QPTICALLY ACTIVE TETRAZOLE ANALOGS
OF AMENO ACIDS

The“: fan Hm Dogma 05 pk. D
fi¢ui GREi S”AT3 JREV“25”W'
Vincent? "‘ E3 012:9

1963

 

THESIS

 

(92/

1CI~IKZ¢N STATE

M
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EAST LANSING,
MICHIGAN GTATF NAUVERSITY

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MICHIGAN

  
  

ABSTRACT

OPTICALLY ACTIVE TETRAZOLE ANALOGS
OF AMINO ACIDS

by Vincent T. D'Orazio

The synthesis of compounds which are similar in structure and
reactivity to compounds that play an important role in metabolic
processes of'living organisms has been the object of extensive study.
The concept of antagonism is defined as a competition between metabo-
lites and structurally similar metabolites, o'r structurally related
non-metabolites (antimetabolites) for an enzyme system. Since many
amino acids are essential for the development and maintenance of
the organism, they have been used as model systems in the synthesis
of potential antimetabolites.

Because of the acidic nature of S-monosubstituted tetrazoles,
their use as potential antimetabolites for amino acids appeared
promising. McManus (l) and Sterken (2) prepared a series of tetrazole
analogs of D, L-amino acids. Since all of the amino acids obtained
from proteins are of the L-configuration, it was considered germane
to extend this series to include the synthesis of the tetrazole analogs
of L-amino acids and determine some characteristic physical properties
for comparison with the corresponding amino acids.

The tetrazole analogs of L-phenylalanine and L-tyrosine were
prepared through interaction of the appropriate acylated aminonitriles
with hydrazoic acid or aluminum azide. The acylated aminonitriles were

synthesized from the corresponding L-amino acids.

Vincent T. D' Orazio

 

 

 

 

z
c6H,(c0)zo, C6H5COC1,
NHr'CH-COOH Or CH3COC1 > RuNH-cH-COOH
R R
1. soc1z

R'-NH-CH-COOH 2' NH3 > R'-NH-CH-C5N

| 3. C6H5803C1 .

R p R

__ HN;1 '

RuNH-pH-c=N ~> R'-NH-CH-fi IFI-H

R or A1(N3)3 N /N

\N /

R2 ~C6H5, -C6H4OH
R': c6H4(c0)z.', C6H5CO-, CH3CO-

Acid hydrolysis of 5-benzoyl and S-acetylaminoalkyltetrazole gave

rise to tetrazole analogs of D, L-phenylalanine and L-tyrosine.

The phthaloylaminoalkyltetrazole was converted to the tetrazole analog

of L-phenylalanine by hydrazinolysis.

Alkylation of ethyl acetamidoeyanoacetate with anisyl chloride

followed by treatment‘with aluminum azide, and subsequent acid hydroly-

sis gave rise to the tetrazoleanalog of O-methyI-D, L-tyrosine.

 

C=-N

. /
CH,O-.-CHZC1 + HC\-NH-COCH3 NaOCZH-‘t

COzcsz’

 

Vinc ent T. D'Orazio

 

3
0
ll
NH-C-CH,
1. A1(N3)3
H - - - - a r
c ,0 Q CH2 <5 G N 2. co... rim ~
cozcsz
IF‘HZ
CH3O- Q - CHZ-CH-c— leI—H
L yN

\N

The structure and configuration of the tetrazole analogs were
verified by conversion to the Z, 5-disubstituted l, 3, 4-oxadiazoles and
subsequent‘ acid hydrolysis afforded the original L-amino acids.

Many of the biological properties of amino acids are related to
their physical properties. The pK values of the tetrazole analogs
were determinedby potentiometric titration and found to be in close
agreement with their amino acid counterparts. Also, the degree of
optical activity is affected by the acidity'or alkalinity of the solution.
The specific rotations of L-phenylalamine and L-tyrosine in pre-
determined amounts of acid and base were compared to those of the

tetrazole analdgs.

LITERATURE CITED

1

l. J. McManus, J. Org. Chem., 51, 1643 (1959).

 

Z. G. Sterken, "Synthesis ‘of Tetrazoles as Amino Acid Analogs, "
Ph.D. thesis, Michigan State University, 1960.

OPTICALLY ACTIVE TETRAZOLE ANALOGS
OF AMINO ACIDS

BY

Vincent T. D' Orazio

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Chemistry

1963

'17

.3 M.

 

ACKNOWLEDGMENT

The writer would like to express his appreciation
to Dr. Robert M. Herbst for his invaluable guidance
and understanding so generously given during the course
of this work.

Also, appreciation is extended to Parke-Davis who
provided financial assistance through their Fellowship
Program during the academic year of 1961-1962.

*****>§<**>§<******

ii

To Connie

for her everenduring patience
and understanding

TABLE OF CONTENTS

INTRODUCTION.......................

HISTORICAL.........................

PART I

T ETRAZOLE ANALOGS OF AMINO ACIDS

DISCUSSION 0 O O C O O O O O O O O O I O O O O O O O O O O 0

EXPERIMENTAL O O O O O O O O O O O O O O O O O O O O O O

The Preparation of D, L-S-(a.-amino-fi-phenylethylhetra-

zole (D,L-Pheny1a1anine Analog) . . . . . . . . . . .

Scheme A

(a) D, L-a-Phthalimido-8-pheny1propionic Acid .
(b) D, L-u-Phthalimido-8-pheny1prbpiony1 Chloride 18
(c) D, L-a-Phthalimido-8-phenylpropionamide . .
(d) D, L-a-Phthalimido-B-phenylpropionitrile . .

(e) D, L- 5 - (a- Phthalimido¥ B-phenylethyl)tetrazole

(f) D, L-S-(a-Amino-8-pheny1ethy1)tetrazole . . .

Scheme B

(a) N-Benzoyl-D,L-phenylalanine. . . . . . .

(b) D, L-Ethyl-a-benzarnido-B-phenylpropionate
(c) D, L-a-Benzamido-oB-phenylpropionamide . .
(d) D, L-a-Benzamido-fi-phenylpropionitrile . .

(e) D, L-S-(a-Benzamido-B-phenylethyl)tetrazole
(f) D, L-5-(a-Amino-fi-phenylethylhetrazole . . .

Scheme C

(a) N-Acetyl-D,L-phenylalanine . . . . . .

iv

10

18

18

18

19
19
20
20

21
21
22
22
22
23

23

TABLE OF CONTENTS - Continued
Page

(b) D,.L-Ethy1-n-acetamido-fi-phenylpropionate. . 24
(c) D, L-a-Acetamido-B-phenylpropionamide . . . 24
(d) D,,L-e-Acetamido-fi-phenylpropionitrile. . . . 24
(e) D,-L-5-(a-Acetamido-[3-phenylethyl)tetrazole . 25
(f) D, L-S-(a-Amino-fi-phenylethylhetrazole . . . 25

The Preparation of L-5-(ti-Amino-B-phenylethyl)tetrazole
(L-Phenylalanine Analog) . . . . . . . . . . . . . . . . 26

Scheme A

(a) L-a-Phthalimido-B-phenylpropionic acid . . . 26
(b) L-a-Phthalimido-B-phenylpropionyl chloride . 26
(c) L-a-Phthalimido-[3-phenylpropionamide. . . . 27
(d)L-a-Phthalimido-8-pheny1propionitrile . . . . 27
(e) L-S-(a-Phthalimido-8-pheny1ethy1)tetrazole. . 28
(f) L-5-(a-Amino-[3-pheny1ethyl)tetrazole . . . . . 28

The Preparation of L-S-[a-Amino-B-(p—hydroxyphenyl)ethyl]-
tetrazole (L-Tyrosine Analog) . . . . . . . . . . . . . 29

Scheme B

(a) L-Tyrosine ethyl ester. . . '. . . . . . . . . . 29
(b) L- “Ethyl-a-benzamido-B -(p-hydroxypheny1)-
propionate...................29
(c) L-a-Benzamido-B- -(p-hydroxypheny1)- .° ‘1- 1
propionamide....‘.............30
(d) L-a-Benzamido-fl- -(p-benzenesulfonyloxyphenyl)-
propionamide.................3Q
(e) L- a- Benzamido- f3- (p-benzene sulfonyloxy-
pheny1)propionitrile . 1 . . . . . . . . . . . . 31
(f) L-S-[e-Benzamido-fi-(p-benzenesulfonyloxy- _
pheny1)ethyl]tetrazole. . .i . . . . . . . . . . 31
(g) L- 5 -[c1-Amino- B~ (p-hydroxyphenyl)ethy1]-
tetrazole......~................ 32

Scheme'C
(a) N-Acetyl-L-tyrosine. . .1... . . . . . . . . . 33

V

TABLE OF CONTENTS - Continued
Page

(b) L-‘Ethyl- a-ac etamido- (3- (p- hydroxyphenyl) -
propionate..................~. 33
(c) L-a-Acetamido-fl-(p-hydroxyphenyl)propion-
amide........1............ 34
(d) L- a-Acetamido-fl-(p-benzenesulfonyloxy-
phenyl)propionitrile . . . . . . . . . . . . . 34
(e) L-S-[a-Acetamido-B-(p-benzenesulfonyloxy-
phenyl)ethyl]tetrazole . . . . . . . . . . . . 35
(f) L- 5 - [a-Amino- B-(p-hydroxypheny1)ethy1]-
tetrazole................... 35

The Preparation of D, L-5v[a-Amino-f3-(p-methoxyphenyl)-
ethy1]tetrazole (O-Methyl-D, L-‘I‘yrosine Analog) . . . 36.

Scheme D

(a) p-Methoxybenzyl chloride (anisyl chloride) . . 36
' (b) D, L-Ethyl-a-acetamido-o-cyano-fi-(p-
methoxypheny1)propionate . . . . . . . . . . 36
(c) D, L-Ethyl—a-acetamido- ap(5-tetrazolyl)-B-
(p-methoxyphenyl)propionate. . . . . . . . . 37
(d) D, L-a-Acetamido-a-(5-tetrazoly1)-B-(p-
methoxyphenyl)propionic acid . . . . . . . . 37
(e) D, L-5-[a-Ac etamido-B-(p-methoxypheny1)-
ethyl]tetrazole. _ . . . . . . . . . . . . . . . 38
(f) D, L-S-[a-Amino-fl— (p-methoxyphenyl)ethy1]-
tetrazole................... 39

PART II

PROOF OF STRUCTURE AND CONFIGURATION OF
TETRAZOLE ANALOGS OF, AMINO ACIDS

DISCUSSION 0 O O O O O O O O. O O O O O O O O O O O O O O O, O O O ‘ 41
EXPERIMENTAL O O O O O O O O O O O O O O O O O O O O O O O . O 48

A. Synthesis of 2-Substituted-5-Pheny1-l, 3, 4-Oxadiazoles
From Tetrazole Analogs of Amino Acids . . . . . . . 48

vi

TABLE.OF CONTENTS - Continued
Page

D,‘L-5-(a-Amino-fl-phenylethyl)tetrazole (D,IL—
Phenylalanine analog) . . . . . . . . . . . . . . . 48

(a) D, L-S-(a-Amino-B-phenylethyl)tetrazole. . . 48
‘ (b) D, L-Z-(l'-Benzamido-2'-phenylethyl)-5-
phenyl-l,3,4-oxadiazole. . . . . . . . . . . 48

'L—5-(a-Amino-fi-phenylethylhetrazole (L-Phenyl-
alanineanalog)................... 49

(a) L-5-(a-Amino-fi-phenylethyl)tetrazole . . . . 49
(b) L-2-(l'-Benzamido-2'-phenylethyl)-5-pheny1-
1,3,4'0xadiaZOleo0.000.000.0000 49

L-S-[a-Amino-fi-(p-hydroxyphenyl)ethy1]tetrazole . . 49

(a) L- 5- [a-Amino-fi- (p-hydroxyphenyl) ethyl]-
tetrazole................... 49

(b) L-2-[1'-Benzamido-2'-(p-benzoyloxyphenyl)-
ethyl]-5-pheny1-l,3,4-oxadiazole . . . . . . 49

B. Acid Hydrolysis of 2-Substituted-5-Pheny1-1, 3, 4-
Oxadiazoles as Proof of Structure and Configuration . 50 -

(a) Hydrolysis of D, L-2-(l'-Benzamido-2'-
phenylethy1)-5-phenyl- 1, 3, 4-oxadiazole. . . 50

(b) Hydrolysis of L-2-(l'-benzamido-2'-pheny1-
ethyl)-.5-phenyl-l,3,4-oxadiazole . . . . . . 51

(c) Hydrolysis of L-2-[l'-benzamido-2'.- (p-
benzoyloxyphenyl) ethyl]-5-phenyl— 1, 3 , 4—
oxadiazole......-.....5....... 51

PART III

OPTICAL ROTATION AND pK DETERMINATIONS OF
TETRAZOLE ANALOGS OF AMINO ACIDS

DISCUSSIONooooooo‘Qooooooo'oooooooooooo 54

vii

TABLE OF CONTENTS - Continued

EXPERIMENTAL O O O O O O O C C O O O O O O O O O O O O O C

A. Specific Rotations of L-Phenylalanine, L-Tyrosine,
and Their Tetrazole Analogs in Various Concen-
trations ofAcidandBase. . . . . . . . . . . . . .

(a) L-Phenylalanine . . . . . . . . . . . . . .
(b)L-Tyrosine... ..............
(c) L-5-(a-Amino-[3-phenylethy1)tetrazole. . .
(d) L-5-[a-Amino-B-(p-hydroxyphenylethy1]-
tetrazole (L-Tyrosine Analog). . . 1 . . .

B. The Determination of the Apparent pK Values of Some

Amino Acids and Their Tetrazole Analogs . . . . .
SUMMARY. 0 O C C O O C O O O O I O O O O O O O O O O O O O O
LITERATURE CITED 0 C O O O O O O O O O O O O O O O I O O 0

APPENDIX 0 O O O O O O O O O O O O O O O O O O O O O O O O 0

viii

Page

64

64
64
64
64

66

67
76
77

80

TABLE

10.

11.

LIST OF TABLES

ix

Page
.Specific Rotations of L-Phenylalanine in NaOH and
HCl. 0 Q I O C O O O O O C O O O C O O O O C O C O O O O O 65
Specific Rotations of L-Tyrosine in NaOH and HCl. . . 65
Specific Rotations of L-5-(a-Amino-fi-phenylethy1)-
tetrazoleinNaOHandHCl............... 66
Specific Rotations of L-S-[a-Aminoi-fl-(p-hydroxypheny1)-
ethyl]tetrazole in NaOH and HCl . . '. . . . . . . . . . 67
Potentiometric Titration of L-S-(a-Amino-B-phenyl-
ethy1)tetrazole with Hydrochloric Acid. . . . . . . . . 69
Potentiometric Titration of L-5-(a-Amino-(3-pheny1e
ethyl)tetrazole with Sodium Hydr0xide . . . . . . . . . 70
Potentiometric Titration of D, L-S-[a-Amino-B-(p-
methoxyphenyl)ethy1]tetrazole with Hydrochloric Acid. 71
, Potentiometric Titration of D, L-5-[a-Amino-8-(p-
methoxyphenyl)ethy1]tetrazole with Sodium- Hydroxide . 72
Potentiometric Titration of L-S-[a-Amino-[S-(p-
hydroxyphenyl)ethy1]tetrazole with Hydrochloric Acid . 73
Potentiometric Titration of L-5-[a-Amino-[3-(p-
hydroxyphenyl)ethyl]tetrazole with Sodium Hydroxide ." 74.
.Apparent pK Values of Some Amino Acids and Their
TetrazoleAnalogs.................... 75

LIST OF FIGURES

FIGURE .Page
1. Complete Ionization Scheme for Tyrosine. . . . . . . . 59
2. Ionization Scheme for the Tetrazole Analog of L-

10.

11.,

12.

13.

