3-Hydroxypyrrole/Pyrrole Ring Pairings
J. Am. Chem. Soc., Vol. 121, No. 50, 1999 11629
recovered as a white powder upon lyophilization of the appropriate
fractions (3.8 mg, 77% recovery). UV (H2O) λmax 246, 312 (66 000);
1H NMR (DMSO-d6) δ 10.34 (s, 1 H), 10.24 (s, 1 H), 10.00 (s, 2 H),
9.93 (s, 1 H), 9.87 (s, 1 H), 9.83 (s, 1 H), 9.4 (br s, 1 H), 9.04 (s, 1 H),
8.03 (m, 3 H), 7.58 (s, 1 H), 7.44 (s, 1 H), 7.42 (s, 1 H), 7.23 (s, 1 H),
7.20 (m, 3 H), 7.12 (m, 2 H), 7.05 (d, 1 H), 7.02 (d, 1 H), 6.83 (s, 1
H), 6.79 (s, 1 H), 3.96 (s, 6 H), 3.90 (s, 3 H), 3.81 (s, 6 H), 3.79 (s, 3
H), 3.75 (d, 6 H), 3.33 (q, 2 H, J ) 5.4 Hz), 3.14 (q, 2 H, J ) 5.4 Hz),
3.08 (q, 2 H, J ) 6.1 Hz), 2.99 (m, 2 H, J ) 5.4 Hz), 2.69 (d, 6 H, J
) 4.2 Hz), 2.31 (m, 4 H), 1.72 (m, 4 H); MALDI-TOF-MS (monoiso-
topic), 1239.6 (1239.6 calcd for C57H71N22O11).
were used without further purification. DNA manipulations were
performed according to standard protocols.15
Construction of Plasmid DNA. The plasmid pAU2 was constructed
by hybridization of the inserts, 5′-GATCATGGACATCGATCTC-
TATGGTCATGCTATGCGATGGGCATATCAGCGTATGGCCAT-
3′ and 5′-AGCTATGGCCATACGCTGATATGCCCATCGCATAGC-
ATGACCATAGAGATCGATGTCCAT-3′. The hybridized insert was
ligated into linear pUC19 BamHI/HinDIII plasmid using T4 DNA
ligase. The resulting construct was transformed into Top10F′ OneShot
competent cells from Invitrogen. Ampicillin-resistant white colonies
were selected from 25 mL Luria-Bertani (LB) medium agar plates
(containing 50 µg/mL ampicillin and treated with XGAL and IPTG).
Large-scale plasmid purification was performed with Qiagen Maxi
purification kits. Dideoxy sequencing was used to verify the presence
of the insert. The concentration of the resulting plamid was determined
at 260 nm using the relationship of 1 OD unit ) 50 µg/mL.
Preparation of 3′-End-Labeled Restriction Fragments. The plas-
mid pAU2 was linearized with PVuII and EcoRI restriction endonu-
cleases and then treated with Klenow fragment, deoxyadenosine 5′-
[R-32P]triphosphate and thymidine 5′-[R-32P]triphosphate for 3′-end-
labeling. The labeled fragments were loaded onto a 7% non-denaturing
preparatory polyacrylamide gel (5% cross-link), and the desired 261
bp fragment was visualized by autoradiography and isolated. Adenosine
sequencing reaction was performed according to published methods.16
Quantitative DNase I Footprint Titrations.9 All reactions were
carried out in a volume of 400 µL. We note explicitly that no carrier
DNA was used in these reactions until after DNase I cleavage. A
polyamide stock solution (or water for reference and intact lanes) was
added to an assay buffer where the final concentrations were 10 mM
Tris‚HCl buffer (pH 7.0), 10 mM KCl, 10 mM MgCl2, 5 mM CaCl2,
and 25 kcpm 3′-radiolabeled DNA. The solutions were allowed to
equilibrate for 12 h at 22 °C. Cleavage was initiated by the addition of
10 µL of a DNase I stock solution (diluted with 1 mM DTT to give a
stock concentration of 1.13 u/mL) and allowed to proceed for 7 min at
22 °C. The reactions were stopped by adding 50 µL of a solution
containing 2.25M NaCl, 150 mM EDTA, 0.6 mg/mL glycogen, and
30 µM base-pair calf thymus DNA, and then ethanol-precipitated (2.1
volumes). The cleavage products were washed with 75% ethanol,
resuspended in 16 µL RNase-free water, lyophilized to dryness, and
then resuspended in 100 mM Tris-borate-EDTA/80% formamide
loading buffer (with bromophenol blue as dye), denatured at 90 °C for
10 min, and loaded directly onto a pre-run 8% denaturing polyacryl-
amide gel (5% cross-link, 7 M urea) at 2000 V for 1 h. The gels were
dried in vacuo at 80 °C and then exposed to a storage phosphor screen
(Molecular Dynamics). Equilibrium association constants were deter-
mined as previously described.3
ImImOpPy-γ-ImOpPyPy-â-Dp (4-Me). ImImOpPy-γ-ImOpPyPy-
