THE NOSYNTHESfi OF TERTHDENYL IN THE
COMM #AREQGLD

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Terry BEE! Waggoner

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ABSTRACT

THE BIOSYNTHESIS OF TERTHIENYL
IN THE COMMON MARIGOLD

by Terry Bill Waggoner

This investigation represents the first attempt to
elucidate the biosynthetic pathway of the naturally
occurring polythiophene, 2,2'; 5',2"-terthienyl. Marigold
plants growing in nutrient solution were fed suspected
precursors containing sulfur-35 or carbon-14. The radio-
isotOpes studies were sodium sulfate-S-35, sodium hydrogen
sulfide-S-BS, L-methionine-S-35, DL-methionine-l-C-lh,
DL-methionine-2-C-lh, succinic acid-2,3-C-14, DL-glutamic
acid-2-C-14, and sodium acetate-l-C-lu. Terthienyl was
isolated from the roots at Specific times from the
initial feeding, and its radioactivity was determined.

Sulfur-35 was incorporated into terthienyl yielding
dilution factors of Specific activity ranging from 77-294
representing approximately 0.06% incorporation of the
original radioactivity administered. Methionine-S-35 gave
a dilution factor of 735, and no incorporation was observed
from sodium hydrogen sulfide-S-35. No incorporation into
terthienyl was detected from feeding DL-methionine-l-C-la,
via stems, DL-glutamic acid-2-C-lu, or succinic

801d-2,3-C-14.

Terry Bill Waggoner

Dilution factors from other carbon-14 compounds were 1,430
from DL-methionine-Q-C-14 and 10,200 to 35,100 from sodium
acetate-l-C-lfl.

From the consideration of the dilution factors, it
was ascertained that sulfur—35 was incorporated best when
supplied as sodium sulfate and to a lesser extent from
methionine-S-35. It was found that carbon-1M was incor-
porated only when supplied as methionine—Q-C-lu. The other
carbon-14 compounds provided no incorporation to a signifi-
cant extent.

Isolation of terthienyl after various time intervals
of uptake of the radioisotOpe indicated that young plants
of 8-10 weeks were most desirable for experimental work.

It was suggested that terthienyl was actively metabolized
and was not a ”storage'I product of plant metabolism. The
"site of synthesis” of terthienyl was designated to be

in the roots as determined from root vs stem feeding pro-
cedures. Sulfur-35 in the form of sodium sulfate and
carbon-14 in the form of methionine were not incorporated .
into terthienyl when fed to the plant via stems, whereas
incorporation was observed when fed via roots.

The lack of incorporation of carbon—14 in the form
of "acetate” and of sulfur-35 in the form of hydrosulfide
suggested that previous hypotheses of the biogenesis of
terthienyl were not necessarily correct. From the experi—
mental evidence obtained, a biosynthetic scheme involving

homocysteine as a possible precursor was presented.

THE BIOSYNTHESIS OF TERTHIENYL

IN THE COMMON MARIGOLD

By

Terry Bill Waggoner

A THESIS

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

DOCTOR OF PHILOSOPHY

College of Natural Science
Department of Chemistry

1963

ACKNOWLEDGMENTS

The author wishes to express his thanks for financial
support from Grant AT (11-1) 1034 from the Atomic Energy
Commission. His gratitude is also given to the Department
of Chemistry for use of equipment and the facilities and
for financial support during the first three quarters of
school in the form of a Graduate Assistantship.

The initial stages of this investigation required
the advice and help from several peOple throughout the
University. Many hours of work were saved with the aid of
H. M. Sell, Professor, Department of Biochemistry, for the
use of his flash evaporator for the concentration of approxi-
mately 200 liters of ethanol extract of marigold blooms
during the summer of 1961. With the permission of R. F.
Stinson, Associate Professor, Department of Horticulture,
the author was able to obtain thousands of marigold blooms
from the campus gardens. Thanks are also given to J. A.
Knierim, Assistant Professor, Department of Entomology, for
his interest in the problem and his permission to pick
marigold roots from his experimental plots located at the
Muck Farm of Michigan State University. Helpful suggestions
of experimental procedures were also given by the late W.

J. Haney, Associate Professor, Department of Horticulture,
and thanks are also given to him for personally growing

most of the marigold plants used in the study.

ii

An appreciation is extended to Floyd Challender,
Foreman, Plant Science Greenhouse, for his pleasant manner
in helping the author raise healthy marigold plants from
seeds, especially in cold weather. R. Lindstrom, Assistant
Professor, Department of Horticulture, also gave helpful
suggestions concerning the experimental procedures in
develOping root systems.

Helpful discussions concerning the initial radioiso-
tOpe experiments with T. Griffith, Department of Chemistry,
Northern Michigan University, were fruitful and stimulating.
His encouragement and support were appreciated.

Discussions with R. U. Byerrum, Dean, College of
Natural Science, were helpful and offered direction at a
crucial time during the course of this work. Thanks are
given for his suggestions, gift of two of the radioisotopes
studied, and time from his schedule for serving on the guid—
ance committee.

The author is thankful to the following faculty
members of the Department of Chemistry for serving on his
guidance committee: W. H. Reusch, Assistant Professor;

J. L. Dye, Associate Professor; and H. A. Eick, Associate
Professor.

The author has the highest regard for his advisor,
R. D. Schuetz, Professor, Department of Chemistry, and
sincerely appreciated the Opportunity of working on the

advisor's research proposal with an unlimited amount of

ill

freedom. The free manner of investigation on the part of
the author and Just the "right" amount of direction on the
part of Doctor Schuetz provided a most satisfying relation-

ship. For this the author is extremely grateful.

iv

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES.
LIST OF SCHEMA
INTRODUCTION.
EXPERIMENTAL.

Plants. . . .
Preparation of the Nutrient Solution.
Administration of RadioisotOpes
Harvest of the Roots . .
Preparation of the Samples for Counting. .
Self- -absorption by Terthienyl- and 5- Acetyl-
terthienyl -S- 35 . . .
Determination of Specific Activities.
IsotOpe Dilution Analysis
Adsorption Chromatography . . . .
Oxidation of 5— (3-Buten- l-yny1)— 2, 2' -dithienyl .
Preparation of 5- Acetyl— 2 2',5', 2"- terthienyl and
5, 5"-Diacetyl- 2 ,2';5', 2'-terthieny1 . .
Oxidation of 5— Acetyl- 2 ,2"-terthieny1 .
Paper Chromatography of 2 ,2 ,5', 2"-terthienyl.
Paper Chromatography of 5- Acety1—2 ,2',5', 2"-
terthienyl and 5, 5"- Diacetyl- 2 ,2'35', 2"— terthienyl
Thin Layer Chromatography . . . . .

RESULTS . .

Sodium Sulfate-S-35

L-Methionine-S-35 .

DL—Methionine-l-C—l4

DL- Methionine- 2— C— 14

Sodium Acetate- 1- C- 14. .

IsotOpe Dilution Ex eriments . . . . . .
Investigation of 5— Buten- 1- yny1)- 2,2'—dithienyl

DISCUSSION
APPENDIX
SELECTED BIBLIOGRAPHY.

Page
vi
viii

ix

Table

10.

ll.

12.

13.

14.

15.

LIST OF TABLES

Structures of Naturally 0ccurring Thiophene
Compounds . . . . . . .

A Simplified Survey of the Family of the
Compositae. . . . . . . .

 

Preparation of 2,5—Disubstituted ThiOphenes
Under Biogenetic Conditions . . .

Self—absorption by 2,2';5',2"—Terthienyl-S-35.

Self- -absorption by 5-Acety1-2,2';5', 2”-
terthienyl- S- 35 . . . .

Comparison of Rf Values of Terthienyl Against
the Amount of Sample . .

Comparison of R Values of Terthienyl Against
Changes in Solvent Composition . .

Rf Values of 5- AcetyL 2 ,2';5', 2”—terthienyl.
and 5, 5"- Diacetyl- 2 ,2';5', 2"— terthienyl
from Paper Chromatography. .

Compounds Investigated by Thin Layer
Chromatography . . .

The Incorporation of Sodium Sulfate —S- 35 into
Terthienyl in Marigold Roots. . . .

The Incorporation of L—Methionine-S-35 into
Terthienyl in Marigold Roots. . .

The Incorporation of DL— Methionine— 1- C- 14 into
Terthienyl in Marigold Roots. . .

The Incorporation of DL-Methionine-2-C-l4 into
Terthienyl in Marigold Roots. . . .

The Incorporation of Sodium Acetate-1-C-14
into Terthienyl in Marigold Roots

The Incorporation of Sulfur- 35 and Carbon- 14
into Terthienyl as Determined by IsotOpic
Dilution Methods. . .

vi

Page

14
23

24

37

38

4O

44

46

48

49

51

53

55

Table Page

16. Weights and Specific Activities of Isolated

Terthienyl in Experiments 23-26 . . . . 56
17. Properties of the Dithienyl Compound

Compared with Terthienyl - Experiment 4 . 58
18. Paper Chromatography of Carboxylic Acids. . 60
19. Specific Activity of Isolated Radioactive

Terthienyl . . . . . . . . . . . 64
20. Per Cent Incorporation of Radioactivity in

Terthienyl . . . . . . . . . . . 66
21. UV Absorption of Some Dithienyl Compounds . 84
22. Weight of Terthienyl Isolated from Various

Species of Marigolds. . . . . . . . 86

vii

LIST OF FIGURES

Figure Page
1. Data from Self-absorption Measurements of
2,2';5',2"-Terthienyl-S—35 and 5-Acetyl—
2,2';5',2"—terthienyl-S-35 . . . . . . 25
2. Incorporation of Sodium Sulfate-S—35 into
Terthienyl. Specific Activity of
Terthienyl-S—35 vs Time . . . . . . . 71

viii

Schema
1.
2.

LIST OF SCHEMA

Challenger's Biogenetic Pathway to Terthienyl.

Sorensen and Sorensen's Suggested Biogenetic
Pathway to Terthienyl

Biogenetic Formation of the Triple Bond.

PrOposed Biogenetic Pathway to Terthienyl from
Homocysteine . . . . . . . .

Biosynthetic Pathway of Sodium Sulfate —S- 35
to Terthienyl. . . . . . .

Biosynthetic Pathway of DL-Methionine-2-C-l4
to Terthienyl. . . . . . . .

Biosynthesis of "Acetate Derived” Compounds
(26).

ix

Page

12

17

68

7a

78

INTRODUCTION

The first naturally occurring polythiOphene com-
pound, 2,2‘;5',2"-terthienyl, was isolated by Zechmeister
and Sease (1) from the blooms of the common marigold,

Taaetes erecta L. Until that time the only other thio-

[Y

phene derivative known to be found in nature was a growth
factor, biotin (2). The number of thiophene derivatives
known to exist in nature has increased in the past eight
years. In 1955 a metabolic product called ”Junipal" was

isolated from a wood-destroying fungus, Daedalena_Juniper—

 

lag, which infests the East African cedar, Juniperus
procera Hochst (3). Sorensen and Sorensen (4) added a
fourth compound to the list with the isolation of 2-phenyl-
5-(alpha-propynyl)-thiophene from the essential oils of

Coreopsis grandiflora, Hogg ex Sweet. Shortly after,

 

Sorensen and Guddal (5) reported the occurrenceand isola—
tion of methyl cis—beta (5-propyny1—2—thienyl)acrylate in

the roots of Chrysanthemum vulgare Bernh. Sorensen and his

 

collaborators reported the isolation and identification of

the trans isomer from the scentless mayweed, Matricaria

 

indora (11). The nematicidal properties of the concentrate
from the roots of African marigolds was investigated by
Uhlenbroek and Bijloo. Two active ingredients were
identified, terthienyl and a new polythiophene, 5-(3-buten-

1-ynyl)-2,2‘-dithienyl (6). In Sorensen‘s paper the

isolation of 5—methyl-5'—butadienyl-2,2'-dithienyl was
reported by Mrs. Liaaen Jensen, in yet unpublished results.
This supposedly is the first naturally occurring dithienyl
to be discovered (11). With other workers Sorensen has
identified another thiophene derivative isolated from the

thistles, Berkheya macrocephala and EchinOps sphaereo-

 

 

cephalus. It was believed to have the structure of com-
pound VIII, Table l. Bohlmann, Bornowski, and Schonowsky
isolated and identified the structures of three compound
containing a single thiophene ring, two of which possessed
an oxygen containing ring: XII, cis and trans isomers

from Anthemus nobilis L. (also, the trans isomer from

 

Artemisia vulgaris L.); XIII, cis and trans isomers from

 

Santolina pinnata Viv; XIV, from Artemisia ludoviciana

 

Nutt (15). The structures of the fourteen naturally
occurring thiophene derivatives which have been reported
are shown in Table 1. A recent summary of the naturally
occurring thiophene compounds was published by Katritzky (12).
The present research was undertaken to obtain
experimental evidence for the biosynthetic pathway of
terthienyl. To understand the scope of the problem, it
is best to first consider the scanty evidence from biogenetic
and biosynthetic studies which have led to some specula-
tions by a few investigators concerning the origin of the

thiophene derivatives which are listed in Table l.

