dichloromethane (15 mL) was added dropwise to the reaction
mixture. After being kept at Ϫ2 ЊC for 4 h the temperature was
allowed to rise to ambient and stirring was continued for 2 days.
The reaction mixture was then recooled to Ϫ5 ЊC and a second
batch of DMAP (5.0 mmol) and EDC (5.0 mmol) in dry DMF
(8 mL)–dry dichloromethane (20 mL) was added. After 3 h, the
reaction mixture was allowed to warm to room temperature
and stirring was continued for 4 days. After removal of solvent
the yellow–brown residue was dissolved in a mixture of ethyl
acetate (100 mL) and distilled water (150 mL). After separation
of layers, the aqueous phase was washed a second time with
ethyl acetate (100 mL). The combined organic layers were
washed successively with 10% aq. citric acid (50 mL), 10% aq.
NaHCO3, and distilled water (2 × 50 mL). After drying over
anhydrous magnesium sulfate, filtration, and removal of solv-
ent, a yellowish solid was usually obtained. The protected cyclic
pentapeptides synthesised by this method were as follows:
Cyclo[-Arg(Mtr)-Gly--Gla(OBut)2-Ser(But)-Lys(Boc)-] as
a yellow solid (82%), tR 14.51 min (78% by integration), on
HPLC (isocratic 50% aq. MeCN) (tR = 9.6 min, linear gradient
5–100% MeCN over a period of 15 min). FAB-MS m/z 1104
(M ϩ Na)ϩ, 1082 (M ϩ H)ϩ. Accurate mass Found:
(M ϩ Na)ϩ, 1104.56180. C50H83N9O15SؒNaϩ requires m/z,
1104.562705.
min for the -Gla analogue. Evaporation of the eluent solvent
initially gave oils, but, on trituration with acetonitrile, white
solids were obtained, with the following physical data.
Cyclo[-Arg(Mtr)-Gly--Gla(OBut)2-Ser(But)-Lys(Boc)-], ES-
MS m/z 1082 (M ϩ H)ϩ, 1105 (M ϩ Na)ϩ; δH (400 MHz;
CDCl3) 1.3 (9 H, s, But), 1.42 (9 H, s, Boc), 1.45–1.46 (4 H,
overlapping, Arg and Lys CH2), 1.47 (18 H, s, Gla But), 1.5–1.6
(4 H, overlapping, Arg and Lys CH2), 1.83 (2 H, m, Lys CH2),
2.13 (3 H, s, Mtr CH3), 2.2–2.4 (2 H, m, Gla CH2), 2.64 (3 H, s,
Mtr CH3), 2.7 (3 H, s, Mtr C3), 3.1 (2 H, br s, Lys εCH2), 3.22–
3.33 (2 H, m, Arg δCH2), 3.5 (2 H, br m, Ser CH2), 3.7 (2 H,
overlapping peaks, Gly αCH and Gla γCH), 3.83 (3 H, s, Mtr
OCH3), 3.9 (1 H, br s, Ser αCH), 4.0 (1 H, s, Gly αCH), 4.4 (1 H,
s, Gla αCH), 4.5 (2 H, br s, Arg and Lys αCH), 5.05 (1 H, s, Lys
εNH), 6.32 (3 H, br s, 3 × Arg NH), 6.5 (1 H, s, Mtr CH), 7.16
(1 H, s, Ser NH), 7.51–7.54 (2 H, d, Arg and Lys NH), 7.96 (1 H,
s, Gla NH), 8.51 (1 H, br s, Gly NH).
