Synthesis of substrate analogs of methyltransferases in the vitamin B12 biosynthetic pathway and characterization of their enzymatic products

Article abstract of DOI:10.1016/j.bmc.2006.01.041

The specificity toward substrate analogs of the first two methyltransferases in the vitamin B₁₂ biosynthetic pathway was probed with 15 synthetic porphyrinogens. Several novel methylated chlorins and isobacteriochlorins were isolated and charac

Full text of DOI:10.1016/j.bmc.2006.01.041

Bioorganic & Medicinal Chemistry 14 (2006) 3904–3922
Synthesis of substrate analogs of methyltransferases
in the vitamin B12 biosynthetic pathway and characterization
of their enzymatic products
Clotilde Pichon-Santander, Patricio J. Santander and A. Ian Scott*
Department of Chemistry, Texas A&M University, PO Box 30012, College Station, TX 77842-3012, USA
Received 7 October 2005; revised 20 January 2006; accepted 20 January 2006
Available online 7 February 2006
Abstract—The specificity toward substrate analogs of the first two methyltransferases in the vitamin B12 biosynthetic pathway was
probed with 15 synthetic porphyrinogens. Several novel methylated chlorins and isobacteriochlorins were isolated and character-
ized, suggesting the same methylation sequence C-2 > C-7 > C-20 as for the natural substrate, uro’gen III. The results allow us
to narrow down possible structural requirements concerning substrate recognition by the methyltransferase enzymes.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
of uro’gen III 1e to its precorrin-2 (3e) or precorrin-3
(4e) analogs.15,16
Over the last 50 years, several laboratories have ex-
plored different aspects of the complex biosynthetic
pathway to vitamin B12,1 which utilizes over 25 enzymes.
Of these, only the earlier ones, responsible for the for-
mation of the tetrapyrrole macrocycle, have been stud-
ied in detail by us and others, and much less is known
about the enzymes implicated in the steps after uropor-
phyrinogen III (1a, uro’gen III) (Scheme 1), including C-
methylations at positions 2, 7, and 20 to give precorrin-1
(2a), precorrin-2 (3a), and precorrin-3 (4a), respectively.
These intermediates were isolated and characterized as
their oxidized forms, factor 1 (6a),2–4 factor 2 (7a),5,6
and factor 3 (8a).6,7 A single enzyme (M-1) is responsible
for the first two methylations, while a second enzyme
(M-2) mediates the third methylation. Both enzymes
are S-adenosylmethionine (SAM) dependent and have
been overexpressed from different sources.8–11 The natu-
ral substrate of M-1 is uro’gen III (1a, Scheme 1); how-
ever, its isomers uro’gen I, II, and IV (1b, 1c, and 1d in
Scheme 2) have also been shown to serve as sub-
strates.12–14 In addition, it has been demonstrated that
cell-free systems from Propionibacterium shermanii and
Pseudomonas denitrificans, both organisms producing
vitamin B12, are able to methylate the 12-methyl analog
These results suggested the possibility of further
exploring the substrate specificity of the first two
methyltransferases in order to gain a better under-
standing of the structural requirements for activity
and to utilize these enzymes in
a semisynthetic
approach to obtain unnatural chlorins and isobacterio-
chlorins, compounds which in recent years have
attracted strong interest as photosensitizers for use in
photodynamic therapy.17,18
2. Porphyrin syntheses
All the analogs tested in this study are minor variations
on the nature and pattern of substitution present on the
naturally occurring porphyrinogens uro’gen I and uro’-
gen III, which exhibit only acetate and propionate side
chains (Scheme 2). Since porphyrinogens are oxidized
rather easily upon air exposure, all these analogs are
more conveniently prepared in their oxidized form
(i.e., porphyrin) and reduced back to the porphyrinogen
form just before the enzymatic incubation.
The series 5f–5h (Scheme 2) corresponds to analogs of
uroporphyrin (uro) I (5b), whose propionate substitu-
ents have been replaced by butyrate (5f), acetate (5g)
or methyl groups (5h). Coproporphyrin (copro) I (5i)
is the known naturally occurring product of decarboxyl-
Keywords: Vitamin B12; Methyltransferase; Chlorin; Isobacteriochlorin.
*
Corresponding author. Tel.: +1 979 845 3243; fax: +1 979 845
5992; e-mail: scott@mail.chem.tamu.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.041

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3905
P
P
A
P
P
A
Me
A
Me
A
5
7
A
A
A
2
A
A
P
P
P
P
A
B
Me
Me
Me
Me
N
N
HN
N
NH
NH
1
HN
HN
HN
HN
HN
M-1
M-2
M-1
10
20
NH
12
HN
NH
NH
HN
A
C
A
A
A
D
A
A
A
15
P
P
P
P
P
P
P
P
precorrin-3 (4a)
uro'gen III (1a)
precorrin-1 (2a)
precorrin-2 (3a)
oxidation
oxidation
oxidation
oxidation
P
A
P
P
A
P
Me
A
Me
A
A
A
A
A
A
P
P
P
P
Me
Me
Me
N
N
HN
N
NH
N
N
HN
N
HN
Me
A
NH
NH
N
NH
HN
N
A
A
A
A
A
A
P
P
P
P
P
P
P
P
factor 2 (7a)
factor 3 (8a)
uro III (5a)
factor 1 (6a)
A=CH2CO2H, P=CH2CH2CO2H
Scheme 1. First three methylations in the biosynthetic pathway of vitamin B12
.
ation of uro I, which has methyl groups in place of all
four acetate side chains. This series of porphyrins was
obtained by tetramerization of their parent 2-hydroxy-
methylpyrroles in buffer pH 7.8–8.0, followed by cycliza-
tion and oxidation, as described in a previous report.19
The preponderance of the type I isomer was over 90%
in all cases, based on NMR analysis.
(Schemes 2 and 3). The appropriate dipyrromethanes,
14 and 15, for porphyrins 5j, 5k, and 5l (Scheme 2),
16, and 17 for porphyrins 5e, 5m and 5n (Scheme 3),
were dissolved in CH2Cl2 + 10% MeOH and treated
with p-toluenesulfonic acid under an inert atmosphere
in the dark for one to two days. After neutralization
with sodium bicarbonate, the coupling products were
fully oxidized under an oxygen atmosphere to porphy-
rins and isolated by preparative TLC, eluting twice with
CH2Cl2 + 2% MeOH. Their RF’s decrease with the num-
ber of carboxylate b-substituents, in the order
hexa ! hepta ! octa-methyl esters. The yields of the
target porphyrins vary from 15% to 31%, thus making
them comparable with those reported using other meth-
ods. Variation in the ratio of dipyrroles utilized (2:1 to
1:2) did not improve the yields of the desired porphyrins
and a 1:1 ratio was routinely used.
A second series of uro analogs 5j–5n and 5e (Scheme 2)
retains the same acetate and propionate substitution
pattern on the ‘Northern part’ of the molecule (rings
A and B) as the natural substrate uro’gen III, but in-
cludes variations on the ‘Southern part’ (rings C and
D). From the preparative point of view 5j–5l, which
have a symmetrical ‘Southern part’, were made using
the MacDonald approach,20,21 consisting of the conden-
sation of
a diformyldipyrromethane (14) with a
dipyrromethane-dicarboxylic acid (15) (Scheme 3). Por-
phyrins 5e, 5m, and 5n, which do not present symmetry
in the ‘Southern part,’ had to be made by a different
strategy. In order to limit the number of preparative
steps by using the building blocks already available in
our laboratory, a modification of the MacDonald meth-
od was devised for their synthesis, in which each of the
dipyrroles carries one aldehyde and one carboxylic acid
(Scheme 4); notice that although mixtures of products
are obtained, these are easily separated thanks to the dif-
ferent chromatographic behaviors of the alternative
products bearing a different number of methyl ester side
chains. The side-products, hexamethylester porphyrins
5o and 5p, were also included in our activity study.
3. Enzymatic study results
The methyltransferase source chosen for the experi-
ments was CR395, a recombinant strain of Escherichia
coli, in which two vitamin B12 enzymes CobA (M-1)
and CobI (M-2) from P. denitrificans are overexpres-
sed.24 The enzymes were used as a clear cell lysate with-
out further purification except for removal of
endogenous porphyrinoids.
The methyl esters of the porphyrin analogs were hydro-
lyzed with 2 N KOH. The pH of the solution was reduced
to 8–9 and the free-acid porphyrins were reduced back to
porphyrinogens by addition of small pieces of 3% sodium
amalgam to the solution until colorless (RT, 2 h). To
avoid any oxidation, the reduction, enzymatic incuba-
tion, work up, and esterification were all performed under
All dipyrromethanes were prepared following published
procedures.22,23 The benzyl esters were hydrogenated in
THF over 10% Pd–C in the presence of Et3N and after
work up, used as such for the coupling reactions

3906
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
Scheme 2. Substrate analogs tested in this study and their enzymatic products.
argon atmosphere in a glove-box. The free-acid porphyri-
nogen solutions were filtered into a degassed Tris-buffer
solution, adjusted to pH 7.5 containing CobA and CobI
lysate, and SAM, and incubated at 37 ꢁC under argon
overnight. The products were isolated by adsorption on
DEAE–Sephadex resin, elution and esterification with
MeOH + 5% H2SO4, neutralization, extraction into
CH2Cl2, and chromatography on preparative TLC (ben-
zene/ethyl acetate/MeOH, 70:27:3).
of the enzyme substrates and products makes it very dif-
ficult to perform kinetic studies. Since the main objective
of this work was to explore the possible use of these en-
zymes for synthesis of new unnatural chlorins and iso-
bacteriochlorins, more emphasis was put in their
isolation and characterization.
1
All products were characterized by H NMR, UV–vis,
and mass spectrometry, and in a few cases by 13C
NMR. Some of the products isolated are known (7a, 7b,
7d, 8a, 8e, 10a, 11a, 12a and 13a) and our NMR data
and assignments are in agreement with the published re-
The results are shown in Table 1 and correspond to the
isolated yields of the products. Instability to oxidation

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3907
PMe
AMe
data and slight differences in their NMR data, they are as-
sumed to be epimerization products at C-3 or C-8, like
those described for factor 1 6a and factor 2 7a.3,27 The
meso protons and methyl H NMR assignments of all
products are reported in Table 2.
PMe
AMe
AMe
PMe
CHO
AMe
PMe
1
NH
HN
HN
OHC
NH
N
14
1) [H+]
2) O2
+
RO2C
R1
CO2R
R1
N
HN
R1
As seen in Table 1, some of the porphyrinogens acted
solely as substrates of the first methyltransferase (1b,
1d, 1f, 1j, and 1n); apart from the natural substrate 1a,
only two porphyrinogens (1e and 1m) served as sub-
strates of the two methyltransferases and for most (1c,
1g, 1h, 1i, 1k, 1l, 1o, and 1p), no methylation products
could be detected. Although cell lysates expressing
simultaneously CobA and CobI were used instead of
pure proteins, control experiments with the natural sub-
strate uro’gen III (1a) show virtually complete conver-
sion to factor 3 (8a) and lactones (12a and 13a), with
no factor 1 (6a) or factor 2 (10a) or respective lactone
being observed, which means that both enzymes were
sufficiently well expressed and active.24 Therefore, it
can be safely concluded that the absence of product for-
mation with some substrates cannot be attributed to the
lack of expression or activity of any of the enzymes, but
to other factors. The results of this study indicate that,
although a majority of the analogs tested were not meth-
ylated by CobA or CobI, these enzymes display a great-
er tolerance than the earlier enzymes on the vitamin B12
biosynthetic pathway, such as PBG deaminase and uro’-
gen III synthase.19
NH
R1
R2
R2
R2
R2
5j R1=R2=AMe
5k R1=R2=PMe
15
5l R1=Me R2=PMe
AMe=CO2Me PMe=CH2CH2CO2Me R=H or tBu
Scheme 3. Synthesis of porphyrins by the MacDonald method.
sults.6,12,14,16,25,26 Their isolation confirms that the gener-
al protocol for enzymatic incubation, and product isola-
tion and purification were adequate. The nature and
methylation level of the other products are based on their
UV–vis, mass, and NMR data. Their structures are de-
rived by correlation with the NMR of the known com-
pounds, using the sp2 or sp3 nature of individual
carbons revealed by their NMR as guidance to determine
the sites of enzymatic methylation. Often, isomers were
also observed by 1H NMR and in most cases, only a par-
tial assignment was possible, since the products could not
be separated from the isomeric mixture and/or were pres-
ent in too small a quantity. Based on their identical mass
PMe
AMe
PMe
AMe
R2
R3
PMe
AMe
AMe
PMe
AMe
R1
R4
AMe
NH
PMe
R4
PMe
HN
NH
NH
NH
N
N
N
OHC
CO2H
1) [H+]
2) O2
16
+
+
+
HO2C
R4
CHO
R1
N
HN
N
HN
N
HN
PMe
R1
R1
AMe
R4
HN
NH
R2
Heptamethyl ester
R2
Hexamethyl ester
PMe
R3
R3
AMe
R3
R2
5b
17
5e R1=Me R2=R3=PMe R4=AMe 5p
5m R1=AMe R2=R3=PMe R4=Me 5o
5n R1=AMe R2=R4=PMe R3=Me 5p
AMe=CO2Me PMe=CH2CH2CO2Me
Scheme 4. Synthesis of porphyrins by a modified MacDonald method.
Table 1. Results of porphyrinogen incubations with CobA and CobI
Substratea
Isolated products and yields (%)
DiMeIBC 7b DiMeIBC TriMeIBC 8b TriMeIBC
DiMeIBC
monolactone 10b dilactone 11b monolactone 12b dilactone 13b
MeC 6b MeC
monolactone 9b
TriMeIBC
1a
1b
1d
1e
1f
—
—
—
30–36%c
3–5%
1%
4%c
4–5%
Tracesd
—
2%c
<1%
—
22%
—
Tracesd
28%
—
8%
—
Tracesd
9%
—
6%
—
—
7%
—
—
—
—
—
19%
2%
Tracesd
2%
—
—
—
—
—
1–3%
5%
—
9%
1j
13%
—
5%
—
—
8–11%
—
—
—
1m
1n
2–3%
10%
—
1%
1%
—
1%
—
2%
1%
a Other porphyrinogens 1c, 1g, 1h, 1i, 1k, 1l, 1o, and 1p were tested, but no product of methylation was detected.
b In most cases, epimeric forms are present and were observed by 1H NMR (500 MHz) (Table 2).
c Incubation run with CobA (M-1) only, instead of CobA (M-1) and CobI (M-2).
d Products were isolated in very small quantities and were not confirmed to be derived from uro’gen IV.

3908
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
1
Table 2. Characteristic H NMR assignments (d in C6D6, 500 MHz) for methylation products
Products
H-15
H-10
H-20
H-5
Me-20
Me(C#)
3.11 (12)
Me-2
Me-7
Ref.a
MeC
6d
10.09
10.08
10.09
10.29
10.10
10.10
10.08
10.08
10.09
10.08
9.91
9.16
9.08
9.18
9.19
9.14
9.16
9.17
9.10
8.97
8.92
9.00
9.06
8.86
8.90
9.00
8.95
2.22
1.85
2.24
2.28
2.20
2.25
2.25
1.85
3 and 4
6e
6f
10.19
10.04
10.00
9.89
6j
6m
6n
3.10 (18)
3.11 (17)
3.20 (17)
9.89
MeC monolactone
9d
10.21
10.19
9.35
8.80
1.72
DiMeIBC
7a
8.85
8.82
8.83
8.84
8.80
8.85
8.83
8.82
8.80
8.78
8.71
7.64
7.52
7.65
7.65
7.55
7.61
7.55
7.59
7.61
7.50
7.69
7.54
7.41
7.53
7.48
7.47
7.55
7.43
7.48
7.35
7.47
7.53
6.78
6.68
6.71
6.80
6.71
6.81
6.72
6.76
6.71
6.68
6.83
1.75
1.84
1.59
1.68
1.79
1.74
1.85
1.71
1.59
1.80
1.75
1.60
1.44
1.53
1.61
1.45
1.63
1.49
1.60
1.52
1.45
1.65
6
7b
12
14
7d
7e
7j
2.63 (12)
2.78 (17)
7n
DiMeIBC monolactone
10a
10b
10j
8.96
8.98
8.95
8.94
8.90
8.86
8.88
8.85
8.79
8.06
7.68
8.08
7.70
7.61
7.97
7.99
7.77
7.69
7.55
7.53
7.48
7.55
7.45
7.39
7.48
7.61
7.57
6.71
6.97
6.64
7.02
6.92
6.56
6.59
6.99
6.95
1.75
1.44
1.67
1.45
1.43
1.51
1.71
1.50
1.46
1.21
1.37
1.22
1.35
1.34
1.20
1.22
1.41
1.39
25
10n
2.81 (17)
2.78 (17)
DiMeIBC dilactone
11a
11b
11j
9.28
8.25
8.28
8.13
8.37
7.75
7.79
7.63
7.86
7.08
7.27
7.07
7.37
1.42
1.42
1.38
1.46
1.31
1.31
1.25
1.36
26
9.25
9.10
9.15
11n
2.93 (17)
TriMeIBC
8a
8.73
9.05
8.80
8.72
8.74
8.81
8.95
8.85
7.50
7.76
7.41
7.41
7.74
7.41
7.79
7.61
6.51
6.99
6.64
6.50
6.56
6.64
6.58
6.67
2.77
2.79
2.85
2.78
2.65
2.85
2.83
2.84
1.66
1.51
1.68
1.85
1.67
1.68
1.71
1.75
1.40
1.39
1.49
1.43
1.49
1.49
1.26
1.46
6
8e
2.56 (12)
2.53 (12)
2.52 (12)
2.56 (18)
2.47 (18)
2.65 (18)
16
8m
TriMeIBC monolactone
12a
12e
12m
8.85
8.95
8.97
7.88
7.79
8.00
6.40
6.58
6.60
2.73
2.83
2.73
1.67
1.71
1.78
1.16
1.26
1.21
2.47 (12)
2.64 (18)
TriMeIBC dilactone
13a
13m
9.46
8.38
8.49
8.29
7.58
7.75
7.74
2.99
2.90
3.17
1.49
1.55
1.60
1.45
1.52
1.46
9.63
9.54
2.84 (18)
2.63 (18)
a In agreement with values reported in the references cited.
4. Discussion
to 1e), methylations for the natural substrate uro’gen
III (1a) have been shown to take place sequentially at
C-2, C-7, and C-20.1–4,6 NMR data for the product of
methylation of uro’gen I (1b) indicate that the sites of
In the series of porphyrinogens which have previously
demonstrated substrate activity with M-1 and M-2 (1a

