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This is to certify that the
thesis entitled
REGIOSPECIFIC SYNTHESIS OF VINYL
DIHYDROXYCHLORIN: MODEL COMPOUNDS FOR HEME d

presented by

EMMANUELLE GUERIN

has been accepted towards fulfillment
of the requirements for

[Md-Vhr’ degree in 6/14““737‘74!

Major professor

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FIG-9.1

REGIOSPECIFIC SYNTHESIS OF VINYL
DIHYDROXYCHLORIN; MODEL COMPOUNDS FOR HEME (I

By

Emmanuelle Guerin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Chemistry

1993

ABSTRACT

REGIOSPECIFIC SYNTHESIS OF VINYL
DIHYDROXYCHLORIN: MODEL COMPOUNDS FOR
HEME (I

By

Emmanuelle Guerin

The structure of green-colored heme d prosthetic group of cytochrome bd
oxidase originally deduced from spectral characterization was believed to be a
vinyl dihydroxychlorin containing a y—lactone ring derived from one hydroxy
group and a geminal propionate side chain. Subsequent studies indicated that
the lactone is not the true form of the heme d and probably arises as a
consequence of the isolation procedures of the demetallated prosthetic group.
Although the free base dihydroxychlorin of the heme d in the form of
dimethyl ester can be obtained by synthesis, the iron complex of this chlorin
has not be available synthetically because the facile lactonization occured
during iron inertion. To satisfy the need for heme d as well as its model
compounds in spectroscopic characterizations, a number of dihydroxychlorins
were synthesized containing vinyl side chains but without a gem-propionic
acid group in order to avoid the y—lactone formation. The synthesis of two
chlorins, 3-ethoxycarbonyl-13,17-diethyl-2,7,12,18-tetramethyl-8-vinyl-12,13-
dihydroxychlorin and 13,17-diethyl-2,7,12,18-tetramethyl-3,8-divinyl-12,13-
dihydroxychlorin, using a selective formation of the diol is described in this

thesis.

To my parents

ACKNOWLEDGMENT

First of all I would like to thank Professor C.l(. Chang for his advice,
helpful guidance and financial support during the course of this work.

I would also like to thank Professor 6.1. Baker and TJ. Pinnavaia for
serving as members on my guidance committee as well as Dr. Long for his
great help with the NOE experiments.

Financial support from Michigan State University in the form of
teaching assistantships is also gratefully acknowledged.

Special thanks are also extended to the members of our group,
particularly Ying Liang and lrene Morrison for their encouragement and
their friendship.

My deepest appreciation is due to my parents for their love,
encouragement and faith in me during the course of my studies.

Finally I would like to thank my friends Celina, Marie-Helene, Pascal,
Bicho for their constant support and particularly George for his love, his

encouragement and his patience.

TABLE OF CONTENTS

LIST OF FIGURES
CHAPTER 1: INTRODUCTION: From porphyrin to Home (1
I _ Structure of porphyrins and chlorins
II _ Previous study on Ileme d
CHART ER 2: RESULT AND DISCUSSION: Synthesis of a model for I-Ieme d
l _ Background
II _ Preparation of the ester porphyrin
III __ NOE study on the ester chlorin
IV - Preparation of l 3,17-diethyl-2.3.12.18-tetramethyl-l 2,13-di-
hydro.\y-3,8-divinylchlorin: model for Home (:1
CHAPTER 3: EXPERIMENTAL
APPENDIX
REFERENCES

iv

Page

v-vi

10
10
14
19

27
44
60

Figure 1.
Figure 2.
Figure 3.
Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

LIST OF FIGURES

Structure of Protoheme and Heme a
Structure of porphyrin and unsaturated porphyrins
Structure of chlorophyll and bacteriochlorophyll

Structure of Heme d1 and Heme d

Absorption spectra at 25°C of the air-oxidized form of the
cytochrome (1 complex

Structure of Meme 0 and Home d, responsible for the
reduction of oxygen to water in Escherichia coli
Arrangement of cytochromes in the respiratory chain of
cells of Escherichia coli in the late exponetial phase of
aerobic growth

Selective hydroxylation of porphyrin in the quadrant

opposite to the electronegative group

Non-regioselective hydroxylation with abscence of electron
withdrawing group

Figure 10. Synthesis of 3-ethoxycarbonyl-l3,] 7-diethyl-2,7,12,18-tetra-

methyl-12,13-dihydroxy-8—vinylchlorin

Figure 11. Synthesis of 3-ethoxycarbonyl—Z-formyl-4,S-dimethylpyrrole17

Figure 12. Synthesis of +(Z-chloroethyl)-3,S-dimethylpyrrole-Z-car-

boxylic acid

Figure 13. Structure of ester chlorin in NOE study

Figure 14. Attempted route for the synthesis of 3-acetyl-8-(2-chloro-

ethyI)-13,17-diethyl-2,7,12,18-tetmmethyl porphyrin

Figure 15. Synthesis of iron chelate 13,1 8-diethyl-2,7,12,1 8-tetramethyl

-3,8-di\'inylchlorin (part 1)

Figure 16. Synthesis of iron chelate 13,18-diethyl-2,7,12,18-tetramethy1

-3,8-divinylchlorin (part 2)

Figure 17. 300 MHZ 1H-NMR spectrum of porphyrin 9

Figure 18. 300 MHZ 1l-l-NMR spectrum of chlorin ll

\.

Page

|\)

3;.th

6

15

18
19

N
I\J

Iv
4;.

25
44
4s

Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.

Figure 34.

300 MHz 1H-NMR spectrum of chlorin 11 and NOE study

300 MHZ Ill-NMR spectrum ofchlorin 18
300 MHZ 1H-NMR spectrum of porphyrin 32
300 MHZ lit-NMR spectrum of porphyrin 10
300 Mill 111-Nl\lR spectrum of chlorin 12
Mass spectrum of porphyrin 9

Mass spectrum of chlorin 1 1

Mass spectrum of porphyrin 10

Mass spectrum of chlorin 12

Mass spectrum of chlorin 35

Mass spectrum of chlorin 35 bis

Mass spectrum of chlorin 36

Visible absorption spectrum of porphyrin 9
Visible absorption spectrum of porphyrin 11
Visible absorption spectrum of chlorin lO

Visible absorption spectrum of chlorin 12

\‘i

56
57
58
58
59
59

CHAPTER 1
INTRODUCTION: FROM PORPHYRIN TO HEME d

I. STRUCTURE OF PORPHYRINS AND CHLORINS

In nature, most heme-containing proteins and enzymes possess a
protoheme prosthetic group made from an iron atom metal coordinated within
a fully porphyrin macrocycle. Porphyrins1 are macrocyclic structures which
contain four pyrroles units connected together by four methene bridges.
These planar aromatic structures are 18 l'I-electron systems. They show an
intense absorption, known as Soret band around 400 nm and four Qbands in
the visible region between 490-640 nm. The intensity of these smaller bands
varies with the nature of the substituents on the porphyrin ring. The
porphyrin metal-complexes also exibit the Soret band but only two Q bands
known as a and [3 bands.

Best known examples of hemoproteins include hemoglobin that play a
vital role in transporting oxygen and myoglobin which is responsible for the
storage of oxygen in muscle tissue. Other examples are cytochromes a,b and c
that are responsible for the electron transfer in animals, plants and
microorganisms. The prosthetic group present in cytochrome aa3 is different
from the normal proteheme (figurel ).

Porphyrins and their metal complexes can be reduce to give diverse
isolable productsz. They are dihydroporphyrins (chlorin) and

tetrahydroporphyrins (bacteriochorins, isobacteriochlorins), all of them

2
resulting from the saturation of peripheral double bonds of the porphyrin

ring (figure 2).
Unlike porphyrins, chlorins usually display a stong absorption for the most
red-shifted Qband around 630 nm. A strong at band of the metal complexe is

also observed.

 

COOH coo}
Protoheme
H3
,CHz-(CHZ -C =C'CH2)3-H

HO-H

  

C00' (300'

Home a

Figure 1: Structure of Protoheme and Heme a

 

Porphyrin Chlorin
H
H
i n 3 H
H H
Bacteriochlorin Isobacteriochlorin

Figure 2: Structure of porphyrin and unsaturated porphyrin
The biological interest of these reduced porphyrins has centered mostly
on the magnesium complexes of chlorins and bacteriochlorins that are the
essential chromophoric units of algae and plant chlorophylls l and

bacteriochlorophylls 2.

