2D correlated (HETCOR) experi-
ments. The FAB mass spectra were obtained with glycerol or
3-nitrobenzyl alcohol as matrix. TLC was performed on silica gel
HF254, with detection by UV light and nynhidrine reagent. Silica
gel 60 (230 mesh) was used for preparative chromatography.
Anhydrous solvents and reagents were freshly distilled under N2
prior to use.
mol-1 All final ensembles were subjected to further TNCG
.
minimization after the search was completed in order to obtain fully
minimized structures. Ensembles generated for each of the scaffolds
were queried for geometric similarities using the XCluster24
program.
Synthesis of Boc-Protected Triamine Templates 8a-8c, 9a-
9c, and 10a-10c). General Procedure. To a cooled (0 °C)
suspension of the corresponding tricarboxylic acid 1-3 (0.5 mmol)
in dry CH2Cl2 was added EDCI (1.65 mmol). The N-Boc-protected
diamine 7(a-c) (1.65 mmol) was then added dropwise, and the
reaction was left to warm to room temperature and stirred overnight.
The mixture was washed with water, and the organic phase was
dried over anhydrous sodium sulfate. Chromatography purification
on silica gel (CH2Cl2/MeOH 3% f 5%) afforded the target
compounds. (See Supporting Information for analytical and spec-
troscopic data).
Synthesis of Maleimide Templates 4a-4c, 5a-5c, and 6a-
6c). General Procedure. The N-Boc-protected triamine [8a-8c,
9a-9c, 10a-10c] (0.2 mmol) was dissolved in MeOH and cooled
to 0 °C, and concentrated HCl (2.2 mL) was added dropwise. The
reaction was stirred at 0 °C until TLC showed lack of substrate.
Solvents were evaporated, and the residue dissolved again in
methanol and neutralized with sodium to pH ) 7.0. After filtration
of the NaCl formed, the filtrate was dried over anhydrous sodium
sulfate and concentrated to afford the corresponding free triamine.
A solution of this triamine (0.23 mmol) in CHCl3 (10 mL) was
cooled to 0 °C, and then tetrabutylammonium hydrogen sulfate (1.38
mmol) and N-methoxycarbonylmaleimide (1.38 mmol) were added.
Triethylamine (97 µL) was then slowly added, and the mixture
stirred at 0 °C for 10 min. After removing the ice bath, saturated
aqueous solution of NaHCO3 (10 mL) was added in one portion.
The reaction was stirred vigorously at room temperature for 3-4
h. Phases were then separated, and the organic layer was then dried
over anhydrous sodium sulfate, concentrated, and purified by
column chromatography on silica gel (CH2Cl2/MeOH 3% f 5%)
to give the target compounds. (See Supporting Information for
analytical and spectroscopic data).
Acknowledgment. The authors thank Nora Green for her
interest in the work, the European Commission (FP6, TRIoH,
LSHG-CT-2003-503480), the Ministerio de Educacio´n y Ciencia
of Spain (CTQ2004-00649/BQU), and the Junta de Andaluc´ıa
(FQM-345) for financial support. Acknowledgment is also made
to the Donors of the American Chemical Society Petroleum
Research Fund, the National Science Foundation under grant
CHE-0211577, and the Thomas F. Jeffress and Kate Miller
Jeffress Memorial Trust for partial support of this work. C.P.
also acknowledges support from the Camille and Henry Dreyfus
Foundation through receipt of a Henry Dreyfus Teacher-Scholar
award. S.R. acknowledges support from the Arnold and Mabel
Beckman Foundation through receipt of a Beckman Scholars
award. Computational resources were provided, in part, by the
MERCURY supercomputer consortium (http://mars.hamilton-
.edu) under NSF grant CHE-0116435.
Supporting Information Available: Data of the compounds
4a-4c, 5a-5c, 6a-6c, 8a-8c, 9a-9c, 10a-10c; 1H and 13C NMR
spectra for compounds 5b, 5c, 6a, 6b, 9b, 9c, and 10b; NOESY
experiments and NOE contacts for compounds 4a, 5c, and 6c; force
field parameter analysis for compounds 5c and 6c; LM:MC
conformational search results on the OPLS2005/GBSA(water)
surface of 4a-4c, 5a-5c, and 6a-6c, and DFT results for 4a. This
materialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.
JO0709293
Computational Methods. The conformational ensembles gener-
ated in this study were calculated using the MacroModel V9.1 suite
of software25 programs. A variety of force fields available in the
MacroModel software package were evaluated for their ability to
reproduce quantum mechanical conformational data obtained at the
B3LYP/6-311G* level using Jaguar V6.5.26 Solvent effects were
included using the Generalized Born/Surface Area (GB/SA)27 and
Self-Consistent Reactive Field (SCRF)28 continuum models for
water, in the molecular and quantum mechanical calculations,
respectively.
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