B. Kçnig et al.
dissolved in a small amount of CHCl3, was slowly added. The reaction
mixture was allowed to warm to RT and was stirred overnight (20 h) at
408C. The reaction progress was monitored by TLC (ethyl acetate).
After completion, the solution was diluted with ethyl acetate and the pre-
cipitated dicyclohexyl urea was filtered off over celite. The solvent was
removed in vacuo, and the crude product was purified by column chro-
matography on silica gel (CHCl3/MeOH 4:1, Rf =0.75, ethyl acetate),
yielding the tris tert-butyl ester of 4 (198 mg, 60%) as a light yellow oil.
1H NMR (600 MHz, CDCl3): d=1.40 (s, 18H; HSQC: C1H3), 1.41 (s, 9H;
HSQC: C25H3), 1.79–1.88 (m, 1H; COSY: C6H2), 1.98–2.07 (m, 1H;
COSY: C6H2), 2.33–2.45 (m, 2H; COSY: C7H2), 2.63 (brs, 2H; COSY:
C11H2), 2.77 (brs, 4H; C12H2), 3.22–3.32 (m, 3H; COSY: C5H, C10H2),
3.36 (d, 2J=17.2Hz, 1H; C 4H2), 3.44 (d, 2J=17.2Hz, 1H; C 4H2), 3.57
(brs, 4H; HMBC: C13H2), 3.61–3.65 (m, 4H; HMBC: C14H2), 3.72–3.76
(m, 4H; HMBC: C15H2), 3.85 (s, 6H; COSY: C22H3), 3.88–3.91 (m, 4H;
HMBC: C16H2), 4.15–4.23 (m, 4H; HMBC: C17H2), 6.88 (brs, 1H;
HSQC: N9H), 7.18 ppm (s, 2H; HSQC: C19H); 13C NMR (150 MHz,
CDCl3): d=28.1 (+, 6C; HSQC: C1), 28.2 (+, 3C; HSQC: C25), 26.3 (À,
1C; HSQC: C6), 32.5 (À, 1C; HSQC: C7), 37.4 (À, 1C; HSQC: C10), 52.5
(+, 2C; HSQC: C22), 54.1 (À, 2C; HMBC: C4), 54.2( À, 3C; COSY: C11,
HSQC: C12), 64.8 (+, 1C; HSQC: C5), 68.8 (À, 2C; COSY: C13), 69.5 (À,
2C; HMBC: C16), 69.7 (À, 2C; HMBC: C17), 70.6 (À, 2C; HMBC: C14),
71.1 (À, 2C; HMBC: C15), 80.7 (Cquat, 2C; HMBC: C2), 81.2(C quat, 1C;
HMBC: C24), 113.7 (+, 2C; HSQC: C19), 125.4 (Cquat, 2C; HMBC: C20),
emission signal; the ammonium moiety of a lysine side
chain does not. Cysteine-containing peptides also trigger
emission signals of 5, but these are instead due to altered
electronic properties of the copper ion cancelling its quench-
ing of the benzocrown emission.
The selectivity and affinity of 5 in its current state may
not be sufficient for direct practical use in peptide sensing,
but these investigations have clearly shown that the interac-
tion of a hybrid compound such as 5 with peptides can be
explained on the basis of established coordination motifs of
copper(II) ions to peptides. This offers the potential for a
more rational design of sequence-selective peptide chemo-
sensors tapping the extensively available knowledge of pep-
tide to metal ion coordination reported over the last 50
years.
Experimental Section
General: Details of the preparation of model peptides and the spectro-
scopic determination of binding constants are given in the Supporting In-
formation.
150.5 (Cquat, 2C; HMBC: C18), 167.7 (Cquat, 2C; HMBC: C21), 170.7 (Cquat
,
2C; HMBC: C3), 171.8 (Cquat, 1C; HMBC: C8), 172.9 ppm (Cquat, 1C;
HMBC: C23); see Supporting Information for compound numbering; IR
(KBr): n˜ =3056, 2976, 2931, 2823, 1724, 1658, 1600, 1520, 1363, 1138,
736 cmÀ1; UV (MeOH): lmax (log e)=202 (3381), 224 (3933), 267 nm
(3423 molÀ1 dm3 cmÀ1); MS (ESI(+), CH2Cl2/MeOH+10 mmol NH4Ac):
m/z (%): 928.5 (100) [M+H]+; HRMS: m/z: calcd for C45H74N3O17:
928.5018; found: 928.5034Æ0.0012; elemental analysis calcd (%) for
C45H73N3O17: C 58.24, H 7.93, N 4.53; found C 58.42, H 8.16, N 4.97.