 

Figure 3: Structure of chlorophyll and bacteriochlorophyll

4

But more recently , it has been shown that a significant number of
organisms contain C-substituted chlorins and isobacteriochlorins. Among
these are Factor I from the B-lZ-producing Clostridium tetanomorphum,4
heme d1 from Pseudomonas aeruginosa 5which is involved in a process known
as clenitrification6 by reducing nitrite to nitrous oxide and heme d of

Escherichia coli7 which catalyzes the reduction of 02 to H20.

 

COOH COOH

heme d1

 

3 4

Figure 4: Structure of Heme d1 and Heme d

The main difference between the substituted chlorins or
bacteriochlorins and the unsubstituted chlorin is that the C or Oil-substituted
compounds can resist dehydrogenation. Therefore they are better suited for
undertaking the redox processes with which they may be associated in vivo.

With the elucidation of the structure of these unique molecules, it has
become timely to investigate their chemistry. Detailed accounts of heme d1S
and heme d7’8 have been reported but an incertitude remains on the real

structure of heme d8.

5
II. PREVIOUS STUDY ON HEME d

In 1928 a green pigment was observed in cells of Escherichia coli and of
Shigella dysen teriae grown aerobically. This appeared to be an unusual

cytochrome with a pronounced red shift of the a band of the visible spectrum

to the 630 nm region9.

 

* 1 r r a T

05- fiOOS

4M

oil-t

 

 

 

 

- 0.04 .-
5 s
5 s
y 0 03 3
a z
‘ 3
é a
0 c 2 L- 002 m
‘ ‘3
°" F - o 0:
C i I i i g L . t"
400 ‘50 500 :50 600 650 '00 75:;

HAVQ’LOQGYN trout

Figure 5: absorption spectra at 25°C of the air-oxidized form

of the cytochrome d complex

Further work on the respiratory electron transport chain of certain
bacteria confirmed the existence of this cytochrome which was originally
called cytochrome 3210. Since about 1970, this cytochrome and its prosthetic
group heme have been termed respectively cytochrome d and heme d 4 to
avoid confusion with cytochrome aa3. This heme has also been detected in
diverse bacteria including Azotobacter, Proeus, Salmonella bacillus,
Pseudomonas and Haemophilus.

By in vivo experiments as well as isolation and characterization of
purified enzymes, the function of cytochrome d was established. The aerobic

respiratory chain of Escherichia coli like many other bacteria is branched“.

6

Under conditions of high aeration, a respiratory chain containing only b-type
cytochromes is predominant: the heme prosthetic group is referred to as
cytochrome 0.

When Escherichia coli is grown under conditions of limited oxygen,
three new cytochromes are induced; cytochromes b553, a1 and d9:12»13. The
Km value for oxygen of cytochrome d is about eight times lower than that of
cytochrome 011(Figure 6). The more efficient utilization of oxygen by
cytochrome d presumably allows the microbes to maintain efficient oxidative
energy conservation over a wide range of oxygen pressures by changing the

relative ratio of the two oxidases.

Dioxygen Reduction Catalyzed by Heme Enzymes
of Bacteria Escherichia Coli

02““;th+ —> 2H20

 

00H COOH COOH c001

Heme O Heme d
Figure 6: Structure of Heme o and Heme d, reponsible for the reduction of
oxygen to water in cytochrome d.

7

The propose branched arrangement of the cytochromes in the
respiratory chain of the cells in the late exponential phase of aerobic growth

is shown in figure 7.

/ CYt b562 ——’ Cyto —" 02(high 02)
Cytbsss _’ Q

\

Figure 7: Arrangement of cytochromes in the respiratory chain of cells of
Escherichia Coli in the late exponential phase of aerobic growth; Q;
ubiquinone

C)" bsss -—> Cyt d ——-> 0200“! 02)

In 1956 Barrett 14 proposed a structure for heme d as the iron chelate of
7,8-dihydroprotoporphyrin IX 5. The site of saturation could not be determine

at that time.

 

This proposal was based upon derivation chemistry as well as
comparisons of chromatographic behavior and visible spectra with models.

The main souce of material was cells of Aerobacter aerogenes.

8
The tentatively formulated structure of Barrett remains unchallenged

in the literature for almost 30 years and has served as model for many other
green hemes found in various bacterial cytochromes.

In 1985, Timkovitch and collaborators7 isolated sufficient amounts of
the heme d prosthetic group from purified Escherichia coli oxidase and
characterized the structure of the metal free, esterified chromophore by
means of H NMR, IR, UV-visible, and mass spectrometry. The proposed
structure comprises an unusual chlorin core with a spiro-y-Iactone group at

the saturated pyrrole ring C 6.

 

This "lactochlorin" structure is certainly unique but as the authors
noted it is not clear whether the lactone ring found in the metal-free
macrocycle is an authentic feature of the heme or whether it is an artifact
formed during isolation. The evidence of the lactone in the metal free heme d
was largely based on an intense IR absorption at 1782 cm'l.

To verify the heme d structure, and to determine whether the lactone is
an artifact or whether it is an authentic feature of the heme d, the

Timkovitch's lactochlorin, its diastereomer and the nonlactonized forms have

9
been synthesized. The results supported that the lactone is not a feature of the

heme d but an artifact of the isolated chromophore formed by prolonged
contact with silica gel during purification.

Also the absence of IR peak at 1782 cm"1 in the extracted but
unesterified heme is a strong argument against the lactone in the vivo
hemels. The true ligand structure of heme d in the enzyme is more likely to be
5,6 dihydroxyprotochlorin IX 4.

Despite the previous success in the synthesis of the dimethyl ester of 5,6-
dihydroxyprotochlorin IX, the iron complex of which was not synthesized
because of the facile lactonization occured during a variety of iron insertion
procedures. Due to the need for heme d or its model compounds in
spectroscopic characterization of the cytochrome d, we decided to synthesize a
number of dihydroxychlorins with conjugated vinyl side chains but without
the propionic acid group so as to avoid the y—lactone formation. The synthesis
of two chlorins, 3-ethoxycarbonyl-13,17-diethyl-2,7,12,18-tetramethyl-12,13-
dihydroxy-S-vinyl-chlorin and 13,17-diethyl-2,7,12,18-tetramethyl-12,l3-
dihydroxy-8,13-vinyl-chlorin, using a selective formation of the diol is

described in this thesis.

CHAPTER 2
RESULTS AND DISCUSSION: SYNTHESIS OF A MODEL FOR
HEMEd

I. BACKGROUND

As we said in the introduction, if a diol chlorin bears a geminal
propionate ester it seems that it leads to the formation of a lactone 7 during the

purification on t.l.c. plates or with the presence of a base16.

 

COOMe

7
To avoid the formation of this lactone and then be able to ensure that
lactone is only an artifact and not a feature of nascent heme d, the object of
this work is the synthesis of a possible model having ethyl groups instead of

propionic ester groups (structure 8).

10

11

 

In general, dihydroxylation of the porphyrin can be accomplished by
osmium tetraoxide addition to double bonds followed by quenching with
hydrogen disulfide. However such a reaction on porphyrin would lead to a
mixture of four different products resulting from the attack of the double bond
at any of the four pyrrole ring (see figure 9). This would give a very poor
yield of the desired chlorin. Furthermore, the mixture of the chlorins would be
very difficult to separate.

Recently K. M. Smith17 reported that introduction of an electron-
withdrawing group directly linked to the macrocycle of the porphyrin may
direct selectively the attack of the osmium tetraoxide at the subunit in the

quadrant opposite to the electronegative group (figure 8).

  
     

O
H
0504
——>
Pyridine craze:2
OH
O H
00MB OOMe COOMe OOMe

Figure 8: Selective hydroxylation of porphyrin in the quadrant opposite to the
electronegative group

12

 

 

Figure 9: Non-regioselective hydroxylation with the abscence of
electron-withdrawing group

13

Thus we describe first the synthesis of two porphyrins: 3-
ethoxycarbonyl-8-( 2-chloroethyl)- 1 3 , 17-diethyl-2,7, 1 2, l 8-tetramethyl-por-
phyrin 9 and 3-acetyI-8-(2-chloroethyl)-13,l7-diethyl-2,7,12,18-tetramethyl-
porphyrin 10 with a withdrawing group placed at a key point on the
macrocycle to lead to the selective formation of the desired chlorins 11 and 13,

respectively.