Phenylalanine....................... 60

Ionization Scheme for the Tetrazole Analog of L-
TerSineoooooeoooo‘ooo0.0.000...no 62

Infrared Spectrum of D, L- a-Acetamido-(S-phenyl-
propionitrileO O O O O O O O O O O O O O O O O O O O O O O 81

Infrared Spectrum of L-5-(d-Phthalimido-fi-phenyl-
ethyl)tetraZOIe O O O O O O O O O O O O O O O O O O O O O O 82

Infrared Spectrum of L-5-(a-Amino-[S-phenylethyl)-
tetraZOIeO O O O O O O O O O O O O O O O O O O O O O O O O 83

Infrared Spectrum of L-a- Benzamido-fi—(p-benzene-
sulfonyloxyphenyl)propionamide. . . . . . . . . . . . . 84

Infrared Spectrum of L-a-Benzamido-B-(p-benzene-
sulfonyloxyphenyl)propionitrile . . . . . . . . . . . . . 85

Infrared Spectrum of L-S-[a- Benzamido-B-(p-benzene-
sulfonyloxyphenyl)ethy1]tetrazole . . . . . . . . . . . . 86

Infrared Spectrum of L-5-[a-Amino-[B-(p-hydroxyphenyl)-
ethy11tetraZOIeo0.0.0000.on...coco... 87

Infrared Spectrum of L-a-Acetamido-B-(p-benzene-
sulfonyloxyphenyl)propionitrile . . . . . . . . . . . . . 88

Infrared Spectrum of L-5-[c1-Acetamido-B-(p-benzene-
sulfonyloxyphenyl)ethy1]tetrazole . . . . . . . . . . . . 89

Infrared Spectrum of D, L-S-[a-Amino-fi-(p-methoxy-
pheny1)ethy1]tetrazole. . . . . . . . . . . . . . . . . . . 90

LIST OF FIGURES - Continued
FIGURE Page

14. Infrared Spectrum of L-Z-(l'-Benzamido-2'-pheny1-
ethy1)-5-phenyl-l,3,4-oxadiazole. . . . . . . . . . . . 91

15. Infrared Spectrum of L-2-[l'-Benzamido-2'-(p-benzoy1-
oxyphenyl)ethyl]-5-phenyl-1,3,4-oxadiazole . . . . . . 92

16. Specific Rotation of L-Phenylalanine in NaOH and HCl. 93
17. Specific Rotation of L-Tyrosine in NaOH and HCl . . . 94

18. Specific Rotation of L-5-(d-Amino-B-phenylethyl)-
tetrazole in NaOH and HCIO O O O O O O O O O O O O O O O 95

19. Specific Rotation of L-5-[a-Amino-B-(p-hydroxypheny1)-
ethyl]tetrazole in NaOH and HCl. . . . . . . . . . . . . 96

20. Potentiometric Titration of L-5-(a-Amino—8-phenyl-
ethy1)tetrazole with Hydrochloric Acid . . . . . . . . . 97

21. Potentiometric Titration of L-5-(a-Amino-(3-phenyl—
ethyl)tetrazole with Sodium Hydroxide . . . . . . . . . 98

22. Potentiometric Titration of D, L-5-(a-Amino-6-(p-
methoxyphenyl)ethyl]tetrazole with Hydrochloric Acid . 99

23. Potentiometric Titration of D, L-5-[a-Amino-8-(p-
methoxyphenyl)ethyl]tetrazole with Sodium Hydroxide . 100

24. Potentiometric Titration of L-S-[a-Amino-fi-(p-
hydroxyphenyl)ethy1]tetrazole with Hydrochloric Acid . 101

25., Potentiometric Titration of L-5-[a-Amino-[3-(p-
hydroxyphenyl)ethy1]tetrazole with Sodium Hydroxide . 102

xi

INTRODUCTION

Amino acids play a vital role in the metabolic processes of
living organisms. A close metabolic relationship exists between
two structurally similar amino acids; phenylalanine, and tyrosine.
Phenylalanine is an essential amino acid, while tyrosine is not,
even though tyrosine serves as a precursor for such physiologically
important compounds as adrenaline and thyroxine. It has been
demonstrated that phenylalanine can be converted to tyrosine in the
body. The concept of antagonism between structurally similar
metabolites, or between a metabolite and a structurally related non-
metabolite is explained on the existence of a competition between
these compounds for an enzyme system. These metabolic antagonists,
or antimetabolites, have been important as chemotherapeutic agents
in the treatment of disease.

Many compounds have been synthesized which are related
chemically and structurally to essential metabolites. Usually these
structural alterations are in the form of adding substituents to the
molecule, or replacing a functional group with one that has a related
structure and reactivity.

McManus (3) and Sterken (19) prepared a series of tetrazole
analogs of D, L-amino acids. Since all amino acids obtained from
proteins are of the L configuration, it was considered cogent to extend
this series to include the synthesis of the tetrazole analogs of
L—amino acids, and determine some characteristic physical properties
of these compounds for comparison with their amino acid counterparts.

The tetrazole analogs of L-phenylalanine and L-tyrosine were

prepared through interaction of the appropriate nitriles with hydrazoic

acid or aluminum azide. The nitriles were synthesized from optically
active amino» acids in which the amino group had been acylated with

a phthaloyl, benzoyl, or acetyl group. It was found that the procedure
which employed the benzoyl group for blocking the amino moiety was
the most convenient preparative method for general use. The tetrazole
analog of O-methyl-D, L-tyrosine was prepared by alkylating ethyl
acetamidocyanoacetate with anisyl chloride. Interaction of the nitrile
with aluminum azide afforded the tetrazole, and subsequent acid
hydrolysis gave rise to the desired product.

In order to verify the structure and configuration of the tetrazole
analogs, the 2, 5-disubstituted 1, 3, 4-oxadiazoles were prepared from
the tetrazole analogs. Subsequent acid hydrolysis of the oxadiazoles
afforded the original L-amino acids.

Many of the biological properties of amino acids are. related to
their physical properties. The ability of an amino acid to act as both
a proton donor and a proton acceptor has long been recognized. The
pK values for the tetrazole analogs were determined by potentiometric
titration and found to be in close agreement with the corresponding
amino acids.

The degree of optical activity of amino acids is affected by the
acidity or alkalinity of the solution. Lutz and Jirgensons (20, 21)
determined the specific rotations of a series of naturally occurring
amino acids in acid and base. From the curves obtained by plotting
the specific rotation against the amount of acid or base, they were able
to establish the configuration of these amino acids. Although the ana-
logous curves for the tetrazole analogs were inverted compared to
those of the amino acids, it appears that the rotation curves of the

I

tetrazole analogs can be utilized for configuration assignment.

HISTORICAL

The first synthesis of a tetrazole reported in the literature
was carried out by Bladin (24, 40) in 1885. 2-Phenyl-S-cyanotetrazole
was synthesized from dicyanophenylhydrazine on treatment with
nitrous acid. Hydrolysis of the cyano group followed by decarboxyla-
tion resulted in the formation of Z-phenyltetrazole. Bladin (25) also
synthesized tetrazole itself from 2-pheny1-5-carboxytetrazole by the

following scheme:

HOOC-CIJ =1? Egg-91> HOOC-(li =1]! ‘
N§ /N-C,H, N§ /N-C,H,Noz
N N
fl“ HOOC-(fi :1? M9 H-(I: =1?
N§N/N-C¢,H4NHZ N§N/NH

The stability of the tetrazole ring was illustrated by its inertness to
potassium. permanganate.

In 1897 Pinner (27) prepared 5-substituted tetrazoles from nitriles
by formation of the iminoether hydrochlorides. Treatment of the
iminoether hydrochlorides with hydrazine gave theamidrazone hydro-
chlorides, which were subsequently converted by diazotization with
nitrous acid to the imidyl azides.

The latter cyclized to form the tetrazoles. The scheme may be

illustrated as follows:

 

NH.HC1 /NH
Ar-CEN CJ'HSOH > Ar-C// 21133-9 Ar-C\/ - HCl
OCZHS N2H3

-H

 

NH
$521.1 114/ _..___1 111',

1'
N3 N N

/
\N/

Ainsworth (28) prepared 5-(2'-aminoethyl)tetrazole from
8-benzamidopropionitrile by three different methods. The first
method was similar to the scheme used by Pinner (27). However,-
amyl nitrite was used as the diazotizing agent in place of sodium
nitrite for the preparation of the imide azide. In the second method
Ainsworth adapted a procedure developed by Mihina and Herbst (29).
fl-Benzamidopropionitrile was heated with hydrazoic acid in xylene.
The third technique involved the reaction of hydrazoic acid in glacial
acetic acid with the appropriate iminoether hydrochloride.

Herbst and Wilson (30) prepared 5-ary1tetrazoles by treating
the aryl cyanide with hydrazoic acid generated i_n _s_i_t_u from sodium
azide and glacial acetic acid in boiling butyl alcohol.

The preparation of tetrazoles by reaction of aluminum. azide
generated i_n -s_i_1_:_u_ with nitriles was reported by Behringer and Kohl (31)
in 1956. The nitrile was refluxed in dry tetrahydrofuran containing
anhydrous aluminum chloride and sodium azide. The yields of the
resulting acylaminoalkyltetrazoles were 80-90%. .

In 1958 Finnegan and co-workers (32) introduced the use of
ammonium azide for the synthesis of tetrazoles. In their procedure
the nitrile, ammonium chloride, and sodium azide were heated in
dimethylformamide. The ammonium salt of the tetrazole was con-

verted to the free tetrazole by treatment with dilute acid. The yields

were higher and the reaction time shorter than the aluminum azide and
hydrazoic acid methods.

McManus (3) prepared the first tetrazole analogs of amino acids
in 1958. The tetrazole analogs of glycine, D, L-alanine, fi-alanine,
D,.L-phenylalanine, D, L-tryptophane, p-aminobenzoic acid, 2-hydroxy-
4-aminobenzoic acid, picolinic acid, nicotinic acid, isonicotinic acid,

2, 4-dichlorophenoxyac etic acid, 2, 4, 5-trichlorophenoxyacetic acid,
3, 4, 5-trimethoxybenzoic acid, and 3-indoleac etic acid were prepared
by three methods.

In the first scheme the a-haloacyl halide was converted to the
N-benzyl-a-haloamide by reaction with benzylamine. The 1-benzy1-5-a-
haloalkyltetrazole was synthesized by the interaction of the a-haloamide
with-phosphorus pentachloride to give the imino chloride followed by
treatment with hydrazoic acid. Reaction with potassium phthalimide
' gave rise to the l-benzyl-5-a-phthalimidoalkyltetrazole and subsequent
treatment with ethanolic hydrazine resulted in the formation of l-benzyl-

a-aminoalkyl tetrazole. The benzyl group was then removed by

 

 

 

 

 

hydrogenolysis.
>.< 0 ’f ?
R-CH-éix CJHiCHZNHZ> R-CH-C-NH-CHz-CbHs
X .
212.31%? > R-cH-fi—N-CHz-chs cthcoL’tNK;
N N
\Né
c,H,(c0)zN-c|:H-fi N-CHz-cm, Eli—9
R N N
\N/
NH, If‘Hz
R-CH 'fi ITI-CHz-Csl-Is %? R-CH-fi—N-H
N N N N
\N// \N/

In the second scheme the amino acid was fused with phthalic
anhydride. The N-phthaloyl amino acid was converted to the nitrile
by way of the acid chloride and amide. Reaction of the nitrile with
aluminum azide followed-by hydrazinolysis of the phthaloyl compound

gave rise to tetrazole analogs of alanine, B-alanine, and D, L-phenyl-
alanine.
NH;

|
R-CH-COOH CEUCCLO; c,H,(CO)Z-N~<':H-COOH

 

 

R
1. soc1z
2. NH; 4 = NaN
3. c6H,soch C6H‘(CO)ZN'?H’C‘N 111(31,a
R

N11z

C6H,(CO)zN-C'3H-fi—Il\l-H 333-14» R-CH-fi—Iil-H
N N N /N
\N// \N/

In a third technique McManus (3) alkylated ethyl-a-acetamidocyano- '
acetate with benzyl chloride and gramine. The cyano compounds were
then converted to the tetrazoles and following hydrolysis the tetrazole

analogs of D, L-phenylalanine and tryptophane were obtained.

 

 

 

/
I
c,H5-CH201 + HC\-NH-C-CH3 NaOCsz. g
COZCZHS
O

NH-c-CH, NH-c-CH,
CbHs'CHz-C - C=N , CbHs‘CHz-Cl3-CN4H

COzCsz COZCsz

NH2
1. Cone. HCl \
2.1NaOH r C6H5’CH2'CH-fi —N-H
N N

\N/

- Sterken (19) prepared the tetrazole analogs of a-aminobutyric
acid, a-aminoisobutyric acid, 'y-aminobutyric acid, valine, leucine,
and C-phenylglycine by methods similar to those employed by McManus.

The formation of a 2, 5-disubstituted-1, 3, 4-oxadiazole from a
5-substituted tetrazole was first reported by Stolle (33) in 1929. On
heating acetic anhydride with 5-aminotetrazole the 2-acetamido-5-
methyl- 1, 3, 4-oxadiazole resulted. Stolle proposed a mechanism
involving the elimination of nitrogen from the 2 and 3 positions of the
tetrazole ring, formation of a nitrogen diradical, followed by the shift
of the acetamido group from the ring carbon to the diradical in a
Curtius type rearrangement to give a carbodiimide. The ca'rbodiimide
then underwent acylation, tautomerization, and ring closure to form
therl, 3, 4-oxadiazole. , .

Huisgen and co-workers (34) synthesized 2-(p-nitropheny1)-:5-
phenyl-l, 3, 4-oxadiaz-ole from the reaction of 5-pheny1tetrazole with
p-nitrobenzoyl chloride and pyridine. Huisgen'extended the Scope of
the reaction by converting a number of‘5-alkyl and 5-ary1tetrazoles
to their respective oxadiazoles by heating with pyridine and a wide
variety of acylating agents such as acetyl, benzoyl, p-toluyl, and
succinyl chlorides as well as acetic and benzoic anhydrides. - Huisgen's
proposed mechanism for the formation of‘the~oxadiazole is based on
the assumption that acylation occurs at the 2 position of the tetrazole
ring. I

The theory of amphoteric dissociation of amino acids has long
been recognized. Hitchcock (35), Bjerrum (36), and Edsall (37, 38)
have determined the ionization constants for many amino acids.
Recently Edsall and co-workers (22) worked out a complete ionization
scheme for tyrosine and proposed the scheme as illustrated in
Figure 1. 'Since each of the three functional groups can exist in either

the ionized or unionized form, there are eight possible forms for

tyrosine in going from the protonated form to the dianion. Edsall
also determined the ionization constants for tyrosine by two different
methods; direct titration, and calculation from "microconstants. "
The micro-constants are the individual interrelationships between the
eight forms.

The pKl and pKz values for the tetrazole analogs of some amino
acids were determined by McManus (3) and Sterken (19) and were found
to be in close agreement with pK values of the respective amino acids.

The influence of pH on the optical rotation of optically active
electrolytes, with the resulting changes in disassociation, has been
extensively studied. . Lutz and Jirgensons (20,- 21) determined the optical
rotations of a series of naturally occurring amino acids dissolved in
various amounts of acid and base. From a plot of the specific rotation
against the moles of acid and base, Lutz and Jirgensons predicted the
configuration of the amino acid. This was done by examining the acid
branch of the optical rotation curve. If, with increasing acid concen-
tration, it was found that the rotation changed toward the positive
direction, the amino acid was the L-antipode. If the rotation became
more negative, the amino acid was assigned the D-configuration.

In this manner configurations were assigned to naturally occurring
glutamic acid, arginine, lysine, ornithine, histidine, alanine, tyrosine,
aspartic acid, leucine, tryptophane, proline, hydroxyproline, and

di oxyphenylalanine .

PART I

TETRAZOLE ANALOGS OF AMINO ACIDS

DISCUSSION

The close metabolic relationship of phenylalanine (I) and tyrosine

(11) might be expected from the similarity in their chemical structure.

