Biosynthesis of porphyrins and related macrocycles. Part 52.1'2 Synthesis of 1-SH11-4Hjporphobilinogen and the (11R)enantiomer for stereochemical studies on hydroxymethylbilane synthase (porphobilinogen deaminase)

Article abstract of DOI:10.1039/a904262h

A synthetic route is devised for the synthesis of (115)-[11-²H,]porphobilinogen la and of the (1 lβ)-enantiomer Ib. Their absolute configurations and enantiomeric purity are established by degradation to a derivative of [2-2H,]glycine of known stereochemistry. Methods are then developed, based on the synthesis of chiral imidate esters, for determination of the configuration of [₂H₁]-labelled aminomethylpyrroles by converting them into [2H,]-labelled amidines followed by analysis using 'H-NMR. The labelled samples of PEG la and Ib serve as substrates for hydroxymethylbilane synthase and the products are trapped as [²H₁]-labelIed aminomethylbilanes 7c and 7d. Their configurations are determined by the NMR assay to demonstrate that as PBG 1 is enzymically converted into the aminomethylbilane 7, there is overall retention of configuration at the aminomethyl carbon. The Royal Society of Chemistry 1999.

Full text of DOI:10.1039/a904262h

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Biosynthesis of porphyrins and related macrocycles. Part 52.1,2
Synthesis of (11-S)-[11-2H1]porphobilinogen and the (11R)-
enantiomer for stereochemical studies on hydroxymethylbilane
synthase (porphobilinogen deaminase)
Werner L. Neidhart, Paul C. Anderson, Graham J. Hart and Alan R. Battersby*
University Chemical Laboratory, Lensfield Road, Cambridge, UK CB2 1EW
Received (in Cambridge, UK) 27th May 1999, Accepted 26th July 1999
A synthetic route is devised for the synthesis of (11S)-[11-2H1]porphobilinogen 1a and of the (11R)-enantiomer 1b.
Their absolute configurations and enantiomeric purity are established by degradation to a derivative of [2-2H1]glycine
of known stereochemistry. Methods are then developed, based on the synthesis of chiral imidate esters, for
determination of the configuration of [2H1]-labelled aminomethylpyrroles by converting them into [2H1]-labelled
amidines followed by analysis using 1H-NMR. The labelled samples of PBG 1a and 1b serve as substrates for
hydroxymethylbilane synthase and the products are trapped as [2H1]-labelled aminomethylbilanes 7c and 7d. Their
configurations are determined by the NMR assay to demonstrate that as PBG 1 is enzymically converted into the
aminomethylbilane 7, there is overall retention of configuration at the aminomethyl carbon.
All the natural porphyrins, chlorins, corrins and their relatives
are biosynthesised3 from the same parent, uroporphyrinogen
III 9, shortened to uro’gen III, Scheme 1. This is built from
porphobilinogen 1, PBG, by the sequential action of two
enzymes. The first, hydroxymethylbilane synthase, E.C.4.3.1.8
(formerly called PBG deaminase) assembles four PBG units
1 head-to-tail to generate hydroxymethylbilane 6. This is
then converted into uro’gen III 9 by a remarkable ring-
closure and rearrangement process catalysed by the second
enzyme, uroporphyrinogen III synthase, usually called
cosynthetase.3
Results and discussion
Synthesis of (11S)-[11-2H1]PBG 1a and its (11R)-enantiomer 1b
The synthetic route to the labelled samples of PBG 1a and 1b is
shown in Scheme 2. The first approach was subsequently super-
seded but it will be briefly described, without experimental
detail, as it led to helpful findings. It involved condensation
of the hydrazine 11a, derived8 from commercially available
(Ϫ)-(1R,2S)-ephedrine, with the deuteriated aldehyde9 12a;
the unlabelled aldehyde 12 had been prepared earlier.10 The
hydrazone 13c was reduced by cyanoborohydride with toluene-
p-sulfonic acid as the proton source to yield the hydrazine con-
taining 14a and 14b. An unlabelled sample 14 prepared in the
same way from the aldehyde 12 gave well separated signals in its
1H-NMR spectrum from the two diastereotopic protons of the
RNHCH2-group. The method for analysis of the products in
the 2H-labelled series was thus available and the foregoing
labelled hydrazine was shown in this way to consist of the two
diastereoisomers 14b and its epimer at C-11 14a in the ratio
4:1. Proof that the major one was 14b will be given later.
The improved synthetic route started with unlabelled hydra-
zone 13a (97% yield from 11a and 12) which was reduced with
cyanoborodeuteride, importantly in the presence of deuteriated
toluene-p-sulfonic acid11 (ArSO3DؒD2O). When ArSO3Hؒ
H2O was used, the product contained 40–50% of unlabelled
material, no doubt due to the reducing agent undergoing rapid
exchange of H for D.12 Analysis of the product from the former
conditions showed surprisingly that it contained equal amounts
of the two diastereoisomers 14a and 14b. It turned out that
this was a result of the larger quantities and so higher concen-
trations used initially for the improved route as compared with
those for the first approach. Study of this concentration effect
led to optimum conditions being selected for the reduction
Earlier studies3 showed that hydroxymethylbilane synthase is
an unusual enzyme in that it binds its substrate covalently and,
surprisingly, that the first PBG unit becomes attached to a novel
dipyrrolic cofactor 2 present in the enzyme and covalently
bound to it. Thus is formed the ES1 complex 3. Three further
molecules of PBG are then added stepwise to generate the ES2,
ES3 and finally the ES4 complex 4 from which hydroxymethyl-
bilane 6 is released so regenerating the unloaded enzyme 2 for a
further cycle. There is strong evidence4 that release of the bilane
6 from the ES4 complex 4 occurs via the azafulvene 5. This then
reacts with water to form the hydroxymethylbilane 6 but it can
also be trapped as 7 or 8 by including the better nucleophiles
ammonia or hydroxylamine, respectively, in the incubation
medium.4,5 In addition, there is support for the view that the
four interpyrrolic methylene groups of uro’gen III 9 are gener-
ated in a stereospecific way6,7 but at none of them is it known
whether the building process from PBG 1 involves overall
retention or inversion of configuration.
The aim of the work described in the present paper was to
determine the overall stereochemistry of the steps by which
hydroxymethylbilane synthase generates the aminomethyl
group of the bilane 7 formed by trapping the azafulvene 5 with
ammonia. The amine 7 was chosen for this initial foray because
it cyclises non-enzymically to uro’gen I 10 much less rapidly
than does the hydroxymethylbilane 6 thus allowing sufficient
quantities of material to be accumulated for study by NMR.
The first requirement was the development of syntheses of
PBG (as 1) having C-11 labelled stereospecifically, or at least
stereoselectively, with an appropriate isotope.
1
which afforded a 92% yield of product shown by H-NMR to
contain only 5% of undeuteriated molecules and of the deuteri-
ated material, 83% was shown later to be 14a and 17% was the
isomer 14b.
A strictly complementary synthesis starting with the alde-
hyde 12 and the hydrazine 11b, prepared from (ϩ)-(1S,2R)-
ephedrine, yielded a mixture of the diastereoisomers 14c and
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This journal is © The Royal Society of Chemistry 1999
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Scheme 1
14d. The former will be shown below to make up 83% of the
2H1]PBG lactam ester 16b which made up 83% of the deuteri-
deuteriated materials which again formed 95% of the total
product. The diastereoselectivity achieved in the two foregoing
reductions was thus amply high enough to allow the problem
outlined in the introduction to be solved. From now on, in
order to avoid constant repetition, it will be taken as under-
stood that in all cases the major enantiomer or diastereoisomer
present is being discussed.
The stereochemical control realised in the foregoing syn-
theses seems to depend on two factors. a) One stereoisomer of
the hydrazone 13a is formed as shown by chromatographic
homogeneity and by NMR; steric arguments suggest that it is
the illustrated E-isomer and b) good stereocontrol in the reduc-
tion probably involves some form of complex between the
reducing agent and the hydrazone, in keeping with the striking
dilution effect, which for 13a directs the transfer of deuteride to
ated product, the rest being the (11S)-enantiomer 16a.
In parallel with the foregoing experiments, we also studied
the use of (S)-1-amino-2-(methoxymethyl)pyrrolidine13
(SAMP) 17 as the chiral auxiliary. The chemistry followed
closely that described above and the reaction sequence via the
hydrazone 18 is shown in Scheme 3. 1H-NMR showed that the
hydrazine 19a contained 95% deuteriated material of which one
diastereoisomer made up 78% and the other 22%. The previous
approach gave a higher stereoselectivity and the hydrazine 19a
proved to be unstable so the effort focused on the route using
ephedrine.
Determination of the absolute configuration of the 2H-labelled
samples of PBG
the si-face of the C᎐N bond.
The plan was to degrade the PBG lactam ester 16a to a
derivative of glycine. Alkaline hydrolysis of 16a yielded (11S)-
[11-2H1]PBG 1a, Scheme 4, which without isolation was con-
verted by two-phase acylation with (Ϫ)-(1S,4R)-camphanyl
chloride 20 into the amide 21a. After treatment of the products
with diazomethane, the diester 22a was spread on silica14 and
oxidised with ozone to break down the pyrrole ring. The
products were esterified with diazomethane and N-camphanyl
glycine methyl ester was isolated from the mixture by chrom-
᎐
Advantage could now be taken of the susceptibility of N–N
bonds to reductive cleavage. When the hydrazone 14a was
hydrogenated over a substantial amount of platinum in acetic
acid–methanol, it was rapidly cleaved to afford the dimethyl
ester of PBG 15a as its acetate salt. Heating this product in
methanol with sodium carbonate caused cyclisation to the
crystalline (11S)-[11-2H1]PBG lactam ester 16a containing ca.
17% of the (11R)-enantiomer 16b. The overall yield of the
lactam esters was 65% from the aldehyde 12.
1
atography. Amarego et al.15 had assigned the H-NMR signals
Similarly, the foregoing hydrazine 14c afforded (11R)-[11-
from the diastereotopic protons of the unlabelled amide 23
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Scheme 2
Scheme 3
showing that HR gives the higher field signal. Analysis of our
product in this way proved it was N-camphanyl-(2S)-[2-2H1]-
glycine methyl ester 23a and therefore the original lactam had
the (11S)-configuration 16a.
The same degradative sequence was carried out on the lactam
16b generated from the enantiomeric ephedrine derivative 11b.
NMR analysis of the glycine ester derived from the amide 22b
gave interlocking strength by proving it was N-camphanyl-(2R)-
[2-2H1]glycine methyl ester 23b. Thus the original PBG lactam
ester was the [11R]-enantiomer 16b.
The foregoing degradative methods (and several unsuccessful
ones) were studied first using unlabelled PBG 1 which was thus
required in substantial amounts. It was best prepared from the
oxime of pyrrole 12 by formation of the oxime hydrochloride
followed by hydrogenation over palladium to yield PBG
dimethyl ester 15 as its hydrochloride salt, Scheme 2. Similar
hydrogenation of the oxime free base gave much lower yields.
The hydrochloride of 15 was then treated with methanolic
Scheme 4
sodium methoxide to afford PBG lactam methyl ester 16, the
precursor of PBG 1, in 63% overall yield from 12.
