Synthesis of Porphyrins from Oxobilane Intermediates

6 Synthesis of Porphyrins from Oxobilane Intermediates P. S. CLEZY and A. H. JACKSON

I. II. III. IV. V. VI. VII.

Introduction Dipyrroketones . . . . . Porphyrins from û-Oxobilanes . Porphyrins from ^-Oxobilanes . Differential Protection of Pyrrole Rings Synthesis of Porphyrins Bearing Labile Groups Conclusions References . . . . . .

265 266 269 274 279 280 284 286

I. INTRODUCTION For a porphyrin synthesis to be of general application and free of all symmetry restraints, it is necessary first to construct a linear tetrapyrrole in a logical fashion from separate pyrrolic units. Only by proceeding through such a well-defined intermediate, which is then cyclized to the porphyrin macrocycle, can many naturally occurring members of this class be prepared. Moreover, such a plan may allow the labeling of specific atoms, and this has considerable potential for studying problems of porphyrin biosynthesis. In view of the methods followed by nature in porphyrin biosynthesis, it is clear that consideration should be given to a linear tetrapyrrole of the bilane type (1) as an intermediate of porphyrin synthesis in the laboratory. A number of procedures1-6 have been devised to link, through a méthylène group, two diversely substituted pyrrolic rings (dipyrromethane). It appeared from early studies that these methods could not be extended beyond the dipyrromethane stage, except in a limited number of cases where the substitution pattern was favorable.4·5 Although more recent work has indicated that this restriction can be overcome by using milder condensing procedures,6 the stability of the 265

266

P. S. CLEZY AND A. H. JACKSON

H

H

a

A O

H

b

t

Bilane 1

H c

H

H

H

H

tf-Oxobilane 2

H

H

^

H

H

6-Oxobilane 3

bilanes was to prove an obstacle to this type of approach. It became clear from work at Liverpool 7 and from other groups 8 that systems like the bilanes, when investigated outside the cellular environment, were too sensitive to oxidation and to attack by electrophilic species (e.g., acids) to be suitable as intermediates in porphyrin synthesis. In particular, they underwent cleavage and redistribution reactions which led to mixtures of porphyrins. Negative groups, as expected, retard electrophilic attack on pyrrolic systems, and, hence, at Liverpool, attention was directed to the chemistry of the oxobilanes which are stabilized by the presence of a carbonyl linkage. Two series of oxobilanes are possible: the a-oxobilanes (2) and the b-oxobilanes (3). Both systems have been utilized as intermediates in porphyrin synthesis. II. DIPYRROKETONES The dipyrroketones (2,2'-dipyrrolyl ketones) were required as intermediates for the formation of certain oxobilanes. In addition, since they resemble the oxobilanes structurally, an understanding of the reactions of these simple ketones is a useful preliminary to following the chemistry of the oxobilanes. Thus, the preparation and properties of the dipyrrolic ketones will be reviewed at this stage. Earlier methods of synthesis 9-11 of the dipyrroketones were not entirely satisfactory for the preparation of diversely substituted members of this class,

267

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

but an adaption of the Vilsmeier-Haack procedure provided the type of dipyrroketone required. 12_12b In this process, a pyrrolic amide (4) activated as its phosphoryl chloride complex was condensed with an α-free pyrrole (5); uv spectroscopy proved to be an ideal method of monitoring the progress of the reaction, and complex formation was recognized by the loss of absorption near 280 nm and the appearance of a new maximum at 380 nm. Reaction of the complex with an α-free pyrrole produced an imine salt (6), characterized by an absorption above 400 nm, and hydrolysis under basic conditions then furnished the dipyrroketone (7). R3

R^

Ä

R1[^CONMe2

H

4

R3

Me

R'[^c/ H

Amftx 280-290 ΠΠ1

R^

R3

R^

R4

H

H H ° Amax 345-370 nm 7

R4

Me

N M e

+

>

I OPOCla "max 355-380 nm

R3

R2

C^Me H 5

R4

Me

H

| H NMe 2 Amax 400-410 nm 6

The dipyrroketones show a high degree of dipolar character which presumably arises from mesomeric contributions of the type 8a <-> 8b.

H

H

o

8a

H

H

I

o-

H

8b

That contributions from structures of type (8b) are important can be clearly seen from both the basicity and the spectroscopic properties of the system. Thus, the ir carbonyl frequency is found below 1600 c m - 1 , n ~ 1 4 and the principal electronic absorption occurs near 350 n m 1 2 ~ 1 2 b * 1 4 (cf. 300 nm for simple pyrrole ketones 1 5 ' 1 6 ). In acidic solutions, in which a meso-hydroxydipyrromethene salt (9) is formed, the absorption maximum is well above 400

268

P. S. CLEZY AND A. H. JACKSON

H

I OH 9

H

i2-i2b xhese basic carbonyl compounds have been regarded as vinylogues of urea, and, as in tropone, the highly polarized carbonyl group allows conjugation of the τΓ-electron system. 12-12b Reduction of the carbonyl group of dipyrroketones is an important reaction from the standpoint of porphyrin synthesis, as at some stage this functional group (introduced to stabilize an intermediate linear tetrapyrrole), has to be reduced out of the system. It is sometimes possible to reduce dipyrroketones to the corresponding dipyrromethane by borohydride ions 12-12b · 17 under vigorous conditions, but in general they are inert to this type of reducing agent, as was predicted from the polar character of the C-O bond. On the other hand, diborane reduces polarized carbonyl groups very efficiently, as it coordinates initially with the oxygen atom, 18 and this reagent has proved most valuable in the reduction of dipyrroketones directly to dipyrromethanes. Dipyrroketones are virtually unaffected by catalytic hydrogénation, so that benzyl esters in this type of environment may still be conveniently converted into the free acids by hydrogénation. 12 Diborane reduction of a dipyrroketone does not stop at the carbinol stage because elimination of the hydroxyl group occurs with formation of the dipyrromethene, which is then immediately reduced by more diborane to the dipyrromethane. The dipyrromethane can be detected spectroscopically in the course of the reaction, and this type of reduction is characteristic not only of dipyrroketones but also of other ketones attached to strongly electron-releasing aromatic nuclei.12a Another aspect of dipyrroketone chemistry important to porphyrin synthesis is the functionalization of the a-positions to allow the chain length of the ketones to be increased. Osgerby and MacDonald 11 have reported that dipyrroketones failed to undergo many of the usual electrophilic substitution reactions characteristic of pyrrolic compounds. This is almost certainly due to the acidic catalysts or reagents employed in this type of reaction. The dipolar character of these ketones favors the formation of oxonium salts (cf. 9) and deactivation results. However, under alkaline conditions, dipyrroketone acids (e.g., 10) can be iodinated with decarboxylation, 12-12b · 19 and Ballantine et al12 have reported that the lithium salt of 10 will condense with pyridinium salts (e.g., 11) to give tripyrrolic derivatives (in this case, 12). Because of these difficulties with electrophilic substitution, the direct formylation of dipyrroketones has not been achieved,11 and an alternative route to this, or another suitable functional group for the a-position, had to be found. nm