TABLE l.--Structures of Naturally Occurring Thiophene

 

 

 

Compounds
Compound Structure
/°\
I Biotin
(CH2) —COOH
,3 4
i l
Terth eny S 5 S
III "Junipal" CH3'CEC.J[Ti§L.CHO
6
IV 2~Phenyl-5- CH3°C:C.JéTIj§L{C:>
(alphapr‘ommgl ) - S
thiophene
v Methyl-cis-beta CH'CEC —1[f_j§—CH=CH°COOCH3
(5-propynyl-2- 5

thienyl) acrylate

VI 5-Methy1-5‘-
butadienyl-2,

2'—dithienyl

VII 5-Methy1-5'- CH
butadienyl-2,

2'-dithieny1

/

VIII 5-(4—Penten-
l-yny1)—2,2'-

dithienyl

CH3

$3

/

\ CH=CH°Ch=CH2

33

\ /
'5 S

/ \

CH=CH°CH=CH2

/\
s

c: C°CH2°CH=CH2

\-/\
s

S.

Table 1. continued:

 

 

 

 

IX

XI

XII

XIII

XIV

2—(3,5,7— (QTTBX—CEC-CH=CH'CH=CH°CH=CH2
octatrien-l- .S

ynyl)-thiophene

2-(7-ketO-3,5- (/ \x-CEC-CH=CH°CH=CH‘CO‘C2H5
nonadien—l-ynyl) S

thiophene

2-(7-ketO-3- A/ \5 CEC'CH=CH°CH2-CH2°CO°C2H5
nonen-l-ynyl)- 5

thiOphene
Methyl-5 CH3—4f_i§LCEC°CH=CH-C00CH3
(5-meth 1—2— 5

thienyl —pent
4-yne-2-enylate

1-Thienyl—4- AZTiEX—CEC'CH=CH _Jé:—:§
S

furanyl-but-l— O
yne-3-ene

—- O
1- -Spiro( (2'-oxo-/
tetra- -hydropyran S O
4-(2"-—thieny1methy—

lene)furan— 2— —ene

 

Compounds II and IV-XIV have in common not only the
thiophene nucleus and unsaturated linkages in the latter
eleven, but all are found in plants of the Natural Order

Compositae. This order includes such plants as the daisy,

 

dandelion, aster, ragweed, and wormwood. These plants
have small flowers or florets borne in dense involucrate
beads which resemble single flowers (7). Of the living

seed plants the Compositae comprise one tenth of the total

 

number including herbs, shrubs, and trees embracing the
most highly developed family in the vegetable kingdom.
Depending upon a personal choice of definition, there exist
today somewhere between 15,000 and 100,000 species. A

simplified summary of the family of the Compositae is given

 

in Table 2. This was taken from Sorensen (11) who based
the data on the most recent botanical treatment of the
family performed by Hoffman in 1889.

Of significance is the fact that all of the plants

of the Compositae which produce the thiophene derivatives

 

also have been the sources of another class of naturally
occurring compounds, the polyacetylenes. Because of the
proximity of polyacetylenes and unsaturated thiophene
derivatives in the same order of plants, an intimate
interrelationship of these two classes of compounds was
suggested. Challenger speculated that the occurrence of
polyacetylenes with low hydrogen content lends support to

them as the origin of terthienyl (2). His suggested

TABLE 2.-—A Simplified Survey of the Family of the

 

 

 

 

Comppsitae
Botanical Common Trivial Tribus Approximate
Tribus Genus Designation No. of Genera
I Vernonieae Veronia Ironweeds 41
II Eupatorieae Eupatorium Thoroughworts 42
III Astereae Aster Aster 99
Solidago) Goldenrods)
Bellis) Daisy)
IV Inuleae Gnaphalium Everlastings 152
Antennaria
(Inula)
V Heliantheae Helianthus Sunflowers 144
(Dahlia)
Cosmos
Bidens
(Coreopsis)
VI Helenieae Helenium Sneezeweeds 55
(Tagetes) (African marigolds)
VII Anthemideae Chrysanthemum Chrysanthemum 49
Artemisia)
Matricaria)
VIII Senecioneae Senecio Groundsel 51
IX Calenduleae Calendula Marigolds 8
X Arctotideae Arctotis 11
XI Cynareae Centaurea Thistles 34
XII Mutisieae Gerbera Gerbera 57
XIII Cichorieae Taraxacum Dandelions 63
(Hieracium) ——’——
Total 806

 

pathway is represented in Scheme 1. As a starting point
the long carbon chain could have its origin a long chain
paraffin or fatty acid. Hydrogen sulfide could possibly
arise from cysteine. In Scheme 1 as well as the others
to be mentioned it is assumed that other processes, very
probably enzymic, such as oxidation, reduction, decarboxy-
lation, etc., are involved. Scheme 1 would also be
facilitated by the presence of olefinic and acetylenic
linkages which could aid in ring closure. A specific
example of such a pathway is the addition of hydrogen
sulfide to l—phenylheptatriyne to give compound IV (Table
1).

Sorensen and Sorensen (4) have considered the
suggestion of Challenger and speculated that hexaacetylene
would be required as a precursor to terthienyl; however,
since polyacetylenes higher than triacetylene leads to
carbonaceous materials, the existence of hexaacetylene
even in dilute solution in the plant cells in the presence
of possible stabilizers seemed highly unlikely. They
suggested that some polyketo precursors were more pro-
bable, and these could serve both as the common source of
polyacetylenes and naturally occurring thiophenes. This
method of formation is represented in Scheme 2. The
original compound consists of a 2,4-diketo grouping with
dehydration and thiophene formation following in a stepwise

manner. Scheme 2 represents a speculative pathway for the

Scheme 1

Challenger's Biogenetic Pathway to Terthienyl

R-C:C-C:C-C:C-C:C -C_=_C- EC-

1

C_=_C ~C:C-C=_-C-C_=_C-C_=;C-R

. I
S

H

R‘Q‘CEC'CEC-CgC-CEC—R
l

/\ /\ /\
5 S S

Scheme 2

Sorensen and Sorensen‘s Suggested Biogenetic
Pathway to Terthienyl

CH
3 . COOH

H. sic -CECWYCOQH
i
<3. CEC‘CEC W COOH
L
M CEC-CEC—COOH

L

/\ /\ /\
~S '8 ~S

lO

formation of terthienyl. A specific example of their
scheme is the conversion to compound V (Table 1) from
the addition of hydrogen sulfide to the dehydromatricaria
ester, XV, which is found to occurr in a closely related

species (4).

XV Dehydromatricaria ester

Recent evidence for the transformation of a
methylene carbon to an acetylene linkage was obtained by
Craig and Moyle (14). The model compound in their
experiments was the methyl ester of acetone dicarboxylic
acid. It was converted to its enol phosphate, and the
conversion of the phosphate to an allene under mild
conditions was achieved (equations 1 and 2). The results
shown in equation 2 indicate that acetylene-allene
conversion occurs with the allene being the stable product.

Craig and Moyle suggested that in nature the triple bond

Equation 1

 

CH3O

// —
\1/\/\ C02CH3 H003 CH3O CH=C=CH—C02CHg
O V '
\

(OEt)2

ll

 

Equation 2.
HCO3
CH2-C3C—COQH 7;;____9 <::>}—CH=C=CH-CO2H

is accompanied by intramolecular transphosphorylation
giving an intermediate, the hemiketal phosphate. The
dehydration could give two different enol phosphates
leading to a stable diyne, found in nature, or a diethynyl-
methane, unknown in nature. Thus, Scheme 2 is supported
by this biogenetic evidence. The formation of the triple
bond as indicated by Craig and Moyle is represented by
Scheme 3.

Sorensen and Sorensen have reported that their
preliminary investigations revealed about a dozen new

compounds from members of the Compositae as being thiOphene

 

derivatives and in part acetylenic thiophenes. Four of
these new compounds have been tentatively identified by
Sorensen and are listed in Table l as compounds VIII-XI (11).
No other published results are available with respect to
these new compounds.

In addition to the above discussion Sorensen
implied that the occurrence of thiophene compounds in the

Compositae may indicate thiOphenes to be precursors to

 

polyacetylenes rather than end products of polyacetylene
metabolism.
There is some indirect evidence concerning the

biosynthesis of terthienyl. Uhlenbroek and Bijloo

12

Scheme 3

Biogenetic Formation of the Triple Bond

-c:c-CH2-cfc-

Unknown

(H
) W
(O 0510
“E) /
u/// (OEt)2
/:;:>Kb:«\\‘czc-
on
(oat)2
Hemiketal - PO(0Et)2
/
//A\W’/)\\C:C-
o
\P=O
Et
(0 >2
-CH2-CEC-C:C—

\y Known

-CH=C=CH—C:C-

Known

13

identified not only terthienyl but compound VI (Table l)
as being produced by the roots of the African variety of

the common marigold (Tagetes erecta). They suspected that

 

terthienyl might arise from the addition of hydrogen
sulfide to compound VI followed by dehydrogenative ring
closure (6). Obviously, this theoretical reasoning
only suggests that compound VI could possibly beapre-
cursor of terthienyl.

Terthienyl was claimed to have been obtained from
the reaction of hydrogen sulfide and 1,4-dithienylbutadiene
in a weakly alkaline medium at 20-60 degrees (13). Other
2,5-disubstituted thiOphenes were also prepared under
mild conditions. The reactions are shown in Table 3.

The products obtained in reactions 3, 4, and 6 (Table

3) , are those found in nature. This biogenetic relation-
ship may be of some significance. If the assumption is
made that polyacetylenes are precursors to terthienyl, the
work of J. D. Bu'Lock and his co-workers represents a
major contribution to the resolving of the biosynthetic
problem.

Observing fungus cultures of Basidiomycetes B.

841, Bu'Lock and Leadbeater (8) concluded that glucose

was metabolized via two different routes, and that
polyacetylenes were further converted to breakdown products.
Besides the reactions producing "normal" metabolites,
glucose was also converted to polyacetylenes under appro-

priate conditions with as much as 7 per cent conversion by

14

TABLE 3.--Preparation of 2,5—Disubstituted Thiophenes
Under Biogenetic Conditions

 

 

m Reactants Products

1. CH3-C_=_C-C;:._C-CH3 +HQS __7 CH3©CH3

:QCE-EPCCC©+HS —‘) H/S\

/:\/\/32\+HS ———>/\/\/\
S s s

= - = - = -' = - 9 ’ = / \ = -
A. CH3C_C c_c C_C CH CH C02H+H28 CH3C_C (S XCH CH COQH

~s . fl
5. CH3CEC-C1C-CEC-Ch20H +1123 CH3C;_-_C s Ch20H

CHécgcQCHQOH + Ivmo2 ’ CHBCEC Q CHO

 

 

15

this "alternate" pathway. Bu'Lock and Gregory (9) fed
acetate-l-C—l4 to the fungus cultures and observed

that 15-20 per cent of the original radioactivity was
incorporated into one particular polyacetylene, nemotinic
acid, XVI. The stepwise degradation of nemotinic acid
revealed that the carboxyl carbon atom was labelled as
well as alternate carbon atoms in the chain. Thus, the
ten carbon acid was formed by head-to-tail linkage of
five acetate units. Bu‘Lock, Allport, and Turner further
showed that acetate-l-C—l4 was incorporated in a head-to-
tail fashion into the matricaria ester, XVII, which is

found both in Compositae and the fungus, Polyporus

 

 

anthraCOphilus, a Basidiomycete which was actually used

 

in the experiments.

OH
H‘ CEO-CECW COQH

XVI Nemotinic acid
CH \\\///\\\ CO2CH
3 / CEC -CEC /\\/ 3
XVII Matricaria ester

The research reported here was undertaken to attempt
to elucidate the biosynthesis of terthienyl in the African
variety of marigold. It was hOped that an organo sulfur
compound could be discovered which was a precursor to
terthienyl in the plant. The relationships of organo
sulfur compounds in plants was considered with respect to
the known metabolic conversions of sulfur compounds in

higher animals.

l6

Initially consideration was given to the possibility
of a four carbon unit as a precursor, namely homocysteine.
This compound contains not only four carbons but also a
sulfur atom, the ingredients of a thiOphene ring system.
Such a pathway involving homocysteine is represented I
in Scheme 4. Because of the uncertainty of the reactions
leading to terthienyl, the pathway cannot include details
of such a conversion.