Synthesis of the (S-aminomethyl )bilane 7a and the (R-amino-
methyl )-isomer 7b; development of the stereochemical assay
The aminomethylbilane 7 had been synthesised previously10,16
in unlabelled form, the building blocks and a key intermediate
24 being shown in Scheme 5. This route was followed here and
the experimental details now reported include some improve-
ments and also fuller characterisations. Replacing unlabelled
PBG lactam ester 16 used in the earlier work by (11S)-[11-
2H1]PBG lactam ester 16a from above led to the (S-amino-
methyl)bilane 7a. Similarly, (11R)-[11-2H1]PBG lactam ester
16b afforded the (R-aminomethyl)-isomer 7b.
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Scheme 5
It was now necessary to attach a chiral residue to the amino-
methyl group of each of the foregoing standard bilanes of
known absolute configuration to set up diastereoisomers for
1
assay by H-NMR. Though simple in principle, this proved to
be extremely difficult in practice because a) the bilanes readily
undergo non-enzymic ring closure to uro’gen I 10 and b) the
derivatisation must be done in water to reproduce the
conditions to be met subsequently with the bilanes produced
enzymically. It is not necessary to report the many unsuccessful
conditions and reagents tested. Success came by taking advan-
tage of the smooth reaction between imidate esters and primary
amines at ca. pH 10 in water to generate amidines; this reac-
tion has been used to modify amino groups of proteins.17
Synthesis and reactions of chiral imidate esters
The key starting material for synthesis of an imidate ester is the
corresponding nitrile and for the four cases studied, Scheme 6,
this was either available or it was prepared from the acid via the
amide which was dehydrated. For synthesis of 26 and its (2R)-
enantiomer 26a, treatment of the nitrile with hydrogen chloride
in dry ethereal methanol afforded the required imidate ester
hydrochloride in good yields. When these conditions were
applied to the nitrile 27, a high yield of the amide 28 was
obtained. However, treatment of this nitrile 27 with methoxide
in dry methanol smoothly yielded the imidate ester 29 as the
free base. These conditions were also successful for preparation
of the ester 31 from the nitrile 30 but a substantial by-product,
C10H16O3, was isolated showing spectroscopic data in agree-
ment with structure 32. A rationalisation of its formation is
shown in Scheme 6.
The fourth imidate ester we required was 34, the correspond-
ing nitrile being (ϩ)-(2R)-mandelonitrile 33. This has been
prepared by acidic hydrolysis of natural (ϩ)-amygdalin18 but
unfortunately the experimental section of the paper does not
report either the solvent used or the concentration of acid. It
was therefore important to demonstrate that the conditions now
described had yielded enantiomerically pure mandelonitrile 33
and that no significant racemisation had occurred during the
formation of the imidate ester 34 or in our use of the latter for
the preparation of amidines. Our concern was reinforced by the
reported racemisation of some chiral amidines.19 The necessary
proof came from reacting the imidate ester 34 with unlabelled
Scheme 6
PBG 1 and with the (11S)-, and (11R)-labelled forms, 1a and 1b
respectively, followed by analysis by NMR; this preparative
work is described more fully below. The argument is best pre-
sented using structure 35, Scheme 6, making a simplifying
assumption that the starred centre and the hydroxy bearing
centre are both enantiomerically pure. The 1H-NMR signal
from the hydrogen at the starred centre (after hydrogens at
exchangeable positions have been replaced by deuterium) will
appear as a singlet. However, if the hydroxy bearing centre has
been fully racemised, two separate singlets of equal size will be
seen and the position where the second signal would appear is
known from the spectrum shown by the amidine derived from
unlabelled PBG 1. Partial racemisation would give a result in
between the foregoing extremes. It will be evident that even
though the labelled samples of PBG 1a and 1b do not contain
100% of one enantiomer but 83%, the assay by NMR can be
done and it showed that 33 and 34 were enantiomerically pure,
within the limits of detection by NMR, and that the final
amidines were configurationally stable.
The next step was to work out a) the best conditions for using
the foregoing imidate esters to form amidines from amino-
methylpyrroles under aqueous conditions and b) which of the
reagents 26, 29, 31 and 34 formed a handleable amidine that in
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Scheme 7
1
addition gave good discrimination by H-NMR between the
diastereotopic protons of the aminomethyl group. Experiments
were carried out on PBG 1, the dipyrromethane 43 and finally,
the aminomethylbilane 7. Since these reactions were carried
out at ca. pH 10 and imidate esters have a pKa of ca. 6,20 the
reagents could be used directly either as free bases or as hydro-
chlorides. First it was shown that the reaction at room temper-
ature of a simple achiral imidate ester 36 with PBG 1, Scheme
7, was complete in 3 h as shown by TLC on cellulose. After
esterification of the disodium salt 37 with dimethyl sulfate,
a mono-ester 38 or 39 could be extracted for spectroscopic
characterisation. In contrast, there was no detectable reaction,
as judged by TLC and NMR, between PBG 1 and the imidate
esters 29 and 31 probably due to steric hindrance. A large
amount of unchanged 29 and 31 was recovered in each case so
the failure was not due to hydrolysis of the reagents. However,
the imidate ester 26 did react with PBG 1 to yield the racemic
amidine (as 40), Scheme 7, and the diastereotopic protons at the
A step up in complexity led to the known dipyrrolic lactam21
42 which was hydrolysed21 to form the aminomethyldipyrro-
methane 43, Scheme 7. This reacted with the racemic imidate
ester 26 under the conditions described above to form the
amidine 44 which showed an AB multiplet in its 1H-NMR spec-
trum from the starred centre. Similarly, 43 was converted by 34
into the amidine 45. Now the starred centre gave a poorly
resolved 1H-NMR signal but, interestingly, the signal from the
interpyrrolic methylene group was a well resolved AB multiplet
despite its being 8 atoms away from the chiral centre.
The foregoing experiments set the stage for work on the
aminomethylbilanes 7, 7a and 7b; syntheses of the latter two
were described above (see also Scheme 5). There was immediate
disappointment in that the imidate ester 26 failed to react with
the bilane 7. However, the other imidate 34 reacted smoothly
and converted the (S-aminomethyl)bilane 7a into the (S)-
amidine 25a and the (R-aminomethyl)bilane 7b into the (R)-
isomer 25b, Scheme 5. Fig. 1(b) shows the 1H-NMR signal from
the starred centre of the R-sample 25b with the large signal to
higher field and the complementary result in Fig. 1(a) shows the
large signal at lower field from the (S)-sample 25a. To ensure
that no external factors had affected the chemical shifts, the
(S)-amidine 25a was mixed with a larger amount of the
(R)-amidine 25b to give the NMR pattern shown in Fig. 2(c). A
thoroughly reliable method of assay was thus available for
determination of the absolute configuration of labelled bilanes
from the enzymic experiments.
1
starred centre were distinguishable by H-NMR at 400 MHz
(AB multiplet). This encouraged study of the reaction of the
(2R)-imidate ester 26a with the (11S)-, and (11R)-labelled
1
forms of PBG, 1a and 1b, respectively. The H-NMR signal
from the starred centre of the (11S)-product 40a appeared at
lower field than that from the (11R)-sample 40b. Finally, the
imidate ester 34 was used to convert PBG 1 into the amidine 41
which showed a well resolved AB multiplet from the starred
centre in its 1H-NMR spectrum. Analogous preparation of the
amidines 41a and 41b from (11S)- and (11R)-labelled PBG 1a
and 1b, respectively, demonstrated that the starred centre of the
Enzymic formation of the labelled bilanes 7c and 7d
(11S)-[11-2H1]PBG 1a was incubated in the presence of 0.2 M
1
(11S)-amidine 41a gave a H-NMR signal at lower field than
that from the (11R)-isomer 41b.
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Scheme 8
ammonium chloride with amply enough hydroxymethylbilane
synthase to convert >95% of 1a in 0.5 h into the aminomethyl-
bilane,22 Scheme 8. This was rendered more stable by raising the
pH to >13 and excess ammonia was then removed by two
rounds of freeze drying. Addition of the imidate ester 34 then
converted the bilane into the amidine which displayed the
1H-NMR signal from the starred centre shown in Fig. 2(a);
the large signal appeared at low field. This matched Fig. 1(a)
proving that this amidine had the (S)-configuration 25c
formed from the (S-aminomethyl)bilane 7c.
Fig. 1 1H-NMR signals (400 MHz) from the starred centre of (a) the
(S)-amidine 25a and (b) the (R)-amidine 25b. Both spectra were run in
CH3OD.
This entire enzymic preparation was repeated starting from
(11R)-[11-2H1]PBG 1b and now the final amidine showed the
large 1H-NMR signal at high field, Fig. 2(b), matching the
standard spectrum in Fig. 1(b). It followed that this was the
(R)-amidine 25d derived from the (R-aminomethyl)bilane 7d. In
order to leave no doubt about the signal assignments, a small
amount of the (R)-amidine 25d was added to the (S)-amidine
25c. This caused the small high field signal seen in Fig. 2(a) to
increase in size and addition of a second portion of the (R)-
amidine 25d resulted in a further increase in the size of this
signal.
These results proved that (11S)-PBG 1a had been converted
into (S)-bilane 7c and the finding that (11R)-PBG 1b had yield-
ed (R)-bilane 7d added complementary strength. It is thus cer-
tain that when hydroxymethylbilane synthase acts on PBG 1
and the tetrapyrrole is trapped as the aminomethylbilane 7, the
overall stereochemical outcome at the aminomethyl centre is
retention of configuration. Further discussion of this finding is
best deferred until the conclusion of the following paper23 when
the results for the aminomethylbilane 7 and the hydroxymethyl-
bilane 6 can be considered together.
Experimental
General
For general directions, see ref. 16.
Synthesis of (11S)-[11-2H1]porphobilinogen lactam methyl ester
16a and the (11R)-[11-2H1]enantiomer 16b
Fig. 2 1H-NMR signals (400 MHz) from the starred centre of (a) the
(S)-amidine 25c from the enzymic experiments, (b) the (R)-amidine 25d
from the parallel enzymic run, and (c) a mixture of the synthetic (S)-
amidine 25a with a larger quantity of the synthetic (R)-amidine 25b.
(a) (؊)-N-Aminoephedrine 11a and (؉)-N-aminoephedrine
11b. To a cooled (0 ЊC) stirred solution of (Ϫ)-ephedrine hydro-
chloride (12 g, 0.06 mol) in water (50 cm3) and 2 M hydrochloric
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acid (25 cm3) was added over a period of 90 min a solution of
sodium nitrite (8.4 g, 0.12 mol) in water (50 cm3). The mixture
was stirred at 0 ЊC for 1 h to complete the precipitation of the
product which was collected and dried to give (Ϫ)-N-nitroso-
ephedrine (11.4 g, 98%), mp 90 ЊC (lit.,8 92 ЊC). νmax (Nujol)/
cmϪ1 3350, 2840 and 1445 (NO); δH (80 MHz) 1.45 (3 H, d,
J 6.9, CHCH3), 2.4 (1 H, br s, OH), 2.93 (3 H, s, NCH3), 4.67
(1 H, dq, J 5 and 6.9, CHCH3), 5.03 (1 H, d, J 5, CHOH), 7.32
(5 H, s, ArH).
