inhibit the enzyme. Here we describe the synthesis of two such
analogues, 6-methyl-PBG 19 and 6,11-ethano-PBG 22.
catalysed cleavage of the benzyl group, iodinative decarboxyl-
ation of acid 15 and then hydrogenolysis of the iodide 16. The
formyl group of 17 was converted to the amine hydrochloride
18 by hydrogenation of the corresponding oxime (69% over
two steps). Finally hydrolysis of the esters with NaOH gave
6-methyl-PBG 19, isolated as its mono-ammonium salt in 91%
yield after treatment with Dowex 50 (NH4ϩ form).
A review of published syntheses of PBG6 suggested that the
easiest route to 6-methyl-PBG 19 would be a modification of the
Knorr pyrrole synthesis route first reported by Kenner et al.7
Thus treatment of the β-ketoadipate diester 9 with NaNO2 in
acetic acid–water, followed by reduction of the resulting oxime
with zinc in acetic acid and concomitant condensation with
pentane-2,4-dione 8 results in the pyrrole 10 (Scheme 3).7
The 6,11-ethano derivative 22 of PBG (Scheme 4) was syn-
Scheme 4
thesised in the same way from β-ketoadipate diester 9 and
cyclohexane-1,3-dione 20, with similar yields all through the
synthesis. The only significant difference was that the oxidation
of the α-methylene group to a ketone was best performed using
ceric ammonium nitrate10 (84% yield) instead of sulfuryl chlor-
ide. In the reduction of the oxime that generates the amine 21,
two diastereoisomers could be formed. In fact only one product
was observed (84% isolated yield), which was proved using a
NOESY spectrum to have the cis orientation of the amino and
ester groups. Presumably the hydrogenation occurs from the
opposite side to the methoxycarbonyl group because of steric
hindrance from this group. The NOESY spectrum showed that
the methoxycarbonyl group is predominantly in an axial pos-
ition, where it avoids an allylic 1,3-interaction with the adjacent
propionate side-chain, and for this reason it would be very
hindering of the top face of the carbocyclic ring.
The two PBG analogues were tested11 as inhibitors of HMBS
from Escherichia coli (kindly supplied by Dr N. P. J. Stamford).
Whereas the 6,11-ethano-PBG 22 showed no inhibition of the
enzyme at concentrations up to 900 µM, 6-methyl-PBG 19
showed significant inhibition in the 2 to 10 µM range. Accord-
ingly assays were performed with a range of concentrations
of both substrate (PBG) and inhibitor (6-methyl-PBG). The
Michaelis–Menten plots of this data show that the inhibitor
both decreases the Vmax value and increases the Km value of the
enzymic reaction. This indicates mixed inhibition, which is not
surprising given the complexity of the reaction mechanism. A
Dixon plot of the data† gave convergent lines with a reasonably
good intersection point corresponding to an apparent Ki value
of 3 µM (cf. Km for PBG = 20–40 µM). Before this the best
reported inhibitor of HMBS was 9-fluoro-PBG with an appar-
ent Ki value of 6 µM.11 It would be interesting to determine
whether this inhibition is primarily due to only one of the two
enantiomers of 6-methyl-PBG 19, as one might expect.
The inhibition caused by 19 might be simply due to non-
covalent binding, but equally well it might be due to covalent
attachment to the dipyrromethane cofactor, in the same way as
PBG normally binds, if this causes subsequent steps to be
slower. To investigate this HMBS was incubated with three
molar equivalents of 6-methyl-PBG 19 for 20 min and the
sample then injected onto a MonoQ FPLC column and eluted
under conditions known11 to separate the various enzyme–
Scheme 3 Reagents: i, NaNO2 then Zn, AcOH; ii, NaBH4 then Ac2O,
pyridine; iii, Me3SiCN, TiCl4; iv, MeOH, HCl; v, SO2Cl2; vi, H2SO4,
TFA; vii, I2, KI, NaHCO3; viii, H2, Pt2O; ix, NH2OH then Pd, HCl;
x, NaOH then Dowex 50 (NH4ϩ).
In Kenner’s synthesis of PBG the acetyl group of 10 was
rearranged to a methoxycarbonylmethyl group using thallium
trinitrate. Here we want to keep the methyl group of the acetyl
side-chain and introduce a methoxycarbonyl group in place of
the carbonyl. This was achieved by initial reduction of the
ketone and acetylation to give the acetate 11 (58%). The key
step then was treatment of 11 with trimethylsilyl cyanide and
titanium tetrachloride, which results in replacement of the
acetoxy group by cyanide in 92% yield, presumably via an SN1
mechanism. The cyano group of 12 was then methanolysed
using hydrogen chloride in methanol to give the methyl ester 13
(73%), with transesterification of the ethyl ester also occurring.
The remainder of the route to 6-methyl-PBG 19 followed
published procedures for the synthesis of PBG and related
compounds.7–9 Thus the α-methyl group of 13 was oxidised to a
formyl group 14 in 74% yield by chlorination with sulfuryl
chloride followed by hydrolysis of the dichloride. The benzyl
ester was removed in three steps and 71% overall yield by acid-
substrate complexes. The elution profile, measuring A280
,
showed clearly the formation of three complexes in addition to
some remaining native holoenzyme (Fig. 1). Enzymic assay
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 – 2 3
22