A R T I C L E S
Hudgins et al.
over MgSO4 and rotary evaporation gave the azide 3 (170 mg crude
product, 81%) with ∼5% HMPA. This intermediate was not purified
for the next step due to its instability even at low temperature: 1H
NMR (400 MHz, CDCl3) δ 1.15-1.25 (2 H, m, CH2), 1.32-1.43 (2
H, m, CH2), 1.51-1.68 (4 H, m, CH2), 3.90 (2 H, s, CH2-N3), 5.17 (1
H, s br, CH); 13C NMR (101 MHz, CDCl3) δ 21.7 (2 C, CH2), 24.2 (2
C, CH2), 58.3 (CH2-N3), 61.9 (CH), 67.0 (Cq) ppm.
A solution of azide 3 (164 mg, 0.99 mmol) in 10 mL of dry THF
was added dropwise to a stirred slurry of lithium aluminum hydride
(76 mg, 2 mmol) in THF (5 mL) under nitrogen. The mixture was
stirred at 68 °C for 6 h, cooled to room temperature, diluted with 10
mL THF, and slowly treated with 15% NaOH until a white, granular
precipitate was formed. The precipitate was removed by filtration and
washed with THF, and the combined filtrates were evaporated. The
residue was dissolved in CH2Cl2 and dried over KOH pellets.
Concentration and flash column chromatography (CH2Cl2/methanol/
NEt3 89:10:1) gave the amine 4 (111 mg, 80%) as a colorless,
hygroscopic wax melting at room temperature: UV (D2O) λmax 369
nm, ꢀ 43 M-1 cm-1 (D2O); 1H NMR (400 MHz, CDCl3) δ 1.18-1.25
(2 H, m, CH2), 1.31-1.48 (4 H, m, CH2), 1.60-1.67 (2 H, m, CH2),
1.78 (2H, br s, NH2), 3.21 (2 H, br s, CH2N), 5.14-5.17 (1 H, m, CH)
ppm; 13C NMR (101 MHz, CDCl3) δ 22.0 (2 C, CH2), 24.0 (2 C, CH2),
49.2 (1 C, CH2N), 61.9 (1 C, CH), 67.2 (1 C, Cq) ppm. The hygroscopic
nature of the pure amine prevented an accurate elemental analysis.
Fmoc), 50.6 (CH Asp), 62.2 (CH), 67.2 (Cq), 67.3 (CH2 Fmoc), 120.0
(2 C, CH Fmoc), 125.2 (2 C, CH Fmoc), 127.1 (2 C, CH Fmoc), 127.7
(2 C, CH Fmoc), 141.3 (2C, Cq Fmoc), 143.8 (2 C, Cq Fmoc), 156.0
(CdO Fmoc), 172.1 (CdO), 172.6 (COOH); FAB+ MS (NBA) 477
(M + H+); 515 (M + K+). HR-MS: calcd 477.2137 (M + H+); found
(+ESI-TOF) 477.2120.
Peptide Synthesis. Polypeptides were made by Affina Immuntechnik
GmbH (Berlin, Germany). The raw polypeptide was precipitated in
diethyl ether and purified by semipreparative HPLC (LC-8A, Shimadzu)
on an RP-18 column at 40 °C (VYDAC No. 218TP101522, 10 µL).
Flow rates of 0.7 (250 × 4.5 column) or 8 mL min-1 (250 × 22 column)
with water containing 0.1% trifluoroacetic or phosphoric acid as eluent
were adjusted to which a gradient of up to 50% acetonitrile containing
0.1% trifluoroacetic or phosphoric acid as coeluent was applied within
20 min. The retention times ranged between 10 and 22 min. The purity
of the polypeptides was >95% as determined by MALDI-MS and
HPLC. UV spectrophotometry confirmed also the presence of the
characteristic chromophores (DBO, Trp). The extinction coefficients
were the same, within error, as those reported for Trp (∼5500 M-1
19
cm-1
)
and DBO (50 M-1 cm-1),12,20 which provides another purity
and sample identity criterion.