Mϩ Ϫ C7H7O, 311.1819. C15H23DN3O4 requires M Ϫ C7H7O,
311.1829, 95 atom% D); νmax/cmϪ1 3300 (br) and 1765; δH (400
MHz) 0.85 (3 H, d, J 6.6, CHCH3), 2.54 (2 H, t, J 6.6,
CH2CH2CO2), 2.55 (3 H, s, NCH3), 2.76 (3 H, m, CH2CH2CO2
and CHCH3), 3.46 (2 H, ABq, J 13.4, CH2CO2), 3.63 and 3.68
(2 × 3 H, 2 × s, 2 × OCH3), 3.9 [0.16 H, s, (11R)-NCHD], 4.03
[0.79 H, s, (11S)-NCHD], 5.2 (1 H, d, J 2, CHOH), 6.5 (1 H, d,
J 2.6, pyrrole-H) 7.3 (5 H, m, ArH) and 8.45 (1 H, br s, NH);
m/z (FD) 419 (Mϩ ϩ 1, 100%) and 418 (Mϩ, 29%).
To a stirred mixture of zinc (18 g) in water (90 cm3) at 0 ЊC
was added dropwise over a period of 30 min a solution of
(Ϫ)-N-nitrosoephedrine (11.64 g, 0.06 mol) in acetic acid (60
cm3). The mixture was stirred for 1 h at room temperature and
then 1 h at 80 ЊC. The precipitate was filtered off, the filtrate
concentrated to about half volume, benzene (90 cm3) was added
and the solution was made alkaline by addition of saturated
aqueous potassium carbonate. The equilibrated phases were
separated, the aqueous phase extracted several times with ben-
zene and the combined organic phases dried. Evaporation of
the solvent gave (Ϫ)-N-aminoephedrine 11a (8.3 g, 76%) as a
yellow oil which was essentially pure and was distilled in vacuo,
bp 138–142 ЊC/8 mmHg (lit.,8 138–150 ЊC/8 mmHg). νmax/cmϪ1
3300 (br) and 2950; δH (80 MHz) 0.82 (3 H, d, J 6.7, CHCH3),
2.57 (3 H, s, NCH3), 2.74 (1 H, d q, J 2.1 and 6.7, CHCH3), 2.3–
3.5 (3 H, br s, OH and NH2), 5.2 (1 H, d, J 2.1, CHOH), 7.3
(5 H, m, ArH).
The enantiomer 14c, prepared from 13b as above, gave
identical spectroscopic data.
The unlabelled hydrazine 14 was prepared as above from 13a
using sodium cyanoborohydride; δH (400 MHz, in part) 3.86
(1 H, d, J 13.4, HS of NCH2) and 4.02 (1 H, d, J 13.4, HR of
NCH2), all other signals as for 14a; m/z (FD) 418 (Mϩ ϩ 1,
100%) and 417 (Mϩ, 40%).
(d) (11S)-[11-2H1]PBG lactam methyl ester 16a and the (11-
R)-enantiomer 16b. All solvents used in this preparation were
deoxygenated and saturated with argon by heating for 2 h under
reflux then distillation, both under an argon stream. The hydra-
zine 14a (2.4 g, 5.74 mmol) in dry THF (10 cm3) was added
by syringe to pre-reduced Adam’s catalyst (0.5 g) in methanol
(100 cm3) and acetic acid (15 cm3) and the mixture was stirred
under hydrogen at room temperature until uptake ceased (ca.
1 h). The filtered solution was evaporated and the residue
in dichloromethane–methanol (9:1, 50 cm3) was shaken with
saturated aqueous sodium carbonate. The aqueous phase was
extracted several times with dichloromethane–methanol (9:1)
and the combined organic layers were dried and evaporated.
The residue in methanol (25 cm3) was heated under reflux for
1 h to form PBG lactam methyl ester which started to precipi-
tate. Crystallisation was completed by keeping the mixture at
Ϫ10 ЊC for 2 h. The product was collected by centrifugation,
washed with methanol and dried to afford the (11-S)-[11-2H1]-
lactam 16a (830 mg, 65%) as off-white prisms, mp 247–249 ЊC
(lit.,24 248–249 ЊC)(Found:Mϩ,223.1048.C11H13DN2O3 requires
M, 223.1067); δH (400 MHz) 2.54 and 2.72 (2 × 2 H, 2 × m,
CH2CH2CO2), 3.41 (2 H, d, J 3.3, CH2CONH), 3.66 (3 H,
s, OCH3), 4.47 (1 H, br s, NCHD), 5.92 (1 H, br s, HNCHD),
6.55 (1 H, d, J 1.8, pyrrole-H) and 7.72 (1 H, br s, NH); m/z 223
(Mϩ, 100%), 222 (62, M Ϫ 1), 192 (11, M Ϫ CH3O) and 150
(65, M Ϫ C3H5O2).
(ϩ)-N-Aminoephedrine 11b was prepared as above from (ϩ)-
ephedrine.
(b) The enantiomeric hydrazones 13a and 13b. The aldehyde
1224 (3.5 g, 13.8 mmol) and (Ϫ)-N-aminoephedrine 11a (5 g,
27.8 mmol) were dissolved in dry benzene (150 cm3). Molecular
sieves (4 Å, 3 g) were added and the solution was heated at
reflux for 2 h under argon. The filtered solution was evaporated
and the product was chromatographed on silica (0.04–0.063
mm, 3 × 20 cm) using diethyl ether as eluant to give the hydra-
zone 13a (5.58 g, 97%) as an oil (Found: Mϩ, 415.2111.
C22H29N3O5 requires M, 415.2107); νmax/cmϪ1 3375 (br) and
1720; δH (100 MHz) 1.06 (3 H, d, J 6.7, CHCH3), 2.68 (4 H, m,
CH2CH2CO2), 2.86 (3 H, s, NCH3), 3.39 (1 H, dq, J 3 and 6.7,
CHCH3), 3.54 (2 H, s, CH2CO2), 3.68 (6 H, s, 2 × OCH3), 4.30
(1 H, br s, OH), 5.20 (1 H, d, J 3, CHOH), 6.51 (1 H, d, J 2.5,
pyrrole-H), 7.3 (6 H, m, ArH and HC᎐N), 8.56 (1 H, br s, NH);
᎐
m/z 415 (Mϩ, 5%), 384 (3), 308 (100, M Ϫ C7H7O).
The enantiomer 13b prepared in the same way from 11b
showed identical spectroscopic properties.
The enantiomer 16b was prepared in the same way from 14c
(Found: Mϩ, 223.1053, C11H13DN2O3 requires M, 223.1067);
all other data as for 16a.
(c) The enantiomeric hydrazines 14a and 14c and the
unlabelled analogue 14. The acidic protons of the hydrazone 13a
were exchanged by treatment of it (2.7 g, 6.5 mmol) in tetra-
hydrofuran (THF, 15 cm3) with D2O (5 cm3, 99.8 atom%) for 30
min. The reaction mixture was evaporated to dryness and the
procedure was repeated once more. A solution of the final resi-
due in THF (2 dm3) together with sodium cyanoborodeuteride
(1 g, 15.2 mmol, 98 atom% D) and a few milligrams of bromo-
cresol green was stirred under argon in a three-necked flask
equipped with a dropping funnel. A solution of O-deuteriated
toluene-p-sulfonic acid mono(deuterium oxide)11 (2.7 g, 14
mmol, 99.2 atom%) in THF (180 cm3) was added at room tem-
perature during 90 min to maintain pH 3.5, indicated by the tan
colour of the indicator. The sodium salt of toluene-p-sulfonic
acid precipitated during the reaction. After the mixture had
stirred for 30 min more, it was filtered, the solvent was removed
in vacuo and the residue in CH2Cl2 (60 cm3) was washed with
saturated aqueous sodium hydrogen carbonate. The residue
from evaporation was chromatographed on silica using ethyl
acetate–diethyl ether (8:2) as eluant to give the hydrazine 14a
(2.5 g, 92%) as a pale yellow oil. This product was stable under
argon in the deep freezer for several months but rapidly
decomposed when exposed to air at room temperature (Found:
Study of (S)-1-amino-2-(methoxymethyl)pyrrolidine (SAMP) 17
as the chiral auxiliary
A solution of the aldehyde24 12 (50 mg, 0.2 mmol) and SAMP13
(52 mg, 0.4 mmol) in benzene (2 cm3) was heated under reflux
for 20 min. The mixture was diluted with dichloromethane,
filtered, dried and the solvent was evaporated. The residue
was purified by PLC using diethyl ether to give the hydrazone
18 as a yellow oil (40 mg, 54%). νmax//cmϪ1 3475 and 1735;
δH (100 MHz) 1.90 (4 H, m, 2 × pyrrolidine-β-CH2), 2.68 (6 H,
m, CH2CH2CO2 and NCH2), 3.38 (3 H, s, OCH3), 3.5 (2 H, s,
CH2CO2), 3.66 (6 H, s, 2 × COOCH3), 3.3–3.8 (3 H, m, OCH2
and CH), 6.5 (1 H, br s, pyrrole-H), 7.23 (1 H, s, NCH), 8.58
(1 H, br s, NH) and a small singlet at 7.36 (~ 10%) rising from
the NCH of the Z-isomer; m/z (FD) 365 (Mϩ for C18H27N3O5,
100%).
The hydrazone 18 (40 mg, 0.11 mmol) in THF (1 cm3) was
treated with D2O (0.1 cm3) for 20 min before evaporating the
solution to dryness; the procedure was repeated once more.
A solution of this hydrazone (40 mg, 0.11 mmol), a trace of
bromcresol green and sodium cyanoborodeuteriide (30 mg,
0.46 mmol) in dry THF (8 cm3) was stirred at room temperature
under argon. A solution of O-deuteriated toluene-p-sulfonic
J. Chem. Soc., Perkin Trans. 1, 1999, 2677–2689
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acid mono(deuterium oxide)11 (80 mg, 0.4 mmol, 99.2 atom%)
in THF (4 cm3) was slowly added at room temperature (45 min)
to maintain pH 3.5, indicated by the tan colour of the indicator;
the sodium salt of toluene-p-sulfonic acid precipitated. The
solution was then filtered under argon, the solvent was evapor-
ated and the residue in dichloromethane (5 cm3) was washed
with saturated aqueous sodium hydrogen carbonate. The
organic solvent was dried and evaporated and the product was
purified by PLC under argon in the dark with methanol–
dichloromethane (7:93) as eluant to give the hydrazine 19a
(20 mg, 49%) as an oil. It was unstable and decomposed at 4 ЊC
under argon within a few days; νmax/cmϪ1 3400 (br) and 1735;
δH (400 MHz) 1.6, 1.75 and 1.87 (1 H, 2 H, 1 H, 3 × m,
2 × pyrrolidine-β-CH2), 2.56 (2 H, m, CH2CH2CO2), 2.70 (2 H,
m, pyrrolidine-α-CH2), 2.76 (2 H, m, CH2CH2CO2), 3.37 (3 H,
s, OCH3), 3.41 (2 H, ABq, CH2CO2), 3.35–3.55 (3 H, m, OCH2
and pyrrolidine-α-CH), 3.64 and 3.66 (2 × 3 H, 2 × s, 2 ×
CH3OCO), 3.96 and 3.93 (2 × s, intensity: 3.7/1, HNCHD),
6.45 (1 H, d, J 2.5, pyrrole-H) and 9.3 (1H, br s, NH). This
spectrum showed the diastereotopic excess to be 57% and com-
parison with the spectrum of unlabelled material below demon-
strated at least 95% of the sample was deuteriated.
The [11R-2H1]isotopomer 22b was prepared in the same way
from 16b (Found: Mϩ, 435.2091. C22H29DN2O7 requires M,
435.2131); δH (400 MHz, in part) 4.33 [0.79 H, d, J 6, (11R)-
NHCHD], 4.38 [0.16H, d, J 6, (11S)-NHCHD], with all other
signals as above; these results are complementary to those for
the [11S] sample.
