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A method for the synthesis of ring-labelled N15 tryptophan

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A method for the synthesis of ring-labelled N15 tryptophan

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Content A METHOD FOR THE SYNTHESIS OF RING-LABELLED
N15 TRYPTOPHAN
A Thesis
Presented to
the Faculty of the Department of Biochemistry
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
*>y
James E. Hood
August 1949
UMI Number: EP41305
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This thesis, w ritten by
...... J . a m e s . . . E ^ . . . H . o . Q . 4.............. *jjr°
under the guidance of hfi*&... Faculty Com m ittee, x
and approved by a ll its members, has been
presented to and accepted by the Council on
Graduate Study and Research in p a rtia l fu lfill
ment of the requirements fo r the degree of
Master of Science
E.S.Bogardms
.........Dean'
Faculty Committee
t Chairman
I*S*J
TABLE OF CONTENTS
CHAPTER PAGE
I. INTRODUCTION AND HISTORICAL REVIEW 1
Isolation and structural elucidation 1
Occurrence and dietary importance 2
The metabolism of tryptophan 3
Breakdown of kynurenine and kynurenic acid 3
The implication of pyridoxine in tryptophan
metabolism 4
Conversion to nicotinic acid 6
Motivation for the present experiment 11
II. METHODS OF SYNTHESIS OF TRYPTOPHAN 12
Syntheses of indole 12
The Heumann synthesis of indigo 14
The Tyson synthesis of indole 16
III. EXPERIMENTAL 18
The Heumann synthesis 18
The preparation of anthranilic acid from
phthalimide 18
The preparation of N-phenylglycine-o-
carboxylic acid from anthranilic acid 19
The preparation of indoxyl from N-phenyl-
glycine-o-carboxylic acid 21
The preparation of indole from indoxyl 22
CHAPTER
iii
PAGE
The Tyson synthesis 24
The nitration of toluene 24
The reduction of nitrotoluene to tolui-
dine 26
The formylation of ortho-toTuidine 27
The formation of indole 28
The enzymatic synthesis of tryptophan from
indole and serine 30
IV. DISCUSSION 35
V. SUMMARY 37
BIBLIOGRAPHY 39
LIST OP TABLES
TABLE PAGE
I. Summary of Data 31
CHAPTER I
INTRODUCTION AND HISTORICAL REVIEW
The amino acid, tryptophan, has presented the biochem
ist, nutritionist, and geneticist a most interesting problem
in its various aspectss its synthesis in plants and micro
organisms, its. metabolism within the animal body and micro
organism, and its dietary significance as an essential amino
acid and precursor of niacin.
I. ISOLATION AND STRUCTURAL ELUCIDATION
Although Its existence was known and its relation to
indole suspected for a number of years by means of its color
reaction with chlorine and bromine, its isolation was finally
achieved In 1901 by Hopkins and Cole (1). They developed
the glyoxylic acid color test, the "Hopkins-Cole Reaction,"
specific for the indole nucleus, and relied on this to guide
them in their subsequent isolation of tryptophan from an en
zymatic digest of casein. Their technic, that of forming the
mercuric sulfate complex has been the standard method of tryp
tophan isolation for a number of years and only recently has
it been challenged by a newer technic, developed In chromato
graphic analysis.
Tryptophan was synthesized by Ellinger and Flammand
in 1907 by means of a condensation reaction of indole aide-
hyde with hippuric acid, followed by hydrolysis,, showing
Its structure to be <*.-amino-3-indolepropionic acid (2).
CO0H
1
CH=CNHCOC6Hr
COOH
^HjNHCOCjH. CHO
Reduction
Hydrolysi
CH
C*HyCOOH
of-am ino - 3 -.m dolepropionic acid
Their work was influenced by an earlier observation of El-
linger that indole, present in the intestine and in putre
fied digests of proteins, probably was derived from trypto
phan.
II. OCCURRENCE AND DIETARY IMPORTANCE
L-tryptophan is found invariably in low concentration
in both animal and vegetable proteins, ranging from 0.5 to
2.5$ in the former; while some proteins are completely lack
ing in the amino acid, notably gelatin, elastin, and zein.
Its preparation from protein is rendered difficult by the
ease of its destruction or alteration in the hydrolysis re
action. It is destroyed by acid hydrolysis of protein and
racemized by alkaline hydrolysis; enzymatic hydrolysis avoids
these disadvantages and assures good yield of the ! I LM isomer.
The character of tryptophan as an indispensible amino
acid was established in early nutritional studies. In 1905
Willeock and Hopkins obtained the tryptophan-deficient protein,
3
zein, from corn by alcohol extraction* They fed this, as
the single source of protein, to growing mice, and observed
a rapid loss of weight, followed by death of the animals (3)*
Restoration of tryptophan to the diet greatly extended the
survival time* The more elaborate studies on rats conducted
by Osborne and Mendel"in 1925 gave final proof of the need
for tryptophan not only for growth but for maintenance of
body weight (4). These findings have been confirmed with a
number of animals, including man, demonstrating in every case
that exclusion of tryptophan from the diet results in a nega
tive nitrogen balance, followed by loss of appetite, decline
in weight, and eventual death of the animals* Secondary ef
fects observable in the rat are a lowering of the blood albu
min and globulin and the inducement of cataract formation*
III. THE METABOLISM OF TRYPTOPHAN :
Breakdown to kynurenine and kynurenic acid. "The ear
liest information of the metabolic pattern for tryptophan was
given us in the work of Ellinger (1904),, whereby It was estab
lished that kynurenic acid, OH
C00H
discovered many years before-by Liebig, was a breakdown pro
duct of tryptophan (5)• Kotake and Iwao in 1931 found that
another product, kynurenine, the existence of which was first
4
reported by Matsuoka and Yoshimatsu in 1925, was the inter
mediate in this breakdown of tryptophan to kynurenic acid
(6), Further investigation indicated the existence of spe
cies differences to complicate the picture. Thus it was
found that rats, guinea pigs, and dogs apparently synthesize
kynurenic acid from L-tryptophan, while man and cats do not*
Kynurenic acid, then, has come to be regarded as an end-pro
duct of tryptophan metabolism in higher animals, although its
formation is probably via a bypath utilized only in certain
species.
