Journal of the American Chemical Society
ARTICLE
Chemical Synthesis. All chemicals were purchased from commer-
cial vendors and used without further purification, unless indicated
otherwise. Rink Amide resin, all Fmoc-protected amino acids, O-ben-
zotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate (HB-
TU), N-hydroxybenzotriazole (HOBt), triisopropylsilane (TIS), and all
other peptide synthesis reagents were purchased from GL Biochem
(China). All nonaqueous reactions were carried out under a nitrogen
atmosphere in oven-dried glassware. Reaction progress was monitored
by TLC on precoated silica plates (Merck 60 F254, 250 μm thickness)
and spots were visualized by Ceric Ammonium Molybdate (CAM),
basic KMnO4, UV light or iodine. Flash column chromatography was
carried out using Merck 60 F254 0.040ꢀ0.063 μm silica gel. The 1H and
13C NMR spectra were taken on a Bruker 300 MHz or DPX-300 MHz or
Bruker Avance 500 MHz NMR spectrometer. Chemical shifts are
reported in parts per million (ppm) referenced with respect to residual
solvent (CDCl3 = 7.26 ppm, (CD3)2SO = 2.50 ppm) or Tetramethylsi-
lane (Si(CH3)4 = 0.00 ppm). 1H NMR coupling constants (J) are
reported in hertz (Hz) and the following abbreviations were used in
reporting spectra: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, dd = doublet of doublets, ddd = doublet of doublets of
doublets, dt = doublet of triplets or overlap of nonequivalent resonances.
Mass spectra were recorded on a Finnigan LCQ mass spectrometer, a
Shimadzu LC-IT-TOF spectrometer or a Shimadzu LC-ESI spectro-
meter. Analytical HPLC was carried out on Shimadzu LC-IT-TOF or
LC-ESI systems equipped with an autosampler, using reverse-phase
Phenomenex Luna 5 μm C18 100 Å 50 ꢁ 3.0 mm columns. Preparative
HPLC was carried out on Gilson preparative HPLC system using
Trilution software and reverse-phase Phenomenex Luna 5 μm C18(2)
100 Å 50 ꢁ 30.00 mm column. A 0.1% TFA/H2O and 0.1% TFA/
acetonitrile were used as eluents for all HPLC experiments. The flow
rate was 0.6 mL/min for analytical HPLC and 10 mL/min for pre-
parative HPLC. Details of synthetic procedures and characterizations
of most compounds were reported in the Supporting Information.
Characterizations of key intermediates and final probes are reported
below.
(E)-9-(4-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
4-tert-butoxy-4-oxobutanamido)phenyl)-1-(4-((4-(dimethy-
lamino)phenyl)diazenyl)phenyl)-1,7,10-trioxo-8-oxa-2,6,11-
triazatridecan-13-oic Acid (17a). HPLC-grade THF was degassed
with Argon for 1 h. Then compound 16a (prepared from Fmoc-
Asp(tBu)ꢀOH in multiple steps; see Scheme S2 in the Supporting
Information) (2.4 g, 2.4 mmol) and PhSiH3 (0.6 mL, 4.8 mmol) were
added, followed by Pd(PPh3)4 (5 wt %, 120 mg) under an argon
atmosphere. The reaction mixture was concentrated in vacuo and purified
by flash chromatography (MeOH/DCM = 1:8) to afford 17a as an
orange compound (1.6 g, 69.2%). 1H NMR (300 MHz, CDCl3) δ 10.19
(s, 1H), 8.67 (s, 1H), 8.01 (d, J = 8.6 Hz, 2H), 7.88ꢀ7.79 (m, 7H), 7.70
(t, J = 7.2 Hz, 3H), 7.57 (t, J = 8.4 Hz, 3H), 7.38 (d, J = 7.2 Hz, 4H),
7.32ꢀ7.28 (m, 2H), 6.84 (d, J = 9.2 Hz, 2H), 5.82 (s, 1H), 4.51 (q, J = 7.9
Hz, 1H), 4.34ꢀ4.19 (m, 3H), 3.07 (s, 6H), 2.72 (m, 1H), 2.55 (m, 1H),
1.68 (t, J = 6.7 Hz, 2H), 1.36 (s, 9H). 13C NMR (75 MHz, CDCl3)
δ 169.7, 169.53, 165.77, 155.46, 154.39, 153.30, 144.27, 144.19, 143.13,
141.17, 139.88, 139.29, 137.90, 135.26, 132.41, 129.40, 128.81, 128.31,
128.11, 127.77, 127.52, 125.73, 125.54, 121.96, 121.84, 120.55, 120.49,
119.68, 112.05, 110.20, 80.76, 74.90, 66.24, 52.68, 47.08, 38.69, 38.01,
37.42, 29.82, 28.23, 28.16. IT-TOF: m/z [M + H]+ calcd 969.41, found
969.40.