(b) Degradation by ozone. The plan was to degrade both
foregoing labelled amides to yield labelled methyl (1S,4R)-
camphanyl glycinate. Therefore an unlabelled sample 23 was
prepared as described by Armarego et al.15 δH (100 MHz): 1.09
and 1.11 (2 × 3 H, 2 × s, 2 × camphanyl-CH3), 0.96 (3 H, s,
camphanyl-CH3), 1.8 (3 H, m, camphanyl CH2 and CH), 2.5
(1 H, m, camphanyl-CH), 3.75 (3H, s, OCH3), 4.06 (2 H,
2 × ABq; J 6 and 18 NCH2) and 6.9 (1 H, br s, NH); δH (250
MHz , in part) 3.98 (1 H, dd, J 5.1 and 18, HR of NCH2), 4.19
(1 H, dd, J 6 and 18, HS of NCH2); all these signals agree
closely with those reported;15 m/z 269, (Mϩ). C13H19NO5
requires M 269.
To a solution of the [11S-2H1]camphanamide 22a (27 mg,
0.062 mmol) in dichloromethane (2 cm3) was added silica (1.5 g,
60–120 mesh) followed by evaporation to dryness. The silica
was cooled to Ϫ70 ЊC and a stream of ozone was passed
through it for 30 min causing the silica to turn blue by adsorbed
ozone. The solid was then warmed to room temperature during
1 h and the degradation products were washed off the silica
with ethyl acetate. The residue from evaporation of the extracts
was dissolved in methanol (3 cm3) and treated with an excess of
ethereal diazomethane. The (mainly) camphanyl-(2S)-[2-2H1]-
glycine methyl ester 23a (3.3 mg, 20%) was isolated by PLC with
ethyl acetate–hexane (1:1) as eluant (Rf 0.4) and identified by
comparison with the foregoing unlabelled material (Found:
Mϩ, 270.1339. C13H18DNO5 requires M, 270.1341) δH (400
MHz, in part) 3.98 [0.79 H, dt, J 5.1 and 2.5 (2S)-NCHD] and
4.19 [0.16 H, dt, (2R)-NCHD]; all other signals were as for the
unlabelled material 23.
The unlabelled hydrazine 19 was prepared as above from 18
using sodium cyanoborohydride δH (400 MHz, in part) 3.93
(2 H, ABq; J 14.4; NCH2) all other signals as for 20a; m/z (FD),
368 (Mϩ ϩ 1, 100%), 367 (Mϩ for C18H29N3O5, 50%).
Determination of absolute configuration of (11S)- and (11R)-
[11-2H1]porphobilinogen 1a and 1b
(a) Preparation of camphanamide derivatives. Unlabelled
PBG lactam methyl ester 16 (50 mg, 0.22 mmol) in aqueous 2 M
potassium hydroxide (0.6 cm3, 2 mmol) was heated at 70 ЊC for
5 min under argon and kept at room temperature for 12 h in the
dark. (1S,4R)-Camphanyl chloride 20 (100 mg, 0.46 mmol) in
toluene (1 cm3) was added dropwise to the foregoing ice cooled
solution as the mixture was vigorously stirred. Stirring was
then continued for 6 h at room temperature while keeping the
pH above 7 by dropwise addition of aqueous 2 M potassium
hydroxide. The mixture was extracted with dichloromethane
(discarded), the aqueous layer was acidified to pH 2 and
extracted several times with dichloromethane–methanol (9:1),
total volume 70 cm3. The dried organic solution was evaporated
and the amide 21 in methanol (5 cm3) was treated with an excess
of ethereal diazomethane. The product was purified by PLC on
silica with dichloromethane–methanol (93:7) as eluant (Rf 0.5)
to give the camphanamide of porphobilinogen dimethyl ester
22 (54 mg, 56%) as an oil (Found: Mϩ, 434.2068. C22H30N2O7
requires M, 434.2053); νmax//cmϪ1 3430, 1790, 1730 and 1660;
δH (400 MHz) 0.78 (3 H, s, camphanyl-CH3), 1.08 and 1.082 (2 ×
3 H, each s, 2 × camphanyl-CH3), 1.66 (1 H, m, camphanyl-
CH), 1.88 (2 H, m, camphanyl-CH2), 2.50 (3 H, m, CH2CH2-
CO2 and camphanyl-CH), 2.74 (2 H, m, CH2CH2CO2), 3.43
(2 H, s, CH2CO2), 3.65 and 3.68 (2 × 3 H, 2 × s, 2 × OCH3),
4.36 (2 H, 2 × ABq, J 6 and 13.4, NHCH2), 6.46 (1 H, d, J 2.6,
pyrrole-H), 7.13 (1 H, t, J 6, NHCH2) and 8.62 (1 H, br s, NH);
irradiation at δ 7.13 converted the 8 signals at 4.36 into one
ABq, with the centres 23 Hz apart; the low field doublet corre-
sponds to HR of NHCH2 and that at high field to HS; the
assignments depend on subsequent work below; m/z 434
(Mϩ, 7%), 361 (M Ϫ C3H5O2, 18), 265 (10), 253 (16), 237 (30)
and 223 (100).
The (mainly) camphanoyl-(2R)-[2-2H1]glycine methyl ester
23b was obtained by degradation of the [11-R-2H1]-isotopomer
22b as described above for 22a (Found: Mϩ, 270.1340.
C13H18DNO5 requires M, 270.1341); δH (400 MHz, in part) 3.98
[0.16 H, dt, (2S)-NCHD] and 4.19 [0.79 H, dt, J 6 and 2.5, (2R)-
NCHD]; m/z 270 (Mϩ, 9%), 224 (30), 184 (5) and 180 (8).
Preparation of PBG lactam methyl ester 16. The aldehyde 12
(102 mg), hydroxylamine (42 mg) and sodium acetate (50 mg)
were stirred in methanol (2 cm3) at 18 ЊC for 2 h and the product
was isolated as described24 to give the oxime (100 mg). This was
dissolved in 2 M methanolic hydrogen chloride (2 cm3) and the
solvent was evaporated. A solution of the residual hydro-
chloride in methanol (8 cm3) was stirred with palladium on
carbon (10%, 80 mg) for 0.5 h under hydrogen at room temper-
ature. The filtered solution was concentrated until the PBG
dimethyl ester hydrochloride (as 15, HCl salt) started to crystal-
lise, a process completed by cooling the mixture to 0 ЊC. The
solid was collected and dried (78 mg); it was shown by TLC
using methanol–chloroform (1:9) to be free from the corre-
sponding lactam (Rf of lactam, 0.5; Rf of product, 0.1). δH (100
MHz) 2.47 (4 H, m, CH2CH2CO), 3.46 (2 H, br s, CH2CO), 3.54
and 3.62 (each 3 H, s, 2 × OCH3), 4.14 (2 H, br s, CH2N), 6.5
(1 H, br s, pyrrole-H), 8.0 (3 H, br s, NH3ϩ) and 10.0 (1 H, br s,
NH). The signals became sharper when the spectrum was
determined in CDCl3–CD3OD (4:1); m/z (FD) 254, (M Ϫ HCl;
C12H18N2O4).
The [11S-2H1]isotopomer 22a was prepared as above via 21a
from the [11S-2H1] lactam 16a (Found: Mϩ, 435.2118. C22H29-
DN2O7 requires M, 435.2131); δH (400 MHz, in part) 4.33 [0.16
H, d, J 6, (11R)-NHCHD], 4.38 [0.79 H, d, J 6, (11S)-NHCHD]
with all the other signals as above; this showed that 95% of the
total material was deuteriated and the enantiomeric excess was
66%; m/z 435 (Mϩ, 100%), 362 (M Ϫ C3H5O2, 38), 276 (5), 254
(7), 238 (23) and 222 (100).
The foregoing crystals in methanol (5 cm3) were treated with
an aliquot (0.2 cm3) from a solution of sodium (0.5 g) in
methanol (10 cm3). After the mixture had been heated under
reflux for 5 min, it was concentrated to low volume when the
lactam ester 16 crystallised; it was collected by centrifugation
(56 mg, 63% overall from 12) and identified by comparison with
authentic material.24
2684
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Synthesis of the bilane 7 and the (S)- and (R)-analogues 7a and
3,8,13,18-Tetrakis(2-methoxycarbonylethyl)-7,12,17-tris-
7b 2H1-labelled at the aminomethyl group
(methoxycarbonylmethyl)-6Ј-oxo-1Ј,2Ј,5Ј,6Ј-tetrahydropyrido-
[3,4-a]biladiene-a,c dihydrobromide 24 and the 2H1-labelled ana-
logues 24a and 24b. This was prepared as earlier10,16 from the
foregoing tripyrrene hydrochloride (294 mg, 0.35 mmol) and
3-(2-methoxycarbonylethyl)-4-methoxycarbonylmethyl-5-
formylpyrrole 12 (92.4 mg, 0.37 mmol). The resultant biladiene
dihydrobromide 24 (386 mg, 100%) was identified by com-
parison with the earlier product and it showed δH (400 MHz)
2.05 (2 H, m), 2.60 (6 H, m), 2.84 (2 H, m), 2.93 (2 H, m) and
3.01 (4 H, m) (4 × CH2CH2CO2), 3.27, 3.37, 3.609, 3.614, 3.67,
3.69 and 3.72 (each 3 H, s, 7 × OCH3), 3.52, 3.80 and 3.86 (each
2 H, s, CH2CO2) 4.98 (2 H, s, CH2NH), 5.30 (2 H, s, methane
CH2), 6.21 (1 H, br, CH2NH), 7.72 and 7.76 (each 1 H, s,
2 × methene CH), 7.78 (1 H, d, J 4.4, 19-H), 13.65, 13.66, 13.72,
and 13.98 (each 1 H, br, 4 × NH).
Using the same procedure, the foregoing [R-aminomethylene-
2H1]tripyrrene hydrochloride (146 mg, 0.176 mmol) and the
unlabelled formylpyrrole 12 (46.3 mg, 0.183 mmol) gave [R-
aminomethylene-2H1]-3,8,13,18-tetrakis(2-methoxycarbonyl-
ethyl-7,12,17-tris(methoxycarbonylmethyl)-6Ј-oxo-1Ј,2Ј,5Ј,6Ј-
tetrahydropyrido[3,4-a]biladiene-a,c dihydrobromide 24b (193
mg, 100%) as a red solid, 131–140 ЊC (decomp.). The 1H-NMR
spectrum was identical to that for unlabelled material except for
δ 4.98 (1 H, br, CHD); m/z (FD) 931 (Mϩ Ϫ HBr2).