The existence of Schemes l, 2, and 4 was also
investigated. Compounds containing sulfur-35 or carbon-l4
which were suspected as likely precursors to terthienyl
were fed to marigold plants. The work reported here
represents the first biosynthetic study of terthienyl or
of any of the known naturally occurring thiOphene derivatives

other than biotin.

17
Scheme 4

Proposed Biogenetic Pathway to Terthienyl from Homocysteine

4:12:24 a:
Q...

[.30

é

EXPERIMENTAL

Plants

The seeds of Tagetes erecta variety were purchased

 

from W. Atlee Burpee Company, Fordhood Farms, Doylestown,
Pennsylvania. The Tagetes were used in all experiments
except two. A petite variety of marigolds with orange
blooms was studied in Experiments 3 and 4. Generally, four
plants were utilized, unless some expired during the

course of the experiment. The actual weight of roots
obtained varied from 0.3 to 4.0 g. with most yields in

the range of 2.0 g. The plant age at the time of the
administration of the radiosotope was 2 to 5 months. Each
plant was transferred from the soil to the nutrient
solution two to four weeks before dispensing the radioisotope.
This period in the nutrient solution was necessary in order
for a large enough root system to develop, since about 80%
of the roots were cut away after the plant was removed

from the soil. In later experiments (25-26) the plants

were seeded and grown in sand prior to the transfer to the
nutrient solution. The roots did not die after the transfer
under these conditions, and therefore were not cut away.
Each plant was placed in a separate Erlenmeyer flask

containing enough nutrient solution to cover the roots.

18

19

Additional solution was required while the new root system

was developing.

Preparation of the Nutrient Solution

Hoagland's #2 solution was utilized as the nutrient
solution (18). Stock solutions of 1N concentration were
prepared: 19.2 g. ammonium dihydrogen phosphate, 50.5 g.
potassium nitrate, 41.0 g. calcium nitrate tetrahydrate,
and 30.0 g. magnesium sulfate were dissolved in separate
volumes of 500 ml. each of distilled water and stored
until needed. For the preparation of 1 l. of nutrient
solution,aliquots of l, 6, 4, and 2 ml., respectively, were
diluted to l l. with distilled water. The solution was

then ready for use.

Administration of Radioisotopes

 

Method A: The plants were removed from the nutrient
solution, and the roots were dried by blotting them with
absorbent paper. The roots were further air-dried for an
additional hour. At this time the solution of radioisotope
(approximately 20 microcuries/ml.) was added with the aid
of a pipette to each plant, so that each one received 5
microcuries distributed over the roots. Nutrient solution
was again added to each plant in one hour following the
initial dispensing of the radioisotope. The above pro-

cedure was based upon that of an earlier publication (21).

20

Method B: It was not required under these circum-
stances to remove the plants from the nutrient solution.

A white cotton thread (Coats and Clark's) was inserted with
the aid of a sewing needle through the stem of each plant
about three inches above the roots. The two ends of the
thread functioned as the wick. A five ml. beaker contain-
ing 0.25 ml. (approximately 5 microcuries) of radioactive
solution was attached below the Junction of the thread and
stem. The solution was taken up in approximately two
hours, and 0.5 ml. of distilled water was added to the
beaker in order to "wash" as much radioisotope as possible
into the plant. At the conclusion of the feeding time the
thread was extracted twice with 100 ml. of distilled water.
The amount of radioactivity extracted from the thread was
subtracted from that amount originally dispensed. The
difference was assumed to be the amount actually taken up
by the plant.

Method 0: An attempt to inject the radioactive
solution directly into the stem with the aid of a needle
and syringe was unsatisfactory. A syringe was situated in
a vertical position with the end of the needle inserted
into the stem, and a weight was applied to the plunger.
None of the solution was taken up by the plant after forty-

eight hours of application.

Harvest of the Roots

 

The plants were removed from the nutrient solution

at the appropriate times. Only the roots were investigated,

21

and they were separated from the remainder of the plant,
washed with 95% Ethanol, and weighed after they were

blotted dry with adsorbent paper. The roots were then
disintegrated in 100 ml. of 95% ethanol for five minutes
with a Waring blendor. A sample of "cold" terthienyl was
added (if desired) to the ethanol mixture immediately

before the blending step. The mixture was filtered through
a soxhlet thimble, and extraction of the residue with the
use of the filtrate was continued for twenty-four hours in
the soxhlet apparatus. Tbefiltrate was evaporated to dry-
ness under reduced pressure, and the residue was redissolved
in petroleum ether (30—60). The petroleum ether solution

of crude terthienyl was chromatographed over activated
alumina. The specific activity of terthienyl was determined.
Further purification was carried out until a constant

specific activity of terthienyl was attained.

Preparation of the Samples for Counting

 

Aliquots of 0.1 to 1.0 ml. of the desired solutions
were evaporated to dryness in aluminuniplanchets using an
infrared lamp as a source of heat. The length of counting
time for each sample was determined by the accuracy desired
and the activity of the sample. All counting was conducted
in a windowless, gas-flow proportional counter: Baird
Associatates—Atomic Instrument Company, Cambridge 39, Massa-

chusettes. The solid samples of terthienyl and its acetyl

22

derivates were obtained from the evaporation of their
ethanol solutions. The addition of several drOps of water
to the solution of sample in the planchet enabled a more
uniform layer of the solid deposited. A thin uniform layer
resulted with no self-absorption by the sample being
observed. The lack of self-absorption is shown by the

data represented in Figure l and Tables 4 and 5.

Self-absorption by Terthienyl—and
5-Acetylterthienyl-S—35

 

 

Terthienyl-S—35 from Experiment 23 was purified by
alumina chromatography and diluted to 25 ml. (concentration =
18.6 mg./ml.). Aliquots of the solution were added to
each of ten aluminum planchets. Enough ethanol was added
to bring the total volume to 1.0 ml. The solvent was
evaporated under an infrared lamp, and the activity was
determined with a SD of'2%. The net activity (cpm) was
plotted against the density of the sample. The same
procedure was followed with the 5-acetyl derivative (con-
centration = 1.84 mg./ml.) also taken from Experiment 23.
The results are summarized in Tables 4 and 5. The data
obtained from each compound were plotted. A straight line
was constructed for each set of points using the method of
least squares. The results are shown graphically in Figure

1.

23

TABLE 4.--Se1f-absorption by 2,2'-5',2”—Terthienyl—
3'35

 

 

Planchet Area = 7.07 cm.2

Sample Terthienyl Ethanol Terthienyl Density- Net
Solution-ml. Volume-ml. Weight-mg. mg./cm.2 cpm

w—

 

1 0.1 0.9 0.0744 0.0105 15
2 0.2 0.8 0.149 0.0211 30
3 0.3 0.7 0.223 0.0316 54
4 0.4 0.6 0.297 0.0420 73
5 0.5 0.5 0.372 0.0525 69
6 0.6 0.4 0.446 0.0631 77
7 0.7 0.3 0.520 0.0735 101
8 0.8 0.2 0.595 0.0841 113
9 0.9 0.1 0.669 0.0946 126
10 1.0 0.0 0.744 0.105 130

 

24

TABLE 5.--Self-absorption by 5-Acety1—2,2'-5',2"—
Terthienyl-S—35

 

Planchet Area = 7.07 cm.2

Sample Terthienyl Ethanol Terthienyl Density- Net
Solution—ml. Volume-ml. Weight-mg. mg./cm.2 cmp

 

1 0.1 0.9 0.0184 0.00260 None
2' 0.2 0.8 0.0368 0.00520 7

3 0.3 0.7 0.0552 0.00781 20
4 0.4 0.6 0.0736 0.0104 41
5 0.5 0.5 0.0920 0.0130 42
6 0.6 0.4 0.110 0.0156 63
7 0.7 0.3 0.128 0.0181 82
8 0.8 0.2 0.147 0.0208 90
9 0.9 0.1 0.165 0.0233 112
10 1.0 0.0 0.184 0.0260 105

 

25

Figure l.—-Data from Self-absorption Measurements of
2,2'-5',2"-Terthienyl-S-35 and 5-Acetyl-
2,2'-5‘,2”—terthienyl—S-35

140

120

100

80

Net

cpm

60

4O

20

 

 

 

 

/// —--— -—-—-5-Acetyl Terthienyl
C)
/ Terthienyl
/A
/
/’Zl
/
/
/£§ 1 l A _L l 4 I 1 4
.2 .3 .4 .5 .6 .7 .8 .9 1.0 1.1 10‘1
3 .6 9 1.2 1.5 1.8 2.1 2.4 2.7 2.0 10'2

mg./cm.2

26

Determination of Specific Activities

 

Quantitative determination of terthienyl was obtained
by measuring its absorption at 350 millimicrons (€‘=24,lOO).
The radioactivity measurements were carried out by
planchet counting of solid samples. Constant specific
activities resulted after several rechromatographic
Operations over alumina. Another method of purification
was the conversion of terthienyl to its acetyl derivatives,
5-acetyl-2,2,2‘;5‘,2”-terthienyl and 5,5”-diacetyl-2,2';5',
2"-terthienyl. The acetyl derivatives were purified to
constant specific activity by adsorption chromatography over
alumina. The 5-acety1 compound was easier to handle of the
two. Its elution from the alumina column was faster.
Qualitative measurements were taken by its absorption
at 391 millimicrons (e = 30,500). The radioactivity was
determined in the same manner as that for terthienyl. The
purity of the above compounds was checked by paper
chromatography and thin layer chromatography. The UV
absorptions were obtained with a Beckman DU spectrophoto—
meter. The statistical treatment of the counting data

was based upon the discussions of Overman and Clark (19).

Isotope Dilution Analysis

 

The procedure was essentially that which was reported
by Mayor and Collins (20). To the crude terthienyl

fraction from the alumina column was added a known quantity

27

of "cold" terthienyl (0.8-10 mg.). The diluted mixture
was divided, and a second quantity of terthienyl, twice
that of the first dilution, was added to one of the divided
portions. The terthienyl from each dilution was converted
to its 5-acetyl derivative and purified to constant
specific activity. The original terthienyl isolated from
the plant and its original specific activity were calcu-
lated from the following relationships:

x = A2D2/Al-A2- (Dl-S) A0 = AlDl/X
X = Weight of terthienyl isolated
A0: Specific activity of X
D1: Dilution 1
D2: Dilution 2
S = Weight of diluted terthienyl taken for analysis
A1: Specific activity of D

1
A2: Specific activity of D2
Isot0pe Dilution Calculations of DL-Methionine-S—35,
Experiment 23:

A1* = Actual sample counted

A2* = Actual sample counted

RS = Net counting rate in cpm
Rb = Background rate in cpm
R Rate of sample and background in cpm

A1* = Two measurements were observed with counting intervals

of 20 minutes each.

Rb (1) 2560 c/20 m = 123 cpm
Rb (average) = 119 cpm
(2) 2310 c/20 m = 115 cpm
Rs+b (1) 4250 c/20 m = 212 cpm
(2) 3800 c/20 m = 190 cpm
RS (1) 212-119 = 93 i 4 cpm SD = 4%
(2) 190-119 = 71 + 4 cpm SD = 6%

Standard deviation of RS was calculated from the equation:

SD=(R

s+b

/t + Rb/t)l/2

Total RS = RS x Dilution factor

B. (1) 93x5
(2) 73x5

A1 = Total RS/.805 mg.

(l) 465 cpm/.805 mg.
(2) 365 Cpm/.805 ms-

A2*: Two measurements

465 cpm
365 cpm

578 cpm/ mg.
493 cpm/ mg.

were observed with counting intervals

of 20 minutes each.

Rb (1) 1470 c/20 m =
(2) 1400 0/20 m =
Rs+b (1) 2110 0/20 m

(2) 2230 c/20 m

33 i
(2) 111-72 = 39 :

RS (1) 105-72

74 cpm
R (average) = 72 cpm
70 cpm b
= 105 cpm
= 111 cpm
3 0pm SD = 9%
3 cpm SD = 8%

Total R8 = Rsx Dilution factor

RS (1) 33 x 2

(2) 39x2

66 cpm
78 cpm

29

Since these samples are'assayed 7 days later than
those of A1, a correction was necessary for the radio-
active decay during this interval.

-2.3 (log 66 - log Rs) = .00778 (7)

Corrected RS (1) 70 cpm

(2) 83 cpm
A2 = Total RS/.705 mg.
A2 (1) 7O cpm/.705 mg. = 99 cpm/mg.
(2) 83 cpm/.705 mg. = 113 cpm/mg.