(s, 2H), 6.62 (s, 2H), 4.29 (s, 2H), 4.15ꢀ4.07 (m, 4H), 2.98 (s, 6H), 2.30
(s, 2H), 2.15ꢀ2.09 (m, 2H), 1.58 (s, 2H), 1.34 (s, 9H). 13C NMR (125
MHz, CDCl3) δ 172.56, 156.56, 156.52, 154.74, 152.62, 143.79, 143.54,
141.09, 128.02, 127.53, 126.97, 125.29, 125.01, 122.03, 119.76, 111.38,
80.84, 67.16, 67.11, 66.31, 65.76, 60.46, 54.87, 49.86, 49.68, 49.52, 49.34,
46.92, 40.11, 35.14, 31.85, 31.51, 29.62, 29.29. IT-TOF: m/z [M + H]+
calcd 983.42, found 983.41.
(E)-9-(4-(Bis(2-nitrobenzyloxy)phosphoryloxy)phenyl)-
1-(4-((4-(dimethylamino)phenyl)diazenyl)phenyl)-1,7,10-
trioxo-8-oxa-2,6,11-triazatridecan-13-oic Acid (35). Com-
pound 34 (prepared based in Scheme S3 in the Supporting Information)
(0.83 g, 0.86 mmol) and PhSiH3 (0.2 mL, 1.7 mmol) dissolved in THF
were added Pd(PPh3)4 (5 wt %, 41 mg) under an argon atmosphere.
The reaction mixture was stirred for 30 min at room temperature
before being concentrated in vacuo and purified by flash chromatography
(MeOH/DCM = 1:8) to afford 35 asanorange product(0.63g, 80%). 1H
NMR (300 MHz, CDCl3) δ 8.64 (broad, NH), 8.11 (d, J = 3.8 Hz, 2H),
8.00 (d, J = 4.3 Hz, 2H), 7.81ꢀ7.73 (m, 6H), 7.65ꢀ7.59 (m, 4H), 7.52
(d, J = 4.3 Hz, 2H), 7.22 (d, J = 4.2 Hz, 2H), 6.82 (d, J = 4.6 Hz, 2H),
5.93 (s, 1H), 5.58 (d, J = 3.7 Hz, 4H), 3.68ꢀ3.53 (m, 4H), 3.30 (broad,
2H), 3.05 (s, 6H), 1.70 (t, J = 6.4 Hz, 2H). 13C NMR (75 MHz, CDCl3)
δ 172.6, 167.9, 165.7, 154.9, 154.0, 152.8, 149.8, 149.7, 146.8, 142.7,
134.8, 134.4, 134.3, 129.6, 129.6, 128.8, 128.3, 125.1, 124.9, 121.5,
119.92, 119.86, 111.5, 73.8, 66.4, 66.3, 38.3, 37.0, 29.4. 31P NMR (121
MHz, MeOD) δ ꢀ6.15. IT-TOF: m/z [M + H]+ calcd 927.26, found
927.26.