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3909
methylation are also acetate termini on contiguous
rings, consistent with the dimethylated isobacteriochlo-
rin (diMeIBC) 7b.12 Uro’gen IV (1d) also gave a product
of dimethylation at C-2 and C-7 7d,14 however the main
product was monomethylation at C-2 to form the meth-
ylchlorin (MeC) 6d. For porphyrinogen 1e, the 12-meth-
yl analog of the natural substrate 1a, the complete series
of mono-, di- and trimethylated products (6e, 7e and 8e)
was isolated, with the trimethylated isobacteriochlorin
(triMeIBC) 8e16 being the major product, and follows
the same methylation sequence as for the natural sub-
strate. The spectroscopic data of all additional products
isolated from incubations of 1a, 1b, 1d, and 1e with
CobA and CobI are consistent with products of epimer-
ization at C-3 or C-8,3,27 or lactonization on ring A and/
or ring B.25,26
quantities of MeC 6f were isolated (Table 1). No meth-
ylation was ever observed at a methyl or propionate ter-
minus, nor if C-3 was substituted by an acetate or
methyl group. In turn, the second methylation on the
adjacent ring B always occurs at C-7 with an acetate ter-
minus next to a propionate terminus at C-8. Only three
porphyrinogens acted as substrates of M-2 for the third
methylation at C-20: uro’gen III (1a), the natural sub-
strate, and porphyrinogens 1e and 1m, analogs of 1a
with a methyl replacing the acetate group on ring C
for 1e and ring D for 1m. From these results, it seems
that the sequence APAPXPPY (A, acetate; P, propio-
nate) with X and Y being A or Me would be necessary
in order to have methylation at C-20.
However, these structural requirements do not explain
all our results. For instance, porphyrinogens 1d and
1n possess the substitution sequence APAP on their
‘Northern part’ and thus, dimethylation was expected,
but although the dimethylated products 7d and 7n
were isolated, the main products in both cases were
the monomethylated chlorins 6d and 6n (Table 1).
Similarly, monomethylation could have been anticipat-
ed for the porphyrinogens 1c, 1k, 1l, 1o, and 1p,
which all have a ring bearing the natural AP substitu-
tion pattern, but no product of methylation was
detected. It should be mentioned that we had in our
hands limited amounts of 1c and 1l, so incubations
with these two porphyrinogens could not be repeated.
As for 1o and 1p, solubility problems with their meth-
yl esters may have led to incomplete ester hydrolysis
and the subsequent porphyrin to porphyrinogen
reductions. Steric hindrance for fitting in the catalytic
site of M-1 could explain the lack of reactivity of 1k.
Thus, it seems that the structural requirements out-
lined above would constitute minimum conditions to
be met for substrate activity with the methyltransfe-
rases CobA and Cob I.
In earlier work with the E. coli M-1 CysG, a dimethyla-
tion product was isolated from incubation with uro’gen
II (1c).14 The NMR data of this methylation product
pointed to the second methylation occurring at C-7 on
a propionate terminus. However, in our studies, incuba-
tions of 1c with CobA and CobI did not give any meth-
ylated product and no evidence of methylation at a
propionate terminus with any of the porphyrinogens
tested was found.
By comparison with the data discussed above, the spec-
troscopic data for the products of the four new por-
phyrinogen, tested in our study (1f, 1j, 1m, and 1n)
indicate a similar methylation site pattern: C-2 > C-
7 > C-20. Although it could be ascertained that the only
methylation product from 1f is monomethylated at an
acetate terminus (6f), the presence of isomers in the
starting porphyrinogen 1f combined with the very low
yield of methylation does not allow determination of
its ‘isomeric type’ (i.e., I–IV). In case of porphyrinogen
1j, which has the same ‘Northern part’ as the natural
product 1a, but only acetate substituents on the ‘South-
ern part,’ it seems likely that methylations also occur at
C-2 and C-7, adjacent to propionate termini on the
‘Northern part,’ since porphyrinogen 1g bearing only
acetate substituents is not a substrate of these methyl-
ases. Finally, for porphyrinogens 1m (18-methyl analog
of uro’gen III 1a) and 1n (17-methyl analog of uro’gen I
1b), NMR data show that the carbon carrying the meth-
yl group originally present remains sp2 in the enzymatic
products, showing that methylations, once again, had
taken place on the ‘Northern’ rings with the natural ace-
tate/propionate substitution pattern. However, the pre-
cise position of the methyl group originally present has
not been assigned and could be either at C-13 or C-18
for the products derived from 1m and either C-12 or
C-17 for the products derived from 1n. Products of epi-
merization on ring A and/or ring B were also observed
with all four porphyrinogens.
In addition, it is interesting to note that porphyrinogen
1e (the 12-methyl analog of uro’gen III) acts as a sub-
strate of CobA and CobI as efficiently as the natural
substrate uro’gen III (1a) (Table 1). Thus, the trimethy-
lated dihydroisobacteriochlorin (triMediHIBC) 4e be-
comes a logical candidate for further methylations.
However, prior experiments with 14C-enriched 1e gave
very low incorporation into cobester,28–30 the stable co-
balt containing intermediate of vitamin B12 obtained
from uro’gen III after eight methylations, one decarbox-
ylation at the C-12 acetate, and one methyl migration
from C-11 to C-12. This would imply that 1e could only
be taken up to a certain point along the enzymatic path-
way of vitamin B12. The block in the pathway could be
at the fifth methylation at C-11 or at the methyl shift
from C-11 to C-12, which takes place after the final
methylation, as these are the only two steps in the path-
way which involve ring C.1
From this study, some electronic factors governing the
methyltransferase CobA and CobI mechanism could
be discerned. The first methylation on ring A always
happens at C-2 with an acetate terminus next to a pro-
pionate substituent at C-3, except for 1f, which bears a
butyrate group at C-3, but for which only very small
5. Conclusion
A number of porphyrinogens have been tested for sub-
strate specificity with the first two methyltransferases

3910
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
of the vitamin B12 biosynthetic pathway. Eight of them
gave methylation products, which have been isolated
and characterized, and showed to follow the same meth-
ylation pattern C-2 > C-7 > C-20 as the natural sub-
strate. This has allowed us to discern the substitution
sequence APAPXPPX (with A, acetate; P, propionate;
X and Y, A or methyl) as necessary for substrate recog-
nition by the methyltransferase enzymes CobA and
CobI. Furthermore, four of the porphyrinogens have
never been tested before and through a semisynthetic
approach yielded a series of novel chlorins and isobacte-
riochlorins, two important classes of compounds in the
field of photodynamic therapy.
8H, 4 CH2CO2Me); 4.31 (m, 8H, 4 CH2CH2CO2Me);
3.83, 3.82, 3.79, 3.78, 3.71, 3.69 (6s, 24H, 8 CO2CH3);
3.31 (m, 8H, 4 CH2CH2CO2Me). 13C NMR d 173.44,
171.81, 8CO2Me; 140.91, 132.79, 16 C porphyrinic;
97.85, 97.75, 97.69, 97.59, 4 meso C; 52.40, 51.73, 8
CO2CH3; 37.01, 36.90, 4 CH2CH2CO2Me; 32.36, 4
CH2CO2Me; 21.72,
CH2CH2CO2Me.
21.68,
21.60,
21.51,
4
6.1.2.2. Uro I (5b). 1H NMR (CDCl3): d 10.7 (s, 4H, 4
meso H); 5.06 (s, 8H, 4 CH2CO2Me); 4.38 (t, J = 7.8,
8H, 4 CH2CH2CO2Me); 3.77, 3.68, (2s, 24H, 8
CO2CH3); 3.33 (t, J = 7.8, 8H, 4 CH2CH2CO2Me). 13C
NMR d 173.44, 171.83, 8 CO2Me; 141.06, 132.96, 16
C porphyrinic; 52.42, 51.75, 8 CO2CH3; 37.05, 4
6. Experimental
6.1. Chemistry
CH2CH2CO2Me; 32.49,
CH2CH2CO2Me.
4
CH2CO2Me; 21.76,
4
6.1.2.3. Uro II (5c). 1H NMR (CDCl3): d 10.16, 10.14
(2s, 4H, 4 meso H); 5.10 (s, 8H, 4 CH2CO2Me); 4.43 (t,
J = 7.3, 8H, 4 CH2CH2CO2Me); 3.79, 3.66 (2s, 24H, 8
CO2CH3); 3.33 (t, J = 7.3, 8H, 4 CH2CH2CO2Me). 13C
NMR d 173.48, 171.82, 8 CO2Me; 141.10, 133.20, 16
C porphyrinic; 98.12, 97.82, 4 meso C; 52.48, 51.78, 8
6.1.1. General procedures. All reagents were purified and
dried when necessary according to standard literature
methods. All starting pyrroles come from a large collec-
tion of monopyrrole building blocks with acetate, propi-
onate, butyrate, and methyl b-substituents previously
made in our laboratory. All reactions were conducted
in anhydrous solvents and under argon atmosphere, un-
less specified otherwise. Column chromatographies were
carried out on E. Merk Kieselgel 60 (100–200 mesh),
TLC, and preparative TLC (PLC) on Analtech silicagel
GF plates.
CO2CH3; 36.99,
CH2CO2Me; 21.74, 4 CH2CH2CO2Me.
4
CH2CH2CO2Me; 32.55,
4
1
6.1.2.4. Uro IV (5d). H NMR (CDCl3): d 10.14 (s,
4H, 4 meso H); 5.10, 5.08 (2s, 8H, 4 CH2CO2Me);
4.42 (t, J = 7.7, 8H, 4 CH2CH2CO2Me); 3.78, 3.77,
3.68, 3.66 (4s, 24H, 8 CO2CH3); 3.35, 3.32 (AB,
J = 8.0, 8H, 4 CH2CH2CO2Me). 13C NMR d 173.50,
173.46, 171.85, 8 CO2Me; 141.12, 133.13, 16 C porphyri-
nic; 97.96, 4 meso C; 52.48, 51.79, 8 CO2CH3; 37.10,
37.01, 4 CH2CH2CO2Me; 32.77, 32.51, 4 CH2CO2Me;
21.86, 21.72, 4 CH2CH2CO2Me.
1H and 13C NMR (500 MHz) were recorded on a Bruc-
ker AM-500 and the coupling constants expressed in
MHz. Concerning the carbon signals of the porphyrin
macrocycle, it should be noted that, as previously postu-
lated by others, the relatively slow exchange on NMR
timescale between tautomeric forms of the free-base por-
phyrin macrocycle leads to the observation of only one
broad and weak signal for the a-carbons, often barely
distinguishable from the baseline noise, and one broad
but sharper signal at higher field for the b-carbons, even
in the absence of complete symmetry in the molecule. It
is also known that the tautomeric mobility is increased
by electron-withdrawing substituents. Thus, observation
of the signals for the a- and b-carbons of the porphyrin
macrocycle varies with the substituents and degree of
symmetry.31
6.1.3. Preparation of the symmetrical porphyrins 5f, 5g,
5h, and 5i. The syntheses of the a-free formylpyrrole pre-
cursors of porphyrins 5f, 5g, 5h, and 5i were performed
following standard procedures.32
6.1.3.1.
2-Formyl-3-methoxycarbonylmethyl-4-(3-
methoxycarbonylpropyl)-pyrrole (precursor of 5f). 1H
NMR (CDCl3): d 10.24 (s br, 1H, NH); 9.55 (s, 1H,
CHO); 6.87 (d, J = 2.9, 1H, H-5); 3.70 (s, 2H,
CH2CO2Me); 3.64, 3.61 (2s, 6H, 2 CO2CH3); 2.44 (t,
J = 7.6, 2H, CH2CH2CH2CO2Me); 2.30 (t, J = 7.4, 2H,
CH2CH2CH2CO2Me); 1.82 (m, 2H, CH2CH2CH2CO2
Me). 13C NMR d 178.03, CHO; 173.79, 171.18, 2 CO2Me;
129.89, 125.67, 125.23, 124.56, 4 C pyrrolic; 52.16, 51.43, 2
CO2CH3; 32.25, CH2CH2CH2CO2Me; 29.43, CH2CO2
Me; 25.24, CH2CH2CH2CO2Me; 23.66, CH2CH2CH2
CO2Me.
UV spectra were obtained on an Ocean Optics USB2000
Miniature Fiber Optic Spectrophotometer and mass
spectra on a PE SCIEX QSTAR quadrupole time-of-
flight hybrid mass spectrometer using an electrospray
ionization source.
6.1.2. Uro III (5a), uro I (5b), uro II (5c), and uro IV (5d).
Uro III (5a) was obtained by incubation of PBG with
PBG deaminase and uro’gen III synthase, and uro I
(5a) by incubation with PBG deaminase as described
in Ref. 19. Uro II (5c) and uro IV (5d) were made for
previous work in our group.14
6.1.3.2. 2-Formyl-3,4-dimethoxycarbonylmethyl-pyr-
role (precursor of 5g). H NMR (CDCl3): d 10.46, (s
1
br, 1H, NH); 9.52 (s, 1H, CHO); 6.95 (d, J = 3.0, 1H,
H-5); 3.73 (s, 2H, CH2CO2Me at C-3); 3.60 (s, 6H, 2
CO2CH3); 3.45 (s, 2H, CH2CO2Me at C-4). 13C NMR
d 178.13, CHO; 171.69, 170.90, 2 CO2CH3; 129.82,
125.88 (2), 117.82, 4 C pyrrolic; 52.03, 51.79, 2 CO2CH3;
30.20, 29.31, 2 CH2CO2Me.
6.1.2.1. Uro III (5a). 1H NMR (CDCl3): d 10.04, 9.97,
9.95, 9.94 (4s, 4H, 4 meso H); 5.06, 5.04, 5.00, 4.95 (4s,

