Minimalist Redesign of l-Fuculose-1-Phosphate Aldolase
FULL PAPER
3J=19.8 Hz, 1H), 4.40 (B of AB system, J=19.7 Hz, 1H), 4.23 (dd, 3J=
2.4, 3.3 Hz, 1H), 4.15 (d, 3J=2.4 Hz, 1H), 4.03 (m, 1H), 3.54–3.43 (m,
2H), 2.35–2.01 (m, 2H), 1.98–1.82 ppm (m, 2H); 13C NMR (101 MHz,
CD3OD): d=213.7, 158.6, 138.2–129.5, 78.7, 73.6, 68.4, 68.2, 61.4, 47.7,
27.5, 24.8 ppm.
volume 48 mL, 26 mg). In each case, when necessary the operation was
repeated until the whole of the crude sample was consumed. Pure frac-
tions were pooled and lyophilised. Physical and NMR spectroscopy data
are listed below. 1H and 13C NMR spectra assignments are given in the
Supporting Information.
(R)-Benzyloxycarbonyl-2-((1R,2R)-1,2,4-trihydroxy-3-oxobutyl)-4-(R)-
hydroxypyrrolidine (8a): The title compound was prepared by the Gener-
al Procedure described above (204 mg, 60%). Major conformation
1H NMR (600 MHz, CD3OD): d=7.39 (m, 5H), 5.15–5.12 (m, 2H), 4.59
(A of AB system, 3J=19.3 Hz, 1H), 4.45 (B of AB system, 3J=19.3 Hz,
1H), 4.33 (d, 3J=2.3 Hz, 1H), 4.30 (m, 1H), 4.26 (brs, 1H), 4.10 (d, 3J=
8.1 Hz, 1H), 3.70 (m, 1H), 3.33 (m, 1H), 2.17 ppm (m, 1H); 13C NMR
(101 MHz, CD3OD): d=210.6, 155.4, 129.2–138.2, 75.4, 69.2, 68.3, 66.7,
66.5, 58.9, 55.2, 32.5 ppm. Minor conformation 1H NMR (600 MHz,
CD3OD): d=7.39 (m, 5H), 5.22–5.17 (m, 2H), 4.54 (A of AB system,
3J=19.3, 95.7 Hz, 1H), 4.38 (B of AB system, 3J=19.4, 95.6 Hz, 1H),
4.32 (m, 1H), 4.33 (m, 1H), 4.32 (m, 1H), 4.22 (d, 3J=1.4 Hz, 1H), 4.05
(d, J=8.4 Hz, 1H), 3.70 (m, 1H), 3.29 (m, 1H), 2.26 ppm (m, 1H);
13C NMR (101 MHz, CD3OD): d=210.7, 155.3, 129.5–138.2, 75.0, 70.4,
69.5, 66.7, 66.5, 58.5, 55.4, 33.1 ppm.
(1R,2S,3R,7aR)-1,2-Dihydroxy-3-(hydroxymethyl)pyrrolizidine (2-epihya-
cinthacine A2, 9a): The title compound was prepared by the General
Procedure described above (51 mg, 49% from the aldol adduct). [a]D22
=
ꢀ26.5 (c=2 in H2O); 1H NMR (500 MHz, D2O): d=4.23 (t, 3J=3.6 Hz,
1H), 3.89 (dd, 3J=8.7, 3.9 Hz, 1H), 3.83 (dd, 3J=11.0, 7.7 Hz, 1H), 3.64
(dt, 3J=12.4, 6.2 Hz, 1H), 3.37 (td, 3J=8.3, 3.6 Hz, 1H), 2.93 (ddd, 3J=
7.9, 6.2, 3.3 Hz, 1H), 2.84 (m, 1H), 2.70 (dt, 3J=11.0, 5.6 Hz, 1H), 1.92
(m, 1H), 1.83 (m, 1H), 1.76 ppm (m, 2H); 13C NMR (101 MHz, D2O):
d=79.4, 75.9, 71.3, 66.2, 62.6, 56.7, 31.7, 26.9 ppm. ESI-TOF: m/z calcd
for C8H16NO3 [M+H]+: 174.1130; found 174.1131.