2-(Bis-tert-butoxycarbonylmethylamino)-pentanedioic acid 1-tert-butyl
ester (2):[21] H-l-Glu
(OBzl)-OtBu·HCl (312mg, 0.95 mmol) was dissolved
R
under nitrogen in DMF (10 mL). DIPEA (0.8 mL, 4.73 mmol) and tert-
butyl bromoacetate (0.6 mL, 3.78 mmol) were added to the stirred solu-
tion. The reaction mixture was heated at 808C overnight with continuous
stirring and monitored by TLC (petrol ether/ethyl acetate 3:1). The solu-
tion was concentrated under reduced pressure, and the residue was taken
up in a small amount of ethyl acetate, filtered and washed several times
with petrol ether/ethyl acetate 3:1 and ethyl acetate until the filter resi-
due was colourless. The filtrate was evaporated, and the crude product
was purified by column chromatography on silica gel (petrol ether/ethyl
acetate 3:1, Rf =0.60), yielding the title compound (418 mg, 84%) as a
light yellow oil. 1H NMR (300 MHz, CDCl3): d=1.42(s, 18H; N-
CH2COOtBu), 1.45 (s, 9H; CaHCOOtBu), 1.82–2.07 (m, 2H; Glu-CH2),
The tris tert-butyl ester of 4 (108 mg, 0.12mmol) was suspended in tri-
fluoroacetic acid (TFA, 4 mL). The reaction mixture was stirred at RT
for 20 h and the reaction progress was monitored by TLC (ethyl acetate).
TFA was evaporated, and the obtained triflate salt was redissolved in
water and lyophilized, yielding 4 (118 mg, 0.12mmol, 100%) as a white
hygroscopic solid. M.p. 60–628C; 1H NMR (300 MHz, D2O): d=1.66–
1.84 (m, 2H; Glu-CH2), 1.98–2.20 (m, 2H; Glu-CH2), 3.13–3.25 (m, 2H;
CH2), 3.27–3.45 (m, 6H; CH2), 3.51–3.94 (m, 28H; CH2, OMe, N-CH2, a-
CH, NH), 3.98–4.08 (m, 2H; CH2), 4.0–4.25 (m, 2H; CH2), 7.05–
7.12ppm (s, 2H; CH); 13C NMR (75 MHz, D2O): d=22.5 (À, 1C; CH2),
31.6 (À, 1C; CH2), 33.9 (À, 1C; CH2), 52.2 (À, 1C; CH2), 53.3 (+, 2 C;
3
2.50–2.77 (m, 2H; Glu-CH2), 3.38 (dd, J=5.5 Hz, 9.8 Hz, 1H; Glu-CaH),
3.43 (s, 4H; N-CH2), 5.08–5.12(s, 2H; Ph-CH 2), 7.26–7.38 ppm (m, 5H;
Ar-H); 13C NMR (75 MHz, CDCl3): d=25.4 (À, 1C; CH2), 28.1 (+, 9C;
CH3), 30.5 (À, 1C; CH2), 53.8 (À, 2C; N-CH2), 64.3 (+, 1C; CH), 66.1
(À, 1C; Ph-CH2), 80.7 (Cquat, 2C; COOtBu), 81.3 (Cquat, 1C; COOtBu),
128.1 (+, 1C; Ar-CH), 128.2 (+, 2C; Ar-CH), 128.5 ( +, 2C; Ar-CH),
CH3), 53.6 (À, 2 C; CH), 54.1 (À, 2C; N-CH2), 63.6 (À, 2 C; CH), 65.7
2
2
(+, 1C; CH), 68.1 (À, 2 C; CH), 69.1 (À, 2 C; CH), 69.8 (À, 2 C; CH),
2
2
2
69.9 (À, 2 C; CH), 112.6 (+, 2C; CH), 116.3 (Cquat, q, 1JC,F =292.1 Hz;
136.2(C quat, 1C; Ar-C), 170.5 (Cquat, 2C; NCH2COOtBu), 171.8 (Cquat
,
2
CF3COOÀ), 124.5 (Cquat, 1C; Ar-C), 124.7 (Cquat, 1C; Ar-C), 149.58 (Cquat
,
1C; COOtBu), 173.5 ppm (Cquat, 1C; COOBn); MS (ESI(+), CH2Cl2/
MeOH+10 mmol NH4Ac): m/z (%): 522.4 (100) [M+H]+, 466.3 (3)
[M+HÀC4H6]+.
