C-Glycosyl Amino Acids
FULL PAPER
H5, Gal), 3.70 (t, 1H, 3JH4;H3 =3JH4;H5 =9.3 Hz, H4, Glc), 3.50–3.34 (m,
2H, H1, Glc {3.39}, H5, Glc {3.43}), 2.33–2.25 (m, 1H, Hb), 2.15, 2.05,
2.03, 2.03, 2.02, 1.97, 1.96 (7ꢃs, 21H, CH3, Ac), 1.68–1.53 (m, 1H, CH2a-
exo), 1.37–1.30 (mc, 1H, CH2b-exo), 0.94 ppm (d, 3H, 3J=6.4 Hz, CH3,
Thr); 13C NMR [BB, HSQC] (100.6 MHz, CDCl3): d=175.10, 170.68,
170.42, 170.19, 170.11, 170.00, 169.14 (C=O, Ac, COOH), 156.33 (C=O,
Fmoc), 143.75, 143.64 (C1a, Fmoc, C8a, Fmoc), 141.27, 141.25 (C4a,
Fmoc, C5a, Fmoc), 128.98, 128.18, 127.78, 127.75, 127.05 (C3, Fmoc, C6,
Fmoc, C2, Fmoc, C7, Fmoc), 125.25, 124.93 (C1, Fmoc, C8, Fmoc),
120.03, 120.01 (C4, Fmoc, C5, Fmoc), 101.07 (C1, Gal), 76.62 (C4, Glc),
76.45 (C5, Glc), 75.82 (C1, Glc), 74.24 (C3, Glc), 72.47 (C2, Glc), 70.96
(C3, Gal), 70.56 (C5, Gal), 69.09 (C2, Gal), 66.94 (CH2, Fmoc), 66.60
(C4, Gal), 62.40 (C6, Glc), 60.79 (C6, Gal), 57.89 (Ca), 47.08 (CH,
Fmoc), 34.72 (CH2-exo), 32.57 (Cb), 20.81, 20.64, 20.60, 20.48 (CH3, Ac),
15.35 ppm (CH3, Thr); MS (ESI): m/z: 980.32 ([M+Na]+, calcd: 980.32);
HR-MS (ESI): m/z: 980.3167 ([M+Na]+, calcd for C46H55NO21:
980.3164). 52b: colorless, amorphous solid; analytical HPLC: tR =
28.2 min (Phenomenex Luna, MeCN/H2O, (50:50) to (65:35), 60 min, l=
214 nm); preparative HPLC: tR =45.9 min (Phenomenex Luna, MeCN/
H2O, (50:50) to (65:35), 60 min, l=214 nm); [a]2D3 = +0.4 (c=1.0,
143.73 (C1a, Fmoc, C8a, Fmoc), 141.43, 141.38 (C4a, Fmoc, C5a, Fmoc),
127.83, 127.78, 127.11, 126.98 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7,
Fmoc), 124.79, 124.77 (C1, Fmoc, C8, Fmoc), 120.14, 120.03 (C4, Fmoc,
C5, Fmoc), 101.90 (C1, Gal), 83.01 (C4, Glc), 75.95 (C5, Glc), 75.04 (C1,
Glc), 74.60 (C3, Glc), 73.23 (C2, Glc), 71.36 (C5, Gal), 70.90 (C3, Gal),
68.61 (C2, Gal), 66.83 (C4, Gal), 66.54 (CH2, Fmoc), 63.16 (C6, Glc),
61.84 (C6, Gal), 57.97 (Ca), 47.16 (CH, Fmoc), 34.66 (CH2-exo), 33.69
(Cb), 20.83, 20.63, 20.60, 20.48, 20.37 (CH3, Ac), 16.88 ppm (CH3, Thr);