From X =A2D2/Al—A2- Dl+ S and using the combination
of the two different values of Al and A2,four values of X

were calculated :

X = 99 x 20/578-99 - 5.0 = 1980/479 - 5.0 = 4.14-5.0 = -.86

x = 99 x 20/441-99 - 5.0 = 1980/342 - 5.0 = 5.80-5.0 = +.80

x = 113 x 20/578-113 - 5.0 = 2260/465 - 5.0 = 4.86-5.0 = —.14
x = 113 x 20/441-113 - 5.0 = 2260/328 - 5.0 = 6.90-5.0 = +1.9g
X (average) = 1.5 mg. which is equivalent to 5.14 Micromoles

Since A0 = AlDl/X, then
A0 = (1) 578 x 10/1.5 = 3380 cpm/mg. = 1.12 x 106 cpm/mM
0.86 x 106 cpm/mM

(2) 441 x 10/1.5 2960 cpm/mg.

AO (average) = 0.99 x 106 cpm/mM

Adsorption Chromatography

 

Terthienyl was best separated from the petroleum
ether solution of the roots by chromatography of the crude

sample over 7.5 g. of alumina (Alcoa, F-20) in a 25 x 1.5 cm.

30

glass column fitted with a st0pcock. The sample size was
approximately 15 ml. Fractions of 10 ml. were collected.
Terthienyl was eluted with petroleum ether (30-60) after
40 to 60 ml. of eluant had been collected. A 2% diethyl
ether solution hastened its movement through the column.
The 5—(3—buten-l-yny1)-2,2'-dithienyl fraction was always
collected prior to the terthienyl fraction. Both fractions
were eluted together if caution was not taken when using
2% diethyl ether as the eluant. The terthienyl fraction
was identified from its blue fluOrescence and instant
violet color with isatin dye in concentrated sulfuric
acid. The dithienyl compound also displayed a positive
reaction with isatin, but the color was wine in appearance
compared to that with terthienyl. Both compounds were
distinguished by their behavior on a paper chromatogram.

A further purification of terthienyl over 5 g. of alumina
resulted in only a partial purification in some cases as
shown by the data presented below.

The acetyl derivatives of terthienyl were best
purified by adsorption chromatography over alumina using
carbon tetrachloride as the solvent. The 5-acetyl deriva—
tive was eluted from the column by a 10% solution of
diethyl ether in carbon tetrachloride. The percentage of
diethyl ether was increased to 60% in order to elute the
5,5”-diacety1 derivative. Terthienyl passed through the

column with carbon tetrachloride. The sample size was not

crucial and could vary from 5 to 75 ml.

alumina and a total of 20 mg.

31

using 5 g. of

of solids in solution.

The results of the purification of terthienyl-S-35

are shown below.

and 3 using sodium sulfate-S—35.

"Cold"

This data was taken from Experiments 2

terthienyl was

added during the step in which the roots were disin-

tegrated in ethanol.

Experiment 2:

 

Ethanol extract

Petroleum ether
solution

First chromato—
SPaPhY

5-Acetyl
derivative

Experiment 3:

 

Ethanol extract

Petroleum ether
solution

First chromato-
graphy

Second chromato-

sraphy

 

 

Total Terthienyl Specific
cpm MicroM Activity
cmp/MicroM
37.2 x 104 274 1350
6.56 x 104 227 289
1.27 x 101+ 271 47
u
.109 x 10 23.3 47
Total Terthienyl Specific
cpm MicroM Activity
cpm/MicroM
65.4 x 104 238 2740
17.1 x 101*L 254 674
1.84 x 104 189 97
1.40 x 104 156 90

32

The data presented for Experiment 3 represents the
actual observed activity, and corrections for the half life
of sulfur-35 were not considered here as they were in
Table 10. The purification to constant specific activity
of added "cold" terthienyl in Experiment 14 is shown

as follows

 

Total Terthienyl Specific
cpm MicroM Activity
cpm/MicroM
First chromato-
graphy 3890 80.7 48
5-Acetyl
derivative 130 8.6 15

Oxidation of 5-(3-Buten-l-ynyl)—
2,2‘-dithienyl

 

 

In trial 1 the fraction from the alumina column
containing the dithienyl compound was evaporated to dryness.
The residue was dissolved in 10 m1. of acetone. Approximately
50 mg. of potassium permanganate and 1 ml. of 2N
sulfuric acid were added. The reaction mixture was stirred
for 45 minutes at room temperature. The product was
isolated according to the procedure of Uhlenbroek and
Bijloo (6). In trial 2 the same procedure was followed,
except the reaction mixture was heated to reflux for 5
minutes and immediately allowed to cool to room tempera—

ture. A gas appeared to be given off.

33
Preparation of 5-Acetyl-2,2';5',2"-terthienyl
and—5,5"-Diacetyl-2,2'35',2"—terthienyl
Several different experimental conditions were
investigated in order to obtain the highest yield of the
5-acetyl compound. Using 100 mg. of terthienyl as start-
ing material, yields ranged from 2-42%. A 100 mg. quantity
(.4 mM) of terthienyl was dissolved in 50 ml. of dry
benzene. The solution was heated to its reflux tempera-
ture, and .056 m1. (.8 mM) of acetyl chloride and 8 drops
(3mM) of stannic chloride were added. After heating it at
reflux temperature for 24 hours the reaction mixture was
poured into 50 ml. of 1N hydrochloric acid and ice. The
mixture was neutralized with sodium bicarbonate and ex-
tracted with diethyl ether. The ether solution was dried
over sodium sulfate and evaporated to dryness. The residue
was dissolved in carbon tetrachloride and chromatographed
over alumina. The unreacted terthienyl was recovered, and
the maximum yield of 5-acetyl compound was 42%. The 5-
acetyl compound was obtained as fine, yellow crystalline
needles, m.p. 168-169 ; UV maximum 391 millimicrons (6::
30,500) and 252 millimicrons. The 5,5”-diacetyl compound
was obtained as yellow needles after sublimation (235 , 6mm.),
m.p. 243 ; UV maximum 406 millimicrons (6:: 39,300) and
253 millimicrons. A yield of 42% was also obtained with
8 mg. of terthienyl as starting matieral dissolved in 20
ml. of dry benzene. Two drops each of stannic chloride and
acetyl chloride added, and the reaction mixture was heated

at its reflux temperature for 15 hours.

34

Oxidation of 5-Acetyl-2,2‘;5',2"-terthienyl

 

A solution of potassium hypochlorite was prepared
following a modified procedure of Newman and Holmes.(24)
A 2.5 g. quantity of HTH (calcium hypochlorite) was
dissolved in 10 ml. of warm water, and a 3 ml. solution
containing 2.1 g. of potassium carbonate and 0.5 g. of
potassium hydroxide was added. The gel which formed was
stirred until it was liquid. The mixture was filtered,
and the filtrate of potassium hypochlorite was saved for
the oxidation procedure. A 10 ml. volume of the hypochlorite
solution was added to approximately 8 mg. of the 5-acetyl
terthienyl previously dissolved in 5 ml. of dioxan. The
homogeneous mixture was stirred until the reaction was
completed. The extent of reaction was followed by the
use of thin layer chromatography. An aliquot of the
reaction mixture was taken at 0, 5, 80, and 155 minutes
after the addition of potassium hypochlorite. At zero
time the 5-acetyl terthienyl was the only compound present
(Rf approximately 0.2). After 5 minutes a new compound
appeared which did not move from the origin. The new
compound at the origin increased in fluorescence (blue),
and fluorescence (green) of the 5—acety1 terthienyl
decreased after 80 minutes. The reaction was complete
at the end of 155 minutes, since the main component was
the compound at the origin, and only a trace of fluorescence

was present due to the acetyl compound.

35

The reaction mixture was diluted to 50 ml. with
distilled water. The solution was acidified to pH 5 and
extracted for 18 hours with diethyl ether in a liquid—
liquid extraction apparatus. The yellow, amorphus residue
remaining was redissolved in a minimum of ethanol.

Paper chromatography of the ethanol solution showed a
blue fluorescent spot at Rf of 0.61 (terthienyl Rf of
0.62) and a blue fluorescent spot at the origin. The
chromatogram was treated with ammonia and sprayed with a
bromothymol blue solution. The compound at the origin
exhibited a yellow spot against a blue background. The
remaining aqueous layer from the liquid-liquid
extraction did not show any acid components when treated
in the same manner, but only one blue fluorescent spot

at Rf of 0.44 (development solvent-methanol:water, 66:34).

Paper Chromatography_of 2,2';5',2”-Terthienyl

 

The ascending method of chromatography was employed
because of its convenience. The development tank consisted
of a 10 x 30 cm. battery jar protected with a glass plate.
Whatman #1 filter paper was cut into rectangular strips,

7 x 25 cm. and suspended from the glass plate by the use

of masking tape. The width of the paper strips was determ—
ined by the number of samples to be chromatographed at one
time. An aqueous methanol solution was utilized as the

development solvent. It was found that a 66-70% methanol

36

solution (aqueous) offered the best results for terthienyl.
The compound was adequately separated from other compounds,
and reproducible Rf values were realized as shown by the
following experiment results.

A stock solution of terthienyl was prepared by
dissolving 25 mg. in 500 ml. of 95% ethanol. Five different
aliquots were taken and applied to the origin of the paper.
The solvent front moved 13.5 cm. after development for one
hour with 66% methanol. The results are shown by sample
numbers 1 through 5 in Table 4. Samples A through C in
Table 6 were treated in the same manner, except the solvent
front traveld 4.0 cm. Six different Chromatograms were
also prepared using a sample size of 2 micrograms. The
movements of the solvent fronts varied from 10.1 to 11.8
cm. yielding Rf values ranging from 0.66 to 0.73. The
latter six values are not shown in the table.

The consequence of varying the percentage composition
of the solvent was not throughly investigated. Some
results of solvent effects are summarized in Table 7. The
sample size was from 2 to 4 micrograms, and the solvent
front traveled 7.5 cm.

Terthienyl was located on the chromatogram by
observing the paper under an ultra violet lamp. The com—
pound was easily distinguished from its brilliant blue
fluorescence. As little as 0.1 microgram was detected

by this method.

37

TABLE 6.—-Comparison of Rf Values of Terthienyl
Against the Amount of Sample

 

 

 

Sample Number Micrograms of Sample Rf Value
1 0.11 0.65
2 0.25 0.65
3 0.35 0.67
4 0.44 0.67
5 0.70 0.68
A approximately 2 0.66
B approximately 4 0.58
C approximately 6 0.55

 

38

TABLE 7.-—Comparison of Rf Values of Terthienyl
Against Changes in Solvent Composition

 

 

 

% Methanol Rf Value
50 0.00
75 0.73
88 0.80

100 1.00

 

39

. A second solvent system was investigated which
involved the use of Whatman #1 filter paper previously
treated with a 5% paraffin solution of benzene. The
development solvent consisted of a 5% ethyl acetate solu-
tion of n-heptane. Rf values were irregular but were mostly
in the range of 0.68 to 0.73. The system was not investi-
gated further.

Paper Chromatography of 5-Acetyl-2,2';5',2"—terthienyl
and 5,5"-Diacetyl-2,2L,5',2"—terthienyl

 

During the preparation of these compounds by
acetylation of terthienyl, it was necessary to check the
purity of the products. Both compounds were treated in the
same manner, and the develOpment of the chromatograms
was identical to that which was followed for terthienyl.
The results are summarized in Table 8. The solvent was
66% methanol (aqueous), and development time was one hour.

The solvent front traveled 13.6 cm.

Thin Layer Chromatography

 

Materials:

The adsorbent used for the preparation of all
chromatostrips was Fisher Alumina, A-540, 80-200 mesh or
Alcoa Activated Alumina, F-20 grade. All solvents were 0.
P. grade and could be used without further purification.
Soluble starch (Nutritional Biochemicals Corporation) or a
commercial plaster of paris was mixed as a binder with the

adsorbent.

40

TABLE 8.--R Values of 5-Acety1-2,2':5',2"-terthienyl
agd 5,5"—Diacetyl-2,2';5',2"-terthienyl
from Paper Chromatography

 

 

 

Compound Ultra Violet Light Rf Valuea
Terthienyl Blue 0.71
5-Acetyl— Yellow 0.46
5,5"-Diacety1- Blue-green 0.16
Mixture:
Terthienyl Blue 0.72
5-Acetyl- Yellow 0.45
5,5"-Diacetyl- Blue—green 0.15

 

a Average value of two chromatograms

41

Preparation of the Chromatostrips:

Two methods were found to be satisfactory for the
separation of compounds containing the thiophene nucleus.
Method l--A1umina and starch in a ratio of 19:1 dry weight
were thoroughly mixed. For each 20 g. of dry mixture 36
ml. of distilled water were added. The slurry was heated
on the steam bath and stirred for 4 minutes. The upper
water layer was poured off leaving a thick, smooth
mixture. This was spread immediately on previously cleaned
microscope slides, 25 x 75 mm. The chromatostrips were
dried in an oven for 30 minutes at 110° C. The dried
chromatostrips were stored in a vacuum desiccator over
potassium hydroxide until needed.