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3911
6.1.3.3. 2-Formyl-4-methyl-3-methoxycarbonylmethyl-
pyrrole (precursor of 5h). H NMR (CDCl3): d 10.52 (s
br, 1H, NH); 9.51 (s, 1H, CHO); 6.82 (d, J = 2.7, 1H,
H-5); 3.69 (s, 2H, CH2CO2Me); 3.62 (s, 3H, CO2CH3);
1.98 (s, 3H, CH3). 13C NMR d 177.81, CHO; 171.16,
CO2Me; 129.63, 126.34, 125.41, 121.17, 4 C pyrrolic;
51.97, CO2CH3; 29.38, CH2CO2Me; 9.25, CH3.
ylethyl)-3,40-dimethoxycarbonylmethyl-2,20-methylene-
dipyrrole-5-carboxylate32 (1.07 g, 1.54 mmol) was
dissolved in THF (25 mL) and hydrogenated over 10%
Pd–C (100 mg) at RT overnight. The solution was fil-
tered through Celite, the catalyst washed with CH2Cl2,
and the filtrate evaporated to dryness. The 50-tertbu-
tyl-30,4-di-(2-methoxycarbonylethyl)-3,40-di-methoxy-
carbonylmethyl-2,20-methylenedipyrrole-5-carboxylic
acid (915 mg, 1.51 mmol, 98%) was used without further
purification in the next step. 1H NMR (CDCl3): d 10.74,
10.49 (2s, 2H, 2NH); 4.07 (s, 2H, CH2 meso) 3.91 (s, 2H,
1
6.1.3.4. 2-Formyl-4-methyl-3-(2-methoxycarbonyleth-
yl)-pyrrole (precursor of copro 5i). H NMR (CDCl3):
1
d 10.41 (s br, 1 H, NH); 9.51 (s, 1 H, CHO); 6.83 (d,
J = 2.8, 1H, H-5); 3.59 (s, 3H, CO2CH3); 2.98 (t,
J = 7.7, 2H, CH2CO2Me); 2.51 (t, J = 7.7, 2H,
CH2CH2CO2Me); 1.97 (s, 3H, CH3). 13C NMR d
177.64, CHO; 172.83, CO2Me; 133.37, 129.12, 125.74,
CH2CO2Me); 3.72, 3.68, 3.64, 3.60 (4s, 12H,
4
CO2CH3); 3.50 (s, 2H, CH2CO2Me); 2.98, 2.76 (2t,
J = 7.6, 6.8, 4H, 2 CH2CH2CO2Me); 2.54, 2.47 (2t,
J = 7.6, 6.8, 4H, 2 CH2CH2CO2Me); 1.47 (s, 9H,
CO2C(CH3)3). 13C NMR d 173.92, 173.70, 172.88,
172.04, 4 CO2Me; 165.18, CO2CMe3; 161.47, CO2H;
133.11, 131.28, 130.22, 121.41, 120.53, 119.85, 117.54,
114.54, 8 C pyrrolic; 81.49, CMe3; 52.25, 51.74, 51.38,
4 CO2CH3; 34.75, 34.57, 2 CH2CH2CO2Me; 30.84,
29.37, 2 CH2CO2Me; 28.21, C(CH3)3; 22.29, CH2 meso;
20.36, 18.89, 2 CH2CH2CO2Me.
120.21,
CH2CO2Me; 18.92, CH2CH2CO2Me; 9.33, CH3.
4
C
pyrrolic; 51.48, CO2CH3; 35.22,
The symmetrical porphyrins were obtained by tetramer-
ization of their parent monopyrroles in buffer as
described in Ref. 19.
6.1.3.5. Porphyrin 5f (main isomer). 1H NMR
(CDCl3): d 10.27 (s, 4H, 4 meso H); 5.12 (s, 8H, 4
The product prepared above was redissolved in TFA
(10 mL) and stirred at RT for 4 h. The solution was
cooled in ice, trimethylorthoformate (5 mL) was added
dropwise, and the reaction was stirred for another 3 h
at RT. Ethyl acetate (30 mL) was poured into the reac-
tion and the solution was washed with H2O (3· 30 mL),
10% NaHCO3 (2· 25 mL), and NaHCO3 until no longer
acidic. After evaporation, the product 14 was purified by
PLC eluting twice in ethyl acetate/hexanes (2/1) (203 mg,
CH2CO2Me);
4.16
(t,
J = 7.5,
8H,
4
CH2CH2CH2CO2Me); 3.73, 3.72 (2s, 24H, 8 CO2CH3);
2.74 (t, J = 6.7, 8H, 4 CH2CH2CH2CO2Me); 2.66 (m,
8H, 4 CH2CH2CH2CO2Me). 13C NMR d 174.04,
172.01, 8 CO2Me; 142.09, 132.68, 16 C porphyrinic;
98.00, 4 CH meso; 52.38, 51.57, 8 CO2CH3; 33.72, 4
CH2CH2CH2CO2Me; 32.54, 4 CH2CO2Me; 28.12, 4
CH2CH2CH2CO2Me; 25.74, 4 CH2CH2CH2CO2Me.
MS (ESI) m/z 999 [M+H]+. HRMS (ESI)
[C52H63N4O16]+, calcd: 999.4239. Found: 999.4217.
1
0.39 mmol, 25%). H NMR (CDCl3): d 10.58, 10.53 (2s
br, 2H, 2 NH); 9.53, 9.51 (2s, 2H, 2 CHO); 4.00 (s, 2H,
CH2 meso); 3.75, 3.71, 3.66, 3.62 (4s, 12H, 4 CO2CH3);
3.61, 3.53 (2s, 4H, 2 CH2CO2Me); 3.00, 2.78 (2t, J = 7.6,
1
6.1.3.6. Porphyrin 5g. H NMR (CDCl3): d 10.86 (s,
4H, 4 meso H); 5.14 (s, 16H, 8 CH2CO2Me); 3.66 (s,
24 H, 8 CO2CH3). 13C NMR d 169.75, 8 CO2Me;
142.05, 135.12, 16 C porphyrinic; 101.15, 4 CH meso;
52.98, 8 CO2CH3; 32.78, 8 CH2CO2Me. MS (ESI) m/z
887 [M+H]+.
6.8, 4H,
2
CH2CH2CO2Me); 2.55 (m, 4H,
2
CH2CH2CO2Me). 13C NMR d 177.64, 177.44, 2 CHO;
174.29, 172.95, 172.75, 171.18, 4 CO2Me; 135.45,
134.46, 133.64, 129.39, 128.72, 125.74, 121.02, 8 C pyr-
rolic; 52.70, 52.31, 52.20, 51.70, 4 CO2CH3; 35.86,
34.17, 2 CH2CH2CO2Me; 29.92, 29.40, 2 CH2CO2Me;
22.48, CH2 meso; 19.03, 18.50, 2 CH2CH2CO2Me.
6.1.3.7. Porphyrin 5h (main isomer). 1H NMR
(CDCl3): d 10.13 (s, 4H, 4 meso H); 5.06 (s, 8H, 4
CH2CO2Me); 3.75 (s, 12H, 4 CO2CH3); 3.66 (s, 12H, 4
CH3). 13C NMR d 174.23, 4 CO2Me; 140.26, 136.12,
16 C porphyrinic; 98.12, 4 CH meso; 52.00, 4 CO2CH3;
29.77, 4 CH2CO2Me; 11.83, 4 CH3. MS (ESI) m/z 655
[M+H]+.
6.1.4.2. 3,30,4,40-Tetramethoxycarbonylmethyl-2,20-
methylenedipyrrole-5,50-dicarboxylic acid (15j).
A
solution of the benzyl-2-methyl-3,4-dimethoxycarbon-
ylmethyl-pyrrole-5-carboxylate (1.08 g, 3.0 mL), pre-
pared following standard procedures,33–35 was
dissolved in AcOH/Ac2O (16 mL/4 mL) and treated with
Pb(OAc)4 (2.0 g, 4.6 mmol) at 80 ꢁC for 4 h, then at RT
overnight. Ethylene glycol (1 mL) was added to react
with the excess of reagent and after 30 min, the reaction
mixture was filtered and partitioned between H2O and
CH2Cl2. The organic phase was evaporated to dryness
by azeotropic distillation with heptane (3· 20 mL).
The pale yellow 2-acetoxymethylpyrrole was recrystal-
1
6.1.3.8. Copro (5i) (main isomer). H NMR (CDCl3):
d 10.06 (s, 4H, 4 meso H); 4.40 (t, J = 7.8, 8H, 4
CH2CO2Me); 3.67 (s, 12H, 4 CO2CH3); 3.64 (s, 12H, 4
CH3); 3.26 (t, J = 7.8, 8H, 4 CH2CH2CO2Me). 13C
NMR d 173.58, 4 CO2CH3; 138.37, 136.67, 16 C por-
phyrinic; 96.59, 4 CH meso; 51.78, 4 CO2CH3; 37.01,
4 CH2CO2Me; 21.89, 4 CH2CH2CO2Me; 11.75, 4
CH3. MS (ESI) m/z 711 [M+H]+.
1
lized from CH2Cl2/hexanes (1.26 g, 3 mmol, 100%). H
NMR (CDCl3): d 9.42 (s br, 1H, NH); 7.38–7.24 (m,
5H, PhH); 5.26 (s, 2H, CH2Ph); 5.04 (s, 2H, CH2O-
COCH3); 3.84 (s, 2H, CH2CO2Me); 3.63, 3.57 (2s, 6H,
2 CO2CH3); 3.49 (s, 2H, CH2CO2Me); 2.03 (s, 3H,
OCOCH3). 13C NMR d 171.48, 171.38, 2 CO2Me and
6.1.4. Preparation of porphyrins 5j, 5k and 5l.
6.1.4.1. 5,50-Diformyl-30,4-di-(2-methoxycarbonylethyl)-
3,40-dimethoxycarbonylmethyl-2,20-methylenedi-pyrrole
(14). The benzyl-50-tertbutyl-30,4-di-(2-methoxycarbon-

3912
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
OCOCH3; 160.42, CO2CH2Ph; 135.80, 122.95, 120.01,
117.52, 4 C pyrrolic; 128.88, 128.51, 128.28, 4 C benzyl-
ic; 66.11, CH2Ph; 56.66, CH2OAc; 52.04, 51.82, 2
CO2CH3; 30.45, 29.69, 2 CH2CO2Me; 20.77, OCOCH3.
3.57, 3.55 (2s, 12H, 4 CO2CH3); 2.83, 2.64 (2t, J = 8.2,
7.4, 8H, 4 CH2CO2Me); 2.43, 2.30 (2t, J = 8.2, 7.4,
8H, 4 CH2CH2CO2Me); 1.40 (s, 18H, 2 C(CH3)3). 13C
t
NMR d 173.35, 173.31, 4 CO2Me; 160.52, 2 CO2 Bu;
129.60, 127.94, 125.79, 119.27, 8 C pyrrolic; 80.42, 2
CMe3; 51.43, 51.18, 4 CO2CH3; 35.21, 35.03, 4
CH2CO2Me; 28.09, 2 C(CH3)3; 22.47, CH2 meso;
20.53, 18.84, 4 CH2CH2CO2Me.
The 2-acetoxymethylpyrrole obtained above was redis-
solved in CH2Cl2 (5 mL) and stirred over Montmoril-
lonite clay (2 g) at RT for 4 days. The solution was
filtered through Celite and the catalyst washed with
CH2Cl2 + 5% MeOH (50 mL). The dibenzyl-3,30,4,40-
tetramethoxycarbonylmethyl-2,20-methylenedipyrrole-5,
50-dicarboxylate was isolated by flash chromatography
(ethyl acetate/hexanes, 1:1) (819 mg, 1.17 mmol, 78%).
1H NMR (CDCl3): d 10.44 (s, 2H, 2 NH); 7.37–7.24
(m, 10H, 2 PhH); 5.20 (s, 4H, 2 CH2Ph); 3.86 (s, 4H,
2 CH2CO2Me); 3.82 (s, 2H, CH2 meso); 3.56, 3.54 (2s,
12H, 4 CO2CH3); 3.49 (s, 4H, 2 CH2CO2Me). 13C
6.1.4.5. Porphyrin 5k. The dipyrromethane 15k
(46 mg, 0.067 mmol) was treated with TFA (500 lL)
for 30 min at RT. A solution of the diformyldipyrro-
methane 14 (33 mg, 0.064 mmol) in CH2Cl2/MeOH
(10/1 mL) was added, then p-TsOHÆH2O (97 mg,
0.51 mmol), and the reaction mixture was stirred in
the dark for 48 h. The work up was conducted as for
5j. Porphyrin 5k was obtained in 15% yield (9 mg,
1
NMR
d
173.27, 171.47,
4
CO2CH3; 160.20,
2
0.01 mmol). H NMR (CDCl3): d 10.18, 10.16, 10.14,
CO2CH2Ph; 135.95, 122.69, 118.90, 114.31, 8 C pyrrolic;
131.89, 128.33, 128.09, 127.75, 8 C benzylic; 65.48, 2
CH2Ph; 52.21, 51.60, 4 CO2CH3; 30.21, 29.45, 4
CH2CO2Me; 22.13, CH2 meso.
10.13 (4s, 4H, 4 CH meso); 5.12 (s, 4H, 2 CH2CO2Me);
4.44 (t, J = 8.7, 12H, 6 CH2CH2CO2Me); 3.77, 3.69,
3.68, 3.67, 3.60 (5s, 24H, 8 CO2CH3); 3.32 (m, 12H,
6 CH2CH2CO2Me). 13C NMR d 173.45, 173.37,
171.87, 8 CO2Me; 147 (m), 144 (m), 140.9 (br), 139.9
(br), 134 (m), 16 C porphyrinic; 97.95, 97.65, 97.32, 4
CH meso; 52.44, 51.79, 8 CO2CH3; 37.47, 37.13,
37.04, 6 CH2CH2CO2Me; 32.58, 2 CH2CO2Me; 21.72,
21.64, 6 CH2CH2CO2Me. MS (ESI) m/z 971 [M+H]+.
HRMS (ESI) [C50H59N4O16]+, calcd: 971.3926. Found:
971.3909.
Part of the dipyrromethane obtained above (126 mg,
0.18 mmol) was redissolved in THF/MeOH (1/1,
10 mL) and hydrogenated over 10% Pd–C (15 mg) at
RT overnight. The solution was filtered and the catalyst
washed with warm THF (50 mL), MeOH (50 mL), and
CH2Cl2 (50 mL) to afford 15j. The compound was pre-
pared immediately before the preparation of porphyrin
5j and used without further purification.
6.1.4.6. 4,40-Dimethyl-3,30-di-(2-methoxycarboxyleth-
yl)-2,20-methylenedipyrrole-5,50-dicarboxylic acid (15l).
The dibenzyl derivative of 15l was prepared as described
for 15j from the benzyl-2-methyl-3,4-di-(2-methoxycar-
bonylethyl)-pyrrole-5-carboxylate (1.57 g, 5 mmol) in
6.1.4.3. Porphyrin 5j. Compounds 15j and 14 (87 mg,
0.17 mmol) were dissolved in CH2Cl2/MeOH (20/2 mL).
p-TsOHÆH2O (264 mg, 1.39 mmol) was added and the
reaction stirred in the dark for 48 h. The solution was
partitioned between CH2Cl2 and aqueous NaHCO3,
dried, and evaporated. The residue was redissolved in
MeOH, the solution was saturated with O2 and stirred
at RT for 24 h. The porphyrin 5j was isolated by PLC
eluting twice with CH2Cl2 + 2% MeOH (34 mg,
0.037 mmol, 22%). 1H NMR (CDCl3): d 9.77, 9.61,
9.60, 9.47 (4s, 4H, 4 H meso); 4.90, 4.89, 4.85, 4.72,
4.66 (5s, 12H, 6 CH2CO2Me); 4.22, 4.05 (2t, J = 7.8,
7.8, 4H, 2 CH2CH2CO2Me); 3.83, 3.82, 3.76, 3.75,
3.73, 3.71, 3.67, 3.66 (8s, 24H, 8 CO2CH3); 3.25, 3.14
(2t, J = 7.8, 7.8, 4H, 2 CH2CH2CO2Me). 13C NMR d
173.38, 173.31, 171.79, 171.73, 171.40, 171.30, 171.26,
8 CO2Me; 142 (m), 140.7 (br), 133.8 (br), 132.8 (br),
16 C porphyrinic; 98.07, 97.67, 97.24, 4 CH meso;
1
55% yield over 2 steps (844 mg, 1.37 mmol). H NMR
(CDCl3): d 9.71 (s, 2H, 2 NH); 7.30–7.21 (m, 10H, 2
PhH); 5.18 (s, 4H, 2 CH2Ph); 3.92 (s, 2H, CH2 meso);
3.57 (s, 6H, 2 CO2CH3); 2.73 (t, J = 7.3, 4H, 2
CH2CO2Me); 2.42 (t, J = 7.3, 4H, 2 CH2CH2CO2Me);
2.26 (s, 6H, 2 CH3). 13C NMR d 173.50, 2 CO2Me;
161.40, 2 CO2CH2Ph; 136.18, 130.91, 120.01, 117.66, 8
C pyrrolic; 128.22, 127.72, 127.62, 127.10, 8 C benzylic;
65.36, 2 CH2Ph; 51.42, 2 CO2CH3; 34.37, 2 CH2CO2Me;
22.31, CH2 meso; 19.12, 2 CH2CH2CO2Me; 10.60, 2
CH3.
The dipyrromethane prepared above (65 mg, 0.1 mmol)
was redissolved in THF (4 mL) and hydrogenated over
10% Pd–C (10 mg) at RT overnight. The solution was
filtered through Celite, the catalyst washed abundantly
with THF, MeOH, CH2Cl2, and the compound 15l
evaporated to dryness. 15l was prepared and used with-
out purification immediately before the preparation of
porphyrin 5l.
52.34, 51.72, 51.68,
8 CO2CH3; 36.89, 36.80, 2
CH2CH2CO2Me; 32.20, 32.12, 31.98, 6 CH2CO2Me;
21.49, 21.26, 2 CH2CH2CO2Me. MS (ESI) m/z 915
[M+H]+. HRMS (ESI) [C46H51N4O16]+, calcd:
915.3300. Found: 915.3307.
6.1.4.4. Ditertbutyl-3,30,4,40-tetra-(2-methoxycarbon-
ylethyl)-2,20-methylenedipyrrole-5,50-dicarboxylate (15k).
The compound 15k was prepared as described for 15j
from the tertbutyl-2-methyl-3,4-di-(2-methoxycarbonyl-
ethyl)-pyrrole-5-carboxylate (706 mg, 2 mmol) in a yield
of 50% over 2 steps (344 mg, 0.5 mmol). 1H NMR
(CDCl3): d 9.25 (s, 2H, 2 NH); 3.85 (s, 2H, CH2 meso);
6.1.4.7. Porphyrin 5l. Compounds 15l (33 mg,
0.076 mmol) and 14 (39 mg, 0.076 mmol) were dissolved
in CH2Cl2/MeOH (10/1 mL) and treated with p-TsOHÆ
H2O (100 mg, 0.525 mmol) at RT in the dark for 48 h.
The reaction was worked up as for 5j. Porphyrin 5l
was obtained in 31% yield (19.6 mg, 0.024 mmol). H
NMR (CDCl3): d 10.02, 10.01, 9.96 (3s, 4H, 4 CH
1

