Synthesis and evaluation of the first cis-cyclobutane-containing receptor for lipid A

Article abstract of DOI:10.1039/b610727c

The first example of a designed receptor containing a cis-1,3-disubstituted cyclobutane ring has been synthesized. This molecule binds diphosphoryl lipid A (a conserved portion of the Gram-(-) bacterial cell membrane, and the causative agent of septic shock) with an affinity comparable to previously described ter-cycloalkane based lipid A-binding compounds. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b610727c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and evaluation of the first cis-cyclobutane-containing receptor for
lipid A†
Kevin M. Bucholtz,c,d Peter C. Gareiss,b,d Stephen G. Tajcb,d and Benjamin L. Miller*a,b,d
Received 25th July 2006, Accepted 13th September 2006
First published as an Advance Article on the web 29th September 2006
DOI: 10.1039/b610727c
The first example of a designed receptor containing a cis-1,3-disubstituted cyclobutane ring has been
synthesized. This molecule binds diphosphoryl lipid A (a conserved portion of the Gram-(−) bacterial
cell membrane, and the causative agent of septic shock) with an affinity comparable to previously
described ter-cycloalkane based lipid A-binding compounds.
The design and synthesis of receptors for carbohydrates and
carbohydrate derivatives represents a key frontier for bioorganic
chemistry.1 In addition to providing new insights into the basic
processes underlying molecular recognition of carbohydrates,2
novel synthetic receptors can serve as important tools in the eluci-
dation and control of carbohydrate-mediated cellular activities, as
components of biosensors3 and, potentially, as therapeutic agents.4
Lipid A, the conserved headgroup of Gram-(−) bacterial
lipopolysaccharide5 and a primary causative agent of bacterial
septic shock,6 is a particularly interesting target for molecu-
lar recognition studies. Several naturally-occurring compounds,
including the well-studied antibiotic polymyxin B,7 are known
to target lipid A. As part of a general effort directed towards
developing receptors for carbohydrates, we initiated a program to
examine the ability of stereoregular oligocycloalkane derivatives to
serve as lipid A receptors. We chose the oligocycloalkane scaffold
in part because of its similarity (in terms of size and anticipated
rigidity) to cholic acid, a compound that has proven useful as
a scaffold for the construction of lipid A binders.8 In contrast
to cholic acid, however, we anticipated that the ter-cycloalkane
scaffold would permit more extensive variation, allowing for subtle
changes on structure and conformation to be examined. As we
reported previously, compound 1 (“TWTCP”) binds lipid A in
buffered aqueous solution with an affinity comparable to lipid A-
binding natural products.9 In this paper, we report the synthesis
and preliminary evaluation of the first structural variant of
TWTCP incorporating a central cis-1,3-disubstituted cyclobutane
ring (“TW545”, 2).
Scheme 1 Oligocycloalkane containing receptors 1 and 2 and precursors.
A binding ability, while potentially displaying subtle differences
in conformational dynamics. While trans-1,3-cyclobutanes have
been examined in the context of molecular recognition by other
researchers,10 to our knowledge the use of a cis-1,3-disubstituted
cyclobutane as a core structural element is unprecedented.
Molecular mechanics analysis of the core scaffold 2 suggests
that it should have a slightly more linear structure than 1,
without dramatically altering the conformational energy profile
(Fig. 1). Therefore, we anticipated that 2 would likely retain lipid
Fig. 1 Left: overlay of minimum-energy conformers of a ter-cyclopentane
scaffold (blue) and
a cyclobutane analog (red). Right: inter-ring
dihedral angle drive (MMFFs force field,11 GB/SA CHCl3,12
Maestro/Macromodel13) for the cyclopentane–cyclopentane (black) and
cyclopentane–cyclobutane (grey) torsion.
aDepartment of Dermatology, University of Rochester, Rochester,
New York, USA. E-mail: Benjamin_Miller@futurehealth.rochester.edu;
Fax: +1 (1)585 273 1346; Tel: +1 (1)585 275 9805
bDepartment of Biochemistry and Biophysics, University of Rochester,
Rochester, New York, USA
Our intent from the outset was to synthesize 2 using a
bidirectional route analogous to that employed previously for the
synthesis of 1 (Scheme 1). Thus, cis-cyclobutane-1,3-dialdehyde
4 represented a key initial synthetic target, by analogy to the
synthesis of 1 from dialdehyde 3. Compounds of this type are
deceptively simple in appearance; however, relatively few examples
of cis-1,3-disubstituted cyclobutanes appear in the literature. The
cDepartment of Chemistry, University of Rochester, Rochester, New York,
USA
dCenter for Future Health, University of Rochester, Rochester, New York,
USA
† Electronic supplementary information (ESI) available: X-Ray data for
compound 7 and additional UV-Vis and fluorescence titration data for
compounds 1 and 2. See DOI: 10.1039/b610727c
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two primary approaches to such compounds have either employed
a one-bond formation (cyclization) strategy,14 or cleavage of
the highly strained (and highly volatile) benzvalene15,16 for the
construction of the cyclobutane ring.