 

Initially we did the synthesis of the ester porphyrin 9 to verify the
account of K. M. Smith, a synthesis that required less steps than the formation
of the acetyl porphyrin. We were hoping to be able to transform the chlorin

11 into our target molecule 8, which unfortunately proved not to be the case.

14
II. PREPARATION OF THE ESTER CHLORIN

The synthetic strategy is shown in figure 10 in which the two
dipyrrylmethenes, "north" 15 and "south" 17 were refluxing together in a
mixture of formic acid and one equivalent of bromine to form the expected
ester porphyrin with a limited yield of 6 to 89613. This low yield is most likely
due to the presence of a withdrawing group which deactivates the cyclization.
This porphyrin was obtained after chromatography on silica gel column. It
was the first pink-purple band eluted by dichloromethane.

Dipyrryl methene 17 was best prepared via the decarboxylation of 5,5'-
bis(ethoxycarbonyl)-4,4'diethyl-3,3'-dimethyl—2,2'dipyrrylmethane 16 and its
bromination with an excess of bromine.

On an other hand the synthesis of 4,3',5,5'-dimethyl-3-ethoxycarbonyl-
4'-(2-chloroethyl)-2,2'-dipyrrylmethene 15 required much more step for its
synthesis. It was prepared by the condensation of ethyl-2-formyl-4,5-
dimethylpyrrole-3-carboxylate 13 and 4-(2-chloroethyl)-3,S-dimethyl
pyrrole-Z-carboxylic acid 14 in methanol and hydrobromic acid. Therefore we
began by the synthesis of these two pyrroles that is summarized in figure 11
and figure 12.

Starting from alanine heated with acetic anhydride in pyridine”, 3-
acetamido-Z-butanone 19 was formed and was allowed to be acidified to give 3-
ammonium-Z-butanone chloride salt 20. By reaction of this salt with diethyl
oxalacetate sodium, the pyrrole 3-ethoxycarbonyl-4,5-dimethylpyrrole-2-
carboxylic acid 21 is formed, which after decarboxylation in molten sodium
acetate and potassium acetate gave the a free pyrrole 22. The formyl pyrrole

23 was then obtained by a classic Vielsmeyer reaction.

COOEt c1
CH0 ”0 H H COOEt

13th l

l. KOH

2. Brz-Formic acid

-H Br

 

0804
Pyridine/CH2C12

DBU

 

DMF

 

Figure 10: Synthesis of 3-ethoxycarbonyl- 1 3, l7-diethyl-2,7,12,1 8-tetramethyl-
1 2,1 3-dihydroxy-8-vinylchlorin

16

The pyrrole 25 was formed in two steps: (1) the reaction of 2,4-
pentanedione and ethyl-Z-chloro-acetate 23 and (2) the reaction of 23 in the
presence of zinc dust with the oxime 24 formed by the reaction of benzyl
acetoacetate with sodium nitrite. Diborane was then used to reduce 25 into
benzyl-4-( 2-hydroxyethyl)-3 , 5-dimethylpyrrole-Z-carboxylate2 1. Substitution
of the hydroxy group by chlorine was carried out with thionyl chloride in
benzene at reflux. The reaction was run under atmosphere of argon to prevent
air oxidation. The benzyl ester group of benzyl-4-(-2-chloroethyl)-3,5-
dimethylpyrrole-2-carboxylate 27 was removed by hydrogenolysis
accomplished under hydrogen atmosphere with 10% palladium on charcoal
catalist. The hydrogenolysis with this pyrrole is very sensitive and failed
several times, but the reaction could be carried out to gived 14 with
quantitative yield.

Once the porphyrin 9 was isolated and characterized, the oxidation of
the porphyrin to prepare the hydroxychlorin could be realized. Reaction of
porphyrin 9 with 0.9 equivalent of 0804 in dichloromethane followed by H28
give 60% of one major chlorin product with 15 to 20% of remaining starting
porphyrin8t16’22v23. Thus we observed the fact that having a withdrawing
group to the porphyrin lead to a more regiospecific reaction.

The products were, isolated by column followed by t.l.c. plates. The major
product was of purple color, contrary to the green color of other
dihydroxychlorin compound without withdrawing group linked to the
macrocycle porphyrin. The dihydroxychlorin was identified by UV-visible
spectra and NMR. NOE study was carried out to ascertain that the oxidation of
the porphyrin takes place at the subunit in the quadrant opposite to the
electron-withdrawing group. More details on the NOE study and the

determination of the chlorin structure are given in the next chapter.

17

 

O
O

 

H.N HCl, Heat 0
’
H3N

19 20

 

’ HOOC KOAc

EtOOC
I m...
5,000 0N3, M NaOAc, 3H20
)
N
H

21

 

EtOOC EtOOC
11 m. » M
DMF OHC

H N N
H H

22 13

Figure 11: Synthesis of 3-ethoxycarbonyl-Z-formyl-4,Sodimethylpyrrole

18

W + c1/\loro\/ ,
IO

 

 

O 23 0
pH
NatNo2
Wp Ph ' ”\OAP"
0 0 Acetic acid 0 24 °

N COOCHzph

 

£ \’

 

 

 

H
25
H Cl
82% SOZCIZ K2m3
THF ArgoT' / \ Benzene ’ I \
N COOCHZPh N COOCHzph
H H
26 27
Cl
H2 Pd/C
» ‘11
THE N COOH
H
14

Figure 12: Synthesis of 4—(2-chlorethyl)-3,5-dimethylpyrrole-Z-carboxylic
acid

19

The chloroethylchlorin was then converted to the vinylchlorin by
elimination of the chlorin with DBU heated in dimethylformamide for two
hours. The structure of this compound was confirmed by NMR and mass
spectra.

The synthesis of this dihydroxychlorin was a success. The same pathway
using an electron-withdrawing group as directing group for the oxidation can

then be utilized in the synthesis of a model for heme d.

III. NOE STUDY ON THE ESTER CHLORIN

By "irradiating" the four different meso peaks a, b, c, and d appeared in
the region of 9 to 10 ppm we can determine their environment and deduce the
structure of the dihydroxychlorin. Thus we can prove that our hydroxy groups

occupy the position 12 and l3.(see figure 13 and figure 19 p. 47)

 

Figure 13: Structure of ester chlorin in NOE study
We can already assume before the measurements that proton a is the
most downfield shifted proton due to its location near the electron-

withdrawing group. Also the peak corresponding to the methyl group geminal

20
to the hydroxy group has to be upfield compared to the three other methyl

peaks. We expect to find it at 2.24 ppm due to the lost of the conjugaison in this
area. Thus by irradiating Ha we can isolated which methyl is in the position 7.

We operated the same way with the meso proton b. This proton enhances
two methyl groups. Hb has to be between the two methyl groups of the
positionsZ and 18. For the peak c, no real signals could be observed. This
suggest that this proton is between the two ethyl groups. Peaks of the ethyl
groups are small so their enhancement by NOE is difficult to see due to the
strong baseline.

From those data we can deduce that the hydroxy groups have to be in
the quadrant opposite to the ester group, since none of the three meso peaks
has NOE effect on the upfield shifted methyl group. This is confirmed by the
irradiation of the last meso peak d. The 2.24 ppm peak is enhanced like
expected.

Thus like predicted by K.M. Smith's article17 the oxidation took place

effectively in the quadrant opposite to the withdrawing group.

Iv. PREPARATION or 13-17-diethyl-2,3,12,18-tetramethyl-12,13-
dihydroxy-3,8-divinylchlorin: MODEL FOR HEME d

We first thought that we could reduce or decarboxylate the ester
porphyrin to obtain a unsubstituted position which can then be acetylated and
transformed later into a vinyl group togive the desired chlorin. But
unfortunately the carboxylester group proved to be very difficult to remove.
The ester group resisted acid hydrolisis as well as alkaline hydrolysis under
conditions that did not eliminate the chlorine on the side chain. Heating the

ester porphyrin in resorcinol melt gave us only products from substitution of

21

the chlorine by resorcinol and other non identified side products; the ester
group remained intact

We then envisaged the synthesis of a new porphyrin starting from
pyrroles, having an acetyl group that can be further reduced to a vinyl group.

The first route attempted to obtain this porphyrin was based on a
strategy similar to a recent publication24 (shown in figure 14). Unfortunatly
the dipyrrylmethene, condensation between the acetyl pyrrole and the chloro
ethyl pyrrole failed possibly for two reasons: the carboxylic acid group of the
chloro compound could not be remove without destruction of the pyrrole and
secondly, the electron—withdrawing acetyl pyrrole hindered such a
condensation.Several alternative methods were tried unsuccessful.

