Organic Process Research & Development
Communication
dried on the filter under vacuum to provide 1.57 kg of the 2-
MeTHF solvate of Fmoc-O-benzylphospho-L-serine. HPLC
assay (Method 1) indicated 91.2 A% and 78.1 wt % purity
(53.8% isolated yield, corrected for wt %). H NMR analysis
indicated 11.9 wt % 2-MeTHF. The product loss to the mother
liquor was 7.0% (of the theoretical yield).
and the cake was washed with ambient temperature MTBE (15
L, 3.0 vol).
The solids were dried at 30 °C on the filter under house
vacuum for 16 h to provide 4.67 kg of Fmoc-O-benzylphospho-
L-serine as the 2-MeTHF solvate (containing 13.2 wt % 2-
MeTHF). When adjusted for the 2-MeTHF, 4.05 kg of Fmoc-
O-benzylphospho-L-serine was isolated (54.5% isolated yield).
The product loss to the mother liquor was 11.8% (of the
theoretical yield).
1
Phosphorylation Procedure (Method B). Volumes
indicated in this procedure are in reference to the weight of
the dry amino acid used. When a hydrated form of Fmoc-L-
serine was purchased (hydrated specified as >0.7 wt % water),
the following azeotropic drying was utilized to remove the
water and increase the yield. A mixture of Fmoc-L-serine (5.04
kg crude mass, 2.8 wt % H2O, 14.97 mol corrected for wt %, 1.0
equiv) and 2-MeTHF (20.0 L, 4 vol) was heated (Tj = 65 °C),
and 2-MeTHF (10.0 L) was removed via distillation at 450
mbar. The solution was diluted with 2-MeTHF (10.0 L), and
this distillation and dilution cycle was repeated two more times.
The final water content of the Fmoc-serine solution was <0.10
wt % H2O (target < 0.7 wt %).
2-MeTHF (35.0 L, 7.0 vol) was cooled to −15 °C, and PCl3
(3.15 kg, 22.9 mol, 1.53 equiv) was charged to the reactor. After
15 min, BnOH (2.90 kg, 26.8 mol, 1.79 equiv) was added over
45 min, and 2-MeTHF (5.0 L, 1.0 vol) was used to rinse the
lines. A maximum temperature of −14 °C was observed.
The solution was then warmed to 0 °C over 30 min. 2,6-
Lutidine (4.82 kg, 45.0 mol, 3.0 equiv) was added to the reactor
over 50 min at <5 °C. A slurry formed during this addition.
2,6-Lutidine (1.61 kg, 15.0 mol, 1.0 equiv) was premixed
with the dried solution of Fmoc-L-serine, and the resulting
mixture was added to the reactor over 1.5 h at <3 °C.
After a 16 h age at 0 °C, an HPLC assay (Method 1) was
taken to confirm reaction completion (<10% starting material
at 225 nm). After confirming reaction completion, H2O (18.0
L, 3.6 vol) was added to the reactor over 30 min with cooling.
An exotherm was observed and the maximum temperature
reached 5 °C. During the addition, all of the solids went into
solution, forming a two-phase mixture.
Desolvation Upgrade (Upgrade 1). Fmoc-O-benzylphos-
pho-L-serine (1.48 kg, 91.2 A%, 78.1 wt %) was mixed with
iPAc (10.4 L, 7.0 vol). The resulting slurry was stirred for 22 h
at 20 °C to allow for digestion of the solids. The slurry was then
cooled to 0−5 °C for 1 h. A supernatant HPLC assay (Method
1) taken at 1 h indicated that the level of product in the mother
liquors was at about 6.7 mg/mL (≤7.0 mg/mL desired).