(1R,2S,3S,7aR)-1,2-Dihydroxy-3-(hydroxymethyl)pyrrolizidine (9b): The
title compound was prepared by the General Procedure described above
(15 mg, 14% from the aldol adduct). [a]2D2 =ꢀ45.3 (c=1.5 in H2O);
1H NMR (500 MHz, D2O): d=4.08 (dd, 3J=4.9, 10.0 Hz, 1H), 4.00 (dd,
3J=5.0, 1.8 Hz, 1H), 3.91 (m, 2H), 3.63 (td, 3J=8.5, 1.7 Hz, 1H), 3.41
(ddd, 3J=10.0, 8.5, 4.5 Hz, 1H), 3.13 (m, 1H), 2.91 (td, 3J=10.7, 5.7 Hz,
1H), 2.25 (m, 1H), 1.99 (m, 1H), 1.73 (m, 1H), 1.58 ppm (m, 1H);
13C NMR (101 MHz, D2O): d=77.3, 72.8, 72.7, 66.7, 61.3, 50.0, 31.6,
28.0 ppm. ESI-TOF: m/z calcd for C8H16NO3 [M+H]+: 174.1130; found
174.1132.
(S)-Benzyloxycarbonyl-2-((1S,2R)-1,2,4-trihydroxy-3-oxobutyl)-4-(R)-hy-
droxypyrrolidine (8b): The title compound was prepared by the General
Procedure described above (251 mg, 49%). Major conformation
1H NMR (600 MHz, CD3OD): d=7.39 (m, 5H), 5.17–5.17 (m, 2H), 4.59
3
3
(A of AB system, J=19.7, 1H), 4.51 (B of AB system, J=19.6 Hz, 1H),
4.39 (s, 1H), 4.39 (m, 1H), 4.23 (d, 3J=4.9 Hz, 1H), 4.19 (m, 1H), 4.14
(m, 1H), 3.63 (d, 3J=11.9 Hz, 1H), 3.42 (dd, 3J=11.6, 4.2 Hz, 1H), 2.32
(m, 1H), 2.03 ppm (m, 1H); 13C NMR (101 MHz, CD3OD): d 213.7,
129.2–138.2, 158.6, 77.0, 71.8, 69.3, 67.0, 66.4, 59.1, 54.3, 34.8 ppm. Minor
conformation 1H NMR (600 MHz, CD3OD): d=7.39 (m, 5H), 5.24–5.11
(1R,2S,3S,6R,7aR)-1,2,6-Trihydroxy-3-(hydroxymethyl)pyrrolizine (9e):
The title compound was prepared by the General Procedure described
above (17 mg, 15% from the aldol adduct). [a]2D2 =ꢀ39.6 (c=1.3 in
H2O); 1H NMR (500 MHz, D2O): d=4.32 (m, 1H), 4.13 (dd, 3J=9.3,
5.4 Hz, 1H), 4.01 (dd, 3J=5.3, 2.7 Hz, 1H), 3.86 (m, 2H), 3.41 (td, 3J=
8.0, 2.5 Hz, 1H), 3.18 (m, 1H), 3.07 (A of AB system, 3J=9.6, 5.8 Hz,
1H), 2.70 (B of AB system, 3J=9.4, 8.0 Hz, 1H), 2.44 (m, 1H), 1.58 ppm
3
(m, 2H), 4.48 (A of AB system, J=19.5 Hz, 1H), 4.34 (B of AB system,
3J=19.6 Hz, 1H), 4.46 (m, 1H), 4.39 (brs, 1H), 4.14 (m, 1H), 4.11 (d,
3J=2.5 Hz, 1H), 3.62 (d, 3J=11.9 Hz, 1H), 3.48 (dd, 3J=11.9, 3.8 Hz,
1H), 3.36 (m, 1H), 2.47 (m, 1H), 1.99 ppm (m, 1H); 13C NMR
(101 MHz, CD3OD): d=213.7, 158.6,129.5–138.2, 77.3, 71.0, 69.3, 67.0,
66.0, 59.1, 54.9, 34.0 ppm.