The implication of pyridoxine in tryptophan metabolism*
Another product of tryptophan became.known thru the work of
Lepkovsky and Nielsen whose interest was aroused by the find
ing of a yellowish-green pigment in the urine of rats main
tained on a pyridoxine-deficient diet (7). They were sub
sequently able to isolate and identify this compound as
xanthurenic acid*
The relationship of tryptophan and xanthurenic acid had been
previously adduced by Musajo, who had noted that the latter,
as well as kynurenic acid, was excreted by rabbits and rats
fed on a high protein diet (8). This was confirmed by Lep
kovsky, who found that xanthurenic acid excretion.ceased when
a low level tryptophan diet was fed to pyridoxine-deficient
V c o o h
rats, and reappeared again when normal anounts of the amino
acid were included (9). Other animals, e.g., dogs, pigs,
and mice (hut not chicks) exhibit the same phenomenon, viz.,
the excretion of xanthurenic acid oh a pyridoxine-free diet.
These results seemed to imply that pyridoxine was directly
concerned with, the metabolism of tryptophan, and that its
absence caused an alternative pathway to be employed, re
sulting in the formation of xanthurenic acid.
Further evidence for the relationship was observed by
Schweiger.t and coworkers in that the level of tryptophan in
the diet had an inverse effect upon the pyridoxine content
of the tissues of the animals (10). In fact, the feeding of
a high protein diet, or the increase in the amount of trypto
phan included in the animals1 rations apparently aggravated
the symptoms arising from the deficiency of the vitamin (11).
Sarma et al., were able to effect a retardation in the growth
of rats on a pyridoxine-deficient diet with the feeding of
indole or dl-tryptophan (12).
- ..... # *
More intensive efforts to^ determine' the exact function
of pyridoxine in the metabolism of tryptophan have yielded
results difficult of.interpretation. Schweigert and Pearson
reported that animals on a pyridoxine-free diet excreted less
niacin, the end-product of tryptophan metabolism (see below)
than those receiving the-vitamin (13). However, the restora
tion of pyridoxine to the experimental diet doesn’t bring
about the expected. Increase in niacin excretion, as shown
by Rosen et al. (14), and by Bell et al. (15). In the latter
study, however, it was found, surprisingly, that the excretion
of N-methylnicotinamide, a known derivative of niacin, did
increase. Hurt et al, found that pyridoxine deficiency has
no apparent short-term effect on the synthesis of niacin in
the liver, although the niacin level of an animal maintained
on a pyridoxine-deficient diet was significantly below that
of the normal animal (16).
Conversion to nicotinic acid. The problem of the
conversion of tryptophan to nicotinic acid had its beginnings
in 1945 when a group of workers, Krehl et al., studying the
role of com In inducing symptoms of nicotinic acid defi
ciency in the rat (retarded growth rate), observed that the
symptoms were directly affected by the level of casein in
the diet they were using (17). Relying on the supposition
that casein supplied certain dietary elements deficient or
absent in corn and that the nicotinic acid content of casein
was inadequate to account for this effect, they were led to
examine the amino acid content of the two sources. The notable
difference was speedily perceived, viz., that corn was almost
entirely lacking in lysine and tryptophan. Subsequent addi
tion of lysine in normal amounts to the corn diet produced
little effect; but the results with tryptophan were startling
(18). The deficiency symptoms were completely overcome and
normal growth was restored.
The initial discovery that tryptophan could substi
tute foh nicotinic acid in the diet of the rat was followed
rapidly by other experiments with the rat (19, 20, 21) and
with other animals: with chicks (22), with pigs (23), with
the dog (24), with horses (25), and with man (26), all re
sults tending to confirm the relationship.' The accumulating
evidence of tryptophan’s efficacy in counteracting the nico
tinic acid syndrome, led Perlzweig to suggest the probability
of its being a precursor of the vitamin, and the idea gained
ready acceptance (26).
Other workers now undertook to explore the pathway of
this postulated tryptophan to niacin transformation. The
first significant report in this connection was that of*
Bonner and Beadle (27). They succeeded in developing three
mutant strains of Heurospora crassa genetically blocked in
the biosynthesis of nicotinic acid. By crossfeeding experi
ments they were able to establish the sequence of the strains
in the synthetic process and to isolate but not identify
(beyond empirical formulae) two precursors of nicotinic acid,
intermediates in the process.
Subsequent investigation with this new tool of ana
lysis, the Heurospora mutant, uncovered other intermediates .
in the chain. Thus, it had been established previously that
kynurenine, i j /AScOGHlCHCOOK
U NH. *H'
was the Immediate breakdown product of tryptophan. Beadle
tested kynurenine for growth stimulation on a mutant which
required tryptophan for growth but was capable of producing
the nicotinic acid it needed from tryptophan, and found it
to be as active as its precursor (28). He thereby established
it as an intermediate.
It is to be noted that the conversion of tryptophan
to nicotinic acid requires a rather extraordinary chemical
rearrangement, for nicotinic acid has a pyridine nucleus
which is not present either in tryptophan or kynurenine. But
kynurenine has been shown to go to kynurenic acid and xanth
urenic acid in the dog, and since both these compounds have
a pyridine nucleus, Mitchell and TSyc thought that they might
be likely precursors of nicotinic acid. However, when these
compounds, together with a number of derivatives were incorp
orated in the medium of the tryptophan-requiring mutant they
were found to be inactive thereby indicating a biological
pathway not involving kynurenine (29).
These workers now undertook a fresh approach to the
problem. Relying upon certain principles of organic chemistry,
there appeared to be adequate reason for supposing that, in
the case of xanthurenic acid, oxidation at the number 8
9
position had preceded formation of the pyridine ring. Pos
tulating that a similar reaction might occur previous to
the formation of the pyridine ring in nicotinic acid, they
reasoned that kynurenine might undergo oxidation in this
case at the number 3 position to form the hydroxy compound,
or with oxidation of the side chain, 3-hydroxy anthranilic
This latter was henceforth synthesized and added to the me
dium of the tryptophan-requiring mutant and its activity was
found to be comparable to nicotinic acid (29). Thus another
link in the chain was exposed.
