Heterocyclic Peptide Nanotube
A R T I C L E S
Table 1. Concentration-Dependent 1H NMR Chemical Shifts
signal
δ at 1.0 mM (Hz)
δ at 0.50 mM (Hz)
δ at 0.25 mM (Hz)
δ at 0.125 mM (Hz)
δ at 0.0625 mM (Hz)
Leu ꢀ CH3
Leu γ CH
292
378
772
1233
1650
1857
1911
2141
3212
296
387
777
1226
1659
1854
1907
2124
3147
300
399
781
1222
1673
1849
1904
2107
3086
303
408
788
1219
1683
1841
1901
2087
a
308
a
a
Leu â CH2 (1)
Phe â CH2 (1)
backbone CH2 (1)
backbone CH2 (2)
Phe R CH
a
1706
1831
1899
2059
a
Leu R CH
amide NH (1)
a Signal was either buried under another peak or not visible above noise at the lowest concentration acquisition.
of the MeCN was removed under vacuum, and the resulting residue
was dissolved in EtOAc (150 mL). The solution was washed with 9:1
satd NH4Cl:NH4OH and then twice with 0.5 N HCl. The organic phase
was dried and concentrated to yield 420 mg (97%) of the desired
product as a white solid, which was used without further purification.
mL, 0.245 mmol) were added to the solution. After stirring for 90 min,
the DMF was removed under vacuum. The resulting residue was
triturated with 4:1 water:acetonitrile (3 × 10 mL). The remaining solid
1
was dried under vacuum to yield 18 mg (65%) of 1. H NMR (500
MHz, DMSO-d6) δ 9.06 (d, J ) 7 Hz, 2H), 8.66 (dd, J ) 4, 8 Hz,
2H), 7.91 (s, 2H), 7.32-7.26 (m, 8H), 7.24-7.18 (m, 2H), 5.33 (t, J
) 7 Hz, 2H), 4.63 (dd, J ) 8, 16 Hz, 2H), 4.45-4.35 (m, 2H), 4.00
(dd, J ) 4, 16 Hz, 2H), 3.00 (dd, J ) 4, 14 Hz, 2H), 2.77 (dd, J ) 11,
14 Hz, 2H), 1.6-1.5 (m, 2H), 1.5-1.4 (m, 2H), 0.9-0.8, (m, 2H),
0.70 (t, J ) 7 Hz, 12H); 13C NMR (125 MHz, DMSO-d6) δ 170.9,
167.7, 146.1, 137.7, 129.1, 128.1, 126.4, 120.4, 60.5, 55.4, 43.1, 37.0,
34.4, 24.0, 22.1, 21.8; MALDI-FTMS (m/z) 683.3774 [M+H]+
(MWcalcd ) 683.3776).
An analytically pure sample was obtained by recrystallization from hot
1
EtOAc/hexanes. [R]22 ) -8.9 (c ) 10 mg/mL in CHCl3); H NMR
D
(500 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.88 (d, J ) 7 Hz and s, total
3H), 7.69 (d, J ) 7 Hz, 2H), 7.40 (t, J ) 7 Hz, 2H), 7.31 (t, J ) 7 Hz,
2H), 5.39 (dd, J ) 4, 11 Hz, 1H), 4.4-4.2 (m, 3H), 3.7-3.1 (broad s,
1H), 2.2-2.1 (m, 1H), 2.0-1.8 (m, 1H), 1.2-1.1 (m, 1H), 0.83 (dd, J
) 7, 23 Hz, 6H); 13C NMR (125 MHz, DMSO-d6) δ 170.7, 156.2,
145.0, 143.9, 140.7, 127.6, 127.1, 125.2, 122.6, 120.1, 65.6, 60.5, 46.7,
36.0, 24.3, 22.6, 20.9 (signal for methylene carbon on isobutyl side
chain is buried under solvent peaks ∼39 ppm and was identified by a
cross-peak observed in an HMQC experiment); ESI-MS (m/z) 435.2
[M+H]+ (MWcalcd ) 434.2).
Linear Peptide 8. (a) Loading of resin: Fmoc-phenylalanine (482
mg, 1.245 mmol) and diisopropylethylamine (0.217 mL, 1.245 mmol)
were dissolved in CH2Cl2 (5 mL) and added to trityl chloride resin
(500 mg, 1.66 mmol/g max loading). The mixture was agitated on a
shaker for 4 h. The vessel was then drained, and the resin was washed
with 8:2:1 CH2Cl2:MeOH:DIEA (2 × 10 min), CH2Cl2 (3 × 1 min),
and Et2O. After drying under vacuum, loading was quantified by UV
quantitation of Fmoc release. Final loading was found to be 1.0 mmol/
g.