THZ TH;
Q -CHz-CH-COOH HO- O -CHz-CH-COOH
1 11

Numerous studies have been carried out to illustrate this relationship.
By use of isotope techniques (48, 10) it was shown that phenylalanine can
be converted to tyrosine in mammals. This finding is in accord with

the earlier work of Rose (44) on amino acids, which are essential to man.
He demonstrated that while phenylalanine was indispensable, tyrosine
was not, and that the requirement for phenylalanine both in the rat and

man is markedly reduced by the inclusion of tyrosine in the diet (11),

Some microorganisms, such as Neurospora can also convert phenyl-

 

alanine to tyrosine (46). In addition, several mutant strains of

Escherichia coli are capable of carrying out a reversible interconversion

 

of phenylalanine and tyrosine, since either amino acid will permit their
growth on an otherwise amino acid-free medium. In the mammal,
tyrosine also serves as the precursor of the hormones adrelin,
noradrelin, and thyroxine., Gurin and Delluva's work (45) using phenyl-
alanine labeled with tritium in the benzene ring and CM in the a-carbon
illustrated that this amino acid is converted to adrenaline in the rat.

The concept of antagonism between structurally similar metabolites
is explained on the basis of the existence of a competition between the

similar species for an enzyme or enzyme system with which both are

10

11

capable of associating. The application of this concept has been extended
to include the antagonism of a metabolite by a structurally related non-
metabolite, or "antimetabolite. " When interaction between the anti-
metabolite and the enzyme is reversible a competitive inhibition in the
utilization of a metabolite results. Non-competitive inhibition occurs
when the antimetabolite-enzyme reaction is irreversible.

Since the amino acids play such a vital and universal role in meta-
bolic processes of living organisms, they have been used as model
systems in the synthesis of potential antimetabolites. Phenylalanine has
been investigated thoroughly in this respect. Various alterations in the
molecular structure of phenylalanine have produced antagonism to
naturally occurring phenylalanine in bacterial growth. These alterations
are in the form of adding substituents to the amino group or the ring,
replacing the benzene moiety with other ring systems, and replacing the ,
carboxyl group with a functional group of related structure and reactivity,
such as the tetrazolyl moiety (43).

McManus (3) and Sterken (19) prepared a series of tetrazole ana-
logs of amino acids. It was considered germane to extend this series
to include optically active tetrazole analogs of phenylalanine and tyrosine,
and determine some characteristic physical properties for comparison
with the corresponding amino acids.

The most common and widely used technique for the synthesis of
5-substituted tetrazoles is through interaction of a nitrile with hydrazoic
acid or one of its salts. Three general schemes were used for the
preparation of nitriles from the corresponding L-amino acids with the
amino group suitably blocked. A fourth scheme employed, consisted of
alkylating ethyl acetamidocyanoacetate. A brief outline of each scheme

is given.

12

Scheme A

This method involved the use of the phthaloyl group as the amino
protecting group. L-phenylalanine was fused with phthalic anhydride in
an oil bathsat 145-1500C. The temperature must be kept below 150°C.
to prevent racemization. The N-phthaloyl amino acid is then converted
to the acid chloride with thionyl chloride, and subsequently to the amide
with ammonium hydroxide. The amide was dehydrated with benzene-
sulfonyl chloride in the presence of pyridine to give the nitrile. It was
possible to use a three to one molar ratio of benzenesulfonyl chloride to
amide without appreciably affecting the yield. A cleaner reaction
resulted by using the smaller amount of benzenesulfonyl chloride than
that used by Peterson and Nieman (2) to prepare the nitrile. Treatment
of the nitrile with aluminum azide, generated in 21‘} from sodium
azide and anhydrous aluminum chloride in dry tetrahydrofuran, gave the
tetrazole. It was noted that difficulty was encountered in obtaining a
clean, solid tetrazole if the aluminum chloride-tetrahydrofuran mixture
became dark. By cooling the tetrahydrofuran, then‘ adding it slowly to
the aluminum chloride cooled in an ice bath, only a slight yellow dis-
coloration appeared. The result was a more workable product. The
aluminum salt of the tetrazole was suspended in 1 N hydrochloric acid
liberating the free tetrazole. The phthaloyl group was removed by
hydrazinolysis giving the tetrazole analog of L-phenylalanine. This

scheme may be outlined as follows:

 

,E\
NH; 11
' \
Q -CHz-CH-COOH 1500C ,

/
0:0
I
\
O
\z
0
=5
0
O
O
I

 

 

 

1. $0012 [2
2. Nikon ‘ \ = A1(N;)3
3. cénssozm ’ .C/N (EH‘C-N

C5H5N H CIHZ

0 II
o
1' AH:
\ 1.N,H, L

.:C/N cH - fi—N-H 2. HCl a CHz-CH fi—lil-H

Attempts to apply the use of the phtholyl group as a suitable amino
blocking group for tyrosine were unsuccessful. Fusions with-L-tyrosine
and phthalic anhydride were attempted at 150°C. , 175°C. , and 200°C.

. for 30-45 minutes. At the higher temperature the reactionrappeared to
take place from the fact that water condensed in the upper part of the
flask” but efforts to obtain the desired product from the melt were with-

out success.

Scheme B

 

The tetrazole analogs of D, 12.-phenylalanine and L-tyrosine were

synthesized from the alph -benzamidonitriles. L-tyrosine was converted

14

to the ester since the benzoylation of the amino group was found to
occur more readily for the ester than the amino acid. Ammonolysis
of the ester gave the amide.

On addition of pyridine to the amide of N-benzoyltyrosine, a

precipitate formed which was believed to be the pyridinium phenoxide

salt.
11
H-lT-C-C6H5
/ O
Q 1 1WD.” ......
\
NH,
‘1
/ H-N- -c,H,

o
-CH,-CH-c

E-Z/
+ ......
9|
/\\

NH,

Since the phenoxide anion is a stronger nucleophile than the amide,
reaction of benzenesulfonyl chloride with the phenoxide anion would
occur first. Reaction between equal molar amounts of benzenesulfonyl
' chloride and N-benzoyltyrosine amide resulted in the formation of
L-a- .benzamido- (3- (p- benzenesulfonyloxypheny1)propionamide. When
the molar ratio of benzenesulfonyl chloride to N-benzoyltyrosine amide
was 3 to 1, L-a-benzamido-fi-(p-benzenesulfonyloxyphenyl)propionitrile
was formed.

Formation of the tetrazole from hydrazoic acid generated 111 £121
from sodium azide and glacial acetic acid, resulted in a cleaner product
while maintaining high yields. Another advantage found in using
hydrazoic acid was that the work up of the product was simplified. The
reaction mixture was evaporated to dryness leaving only the tetrazole
and its sodium salt in the residue. Acidification resulted in a tetrazole

of high purity. The free tetrazole was obtained by acid or base hydrolysis

15

of the benzamido moiety. These compounds were found to be difficultly
hydrolyzed, requiring 24-48 hours of refluxing. Hydrolysiswith con-
centrated hydrochloric acid is preferred to basic hydrolysis, since the
progress of the reaction can be followed by formation of the soluble
amine hydrochloride, and the possibility of racemerization is precluded.
Refluxing is continued until all of the benzamido tetrazole goes into

solution. The scheme is outlined as follows:

 

NH; RIHZ
I c,H50H
HO- -CH,-CH-COOH > 140- -CH,-CH-CO,C,H5
H,so,
O
H
HN-C-C6H5

 

 

C§5COC1> HO-</ \>-CH,-CH.-co,c,H5 NH‘LOH \

 

 

 

 

TH-C-C6H5
0
Ho- / \ -CH,-CH-c% CéH-ficlzllm ;
__ \NH2 5 5
o
H
HN-C-C6H5
I _ HN
-so,o- -CH,-CH-C=N —3-—>
fi
NH-C-C6H5
1. Cone. HC1\
”-SOzO—-CH,-CH-fi N-H 2. NH,0H 4,
N N
/
\N/
Na
Ho-.-CH,-CH-lcl: NH
N N

16

Scheme C

This method is essentially the same as scheme B except that the
acylation of the amino group is accomplished with acetic anhydride.
The scheme was found to be readily adaptable for the preparation of the
tetrazole analog of D, L-phenylalanine, but when applied to the synthesis
of the tetrazole analog of L-tyrosine, many difficulties were encountered.
The acetylated derivatives of L-tyrosine were found to be very water
soluble and difficult to crystallize, except for N-acetyl tyrosine ethyl
ester. A great deal of difficulty was experienced in trying to get the
acetylated nitrile to crystallize. In this respect the benzoylated com-
pounds are more desirable for synthetic purposes. However, the
5-acetylaminoalkyltetrazoles were more easily hydrolyzed and the acetic

acid produced can be removed by evaporation.

Scheme D

The alkylation of ac etylaminomalonic ester and ethyl acetamidocyano-
acetate has been a classical method for the synthesis of amino acids.
However, its application is limited to the synthesis of D, L-amino acids.

A In the synthesis of tetrazole analogs of amino acids by the alkylation of
ethyl acetamidocyanoacetate, only a minimum number of steps is

required. The procedure is outlined as follows:

 

in) I?
NH-C-CH3 'HN-C-CH3
l __ Naoc,H '
CH,O- -CHz-Cl + H? - C=N —-———; CH3O- -CHz-Cl:-C-:-N
COzCsz COZCsz
O
H
H-IiI-C-CHE;
A1(N) 1. Cone. HC
__._JJ_> _ ... - .— __ _
C N /N

/
o/ ‘oc,H5 \N/

17

NH,

|
CH3O-.-CH,-CH-|CIJ————Il\1—H

N\NéN

Anisyl chloride is prepared by the interaction of concentrated
hydrochloric and anisyl alcohol. The chloride should be used as quickly
as possible ;and not stored. Several minor explosions have been reported
on storing the material overnight (50). Following alkylation of ethyl
acetamidocyanoacetate with anisyl chloride, the product was converted
to the tetrazole with aluminum azide. Either a stepwise hydrolysis of
the functional groups, or a single step acid hydrolysis, gave rise to the
tetrazole analog of O-methyl-D, L-tyrosine. Attempts to obtain the
tetrazole analog of D, L-tyrosine by refluxing the O-methyl compound

with 57% hydroiodic acid were unsuccessful.

EXPERIMENTAL ‘1 Z» 3

The Preparation of D, L-5-(ti-Amino-B-phenylethyl)tetrazole
(D, L-Phenylalanine Analog)

Scheme A

(a) D, L-a-Phthalimido-8-phenylpropionic Acid

 

A mixture of 25. 0 g. (0. 152 mole) of D, L-phenylalanine and
22.4 g. (0. 152 mole) of phthalic anhydride was fused in an oil bath at a
temperature of 1800 for a period of forty-five minutes. After allowing
the resulting melt to cool to room temperature, it was dissolved in
100 m1. of methyl alcohol, filtered, and 100 m1. of hot water added.
The solution was then heated and allowed to crystallize. The solid
phthalimido derivative was filtered and dried to give 27. l g. (92% of
theory) of product, m.p. 175-1760 C.

J. Sheenan and V. Frank (1) reported this compound to melt at

177.5-179° C.

(b) D, L-a-Phthalimido-fi-phenylpropionLl Chloride

 

To a refluxing solution of 25. 0 g. (0. 0848 mole) of D, L-a-phthalimido-
B-phenylpropionic acid suspended in 250 m1. of dry benzene was added
10.7 g. (0.09 mole) of thionyl chloride with stirring over a period of ten

minutes. Refluxing was continued for an additional hour and during this

 

1Microanalysis by Microtech Laboratories, Skokie, Illinois.

zMelting points were taken in Open capillary tubes and are
uncorrected.

3All infrared spectra taken as Nujol mulls.

l8

19

period 5. 0 g. of thionyl chloride was added to insure complete reaction.
The reaction mixture becomes homogeneous since the acid chloride is
soluble in benzene. The benzene and excess thionyl chloride were removed
under reduced pressure on a steam bath; 100 ml. of fresh benzene was
added to the residue and again evaporated. The yield of crude acid
chloride was 26. 0 g. (98% of theory), m.p. 133-1360.

Sheenen and Frank (1) reported a melting point of 124-1260 C. for

the acid chloride.

(c) D, L-a- Phthalimido-p-phenylpr opi onamide

 

Twenty—five grams (0. 0794 mole) of D, L-a-phthalimido-[B-phenyl-
propionyl chloride was added to 100 ml. of cold ammonium hydroxide
and stirred for thirty minutes. The solid was filtered, washed with cold
water, and dried to give 21.1 g. of product (92% of theory), m.p.
232-233° C.

This compound was reported by Peterson and Nieman (2) to melt

at 236-2370 C.

(d) D, L- a- Phthalimido- kphenLlpropionitrile

 

To 20. 0 g. (0. 068 mole) of D, L-a-amino-B-phenylpropionamide
suspended in 72. 5 ml. of pyridine was added 36.0 g. (0. 204 mole) of
benzenesulfonyl chloride and the resulting mixture Was refluxed for ten
minutes. After cooling to room temperature, the solution was poured
into ice water. The precipitate which formed was filtered, washed with
cold water, and dried. The crude nitrile weighed 18.2. g. (97% yield),
m.p. 127-1290 C. After recrystallization from methyl alcohol, the
melting point was raised to 134-1350 C. ,

Peterson and Nieman (2) reported a melting point of 134-1350 C.

for this compound.

20

(e) D, L-5- (a- PhthalimichB-Jahenylethyl)tetrazole
To 17.0 g. (0.0616 mole) of D,~L-a.-phthalimido-fi-phenyl-

 

propionitrile and 12. 0 g. (0. 185 mole) of sodium azide placed in a

300 m1. 3-necked round bottom flask equipped with stirrer and con-
denser was added 9. 1 g. (0. 068 mole) of anhydrous aluminum

chloride dissolved in 150 m1. of dry tetrahydrofuran. The dissolution of
aluminum chloride in tetrahydrofuran is best accomplished by slowly
adding cold tetrahydrofuran to the aluminum chloride which is in a

flask placed in an ice bath. The nitrile, sodium azide, aluminum
chloride mixture was refluxed for a period of twenty-four hours.

The tetrahydrofuran was then distilled out of the reaction mixture and
water added at such a rate that the volume of the mixture remained
constant. The solid residue was filtered, washed with water, and dried.
The finely ground solid was suspended in 175 m1. of water, and 20 m1.-
of concentrated hydrochloric acid added. The precipitate was filtered,
washed with water, and dried. The yield of tetrazole was 19. 2 g.

. (98% of theory), m.p. 213-214°C.

This compound, prepared in a similar manner is reported by

McManus (3) to have a melting point of 212. 5-213° C.

(f) D, L-5- (a-Amino-[3-phenjlethyl)tetrazole,
To 18. 0 g. (0.0565 mole) of D, L-5-(a-phthalimido-B-phenylethyl)-

 

tetrazole suspended in 100 ml. of absolute ethanol was added 56. 5 ml.
of 1 M ethanolic hydrazine solution. The mixture was refluxed for two
hours, cooled to room temperature, then evaporated to dryness. The
residue was treated with 140 ml. of 2 N hydrochloric aCid and warmed.
on a steam bath at 50-550 C. for ten-minutes. The mixture was
filtered and the filtrate was neutralized to-a pH of 5 withconcentrated
ammonium hydroxide. The resulting precipitate was filtered, washed
with cold water, and dried. The product weighed 8. 5 g. (80% yield),

m.p. 270-2710 C. with decomposition.

21

‘McManus (3) reported a melting point of 271-2720 C. with

decomposition for this compound.

. Scheme B

(a) N - Benzoyl-D, L-phenylalanine

 

To 32. 0 g. (0. 194 mole) of D, L-phenylalanine placed inAa
3-necked 300 m1. round bottom flask equipped with a stirrer was
added 194 ml. of 1 N sodium hydroxide. The flask was immersed in
an icewater bath to maintain the temperature at 10 C. or lower.
Benzoyl chloride (27. 3 g. , 0. 194 mole) was added and, simultaneously,
at a rate four times faster 97 ml. of 2 N sodium hydroxide was added.
After complete addition of the reagents, the mixture was stirred for an
additional thirty minutes. If an appreciable amount of solid material
separated, a little Water was added. The solution was thenfiltered
and the filtrate acidified with cooling to Congo red. The precipitate
was filtered, washed with water, and dried i_n m over phosphorus
pentoxide. The yield was 45. 5 g. (87% of theory), m.p. 184--186o C.

