6432
H. Herzner, H. Kunz / Tetrahedron 63 (2007) 6423–6436
NH4OAc)/40% CH3CN/60% H2O (0.1 N NH4OAc) in
50 min, tR¼31.8 min. 1H NMR (600 MHz, DMSO), 1H–1H
COSY, 1H–1H NOESY: d 0.77 (d, 3H, JH-da,H-g¼6.8 Hz, H-
da Leu); 0.83 (d, 3H, JH-db,H-g¼6.5 Hz, H-db Leu); 1.01 (d,
For detachment of the peptide, the resin was shaken with
a mixture of CH2Cl2 (8 mL), TFA (8 mL), and water
(0.2 mL) for 1 h at room temperature. After a few minutes
a ruby color occurred. After filtration, the resin was washed
with TFA/CH2Cl2 (1:9), the combined organic layers were
concentrated in vacuo and co-distilled several times with tol-
uene to dryness. The crude product was dissolved in 4 mL of
dichloromethane and precipitated by dropwise addition of
20 mL of dry diethyl ether. Purification of the precipitate
was achieved by preparative RP-HPLC (column: Eurospher
C8, gradient: 40% H3CCN/60% H2O/90% CH3CN/10%
H2O in 70 min, tR¼46.6 min). Yield: 207 mg (0.148 mmol,
45% related to the loading glutamic acid), colorless amor-
phous solid; Rf¼0.47 (CH2Cl2/MeOH/AcOH 8:1:0.1);
[a]2D2 ꢂ24.9 (c 1, CHCl3); analyt. HPLC: tR¼30.9 min (col-
umn: Eurospher C8, gradient: 1% CH3CN/99% H2O/
3H, JH-g,H-b¼6.2 Hz, H-g Thr); 1.16 (d, 3H, JH-b,H-a
¼
7.1 Hz, H-b Ala); 1.42 (t, 2H, JH-a,H-b¼JH-b,H-g¼7.2 Hz,
H-ba Leu, H-bb Leu); 1.58 (mc, 1H, H-g Leu); 1.77 (m,
2H, H-bb Glu1, H-bb Glu2); 1.81 (s, 3H, CH3CO–); 1.89
(m, 2H, H-ba Glu1, H-ba Glu2); 2.37 (m, 4H, H-g Glu1, H-
g Glu2); 2.67 (dd, 1H, JH-bb,H-ba¼14.1 Hz, JH-bb,H-a
¼
10.3 Hz, H-bb Phe2); 2.70 (dd, 1H, JH-bb,H-ba¼16.4 Hz,
JH-bb,H-a¼6.8 Hz, H-bb Asp); 2.76 (dd, 1H, JH-bb,H-ba
¼
13.2 Hz, JH-bb,H-a¼8.8 Hz, H-bb Phe1); 2.83 (dd, 1H,
JH-ba,H-bb¼16.1 Hz, JH-ba,H-a¼6.2 Hz, H-ba Asp); 2.95 (d,
1H, JH-ba,H-bb¼11.2 Hz, H-ba Phe2); 3.02 (d, 1H, JH-ba,H-bb
¼
10.9 Hz, H-ba Phe1); 3.60 (s, 2H, –CH2SO3H); 3.62 (s,
2H, –CH2SO3H); 3.92 (mc, 1H, H-b Thr); 4.25 (q, 1H,
JH-a,H-bazJH-a,H-bbzJH-a,NH¼7.1 Hz, H-a Glu1); 4.28–
4.35 (m, 4H, H-a Leu, H-a Ala, H-a Thr, H-a Glu2); 4.36
(d, 1H, Jgem¼11.7 Hz, –CH2Bn ether); 4.43 (d, 1H,
Jgem¼11.7 Hz, –CH2Bn ether); 4.47 (mc, 1H, H-a Phe2);