Method 2--A1umina and plaster of paris were
thoroughly mixed dry in a ratio of 7:3 dry weight. For
each 10 g. of dry mixture 8 ml. of distilled water were
added. For increased hardness of the surface 2% sodium
hydroxide was substituted in place of the water. For a
thinner slurry water was added enabling an easier spread-
ing of the mixture. The chromatostrips were dried from 2-5
hours in an oven at 75° C. The chromatostrips were stored
in the same manner as that in Method 1.

For every 10 g. dry weight of adsorbent plus binder,
approximately eight chromatostrips were obtained. The
surface of the adsorbent was slightly pitted and was

smoothed by rubbing it lightly with tissue paper. Immediately

42

before using each chromatostrip, a border of at least 1mm.
in width was made by scraping the adsorbent from the edge
of the slide. This enabled an even flow of solvent during

development.

Application of Sample:

The sample was applied either as a single spot or
as a line at the origin. The origin was one half inch
from one end of the chromatostrip. Solutions of sample
ranging from 0.01 to 1.0 ml. were applied taking normal
precautions in order that spreading of the spot was kept
to a minimum. The use of a hair dryer was excellent for

this procedure.

DevelOpment of the Chromatostrip:

A nonpolar and polar solvent were required for the
develOpment of the samples. Most compounds were separated
by develOpment with a solvent mixture which was from 1 to
10% composition of the polar solvent. Nonpolar solvents
included petroleum ether (30—60), n—hexane, n-heptane,
or benzene. The polar solvents comprised diethyl ether,
ethyl acetate, or acetone. A n-hexane solution of 5%
diethyl ether provided the best over-all separation of the
compounds investigated.

After the application of the sample, the chromato-
strip was placed in a test tube, 3 x 12 cm., containing 1—2

ml. of the desired development solvent. The solvent

43

immediately commenced to ascend. The time required for
the solvent to travel the distance of the chromatostrip
was less than 5 minutes. The chromatostrip was removed
from the solvent and dried either at room temperature or

in a stream of warm air.

Location of the Compounds:

All the compounds incorporating a thiophene nucleus
were detected by very quickly immersing the dried chromato-
strip into concentrated sulfuric acid containing 1% isatin
dye. Characteristic areas of color were observed for each
compound studied with a few exceptions. If the chromato-
strip was immersed longer than two seconds in the isatin
solution, the surface of the adsorbent was usually destroyed.

The presence of functional groups was also exploited
by the use of standard qualitative reagents. Aldehydes
were located by immersing the chromatostrip into 2,4-
dinitrophenylhydrazine or Tollen's reagent. A few
compounds were located by heating the chromatostrip for
several minutes at 1003 C after it was immersed in an
aqueous 2% potassium permanganate solution. The easiest
method of locating the compounds was from observing the
chromatostrip under an ultra violet light. Most compounds
resulted in an easily visible fluorescent spot. A summary
of the compounds investigated by thin layer chromatography

is given in Table 9.

44

TABLE 9.--Compounds Investigated by Thin Layer

 

 

 

Chromatography
Name Isatin Fluorescence

Thiophene Blue None
2-Thi0phenaldehyde Yellow None
2-Acetylthiophene Reda None
2,2'-Dithienyl Green None
5—Formyl-2,2'-dithienyl Yellow Blue
2,2'-Dithienyl-5-carboxylic acid Brown Trace
5—Thiocarbethoxy-2,2'—dithienyl Colorless Blue
2,2';5',2"-Terthieny1 Violet Blue
2,2‘;5',2”-Terthienyl-5-carboxylic

acid - Blue
5-Acetyl-2,2‘;5',2”—terthienyl Violet Green
5,5"-Diacetyl-2,2‘;5‘,2"-terthienyl Red Green

 

a 2,4-dinitr0phenylhydrazine

RESULTS

Sodium Sulfate-S-35

 

Experiments 1 through 5 were conducted in order to
determine the duration required for maximum incorporation
of sulfur-35 into terthienyl as measured from the initial
time of administration of the radioisotOpe. Both feeding
methods were investigated, and terthienyl was radioactive
only in those experiments where feeding was via roots. The
total per cent incorporation, found by dividing the total
radioactivity in terthienyl by the total originally fed,
ranged from 0.04-0.08%. Dilution factors, found by dividing
the original Specific activity of terthienyl by the specific
activity of the administered radioisotope, varied from 77—294.

With Tagetes erecta the dilution factor varied from 294

 

at two days duration to 260 at fifteen days duration, while
the per cent of activity incorporated was 0.06% and 0.08%,
respectively. With the dwarf marigolds (not a Tagetes
species) the dilution factor was 161 and decreased to 77 at
five and ten days, reSpectively, while the per cent of
activity incorporated increased from 0.04 to 0.06%. The age
of the Tagetes and dwarf plants was 5 and 1.5 months. The

results of the above experiments are summarized in Table 10.

45

46

TABLE 10.--The Incorporation of Sodium Sulfate-S-35
into Terthienyl in Marigold Rootsd

 

 

 

 

 

 

Total
Method Weight Total Activity
Experiment Time, of of Terthienyl, DiSpensed,
Number Days Feeding Roots g. mg. 0pm
1 2 Stem 0.45 18 3.58 x 107
2 2 Root 1.55 67 1.99 x 107
3C 5 Root 2.30 57 8.94 x 107
4c 10 Root 2.73 28 11.8 x 107
5 15 Root 3.91 21 4.21 x 107
Total Activity (cpm)
Terthienyl,cpm
Experiment Ethanol Petroleum Nutrient
Number Extract Ether Extract Solution Total /g.Root
1 61200 3800 None None --
2 372000 65600 b 12700 8190
3 929000 243000 a 30400 12300
4 1690000 a 2.64 x 106 70400 25800
5 a 252000 6.67 x 106 32300 8260

 

aNot measured for radioactivity.

b2ml. aliquot was too ”hot” for radioactive

assay.

CDwarf variety of plants, exact species unknown.

dExperiments l, 2, and 5 were Tagetes, age 21 weeks; Experi-
ments 3 and 4 were dwarfs, age 7 weeks.

47

L-Methionine-S—35

 

The purpose of Experiments 6 through 10 was to see if
sulfur from methionine-S-35 was incorporated into terthienyl,
to observe the effect of the age of the plant on the incor-
poration, and to compare the feeding via stems with root
feedings.

The older plants took more radioactive sulfur into the
roots than the younger plants as shown by the total activity
in the ethanol extracts. No radioactivity was found in
terthienyl until 17 days had elapsed from the original feeding.
This amount of radioactive terthienyl represented an incorpor-
ation of 0.003% of the activity, and the diultion factor was
60,200. The results of these experiments are summarized in

Table 11.

DL-Methionine-l-C—l4

 

The first carbon compound investigated as a possible
precursor to terthienyl was methionine labelled in the Cl
position. These studies are represented in Experiments 11
through 13. The labelled compound was fed via stems. None
of the radioactivity was found in terthienyl after 10-19 days
from the original feeding. This data is summarized in

Table 12.

DL—Methionine-2-C—14

 

Experiments 14 through 16 represent studies to deter-

mine if carbon-14 of methionine located in the 02 position

48

TABLE ll.——The Incorporation of L-Methionine-S-35

into Terthienyl in Marigold Rootsa

 

 

 

 

 

Experi- Weight Terthienyl Added
ment Time, Plants of to Total Cpm
Number Days Age,Mo. Roots,g. Ethanol Extract Dispensed
6 2 4 3.14 20 mg. 4.58 x 107
7 2 1.5 2.45 20 mg. 5.03 x 107
8 6 1.5 2.61 25 mg. 4.02 x 107
9 12 1.5 2.22 None 3.02 x 107
10 17 1.5 2.16 None 2.91 x 107
Tot 1 A t t ‘
Experi- a C 1V1 y (0pm) Corrected Total cpm
ment Ethanol Nutrient Activity in
Number Extract Solution Thread Dispensed Terthienyl
6 72200 82800 1.22x107 3.36x107 None
7 34100 30700 2.72x107 2.31x107 None
8 131000 11400 2.69x107 1.33x107 None
9 2390000 26400 1.99x107 1.03x107 None
10 37500 163000 1.12x107 1.79x107 600

 

aAll experiments were stem feedings.

49

TABLE 12.—-The Incorporation of DL—Methionine—l-C—l4
into Terthienyl in Marigold RootsC

 

 

Experiment Time, Weight of Total Total cpm
Number Days Roots, g. Terthienyl, mg. Dispensed
11 10 4.03 25.5d 2.71 x 107

12 15 3.02 20.8a 2.07 x 107

13 19 0.35 20.8a 2.07 x 107

 

Total Activity (cpm)
Experiment Ethanol Petroleum Nutrient Total Cpm in

 

 

Number Extract Ether Extract Solution Terthienyl
11 22200 40 8700 None
12 b b 66100 None
13 47000 2 16800 None

 

8Amount in fraction from alumina chromatography.

bActivity not determined.

CStem feedings.

dEthanol extract.

50

is a precursor to terthienyl when fed via roots instead of
the stems. Radioactivity was found in terthienyl only in

the two day experiment. All of the activity of terthienyl
was eliminated after this time. The dilution factor obtained
for methionine—2-C-l4 for the two day experiment was 1,430
which represented a total incorporation of 0.01%. The total
radioactivity of the roots as shown by the activity in the
ethanol extracts, petroleum ether extracts, and the crude
terthienyl fractions decreased with time. The results of
these experiments are summarized in Table 13.

The diethyl ether extract of the aqueous nutrient solu-
tion was investigated further. A portion of the radioactive
ether solution was analyzed by chromatostrip chromatography.
One blue fluorescent area was extracted from the chromato-
strip. UV absorption was detected with the maxima at 335
and 251 millimicrons. No radioactivity was found in this
unknown compound. Paper chromatography of the compount in
70% aqueous methanol resulted in an Rf—ratio with terthienyl

of 1.12.

Sodium Acetate-l—C-14

 

Sodium acetate labelled in the carboxyl position was
fed to the plants by both feeding methods: Experiments 17
through 19 via stems and Experiments 20 through 22 via roots.
Again, it was shown that the accumulated radioactivity in the

ethanol extract was less with the stem feedings. Also, from

51

TABLE 13.--The Incorporation of DL-Methionine-2-C-14
into Terthienyl in Marigold RootsZ

‘
L

 

 

 

 

 

Experiment Time, Weight of Crude Terthienyl Total cpm
Number Days Roots, g. Isolated, mg. DiSpenseda
14 2 3.39 0.104 1.78 x 107
15 10 3.22 0.134 1.33 x 107
16 20 3.13 0.032 0.88 x 107
Total Activity (cpm)
Experiment Ethanol Nutrient Crude Terthienyl, cpm
Number Extract Solution Terthienyl Total /g.Root
14 1600000 37900 10800 1210 357
15 872000 27800 3250 None --
16 312000 c 200 d --

 

aThe total activity was determined by assuming 40% counting
efficiency and indicating the activity fed as 0pm.

bDiethyl ether extract of the aqueous nutrient solution.

CActivity not determined.

dThe purified sample was lost.

52

the activity of the ethanol extracts in Experiments 20
through 22, the decrease of activity with time was observed.
This is seen in the decreasing amount of activity of the
crude terthienyl fraction. Radioactivity in terthienyl was
found only with the root feedings and only after ten days
from the initial feeding. The results are summarized in
Table 14. After ten days duration the amount of radioactivity
incorporated was 0.002%, and the dilution factor was 35,100.
At the end of 20 days the radioactivity incorporated doubled
to 0.004% while the dilution factor decreased to 10,200.

The nature of the radioactivity found in the diethyl
ether extract of the nutrient solution in Experiments 20-22
was investigated. Paper chromatography with 70% aqueous
methanol showed two compounds which moved from the origin.
One compound which showed a yellow fluorescence on paper
gave an Rf-ratio with terthienyl which varied from 0.63 to
0.91. The second compound which moved from the origin
showed a blue fluorescence on paper and had a relatively
constant Rf-ratio with terthienyl of 1.13. A qualitative
determination of radioactivity in the ten day experiment
indicated that the yellow fluorescent compound contained
only a trace, whereas the blue fluorescent compound con-
tained a significant amount of radioactivity (about 100 cpm
in an aliquote of the ether). This was determined by
eluting the spot from the paper chromatogram into a planchet
and counting the residue left from the evaporation of the

solvent.