Another way to get this porphyrin was to follow the same pathway as in
the ester porphyrin 9, using an acetyl pyrrole instead of the starting

ethoxycarbonyl pyrrole l3.

/\

H
28

OH

But unlike the formylation of the pyrrole 22, 3-acetyl-2-formyl-4,5-
dimethyl pyrrole 28 could not be obtained by a Vielsmeyer reaction on 3-
acetyl-4,5-dimethyl pyrrole. We were forced then to prepare the
acetylporphyrin in a stepwise manner. The strategy is shown in figure15 and
16.

As we did for the preparation of the "north" dipyrrole of the ester

porphyrin, we reacted the two pyrroles 14 and 29 with hydrobromic acid in

22

acetic acid. We chose acetic acid as solvent instead of methanol to improve

solubility and also in order to avoid ester formation.

c1
OA+ 002i,
820 C H
H l H

l

O

OH (CH3)2 H
+

 

Figure 14: Attempted route for synthesis of 3-acetyl-8-(2-chloroethyl)—13,17-
diethyl-2,7,12,18-tetramethyl porphyrin

Pyrrole 29 was obtained from 14 after saponification of the ester with

potassium hydroxide in ethanol.

EtOOC K H HOOC

N Ethanol OHC N
H H

29

 

23
The dipyrrylmethene 30 was then refluxed with 17 in a mixture of

formic acid and bromine to form the porphyrin. During the reaction the
porphyrin was decarboxylated and the carboxylic group was replaced by
bromine (major product) or hydrogen. The total yield was about 6%, poor yield
due to the withdrawing group13.

Since the major product of this reaction is the bromoporphyrin 31 we
reduced this porphyrin by hydrogenation under one atmosphere of hydrogen
with palladium on charcoal. Some porphynogen was formed during the
hydrogenation, which must be oxidized by DDQ, In addition, byproducts were
present and the debromination was never complete. The hydrogen porphyrin
32 after purification on silica gel column gave a yield of 49%.

Iron was inserted into the porphyrin26 with ferric bromide and metal
complex was acetylated by a Friedel and Craft reaction with acetic anhydride
and stannic chloride27. The iron porphyrin was then demetallated and the
acetyl porphyrin 10 was obtained with a yield of 51%. After recrystallization
in dichloromethane and methanol, the porphyrin was analyzed by H-NMR,
mass spectroscopy, and UV visble spectroscopy. Like in the case of the ester
porphyrin we observed, due to the acetyl group, a rhodo-type visible spectrum
with the second Qband more intense than the other peaks.

The formation of the dihydroxychlorin 12 was carried out in the same
condition as the oxidation of the ester porphyrin. Reaction of 1.5 eqivalent of
osmium tetraoxide in dichloromethane followed by quenching with hydrogen
sulfide gave the expected chlorin. The reaction was as selective as with the
ester and gave only one major product of purple color also. A larger amount of
osmium tetraoxide had to be used, the acetyl group being more withdrawing,

thus more ring deactivating for the porphyrin.

24
COOH C1
,2} + 0023’?
CHO H
H H
29 14

 

HBr 48%

Acetic Acid
OOH

C1
N + N +
HBr' H Br HBr’ H Br
30 17
L Brill-{COOH
H2
Pd/C THF

FeB r2

THF
/ V

33

 

 

FigurelS: Synthesis of iron chelate 13,18-diethy1-2,7,12,18-tetramethyl-3,8-
divinylchlon'n (part 1)

25

 

 

 

 

 

Acetic

Anhydride V

SnCl4

0504 DBU
Pyridine/Ch20|2 DMF V

 

1 . NaBH.,,

2. DMF, Heat

 

FeBr2

 

Figure16: Synthesis of iron chelate 13,18-diethyl-3,8-divinyl-2,7,12,18-
tetramethylchlorin (part 2)

26
Elimination of the chlorine with DBU in dimethylformamide gave the

acetyl vinyl chlorin 35. The structure of this chlorin was verified by the
presence of vinyl peaks near 6 and 8 ppm in the H—NMR spectrum.

To obtain the divinyl chlorin 36, the acetyl group was reduced in
alcohol using sodium borohydride in a mixture of tetrahydrofuran and
methanol. With the reduction of the acetyl group to eliminate the electron
withdrawing group, the chlorin color turned from red-puple to green
(characteristic color of chlorin and heme d). This hydroxyethyl side chain
could be converted into the second vinyl group by heating the chlorine for 10
minutes under argon in dimethylformamide. Mass spectrum of the product
clearly indicated the formation of the divinyl dihydroxychlorin 36.

At this point, our overall yield was so low that there was not enough
material on hand to carry out the iron insertion to obtain the final target
chlorin-heme. Worse, the small amount of 36 appeared to be unstable in
solution to preclude a complete analysis of this compound.

In conclusion, this work demonstrated a regiospecific synthesis of the
dihydroxychlorin as well as the general validity of the protection-
deprotection strategy to incorporate the vinyl groups on the chlorin core

structure.

CHAPTER3
EXPERIMENTAL

GENERAL

1H Nuclear Magnetic Resonance spectra were obtained on a Varian
Gemini 300 or VRX 300 instrument in CDCl3 or solvents otherwise indicated.
UV-visible spectra were measured on a Schimadzu 160 spectrophometer. Mass
spectra were obtained on a VG trio-1 spectrometer. Melting points were
obtained on a capillary melting point apparatus Unimelt and are uncorrected.
Preparative TLC plates were purchased from analtech (silicagel GF, 1000 or

1500 mm).

3-Acgtamido-2-butangng 19

35.1 g (0.39 mol) of D,L-alanine, 159 ml (1.98 mol) of pyridine and 224 ml
(2.35 mol) of acetic anhydride were mixed together and heated over a steam
bath with stirring until a clear solution was obtained. This orange-brown
solution was then heated for six more hours. After this period of time, the
excess of solvents, pyridine, acetic anhydride and formed acetic acid were
removed under reduced pressure and low heating. The dark residue (about 50
ml) was distillled through a packed column with helices under vaccum to give
the crude product boiling at ll()-125°C/3mm Hg. Redistillation gave 41g of a

light yellow liquid; yield 88%.

27

28
lemmeniunL-Zchutanenublsm'dc 20

A mixture of 41g (0.31 mol) of 3-acetamido—2-butanone 19 and 410 ml of
a 35% solution of HCl was refluxed for 8 hours. After it was cooled down at 20°C,
it was filtered and the filtrate was evaporated under vaccum with low heating
until dryness. The brown yellow residue was filterd off and washed with
plenty of acetone to give 34.5g of a white powder; yield 88%; mass spectrum

m/e (rel. intens.) 123 (35%) (molecular ion), 97 (44%), 82 (59%), 44 (100%).

—Eh x arbon 1-4 5- im th 1 rrol -2- rbox 11 i 21

16g (0.13 mol) of the above 3- ammonium-Z-butanone chloride 20 were
dissolved in 22.4 ml of water and 1 1.2 ml of a 10 % solution of IICI was added to
it. This solution was slowly added (4 to 5 hours) to a stirred solution of 10g of
diethyl oxalacetate sodium salt in 9 ml of water and 15 ml of 10% aqueous
sodium hydroxide (this solution could be obtained only under heating on a
steam bath). During the addition, 3—ethoxycarbonyl-4,S-dimethylpyrrole—Z-
carboxylic acid precipitated out. The solution was further diluted with 50 ml of
water and acidified with a 10% 11C] solution until all the pyrrole precipitated
out. The white solid was collected by filtration, washed with plenty of water
and dried in air.; yield 37%; m.p. 203-205°C; NMR(in CDCL3), 8 1.42 (t, 3H,
CflfiCl-Izco-L 2.18 (s, 31-1, CH3), 2.26 (s, 31-1, CH3), 4.44 (q, 2H, CI-l3QH2CO—), 10.22 (s,
1H, N11); mass spectrum, m/e (rel. intcn.) 211 (35.5%) (molecular ion), 165

(100%), 147 (62%), 121 (58%)

29
.idEunnaflaulsnnd:4£&sunaahudnxnune 22

2g of 3-ethoxycarbonyl-4,5-dimethylpyrrole-Z-carboxylic acid 21 were
mixed with 5g of sodium acetate trihydrate and 5g of potassium acetate in a
round bottom flask. This powdery mixture is heated with an oil bath between
120 and 160°C until it was completed melted. It was cooled down, stirred with
water (20 ml) filtered and washed with plenty of water to give 1.46g of a pink-
brownish powder.; yield 93% ; m.p. 102-104°C; NMR (in CDCL3) 6 1.32 (t, 3H,
-OCH2§113), 2.17, 2.20 (s, 311, -CH3), 4.26 (q, 211, 09113013), 8.23 (s, 1H, a proton),
8.86 (s, 111, N11); mass spectrum, m/e (rel. inten.) 167(62%) (molecular ion),

138(67%), 122( 100%).