Crystal Data Collection and Structure Determination. Crystals
of 1 were prepared by slow evaporation of a saturated in solution in
ethanol. Each crystal was mounted on a cryo-loop with paratone-N
oil. Data were collected on an Raxis IV image plate detector equipped
with Osmic confocal mirrors and Xstream cryo-device (100K) using
Cu KR radiation (λ ) 1.5418 Å) from a Ru200 X-ray generator operated
at 50 kV, 100 mA. Data were processed using MSC Crystal Clear.
The space group was determined to be P1 with cell dimensions a )
5.51 Å, b ) 12.59 Å, c ) 14.71 Å, R ) 83.30°, â ) 72.61°, γ )
81.88°. The data set was 96% complete and included 2356 reflections
of which 320 were unique. The scaling and averaging gave an Rmerge
of 7.4%. The mean I/σ was 6.7, and the average multiplicity for the
data set was 7.3. Each cell contains 1‚EtOH (C36H46N10O4‚C2H6O).
(b) Peptide Synthesis: The resin (150 mg, 0.100 mmol) prepared
above was placed in a sintered glass peptide synthesis vessel and
swollen in CH2Cl2 for 45 min. The resin was washed twice with DMF,
treated with 20% piperidine/DMF (2 × 8 min) to remove the Fmoc
group, and washed again with DMF (3×). A solution of 6 (241 mg,
0.555 mmol), diisopropylcarbodiimide (70 mg, 0.555 mmol), and
HOBT‚H2O (85 mg, 0.555 mmol) in DMF (2 mL) was added to the
vessel. The resin suspension was agitated for 30 min, drained, and
washed with DMF (3×). After Fmoc deprotection and washing as
above, a solution of Fmoc-phenylalanine (233 mg, 0.602 mmol), HBTU
(210 mg, 0.553 mmol), and diispropylethylamine (0.263 mL, 1.501
mmol) in DMF (2 mL) was added. The coupling was carried out for
30 min followed by washing and Fmoc deprotection. The final coupling
of 6 was carried out under the same conditions as the first coupling.
After removal of the terminal Fmoc, the resin was washed with DMF
(3×) and then CH2Cl2 (3×). Product was cleaved from the resin by
treatment with 5% TFA in CH2Cl2 (5 × 3 mL) followed by washing
with CH2Cl2 and MeOH. The acid solution and subsequent washes were
combined and concentrated under vacuum. Water was added to the
oily residue, and the resulting suspension was frozen and lyophilized.
The crude product was purified by preparative reverse-phase HPLC
on a C18 column to yield 46 mg of 8 (66% based on resin loading).
MALDI-FTMS (m/z) 701.3886 [M+H]+ (MWcalcd ) 701.3882).
The structure was solved by molecular replacement and restrained
refinement using the CCP4 program suite.13 The search model was
generated from an energy-minimized structure of 1 calculated in the
Discover module of InsightII. Geometric restraints for the triazole
portion were assembled from a survey of 1,4-disubstituted 1,2,3-triazoles
in the Cambridge Structural Database. Hydrogen atoms were used in
the refinement but were fixed to moving C, N, and O atoms. After
several cycles of restrained refinement, electron density for the ethanol
was located in a |Fo| - |Fc| map and modeled. The final model gave
an Rfactor of 16.7% for all data and an Rfree value of 18.0% for 10% of
the data set aside throughout structure solution and refinement. The
mean B value for the model was 17.8 Å2 with rms deviations from
ideal bond lengths and angles of 0.02 Å and 2.6°, respectively.
1
Variable-Concentration H NMR. A 1.0 mM stock solution of 1
was prepared in CDCl3. Serial 2-fold dilutions were carried out to
produce 0.5, 0.25, 0.125, and 0.0625 mM stocks. A 0.6 mL amount of
each solution was transferred to separate NMR tubes. These tubes were
1
placed on an autosampler, and HNMR spectra were acquired on a
Varian 400 MHz instrument. As all peaks are broad, each chemical
shift was recorded as the midpoint of the observed signal. The recorded
data is summarized in Table 1.
Macrocycle 1. Linear precursor 8 (29 mg, 0.041 mmol) was
dissolved in DMF (29 mL). PyBOP (32 mg, 0.061 mmol), HOAT
(0.123 mL of 0.5 M solution in DMF, 0.061 mmol), and DIEA (0.043
(13) Software used in structure solution: (a) CCP4: Collaborative Computational
Project Number 4. Acta Crystallogr. 1994, D50, 760-763. (b) Xtalview:
McRee, D. E. J. Mol. Graphics 1992, 10, 44-46.
9
J. AM. CHEM. SOC. VOL. 125, NO. 31, 2003 9375