Because neither of these approaches seemed ideal, we initially
sought to adapt an intramolecular photochemical cycloaddition
of diacrylimides (Scheme 2). An analogous system derived from
methacrylimide was reported by Lalonde and Aksentijevitch as
providing the cis-1,3 isomer (6).17 This assertion was subse-
quently confirmed spectroscopically by Bellus and coworkers.18
Preparation of the required diacrylimide (5) by alkylation of
acryloyl chloride and phenethyl amine proceeded smoothly in
73% yield. Subsequent photolysis and hydrolysis of the imide to a
monoamide–monocarboxylic acid provided a single cyclobutane
product; however, single crystal X-ray analysis‡ revealed that this
was not the desired 1,3-substituted compound, but rather the
cis-1,2-cycloadduct 7.19 Presumably, the reduced steric demand
of 5 (X = H) switches the mode of the cycloaddition, and we
are currently exploring alternative syntheses employing removable
groups at the a-position of the acryloyl functionality to overcome
this problem.
Scheme 3 (a) 1 M BH3–THF (5 eq), THF, 12 h, RT, 83%; (b) PCC (3
eq.), CH2Cl2, RT, 4 h; (c) (i-PrO)2POCH2COCH2CH2Ph (2.4 eq.), DBU
(2.4 eq.), LiCl (2.0 eq.), CH3CN, RT, 8 h, 32% (over two steps); (d) Me3Al
(0.05 eq.), AlCl3 (0.5 eq.), CH2Cl2, 0 ◦C, 1 h, then cyclopentadiene (10
eq.), CH2Cl2, 4 ◦C, 12 h, 60%; (e) LiAlH4 (6 eq.), THF, RT, 4 h; (f) BzCl
(8 eq.), Et3N (9.25 eq.), CH2Cl2, RT, 4 h, 61% (over two steps); (g) O3,
MeOH–CH2Cl2 (1 : 1), −78 ◦C, 1 h, then NaBH4 (14 eq.), RT, 6 h, 58%;
(h) Boc–L-Trp (5.5 eq.), DCC (6.4 eq.), DMAP (3.6 eq.), CH2Cl2–DMF
(7 : 3) RT, 12 h; (i) TFA, triethylsilane, CH2Cl2, RT, 12 h, 14% (over two
steps).
13C NMR spectra clearly show doubling of several resonances,
indicating that 11 is most likely a mixture of meso and pseudo
C2-symmetric diastereomers. Because the primary goal of this
study was simply to demonstrate that compounds incorporating
the cis-1,3-disubstituted cyclobutane are able to bind lipid A, we
set the issue of diastereomeric purity aside for the time being
and proceeded with the synthesis. Subsequent reduction of 11
and derivatization with benzoyl chloride afforded 12 in 61%
yield over two steps. Ozonolysis of 12 followed by reductive
workup with NaBH4 provided a 58% yield of the tetra alcohol,
13. Finally, DCC-mediated coupling of N-Boc-L-tryptophan and
global deprotection with trifluoroacetic acid provided the desired
receptor 2 in 14% yield over two steps.
Like 1, 2 is sufficiently soluble in buffered aqueous solution
to permit binding analysis by spectrophotometric methods. We
evaluated its affinity for diphosphoryl lipid A (14, from E. coli
F583, Rd mutant, and 15, from Salmonella minnesota Re595) by
UV-Vis titration in HEPES buffer at pH 7.0. An example titration
is shown in Fig. 2. We also confirmed the affinity of 1 and 2 for E.
coli F583, Rd mutant lipid A by fluorescence titration. In all cases,
Scheme 2 Intramolecular photocycloaddition products.
We next turned to the literature synthesis of cis-1,3-cyclobutane
carboxylic acid 8, originally reported by Allinger and coworkers.20
While not expedient (9 steps), this route permitted sufficient pro-
duction of cyclobutane 8 to permit carrying the synthesis forward
(Scheme 3). Reduction of the cis-1,3-cylobutane dicarboxylic acid
8 with a borane–THF complex afforded the diol (9) in 83% yield.
Conversion of diol 9 to a dialdehyde was accomplished via PCC
oxidation. Subsequent Horner–Wadsworth–Emmons olefination
by analogy to our previous efforts yielded several products;
separation and HPLC analysis indicated that a substantial fraction
of the material had epimerized under the reaction conditions to
provide a mixture of the cis- and trans-1,3-cyclobutane isomers.