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3913
meso); 5.05, 5.04 (2s, 4H, 2 CH2CO2Me); 4.37 (m, 8H, 4
CH2CH2CO2Me); 3.78, 3.76, 3.71, 3.70, 3.65 (5s, 18H, 6
CO2CH3); 3.60, 3.59 (2s, 6H, 2 CH3); 3.28 (m, 8H, 4
CH2CH2CO2Me). 13C NMR d 173.49, 172.00, 171.87,
6 CO2Me; 143.3 (m), 140.4 (br), 138.6 (br), 137.2 (br),
132.4 (br), 16 C porphyrinic; 97.71, 97.22, 97.05,
96.39, 4 CH meso; 52.50, 52.33, 51.85, 51.78, 51.68,
51.57, 6 CO2CH3; 37.14, 36.79, 4 CH2CH2CO2Me;
32.54, 32.49, 2 CH2CO2Me; 21.74, 4 CH2CH2CO2Me;
11.63, 2 CH3. MS(ESI) m/z 827 [M+H]+. HRMS (ESI)
[C44H51N4O12]+, calcd: 827.3503. Found: 827.3463.
CH2 meso); 3.82 (s, 2H, CH2CO2Me); 3.66, 3.65, 3.60
(3s, 9H, 3 CO2CH3); 2.86, 2.74 (2t, J = 7.5, 7.9, 4H, 2
CH2CH2CO2Me); 2.49, 2.29 (2t, J = 7.5, 7.9, 4H, 2
CH2CH2CO2Me); 2.25 (s, 3H, CH3). 13C NMR d
176.18, CHO; 173.13, 173.00, 172.13, 3 CO2Me;
165.12, CO2H; 137.91, 134.64, 130.96, 128.88, 123.64,
121.30, 121.06, 118.93, 8 C pyrrolic; 51.87, 51.67,
51.59, 3 CO2CH3; 35.04, 34.67, 2 CH2CH2CO2Me;
30.59, CH2CO2Me; 22.44, CH2 meso; 19.54, 19.11, 2
CH2CH2CO2Me; 9.01, CH3.
6.1.5.3. Porphyrin 5e and 5p. The dipyrromethanes
17e (22 mg, 0.046 mmol) and 16 (25 mg, 0.046 mmol)
were dissolved in CH2Cl2/MeOH (10/1 mL). p-TsOHÆ-
H2O (70 mg, 0.37 mmol) was added and the reaction
mixture was stirred at RT in the dark for 48 h. The work
up was done as for 5j. Three products were isolated by
PLC. The symmetrical porphyrin 5p (R1 = Me,
R2 = R3 = CH2CH2CO2Me, R4 = CH2CO2Me) (2 mg,
0.002 mmol, 9%) has the highest RF: 1H NMR (CDCl3):
d 10.16, 10.09 (2s, 4H, 4 CH meso); 5.07 (s, 4H, 2
6.1.5. Preparation of porphyrins 5e, 5m, 5n, 5o, and 5p
6.1.5.1. 50-Formyl-30,4-di-(2-methoxycarbonylethyl)-
3,40-dimethoxycarbonylmethyl-2,20-methylenedipyrrole-
5-carboxylic acid (16). The benzyl ester of 16 was
prepared as in Ref. 32. Part of it (29 mg, 0.046 mmol)
was dissolved in THF (3 mL), Et3N (200 lL) was added
and the solution hydrogenated over 10% Pd–C (5 mg) at
RT overnight. The solution was filtered through Celite,
the catalyst washed with CH2Cl2 (10 mL), and the fil-
trate neutralized by washing with 0.1 N HCl. 16 was
prepared and used without purification immediately
prior to the preparation of porphyrins 5e, 5m, 5n, 5o,
CH2CO2Me); 4.45, 4.41 (2t, J = 7.7, 7.8, 4H,
2
CH2CH2CO2Me); 3.77, 3.70, 3.67 (3s, 18H, 6 CO2CH3);
3.64 (s, 6H, 2 CH3); 3.34, 3.28 (2t, J = 7.8, 7.7, 4H, 2
CH2CH2CO2Me). 13C NMR d 173.64, 173.39, 172.09,
6 CO2Me; 148.0 (m), 142.6 (br), 139.0 (m), 137.0 (br),
135.2 (br), 134.5 (br), 16 porphyrinic C; 97.25, 97.17; 4
CH meso; 52.42, 51.78, 6 CO2CH3; 37.26, 36.88, 4
CH2CH2CO2Me; 32.76, 2 CH2CO2Me; 21.91, 21.76, 4
CH2CH2CO2Me; 11.60, 2 CH3. MS (ESI) m/z 827
[M+H]+. HRMS (ESI) [C44H51N4O12]+, calcd:
827.3503. Found: 827.3494. The middle band contained
the desired porphyrin 5e (7.6 mg, 0.009 mmol, 20%):36
1H NMR (CDCl3): d 10.11, 10.06, 10.05, 9.97 (4s, 4H,
4 CH meso); 5.13, 5.06, 4.98 (3s, 6H, 3 CH2CO2Me);
4.43, 4.39, 4.34 (3m, 8H, 4 CH2CH2CO2Me); 3.79,
3.78, 3.76, 3.70, 3.68, 3.63 (6s, 21H, 7 CO2CH3); 3.62
(s, 3H, CH3); 3.33, 3.26 (2m, 8H, 4 CH2CH2CO2Me).
13C NMR d 173.54, 173.49, 173.37, 171.94, 171.86,
171.77, 7 CO2Me; 147.34–131.42 (m), 16 porphyrinic
C; 97.58, 97.45, 96.84, 4 CH meso; 52.39, 51.72, 7
CO2CH3; 36.95, 36.61, 4 CH2CH2CO2Me; 32.43, 32.30,
32.23, 3 CH2CO2Me; 21.59, 21.47, 4 CH2CH2CO2Me;
11.41, CH3. MS (ESI) m/z 885 [M+H]+, 907 [M+Na]+.
The lowest band was 5b (9 mg, 0.01 mmol, 21%).
1
and 5p. H NMR (CDCl3): d 11.40, 10.61 (s br + s,
2H, 2 NH); 9.30 (s, 1H, CHO); 3.94 (s, 2H, CH2 meso);
3.67, 3.62, 3.61, 3.56 (4s, 12H, 4 CO2CH3); 3.63,
3.48 (2s, 4H, 2 CH2CO2Me); 2.96, 2.79 (2t, J = 7.4,
7.7, 4H,
2
CH2CH2CO2Me); 1.77 (m, 4H,
4
CH2CH2CO2Me). 13C NMR d 177.39, CHO; 173.53,
173.33, 172.49, 170.90, 4 CO2Me; 164.66, CO2H;
136.15, 131.64, 131.44, 129.03, 127.80, 121.55, 117.90,
114.85, 8 C pyrrolic; 52.13, 51.60, 51.27, 3 CO2CH3;
34.51, 34.44,
CH2CO2Me; 22.52, CH2 meso; 20.39, 18.68,
CH2CH2CO2Me.
2
CH2CH2CO2Me; 29.72, 29.44,
2
2
6.1.5.2.
50-Formyl-40-methyl-3,30-di-(2-methoxycar-
bonylethyl)-4-methoxycarbonylmethyl-2,20-methylene di-
pyrrole-5-carboxylic acid (17e). The benzyl ester of
dipyrromethane 17e (867 mg, 1.53 mmol) was prepared
following standard procedures described in Refs. 32–35
from the monopyrroles tertbutyl-2,4-dimethyl-3-(2-
methoxycarbonylethyl)-pyrrole-5-carboxylate and ben-
zyl-2-methyl-3-(2-methoxycarbonyl-ethyl)-4-(methoxy-
carbonylmethyl)-pyrrole-5-carboxylate.
1H
NMR
(CDCl3): d 10.88, 10.54 (s br + s, 2H, 2 NH); 9.25 (s,
1H, CHO); 7.18–7.07 (m, 5H, PhH); 5.09 (s, 2H, CH2Ph);
3.92 (s, 2H, CH2 meso); 3.70 (s, 2H, CH2CO2Me); 3.55,
3.52, 3.46 (3s, 9H, 3 CO2CH3); 2.68 (m, 4H, 2
CH2CH2CO2Me); 2.32 (m, 4H, 2 CH2CH2CO2Me);
2.12 (s 3H, CH3). 13C NMR d 176.55, CHO; 173.18,
173.08, 171.75, 3 CO2Me; 160.52, CO2CH2Ph; 136.71,
135.84, 132.97, 130.02, 122.74, 121.04, 120.57, 118.71, 8
C pyrrolic; 128.47, 128.12, 127.98, 127.72, 4 C benzylic;
65.40, CH2Ph; 51.53, 51.42, 3 CO2CH3; 34.77, 34.17, 2
CH2CH2CO2Me; 30.54, CH2CO2Me; 22.36, CH2 meso;
19.07, 18.92, 2 CH2CH2CO2Me; 8.63, CH3.
6.1.5.4. 50-Formyl-4-methyl-3,30-di-(2-methoxycarbon-
ylethyl)-40-(methoxycarbonylmethyl)-2,20-methylene di-
pyrrole-5-carboxylic acid (17m). The benzyl ester of the
dipyrromethane 17m (753 mg, 1.33 mmol) was prepared
as for 17e from the monopyrroles benzyl-2-methyl-4-(2-
methoxycarbonylethyl)-3-methoxycarbonyl methyl-pyr-
role-5-carboxylate and benzyl-2,4-dimethyl-3-(2-meth-
oxycarbonylethyl)-pyrrole-5-carboxylate. 1H NMR
(CDCl3): d 10.82, 10.18 (s br + s, 2H, 2NH); 9.39 (s,
1H, CHO); 7.28–7.18 (m, 5H, PhH); 5.18 (s, 2H,
CH2Ph); 3.99 (s, 2H, CH2 meso); 3.66, 3.64, 3.62, 3.58
(4s, 12H, 4 CO2CH3); 2.75 (m, 4H, 2 CH2CH2CO2Me);
2.40 (m, 4H, 2 CH2CH2CO2Me); 2.24 (s, 3H, CH3). 13
C
The dipyrromethane prepared above was hydrogenated
as for preparation of 16 to give 17e and used without
further purification: H NMR (CDCl3): d 11.85, 10.79
(s br + s, 2H, 2 NH); 9.13 (s, 1H, CHO); 3.97 (s, 2H,
NMR d 177.35, CHO; 173.48, 173.25, 171.01, 3 CO2Me;
161.33, CO2CH2Ph; 136.16, 129.74, 128.71, 121.36,
120.28, 117.93, 8 C pyrrolic; 128.22, 127.71, 127.57,
127.04, 4 C benzylic; 65.30, CH2Ph; 52.14, 51.53, 2
1

3914
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
CO2CH3; 34.40, 34.32,
CH2CO2Me; 22.47, CH2 meso; 19.23, 18.72,
CH2CH2CO2Me; 10.58, CH3.
2
CH2CH2CO2Me; 29.58,
2
dipyrromethane 17n (598 mg, 1.06 mmol) was prepared
as for 17e from the monopyrroles benzyl-2-methyl-4-
(2-methoxycarbonylethyl)-3-methoxycarbonylmethyl-pyr-
role-5-carboxylate and benzyl-2,3-dimethyl-4-(2-meth-
oxycarbonylethyl)-pyrrole-5-carboxylate. 1H NMR
(CDCl3): d 11.15, 10.50 (s br + s, 2H, 2 NH); 9.32 (s,
1H, CHO); 7.18–7.06 (m, 5H, PhH); 5.11 (s, 2H,
CH2Ph); 3.85 (s, 2H, CH2 meso); 3.58, 3.55, 3.51 (3s,
The dipyrromethane prepared above (36 mg,
0.058 mmol) was hydrogenated as for preparation of
1
16 to afford 17m. H NMR (CDCl3): d 11.80, 10.54 (s
br + s, 2H, 2NH); 9.27 (s, 1H, CHO); 3.97 (s, 2H,
CH2 meso); 3.67 (s, 2H, CH2CO2Me); 3.62, 3.61, 3.60
(3s, 9H, 3 CO2CH3); 2.85, 2.70 (2t, J = 7.5, 7.7, 4H, 2
CH2CH2CO2Me); 2.47, 2.30 (2t, J = 7.5, 7.7, 4H, 2
CH2CH2CO2Me); 2.23 (s, 3H, CH3). 13C NMR d
177.26, CHO; 173.11, 172.90, 170.75, 3 CO2Me;
165.52, CO2H; 137.23, 130.67, 128.95, 128.29, 121.55,
120.58, 117.78, 8 C pyrrolic; 52.11, 51.42, 51.36, 3
9H, 3 CO2CH3); 2.91, 2.69 (2t, J = 8.0, 7.8,
CH2CH2CO2Me); 2.38, 2.30 (2t, J = 8.0, 7.8,
2
2
CH2CH2CO2Me); 1.95 (s, 3H, CH3). 13C NMR d
177.49, CHO; 173.41, 173.08, 170.91, 3 CO2Me;
160.88, CO2CH2Ph; 136.79, 135.91, 130.12, 129.35,
128.60, 121.52, 117.47, 116.82, 8 C pyrrolic; 128.17,
127.65, 4 C benzylic; 65.47, CH2Ph; 52.13, 51.46,
51.20, 3 CO2CH3; 34.52, 34.40, 2 CH2CH2CO2Me;
29.54, CH2CO2Me; 22.53, CH2 meso; 20.63, 18.77, 2
CH2CH2CO2Me; 8.61, CH3.
CO2CH3; 34.62, 34.52,
CH2CO2Me; 22.37, CH2 meso; 19.47, 18.75,
CH2CH2CO2Me; 10.48, CH3.
2 CH2CH2CO2Me; 29.63,
2
6.1.5.5. Porphyrin 5m and 5o. The dipyrromethanes
17m (28 mg, 0.058 mmol) and 16 (31 mg, 0.058 mmol)
were dissolved in CH2Cl2/MeOH (10/1 mL). p-TsOHÆ-
H2O (88 mg, 0.46 mg) was added and the reaction mix-
ture was stirred at RT in the dark for 48 h. The reaction
was worked up as for 5j. Three products were isolated
by PLC. The symmetrical 5o (R1 = CH2CO2Me,
R2 = R3 = CH2CH2CO2Me, R4 = Me) has the highest
RF (6.7 mg, 0.008 mmol, 28%): 1H NMR (CDCl3): d
10.14, 10.11 (2s, 4H, 4 CH meso); 5.05 (s, 4H, 2
The dipyrromethane prepared above (40 mg,
0.071 mmol) was hydrogenated as for preparation of
16 to afford 17n. H NMR (CDCl3): d 11.98, 10.74 (s
1
br + s, 2H, 2 NH); 9.26 (s, 1H, CHO); 3.91 (s, 2H,
CH2 meso); 3.68 (s, 2H, CH2CO2Me); 3.65, 3.63, 3.60
(3s, 9H, 3 CO2CH3); 3.00, 2.85 (2t, J = 7.9, 7.7, 4H, 2
CH2CH2CO2Me); 2.51, 2.45 (2t, J = 7.9, 7.7, 4H, 2
CH2CH2CO2Me); 2.00 (s, 3H, CH3). 13C NMR d
177.38, CHO; 173.55, 172.98, 170.80, 3 CO2Me;
165.40, CO2H; 137.25, 131.23, 130.55, 128.97, 121.68,
117.44, 117.38, 8 C pyrrolic; 52.26, 51.55, 51.30, 3
CH2CO2Me); 4.44, 4.39 (2t, J = 7.6, 7.8, 8H,
4
CH2CH2CO2Me); 3.77, 3.68 (2s, 18H, 6 CO2CH3);
3.62 (s, 6H, 2 CH3); 3.24, 3.27 (2t, J = 7.8, 7.6, 8H, 4
CH2CH2CO2Me). 13C NMR d 173.64, 173.38, 172.18,
6 CO2CH3; 148 (m), 142.6 (br), 139.0 (m), 137.0 (br),
135.3 (br), 134.4 (br), 16 porphyrinic C; 97.41, 97.02, 4
CH meso; 52.43, 51.75, 6 CO2CH3; 37.12, 36.73, 4
CH2CH2CO2Me; 32.72, 2 CH2CO2Me; 21.84, 21.65, 4
CH2CH2CO2Me; 11.53, 2 CH3. MS (ESI) m/z 827
[M+H]+, 849 [M+Na]+. HRMS (ESI) [C44H51N4O12]+,
calcd: 827.3503. Found: 827.3542. The middle band
was porphyrin 5m (11.3 mg, 0.013 mmol, 22%):37 1H
NMR (CDCl3): d 9.99, 9.93, 9.92 (3s, 4H, 4 CH meso);
5.03, 4.99, 4.96 (3s, 6H, 3 CH2CO2Me); 4.38, 4.32
(t + m, J = 5.2, 8H, 4 CH2CH2CO2Me); 3.79, 3.78,
3.72, 3.71, 3.70, 3.64 (6s, 21H, 7 CO2CH3); 3.58 (s,
CO2CH3; 34.80, 34.55, 2 CH2CH2CO2Me; 29.72,
CH2CO2Me; 22.51, CH2 meso; 20.64, 18.89,
CH2CH2CO2Me; 8.80, CH3.
2
6.1.5.7. Porphyrins 5n and 5p. The dipyrromethanes
17n (34 mg, 0.071 mmol) and 16 (38 mg, 0.071 mmol)
were dissolved in CH2Cl2/MeOH (10/1 mL). p-TsOHÆ-
H2O (108 mg, 0.57 mmol) was added and the reaction
stirred at RT in the dark for 48 h. The reaction was
worked up as for 5j. Three products were isolated by
PLC. The compound with the highest RF was the
symmetrical porphyrin 5p (R1 = CH2CO2Me, R2 =
R4 = CH2CH2CO2Me, R3 = Me) (11 mg, 0.13 mmol,
18%). The middle band was porphyrin 5n (16 mg,
0.018 mmol, 26%). 1H NMR (CDCl3): d 9.91, 9.87,
9.86, 9.79 (4s, 4H, 4 CH meso); 5.01, 4.96, 4.86 (3s,
3H, CH3); 3.31, 3.24 (m + t, J = 5.2, 8H,
4
CH2CH2CO2Me). 13C NMR d 173.54, 173.50, 173.38,
172.13, 172.02, 171.93, 171.76, 7 CO2Me; 146.4–133.5
(m), 16 porphyrinic C; 97.64, 97.56, 97.27, 96.97, 4
CH meso; 52.40, 51.73, 7 CO2CH3; 37.03, 36.98, 36.64,
4 CH2CH2CO2Me; 32.52, 32.44, 32.33, 3 CH2CO2Me;
21.66, 21.55, 4 CH2CH2CO2Me; 11.47, CH3. MS (ESI)
m/z 885 [M+H]+. 5m structure was confirmed by isola-
tion of the first product of decarboxylation of uro’gen
III (1a) during its enzymatic production from PBG in
the presence of PBG deaminase and uro’gen III synthase
lysates containing decarboxylases. The product isolated
6H,
3
CH2CO2Me); 4.30, 4.25 (2m, 8H,
4
CH2CH2CO2Me); 3.80, 3.79, 3.77, 3.74, 3.72, 3.71,
3.68 (7s, 21H, 7 CO2CH3); 3.56 (s, 3H, CH3); 3.30,
3.25 (2m, 8H, 4 CH2CH2CO2Me). 13C NMR d 173.49,
173.37, 171.94, 171.87, 171.75, 7 CO2Me; 147-131 (m),
16 porphyrinic C; 97.61, 97.47, 96.33, 4 CH meso;
52.37, 51.73,
7 CO2CH3; 37.07, 36.98, 36.77, 4
CH2CH2CO2Me; 32.41, 32.27, 3 CH2CO2Me; 21.72,
21.60, 4 CH2CH2CO2Me; 11.45, CH3. The lowest band
was 5b (15 mg, 0.016 mmol, 23%).
1
from this incubation has identical H- and 13C NMR.
6.2. Enzymatic studies
The lowest band contained 5b (6 mg, 0.006 mmol, 22%).
6.2.1. Enzymes. Pseudomonas dinitrificans cobA and
cobI genes were simultaneously overexpressed in an
engineered strain of E. coli, providing a source of uro’-
gen III methyl transferase and precorrin-2 methyltrans-
6.1.5.6. 50-Formyl-3-methyl-30,4-di-(2-methoxycarbon-
di-
pyrrole-5-carboxylic acid (17n). The benzyl ester of
ylethyl)-40-methoxycarbonylmethyl-2,20-methylene

