Bioconjugate Chemistry
ARTICLE
suc-AlaAlaProPhe-Rh110-MC; sucAAPF-Rh110-MC. TBTU
(N,N,N0,N0-tetramthyl-o-(benzotriazol-1-yl)uranium tetrafluoro-
borate) was obtained from Novabiochem and Boc-(L)-AlaAla-
Pro-OH from Bachem. A mixture of TBTU (215 mg, 0.7 mmol)
and DIPEA (350 μL, 2 mmol) was dissolved in 1 mL of dry DMF
and added to Boc-L-Phe-OH (180 mg, 0.7 mmol) dissolved in
2 mL of dry CH2Cl2 under Ar. MC-Rh110 (50 mg, 0.1 mmol) was
dissolved in 1 mL dry CH2Cl2 and slowly added to the reaction.
After 17 h of stirring, the solvents were evaporated and the crude
mixture was directly purified by HPLC using an acetonitrile/water
gradient (12ꢀ100% ACN). Yield: 40% (30 mg, 0.04 mmol) Boc-
Phe-Rh110-MC. MS (ESI+): 692 (MH+).
Deprotection of Phe was achieved by slow addition of 50/50
(v/v) TFA/CH2Cl2 (3 mL) to Boc-Phe-Rh110-MC (30 mg,
0.04 mmol) in CH2Cl2 (1.5 mL). After 3 h, the reaction was
stopped and concentrated under vacuum. MS (ESI+): 592
(MH+).
suc-AAPF-Rh110-MC. Deprotection and succinylation was per-
formed as described above. The purified product was eluted from
HPLC using 35% ACN in water (isocratic). MS (ESI+): 692
(MH+). 1H NMR (400 MHz, DMSO): δ (ppm) 10.51 (s, 1H),
8.78 (s, 1H), 8.39 (d, J = 6.5 Hz, 2H), 7.94 (d, J = 7.8 Hz, 1H),
7.90 (s, 1H), 7.72 (t, J = 7.3 Hz, 1H), 7.65 (t, J = 7.3 Hz, 1H), 7.59
(S, 1H), 7.38ꢀ7.35 (m, 1H), 7.21ꢀ7.17 (m, 5H), 7.12ꢀ7.09 (m
2H), 6.64 (Sbr, 2H), 6.60 (t, J = 8.3 Hz, 4H), 4.50ꢀ4.48 (m, 1H),
3.54 (t, J = 4.3 Hz, 5H), 3.37 (t, J = 4.8 Hz, 5H), 3.07 (dd, J = 4.0
Hz, J = 13.7 Hz, 2H), 2.80 (dd, J = 9.8 Hz, J = 13.4 Hz, 2H), 2.6
(s, 1H), 2.26ꢀ2.01 (m, 5H).
Boc-Phe-Rh110; Boc-F-Rh110. During the synthesis of (Boc-
Phe)2-Rh110, monosubstituted Boc-F-Rh110 was obtained as a
side product after silica chromatography. It was further purified
by HPLC using an ACN/water gradient (10ꢀ100% ACN). MS
1
(ESI+): 579 (MH+). NMR: H NMR (600 MHz, CDCl3): δ
(ppm) 7.99 (d, J = 7.5 Hz, 1H), 7.68ꢀ7.56 (m, 3H), 7.30 (d, J =
6.8 Hz, 1H), 7.29ꢀ7.21 (m, 3H), 7,14 (d, J = 6.8 Hz, 1H), 6.79
(sb, 1H), 6.65 (d, J = 8.3 Hz, 1H), 6.55ꢀ6.51 (m, 2H), 6.34 (d, J =
7.9 Hz, 1H), 5.10 (s, 1H), 4.44 (s, 1H), 3.90 (s, 2H), 3.15 (s, 2H),
1.42 (s, 9H). 13C NMR (125 MHz, CDCl3): δ (ppm) 177.6 (C),
169.8 (C), 153.4 (C), 152.6 (C), 152.0 (C), 149.0 (C), 139.2
(C), 135.0 (CH), 129.7 (CH), 129.4 (CH), 129.3 (CH), 129.1
(CH), 128.7 (CH), 127.4 (CH), 127.0 (C), 125.1 (CH), 124.1
(CH), 115.2 (CH), 111.8 (CH), 108.6 (C), 108.0 (CH), 101.6
(CH), 100.1 (C), 83.5 (C), 56.8 (CH), 38.1 (CH2), 28.4 (CH3).