- -2- r - s- i l 13

4g of 3-ethoxycarbonyl-4,5-dimethylpyrrole was dissolved in 35 ml of
dimethylformamide 22. The solution was cooled in an ice bath while argon gas
was running through the solution. 3.5 ml of phosphoryl chloride were added
dropwise to the stirred cooled solution over a period of 15 minutes. The
internal temperature was kept below 10°C and then further stirred for two
hours at room temperature. 35 ml of benzene were added and stirred for 30
minutes and the mixture was left standing for a while. Addition of water and a
small amount of 10% sodium hydroxide made the product precipitate out. The
white precipitates were filtered, washed with water and dried in air; yield 56%,
m.p. 125-127°C; NMR (in CDC13), 6 1,38 (t, 3H, Q;I3Cl-12-), 2.22 (s, 3H, CH 3), 2.28 (s,
3H, CH3), 4.37 (q, 211, CIRCLE-L 10.04 (s, 1H, 4CHO), 10.46 (s, 1H, —NH); mass

spectrum, m/e (rel. inten.) l95(71%) (molecular ion), 148(81%), 121(IOO%).

3O
-- --x 23

To 500 ml of 2-butanone were added with stirring 150 g of 2-4-
pentadione (1.5mol), 184 g of ethyl chloroacetate (1.5 mol), 107 g of anhydrous
potassium carbonate (1.5 mol) and 45.15 g of potassium iodide (0.27 mol). The
reaction was brought to reflux. The reaction became violent because of its
exothermic character and the heating mantle had to be removed. When the
reaction slowed down, it was refluxed for several hours with a hot water bath
until a yellow liquid mixed with white solids were resulted. The reaction
mixture was diluted with acetone and the salts were filtered off and washed
further with acetone. The filtrates were concentrated in vacuo and the
residual oil was distilled under reduced pressure; yield 51%; b.p. 126°C (8-9 mm
Hg) to 139°C (13 mm Hg); NMR (in CDC13) 8 keto form 1.09 (t, 3H, -OCHzQH3), 2.11
(s, 6H, -CO§113), 2.72 (s, 2H, -COQL]2), 3.10 (s, 111, -CI~I), 3.96 (q, 211,-OQ;13CH3); mass
spectrum m/e (rel. intens.) 186 (10%) (molecular ion), 185 (100%), 141 (61%),
43 (97%).

Benzyl-4-ethoxycarbonylmethyl-3,,5-g1imethylpyrrole-Z-carboxylate 25

A stirred solution of 288 g of benzyl acetoacetate (1.5 mol) in 300 ml of
glacial acetic acid was cooled in an ice bath and a solution of 105 g of sodium
nitrite diluted in 150 ml of water was added dropwise over a period of 2 hours.
The orange solution was let stand overnight.

The resulting solution of benzyl a-oxoaminoacetate 24 was added dropwise,
along with small portions of zinc dust (301 g, 4.6 mol), to a stirred solution of
279 g of ethyI-3—acetyl—4—oxopentanoate 23 (1.5 mol) in 500 ml of acetic acid

over a period of about 45 minutes. The temperature was kept below 80°C. After

31

the completion of the addition the mixture was refluxed for an hour. It was
cooled to room temperature, poured into 2 liters of water and stood for a while
to grow crystals. The solid was then filtered off and washed with plenty of
water. The product was recrystallized in methanol/water; yield 45% ; m.p. 79-
80°C; NMR (in CDCI3) 5 1.23 (t, 31-], 00129113), 2.21 (s, 3H,-C113), 2.29 (s, 3H, CH3),
4.11 (q, 211,-0§1J'_)Cll3), 5.38 (s, 211, -0C1]3Ph), 7.39 (m, 511, phenyl protons), 8.68

(s, 1H, NH); mass spectrum m/e (rel. intens.) 315 (39%) (molecular ion), 242

(58%), 91 (100%).

 

63 g of benzyl-4-ethoxycarbonylmethyl-3,5-dimethy1pyrrole-2-
carboxylate 25 and 9.45 g of sodium borohydride were dissolved in 150 ml of
dry tetrahydrofuran under argon in a round bottom flask with stirring.
Trifluoride-ether complex (42.5 ml) was added dropwise to the solution over a
period of 25 minutes. The mixture reached reflux spontaneously and had to be
cooled externally with an ice bath. After further stirring for one hour 25 m1 of
glacial acetic acid were caustiously added dropwise to the mixture followed by
water until gas evolution ceased, The mixture was diluted with 200 ml of
dichlomethane and washed with 500 ml of water. The organic phase is filtered
off to removed the boric acid and evaporated in vacuo. Acetic acid still present
in the organic layer was removed by addition of toluene that was evaporated
and itself removed by methanol. The residue was recrystallized in 125 ml of
methanol and in 125 ml of water. The crystals were filtered off the next day;
yield 92%;. m.p. 115-116“’C :NMR (in coc13) s 2.21 (s, 3H, £113), 2.29 (s, 3H, -CH3),
2.65 (t, 211, {1120 12011), 3.66 (t, 21-l,-C1~12QJ20H), 5.29 (s, 2H,-0§;1;13Ph), 7.39 (m,

32
SH, phenyl protons), 8.8(-)(s, 111, N11); mass spectrum m/e (rel. intens.) 273

(80%) (molecular ion), 242 (100%), 134 (49%), 91 (100%).

mmmmemmsdmmummmmmv

45 g (0.16 mol) of benzyl-4-(2-hydroxyethyl)-3,5-dimethylpyrrole-2
carboxylate 26 were mixed with 90 g (0.65 mol) of anhydrous potassium
carbonate and dissolved in 900 ml of benzene and 28 ml (0.38 mol) of thionyl
chloride. This solution was refluxed for 3 hours under argon. It was cooled and
the solid was filtered off. The filtrate was evaporated to dryness to obtain a
brown crude product which was purified on silica gel column. The product was
eluted by dichloromethane. The pure pyrrole was the light yellow fraction
that was washed out after the dark yellow-orange impurities. The solvent was
removed and the product was recrystallized from a mixture of hexane and
(311202.; yield 86%; m.p. 119-120‘C; NMR (in CDC13) 8 2.21 (s, 3H, CH3), 2.30 (s, 3H,
CH3), 2.84 (t, 211, -Cfl3CHzCl), 3.51 (t, 2H, —CHZCL12C1), 5.29 (s, 2H, -0§113Ph), 7.39

(m, 5H, phenyl protons), 8.75 (s, 111, N11); mass spectrum m/e (rel. intens.) 291

(12%) (molecular ion), 242 (21%),91 (100%).

 

22 g (0,075%) of benzyl-4-(2-chloroethyl)-3,5-dimethylpyrrole-2
carboxylate 27 were dissolved in 250 ml of tetrahydrofuran in a l-L round
bottom flask of and stirred with a magnetic stirrer. To this solution 2.3 g of 10%
palladium on charcoal was added under argon as well as 10 drops of
triethylamine. The mixture was then stirred under hydrogen (1 atm., room

temperature) until the expected volume of hydrogen (1.66 liter) necessary to

33

the reduction was absorbed by the mixture. After complete hydrogenation,
argon was bubbled for half an hour and palladium charcoal was filtered off
and washed further with dichloromethane. The solvent was removed. The pink
product became hard and very dark with time and light. [Note: the reaction is
often unreproducible and can either failed to give back 100% of the starting
material or succeed to give a quantitative yield] m.p. 114-116°C; NMR (in CDC13)
8 1.13 (s, 1H, -COOH), 2.23 (s, 311,-C113), 2.29 (s, 3H, -CH3), 2.73 (t, 2H, -Q:12CHzC1),
3.51 (t, 2H, 01291130), 8.70 (s, 111, NH); mass spectrum m/e (rel. intens.) 201

(18%) (molecular ion), 152 (51%), 134 (100%).