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3915
ferase. 4· 1 L Luria–Bertani (LB) medium containing
50 mg/L ampicillin was inoculated and incubated for
16 h in a shaker set at 37 ꢁC and 200 rpm. The cells were
collected by centrifugation and resuspended in 150 mL
buffer (50 mM Tris–HCl, 75 mM KCl, and 12 mM
MgCl2ÆH2O, pH 7.5). The cells were lysed by sonication
maintaining the temperature below 20 ꢁC, the cell debris
removed by centrifugation (12,000g for 30 min) and the
lysate was then treated with 0.5 g DEAE–Sephadex A-
25 to remove endogenous porphyrinoids. After addition
of 30 mL glycerol, the lysate was kept frozen at ꢀ20 ꢁC
until use, remaining active for several months.
tant 1H NMR signals, corresponding to the meso
protons and methyl groups, are reported in Table 2.
6.2.3. Product characterizations
6.2.3.1. From uro’gen III (1a). (a) When Cob A was
used instead of CobA and CobI, 3 products were isolat-
ed: factor 2 methyl ester (7a) (30–36%), factor 2 mono-
lactone methyl ester (10a) (4%), and factor 2 dilactone
methyl ester (11a) (2%). Factor 2 7a (major isomer):6
1H NMR (C6D6): d 8.85 (s, 1H, H-15); 7.64 (s, 1H, H-
10); 7.54 (s, 1H, H-20); 6.78 (s, 1H, H-5); 4.14 (m, 6H,
2 CH2CO2Me at C-12, C-18 and H-3, H-8); 3.79 (t,
J = 7.6, 4H, 2 CH2CH2CO2Me at C-13 and C-17);
3.42, 3.37, 3.34, 3.33, 3.25 (2), 3.22, 3.16 (7s, 24H, 8
CO2CH3); 2.98 (t, J = 7.6, 4H, 2 CH2CH2CO2Me at
C-13 and C-17); 2.62, 2.53 (AB, J = 16.3, 2H,
CH2CO2Me at C-2); 2.52 (d, J = 9.1, 2H, CH2CO2Me
at C-7); 2.47 (t, J = 8.8, 2H, CH2CH2CO2Me at C-8);
2.25, 2.11, 1.92 (3m, 6H, CH2CH2CO2Me at C-3 and
CH2CH2CO2Me at C-8); 1.75, 1.60 (2s, 6H, 2 CH3).
UV kmax (MeOH) 280 (0.23), 368 (sh, 0.65), 373 (1),
405 (sh, 0.55), 505 (0.12), 543 (0.19), 585 (0.27), 627
(0.03) nm. MS (ESI) m/z 975 [M+H]+. Starting with
1a labeled at C-4, C-5, C-9, C-10, C-14, C-15, C-16,
6.2.2. Enzymatic reactions. General procedures
6.2.2.1. Hydrolysis and reduction of the porphyrin
methyl esters. Each porphyrin methyl ester (5–25 mg) was
suspended in THF (1 mL per 10 mg substrate) and hydro-
lyzed with 2 N KOH (1 mL per 10 mg substrate) at RT
overnight. The solvent was evaporated and the pH of
the residual aqueous solution was decreased by addition
of 1 N HCl (1.5 mL per 10 mg substrate). The solution
was placed under argon atmosphere and transferred to
an argon-purged glove-box. Pieces of 3% sodium amal-
gam were added to the stirred free-acid porphyrin solu-
tion until it became colorless (ca. 2 h) to provide the
porphyrinogen.
1
and C-20 allows a partial H and 13C NMR assignment
for the 3 isomers in agreement with published results.6
The major isomer of 7a was isolated in the band with
6.2.2.2. Incubations. A solution of buffer (150 mM
Tris–HCl, 100 mM KCl, and 20 mM MgCl2Æ6H2O, pH
7.5; 100 mL per 10 mg substrate) containing S-adeno-
syl-L-methionine (100 mg per 10 mg substrate) was de-
gassed by several cycles of high vacuum and argon
bubbling. The CobA and CobI lysate (20 mL per
10 mg substrate) was added with antifoam 204 (Sigma,
0.5 mL). After several additional cycles of degassing,
the buffer solution was transferred to the glove-box.
The porphyrinogen solution was filtered through a cot-
ton-plugged pipette into the buffer solution. After a few
additional cycles of degassing, the flask was sealed and
incubated for 20 h at 37 ꢁC in the dark.
the lowest RF: 1H NMR (C6D6):
d
8.85 (d,
JCH = 152.4, 1H, H-15); 7.64 (d, JCH = 154.5, 1H, H-
10); 7.53 (d, JCH = 153.3, 1H, H-20); 6.77 (d,
JCH = 155.0, 1H, H-5); 4.15 (s, 2H, CH2CO2Me at C-
18); 4.13 (AB + m, J = 13, 4H, CH2CO2Me at C-12
and H-3, H-8); 3.78 (m, 4H, 2 CH2CH2CO2Me at C-
13, C-17); 3.42, 3.37, 3.34, 3.33, 3.25 (2), 3.22, 3.15 (8s,
24H,
8
CO2CH3); 2.98 (t, J = 7.5, 4H,
2
CH2CH2CO2Me at C-13, C-17); 2.62, 2.53 (AB,
J = 15, 2H, CH2CO2Me at C-2); 2.52 (d, J = 9.0, 2H,
CH2CO2Me at C-7); 2.47 (t, J = 7.9, 2H,
CH2CH2CO2Me at C-18); 2.30, 2.25, 1.90 (3m, 6H,
CH2CH2CO2Me at C-3 and CH2CH2CO2Me at C-18);
1.75 (s, 3H, CH3 at C-2); 1.59 (s, 3H, CH3 at C-7).
13C NMR d 164.87 (d, JCH = 71.5, C-4); 152.58 (d,
JCH = 77.9, C-9); 138.04 (d, JCH = 72.2, C-14); 135.68
(d, JCH = 69.9, C-19); 108.60 (t, JCH = 71.4, C-15);
95.89 (d, JCH = 77.0, C-10); 94.17 (s, C-20); 89.57 (d,
JCH = 75.5, C-5). The middle band contained some of
the major isomers and 2 epimers in a 3:2 ratio. The band
with the highest RF contained only the 2 epimers in the
inverted ratio 2:3. Analysis of the two bands allowed the
following tentative assignments for the 2 epimers by cor-
6.2.2.3. Isolation and purification of the products. The
incubation mixture was centrifuged to remove precipi-
tated protein. The solution was passed slowly through
DEAE–Sephadex A-25 (300 mg per 10 mg of substrate)
to trap the porphyrinoid compounds. After washing
with argon saturated acetone (20–50 mL), the resin
was dried by passing argon through it, then the por-
phyrinoids were eluted with MeOH + 5% H2SO4
(20 mL per 10 mg substrate) and esterified for 18 h.
The solution was diluted with CH2Cl2 (20–30 mL) and
H2O (50 mL), then neutralized with saturated NaHCO3
and extracted in CH2Cl2 (3· 25 mL). After evaporation
of the solvent to dryness, MeOH (2–3 mL) was added to
the crude material and the soluble fraction was filtered
off the solid residue, which was washed with more
MeOH (3· 1 mL). The solid residue contained oxidized
unreacted starting material (porphyrins). The MeOH-
soluble fractions contained the methylation products,
chlorins and isobacteriochlorins. They were collected,
evaporated, and chromatographed on PLC eluting 2–3
times with benzene/ethyl acetate/MeOH (70:27:3). Prod-
ucts and yields are reported in Table 1. The most impor-
1
relation to the data in Refs. 3 and 4. 7a epimer 1: H
NMR (C6D6): d 8.82 (d, JCH = 152.6, 1H, H-15); 7.52
(d, JCH = 153.5, 1H, H-10); 7.41 (d, JCH = 134.5, 1H,
H-20); 6.68 (d, JCH = 155.29, 1H, H-5); 4.13, 4.11
(2s + 2m, 6H, 2 CH2CO2Me at C-12, C-18 and H-3,
H-8); 3.78 (m, 4H, 2 CH2CH2CO2Me at C-13 and C-
17); 3.43, 3.37, 3.36, 3.32, 3.25, 3.24 (2), 3.15 (7s, 24H,
8 CO2CH3); 2.98–2.84 (m, 4H, 2 CH2CH2CO2Me at
C-13 and C-17); 2.65–2.50 (m, 4H, 2 CH2CO2Me at
C-2 and C-7); 2.48–2.35 (m, 2H, CH2CH2CO2Me at
C-8 or C-3); 2.24, 2.08 (m, 6H, CH2CH2CO2Me at C-
3 or C-8 and CH2CH2CO2Me at C-3 or C-8); 1.84 (s,
3H, CH3 at C-2); 1.44 (s, 3H, CH3 at C-7). 13C NMR

3916
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
d 166.74 (d, JCH = 70.8, C-4); 150.65 (d, JCH = 78.2, C-
9); 140.11 (d, JCH = 73.2, C-14); 134.15 (d, JCH = 70.9,
C-19); 109.08 (t, JCH = 71.3, C-15); 96.05 (d,
JCH = 80.3, C-10); 95.02 (s, C-20); 89.42 (d,
(s, 3H, CH3 at C-2); 1.31 (s, 3H, CH3 at C-7). UV kmax
(MeOH) 279 (0.26), 357 (0.48), 374 (1), 385 (sh, 0.76),
405 (0.62), 487 (0.12), 540 (0.14), 578 (0.16), 624
(0.12). MS (ESI) m/z 943 [M+H]+.
1
JCH = 73.2, C-5). 7a epimer 2: H NMR (C6D6): d 8.83
(d, JCH = 152.1, 1H, H-15); 7.65 (d, JCH = 140.3, 1H,
H-10); 7.53 (d, JCH = 155.5, 1H, H-20); 6.71 (d,
JCH = 155.6, 1H, H-5); 4.13, 4.12 (2s, 4H, 2 CH2CO2Me
at C-12 and C-18); 4.13, 4.11 (2m, 2H, H-3 and H-8);
3.78 (m, 4H, 2 CH2CH2CO2Me at C-13 and C-17);
3.41, 3.38, 3.35, 3.34, 3.26, 3.23, 3.22, 3.00 (8s, 24H, 8
CO2CH3); 2.98–2.84 (m, 4H, 2 CH2CH2CO2Me at C-
13 and C-17); 2.65–2.50 (m, 4H, 2 CH2CO2Me at C-2
and C-7); 2.48–2.35 (m, 2H, CH2CH2CO2Me at C-8 or
C-3); 2.24, 2.08 (m, 6H, CH2CH2CO2Me at C-3 or C-
8 and CH2CH2CO2Me at C-3 or C-8); 1.59 (s, 3H,
CH3 at C-2); 1.53 (s, 3H, CH3 at C-7). 13C NMR d
161.11 (d, JCH = 70.1, C-4); 154.55 (d, JCH = 79.1, C-
9); 138.73 (d, JCH = 73.1, C-14); 135.25 (d, JCH = 70.6,
C-16); 109.08 (t, JCH = 71.3, C-15); 96.99 (d,
JCH = 77.2, C-10); 93.00 (s, C-20); 90.04 (d,
JCH = 78.6, C-5).
(b) Incubation of CobA and CobI with uro III (1a) gave
3 isolated products: factor 3 methyl ester (8a) (22%), fac-
tor 3 monolactone methyl ester (12a) (8%), and factor 3
dilactone methyl ester (13a) (6%). Factor 3 8a (major
isomer):6 1H NMR (C6D6): d 8.73 (s, 1H, H-15); 7.50
(s, 1H, H-10); 6.51 (s, 1H, H-5); 4.21 (d, J = 19.7, 2H,
CH2CO2Me at C-18); 4.06, 4.04 (2m, 2H, H-3 and H-
8); 4.03 (AB, J = 12.8, 2H, CH2CO2Me at C-12); 3.69
(m, 4H, 2 CH2CH2CO2Me at C-13 and C-17); 3.43,
3.38, 3.37, 3.31, 3.27, 3.26, 3.24, 3.22 (8s, 24H, 8
CO2CH3); 2.92 (2m, 4H, 2 CH2CH2CO2Me at C-13
and C-17); 2.77 (s, 3H, CH3 at C-20); 2.67, 2.50 (AB,
J = 9.1, 2H, CH2CO2Me at C-2 or C-7); 2.65 (s, 2H,
CH2CO2Me at C-2 or C-7); 2.59 (m, 2H,
CH2CH2CO2Me at C-8); 2.29, 2.18, 2.09 (3m, 6H,
CH2CH2CO2Me at C-3 and CH2CH2CO2Me at C-8);
1.66 (s, 3H, CH3 at C-2); 1.40 (s, 3H, CH3 at C-7).
UV kmax (MeOH) 275 (0.24), 361 (sh, 0.59), 381 (1),
410 (sh, 0.52), 500 (0.1), 544 (0.15), 574 (0.17), 633
Factor 2 monolactone 10a (major isomer). 1H NMR
(C6D6): d 8.96 (s, 1H, H-15); 8.06 (s, 1H, H-10); 7.55 (s,
1H, H-20); 6.61 (s, 1H, H-5); 4.16, 4.04 (s + d, J = 2.5,
4H, 2 CH2CO2Me at C-12 and C-18); 4.15 (m, 1H, H-3
or H-8); 3.83 (m, 4H, 2 CH2CH2CO2Me at C-13 and C-
17); 3.43, 3.35, 3.33, 3.32, 3.25, 3.19, 3.16 (7s, 21H, 7
1
(0.07) nm. MS (ESI) m/z 989 [M+H]+. 8a epimer: H
NMR (C6D6): d 9.05 (s, 1H, H-15); 7.76 (s, 1H, H-10);
6.99 (s, 1H, H-5); 4.40, 4.31 (AB, J = 18.2, 2H,
CH2CO2Me at C-12 or C-18); 4.36, 4.05 (2m, 2H, H-3
and H-8); 4.14, 4.03 (AB, J = 12.8, 2H, CH2CO2Me at
C-12 or C-18); 3.87 (m, 4H, 2 CH2CH2CO2Me at C-13
and C-17); 3.43, 3.39, 3.35, 3.27 (2), 3.25, 3.15 (7s,
24H, 8 CO2CH3); 2.98 (m, 4H, 2 CH2CH2CO2Me at
C-13 and C-17); 2.79 (s, 3H, CH3 at C-20); 2.64 (s,
2H, CH2CO2Me at C-2 or C-7); 2.62, 2.44 (AB,
J = 18.2, 2H, CH2CO2Me at C-2 or C-7); 2.29–2.00
(m, 8H, 2 CH2CH2CO2Me at C-3 and C-8); 1.51 (s,
3H, CH3 at C-2); 1.39 (s, 3H, CH3 at C-7).
CO2CH3); 3.01, 2.93 (2t, J = 9.1, 9.1, 4H,
2
CH2CH2CO2Me at C-13 and C-17); 2.80, 2.53 (AB,
J = 18.2, 2H, CH2CO2Me at C-2 or C-7); 2.36 (d,
J = 18.0, 2H, CH2CO2Me at C-7 or C-2); 2.62 (m, 2H,
CH2CH2CO2Me at C-3 or C-8); 2.20–2.00 (m, 6H,
CH2CH2CO2Me at C-3 or C-8 and CH2CH2CO2Me at
C-3 or C-8); 1.75 (s, 3H, CH3 at C-2); 1.21 (s, 3H, CH3
at C-7). UV kmax (MeOH) 280 (0.25), 356 (sh, 0.58), 375
(1), 404 (0.48), 504 (0.1), 538 (0.17), 575 (0.19), 629
1
1
(0.06) nm. MS (ESI) m/z 959 [M+H]+. 10a epimer: H
Factor 3 monolactone 12a. H NMR (C6D6): d 8.85 (s,
NMR (C6D6): d 8.98 (s, 1H, H-15); 7.68 (s, 1H, H-10);
7.53 (s, 1H, H-20); 6.97 (s, 1H, H-5); 4.21 (m, 1H, H-3
or H-8); 4.16, 4.12 (2d, J = 8.2, 8.1, 4H, 2 CH2CO2Me
at C-12 and C-18); 3.84, 3.67 (t + m, J = 7.0, 4H, 2
CH2CH2CO2Me at C-13 and C-17); 3.41, 3.34, 3.33,
3.32, 3.25, 3.23, 3.19 (7s, 21H, 7 CO2CH3); 2.99 (m, 4H,
2 CH2CH2CO2Me at C-13 and C-17); 2.54, 2.30 (AB,
J = 17.5, 2H, CH2CO2Me at C-2 or C-7); 2.52 (s, 2H,
CH2CO2Me at C-2 or C-7); 2.59, 2.37 (2m, 2H,
CH2CH2CO2Me at C-3 or C-8); 2.44 (t, J = 7.0, 2H,
CH2CH2CO2Me at C-3 or C-8); 2.21, 2.09, 2.00 (3m,
4H, 2 CH2CH2CO2Me at C-3 and C-8); 1.44 (s, 3H,
CH3 at C-2); 1.37 (s, 3H, CH3 at C-7).
1H, H-15); 7.88 (s, 1H, H-10); 6.40 (s, 1H, H-5); 4.19
(AB, J = 17.5, 2H, CH2CO2Me at C-18); 4.11 (m, 1H,
H-3 or H-8); 3.94 (AB, J = 7.7, 2H, CH2CO2Me at C-
12); 3.73, 3.64 (2m, 4H, 2 CH2CH2CO2Me at C-13
and C-17); 3.42, 3.40, 3.37, 3.27, 3.24, 3.19, 3.17 (7s,
21H, 7 CO2CH3); 2.99 (d, J = 17.5, CH2CO2Me at C-
7); 2.94, 2.88 (2 AB, J = 6.9, 2 CH2CH2CO2Me at C-
13 and C-17); 2.73 (s, 3H, CH3 at C-20); 2.64 (s, 2H,
CH2CO2Me at C-2); 2.38 (d, J = 18.2, 2H,
CH2CH2CO2Me at C-3 or CH2CH2CO2Me C-8); 2.42,
2.31 (AB, J = 18.0, 2H, CH2CH2CO2Me at C-3 or C-
8); 2.18, 2.00 (2m, 4H, 2 CH2CH2CO2Me at C-3 and
C-8); 1.67 (s, 3H, CH3 at C-2); 1.16 (s, 3H, CH3 at C-
7). UV kmax (MeOH) 274 (0.24), 360 (0.55), 379 (1),
407 (0.52), 500 (0.10), 537 (0.16), 580 (0.17), 631 (0.07)
nm. MS (ESI) m/z 973 [M+H]+.
1
Factor 2 dilactone 11a. H NMR (C6D6): d 9.28 (s, 1H,
H-15); 8.25 (s, 1H, H-10) ; 7.75 (s, 1H, H-20); 7.08 (s,
1H, H-5); 4.24, 4.11 (d + s, J = 2.8, 4H, 2 CH2CO2Me
at C-12 and C-18); 4.06, 3.93, 3.82 (3m, 4H, 2
CH2CH2CO2Me at C-13 and C-17); 3.45, 3.32, 3.26,
3.23, 3.19 (2) (6s, 18H, 6 CO2CH3); 3.04, 2.98 (2t,
J = 8.9, 8.9, 4H, 2 CH2CH2CO2Me at C-13 and C-17);
2.64, 2.37 (AB, J = 17.9, 2H, CH2CO2Me at C-2 or C-
7); 2.53 (s, 2H, CH2CO2Me at C-2 or C-7); 2.59, 2.20–
2.00 (2m, 8H, 2 CH2CH2CO2Me at C-3 and C-8); 1.42
1
Factor 3 dilactone 13a. H NMR (C6D6): d 9.46 (s, 1H,
H-15); 8.38 (s, 1H, H-10); 7.58 (s, 1H, H-5); 4.49 (AB,
J = 18.9, 2H, CH2CO2Me at C-18); 4.16 (s, 2H,
2
CH2CO2Me at C-12); 4.02-3.91 (m, 4H,
CH2CH2CO2Me at C-13 and C-17); 3.43 (2), 3.26,
3.25, 3.20, 3.16 (6s, 18H, 6 CO2CH3); 3.06, 3.02 (2t,
J = 6.8, 4H, 2 CH2CH2CO2Me at C-13 and C-17);