4.53 (m, 2H, H-a Asp, H-a Phe1); 4.98–5.11 (m, 6H,
–CH2Bn ester); 7.77 (d, 1H, JH-a,NH¼7.6 Hz, –NH–); 7.90
1
100% CH3CN in 40 min). H NMR (400 MHz, CHCl3),
d 0.80 (d, 3H, JH-da,H-g¼5.3 Hz, H-da Leu); 0.94 (d, 3H,
JH-db,H-g¼5.9 Hz, H-db Leu); 1.46–1.67 (m, 3H, H-ba Leu,
H-bb Leu, H-g Leu); 1.72–1.94 (m, 4H, H-ba Pro, H-bb
Pro, H-ga Pro, H-gb Pro); 1.98–2.49 (m, 8H, H-ba Glu, H-
bb Glu, H-ba Glu–NH2, H-bb Glu–NH2, H-ga Glu, H-gb
Glu, H-ga Glu–NH2, H-gb Glu–NH2); 2.50–2.63 (m, 2H,
–CH2COOH); 2.75–2.84 (m, 2H, Hba Asn, H-bb Asn);
2.90–3.01 (m, 1H, H ba Phe); 3.02–3.11 (H-bb Phe); 3.20–
3.39 (m, 2H, H-da Pro, H-db Pro); 3.40–3.47 (m, 2H,
–NH–CH2CH2–O–); 3.47–3.63 (m, 14H, –CH2–O–); 3.64–
3.75 (m, 2H, –O–CH2–CH2–COOH); 3.97–4.22 (m, 3H,
H-a Pro, H-9 Fmoc, –CH2a–Fmoc); 4.28–4.42 (m, 3H,
–CH2b–Fmoc, H-a Glu, H-a Glu–NH2); 4.53–4.69 (m, 2H,
H-a Phe, H-a Asn); 4.78–4.88 (m, 1H, H-a Leu); 4.99–
5.10 (m, 2H, –CH2Bn); 5.66 (a-NH Phe); 6.81 (s, 2H,
–CO–NH2); 7.05–7.40 (m, 19H, H–Ar); 7.43–7.51 (m, 2H,
H–Ar); 7.60–7.92 (m, 4H, 2ꢀH–Ar, 2ꢀ–NH–); 8.02–8.11
(s, 1H, –NH–). 13C NMR (100.3 MHz, CHCl3), BB,
DEPT, d 21.58 (C-da Leu); 23.27 (C-db Leu); 24.63 (C-g
Leu); 25.26 (C-g Pro); 25.91, 26.39 (C-b Glu, C-b Glu–
NH2); 28.38 (C-b Pro); 30.89, 31.12 (C-g Glu, C-g Glu–
NH2, –O–CH2–CH2–CO–); 36.83, 38.67 (C-b Asn, C-
b Phe); 39.40 (–CH2–NH–); 40.98 (C-b Leu); 47.15 (C-9
Fmoc); 47.32 (C-d Pro); 49.46 (C-a Leu); 51.96, 53.04,
55.48, 55.71, 61.24 (C-a Asn, C-a Glu, C-a Glu–NH2, C-
a Pro, C-a Phe); 66.18, 66.80, 66.99 (–O–CH2CH2COOH,
–CH2Bn, –CH2–Fmoc); 69.43, 70.13, 70.34, 70.45, 70.49
(–O–CH2–glycol); 119.84 (C-4 Fmoc, C-5 Fmoc); 125.02,
125.11, 126.78, 126.97, 127.59, 127.90, 128.01, 128.34,
128.43, 128.56, 129.38 (C–Ar); 135.43, 135.98, 136.41
(C-ipso Bn); 143.77, 141.19 (C-4a Fmoc, C-4b Fmoc, C-
8a Fmoc, C-9a Fmoc); 155.72 (–O–CO–NH–Fmoc);
170.60, 170.96, 171.31, 172.06, 172.62, 172.91, 173.32,
173.97 (–CO–). MALDIMS: m/z¼1398.1 [M+H]+, 1420.3
[M+Na]+, 1436.3 [M+K]+, 1442.1 [2MꢂH+2Na]+, 1458.1
[MꢂH+Na+K]+, 1474.1 [Mꢂ2H+2 K]+, calcd: 1397.6.
