Photoreactive Activity-Based Probes of Metalloproteases
tracted twice with 0.1 aq. HCl (50 mL) and brine, dried with
MgSO4 and concentrated under reduced pressure. The resulting
mixture was purified by column chromatography (1.5% EtOAc/
petroleum ether) and the product was obtained as a yellow solid
(yield: 1.65 g, 2.31 mmol, 88%). 1H NMR (500 MHz, CDCl3): δ =
7.30 (d, J = 8.5 Hz, 2 H, Ph), 7.16 (d, J = 8.0 Hz, 2 H, Ph), 3.62–
3.56 (m, 1 H, CHα), 3.33 (dd, J1 = 18.5, J2 = 9.5 Hz, 1 H, 0.5ϫ
PhCH2), 2.23 (dd, J1 = 13.5, J2 = 6.5 Hz, 1 H, 0.5ϫ PhCH2), 3.07–
4.38–4.36 (m, 1 H, NCHα), 3.00 (dd, J1 = 13.6, J2 = 5.2 Hz, 1 H,
0.5ϫ PhCH2), 2.98–2.92 (m, 1 H, CHα), 2.82–2.75 (m, 2 H, 2ϫ
0.5ϫ PhCH2), 2.62 (dd, J1 = 13.6, J2 = 5.6 Hz, 1 H, 0.5ϫ PhCH2),
2.57 (d, 3 H, NCH3), 2.11 (dd, J1 = 14.8, J2 = 7.2 Hz, 1 H, 0.5ϫ
CH2,α), 1.94 (dd, J1 = 14.6, J2 = 7.4 Hz, 1 H, 0.5ϫ CH2,α) ppm.
13C NMR (100 MHz, [D6]DMSO): δ = 173.7, 172.0, 168.3, 142.8,
139.0, 130.8, 130.0, 129.0, 127.1, 127.0, 126.2, 55.0, 44.1, 38.2, 37.8,
35.1, 26.4 ppm. [α]2D3 = –10.8 (c = 1, DMSO). LC-MS (system A):
2.99 (m, 2 H, CH2,α), 1.54 (s, 9 H, 3ϫ CH3), 0.99 (s, 9 H, 3ϫ gradient 10% Ǟ 90% ACN/(0.1% TFA/H2O): Rt (min): 7.20 [ESI-
CH3), 0.14 (s, 3 H, SiCH3), 0.12 (s, 3 H, SiCH3) ppm. 13C NMR MS: m/z = 492.07 [M + H+]]. HRMS: calcd. for C23H24F3N5O4
(125 MHz, CDCl3): δ = 170.5, 169.6, 151.9, 141.1 (dd, J1 = 250 H,
J2 = 9.0 Hz, o-CF PFP), 139.5 (dt, J1 = 264, J2 = 13.5 Hz, p-CF3
PFP), 139.3, 137.8 (dt, J1 = 249, J2 = 13.5 Hz, m-CF3 PFP), 129.5,
127.9, 126.8, 125.0 (t, J2 = 12.8 Hz, C-O PFP), 122.1 (q, J =
273 Hz, CF3), 84.9, 42.4, 38.7, 37.1, 28.3 (q, J = 40.1 Hz, CNN),
27.8, 25.6, 18.1, –5.2, –5.3 ppm. 19F NMR (188 MHz, CDCl3): δ =
–65.8 (s, 3 F), –152.4 (d, J = 18.6 Hz, 2 F), –158.6 (t, J = 21.1 Hz,
1 F), –163.0 (t, J = 21.4 Hz, 2 F) ppm. [α]2D3 = +8.8 (c = 1, CHCl3).
HRMS: calcd. for C30H33F8N3O6Si [M + Na]+ 734.19031; found
734.19061.
(2R)-N4-(tert-Butoxycarbonyl)-N4-(tert-butyldimethylsilyloxy)-N1-
[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl]-2-{4-[3-(trifluoro-
methyl)-3H-diaziren-3-yl]benzyl}succinamide (26): -phenylala-
ninemethylamide[43] (25, 21 mg, 120 µmol) was added to a solution
of compound 24 (85 mg, 120 µmol) in DMF (2 mL). The reaction
was stirred at room temp. for 24 h after which no more starting
material was consumed (followed by LC-MS analysis). Et2O
(10 mL) and 0.1 aq. HCl solution (10 mL) were added and the
layers were separated. The aq. layer was extracted with Et2O
(10 mL) and the combined organic layers were extracted with a
[M + H]+ 492.18532; found 492.18494.