3
(dt, J=12.9, 7.8 Hz, 1H); 13C NMR (101 MHz, D2O): d=77.9, 73.0, 72.8,
70.7, 67.2, 61.8, 55.2, 39.2 ppm; ESI-TOF: m/z calcd for C8H16NO4
[M+H]+: 190.1079; found 190.1077.
(1S,2S,3R,7S,7aS)-1,2,7-trihydroxy-3-(hydroxymethyl)pyrrolizine
(9 f):
(S)-Benzyloxycarbonyl-2-((1S,2R)-1,2,4-trihydroxy-3-oxobutyl)-3-(S)-hy-
droxypyrrolidine (8c): The title compound was prepared by the General
Procedure described above (149 mg, 44%). 1H NMR (600 MHz,
CD3OD): d=7.41 (m, 5H), 5.19 (m, 2H), 4.62 (A of AB system, 3J=
20.0 Hz, 1H), 4.52 (B of AB system, 3J=19.8 Hz, 1H), 4.48 (brs, 1H),
4.17 (s, 1H), 3.85 (d, 3J=9.5 Hz, 1H), 3.66 (d, 3J=9.3 Hz, 1H), 3.64 (m,
1H), 3.52 (t, 3J=9.9 Hz, 1H), 2.18 (tt, 3J=13.5, 6.6 Hz, 1H), 1.94 ppm
(dd, 3J=13.7, 7.0 Hz, 1H); 13C NMR (101 MHz, CD3OD): d=211.9,
157.7, 129.2–138.2, 76.2, 71.5, 72.0, 67.5, 67.3, 67.0, 44.9, 31.0 ppm.
The title compound was prepared by the General Procedure described
above (3 mg, 3% from the aldol adduct). 1H NMR (500 MHz, D2O): d=
4.57 (s, 1H), 4.39 (dd, 3J=8.7, 3.8 Hz, 1H), 4.31 (t, 3J=3.3 Hz, 1H), 3.91
(A of AB system, 3J=11.6, 6.2 Hz, 2H), 3.81 (B of AB system, 3J=11.7,
3
7.2 Hz, 1H), 3.75 (m, 1H), 3.57 (m, 1H), 3.25 (A of AB system, J=12.5,
3.1 Hz, 1H), 3.12 (B of AB system, 3J=12.1 Hz, 1H), 2.26 (m, 1H),
3
2.04 ppm (d, J=13.6 Hz, 1H); 13C NMR (101 MHz, D2O): d=79.6, 75.3,
75.2, 73.3, 69.8, 62.9, 61.7, 38.5 ppm; ESI-TOF: m/z calcd for [M+H]+
C8H16NO4: 190.1079; found 190.1076.
Removal of Cbz group and reductive amination: The aldol adducts ob-
tained (0.40–0.74 mmol) were dissolved in ethanol (5–10 mL), followed
by the addition of plain water (20–45 mL). Pd/C (200 mg) was added.
The reaction mixture was shaken under hydrogen (50 psi) overnight at
room temperature. After removal of the catalyst by filtration through de-
activated aluminium oxide, the pH of the filtrate was adjusted to pH 5.5
with formic acid (1m), the solvent was removed under reduced pressure,
and the product was lyophilised.