MS (ESI): m/z: 938.30 ([M+Na]+, calcd: 938.31). 53b: colorless, amor-
phous solid; analytical HPLC: tR =22.2 min (Phenomenex Luna, MeCN/
H2O, (50:50) to (65:35), 60 min, l=214 nm); preparative HPLC: tR =
37.9 min (Phenomenex Luna, MeCN/H2O, (50:50) to (65:35), 60 min, l=
214 nm); [a]2D3 = +9.2 (c=1.0, CHCl3); 1H NMR [1H1H-COSY]
(400 MHz, CDCl3): d=7.77 (d, 2H, 3J=7.5 Hz, HAr, Fmoc), 7.59 (t, 2H,
3J=7.8 Hz, HAr, Fmoc), 7.41 (t, 2H, 3J=7.3 Hz, HAr, Fmoc), 7.35–7.28
3
(m, 2H, HAr, Fmoc), 5.37 (sb, 1H, H4, Gal), 5.24 (dd, 1H, JH2;H1 =8.1 Hz,
3JH2;H3 =10.2 Hz, H2, Gal), 5.00 (db, 1H, 3JH3;H2 =10.2 Hz, H3, Gal), 4.73
(t, 1H, 3JH2;H1 =3JH2;H3 =8.9 Hz, H2, Glc), 4.57–4.48 (m, 2H, H1, Gal
{4.54}, Ha {4.52}), 4.43 (dd, 1H, 2J=10.1 Hz, 3J=7.4 Hz, CH2a, Fmoc),
4.35 (dd, 1H, 2J=10.6 Hz, 3J=7.2 Hz, CH2b, Fmoc), 4.32–4.20 (m, 2H,
H6a, Glc {4.30}, CH, Fmoc {4.23}), 4.17–4.06 (m, 2H, H6a, Gal, H6b,
Gal), 4.04–3.97 (m, 2H, H5, Gal {4.01}, H6b, Glc {4.00}), 3.73 (t, 1H,
3JH3;H2 =3JH3;H4 =8.1 Hz, H3, Glc), 3.61–3.52 (m, 2H, H1, Glc, H5, Glc),
3.51–3.43 (m, 1H, H4, Glc), 2.46 (sb, 1H, Hb), 2.16, 2.08, 2.07, 2.03, 1.98,
1.95 (6ꢃs, 18H, CH3, Ac), 1.63–1.53 (m, 1H, CH2a-exo), 1.44–1.35 (m,
1H, CH2b-exo), 0.92 ppm (d, 3H, 3J=6.7 Hz, CH3, Thr); 13C NMR [BB,
HSQC] (100.6 MHz, CDCl3): d=175.27, 170.94, 170.51, 170.06, 169.99,
169.50 (C=O, Ac, COOH), 156.47 (C=O, Fmoc), 143.60 (C1a, Fmoc, C8a,
Fmoc), 141.27, 141.25 (C4a, Fmoc, C5a, Fmoc), 127.80, 127.10 (C3, Fmoc,
C6, Fmoc, C2, Fmoc, C7, Fmoc), 125.04, 124.96 (C1, Fmoc, C8, Fmoc),
120.05 (C4, Fmoc, C5, Fmoc), 101.97 (C1, Gal), 83.18 (C4, Glc), 75.31
(C5, Glc), 74.66 (C1, Glc), 74.37 (C3, Glc), 73.45 (C2, Glc), 71.37 (C5,
Gal), 70.83 (C3, Gal), 68.64 (C2, Gal), 67.26 (CH2, Fmoc), 66.77 (C4,
Gal), 63.03 (C6, Glc), 61.72 (C6, Gal), 55.79 (Ca), 47.06 (CH, Fmoc),
34.68 (CH2-exo), 31.61 (Cb), 20.74, 20.59, 20.47, 20.38 (CH3, Ac),
15.72 ppm (CH3, Thr); MS (ESI): m/z: 938.29 ([M+Na]+, calcd: 938.31).