' '-'Dn‘1 -2.» I. 0101 - '- o org - 500.1“ 'l'l.ll00l10‘ 15

6 g (29.8 mmol) of 4-chloroethyl-3,5-dimethylpyrrole carboxylic acid 14
and 5.81 g (29.8mmol) of 2-formyl-3-ethoxycarbonyl-4,5-dimethylpyrrole 13
were dissolved in 80 ml of methanol. 8 ml of 48% hydrobromic acid were added
dropwise to the stirred solution and the mixture was heated up for 10 minutes
on the steam bath. Some red crystals formed immediately. It was then left stand
for two hours at room temperature. The precipitate was filtered, washed with
methanol containing few drops of hydrobromic acid and rinsed with a little bit
of ether. 11.35g of nice red powder were obtained; yield 92%; m.p. 199-201°C;
NMR (in CDCL3) 8-1.50 (s, 111, -N11), -1,32 (s, 1H, -NH), 1.44 (t, 3H, -CH2Q:13), 2.27
(s, 3H, ~Cll3), 2.37 (s, 311, -Clrl3), 2.70 (s, 311, £113). 2.79 (s, 3H, -CH3), 2.94 (t, 2H,
-CB2CHzCl), 3.60 (t,2H, ~C112C113Cl), 4.41 (q, 211, -CH3CH3), 8.44 (s, 1H, bridge
proton); mass spectrum, m/e (rel. inten.) 80 (100%), 198 (61.72%), 261 (72.66%),

334 (24%) (molecular ion -IlBr).

34
4 O- o -3 3'- cm -7 2.- o l 15 b

30 g of 5,5'-bis(ethoxycarbonyl)-4,4'-diethyl-3,3'-dimethyl-2,2'-
dipyrrylmethane 16 were dissolved in 100 ml of hot ethanol and brought to a
gently reflux. A solution of 15 g of potassium hydroxide dissolved in 25 m1 of
water was added through the condenser and refluxed for two hours. After this
period of time the condenser was removed and the solution was evaporated to
one third. 38 ml of water were added with few drops of hydrazine. The mixture
was refluxed for twelve hours without interruption. At the end of this period, a
layer of brown oil was separated. The mixture was allowed to cool at room
temperature and the solidified material was filtered off and washed with water.
The fresh product had a dark brown shiny color and can be storred
indefinitely in a refrigerator; quantitative yield; m.p. 49-50°C; NMR (in CDCI3)
8 l.12(t, 6H, —Cl-12C|l3). 2.03 (s, 611, C113), 2.47 (q, 411,-CH2CH3), 3.79 (s, 2H,-CH 2),

6.35 (m, 211, 5-11), 7.26 (s, 211, N11); mass spectrum m/e (rel. intens.) 230 (68%)

(molecular ion), 201 (21%), 121 (100%).

 

9.45 g of bromine were added to 60 ml of a stirred solution of formic acid. To
this solution under stirring 4,4'-diethyl-3,3'-dimethyl-2,2'-dipyrrylmethane
15 b was added in small portions. The mixture was stirred for an additional 20
minutes during which precipitation occured. The mixture was then evaporated
in vacuo and the residue was triturated in 10 ml of ether/methanol. The
product was collected by filtration and dried in air; mass spectrum m/e (rel.

intens.) 386 (23%) (molecular ion —lllir), 276 (100%), 198 (81%), 80 (68%)

- to. ._ not -- 1M; 1- 3 -o"1l- ._ °-‘t-1‘lnn "1 ll

11 g (0.26 mmol) of 4,3',5,5'-dimethyl-3-ethoxycarbonyl-4'-chloroethy1-
2,2'-dipyrrylmetheniumbromide 15 and 12.38 g (0.26 mmol) of 5,5'-dibromo-
3,3'-diethyl-4,4'-dimethyl-2,2'-dipyrrylmetheniumbromide 16 were dissolved
in 125 m1 of formic acid and treated with 1.5 ml (0.26 mmol) of bromine. The
mixture was refluxed in an oil bath for two and half hours . The solvent was
then allowed to boil off over a period of an hour (until dryness). The residue
was redissolved in dichloromethane and chromatographed on a silica gel
column. The first purple band was the expected porphyrin mixed with a little
bit of bromoethylporphyrin. It was then recrystallized from dichloromethane
and methanol to give 950 mg of porphyrin.; yield 6.5%; m.p. 245-247°C; NMR
(in CDCL3) 8 -3.7() (s, 211, -Nll), 1.92 (t, 911, {1121113 and -0Cl-12§H3), 3.52, 3.64,
3.66, 3.94 (4s, 1211, -Cll3), 3.98, 4.08 (2q, 411, -QI;13C113), 4.28, 4.50 (2t, 411,
MCI), 4.90 (q, 211, 41912013), 11.10, 10.15, 9.95 and 9.85 (s, 4H, meso
protons); UV-vis 1(0) 630 (5700), 573 (11300), 545 (17000), 506 (15000), 405.5
(182200); mass spectrum, m/e (rel.inten.) 600 (10.9%) (bromoporphyrin), 556

(19.4%) (molecular ion), 520 (40.5%), 108 (100%).

- 10 .uo -- hlou-r h 1-131 -i h 1-2 121—Hrm h H l-iih . . . -
chlorin 11

To a solution of 60 mg (0.107 mmol) of 3-ethoxycarbonyl-8-chloroethyl-
13,17-diethyl-2,7,12,18-tetramethyl-porphyrin 9 in 15ml of dichloromethane
and one drop of pyridine, 25 mg (0.0963 mmol, 0.9 eq.) of osmium tetraoxide
were added. The stirred solution was left under argon at room temperature in

the dark and monitored by thin layer chromatography. After 30 hours the

36

reaction was diluted with 2ml of methanol and quenched by 1125 for twenty
minutes. Osmium was removed by filtration and the solvent was evaporated
under reduced pressure. The mixture of starting material and the product were
chromatographed on t.l.c. plates with C112Cl2/3%Me011. The porphyrin was
eluted first while the major chlorin product of purple color was the second
band.; yield 60%; m.p.>260°C; NMR(in CDCL3) 8 -2.26 (s, 211, -Nll), 1.70 (t, 6H,
Olmmd 0012013), 2.24 (s 311, -Cll3), 2.44 (q, 2H, (112013), 3.20 (s, 3H, CH3),

3.40 (s, 311, -C1-13), 3.51 (s, 311, -C113), 3.80-4.16 (m, 611, 0120-120, CH 2012C] and
(112013), 4.68 (q, 211, -0 (112013), 8.78, 8.98, 9.57 and 10.38 (s, 411, meso protons);
UV-vis 1(e) 640 (18700), 587 (6200), 545 (8100), 510 (9300), 413 (92000); mass
spectrum, m/e (rel. intens.) 634 (11%) (molecular ion, bromoethylchlorin),

616 (63%), 590 (13%) (molecular ion), 572 (87%), 536 (92%), 450, (100).

-1ox -1101 -I3 -o"h 1-2 71-1“ ‘ --vin - 3-'1 0.10 - 1011

18

To a stirred solution of 100 mg of 3-ethoxycarbonyl-8-chloroethyl-13,17-
diethyl-2,7,12,18-tetramethyl-12,l3-dihydroxychlorin 11 dissolved in 20 ml of
dimethyl formamide, 0.2 ml of 0811 was added. (DBU was opened under argon to
avoid moisture but the reaction does not require either argon or nitrogen).
This solution was heated on the steam bath at 80°C for two hours. After cooling
to room temperature, 25 ml of water were added and the mixture was stirred for
a further hour. It was then filtered on celite, rinsed with water and few drops
of methanol. The product was extracted from celite with dichloromethane.
Solvent was removed; yield 90%; m.p. 210-212°C; NMR (in CDCI3) 8 -2.70 (-NH),
1.58, 1.72 (t, 611, ~C112C113,-0C112C1|3). 2.22 (s, 311,-C113), 3.08 (s, 6H, -CH3), 3.28 (s,
3H,-C113), 3.70-4.10 (m. 611, -C112Cl 12C], -Cl‘1 2013), 4.57 (q, 211, -0CH2C113), 6.15 (dd,

37
2H,-CI'I=C112), 7.90 (dd, lll, ~Cll=C112). 8.96, 9.15, 9.65 (s, 411, meso protons); UV-

vis 1(e) 641 (19800), 587 (6500), 545 (8700), 510 (9800), 413 (95500); mass

spectrum, m/e (rel. intens.) 554 (85%) (molecular ion), 536 (98%), 44 (100%)

 

9 g of 2-formyl-3-ethoxycarbonyl-4,5-dimethylpyrrole 13 were
dissolved in 180 ml of a solution of 15% sodium hydroxide and 90 ml of ethanol
heated on a steam bath. It was heated for two and half hours and then cooled in
a ice bath. The solution was acidified by addition of a solution of 15%
hydrochlorhydric acid until pink fine crystals precipitated out. They were
filtered, washed with a lot of water and dried in air.; yield 93%; m.p. >260°C;
NMR (in C505N) 8 2.22 (s, 311, -C113), 2.45 (s, 311, -CH3), 10.80 (s, 111, ~C110), 11.15
(s, 111, -N11) ; mass spectrum m/e (rel. intens.) 167 (96.5%) (molecular ion), 149

(93%), 138 (87%), 121 (100%).

