938
M. Keller et al.
LETTER
After 7h at 80 °C, only one isomer could be observed by
(7) Wöhr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun,
X.; Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218-9227, and
ref. cited therein.
HPLC. Heating at 80 °C was continued for another 10 h; very
weakly, a second peak close to the peak assigned to the (R)-
epimer 5b was observed. To the yellowish liquid was added
EtOAc (20 ml), and the solution washed with Na2CO3 (10%,
20 ml) and water (20 ml) before drying the organic layer over
MgSO4. Deprotection of the methylester was achieved using
LiOH (3.5 equiv in THF/H2O = 4:1, 5 ml, 2 h). The epimers
now were separated on the same gradient as above. The
relation between the 2-C (R) and (S)-iomer only changed
slightly to 8:92. (S)-epimer 3a: tR = 15.13 min, (R)-epimer
3b: tR = 15.84 min (50-100% B, 20 min, C18). The product was
purified by flash chromatography over silica using CHCl3/
CH3OH (100:15) as eluent to obtain 2-C(S) Fmoc-NMeIle-
Thr(ΨΗ,pmppro)-OH. C35H38N2O7 = 586.7. Yield:56 mg, 96%.
MS-ESI (m/z) 587.6 [M+H]+. Co-injection of the purified 2-
C(S) compound with the isolated kinetic product of the O-
benzyl reaction gave one single peak in HPLC.
(8) Experimentals to Scheme 2: Fmoc-NMeIle-Thr(ΨΗ,pmppro)-
OBn (2) C41H44N2O7 = 676.8 Fmoc-NMeIle-Thr-Bn (1; 50
mg, 0.09 mmol) was dissolved in THF (2 ml). PPTS (6.8 mg,
0.3 equiv) and anisaldehyde dimethylacetal (0.096 ml, 5
equiv) added and heated under reflux for 7h. Samples were
taken after 1h, 3h and 7h in order to follow the reaction by
HPLC: isomer 2-C(S) 2a: rt = 22.48 min, isomer 2-C(R) 2b:
rt = 23.47 min, 50-100% B, C18 (A = water containing 0.09%
TFA; B = acetonitrile for HPLC-R containing 10% water and
0.09% TFA; cf. Scheme 2 in lit.4). Complete conversion to 2-
C(R) 2b was stated after 7 hours. The solvent was evaporated
and replaced by AcOEt (20 ml) and washed subsequently with
aqueous Na2CO3 (0.5 M, 20 ml, 3x) and water (20 ml, 3x). The
organic layer was dried over MgSO4, filtered and the solvent
removed in vacuo. To the slightly yellow oily residual was
added methanol (0.10 ml), before addition of ether (5 ml) to
yield oxazoline 2b as a white precipitate (98%), which was
collected on a glass filter and recrystallized from methanol
(0.1 ml) / ether (5 ml). HPLC: 23.52 min (95% purity; no other
isomer detected). MS-ESI (m/z): 676.8 [M+H]+. Separation of
the diastereomers: In a separate reaction, the same conditions
as above described were used, but the reaction was stopped
after 1.5 h and the isomers separated by means of reversed