This epimerization had not been a complicating factor in our
prior synthesis of TWTCP. One potential factor in this difference
is the higher s-character in the exocyclic bonds of cyclobutane
rings relative to cyclopentane rings, postulated as a factor in the
increased acidity of cyclobutyl protons in other studies,21 may
allow for more facile deprotonation of this system. Substitution
of Roush–Masamune conditions22 in the olefination reaction
provided the desired cis compound (10) in 32% yield over two
steps from 9.
changes in the absorbance (kmax = 280 nm) or fluorescence (kem
=
357 nm) of indole substitutents as a function of added lipid A were
observed. Both UV-Vis and fluorescence titration measurements
were corrected for sample dilution; UV-Vis titrations were also
corrected for the concentration-dependent absorbance of lipid
A. As shown in Table 1, 2 binds lipid A with affinity comparable to
1. We observed only a slight difference in affinity between E. coli
and S. minnesota derived lipid A; these structures differ only in
the number of lipid chains (6 vs 7, respectively). This observation
supports a binding model in which 2 interacts primarily with
the diphosphoryl disaccharide headgroup of lipid A, rather than
with the lipid tails. Analysis of the interaction using the method
of continuous variation (Job plot) showed an inflection point
at approximately 50% mole fraction of 2; this provides strong
support for the existence of a 1 : 1 binding interaction (Fig. 3).
This also suggests that the lipid A binding constants for the two
Double Diels–Alder reaction of 10 proceeded smoothly to
provide 11 (two inseparable diastereomers, only one of which
is shown) in 60% yield following chromatography. Interestingly,
compound 11 (and all subsequent compounds in the synthesis)
elutes as a single peak on normal phase and gradient reverse-
phase HPLC, as well as on several chiral HPLC columns. However,
‡ CCDC reference number 616053. For crystallographic data in CIF
format see DOI: 10.1039/b610727c
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Table 1 UV-Vis titrations of lipid A into 1 and 2. Binding constants (KD)
are in lM
E. coli (14)a
S. minnesota (15)a
1
2
3.0 0.8
5.9 1.5; 3.5 0.3 (fluorescence)
6.0 1.6
6.5 1.1
a Conditions: 20 mM HEPES, pH. 7.0.
Fig. 2 UV-Vis titration of 6.0 lM TW545 (2) with 300 lM E. coli
diphosphoryl lipid A in 20 mM HEPES at pH 7.0. Extracted A280 values
are corrected for lipid A absorbance and dilution of 2. A Kd of 5.9 lM is
derived from nonlinear least-squares fit (dashed line) to a one-site binding
model.
diastereomers of 2 are similar, since one would expect to see
deviation from 1 : 1 binding behavior if the two diastereomers
present in the mixture had substantially different affinities for
lipid A.
Fig. 3 Job plot of E. coli diphosphoryl lipid A into 2 (total concentration
12.0 lM in HEPES, pH 7.00) is consistent with a 1 : 1 binding
stoichiometry.
TWTCP (1). This represents the first example of the synthesis of
a designed receptor incorporating a cis-1,3-disubstituted cyclobu-
tane moiety. The affinity of 2 for diphosphoryl lipid A in aqueous
solution is similar to that observed for TWTCP, suggesting that
this subtle structural modification does not diminish the binding
ability of the receptor. Additional experiments designed to explore
structural and conformational differences between TWTCP and
TW545, as well as synthetic studies designed to improve the
diastereoselectivity of the bidirectional Diels–Alder reaction, are
in progress.
Experimental
All nonaqueous reactions were conducted in flame dried glassware,
under an atmosphere of N2, and were stirred with a Teflon coated
magnetic stir-bar, unless otherwise stated. Air-sensitive reagents
and solutions were transferred via syringe (unless otherwise stated)
and were introduced to the reaction vessel through a rubber
septum. Room temperature (RT) refers to ambient temperatures,
which is approximately 25 ◦C. Unless otherwise stated, tempera-
tures other than RT denote the temperature of the cooling/heating
bath. All distillations were performed under an N2 atmosphere, or
under at reduced pressure aspirator (15–30 mm Hg), or vacuum
pump (ꢀ1 mm Hg). The phrase “reduced or concentrated in
vacuo” refers to removal of solvent by means of a Buchi rotary-
evaporator attached to an aspirator pump (10 mm Hg).
Conclusions
Purification by flash chromatography was performed following
the procedure of Still23 using the indicated solvent systems on
EM Reagents silica gel 60 (230–400) mesh. Analytical thin layer
In conclusion, we have reported the first synthesis of 2, a cis-1,3-
cyclobutane analog of our previously disclosed lipid A receptor,
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chromatography (TLC) was performed using EM silica gel 60 F-
254 pre-coated glass plates (0.25 mm). Visualization was effected
by short-wave UV illumination and/or by dipping the plate into
a solution of KMnO4, ceric ammonium molybdate (CAM), or
2,4-dinitrophenyl hydrazine (2,4-DNPH), followed by heating on
a hot plate.