After 75 min, the slurry was filtered and washed with room
temperature iPAc (3.0 L, 2.0 vol) without allowing the cake to
crack. The solids were dried on the filter under house vacuum
at ambient temperature to provide 1.10 kg of product (50%
yield from Fmoc-L-serine, corrected for wt % and for the
amount of solvate used). HPLC assay (Method 1) indicated
99.4 A% and 97.3 wt % purity (versus a recrystallized standard,
>100 wt % versus commercially available Fmoc-O-benzylphos-
pho-L-serine). Chiral HPLC assay (Method 2) indicated 100%
ee. 1H NMR analysis indicated 1.0 wt % 2-MeTHF and 0.40 wt
% IPAc. KF Fischer analysis indicated 0.25 wt % H2O. 1H NMR
(400 MHz, DMSO): 7.88 (m, 3H), 7.74 (d, J = 8.03 Hz, 2H),
7.25−7.45 (m, 9H), 4.94 (d, J = 7.52 Hz, 2H), 4.05−4.40 (m,
6H). 13C NMR (400 MHz, DMSO): 170.7 (s, 1C), 155.9 (s,
1C), 143.7 (s, 2C), 140.6 (s, 2C), 136.7 (d, 1C), 128.3 (s, 2C),
127.9 (s, 1C), 127.6 (s, 2C), 127.5 (s, 2C), 127.0 (s, 2C), 125.2
(s, 2C), 120.0 (s, 2C), 67.4 (d, 1C), 65.8 (s, 1C), 65.1 (d, 1C),
54.3 (d, 1C), 46.5 (s, 1C). 31P NMR (400 MHz, DMSO):
−1.50. HRMS: [MH+] = 498.1306 (calc = 498.1312). Specific
Rotation: −9.69°, c = 0.0509 g/mL in DMF.
ASSOCIATED CONTENT
Solid NaBr (3.54 kg, 34.4 mol, 2.30 equiv) was then added to
the two-phase mixture in the reactor at 0 °C over 20 min. This
was followed by addition of solid NaBrO3 (1.08 kg, 7.16 mol,
0.48 equiv) over 20 min. After the addition, an exotherm was
observed, and the jacket was used to maintain an internal
temperature of 0 5 °C. After a minimum of 2.5 h, HPLC
assays (Method 1) were taken to confirm reaction completion
(completion defined as phosphite intermediate <4.0 A% at 225
nm). When reaction completion was confirmed, solid Na2S2O5
(510 g, 2.68 mol, 0.18 equiv) was added to the reactor at 0 °C
to quench any remaining oxidant.
■
S
* Supporting Information
1H, 13C, and 31P NMR, HPLC methods, and additional
experimental details. This material is available free of charge via
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
The batch was then warmed to 20 °C, and after allowing the
layers to settle, the aqueous layer was drained from the reactor.
The organic layer was washed with aqueous NaCl (6.3 kg,
diluted with 35 L of H2O).
ACKNOWLEDGMENTS
■
We thank Emily Kordwitz, Mark Olsen, Chris Neville, and Greg
Gilmartin for their assistance in gathering analytical data
throughout this process development. We thank Daniel
Patience for his assistance in developing the crystallization
parameters. We also thank Sebastien Rose for commercial
process validation of Method B at Laboratoires Mitry-Mory,
and Michael Christie for helpful discussions and support in the
scale-up of Method A.
Solvent was then removed from the resulting organic layer
via vacuum distillation with the jacket temperature at 40
5
°C. When the total volume in the reactor was reduced to 25.0
L, fresh 2-MeTHF (35.0 L, 7 vol) was added, and the
distillation was continued. Upon reaching 25.0 L, the solution
was again diluted with fresh 2-MeTHF (35.0 L, 7 vol) and
distilled one final time to 25.0 L.
The resulting solution was held at 20 °C while MTBE (8.0 L,
1.6 vol) was added over 40 min. This mixture was allowed to
stir for 16 h to allow the seed bed to mature and was then
cooled to −10 °C. After 6 h at −10 °C, the slurry was filtered
REFERENCES
■
(1) Broncel, M.; Wagner, S.; Paul, K.; Hackenberger, C.; Koksch, B.
Org. Biomol. Chem. 2010, 8, 2575−2579.
1864
dx.doi.org/10.1021/op300233g | Org. Process Res. Dev. 2012, 16, 1861−1865