This compound was prepared in a similar manner by Steiger (4)

who. reported a melting point of 1860 C.

(b) D, L-Ethyl-a-benzamido-p-phenjlp_ropionate ‘

 

N-Benzoyl-D, L-phenylalanine (42.0 g. , 0. 156 mole) was suspended
in 200 m1. of absolute ethanol, and 3 ml. of concentrated sulfuric acid
was added. 1 After refluxing for two hours, the solution was filtered
and the filtrate was evaporated to dryness under reduced pressure.

The solid product was then washed several times with cold water and
recrystallized from 50% ethanol. - After drying at 70°C. , the ester
weighed 45.6 g. (99% of theory), m.p. 94-95. 50 C. .

- Max (5) reported this compound to melt at 95-95. 50 C.

22

(c) D, L-c-Benzamido-8-phenylgropionamide
To 40.0 g. (0. 135 mole) of N-benzoyl-D,L-phenylalanine ethyl

 

ester dissolved in 50 ml. of acetone was added 100 m1. of concentrated
aqueous ammonium hydroxide. The solution was allowed to stand at
room temperature for two days with occasional stirring. - The solution
was then evaporated to dryness and the‘residue washed several times
with cold water. The crude product weighed 32. 5 g. (93% of theory),
m.p.. 195-1970 C. Recrystallizationfrom chloroform raised the melting
point to 1980 C. "

A melting point of 1980 C. for this compound was reported by
Max (5).

(d) D, L-a-Benzamido-fi-phenylpropionitrile

 

To 20. 0 g. (0. 0747 mole) of D, L-a— benzamido-0-pheny1propionamide
and 105 m1. of pyridine placed in a 250 m1. flask, was added 39.5 g.
(0. 244 mole) of benzenesulfonyl chloride and the mixture refluxed gently
for ten minutes. The resulting solution was allowed to cool to room
temperature and poured into ice water. Upon standing in the refrigerator
overnight, a yellow precipitate resulted which was filtered, washed
withcold water, and dried. The crude nitrile weighed 1'8. 1 g. (97%
yield) and had a melting point of 130-134°C. Recrystallization from
chloroform-petroleurn ether raised the melting point to 1480 C.

Kjaer (6) reported the melting point for theanitrile to be 1500 C.

(e) D, L-5- (a- Benzamido-p-phenylethy1)tetrazole
To 10.0 g. (0.040 mole) of D,.Lea-benzamido-B-phenyl-

 

propionitrile and 7. 8 g1- (D. 12 mole) of sodium azide was added 5.9 g.
(0.044 mole) of anhydro‘us aluminum chloride dissolved in 150 m1. of
dry tetrahydrofuran. »After refluxing the solution for a-period of
twenty-four hours, the tetrahydrofuran was distilled outof the reaction

mixture while water was added at such a rate that the volume of the

23

mixture remained constant. The solid was filtered, washed with water,
and dried. The finely ground solid was suspended in 175 m1. of water
and 20 ml. of concentrated hydrochloric acid added. The precipitate
was then filtered, washed with water, and dried. - The product
weighed 10.8 g. (92% of theory) and melted at 232-2330 C.

McManus (3) reported this compound to have a melting point of

233-234° C.

(f) D, L-5- (ti-Amino-fi-phenylethylhetrazole
To 8. 0 g. (0. 0273 mole) of D, L-5-(o.-benzamido-fl-phenylethyl)-

 

tetrazole was added 100 m1. of concentrated hydrochloric acid and the
mixture refluxed for twenty-four hours. The clear solution was allowed
to cool to 00 C. and the resulting precipitate filtered; 3.26 g. of
material having a melting point of 119-1200 C. was collected. When
mixed with an authenic sample of benzoic acid, the melting point of
the product showed no depression. The filtrate was adjusted to pH 5
by addition of aqueous ammonium hydroxide and the resulting precipitate
filtered, washed with water, and dried. The weight of the tetrazole was
4.2 g. (81% of theory); m.p. 272-273° C. with decomposition.

A mixture melting point with a sample of D, L-S-(a-amino-B-

phenylethyl)tetrazole prepared by Scheme A showed no depression.

Scheme C

 

(a) N-Acetyl-D, L-Lphenylalanine ' _
D, L-Phenylalanine (30. 5 g. , 0. 1845 mole) was dissolved in

 

92.5 ml. of 2 N sodium hydroxide and 100 ml. of water. The solution
was cooled in an ice bath and 48. 8 ml. of acetic anhydride and 488 m1.
of 2 N sodium hydroxide were added in eight equal portions with

vigorous shaking and cooling between additions. The mixture was then

allowed to stand at room temperature for forty-five minutes.

24

Addition of 182 ml. of 6 N sulfuric acid caused precipitation of the
product which was filtered, washed with water, and dried, yielding
33.7 g. (89% of theory) of N-acetyl-D,‘L-pheny1alanine,. m.p. 150-
152° C.

The compound is reported by du Vigneaud and Meyer (7) to have
a melting point of 151-152° C.

(b) D, L-Ethyl- a- ac etamido-p-pheny1pr0pionate

 

To a solution of 30. 0 g. (0. 145 mole) of N-acetyl-D, L-phenyl-
alanine dissolved in 200 m1. of absolute ethanol was added 3 m1. of
concentrated sulfuric acid. After refluxing the mixture for two hours,
the alcohol was evaporated under reduced pressure and the solid
residue was washed thoroughly with cold water, filtered, and dried
over phosphorus pentoxide i_n w. The yield was 34.6 g- (91%),
m.p. 60-61%.

The same compound prepared in another manner was reported

to have a melting point of 670 C. by Ashley and Harington (8).

(c) D, L-a-Ac etamido-p-phenylgoflonamide
To 29. 2 g. (0.125 mole) of N-Acetyl-D, L-phenylalanine ethyl

 

ester dissolved in 100 ml. of acetone was added 200 ml. of concen-
trated aqueous ammonium hydroxide. The mixture was allowed to
stand at room temperature for two days with occasional stirring.

The solution was then evaporated to dryness and the residue washed
with cold water. The yield of amide was 18.0 g. (70%), m.p. 143-
1450 C. The melting point was raised to 159-1620- C. after recrystal-
lization from 50% ethanol. A melting point of 161. 50 C. was reported

by Bergmann (l 3) .

(d) D, L-c-Ac etamido-g-Rhflylpropionitrile

 

To 16. 0 g. (0.0778 mole). of D, L-a-acetyl-B-phenylpropionamide
and 116 m1. of pyridine was added 41. 0 g. (0. 233 mole) benzenesulfonyl

25

chloride and the mixture refluxed for ten minutes. After cooling to
room temperature, the solution was poured into water and allowed

to stand in the refrigerator overnight. The resulting precipitate was
filtered, washed with water and dried. The yield of crude nitrile
was 12.8 g. (88% of theoretical), m.p. 99-100o C. After several
recrystallizations from chloroform-petroleum ether, the melting
point was raised to 108.5-109.5° C.

Analysis.

Calculated for CnleNzO: C, 70.19; H, 6.43; N, 14.89.

Found: C, 70. 03; H, 6. 33; N, 15.11.

(e) D, L-5-(a-Acetamido-8-pheny1ethy1)tetrazole

To 10. 0 g. (0.0533 mole) of D, L-5-a-acetamido-fi-phenylpropionitrile
and 10.4 g‘. (0.160mole) of sodium azide was added 7.9 g. (0.059 mole)
of anhydrous aluminum chloride dissolved in 150 m1. of dry tetrahydro-
fiiran. After refluxing for twenty-four hours, the tetrahydrofuran
was distilled out of the reaction mixture while water was added at such a
rate that the volume of the mixture remained constant. The aluminum
salt of the tetrazole, which separated as a solid, was filtered and dried.
The finely ground crude tetrazole salt was suspended in 175 ml. of water,
and 20 ml. of concentrated hydrochloric acid added. The precipitate
was filtered, washed'with water, and dried. The yield of tetrazole was
10. 1 g- (82% of theory), m. p. 226-2270 C.

McManus reported this compound to have a melting point of

224.5-225.5°‘C.7

(f) D, L-5-(a:Amino-p-phenylethyl)tetrazole

T08. 0 g.. (0. 0346 mole) of D, L-S-(Cl-acetamido-B-phenylethyl)-
tetrazole was added 100 m1. of concentrated hydrochloric acid and the
mixture refluxed for twelve hours. The clear solution was filtered.

The filtrate was adjusted to pH 5 by addition of concentrated ammonium

26

hydroxide and the resulting precipitate was filtered, washed with water,
and dried to give 5. 0 g. (78% of theory) of product, m.p.. 270-2710 C.
with decomposition.

A mixture melting point with an authenic sample of D, L-5-(a-amino-

8-pheny1ethy1)tetrazole showed no depression.

The Preparation of L-5-(ii-Amino-(3-phenylethyl)tetrazole
(L-Phenylalanine Analog)

Scheme A.

 

(a) L-a-Phthalimido-p—phenylpropionic acid

 

A mixture of 16. 5 g. (0.1 mole) of L-phenylalaninel' z and 14.8 g.
(0. 1 mole) of phthalic anhydride was fused in an oil bath at a temperature
of 145—1500 C. for thirty minutes. The melt was then allowed to cool to
room temperature and dissolved in 50 ml. of methanol. A small amount
of unreacted amino acid was filtered off and 50 ml. of water added to the
filtrate. The solution was then heated and allowed to crystallize. A total
of 27. 3 g. (93% of theory) of product was obtained which had a'melting
point of 177-1790 C. ,‘ [“13 .= -2060, C = 1.92 in absolute ethanol.

This compound was reported by Sheehan (9) to have a melting

point of 183-1850 C., [a]: = -212°, c = 1.92 in absolute ethanol.

(b) L-a-Phth’alimido-p-phenylpropionyl chloride
To 1a refluxing suspension of 25.0 g. (0. 0848 mole) of L-a-
phthalimido-8-phenylpropionic acid in 100 ml. of dry benzene was added

11. 9 g. (0. 1 mole) of thionyl chloride with stirring over a period of

 

lNutritional Biochemicals Corp.

‘.[a]‘D5 = -34.9°, C = 1.936 in H,o.

27

ten minutes. The reaction mixture was heated at reflux temperature

for an additional hour during which 5. 0 g. more thionyl chloride was

added to insure complete reaction. The benzene and excess thionyl

chloride were removed from the clear solution under reduced pressure.

The residue was dissblved in 50 m1. of fresh benzene which was again

removed under reduced pressure. The crude acid chloride weighed

26. 3 g. (99% of theory) and had a melting point of 83-850 C. ,

' [a]? = ~198o, C’: 2.256 in benzene. ,
Sheenan (9) reported a melting point of 83-840 0., [egg-5 = -l97,°

C = 2. 256 in benzene.

(c) L- a- Phthalimido- B-Rhenylpropionamide

 

L-a-PhthalimidoeB-phenylpropionyl chloride (25. 0 g. , 0. 080 mole)
was added to 200 ml. of cold concentrated ammonium hydroxide and stirred

for thirty minutes. The solid was filtered, washed with cold water, and

.dried to give 21.6 g. (92% of theory) of amide, m.p. 214-2160 C.

b‘
Recrystallization from 95% ethanol raised the melting point to 217-218 C.

Peterson and Nieman (2) reported the melting point to be 226-227. 50 C.

(d) L—a-PhthalimidojS-phenylpropionitrile

To 20.0 g. (0.0681 mole) of L-a-phthalimido-8-pheny1propionamide
suspended in 72. 5 m1. of pyridine was added 36. 0 g. (0. 204 mole) of
benzenesulfonyl chloride and the resulting mixture was refluxed gently
for ten minutes. After cooling to room temperature, the solution was
poured into ice water. The precipitate which formed was filtered,
washed with cold water, and dried. The crude nitrile weighed 18. 7 g.
(99% of theory), m.p. 146-1490 C. Recrystallization from methanol
raised the melting point to 150-151° c., [egg a -102°, C = 2 in CHCl,.

A melting point of 150-153.2° C. and [er-I; e 403?, C = 2% in CHC1,,

was reported by Peterson and'Nieman (2).

28

(e) 1.1-5-(o-Phthalimido-Q-Lphenylethyl)tetrazole

T016. 2 g. (0. 059 mole) of L-a-phthalimido-fi-phenylpropionitrile
and 11.5 g- (0.177 mole) of sodium azide was added 8.7 g. (0.065 mole)
of anhydrous aluminum chloride dissolved in 150 ml. of dry tetra-
hydrofuran. After refluxing for twenty-four hours, the tetrahydrofuran
.was distilled out of the reaction flask and water added at such a rate
that the volume remained constant. The solid product was filtered and
dried. The finely ground crude tetrazole salt was suspended in 175 ml.
of water and 20 m1. of concentrated hydrochloric acid added. A The pre-
cipitate was filtered; washed with water, and dried. . The crude product
weighed 16.9 g. (90% of theory) and melted at 170-1780 C. The melting
point was raised to 182-1830 C. after recrystallization from chloroform-

petroleum ether. [0.]2D5 = -139. 90,1 C = 4 in absolute ethanol.

Analysis.
Calculated for C17H13N502: C, 63.94; H, 4. 10; N, 21.93.
Found: C, 63.81; H, 3.88; N, 22.17.

(f) L-5-(o-Amino-fi-phenylethyl)tetrazole

To 14.0 g. (0. 0439 mole) of L-5-(o-phthalimido-8-phenylethy1)-
tetrazole suspended. in 100 ml. of absolute ethanol was added 43. 9 ml.
of a 1 M ethanolic hydrazine solution. The mixture was refluxedefor
two hours, cooled to room temperature, then evaporated todryness.
The residue was treated with 109 ml'.. of 2 N hydrochloric acid and
A warmed on a steam bath at 50-550 C. for ten minutes. The mixture
was filtered and the filtrate adjusted to pH 5 with concentrated ammonium
hydroxide. The resulting precipitate was filtered, washed with cold water,
and dried. The product weighed 6. 0 g. (72% of theory), m.p. 2870 C.
withdecomposition, [71]}; = +38.0‘,’. C = 0_. 945 in H,o.

Analysis.

Calculated for c.,HnNs': C, 57. 12; H, 5.86; N, 37.02.

Found: C, 57.20; H, 5.85; N,"36.88.

29

The Preparation of L-5-[ti-Amino-[Mp-hydroxyphenyl)ethy1]tetrazole
' (L-Tyrosine Analog)

Scheme B

 

(a) L-Tyrosine etlfil ester

 

One hundred grams (0. 552 mole) of L-tyrosine. was placed in a
500 ml. round bottom flask containing 300 ml. of absolute ethanol.
The suspension was treated with hydrogen chloride gas until all of the
tyrosine dissolved. After addition of 3 ml. of concentrated sulfuric
acid, the solution was refluxed for 6 hours. The excess ethanol was
removed under reduced pressure and the residue dissolved in 200 m1.
of water; the solution was filtered, the filtrate adjusted to p118 with
concentrated ammonium hydroxide. The ester whichseparated as a
crystalline solid was filtered with suction, washed several times with
cold water, and dried at 80° C. The crude ester weighed 105.0 g.
. (92% of theory), m.p. 99-102o C. Recrystallization from ethyl acetate-
petroleum ether, gave a melting point of 103-1040C. , [a]? = +17.40,°
C = 4.0 in absolute ethanol.

Fischer (12) reported the ester to have a melting point of 108-
109° c., {or}; = +20.40,° C = 4.85% in absolute ethanol.

(b) L- Ethyl-o- benzamido- p- (p-hydroxypheny1)propionate

 

A solution of 103.4 g. (0.495 mole)..of L-tyrosine ethyl ester in
750 m1- of chloroform was cooled to 00 C. Benzoyl chloride (7.0. 3 g. ,

0. 50 mole) was added in seven portions with vigorous shaking between
additions. ~ The temperature was maintained below 10° C. After all of
the benzoyl chloride had been added, 250 m1. of 2 N sodium carbonate
(0.50 eq.) was added and the mixture shaken until no more carbon
dioxide was Evolved. The chloroform layer was then separated, washed

several times with cold'water, and evaporated to dryness. The yield

30

of crude product was 149.6 g- (96% of theory), m.p. 115-1180 C.
Recrystallization from chloroform-petroleum ether raised the melting
point to 120--121o C., [a]? = -22.50, C = 3.7 in absolute ethanol.
Bergel and Lewis (14) reported a melting point of 122-1230- C. and
[a]? = -23.70, C = 3.7 in absolute ethanol.