HA-succ(tmd)-Phe-Ahx-Lys(Biotin)-NH2 (2a): This compound was
synthesized on solid support on 20 µmol scale [based on the load-
ing of Fmoc-Lys(Biotin)] following the general procedure for solid-
phase synthesis. The final coupling step involved the addition of
compound 24 (70 µmol, 50 mg) and DIEA (40 µmol, 90 µL 0.45
in NMP) in NMP (0.40 mL) to the resin and shaking for 2 h. The
compound was purified by HPLC [system B, gradient 10% Ǟ 90%
ACN/(0.1 % TFA/H2O)] and was obtained as a colourless solid
(yield: 4.3 mg, 6.0 µmol, 30% after 3 coupling steps). 1H NMR
(400 MHz, [D6]DMSO): δ = 10.38 (s, 1 H, NHOH), 8.25 (br. s, 1
H, NHOH), 8.13 (t, J = 8.72, 8.72 Hz, 1 H, NH-Phe), 7.80–7.72
(m, 4 H, NH-Ahx + α-NH-Lys), 7.65 (t, J = 5.58 Hz, 1 H, ε-NH-
Lys), 7.37–7.13 (m, 8 H, Ph), 7.10 (t, J = 7.74 Hz, 2 H, Ph), 6.94
(br. s, 1 H, 0.5ϫ NH2), 6.42 (br. s, 1 H, NH-Biotin), 6.36 (br. s, 1
H, NH-Biotin), 4.35–4.32 (m, 2 H, CHα-Phe + NCH-Biotin), 4.18–
4.09 (m, 2 H, CHα-Lys + NCH-Biotin), 3.09 (dd, J1 = 12.83, J2 =
6.04 Hz, 1 H, SCH), 3.03–2.87 (m, 6 H, 0.5ϫ CHα + 0.5ϫ PhCH2
+ NCH2-Ahx + NCH2-Lys), 2.82 (dd, J1 = 12.40, J2 = 5.07 Hz, 1
H, 0.5ϫ CH2S), 2.78–2.72 (m, 1 H, 0.5ϫ CHα), 2.68–2.66 (m, 1
0.1 aq. HCl solution (2ϫ10 mL) and brine, dried with MgSO4 H, 0.5ϫ PhCH2), 2.64–2.60 (m, 1 H, 0.5ϫ PhCH2), 2.60 (d, J =
and concentrated under reduced pressure. The resulting mixture
was purified by column chromatography (10% Ǟ 50% EtOAc/pe-
troleum ether) and the product was obtained as a colourless oil
12.4 Hz, 1 H, 0.5ϫ CH2S), 2.13 (t, J = 7.35 Hz, 2 H, CH2,α-Ahx),
2.07 (t, J = 7.60 Hz, 2 H, CH2,α-Biotin), 2.02–1.96 (m, 1 H, 0.5ϫ
CH2,α) 1.91 (dd, J1 = 13.98, J2 = 7.50 Hz, 1 H, 0.5ϫ CH2,α), 1.65–
(yield: 38 mg, 54 µmol, 45%). 1H NMR (400 MHz, CDCl3): δ = 1.56 (m, 1 H, 0.5ϫ CH2-Lys), 1.52–1.40 (m, 9 H, 0.5ϫ CH2-Lys
7.29–7.17 (m, 7 H, Ph), 7.06 (d, J = 8.0 Hz, 2 H, Ph), 6.14 (d, J =
+ 2ϫ CH2-Ahx+ 2ϫ CH2-Biotin), 1.40–1.22 (m, 6 H, 2ϫ CH2-
8.0 Hz, 1 H, NH), 5.29–5.28 (m, 1 H, NH), 4.50 (q, J = 6.9 Hz, 1 Lys, CH2-Biotin), 1.20–1.12 (m, 2 H, CH2-Ahx) ppm. LC-MS (sys-
H, NCHα), 3.21–3.11 (m, 2 H, CHα + 0.5ϫ PhCH2), 2.97–2.85 (m, tem A): gradient 10% Ǟ 90% ACN/(0.1% TFA/H2O): Rt (min):
4 H, PhCH2 + 0.5ϫ PhCH2 + 0.5ϫ CH2,α), 2.77–2.69 (m, 1 H, 6.45 (ESI-MS: m/z = 945.53 [M + H+]). HRMS: calcd. for
0.5ϫ CH2,α), 2.57 (d, J = 4.8 Hz, 3 H, NCH3), 1.54 (s, 9 H, 3ϫ C44H59F3N10O8S [M + H]+ 945.42629; found 945.42682.