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES Me

Et

Me

Et

Me

269

Et

o 10

11 Me

PhCH202C

Et

Me

H

Et H

Me H

o

12

Et H

The functionalization of an α-methyl group is an important reaction in pyrrole chemistry, and in this connection extensive use has been made of lead tetraacetate, 20 bromine, 21 sulfuryl chloride, 21a or sometimes a combination of these reagents.22 The resulting acetoxymethyl- or halomethylpyrroles have been widely employed in the preparation of dipyrromethanes. Sulfuryl chloride has been used to convert α-methylpyrroles into di- or trichloromethyl derivatives from which formyl 22a or carboxylic acid pyrroles 22b can be derived. Although these reagents have occasionally been employed 9,19 ' 23 to oxidize α-methyldipyrroketones, their application has not been as uniformly successful as with simple pyrroles, possibly because of the cleavage of the dipyrroketone system and its conversion into dipyrromethenes. 23 ' 23a A search for a milder procedure to functionalize the α-methyl group of dipyrroketones revealed that tert-buty\ hypochlorite, when used under carefully controlled conditions, was a useful reagent.12_12b*24 For example, the ketone (13) gave the monochloro- (14) or the dichloromethyl derivative (15) under appropriate conditions. By treatment of the latter species with dimethylamine and subsequent hydrolysis, the formyldipyrroketone (16) was obtained.12-1215 Me

Et

Me

Et

PhCH2o2ctNJLANJR o 13 R = Me 14 R = CH2C1

15 R = CHC12 16 R = CHO

III. PORPHYRINS FROM a-OXOBILANES7 The first porphyrin of real significance prepared by the a-oxobilane procedure was mesoporphyrin dimethyl ester.7 The synthesis of this compound,

270

P. S. CLEZY AND A. H. JACKSON

which is not symmetrical, offered sufficient challenge to test the application of the procedure without requiring the handling of difficult substituent groups. In addition, the product is a well-characterized porphyrin, so that the synthetic material could be readily compared with authentic mesoporphyrin. Initially, an attempt was made to construct an ö-oxobilane suitable for cyclization to mesoporphyrin by the acid-catalyzed condensation of the dipyrroketone aldehyde (16) with an a-free dipyrromethane (17). Although there was spectroscopic evidence to suggest that the reaction had been successful, a crystalline product could not be obtained either directly, or after borohydride reduction. Characterization of the intermediate linear tetrapyrrole was essential if the logical progress of the synthesis was to be established. Hence, another route to the a-oxobilane was sought. ΡΜβ

Me

ΡΜβ

Me

OLX1

Me0 2 C I

H

PMe

17 = CH2CH2C02Me

H

The chloromethyldipyrroketone (14) was converted into the pyridinium salt (18) which, when condensed with the lithium salt (19), furnished the crystalline û-oxobilane (21) in good yield. Its structure was confirmed by combustion data and spectroscopic studies. Further, the tetrapyrrolic aldehyde (22) was prepared in a similar manner from 18 and 20. Me

Et

Me

Et

PhCH2o2ctN J L 1 N J C H 2 P V H

Me

T O 18

+

H

PMe

Me

UÖ,CILNA^NJR H

H

19 R = C02CH2Ph 20 R = CHO

Et

H

PMe

Me

Et

Me

II

O

21 R1 22 R1 23 R1 24 R1

Me

H = = = =

R2 = COaCH2Ph C02CH2Ph, R2 = CHO C0 2 H, R2 = CHO R2 = C0 2 H

Me H

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

271

Both linear tetrapyrroles (21 and 22) contained the substituents of mesoporphyrin arranged in the correct order. What remained was the cyclization step. Attempts to cyclize the aldehydo acid (23), derived by hydrogenolysis of (22), failed, as did efforts to complete the macrocycle by use of one carbon units (e.g., formaldehyde, formic acid, methyl orthoformate) with the diacid (24). Such results were not altogether unexpected, owing to the inertness of dipyrroketones towards electrophilic attack. As discussed above, this is due to the formation of the conjugate acid of the dipyrroketone system under acidic conditions. Indeed, during attempts to cyclize the oxobilanes (23 and 24), the formation of the conjugate acids, which have dipyrromethene-like chromophores, was recognized spectroscopically (Amax 428 nm). Clearly then, it was necessary to reduce the carbonyl group of the Û-OXObilane before cyclization could be effected. As has been already pointed out, diborane was the reagent of choice for dipyrroketone reduction, and it seemed likely that the carbonyl group in an oxobilane would be similarly reduced. However, the problem of how the other carbonyl groups in these molecules would survive treatment with diborane remained to be answered. In the event, nuclear esters proved to be largely inert to this reagent, while side chain esters were slowly reduced.7 Fortunately, the latter reduction was minimized by the addition of ethyl acetate to the main solvent, tetrahydrofuran. The stability of nuclear esters toward diborane was surprising, since other carbonyl groups attached to pyrrolic systems are readily attacked. For example, formylpyrroles are reduced by diborane almost as readily as the dipyrroketone system,12-1213 which, of course, meant that intermediates such as 22 were of little use for porphyrin synthesis. (Possible reasons for the stability of esters attached to aromatic nuclei toward diborane have been discussed by Biswas and Jackson. 12b ) Attention was, therefore, directed to the dibenzyl ester (21), but as diborane reduces carboxylic acids,25 it was essential that reduction of the oxobilane system preceded hydrogenolysis of the benzyl esters; the hydrogenolysis product was presumably the bilane (25). However, attempts to cyclize this intermediate with trimethyl orthoformate in the presence of trichloroacetic acid resulted in the formation of a mixture of porphyrins in only low yield. Such a result was not altogether surprising owing to the instability of bilanes to electrophilic reagents (which has been mentioned above). Therefore, the saturated intermediate was oxidized to the bilene-Z? salt (26) prior to subjecting the system to the acidic conditions required for cyclization. It seemed reasonable that the bilane (25) would yield a bilene-è salt upon oxidation, since the two outside méthylène groups would be made less sensitive to oxidation by conjugation with the terminal acidic functions. In addition, it seemed likely that the planar cisoid conformation of the methene moiety would aid cyclization, rather than random polymerization.