Synthesis, Purification, and Characterizations of the Pro-
tease Probes (P1ꢀP4, C1, and C2) and the PTP Probes
(P5ꢀP7). First, Fmoc-Lys(Biotin)ꢀOH (4 equiv) was synthesized
following previously published procedures.28 To load it onto rink amide
AM resin, it was dissolved in dry DMF (1.5 mL) together with HBTU
(4 equiv), HOBt (4 equiv), and DIEA (8 equiv). The resin (50 mg,
loading ∼0.5 mmol/g, premixed in dry DMF) was added and the
resulting mixture was shaken for 3 h at room temperature. The resin was
filtered and washed thoroughly with DMF (3ꢁ), DCM (3ꢁ), and DMF
(3ꢁ) until the filtrate became colorless. Any unreacted resin was capped
with a solution of Ac2O (10 equiv), DIEA (20 equiv) in DCM, and the
reaction mixture was allowed to react for 2 h at room temperature,
followed by extensive washes with DCM (3ꢁ) and DMF (3ꢁ). Next,
the Fmoc-protected amino-functionalized resin was treated with 20%
piperidine in DMF for 1 h at room temperature, then washed with DMF
(3ꢁ), DCM (3ꢁ), and DMF (3ꢁ). The coupling of an Fmoc-protected
amino acid was carried out using the following general procedure. Fmoc-
amino acid (4 equiv), HBTU (4 equiv), and HOBt (4 equiv) were
dissolved in dry DMF (1.5 mL) and DIEA (8 equiv) was added and
agitated for 5 min. This preactivated Fmoc-amino acid solution was
added to the resin and shaking was continued for 3 h at room
temperature. The resin was filtered and washed with DMF (3ꢁ),
DCM (3ꢁ), and DMF (3ꢁ). To couple 17a and 17b, the compound
(4 equiv), HATU (4 equiv), and HOAt (4 equiv) were dissolved in dry
DMF (1.5 mL). DIEA (8 equiv) was added and the resulting mixture
was agitated for 5 min. This preactivated unnatural amino acid solution
was added to the resin and shaking was continued overnight at room
temperature. The resin was filtered and washed with DMF (3ꢁ), DCM
(3ꢁ), and DMF (3ꢁ). The procedure for the coupling of Fluorescein-
(OAc)-NHS (step g in Scheme S2) was the following. First, the resin
was swollen in dry DCM for 30 min, then treated with 1% TFA, 5% TIS
in DCM (1.5 mL) for another 30 min. Successful Mtt deprotection was
indicated by ninhydrin test. The resin was washed with DMF (3ꢁ),
DCM (3ꢁ) and DMF (3ꢁ), then added into an DMF solution con-
taining Fluorescein(OAc)-NHS (4 equiv) and DIEA (8 equiv) (1.5 mL)
followedbyshakingovernight atroom temperature. Theresin was filtered,
then washed with DMF (3ꢁ), DCM (3ꢁ), and DMF (3ꢁ), followed by
capping with with Ac2O (10 equiv) and DIEA (20 equiv) in DCM for 2 h
9-(4-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-
4-carboxybutanamido)phenyl)-1-(4-((E)-(4-(dimethylamino)
phenyl)diazenyl)phenyl)-1,7,10-trioxo-8-oxa-2,6,11-triazatri-
decan-13-oic Acid (17b). Obtained similarly from compound 16b
(prepared from Fmoc-Glu(tBu)ꢀOH as shown in Scheme S2). Yield:
61.8%. 1H NMR (500 MHz, CDCl3) δ 7.77 (s, 2H), 7.71 (s, 2H), 7.62
(s, 4H), 7.41 (s, 4H), 7.38ꢀ7.37 (d, J = 4.9 Hz, 2H), 7.26 (s, 4H), 7.15
12017
dx.doi.org/10.1021/ja200808y |J. Am. Chem. Soc. 2011, 133, 12009–12020