The unlabelled bilane 7 had been synthesised earlier16 and the
same approach was used here. However, some changes in pro-
cedure are described below and new data from a fuller spectro-
scopic study are reported.
tert-Butyl
5-acetoxymethyl-3-(2-methoxycarbonylethyl)-4-
methoxycarbonylmethylpyrrole-2-carboxylate. Freshly distilled
sulfuryl chloride (4 g, 29.6 mmol) was added to an ice-cooled
solution of tert-butyl 5-methyl-3-(2-methoxycarbonylethyl)-
4-methoxycarbonylmethylpyrrole-2-carboxylate25 (10 g, 29.5
mmol) in dichloromethane (125 cm3) containing finely divided
calcium carbonate (10 g). The mixture was stirred at 5 ЊC for
2 h, with protection from moisture then the solvent was evapor-
ated below room temperature. The residue was redissolved in
dichloromethane (150 cm3) and the filtered solution was
evaporated. The residue with sodium acetate (7.5 g, 91 mmol)
in glacial acetic acid (200 cm3) was stirred at 60 ЊC for 30 min
then evaporated. The residue was partitioned between water
(50 cm3) and dichloromethane (50 cm3, 2 × 25 cm3). The
combined organic extracts were dried and evaporated; the
residue by flash chromatography using 3.5:6.5 ethyl acetate–
hexane gave the acetoxymethylpyrrole (9.33 g, 80%) as an oil
1
which crystallised on standing. The product gave an H-NMR
Also by the same procedure, the foregoing [S-amino-
methylene-2H1]tripyrrene hydrochloride (148 mg, 0.178 mmol)
and unlabelled formylpyrrole 12 (47.9 mg, 0.189 mmol) gave the
[S-aminomethylene-2H1]-analogue as its dihydrobromide 24a
(201 mg, 100%) mp 131–140 ЊC (decomp.). The 1H-NMR spec-
trum was identical to that for unlabelled material except for
δ 4.97 (1 H, br, CHD); m/z (FD) 931 (Mϩ Ϫ HBr2).
spectrum identical to that from an earlier preparation25 of it
by a different method.
tert-Butyl
2,7,12-tris(2-methoxycarbonylethyl)-3,8-bis-
(methoxycarbonylmethyl)-6Ј-oxo-1Ј,2Ј,5Ј,6Ј-tetrahydropyrido-
[4,3-a]tripyrrene-b-1-carboxylate hydrochloride and the 2H1-
labelled analogues.
A solution of hydrogen chloride in
dichloromethane (0.2 M, 4 cm3) was added to a stirred solution
of porphobilinogen lactam methyl ester 16 (91.9 mg, 0.41
mmol) and formyldipyrromethane25 illustrated in Scheme 5
(237 mg, 0.4 mmol) in dichloromethane (26 cm3) and meth-
anol (2 cm3). After 30 min, dry benzene (40 cm3) was added,
the solution was evaporated to dryness at room temperature
and this process was repeated. Trituration of the residue with
benzene–diethyl ether gave a solid which was collected by
centrifugation, washed with diethyl ether (3 × 10 cm3) and
dried in vacuo to yield the orange tripyrrene hydrochloride
(313 mg, 94%), 123–126 ЊC (decomp.). δH (400 MHz) 1.54
(9 H, s, C(CH3)3), 2.39 (2 H, t, J 7.6), 2.53 (2 H, t, J 7.5),
2.61 (2 H, t, J 6.8), 2.78 (2 H, t, J 7.6), 2.94 (2 H, t, J 8.3)
and 3.00 (2 H, t, J 6.8), (3 × CH2CH2CO2), 3.51 (2 H, s,
CH2CO2), 3.56, 3.62, 3.63, 3.67, and 3.71 (each 3 H, s,
5 × OCH3), 3.78 (2 H, s, CH2CO2), 4.38 (2 H, s, methane
CH2), 4.87 (2 H, s, CH2NH), 6.01 (1 H, s, CH2NH), 7.64
(1 H, s, methene CH), 10.72 (2 H, br, 2 × pyrromethene NH);
m/z (FD) 794 (Mϩ Ϫ HCl). This tripyrrene had been pre-
pared earlier as its hydrobromide25 and the present sample
was correlated with it spectroscopically.
3,8,13,18-Tetrakis(2-methoxycarbonylethyl)-7,12,17-tris-
(methoxycarbonylmethyl)-6Ј-oxo-1Ј,2Ј,5Ј,6Ј-tetrahydropyrido-
[3,4-a]bilane and the 2H1-labelled analogues. This was prepared
as earlier10,16 by Method B from the biladiene dihydrobromide
24 (374 mg, 0.342 mmol) to afford the bilane (lactam of
esterified 7) (229 mg, 72%) as a pale brown powder, 229–235 ЊC
(decomp.) (lit.,10,16 mp 225–228 ЊC (decomp.)). δH (400 MHz)
2.33 (2 H, m), 2.49 (6 H, m) and 2.70 (8 H, m) 4 × CH2CH2-
CO2), 3.32, 3.42, and 3.44 (each 2 H, s, 3 × CH2CO2), 3.36
(2 H, s, CH2CONH), 3.56, 3.58, 3.60, 3.62, 3.64, 3.65, and
3.69 (each 3 H, s, 7 × OCH3), 3.68, 3.72 and 3.74 (each 2 H, s,
3 × methane CH2), 4.38 (2 H, br, CH2NH), 5.81 (1 H, br,
CH2NH), 6.38 (1 H, br, 19-H), 8.61 (1 H, br, NH), 9.00 (2 H, br,
2 × NH) and 9.17 (1 H, br, NH); m/z (FD) 957 (Mϩ ϩ H ϩ Na)
and 934 (Mϩ ϩ H). This product was shown to be identical to
the earlier10,16 fully characterised sample.
Using the same procedure, [R-aminomethylene-2H1]biladiene
dihydrobromide 24b (162 mg, 0.148 mmol) gave [R-amino-
methylene-2H1]-3,8,13,18-tetrakis(2-methoxycarbonylethyl)-
7,12,17-trismethoxycarbonylmethyl-6Ј-oxo-1Ј,2Ј,5Ј,6Ј-tetra-
hydropyrido[3,4-a]bilane (lactam of esterified 7b) (67.4 mg,
48%), mp 224–229 ЊC (decomp.). The 1H-NMR spectrum
was identical to that for unlabelled material except for δ 3.36
(2 H, d, J 3.4, CH2CONH), 4.38 (1 H, br, CH DNH) and 6.38
(1 H, d, J 2.4, 19-H). m/z (FD) 935 (Mϩ ϩ H).
Using the same procedure, (11-R)-[11-2H1]-porphobilinogen
lactam methyl ester 16b (44.7 mg, 0.2 mmol) and the same
formyldipyrromethane (118 mg, 0.2 mmol) gave tert-butyl [R-
aminomethylene-2H1]-2,7,12-tris(2-methoxycarbonylethyl)-3,8-
bis(methoxycarbonylmethyl)-6Ј-oxo-1Ј,2Ј,5Ј,6Ј-tetrahydro-
pyrido[4,3-a]tripyrrene-b-1-carboxylate hydrochloride (151
mg, 91%), as an orange solid, mp 123–126 ЊC (decomp.). The
1H-NMR spectrum was identical to that for unlabelled material
except for δ 4.87 (1 H, br, CHD); m/z (FD) 795 (Mϩ Ϫ HCl).
Also by the same procedure, (11-S)-[11-2H1]porphobilinogen
lactam methyl ester 16a (45.4 mg, 0.2 mmol) and the formyldi-
pyrromethane (119 mg, 0.2 mmol) gave the [S-aminomethylene-
2H1]-analogue as its hydrochloride (155 mg, 92%), an orange
Also by the same procedure, [S-aminomethylene-2H1]-
biladiene dihydrobromide 24a (185 mg, 0.169 mmol) gave the
[S-aminomethylene-2H1]-analogue (lactam of esterified 7a)
(85.2 mg, 54%) mp 226–235 ЊC (decomp.). The 1H-NMR spec-
trum was identical to that for R-labelled material; m/z (FD) 935
(MϩϩH).
Synthesis of chiral imidate esters including single enantiomers
1
solid, mp 123–126 ЊC (decomp.). The H-NMR spectrum was
(2RS)-2-Phenylbutyramide. Aqueous ammonia (17.5%, 5
cm3) was added to a stirred solution of (2RS)-2-phenylbutyryl
chloride (1 g, 5.45 mmol) in diethyl ether (10 cm3). After 5 min,
identical to that for unlabelled material except for δ 4.87 (1 H,
br, CHD); m/z (FD) 795 (Mϩ Ϫ HCl).
J. Chem. Soc., Perkin Trans. 1, 1999, 2677–2689
2685

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the layers were separated and the organic solution was washed
with water (5 cm3), dried, and evaporated. The product was
recrystallised from diethyl ether to give amide (694 mg, 78%) as
needles, mp 82–83.5 ЊC (lit.,26 mp 85–86 ЊC) (Found: C, 73.6; H,
8.1; N, 8.5. C10H13NO requires C, 73.6; H, 8.0; N, 8.6%); νmax
(CHCl3)/cmϪ1 3480, 3370, 2920, 1660; δH (250 MHz), 0.88 (3 H,
t, J 7.4, CHCH2CH3), 1.80 (1 H, m, CHCHHCH3), 2.20 (1 H,
m, CHCHHCH3), 3.28 (1 H, dd, J 8.1 and 7, CHCH2CH3),
5.44 and 5.50 (each 1 H, br, 2 × NH) and 7.23–7.37 (5 H, m,
ArH); m/z (EI) 163 (Mϩ, 16%), 135 (M Ϫ C2H4, 20%), 119
(M Ϫ CONH2, 20%) and 91 (C7H7ϩ, 100%).
CF3COCH3), 3.80 (3 H, s, CNHOCH3), 7.41 (5 H, m, ArH) and
8.30 (1 H, br, NH); δC (100 MHz) 54.1 and 54.4 (each s,
2 × OCH3), 123.9 (q, J 290, CF3), 127.8, 128.5 and 129.4 (each
d, o, m, p ArH), 132.3 (s, Ar–C–C), 166.9 (s, C᎐NH), signal for
᎐
CH3OCCF3 too weak to observe; m/z (EI) 247 (Mϩ, 11%), 233
(M Ϫ CH3, 20), 189 (C6H5C(CH3)OCF3ϩ, 19), 170 (C6H5ϩ, 42),
69 (CF3ϩ, 23) and 58 (HNCOCH3ϩ, 100).
Attempted preparation of the foregoing imidate as its hydro-
chloride by the standard method as follows, led to a different
product. An ice cooled solution of nitrile 27 (420 mg, 1.95
mmol) in diethyl ether (10 cm3) containing methanol (0.5 cm3)
was saturated with dry hydrogen chloride gas. The solution was
kept at 0 ЊC overnight then treated with more hydrogen chloride
gas and after a further 24 h at 0 ЊC, nitrogen was passed
through the solution (there was no precipitate) to remove HCl.
It was then evaporated below room temperature to give (2RS)-
2-methoxy-2-trifluoromethylphenylacetamide 28 (405 mg, 89%)
mp 77–78.5 ЊC from diethyl ether–hexane (Found: C, 51.7; H,
4.3; N, 6.0. C10H10F3NO2 requires C, 51.5; H, 4.3; N, 6.0%);
νmax (CHCl3)/cmϪ1 3495 , 3375, 2960, 1700; δH (250 MHz), 3.44
(3 H, q, J 1.6, OCH3), 5.99 and 6.68 (each 1 H, br, NH2), 7.38–
7.44 (3 H, m, m, p-ArH) and 7.44–7.58 (2 H, m, o-ArH); m/z
(EI) 233 (Mϩ, 6%), 190 (M Ϫ CONH, 80%), 189 (M Ϫ
CONH2, 90%), 175 (M Ϫ CONHCH3, 45%), 105 (C6H5CO,
100%) and 77 (C6H5, 50%).