272

P. S. CLEZY AND A. H. JACKSON Me H02C

C

Et

Me

Et

ΡΜβ

Me

ΡΜβ

Me

l Jc02H 25 [O]

Me

Et

Me

Et

P Me

Me

P Me

Me

H02Cl H

H

H

H

26

Oxidation of 25 with tert-butyl hypochlorite gave rather better results than aerial oxidation or, the use of iodine, and, in this manner, the bilene-è salt (26) was obtained crystalline, although it proved difficult to characterize thoroughly either by elemental analysis, or by spectroscopic means. However, the crude bilene-ft when treated with trimethyl orthoformate-trichloroacetic acid (which proved to be the most efficient reagent for cyclization) yielded mesoporphyrin dimethyl ester (27) after aeration. The product was identical, in all respects, to an authentic sample of mesoporphyrin dimethyl ester. Although

Me 27 R 1 28 R 1 29 R 1 30 R 1 31 R 1 32 R 1

= = = = = =

R3 R3 R4 R3 R2 R3

= = = = = =

Me, R 2 = R 4 = Et Me, R 2 = R 4 = P Me PMe, R 2 = R 3 = M e Me, R 2 = Et, R 4 = P Me R 3 = Me, R 4 = Et R 4 = Me, R 2 = Et

a number of largely uncharacterized intermediates were involved in the conversion of oxobilane into porphyrin, the overall yield was quite good (about 257o based on oxobilane) and the product was uncontaminated by other porphyrinic material. The progress of the reactions could be conveniently monitored by following the changes in electronic spectra, examples of which are shown in Scheme 1.

273

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES Oxobilane dibenzyl ester

BaHe

* Bilane dibenzyl ester

Amax 354 nm

^max 286 nm H2/Pd-C fm-BuOCl

Bilene-6 diacid

Bilane diacid Amax 282 nm

^max 284, 505 nm 4

1. (MeO)3CH/H 2. Oa

c

Porphyrin 408, 498, 530, 570, 625 nm Scheme 1

In another series of experiments, coproporphyrin III and coproporphyrin IV were prepared as their tetramethyl esters 26 (28, 29, respectively) through a-oxobilane intermediates following very much the same plan as outlined for mesoporphyrin. The pyrrolic amides, (33 and 34), were condensed separately as their phosphoryl chloride complexes with the α-free pyrrole (35) to give the dipyrroketones (36 and 37). Chlorination with tert-butyl hypochlorite and reaction of the intermediate chloromethyl derivatives with pyridine gave the required pyridinium salts (38 and 39), which were coupled separately with the lithium salt (19) to furnish the a-oxobilanes (40 and 41), respectively. Then followed the cyclization procedure already described: diborane reduction to the bilane, hydrogenolysis of the benzyl esters, oxidation to the bilene-Z? salt, cyclization with trimethyl orthoformate-trichloroacetic acid, and, finally, aeration to yield the porphyrin. The coproporphyrins obtained were isomerically pure in each case and were identical with samples prepared previously in other laboratories. 3,27 · 28

PhCH202C 1

1

PhCH202C

CONMe2 H

H

33

34

CONMe2

PMe

PhCH;

36 37 38 39

R1 R1 R1 R1

= = = =

R 3 = Me, PMe, R2 = Me, R 2 = PMe, R2 =

n o

N H 35

Me

H

PMe

Me

Me

Me

H

R2 = PMe R 3 = Me P M e , R 3 = CH 2 Py + Me, R 3 = CH 2 Py +

Me

274

P. S. CLEZY AND A. H. JACKSON R

PhCH2C

2

V

Me

P Me

Me

PMe

Me

x ^ L N / C02CH2Ph

o

H 40 R1 = Me, R2 = PMe 41 R1 = PMe, R2 = Me

Other porphyrins synthesized by way of û-oxobilane intermediates are shown in Table 1. IV. PORPHYRINS FROM Ä-OXOBILANES29 The a-oxobilane route, outlined in the previous section, suffers from the disadvantage that the location of the stabilizing carbonyl function necessitates that this group be removed before cyclization can be effected. Moreover, once the carbonyl group of the oxobilane has been reduced, the resulting bilane must be reoxidized to a bilene-è salt to prevent rearrangement reactions occurring. These complications can be avoided by the utilization of boxobilanes (3) as intermediates in porphyrin synthesis. This species has a similar stability to the a-oxobilane and is capable of direct transformation into the macrocycle, at which stage the carbonyl group can be removed from the nucleus. Initially, coproporphyrin III tetramethyl ester and mesoporphyrin dimethyl ester, both compounds of biological significance, were prepared by the boxobilane route.29 To construct the required linear tetrapyrrolic intermediates, the dipyrromethane amides (42 and 43) were needed and were accessible by the pyridinium salt procedure,4 as illustrated in Scheme 2. Me

Me PhCH; O a c l

JcH 2 Py +

Me PhCH 2 0 2 C

i

R1

+

Li+ -O a c!^

Me

R2

J

42 R1 = R2 = Et 43 R1 = R2 = PMe Scheme 2

CONMe2

CONMe2

Me Me Me Me Me Me Me Me Me Me Me

Et Me Et Et CH2CH2OAc CH2CH2OAc

(Br)d C0 2 Me

pMe

pMe

pMe

5

4 pMe pMe H b pMe ρΜθ c

DMe ρΜθ ρΜθ c pMe

pMe

Et

Et

pMe

pMe

pMe

pMe

pMe

pMe

(P )

Me Me Me Me Me Me Me Me Me Me Me

ρΜθ

ρΜθ pMe

Et

8

7

6

Dipyrromethane

48

—

44 35 48 32 44 42 32 35 38

a-Oxobilane 29 24 24 28 26 30 23 23 24 5e 30

Porphyrin (methyl esters)

Yields (7o)

27, Meso32 31 Meso-Xl 64 (63, Proto-) 30 28, Copro III 29, Copro IV (68, Isopempto)

Text no. and trivial name of porphyrina

b

Names and numbers in brackets refer to porphyrins obtained after further transformation of the side-chains. Derived from dipyrromethane (58b). c Also using PH [derived from dipyrromethane (58a)]. d Br removed during hydrogénation of a-oxobilane. e Crude oxobilane used directly.