Bonner, in continuation of the analysis of one of the
two intermediates isolated by Bonner and Beadle (above), suc
ceeded in Identifying It with 3-hydroxy anthranilic acid, con
firming the work of Mitchell and Nyc (30). The other inter
mediate was found to possess a pyridine nucleus, indicating
that Its position is farther along in the chain of conversion,
i.e., hearer nicotinic acid. Its identity Is as yet unknown.
This laboratory in recent years had been interested
in the problem of the conversion of tryptophan to niacin and
certain experiments have been conducted to Investigate the
process. Whereas Mitchell and Nyc had examined the effect of
3-hydroxy anthranilic acid on the growth of rats, Albert et
acid.
XCOOH
OH
10
al* have shown that the feeding of this compound to the
animals results directly in the increased excretion of nico
tinic acid and N-methylnicotinamide (31). Hurt et al. have
established that the liver is one of the sites of the con
version, if not the principal one (16).
A summary of the intermediary metabolism of trypto
phan is as follows:
H H
'°"mt
c h 4 c h c o o h
KlHt
CH»CHC00H
Tryptophan
2.- Hydroxy Tryptophan
%|COCHtCHCOOH
J.... Ah,
COOH
OH
Kyna renin e
Kynurenic Acid
COOH
OH
X anthurenic Acid 3 -Hydroxyanthm nihe Acid
N‘ ~ Methyl nicotinamide
%comx
COOH
Hicofinamide Nicotinic Acid
11
IV. MOTIVATION FOR THE PRESENT EXPERIMENT
While the evidence that tryptophan is a precursor of
nicotinic acid has been, to this point, based upon feeding
experiments, and this is indirect (albeit convincing), re
cently investigation has been undertaken using a different
technic, that of labelling tryptophan and isolating the pro
ducts of its metabolism. Lepkovsky et al., using tryptophan
with C14 in the number 3 position of the indole nucleus ob
tained nicotinic acid with the C^4 in the carboxy group (32).
Some time ago this laboratory became interested in the
possibility of preparing labelled tryptophan. Our attention
was directed to the question of the fate of the nitrogen in
tryptophan. An examination of the structural formulas of
tryptophan, niacin, and the presently known intermediates
suggested superficially that this nitrogen in the indole nuc
leus of tryptophan became successively the primary amine of
anthranilic acid and the ring nitrogen of the nicotinic acid
molecule. Inasmuch as It is rather difficult, on the basis
of known organic reactions, to conceive of the mechanism for
the transformation of an aniline derivative to a pyridine,
the question was deemed worthy of investigation by this lab
oratory. It was felt that the problem could best be attacked
15
by preparing tryptophan with N in the indole nucleus. This,
then, became the project undertaken by this investigator and
the following results are herewith submitted.
CHAPTER II
METHODS OF SYNTHESIS OF TRYPTOPHAN
A number of syntheses of dl-tryptophan and its de
rivatives have been reported and among these are the tech
nical processes employed in md£ ing it commercially available.
However, recent studies of the enzymes involved in its bio
synthesis have suggested the possibility of an enzymatic syn
thesis, which would simplify the chemistry Involved and, in
addition, avoid the racemization obtained otherwise, i.e.,
it would permit preparation of the isomer alone.
In December of 1947 Umbreit et al. reported the pre
paration of an enzyme fraction which catalyzed the reaction:
indole + serine — -» tryptophan (33). They were unable to
obtain it in pure form, but their analysis showed that pyri-
doxal phosphate was required as a coenzyme. Mitchell and
Gordon undertook the work of isolation and eventually were
successful in obtaining a highly active preparation. Their
generosity in furnishing this worker with the details of their
procedure has enabled him to reduce the problem to the less
difficult one of the synthesis of N^-labelled indole.
I. SYNTHESES OF INDOLE
The choice of a synthesis of Indole was determined by
three considerations: 1) the possibility of introducing
13
isotopic nitrogen, using the compounds in which it is made
available, 2) the yields obtainable by a given-method, and
3) the limits of laboratory facilities. The many syntheses
which have been reported, approximately forty according to a
recent review (34), invariably employ a starting compound in
which the nitrogen is already incorporated. Thus the enquiry
is directed to the means of introducing the particular nitro
gen group into the compound.
An investigation of the methods for effecting nitrogen
substitution of an aromatic ring disclosed three possibilitiesx
first, nitration using concentrated nitric acid alone or in
combination with concentrated sulfuric acid; secondly, direct
amination under conditions of high temperature and pressure,
employing a nickel catalyst; and finally, formation of the
imide of an aromatic anhydride, followed by the Hofmann de
gradation to yield the amino-carboxylic acid.
At the time the work was undertaken isotopic nitrogen
was unavailable in the form of nitric acid and hence, any
synthesis which started with a nitro compound or one derived
from a nitro compound could not be considered. The second
method, i.e., direct amination called for the use of high
pressure equipment not available to us at the time, and this
method likewise had to be rejected. There remained, then,
only the third possibility, that of obtaining amination through
formation of the imide and subsequent degradation to the amind
14
compound. This procedure, fortunately, could be accomplished
tinder relatively mild conditions with good yield, and from
these considerations furnished the only feasible method at
our disposal.
While none of the possible syntheses which employ a
starting compound having an amino group mentioned the use
of this method nor appeared to be particularly adapted to the
use of it, it forms an integral step in an industrial process
for the manufacture of indigo. Examination of this process,
i.e., the Heumann synthesis of indigo, disclosed that it could
be applied to the preparation of indole by a modification of
the last step in the procedure. Moreover, phthalimide, the
product of the first step in the process, is one of the avail
able compounds having isotopic nitrogen and so it could be
used as the starting compound to reduce the nuMber of steps
in the procedure. These advantages seemed to render it es
pecially suited to our problem and hence was adopted for our
synthesis of indole.