Analytical purification using high pressure liquid chromatogra-
phy (HPLC) was performed on a Shimadzu LC-2010A (reverse
phase) and a Beckman 112 Solvent Delivery System and Waters
450 Variable Wavelength Detector set at 254 nm (normal phase).
Reagent-grade solvents were used without further purification
for all extractions and work-up procedures. Deionized water was
used for all aqueous reactions, work-ups, and for the preparation
of all aqueous solutions. Reaction solvents diethyl ether (Et2O),
tetrahydrofuran (THF), methylene chloride (CH2Cl2), triethy-
lamine (TEA), and dimethyl formamide (DMF) were obtained
from a Glass Contour solvent purification system.
addition of 50 ml of 10% HCl. The organic layer was separated
and washed with 3 × 50 ml of 10% HCl, 1 × 50 ml water, 3 × 50 ml
saturated sodium bicarbonate solution. The organic layer was then
washed with brine solution, dried with magnesium sulfate, filtered,
and concentrated in vacuo to give a yellow oil. Purification of the
yellow oil via flash chromatography (silica, 75 : 25 hexanes–ethyl
acetate) yielded 5 as a light yellow oil (3.34 g, 14.59 mmol, 73%
yield). mmax/cm−1 (film) 3023, 1680, 1651, 1620, 1401, 1150; dH
(400 MHz, CDCl3) 7.32 (2H, t); 7.28–7.22 (3H, m); 6.59 (2H, dd,
J = 3.2, 13.6 Hz); 6.42 (2H, d, J = 9.2 Hz); 5.79 (2H, d, 5.8 Hz);
4.00 (2H, t, J = 7.6 Hz); 2.93 (2H, t, J = 7.6 Hz); dC (75 MHz,
CDCl3) 168.8, 138.1, 130.5, 129.9, 128.9, 128.6, 126.7, 46.3, 35.2;
HRMS m/z (EI) 229.1098 (M+. C14H15NO2 requires 229.1103).
1,2-Monocarboxylic acid, mono N-phenethyl amide cyclobutane
(7)
Proton nuclear magnetic resonance (NMR) spectra were ob-
tained on a Bruker WH-400 (400 MHz), or an Avance 400
(400 MHz), or a Bruker-500 (500 MHz) instrument. Carbon
NMR spectra were obtained on an Avance 400 (75 MHz), or
on a Bruker-500 (75 MHz). Chemical shifts are reported in ppm
(d) relative to the appropriated deuturated solvent. Multiplicity
was designated by the following abbrevations and combinations
thereof: singlet (s), doublet (d), doublet of doublet (dd), doublet
of triplets (dt), multiplet (m). Infrared (IR) spectra were recorded
on a Perkin Elmer 1610 FT-IR spectrophotometer and are
reported in wavenumbers (cm−1) with a polystyrene standard.
High resolution mass spectrometry (HRMS) was performed at
the mass spectrometry facility at the State University of New York
at Buffalo, Buffalo, NY. The X-ray crystallographic data‡ were
collected on a standard Siemens SMART CCD Area Detector
System equipped with a normal focus molybdenum-target X-ray
tube operated at 2.0 kW (50 kV, 40 mA).
Photochemical irradiations were carried out using a 450 W
Hanovia medium pressure mercury vapor arc lamp. The lamp
operates at an internal pressure between 1 to 10 atm and emits
radiation over the region of 200–1400 nm. Particularly intense
emissions are at 254, 313, 366, 435.8, and 546.1 nm. All solvents
used were degassed via bubbling nitrogen gas through the solution
for 30 min. The irradiations were carried out in immersion well-
type reaction vessels. The outer well was a three neck flask that held
250 ml of solvent. For larger scale reaction a 2000 ml graduated
cylinder was used. The inner well was a quartz glass immersion
well with a water cooled jacket. To select the lower wavelength
output from the lamp only quartz glassware was used. To filter
out 254 nm light and allow only wavelengths greater than 300 nm
a pyrex filter was used that surrounded the lamp.
N-Phenethyl diacrylimide (5) (1.661 g, 7.22 mmol) was dissolved
in 1400 ml of acetonitrile and was then degassed by bubbling
nitrogen through the solution. The solution was poured into a
2000 ml graduated cylinder and a quartz cooled water jacketed
photochemical immersion well was submerged into the solution.
A 450 W Hanovia medium pressure mercury vapor lamp was used
and the solution was irradiated for 18 h. The solution was then
concentrated in vacuo to yield a dark brown oil, and 75 ml of 3.5 M
KOH was added. The reaction was stirred at RT for 20 min and
then the solid was filtered off with suction filtration. The solid was
then washed with 3 × 25 ml of 3.5 M KOH to yield a light tan solid.