Cyclo[-Arg(Mtr)-Gly--Gla(OBut)2-Ser(But)-Lys(Boc)-],
ES-MS m/z 1082 (M ϩ H)ϩ, 1105 (M ϩ Na)ϩ; δH (400 MHz;
CDCl3) 1.23 (9 H, s, But), 1.42 (9 H, s, Boc), 1.45–1.46 (4 H,
overlapping, Arg and Lys CH2), 1.47 (18 H, s, Gla But), 1.53–
1.63 (4 H, overlapping, Arg and Lys CH2), 1.83 (2 H, d, Lys
CH2), 2.13 (3 H, s, Mtr CH3), 2.2–2.4 (2 H, m, Gla CH2), 2.64 (3
H, s, Mtr CH3), 2.7 (3 H, s, Mtr CH3), 3.1 (2 H, br s, Lys εCH2),
3.22–3.33 (2 H, m, Arg δCH2), 3.5 (2 H, br m, Ser CH2), 3.7
(2 H, overlapping peaks, Gly αCH and Gla γCH), 3.83 (3 H, s,
Mtr OCH3), 3.9 (1 H, br s, Ser αCH), 4.0 (1 H, s, Gly αCH),
4.35 (1 H, s, Gla αCH), 4.5 (2 H, br s, Arg and Lys αCH), 5.05
(1 H, s, Lys εNH), 6.32 (3 H, br s, 3 × Arg NH), 6.5 (1 H, s, Mtr
CH), 7.16 (1 H, s, Ser NH), 7.51–7.54 (2 H, 2s, Arg and Lys
NH), 7.96 (1 H, s, Gla NH), 8.51 (1 H, br s, Gly NH).
Cyclo[-Arg(Mtr)-Gly--Gla(OBn,NC5H10)-Ser(But)-Lys-
(Boc)-] as a yellow solid (94% crude yield), tR split peak 17.4–
17.7 min (73% by integration) on HPLC (gradient 5–70%
MeCN over a period of 15 min). Electrospray MS m/z 1127.
C54H82N10O14S requires M, 1127.4; δH (400 MHz; d6-DMSO)
1.09 and 1.35 (18 H, 2s, Boc and But), 1.2–1.7 (16 H, br m,
piperidide CH2, Lys βγδCH2, Arg βγCH2) 2.04, 2.49, 2.58 (9 H,
m, 3 × Mtr CH3), 2.50 (2 H, m, Gla βCH2), 2.8 (2 H, m, Lys
εCH2), 3.0 [6 H overlapping signals Arg δCH2, 2 × CH2
(NC5H10)], 3.43 (2 H, m, Ser CH2), 3.46 (1 H, m, Gly αCH),
3.78 (3 H, s, Mtr OCH3), 4.2–4.4 (3 H, overlapping, Arg αCH,
Ser αCH, Gla αCH), 5.1–5.2 (2 H, m, CH2Ph), 6.4 (1 H, br s,
Arg εNH), 6.7 (3 H, m/s, Lys ε NH, Mtr CH), 7.2–7.4 (5 H, m,
ArH), 7.8–8.4 (5 H, 5m, amide NH). Exact positions of a Gly
αCH and Gla γCH could not be correlated.
Removal of side-chain-protecting groups. The protected
cyclopentapeptides (0.2 mmol) were dissolved, either in 99%
TFA or 95% TFA (10 mL), for periods of between 2 and 12 h at
room temperature, until all protecting groups (especially the
Mtr group which was the most difficult to remove) had been
removed as monitored by HPLC. Removal of TFA on a rotary
evaporator usually gave a brown gum, which was then washed
several times with diethyl ether to give quantitative yields of the
crude cyclopentapeptides as solid, off-white residues. Each of
the residues had to be purified by collecting fractions off the
HPLC columns at tR-values reported for each compound.
Cyclo[-Arg-Gly--Gla-Ser-Lys-] as an off-white solid (36%),
tR 3.61 min on semi-preparative HPLC (linear gradient 5–70%
aq. MeCN during 15 min); FAB-MS m/z 602 (M ϩ H)ϩ.
C23H39N9O10 requires M, 601.28; δH (400 MHz; D2O) 1.48
(2 H, Lys CH2), 1.64–1.66 (4 H, m, Arg CH2), 1.72–1.89 (4 H,
m, Lys CH2), 2.5 (2 H, m, Gla CH2), 3.02 (2 H, m, Lys εCH2),
3.09 (1 H, m Lys αCH), 3.24 (2 H, m, Arg δCH2), 3.94 (2 H, br
m, Ser CH2), 3.7–4.3 (2 H, overlapping peaks, Gly αCH2),
4.07 (1 H, br s, Ser αCH), 4.12 (1 H, m, Arg αCH), 4.40 (2 H,
m, Gla α- and γ-CH) (NH signals very weak as sample was
in D2O).