â-Pam-resin was synthesized in a stepwise fashion by machine-assisted
solid-phase methods from Boc-â-Pam-resin (0.66 mmol/g) as described
for ImImPyPy-γ-ImOpPyPy-â-Dp (2Me). A sample of ImImOpPy-γ-
ImOpPyPy-â-Pam-resin (400 mg, 0.40 mmol/gram) was placed in a
glass 20 mL peptide synthesis vessel and treated with neat dimethyl-
aminopropylamine (2 mL) and heated at 55 °C with periodic agitation
for 16 h. The reaction mixture was then filtered to remove resin, 0.1%
(wt/v) TFA added (6 mL) and the resulting solution purified by reversed
phase HPLC. ImImOpPy-γ-ImOpPyPy-â-Dp was recovered upon
lyophilization of the appropriate fractions as a white powder (97 mg,
1
49% recovery). UV (H2O) λmax 246, 316 (66 000); H NMR (DMSO-
d6) δ 10.46 (s, 1 H), 10.00 (s, 1 H), 9.88 (s, 1 H), 9.30 (s, 1 H), 9.23
(s, 1 H), 9.13 (s, 1 H), 8.99 (s, 1 H), 8.06 (m, 3 H), 7.62 (s, 1 H), 7.50
(s, 1 H), 7.46 (s, 1 H), 7.28 (s, 1 H), 7.24 (d, 1 H), 7.22 (s, 1 H), 7.16
(s, 1 H), 7.15 (d, 2 H), 7.08 (d, 1 H), 7.03 (d, 1 H), 6.92 (s, 1H), 6.86
(s. 1 h), 3.99 (s, 9 H), 3.96 (s, 3 H), 3.85 (s, 6 H), 3.82 (s, 3 H), 3.79
(s, 9 H), 3.15 (q, 2 H, J ) 7.5 Hz), 3.05 (q, 2 H, J ) 6.2 Hz), 2.98 (q,
2 H, J ) 4.2 Hz), 2.84 (m, 2 H, J ) 4.2 Hz), 2.74 (s, 3 H), 2.73 (s,
3H), 2.34 (m, 4 H), 1.75 (m, 4 H); MALDI-TOF-MS (monoisotopic),
1282.61 (1282.59 calcd for C59H74N22O12).
ImImHpPy-γ-ImHpPyPy-â-Dp (4). A sample of ImImOpPy-γ-
ImOpPyPy-â-Dp (5 mg, 3.9 µmol) was treated with sodium thiophe-
noxide and purified by reversed phase HPLC as described for
ImImPyPy-γ-ImHpPyPy-â-Dp 2. ImImHpPy-γ-ImHpPyPy-â-Dp is
recovered as a white powder upon lyophilization of the appropriate
fractions (3.8 mg, 77% recovery). UV (H2O) λmax 246, 312 (66 000);
1H NMR (DMSO-d6) δ 10.34 (s, 1 H), 10.24 (s, 1 H), 10.00 (s, 2 H),
9.93 (s, 1 H), 9.87 (s, 1 H), 9.83 (s, 1 H), 9.4 (br s, 1 H), 9.04 (s, 1 H),
8.03 (m, 3 H), 7.58 (s, 1 H), 7.44 (s, 1 H), 7.42 (s, 1 H), 7.23 (s, 1 H),
7.20 (m, 3 H), 7.12 (m, 2 H), 7.05 (d, 1 H), 7.02 (d, 1 H), 6.83 (s, 1
H), 6.79 (s, 1 H), 3.96 (s, 6 H), 3.90 (s, 3 H), 3.81 (s, 6 H), 3.79 (s, 3
H), 3.75 (d, 6 H), 3.33 (q, 2 H, J ) 5.4 Hz), 3.14 (q, 2 H, J ) 5.4 Hz),
3.08 (q, 2 H, J ) 6.1 Hz), 2.99 (m, 2 H, J ) 5.4 Hz), 2.69 (d, 6 H, J
) 4.2 Hz), 2.31 (m, 4 H), 1.72 (m, 4 H); MALDI-TOF-MS (monoiso-
topic), 1254.57 (1254.55 calcd for C57H70N22O12).
Acknowledgment. We are grateful to the National Institutes
of Health (GM-27681) for research support, National Institutes
of Health for a research service award to J.W.S and S.W., J.
Edward Richter for an undergraduate fellowship to J.M.T., the
National Science Foundation for a predoctoral fellowship to
A.R.U., and the Howard Hughes Medical Institute for a
predoctoral fellowship to E.E.B. We thank G. M. Hathaway
for MALDI-TOF mass spectrometry.
DNA Reagents and Materials. Enzymes were purchased from
Boehringer-Mannheim and used with their supplied buffers. Deoxy-
adenosine and thymidine 5′-[R-32P] triphosphates were obtained from
Amersham. Calf thymus DNA (sonicated, deproteinized) and DNase I
(7500 u/mL, FPLC pure) were obtained from Pharmacia. Tris-HCl,
dithiothreitol (DTT), RNase-free water (used for all footprinting
reactions), and 0.5 M EDTA were purchased from US Biochemicals.
Xgal and IPTG were from ICN Biomedicals. Ampicillin trihydrate was
acquired from Sigma. Ethanol (200 proof) was purchased from Equistar.
Calcium chloride, potassium chloride, and magnesium chloride were
from Fluka. Formamide and premixed tris-borate-EDTA (Gel-Mate)
were from Gibco. Bromophenol blue was from Acros. All reagents
JA9930667
(15) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning; Cold
Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989.
(16) Iverson, B. L.; Dervan, P. B. Nucleic Acids Res. 1987, 15, 7823-
7830.