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3917
2.99 (s, 3H, CH3 at C-20); 2.84, 2.60, 2.73, 2.36 (2AB,
J = 18.0, 18.2, 4H, 2 CH2CO2Me at C-2 and C-7);
2.80, 2.54 (2m, 2H, CH2CH2CO2Me at C-3 or C-8);
2.20–2.00 (m, 6H, CH2CH2CO2Me at C-3 or C-8 and
CH2CH2CO2Me at C-3 or C-8); 1.49 (s, 3H, CH3 at
C-2); 1.45 (s, 3H, CH3 at C-7). UV kmax (MeOH) 282
(0.14), 359 (sh, 0.33), 377 (1), 387 (sh, 0.53), 407
(0.69), 489 (0.07), 535 (0.08), 578 (0.1), 624 (0.12) nm.
MS (ESI) m/z 957 [M+H]+.
3.32, 3.27, 3.25, 3.19 (7s, 21H, 7 CO2CH3); 3.00, 2.88
(2t, J = 6.6, 6.6, 2 CH2CH2CO2Me at C-13 and C-
18); 2.67, 2.37 (AB, J = 24.6, 2H, CH2CO2Me at C-2
or C-7); 2.51 (s, 2H, CH2CO2Me at C-2 or C-7); 2.45
(t, J = 7, 2H, CH2CH2CO2Me at C-3 or C-8); 2.20–
2.00 (m, 6H, CH2CH2CO2Me at C-3 or C-8 and 2
CH2CH2CO2Me at C-3 and C-8); 1.47 (s, 3H, CH3
at C-2); 1.35 (s, 3H, CH3 at C-7). UV kmax (MeOH)
278 (0.25), 362 (sh, 0.60), 379 (1), 406 (0.57), 505
(0.11), 542 (0.16), 586 (0.21), 638 (0.05) nm. MS
(ESI) m/z 959 [M+H]+, 981 [M+Na]+. HRMS (ESI)
[C49H58NaN4O16]+, calcd: 981.3746. Found: 981.3188.
6.2.3.2. From uro’gen I (1b). Three products were iso-
lated: diMeIBC methyl ester 7b (3–5%), diMeIBC
monolactone methyl ester 10b (4–5%), and diMeIBC dil-
actone 11b (<1%). DiMeIBC 7b (major isomer):12 1H
NMR (C6D6): d 8.84 (s, 1H, H-15); 7.65 (s, 1H, H-10);
7.48 (s, 1H, H-20); 6.80 (s, 1H, H-5); 4.32, 4.07 (2m,
2H, H-3 and H-8); 4.27, 4.16 (2m, 4H, 2 CH2CO2Me
at C-12 and C-17); 3.75 (m, 4H, 2 CH2CH2CO2Me at
C-13 and C-18); 3.42, 3.37, 3.34, 3.33, 3.32, 3.26, 3.22,
DiMeIBC dilactone 11b. 1H NMR (C6D6): d 9.25 (s, 1H,
H-15); 8.28 (s, 1H, H-10); 7.79 (s, 1H, H-20); 7.27 (s, 1H,
H-5); 4.41, 4.15 (s + AB, J = 1.8, 4H, 2 CH2CO2Me at
C-7 and C-12); 3.91, 3.83, 3.72 (3m, 4H,
2
CH2CH2CO2Me at C-13 and C-18); 3.44, 3.38, 3.26
(2), 3.20 (2), (4s, 18H, 6 CO2CH3); 3.00, 2.90 (2t,
J = 6.8, 6.8, 2 CH2CH2CO2Me at C-13 and C-18);
2.78, 2.46 (AB, J = 21.1, 2H, CH2CO2Me at C-2 and
C-7); 2.56 (t, J = 9.2, 2H, CH2CH2CO2Me at C-3 or
C-8); 2.68, 2.27 (2m, 2H, CH2CH2CO2Me at C-3 or
C-8); 2.54 (s, 2H, CH2CO2Me at C-2 or C-7); 2.15,
2.03 (2m, 4H, 2 CH2CH2CO2Me at C-3 and C-8); 1.42
(s, 3H, CH3 at C-2); 1.31 (s, 3H, CH3 at C-7). UV kmax
(MeOH) 281 (0.20), 355 (sh, 0.43), 375 (1), 384 (sh,
0.75), 405 (0.75), 487 (0.09), 543 (0.10), 582 (0.13), 625
(0.12) nm. MS (ESI) m/z 943 [M+H]+, 965 [M+Na]+.
HRMS (ESI) [C48H54NaN4O16]+, calcd: 965.3433.
Found: 965.3245.
3.16 (8s, 24H,
8
CO2CH3); 2.97 (m, 4H,
2
CH2CH2CO2Me at C-13 and C-18); 2.59, 2.52 (2 AB,
J = 13.3, 10.6, 4H, 2 CH2CO2Me at C-2 and C-7);
2.46 (t, J = 7.9, 2H, CH2CH2CO2Me at C-8); 2.30,
2.26, 2.01 (3m, 6H, CH2CH2CO2Me at C-3 and
CH2CH2CO2Me at C-8); 1.68 (s, 3H, CH3 at C-2);
1.61 (s, 3H, CH3 at C-7). UV kmax (MeOH) 277 (0.27),
359 (sh, 0.67), 377 (1), 392 (sh, 0.86), 507 (0.12), 545
(0.18), 588 (0.28), 645 (0.07) nm. MS (ESI) m/z 975
[M+H]+, 997 [M+Na]+. HRMS (ESI) [C50H63N4O16]+,
calcd: 975.4239. Found: 975.3826. Starting with 1b
labelled at C-4, C-5, C-9, C-10, C-14, C-15, C-19 and
C-20 allows for a partial 13C NMR assignment for the
major isomer of 7b in agreement with Ref. 12. 13C
NMR d 164.57 (d, JCH = 63.8, C-4); 152.94 (d,
JCH = 79.3, C-9); 144.69 (d, JCH = 65.4, C-19); 137.94
(d, JCH = 71.0, C-14); 108.30 (d, JCH = 71, C-15); 95.93
(d, JCH = 74.5, C-10); 93.89 (d, JCH = 67.5, C-20);
89.55 (d, JCH = 71.8, C-5).
6.2.3.3. From uro’gen IV (1d). Three products were
isolated: MeC methyl ester 6d13 (19%), MeC monolac-
tone methyl ester 9d (7%), and diMeIBC methyl ester
7d14 (1%). The product MeC 6d could not be obtained
free of the other isomer. However, fractions containing
different ratios of the isomers could be analyzed and al-
lowed these tentative assignments by correlation to the
data in Refs. 3 and 4. 6d (major isomer): 1H NMR
(C6D6): d 10.09 (s, 2H, H-10 and H-15); 9.16 (s, 1H,
H-20); 8.97 (s, 1H, H-5); 4.98 (t, J = 7.5, 1H, H-3);
4.72, 4.71 (m + s, 6H, 3 CH2CO2Me at C-7, C-13 and
C-18); 4.33-4.24 (m, 6H, 3 CH2CH2CO2Me at C-8, C-
12 and C-17); 3.47, 3.41(2), 3.29, 3.28, 3.27, 3.26, 3.10
(8s, 24H, 8 CO2CH3); 2.95, 2.75 (AB, J = 15.9, 2H,
CH2CO2Me at C-2); 3.23, 3.25 [overlapping with signals
of methyl esters] (t + m, 6H, 3 CH2CH2CO2Me at C-8,
C-12 and C-17); 2.46 (m, 2H, CH2CH2CO2Me at C-3);
2.19 (m, 2H, CH2CH2CO2Me at C-3); 2.22 (s, 3H,
1
DiMeIBC monolactone 10b (major isomer). H NMR
(C6D6): d 8.95 (s, 1H, H-15); 8.08 (s, 1H, H-10); 7.48
(s, 1H, H-20); 6.64 (s, 1H, H-5); 4.29, 4.08 (m + AB,
J = 6.7, 4H, 2 CH2CO2Me at C-12 and C-17); 4.05
(m, 1H, H-3 or H-8); 3.83, 3.78, 3.72 (3m, 4H, 2
CH2CH2CO2Me at C-13 and C-18); 3.43, 3.37, 3.34,
3.32, 3.31, 3.21, 3.16 (7s, 21H, 7 CO2CH3); 2.96 (AB,
J = 5.7, 4H, 2 CH2CH2CO2Me at C-13 and C-18);
2.80, 2.37 (AB, J = 18.3, 2H, CH2CO2Me at C-2 or
C-7); 2.62 (t, J = 7.3, 2H, CH2CH2CO2Me at C-3 or
C-8); 2.53 (d, J = 3.8, 2H, CH2CO2Me at C-2 or C-
7); 2.20, 2.03 (2m, 6H, 2 CH2CH2CO2Me at C-3 and
C-8 and CH2CH2CO2Me at C-3 or C-8); 1.67 (s, 3H,
CH3 at C-2); 1.22 (s, 3H, CH3 at C-7). UV kmax
(MeOH) 279 (0.26), 356 (sh, 0.61), 377 (1), 403
(0.58), 500 (0.11), 537 (0.16), 577 (0.19), 639 (0.07)
nm. MS (ESI) m/z 959 [M+H]+, 981 [M+Na]+. HRMS
(ESI) [C49H59N4O16]+, calcd: 959.3926. Found:
959.3205. 10b epimer: 1H NMR (C6D6): d 8.94 (s,
1H, H-15); 7.70 (s, 1H, H-10); 7.55 (s, 1H, H-20);
7.02 (s, 1H, H-5); 4.32 (m, 1H, H-3 or H-8); 4.30 (m,
2H, CH2CO2Me at C-12 or C-17); 4.14 (AB, J = 9.9,
1
CH3 at C-2). 6d epimer: H NMR (C6D6): d 10.08 (s,
2H, H-10 and H-15); 9.08 (s, 1H, H-20); 8.92 (s, 1H,
H-5); 4.73, 4.70 (s + m, 6H, 3 CH2CO2Me at C-7, C-
13 and C-18); 4.62 (m, 1H, H-3); 4.33–4.24 (m, 6H, 3
CH2CH2CO2Me at C-8, C-12 and C-17); 3.58, 3.51
(AB, J = 15, 2H, CH2CO2Me at C-2); 3.37, 3.36, 3.31
(2), 3.28, 3.27, 3.26, 3.10 (8s, 24H, 8 CO2CH3); 3.15,
3.25 [overlapping with signals of methyl esters] (2m,
6H, 3 CH2CH2CO2Me at C-8, C-12 and C-17); 2.60,
2.11 (2m, 4H, CH2CH2CO2Me at C-3); 1.85 (s, 3H,
CH3 at C-2). UV kmax (MeOH) 282 (0.12), 354 (sh,
0.23), 396 (1), 495 (0.1), 589 (0.05), 645 (0.29) nm. MS
(ESI) m/z 959 [M+H]+, 981 [M+Na]+, 997 [M+K]+.
CH2CO2Me at C-12 or C-17); 4.06 (m, 4H, 2
CH2CH2CO2Me at C-13 and C-18); 3.41, 3.37, 3.33,