(2RS)-2-Phenylbutyronitrile. A solution of the foregoing
amide (452 mg, 2.55 mmol) in thionyl chloride (3 cm3) was
heated at reflux for 2 h, then evaporated and flash chrom-
atography of the residue using 1:9 ethyl acetate–hexane
followed by Kugelrohr distillation (110 ЊC/19 mmHg) gave the
nitrile (314 mg, 77%) (lit.,27 bp 107 ЊC/7 mmHg) (Found: C,
82.9; H, 7.7. C10H11N requires C, 82.7; H, 7.6%); νmax (neat)/
cmϪ1 3100–2850, 2255; δH (250 MHz) 1.07 (3 H, t, J 7.4,
CHCH2CH3), 1.93 (2 H, m, CHCH2CH3), 3.73 (1 H, t, J 7.2,
CHCH2CH3) and 7.27–7.41 (5 H, m, ArH); δC (100 MHz) 11.2
(q, CH3), 29.0 (t, CH2), 38.6 (d, CH), 120.8 (s, CN), 127.1,
127.8, and 128.8 (each d, o, m, and p-ArH), 135.9 (s, Ar-C-C);
m/z (EI) 145 (Mϩ, 32%), 117 (M Ϫ C2H4, 100%), 116
(M Ϫ C2H5, 74%), 90 (C7H6ϩ, 27%), 89 (C7H5ϩ, 30%) and 69
(CH3CH2CHCNHϩ, 26%).
(1R,4S)-4,7,7-Trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-
carboxamide. Aqueous ammonia (17.5%, 20 cm3) was added to
a vigorously stirred solution of (ϩ)-(1R,4S)-camphanyl chlor-
ide (1 g, 4.6 mmol) in dichloromethane (20 cm3) and after 45
min, the separated aqueous phase was extracted with dichloro-
methane (2 × 10 cm3). The combined organic solution was
washed with aqueous 2 M hydrochloric acid (20 cm3), dried and
evaporated; recrystallisation of the residue from hexane–ethyl
acetate gave the amide (821 mg, 90%) as needles, mp 209–211 ЊC
(Found: C, 60.6; H, 7.7; N, 7.1. C10H15NO3 requires C, 60.9; H,
7.7; N, 7.1%); νmax (CHCl3)/cmϪ1 3490, 3380, 2920, 1775, 1680;
δH (250 MHz), 0.95, 1.10 and 1.11 (each 3 H, s, 3 × CH3), 1.68,
1.94 and 2.51 (1 H, 2 H, 1 H, each m, CH2CH2), 5.75 and 6.39
(each 1 H, br, NH2); m/z (EI) 197 (Mϩ, 10%), 169 (M Ϫ C2H4,
15%), 151 (M Ϫ CO2H2, 90%) and 83 (C6H11ϩ, 100%).
Methyl (2RS)-2-phenylbutyrimidate 26 and the corresponding
(2R)-enantiomer 26a. An ice cooled solution of the foregoing
nitrile (187 mg, 1.06 mmol) in diethyl ether (5 cm3) containing
methanol (0.25 cm3) was saturated with dry hydrogen chloride
gas. After 12 h at 0 ЊC, the solvent was evaporated below room
temperature, the residue was washed with diethyl ether and
dried at high vacuum to afford the imidate ester hydrochloride
26 (243 mg, 100%) as a hydroscopic solid, mp 95–101 ЊC.
δH (250 MHz) 0.95 (3 H, t, J 7.3, CHCH2CH3), 2.02 and 2.12
(each 1 H, sextet, J 7.5, CHCH2CH3), 4.30 (4 H, m, overlapping
3 H, s, CHCH2CH3, OCH3), 7.32 (3 H, m, m, p-ArH), 7.47 (2 H,
m, o-ArH), 11.71 and 12.04 (each 1H, br, NH2); m/z (FD) 178
(M Ϫ Cl) and 162 (M Ϫ HCl Ϫ CH3).
Part of the foregoing imidate hydrochloride was converted
into the free base by extraction with dichloromethane from
aqueous base (Found: Mϩ 177.1150. C11H15NO requires M
177.1154); νmax (neat)/cmϪ1 3310, 2960, 1640; δH (250 MHz)
0.86 (3 H, t, J 7.3, CH3), 1.81 and 1.99 (each 1 H, m, CH2CH3),
3.35 (1 H, dt, J 7.9 and 0.9, CHCH2CH3), 3.68 (3 H, s, OCH3),
7.07 (1 H, br, NH) and 7.18–7.28 (5H, m, ArH); m/z (EI) 177
(Mϩ, 33%), 162 (M Ϫ CH3, 58%), 91 (C7H7ϩ, 100%) and 58
(CH3OCNHϩ, 34%).
Commercially available (2R)-2-phenylbutyric acid was carried
through exactly the same steps to yield methyl (2R)-2-phenyl-
butyrimidate hydrochloride 26a, identified by spectroscopic
comparison with the foregoing sample.
(1R,4S)-4,7,7-Trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-
carbonitrile 30. A solution of the foregoing amide (269 mg, 1.36
mmol) in toluene (5 cm3), containing phosphorus pentoxide
(0.25 g) was heated at reflux for 5 h then cooled, poured into
saturated aqueous sodium hydrogen carbonate (50 cm3) and
extracted with dichloromethane (3 × 20 cm3). The combined
organic solution was dried and evaporated. The residues by
flash chromatography using 3:7 ethyl acetate–hexane and
recrystallisation of the product from hexane–diethyl ether, gave
nitrile 30 (162 mg, 66%) as needles, mp 134–136 ЊC (sealed
capillary) (Found: C, 67.8; H, 7.4; N, 7.8. C10H13NO2 requires
C, 67.0; H, 7.3; N, 7.8%); νmax (CHCl3)/cmϪ1 2930, 1790; δH (250
MHz) 1.06, 1.13, 1.14 (each 3 H, s, 3 × CH3), 1.72 (1 H, ddd,
J 13.5, 8.6 and 5.0), 1.93 (1 H, ddd, J 1.3, 10.3 and 4.8) and
2.16–2.37 (2 H, m, CH2CH2); δC (100 MHz) 9.6, 16.1 and 16.7
(each q, 3 × CH3), 28.0 and 31.9 (each t, 2 × CH2), 52.6 and
54.5 (each s, 2 × quat.-C), 81.6 (s, OCCN), 114.3 (s, CN) and
Methyl (2RS)-2-methoxy-2-trifluoromethylphenylacetimidate
29. Sodium methoxide (1.7 g, 31.5 mmol) was added to a stirred
solution of the commercially available (2RS)-nitrile 27 (1.7 g,
7.9 mmol) in dry methanol (15 cm3). After 2 h at room temper-
ature, the solution was poured into saturated aqueous sodium
hydrogen carbonate (200 cm3) and extracted with dichloro-
methane (100 cmϪ3, 3 × 50 cm3). The combined organic solu-
tion was dried and evaporated. Flash chromatography of the
residue using first 1:9 then 3:7 ethyl acetate–hexane gave, after
Kugelrohr distillation (95 ЊC/19 mmHg) recovered nitrile 27
(855 mg, 50%) and later (125 ЊC/19 mmHg) the imidate ester 29
(825 mg, 42%) as an oil (Found: C, 53.2; H, 4.3; N, 6.0.
C11H12F3NO2 requires C, 53.4; H, 4.5; N, 6.0%); νmax (CHCl3)/
cmϪ1 3310, 2940, 1660; δH (250 MHz) 3.29 (3 H, d, J 1.3,
176.4 (s, C᎐O); m/z (EI) 179 (Mϩ).
᎐
Methyl
(1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]-
heptane-1-carboximidate 31. Sodium methoxide (350 mg, 6.42
mmol) was added to a stirred solution of nitrile 30 (569 mg,
3.17 mmol) in dry methanol (10 cm3) under argon. After 30
min, the solution was poured into saturated aqueous sodium
hydrogen carbonate (25 cmϪ3) and extracted with dichloro-
methane (3 × 20 cm3). The combined organic solution was
dried and evaporated. Flash chromatography of the residue
using first 3:7 then 1:1 ethyl acetate–hexane gave imidate ester
2686
J. Chem. Soc., Perkin Trans. 1, 1999, 2677–2689

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31 (355 mg, 53%) mp 69–70.5 ЊC from diethyl ether–hexane
(Found: C, 62.7; H, 8.2; N, 6.7. C11H17NO3 requires C, 62.5;
H, 8.1; N, 6.6%); νmax (CHCl3)/cmϪ1 3310, 2840, 1770, 1645;
δH (250 MHz) 0.85, 1.01 and 1.09 (each 3 H, s, 3 × CH3), 1.65–
1.73 (1 H, m), 1.82–1.96 (2 H, m), 2.39 (1 H, m, CH2CH2), 3.80
(3 H, s, OCH3) and 7.79 (1 H, br, NH); δC (100 MHz) 9.5, 16.3
and 16.7 (each q, 3 × CH3), 28.9 and 29.8 (each t, 2 × CH2),
53.0 (q, OCH3), 53.0 and 54.9 (each s, 2 × quat.-C), 91.7 (s,
of sodium chloride. δH (CD3OD, 100 MHz) 2.2–2.9 (4 H, m,
CH2CH2CO2), 3.27 (2 H, s, C6H5CH2), 3.3 (2 H, s, CH2CO2),
4.48 (2 H, s, CH2N), 6.45 (1 H, d, pyrrole-H), 7.5 and 7.64 (3H
and 2 H, 2 × m, ArH). This product gave no Mϩ by mass spec-
trometry, even using FD with application from a solution in
acetic acid.
A solution of the foregoing amidine 37 (10mg) in methanol
(3 cm3) was treated with dimethyl sulfate (0.4 cm3) and stirred
for 0.5 h at room temperature, with monitoring by TLC; Rf of
product, 0.9; Rf of starting material 0.5 using isopropanol–
water (15:4). The reaction mixture was diluted with water (final
pH was 2), the product was extracted with dichloromethane
and the dried extracts were evaporated to give a monomethyl
ester 38 or 39 (7 mg, 70%) which was pure by NMR and TLC,
Rf in CH3OH–CH2Cl2 (1:9) 0.15. δH (CDCl3–CD3OD, 100
MHz) 2.55 (4 H, m, CH2CH2CO2), 3.46 (2 H, s, CH2CO2), 3.58
(2 H, s, CH2 CN), 3.62 (3 H, s, OCH3), 4.56 (2 H, s, CH2N), 6.5
(1 H, d, J 3, pyrrole-H) and 7.6 (5 H, m, ArH); the NH groups
had exchanged. δH (CDCl3, 100 MHz) 2.62 (4 H, m, CH2-
CH2CO2), 3.54 (2 H, s, CH2CO2), 3.66 (3 H, s, OCH3), 3.97
(2 H, s, CH2CN), 4.77 (2 H, d, J 5.5, CH2NH), 6.52 (1 H, br s,
pyrrole-H), 7.55 and 7.75 (1 × 3 H, 1 × 2 H, both m, ArH) and
8.1, 8.92 and 9.15 (each 1H, 3 × br s, 3 × NH); m/z (FD) 358
(Mϩ, 100%); C19H24N3O4 requires 358.
O-C-C᎐O), 167.1 (s, C᎐NH) and 178.1 (s, C᎐O); m/z (EI) 211
᎐
᎐
᎐
(Mϩ, 17%), 183 (M Ϫ CO, 80%), 167 (M Ϫ CO2, 40%), 155
(M Ϫ COC2H4, 75%) and 83 (C6H11ϩ, 75%).