53

TABLE l4.-—The Incorporation of Sodium Acetate—l—C—l4
into Terthienyl in Marigold Roots

 

Experiment Time, Weight of Crude Terthienyl Total cpm

 

 

 

 

 

Number Days Roots, g. Isolated, mg. Dispenseda
17 2 4.87 0.263 0.95 x 107
18 9 3.61 Unknown 0.98 x 107
19 17 3.82 Unknown 0.95 x 107
20 2 2.80 0.230 1.77 x 107
21 10 2.45 0.032 1.33 x 107
22 20 2.60 0.198 1.33 x 107

Total Activity (cpm)

Experiment Ethanol Nutrient Crude Terthienyl,cpm
Number Extract Solution Terthienyl Total /g.Root
17 15500 1470000 300 None -—

18 14800 2300 None None --

19 4700 7400 300 b -—

20 940000 22400 3600 None -—

21 186000 5400 1250 215 88
22 132000 6500 625C 785d 302

 

8Correction for activity remaining in the cotton thread in

17-19.
bTerthienyl not further purified.

CThe %SD of the sample counted was + 40: the value given
could range from 375—875 cpm.

dThe %SD of the sample counted was i 6.

54

Isot0pe Dilution Experiments

 

Several experiments were conducted in order to deter-
mine the original specific activity of terthienyl isolated
from the roots. The method chosen was constructed by Mayor
and Collins (20) and consisted of a double dilution method.
See the experimental section for details of the procedure.

Four different compounds were studied in this investi—
gation: DL-methionine-S-35, sodium hydrogen sulfide-S-35,
succinic acid-2,3-C-l4, and Dl—glutamic acid-2-C-l4. The
highest incorporation of activity into the roots, as shown
by the activity in the ethanol extracts, occurred with
methionine as the source of sulfur. The highest incorpor-
ation into the roots with carbon-l4 compounds was observed
with succinic acid. Only a trace of activity was found in
terthienyl from sodium hydrogen sulfide. None of the
activity from the carbon—l4 compounds was incorporated into
terthienyl. The per cent incorporation from methionine
amounted to 0.03%, and the dilution factor was 4,420. The
results are summarized in Table 15. The calculated specific

activity of terthienyl is shown in Table 16.

Investigationof 5—(3éButen—l-ynyl)-2,2'—dithienyl

 

 

Evidence of the presence of another known radioactive
compound, 5-(3-Buten-1—ynyl)-2,2'-dithienyl, was observed in
Experiment 4. Terthienyl was collected in fractions 3 and 4
from the alumina column, and the dithienyl compound was

collected in fraction 2 prior to terthienyl. The pr0perties

TABLE l5.--The Incorporation of Sulfur-35 and Carbon-14
into Terthienyl as Determined by Isot0pic

Dilution Methods

 

Activity (cpm)

 

 

Experiment Time, Weight of Ethanol Nutrient Total 0pm
Number Hours Roots, g. Extract Solution Dispensed

23 32 1.98 1150000 0.50x107 3.80x107

24C 7

(a) 40 1.32 29200 2.42x10 3.33x107

(b) 30 7.85 5280000 3.00x107 5.96x107

25 44 0.84 297000 0.37x107 2.22x107

26 49 1.11 19500 0.58x107 0.70x107

 

CTwo different experiments.

56

TABLE l6.-—Weights and Specific Activities of Isolated
Terthienyl in Experiments 23—26

 

 

Experi— Specific
ment Terthienyl Activity Terthienyl
Number Compound MicroM.C cpm/mMoleC opm/g.Root
6
23 Methionine 5.14 .99 x 10 2600

24d Sodium bisulfide

(a) -- -- --

(b) -- -- -_
25 Succinic acid -- -_ __

26 Glutamic acid -- -- --

 

CCalculated according to the double dilution method.

dTwo different experiments.

57

are listed in Table 17 with terthienyl as a comparison,
The ditheinyl compound from this experiment was
believed to correSpond in structure to that of compound

XVIII (6). Further evidence was

/ \ \ C:C-CH=CH2

S S

Compound XVIII 5-(3-Buten-l-yny1)-2,2'-dithienyl

presented from the L-methionine-S-35 experiments, when a
compound was eluted from the alumina column of the petroleum
ether solution which absorbed in the UV range, 343-344 milli-
microns in petroleum ether. The behavior on a paper chroma-
togram yielded the same relative Rf value to terthienyl as
that shown in Table 17.

In the two day experiment of L—methionine-S—35, the
total radioactivity found in the fraction containing compound
XVIII was 500 cpm. Whether the activity was due to the
dithienyl compound or impurities was not determined. The
evidence obtained from the sodium sulfate-S-35 experiment,
Table 17, suggests the incorporation of sulfur—35 into com-
pound XVIII.

Uhlenbroek and BiJloo previously reported that the
oxidation of compound XVIII produced a diacid, XXI, with

potassium permanganate in acetone. The fraction believed

58

TABLE 17.-—Pr0perties of the Dithienyl Compound Compared
with Terthienyl - Experiment 4

 

Potassium UV

3
Frac- Permang— Ethanol Rf Activity
tion Compound Isatin anate milli- Value 0pm
crons

 

2 Dithienyl- Violet Positive 346,254a 0.84 75300

3&4 Terthienyl Violet Negative 350,252 0.71 70400

 

aFrom another isolation experiment of marigold blooms, the
dithienyl compound was purified by chromatostrip chromato—
graphy, produced a violet color with isatin, and absorbed
in the UV at 347 millimicrons in ethanol and 341 milli-
microns in petroleum ether (30-60).

59

to contain the dithienyl compound was oxidized according to
their procedure. Because of the micr0gram quantity of
starting material, the identification of the diacid was shown
by its Rf value on paper chromatograms compared to known com-
pounds.(l7) Compounds XIX-XXI were chromatOgraphed with the

unknown sample. The results are given in Table 18.

Q COBH MCOQH H0260. COQH

XIX XX XXI

The 5—(3-buten-l-yny1)-2,2'—dithienyl fraction from
the alumina chromatOgraphy of the petroleum ether solution
was identified in experiments 20—22 by a positive isatin
test and paper chromatography with known terthienyl. The
Rf-ratio with terthienyl was 1.10. No radioactivity was
found in the 2 day and 20 day experiments. The dithienyl
fraction in the 10 day experiment was not measured for radio—
activity. Instead, the oxidation to the known 2,5—thiophenedi—
carboxylic acid was attempted.

Two trials of the oxidation from two different isola—
tions were completed. The starting material of the first
trial absorbed in the UV at 343 and 256 millimicrons (ethanol),
produced a violet color with isatin, and showed one Spot on

a paper chromatogram with an Rf value of 0.83 (terthienyl Rf

60

TABLE l8.--Paper Chromatography of Carboxylic Acids

 

Whatman #1 filter paper, ascending method

Reagent- 2,6-dichlorobenzenone-ind0phenol, pink Spots on
blue surface

 

 

Solvent A: butanol, pyridine, ethanol, and water (3:1:1:
Solvent B: methanol and water (7:3)

Compound Fizgggs' Acid Spot Solvent Fi88tfi8m. Rf

XIX None Pink A 11.5 0.81
XIX None Pink B 12.1 0.89
XX None Pink A 11.5 0.73
XX Blue None B 12.1 0.74
XXI None Pink A 11.5 0.41
XXI None Pink B 12.1 0.14

Unknown

trial 1 None Pink A 12.1 0.42
trial 2 None Pink A 11.5 0.05

 

61

of 0.75 or R —ratio to terthienyl of 1.10). The UV absorp-

f
tion of the crude oxidation product resulted in two shoulders
and one broad maxima at 333, 222, and 213 millimicrons,
respectively. The starting material from the second trial
produced a wine color with isatin and showed one Spot on a
paper chromatogram with an Rf value of 0.84 (terthienyl Rf

of 0.76 or Rf-ratio to terthienyl of 1.10). Paper chromato-
graphy of the starting material before oxidation showed no
acids to be present as determined from an aliquot. Paper
chromatography with solvent A, Table 18, of the crude product
of trial 1 showed a very faint pink spot with a Rf value of
0.42 and a blue fluorescent spot at the solvent front. Paper
chromatography of the oxidation products from trial 2 showed
a faint pink spot with an Rf value of 0.05.

An aliquot of the reaction mixture from trial 1 was
applied to a chromatostrip. Development was carried out
using 50% diethyl ether in n-hexane. Three different com-
pounds were present as shown by their blue fluorescence: A,
at the origin; B, intermediate between the origin and solvent
front; 0, at the solvent front. Each was eluted from the
chromatostrip with ethanol and diluted to 10 ml. The UV ab-
sorption for each compound was recorded: A, green fluores-
cence and gradual absorption from 250—400 millimicrons; B,
blue fluorescence and maxima at 243, 248, 254, and 260 milli-
microns; 0, blue fluorescence and two shoulders at 273 and

255 millimicrons.

62

In Experiments 14-16 the 5—(3—buten-l-ynyl)-2,2'-dith-
ienyl fraction from the alumina chromatography of the petroleum
ether solution was identified by its wine color with isatin,

Rf value from paper chromatography, and the UV absorption
which varied from 340—344 millimicrons. N0 radioactivity
was detected in the fraction in the 2 day experiment or the
10 day experiment, but a small amount was found after 20

days (400 cpm).

DISCUSSION

A convenient way of representing the incorporation of
suSpected precursors into terthienyl was the use of a "dilu—
tion factor” of specific activity. This was calculated by
dividing the Specific activity of the compound fed to the
plant by the Specific activity of the isolated terthienyl.
The data in Table 19 show the dilution of sulfur—35 adminis-
tered either as sodium sulfate or methionine varied from 77
to 4,420. The dilution factors obtained from the carbon-l4
compounds were from 1,430 to 35,100. Since the least dilu-
tion was received with sodium sulfate-S-35, the sulfur was
incorporated near the final step in the biosynthetic pathway
as far as the sulfur atom is concerned.

However, the data does not indicate that the carbon—14
precursors have been conclusively demonstrated. The least
dilution of methionine—2-C-l4 (1,430) showed it to be closer
to the final step of the carbon-l4 pathway than acetate-l—C—14
(10,200-35,100). The difference in the Specific activities
of isolated terthienyl from the sulfur-35 compounds as com-
pared to the carbon—l4 compounds differed by a factor of 102
(sulfur-35 greater than carbon—l4), and the dilution factors
also differed by 102. The similarity in the differences of

the dilution factors and Specific activities of the isolated

63

64

TABLE l9.—-Specific Activity of Isolated Radioactive
. Terthienyl

 

Assumption: Terthienyl concentration = 0.444 microM/g.Root

 

 

Experi- Specific
ment Feeding Activity Time, Terthienyl Dilution
Number Method Isot0pe cpm/mM Days cpm/mM Factor
1 Stem sou —- 2 . 0 __
2 Root s04 5.42x109 2 1.84x107 294
3 Root sou 4.46x109 5 2.77x107 161
.4 Root so“ 4.46x109 10 5.80x107 77
5 Root sou 4.83x109 15 1.86x107 260
10 Stem Me-S 37.6 x109 17 6.25x105 60,200
13 Stem Me-Cl -- l9 0 --
14 Root Me—C2 1.13x109 2 7.88x105 1,430
16 Root Me—Cg -— 20 0 ~—
19 Stem Ac—Cl —- 10 0 —-
20 Root Ac-Cl -- 2 0 ——
21 Root Ac—Cl 6.96x109 10 1.98x105 35,100
22 Root Ac—Cl 6.96x109 20 6.80x105 10,200
23 Root Me~S 4.38x109 1.3 .99x106 4,420a

 

aValue from isot0pic dilution experiment.

65

terthienyl indicated that both methods of showing incorpora-
tion were giving similar results as to what was actually
occurring in the plant.