This was dissolved in 50 ml of 10% HCl, concentrated, and dried
in vacuo to yield a light tan solid (1.159 g, 4.693 mmol, 65% yield
over two steps). Crystals suitable for X-ray analysis‡ were obtained
by dissolution in methanol followed by slow evaporation of the
solvent. mmax/cm−1 (film) 3323, 2940, 1718, 1615, 1548, 1168; dH
(400 MHz, CDCl3) 7.32 (2H, t); 7.28–7.20 (3H, m); 5.63 (1H, bs);
3.58 (2H, m); 3.42 (1H, m); 3.30 (1H, m); 2.84 (2H, t, J = 5.8 Hz),
2.49 (1H, m); 2.30–2.17 (3H, m); dC (75 MHz, CDCl3) 172.5, 169.6,
134.7, 124.8, 124.7, 122.5, 38.4, 36.8, 36.7, 31.5, 18.7, 18.2; HRMS
m/z (EI) 247.1197 (M+. C14H17NO3 requires 247.1208).
Ester 10
Pyridinium chlorochromate (3.724 g, 17.28 mmol) was dissolved
in 35 ml of CH2Cl2 and the resulting orange slurry was stirred
vigorously. Diol 9 (668 mg, 5.76 mmol) was dissolved in 6 ml
of CH2Cl2 and was then added dropwise to the solution over
20 min. The reaction was allowed to run for 4 h, at which time
monitoring via TLC indicated disappearance of starting material
and formation of an aldehyde (2,4-DNPH stain). The reaction was
then quenched with 150 ml of diethyl ether, and was stirred for 1 h.
The ether layer was decanted away from the black, tarry chromium
residue, filtered through a pad of Florisil, and flushed with 5 ×
50 ml of ether. The solution was then concentrated very carefully
in an ice bath in vacuo to remove the majority of solvent. When
the total weight of the crude mixture became ∼1 g, concentration
was stopped, and carried on to the next step.
N-Phenethyl diacrylimide (5)
Acryloyl chloride (4.63 ml, 56.97 mmol) was added to 55 ml of dry
methylene chloride at 0◦ C. To this cooled and stirred solution was
added dropwise over 20 min 2,6 lutidine (7.29 ml, 62.67 mmol).
The solution became orange in color. Phenethylamine (2.50 ml,
19.99 mmol) was then added dropwise via an addition funnel over
1 h. After the addition was complete, the reaction was stirred at 0◦
C for 1 h, and then allowed to warm to room temperature. After
16 h at room temperature, the reaction was quenched with the
Reagent quantities for the olefination step were calculated based
on an assumption that the maximum yield in the foregoing
reaction was 75%. To a stirred suspension of lithium chloride
(440 mg, 10.37 mmol) in 100 ml of dry acetonitrile was added
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(i-PrO)2POCH2COCH2CH2Ph (5.10 g, 10.37 mmol) and then
10 min later was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
(1.29 ml, 8.64 mmol). The solution was stirred for 10 min, and the
crude cyclobutane cis-1,3-dialdehyde (∼484 mg, 4.32 mmol) was
then added. After the addition of all of the reagents the lithium
chloride began to dissolve. The reaction was monitored via TLC
and after 8 h was complete. The reaction solution was quenched
with the addition of water (20 ml). The solution was extracted
with diethyl ether (4 × 100 ml). The organics were pooled and
washed with brine solution, dried with magnesium sulfate, filtered
and concentrated in vacuo to yield a yellow oil. Purification of
the yellow oil via flash column chromatography (silica, 80 : 20
hexanes–diethyl ether) afforded 10 as a light yellow oil (743 mg,
1.84 mmol, 32% over two steps, E,E : E,Z > 20 : 1). mmax/cm−1
(film) 2977, 1703, 1646, 1465, 1380, 1303, 1266, 1158, 1093; dH
(400 MHz, CDCl3) 7.31 (4H, t); 7.23 (6H, m); 7.10 (1H, dd, J =
15.6 Hz); 6.94 (1H, dd, J = 15.6 Hz); 5.75 (2H, dd, J = 15.6 Hz
); 4.35 (4H, q, ); 3.11 (1H, q); 3.03 (5H, m); 2.42 (1H, q); 2.21
(2H, t ); 1.88 (1H, q); dC (75 MHz, CDCl3) 166.7, 151.7, 151.4,
137.9, 128.9, 128.5, 126.6, 119.9, 119.7, 64.9, 35.2, 33.8, 33.6, 32.0;
HRMS m/z (EI) 404.1975 (M+. C26H28O4 requires 404.1988).
(0.14 ml), 15% aq. NaOH (0.14 ml), and more water (0.42 ml).
