Chemical Research in Toxicology
Article
concentrated under vacuum, and redissolved in EA for a basic wash
layer was dried with Na SO , filtered, and concentrated for column
2
4
(
0
5×, saturated NaHCO /brine), followed by an acidic wash (2×, brine
chromatography. (The column was packed with 1% HCO H/EA and
3
2
.1 M HCl, pH 1). The organic layer was concentrated to a sticky
then eluted with EA to 10% MeOH/DCM.) Depending on the purity
of the product, an optional second column chromatography was
performed (CHCl to 5% MeOH/CHCl ). Fractions of pure product
white foam (5.4 g, 37% yield) and redissolved in minimal DCM for
precipitation into vigorously stirring hexane. The supernatant was
3
3
removed following centrifugation and the residue lyophilized to a
were identified by ESI, pooled, and concentrated to a white foam (1 g,
55% yield) that was redissolved in DCM and precipitated by dripping
the solution into hexanes. The solution was centrifuged, the solvent
decanted, and the residue lyophilized over 1 week into a white powder.
Some Fmoc cleavage was detected in the powder and confirmed by
1
white powder. H NMR (CDCl ): δ 1.10−1.48 (m, 9H), 2.60−3.30
3
(
br, m, 9H), 4.13 (br, m, 4H), 4.38 (br, m, 5H), 4.56 (br, s, 4H),
13
7
2
1
1
.24−7.74 (m, 32H). C NMR (CDCl ): δ 11.4, 14.2, 18.8, 20.7,
3
2.6, 25.3, 29.1, 31.6, 34.5, 34.7, 36.1, 47.1, 47.3, 47.36, 47.44, 77.2,
19.9, 120.0, 124.5, 124.6, 124.7, 124.9, 125.1, 127.0, 127.1, 127.7,
27.8, 141.31, 141.34, 141.37, 141.39, 143.8, 143.9, 156.5, 156.6. IR
+
+
ESI/MS (1363.8 m/z = [M-Fmoc + Na] 1363.6). HRMS calcd for
+
C H N O NaS (M + Na ) 1563.7309; found, 1563.7302;
9
1
103
8
14
−1
(film): 3292 (br), 2927, 1703, 1644, 1553 cm . HRMS calcd for
confirmed the desired product.
+
C H N O (M + H ) 1135.4852; found, 1135.4887.
Fmoc-cleavage of the protected form of 2 (200 mg, 128 μmol) was
carried out in 50% cyclohexylamine/DCM (15 mL). After 15 min, the
reaction was diluted with DCM (20 mL) and water (100 mL). After
increasing the pH of the aqueous layer to 12 with NaOH (4 M), it was
washed with DCM (5 × 100 mL). The aqueous layer was filtered
through a 0.2 μm syringe filter, concentrated under vacuum, and
azeotropically dried with toluene (3 × 50−100 mL), to afford 2 as a
71
67
4
10
Synthesis of 5. A THF solution of DMTrCl (1.4 g, 4 mmol, 0.2
M) was added dropwise to a vigorously stirred solution of diamine 4
48 mmol, 10.6 mL) in THF (20 mL). Additional DMTrCl/THF
(
solution (6.7 g, 20 mmol, 55 mL THF) was added in 5 mL portions
for a total of 30 mL. Tritylation was very rapid. The appropriate
amount of DMTrCl added was followed by TLC (30% EA/HEX) to
maximize monotritylation and minimize ditritylation. Volatiles were
removed in vacuo, and the residue diluted with DCM (200 mL),
washed with brine (3 × 200 mL), dried with Na SO , filtered, and
pale yellow, amorphous solid that was difficult to completely dry (264
1
mg, > 100% recovery). H NMR (D O): δ 1.33 (br, m, 6H), 1.45−
2
1.56 (m, 13H), 2.17−2.19 (t, J = 4 Hz, 2H), 2.37−2.54 (m, 10H),
2.68−2.71 (d, J = 12 Hz, 1H), 2.89−2.93 (dd, J = 4, 8 Hz, 1H), 3.03−
3.07 (t, J = 6 Hz, 1H), 3.16−3.19 (t, J = 6 Hz, 4H), 3.20−3.26 (m,
1H), 3.49 (t, J = 6 Hz, 4H), 3.58−3.61 (m, 8H), 4.32−4.35 (dd, J = 6,
2
4
concentrated for column chromatography (isocratic elution: 5% TEA/
1
DCM), affording 7.7 g (91% yield) of 5 as a clear yellow oil. H NMR
(
=