II. THE HEUMANN SYNTHESIS OF INDIGO
With BaeyerTs original laboratory preparation of in
digo in 1870 the prospect of obtaining the dye synthetically
on an industrial scale evoked the efforts of a number of able
synthetic chemists. In 1897 Heumann obtained a patent on a
process, using naphthalene as a starting material (35).
15
This synthesis became the principal method for manufacture
of the dye and maintained this preeminence for many years,
eventually being superceded by a process, quite similar to
it, involving aniline as a starting material. The essential
steps in the Heumann synthesis are:
COOH
> 4 aS0u
Naphthalene
SO^SO
^ A ^ COOH
P hth alic Acid
" HtO
(3;
NaOO
Phthalic Anhydride PhThalii’ ntdg
COOH
NaOH
C4)
COOH S
n C1CH.COOH y '
V / NHx Nai C C ^ 'V ^N C H xC O O H
Anthranilic. Acid . ,H
H~pHenylqlycme-o-carbaxylit Acid
♦
It will be noted that indoxyl is formed in step 5,
In 1904 Vorlander and Apelt succeeded In reducing this com
pound to indole with sodium amalgam and with zinc dust and
alkali (36). The reduction may also be accomplished cataly-
tically with very good yield (37). However, the Vorlander
method was used here since It appeared to offer the simplest
means In accord with our facilities.
III. THE TYSON SYNTHESIS OF INDOLE
16
When the work on the ahove synthesis was nearing com
pletion information was received that nitric acid with iso
topic nitrogen was shortly to be obtainable. This revived
the possibility of employing other synthetic methods for the
preparation of indole and inasmuch as a number of difficulties
were being encountered in the last step in the above pro
cedure it was decided to abandon further efforts toward its
completion in favor of attempting a new synthesis, using
nitric acid.
mising from the standpoint of simplicity and overall yield
was the one worked out by Tyson (38) and presented here in
brief:
o -CH3C6H„NHCHO -*-(C H ^C O K — ► - * ■
o ~ FormoToluide
Of the many syntheses making use of a starting com
pound obtained originally through nitration, the most pro-
o-O 4,C 4H,NCcH0)K *0 - CH,C*M1(NHK + CO
O -C H ,C 6H^NHK + o-CHjC^H^NCcHO)^
Indole
t KOH
17
The starting material here is o-toluidine. It could
he obtained readily from e-nitrotoluene by reduction, and
this, in turn, could be most easily prepared by direct nit
ration of toluene* The isomers could then be separated by
fractional distillation*
The yield in the nitration reaction approximates 63$
of theoretical, while that in the next reaction, the reduc
tion of the nitrotoluene, is nearly 80$* Tyson reported
that the formylation of toluidine could be carried out with
a yield of 90$ of theoretical or better and for the final
step in the synthesis, that whereby indole was produced from
the formyl derivative of toluidine, a 79$ was claimed. On
the basis of these figures the overall yield is approximately
35$, compared to that of the previous synthesis where the
maximum overall yield was of the order of 60$. However, the
ultimate advantage in the method resided in its reliability
for the individual steps in the process had been adequately
tested.
CHAPTER III
EXPERIMENTAL
I. THE HEUMANN SYNTHESIS
Each step in the process was regarded as an individual
synthesis and therefore the discussion is most appropriately
presented in terms of individual steps.
The preparation of anthranilic acid from phtha1imide.
This reaction was performed largely as detailed in Werthelm*s
”Laboratory Manual of Organic Chemistry” (39), with modi
fication that sodium hypochlorite was substituted for the
hypqbromite. This change was made in accordance with infor
mation given in a publication of E. Wallis and J. Lane (40)
reporting the higher yield obtainable with use of the hypo
chlorite.
A liter of 0.5 N sodium hypochlorite was prepared by
dissolving chlorine in a chilled 10$ sodium hydroxide solu
tion (1 liter).. The chlorie was generated by allowing 210 cc.
of concentrated hydrochloric acid contained in a separatory
funnel to drop at a steady rate oh' 16.1 g. of potassium per
manganate. The strength of the hypochlorite solution, so
prepared, was determined by taking a small sample, adding an
excess of potassium iodide solution and acidifying with sul
furic acid, then adding a few drops of starch solution as an
19
indicator and titrating with a standardized sodium thiosul-
fate solution (0.01 N)•
A 500 cc. beaker containing 150 cc. of the 0.5 N sod
ium- hypochlorite was placed in an ice-salt bath and "6 g* of
phthalixnide was dissolved with stirring, to be followed by
the addition of 15 g. of sodium hydroxide. The temperature
began to rise, and when solution waa complete the beaker was
removed from the ice bath and warmed to 80° for two minutes.
The solution was cooled again in the ice "bath and neutralized
with concentrated hydrochloric acid. The anthranilic was
precipitated with glacial acetic acid (about 11 cc. were
required), and after standing overnight in the icebox, the
product was collected by filtration, washed with water, and
recrystallized from hot water.
The article of Wallis and Lane (40) reported a yield
of 95/o of theoretical. The best yield obtained by this in
vestigator was 92$ of theoretical before recrystallization,
89$ after recrystallization.
The preparation of N-phenylglycine-o-carboxylic acid
from anthranilic acid. The conditions for performing this
step were thoroughly investigated by Haller in 1922 (41).
His findings as to the best concentrations of reactants,
temperature and time of reaction, and other conditions were
faithfully followed.
Four g. of anthranilic acid, 1.38 g. of chloroacetic
20
acid, 3.6 g. of sodium carbonate, and 32 cc. of water were
mixed in a three-necked 600 cc. round bottom flask. The
flask was fitted with a thermometer^ a reflux condenser', and
a mechanical stirrer. The solution in the flask was rapidly
heated to 90° in a water bath and maintained at that tempera
ture with stirring for an hour. At the end of this period
the water bath was removed and the stirring continued for
another ten to fifteen minutes while the solution cooled;
after which it was poured into a beaker and acidified with
hydrochloric acid. Upon standing 24 hours the precipitate
which formed was filtered off and washed with water, keeping
the washings separate from the mother liquor. The precipitate
was dried and weighed.