phase HPLC, using an isocratic gradient 40% A and 60% B to
100% B, to obtain 20 mg of each epimer (2a and 2b)
(9) Experimentals to Scheme 3: Fmoc-Pro-Thr(ΨH,p-F-Phpro)-
OBn (7) C38H35N2O6F = 634.7. Fmoc-Pro-Thr-OBn (6; 1.3g,
2.05 mmol) was dissolved in CH2Cl2 (130 ml) before adding
para-fluorobenzaldehyde dimethylacetal 2.1 ml, 10 equiv)
under nitrogen atmosphere. To the clear solution was added
BF3.OEt2 (965 ml, 7.8 mmol) and stirred. The solution first
turned to yellow and after two minutes to bordeaux red. After
20 min, the reaction was stopped by adding a solution of
Na2CO3 (10%, 50 ml). The organic layer was washed with
water (50 ml, 2) and dried over MgSO4. All solvent was
evaporated and replaced with CH3OH. To this solution of
crude acetal 7, Pd-C (130 mg) was added and hydrogen
bubbled through the solution for 1.5 h. After filtration over
Celite and evaporation of the filtrate, a yellowish oil was
reconstituted, which was purified on silica using CHCl3/
CH3OH/HOAc (100/10/1 ml) as eluent. 660 mg (1.21 mmol,
49%) of a white solide was isolated and identified as Fmoc-
Pro-Thr(ΨH,p-F-Phpro)-OH (8b; assigned to 2-C(R) based on
a preliminary 1H 2D NMR NOESY experiment)
separately. Fmoc-NMeIle-Thr(ΨΗ,pmppro)-OH (3)
C35H38N2O7 = 586.7 Deblocking of the benzyl protecing
group was achieved under hydrogen atmosphere in methanol
(5 ml) using Pd/C as catalyst. After completion of the
deprotection, the suspension was filtered over Celite and all
solvent evaporated. HPLC: 2-C(R) epimer 3b 15.9 min (95%
purity; no other isomer), 2-C(S) epimer 3a 15.1 min (single
peak), 50-100% B, C18; MS-ESI m/z) 559.8 [M+H]+. 1H
NMR(400MHz, 10mg/ml, CDCl3, 300 K):2-C(R)-stereo-
isomer (3b; all-trans in CDCl3): δ (ppm) 7.8 (d, 2H, J = 7.6
Hz, Fmoc), 7.62 (d, 2H, J = 7.6 Hz, Fmoc), 7.55 (d, 2H,
J = 8.8 Hz, o-pmp), 7.42 (d×d, 2H, Fmoc), 7.35 (d×d, 2H,
Fmoc), 7.28 (s, CHCl3), 6.94 (d, 2H, J = 8.8 Hz, m-pmp), 6.73
(s, 1H, 2-H), 4.6 (d, 1H, β-Thr), 4.59 (d, 1H, Fmoc-CH), 4.46
(m, 1H, Fmoc-CH2), 4.4 (m, 1H, β-Ile), 4.3 (m, 1H, Fmoc-
CH2’), 4.29 (d, 1H, J = 8 Hz, α-Thr), 3.835 (s, 3H, OMe), 2.88
(s, 3H, NMe), 2.03 (m, 1H, β-Ile), 1.46 (d, 3H, J = 6 Hz, β-
Thr), 0.721 (m, 3H, δ-Ile), 0.472 (d, 3H, J = 6.8Hz, γ-Ile). 2-
C(S)-stereoisomer (3a; 40% cis and 60% trans in DMSO-d6):
7.8 (d, 1H, J = 6.8 Hz, m-pmp trans), 7.74 (d, 1-2H, J = 6.0
Hz, m-pmp cis), 7.6 (d, 1H, J = 6.2 Hz, o-pmp trans), 7.52 (d,
1-2H, J = 6.4 Hz, o-pmp cis), 7.29-7.33 (m, 4-6H, Fmoc cis
and trans), 7.04 (d, 1-1.5H, J = 8.8 Hz, Fmoc), 6.86 (d, 1-
1.6H, J = 8.8 Hz, Fmoc), 6.15 (s, 0.2-0.5H, α-Thr trans), 6.09
(s, 1-2H, α-Thr cis), 5.89 (s, 1-1.5H, 2-H trans), 5.49 (s, 0.8-