' '-D11‘ 1- '- tloror 5’ _oo'-i n 11‘ “11.110 0110'-3- ._ m . '— '° 30

6 g (0.036 mol) of 2-formyl—4,5-dimethyl-3-carboxylic acid pyrrole 29
and 7.22 g (0.036 mol) of 4-chloroethyl-3,5-dimethylpyrrole carboxylic acid 14
were dissolved in 200 ml of acetic acid by heating on the steam bath. 20 m1 of
48 % hydrobromic acid to this warm solution and heating on the steam bath
was continued for 10 more minutes. Dark red crystals formed during this time.
The mixture was allowed to stand for an hour and the precipitates were filtered
off and washed with a little bit of ether.; yield 90%; m.p.208-210°C; NMR (in
CD03) 8 2.31 (s, 311,-C113), 2.36 (s, 311, £18). 2.68 (s, 311, -C113), 2.77 (s, 3H, -C113),
2.92 (t, 211, -C112C112C1), 3.60 (t, 211, —CII2C112C1), 8.40 (s, 111, bridge proton); mass

38
spectrum m/e (rel. intens.) 306 (44%) (molecular ion -llBr), 261 (100%), 198

(82%).

-' one - -1 02‘1 - 3 -0‘. l - _I-" 1211‘ OOIOh .131

10 g of 2,3',5,5'-dimethyl-2'-chloroethyl-2,2'-dipyrrylmetheniumbromi
de-3-carboxylic acid 30 and 12 g of 5,5'-dibromo-3,3'-diethyl-4,4'-dimethyl-
2,2'-dipyrrylmetheniumbromide 15 were dissolved in 120 ml of formic acid and
treated with 1.5 ml of bromine. The mixture was refluxed in an oil bath for two
and half hours . The solvent was then allowed to boil off over a period of an
hour (drynees) during which the porphyrin was formed. The residue was
redissolved in dichloromethane and was chromatographed on a silica gel
column. The first purple band was the expected porphyrin mixed with a little
bit of the bromoethylporphyrin. It was then recrystallized from
dichloromethane and methanol to give 850 mg of porphyrin.; yield 6%; NMR
(in CDCL3) 8 -4.05 (s, 211, -N11), 1.85 (t, 911, -C112QLI3 and -0C112Cl;13), 3.52 to 3.64
(43, 1211, ~C113), 4.02, 4.12 (2q, 411, £12013), 4.26, 4.50 (2t, 411, MCI), 9.88,
9.96, 10.03 and 10.15 (s, 411, meso protons); lIV-vis l(e) 620 (5300), 566 (8000), 535
(10692), 500 (12150), 400 (109000); mass spectrum, m/e (rel.inten.) 608 (35%)

(bromoethylporphyrin), 562 (78%) (molecular ion), 484 (100%).

8-1 Z-Chlgrggthyl )- 1 3. ,1 Z-diethyl-2,Z,12, l fl-tetrnmgmyl-pgrphyrig 32

600 mg of 3-bromo-8-(2-chlorocthyl)-l3,17-diethyl-2,7,12,18-
tetramethylporphyrin 31 were dissolved in tetrahydrofuran and 100 mg of
10% palladium on charcoal were added to the solution under argon with few

drops of triethylamine. The mixture was stirred under hydrogen (1 atm, room

39

temperature) for 4 hours. After this time argon was bubbled into the solution
for half an hour and the catalist was filtered off. Some of the porphyrin was
over hydrogenated to become porphyrinogen (monitored by t.l.c.). Therefore
DDQ was added to the solution until most of the porphyrinogen was reoxidized.
The reaction was not totally complete and the product had to be
chromatographed on silica gel column. Bromoporphyrin and hydroporphyrin
could be separated with a 40/60 mixture of hexane/dichloromethane; yield
49%; NMR (in CDCI3) 8—3.80 (s, 211, N11), 1.82 (t, 611, -CI12C113), 3.59 (s, 9H, 3 CH 3),
3.72 (s, 3H, C113), 4.06 (q, 411, -C112C113), 4.29 (t, 211, -CH2CH2C1), 4.48 (t, 2H,
-CH2012C1), 9.06 (s, 111, 3-1-1), 9.95, 10.01, 10.07, 10,10 (43, 411, meso protons); UV-
vis l(e) 618 (6400), 566 (8300), 530 (10500), 497 (14550), 397 (140000) ; mass

spectrum m/e (rel. intens.) 528 (38%) (bromoethylporphyrin), 484 (84%)

(molecular ion), 448 (100%), 108 (95%).

 

Iggn insggign

A solution of 200 mg of 8-(2-chloroethyl)-13,17-diethyl—2,7,12,18-
tetramethylporphyrin 32 in 60 ml of benzene, 100 ml of tetrahydrofuran and
20 drops of collidine was aerated by bubbling argon in it for 20 minutes. Then
ferric bromide (400 mg) was quickly added and argon was bubbled for 10 more
minutes. The mixture was kept under of argon and brougth to a gentle reflux
until the fluorescence disappeared totally (about 15 minutes). The solution was
evaporated and washed with water and 10% 110. The metallated porphyrin was
collected after evaporation of the solvent.; quantitative yield; UV-vis. A 637,
503, 381; mass spectrum, m/e (rel. intens.) 538 (52%) (molecular ion), 504

(100%), 490 (65%).

40

Ammmn

250 mg of the iron porphyrin were dissolved in 25 ml of acetic
anhydride and cooled down in an ice bath. To this solution 2 ml of stannic
chloride was added under stirring and left for 5 minutes in an ice bath. Then
the ice bath was removed and the solution was stirred for an additional ten
minutes. The recation was quenched with ice-water added dropwise at first.
The mixture was filtered and the solid was redissolved in dichloromethane,
dried with magnesium sulfate and evaporated. The presence of iron acetyl
porphyrin was verified by t.l.c. and mass spectrum; mass spectrum m/e (rel.

intens.) 580 (67%) (molecular 1011), 504 (47%), 207 (100%).

Dcmclallatjsm

The iron porphyrin was dissolved in 70 ml of acetic acid and was aerated
by bubbling argon for 20 minutes at room temperature. 10 ml of 11C] and 100
mg of ferric sulfate was added and the resulting mixture was left under argon
with stirring for 30 minutes. The solution was then diluted with 100 ml of
dichloromethane and washed twice with water and once with a solution of 30%
of sodium acetate. The solvent was evaporated and the products were
chromatographed on a silica gel column with dichloromethane as eluent; yield
51%; m.p. >260’C; lIV-vis l(e) 634 (5000), 575 ( 11000), 547 (15300), 509 (13694),
407.5 (165000); NMR (in (2003) 8 ~3.65 (s, 211, N11), 1.92 (2t, 6H, £12013), 3.28
(s,3H, {0013), 3.52 (s, 311,-C113), 3.62 (s, 31], -C113), 3.66 (s, 311,-C113), 3.84 (s, 3H,
-CH3), 3.98 (t, 211, £12013), 4.09 (t,21l, -CI2CI-l3), 4.27 (t, 211, -Q2C12Cl), 4.50
(t,2H, £12020), 9.83, 9.97, 10.08, 10.78 (48, 411, meso protons), mass spectrum
m/e (rel. intens.) 570 (24%) (bromoethyl porphyrin), 526 (100%) (molecular

ion), 490 (42%), 44 (81%).