α

Me Me

pMe

Et Et Me

Me Me Me Me Me Me Me Me

3

Me Me Me ρΜθ Me Me Et CH2CH2OAc Me Me Et ρΜθ Me Me Me CH2CH2OAc Me Et Me

2

1

Dipyrroketone

Substituents in starting materials

TABLE 1 Syntheses of Porphyrins by the a-Oxobilane Route

7 7 7 32 38 38 26 26 26 39 45a

Refs.

276

P. S. CLEZY AND A. H. JACKSON

ΡΜβ

Me

ΡΜβ Me

ULJ1.

I C02CH2Ph

H

H

44

With phosphoryl chloride, the dipyrromethane amides (42 and 43) furnished complexes which reacted slowly with the a-free dipyrromethane (44) to produce the imine salts, (45 and 46), respectively. Alkaline hydrolysis of these salts gave the ft-oxobilanes (47 and 48) respectively, which were crystalline and amenable to thorough characterization. Hydrogenolysis of the terminal

Ut<

\

PhCH202C PhCH202C

ττ^

—NH HN—

Y V=x

—NH HN — ^>Me Me^

J^^\^

45 46 47 48

R R R R

= = = =

Et, X = NMe2 PMe, X = NMe2 Et, X = O PMe, X = 0

benzyl esters followed by cyclization of the "linear" system with trimethyl orthoformate in the presence of trichloroacetic acid (as developed for the aoxobilane series) gave the oxophlorins (or oxyporphyrins) (49 and 50), after aeration.

49 R = Et 50 R = PMe

These oxophlorins are an interesting and important group of porphyrin derivatives, and their chemistry will be reviewed in some detail in a later chapter (Volume II, Chapter 4). For the present, it is sufficient to say that they are tautomeric substances; the keto isomer is dominant, although the enolic tautomer can be isolated as ester or ether derivatives, or as the dicationic salts or metal complexes.30

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

277

To complete the porphyrin synthesis, removal of the carbonyl function from the meso position must be achieved. Following a method used for xanthoporphyrinogen reduction, 31 sodium amalgam and acetic acid can be employed to eliminate the meso-oxygen function directly from the oxophlorins. Presumably, an intermediate alcohol is formed which readily dehydrates to give the aromatic porphyrin system. Alternatively, catalytic reduction furnishes a macrocyclic ketone which, after reduction by diborane, can be reoxidized to porphyrin by air or iodine. However, neither process is very convenient for large-scale preparative work. Reoxidation of the macrocyclic ketone obtained by catalytic reduction of the oxophlorin regenerates the original tetrapyrrolic system. This lack of activity of the macrocyclic carbonyl group is reminiscent of the behavior of the structurally similar dipyrroketones under comparable conditions. Acetylation of the oxophlorins (49 and 50) with acetic anhydride in the presence of pyridine produced the enol acetate derivatives, (51 and 52),

51 R = Et 52 R = P Me

pMe

pMe

respectively. Hydrogénation over palladium on charcoal yields the colorless acetoxyporphyrinogens from which the acetoxy group is lost by elimination, or perhaps, by hydrogenolysis. Dehydrogenation in the presence of iodine, or oxygen, restores the porphyrin chromophoric system; DDQ has recently also been used and gives better yields.31a Porphyrin synthesis via the meso-acetoxy derivative is usually the most convenient preparative procedure and has proved to be the method of choice for removal of the ra^ö-oxygen function. This route also has the advantage that it is not usually necessary to isolate the oxophlorin itself; the acetates are generally more stable and are easier to characterize. The overall yield from the dipyrromethanes is usually of the order of 20-30% (cf. Table 2). Mesoporphyrin dimethyl ester (27) and coproporphyrin III tetramethyl ester (28) prepared by this route had physical constants and spectral characteristics which corresponded well with samples made by other synthetic processes. The porphyrin (53)29 has also been made by the ό-oxobilane route, and other examples are given later.

pMe

pMe

Me

AE t

Me

AM e pMe

Me Me Me Me

pMe AM e

AM e pMe

4

pMe

Et

pMe

pMe

Et Me Et Me pMe Me Et Me Me CH2CH2OAc Me Et Me CH2CH2OAc Me Et Me CH2CH2OAc pMe Me Me CH2CH2OAc Me Et COaMe Me C0 2 Me Me CH2CH2OAc Me Me Et C0 2 Me Me C0 2 Bu l Me C0 2 Bu l Me pMe Me

5 Me Me Me Me Me Me Me Me Me Me Me Me Et Et Me Me Me Me Me Me Me Me Me Me Me Me

8

pMe

pMe

pMe

pMe

pMe

pMe

Me

pEt

pMe pMe

ΑΜβ

pMe

AM e pMe

Me Me Me Me

(c\y

AM e ΑΜβ

A

Me

pMe

ρΜθ

pMe pMe pMe

pMe

pMe pMe

pMe

Et Et CH2CH2OAc

pMe

pMe pMe

Et Me Me C0 2 Me

pMe

pMe

pMe pMe

Me Me Me Me Me Me Me Me

pMe pMe

pMe pMe

Me Me Me Me Me

pMe pMe

pMe

pMe

pMe

pMe

pMe

Me Me

pMe

Et

pMe

Et

pMe

pMe

pMe

«-free dipyrromethane 6 7

—

38" 56b 36b 50b 43 36 23 40 64 40

45 62 48 49" 48b 57" 46b 44 41 60" 60" 60" 42" 39b 39b

bilane

b_Qxo_

c

b

Qxo_

— — — — — — — — — — — — — — — — — — — —

88 76 65 22 16 24

—

47 39 29 20 15 21 50 51 38 2d 5 36 38 30 26 24 12 28 28 20

70 77 55 24 48

porphyrins

Acetoxy.

. ,. , „ .

i îeias i / 0)

v

phlorin

Names and numbers in brackets refer to porphyrins obtained after further transformations of the side chains. Not obtained in crystalline form. Halogen removed during hydrogenolysis of oxobilane. d Crude oxobilane converted directly into acetoxyporphyrin.

a

pEt

pMe

DMe AM e

pMe pMe

C0 2 Me

AM e

pMe

CH 2 CH 2 OAc Me CH2CH2OAc (Br)c (Br)c CH2CH2OAc

pMe

pMe

pMe

Et

CH2CH2OAc C0 2 Me Me Me Me (Br)<

1

Me Me Me Me Me Me Me Me Me Me Et Et CH2CH2OAc Me

pMe

Me Me Me Me Me

Dipyrromethane amide 2 3

Substituents in starting materials

TABLE 2 Syntheses of Porphyrins by the 6-oxobilane Route

53 71 69 47 36 69 50

— — — —

66 76

— 79

51 51 40 54

— 86

64 63 64 70 73

Porphyrins

j e xt n

(76"S-411") 59c 59e 59a 59f 59g, Isocopro 59d

79, Rhodo XV 80 81

(67, Pempto) (69, Hardero) (70, Isohardero)

(78, Proto I)

27, Meso 53 28, Copro III Meso XI (63, Proto)

of porphyrin °

trivial

29 29 29 32 38 52 31a 39 39 40,41a 40,41a 45a 45a 45a 45b 47 47 48 48 44 54 55 55 55 56 32c

Refs.

