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This is to certify that the

dissertation entitled

STRUCTURE-ACTIVITY RELATIONSHIPS [N THE
OXIDATION OF QUATERNARY AMMONIUM SALTS
OF AMINOALCOHOLS WITH
HORSE LIVER ALCOHOL DEHYDROGENASE

presentedby

SHE KrHONG' HERMAN LAU

has been accepted towards fulfillment
of the requirements for

PhoDo degreein ChemiStry

 

 

Gerosimos J. Karabatsos

 

Major professor

Date 6'8'31

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

 

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LIBRARIES
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RETURNING MATERIALS:
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remove this checkout from
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STRUCTURE-ACTIVITY RELATIONSHIPS IN THE
OXIDATION OF QUATERNARY AMMONIUM SALTS
OF AMINOALCOHOLS WITH
HORSE LIVER ALCOHOL DEHYDROGENASE

By

Shek-Hong Herman Lau

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Chemistry

1981

ABSTRACT

STRUCTURE-ACTIVITY RELATIONSHIPS IN THE
OXIDATION OF QUATERNARY AMMONIUM SALTS
OF AMINOALCOHOLS NITH
HORSE LIVER ALCOHOL DEHYDROGENASE

By

Shek-Hong Herman Lau

Hydrophobic, hydrophilic, and electronic interactions
have long been recognized as important in binding substrate
to enzyme, and in facilitating enzymatic reactions. Under-
standing individual as well as integral effects of these
interactions on enzymatic reactions can provide valuable
information about structure-activity relationships between
substrate and enzyme and about the mechanism of the reaction.

Horse liver alcohol dehydrogenase (HLADH) (E.C.1.1.1.1.),
is an interesting enzyme for investigation of the effect of
hydrOphobic interactions on the enzymatic reaction. The
active site of the enzyme has a catalytic zinc atom at the

bottom of a deep hydrophobic substrate binding pocket about

Shek-Hong Herman Lau

20 g from the surface of the enzyme. Hansch and coworkers
have discussed hydrophobic interaction in the binding of small
molecules to the enzyme. Their work and that of others have
led to the conclusion that binding of the substrate or the
inhibitor to the enzyme is enhanced by increasing the length
of the alkyl chain of the molecule (up to fifteen carbons).

To provide further data on the effective size and length
of the substrate binding pocket, quaternary ammonium salts of
aminoalcohols with a bulky group at one end of the molecule
and a variable alkyl chain, 1, were synthesized, and their

reactions with HLADH and NAD+ were studied.

+ -
R3N -(CH2)n -OH X

I (R CH3, CH3CH2; n = 2 to 10;

X

I or CI)

The initial rates and equilibrium constants for the oxi-
dation of the quaternary ammonium salts of these aminoalcohols
with HLADH and NAD+ at six pHs at room temperature were deter-
mined. Both were found to depend on the pH and the length of
the alkyl chain of the substrate. In general, the initial
rates and the equilibrium constants increase at all pHs, as
the alkyl chain length increases. For example, there is an
8-fold increase in initial rates going from n = Zrto the maxi-
mum at n = 7 in the N,N,N-trimethylaminoalcohol series at pH
8.0 compared to a 33-fold increase in the N,N,N-triethylamino-
alcohol series. The initial rates vary with the pH and a 21-

fold increase is observed going from pH 6.5 to pH 10.0 for

Shek-Hong Herman Lau

n = 7 in the trimethyl series and a 30-fold increase from pH
7.0 to pH 10.0 for n = 7 in the triethyl series. The ratio of
the equilibrium constants for n = 2 and n = 10 in the trimethyl
series at 36 hoUrs at pH 8.0 is 1:1700 and that for the tri-
ethyl series is 1:600. This finding is consonant with the

idea that hydrophobic interactions between substrate and enzyme
at the binding pocket increase the binding of the substrate to
the enzyme and enhance its reactivity. The pH effect on the
initial rates suggests that the enzyme undergoes a conforma-
tional change as the pH changes and that the substrate binding
pocket perhaps becomes more constricted at lower pHs. Conse-
quently, the enzyme becomes more selective with respect to the

size and length of the substrate.

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation
to Professor Gerasimos J. Karabatsos for his guidance
throughout this investigation.

A special thanks goes to Professor John C. Speck, Jr.
without whom this would not be possible.

The author would also like to thank his wife, Amy;
his parents and her parents; and members of the family for
their support and patience throughout the years; and last
but not least, to his daugher, Bonnie, for making the past
couple of years more enjoyable.

Financial support provided by the Department of
Chemistry, Michigan State University, is gratefully

acknowledged.

ii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES

INTRODUCTION .
RESULTS AND DISCUSSION .

Preparations of the quaternary ammonium
salts of aminoalcohols

Initial rate studies of'the quaternary
ammonium salts of aminoalcohols '

Equilibrium studies of the quaternary
ammonium salts of aminoalcohols with .
horse liver alcohol dehydrogenase .

EXPERIMENTAL .
Instrumentation .
Materials
Synthesis

A. Preparation of Chlorohydrins
I. 4-Chloro-1-Butanol . .
II. Other chlorohydrins from
the diols. .

B. Preparation of N ,N-dialkyl-
aminoalkanols . .

I. 4- Dimethylamino- 1- butanol
II. 5-Dimethylamino-1-pentanol
III. N,N-Diethylaminoalkanols
C. Preparation of quaternary ammonium

halides of aminoalcohols
I. Quaternary ammonium chlorides
II. Quaternary ammonium iodides

iii

16

27

36
67
67
67
68

68
68

69'

Page

Qualitative identification of
quaternary ammonium salts . . . . . . . . . 77

Activity of quaternary ammonium halides
of aminoalcohols with horse liver
alcohol dehydrogenase . . . . . . . . . . . 85
APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . 88

Nuclear Magnetic Resonance Studies of
Cadmium-lll-substituted horse liver
alcohol dehydrogenase . . . . . . . . . . . 88

LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . 91

iv

Table
1.