Anal. Calcd for C74H92N8O19$H2O (1397.6$H2O): C,
62.83; H, 6.70; N, 7.92. Found: C, 62.49; H, 6.69; N, 7.91.
(d, 2H, JH-a,NH¼8.5 Hz, –NH–); 8.06 (d, 1H, JH-a,NH
¼
7.1 Hz, –NH–Phe2); 8.11 (d, 1H, JH-a,NH¼7.1 Hz, –NH–);
8.20 (d, 1H, JH-a,NH¼8.2 Hz, –NH–); 8.25 (d, 1H, JH-a,NH
¼
7.9 Hz, –NH–Glu1). 13C NMR (100.6 MHz, DMSO),
1
1H–13C COSY, H–13C HSQC: d 17.1 (C-g Thr); 18.7 (C-
b Ala); 22.3 (C-da Leu); 23.0 (CH3CO–); 23.7 (C-db Leu);
24.8 (C-g Leu); 28.4 (C-b Glu); 30.5 (C-g Glu); 36.9 (C-
b Asp); 37.6 (C-b Phe); 41.8 (C-b Leu); 49.0, 51.7 (C-a
Leu, C-a Ala); 49.5 (C-a Asp); 52.1 (C-a Glu2); 52.5 (C-
a Glu1); 54.2 (C-a Phe1); 54.5 (C-a Phe2); 57.3 (C-a Thr);
58.1 (–CH2SO3H); 2ꢀ66.1, 66.6 –CH2Bn ester); 71.1
(–CH2Bn ether); 75.3 (C-b Thr).
MALDIMS (dhb, C77H92N8O23S2): m/z¼1584.9 [M+Na]+,
1606.9 [M+2Na–H]+, 1629.0 [M+3Na–2H]+, 1650.9
[M+4Na–3H]+, calcd: 1561.7.
4.1.15. N-Fluorenyl-9-methoxycarbonyl-L-phenylalanyl-
L-leucyl-L-prolyl-L-(5-O-benzyl)-glutamyl-L-N4-(14-hy-
droxycarbonyl-3,6,9,12-tetra-oxa-tetradecyl)aspara-
ginyl-L-(5-O-benzyl)-glutaminic acid amide 17. (Fmoc–
Phe-Leu-Pro-Glu(OBn)-Asn(glycol4-COOH)-Glu(OBn)–
NH2.
Rink-amide AM resin (200–400 mesh, Novabiochem)
containing 0.64 mmol/g Fmoc Rink-linker (520 mg,
0.333 mmol) was swollen in DMF (10 mL) in a 100-mL
Merrifield solid-phase reactor. The procedures for Fmoc
removal, coupling reactions and capping reactions were
carried out as described for the synthesis of 16.
Applied amounts of amino acids and coupling times: Fmoc–
Glu(OBn)–OH: 651 mg (1.42 mmol), 15 h; Fmoc–Asn(gly-
col4-COO-t-Bu)–OH:27 751 mg (1.14 mmol), 16 h; Fmoc–
Glu(OBn)–OH: 643 mg (1.40 mmol), 16 h, this coupling
was repeated; Fmoc–Pro–OH: 388 mg (1.15 mmol), 16 h;
according to photometric determination, the remaining
content of Fmoc amounted to 0.267 mmol at this stage of
the synthesis; Fmoc–Leu–OH: 406 mg (1.15 mmol), 15 h;
Fmoc–Phe–OH: 445.5 mg (1.15 mmol), 15 h.
4.1.16. N-Fluorenyl-9-methoxycarbonyl-L-phenylalanyl-
L-leucyl-L-prolyl-L-(5-O-benzyl)-glutamyl-N4-{14-(2-
acetamido-2-deoxy-6-O-benzyl-3-O-(a-L-tri-O-benzyl-
fucopyranosyl)-4-O-(2,4-di-O-acetyl-6-O-benzyl-[methyl
5000-acetamido-4,7,8,9,-tetra-O-acetyl-3000,5000-dideoxy-a-
D-galacto-2000-nonulopyranosylate]-b-D-galactopyrano-
syl)-a/b-D-glucopyranosyl)-aminocarbonyl-3,6,9,12-