CDCl ): δ 1.70−1.80 (m, 7H), 2.2 (t, J = 8 Hz, 2H), 2.76−2.79 (t, J
3
1
3
8 Hz, 2H), 3.49−3.59 (m, 12H), 3.75 s, 6H), 6.76−6.79 (m, 4H),
2 Hz, 1H), 4.51−4.54 (dd, J = 4, 2 Hz, 1H). C NMR (D O): δ 22.4,
2
7
2
7
1
5
.13 (m, 1H), 7.21−7.26 (m, 2H), 7.33−7.35 (m, 4H), 7.42−7.45 (m,
22.6, 25.2, 25.4, 25.7, 25.8, 27.8, 29.1, 29.2, 33.0, 33.6, 36.1, 36.1, 37.2,
42.0, 43.5, 45.68, 52.9, 57.67, 59.2, 59.5, 65.8, 65.9, 66.8, 66.9, 67.01,
67.02, 162.7, 165.9, 173.8, 173.9. HRMS calcd for C H N O NaS
H). 13C NMR (CDCl ): δ 30.8, 33.3, 39.8, 41.3, 55.4, 69.7, 70.1, 70.3,
3
0.37, 70.44, 70.8, 70.9, 113.2, 126.2, 127.9, 128.7, 129.9, 139.0, 147.1,
3
1
62
8
6
+
+
57.9. HRMS calcd for C H N O Na (M + Na ) 545.2986; found,
(M + Na ) 697.4405; found, 697.4429.
31
42
2
5
45.3007.
Synthesis of 6. A mixture of biotin (770 mg, 3.2 mmol), DCC
710 mg, 3.4 mmol), and HOBt (465 mg, 3.4 mmol) was dried in
Solid Phase Synthesis of 9 and 11−13. For the preparation of
11−13, the arginine(s) and lysine residues were added via automated
SPPS from 10 (∼150 mg, 0.45 mmol/g, ∼70 μmol-scale). Fmoc
cleavage was performed with 20% piperidine/NMP. All residues were
coupled (2 × 30 min) with amino acid (∼0.2 M, 5 equiv), HBTU
(∼0.2 M, 5 equiv), and DIPEA (∼0.4 M, 10 equiv) in NMP. The final
lysine residue was coupled in its Boc protected form. Capping was
performed with acetic anhydride (100%). Resin was washed with
DCM, followed by NMP between each step. The remaining portions
of the syntheses of 11−13 and all of 9 were carried out manually as
follows. The resin was swollen in DCM with Ar bubbling for 5−10
min. The liquid was drained by aspiration and the resin washed with
DMF. Alloc group cleavage was performed with Me NH·BH (6 eq 24
(
vacuo for 30 min prior to suspension in 5% TEA/DMF (10 mL) under
Ar at 40 °C. After 30 min, the suspension was added to 5 (1.53 g, 2.94
mmol) (predried by azeotropically drying from pyridine) in 5% TEA/
DMF (10 mL). After 6−8 h, the precipitate was removed by passage
through Celite. DMF was evaporated in vacuo and the residue
partitioned between brine and DCM. The precipitate was again
removed by passage through Celite, and the filtrate was concentrated
for column chromatography (5% TEA/DCM to 5% TEA/5% MeOH/
1
DCM), affording 2.2 g (100%) of 6. H NMR (CDCl ): δ 1.20−1.23
3
(
m, 2H), 1.42−1.76 (m, 8H), 2.16−2.19 (m, 4H), 2.80−2.84 (m, 3H),
2
3
3
.10−3.15 (m, 1H), 3.32−3.34 (m, 1H), 3.52−3.61 (m, 12H), 3.77 (s,
H), 4.26−4.29 (dd, 1H), 4.43−4.50 (dd, 1H), 5.02 (br, s, 1H), 5.72
mg, 0.4 mmol) and (Ph P) Pd(0) (0.05 equiv, 4 mg, 3.4 μmol, 15 min,
3 4
6
(
3×), where the former was added first to a DMF suspension of resin
with Ar bubbling. Following washing as described below, Fmoc-
protected aminocaproic acid (3 equiv) was then preactivated with
PyBop (3 equiv) and TEA (6 equiv) in DMF (≥0.1 M) for 1 h with Ar
bubbling. A second coupling was repeated with 1.5× of reagents. The
Fmoc group was removed by treating the preswollen resin with 20%
piperidine/DMF (5 min, 3×). Biotin was then coupled in a similar
manner. Unreacted amine was acetylated by treatment with 50%
13
br, s, 1 H), 6.49 (bd s, 1H), 6.77 (d, 4H), 7.15−7.44 (m, 9H). C
NMR (CDCl ): δ 24.9, 25.58, 25.63, 28.1, 28.2, 28.8, 33.2, 33.9, 35.9,
3
3
7
7
7.6, 39.6, 40.5, 55.6, 60.1, 61.8, 69.5, 69.9, 70.0, 70.1, 70.5, 76.7, 77.0,
+
7.2, 77.3, 163.8, 173.1. HRMS calcd for C H N O S (M + H )
41
57
4
7
49.3942; found, 749.3968.