Twelve g. of sodium acetate were dissolved in the
mother liquor with stirring. After standing overnight, the
excess anthranilic acid, which was thus precipitated was fil
tered off, washed with water, and dried at 100°. To the fil
trate and washings about 2.5-g. of sodium acetate and an ex
cess of saturated copper acetate solution were added. After
standing for five or six hours the precipitated copper anth-
ranilate was filtered off, washed and dried.
The proportions of anthranilic acid, chloroacetic acid,
and sodium carbonate prescribed for this reaction were in
molar ratios of 2:1:2.33, respectively. Since the anthranilic
acid here is in excess with respect to the chloroacetic acid,
21
the conversion of anthranilic acid to product is less than
50$; however, approximately 80$ of the excess anthranilic
acid can he recovered and reused. Haller’s article claims
an 86$ yield of N-phenylglycine-o-carboxylic acid. This
investigator obtained a yield of 82$ of the theoretical.
The preparationCof indoxyl from U-phenylglycine-o-
carboxylic acid. The alkali fusion was performed in accor
dance with directions found in the report of Phillips (42),
who had made a detailed study of the reaction. In his ex
periments he used an apparatus, which consisted of a battery
of enclosed, metal vessels, containing the reactants, sub
merged in molten solder and mechanically rotated therein
during the reaction period. In place of this elaborate ap
paratus, a piece of equipment was constructed which is es
sentially similar in operation, but of simpler design. An
oil bath was employed in place of the solder.
Pour g. of N-phenylglyeine-o-carboxylic acid and
18.4 g. of potassium hydroxide (the procedure calls for a
molar ratio of 12 to 16 moles of the hydroxide per 1 mole of
the acid) was placed in the reaction vessel, which had pre
viously been flushed out with nitrogen gas so as to exclude
the atmosphere. The oil bath was previously brought to a
temperature of about 270° and the vessel and apparatus sub
merged in it. The rotation of the vessel was started and
the reaction continued for ten minutes. It had been found
22
that the insertion of the apparatus into the bath causes a
fall in the temperature of the latter of about 10° to the
desired temperature of 260°.
At the conclusion of the reaction the vessel was
cooled and opened; the fused mass was. broken up and dumped
into a large, specially-adapted test tube, preparatory to
the next operation. The material which adhered to the walls
of the reaction vessel was dissolved away by boiled water
and the washings were likewise added to the test tube.
There was no attempt at this point to separate the
indoxyl formed because of the ease with which it is oxidized
to indigo and consequently lost. Accordingly, the next step
was carried out without interruption and no data were ob
tained on the yields from this operation. However, the
yields reported by Phillips were of the order of 80$ of theory
or slightly higher.
The preparation of indole from indoxyl. The reduction
of indoxyl to indole was carried out substantially as out
lined. in the original article of Vorlander and Apelt, employ
ing sodium amalgam as the reducing agent (36).
The test tube described in the last operation, to
which had been transferred the indoxyl and washings, was
fitted with a three-hole stopper and through this were in
serted, respectively, a tube for passing nitrogen into the
solution to maintain anaerobic conditions, a reflux condenser,
25
and a mechanical stirrer* The vessel was placed in an oil
bath heated to 70° and maintained between 60° and 70° through
out the reaction*
A 5% sodium amalgam wa3 prepared in accordance with
directions given in Fieser’s ’ ’Experiments in Organic Chem
istry” (43). A small amount of the amalgam was added re
gularly every fifteen minutes during the reaction, and al
together a total of 60 g. of the amalgam were consumed. To
ward the end of the experiment small samples of the solution
were withdrawn and tested to determine the extent of the re
duction* Incomplete reduction was indicated if the sample
turned blue upon exposure to the atmosphere, i.e., it was
oxidized to indigo.
The results of two experiments carried out in the
above manner were negative, however, for there was no evi
dence of reduction as shown by the exposure of small sample s
to air, nor when the solution was steam distilled, when any
indole which had formed would have appeared in the distillate.
Inasmuch as the article hadn’t mentioned the concentration of
the sodium amalgam used (nor the time of reaction) it was
thought that a stronger concentration of the reducing agent
was called for. Accordingly, a 10% sodium amalgam was tried
but this proved no better.
Upon comparing,our experiment with the original of
Vorlander and Apelt, a significant difference was noted.
24
Whereas they employed Indoxyl, which had been obtained by
a different method from indoxylic acid, our Ind'oxyl, prepared
by alkali fusion, was in combination with a large excess of
potassium hydroxide. It occurred to us that this might be
hindering the reduction, and so an attempt was made to get
rid of the excess alkali by taking up the indoxyl in alcohol
and passing carbon dioxide into the solution to form the
alcohol-insoluble bicarbonate (and some of the equally inso
luble carbonate). When this was tried there was some evi
dence of formation of the bicarbonate, but unexpectedly, the
whole solution jelled and there were signs of extensive
oxidation. This latter reaction occurred in spite of all
precautions to maintain anaerobic conditions. Because of
the adverse results further experimentation was discontinued.
II. THE TYSON SYNTHESIS
The discussion of the experimental work will be treated
here in accordance with the arrangement adopted for the pre
vious discussion, i.e., in terms of the individual steps.
The nitration of toluene. The directions for this
reaction were taken from W. J. Hickenbottom’s ’ ’Reactions of
Organic Compounds” (44).
The mixed acid used in the nitration was prepared by
adding 50 g. of concentrated nitric acid in 10 cc. portions
to 75 g. of concentrated sulfuric acid, cooling the mixture
25
after each addition. Fifty g. of toluene was placed in a
liter round-bottomed flask, and the latter was fitted with
a mechanical stirrer, a thermometer, and a 250 cc. separatory
funnel. The acid was placed in the separatory funnel and ad
mitted to the flask with stirring, at a rate which did not
allow the reaction temperature to exceed 50°. Cooling was
effected by immersing the flask in an ice bath.