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

279

V. DIFFERENTIAL PROTECTION OF PYRROLE RINGS All the porphyrin syntheses so far discussed have utilized the dipyrromethane (54) or its decarboxylated derivative (44). These products were obtained by the semihydrogenolysis of the symmetrical dibenzyl ester (55), which is not a particularly convenient route to this type of intermediate. ΡΜθ

Me PhCH 2 0 2 C H

ΡΜβ

Ο^

Me

H

56 R = co 2 c 6 ci 5 57 R = COaBu*

54 R = C 0 2 H 55 R = C0 2 CH 2 Ph

Procedures of more general application make use of dipyrromethanes substituted in the 5- and 5'-positions with esters, which can be cleaved in a selective manner. By employing benzyloxycarbonyl, ter/-butyloxycarbonyl, or pentachlorophenyloxycarbonyl functions dipyrromethane~5,5'-diesters, can be constructed which can be selectively uncovered by treatment with hydrogen, acid or base, respectively.32 Thus, the dipyrromethane (54) can be conveniently prepared as the triacid benzyl ester (58a) by the action of base on 56.32 Alternatively, the dipyrromethane (44) can be derived from 57 by treatment with trifluoroacetic acid.32 Making use of special pyrrolic esters of this type which allow selective cleavage, dipyrromethane intermediates have been prepared from which mesoporphyrin XI dimethyl ester has been obtained by both the a and boxobilane routes.32 Me

ocI

R

PhCH22022C IL _ X.

PH A

V I

Me J C0 2 H

H H 58a R = CH2CH2C02H 58b R = Et

280

P. S. CLEZY AND A. H. JACKSON

Recent work with appropriately substituted 5-benzyloxycarbonyl-5'-ter/butyloxycarbonyldipyrromethanes has allowed the ft-oxobilane route to be utilized in the synthesis of several porphyrins related to intermediates between uroporphyrinogens I and III and the corresponding coproporphyrinogens. As a result of this work, it was shown32a that the natural route from uroporphyrinogen III to coproporphyrinogen III is highly specific, and involved the "clockwise" decarboxylation of the four acetic acid residues starting with that on ring D. On the other hand, the pathway from uroporphyrinogen I to coproporphyrinogen I was shown to be nonspecific, both possible type I hexacarboxylic porphyrins (59a, 59b) being found to occur naturally.32b The synthesis of the type III heptacarboxylic porphyrin with a D-ring methyl group (59c) has been completed at Cardiff32* and at Sydney320 by the è-oxobilane route. By the same procedure, the preparation of the analogous pentacarboxylic porphyrin (59d) has also been achieved,320 as have the type I hepta-, hexa-, and pentacarboxylic porphyrins (59e, 59a, and 59f, respectively).3215 Isocoproporphyrin (59g), an abnormal degradation product of the natural type III pentacarboxylic acid (59h) derived from uroporphyrinogen III, has also been synthesized by the 6-oxobilane route.32d Details of these syntheses are summarized in Table 2.

59a R 1 59b R 1 59c R 1 59d R 1 59e R 1 59f R 1 59g R 1 59h R 1

= = = = = = = =

R5 R4 R3 R3 R3 R3 R3 R3

= = = = = = = =

A M e ; R 2 = R e = P Me ; R 3 = R 4 = Me Me; R 2 = R e = P Me ; R 3 = R 5 = A Me R 4 = A M e ; R 2 = R 5 = P Me ; R e = Me R 4 = Me; R 2 = R 5 = P Me ; R e = A Me R 5 = A M e ; R 2 = R e = P Me ; R 4 = Me R 4 = Me; R 2 = R 6 = P Me ; R 5 = A Me R6 = Me; R 2 = Et; R 4 = A M e ; R 5 = P Me R e = Me; R 2 = R 5 = P Me ; R 4 = A Me

VI. THE SYNTHESIS OF PORPHYRINS BEARING LABILE GROUPS The syntheses discussed so far have all involved porphyrins bearing alkyl or substituted alkyl groups. However, many important porphyrins have functional groups attached directly to the periphery of the ring system, e.g.,

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

281

vinyl, formyl, and ester groups. The presence of these substituents imposes certain difficulties with regard to porphyrin synthesis. Substituents such as the formyl or ester groups are strongly deactivating towards electrophilic attack, and since the pyrrolic units are usually linked together by electrophilic substitution reactions, the presence of these electronegative groups does not facilitate porphyrin synthesis. For example, while a number of variously substituted dipyrromethane amides of a type suitable for è-oxobilane synthesis can be prepared by the same method as that shown for (42 and 43) (Section IV), it has also been noted that the process becomes difficult when R2 is a strongly electron-withdrawing group. 6,29 The vinyl group is a common constituent of porphyrins of natural origin, but its lability precludes it being carried through many steps during construction of the porphyrin nucleus. It is, therefore, necessary to introduce this function in the form of a suitable precursor from which the vinyl group can be generated at a later stage. The acetyl group 326,33 and the 2-aminoethyl substituent 32e,34~37 have been used as progenitors of the vinyl substituent, but the Liverpool group sought another path to this olefinic function which was more compatible with their oxobilane procedures. Finally, the 2'hydroxyethyl substituent was selected to serve this purpose, and, by way of intermediate porphyrins bearing this group, the biologically important protoporphyrin dimethyl ester has been synthesized by both the a-oxobilane and 6-oxobilane routes. 38 The bis(2-acetoxyethyl)porphyrin (60) was a common intermediate of both routes, and was obtained by similar procedures 38 to those already discussed in detail above. The porphyrin (60) was converted to the diol (61) by acidcatalyzed methanolysis. Treatment of the product (61) with either mesyl chloride in pyridine, or thionyl chloride in dimethylformamide, gave the bischloroethylporphyrin (62). Dehydrochlorination of 62 was effected by reaction of its zinc complex in tetrahydrofuran with potassium ter/-butoxide.38 The protoporphyrin dimethyl ester (63), obtained in this way, was identical with material of natural origin. Recent work has shown that benzoyl chloride/dimethylformamide38a is a useful reagent for the conversion of 2'-hydroxyethylporphyrins into the chloroethyl analogues, while sodium hydroxide in aqueous pyridine effectively achieves the dehydrochlorination of the latter function. Other vinylporphyrins prepared by the oxobilane procedures, and making use of the 2,-hydroxyethyl substituent as a precursor of the vinyl group, include pemptoporphyrin dimethyl ester (67) 39 and its isomer (68),39 harderoporphyrin dimethyl ester (69) 40,41,41a and its isomer (70) 40,41,41a and Spirographis porphyrin dimethyl ester (72).39 Recently, the preparation of protoporphyrin I dimethyl ester (78) 31a has been reported, and, during the exploratory stages of the development of the 2'-hydroxyethyl substituent