The DBO probe and the Fmoc-DBO amino acid are fully compatible
with standard Fmoc solid-phase peptide synthesis. No complications
were found in coupling, and there was no apparent degradation of DBO
during cleavage with 95% trifluoroacetic acid and HPLC purification.
No special scavengers21 or protecting groups are required for the DBO
residue during synthesis and cleavage.
Synthesis of Fmoc-DBO. Amine 4 (80 mg, 0.575 mmol), Fmoc-
Asp-Ot-Bu (240 mg, 0.583 mmol) and 184 mg of EEDQ (0.75 mmol)
were stirred in 15 mL of dry CH2Cl2 under argon for 2 days. The
mixture was diluted to 50 mL, washed successively with 5% citric acid,
water, saturated NaHCO3, water, and brine, and dried over MgSO4.
Concentration and flash column chromatography (CH2Cl2 with 2%
MeOH) gave the amide 5 (272 mg, 89%) as a colorless solid with mp
178-180 °C: 1H NMR (CDCl3, 500 MHz) δ 1.06-1.16 (2 H, m, CH2),
1.24-1.35 (2 H, m, CH2), 1.47 (9 H, s, CH3), 1.45-1.66 (4 H, m,
CH2), 2.78 (1 H, dd, J ) 16.0, 4.4 Hz, â-CH2 Asp), 2.95 (1 H, dd, J
) 16.0, 4.4 Hz, â-CH2 Asp), 3.82 (2 H, d, J ) 6.1 Hz, CH2N), 4.21 (1
H, t, J ) 7.4 Hz, CH Fmoc), 4.28 (1 H, dd, J ) 10.4, 7.4 Hz, CH2
Fmoc), 4.39 (1 H, dd, J ) 10.4, 7.4 Hz, CH2 Fmoc), 4.48-4.52 (1 H,
m, R-CH Asp), 5.20 (1 H, br s, CH), 6.05 (1 H, br d, J ) 8.5 Hz,
urethane NH), 6.55 (1 H, br t, J ) 6.1 Hz, NH), 7.30 (2 H, t, J ) 7.4
Hz, CH Fmoc), 7.40 (2 H, t, J ) 7.4 Hz, CH Fmoc), 7.58-7.62 (2 H,
m, CH Fmoc), 7.76 (2 H, d, J ) 7.4 Hz, CH Fmoc) ppm; 13C NMR
(CDCl3, 126 MHz) δ 21.7 (2 C, CH2), 24.1 (2 C, CH2), 27.9 (3 C,
CH3), 38.0 (CH2 Asp), 45.3 (CH2N), 47.1 (CH Fmoc), 51.3 (CH Asp),
62.0 (CH), 66.8 (Cq), 67.2 (CH2 Fmoc), 82.3 (Cq), 119.9 (2 C, CH
Fmoc), 125.2 (2 C, CH Fmoc), 127.0 (2 C, CH Fmoc), 127.7 (2 C,
CH Fmoc), 141.3 (2 C, Cq Fmoc), 143.8 (Cq Fmoc), 143.9 (Cq Fmoc),
156.1 (CdO Fmoc), 170.0 (CdO), 170.3 (CdO); FAB+ MS (NBA)
533 (M + H+), 571 (M + K+). Anal. Calcd for C30H36N4O5‚0.2CH2-
Cl2: C, 66.00; H, 6.68; N, 10.19; O, 14.55. Found: C, 65.81; H, 6.82;
N, 10.10; O, 14.42.