1C; Ar-C), 149.62(C
,
1C; Ar-C), 162.7 (Cquat
CF3COOÀ), 169.2(C quat, 1C; COOMe), 169.3 (Cquat, 1C; COOMe), 170.7
(Cquat, 2C; NCH2COOH), 171.2(C quat, 1C; CONH), 174.9 ppm (Cquat
,
q, 2JC,F =35.5 Hz,
quat
,
The NTA bis-ester (624 mg, 1.2 mmol) was dissolved in EtOH (20 mL),
to which a spatula tip of Pd/C (10%) was added. The reaction mixture
was stirred in an autoclave under 20 bar H2 pressure for 18 h. The sus-
pension was filtered twice and the filtrate was concentrated under re-
duced pressure, yielding compound 2 (513 mg, 99%) as a colourless oil.
1H NMR (300 MHz, CDCl3): d=1.43 (s, 18H; N-CH2COOtBu), 1.45 (s,
9H; CaHCOOtBu), 1.78–2.07 (m, 2H; Glu-CH2), 2.47–2.78 (m, 2H; Glu-
CH2), 3.36 (dd, 3J=5.5 Hz, 10.1 Hz, 1H; Glu-CaH), 3.44 ppm (s, 4H; N-
CH2); 13C NMR (75 MHz, CDCl3): d=25.4 (À, 1C; CH2), 28.1 (+, 9C;
CH3), 31.1 (À, 1C; CH2), 54.0 (À, 2C; N-CH2), 64.7 (+, 1C; a-CH), 81.3
1C; CHCOOH); IR (KBr): n˜ =2957, 2914, 2360, 2341, 1732, 1642, 1528,
1428, 1351, 1200, 814, 719 cmÀ1; UV (MeOH): lmax (log e)=202 (3860),
224 (4070), 267 nm (3587 molÀ1 dm3 cmÀ1); MS (ESI(+), CH2Cl2/
MeOH+10 mmol NH4Ac): m/z (%): 796.4 (100) [MÀ2H++K+]À, 780.5
(10) [MÀ2H++Na+]À, 758.5 (3) [MÀH+].
Glutamic acid copper NTA crown ether (Cu-NTA CE, 5): Compound 4
(164 mg, 0.17 mmol), NaHCO3 (28 mg, 0.3 mmol) and Cu2(OH)2CO3
(18.4 mg, 0.08 mmol) were dissolved in water (5 mL). The mixture was
stirred at room temperature overnight, and was subsequently heated at
reflux for 3 h and filtered immediately. The resulting greenish-blue solu-
tion was concentrated under reduced pressure and lyophilized, yielding 5
(185 mg, 98%) as a turquoise solid. M.p >1758C (decomp); IR (KBr):
n˜ =2929, 2357, 2341, 1682, 1629, 1523, 1437, 1351, 1295, 1204, 1130, 801,
721 cmÀ1; MS (ESI(+), H2O/MeCN): m/z (%): 819.4 (100) [M]À, 731.4
[MÀ2CO2]À, 775.3 [MÀCO2]À, 901.4 [M+2MeCN]À. The purity of the
compound was determined by HPLC, diode array detection at 226 nm,
and found to be >97%.
(Cquat, 2C; COOtBu), 81.7 (Cquat, 1C; COOtBu), 170.6 (Cquat, 2 C;
NCH2COOtBu), 171.3 (Cquat, 1C; COOtBu), 176.2(C quat, 1C; COOH);
MS (ESI(+), CH2Cl2/MeOH+10 mmol NH4Ac): m/z (%): 432.3 (100)
[M+H]+, 454.3 (3) [M+Na]+.
Compound 4: Compound 2 (150 mg, 0.35 mmol) was dissolved under N2
in a small amount of CHCl3 and mixed with ice cooling with DIPEA
(0.3 mL, 1.74 mmol), DCC (86 mg, 0.42mmol) and HOBt (56 mg,
0.42mmol). Then, crown ether dihydrochloride 3 (245 mg, 0.42 mmol),
2540
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2536 – 2541