2
1
(s), 88.8 (d, JC–P 51.7, PCCH), 73.9 (d, JC–P 294.7, PCCH),
1719 (CvO), 1508, 1451, 1247 (PvO), 998 (P–O); δH
(500 MHz, CDCl3): 8.09 (1H, s, Tz-H5), 7.70 (2H, d, J 7.38, 2 ×
Fmoc-H5), 7.51 (2H, m, 2 × Fmoc-H2), 7.39–7.17 (14H, m, 2 ×
Fmoc-H4 + 2 × Fmoc-H3 + 10 × Ph-H), 6.30 (0.7H, d, J 7.0,
NH), 5.20–5.00 (4H, m, 2 × PO(OCH2Ph)), 4.98–4.72 (3H, m, 2
× CHβ + CHα), 4.36 (1H, m, OCHH′C), 4.26 (1H, m, OCHH′C),
4.12 (1H, t, J 7.03, OCH2CH); δC (75MHz, CDCl3):172.0 (s,
COOH), 155.3 (s, NHCOO), 143.0 (d, J 4.3, Ph-C1), 140.4 (s,
Fmoc-C1), 135.8 (d, J 241.8, Tz-C4), 131.4 (d, J 33.9, Tz-C5),
127.4 (s), 127.6 (s), 127.2 (s), 127.0 (s) 126.4 (s), 124.4 (s),
119.1 (s), 67.5 (d, J 5.5, POCH2Ph), 66.1 (s, Fmoc-CH2), 53.5
(s, Cα), 49.9 (s, Cβ), 46.4 (s, Fmoc-CH); δP (121MHz, CDCl3):
8.03 (p, J 7.9); [α]2D2 +23.6 (c 1.3, CHCl3); HRMS Found MH+:
639.2012 C34H32N4O7P requires 639.2003; found MNa+
661.1844, C34H31N4NaO7P requires 661.1823.
2
68.8 (d, JC–P J 5.3, OCH2Ph); δP (121 MHz, CDCl3): −7.81
(dp, JP–H 8.6, JP–H 13.5); HRMS Found MH+: 287.0834,
3
3
C16H16O3P requires 287.0832.
(2S)-3-[4-(Di-tert-butyl-phosphoryl)-[1,2,3]triazol-1-yl]-2-
(9H-fluoren-9-ylmethoxycarbonyl)aminopropionic acid (14)
(Fmoc-pTz(OtBu)2-OH)
A freshly prepared solution of copper(II) sulphate (7 mg,
0.04 mmol) and sodium ascorbate (25 mg, 0.24 mmol) in H2O
(2 mL) was added to a stirred solution of di-tert-butyl ethynyl-
phosphonate 16 (90 mg, 0.41 mmol) in THF (2 mL). After
1 min, a solution of Fmoc-azidoalanine (160 mg, 0.45 mmol) in
1 : 1 THF : H2O (6 mL) was added in one portion and the
mixture stirred at rt for 3 h, at which time TLC showed complete
consumption of the azide. The reaction mixture was concentrated
in vacuo to a yield a pale green oil, column chromatography
eluting with DCM–MeOH–AcOH (94.5 : 5 : 0.5 then 94 : 5 : 1)
yielded the title compound 14 as an amorphous colourless solid
(204 mg, 70%). Rf (DCM–MeOH–AcOH 94 : 5 : 1) 0.06; νmax
(solid)/cm−1 3333 (NH), 3140 (COOH), 1726 (CvO), 1673,
1538, 1451, 1219 (PvO), 1054 (P–O); δH (500 MHz, CDCl3):
8.24 (0.73H, s, Tz-H5), 7.73 (2H, d, J 7.5, Fmoc-Ar-H), 7.56
(2H, t, J 7.9, Fmoc-Ar-H), 7.60–7.52 (2H, m, Fmoc-Ar-H),
7.32–7.26 (2H, m, Fmoc-Ar-H), 6.16 (0.8H, s(b), NH),
5.11–4.90 (2H, m, CHβ + CHβ′), 4.85 (1H, app s(b), CHα),
4.42–4.34 (1H, m, OCHH′C), 4.32–4.23 (1H, m, OCHH′C),
4.17 (1H, t, J 6.74, OCH2CH), 1.47 (18H, s, 2 × C(CH3)3)); δC
(75MHz, CDCl3): 171.2 (COOH), 155.9 (OCONH), 143.7 (s),
141.2 (s), 140.7 (d, J 251.1, Tz-C4), 131.2 (d, J 35.2, Tz-C5),
127.7 (s), 127.1 (s), 125.2 (s), 119.9 (s), 85.2 (d, J 8.5, C
(CH3)3), 67.4 (s, Fmoc-CH2), 54.1 (s, Cα), 51.0 (s, Cβ), 47.0 (s,
Fmoc-CH), 30.4 (d, J 3.6, C(CH3)3); δP (121MHz, CDCl3):
−2.09 (s); [α]2D2 +2.8 (c 0.7, MeOH); HRMS Found MH+:
571.2291, C28H36N4O7P requires 571.2316, found MNa+
593.2126, C28H35N4NaO7P requires 593.2136.