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as a satisfactory precursor for the vinyl group, the synthesis of the model porphyrin (64) was also accomplished.38 In the preparation of porphyrins having an unsubstituted peripheral

60 61 62 63 64 65 66 67 68

R1 R1 R1 R1 R1 R1 R1 R1 R1

= = = = = = = = =

R2 = CH2CH2OAc R2 = CH2CH2OH R2 = CH2CH2C1 R2 = CH = CH2 Et, R2 = CH = CH2 H, R2 = CH2CH2OAc H, R2 = CH2CH2C1 H, R2 = CH = CH2 CH = CH2, R2 = H

69 70 71 72 73 74 75 76 77

R1 R1 R1 R1 R1 R1 R1 R1 R1

= = = = = = = = =

CH = CH2, R2 = PMe PMe, R2 = CH = CH2 CHO, R2 = CH2CH2C1 CHO, R2 = CH = CH2 CHO, R2 = Et CHO, R2 = PMe PMe, R2 = CHO CH = CHC02Me, R2 = PMe PMe, R2 = CH = CHC0 2 Me

position (67 and 68), it was beneficial to protect this vacant site by a bromine atom during the initial stages of the synthesis.39 Thus, pemptoporphyrin dimethyl ester (67) was constructed by the Z?-oxobilane method, the bromine atom being hydrogenolyzed simultaneously with the benzyl esters. The porphyrin (65) obtained at the end of the Z?-oxobilane sequence was converted into the chloroethyl derivative (66), and dehydrochlorination by the procedure used to synthesize protoporphyrin then furnished pemptoporphyrin (67). The synthesis of Spirographis porphyrin (chlorocruoroporphyrin) (72) posed a new problem, as this tetrapyrrole contained a formyl substituent. The problem was solved by introduction of the aldehydic function by electrophilic substitution after the porphyrin nucleus had been constructed. Following the procedure of Fischer and Schwartz,42 treatment of the iron CH = CH2 Me CH2

78

283

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

complex of 66 with methyl dichloromethyl ether in the presence of stannic chloride gave the formylporphyrin (71) after removal of the metal. Dehydrochlorination gave Spirographis porphyrin dimethyl ester (72), although it was necessary to protect the labile formyl group as an acetal during this step. 39 In preliminary model experiments, the formyl porphyrin (73) was also prepared. 39 As part of the structural investigation of a porphyrin isolated from calf meconium, the formylporphyrins (74 and 75) have recently been prepared as intermediates in the synthesis of the acrylate esters (76 and 77). 43,44 The porphyrin nucleus of 74 was constructed by the Z>-oxobilane route and that of 75 by a variant of the MacDonald route, 28 and the formyl group introduced at a late stage by electrophilic substitution.42 Condensation with malonic acid gave 76 and 77, after esterification,44 and the former proved to he identical with the meconium porphyrin. The Liverpool group have also completed the synthesis of rhodoporphyrin XV dimethyl ester (79), its 2-vinyl derivative (80), and the 2,4-divinyl derivative (81) by the 6-oxobilane pathway 45_45b (Table 2). Conversion of the

79 R1 = R2 = Et 80 R1 = CH = CH2, R2 = Et 81 R1 = R2 = CH = CH2

C0 2 Me

V-COaMe

/

C0 2 H

-* rco-\ X

=N

rK

V-coci

\ - COCH2C02Me

A

C0 2 Me V-COCH \f \:θ2Βιι' Scheme 3

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P. S. CLEZY AND A. H. JACKSON

nuclear ester into the strongly enolized ß-keto ester derivative has been achieved by two procedures, 45-46 as illustrated in Scheme 3. It has been shown in vitro that cyclization of the ß-keto ester system gives pheoporphyrin derivatives. 45-46 Preliminary results with meso-triüated magnesium complexes of porphyrin ß-keto esters indicate that it is likely that such compounds are also biosynthetic intermediates in the formation of chlorophyll derivatives.4545b Synthesis of porphyrins related to heme-a, and bearing nuclear carboxylic ester substituents have also been accomplished in Liverpool 47 · 48 (Table 2).

VII. CONCLUSIONS The a- and è-oxobilane routes to the porphyrin nucleus are both versatile processes, and a variety of porphyrins have been prepared by one or both of these pathways. In principle, specific labeling of any position within the molecule could be achieved. Recently, there has been a great deal of interest in the preparation of specifically labeled porphyrins for biogenetic49 and other reasons, 50 and several such oxophlorins and porphyrins have been prepared by the 6-oxobilane procedures.38·43*45-4515,51 This pathway permits very ready exchange of the meso proton opposite the ring ketone at the oxophlorin stage of the synthesis, and this aspect of oxophlorin chemistry will be referred to in another chapter. For the present, it is sufficient to relate that advantage of this has been taken to tritiate protoporphyrin at the δ-position,38 as shown in Scheme 4. This was achieved by reaction of the oxophlorin (82) with tritiated acetic acid to give the δ-tritiated derivative (83), which was converted into labeled protoporphyrin dimethyl ester (84) by the usual sequence of reactions. The tritium in the labeled ß-keto esters referred to above was also introduced in the same way.38 Some loss of label was encountered during the hydrogénation and aeration step by which the mesooxygen atom is removed, but this loss was minimized by the primary isotope effect. The magnesium complex of this tritiated protoporphyrin was shown to be incorporated into chlorophyll in experiments with isolated chloroplasts. 45-45b y-Tritiated α-oxymesoporphyrin and δ-tritiated ß-oxymesoporphyrin (Table 2) were also prepared 52 by the 6-oxobilane/oxophlorin procedure, and the iron complex of the former was shown to be metabolized to bile pigments in the rat 51 more efficiently than the latter. The 14C-labeled acrylate ester (76) has also been prepared via the £-oxobilane synthesis as described above,44 and using the monomethyl ester of [2-14C]malonic acid in the final stage (74 -> 76). Catalytic reduction of this acrylate porphyrin (76) gave coproporphyrin III tetramethyl ester, which was mixed with mesotritium labeled material, hydrolyzed, and reduced with sodium amalgam to the double-labeled porphyrinogen.43 Incubation with a hemolysate of chicken