The reaction formed a white precipitate, which stirred vigorously
for an additional 2 h at RT. The reaction mixture was filtered
through a plug of Celite and subsequently washed with ether (5 ×
10 ml). The filtrate was dried over magnesium sulfate, filtered,
and reduced in vacuo to give a yellow oil that was used without
further purification. The crude oil was an inseparable mixture of
pheneythyl alcohol (1.258 mmol) and the desired diol. The diol was
slurried into 6 ml of CH2Cl2 at RT. Sequential addition of benzoyl
chloride (706 mg, 5.032 mmol), and Et3N (0.80 ml, 5.832 mmol) at
RT caused the reaction to become a pale yellow. After stirring for
1 h at RT, the reaction began to form a white precipitate. After 4 h,
the reaction mixture was quenched with water (5 ml), and poured
into diethyl ether (20 ml). The layers were separated, and the
organic layer was washed sequentially with water (2 × 20 ml), 10%
HCl (3 × 20 ml), water (1 × 20 ml), and saturated aq. Na2CO3 (3 ×
20 ml). The organic layers were combined, washed with brine, dried
over magnesium sulfate, filtered, and reduced in vacuo to afford
a tan oil. Purification of the oil via flash column chromatography
(silica, 95 : 5 hexanes–ether) yielded 12 (194 mg, 0.382 mmol, 61%
yield over two steps) as a pale yellow oil mmax/cm−1 (film) 2961,
1711, 1445, 1385, 1272, 1114; dH (400 MHz, CDCl3) 8.07 (4H, t);
7.62–7.40 (6H, m); 6.30–6.15 (2H, m) 6.10–6.03 (2H, m); 4.32–4.06
(2H, m); 3.97–3.81 (2H, m); 2.90–2.70 (2H, m); 2.56–2.20 (2H, m);
2.11 (1H, m), 2.03–1.85 (5H, m); 1.47–1.37 (5H, m); 1.31 (1H, m),
1.10 (1H, dd); 0.86 (1H, dd); dC (75 MHz, CDCl3) 162.7, 134.5,
129.8, 128.9, 126.5, 125.6, 124.4, 64.9, 64.8, 46.6, 45.1, 45.1, 42.3,
42.1, 41.2, 40.6, 40.5, 40.4, 40.2, 32.7, 30.1, 28.0; HRMS m/z (EI)
508.2607 (M+. C34H36O4 requires 508.2614).
Diels–Alder adduct 11
Bis-dienophilic ester 10 (565 mg, 1.40 mmol) was dissolved into
5 ml methylene chloride. The solution was cooled to 0 ◦C for
15 min. Addition of (CH3)3Al (0.045 ml, 0.07 mmol, 2.0 M
in hexanes) produced the evolution of small amounts of gas.
After allowing this mixture to stir at 0 ◦C for 10 min, AlCl3
(0.70 ml, 0.70 mmol, 1.0 M in CH3NO2) was then added to the
solution via syringe, and the reaction was stirred for 5 min at
0 ◦C. Cyclopentadiene (924 mg, 14 mmol, 4.0 M in CH2Cl2) was
added via a syringe over 30 min (0.5 ml portions every 5 min). The
Alcohol 13
The dibenzoate 12 (158 mg, 0.311 mmol) was dissolved into 3 ml of
a solution of 1 : 1 CH2Cl2–MeOH. This solution was then cooled
to −78 ◦C for 10 min. O3 was bubbled through the solution at
a moderate rate, and after 1 h the solution became a persistent
blue color. After another 20 min of O3 treatment, O3 production
was discontinued, and O2 was bubbled through the solution for
approximately 5 min to remove any excess O3. After the blue color
disappeared, the reaction was warmed from −78 to 0 ◦C and
maintained at 0 ◦C for 10 min. NaBH4 (165 mg, 4.354 mmol)
was added slowly. (Caution: vigorous evolution of H2 gas.) After
all of the NaBH4 had been added, the reaction was stirred and
warmed slowly to RT over 30 min. Once at RT, the reaction was
stirred for an additional 6 h. 10% aq. HCl was then added until a
pH of 1.0 was reached, as measured by pH paper. The mixture
was diluted with ethyl acetate, and the layers were separated.
The aqueous layer was extracted with more ethyl acetate (3 ×
20 ml) and the organic extracts were combined and then washed
sequentially with water (20 ml), sat. aq. Na2CO3 (3 × 20 ml), and
brine solution (1 × 20 ml). The organic solution was then dried
over magnesium sulfate, filtered, and reduced in vacuo to afford
a white solid. The solid was purified via flash chromatography
(silica, 95 : 5 CH2Cl2–MeOH) to afford 13 (105 mg, 0.181 mmol,
58% yield) as a colorless oil mmax/cm−1 (film) 3400, 2923, 1715,
1558, 1272, 668, 609; dH (400 MHz, CDCl3) 8.17–8.02 (4H, m);
7.77–7.61 (2H, m); 7.60–7.48 (4H, m); 4.51–4.11 (4H, m); 3.83–
3.40 (8H, m); 2.61–2.42 (1H, m); 2.42–2.10 (7H, m); 2.10–1.73 (7H,
m); 1.608–1.57 (1H, m); 1.50–1.30 (2H, m); dC (75 MHz, CDCl3)
◦
reaction was then warmed to 4 C and allowed to proceed with
stirring for 12 h. The reaction was then quenched with pyridine
(0.2 ml) and warmed to RT. The resulting white slurry was filtered
through a plug of silica gel, and washed with diethyl ether (5 ×
20 ml). The organics were reduced in vacuo. The pyridine and
CH3NO2 were removed azeotropically via treatment with heptane
(4 × 10 ml). Final concentration in vacuo yielded a yellow residue.