Also obtained from this reaction was methyl (1S)-1,2,2-tri-
methyl-3-oxocyclopentane carboxylate 32 (232 mg, 40%) as an
oil which crystallised (mp 24–26 ЊC) after Kugelrohr distillation
(100 ЊC/19 mmHg) (Found: C, 65.4; H, 8.6. C10H16O3 requires
C, 65.2; H, 8.6%); νmax (CHCl3)/cmϪ1 2980, 1720; δH (250 MHz)
0.90, 1.01 and 1.20 (each 3 H, s, 3 × CH3), 1.80 (1 H, m) and
2.20–2.58 (3 H, m, CH2CH2) and 3.66 (3 H, s, OCH3); δC (100
MHz) 19.1, 19.2, and 19.7 (each q, 3 × CH3), 28.8 and 33.5
(each t, 2 × CH2), 51.4 (q, OCH3) 51.5 and 52.3 (each s,
2 × quat.-C), 176.2 and 176.3 (each s, 2 × C᎐O); m/z (EI) 184
᎐
(Mϩ) and 156 (M Ϫ CO).
Methyl (2R)-2-phenyl-2-hydroxyacetimidate hydrochloride 34.
(ϩ)-Amygdalin (10 g) in distilled water (30 cm3) was treated
with concentrated sulfuric acid (11 cm3) and the solution was
heated at 85–90 ЊC (bath temperature) for 50 min during which
time a brown oil separated. Water (50 cm3) was added to the
cooled mixture and the mandelonitrile was extracted thrice
with benzene. The combined organic layers were washed once
with water, dried for 10 min over sodium sulfate and evaporated
to give the single enantiomer (R)-mandelonitrile 33 (2.11 g).
This material is easily racemised so it was immediately con-
verted into the following imidate hydrochloride.
To the (R)-mandelonitrile 33 (2.11 g, 15.9 mmol) in dry
diethyl ether (7 cm3) was added dropwise with ice cooling, a
solution of hydrogen chloride in dry methanol (3 cm3, contain-
ing 1.5 g (3 equiv.) of CH3OH and 1.5 g (2.6 equiv.) of hydrogen
chloride). After the mixture had been stirred for 2 h at 0 ЊC, the
precipitate was collected by centrifugation, washed twice with
dry diethyl ether and dried in vacuo to give the imidate hydro-
chloride 34 (2.1 g, 65%) (Found: Mϩ Ϫ HCl, 165. C9H12NO2Cl
requires (M Ϫ HCl), 165); δH (CD3OD, 100 MHz) 4.1 (3 H, s,
OCH3), 5.5 (1 H, s, CHOH) and 7.4 (5 H, m, ArH); m/z 165
(M Ϫ HCl, 55%), 108 (55) and 107 (M Ϫ HCl Ϫ C2H4ON, 88);
C9H12NO2Cl requires for M Ϫ HCl, 165.
The racemic amidine (as 40). A solution of the (RS)-imidate
hydrochloride 26 (47.5 mg, 0.21 mmol) in 0.2 M carbonate
buffer (pH 10, 4 cm3) containing DMF (0.5 cm3) was added to
a solution of PBG 1 (18.2 mg, 0.08 mmol) in the same buffer
(0.5 cm3). The mixture was stirred for 4 h at room temperature
in the dark under argon then adjusted to pH 7–8 with 1 M
hydrochloric acid and the excess imidate was extracted into
chloroform (3 × 3 cm3). The aqueous solution was evaporated
to dryness at <40 ЊC under vacuum and the residue was redis-
solved in D2O (2 cm3). The solution was evaporated to dryness
and the process was repeated before dissolving the final residue
in CD3OD (1.5 cm3). This solution was centrifuged and filtered
twice until perfectly clear for NMR analysis. The signal from
the NCH2-pyrrole group of the amidine (as 40) was an ABq
with the four signals at δ 4.237, 4.298, 4.309 and 4.370.
The chloroform washings were worked up to give recovered
imidate as the free base (26 mg, 71%).
Conversion of (11-R)-[11-2H1]PBG 1b and (11S)-[11-2H1]-
PBG 1a into the amidines 40b and 40a. (11-R)-[11-2H1]PBG
lactam methyl ester 16b (19.7 mg, 0.088 mmol) was heated at
70 ЊC for 5 min, under argon, in argon saturated aqueous 2 M
potassium hydroxide (0.6 cm3) then the resulting solution was
kept at room temperature in the dark under argon for 18 h. The
solution was adjusted to pH 7–8 with 1 M hydrochloric acid
followed by 0.2 M sodium carbonate buffer (pH 10, 0.4 cm3). To
this was added a solution of the (2R)-imidate hydrochloride 26a
(58 mg, 0.27 mmol) in the same carbonate buffer (4 cm3) con-
taining DMF (0.5 cm3). The remaining steps were as above
to give a solution in CD3OD of the amidine 40b having the
(R)-configuration at the starred centre.
Conversion of PBG into amidine derivatives
The amidine 37. All the experiments that follow involved
fragile products that were highly polar and water soluble.
Therefore, the chemistry to be applied eventually to bilanes was
developed using similar, though simpler, starting materials; the
generation of an amidine was studied first on PBG.
A solution of PBG 1 (35 mg, 0.15 mmol) in aqueous 0.2 M
sodium hydrogen carbonate buffer (pH10) was treated with a
solution of methyl 2-phenylacetimidate hydrochloride 36 (70
mg, 0.4 mmol) in the same buffer (7 cm3), the pH of the final
solution being 9.4. This was stirred at room temperature, the
reaction being complete in 3 h as monitored by TLC on a cellu-
lose plate using isopropanol–water–acetic acid (15:5:1); Rf of
product, 0.75; Rf of PBG, 0.5. The mixture was extracted twice
with chloroform to remove excess reagent, the pH was adjusted
to 7.5 with 1 M hydrochloric acid and the water was evaporated
at high vacuum. The residue was extracted twice with methanol
(2 × 2 cm3), the extracts were evaporated and the residue was
dissolved in [2H4]methanol (1 cm3). The solution was evapor-
ated and the residue dried at high vacuum for 3 h. This material,
the disodium salt 37, was homogeneous by NMR and TLC, Rf
in isopropanol–water (15:4) 0.5, apart from a small amount
Using the same procedure, (11-S)-[11-2H1]PBG lactam ester
16a (19.8 mg, 0.088 mmol) and the (2R)-imidate ester hydro-
chloride 26a (54.3 mg, 0.25 mmol) afforded a solution in
1
CD3OD of the amidine 40a. H-NMR showed that the signal
from the starred centre of the sample having the (S)-configur-
ation at that site appeared at lower field (δ 4.33) and that from
the other diastereoisomer, at higher field (δ 4.26).
The amidine 41. PBG (35 mg, 0.15 mmol) in 0.2 M sodium
hydrogen carbonate buffer (1 cm3, pH 10) was treated with the
imidate hydrochloride 34 (80 mg, 0.4 mmol) in the same buffer
(8 cm3) and the solution (final pH 9.4) was stirred at room
temperature until complete reaction (2 h), as shown by TLC on
cellulose. The product showed Rf 0.7 and starting material
J. Chem. Soc., Perkin Trans. 1, 1999, 2677–2689
2687

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showed Rf 0.5 using isopropanol–water–acetic acid (15:5:1).
The solution was extracted twice with chloroform, the pH
adjusted to 7.5, the water evaporated and the residue was dried
for 3 h at high vacuum. It was then extracted with dry methanol
(2 × 2 cm3), the extracts were clarified by centrifugation and
same buffer (4 cm3). The solution, final pH 9.4, was stirred at
room temperature for 2 h until the conversion was complete as
shown by TLC on cellulose using isopropanol–water (15:5): Rf
of product, 0.6; Rf of starting material, 0.5. The solution was
extracted twice with chloroform, the pH was adjusted to 8 and
then evaporated to dryness and further dried at high vacuum.
The residue was extracted with dry methanol (2 × 3 cm3), the
extracts were centrifuged and the clear solution was evaporated.
The product was dissolved in CD3OD (0.5 cm3), centrifuged
clear and the solvent was evaporated to give the amidine 45
which was homogeneous by NMR and TLC. δH (CD3OD, 250
MHz) 2.4 (4 H, m, CH2CH2CO2), 2.77 (4 H, m, CH2CH2CO2),
3.28 and 3.36 (2 × 2 H, 2 × s, 2 × CH2CO2), 3.75 (2 H, ABq,
J 16.7, 5-H2), 4.34 (2 H, br s, NCH2), 5.38 (1 H, s, CHOH),
6.38 (1 H, s, pyrrole-H) and 7.25 and 7.4 (5 H, m, ArH). When
the spectrum was measured at 400 MHz, the interpyrrolic CH2
(5-H2) appeared as an ABq with ∆ν = 24 Hz whilst the signal
from the NCH2 group was not clearly resolved.
2
evaporated. The residue in H4-methanol (1 cm3) was centri-
fuged and the clear solution was evaporated to give 41 as its
disodium salt which was pure by NMR and TLC. δH (CD3OD,
400 MHz) 2.38 (2H, m, CH2CH2CO2), 2.7 (2H, m, CH2CH2-
CO2), 3.3 (2H, s, CH2CO2), 4.37 (2H, ABq, J 15.2; the high field
part of this quartet corresponds to HS of NHCH2), 5.36 (1H,
s, CHOH), 6.54 (1H, s, pyrrole-H), 7.3 and 7.5 (5H, 2 × m,
ArH). It was not possible by FD-MS to generate Mϩ under any
conditions tested.
Conversion of (11S)-[11-2H1]PBG 1a and (11-R)-[11-2H1]-
PBG 1b into the amidines 41a and 41b, respectively. A suspen-
sion of the lactam ester 16a in aqueous 2 M potassium hydrox-
ide (0.33 cm3) was heated at 70 ЊC for 5 min and the solution
was kept for 18 h at room temperature in the dark. It was
adjusted to pH 8 with 1 M hydrochloric acid and aqueous
0.2 M sodium hydrogen carbonate buffer (0.2 cm3, pH 10) was
added followed by the imidate hydrochloride 34 in the above
buffer (2.2 cm3). The solution (final pH 9.4) was stirred for 2
h at room temperature and the amidine 41a having mainly
the (S)-configuration at the starred centre was isolated as for
the unlabelled analogue 41. δH (CD3OD, in part, 400 MHz) 4.32
[0.16 H, s, (11R) NHCHD], 4.39 [0.79H, s, (11S) NHCHD];
the other signals were as for the unlabelled analogue 41.
The lactam 16b having mainly the (11R)-configuration was
used in the same way to afford the amidine 41b having largely
the (R)-configuration at the starred centre. δH (CD3OD, in part,
400 MHz) 4.32 [0.79 H, s, (11R)-NHCHD], 4.39 [0.16 H, s,
(11S)-NHCHD]; the rest of the spectrum was identical to that
from the unlabelled 41.
Synthesis of the standard unlabelled amidine 25 derived from
bilane 7 and the analogous [2H1]-labelled amidines 25a and 25b.