When all of the acid had been added, the ice bath was
exchanged for a warm water bath and the solution was brought
to 50° and maintained at that temperature, with stirring,
for an hour. The solution was then transferred to a separatory
funnel and the lower acid layer drawn off. The nitrotoluene
was washed successively with 100 cc. of water, 100 cc. of a
2$ sodium hydroxide solution, and 50 cc. of water. It was
then placed in an oven at 40° overnight.
The excess acid left from the nitration was distilled
under reduced pressure to reclaim the nitric acid. The dis
tillate was titrated with a 0.1 N sodium hydroxide solution
and showed a recovery of 0.205 equivalents, or about 81$ of
the theoretical.
The isomers of nitrotoluene were separated by fractional
distillation, the ortho isomer distilling first at around
220°. The results of two experiments were:
43.9 g. of o-nitrotoluene (59.0$ of the theoretical)
45.6 g. of o-nitrotoluene (61.4$ of the theoretical)
26
The toluene used intiie first experiment was of a technical
grade, while a purified material was employed in the second.
Whether this would account for the difference in yields is
not known.
The reduction of nitrotoluene to toluidine. Inasmuch
as no specific directions for the reduction of nitrotoluene
could be found, it was decided to follow the procedure given
by Fieser's ’ ’Experiments in Organic Chemistry” (45:) for the
preparation of aniline from nitrobenzene.
Seventy-five g. of granulated tin and 41,1 g, (0,3
mole) of nitrotoluene were placed in a 2 liter round-bottomed
flask. The latter was fitted with a reflux condenser and a
mechanical stirrer, Next, 165 g, of concentrated hydrochloric
acid was slowly added through the condenser. The manner of
addition was such as to initiate And maintain the reaction
(vigorous ebullition) while avoiding the admission of amounts
such as would cause violent boiling. As a precaution against
the latter eventuality a pan of ice water was kept near at
hand into which the flask could be immersed to suppress the
reaction. After the acid had 'all been added the mixture
was heated on the steam bath for one-half hour and then cooled
before the next reaction.
A solution of 115 g. of sodium hydroxide in 225 cc. of
water was added to the mixture as rapidly as could be done
without causing excessive heating. The mixture was then
27
steam distilled, collecting an additional 200 cc. of the
distillate after the latter had ceased to exhibit turbidity*
The distillates was saturated with sodium chloride,
about 25 g. of the latter being required for each 100 cc. of
the solution and then extracted with ether. The ethereal
extract was dried for about 4 hours with sodium hydroxide
pellets, after which the ether was removed by distillation*
The crude o-toluidine residue had a weight in two experiments
of 21.6 g. (67.2$ of theoretical) and 23.76 g. (74.1$ of
theoretical)•
The p-nitrotoluene which remained from the previous
experiment was reduced similarly and then treated in accor
dance with the method used for carrying out a Kjeldahl deter
mination for nitrogen. In this way the nitrogen was recovered
as ammonia and the results for the two experiments are as
follows:
0.014 equivalents of (78.1$ recovery)
0.0154 equivalents of MHg (86.1$ recovery)
The formylation of ortho-1oluidine. The procedure
here, followed the one outlined in Tyson's article (38).
Twenty g. of o-toluidine was added to 9.5 g. of 90$
formic acid in a 250 cc. round-bottomed flask. The flask
was fitted with reflux condenser and heated on a steam bath
for three hours, after which the reaction mixture was allowed
to stand overnight.
28
The mixture was fractionated under reduced pressure
and the yields for two experiments were 19.6 g. of o-formo-
toluide (77.5$ of theoretical) and 20.8 g. of o-formotoluide
(82.6$ of theoretical).
The formation of indole. The conditions set forth in
the Tyson article were adhered to as closely as possible.
However, the arrangement of the equipment was adapted to suit
the particular purposes of the project. For example, it was
necessary to recover the toluidine which distilled over in
the course of the experiment, since otherwise there would
have been a loss of isotopic material. Accordingly, provision
was made for effectively trapping the toluidine, by insuring
adequate cooling of the ditillate. Again, for the metal bath
described in the experiment baths of sand and of salt were
substituted, since the former could not easily be obtained.
A one liter three-necked, round-bottomed flask was
fitted with a reflux air condenser and a glass inlet tube
connected to a cylinder of nitrogen. The third opening was
closed by a stopper. A short water condenser was fitted to
the top of the reflux condenser, and the former emptied into
a 500 cc. suction flask through an adapter. The flask, in
turn, was connected to another 250 cc. suction flask. This
second flask contained 100 cc. of paraffin oil and the inlet
tube extended slightly below the surface of the oil. This
system functioned as an air trap. In addition, the first
29
suction flask was surrounded by an ice bath to effect con
densation of any vapors that distilled.
In the reaction flask was placed 180 cc. of tert.-butyl
alcohol,-and the air'in the.flask was displaced by dry nitro
gen gas. Next 8.6 g. of metallic potassium was added, in
portions,, to the alcohol. After the potassium had dissolved
20 g. of o-formotoluide was added and brought into solution.
A salt bath was prepared by mixing 600 g. of sodium nitrate
and 860 g. of potassium nitrate. The reaction vessel was
placed in the bath and the latter was heated. The excess
alcohol distilled over and was collected in the trap and
when no more alcohol came over, another 500 cc. suction flask
was substituted. As the temperature rose the toluidine formed
in the reaction was collected in this new flask.
The residue was heated to a temperature of 350°-360°
for about twenty minutes and then allowed to cool in a stream
of nitrogen. The residue was subsequently decomposed by ad
dition of 90 cc. of water, and the mixture was steam-distilled
to remove the indole. The distillate was extracted with ether
and the ethereal extract was shaken with cold dilute 5% hydro
chloric acid to remove small amounts of o-toluidine. The
extract was then washed with about 50 cc. of water, followed
by 50 cc. of 1 5 % sodium carbonate solution, and was dried over
7 g. of sodium sulfate. The ether was removed by distillation,
and the residue distilled under reduced pressure. The yields
30
of Indole from two experiments were 2.14 g. (24.7$ of theo
retical) and 5.32 g. (61.3$ of theoretical).