6. PORPHYRINS FROM OXOBILANE INTERMEDIATES

pMe

pMe

pMe

82

285

pMe

83

MeOH/H +

Scheme 4

erythrocytes afforded protoporphyrin which had only half the tritium present in the original coproporphyrin III, and, thus, showed that the final oxidation to porphyrin is a stereospecific process in nature. 43 As part of a program to assign resonances in the paramagnetic nmr spectra of low spin iron (III) porphyrins 53 and hemoproteins,54 the synthesis of protoporphyrin dimethyl ester as the hexadeuterio derivatives (85), (86), and (87) has been accomplished.53,55 The preparation of l,8-bis(trideuteriomethyl)protoporphyrin dimethyl ester (87) was completed by the è-oxobilane procedure commencing with appropriately deuterated pyrroles. The success of this synthesis highlights the value of this method in producing specifically labeled porphyrins. The oxobilane procedures, as with most porphyrin syntheses, become more difficult as the number of negative groups in the molecule increases. As pyrrolic ring systems are π-excessive in character, the linkage of the four individual units to yield ultimately the porphyrin nucleus inevitably involves

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pMe

pMe

85 R 1 = R2 = CH 3 , R 3 = R4 = CD 3 86 R 1 = R2 = CD 3 , R3 = R4 = CH 3 87 R 1 = R4 = CD 3 , R2 = R3 = CH 3

electrophilic substitution reactions of one type or another. Naturally, such procedures are not favored by an overabundance of electron-withdrawing substituents. Both oxobilane routes require the presence of ester groups at the terminal positions of the oxobilane. This, in turn, minimizes the number of negative groups which can be located around the periphery of molecules prepared by these sequences. This was one reason why the acetyl group was rejected as a precursor of the vinyl substituent in this series of porphyrin syntheses. REFERENCES 1. H. Fischer, F. Baumann, and H. J. Riedl, Justus Liebigs Ann. Chem. 475, 237 (1930). 2. A. Hayes, G. W. Kenner, and N. R. Williams,/. Chem. Soc. p. 3779 (1958). 3. E. J. Tarlton, S. F. Macdonald, and E. Baltazzi,/. Am. Chem. Soc. 82, 4389 (1960). 4. A. H. Jackson, G. W. Kenner, and D. Warburton,/. Chem. Soc. p. 1328 (1965). 5. P. S. Clezy and A. J. Liepa, Aust. J. Chem. 23, 2443 (1970). 6. J. A. S. Cavaleiro, A. M. d'A Rocha Gonsalves, G. W. Kenner, and K. M. Smith, /. Chem. Soc.t Perkin Trans. 1 p. 2471 (1973). 7. A. H. Jackson, G. W. Kenner, and G. S. Sach,/. Chem. Soc. C p. 2045 (1967). 8. D. Mauzerall,/. Am. Chem. Soc. 82, 2601 and 2605 (1960). 9. H. Fischer and H. Orth, Justus Liebigs Ann. Chem. 489, 62 (1931); 502, 237 (1933). 10. A. Treibs and K. H. Michl, Justus Liebigs Ann. Chem. 577, 129 (1952). 11. J. M. Osgerby and S. F. MacDonald, Can. J. Chem. 40, 1585 (1962). 12. J. A. Ballantine, A. H. Jackson, G. W. Kenner, and G. McGillivray, Tetrahedron 22, Suppl.7,241(1966). 12a. K. M. Biswas, L. E. Houghton, and A. H. Jackson, Tetrahedron 22, Suppl. 7, 261 (1966). 12b. K. M. Biswas and A. H. Jackson, Tetrahedron 24, 1145 (1968). 13. H. Rapoport and C. D. Willson,7. Am. Chem. Soc. 84, 630 (1962). 14. P. S. Clezy and A. W. Nichol, Aust. J. Chem. 18, 1977 (1965). 15. G. H. Cookson, J. Chem. Soc. p. 2789 (1952). 16. U. Eisner and P. H. Gore, / . Chem. Soc. p. 922 (1958). 17. R. Chong, P. S. Clezy, A. J. Liepa, and A. W. Nichol, Aust. J. Chem. 22, 229 (1969).