1.2H, 2-H cis), 4.28 (d, 1H, J = 7.8 Hz, β-Thr), 4.11 (m, 0.5-
1H, α-Ile cis), 4.05 (m, 0.3-0.7H, α-Ile trans), 3.87 (m, 1-2 H,
Fmoc-CH cis and trans), 3.79 (s, 3H, NMe), 3.74 (s, 3H,
OMe), 3.66 (m, 2-3H, Fmoc-CH2), 3.3-3.5 (s, H2O), 2.65 (m,
1H, β-Ile cis), 2.65 (m, 1H, β-Ile trans), 2.49 (s, DMSO-d6),
1.79 (m, 2-3H, β-Ile-CH3, cis), 1.72 (m, 3-4H, β-Ile-CH3
trans), 1.43 (d, 3-4H, J = 5.6 Hz, β-Thr-CH3 trans), 0.91 (d, 2-
3H, β-Thr-CH3 cis), 0.8 (m, 3-4H, γ-Ile-CH3 trans), 0.72 (d, 2-
3H, J = 6.8 Hz, γ-Ile-CH3 cis). Fmoc-NMeIle-
C31H29N2O6F = 544.7. MS-ESI (m/z) 545.2 M+H+. HPLC
tR = 12.38 min (50-100% CH3CN), 93% purity (only one
isomer). 1H NMR (400 MHz, CDCl3, 10 mg/ml, 295 K; two
conformers; 80% ω-trans): δ (ppm) 7.8 (d, 2H, aromatic
Fmoc), 7.65 (dd, 2H, ortho H-p-F-Ph), 7.6 (t, 2H, aromatic
Fmoc), 7.4 (t, 2H, aromatics Fmoc), 7.3 (t, 2H, aromatic
Fmoc), 7.27 (s, CDCl3), 7.1 (t, 2H, meta H-p-F-Ph), 6.7 (s, 1H,
2-H oxazolidine; ω-trans), 6.45 (s, 0.08H, 2-H oxazolidine;
further conformational isomer), 6.0 (s, 0.17H, 2-H
oxazolidine, ω-cis), 4.5 (t, 1H, β-Thr), 4.4 (dd, 1H, α-Thr and
Fmoc-CH), 3.6 (m, 1H, γ-Pro), 3.5 (m, 1H, γ-Pro), 2.1 (m, 1H,
δ-Pro), 1.95 (m, 1H, δ’-Pro), 1.75 (m, 1H, β’-Pro), 1.5 (m, 1H,
β’-Pro), 1.45 (d, 3H, β-CH3-Thr; ω-trans), 1.25 (d, 0.8H, β-
CH3-Thr; ω-cis).
(10) Experimentals to Scheme 4: Solid Phase Synthesis of Ac-Pro-
Thr(H,para-F-PhPro)4-OH (9). Standard protocols for Fmoc-
chemistry on Sasrin resin were used. Commercially available
Fmoc-Pro-Sasrin (0.64 mmol/g resin, BACHEM, Bubendorf,
Switzerland) was dried in vacuo over night before use. 50
mmol/g resin (ca. 0.03 mmol) was swelled in DMF
(dimethylformamide) for 30 min and washed with CH3OH (10
ml, 1x), CH2Cl2 (10 ml, 3x) and DMF (10 ml, 3x). Fmoc
deprotection was carried out using piperidine in DMF (20%,
3 × 5 min). Coupling reagent HBTU (3 equiv, 35 mg, ALEXIS,
Läufelfingen, Switzerland), deprotection base DIEA
(diisopropylethylamine, 6 equiv, 35 ml). For each coupling, 3
equivalents of Fmoc-Pro-Thr(H,para-F-PhPro)-OH (50 mg) were
taken. Solvent for the couplings was DMF (5 ml). Couplings
(1.5 h) were verified upon their completion by reversed phase
HPLC of a small aliquot cleaved from the resin be 2% TFA in
Thr(ΨΗ,pmppro)-OMe (5) C36H40N2O7 = 600.8. Fmoc-NMe-
Ile-Thr-OMe (4; 50 mg, 0.083 mmol), PPTS (7.8 mg, 0.3
equiv) and anisaldehyde dimethylacetal (0.11 ml, 5 equiv) in
THF (2.5 ml) were heated under reflux for 16 h. The reaction
was followed by HPLC (Gradient 50-100% B, 20 min, C18).
Synlett 1999, S1, 935–939 ISSN 0936-5214 © Thieme Stuttgart · New York