1
CHCl3); H NMR [1H1H-COSY, TOCSY] (400 MHz, CDCl3): d=7.76 (d,
2H, 3J=7.5 Hz, HAr, Fmoc), 7.59 (t, 2H, 3J=7.3 Hz, HAr, Fmoc), 7.40 (t,
2H, 3J=7.4 Hz, HAr, Fmoc), 7.35–7.28 (m, 2H, HAr, Fmoc), 5.37 (d, 1H,
3JNH;Ha =8.9 Hz, NH), 5.34 (d, 1H, 3JH4;H3 =3.1 Hz, H4, Gal), 5.19 (t, 1H,
3
3JH3;H2 =3JH3;H4 =9.0 Hz, H3, Glc), 5.11 (dd, 1H, 3JH2;H1 =8.0 Hz, JH2;H3
=
10.4 Hz, H2, Gal), 4.95 (dd, 1H, 3JH3;H2 =10.4 Hz, 3JH3;H4 =3.4 Hz, H3,
Gal), 4.75 (t, 1H, JH2;H1 =3JH2;H3 =9.5 Hz, H2, Glc), 4.56–4.38 (m, 4H, Ha
3
{4.51}, H1, Gal {4.47}, H6a, Glc {4.44}, CH2a, Fmoc {4.42}), 4.34 (dd, 1H,
2J=10.5 Hz, 3J=7.4 Hz, CH2b, Fmoc), 4.22 (t, 1H, 3J=7.0 Hz, CH,
Fmoc), 4.15–4.01 (m, 3H, H6a, Gal, H6b, Gal, H6b, Glc), 3.86 (t, 1H,
3JH5;H6a =3JH5;H6b =7.0 Hz, H5, Gal), 3.71 (t, 1H, 3JH4;H3 =3JH4;H5 =9.4 Hz,
H4-Glc), 3.66–3.57 (m, 2H, H1-Glc, H5-Glc), 2.45 (sb, 1H, Hb), 2.08,
2.05, 2.03, 2.01, 1.96, 1.90 (6ꢃs, 21H, CH3, Ac), 1.62–1.51 (m, 1H, CH2a-
exo), 1.42–1.31 (m, 1H, CH2b-exo), 0.90 ppm (d, 3H, 3J=6.4 Hz, CH3,
Thr); 13C NMR [BB, HSQC] (100.6 MHz, CDCl3): d=175.52, 170.73,
170.41, 170.21, 170.12, 169.86, 169.07 (C=O, Ac, COOH), 156.48 (C=O,
Fmoc), 143.71, 143.53 (C1a, Fmoc, C8a, Fmoc), 141.26, 141.23 (C4a,
Fmoc, C5a, Fmoc), 128.99, 128.18, 127.79, 127.08 (C3, Fmoc, C6, Fmoc,
C2, Fmoc, C7, Fmoc), 125.03, 124.95 (C1, Fmoc, C8, Fmoc), 120.02 (C4,
Fmoc, C5, Fmoc), 100.94 (C1, Gal), 76.73 (C4, Glc), 76.52 (C5, Glc),
74.90 (C1, Glc), 74.08 (C3, Glc), 72.70 (C2, Glc), 70.97 (C3, Gal), 70.60
(C5, Gal), 69.06 (C2, Gal), 67.25 (CH2, Fmoc), 66.62 (C4, Gal), 62.51
(C6, Glc), 60.82 (C6, Gal), 55.78 (Ca), 47.06 (CH, Fmoc), 34.84 (CH2-
exo), 31.59 (Cb), 20.81, 20.73, 20.61, 20.58, 20.50 ppm (CH3, Ac), 15.61
(CH3, Thr); MS (ESI): m/z: 980.32 ([M+Na]+, calcd: 980.32); HRMS
(ESI): m/z calcd for C46H55NO21: 980.3164 [M+Na]+; found: 980.3167.
Side products with missing acetyl group: N-(Fluoren-9-ylmethoxycarbon-
yl)-C-[2,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl)-
b-d-glucopyranosyl]-threonine; 53a: colorless, amorphous solid; analyti-
cal HPLC: tR =25.2 min (Phenomenex Luna, MeCN/H2O, (50:50) to
(65:35), 60 min, l=214 nm); preparative HPLC: tR =41.9 min (Phenom-
Amino-4,7,10-trioxadodecanylamido-N-l-prolyl-l-alanyl-l-histidyl-l-
glycyl-l-valyl-l-threonyl-l-seryl-l-alanyl-l-prolyl-l-aspartyl-l-threonyl-l-
arginyl-l-prolyl-l-alanyl-l-prolyl-l-glycyl-C-[2,3,4-tri-O-benzyl-b-l-fuco-
pyranosyl]-l-tyrosyl-l-threonyl-l-alanyl-l-prolyl-l-prolyl-l-alanine (54):
The synthesis was carried out in an Applied Biosystems ABI 433A pep-
tide synthesizer starting with Tentagel R resin (563 mg) pre-loaded with
Fmoc-Ala-O-Trt (0.09 mmol, loading 0.16 mmolgÀ1). For the coupling re-
actions, the amino acids Fmoc-Ala-OH, Fmoc-Arg
Asp-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Ser-
(tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Val-OH were employed. In
ACHTUNGTREN(NGNU Pmc)-OH, Fmoc-
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