Fluorescence Spectroscopy. All measurements were performed in
aerated D2O at ambient temperature. Fluorescence lifetimes were
measured on a laser flash photolysis (LFP) setup (LP900, Edinburgh
Instruments, Edinburgh, Scotland) with 7-mJ, 355-nm pulses of 4-ns
width from a Nd:YAG laser (Minilite II, Continuum, Santa Clara, CA),
and with a time-correlated single-photon counting (SPC) fluorometer
(FLS900, Edinburgh Instruments) using a 1.5-ns pulse-width H2 flash
lamp at 370 nm. The FLS900 instrument was also used for the steady-
state fluorescence (SSF) spectra (λexc ) 365 nm). Fluorescence was
detected at 430 nm on both time-resolved setups. The resulting data
were analyzed with the Edinburgh software of the LP900 and FLS900
setup by means of monoexponential or biexponential decay functions
and a reconvolution function for the excitation light pulse. Intermo-
lecular quenching experiments were performed with 10 µM solutions
of DBO and varying quencher concentrations up to 50% quenching
effect or up to the solubility limit of the quencher (4-5 data points).
Typical concentrations of polypeptides were 10 µM for LFP and 100
µM for SPC experiments. The polypeptides were measured over a
concentration range of 1 µM-1 mM by LFP and 10 µM-1 mM by
SPC. The fluorescence lifetimes remained constant within error within
this concentration range. In the case of SSF measurements, a linear
increase of the intensity with concentration (200 µM-1 mM, 5 data
points) was found.
Amide 5 (181 mg, 0.340 mmol) in 5 mL of dry CH2Cl2 was
converted to the free carboxylic acid by adding 3 mL of TFA to the
ice-cooled solution and subsequent stirring at room temperature for 3
h. Rotary evaporation of the mixture and coevaporation with toluene
and acetonitrile gave Fmoc-DBO (160 mg, 98%) as a colorless solid,
which was used directly for peptide synthesis: 1H NMR (CDCl3, 500
MHz) δ 1.07-1.19 (2 H, m, CH2), 1.26-1.39 (2 H, m, CH2), 1.50-
1.57 (2 H, m, CH2), 1.61-1.68 (2 H, m, CH2), 2.81 (1 H, dd, J )
15.6, 8.1 Hz, â-CH2 Asp), 3.03 (1 H, dd, J ) 15.6, 2.7 Hz, â-CH2
Asp), 3.74-3.88 (2 H, m, CH2N), 4.20 (1 H, t, J ) 7.2 Hz, CH Fmoc),
4.30-4.40 (2 H, m, CH2 Fmoc), 4.52-4.57 (1 H, m, R-CH Asp), 5.20
(1 H, br s, CH), 6.23 (1 H, br d, J ) 4.7 Hz, urethane NH), 7.30 (2 H,
t, J ) 7.5 Hz, CH Fmoc), 7.39 (2 H, t, J ) 7.5 Hz, CH Fmoc), 7.44
(1 H, br t, J ) 11 Hz, NH), 7.56-7.60 (2 H, m, CH Fmoc), 7.75 (2 H,
d, J ) 7.5 Hz, CH Fmoc); 13C NMR (CDCl3, 126 MHz) δ 21.7 (2 C,
CH2), 24.2 (CH2), 24.3 (CH2), 37.8 (CH2 Asp), 45.7 (CH2N), 47.0 (CH
Results
Quenching by Amino Acids and Denaturing Agents. The
photophysical methodology outlined in Scheme 1 required the
selection of a quencher for the excited DBO with an efficient,
preferably diffusion-controlled, quenching rate constant. It was
appealing to select a natural amino acid as an intrinsic
quencher.10 For this purpose, the quenching rate constants of
the parent DBO by the 20 natural amino acids were measured
in D2O, H2O, and pH 7.0 phosphate buffer. Table 1 reports the
data for six amino acids that gave rise to significant quenching
(19) Luisi, P. L.; Rizzo, V.; Lorenzi, G. P.; Straub, B.; Suter, U.; Guarnaccia,
R. Biopolymers 1975, 14, 2347-2362.
(20) Nau, W. M. EPA Newsl. 2000, 70, 6-29.
(21) Guy, C. A.; Fields, G. B. Methods Enzymol. 1997, 289, 67-83.
9
558 J. AM. CHEM. SOC. VOL. 124, NO. 4, 2002