Purification via flash chromatography (silica, 95 : 5, hexanes–
diethyl ether) afforded 11 as a yellow oil (449 mg, 0.84 mmol,
60% yield) mmax/cm−1 (film) 2961, 1721, 1468, 1380, 1168, 1091;
dH (400 MHz, CDCl3) 7.35–7.29 (4H, m); 7.26–7.22 (6H, m); 6.23
(2H, m); 5.83 (2H, apparent q); 4.35–4.21 (4H, m); 3.07 (2H, bs);
2.93 (4H, t, J = 7 Hz); 2.50 (2H, bs); 2.32 (2H, m); 2.17–2.04
(2H, m); 1.93–1.83 (3H, m); 1.81 (1H, m); 1.67 (1H, m); 1.57 (1H,
m); 1.48–1.33 (4H, m); dC (75 MHz, CDCl3) 174.8, 174.5, 138.7,
138.5, 137.9, 133.5, 128.8, 128.4, 126.4, 64.6, 50.3, 49.5, 49.4, 46.2,
46.2, 46.1, 45.8, 45.0, 36.8, 36.1, 35.1, 33.0, 31.1; HRMS m/z (EI)
536.2908 (M+: C36H40O4 requires 536.2927).
Dibenzoate 12
LiAlH4 (140 mg, 3.774 mmol) was slurried into 12 ml of THF at
RT. Ester 11 (337 mg, 0.629 mmol) in 2 ml of THF was added
dropwise via syringe to this slurry over 15 min at RT. During the
addition, hydrogen gas was evolved. The mixture was stirred for
4 h at RT. It was then carefully quenched sequentially with water
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161.7, 132.8, 130.2, 130.1, 129.1, 128.2, 66.2, 65.6, 65.4, 65.2, 65.1,
62.1, 62.1, 49.9, 49.8, 48.8, 48.7, 46.1, 45.9, 44.1, 43.9, 43.7, 43.5,
43.4, 43.2, 42.9, 42.6, 37.1, 36.9, 36.6, 36.5, 32.7, 32.6, 31.7, 29.7;
HRMS m/z (MALDI) 603.2903 (M + Na+. C34H44O8Na requires
603.2953).
at ambient temperature in a Shimadzu 1600-PC UV spectropho-
tometer. Tryptophan indole absorbance was monitored at 280 nm
to ensure a completely equilibrated solution had been obtained.
Aliquots of 2.0 ll of a 150 lM diphosphoryl lipid (prepared as
described above) were sequentially added to the cuvette. After each
addition, the solution was allowed to equilibrate for a minimum
of 10 min, then scanned for absorbance from 500–220 nm. The
control experiment of diphosphoryl lipid A titrated into HEPES
buffer was subtracted from each of the titration experiments. Other
titrations discussed in the text were carried out analogously.
Receptor 2
Alcohol 13 (32 mg, 0.055 mmol) was dissolved into 1 ml of
DMF. Boc–L-tryptophan (92 mg, 0.303 mmol) was added to the
solution, followed 10 min later by DCC (73 mg, 0.352 mmol), and
by DMAP (24 mg, 0.196 mmol) after another 10 min. This solution
was stirred at RT for 21 h, at which point a significant amount of
white solid had precipitated. The mixture was filtered through
Celite to remove the precipitant, and the pad was washed with
ethyl acetate (5 × 10 ml). The organics were combined, and were
washed sequentially with 1% HCl (4 × 10 ml), water (1 × 10 ml),
sat. aq. NaHCO3 solution (4 × 10 ml), and brine solution, dried
over sodium sulfate, and then filtered through filter paper. The
solution was then filtered through a pad of Celite to remove any
remaining white solid, and was reduced in vacuo affording a light
tan residue (117 mg) that was used without further purification.
The crude Boc-protected tetratryptophaninate was dissolved into
0.84 ml of CH2Cl2. In a separate vial, 0.361 ml of trifluoroacetic
acid, 0.180 ml of triethyl silane, and 0.3 ml of CH2Cl2 were mixed
and then added dropwise via a syringe over 15 min to the Boc-
tryprophaninate solution. The reaction was allowed to run for
12 h and was then concentrated in vacuo to afford a yellow oil.