-. ' --7-1o 01‘1 - 3 -c"h -7 t‘ 111' - 3-°1o o 1011

12

To a solution of 60 mg (0.11 mmol) of 3-acetyl-8-(2-chloroethy1)-13,17-
diethyl-2,7,12,l8-tetramethylporphyrin 10 in 15ml of dichloromethane, 45 mg
(0.17 mmol, 1.5 eq.) of osmium tetraoxide were added. The stirred solution was
left under argon at room temperature in the dark and monitored by thin layer
chromatography. After 30 hours the reaction was diluted with 2ml of methanol
and quenched by 1128 for twenty minutes. Osmium sulfide was removed by
filtration and the solvent was evaporated under reduced pressure. The mixture
of starting material and the product was chromatographed on t.l.c. plates with
01202/3% MeOll. The porphyrin was eluted first while the major chlorin
product of purple color was the second band.; yield 55%; NMR(in CDCL3) 82.21
(s, 211,-N11), 0.78 (t, 311,-012013), 1.75 (t, 611, £11202), 2.24 (s 311, -Cll3), 2.43 (q,
2H, £12013), 3.14 (s, 311, -COC11 3), 3.39 (s, 311, C113), 3.48 (s, 311, -CI-l3), 3.61 (s, 311,
-CH3), 3.82 (q, 211, £12013), 3.99 (t, 211,-§;12C112C1), 4.16 (t, 211, C11 2020), 8.88,
9.11, 9.76 and 10.38 (s, 411, meso protons); UV-vis 1(8) 641 (16700), 587 (5900), 546
(7900), 511 (8900), 413 (89000); mass spectrum, m/e (rel. intens.) 586 (19%)
(bromoethyl chlorin, loss of water), 560 (15%) (molecular ion), 542 (95%), 108

(100%).
- ‘ - 3 -0‘.1l- 71- ' r'n ' l--vin 1-1213-° o. o 10 '135

To a stirred solution of 30 mg of 3-acetyl-8-(2-chloroethyl)-13,17-
diethyl-2,7,12,18-tetramethyl-12,13-dihydroxyt‘hlorin 12 dissolved in 7ml of
dimethyl formamide,0.7ml of DBU was added. This solution was heated on the

steam bath at 80°C for two hours. After it was cooled to room temperature, 9 m1

42

of water were added and the mixture was stirred for another hour. It was then
filtered on celite, rinsed with water and few drops of methanol. The product
was extracted from celite with dichloromethane. Solvent was then removed.;
yield 90%; UV-vis l(e) 641 (19000), 587 (6500), 545 (8900), 510 (9500), 413
(99500); mass spectrum, m/e (rel. intens.) 524 (48%) (molecular ion), 506

(100%), 490 (40%), 44 (100%).

”1'0.‘ -3 -0‘1i-7 7 --°rt'1.—1- 01.0.1011

35b

30 mg of 3-acetyl-l3,17-diethyl-2,7,l2,18-tetramethyl-8-vinyl-12,13-
dihydroxychlorin 35 were dissolved in a mixture of
methanol/tetrahydrofuran. At ()"C this solution was treated with 150 mg of
sodium borohydride dissolved in cold methanol. It was stirred for 10 minutes in
an ice bath before 0.3 ml of acetic acid was carefully added to quench the
excess of borohydride. The acidic solution was then diluted with 30 ml of
dichloromethane and washed 3 times with water and once with an ammonium

hydroxide solution. Organic layer was dried and the solvent was removed;

yield; mass spectrum m/e (rel. intens.) 508 (10%) (molecular ion), 490 (80%),

44 (100%)

 

3-(1-hydroxyethyl)-l3,17-diethy1-2,7,12,18-tetramethyl-8—vinyl-12,13—
dihydroxychlorin 35 b was dissolved in dimethylformamide and was brought
to 110°C in an oil bath under argon. It was left for 5 minutes then cooled to

room temperature and quenched with water. The product was extracted with

43

dichloromethane and the solvent was evaporated. The major green product was
purified by chromatography on plate; mass spectrum m/e (rel. intens.) 490

(82%) (molcular ion), 461 (45%), 44 (100%)

APPENDIX

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REFERENCES

10.
11.

13.

REFERENCES

An exhaustive compilation on the chemistry, physics, and biology of
porphyrins appeared as the multivolume set entitled "The
Porphyrins",DoIphin, D., ed., Academic Press, New York, 1978.

Shceer, H. In " The Porphyrins", Vol. II, Part B, Dolphin, D, ed., Academic
Press, New York, 1978, chapter 1, 2.

(a) Katz, ,1. 1., In "Inorganic Biochemistry", vol.ll, Eichhorn, G. L, Ed.,
Elserier Publishing Co., Amsterdam, 1973, chapter 29.

(b) Clayton, R. K.; Sistrom, W.R.. "T he Photosynthetic Bacteria", Plenum
Press, New York, 1978.

(a) Imfield, I\I.; Arigoni, D; Deeg, R.; Muller, G. In "vitamin B12"; Zagalac,
B. 1.; Friedrich, W. Eds; de Gruyter, Berlin, 1979; pp.315.

(b) Turner, S. P. D.; Block, M. II.; Sheng, Z-C. ; Zimmerman, S. C. ;
Battersby, A. R. 1. Chem. Soc, Chem. Commun. 1985, 583.

(a) Timkovitch, R.; Cork, M. 8.; Taylor, P. V. .1. Biol. Chem. 1984, 259, 1577.
(b) Chang, C. K. 1. Biol. Chem. 1985, 260, 9520.

(c) Chang, C. K.; Wu, W. 1. Biol. Chem. 1986, 261, 8593.

(d) Chang, C. K.; Timkovitch, R.; Wu, W. Biochemistry 1986, 25, 8447.
(e) Wu, W.; Chang, C. K. J. Am. Chem. Soc. 1987, 109, 3149.

(a) Payne, W. J. "Denitrification";Wiley: New York, 1981.

(b) Delwiche, C. C. "Denitrification, nitrification, and Atmospheic
Nitrous Oxide"; Wiley: New York, 1981.

Timkovith, R.; Cork, M. 8.; Gennis, R. B.; Johnson, P.Y. J. Am. Chem. Soc.
1985, 107, 6069.

Sotiriou, (3.; Chang, C. K. ,1. am. Chem. Soc. 1988, I 10, 2264.

A review of the history of cytochromes d with references to the original
literature may be found in the following: Lemberg, R.; Barrett, J.
"Cytochromes"; Academic Press: New York, 1973; pp 8-14 and 233-240.
Keilin, D. Nature 1933, 132, 783.

Kita, K.; Konishi, K.; Anraku, Y. J. Biol. Chem. 1984, 259, 3375.

Miller, M. ,l.; Gennis, R. B. 1. Biol. Chem. 1983, 258, 9159.

Haddock, B. A.; Jones, C. W. Bacterial. Rev. 1977, 41, 47.

6O

14.
15.

16.

17.

61

Barrett, J. Biochem. J. 1956, (.4, 626-639.

Varra, M. R.; Timkovitch, R.; Yap, F.; Gennis, R. B. Arch. Biochem.
Biophys. 1986, 250, 461.

Andersson, L. A.; Sotiriou, C; Chang, C. K.; Loehr, T. M. J. Am. Chem. Soc.
1987, 109. 258

Pandey, K. D.; Shiau, F-Y.; Isaac, M.; Ramaprasad, 8.; Dougherthy,
T.J.;Smith, K. M. Tetrahedron Letters 1992, 33, 7815

Paine, J. B. III; Chang, C. K.; Dolphin, D. IIeterocycles 1977, 7, 813
Wiley, R. Il.; Borum, O. H. Org. Synthesis Vol. IV pp 5

Carr, R. R; Jackson, Q. 1].; Kenner, G. W.; Jackson, G. S. J. Chem. Soc. (C)
1971, 487

Games, D. 15.; Jackson, A.Il.; ()‘llanlon, P.J. J. Chem. Soc. Perkin l 1976,
2501

Chang, C. K.; Sotiriou, C. J. Org. Chem. 1985, 50, 4989
Chang, C. K.; Sotiriou, C. J. lleterocyclic Chem. 1985, 22, 1739

Schlabach, M.; Ijmbach, ll-M.; Bunnemberg, 13.; Shu A. Y. L; ToIf B. R.;
Djerassi, C. J. Am. Chem. Soc. 1993, 1 15, 4554

Fisher, ll.; Beller, l~l. Angewandte Chemie, 1925, 444, 244
Chang, C. K.; Young, R. J. Am. Chem. Soc. 1985, 107, 898

Barrett, J. Nature 1959, 183, 1185

   

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