(1S,3R,6R,7aR)-1,6-Dihydroxy-3-(hydroxymethyl)pyrrolizine (9g): The
title compound was prepared by the General Procedure described above
(4 mg, 4% from the aldol adduct). [a]2D2 =ꢀ22.2 (c=0.09 in H2O);
1H NMR (500 MHz, D2O): d=4.37 (m, 1H), 4.30 (d, 3J=5.4 Hz, 1H),
3
3
3.85 (A of AB system, J=12.3, 5.2, 1H), 3.80 (B of AB system, J=12.3,
8.1 Hz, 1H), 3.71 (s, 1H), 3.64 (m, 1H), 3.20 (d, 3J=8.2 Hz, 1H), 2.79 (t,
3J=9.1 Hz, 1H), 2.52 (m, 1H), 2.13 (td, 3J=13.1, 5.4, 1H), 1.87 (m, 1H),
1.59 ppm (dt, 3J=13.3, 8.1 Hz, 1H); 13C NMR (101 MHz, D2O): d=77.7,
75.6, 71.9, 65.8, 61.7, 53.9, 38.6, 35.4 ppm; ESI-TOF: m/z calcd for
[M+H]+ C8H16NO3: 174.1130; found 174.1126.
Purification by ion exchange chromatography: The polyhydroxylated pyr-
rolizidine derivatives 9a, 9b and 9e–9j were separated by ion exchange
chromatography with a FPLC system by a procedure described by us.[6,7]
+
(1S,2S,3S,6R,7aS)-1,2,6-Trihydroxy-3-(hydroxymethyl)pyrrolizine
(9h):
CM-Sepharose CL-6B (Amersham Pharmacia) in NH4 form stationary
The title compound was prepared by the General Procedure described
above (49 mg, 35% from the aldol adduct). [a]2D0 =ꢀ21.0 (c=1.4 in H2O)
[lit. (enantiomer)[22] [a]2D0 =+16.0 (c=0.2 in H2O); lit.[21] [a]2D0 =ꢀ5.8 (c=
0.18 in H2O)]; 1H NMR (500 MHz, D2O): d=4.59 (m, 1H), 3.87 (t, 3J=
7.3 Hz, 1H), 3.84 (t, 3J=8.5 Hz, 1H), 3.79 (A of AB system, 3J=11.7,
phase was packed into a glass column (450ꢉ25 mm) to provide a final
bed volume of 220 mL. The flow rate was 4 mLminꢀ1. The CM-Shephar-
+
ose-NH4 was washed initially with H2O. An aqueous solution of the
crude material at pH 7 was then loaded onto the column. Minor coloured
impurities were washed away with H2O (150 mL, 3 bed volumes). The re-
tained compounds were eluted with aqueous NH4OH (0.01m): com-
pounds 9a and 9b (load 150 mg), 9a (elution volume, 584 mL, 51 mg),
9b (elution volume, 992 mL, 15 mg); compounds 9e–9g: 9e (elution
volume 260 mL, 17 mg), 9 f (elution volume 376 mL, 3 mg), 9 f (elution
volume 512 mL, 4 mg); compound 9c (elution volume 376 mL, 49 mg);
compounds 9g and 9h: 9g (elution volume 416 mL, 10 mg), 9h (elution
3
3.7 Hz, 1H), 3.64 (B of AB system, J=11.8, 6.7 Hz, 1H), 3.45 (q, J=7.3,
3
7.2 Hz, 1H), 3.05 (A of AB system, J=11.7, 3.0 Hz, 1H), 2.97 (B of AB
system, 3J=11.8, 4.5 Hz, 1H), 2.82 (m, 1H), 2.09 (m, 1H), 2.00 ppm (m,
1H); 13C NMR (101 MHz, D2O): d=82.6, 79.9, 74.6, 72.7, 68.2, 64.4, 64.2,
40.1 ppm; ESI-TOF: m/z calcd for [M+H]+ C8H16NO4: 190.1079; found
190.1079.
Chem. Eur. J. 2010, 16, 10691 – 10706
ꢆ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10703