Boc-AlaAlaProPhe-Rh110; Boc-AAPF-Rh110. For the synth-
esis of this compound, the coupling of Boc-AlaAlaPro-OH to
Phe-Rh110 was not possible, as the peptide might also react with
the second amino group of Rh110. Instead, the 4 amino-acid-
long peptide Boc-AlaAlaProPhe-OH was coupled to Rh110
directly. In the first step, the peptide Boc-AlaAlaProPhe-OH
was synthesized. Boc-AlaAlaPro-OH (200 mg, 0.6 mmol) was
dissolved in 10 mL of anhydrous THF at 0 °C. To this solution, a
mixture of TBTU (180 mg, 0.6 mmol) and DIPEA (130 μL, 0.7
mmol), dissolved in 2 mL dry DMF, was added. After 10 min, the
cooling bath was removed and the reaction mixture was allowed
to warm to room temperature. Phe-OMe (160 mg, 0.7 mmol),
dissolved in 4 mL dry DMF, was added slowly to the above
mixture. After 20 h of stirring, the solvents were evaporated and
the crude mixture was purified on a silica gel column using
CH2Cl2/MeOH (9:1) as the eluent. Removal of the methyl
group was carried out in CH2Cl2 and 0.3 M NaOH. After that,
the reaction was neutralized by HCl and again purified on a silica
gel column using CH2Cl2/MeOH (9:1) as the eluent. Yield: 83%
(250 mg, 0.5 mmol). MS (ESI+): 527 (MNa+).
Boc-AlaAlaProPhe-OH (53 mg, 0.1 mmol), TBTU (41 mg,
0.1 mmol) and DIPEA (75 μL, 0.4 mmol) were dissolved in 2 mL
of dry DMF. To this mixture, Rhodamine 110 chloride (22 mg,
0.06 mmol in 1 mL dry DMF) was added and the solution was
stirred overnight at 37 °C. The resulting mixture was evaporated
and purified by HPLC (40% ACN in water; isocratic). The yield
was only 2% (1.2 mg). MS (ESI+): 818 (MH+). This low yield
was expected, as it has been shown before that the coupling of the
complete 4 amino-acid-long peptide is much less efficient than
the sequential coupling of amino acids in a multistep reaction.18
Kinetic Measurements. Chymotrypsin (α-Chymotrypsin
from bovine pancreas; Fluka) was dissolved in PBS (10 mM
phosphate pH 7.4, 138 mM NaCl, 2.7 mM KCl; Sigma).
Substrates were prepared as stock solutions in DMSO. Reactions
were started by adding 190 μL of chymotrypsin in PBS to
10 μL of a substrate solution in the cuvette. The final enzyme
TBTU (32 mg, 0.1 mmol) and DIPEA (40 μL, 0.2 mmol)
were dissolved in 1 mL of dry DMF and added to a solution of
Boc-(L)-AlaAlaPro-OH (35 mg, 0.1 mmol in 3 mL dry THF),
kept at 0 °C under Ar. The reaction mixture was stirred for 10
min after which it was left to warm to room temperature. The
Phe-Rh110-MC was dissolved in 1 mL of dry THF and added
to the above mixture. After stirring the reaction mixture at
37 °C for 12 h, the solvents were evaporated and the mixture
was purified by HPLC (45% ACN in water; isocratic). Yield:
52% (21 mg, 0.02 mmol) Boc-AlaAlaProPhe-Rh110-MC. MS
(ESI+): 931 (MH+).