Cyclo[-Arg(Mtr)-Gly-Asp(OBut)-Phe-Phe-] as a yellow solid
(68% yield), tR 5.65 min (59% by integration) on HPLC
(gradient 50–100% MeCN over a period of 15 min), further
purified (55% yield) to 98% purity by repeated collection
of fractions at tR 5.63 min; FAB-MS Found: (M ϩ H)ϩ,
891.404913. C44H59N8O10S requires m/z 891.407488; δH (400
MHz; d6-DMSO) 1.39 (9 H, s, But), 1.35–1.52 (4 H, m, 2 × Arg
CH2), 2.0, 2.55, 2.6 (9 H, 3s, 3 × Mtr CH3), 2.4–2.6 (2 H, m,
Asp CH2), 3.15 (3 H, m, Arg δCH2), 3.8 (3H, s, Mtr OCH3),
3.15, 3.34 (2 H, m, PhCH2), 3.45, 3.9 (2 H, m, 2 × Gly αH),
3.88–3.92 (2 H, m, PhCH2), 4.05 (1 H, m, Phe αCH), 4.1 (1 H,
m, Arg αCH), 4.25 (1 H, m, Phe αCH), 4.5 (1 H, m, Asp αCH),
6.68 (1 H, s, Mtr CH), 7.1–7.4 (10 H, m, ArH), 7.9 (1 H, m, Phe
NH), 8.26 (1 H, m, Arg NH), 8.32 (1 H, m, Asp NH), 8.4 (1 H,
m, Phe NH), 8.7 (1H, m, Gly NH).
Method B.24 The linear pentapeptide precursor (0.1 mmol),
HATU (0.11 mmol) and DIPEA (0.3 mmol) in DMF (100 mL)
– dichloromethane (300 mL) (to bring overall concentration to
2.5 µM) were stirred at 0 ЊC for 1 h and then at room temper-
ature for 1 h. The solvent was removed in vacuo, and the
residual yellow product was re-dissolved in dichloromethane
(100 mL), which solution was washed successively with 10% aq.
citric acid (50 mL), aq. sodium bicarbonate (1 M; 50 mL) and
water (2 × 50 mL). After drying, the organic layer on evapor-
ation yielded a yellowish product in approx. 75% yield.
Purification of both protected cyclic peptides produced by
this method was carried out by preparative HPLC using a
reversed-phase column (PL-32 × 2.75 cm), flow rate 10 mL
minϪ1, with gradient elution 31–70% aq. MeCN during 90 min.
Pure cyclic pentapeptides were collected from fractions corre-
sponding to peaks at tR 76.5 min for the all--form and tR 81
Cyclo[-Arg-Gly--Gla(OBn,NC5H10)-Ser-Lys-] as a white
solid, (30%), tR 4.1 min on semi-preparative HPLC (non-linear,
exponent 2 gradient 5–40% aq. MeCN during 15 min). Electro-
spray MS m/z 759 (M ϩ H)ϩ, 380 (M ϩ 2H)2ϩ. C35H54N10O9
requires M, 758.4; δH (400 MHz; d6-DMSO) 1.10–1.70 (16 H,
m, piperidide CH2, Lys βγδCH2, Arg βγCH2), 2.2 (1 H, m, Gla
βCH), 2.8 (4 H, m, 2 × CH2 from NC5H10), 3.0 (4 H, over-
lapping signals, Arg δCH2, Lys εCH2) 3.5 (2 H, m, Ser CH2), 3.9
(2 H, m, Gly αCH, 1 × unknown H), 4.2–4.4 (3 H, overlapping,
Arg αCH, Ser αCH, Gla αCH), 5.1–5.2 (2 H, m, CH2Ph), 7.2–
7.4 (5 H, m, ArH), 7.8–8.4 (5 H, br signals, amide NH). Some
peaks were obscured by DMSO/H2O signals).
Cyclo[-Arg-Gly-Asp-Phe-Phe-] as a white solid (97%), tR 9.7
min on semi-preparative HPLC (linear gradient 5–50% aq.
MeCN over 15 min); FAB-accurate mass MS Found: (M ϩ
H)ϩ, 623.294200. C30H39N8O7 requires m/z, 623.294171. For
2914
J. Chem. Soc., Perkin Trans. 1, 2000, 2907–2915