Synthesis of 7. A methanolic solution (10 mL) of 6 (1.3 g, 1.8
mmol) was detritylated within minutes following the addition of 1 M
HCl/MeOH (20 mL). The reaction was diluted with water (100 mL),
washed with DCM (3 × 100 mL), and the pH adjusted to 4 with 4 M
NaOH. DCM washes were repeated, the pH adjusted to 12, and the
solvent evaporated to dryness. The product was extracted from the
residue triturating with DCM (5×), followed by filtration over a glass
Ac O/DCM 3 times for 3, 3, and 7 min, with DCM washing in
2
between each treatment. After each coupling, the resin was washed
sequentially with DMF, DCM, MeOH, dry DCM, and dry DMF (2×).
The lysine residue in 9 was coupled as described above for
aminocaproic acid prior to the removal of the alloc group. The
amino group was quantified indirectly by the released fulvene
frit. The filtrate was concentrated to provide 7 as a pale yellow
1
3
−1
−1
amorphous solid (480 mg, 60% yield). H NMR (CDCl ): δ 1.42−
chromophore (ε300 7.8 × 10 M ·cm ).
3
1
2
1
1
.46 (m, 3H), 1.63−1.78 (m, 13H), 2.17−2.20 (t, J = 6 Hz, 2H),
.71−2.80 (m, 4H), 2.88−2.93 (dd, J = 6.7, 2 Hz, 1H), 3.14−3.15 (m,
H), 3.32−3.36 (q, J = 5.3 Hz, 3H), 3.53−3.65 (m, 14H), 4.31 (m,
Peptide cleavage/deprotection was performed with a cleavage
cocktail (88% TFA, 2% TIPS, 5% H O, and 5% phenol). HPLC
2
purification was performed on a C18 semipreparatory column (Waters
H), 4.48 (m, 1H), 5.21 (s, 1H), 6.09 (s, 1H), 6.79 (m, 1H). HRMS
300 × 7.8 mm I.D.) using H O (solvent A) and acetonitrile (solvent
2
+
calcd for C H N O S (M + H ) 447.2636; found, 447.2651.
B) with 0.1% TFA in an elution gradient optimized for each probe at 3
mL/min. Probe-containing fractions were lyophilized, redissolved in
water and titrated to pH 9 with 4 M NaOH, and analyzed by MS in
the positive mode. The following gradients (time, % B) were used. 9:
20
39
4
5
Synthesis of 2. A solution of 7 (482 mg, 1.1 mmol), EDCI (345
mg, 2.2 mmol), and HOBt (300 mg, 2.2 mmol) in DMF (5 mL) under
Ar was added dropwise to 3 (1.7 g, 1.5 mmol) in DMF (10 mL). After
+
16 h, the reaction was quenched with a solution of brine and 0.1 M
0, 0; 5, 0; 30, 20. Ret. time: 10 min. ESI-MS [M + H] : calcd, 501.2;
HCl (100 mL) and extracted with DCM (5 × 100 mL). The organic
obsd, 501.2. 11: 0, 0; 5, 0; 10, 20; 20, 20. Ret. time: 11.5 min. ESI-MS
1
229
dx.doi.org/10.1021/tx500120p | Chem. Res. Toxicol. 2014, 27, 1227−1235