Synthesis of 3′-Functionalized Cephalosporines
J . Org. Chem., Vol. 62, No. 26, 1997 9105
Exp er im en ta l Section
Im m obiliza tion of th e DAO a n d GA on Aga r ose Bea d s.
Activation of agarose gel by reaction with ethylenediamine was
carried out as previously described.22 The aminated gel was
then reacted with 10% glutaraldehyde solution for 12 h at pH
7 and room temperature. The activated gel was incubated
under mechanic stirring with DAO and GA at pH 6 and room
temperature as previously reported18 to obtain the enzyme
derivatives of DAO (100 IU/mL) and GA (250 IU/mL).
Deter m in a tion of th e Ester a se a n d Am id a se Activity
of th e P GA (Sch em e 2). The esterase and amidase activities
have been evaluated by measuring the initial hydrolysis rate
of the (tetrazol-1-yl)acetic acid methyl ester (4) and cefazolin
(3), respectively. The enzyme derivative of PGA (100 IU) was
added to 20 mL of substrate solution (10 mM) in phosphate
buffer 10 mM under magnetic stirring (200 rpm) and at
different pH and temperature. During the reaction the pH
was kept constant by automatic titration and the reaction rate
(µmol/min × mL of enzyme derivative) was calculated from
the NaOH consumption.
Gen er a l Meth od s. Commercially available reagent-grade
solvents were used without purification. Semipurified extracts
of DAO from Trigonopsis variabilis, GA from Acetobacter sp.,
and PGA from E. coli A were from Antibioticos S.A. (Leon,
Spain), while PGA from E. coli B immobilized on Eupergit C
was commercially available (Recordati, Milano, Italy). Cross-
linked 6% agarose beads were donated by Hispanagar (Burgos,
Spain), while Eupergit C was from R o¨ hm Pharma (Darmstadt,
Germany). Melting points were measured on a Kofler hot-
stage apparatus and were not corrected. The elemental
analysis was performed on a Carlo Erba 1106 elemental
1
13
analyzer. H and C NMR spectra were recorded with a
Bruker AC 250 spectrometer, and chemical shifts are reported
in ppm. For NMR analysis, â-lactam compounds were dis-
solved in D
2
O/sodium bicarbonate, while NMR spectra of the
as solvent. GC-MS in
ester 4 were recorded using DMSO-d
6
EI analysis of compound 4 was performed with a Finnigan ITD
instrument. The GC oven was fitted with a Mega (Legnano,
Italy) SE 52 column (15 m × 0.25 mm i.d). The oven was
programmed as follows: starting with 80 °C (for 2 min) and
then increased to 270 °C at 10 °C/min. The injector was in
the splitless mode at 220 °C. The detector temperature was
held at 280 °C, and the electron energy was at 70 eV. HPLC
analyses were run on a Merk-Hitachi L-7100 equipped with
UV detector L-7400. The column was a LiChroCART 250-4
RP select-B (Merck, Darmstadt, Germany) and analyses were
run at 25 °C by using a L-7300 column oven. The LC
conditions are described for each compound. The pH of the
solutions during the enzymatic hydrolysis and synthesis
reactions were kept constant by using an automatic titrator
En zym a tic Syn th esis of Cefa zolin (3) (Sch em e 3).
Following a general procedure, the ester 4 was dissolved into
a solution (20 mL in phosphate buffer 10 mM) of 7-ZACA (2).
The mixture was cooled to 4 °C and pH adjusted at 7.5. The
enzyme derivative of PGA from E. coli A or E. coli B (100 IU)
was then added to the solution under magnetic stirring (200
rpm), and during the reaction course the pH was kept constant
by automatic titration. The reaction was monitored by HPLC
analysis (10% acetonitrile, phosphate buffer 10 mM, pH 3.2,
flow 1 mL/min; UV detector 274 nm). When the maximum
yield was achieved, the reaction mixture was filtered and the
acylation product 3 (tR ) 10.6 min) was purified from the
unreacted â-lactam nucleus 2 on a Dowex 50 column eluted
with water. After lyophilization, compound 3 was identified
7
18 Stat Tritino from Metrohm (Herisau, Switzerland).
1H-Tetr azol-1-yl)-2-acetic Acid Meth yl Ester (4). TZAM
4) was prepared from (1H-tetrazol-1-yl)-2-acetic acid which,
in turn, was prepared by reaction of glycine with sodium azide
and ethyl orthoformate in acetic acid.21 Esterification was
accomplished with diazomethane.
1
13
(
by NMR analysis. The HPLC and H and C NMR analytical
data of the synthesized cefazolin (3) were in agreement with
those of an authentic sample.
(
Deter m in a tion of V
of Differ en t â-La cta m ic Nu clei (Sch em e 4). The V
S
a n d V 1
h
in th e Acyla tion Rea ction
/V 2
S
h
(
1H-Tetr a zol-1-yl)-2-a cetic a cid (TAA): mp 124-126 °C
ratios in the PGA-catalyzed acylation of 7-ACA (1) and 7-ZACA
(2) have been evaluated by measuring the initial rates of
synthesis (V ) and TZAM (4) hydrolysis (Vh2), respectively.