3918
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
HRMS (ESI) [C49H50NaN4O16]+, calcd: 981.3746.
Found: 981.3588.
C-8); 1.85 (s, 3H, CH3 at C-2); 1.49 (s, 3H, CH3 at C-
7). UV kmax (MeOH) 279 (0.27), 357 (sh, 0.76), 381
(1), 405 (sh, 0.70), 500 (0.14), 540 (0.17), 585 (0.22).
MS (ESI) m/z 939 [M+Na]+. HRMS (ESI) [C48H60Na-
N4O14]+, calcd: 939.4004. Found: 939.3731.
1
MeC monolactone 9d. H NMR (C6D6): d 10.21 (s, 1H,
H-15); 10.19 (s, 1H, H-10); 9.35 (s, 1H, H-20); 8.80 (s,
1H, H-5); 4.75 (s, 2H, CH2CO2Me at C-13); 4.70, 4.58
(2 AB, J = 11.4, 8.5, 4H, 2 CH2CO2Me at C-7 and C-
18); 4.33 (AB, J = 6.6, 2H, CH2CH2CO2Me at C-8);
4.30, 4.06 (2t, J = 7.4, 6.2, 4H, 2 CH2CH2CO2Me at
C-12 and C-17); 3.52, 3.40, 3.34, 3.26 (2), 3.21, 3.20
(6s, 21H, 7 CO2CH3); 3.29 (m, 6H, 3 CH2CH2CO2Me
at C-8, C-12 and C-17); 2.97, 2.70 (AB, J = 18.0, 2H,
CH2CO2Me at C-2); 2.50 (m, 2H, CH2CH2CO2Me at
C-3); 2.19 (m, 2H, CH2CH2CO2Me at C-3); 1.72 (s,
3H, CH3 at C-2). UV kmax (MeOH) 285 (0.14), 347
(sh, 0.23), 393 (1), 495 (0.1), 585 (0.05), 640 (0.23) nm.
MS (ESI) m/z 943 [M+H]+, 965 [M+Na]+, 981
[M+K]+. HRMS (ESI) [C48H54NaN4O16]+, calcd:
965.3433. Found: 965.3349.
TriMeIBC 8e (major isomer)16. 1H NMR (C6D6): d 8.80
(s, 1H, H-15); 7.41 (d, J = 0.7, 1H, H-10); 6.64 (s, 1H, H-
5); 4.27 (AB, J = 17.4, 2H, CH2CO2Me at C-18); 4.19 (t,
J = 6.7, 1H, H-3); 4.13 (AB, J = 3.9, 1H, H-8); 3.80,
3.75, 3.63 (3m, 4H, 2 CH2CH2CO2Me at C-13 and C-
17); 3.44, 3.34, 3.32, 3.28, 3.27, 3.23, 3.21 (7s, 21H, 7
CO2CH3); 2.98 (m, 2H, CH2CH2CO2Me at C-17); 2.85
(s, 3H, CH3 at C-20); 2.74 (t, J = 7.5, 2H,
CH2CH2CO2Me at C-13); 2.70 (AB, J = 15.3, 2H,
CH2CO2Me at C-7); 2.56 (s, 3H, CH3 at C-12); 2.54
(AB, J = 14.9, 2H, CH2CH2CO2Me at C-8); 2.49 (m,
2H, CH2CO2Me at C-2); 2.30, 2.23, 2.10 (3m, 6H,
CH2CH2CO2Me at C-3 and CH2CH2CO2Me at C-8);
1.68 (s, 3H, CH3 at C-2); 1.49 (s, 3H, CH3 at C-7).
13C NMR d 173.27, 173.17, 172.98 (2), 172.26, 171.53,
171.29, 7 CO2CH3; 170.17, C-6; 159.46, C-4 and C-9;
153.34, C-1; 151.14, C-19; 143.73, C-11; 141.65, C-17;
136.81, C-13; 135.57, C-16; 131.55, C-14; 125.19, C-18;
123.82, C-12; 107.35, C-15; 104.82, C-20; 94.94, C-10;
90.05, C-5; 55.60, C-3; 51.81, 51.59, 51.10 (2), 51.01,
50.72, 50.56, 7 CO2CH3; 44.40, CH2CO2Me at C-7;
The product DiMeIBC 7d14 was obtained in too small a
quantity to allow a full H NMR assignment, thus only
1
the most important characterization data are reported:
1H NMR (C6D6): d 8.85 (s, 1H, H-15); 7.61 (s, 1H, H-
10); 7.55 (s, 1H, H-20); 6.81 (s, 1H, H-5); 1.74 (s, 3H,
CH3 at C-2); 1.63 (s, 3H, CH3 at C-7). UV kmax (MeOH)
282 (0.28), 379 (sh, 0.77), 397 (1), 500 (0.11), 545 (0.1),
589 (0.27) nm. MS (ESI) m/z 975 [M+H]+. HRMS
(ESI) [C50H63N4O16]+, calcd: 975.4239. Found:
975.3980.
41.90, CH2CO2Me at C-7; 36.85, 36.32,
2
CH2CH2CO2Me at C-13 and C-17; 33.95, CH2CO2Me
at C-18; 32.71, 32.15, 2 CH2CH2CO2Me at C-3 and C-
8; 25.87, 25.22, 2 CH2CH2CO2Me at C-3 and C-8;
21.48, 21.20, 2 CH2CH2CO2Me at C-13 and C-17;
20.58, CH3 at C-7; 19.97, CH3 at C-2; 19.13, CH3 at
C-20; 10.01, CH3 at C-12. UV kmax (MeOH) 274
(0.25), 359 (sh, 0.58), 376 (1), 390 (sh, 0.68), 409
(0.59), 500 (0.09), 550 (0.16), 594 (0.21), 636 (0.08) nm.
MS (ESI) m/z 931 [M+H]+. HRMS (ESI)
[C49H63N4O14]+, calcd: 931.4341. Found: 931.4225. 2
epimers of 8e were isolated together and only partial
6.2.3.4. From porphyrin (1e). After 2 series of PLC, 4
products were isolated: MeC methyl ester 6e (2%), diM-
eIBC methyl ester 7e (2%), triMeIBC methyl ester and
epimers 8e (28%), and triMeIBC monolactone methyl
1
ester 12e (9%). MeC 6e: H NMR (C6D6): d 10.09 (s,
1H, H-15); 9.91 (s, 1H, H-10); 9.18 (s, 1H, H-20); 9.00
(s, 1H, H-5); 5.00 (m, 1H, H-3); 4.91 (AB, J = 12.2,
2H, CH2CO2Me at C-7); 4.72 (s, 2H, CH2CO2Me at
C-18); 4.43, 4.31, 4.19, 4.06 (4m, 6H, 3 CH2CH2CO2Me
at C-8, C-13 and C-17); 3.40, 3.39, 3.31, 3.29, 3.27, 3.24,
3.22 (7s, 21H, 7 CO2CH3); 3.11 (s, 3H, CH3 at C-12);
3.29 [overlapping with signals of methyl esters], 3.16
(2m, 6H, 3 CH2CH2CO2Me at C-8, C-13 and C-17);
2.95, 2.76 (AB, J = 17.5, 2H, CH2CO2Me at C-2); 2.46
(m, 2H, CH2CH2CO2Me at C-3); 2.24 (s, 3H, CH3 at
C-2); 2.20 (m, 2H, CH2CH2CO2Me at C-3). UV kmax
(MeOH) 289 (0.10), 348 (sh, 0.21), 393 (1), 496 (0.10),
536 (0.05), 570 (0.04), 649 (0.19) nm. MS (ESI) m/z
901 [M+H]+. HRMS (ESI) [C47H57N4O14]+, calcd:
901.3871. Found: 901.3438.
1
assignments for H NMR were possible. 8e epimer 1:
1H NMR (C6D6): d 8.72 (s, 1H, H-15); 7.41 (s, 1H, H-
10); 6.50 (s, 1H, H-5); 2.78 (s, 3H, CH3 at C-20); 2.53
(s, 3H, CH3 at C-12); 1.85 (s, 3H, CH3 at C-2); 1.43
1
(s, 3H, CH3 at C-7). 8e epimer 2: H NMR (C6D6): d
8.74 (s, 1H, H-15); 7.74 (s, 1H, H-10); 6.56 (s, 1H, H-
5); 2.65 (s, 3H, CH3 at C-20); 2.52 (s, 3H, CH3 at C-
12); 1.67 (s, 3H, CH3 at C-2); 1.49 (s, 3H, CH3 at C-7).
TriMeIBC monolactone 12e. 1H NMR (C6D6): d 8.95 (s,
1H, H-15); 7.79 (s, 1H, H-10); 6.58 (s, 1H, H-5); 4.27
(AB, J = 19.0, 2H, CH2CO2Me at C-18); 4.08 (m, 1H,
H-3 or H-8); 3.79, 3.60 (2m, 4H, 2 CH2CH2CO2Me at
C-13 or C-17); 3.43, 3.35, 3.30, 3.27, 3.21, 3.18 (6s, 18H,
6 CO2CH3); 3.05 (m, 2H, CH2CH2CO2Me at C-17);
2.99 (AB, J = 8.1, 2H, CH2CO2Me at C-7); 2.83 (s, 3H,
CH3 at C-20); 2.70 (t, J = 7.0, 2H, CH2CH2CO2Me at
C-13); 2.65, 2.48 (AB, J = 13.4, 2H, CH2CO2Me at C-
2); 2.47 (s, 3H, CH3 at C-12); 2.44, 2.35 (AB, J = 15.9,
2H, CH2CH2CO2Me at C-8); 2.25–2.00 (m, 6H,
CH2CH2CO2Me at C-3 and CH2CH2CO2Me at C-8);
1.71 (s, 3H, CH3 at C-2); 1.26 (s, 3H, CH3 at C-7). 13C
NMR d (most important signals) 107.47 (C-5), 104.15
(C-20), 96.88 (C-10), 94.83 (C-3 or C-8), 90.14 (C-5),
DiMeIBC 7e. 1H NMR (C6D6) d 8.83 (s, 1H, H-15); 7.55
(s, 1H, H-10); 7.43 (s, 1H, H-20); 6.72 (s, 1H, H-5); 4.30,
4.05 (2m, 2H, H-3 and H-8); 4.15 (m, 2H, CH2CO2Me
at C-18); 3.81, 3.67 (2m, 4H, 2 CH2CH2CO2Me at C-
13 and C-17); 3.37 (2), 3.33, 3.25, 3.23, 3.21, 3.16 (7s,
21H, 7 CO2CH3); 3.00, 2.71 (2t, J = 6.8, 6.7, 4H, 2
CH2CH2CO2Me at C-13 and C-17); 2.70 (m, 2H,
CH2CH2CO2Me at C-8); 2.63 (s, 3H, CH3 at C-12);
2.63 (d, J = 6.8, 2H, CH2CO2Me at C-7); 2.67, 2.54
(AB, J = 13.5, 2H, CH2CO2Me at C-2); 2.30–2.05 (2m,
6H, CH2CH2CO2Me at C-3 and CH2CH2CO2Me at

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3919
9.75 (CH3 at C-12). UV kmax (MeOH) 277 (0.25), 358 (sh,
0.50), 377 (1), 393 (sh, 0.70), 412 (0.73), 500 (0.10), 539
(0.13), 583 (0.17), 629 (0.09) nm. MS (ESI) m/z 915
[M+H]+. HRMS (ESI) [C48H59N4O14]+, calcd:
915.4028. Found: 915.3973.
isolated together in too small a quantity to allow a full
1H NMR assignment, thus only the most important
characterization data are reported. 7j epimer 1: 1H
NMR (C6D6): d 8.80 (s, 1H, H-15); 7.61 (s, 1H, H-10);
7.35 (s, 1H, H-20); 6.71 (s, 1H, H-5); 4.02, 3.79 (2m,
2H, H-3 and H-8); 1.59 (s, 3H, CH3 at C-2); 1.52 (s,
1
6.2.3.5. From porphyrin (1f). Only one product was
isolated in very small quantities: the methyl ester MeC
3H, CH3 at C-7). 7j epimer 2: H NMR d 8.78 (s, 1H,
H-15); 7.50 (s, 1H, H-10); 7.47 (s, 1H, H-20); 6.68 (s,
1H, H-5); 4.09, 3.69 (2m, 2H, H-3 and H-8); 1.80 (s,
3H, CH3 at C-2); 1.45 (s, 3H, CH3 at C-7). UV kmax
(MeOH) 278 (0.28), 359 (sh, 0.69), 369 (1), 394 (sh,
0.77), 512 (0.13), 546 (0.21), 589 (0.35). MS (ESI) m/z
947 [M+H]+, 969 [M+Na]+. HRMS (ESI) [C48H58Na-
N4O16]+, calcd: 969.3746. Found: 969.3166.
1
6f (1–3%). H NMR (C6D6): d (major isomer) 10.29 (s,
1H, H-15); 10.19 (s, 1H, H-10); 9.19 (d, J = 7.0, 1H,
H-20); 9.06 (d, J = 5.2, 1H, H-5); 5.03 (m, 1H, H-3);
4.84 (m, 2H, CH2CO2Me at C-7); 4.72, 4.68 (AB + d,
J = 4.9, 13.8, 4H, 2 CH2CO2Me at C-12 and C-18);
4.05, 3.98 (2m, 6H, 3 CH2CH2CH2CO2Me at C-8, C-
13 and C-17); 3.45, 3.44 (3), 3.43 (2), 3.42, 3.31 (8s,
24H,
8
CO2CH3); 3.42, 3.41, 3.11 (3m, 6H,
3
DiMeIBC monolactone 10j (major isomer). 1H NMR
(C6D6): d 8.90 (s, 1H, H-15); 7.61 (s, 1H, H-10); 7.45
(s, 1H, H-20); 6.92 (s, 1H, H-5); 4.41, 4.39, 4.28, 4.20
(s + AB + s + AB, J = 11.8, 14.1, 8H, 4 CH2CO2Me at
C-12, C-13, C-17 and C-18); 4.18 (m, 1H, H-3 or H-8);
3.40, 3.27, 3.33 (4), 3.19 (4s, 21H, 7 CO2CH3); 2.52
(m, 4H, CH2CO2Me at C-2 and C-7); 2.44 (t, J = 6.7,
2H, CH2CH2CO2Me at C-3 or C-8); 2.37–1.97 (m, 6H,
CH2CH2CO2Me at C-3 or C-8 and CH2CH2CO2Me at
C-3 or C-8); 1.43 (s, 3H, CH3 at C-2); 1.34 (s, 3H,
CH3 at C-7). UV kmax (MeOH) 279 (0.22), 362 (sh,
0.59), 383 (1), 404 (sh, 0.77), 506 (0.11), 542 (0.17),
583 (0.20), 642 (0.05) nm. MS (ESI) m/z 953 [M+Na]+.
HRMS (ESI) [C47H54NaN4O16]+, calcd: 953.3433.
Found: 953.3226. 2 epimers of 10j were also isolated.
CH2CH2CH2CO2Me at C-8, C-13 and C-17); 3.08,
2.92 (AB, J = 14.9, 2H, CH2CO2Me at C-2); 2.61 (m,
6H, 3 CH2CH2CH2CO2Me at C-8, C-13 and C-17);
2.51 (m, 4H, CH2CH2CH2CO2Me at C-3); 2.28 (s, 3H,
CH3
at
C-2);
2.17
(AB,
J = 7.5,
2H,
CH2CH2CH2CO2Me at C-3). 13C NMR d (partial
assignment) 101.60 (C-15 and C-20), 94.57 (C-10),
92.17 (C-5). UV kmax (MeOH) 288 (0.10), 344 (sh,
0.17), 393 (1), 494 (0.08), 591 (0.03), 643 (0.29) nm.
MS (ESI) m/z 1015 [M+H]+, 1037 [M+Na]+. HRMS
(ESI) [C53H67N4O16]+, calcd: 1015.4552. Found:
1015.4343.
6.2.3.6. From porphyrin (1j). Four products were iso-
lated: MeC methyl ester 6j (5%), diMeIBC methyl ester
7j (13%), diMeIBC monolactone methyl ester 10j (9%),
and diMeIBC dilactone methyl ester 11j (5%). MeC 6j:
1H NMR (C6D6): d 10.10 (s, 1H, H-15); 10.04 (s, 1H,
H-10); 9.14 (s, 1H, H-20); 8.86 (s, 1H, H-5); 4.99 (m,
1H, H-3); 4.88, 4.83, 4.78, 4.72 (4s, 10H, 5 CH2CO2Me
at C-7, C-12, C-13, C-17 and C-18); 4.27 (m, 2H,
CH2CH2CO2Me at C-8); 3.47, 3.41 (3), 3.40, 3.32,
3.28, 3.12 (8s, 24H, 8 CO2CH3); 3.14 (m, 2H,
CH2CH2CO2Me at C-8); 2.94, 2.75 (AB, J = 1.51, 2H,
CH2CO2Me at C-2); 2.56, 2.46, 2.28 (3m, 4H,
CH2CH2CO2Me at C-3); 2.20 (s, 3H, CH3 at C-2). UV
kmax (MeOH) 288 (0.16), 358 (sh, 0.25), 396 (1), 496
(0.11), 542 (0.07), 585 (0.09), 645 (0.29) nm. MS (ESI)
m/z 937 [M+Li]+. HRMS (ESI) [C47H54LiN4O16]+,
calcd: 937.2935. Found: 937.3514.
1
10j epimer 1: H NMR (C6D6): d 8.86 (s, 1H, H-15);
7.97 (s, 1H, H-10); 7.39 (s, 1H, H-20); 6.56 (s, 1H, H-
5); 4.32, 4.29, 4.22, 4.17 (AB + s + 2AB, J = 8.7, 16.6,
4.8, 8H, 4 CH2CO2Me at C-12, C-13, C-17 and C-18);
3.73 (m, 1H, H-3 or H-8); 3.44, 3.37, 3.36, 3.35, 3.34,
3.31, 3.23 (7s, 21H, 7 CO2CH3); 2.79, 2.38 (AB,
J = 18.2, 2H, CH2CO2Me at C-2 or C-7); 2.56 (s + m,
4H, CH2CO2Me at C-2 or C-7 and CH2CH2CO2Me at
C-8); 2.20, 1.97 (2m, 6H, CH2CH2CO2Me at C-3 or
C-8 and 2 CH2CH2CO2Me at C-3 and C-8); 1.51 (s,
3H, CH3 at C-2); 1.20 (s, 3H, CH3 at C-7). UV kmax
(MeOH) 279 (0.29), 358 (sh, 0.63), 378 (1), 405 (sh,
0.6), 503 (0.13), 539 (0.17), 581 (0.23), 640 (0.1). MS
(ESI) m/z 931 [M+H]+, 953 [M+Na]+. HRMS (ESI)
[C47H54NaN4O16]+, calcd: 953.3433. Found: 953.3249.
1
10j epimer 2: H NMR (C6D6) d 8.88 (s, 1H, H-15);
7.99 (s, 1H, H-10); 7.48 (s, 1H, H-20); 6.59 (s, 1H, H-
5); 4.33, 4.30, 4.22, 4.17 (2AB + s + AB, J = 17.7, 2.6,
17.3, 8H, 4 CH2CO2Me at C-12, C-13, C-17 and C-
18); 4.14 (m, 1H, H-3 or H-8); 3.42, 3.37, 3.35 (2),
3.34, 3.32, 3.17 (7s, 21H, 7 CO2CH3); 2.79, 2.36, 2.52
(2AB, J = 18.2, 14.3, 4H, 2 CH2CO2Me at C-2 and C-
7); 2.67, 2.59, 2.19 (3m, 8H, 2 CH2CH2CO2Me at C-3
and C-8); 1.71 (s, 3H, CH3 at C-2); 1.22 (s, 3H, CH3
at C-7). UV kmax (MeOH) 279 (0.27), 355 (sh, 0.60),
377 (1), 404 (sh, 0.47), 500 (0.12), 538 (0.17), 580
(0.21), 639 (0.08) nm. MS (ESI) m/z 931 [M+H]+, 953
[M+Na]+. HRMS (ESI) [C47H54NaN4O16]+, calcd:
953.3433. Found: 953.3176.
DiMeIBC 7j (major isomer). 1H NMR (C6D6): d 8.82 (s,
1H, H-15); 7.59 (s, 1H, H-10); 7.48 (s, 1H, H-20); 6.76 (s,
1H, H-5); 4.35, 4.32, 4.29, 4.24 (s + AB + s + AB,
J = 7.4, 12.6, 8H, 4 CH2CO2Me at C-12, C-13, C-17
and C-18); 4.10, 4.06 (t + m, J = 6.7, 2H, H-3 and H-
8); 3.41, 3.38, 3.36, 3.35, 3.34, 3.33, 3.23, 3.16 (8s,
24H, 8 CO2CH3); 2.61, 2.52 (AB, J = 15.1, 2H,
CH2CO2Me at C-2); 2.52 (AB, J = 7.9, 2H, CH2CO2Me
at C-7); 2.45 (m, 2H, CH2CH2CO2Me at C-8); 2.32,
2.25, 2.21 (3m, 6H, CH2CH2CO2Me at C-3 and
CH2CH2CO2Me at C-8); 1.71 (s, 3H, CH3 at C-2);
1.60 (s, 3H, CH3 at C-7). UV kmax (MeOH) 280 (0.28),
357 (sh, 0.65), 379 (1), 393 (sh, 0.85), 504 (0.14), 546
(0.18), 589 (0.29) nm. MS (ESI) m/z 947 [M+H]+, 969
[M+Na]+. HRMS (ESI) [C48H59N4O16]+, calcd:
947.3926. Found: 947.3288. 2 minor epimers of 7j were
DiMeIBC dilactone 11j. 1H NMR (C6D6): d 9.10 (s, 1H,
H-15); 8.13 (s, 1H, H-10); 7.63 (s, 1H, H-20); 7.07 (s, 1H,
H-5); 4.43, 4.36, 4.30, 4.19 (d + 3AB, J = 0.9, 17.5, 5.7,

