Anti-Infectives from Nature - Natural Products Chemistry: Sources, Separations, and Structures - page 97

R 2

H

R 1

N

H

R

S

CH 3

R 3

N

O N CH 3

O

O

COOH
Carbapenem

COOH

Penicillin

FIGURE 9.15 Structural features of the rings comparing carbapenem to penicillins.

9.11 STRUCTURE DETERMINATION AND STABILITY
OF CARBAPENEM
The reasons for the higher reactivity of the β-lactam ring in carbapenems is explained
by differences in their structure shown in Figure 9.15. The carbon atom substituted
sulfur bonding in penicillin results in a more rigid ive-membered ring. When a
double bond is formed between C2 and C3 and conjugated with the lone pairs on the
nitrogen atom, this conjugation forces the original “envelope-like” bicyclic system to
be on the same plane. This leads to further ring strain, which in turn increases the
β-lactam activity. However, as noted, a problem of stability occurs. For final struc-
ture determination a stable derivative of thienamycin was prepared of the N-acetyl
methyl ester by crystallization from acetonitrile-benzene-hexane and suitable for
X-ray crystallographic analysis.

9.12 ISOLATION OF THIENAMYCIN
The most difficult part of isolating thienamycin from fermentation broth is the hydro-
lytic instability of the carbapenem nucleus. The pKa of the carboxylate is around 3.1,
while the amino group is larger than 8. It is zwitterionic at neutral pH. Under acidic
conditions (pH 2 to 5) instability is similar to penicillin G. As the pH increases, the
hydrolysis rate of the lactam bond immediately accelerates.
In the isolation of thienamycin, UV monitoring coupled to high-performance liq-
uid chromatography (HPLC) was effective in detecting the presence of three carbap-
enems with the most abundant being thienamycin (UV λ max 297nm).
The researchers took advantage of the zwitterionic nature of this carbapenem and
applied ion exchange column chromatography. The application of a weak acid anion
exchange column Dowex 1x2 (HCO 3 - ) with elution using cold carbonic acid as the
eluent at a pH ~5 was successful.
Another approach, since the target compound is zwitterionic, is to use an ionic
extraction for the first step from the fermentation broth. The advantage of ionic extrac-
tion with an ion-pairing reagent is the feasibility of large-scale processing using very
short residence times for thienamycin at unfavorable pH values. Furthermore, ion
exchange chromatography is not practical for large-scale isolation due to the cost.
The ion-pairing extraction provides a cheaper and even more effective way in the iso-
lation of thienamycin. The ion-pairing reagent chosen was dinonylnaphthalene sul-
fonate (DNNS) from acidic broth and methyl tricapryl ammonium bicarbonate from
basic broth.