The residue was purified via reverse-phase HPLC using gradient
elution (linear gradient of 90 : 10 0.1% TFA in water–0.1% TFA
in CH3CN to 100% of 0.1% TFA in CH3CN over 30 min) to give
2 as a light tan–yellow viscous oil (10 mg, 0.0076 mmol, 14% yield
over two steps) mmax/cm−1 (film) 3357, 2924, 1684, 1272, 1204, 1134,
1025; dH (400 MHz, CD3OD) 8.00 (4H, bs); 7.67–7.32 (15H, m);
7.25 (11H, m); 4.44–4.24 (6H, m); 4.24–3.87(12H, m); 3.87–3.77
(2H, m); 3.71–3.65 (2H, unresolved t); 3.63–3.54 (2H, m); 3.39
(8H, bs); 2.43–2.25 (2H, m); 2.14–1.89 (8H, m); 1.88–1.47 (12H,
m); 1.44–1.27 (4H, m); 1.04–0.86 (1H, bm), 0.75–0.61 (1H, bm); dC
(75 MHz, CD3OD) 169.2, 166.4, 136.8, 136.8, 133.1, 129.9, 129.9,
129.2, 129.1, 128.3, 126.8, 124.2, 124.0, 121.6, 118.9, 117.5, 111.4,
111.4, 66.4, 66.2, 66.2, 53.3, 43.9, 43.5, 43.2, 43.0, 41.8, 40.1, 40.1,
39.6, 39.4, 36.2, 36.0, 29.3, 28.5, 26.9, 26.6, 26.5; LRMS m/z (ES)
1325.4 (M + H+. C78H85N8O12 requires 1325.6).
General procedure for fluorescence titrations
A 200 ll, 12.0 lM solution of 2.1 or 2.14 in 20.0 mM HEPES, pH =
7.00 was allowed to equilibrate in a 0.3 ml quartz fluorescence cell
at ambient temperature in a Aminco-Bowman Series 2 Lumines-
cence Spectrometer Fluorometer. Tryptophan indole emission was
monitored at 357 nm to ensure a completely equilibrated solution
had been obtained. Aliquots of 1.0 ll of a 150 lM diphosphoryl
lipid A were sequentially added to the cuvette. After each addition,
the solution was allowed to equilibrate for a minimum of 10 min,
then excited at 280 nm and emission was monitored between
300–400 nm. The control experiment of diphosphoryl lipid A
titrated into HEPES buffer showed zero fluorescence; thus, was
not subtracted from the fluorescence data. Other fluorescence
titrations discussed in the text were carried out analogously.
We thank Dr Rene Lachicotte for carrying out X-ray crystallo-
graphic analysis of compound 7. This research was supported by
the NIH-NIGMS (5RO1-GM62825). S. G. T. was supported via
an NIH training grant in Dermatology (T32AR007472).
References
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Diphosphoryl lipid A Escherichia coli F583 (Rd Mutant) or LPS,
was obtained from Sigma Chemical Company and used without
purification. The diphosphoryl lipid A was first suspended in
doubly distilled water, then diluted by half in 40.0 mM HEPES
buffer, pH 7.00. The diluted diphosphoryl lipid A sample was
then sonicated for 30 min at room temperature. Finally, the
diphosphoryl lipid A sample was heat cycled no fewer than
four times between 4 and 70 ◦C, then stored at 4 ◦C for 12 h
before use.
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General procedure for UV-vis titrations
A 600 ll, 12.0 lM solution of 2.1 or 2.14 in 20.0 mM HEPES,
pH = 7.00 was allowed to equilibrate in a 1.0 ml quartz UV cell
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˚
˚
13 All molecular mechanics calculations and conformational analyses
were conducted using Maestro 4.1.012/Batchmin 7.2 (Schroedinger,
Inc.).
of unit cell dimensions a = 11.7699(8) A, b = 9.9771(7) A, c =
◦
◦
◦
˚
21.9102(15) A, and angles a = 90 , b = 90 , and c = 90 . Unit cell
3
˚
volume was 2572.9(3) A . Data were collected at 292 K. The, crystal
14 N. L. Allinger and L. A. Tushaus, J. Org. Chem., 1965, 30, 1945; P.
Pecquet, F. Huet, M. Legraverend and E. Bisagni, Heterocycles, 1992,
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2405.
was found to be of space group Pca2(1), with two molecules in the unit
cell. Linear absorption coefficient was 0.090 mm−1. 10446 reflections
were collected, 2830 of which were independent. Final R indices [I >
2r(I)] were R1 = 0.0539, wR2 = 0.1474, while R indices (all data) are
R1 = 0.0712, wR2 = 0.1582.
20 N. L. Allinger and L. A. Tushaus, J. Org. Chem., 1965, 30, 1945.
21 J. A. Chang, Y. Chiang, J. R. Keeffe, A. J. Kresge, V. A. Nikolaev and
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23 W. C. Still, M. Kahn and A. Mitra, J. Org. Chem., 1978, 43, 2923.
19 Selected X-ray crystallographic data: compound 7, C14H17NO3, for-
mula weight 247.29, crystallized in an orthorhombic crystal system
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The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3973–3979 | 3979
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