every coupling cycle, the N-terminal Fmoc group was removed by treat-
ment of the resin with a solution of piperidine (20%) in NMP for at least
3ꢃ2.5 min. The coupling of Fmoc amino acids (1 mmol or 10 equiv based
on the loaded resin) was carried out with HBTU (1 mmol), HOBt
(1 mmol), and diisopropylethylamine (DIPEA, 2 mmol) in DMF (20–
30 min vortex). After every coupling step, unreacted amino groups were
capped by treatment with a mixture of Ac2O (0.5m), DIPEA (0.125m),
and HOBt (0.015m) in NMP (10 min vortex). After the coupling of the
first two proline-building blocks, Ala4 was added in a double coupling
step. Coupling of C-glycoside 39 (87 mg, 0.106 mmol, 1.18 equiv based on
the loaded resin) was performed by using HATU (48.3 mg, 0.127 mmol,
1.20 equiv with respect to 39), HOAt (17.4 mg, 0.128 mmol, 1.21 equiv)
and N-methylmorpholine (NMM, 30 mL, 0.273 mmol, 2.57 equiv) for acti-
vation (8 h, vortex). The following glycine-building block was added in a
double coupling step. After coupling of the remaining amino acids by the
standard procedure, a triethylene glycol spacer was added by using N-
enex Luna, MeCN/H2O, (50:50) to (65:35), 60 min, l=214 nm); [a]D23
=
À1.1 (c=1.0, CHCl3); 1H NMR [1H1H-COSY] (400 MHz, CDCl3): d=
7.77 (d, 2H, 3J=7.5 Hz, HAr, Fmoc), 7.61–7.56 (m, 2H, HAr-Fmoc), 7.40
(t, 2H, 3J=7.4 Hz, HAr-Fmoc), 7.31 (t, 2H, 3J=7.4 Hz, HAr, Fmoc), 5.65
3
3
(d, 1H, JNH;Ha =8.9 Hz, NH), 5.37 (d, 1H, JH4;H3 =3.3 Hz, H4, Gal), 5.27
(dd, 1H, 3JH2;H1 =8.4 Hz, 3JH2;H3 =10.2 Hz, H2, Gal), 5.01 (dd, 1H,
3JH3;H2 =10.4 Hz, 3JH3;H4 =3.4 Hz, H3, Gal), 4.66 (t, 1H, 3JH2;H1 =3JH2;H3
=
9.6 Hz, H2, Glc), 4.59–4.52 (m, 2H, H1, Gal {4.57}, CH2a, Fmoc {4.55}),
4.43 (dd, 1H, 2J=10.6 Hz, 3J=6.1 Hz, CH2b, Fmoc), 4.38–4.33 (mc, 1H,
Ha), 4.22 (mc, 1H, CH, Fmoc), 4.18–4.08 (m, 3H, H6a, Glc {4.15}, H6a,
Gal, H6b, Gal
3.85 (dd, 1H, 2J=11.5 Hz, 3J=6.5 Hz, H6b, Glc), 3.66 (t, 1H, JH3;H2
ACHTUNGTRENNUNG
{4.12}), 4.00 (t, 1H, 3JH5;H6a =3JH5;H6b =6.4 Hz, H5, Gal),
3
(9H-Fluoren-9-yl)-methoxycarbonylamido-4,7,10-trioxadecanic
acid[38]
=
3JH3;H4 =8.7 Hz, H3, Glc), 3.38 (t, 1H, 3JH4;H3 =3JH4;H5 =8.8 Hz, H4, Glc),
3.29–3.17 (m, 2H, H1, Glc {3.24}, H5, Glc {3.21}), 2.26–2.18 (m, 1H, Hb),
2.17, 2.10, 2.05, 2.03, 1.99, 1.87 (6ꢃs, 18H, CH3, Ac), 1.62–1.51 (m, 1H,
CH2a-exo), 1.40–1.30 (m, 1H, CH2b-exo), 1.01 ppm (d, 3H, 3J=6.7 Hz,
CH3, Thr); 13C NMR [BB, HSQC] (100.6 MHz, CDCl3): d=170.92,
170.57, 170.06, 169.97 (C=OAc, COOH), 156.24 (C=O, Fmoc), 143.88,
(444 mg, 0.100 mmol, 1.00 equiv based on the loaded resin) by using
HATU (456 mg, 1.20 mmol, 1.20 equiv), HOAt (164 mg, 1.20 mmol,
1.20 equiv), and NMM (0.28 mL, 2.50 mmol, 2.5 equiv) for activation
(8 h, vortex). The N-terminal Fmoc group was removed by using piperi-
dine (20%) in NMP. The peptide was detached from the resin with simul-
taneous removal of all side-chain protecting groups by shaking with TFA
Chem. Eur. J. 2013, 00, 0 – 0
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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