S
1
(
2-propanol); H NMR (DMSO-d
Anal. Calcd for C C, 28.13; H, 3.15; N, 43.74.
6
) 5.45 (s, 2H), 9.29 (s, 1H).
3
H
4
N
4
O
2
:
Found: C, 28.27; H, 3.39; N, 43.27. HPLC analysis: 10%
acetonitrile, phosphate buffer 10 mM, pH 3.2, flow 1.0 mL/
Following a general procedure, the ester 4 (0.0321 g) was
dissolved into a solution of the appropriate â-lactam nucleus
(50 mM) in 20 mL in phosphate buffer 10 mM. The enzyme
derivative of PGA (100 IU) was added into the substrate
solution at 4 °C under magnetic stirring (200 rpm). During
the reaction, the desired pH was kept constant by automatic
titration, and the reaction rates (µmol/min × mL of enzyme
derivative) were calculated from the NaOH consumption, the
min, UV detector 215 nm, t
R
) 2.99 min.
(
1H-Tetr a zol-1-yl)-2-a cetic Acid Meth yl Ester (4). The
acid (1.5 g) was dissolved in dry methanol (50 mL) and then
reacted with diazomethane to obtain, after solvent evaporation,
pure methyl ester 4 (1.63 g) in 73% overall yield: mp 50-52
1
°
C; H NMR (DMSO-d
6
) 3.81 (s, 3H), 5.66 (s, 2H), 9.48 (s, 1H);
+
Vh1, while the formation of the acylation products was evalu-
MS m/z (relative abundance) 142 (M , 84), 87 (61), 61 (12), 55
100). Anal. Calcd for C : C, 33.81; H, 4.26; N, 39.42.
Found: C, 33.87; H, 4.38; N, 39.35. GC analysis: t ) 14.03
min. HPLC analysis: 10% acetonitrile, phosphate buffer 10
ated by HPLC analysis: 2.5% acetonitrile phosphate buffer
(
4 6 4 2
H N O
1
1
)
0 mM, pH 3.2, flow 1.5 mL/min; UV detector 220 nm; tetrazol-
-ylacetic acid t ) 1.64; TZAM (4), t ) 4.47; cefazolin (3), t
25.12; compound 5, t ) 9.99.
En zym a tic Dea cyla tion of Cep h a losp or in C to 7-Am i-
R
R
R
R
mM, pH 3.2, flow 1.5 mL/min, UV detector 215 nm, t ) 4.13
R
R
min.
n ocep h a losp or a n ic Acid (1) (Sch em e 5). To a solution (50
mM) of cephalosporin C (0.415 g; 1 mmol) in 50 mL of
phosphate buffer 10 mM at pH 8 and 25 °C were added 0.2
mL of the enzyme derivative of DAO (20 IU) and 0.15 mL of
the enzyme derivative of GA (40 IU) under magnetic stirring
P r ep a r a tion of th e En zym e Der iva tives. Im m obiliza -
tion of P GA fr om E. coli A on Aga r ose Bea d s. Activation
of agarose gel by etherification with glycidol and oxidation with
periodate, and the further control of the PGA (amine)-agarose
(
aldheyde) multiple-point attachment, were performed as
1
6
2
(200 rpm). During the reaction, a continuous flow of O was
previously described by reaction at pH 10 and final reduction
of the imino double bonds with sodium borohydride.
maintained and the pH was kept constant by automatic
titration. The reaction was monitored by HPLC (2% acetoni-
trile, ammonium acetate buffer 20 mM, pH 3.2, flow 1.5 mL/
min; UV detector 274 nm). After 2 h, the complete deacylation
Im m obiliza tion of P GA fr om E. coli A on Eu p er git C.
Wet Eupergit C (2 g) was suspended under mechanic stirring
into 90 mL of phosphate buffer (1 M) at room temperature
and pH 8. A solution (10 mL) of PGA (200 IU/mL) was then
added to the suspension, and the reaction mixture was stirred
for 24 h. After filtration, the immobilized enzyme was washed
with distilled water and resuspended into 100 mL of a solution
of ethanolamine (0.1 M) in phosphate buffer (0.1 M) at pH 8
and room temperature. After 2 h under mechanical stirring,
the suspension was filtered and the enzyme derivative washed
with distilled water.
of cephalosporin C to 7-ACA (1) (t
and the reaction mixture was filtered. The solution of crude
-ACA (1) was used for the next reaction without purification.
R
) 3.24 min) was achieved,
7
For identification product 1 was isolated by acidification to
pH 4.5 and filtration of the white crystals obtained. The HPLC
1
13
and H and C NMR analytical data of the synthesized 7-ACA
1) corresponded with those of an authentic sample.
(
(22) Fern a´ ndez-Lafuente, R.; Rossel, C. M.; Rodriguez, V.; Santana,
C.; Soler, G.; Bastida, A.; Guis a´ n, J . M. Enzyme Microb. Technol. 1993,
15, 546.
(21) Takshi, K.; Yoshihisa, S. U.S. Patent 3,767,667, 1973.