3920
C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
11.0, 8H, 4 CH2CO2Me at C-12, C-13, C-17 and C-18);
3.43, 3.37, 3.34, 3.33, 3.27, 3.19 (6s, 18H, 6 CO2CH3);
2.60, 2.35 (AB, J = 18.4, 2H, CH2CO2Me at C-2);
2.55, 2.23 (AB, J = 18.3, 2H, CH2CO2Me at C-7);
2.59, 2.41 (2m, 4H, 2 CH2CH2CO2Me at C-3 and C-
8); 2.21, 2.08 (2m, 4H, 2 CH2CH2CO2Me at C-3 and
C-8); 1.38 (s, 3H, CH3 at C-2); 1.25 (s, 3H, CH3 at C-
7). UV kmax (MeOH) 278 (0.20), 358 (sh, 0.46), 373
(1), 384 (sh, 0.79), 405 (0.63), 491 (sh, 0.09), 542
(0.14), 582 (0.15), 628 (0.10) nm. MS (ESI) m/z 921
[M+Li]+, 937 [M+Na]+. HRMS (ESI) [C46H50Na-
N4O16]+, calcd: 937.3120. Found: 937.2852.
3H, CH3 at C-2); 1.46 (s, 3H, CH3 at C-7). UV kmax
(MeOH) 278 (0.23), 359 (sh, 0.57), 380 (1.0), 405 (sh,
0.80), 500 (0.12), 542 (0.15), 582 (0.17), 630 (0.09) nm.
MS (ESI) m/z 937 [M + Li]+. HRMS (ESI) [C49H62Li-
N4O14]+, calcd: 937.3663. Found: 937.4632.
The TriMeIBC monolactone 12m was obtained in too
small a quantity to allow a full H NMR assignment,
thus only the most important characterization data are
1
1
reported: H NMR (C6D6): d 8.97 (s, 1H, H-15); 8.00
(s, 1H, H-10); 6.60 (s, 1H, H-5); 3.43, 3.41, 3.37, 3.27,
3.25, 3.16 (6s, 18H, 6 CO2CH3); 2.73 (s, 3H, CH3 at
C-20); 2.64 (s, 3H, CH3 at C-18); 1.78 (s, 3H, CH3 at
C-2); 1.21 (s, 3H, CH3 at C-7). UV kmax (MeOH) 278
(0.22), 359 (sh, 0.53), 380 (1), 405 (0.74), 500 (0.11),
535 (0.15), 576 (0.16), 629 (0.07) nm. MS (ESI) m/z
915 [M+H]+. HRMS (ESI) [C48H59N4O14]+, calcd:
915.4050. Found: 915.4260.
6.2.3.7. From porphyrin (1m). Four products were iso-
lated: MeC methyl ester 6m (2–3%), triMeIBC methyl
ester and epimers 8m (8–11%), triMeIBC monolactone
methyl ester 12m (1%), and triMeIBC dilactone methyl
1
ester 13m (1%). MeC 6m: H NMR (C6D6): d 10.10 (s,
1H, H-15); 10.00 (s, 1H, H-10); 9.16 (s, 1H, H-20);
8.90 (s, 1H, H-5); 4.99 (m, 1H, H-3); 4.76, 4.72 (2s,
4H, 2 CH2CO2Me at C-2 and C-7); 4.32, 4.06 (2m,
6H, 3 CH2CH2CO2Me at C-8, C-13 and C-17); 3.47,
3.34, 3.31, 3.28, 3.27 (2), 3.26 (7s, 21H, 7 CO2CH3);
3.10 (s, 3H, CH3 at C-18); 3.26, 3.25 [overlapping with
methyl ester signals], 2.97 (2m + t, J = 8.4, 6H, 3
CH2CH2CO2Me at C-8, C-13 and C-17); 2.96, 2.78
(AB, J = 8.3, 2H, CH2CO2Me at C-2); 2.47 (m, 2H,
CH2CH2CO2Me at C-3); 2.25 (s, 3H, CH3 at C-2);
2.04 (m, 2H, CH2CH2CO2Me at C-3). UV kmax (MeOH)
281 (0.1), 350 (sh, 0.21), 396 (1.0), 500 (0.09), 531 (0.04),
587 (0.04), 643 (0.19) nm. MS (ESI) m/z 901 [M+H]+,
923 [M+Na]+. HRMS (ESI) [C47H56NaN4O14]+, calcd:
923.3691. Found: 923.3301.
Two epimers of TriMeIBC dilactone 13m were isolated
together and only partial assignment for H NMR was
1
possible. 13m epimer 1: 1H NMR (C6D6): d 9.63 (s,
1H, H-15); 8.49 (s, 1H, H-10); 7.75 (s, 1H, H-5); 2.90
(s, 3H, CH3 at C-20); 2.84 (s, 3H, CH3 at C-18); 1.55
(s, 3H, CH3 at C-2); 1.52 (s, 3H, CH3 at C-7). 13m epi-
mer 2: 1H NMR d 9.54 (s, 1H, H-15); 8.29 (s, 1H, H-10);
7.74 (s, 1H, H-5); 3.17 (s, 3H, CH3 at C-20); 2.63 (s, 3H,
CH3 at C-18); 1.60 (s, 3H, CH3 at C-2); 1.46 (s, 3H, CH3
at C-7). UV kmax (MeOH) 286 (0.14), 361 (sh, 0.37), 378
(1), 389 (sh, 0.65), 409 (0.86), 497 (0.08), 536 (0.08), 578
(0.09), 624 (0.12) nm. MS (ESI) m/z 899 [M+H]+, 921
[M+Na]+, 937 [M+K]+. HRMS (ESI) [C47H54 Na
N4O14]+, calcd: 921.3535. Found: 921.3495.
1
TriMeIBC 8m. H NMR (C6D6): d 8.81 (s, 1H, H-15);
7.41 (s, 1H, H-10); 6.64 (s, 1H, H-5); 4.27 (AB,
6.2.3.8. From porphyrin (1n). Four products were iso-
lated: MeC methyl ester 6n (10%), diMeIBC methyl ester
7n (2%), diMeIBC monolactone methyl ester 10n (1%),
and diMeIBC dilactone methyl ester 11n (1%). MeC
J = 17.8, 2H, CH2CO2Me at C-12); 4.18, 4.07 (2m,
2H, H-3 and H-8); 3.77, 3.63 (2m, 4H,
2
1
CH2CH2CO2Me at C-13 and C-17); 3.44, 3.34, 3.32,
3.29, 3.28, 3.23, 3.20 (7s, 24H, 7 CO2CH3); 3.00 (m,
2H, CH2CH2CO2Me at C-17); 2.85 (s, 3H, CH3 at
C-20); 2.74 (m, 2H, CH2CH2CO2Me at C-13); 2.70
(AB, J = 6.5, 2H, CH2CO2Me at C-7); 2.56 (s, 3H,
CH3 at C-18); 2.54 (AB, J = 7.5, 2H, CH2CO2Me at
C-2); 2.50, 2.31 (2m, 2H, CH2CH2CO2Me at C-8);
2.22, 2.10, 2.04 (3m, 6H, CH2CH2CO2Me at C-3
and CH2CH2CO2Me at C-8); 1.68 (s, 3H, CH3 at C-
2); 1.49 (s, 3H, CH3 at C-7). UV kmax (MeOH) 276
(0.32), 358 (sh, 0.61), 378 (1.0), 405 (sh, 0.81), 500
(0.12), 540 (0.16), 591 (0.18), 638 (0.07) nm. MS
(ESI) m/z 931 [M+H]+, 953 [M+Na]+. HRMS (ESI)
[C49H63N4O14]+, calcd: 931.4363. Found: 931.4625. 2
minor epimers of 8m were isolated together in too
6n (major isomer): H NMR (C6D6): d 10.08 (s, 1H,
H-15); 9.89 (s, 1H, H-10); 9.17 (s, 1H, H-20); 9.00 (s,
1H, H-5); 4.99 (m, 1H, H-3); 4.72, 4.69 (2s, 6H, 2
CH2CO2Me at C-7 and C-12); 4.27, 4.18 (m + t,
J = 7.4, 6H, 3 CH2CH2CO2Me at C-8, C-13 and C-
18); 3.47, 3.40, 3.32, 3.28, 3.27, 3.24, 3.23 (7s, 21H, 7
CO2CH3); 3.27, 3.15 [overlapping the methyl ester sig-
nals] (m + t, J = 7.3, 6H, 3 CH2CH2CO2Me at C-8, C-
13 and C-18); 3.11 (s, 3H, CH3 at C-17); 2.95, 2.76
(AB, J = 15.1, CH2CO2Me at C-2); 2.47 (m, 2H,
CH2CH2CO2Me at C-3); 2.25 (s, 3H, CH3 at C-2);
2.24, 2.19 (2m, 2H, CH2CH2CO2Me at C-3). The 6n epi-
mer could not be obtained free of the major isomer and
1
1
only partial assignment for H NMR was possible: H
NMR d 10.08 (s, 1H, H-15); 9.89 (s, 1H, H-10); 9.10
(s, 1H, H-20); 8.95 (s, 1H, H-5); 4.65 (m, 1H, H-3);
3.73, 3.54 (AB, J = 16.9, 2H, CH2CO2Me at C-2); 3.20
(s, 3H, CH3 at C-17); 1.85 (s, 3H, CH3 at C-2). UV kmax
(MeOH) 282 (0.09), 346 (sh, 0.21), 394 (1.0), 495 (0.09),
535 (0.04), 592 (0.04), 650 (0.24) nm. MS (ESI) m/z 901
[M+H]+, 923 [M+Na]+. HRMS (ESI) [C47H57N4O14]+,
calcd: 901.3893. Found: 901.3491.
1
small a quantity to allow a full H NMR assignment,
thus only the most important characterization data
are reported. 8m epimer 1: 1H NMR (C6D6): d 8.95
(s, 1H, H-15); 7.79 (s, 1H, H-10); 6.58 (s, 1H, H-5);
3.43, 3.34, 3.30, 3.27, 3.25, 3.19, 3.16 (7s, 21H, 7
CO2CH3); 2.83 (s, 3H, CH3 at C-20); 2.47 (s, 3H,
CH3 at C-18); 1.71 (s, 3H, CH3 at C-2); 1.26 (s,
3H, CH3 at C-7). 8m epimer 2: 8.85 (s, 1H, H-15);
7.61 (s, 1H, H-10); 6.67 (s, 1H, H-5); 2.84 (s, 3H,
CH3 at C-20); 2.65 (s, 3H, CH3 at C-18); 1.75 (s,
The product DiMeIBC 7n was obtained in too small a
quantity to allow a full H NMR assignment, thus only
1

C. Pichon-Santander et al. / Bioorg. Med. Chem. 14 (2006) 3904–3922
3921
5. Scott, A. I.; Irwin, A. J.; Seigel, L. M.; Shoolery, J. N.
J. Am. Chem. Soc. 1978, 100, 316–318.
6. Gonzalez, M. D.; Williams, H. J.; Santander, P. J.; Ozaki,
S.; Stolowich, N. J.; Scott, A. I. Tetrahedron 1992, 48,
5951–5960.
the most important characterization data are reported:
1H NMR (C6D6): d 8.71 (s, 1H, H-15); 7.69 (s, 1H, H-
10); 7.53 (s, 1H, H-20); 6.83 (s, 1H, H-5); 2.78 (s, 3H,
CH3 at C-17); 1.75 (s, 3H, CH3 at C-2); 1.65 (s, 3H,
CH3 at C-7). MS (ESI) m/z 917 [M+H]+. HRMS (ESI)
[C48H61N4O14]+, calcd: 917.4206. Found: 917.3866.
7. Muller, G.; Gneuss, K. D.; Kriemler, H.-P.; Irwin, A. J.;
¨
Scott, A. I. Tetrahedron 1981, 37, 81–90.
8. Warren, M. J.; Roessner, C. A.; Santander, P. J.; Scott, A.
I. Biochem. J. 1990, 265, 725–729.
9. Blanche, F.; Debussche, L.; Thibaut, D.; Crouzet, J.;
Cameron, B. J. Bacteriol. 1989, 171, 4222–4231.
10. Warren, M. J.; Roessner, C. A.; Ozaki, S.; Stolowich, N.
J.; Santander, P. J.; Scott, A. I. Biochemistry 1992, 31,
603–609.
11. Thibaut, D.; Couder, M.; Crouzet, J.; Debussche, L.;
Cameron, B.; Blanche, F. J. Bacteriol. 1990, 172, 6245–
6251.
12. Scott, A. I.; Williams, H. J.; Stolowich, N. J.; Karuso, P.;
Gonzalez, M. D.; Blanche, F.; Thibaut, D.; Muller, G.;
¨
Savvidis, E.; Hlineney, K. J. Chem. Soc. Chem. Commun.
1989, 1, 522–525.
13. Blanche, F.; personal communications; 1989.
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Stolowich, N. J.; Scott, A. I. J. Am. Chem. Soc. 1990,
112, 5343–5345.
15. Battersby, A. R.; Bladon, C.; Huang, J.-J.; Seo, S. J.
Chem. Soc. Chem. Commun. 1986, 1, 1492–1495.
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Leeper, F. J.; Battersby, A. R. J. Chem. Soc. Chem.
Commun. 1988, 1, 1117–1119.
17. Pandey, R. K.; Zheng, G. In The Porphyrin Handbook;
Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Photo-
sensitizers in Photodynamic Therapy; Academic Press:
San Diego, CA, 2000; Vol. 6, pp 157–230.
DiMeIBC monolactone 10n. Compound 10n and one
epimer were isolated together and only partial assign-
1
ment for H NMR was possible. 10n (major isomer):
1H NMR (C6D6) d 8.85 (s, 1H, H-15); 7.77 (s, 1H, H-
10); 7.61 (s, 1H, H-20); 6.99 (s, 1H, H-5); 2.81 (s, 3H,
CH3 at C-17); 1.50 (s, 3H, CH3 at C-2); 1.41 (s, 3H,
1
CH3 at C-7). 10n epimer: H NMR d 8.79 (s, 1H, H-
15); 7.69 (s, 1H, H-10); 7.57 (s, 1H, H-20); 6.95 (s, 1H,
H-5); 2.78 (s, 3H, CH3 at C-17); 1.46 (s, 3H, CH3 at
C-2); 1.39 (s, 3H, CH3 at C-7). UV kmax (MeOH) 280
(0.22), 351 (sh, 0.54), 386 (1), 405 (sh, 0.85), 500
(0.13), 540 (0.15), 582 (0.17), 644 (0.12) nm. MS (ESI)
m/z 901 [M+H]+, 923 [M+Na]+. HRMS (ESI)
[C47H56NaN4O14]+, calcd: 923.3691. Found: 923.3378.
DiMeIBC dilactone 11n. 1H NMR (C6D6): d 9.15 (s, 1H,
H-15); 8.37 (s, 1H, H-10); 7.86 (s, 1H, H-20); 7.37 (s, 1H,
H-5); 4.16 (s, 2H, CH2CO2Me at C-12); 3.90, 3.83, 3.70,
3.59 (4m, 4H, 2 CH2CH2CO2Me at C-13 and C-18);
3.44, 3.25, 3.21, 3.20, 3.16 (5s, 15H, 5 CO2CH3); 2.93
(s, 3H, CH3 at C-17); 2.93, 2.71 (2t, J = 8.0, 7.3, 4H, 2
CH2CH2CO2Me at C-13 and C-18); 2.81, 2.49 (AB,
J = 18.3, 2H, CH2CO2Me at C-2); 2.72 (m, 2H,
CH2CH2CO2Me at C-8); 2.60, 2.28 (AB, J = 18.5, 2H,
CH2CO2Me at C-7); 2.57 (m, 2H, CH2CH2CO2Me at
C-3); 2.28, 2.13, 2.02 (3m, 4H, 2 CH2CH2CO2Me at
C-3 and C-8); 1.46 (s, 3H, CH3 at C-2); 1.36 (s, 3H,
CH3 at C-7). UV kmax (MeOH) 282 (0.15), 357 (sh,
0.34), 375 (1.0), 383 (sh, 0.65), 405 (0.81), 485 (0.09),
576 (0.11), 621 (0.16) nm. MS (ESI) m/z 885 [M+H]+,
907 [M+Na]+. HRMS (ESI) [C46H52Na N4O14]+, calcd:
907.3378. Found: 907.3082.
18. Chen, Y.; Li, G.; Pandey, R. K. Curr. Org. Chem. 2004, 8,
1105–1134.
19. Pichon-Santander, C.; Scott, A. I. Tetrahedron Lett. 2005,
46, 8669–8672.
20. Vicente, M. G. H.; Smith, K. M. Curr. Org. Chem. 2000, 4,
139–174.
21. Shanmugathasan, S.; Edwards, C.; Boyle, R. W. Tetrahe-
dron 2000, 56, 1025–1046.
22. Pichon, C.; Atshaves, B. P.; Stolowich, N. J.; Scott, A. I.
Bioorg. Med. Chem. 1994, 2, 153–168.
23. Pichon, C.; Atshaves, B. P.; Xue, T.; Stolowich, N. J.;
Scott, A. I. Bioorg. Med. Chem. Lett. 1994, 4, 1105–1110.
24. Roessner, C. A.; Park, J.-H.; Scott, A. I. Bioorg. Med.
Chem. 1999, 7, 2215–2219.
25. Battersby, A. R.; Jones, K.; McDonald, E.; Robinson, J.
A.; Morris, H. R. Tetrahedron Lett. 1977, 25, 2213–
2216.
26. Battersby, A. R.; McDonald, E.; Morris, H. R.; Thomp-
son, M.; Williams, D. C. Tetrahedron Lett. 1977, 25, 2217–
2220.
Acknowledgments
We thank the NIH (NIDDK) for financial support and
Dr. Mario D. Gonzalez for uro II and IV samples. Use
of the TAMU/LBMS-Applications Laboratory is also
acknowledged.
References and notes
27. Battersby, A. R.; McDonald, E.; Neier, R.; Thompson, M.
J. Chem. Soc. Chem. Commun. 1979, 1, 960–961.
28. Scott, A. I.; Georgopapadakou, N.; Ho, K. S.; Klioze, S.;
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the two pathways of vitamin B12 biosynthesis; Academic
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¨
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¨
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