The Boc group was removed as described above and the
deprotected product was succinylated in 4 mL THF using
triethylamine (25 μL, 0.2 mmol) and succinic anhydride (10
mg, 0.1 mmol) at 37 °C for 10 h. The mixture was purified by
HPLC (35% ACN in water; isocratic). Yield: 67% (14 mg,
0.015 mmol). MS (ESI+): 931 (MH+), 953 (MNa+). 1H NMR
(300 MHz, DMSO): δ (ppm) 9.98 (d, J = 5.4 Hz, 2H), 8.86 (s,
2H), 8.09ꢀ8.00 (m, 8H), 7.86 (dd, J = 1.7 Hz, J = 4.9 Hz, 2H),
7.80 (t, J = 7.3 Hz, 2H), 7.73 (t, J = 7.3 Hz, 2H), 7.68 (d, J = 1.9
Hz, 2H), 7.30ꢀ7.14 (m, 20H), 6.74 (d, J = 8.7 Hz, 2H), 6.66
(d, J = 8.7 Hz, 2H), 4.58 (dd, J = 1.7 Hz, J = 4.9 Hz, 2H), 4.51
(t, J = 7.0 Hz, 2H), 4.28ꢀ4.21 (m, 4H), 3.62 (d, J = 1.9 Hz,
2H), 7.30ꢀ7.14 (m, 20H), 6.74 (d, J = 8.7 Hz, 2H), 3.15 (dd,
J = 4.7 Hz, J = 14.7 Hz, 2H), 2.93 (dd, J = 9.4 Hz, J = 13.7 Hz,
2H), 2.40ꢀ2.31 (m, 8H), 1.99ꢀ1.92 (m, 2H), 1.81ꢀ1.76 (m,
4H), 1.68ꢀ1.63 (m, 2H), 1.19ꢀ1.14 (m, 14H).
(suc-Phe)2-Rh110; (suc-F)2-Rh110. (Boc-Phe)2-Rh110 was
obtained as the product of the first step of the synthesis of (suc-
AAPF)2-Rh110. It was deprotected and succinylated as described
above. After purification by HPLC (37% ACN in water; iso-
cratic), the desired (suc-F)2-Rh110 was obtained. MS (ESI+):
826 (MH+). 1H NMR (600 MHz, DMSO): δ (ppm) 10.39 (s,
2H), 8.38 (d, J = 7.9 Hz, 2H), 8.02 (d, J = 7.5 Hz, 1H), 7.88 (2s,
2H), 7.79 (t, J = 7.5 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 7.28ꢀ7.21
(m, 10H), 7.20ꢀ7.18 (m, 2H), 6.73 (d, J = 8.7 Hz, 2H), 4.62 (s,
2H), 3.07 (d, J = 9.4 Hz, 2H), 2.87 (t, J = 9.7 Hz, 2H), 2.38ꢀ2.24
(m, 8H). 13C NMR (125 MHz, DMSO): δ (ppm) 173.8 (C),
171.3 (C), 170.8 (C), 168.5 (C), 150.6 (C), 140.8 (C), 137.4
(C), 135.4 (CH), 130.0 (CH), 128.9 (CH), 128.2 (CH), 127.9
(CH), 126.2 (CH), 125.6 (C), 124.7 (CH), 123.7 (CH), 115.5
(CH), 113.1 (C), 106.4 (CH), 81.6 (C), 54.8 (CH), 37.3 (CH2),
30.0 (CH2), 29.7 (CH2).
suc-Phe-Rh110-MC; suc-F-Rh110-MC. Boc-Phe-Rh110-MC
was obtained as the product of the first step of the synthesis of
1934
dx.doi.org/10.1021/bc2001038 |Bioconjugate Chem. 2011, 22, 1932–1938