Peptides
Peptides were synthesised according to standard solid phase syn-
thesis protocols. Couplings of standard amino acids (Fmoc-Gly-
OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Thr(tBu)-OH, and Fmoc-Met-OH) were carried out using
5 equiv. (with respect to resin loading) Fmoc-amino acid, 4.9
equiv. HCTU and 10 equiv. DIPEA. Coupling of Fmoc-pTz
(OBn)2-OH was carried out with two sequential coupling steps
using 1 equiv. Fmoc-pTz(OBn)2-OH, 1 equiv. HATU and 2
equiv. DIPEA followed by 1.7 equiv. Fmoc-pTz(OBn)2-OH, 1.7
equiv. HATU and 3.4 equiv. DIPEA (peptide 33). Full details of
which can be found in the ESI† along with a detailed peptide
synthesis procedure, mass spec and HPLC data.
H-CGAGAG(pTz)GAGAG-OH (33)
The peptide was prepared as described in the ESI† and lyophi-
lised to yield a flocculent yellow solid (63 mg, 51% yield). δH
(300 MHz, D2O): 8.09 (1H, s, pTz-TzH5), 5.02–4.85 (2H, m,
pTz-CH2), 4.84–4.72 (1H, m, pTz-CH), 4.39–4.19 (5H, m, 4 ×
Ala-CHCH3 + Cys-CHCH2SH), 4.00–3.76 (12H, 6 × Gly-CH2),
3.02 (2H, d, J 5.6, Cys-CHCH2SH), 1.39–1.25 (12H, m, 4 ×
Ala-CHCH3); δP (121MHz, D2O): 0.81 (s); HRMS Found MH−:
964.3080, C32H51N15O16PS requires 964.3102
(2S)-3-[4-(Dibenzyl-phosphoryl)-[1,2,3]triazol-1-yl]-2-
(9H-fluoren-9-ylmethoxycarbonyl)aminopropionic acid (13)
(Fmoc-pTz(OBn)2-OH)
A freshly prepared solution of copper(II) sulphate (6 mg,
0.04 mmol) and sodium ascorbate (24 mg, 0.24 mmol) in H2O
(2 mL) was added to a stirred solution of dibenzyl ethynylphos-
phonate 15 (112 mg, 0.391 mmol) in THF (2 mL). After 1 min,
a solution of Fmoc-azidoalanine (150 mg, 0.4533 mmol) in 1 : 1
THF–H2O (6 mL) was added in one portion and the mixture
stirred at rt for 3 h, at which time TLC showed complete con-
sumption of the azide. The reaction mixture was diluted with
10% Na2CO3 aq (5 mL) and extracted with ether (2 × 10 mL).
The aqueous phase was acidified to pH 1 by dropwise addition
of conc. HCl(aq) and extracted with EtOAc (5 × 10 mL). The
EtOAc extracts were combined, dried (MgSO4) and concentrated
in vacuo to yield an off-white foam. Column chromatography
(DCM–MeOH–AcOH, 94 : 5 : 1) yielded the title compound 13
as a colourless oil (143 mg, 85%). Rf (DCM–MeOH–AcOH
94 : 5 : 1) = 0.08; νmax (film)/cm−1 3310 (NH), 3034 (COOH),
H-GMTS(pTz)AA-NH2 (34)
The peptide was prepared and purified as described in the ESI†:
34 was obtained as a colourless solid (4.7 mg, 19% yield after
purification), HRMS Found MH−: 752.2539, C25H43N11O12PS
requires 752.2556.
Acknowledgements
We acknowledge funding from BBSRC (BB/G004145/1) and
EPSRC. (EP/I013083/1) T. E. McAllister was supported by the
EPSRC through the White Rose Doctoral Training Centre (EP/
G500010/1). We would like to thank Christian Hedberg, Stuart
Warriner and Bruce Turnbull for productive discussions.
4048 | Org. Biomol. Chem., 2012, 10, 4043–4049
This journal is © The Royal Society of Chemistry 2012