(c) L-a-Benzamido-fi-(p-hydroxyphenyl)propionamide

To a solution of 143.0 g. (0.457 mole) of L-ethyl-B—benzamido-
0-(p-hydroxypheny1)pr'opionate dissolved in 200 m1. of acetone was
added 300 ml. of concentrated aqueous ammonia. The solution was
allowed to stand at room temperature for two days withroccasional
stirring. The precipitate which formed was collected and the filtrate
was evaporated to dryness i_n 1151323 without heating. The combined solid
products were suspended in 500 ml. of cold water and the pH‘adjusted
to 4 with concentrated hydrochloric acid. The crude amide was filtered,
dried, , and weighed; the yield was 98. 0 g. (76% theory), m. p. 198-2000 C.
A melting point of 204-2050 C. was obtained after recrystallization
from 50% ethanol. [0.]? = -22.90, C = 1 in methanol.

Ber'gmaniand Fruton (15) reported this compound to have a melting

point of 198°C- and [a]? = -24.6°,. C = 4.6% in methanol.

Analysis.
Calculated for C16H16N203: C, 67.59; H, 5.67; N, 9.86.
Found: C, 67.51; H, 5.68; N, 10.07.

(d) L-a-Benzamido-[S-(JL- benzenesulfgnyloxyphenylmropionamide
L-a-Benzamido-B-(p-hydroxpheny1)propionamide (14. 0 g. ,
0.04931mole) was dissolved in 50 ml. of pyridine. A precipitate of
the pyridinium phenoxide salt quickly formed. 1 Benzenesulfonyl chloride
(9. 5 g. , 0. 054 mole)‘.was added, the mixture shaken vigorously for 10

minutes, and refluxed gently for 10 minutes. After allowing the solution

31

to cool to room temperature, it was poured intorcold water and placed
in a refrigerator overnight. The precipitate-which formed was
collected on a filter, washed with cold water, and dried. The product
weighed 10.5 g. (50% of theory), m.p. 210-2130 C. Recrystallization
from 95% ethanol raised the melting point to 218-219o-C. The product
was insoluble in aqueous sodium hydroxide and gave a positive quali-

tative test for sulfur.

Analysis.
Calculated for C22H20N205S: N, 6. 60.
Found: N, 6.82.

(e) L-a- Benzamido-[S-(p-benzenesulfonyloxyphenyl)propionitri1e

To-a suspension of 60. 0 g. (0. 2115 mole) of L-‘acbenzamido-[S-
(p-hydroxypheny1)propionamide in 90 m1. of pyridine, 111. 6 g. (0. 63
mole) of benzenesulfonyl chloride was added. - The mixture was refluxed
for ten minutes, cooled to room temperature, then poured into cold
water and allowed to stand overnight in the refrigerator. -The solid
that had separated was dissolved in 60% ethanol and was cooled overnight
in the refrigerator. The precipitate was collected on a filter and dried,
m.p. 118-1190 C. The yield of crude nitrile was 54. 3 g. (63% of theory).
Several recrystallizations from 60% ethanol raised the melting point to
124-125? C. ,. [of]; = -8. 30,. C = 4.0 in chloroform. .A positive quali-
tative test for sulfur was obtained and the compound was insoluble in

aqueous sodium hydroxide.

.Analysis.
Calculated for C,,H,,N,o.s: C, 65.01; H, 4.46; N, 6.89; S, 7.89.
Found: - C, 64.93; H, 4.63; N, 6.84;.S, 7.77.

(f) L- S-Ia- Benzamido- Q- (p-benzenesulfonyloxyphenyl) ethyljtetrazole
L-a- Benzamido- (3- (p-benzenesulfonyloxyphenyl)propionitrile

(34. 2 g. ,. 0.084 mole) was placed in a 500 ml. 3-necked round bottom

32

flask equipped with a condenser-and a stirrer. Sodium azide (10.4 g. ,
0. 16 mole), glacial acetic acid (9. 6 g. , 0. 16 mole), and n-butyl
alcohol (150 ml.) were added and the mixture refluxed for two days.
The mixture was then evaporated under reduced pressure in the hood.
- The residue was suspended in 500 m1. of cold water], and then acidified
to Congo red with hydrochloric acid. The precipitate was collected
and washed several times withacold water. The product gave a positive
qualitative test for sulfur and was soluble in sodium hydroxide solution.
The yield of crude tetrazole was 36.6 g- (97%), m. p. 204-60 C.
» Recrystallization from ethyl acetate containing a small amount of ethanol
gave a product melting at 209. 5-210. 50 C. , [a]? = -55.5°, C = 2.0 in
1 M sodium hydroxide. 1
.Analysis.
Calculated for szH19N504S: C, 58.78; H, 4.26; N, 15.58; S, 7.13.
Found: C, 58. 74; H, 4. 31; N, 15.44; S, 7. 20.

(g) L-5-[o-Amino-[3-(Jo-hydroxyphenyl)ethyl]tetrazole

A suspension of 33.4 g. (0. 0744 mole) of L-5-[c-benzamido-8-
*(p-benzenesulfonyloxyphenyl)ethy1]tetrazole in 250 m1. of concentrated
hydrochloric acid was boiled under reflux for forty-eight hours. » The
clear solution was cooled slowly toOo C. and the precipitate of benzoic
acid collected. The precipitate weighed 8. 8 g. (97% of theory), m. p.
119-1200 C. A mixture melting point with-an authenic sample of benzoic
acid; showed no depression. The filtrate was evaporated to dryness in an
-air stream, dissolved in 200 ml. of cold water, and adjusted to pH 6
with concentrated ammonium hydroxide. The yield of product was 14. 8
g. (97% of theory), m.p. 2950 C. with decomposition. Recrystallization
from water raised the melting point of the product to. 302° C. with

decomposition, [1113 = + 33.80,. C = 4.0 in 2 N hydrochloric acid.

33

Analysis.

Calculated for C9H11N50: C, 52.67; H, 5.40; N, 34. 13.

Found: , C, 52.20; H, 5.41; N, 33.88.
. Scheme C

 

(a) N-Acetyl-L-tyrosine
L-Tyrosine (45.0 g., 0.2485 mole) was dissolved in 127.5 m1. of

 

2 N sodium hydroxide and 75 ml. of water. The solution was cooled in
an ice bath and 60 m1. of acetic anhydride and 600 ml. of 2 N sodium
hydroxide were added in eight equal portions with vigorous shaking.
After standing at room temperature for forty-five minutes, 251. 7 ml.
of 6N sulfuric was added. The solution was then evaporated to dryness
and the residue extracted with 80% acetone. The sodium sulfate was
filtered off and the acetone extract evaporated i_n w to a thick syrup.
A small amount of water was added to the syrup (about 25 m1.) and the
acetyltyrosine crystallized on standing in the refrigerator overnight.
The yield of product was 12. 1 g. (66% of theory), m.p. 147-1490 C.

du Vigneaud (7) reported a melting point of 152-1540 C.

(b) L- Ethyl-a- ac etamido-p-.(_Ehydroxyphenyl) propionate

N-Acetyl-L-tyrosine (11. 0 g. , 0. 0494 mole) was dissolved in
100 ml- of absolute ethanol, 1 m1. of concentrated sulfuric acid was
added, and the solution heated under reflux for two. hours. The solution
was then evaporated to a thick syrup, 200 ml. of water added, and the
mixture thoroughly shaken. On standing in the refrigerator for» several
hours a precipitate formed. The solid was filtered, washed several
times with cold water, and dried i_n 17352.2 over phosphorus pentoxide.
The crude ester melted at 76-790 C. and the yield was 7.8 g. (64% of
theory). . A melting point of 79-1800 C. was reported by Kaufman and

co-workers (16) for this compound.

34

(c) L-a-Ac etamido-fi-(p-hydroxyphenyl)p_rgpionamide

L-Ethyl-a-acetamido-B-(p-hydroxypheny1)propionate (7.0 g. ,
0.0284 mole) was dissolved in 25 m1. of acetone and 100 m1. of concen-
trated aqueous ammonium hydroxide added. After allowing the mixture
to stand at room temperature for two days with occasional stirring,
the solution was then evaporated to dryness i_n w without heating.
The residue was suspended in water and adjusted to pH 3. The yield of
amide was 4.9 g. (80% of theory), m.p. 220-2210 C. Recrystallization
resulted in a product melting at 221-222° C.

Analysis.
Calculated for CnHuNzO3: C, 59.44; H, 6.35; N, 12.60.
.Found: C, 59.43; H, 6.50; N, 12.62.

This compound was reported by Kaufman and Neurath (17) to have
a melting point of 222-2240 C.

(d) L-o-Ac etamido-p-(p-benzenesulfonyloxyphenyl)propionitrile

L-d-Ac etamido-[S-(p:-hydroxyphenyl)propionamide (4. 0 g. ,9 '0. 018
mole) was suspended in 13. 3 m1. of pyridine and 9. 5 g. (0.054 mole)
of benzenesulfonyl chloride was added. — The mixture was refluxed gently
for ten minutes, cooled to room temperature, and poured into cold
water. After standing in the refrigerator overnight, the water was
decanted and the residue was dissolved in 70% ethanol and refrigerated
overnight. The crystalline solid was filtered and recrystallized from
70% ethanol. . Four and a half grams (73% yield) of nitrile was obtained.
Its melting point was 84-850 C. A positive qualitative test for sulfur

indicated that the benzenepsulfonyate had been formed.

Analysis.
Calculated iorc,,H..N,o.s:' C, 59.30; H, 4.68;.N, 8.13; s, 9.30.

Found: C, 59.22; H, 4.64; N, 7.93; S, 9.39.

35

(e) L-5-ju-Ac etamido-p-(pabenzenesulfonyloxyphenyl)ethyljtetrazole

L-e-Acetamido-B-(p-benzenesulfonyloxyphenyl)propionitrile (4. 0 g. ,1
0. 0116 mole) was placed in a 100 ml- round bottom flask equipped with-a
condenser and a stirrer. After addition of 1. 51 g. (0.0232 mole) of 1,
sodium azide, 1. 39 g. (0.0232 mole) of glacial acetic acid, and 50 m1.
of n-butyl alcohol, the mixture was refluxed for two days. The reaction
.mixture was evaporated to dryness under reduced pressure in the hood,
the residue suspended in 200 ml. of cold water, and thenacidified with
hydrochloric acid to Congo red. - The precipitate of crude tetrazole was
collected on a filter, washed several times with cold water, and dried.
The yield of crude tetrazole was 4. 5 g. . (100% of theory), m. p. 205-2070 C.
Recrystallization from ethyl acetate containing 25% ethanol raised the

melting point to 210-2110 C. A positive qualitative test for sulfur was

obtained.
Analysis.
Calculated for C17H17N504S: C, 52.70; H, 4.42; N, 18.08; S, 8.28.
Found: C, 52.71; H, 4.42;. N, 18. 02;.S, 8.19.

(f) L- 5 - [d-Aminoj3- (p -hydroxyphenyl) ethyl]tetrazole

A suspension of 4. 0 g. (0.0103 mole) of L-5-[a-acetamido-8-
(p-benzenesulfonyloxyphenyl)ethy1]tetrazole in 100 ml. of concentrated
hydrochloric acid was heated under reflux for twodays. , The clear
solution was filtered and the filtrate was evaporated to dryness, dis-
solved in 100 m1. of water, and adjusted to pH 6 with aqueous ammonia.
. The product separatedfrom the solution as a crystalline solid; the
yield was 2.0 g. (94% of theory), m. p. 2970 C. with decomposition.
~Recrystallization of this material from water raised the melting point to
3020 C. with decomposition. r Mixture melting point withra pure sample
of 5-.-[o.-amino-fi-(p-hydroxyphenyl)ethy1]tetrazole prepared by an

alternate method showed no depression. .Also, the infrared spectrum

36

was identical with that of authenic 5-[a-amino-B—(p-hydroxypheny1)-

ethy1]tetrazole.

The Preparation of D, L-5[a-Amino-[3-(p-methoxyphenyl)ethy1]tetrazole
(O-Methyl-D, L-Tyrosine Analog)

Scheme D

 

(a) p-Methoxyhenzjl chloride (anisyl chloride)

In a 100 m1. round bottom flask fitted with a stirrer, was placed
13.8 g. , 12.5 ml. (0. 1 mole) of anisyl alcohol and 24.8 ml. (0. 3 mole)
of concentrated hydrochloric acid. After stirring for ten minutes, (the
solution is transferred to a separatory funnel and the lower layer of
anisyl chloride separated, dried over 20 g. of magnesium sulfate for
thirty minutes, and filtered. This material cannot be stored for pro-
longed periods and should be used as quickly as po§ssible. The procedure
for preparing anisyl chloride is similar to that described by Rorig and
.co-workers (18). Without further purification the crude anisyl chloride
was used for the preparation of ethyl-a-acetamido-o-cyano-(3-(p-

methoxyphenyl) pr opionate .

(b)a D, L- Ethyl-a- ac etamido- d-cyano-j-jp -methoxypheny1) propionate

 

Toia solution of 2.4 g. (0. 1 mole) of sodium in 100 ml. of dry
absolute ethanol was added 17. 0 g. (0. 1 mole) of ethyl acetamido-

c yanoac etate . l

p-Methoxybenzyl chloride (15.6 g., 0. 1 mole) was

added dropwise over a period of fifteen minutes with stirring. Refluxing

. and stirring were continued for an additional two hours. The thick tan

, suspension was poured into cold water and the tan solid which precipitated
was filtered, washed with water, and dried yielding 24. 8 g. (86% of

theory) of crude product, m.p. 160-1630 C. Recrystallization from

 

lAldrichiChemical Co. .

37

50% ethanol afforded a white, crystalline material which melted at

l65.5-166° C.

Analysis.
Calculated for C,,H,,N,o,: C, 62.05; H, 6.25; N, 9.70.
Found: C, 62.04; H, 6.16;N, 9.36.

(c) D,L-Ethy1-o- ac etamido- a- (5 -tetrazoly1) -8- (p-methoxy-
phenyl)propionate

, To 26.1 g. (0.09 mole) of ethyl-a-acetamido-a-cyano-8-(p-
methoxyphenyl)propionate and 17. 5 g. (0. 27 mole) of sodium azide was
added 13. 3 g. (0. 10 mole) of anhydrous aluminum chloride dissolved in
150 m1. of dry tetrahydrofuran. After the resulting mixture was
refluxed for twenty-four hours, the tetrahydrofuran was distilled from
the reaction mixture and water was added at such-a rate as to keep the
volume constant. The aluminum salt of the tetrazole that had separated
from the aqueous solution was filtered and dried. The filtrate was
acidified and on cooling in the refrigerator overnight the small amount
of tetrazole which precipitated was filtered, washed with water, and
dried.

The aluminum salt of the tetrazole was finely ground and sus-
pended in 175 m1. of water and 20 ml. of concentrated hydrochloric acid.
. The tetrazole was filtered, washed with cold water, and dried. The 1
combined products weighed 23. 0 g. (77% of theory). Recrystallization

from benzene gave a melting point of 137. 5-138o C.

, Analysis.
Calculated for C15H19N504: C, 54.04; H, 5.75; N, 21.01.
Found: C, 53.99; H, 5.64; N, 20.93.