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18. H. C. Brown and B. C. Subba R a o , / . Am. Chem. Soc. 82, 681 (1960). 19. P. S. Clezy, A. J. Liepa, A. W. Nichol, and G. A. Smythe, Ausi. J. Chem. 23, 589 (1970). 20. E. Bullock, A. W. Johnson, E. Markham, and K. B. Shaw, / . Chem. Soc. p. 1430 (1958); A. W. Johnson, I. T. Kay, E. Markham, R. Price, and K. B. Shaw, ibid. p. 3416(1959). 21. H. Fischer and H. Orth, "Die Chemie des Pyrrols," Vol. I, p. 85. Akad. Verlagsges., Leipzig, 1934 (Johnson Reprint Corporation, New York and London, 1968). 21a. H. Fischer and H. Orth "Die Chemie des Pyrrols," Vol. I, p. 77. Akad. Verlagsges., Leipzig, 1934. 22. R. Grigg, A. W. Johnson, and J. W. F. Wasley, / . Chem. Soc. p. 359 (1963). 22a. H. Fischer and H. Orth "Die Chemie des Pyrrols," Vol. I, p. 147. Akad. Verlagsges., Leipzig, 1934. 22b. H. Fischer and H. Orth "Die Chemie des Pyrrols," Vol. I, p. 235. Akad. Verlagsges., Leipzig, 1934. 23. P. S. Clezy and A. W. Nichol, Aust. J. Chem. 18, 1835 (1965). 23a. H. Fischer and H. Orth, "Die Chemie des Pyrrols," Vol. I, p. 363. Akad. Verlagsges., Leipzig, 1934. 24. R. Bonnett, M. J. Dimsdale, and G. F. Stephenson, / . Chem. Soc. C p. 564 (1969). 25. H. C. Brown, "Hydroboration." Benjamin, New York, 1962. 26. A. H. Jackson, G. W. Kenner, and J. Wass, / . Chem. Soc, Perkin Trans. / , p. 1475 (1972). 27. F. Morsingh and S. F. MacDonald, / . Am Chem. Soc. 82, 4377 (1960). 28. G. P. Arsenault, E. Bullock, and S. F. MacDonald, / . Am. Chem. Soc. 82, 4384 (1960). 29. A. H. Jackson, G. W. Kenner, G. McGillivray, and K. M. S m i t h , / . Chem. Soc. C p. 294(1968). 30. A. H. Jackson, G. W. Kenner, and K. M. S m i t h , / . Chem. Soc. C p. 302 (1968). 31. H. Fischer and A. Treibs, Justus Liebigs Ann. Chem. 457, 209 (1927). 31a. J. A. S. Calaveiro, G. W. Kenner, and K. M. S m i t h , / . Chem. Soc, Perkin Trans 1 p. 2478(1973). 32. P. J. Crook, A. H. Jackson, and G. W. Kenner, / . Chem. Soc. C p. 474 (1971). 32a. A. H. Jackson, H. A. Sancovich, A. M. Ferramola, N . Evans, D . E. Games, S. A. Matlin, G. H. Elder, and S. G. Smith, Phil. Trans. Roy. Soc. Lond. B273, 191 (1976). 32b. A. H. Jackson, D. M. Supphayen, K. R. N . Rao, and S. G. Smith, in press. 32c. P. S. Clezy, T. T. Hai, and P. C. Gupta, Aust. J. Chem. 29, 393 (1976). 32d. A. H. Jackson and D. J. Ryder, unpublished work; D. J. Ryder, Ph.D. Thesis, University College, Cardiff, 1977. 32e. H. Fischer and H. Orth, "Die Chemie des Pyrrols," Vol. II, Part 1. Akad. Verlagsges. Leipzig, 1937. 33. P. S. Clezy, A. J. Liepa, and N. W. Webb, Aust. J. Chem. 25, 1991 (1972). 34. R. B. Woodward, W. A. Ayer, J. M. Beaton, F. Bickelhaupt, R. Bonnett, P. Buchschacher, G. L. Closs, H. Dutler, J. Hannah, F. P. Hauck, S. Itô, A. Langemann, E. le Goff, W. Leimgruber, W. Lvowski, J. Sauer, Z. Valenta, and H. Volz, / . Am. Chem. Soc 82, 3800 (1960). 35. A. M. Fargal, R. P. Evstigneeva, I. V. Khaidy, and N . A. Preobrazhenskii, Zh. Obsch. Khim., 34, 893 (1964). 36. P. Bamfield, R. Grigg, A. W. Johnson, and R. W. K e n y o n , / . Chem. Soc. C p. 1259 (1968); R. Grigg, A. W. Johnson, and M. Roche, ibid. p. 1928 (1970).

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37. G. L. Collier, A. H. Jackson, and G. W. Kenner, / . Chem. Soc. C p. 66 (1967). 38. R. P. Carr, A. H. Jackson, G. W. Kenner, and G. S. Sach, /. Chem. Soc. C p. 487 (1971). 38a. P. S. Clezy and C. J. R. Fookes,/. Chem. Soc, Chem. Commun, p. 707 (1975). 38b. P. S. Clezy and C. J. R. Fookes, Aust. J. Chem. 30, 217 (1977). 39. A. H. Jackson, G. W. Kenner, and J. Wass, Chem. Commun, p. 1027 (1967);/. Chem. Soc, Perkin Trans. 1 p. 480 (1974). 40. G. Y. Kennedy, A. H. Jackson, G. W. Kenner, and C. J. Suckling, FEBS Lett. 6, 9 (1970); 7, 205 (1970). 41. J. A. S. Cavaleiro, G. W. Kenner, and K. M. Smith,/. Chem. Soc, Chem. Commun. p. 183 (1973). 41a. A. H. Jackson, G. W. Kenner, K. M. Smith, and C. J. Suckling, Tetrahedron 32, 2757 (1976). 42. H. Fischer and A. Schwartz, Justus Liebigs Ann. Chem. 512, 239 (1934). 43. A. H. Jackson, D. E. Games, P. W. Couch, J. R. Jackson, R. V. Belcher, and S. G. Smith, Enzyme 17, 81 (1974). 44. P. W. Couch, D. E. Games, and A. H. Jackson, /. Chem. Soc, Perkin Trans. 1 p. 2492(1976). 45. M. T. Cox, T. T. Howarth, A. H. Jackson, and G. W. Kenner, /. Am. Chem. Soc. 91, 1232 (1969); /. Chem. Soc., Perkin Trans. 1 p. 512 (1974). 45a. T. T. Howarth, A. H. Jackson, and G. W. Kenner, /. Chem. Soc, Perkin Trans. I p. 502 (1974). 45b. M. T. Cox, A. H. Jackson, G. W. Kenner, S. W. McCombie, and K. M. Smith, /. Chem. Soc, Perkin Trans. 7 p. 516 (1974). 46. G. W. Kenner, S. W. McCombie, and K. M. Smith,/. Chem. Soc, Chem. Commun. p. 844(1972). 47. R. Fletcher, A. H. Jackson, and G. W. Kenner, unpublished work. 48. T. Lewis, A. H. Jackson, and G. W. Kenner, unpublished work. 49. A. R. Battersby, G. L. Hodgson, M. Ihara, E. McDonald, and J. Saunders,/. Chem. Soc, Chem. Commun, p. 441 (1973); A. R. Battersby, E. Hunt, and E. McDonald, ibid. p. 442; A. R. Battersby, G. L. Hodgson, M. Ihara, E. McDonald, and J. Saunders,/. Chem. Soc, Perkin Trans. 1 p. 2923 (1973). 50. G. W. Kenner and K. M. Smith, Ann. N. Y. Acad. Sei. 206, 138 (1973). 51. D. C. Nicholson, T. Kondo, A. H. Jackson, and G. W. Kenner, Biochem. J. 121, 601 (1971). 52. P. J. Crook, A. H. Jackson, and G. W. Kenner, Justus Liebigs Ann. Chem. 748, 26 (1971). 53. J. A. S. Cavaleiro, A. M. d'A. Rocha Gonsalves, G. W. Kenner, K. M. Smith, R. G. Shulman, A. Mayer, and T. Yamane,/. Chem. Soc, Chem. Commun, p. 392 (1974). 54. A. Mayer, S. Ogawa, R. G. Shulman, T. Yamane, J. A. S. Cavaleiro, A. M. d'A. Rocha Gonsalves, G. W. Kenner, and K. M. Smith,/. Mol. Biol. 86, 749 (1974). 55. J. A. S. Cavaleiro, A. M. d'A. Rocha Gonsalves, G. W. Kenner, and K. M. Smith, /. Chem. Soc, Perkin Trans. 1 p. 1977 (1974).