The o-toluidine, which was regenerated in the reaction
distilled'as the 220° temperature was reached and was caught
in a flask, along with a small amount of the tert.-butyl
alcohol. The latter was removed by distilling under vacuum
and the amount of o-toluidine reclaimed in the second ex
periment was 5.56 g. (70.2$ of theoretical). In the first
experiment the equipment had not been properly arranged to
collect the o-toluidine and much of the compound was lost,
so that there was no attempt to determine the recovery.
It is believed that the low yield obtained in the
first experiment was due to the use of a sand bath for heating.
It required over an hour to bring the bath up to the prescribed
temperature (350°) and this caused extensive charring of the
material.
III. THE ENZYMATIC SYNTHESIS OF TRYPTOPHAN
FROM INDOLE AND SERINE
The procedure for the enzymatic synthesis consisted of
three operations, viz., culturing the organism,; Neurospora
crassa, from which the enzyme was obtained, the preparation
of the enzyme, and, finally, the synthetic reaction.
The Neurospora was grown in solution of a standard
medium made up according to the directions given in the
article of N. Horowitz and G. Beadle (46) and contained the
TABLE I
31
SUMMARY OP DATA
Reaction Experiment I Experiment II
Weight % Weight %
1. Formation of o-nitro-
toluene 43.9 g. 59 45.6 g. 61.4
2. Reduction of o-nitro-
toluene 21.6 g. 67.2 23.76 g. 74.1
3. Recovery of NH^ from
p-nitrotoluene 78.1 86.1
4. Formylation of tolui
dine 19.6 g. 77.5 20.8 g. 82.6
5. Formation of indole
from o-formotoluide 2.14 g. 24.7 5.32 g. 61.3
6. Recovery of o-tolui-
dine
* « S *
5.56 g. 70.7
7. Overall yield on basis
of recovered N 16.8 30.3
# No data taken.
32
following in g. per liter:
ammonium tartrate
ammonium nitrate
monobasic potassium phosphate
magnesium sulfate*7HgO
sodium chloride
calcium chloride
sucrose
5.0 g.
1.0 g.
1.0 g.
0.5 -g.
0.1 g.
0.1 g.
biotin 5x10 g.
20^0 g.
i n o r .
In addition, it contains the following trace elements,
added as salts, in mg. per liter: B 0.01, Mo 0.02, Fe 0.2,
Cu 0.1, Mn 0.02, Zn 2.0.
Eight liters of the medium were made up in a 12 liter
flask and the whole was sterilized for 45 minutes in an
autoclave. The flask was fitted with a 2-hole rubber stop
per through which were inserted an inlet and an outlet tube,
the former extending below the surface of the medium. An
air filter, consisting of a drying tube which had been packed
solidly with cotton, was attached with rubber tubing to the
inlet and the other end was similarly connected to an air
line. All of these fittings were sterilized. After the me
dium had been inoculated with the organism, air was admitted
at a rate such as would maintain vigorous churning of the
solution to insure thorough aeration.
free of the solution, forming a cake on the filter pad. This
was rapidly cut into small chunks and placed in a flask.
The flask was then stoppered, chilled rapidly in a carbon-
After three days of growth the mycelium was filtered
33
dioxide-ether bath, and placed in the deep freezer for sto
rage until ready for use.
The other operations, i.e., the preparation of the
enzyme, and the performance of the enzymatic reaction, were
not undertaken because the pyridoxal phosphate required in
the latter reaction was not available to us at the time.
However, the procedures will be described here.
The frozen mycelium is ground up with sand and a one-
tenth molar phosphate buffer solution, having a pH of 7.8*
Serine is then added to the solution and the proportions of •
the ingredients are adjusted so that there are 1 mg. of serine
and 3 cc. of buffer solution to 1 g. of the frozen mycelium.
The solution is centrifuged for twenty minutes and the pre
cipitate is discarded. The enzyme is present in turbid solu
tion which remains.
Three solutions are made up of the following composi
tions; 1.17 mg. of indole per cc., 15 mg. of serine per cc.,
and lOOY" of pyridoxal phosphate per cc. These are mixed in
equal proportions and 1 cc. of the resulting solution is
taken per 1 cc. of the extract for the reaction mixture.
The solution is incubated for three hour3.
The tryptophan is precipitated with the standard mer
curic sulfate-sulfuric acid solution, that is, enough sulfuric
acid is added to make a 5% solution and then mercuric sulfate
in 5% sulfuric acid is added on an equimolar basis with the
34
indole used in the reaction. After 24 hours, the yellow
precipitate is filtered off and suspended in water. The
mercuric sulfate complex is decomposed with hydrogen sulfide
and harium hydroxide. The mixture is filtered and sulfuric
acid added to the filtrate to precipitate the barium. After
filtration the filtrate is concentrated under vacuum and re
crystallized from Q0% alcohol.
CHAPTER IV
DISCUSSION
The synthesis of L-tryptophan was undertaken primarily
with the view toward providing the isotopieally labelled amino
acid for study of the conversion of tryptophan to niacin.
It was thought that some evidence could be obtained in re
gard to the fate of the ring nitrogen, and in addition, there
was the possibility that certain of the intermediates in the
process might be isolated. As regards the general problem
of the metabolism of tryptophan it was expected that the N^5
labelled tryptophan could supplement the work which is being
carried on at present with the C14 compound. Some indication
of the amount of work which remains to be undertaken can be
gained from the fact that of the total amount of tryptophan
ingested in the animal body only about 5% can be accounted
for at the present time.
Of the two methods of synthesis here presented, it
was felt that the first one, involving the Heumann synthesis,
offered the better means of obtaining the compound from the
standpoint of overall yield. The final step in the synthsis
remained to be worked out, but we feel that this could be
ultimately accomplished with use of other equipment. At the
time the work on this synthesis was discontinued a catalytic
process for the reduction and dehydration of indoxyl to yield
36
indole was -under consideration. This involved hydrogenation
under high pressure, but the yield from the operation was of
the order of 90$ or better.