((1) D, L-a-Acetamido-c-(S- tetrazolyl)- -8- -(p-methoxyphen l)-
propionic acid

D, L- Ethyl-d-ac etamido-e- (5-tetrazoly1) - 8- (p-methoxypheny1)-

propionate (5. 0 g. , 0. 015 mole) was added to 48 ml. of water containing

38

2.4 g., (0.06 mole) of sodium hydroxide and the mixture refluxed for
one hour. The resulting solution was filtered, the filtrate cooled,
and acidified to Congo red. The mixture was cooled overnight in the
refrigerator and the precipitate which formed was filtered and dried.-
The crude acid had a melting point of 109-1110 C. with (gas evolution,
solidified, and remelted at 162--163o C. Recrystallization attempts
withiethyl acetate-petroleum ether caused a lowering of the melting
point. , Further attempts at recrystallization gave a product having a
melting point of 155-157° C. which was shown to be identical with
~D, L-5-[a-acetamido-8-(p-methoxyphenyl)ethy1]tetrazole by mixture
melting point and infrared spectrum.

In view of the difficulty encountered in recrystallization of the
material, apparently due to decarboxylation, a sample of crude product
was submitted for analysis after drying at room temperature in a

desiccator over‘ P205.

Analysis.
Calculated for C,,H,,N_.,o.-2H,o: N, 20. 52; H,o, 11.43.
Found: N, 20. 56; H20, 11. 34.

The amount of water of hydration in the crude product was
determined by drying a sample to constant weight at 78° C. under re-
duced pressure over phosphorus pentoxide. The dried material melted

at 104°C.

(e) D, L- S-Ia-Acetamido-p- (p-methoxjghenylbthylltetrazole

 

D, L-d-Ac etamido-ob (5 -tetrazolyl) - 8- (p-methoxyphenyl)propionic
acid (2. 0 g. ,. 0. 0066 mole) was heated in an oil bath at 1400 C. with gas
evolution for fifteen minutes. The residue weighing 1. 7 g. (100% of
theory) had a melting point of 162-1630 C. Recrystallization from

aqueous ethanol resulted in‘a melting point of 156-1570 C.

39

Analysis.
Calculated for C12H15N502: C,.55. 16; H, 5.79; N, 26.81.
Found: C, 55.25; H, 5.97;;N, 26.89.

(f) D,'L-5-[a-Amino-8-(p-methoxyphenyl)ethyljtetrazole

D,-‘L-Ethy1-o.-~ac etamido-a- (5 -tetrazoly1) - 8- (p -methoxyphenyl) -
propionatee(18.0 g. , 0.0541 mole) was refluxed with 200 m1. of concen-
trated hydrochloric acid for three hours. The solution wasevaporated
almost. todryness. then dissolved in 100' ml. of water. The; solution
was then filtered and the filtrate adjusted to pH 6 with concentrated
ammonium hydroxide. The precipitate was filtered and dried yielding
9. 2 g. (78% of theory) of product, m.p. 273° C. with decomposition.
-Recrystallizationrfrom water raised the melting point to 298Q C. with

decomposition.
Analysis.
Calculated for CmHnNsO: C, 54.78; H, 5.98; N, 31.95.
Found: C, 54.73; H, 6.05; N, 32.22.

PART II

PROOF OF STRUCTURE AND CONFIGURATION OF
TETRAZOLE ANALOGS OF AMINO ACIDS

40

DISCUSSION

Huisgen (34) and co-workers found that 5-substituted tetrazoles
would react, in the presence of pyridine, with a variety of acyl halides

to form 2, 5-disubstituted 1, 3, 4-oxadiazoles.

 

 

/? fi' :17
R -ng + R-C III-H M R'-C\ /C-R +
N /N 0
\N/ N, + HCl

The oxadiazole was then degraded by refluxing with concentrated

hydrochloric acid.

N

N
H | ,
C C-R Conc. HCL;

 

 

R'-COOH + R-COOH + NszHCl

It therefore appeared fruitful to synthesize the oxadiazoles from the
tetrazole analogs of the amino acids prepared in Part 1. Their subse-
quent hydrolysis would give the original amino acids used as starting
materials. In the case of the Optically active amino acids, this technique
would be a method to prove their configuration, since the amino acids
recover ed from the tetrazoles should have the same rotation as the

original amino acids .

41

42

 

 

 

 

 

 

Formation
fi
IINHZ C6H5-C- H E—fi
2C6H5COC1
C -CH -C- N-H > C -CH -C - C-C H
ans 2 | f]: l CsHsN 6H5 z ‘ \0/ 6 5
H N N
/ H
\N/
ff
NH; fl C6H5-C-NH
_ 3C H COCl
HO-C H -CH -c—c -H ———¢—5——-> c -c-o- -CH -c-
6 s z I "A? CsHsN 6H5 z I
H N N H
\N/
ff if
“C C-C
\0/ .n.
Hydrolysis
l
CsHs-C-NH N N Conc. NHz-HC1
I H H HCI I
CbHs'CHz’C - C C~06H5 "»""_"—'> C6H5-CH2*$-COOH +
l \O/
H ‘ H
NHz-NHanCl + 2C6H5COOH
0
ll

0 C6H5-C-NH N—N Conc. NHz-HCI

| ' H l HCl ,1

c.,Hs-c-o- ~CHz-C - c c--c,,H5 9 Ho-c,H,-CHz-- -COOH +
H \o/ ILI

NHz—NHZ- HCl + 3C6H5COOH

 

43

The amino acids obtained on hydrolysis of the oxadiazoles prepared
from the tetrazole analog of L-phenylalanine and L-tyrosine did have
the same sign of rotation indicating that the tetrazole analogs were of
the L-configuration, but their rotations were not of the same magnitude
as those of the original amino acids. It appeared that during the form-
ation of the oxadiazole, some racemization may have occurred.

It is known :that in the presence of base, the hydrogen at the asymmetric
carbon of optically active acylated amino acids is labile and racemeri-
zation can occur (7). A similar situation could account for the partial
racemerization of the optically active tetrazoles or oxadiazoles in
pyridine. The melting points of the recrystallized amino acids,
obtained on hydrolysis of the oxadiazoles, were lower than both the L
and D, L amino acids. However, their infrared spectra were identical
with those obtained from authenic samples of L-phenylalanine and
L-tyrosine.

Stolle (33) and Huisgen (34) have proposed mechanisms for the
formation of 2, 5-disubstituted 1, 3,4-oxadiazoles from 5-substituted
tetrazoles. Stolle's mechanism was proposed to explain the formation
of 2-acetamid’o-5-methy1-l, 3, 4-oxadiazole from the interaction of
acetic anhydride with 5-aminotetrazole. It is based on the assumption
that acylation occurs at the one position of the tetrazole ring. These
mechanisms, as adapted to the formation of 2, 5-disubstituted 1, 3, 4-
oxadiazoles from tetrazole analogs of phenylalanine and tyrosine, are
as follows. i

In the first step of Stolle's mechanism the amino group is

 

acylated. ('9
* NH; c,H5-<l:-I‘~JH
R-CHz-C - fi—NH chgicom) R-CHz-C - c —NH
I I 5H5N I II I
H N _ N H N N
\Ny \N//

R: -C6H5, -C6H,0H

44

This is followed by acylation at the one position of the tetrazole ring

by a second molecule of benzoyl chloride.

 

 

I? ' no
I
CbHs-C-NH C6H5-C-NH R
I ,
.R-CHz-CIJ - fi NH CbHicocn: R-CHz-(ll - Ci—N-C-Csns
H N H N
\N¢N \N¢

Next, a diradical would result from the rupture of the bond between the
number one and two nitrogens followed by the elimination of number two
and three nitrogens as a nitrogen molecule.

0
II H WINE .

 

 

 

 

 

CbHs-C-III I II
R-CHz-(ll - ICI N-C-C6H5 —> R-CHz-C - 'Cr N-C-C6H5
H N ' H N
\NéN \NzN-
fi
C6H5-C- H 0
I u
——"> R-CHz-Clz " ' N-C-C6H5 " N2 \
H N
- n
GEN-
f?
C6H5-C-IFH fl)
R-CHz-C - C N-C-C6H5

This nitrogen diradical would then give rise to the 1, 3, 4-oxadiazole.

o ‘ o
l I

O 'I
C6H5-C-NH N '3 CbHs-C-NH N—N
R CH 6 Id “N, R CH 6 rd 2 C
- - - —— —-> , - .. - - H
2 I B? _ z | \O/ 6 5
H O—C-C6H5 H

45

The mechanism proposed by Huisgen (34) assumes acylation

takes place at the number two nitrogen.

C
II
C6H5-C-NH
N o

R-CHz-C - C—
I I I ll

H N N-C-CH
§N/ 6 5'

 

Bond cleavage occurs between the number twoand three nitrogens and
this is followed by elimination of a molecule of nitrogen from the

number three and four nitrogens.

 

 

I?
C,H,-C-NH 0 ' o\
' “ C-C6H5
R-CHz-C': - C NCICI) C6H5-C.NH II
N
H‘:N N-C--CH-—->RCH-C- CZN"
H +N
III
N

Elimination of nitrogen, formation of the carbine, and cyclization

would give rise to the oxadiaiole.

Q o

o
CHngNH \fiC‘Hs CH gNH {N'°N
6 5 61_ g” - N 6 5 I 6“) \J\
R- “CH; -C- N —L> R-CHz-C --C: Q,C-C,,H5
H +III H
N 0

II
C6H5-C- -NH III—ll
—————> R-CH -C - C -C-:H
z I \O/c 6 5

H

46

A concerted mechanism can be postulated for the formation of

oxadiazole from tetrazoles, acylated at either the one or two positions.

 

C,,H5 0
II O\\ / II
c.,Hs-C-NH C,,H5 --C NH
l ”(I /0\
R‘CHz-C II-I -$ ND —"'9 R- ”CH2 ' (g E- C6H5
N‘jg—r/III H N——N +sz

 

 

The degradation of 2-acylaminoalkyloxadiazoles with concentrated
hydrochloric acid to give amino acid hydrochlorides, may proceed in
the following manner. Protonation of the nitrogen, attack of a water
molecule, followed by elimination of a hydrogen ion would give rise

to a diacylhydrazine.

 

 

 

+
N N NH
.2 5.. H* II Ian
' " '6 5 ——'> "' 6 5
N—IFIH + N NH
Hio R_C C,,H5 -H R_ C'\ C,H5

 

 

47

'A repetition of the same sequence of reactions as previously out-

lined would rIesult in the formationaof the hydrazide and benzoic acid.

+
H —NH HN—NHz
I H+
R-fi fi-CbHs -————-> R-fi I-C6H5
O O O
+
HIP—IIIHZ
Hzo R_ c/C6H5
' P35”
0 O I
H
+
HN , O
I Ig/IZC6H5 II
R-C \ ——> R-C-NH-NH,+C,H5C00H

Protonation of the hydrazide with subsequent attack by water would

give rise to hydrazine hydrochloride and the amino acid hydrochloride.

0 0
II H+ II I' H O
.R-C-NH-NH, ————> .R-C-NH'z-NHZ ——1-f-—>

<3. . 3;». a

R-C-NHz-NHZ ——-> R-CIJ-gHz-NHZ ——-> R-C-OH + NHz-NHz-HCI

35% OH“

EXPERIMENTAL 1

A. Synthesis of 2-Substituted-5-Pheny1-1, 3, 4-Oxadiazoles
From Tetrazole Analogs of Amino Acids

D, L-5-(o-Amino-8-pheny1ethy1)tetrazole
(D, L-Phenylalanine analog)

(a) D, L-S-(a.-Amino-Q-Lheny1ethy1)tetrazole

This compound was prepared from D, L-phenylalanine by converting
the amino acid to o-phthalimido-B-phenylpropionitrile. The nitrile was
then converted to D, L-S-(o-amino-fi-phenylethylhetrazole according to
the procedure described in Part I. The crude product was recrystal-

lized from water after which it melted at 2780 C. with decomposition.

(b) D, L-2-(1'-Benzamido-Z'-pheny1ethy_l)-5-phenyl- 1, 3, 4-
ozadiazole

 

A mixture of 3. 78 g. (0. 02 mole) of D, L-5-(o-amino-fl-phenylethyl)-
tetrazole, 5.60 g. (0.04 mole) of benzoyl chloride, 16 ml. (0.2 mole)
of pyridine, and 25 ml. of dry benzene was refluxed for ten hours.
A small amount of oxadiazole precipitated from the benzene solution.
The solution was evaporated to dryness and the residue recrystallized
several times from absolute ethanol. Six grams (81% of theory) of

oxadiazole was collected, m.p. 167. 5--168o C.

Analysis
Calculated for C23H19N3023 .C, 74. 78; H, 5. 18; N, 11.38.
Found: C, 74.78; H, 5.16; N, 11.30.

1All infrared spectra taken as Nujol mulls.

48

49

- L-5-(a-Amino-[S-phenylethy1)tetrazole

(L-Phenylalanine analog)

(a) L-SY-(o-Amino-(3—phenylethy1)tetrazole

The procedure used for the preparation of L-S-‘(o-amino-fi-phenyl-
ethy1)tetrazole was the same as that used for the preparation of the
D,‘L-5-(o-amino-fi-phenylethyntetrazole. The melting point of pure
tetrazole was 2870 C. with decomposition, [a]? = +38.0°, C = .945
in HzO.

(b) L- 2 - (1' - Benzamido-Z' Thenylethyl) - 5 -pheny1- 1,13 ,4 -oxadiazole

 

A mixture of 1. 50 g. (0. 008 mole) of L-5-(o-amino-[S-phenylethyl)«-
tetrazole, 2. 53 g. (0.018 mole) benzoyl chloride, 6.4 ml. of pyridine,
and 25 m1. of benzene was refluxed for three hours. The solution was
evaporated to dryness and the residue recrystallized from absolute

ethanol, m.p. 191--l92o C., {or}; = -14.90, C = 5.0 in chloroform.

Analysis.
Calculated for CZ3H19N3OZ: C, 74.78; H, 5.18; N, 11.38.
Found: C, 75.03; H, 5.28; N, 11.36.

L- 5 -[a-Arnino-8- (p -hydroxypheny1)ethy1]tetrazole

(L-Tyrosine Analog)

(a) L- 5- [o-Amino- [3- (p-hydroxyphenyl) ethylltetrazole

This compound was prepared as described in Part I by converting
'L-tyrosine to the N-benzoyl nitrile and subsequently to the tetrazole
followed by removal of the protecting benzoyl group by hydrolysis.
The pure tetrazole had a melting point of 3020 C. with decomposition,

.[ajg = + 33.80, C 54.0 in 2 N hydrochloric acid.

(b) L- 2 - [1' - Benzamido- Z'fi -benzoLloxypheny1)ethy1L- 5 -pheny1-
1, 3,, 4- oxadiazole

 

A mixture of 2.05 g. (0.01 mole) of L-5-[o-amino-fl-(p-hydroxy-
pheny1)ethy1]tetrazole, 4.60 g. (0.033 mole) benzoyl Chloride, 8 m1. of

50

pyridine, and 30 m1. of dry benzene was refluxed for five hours. The
solution was then evaporated to dryness and the residue washed with
cold absolute ethanol. The product was insoluble in sodium hydroxide.
The yield of oxadiazole was 3.8 g. (80% of theory), m.p. 202-2030 C.
after recrystallization from absolute ethanol. {or}; = -19.8°,. C = 2.0

in methylene chloride.

Analysis.
Calculated for C30H33N3O‘: C, 73.61; H, 4. 74; N, 8.58.
Found: C, 73.73; H, 4.87; N, 8.74.

B. Acid Hydrolysis of 2-Substituted-5-Pheny1-1, 3,4-Oxadiazoles
as Proof of Structure and Configuration

(a) Hydrollsis of D, L-2-(1'-Benzamido-2'-pheny1ethy1)-5:phenyl-
1, 3, 4-oxadiazole

Three grams (0. 0082 mole) of D, L-2-(l'-benzamido-2'-pheny1-

 

 

ethy1)-5-phenyl-l, 3,4-oxidiazole was added to 25 m1. of concentrated _
hydrochloric acid and the mixture refluxed for twelve hours. The clear
solution was cooled to Go C. and the precipitate collected on a filter.