Dilution factors were given by Griffith and Byerrum
(22) from the incorporation studies of acetate—l-C-l4,
acetate-2—C-l4, pyruvate-l-C—l4, and pyruvate-3-C-l4 into
nicotine in tobacco plants. The least dilutions occurred
with acetate-2—C-14 and pyruvate-3—C-l4 and ranged from
243-550. The highest dilution was received from pyruvate—
C—l4 (8,370). They stated that incorporation was small for
the compound with the dilution factor of 8,370. An inter-
mediate value was obtained with acetate-l-C-l4 (911 and 981);
thus, acetate was incorporated when labelled at either the
first or second carbon position. The significant conclusion
to be made from a consideration of the data of Griffith and
Byerrum in relation to the present work is that the dilution
factors obtained from sodium sulfate-S-35, methionine-S-35,
and methionine-2-C—l4 indicate incorporation into terthienyl.
The high dilution factors of acetate—l-C-14 suggest that no
incorporation was achieved, at least as a direct precursor
is concerned. The small amount of radioactivity in terthienyl
could arise from randomization of the acetate carbon atoms.
The summary of data in Table 20 shows that acetate-l-C-l4 was
incorporated to an extent of 0.002% after 10 days and increased
to 0.004% after 20 days. The present data does not conclu-

sively show that randomization has occurred. The question

66

TABLE 20.--Per Cent Incorporation of Radioactivity
in Terthienyl

 

 

% Incorporation

Terthienyl cpm / 0pm fed x 100

 

 

Experi-
ment Total cpm Total cpm in Incorporation
Number Compound DiSpensed Terthienyl
1 son: 3.58 x 107 0 0
2 SC“: 1.99 x 107 12700 0.06
3 SC),: 8.94 x 107 30400 0.04
4 sou: 11.8 x 107 70400 0.06
5 S04= 4.21 x 107 32300 0.08
10 Me-S 1.79 x 107 600 0.003
13 Me—Cl 2.07 x 107 0 0
14 Me—C2 1.78 x 107 1210 0.007(0.01)C
16 Me-C2 0.88 x 107 a 0
19 Ac-Cl 0.95 x 107 b 0
20 Ac—Cl 1.77 x 107 0 0
21 Ac—Cl 1.33 x 107 215 0.002
22 Ac-Cl 1.33 x 107 785 0.004
23 Me-S 3.80 x 107 5200 0.015(0.03)C

 

aSee Table 13.

bSee Table 14.

cPer cent incorporation

was not utilized.

was doubled assuming that the D-form

67

could be solved by conducting further time studies or by de-
grading terthienyl-C-l4 to locate the position of the labelled
carbon atoms when acetate-l-C-l4 is fed.

The maximum time of incorporation of sodium sulfate—S-
35 into terthienyl was not a simple question to answer as
seen from the results summarized in Table 10, 19, and 20.
First, using Tagetes plants of age 5 months, the incorpora-
tion increased in a period of 2-lf3days from 0.06% to 0.08%.
The dilution factors of 294 and 260, respectively, represented
about equal dilutions of specific activity. If terthienyl
were synthesized and stored in the plant, an increase of
Specific activity would be expected; however, it was found
that the value of the Specific activity of terthienyl was
practically identical after 15 days. One must conclude
that 92.22X2 synthesis of terthienyl was accompanied by a
breakdown to further metabolic products. The rate of syn-
thesis was equal to the rate of breakdown under the experi—
mental conditions, since a constant specific activity was
observed at the two time intervals. The conversion is
diagramatically represented in Scheme 5.

The symbol, k1, forward rate of synthesis, may include
an indefinite number of steps: k2, reverse of the forward
rate, may also represent the same number of steps: k3 repre-
sents the irreversible breakdown of terthienyl. If radio—
active sulfur is readily available for incorporation, after

administration of the radioisotOpe, k1 is greater than

68

Scheme 5

Biosynthetic Pathway of
Sodium Sulfate-S-35 to Terthienyl

804:

/\ /\ /\
3 .s .5

k3

Metabolic Products

69

k2 + k3 with reSpect to sulfur-35. If sulfur-35 has
"saturated" the biosynthetic pathway after the period of
"rapid" incorporation, the breakdown of terthienyl-S-35
equals it synthesis, kl = k2 + k3, as shown by equal Specific
activities in Table 19. This leads to the conclusion that
terthienyl is not stored as an inert compound in 5 months

old Tagetes plants, and that the time of the maximum peak of
incorporation of sodium sulfate-S-35 into terthienyl does

not exceed two days.

The results of the incorporation using younger plants,
but not a Tagetes variety, were quite different from the
previous results. At the end of 5 days after feeding, the
dilution factor of terthienyl was 161 and decreased approxi-
mately by half at the end of ten days to 77. The per cent
incorporation increased from 0.04% to 0.06%. These results
can be explained by considering Scheme 5. Since the Specific
activity of terthienyl doubled when the time of incorporation
was doubled, kl is greater than k2 + k3. Either terthienyl-
S-35 was being stored in the younger plants, or k3 was con-
siderably slower at this time. The results still are in
agreement with the above conclusion that terthienyl is an
"active” compound, capable of undergoing further metabolic
transformations. The high increase of radioactivity may be
due strictly to the changes in rates of over-all synthetic VS-

breakdown pathways, kl vs k2 and k3.

70

The data does not reveal precisely the ideal conditions
for maximum incorporation. Young plants are more suitable
because of their metabolic activity as evidenced from the
high incorporation of sodium sulfate-S-35 into terthienyl
with time.

It is possible that the peak of highest Specific activ-
ity of terthienyl-S-35 in young plants is near 10 days, and
that breakdown of terthienyl-S-35 may begin to equilibrate
and attain equilibrium conditions where k1 = k2 + k3. Such
a condition can be represented by the curves in Figure 2
taken from the results previously described. If the rate of
synthesis of terthienyl predominates over the rate of break-
down, a straight line will result as shown in both curves.
If the younger plants are synthesizing terthienyl more
actively than the older plants as is shown in Figure 2, a
higher specific activity would result. As the age of the
plants increases, the amount of terthienyl—S—35 should be
less at equilibrium, because of k1 becoming slower and/or
k2 and k3 increasing, so the equilibrium condition of kl =
k2 + k3 is attained.

The feeding method was important, since feeding via
stems did not result in any incorporation into terthienyl.
Even the amount of activity incorporated into the ethanol

extract was less in the 2 day experiments; 13.6 x 104 cpm

17

via stems and 24.0 x 10 cpm via roots per g. of root

extracted after feeding sodium sulfate-S—35.

71

Figure 2.--Incorporation of Sodium Sulfate-S-35

 

 

into Terthienyl. Specific Activity of
Terthienyl-S-35 vs Time

-’-’-
“

/(—————- Dwarf (1.5 months)

Tagetes (5 months)

 

@_._ .—

72

The importance of the feeding method was demonstrated
using L-methionine-S—35. A small incorporation of sulfur-35
'into terthienyl was observed; however, the dilution factor
was 60,500. This indicated that sulfur-35 from methionine
which was present in the upper portion of the plant was not
readily available for introduction into the biosynthetic
pathway. The higher dilution factor can be compared to that
obtained from DL-methionine-S-35, which was administered via
roots yielding a value of 4,420. A comparison of plant ages
was observed in Experiments 6 and 7 which showed that more
than twice the activity was found in the ethanol extract of
the roots at the end of 2 days in the 4 month old plants.
The activity of the ethanol extract in Experiment 9 cannot
be explained, except that an error in the radioactivity
assay was possible.

Since root feeding was necessary in order that
sulfur-35 could be incorporated into terthienyl, it was con-
.cluded that the active Site of synthesis of terthienyl was
in the roots. Stem feedings in Experiments 1 and 6-10
resulted in essentially none of the sulfur-35 being incor-
porated regardless of the original form, sodium sulfate or
methionine. Incorporation of sulfur-35 was observed from
sodium sulfate and methionine from root feedings as seen in
Experiments l-lO and 23, the movement of sulfur-35 from the
stems to the roots was less than from the external medium

(nutrient solution) into the roots.

73

A comparison of root vs stem feedings was also observed
with carbon-l4 which was administered as DL-methionine-l—C-l4
via stems and DL-methionine-2—C-l4 via roots. None of the
radioactivity was incorporated into terthienyl from stem
feedings as seen in Experiments 11-13. Little of the carbon-
14 was present in the ethanol extract of the roots after 10
days, although the amount of activity approximately doubled
at the end of 19 days. Even then, no carbon—l4 was detected
in terthienyl. This would be expected if the carbon-l4 com-
pound did not reach the site of synthesis. In contrast, the
results obtained from root feedings was different. After two
days carbon-l4 was incorporated into terthienyl giving a
dilution factor of 1,430. At 10 and 20 day intervals no
incorporation was observed, thus, supporting the conclusions
from the sulfur-35 experiments.

If one considers a biosynethtlc pathway with respect
to carbon-l4, Scheme 6 can be constructed. The symbols,
k1, kg, and k3, have the same definitions as those given for
Scheme 5. One must assume that methionine—2-C—l4 is metabo—
lized by the plant either to terthienyl-C—l4 or other products;
The lack of any activity in terthienyl after 10 and 20 days
indicates that terthienyl was not stored but actively metabo—
lized. Introduction of methionine-2-C—l4 in Scheme 6 results
in a "wave" of activity traveling through the entire pathway.
The evidence of the "wave" is seen by the specific activity

of terthienyl at the end of two days and declining until all

74

Scheme 6

Biosynthetic Pathway of DL-Methionine—
2-C—l4 to Terthienyl

Metabolic Products

75

of the terthienyl—C-l4 is further metabolized. If methion—
ine-2-C-l4 were available for incorporation all the time, a
leveling of Specific activity would be seen as discussed
above for the sodium sulfate experiments. The assumptions
that sulfur—35 is constantly available from sodium sulfate-
S-35 and that carbon-l4 from methionine is only available
during the initial period of incorporation seems reasonable.

In Experiments 17—22 carbon-l4 from sodium acetate—
l-C-l4 was not incorporated into terthienyl when fed via
stems. Some incorporation into terthienyl was observed when
administered via roots (0.002-0.004%). The activity of the
ethanol extracts of the roots is approximately 10 times
higher from the root feedings, indicating that carbon-l4
in the stems does not move toward the roots as fast as the
carbon—l4 from the external medium (nutrient solution). If
the assumption is made that acetate-l-C-l4 is metabolized
quickly as assumed with methionine, then the same pattern
of specific activity of terthienyl could be expected. No
incorporation after two days and only trace incorporation
after 10 and 20 days indicated that acetate-l-C-l4 was prob—
ably not a direct precursor, and that the activity in
terthienyl resulted from a spreading of activity throughout
the metabolites of the plant.

However, incorporation of radioactivity from acetate—
1—C—14 into the terthienyl fraction is worthy of further

discussion. Horn and Lamberton (16) reported the isolation

76

of glycerides of unsaturated fatty acides in their terthienyl
fractions from the roots of marigolds. They eliminated the
impurities by a saponification step before purification of
terthienyl by chromatography. Saponification was not utilized
in the current isolations, and the radioactivity in the crude
terthienyl may have resulted from acetate incorporation into
the unsaturated glycerides. The disappearance of radio-
activity in the 10 and 20 day experiments of the crude
terthienyl fraction and the increase of radioactivity in the
purified terthienyl may have some correlation to each other.
If acetate was a precursor to terthienyl and passed through
unsaturated fatty acids in the biosynthetic scheme, such an
observation as this is not unreasonable. 0n the other hand,
the maximum amount of radioactivity in terthienyl was 0.004%
and randomization of the carbon-l4 could be reSponsible for
this small amount of radioactivity which was found.

The results of the acetate experiments suggests further
investigations. Malonic acid—C-l4 may serve as a better
precursor to terthienyl if the true pathway proceeds through
an "acetate derived” carbon compound such as a fatty acid or
a derivative. Bu'Lock and his coworkers (25) have shown that
diethyl malonate-2-C-l4 is incorporated by 6% into oleic and
palmitic acids and 2—6% into an aromatic, 6-methyl salicylic

acid, in fungus cultures of Penicillium urticae. The two

 

fatty acids and the aromatic compound are "acetate derived"

and were observed to incorporate the activity from the

77

malonic acid derivative. Bu'Lock and Smalley have extended
their work to the polyacetylenes by feeding diethyl malonate-

2—0-14 to fungus cultures of a Basidiomycetes, Tricholoma

 

grammOpodium. An incorporation of 0.1% was observed into

 

the compound, dec-2-en-4,6,8-triyne—l-ol.

CH - C=C - CEC - 030
3 " ‘\\5‘V\‘CH20H

Compound XIX Dec-2-en—4,6,8-triyne-l-ol

The activity located in 01-08 was 97% which indicated that

09 and 010 served as the "starter group" with the remainder
of the compound being formed from malonate.(26) A scheme

was suggested by Bu'Lock for the formation of the three dif-
ferent "acetate derived" compounds and is shown as Scheme 7.
It provides a method of forming triple bonds which has also
been postulated by Jones (27) and demonstrated biogenetically
in the laboratory by Fleming and Harley-Mason (28, 29). The
same type of malonate conversion has been observed by Bentley
and Keil (34) in the biosynthesis of penicillic acid in the

fungus, Penicillium cyclopium.