CH3), 0.99 (s, 9 H, 3ϫ CH3), 0.14 (s, 3 H, SiCH3), 0.11 (s, 3 H,
(2R)-N1-[(2S)-1-{6-[(S)-1,6-Diamino-1-oxohexan-2-ylamino]-6-
SiCH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 173.6, 170.8,
oxohexylamino}-1-oxo-3-phenylpropan-2-yl]-N4-hydroxy-2-{4-[3-(tri-
170.7, 151.8, 140.7, 136.7, 129.5, 128.6, 127.5, 126.9, 126.5, 122.1
fluoromethyl)-3H-diazirin-3-yl]benzyl}succinamide or HA-succ-
(tmd)-Phe-Ahx-Lys-NH2 (30): This compound was synthesized on
(q, J = 273 Hz, CF3), 84.8, 54.4, 44.5, 39.9, 37.9, 37.6, 29.7, 28.3
(q, J = 40.1 Hz, CNN), 28.0, 26.0, 25.7, 18.1, –4.9 ppm. [α]2D3
=
solid support on 175 µmol scale [based on the loading of Fmoc-
Lys(Boc)] following the general procedure for solid-phase synthesis.
The final coupling step involved the addition of compound 24
(1.12 mmol, 800 mg) and DIEA (750 µmol, 124 µL 0.45 in NMP)
in NMP (10 mL) to the resin and shaking for 2 h. The compound
was purified by HPLC [system B, gradient 10% Ǟ 90% ACN/
(0.1% TFA/H2O)] and was obtained as a colourless solid (yield:
+5.6 (c = 1, CHCl3). LC-MS (system A): = gradient 50 Ǟ 90 ACN/
(0.1 TFA/H2O): Rt (min): 9.81 [ESI-MS: m/z = 705.87 [M + H+]].
HRMS: calcd. for C34H46F3N5O6Si [M + H]+ 706.32422; found
706.32419.
(2R)-N4-Hydroxy-N1-[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-
2-yl]-2-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}succinamide
(27): TFA (1 mL) and deionised H2O (50 µL) were added to a solu-
tion of compound 26 (21 mg, 30 µmol) in DCM (1 mL). After 1 h
of stirring LC-MS and TLC analysis indicated complete conversion
of the starting material. Toluene was added and the mixture was
concentrated under reduced pressure. In order to remove excess
TFA the mixture was co-evaporated twice with toluene. The re-
sulting crude mixture was purified by HPLC [system B, gradient
1
45 mg, 54 µmol, 30% after 3 coupling steps). H NMR (400 MHz,
CD3OD): δ = 7.69 (t, J = 5.62 Hz, 1 H, NH), 7.38–7.34 (m, 1 H,
NH), 7.29–7.16 (m, 9 H, Ph + 2ϫ NH), 7.10 (d, J = 8.03 Hz, 2 H,
Ph), 4.43 (t, J = 7.49 Hz, 1 H, NCHα-Phe), 4.35 (dd, J1 = 8.89, J2 =
5.32 Hz, 1 H, NCHα-Lys), 3.15–3.05 (m, 3 H, NCH2-Ahx + 0.5ϫ
PhCH2-Phe), 3.04–2.99 (m, 1 H, CHα), 2.98–2.83 (m, 4 H, NCH2-
Lys + 0.5ϫ PhCH2-Phe + 0.5ϫ PhCH2), 2.70 (dd, J1 = 13.62, J2
40% Ǟ 65% ACN/(0.1% TFA/H2O)]. The title compound was ob- = 6.17 Hz, 1 H, 0.5ϫ PhCH2), 2.33–2.24 (m, 3 H, CH2,α-Ahx +
tained as a colourless solid (yield: 10.2 mg, 21 µmol, 69%). 1H
NMR (400 MHz, [D6]DMSO): δ = 10.44 (s, 1 H, NHOH), 8.78
0.5ϫ CH2,α), 2.11 (dd, J1 = 14.82, J2 = 6.37 Hz, 1 H, 0.5ϫ CH2,α),
1.87 (ddd, J1 = 14.60, J2 = 7.64, J3 = 4.70 Hz, 1 H, 0.5ϫ CH2-
(br. s, 1 H, NHOH), 8.17 (d, J = 8.0 Hz, 1 H, NH), 7.65–7.58 (m, Lys), 1.78–1.68 (m, 3 H, 0.5ϫ CH2-Lys + CH2-Lys), 1.57 (dd, J1
1 H, NH), 7.30–7.21 (m, 7 H, Ph), 7.14 (d, J = 8.0 Hz, 2 H, Ph), = 14.95, J2 = 7.45 Hz, 2 H, CH2-Ahx), 1.55–1.45 (m, 2 H, CH2-
Eur. J. Org. Chem. 2010, 2100–2112
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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