The solid weighed 1. 95 g. (98% of theory) and had a melting point of
119-1200 C. Recrystallization from water raised the melting point to
120-1210 C. and. a mixture melting point with an authenic sample of
benzoic acid showed no depression. The filtrate was then evaporated to
dryness, the residue dissolved in 25 ml. of water, and the resulting solu-
tion neutralized to pH 6 with concentrated ammonium hydroxide. The
precipitate was filtered, dried, and weighed. The yield of D, L-phenyl-
alanine was 1.35 g. (80% of theory), m.p. 310-3140 C. with decomposition.
Its infrared spectrum was identical with that of an authenic sample of

D, L-phenylalanine.

51

(b) Hydrolysis of L-2-(l'-benzamido-2'-pheny1ethy1)-5-pheny1-
1, 3, 4-oxadiazole

 

 

L-2-(1'-benzamido-2'-pheny1ethy1)-5-pheny1- 1, 3 , 4-oxadiazole
(2.0 g. , 0.0054 mole) was suspended in 25 m1. of concentrated hydro-
chloric acid and refluxed for twelve hours. The clear solution was
cooled to 00 C. and the precipitate collected on a filter. The solid
weighed 625 mg. (95% of theory), m.p. 117-119° C. Recrystallization
from water raised the melting point to 119-1200 C. , and when mixed
with authenic benzoic acid, no depression of the melting point was
observed. The filtrate was evaporated to dryness, the residue was
dissolved in 25 m1. of water, and the solution was neutralized to pH 6
with concentrated ammonium hydroxide. After cooling overnight in the
refrigerator, the precipitate was collected on a filter, dried, and
weighed. The yield of L-phenylalanine, m.p. 234° C. with decompo-
sition, was 615 mg. (69% of theory). After three recrystallizations
from water, the melting point was raised to 2540 C. with decomposition,
[a]: = -4. 1°, C = 1.096 in water. Its infrared spectrum was identical

with authenic L- phenylalanine .

(C) Hydrolysis of L-2-[1'-benzamido-2'-(p-benzoyloxypheny1)-
ethy1]- Sghenyl- 1, 3 , 4-oxadiazole

To 2.4 g. (0. 0049 mole) of L-2-[1'-benzamido-2'-(p-benzoyloxy-

 

 

phenyl) ethy1]-5-pheny1-1, 3,4-oxadiazole was added 50 m1. of concen-
trated hydrochloric acid and the mixture refluxed for forty-eight hours.

The clear solution was cooled to 09 C. and the resulting precipitate
collected on a filter, washed with cold water, and dried. The solid

weighed 1. 78 g. (98% of theory) and melted at 118--119o C. After recrystal-
lization from water, the melting point was raised to 120-1210 C. There
was no depression in melting point when the product was mixed with an
authenic sample of benzoic acid. The filtrate was evaporated to dryness,

the residue dissolved in 50 m1. of water, and the pHof the solution

52

adjusted to 6 with concentrated ammonium hydroxide. The precipitate
was filtered, washed with cold water, and dried. The yield of
L-tyrosine was 775 mg. (82% of theory), m.p. 269-2720 C. with
decomposition. After recrystallization from water; the melting point
was 285-287° C. with decomposition, [e133 = -6.9°, C = 3.54 in 1 N
hydrochloric acid. Its infrared spectrum was identical with that of an

authenic sample of L-tyrosine.

PART III

OPTICAL ROTATION AND pK DETERMINATIONS OF
TETRAZOLE ANALOGS OF AMINO ACIDS

53

DISCUSSION

Many of the biological properties attributed to amino acids are
related to their physical properties. Two of these physical properties,
which play an important role, are the amphoteric nature of amino
acids, and the fact that all naturally occurring amino acids, except
glycine, are optically active.

One of the most important influences, which affect the degree of
optical activity of amino acids, is the acidity or alkalinity of the solution.
The influence of pH on the rotation of Optically active electrolytes, with
the resulting Changes in dissociation, has been extensively studied.
Usually the specific rotation of the neutral molecule differs from that
of the free ions and in some instances it has been possible to correlate
the rotation curves with the dissociation: constants of the substance.

Lutz and Jirgensons (20, 21) determined the specific rotations of
a series of naturally occurring amino acids dissolved in predetermined
amounts of acid and base. From a plot of the specific rotation and
the moles of acid or base, Lutz and Jirgensons predicted the configur-
ation of the amino acids by examining the acid branch of the optical
rotation curve. If, with increasing acid concentration, it was found that
the rotation changed toward the positive direction, the amino acid was
assigned the L-configuration. If the rotation became more negative,
the D-configuration was assigned to the amino acid. In this way con-
figurations were assigned to naturally occurring glutamic acid, arginine,
lysine, ornithine, histidine, alanine, tyrosine, aspartic adid, leucine,
tryptophane, proline, hydroxyproline, and dioxyphenylalanine.

In an effort to apply this theory to the tetrazole analogs of

L-phenylalanine and L-tyrosine, their rotations were determined in

54

55

specified amounts of acid and base; the results are summarized in
Tables 3 and 4. A plot of the specific rotation against the amounts of
acid and base was made and represented in Figures 18 and 19. For
comparison, the rotations of L-phenylalanine and L-tyrosine in specified
amounts of acid and base were also determined; the results summarized
in Tables 1 and 2. Figures 16 and 17 represent the plot of the specific
rotation against the amounts of acid and base for the amino acids.

-Severa1 intrinsic difficulties were encountered. Tyrosine has a
very low water solubility and the measurement of its rotation in water
was not possible. Also, tyrosine was only incompletely soluble in less
than equal molar quantities of acid or base, so that its rotation under
these conditions could not be determined.

From the characteristic features of the curves, some conclusions
were drawn. The curves of the tetrazole analogs of L-phenylalanine
and L-tyrosine were essentially of the same shape, having a-maximum
dextro rotation in water and having a minimum dextro rotation in the
acid portion, when the molar ratio of tetrazole to acid was 1 to 1.
However, in the base portion of the curve, the tetrazole analog of phenyl-
alanine reached a minimum dextro rotation when the molar ratio of
tetrazole to base was 1 to 1, while in the Case of the tetrazole analog of
tyrosine, the minimum dextro rotation was reached when the molar '
ratio of tetrazole to base was 1 to 2. This last observation is anomalous,
since the second mole of base would be expected to react with the phenol
.to form the phenoxide anion, which‘is removed from theasymmetric

center and shouldn't affect the rotation appreciably.

 

 

TH; - NaOH RIB;
‘ .7 1 -
Ho-.-CH,-’<E-~C NH I 5“ “mm; HO-.-CHz-C-C——N

56‘

 

 

1:11-12 NaOH .5 Isz
WO-WC-I I‘ ‘2“dm°1°’>°-O-W*fi—I
H N N H N N
\N/ \N//

If written as the dipolar ion, the overall result is the same.

 

 

 

 

 

+
NH, NaOH NH2
Ho-.—CH,-CH-ic N - ”St mole)% HO-.-CHz-CH-C I]
I I II
N /N N /N
\N/ \N/
NH; NaOH - TH;
I " (2nd mole)\ -
HO- -CHz-CH-E N , o- -CHz-CH-IC|J —-—l|\l
II N N
\Ny \Ny

The curves obtained from the plot of the specific rotations of
L—phenylalanine and L-tyrosine against the moles of acid and base were
similar to those of Lutz and Jirgensons. A maximum levo rotation
‘ was observed for L-phenylalanine in water, and the solutions became
less 'levo rotatory to the point in the acid portion of the curve, where
the molar ratio of amino acid to acid was 1 to 1. Thereafter, the levo
rotation decreased slightly with increased acid concentration. This is
consistent with the theory of Lutz and Jirgensons for L-amino acids.
In the base portion of the curve for L-phenylalanine, a decrease in levo
rotation was also observed from the maximum levo rotation in water,
to the point where the ratio of amino acid to base was 1 to 1. There-

after, the rotation remained constant with increasing base concentration.

57

The curve for L-tyrosine was essentially of the same pattern as that
of L-phenylalanine except that the minimum levo rotation in base was
reached when the molar ratio of sodium hydroxide to tyrosine was

2 to 1.

In comparing the rotation curves for L-phenylalanine and
L-tyrosine with their tetrazole analogs, an inverse relationship was
noted. A maximum levo rotation was observed for the amino acids in.
water solutions, while a maximum dextro rotation for water solutions
of the tetrazole analogs was noted. Lutz and'Jirgensons used the
characteristic change in rotation toward the positive direction in the
acid portion of the curves, to assign optically active amino acids to the
'L-configuration. It appears that the characteristic change in rotation
toward the negative direction in the acid portion of the curves of the
tetrazole analogs, can be used to assign them to the L-configuration.

Conversion of the tetrazole analogs of L-phenylalanine and
L-tyrosine to the oxadiazoles, and the subsequent degradation to the
amino acids established that the tetrazole analogs belong. to the 'L-series.

The amphoteric nature of amino acids and proteins account for
many of their biological properties. The ability of an amino acid or
protein to act as both a proton donor and a proton acceptor has long
been recognized.

In the case of simple amino acids, that is, those that have one

carboxyl group and one amino group, the ionization is as follows:

 

+
+ NH,
l
NH’ __r __.. R-C-COON 1sz
/’ '-
R-C-COOH 1'1 R- -coo
\ 1?le / H
R- -COOH

58

However, for the polyvalent ampholytes such as tyrosine, the
ionization scheme is more complex. Recently Edsall and co-workers
(22) determined the complete ionization scheme for tyrosine as
illustrated in Figure 1. Since each of the three functional groups can
exist in either the ionized form, or the unionized form, there are
eight possible forms for tyrosine in going from the protonated form to
the dianion.

The ionization constants were determined by two different methods;
spectrophotometric measurements and calculation from "microconstants. "
These microconstants are the individual interrelationships between
the eight forms of tyrosine in Figure l. The carboxyl group is by far
the strongest acidic group and the ionic form of the amino and the
hydroxyphenyl groups do not affect K1. Therefore, it was determined by
direct titration. However, since the amino and hydroxyphenyl groups
are of comparable acidity, the ionization of one will be dependent on the
form of the other. The distribution of charges will affect the intensity
of electrostatic interactions between the two groups. The pK values
for the model compounds O-methyl-tyrosine, tyrosine ethyl ester,
tyrosine amide, and phenylalanine were determined by titration, and by
use of mathematical treatments, the microconstants for the ionization
of tyrosine were calculated. The pKz and pK3 values for tyrosine were
calculated from these microconstants. These values were found to be
in close agreement to those obtained by Winnek andSchmidt (26).

The tetrazole analogs of the amino acids are also amphoteric and
would be expected to exhibit the same equilibria as their amino acid
counterparts. In the case of the tetrazole analog of L-phenylalanine,
the ionization is simple and is represented in Figure 2. The tetrazole
analog of tyrosine presents a more complex case. However, it was
noted that the ammonium groups} of the tetrazole analogs of amino acids

were ten times more acidic than those of the corresponding amino acids (3).

-000-m0-Nm0-O

_
Nmz

.mcwmofah. no“ ogonom aOSMNEOH vuoamgou .H onsmwh

$000- $0. N$0.0 0-0 Alllmoou-mo- "$0-0

0\ £2 O-VA «m2

.0 1000-m0-m0-O mooo-m0-Nm0-O

I Nmz 0x mmz

-000- m0- £90 ...0 All II000 mw- £90
.
£2 £2

+ +

o/ ..

0-0 Allm000- m0- £0- / \ -0m

\+ «mm.

0103

60

.mfismfimgaosmud mo mo~ma< 3059308 23 mo ogoaom coflmuficoH .N ouzwfim

 

 

61

Therefore, the form of one group would have less of an effect on the
ionization of the other. The ionization scheme for the tetrazole analog
of tyrosine is represented in Figure 3.

The pK values for the tetrazole analogs of L-phenylalanine,

. L-tyrosine, and O-methyltyrosine were determined by titration of

these compounds in aqueous solution with standard solutions of hydro-
chloric acid and sodium hydroxide. A summary of the results, including
a comparison of the pK values for the corresponding amino acids, is
given in Table 11. The data for the titration of each-amino acid and its
titration curve are found in Part III of the Experimental.

An examination of Table 11 shows that there is a close agreement
in the pK values for the amino acids and their tetrazole analogs. This
comparison is facilitated by taking the antilogarithm of the difference
in the pK value between the amino acid and the corresponding tetrazole,
and is a measure of the relative strength of the acidity'of the two
species. For example, K1 for L-phenylalanine, which is a measure of
the acidity of the carboxyl group, is about 10 times larger than K1 for
the tetrazole analog of II-phenylalanine, where-K1 is a measure of the
acidity of the tetrazole group. Similarly, K2, which is a measure of
the acidity of the ammonium group in both compounds, is about 11. 5
times larger for the tetrazole analog than for L-phenylalanine. The
hydroxyphenyl group of the tetrazole analog is slightly more acidic
than that of the amino acid as, evidenced by a comparison of the pK3
values.

The acidity of the carboxyl group would be expected to be greater
than that of the tetrazole based on the comparative electronegativities
of oxygen-and nitrogen. Since the tetrazolyl group is a weaker acid
than the carboxyl, the tetr‘azolate anion is a stronger base than the
carboxylate anion, and it- exerts a stronger electron withdrawal effect

on the ammonium group‘. This would account for the higher acidity of

62

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63

the ammonium moiety in the tetrazole analog as compared to the
amino acid. Also; the slightly higher acidity of the hydroxyphenyl
group of the tetrazole analog of tyrosine could be explained by the
increased electron withdrawal effect of the tetrazolate anion com-
pared to the carboxylate anion.

The reason for the greater acidity of phenylalanine and its
tetrazole analog as compared to tyrosine and O-methyltyrosine and
their tetrazole analogs is not so apparent. Perhaps the answer may
be that the electron release effect of thehydroxyphenyl and methoxy-
phenyl groups increase slightly the proton affinity of the ammonium

and carboxyl groups, thereby making them less acidic (49).

EXPERIMENTAL

A. Specific Rotations of L-Phenylalanine,- L-Tyrosine, and
Their Tetrazole Analogs in Various Concentrations
of Acid and Base1

(a) L- Phenylalanine

 

Samples of L--phenylalaninez (1.65 g. , 0.01 mole) were dissolved
in predetermined amounts of 1 M sodium hydroxide gand- l M hydro-
chloric acid, and the rotation of the solutions determined. Specific
rotations calculated for each concentration of acid and base are given

in Table 1.

(b) L- Tyrosine

 

Samples of L-tyrosine3 (0.905 g. , 0.005 mole) were dissolved in
predetermined amounts of l M sodium hydroxide and 1 M hydrochloric
acid and the rotation of each solution was determined. Specific rotations

calculated for each concentration of acid and base are given in Table 2.

(c) L- 5 - (a-Amino- p-phenylethylhetrazole
(la-Phenylalanine Analog)

 

This compound was prepared by converting L-phenylalanine to
L-a-phthalimido-B-phenylpropionitrile. The tetrazole was then
synthesized from the nitrile according to the procedure outlined in
Part I. The pure tetrazole had a melting point of 287° C. with decompo-

sition, [(112135 = +38.o°, c = .945 in Hzo.

_!_

1All measurements were carried out on a Rudolph Precision oPolari-
’meter, Model 70, using a 1 dm. tube and sodium light at 24°- C.

zNutritional Biochemicals Corp.
3Nutritional Biochemicals Corp.

64

65

Table ‘1. Specific Rotations of L- Phenylalanine in NaOH and HCl.
Using 1 M NaOH and 1 M HCl at 24° C. 1.65 g. (0. 01 mole)
.‘ of L- -pheny1alanine.

