Strain Preparation
3. Conclusions
The A. aceti MIM 2000/28 strain was routinely maintained on GYC
agar plates [glucose (50 gLÀ1), yeast extract (10 gLÀ1), CaCO3
(30 gLÀ1), agar (15 gLÀ1), pH 6.3] at 288C. Strain was inoculated
into 100 mL Erlenmeyer baffled flask containing GLY medium
[yeast extract (10 gLÀ1) and glycerol (25 gLÀ1), pH 5; 20 mL]. After
growth for 24 h (shaking at 150 rpm, 288C), the liquid culture was
entirely used to inoculate a 1 L Erlenmeyer baffled flask containing
GLY medium (150 mL). Flasks were grown for 24 h at 288C, with
shaking at 150 rpm.
The multistep flow synthesis presented here enabled captopril
to be obtained in an overall yield of 50% after crystallization.
Biocatalytic oxidation facilitated the conversion of a prochiral
substrate into a chiral intermediate with high enantiomeric
excess. Three chemical transformations were performed with-
out isolation of the intermediates. The separation of co-prod-
ucts, by-products, and excess reagents was achieved in-line.
These results clearly highlight the benefits of performing multi-
step chemical synthesis in a flow environment. As a further ad-
vantage, our synthetic protocol benefits from the use of an en-
vironmentally friendly biocatalytic oxidation, avoiding the use
of toxic chemical oxidants. The bioprocess might be further
linked to the chemical transformations by optimizing the ex-
traction of acid (2) with organic solvents, for example, using
specific organic-acid-complexing carriers.[19] In light of a future
scale-up, whereas none of the chemical steps have foreseen
limitations, further efforts should be directed toward increasing
the productivity of the first biocatalyzed step, which at present
represents a bottleneck in the process. The new proposed pro-
tocol for the synthesis of captopril represents a powerful inte-
gration of biocatalysis and flow-chemistry technology.
Preparation of Dry Alginate Beads
Gel beads were prepared by ionotropic gelation, by following
a protocol previously developed by us:[17] a 4% (w/v) sodium algi-
nate solution was prepared in distilled water and stirred until a ho-
mogeneous clear solution was formed. The solution was allowed
to settle for 2 h in order to eliminate the air bubbles. The alginate
solution was then gently mixed in a 1:1 (w/w) ratio with a suspen-
sion of A. aceti cells (40 ODmLÀ1) in sodium acetate buffer (20 mm,
pH 6). The resulting mixture was then pumped dropwise into
a slightly agitated CaCl2 solution (0.2m). Calcium alginate beads
were agitated for 20 min, then filtered, washed with deionized
water and dried at 258C for 16 h.
Synthesis of (R)-3-Hydroxy-2-methylpropanoic Acid (2)
Experimental Section
Dry alginate beads (400 mg) were packed into a glass column (i.d.
15 mm) and swelled until their volume tripled by flowing acetate
All reagents and solvents were purchased from Sigma–Aldrich. The
continuous flow reactions were performed using a commercial
R2C/R4 flow reactor (Vapourtec, Bury St. Edmunds, Suffolk, UK)
equipped with Omnifit glass columns (15 mm i.d. ꢁ 100 mm
length) and PFA reactor coils (2 and 10 mL, respectively). The R2C
unit is the pumping unit that contains two adapted Knauer
pumps, which are able to pump highly concentrated and corrosive
acids. R4 is the heating unit with four heating positions. The addi-
tional HPLC pumps necessary to perform the overall synthesis
were provided by another R2+/R4 flow reactor (Vapourtec) and
by two external pumps (ThalesNano). The temperature sensor sits
on the wall of the PFA tubing. The pressure was controlled by
using two 100 psi BPRs. In-line liquid–liquid extractions were per-
formed using a Zaiput separator. 1H NMR and 13C NMR spectra
were recorded with a Varian Mercury 300 (300 MHz) spectrometer.
Chemical shifts (d) are expressed in ppm, and coupling constants
(J) are expressed in Hz. The molar conversion of the biotransforma-
tion was determined by HPLC analysis using a Luna NH2 100 ꢂ
column (250 mmꢁ4.6 mm, particle size 5 mm, Phenomenex,
Aschaffenburg, Germany) and a Biorad refractor index detector
with an acidic aqueous KH2PO4 buffer (20 mm, pH 2.7) as the
mobile phase (flow rate 0.2 mLminÀ1). The samples (40 mL) were in-
jected as soon as collected and without further treatment. The
enantiomeric composition of 2 was determined by gas chromato-
graphic analysis of the corresponding methyl ester [methyl (R)-3-
hydroxy-2-methylpropionate: tr =3.6 min], obtained after treat-
ment with diazomethane, using a chiral capillary column (diameter
0.25 mm, length 25 m, DMePeBeta-CDX-PS086, MEGA, Legnano,
Italy). Optical rotation determinations were performed using
a Jasco P-1010 spectropolarimeter coupled with a Haake N3-B ther-
mostat. MS analyses were performed on a Varian 320-MS triple
quadrupole mass spectrometer with an electrospray ionization
(ESI) source. Microanalyses (C, H, N) were within Æ0.4% of theoret-
ical values.
buffer (20 mm, pH 6) through the column (flow rate: 400 mLminÀ1
,
60 min). The final volume of the bed was 5.1 mL. Air was delivered
at 17 psi; its flow was measured using the method described in
Ref. [20]. To ensure a constant flow, a BRP (40 psi) was applied
before the air tank. An aqueous solution of 1 (1 gLÀ1, 40 mL) was
pumped at 60 mLminÀ1, joining the airflow at the T-junction,
before entering the column in which the oxidation occurs in ap-
proximately 10 min. The exiting flow stream was directed into a de-
compression column. A BPR (5 psi) ensured a constant and con-
trolled flow of aqueous phase leaving the column. The aqueous
stream was directed into a column filled with Ambersep 900 OH
resin (2 g) and, after washing the column with water (20 mL,
0.5 mLminÀ1), the trapped acid was released by flowing HCl (1n,
5 mL). After lyophilization, compound 2 was isolated as pale yellow
oil (42 mg).
To obtain a larger amount of 2, 1.6 g of alginate beads were used,
with a reactor volume of 20 mL. At this scale, 170 mg of com-
pound 2 was isolated (91% yield). [a]2D0 =À11.55 (c=1.00 in EtOH);
1H NMR (300 MHz, CDCl3): d = 1.22 (d, J=7.4 Hz, 3H), 2.10 (s, 1H),
2.68–2.80 (m, 1H), 3.75 (d, J=6.0 Hz, 2H), 5.70 ppm (brs, 1H);
13C NMR (75 MHz, CDCl3): d = 13.2, 41.6, 64.0, 180.1 ppm; MS (ESI):
m/z: 102.9 [M–H]À;[21] HPLC analysis: 1, tr =22 min; 2, tr =18 min.
Synthesis of (R)-3-Chloro-2-methylpropanoyl Chloride (3)
A solution of 2 (52 mg, 0.5 mmol) was prepared in anhydrous tolu-
ene (410 mL). Imidazole (3.5 mg, 0.1 equiv) and DMF (50 mL) were
added to the solution. A second solution of thionyl chloride
(125 mL, 3.5 equiv) was prepared in anhydrous toluene (375 mL).
The two solutions were mixed into a T-piece and flowed through
a 10 mL reactor coil according to the conditions reported in
Table 1. A 200 psi backpressure regulator was applied to the
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