 

Bu'Lock and his workers have followed the rate of
synthesis from different precursors of polyacetylene anti-
biotics in Basidiomycetes B. 84l.(30) Using acetate and
ethanol as precursors, it was estimated that 90% of the
ethanol was utilized to polyacetylenes by the way of acetate.

Glucose was also consumed at equivalent rates, but the

78

Scheme 7
Biosynthesis of ”Acetate Derived” Compounds (26)

Acyl thiolester + Malonyl-Co A

l

Enol derivative é—-' Acyl malonyl thiolester
[ ‘COQ
Beta-ketoacyl thiolester Cylization

v ’00? i

Polyacetylenes Fatty Acids ' Polyketides

 

79

conversion to polyacetylenes was almost six times less than
that of ethanol. They concluded that the conversion of
glucose to polyacetylenes consisted mainly of a pathway not
involving acetate. Feeding experiments are currently being
conducted in these laboratories with marigold plants using
uniformly labelled glucose as a suspected precursor to
terthienyl.

The isot0pe dilution experiments were completed only
with DL-methionine-S—35. The isolated terthienyl was calcu-
lated by this method to establish the original Specific
activity. The quantity of terthienyl isolated as shown in
Table 16 is high in comparison to the previous values,
Table 20. Even for this high amount, the specific activity

of approximately 106

cpm/mM produced a dilution factor of
4,420. It was concluded that sulfur-35 was incorporated
when supplied as methionine, but the incorporation is about
1/10 that of sodium sulfate. It was surprising that sulfur-
35 was not incorporated when fed as sodium hydorgen sulfide-
S-35. One explanation is that sulfur fed in the reduced
form was not available at least during the short term experi-
ments (30 hours or less).

No carbon-l4 was incprorated from succinic acid-2,3-C-l4
or DL-glutamic acid-2-C-l4. The lack of incorporation from
succinate appears to eliminate compounds of the tricarboxylic

acid cycle as precursors to terthienyl. Glutamic acid was

investigated because it was readily available.

80

With reSpect to Schemes l and 2, the results obtained
in this investigation do not lend support to either. It is
better perhaps to speak of these schemes as involving an
"acetate" pathway, since some polyacetylenes have been shown
by Bu'Lock to be composed of head-to—tail condensations of
acetate. An "acetate" pathway does not distinguish if
polyacetylenes or polyketones are involved in the biosyn-
thesis of terthienyl.

There is some support for a type of pathway represented
in Scheme 4. The results show that sulfur-35 and carbon-14
when supplied as methionine were not incorporated to the
same extent. The dilution factor of methionine—S-35 calcu-
lated from the isot0pe dilution method was 4,420, but was
750 when calculated in the same manner as Experiments’245,
that is, assuming the concentration of terthienyl as 0.44
micromoles per g. of root. The value of 750 is still about
three times greater than that of sodium sulfate-S-35. It
could be that sulfur-35 is a precursor to terthienyl through
methionine (or homocysteine) and also by another pathway.
This would explain the higher dilution factor resulting from
methionine-S-35. Experiments l4 and 23 support Scheme 4.
Additional evidence is needed and should be obtained by
feeding methionine-2-C-l4, isolating terthienyl—C-l4, and
degradating the compound to locate the position of the label.

5-(3—Buten-1-ynyl)-2,2'—dithieny1 was identified from

its UV absorption, color test with isatin, positive test with

81

permanganate, and the identification of its oxidation product
by treatment with potassium permanganate in acetone. The UV
absorption obtained in this work was usually 2-6 millimicrons
higher than that reported in the literature. When the
dithienyl compound was isolated from several kg. of blooms
during the summer, absorption in petroleum ether was 341
millimicrons, and this shifted to 346 millimicrons in ethanol.
Two explanations could account for this behavior; first, sol-
vent effects of the polar ethanol compared to the nonpolar
petroleum ether, and Second, a reaction which occurred such
as polymerization was seen by the appearance of small, yellow
balls which were present when the petroleum ether was evapor-
ated and replaced with ethanol. These yellow balls, insoluble
in ethanol, were also reported by Horn and Lamberton.(l6)
Therefore, the shift to longer wave-lengths may have resulted
from a change of structure and not solvent effects.

The identification of the thiOphene 2,5-dicarboxy1ic
acid was not conclusively shown. The oxidation product was
Shown to be present in trial 1 but could not be detected in
trial 2. If the dithienyl compound polymerized before the
oxidation procedure, then the acid probably would not be
formed. Uhlenbroek and Bijloo previously identified the
product from its UV absorption, but could not investigate
the compound further because of the small amount available.

Experiment 4 using sodium sulfate-S-35 showed about the

same amount of radioactivity in the dithienyl compound as in

82

terthienyl, Table 14. The Specific activity was not deter-
mined. A trace of activity, 500 cpm, was observed in the
dithienyl fraction in the two day experiment of L-methionine-
S-35 when fed through the stems. The activity was not im-
portant, because the purity of the fraction was unknown with
reSpect to the radioactivity. A trace of activity, 400 cpm,
was found after 20 days from the feeding of DL-methionine-2-
C-l4 via roots. A randomization of carbon-l4 could account
for this small amount of radioactivity.

From the results shown in Table 17, it is seen that
5-(3-buten-l-ynyl)-2,2'-dithienyl might be relatively near
terthienyl in the metabolic scheme. It could be only one
step away. Further work is needed to determine whether it
is in the biosynthetic pathway, or if it is a degradative
product of terthienyl.

It has been shown by Uhlenbroek and Bijloo (23) that
one of the two active nematicidal principles of the Tagetes
is terthienyl. They reported that cultivation of Tagetes

reduced the populations of the nematode, Pratylenchus spp.,

 

and that concentrates from the roots showed higher activity
than any other part of the plant. Oostenbrink and his 00-
workers (31) reported the cultivation of Tagetes for short
periods in the Spring or autumn had no effect on nematodes
in the soil, whereas a period of 3-4 months cultivation was
effective against nematodes. These results were confirmed

in the laboratory by 0midvar who leached roots of 8-10 week

83

 

 

old plants of Tagetes minuta, Tagetes florida, and Tagetes
signata. (32) He saved the diffusates and observed no
nematocidal effect. His short term experiments were consid-
ered ineffective because of the low amounts of terthienyl
being produced.

If Tagetes are able to reduce p0pulations of nematodes
by the cultivation of the plants, it must be assumed that
terthienyl is liberated by the roots into the soil. The
presence of terthienyl in the nutrient solution was investi-
gated. If terthienyl was present in the nutrient solution,
it was not detected even by paper chromatography. Instead,
a blue fluorescent compound was discovered which absorbed in
the UV in such a way as to indicate a dithienyl derivative,
335 and 251 millimicrons). The dithienyl type structure was
also suSpected because of the compounds behavior on a paper
chromatOgram using 70% methanol as solvent (Rf-ratiO'with
terthienyl of 1.13). The compound was radioactive after 10
days when acetate-l-C-l4 was administered.

It is possible that its structure is similar to thatcm‘
5 -(3-buten-l-yny1)—2,2'-dithieny1. In Sorensen's attempt
to identify this or a similar compound, a number of deriva—
tives were synthesized, and their UV absorptions were observed,
Table 21.(33) The unknown compound in the nutrient solution
could have a 4 or 5 carbon chain as a substituent group and
unsaturation which might be similar to one of the first 4 R-

groups listed in Table 21. When Tagetes are cultivated in the

84

TABLE 21.--UV Absorption of Some Dithienyl Compounds

R—Group

Millimicrons

340

335

339

Unknown

343

85

soil, it is conceivable that the above compound is converted
to terthienyl or some other nematocidal compound by soil
bacteria or by the nematodes themselves. Further identifi-
cation of the compound may prove interesting. The Specific
action of the effect of Tagetes on nematodes remain specula-
tive according to the literature in that field.

The concentration of terthienyl is variable among
difference varieties, ages of plants, and apparently whether
isolated from petals or roots as shown by the results in
Table 22. Zechmeister noted at the time he first isolated
terthienyl in 1947, that none could be obtained from another
Species of Tagetes. Uhlenbroek and Bijloo suggested that he
did not investigate the roots, but only the petals. Concen-
tration differences are seen from the data in Table 22,
particularly in the dwarf variety and in the two Tagetes
species. Another factor must be considered with respect to
the dwarf variety, that is, the time of the year that terth-
ienyl was isolated. It is possible that terthienyl may be
stored in the plant as the growing season enters the later
maturation of the plant. The data in Table 22 Show the
higher concentrations or terthienyl in the roots compared to
the petals.

The conditions which were present in this work were
similar to that of the isolation from 2 month old Tagetes
erecta yielding approximately 112 microg. per g. of root as

given in Table 22.

86

TABLE 22.--Weight of Terthienyl Isolated from Various
Species of Marigolds

 

 

 

 

 

 

 

Time Weight of Terthienyl Refer-
Species Harvested Roots, kg. Microg./g.Root ence
Tagetes erecta Unknown 14, petals 15 (l)
Tagetes erecta Unknown 24, roots 23 (6)
Tagetes minuta Unknown 0.9, roots 200 (16)
Tagetes erecta 2 mo. 0.0004,roots 112 This work
Dwarf June 5.5, petals 0.7 This work
Dwarf August 6.7, petals 22 This work

 

87

The investigation of dandelion blooms was initiated in
order to isolate terthienyl. The results showed that terth-
ienyl was present in very low quantities. The low amount in
the fraction from the alumina column prevented further iden—
tification of the compound suspected to be terthienyl; there-
fore, the presence of terthienyl in dandelion blooms was not

conclusively proven.

APPENDIX

Investigation of Dandelion Blooms

 

In the Spring of 1962, 2.7 kg. of yellow dandelion
blooms were picked from the lawns of the Michigan State
University campus. They were immediately covered with 20 1.
of 95% ethanol in a large crock and allowed to stand for
13 days. The ethanol was evaporated under reduced pressure
leaving a dark, vicous residue. The solid was redissolved
in 1.Z. of methanol with 50 g. of potassium hydroxide, and
the solution was refluxed for 60 hours. The saponification
mixture was diluted with water and extracted with petroleum
ether. The ether solution was concentrated to 50 m1. and
dried over sodium sulfate. The dried ether solution was
chromatographed over an alumina: celite (25: 2.5 g.)
column. Fractions of 25 ml. each were collected.

Fractions 3 and 4 each gave a red color with isatin.
Paper chromatography of each fraction with a known sample
of terthienyl indicated that two different compounds were
present. The first compound was eluted in fraction 3, and
a trace of it was seen in fraction 4. The Rf value from
66% methanol was 0.78, terthienyl = 0.64. The second com-

pound in fraction 4 gave an Rf value of 0.64 with paper

88

89

. chromatography. Both compounds displayed a blue fluroes-
cence on paper under an ultra violet lamp. Further investi-
gation was not continued because of the minute quantities
which were available.

Three other compounds were eluted with 5-20% diethyl
ether eluant: 1, fraction 10 - colorless crystals, m.p. 73°
C; 2, fraction 14 — colorless crystals, m.p. l48—150° C; 3,
fraction 16 - yellow needles, m.p. l30-l33° 0. Compounds 1
and 2 were crystallized from methanol: water.

The compounds in fractions 3 and 4 showed similar
characteristics to thosecfi‘5*C3buten-l—ynyl)-2,2'-dithienyl
and terthienyl such as the elution time from the alumina
column, reaction with isatin, and their behavior on paper
chromatograms. Further investigation as to the identifica-
tion of these two compounds may prove interesting, since
Sorensen has stated that polyacetylenes and thiOphenes have

not been found in members of the Compositae which are "milk

 

containing” as was observed in this dandelion Species.

10.

11.

12.

13.
14.

15.

16.

17.

A SELECTED BIBLIOGRAPHY

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and J. W. Sease, J, Am, Chem. Soc. 69,

 

Birkinshaw, J. H. and P. Chaplan, Biochem. J. 60, 255
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Sorensen, J. S. and N. A. Sorensen, Acta. Chem. Scand.

12, 771 (1958)-

Sorensen, N. A. and E. Guddal, Acta Chem. Scand. 13,
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Uhlenbroek, J. H. and J. D. Bijloo, Rec. trav. chim.
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Jones, E. R. H. Proc. Chem. Soc. 199 (1960).

 

 

 

 

 

Bu'Lock, J. D. and E. F. Leadbeater, Biochem. J, 62,
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Bu'Lock, J. D. and H. Gregory, Biochem. J, 72, 332
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Bu'Lock, J. D., D. C. All ort, and W. B. Turner, J,
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Sorensen, N. A. Pure and Appl. Chem. 2, 569 (1926).

 

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Private conversation with the late Professor W. J. Haney
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FEB105

WHY WHY

 

 

 

 

"MMAMI