An alkaline solution of the aminomethylbilane 7 was prepared
by hydrolysis as usual16 of the corresponding lactam ester (24
mg). The solution was adjusted to pH 8 with 1 M hydrochloric
acid and aqueous 0.2 M potassium hydrogen carbonate buffer
(0.6 cm3, pH 10) was added followed by the imidate hydro-
chloride 34 (20 mg) in the same buffer (2 cm3). The final solu-
tion (pH 9.4–9.7) was stirred until the conversion was complete
(2.25 h) as shown by TLC using isopropanol–water–acetic acid
(15:4:1); Rf of product, 0.78; Rf of starting material, 0.66. The
solution was extracted twice with dichloromethane (each 20
cm3), then the pH was adjusted to 8 and the water was evapor-
ated. After the powdered residue had been dried for 3 h at high
vacuum, it was extracted twice with methanol (each 3 cm3),
the extracts were centrifuged until clear and the solvent was
removed. The residue was dissolved in [2H4]methanol (1 cm3),
the solution was evaporated and the solid was extracted again
with [2H4]methanol (1 cm3), the solution being centrifuged until
clear ready for NMR analysis of the amidine 25. δH (CD3OD,
400 MHz) 2.3 and 2.7 (each 8 H, CH2CH2CO2), 3.3 (8 H, m,
CH2CO2), 3.6–3.8 (6 H, m, (pyrrole)2CCH2), 4.38, (2H, ABq,
J 16.7, ∆ν ≈ 20 Hz NHCH2; high field part, HS), 5.5 (1 H, s,
CHOH), 6.35 (1 H, s, pyrrole-H) and 7.25 and 7.5 (5H, m,
ArH).
Formation of amidines from dipyrromethanes
Importantly, the solvents used for the next experiment and all
those that follow it were deoxygenated and were then saturated
with argon (see earlier for methods used).
Reaction of (2RS)-2-phenylbutyrimidate hydrochloride 26 with
the aminomethyl dipyrromethane 43. The lactam21 42 (14.5 mg,
0.035 mmol) was treated overnight in the dark under argon with
argon saturated aqueous 0.2 M potassium hydroxide (0.3 cm3)
then the solution was adjusted to pH 7–8 by the addition of
hydrochloric acid followed by carbonate buffer (pH 10, 0.5
cm3). A solution of imidate hydrochloride 26 (57.5 mg, 0.25
mmol) in the same buffer (4 cm3) containing DMF (0.5 cm3)
was added to the product 43 and the resulting solution was
stirred for 2 h at room temperature in the dark under argon.
The solution was extracted with chloroform (3 × 2 cm3) and the
aqueous solution was adjusted to pH 7–8 with 1 M hydro-
chloric acid before evaporation to dryness. The residue was
redissolved in D2O (2 cm3) and evaporated to dryness; this was
repeated. The residue was extracted with CD3OD (1.5 cm3), the
solution was centrifuged and filtered (twice) to give a clear solu-
tion for NMR analysis. The signal from the N-CH2-pyrrole
group of the amidine 44 was an ABq with the four signals at
4.233, 4.271, 4.283 and 4.321.
The labelled amidines 25a and 25b. Each labelled bilane 7a
and 7b was prepared by hydrolysis of the corresponding lactam
and converted into the corresponding amidine 25a and 25b as
described for the unlabelled series. The mainly (S)-amidine 25a
showed δH (CD3OD, 400 MHz) 4.42 (0.16 H, s, (R)-CHDNH),
4.43 (0.79 H, s, (S)-CHDNH). The mainly (R)-amidine 25b
showed δH (CD3OD, 400 MHz) 4.40 [0.79 H, s, (R)-CHDNH],
4.428 [0.16 H, s, (S)-CHDNH]. The rest of the signals matched
in the two samples and also with those from the authentic
unlabelled sample 25. These spectra were recorded one after the
other on the same instrument. Though the large signal at ca.
δ 4.4 from the sample having mainly the (R)-CHDN group was
clearly at higher field than that from the material having mainly
the (S)-CHDN residue, slight differences in the preparation of
the samples prevented the absolute values of the chemical shifts
coinciding exactly. Therefore, the foregoing assignments were
confirmed by mixing the (R)-amidine 25b with a smaller quan-
tity of the (S)-isomer 25a to demonstrate coincidence of the
small NMR signal from the CHDNH group of one with the
large signal from the corresponding position of the other,
Fig. 2(c).
The chloroform extracts yielded recovered imidate free base
(40.2 mg).
The amidine 45. The dipyrromethane 42 was hydrolysed by
stirring it (23 mg) with aqueous 2 M KOH (0.4 cm3) for 3 h at
room temperature under argon and then keeping the solution
for 18 h in the dark to complete the hydrolysis. The solution was
adjusted to pH 7–8 with 1 M hydrochloric acid and aqueous
0.2 M sodium hydrogen carbonate buffer (0.5 cm3, pH 10) was
added followed by the imidate hydrochloride 34 (50 mg) in the
2
Enzymic preparation of H1-labelled aminomethylbilanes and
determination of their configuration by formation of amidines.
The (11S)-PBG 1a prepared as above by hydrolysis of the
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View Article Online
corresponding lactam 16a (39 mg) was converted enzymically22
into the aminomethylbilane 7a by first adjusting the hydrolysate
to pH 9 with 0.1 M phosphoric acid. This solution was made
0.2 M with respect to NH4Cl and then was at pH 8.05.
Hydroxymethylbilane synthase22 (130 000 units) was added and
the mixture (20.8 cm3) was shaken at 37 ЊC for 30 min. The
solution was cooled on ice, adjusted to pH 12.2 with aqueous
1 M sodium hydroxide and sodium dithionite (50 mg) and 2 M
potassium hydroxide (1.6 cm3) were added. This solution was
freeze dried and the residue in aqueous 0.2 M sodium hydrogen
carbonate buffer (1 cm3, pH 10) was again freeze dried to
remove all the ammonia. A solution of the final residue in dis-
tilled water at pH > 11 was mixed with aqueous 0.2 M sodium
hydrogen carbonate buffer (0.8 cm3, pH 10) and adjusted to pH
9.7 with 1 M hydrochloric acid. The imidate hydrochloride 34
(25 mg) in the above buffer (2 cm3) was added and the solution
was stirred at room temperature in the dark for 2 h, monitoring
formation of the amidine 25c by TLC as described above for the
unlabelled analogue 25. The rest of the work-up of 25c through
to the NMR analysis followed exactly that used above for the
synthetic sample 25.
3 Reviewed by A. R. Battersby and F. J. Leeper, Chem. Rev., 1990, 90,
1261.
4 A. R. Battersby, C. J. R. Fookes, G. W. J. Matcham, E. McDonald
and R. Hollenstein, J. Chem. Soc., Perkin Trans. 1, 1983, 3031.
5 R. Radmer and L. Bogorad, Biochemistry, 1972, 11, 904; R. C.
Davies and A. Neuberger, Biochem. J., 1973, 133, 471.
6 C. Jones, P. M. Jordan and M. Akhtar, J. Chem. Soc., Perkin Trans.
1, 1984, 2625.
7 A. H. Jackson, W. Lertwanawatana, G. Procter and S. G. Smith,
Experientia, 1987, 43, 892.
8 H. Takahashi, H. Noguchi, K. Tomita and H. Otamasu, Yakugaku
Zasshi, 1978, 98, 618; D. L. Trepanier, V. Spanemaris and K. E.
Wiggs, J. Org. Chem., 1964, 29, 668.
9 A. D. Abell, B. K. Nabbs and A. R. Battersby, J. Am. Chem. Soc.,
1998, 120, 1741.
10 A. R. Battersby, E. Hunt, E. McDonald, J. B. Paine III and
J. Saunders, J. Chem. Soc., Perkin Trans. 1, 1976, 1008; A. R.
Battersby, C. J. R. Fookes, M. J. Meegan, E. McDonald and
H. K. W. Wurziger, J. Chem. Soc., Perkin Trans. 1, 1981, 2786.
11 D. E. Horning and J. Muchowski, Can. J. Chem., 1968, 46, 3665.
12 M. M. Kreevoy and J. E. C. Hutchins, J. Am. Chem. Soc., 1969, 91,
4329.
13 D. Enders and H. Eichenaur, Chem. Ber., 1979, 112, 2933.
14 H. Klein and A. Steinmetz, Tetrahedron Lett., 1975, 4249.
15 W. L. F. Armarego, B. A. Milloy and W. Pendergast, J. Chem. Soc.,
Perkin Trans. 1, 1976, 2229.
16 A. R. Battersby, C. J. R. Fookes, K. E. Gustafson-Potter, E.
McDonald and G. W. J. Matcham, J. Chem. Soc., Perkin Trans. 1,
1982, 2413.
17 M. J. Hunter and M. L. Ludwig, J. Am. Chem. Soc., 1962, 84, 3491.
18 I. A. Smith, Chem. Ber., 1931, 64, 427; cf. J. W. Walker and
V. K. Krieble, J. Chem. Soc., 1909, 1369.
The (11R)-PBG 1b prepared from the lactam 16b was enzym-
ically converted into the aminomethylbilane 7d and then into
1
the amidine 25d by exactly the same procedure. The H-NMR
spectra (CD3OD, 400 MHz) of the amidines 25c and 25d were
identical, respectively, with the synthetic samples 25a and
25b, apart from the expected differences due to 2H at the
interpyrrolic methylene groups. The signals from the CHDN
residues of each sample are shown in Fig. 2.
19 D. E. Neilson, D. A. V. Peters and L. H. Roach, J. Chem. Soc., 1962,
2272.
It was essential to carry out all the foregoing steps at as low a
temperature and as quickly as possible. Otherwise, the back-
ground hump evident in Fig. 2(b) became much larger; even
then, however, the signals which are quite clear in Fig. 2(a) and
2(b) could still be observed.
20 G. Hafelinger, The Chemistry of Amidines and Imidates, ed. S. Patai,
Wiley, New York, 1975, p. 15.
21 A. R. Battersby, D. A. Evans, K. H. Gibson, E. McDonald and
L. Nixon, J. Chem. Soc., Perkin Trans. 1, 1973, 1546.
22 A. R. Battersby, C. J. R. Fookes, G. W. J. Matcham, E. McDonald
and R. Hollenstein, J. Chem. Soc., Perkin Trans. 1, 1983, 3031.
23 J.-R. Schauder, S. Jendrezejewski, W. L. Neidhart, G. J. Hart and
A. R. Battersby, J. Chem. Soc., Perkin Trans. 1, 1999, 2691, following
paper.
Acknowledgements
24 A. R. Battersby, C. J. R. Fookes, K. E. Gustafson-Potter, E.
McDonald and G. W. J. Matcham, J. Chem. Soc., Perkin Trans. 1,
1982, 2427.
25 A. R. Battersby, S. Kishimoto, E. McDonald, F. Satoh and H. K. W.
Wurziger, J. Chem. Soc., Perkin Trans. 1, 1979, 1927.
26 D. A. Shirley, M. J. Danzig and F. C. Canter, J. Am. Chem. Soc.,
1953, 75, 3278.
Grateful acknowledgement is made to the NSERC (Canada)
for a Postdoctoral Fellowship (to P. C. A.), Professor D. Enders
(Aachen) for gifts of SAMP and the SERC for financial
support. We also thank Dr F. J. Leeper for his help.
References
27 P. E. Gagnon, J. L. Boivin and C. Haggart, Can. J. Chem., 1956, 34,
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1 Part 51. K. Ichinose, M. Kodera, F. J. Leeper and A. R. Battersby,
J. Chem. Soc., Perkin Trans. 1, 1999, 879.
2 Part of this work has been published in preliminary form:
W. L. Neidhart, P. C. Anderson, G. J. Hart and A. R. Battersby,
J. Chem. Soc., Chem. Commun., 1985, 924.
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