The second synthesis was undertaken as an expedient
to demonstrate the feasibility of a laboratory synthesis.
While the overall yield from the method was not high, it
still could be considered as a practical laboratory prepara
tion, and there was the expectation that the yield could be
improved by more efficient performance of some of the steps.
CHAPTER V
SUMMARY
The conversion of tryptophan to niacin in organisms
presents the problem of the steps involved in the chemical
rearrangement, and particularly with respect to the ring
nitrogen of tryptophan. The most plausible theory is that
this is transformed into the ring nitrogen of the vitamin.
A convenient method of studying the problem is with the use
of L-tryptophan having labelled ring nitrogen and the
preparation of such a compound became the subject of this
investigation.
Since L-tryptophan can be produced enzymatically from
indole and serine the problem was further reduced to the pre-
15
paration of indole with N • Two syntheses were presented.
The first, which was largely adapted from an industrial pro
cess for the manufacture of indigo, was uncompleted because
of certain difficulties encountered in the last step. How
ever, the overall yield to that point was good and there
remains the possibility that the obstacles in the last step
could be overcome by a new approach.
The second synthesis represented a combination of the
Tyson method for preparation of indole with other proven re
actions. The overall yield of indole on the basis of two
experiments was low, but there is the expectation that this
38
could be improved by further trial and much of the excess
N ^ can be recovered as NH^.
BIBLIOGRAPHY
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13. Schweigert, B. and Pearson, P., "Effect of Vitamin
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40
Tryptophane to N’-Methylnicotinamide and Nicotinic Acid,”
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14 Rosen, F., Huff, J., and Perlzweig, W., ”The Role
of Bg-Deficieney in the Tryptophane-Niacin Relationships in
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15 Bell, G., Scheer, B., and Deuel, H., Jr., ”Niacin
Excretion in the Rat in Relation to Tryptophan, Pyridoxin,
and Protein Content of the Diet,” J. Nutrition 35: 239 (1948).
16 Hurt, W., Scheer, B., and Deuel, H., Jr., ”The
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17 Krehl, W., Teply, L., Sarma, P., and Elvehjem, C.,
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18 Krehl, W., Teply. L., Sarma, P., and Elvehjem, C.,
"Growth-Retarding Effect of Corn in Nicotinic Acid-Low Rations
and Its Counteraction by Tryptophane, ” Science 101: 489 (1945).
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20 Rosen, F., Huff, J., and Perlzweig, W., ”The
Effect of Tryptophane on the Synthesis of Nicotinic Acid in
the Rat,” J. Biol. Chem. 163: 343 (1946).
21 Singal, S., Briggs, A., Sydenstricker, V., and
Littlejohn, J., "The Effect of Tryptophane on the Urinary
Excretion of Nicotinic Acid in Rats, ” J. Biol. Chem. 166:
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22 Briggs, G., Groschke, A., and Lillie, R., "Effect
of Proteins Low in Tryptophane on Growth of Chickens and on
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23 Luecke, R., McMillen, W., Thorp, F., Jr., and Tull,
C., "The Relationship of Nicotinic Acid, Tryptophane and
Protein in the Nutrition of the Pig,” J. Nutrition 33; 251
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24 Kade Jr., C., Houston, J., Krauel, K., and Sahyun, M.,
"The Maintenance of Nitrogen Equilibrium in Dogs by Intra
venous Alimentation with an Acid Hydrolysate of Casein
41
Fortified with Tryptophane,” J. Biol. Chem. 163: 185 (1946).
25 Sehweigert, B., Pearson, P., and Wilkening, M.,
"The Metabolic Conversion of Tryptophane to Nicotinic Acid
and to Nf-Methylnicotinamide,” Arch. Biochem. 12: 139 (1947).
26 Perlzweig, W., Rosen, F., Levitas, N., and
Robinson, J., ”The Excretion of Nicotinic Acid Derivatives
after Ingestion of Tryptophane by Man," J. Biol. Chem. 167:
511 (1947). ~ "
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Neurospora," Arch. Biochem. 11: (1946).
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as an Intermediat in the Formation of Nicotinic Acid from
Tryptophane by Neurospora,” Proc. Nat. Acad. Sci. 33: 155 (1947).
29 Mitchell, H. and Nyc, J., "Hydroxyanthranilic Acid
as a Precursor of Nicotinic Acid in Neurospora," Proc. Nat.
Acad. Sci. 34: 1 (1948).
30 Bonner, D., "The Identification of a Natural Pre
cursor of Nicotinic Acid,” Proc. Nat. Acad. Sci. 34: 5 (1948).
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Effect of 3-Hydroxy Anthranilic Acid on the Excretion of
Niacin by the Rat," J. Biol. Chem. 175: 479 (1948).
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"Concerning the Mechanism of the Mammalian Conversion of
Tryptophan into Nicotinic Acid," J. Biol. Chem. 176: 1461 (1948).
33 Umbreit, W., Wood, W., and Gunsalus, I., "The
Activity of Pyridoxal Phosphate in Tryptophane Formation by
Cell-Free Enzyme Preparations," J. Biol. Chem. 165: 731 (1946).
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30: 69 (1942). “
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Ber. 37: 1134 (1904).
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42
38 Tyson, F., "Indole”, Organic Syntheses 23: 42
(1943).
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A Laboratory Guide for Organic Chemistry, 2nd Edition (1940),
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of Organic Compounds, 2nd Edition, (1948), p. 54.
45 Fieser, L., "Experiment 29 Aniline,” Experiments
in Organic Chemistry, 2nd Edition, (1941), p. 147.
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o f © to a tto rw GuOJSte./'iolo .wi.

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Asset Metadata

Creator Hood, James E (author)

Core Title A method for the synthesis of ring-labelled N15 tryptophan

Degree Master of Science

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Visser, Donald (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776937

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