\

m r-

 

 

 

Moles‘ of Moles‘ of

Amino ‘Acid Acid or Base (1 [a]3

1.65 g. (0.01 mole) in 100 m1. H30 -_1. 107° -33.5°
1 0.5 mole NaOH -1. 165° -17.7°
1 1.0 mole NaOH —o.19o° -1.44°
1 2.0 moles NaOH -0. 202° -1.s3°
1 4.0 moles NaOH -0.338° -2.56°
1 0. 5 mole HCl -1.618° -34.4°
1 . 1.0 m’ole HCI -1.447° -11.o°
1 2.0 mole HCl -1. 118° -8.5°

 

Table 2. Specific Rotations of L- Tyrosine in NaOH and HCl. Using
1 M NaOH and 1 M HCl at 24° C., 0.905 g. (0.005 mole) of

 

 

 

L- -Tyrosine.
___.______.L __ :—--__._-_--—-___—===-—-==
Moles of r Moles of
Amino Acid Acid or Base 0. [11]?)
o o

l 1 mole NaOH -0.0_59 -3Z.6

1 2 moles NaOH -0.949o -l3.8°

1 1 moletHCl -0. 165° -45.5°

1 2 moles HCl -0.890 -12. 3°

1 14 moles HCl -0.789 -10.9

 

66

Solutions containing 0.4725 g. (0. 0025 mole) of L-5-(a-amino-;3-

phenylethy1)tetrazole and predetermined amounts of 0. 5 M hydrochloric

acid and 0. 5 M sodium hydroxide were prepared and their rotations

determined. The specific rotations calculated for each solution are

given in Table 3.

Table 3.

Specific Rotations of L-5-(o-Amino-fl-phenylethyl)tetrazole in
NaOH and HCl (L-ghenylalanine Analog), Using0.5 M NaOH
andQ.5 M HCl at 24 C.,0.4725 g. (0.0025 mole) of L-5-

(CL-Amino-fi-phenylethylhetrazole.

 

 

Moles of Moles of

Tetrazole Acid or Base (1 [a]:

0.4725‘g. (0.0025 mole) o o

in 50 ml. H30 +0.71? +38.0

1 0. 5 mole NaOH +0. 544° +28.8°
1 1.0 mole NaOH +0.724° 4.19.10
1 2.0 moles NaOH +0.733° +19.4°
1 4. 0 moles NaOH +0.737° +19.5°
1 0.5 mole HCl +0.334° +35.4°
1 1.0 mole HCl +1.277° +33.8°
1 2. 0 moles HCl +1.246° +33.0°
1 4. 0 moles HCl +1.262° +33.5°

 

(d) ~L- 5 -[a- -Amino-fi_-( ~11LdroxMegy1) ethylltetrazole

 

(L-Tyrosine Analog)

This compound was prepared by converting L-tyrosine into L-a-

benzamido- 0- (p- benzene sulfonyloxy)phenylpropionitrile and subsequently

to its tetrazole as described in Part I. The pure tetrazole had armelting

point of 302° c. with decomposition, {or}; = +33. 8°, c = 4.0 in 2 N H61.

67

Solutions of 0. 5125 g. (0.0025 mole) of L-5-[a-amino-B-
(p-hydroxyphenyl)ethyl]tetrazole containing predetermined amounts of
0. 5 M sodium hydroxide and 0. 5 M hydrochloric acid were prepared
and their rotations determined. The specific rotations calculated

for each-solution are given in Table 4.

Table 4. -Specific Rotations of L-5-[a-Amino-[3-(p-hydroxypheny1)ethy1]-
tetrazole in NaOH and HCl (L-Tyrosine Analog). Using 0. 5 M
NaOH and 0.5 M HCl at 24°C., 0.5125 g- (0.0025 mole) of
L- 5 -[a-Amino- [3- (p- hydroxyphenyl) ethyl]tetrazole .

 

Moles of Moles of

 

 

Tetrazole Acid or Base 0. [a];

0.5125 g. (0.0025 mole) in 1000 m1. Hzo +0.042° +41.0°
1 0. 5 mole NaOH +0. 056° +27.4°
1 1. 0 mole NaOH +0.610° +14. 9°
1 2. 0 moles NaOH +0.073° +1.8°
1 4.0 moles NaOH +0. 171° +4. 2°
1 1.0 mole HCl +0.713° '+34.8°
1 2.0 moles HCl +1.430° +34.9°
1 4.0 moles HCI +1.437° +35.0°

 

B. The Determination of the Apparent pK Values of Some
Amino Acids and Their Tetrazole Analogs

The apparent pK values of the 5-aminoalky1tetrazoles were
determined by titration of weighed samples of the tetrazole in aqueous
solution withstandard sodium hydroxide and standard hydrochloric acid

solutions. The weighed samples were placed in volumetric flasks and

68

dissolved in the specified amounts of water. They were then trans-
ferred to a flask and titrated in a thermoregulated bath at 250 21‘- 10 C.
The pH was determined after each‘addition of titrant with a Beckman
pH meter, model H-2.

The pKl values were determined by taking the pH of the solution
at the point of half neutralization, as calculated from the normality of
the acid used as a titrant, and the molecular weight of the tetrazole.

The pKz values were determined by taking the pH of the solution
at the point of half neutralization, as calculated from the normality of
the base used as a titrant, and the molecular weight of the tetrazole.

The pK3 value for the hydroxyphenyl group of the L-iyrofiihe
analog was determined by taking the pH of the solution at the half
neutralization point between the addition of one and two equivalents of
base, as calculated from the normality of the base, and the molecular
weight of the tetrazole.

The pK values determined in this fashion were in close agreement
with those determined by plotting the pH versus the milliliters of.‘
titrant, finding the midpoint of the best straight line drawn at the point
of neutralization, and noting the pH at the point at which one-half the
amount of titrant required for neutralization was added. The titration
curves exhibited a pattern similar to that of most amino acids. The
results are recorded in Tables 5-10 and shown graphically in Figures
20-25. Table 11 gives comparative values for the amino acids and

their tetrazole analogs .

69.

Table 5. (Potentiometric Titration of L-5-(a-Amino-fi-phenylethyl)-
tetrazole with Hydrochloric Acid (L-Phenylalanine Analog)

 

Ml . of Apparent

 

Concentration pH 0. 10156 N HCl pKl
0.140 g./100 m1. of 6.00 0
aqueous solution 3. 88 0. 5 2. 83
3.51 1.0
3.30 1.5
3.18 2.0
2. 95 3.0
2.80 4.0
2.68 5.0
2.58 6.0
2.49 7.0
2.42 8.0
2.34 9.0
2.29 10.0

2.19 12.0

 

70

Table 6. Potentiometric Titration of L-5-(o-Amino-fi-phenylethy1)-
tetrazole with Sodium Hydroxide (L-Phenylalanine Analog)

 

 

 

M1. of Apparent

Concentration pH 0.10036 N NaOH pKz
0.140 g./100 ml. of 6.00 0
aqueous solution 6.97 0.5 8.07

7.30 1.0

7.49 1.5

7.66 2.0

7.92 3.0

8.18 4.0

8.42 5.0

8.80 6.0

9.70 7.0

10.70 8.0

11.00 9.0

11.19 10.0

11.29 11.0

11.35 12.0

11.50 15.0

 

71

Table 7. .Potentiometric Titration of D, L-5—[a-Amino-8-(p-methoxy-
phenyl) ethyl]tetrazolewith'Hydrochloric Acid (O-Methyl-
D, L-Tyrosine Analog)

j
J

(I

 

 

M1. of Apparent

Concentration pH 0. 10156 N HCl pK,
0.150 g./250 ml. of 6.40 0 3.20 F
aqueous solution 4. 15 0. 5

3.88 "1.0

3.62 1.5

3.51 2.0

3.28 3.0

3.12 4.0

3.00 5.0

2.90 6.0

2.78 7.0'

2.70 8.0

2.62 9.0

2.59 10.0

2.50 12.0

2.39 15.0

2.25 20.0

 

72

Table 8. , Potentiometric Titration of D,- L-5-[c-Amino-8-(p-methoxy)-
phenylethyl]tetrazole with Sodium Hydroxide (O-Methyl-D, L-
Tyro-s'ine Analog)

 

 

M1. of Apparent

Concentration pH 0. 10036 N NaOH pK;
0.150 g./250 m1. of 6.40 0 8.08
aqueous solution 7.11 0.5

7.39 1.0

7.59 1.5

7.73 2.0

8.00 3.0

8.25 4.0

8.51 5.0

8.83 6.0

9.23 7.0

9.60 8.0

9.85 9.0

10.06 10.0

10.21 11.0

10.39 12.0

10.78 15.0

 

73

Table 9. Potentiometric Titration of L-5-[a-‘Amino-8-(p-hydroxypheny1)-
ethy1]tetrazole with Hydrochloric Acid (L-Tyrosine Analog)

 

M1. of Apparent

Concentration pH 0. 10156 N HCl pK,
0.1051 g./250 m1. of 6.40 0 3.28
aqueous solution 4.03 0.5

3.70 1.0

3.52 1.5

3.39 2.0

3.29 3.0

3.03 4.0

2.84 6.0

2.70 8.0

2.60 10.0

2.51 12.0

2.41 14.0

2.31 16.0

 

Table 10. Potentiometric Titration of L-S-[a-Amino-(S-(p-hydroxy-
phenyl) ethyl]tetrazole with Sodium Hydroxide (L-Tyrosine

 

74

 

Analog):
Ml. of Apparent
Concentration pH 0. 10036 N NaOH pKz
0.1051 g./250 m1. of 6.40 0
aqueous sohnion 7.38 0. 8.17
7.68 1.0
7.83 1.5 ng
8.00 2.0 10.05
8.17 2.5
8.31 3.0
8.49 3.5
8.62 4.0
8.88 4.5
9.11 5.0
9.34 5.5
9.55 6.0
9.72 6.5
9.90 7.0
10.00 7.5
10.11 8.0
10.22 8.5
10.30 9.0
10.38 9.5
10.47 . 10.0
10.51 10.5
10.59 11.0
10.70 12.0
11.00 15.0
11.27 20.0

 

75

 

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SUMMARY

1. The tetrazole analogs of D,L_—pheny1alanine,r L-phenylalanine,

and L-tyrosine were prepared by three different routes.

2. The tetrazole analog of O-methyl-D, L-tyrosine was prepared

by alkylation of ethyl acetamidocyanoacetate with anisyl chloride.

3. 2-Substituted-5-pheny1-1,13, 4-oxadiazoles were synthesized
from the tetrazole analogs of D, L-phenylalanine, L-phenylalanine,

and L-tyrosine.

4. Proof of structure and configuration of the tetrazole analogs
of L-phenylalanine and L-tyrosine were accomplished by hydrolysis

of their oxadiazoles to L-phenylalanine and L-tyrosine.

5. The apparent pK values for L-phenylalanine, -L-tyrosine, and
O-methyl-D, L-tyrosine were determined by potentiometric titration
and were found to be analogous to those of the corresponding amino

acids.

6. The specific rotations of L-phenylalanine, L-tyrosine, and
their tetrazole analogs were determined in various concentrations of

acid and base.

7. The infrared spectra of a number of new compounds are

reported.

76

10.

11,

12.

13.

14.

150'

16.

17.

18.

LITERATURE CITED

J.-Sheenan and V. Frank, J. Am. Chem. Soc., xvii, 1856 (1949).

 

P.-Peterson and C. Nieman, J. Am. Chem. Soc., ‘73“, 1389 (1957).

 

J. McManus, J. Org. Chem., ‘23" 1643 (1959).

. R. 13.-Steiger,» J. Org. Chem.,vz, 396 (1944).

Jules Max, Ann., 369, 276-86 (1909).
— www

Anders Kjaer, Acta. Chemica Scand.,\6d 1381 (1952).

Vincent du Vigneaud and C. E. Meyer, J. Biol. Chem., 93, 304 (1932).

 

 

Julius N. Ashley and Charles R. Harington, Biochem. J., 30:33 1180 (1929).

 

J. Sheehan, D. W. Chapman, and R. W. Roth, J. Am. Chem. Soc.,
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S. Udenfriend and S. P. Bessman, J. Biol. Chem., 203,, 961 (1953).

 

 

W. C. Rose and R. L. Wixom, J. Biol. Chem., 217, 95 (1955).

Emil Fischer, Ber., 33‘” 451 (1901).

Max Bergmann, Ferdinand Stern, and Charlotte Witte, _ Ann. , 442,,
299 (1926). .

F. Bergel and G. E. Lewis, J. Chem. Soc., 1816-19 (1957).

 

Max Bergmann and Jos.' Fruton, J. Biol. Chem., 124 321 (1938).

 

Seymour Kaufman, Hans Neurath, and Geo. W. Schwert, J. Biol. Chem. ,1
1,11, 795 (1949).

 

S. Kaufman and H. Neurath, J. Biol. Chem., 189, 181 (1949).

 

Kurt Rorig, J. Derland Johnston, 'Robt. W. Hamilton, and Th'omas
J. Telinski, Or rganic Syntheses, Vol. 36, John Wiley and Sons, Inc.
New York, N. Y., 1956, p. 50.

 

77

78 "

19. G. Sterken, Synthesis of Tetrazoles as Amino Acid Analogs,
Ph. D. thesis, Michigan State University, 1960.

 

20. O.vLutz and B. Jirgensons, Ber., 63, 448 (1930).
21. O. Lutz and B. Jirgensons, Ber., 64¢ 1221 (1931).

22. R. B. Martin, John T. Edsall, D. B. Wetlaufer, and B. R.
Hollingworth, J. Biol. Chem., 233, 1429 (1958).

 

23. E. Brand, W. H. Goldwater, and L. Zucker, Abstracts 106th
Meeting of the American Chemical Society, New York, American
Chemical Society News Service, Washington, 1944, ~p. 19B.

 

 

24. J. A. Bladin, Ber., ‘12, 2598 (1886).
25. J. A. Bladin, Ber., 2,5, 1411 (1892).

26. P. S. Winnek and C. L. A. Schmidt, J. Gen. Physiol., 18,,889
(1935).

 

27. A. Pinner, Ann., m 229 (1897).

28. C. Ainsworth, J. Am. Chem. Soc., ,75, 5728 (1953).

 

29. J. Mihina and R. Herbst, J. Org; Chem., 15, 1082 (1950).

 

30. R. Herbst and K. Wilson, J. Org. Chem., 22, 1142 (1957).
31. H. Behringer and K. Kohl, Ber., 82" 2648 (1956).

32. W. Finnegan, R. Henry, and R. Loftquist, J. Am. Chem. Soc.,
80' 3908 (1958). '

 

.33. R. Stolle, Ber., 62, 1118 (1929).

 

34. R. Huisgen, J. Sauer, and H. Strum, Angew. Chem., 30' 272 (1958).

35. D. I. Hitchcock, J. Gen. Physiol.,v‘q) 747 (1924).

 

36. N. Bjerrum, Z. Rhysik. Chem., 104, 147 (1923).

 

37. E. J. Cohn and J. T. Edsall, Proteins, Amino Acids, and Peptides,
Reinhold Publishing Co., New York, N. Y., 1943, Chapt. 4.

 

38.

39.

40.

41,

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

79

J. T. Edsall and J. Wyman, BiophysicalflChem‘istry, Vol. 1,
Academic Press Inc., New York, N. Y., 1958, pp. 463-4.

 

Carl L. A. Schmidt, The Chemistry of AminoAcids and Proteins,
Charles C. Thomas Co., Springfield, Illinois, 1938, pp. 575-8.

 

Frederic Benson, Chem. Revs., 41, 1 (1947).

 

A, Albert, Biochem. J., 50, 690 (1952).

 

Jos. S. Fruton and Sophia Simmons, GeneralgBiochemistry,
John Wiley and Sons, Inc., New York, N. Y., 1958, pp, 824-35.

 

D. Wolley, A Study of Antimetabolites, John Wiley and Sons, Inc. ,
New York, N. Y., 1952, p. 52.

 

 

M. Womack and W. C. Rose, J. Biol. Chem., 107, 449 (1943).

S. Gurin and A. M. Delluva, J. Biol. Chem., 170, 545 (1947).

 

R. W. Barratt at 11., J. Bact., ‘11" .108 (1956).

S. Graff, Essays in Biochemistgy, John Wiley and Sons, Inc. ,
New York, N. Y., 1956.

 

A. R. Moss and R. Schoenheimer, J. Biol. Chem., 135, 415 (1940).

 

C. K. Ingold, . Structure and Mechanism in Oflanic Chemistry,
Cornell University Press, Ithaca, New York, 1953.

 

 

"Anisyl Chloride Storage, " Chemical and Engineering News,
Oct. 24, 1960, p. 5. '

P. S. Winnek and C. L. A. Schmidt, J. Gen. Physiol., “1‘8” 889
(1935).

 

APPENDIX

80

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