10.

11.

12.

13.

LIST OF TABLES

Effects of hydrophobic and hydrophilic
groups on product stereospecificity

Initial rates for the oxidation of
N, N, N— —trimethylaminoalcohols at six pHs
at room temperature . . .

Initial rates for the oxidation of
N, N, N- -triethylaminoalcohols at six pHs
at room temperature . . . .

Relative rates for the oxidation of
N,N,N-trimethylaminoalcohols at six pHs

Relative rates for the oxidation of
N,N,N-triethylaminoalcohols at six pHs.

Equilibrium constants for the oxidation
of N,N,N-trimethylaminoalcohols at six
pHs at room temperature . . . . .

Equilibrium constants for the oxidation
of N,N,N-triethylaminoalcohols at six
pHs at room temperature . . . . .

Yields and boiling points of chlorohydrins

Boiling points and percentage yields of
N, N-Dialkylaminoalkanols . . .

Melting points and percentage yields of
quaternary ammonium halides of
aminoalcohols . . . . .

250 MHz proton NMR spectral data for
N,N,N-trimethylaminoalcohols

250 MHz proton NMR spectral data for
N,N,N-triethylaminoalcohols

Chemical Ionization Mass spectral data
for N,N,N-trimethylaminoalcohols

V

Page

28

29

3O

31

37

43

71

74

78

79

80

81

Table Page

14. Chemical Ionization Mass spectral data
for N,N,N-triethylaminoalcohols . . . . . . . . 83

vi

LIST OF FIGURES

Figure

1. Modified Theorell-Chance mechanism of
horse liver alcohol dehydrogenase .

2. Structure of reduced nicotinamide adenine
dinucleotide . . . . . .

3. Stereospecificity of hydrogen transfer
between substrate and coenzyme

4. Prelog's model

5. Karabatsos' model

6. Graves model, and Cervinka and Hub Model

7. Structure of oxidized nicotinamide adenine
dinucleotide analogs

8. Possible spatial arrangements of the oxidized
nicotinamide adenine dinucleotide
analogs . . . .

9. Active site of horse liver alcohol
dehydrogenase as observed from x-ray
studies — . . . . . .

10. Mechanistic arrangements in the ternary
complex of horse liver alcohol
dehydrogenase . . . . . .

11. Structure of quaternary ammonium halides
of primary aminoalcohols

12. Chemical ionization mass spectrum of
N, N, N- -trimethyl- 10- -hydroxydecyl
ammonium chloride . .

13. Chemical ionization mass spectrum of

N, N, N- triethyl- 10- hydroxydecyl
ammonium iodide . . .

vii

Page

\ONONU'I

10

11

12

14

20

21

Figure

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

250 MHz proton NMR spectrum of N,N,N-tri-
methyl-Z-hydroxyethyl ammonium iodide

250 MHz proton NMR spectrum of N,N,N-tri-
methyl-lo-hydroxydecyl ammonium chloride

250 MHz proton NMR spectrum of N,N,N-tri-
ethyl-Z—hydroxyethyl ammonium iodide

250 MHz proton NMR spectrum of N,N,N-tri-
ethyl-IO-hydroxydecyl ammonium iodide .

Initial rates for the oxidation of N,N,N-
trimethylaminoalcohols as a function
of n and pH at room temperature .

Initial rates for the oxidation of N,N,N-
triethylaminoalcohols as a function
of n and pH at room temperature .

Equilibrium constants for'the oxidation of
N,N,N-trimethylaminoalcohols as a
function of n and pH at 36 hours at room
temperature . . . . . . .

Equilibrium constants for the oxidation of
N,N,N-trimethylaminoalcohols as a function
of n and time at pH 6.5 at room temperature .

Equilibrium constants for the oxidation of
N,N,N-trimethylaminoalcohols as a function
of n and time at pH 7.0 at room temperature .

Equilibrium constants for the oxidation of
N,N,N-trimethylaminoalcohols as a function
of n and time at pH 7.5 at room temperature .

Equilibrium constants for the oxidation of
N,N,N-trimethylaminoalcohols as a function
of n and time at pH 8.0 at room temperature

Equilibrium constants for the oxidation of
N,N,N-trimethylaminoalcohols as a function
of n and time at pH 8.8 at room temperature

Equilibrium constants for the oxidation of
N,N,N-trimethylaminoalcohols as a function

of n and time at pH 10.0 at room temperature .

Equilibrium constants for the oxidation-of
N,N,N-triethylaminoalcohols as a function
of n and pH at 36 hours at room temperature

viii

Page

23

24

25

26

32

33

. 49

50

51

52

53

54

55

56

Figure

28.

29.

30.

31.

32.

33.

34.

Equilibrium constants for the oxidation of
N,N,N-triethylaminoalcohols as a function
of n and time at pH 6.5 at room temperature

Equilibrium constants for the oxidation of
N,N,N-triethylaminoalcohols as a function
of n and time at pH 7.0 at room temperature

Equilibrium constants for the oxidation of
N,N,N-triethylaminoalcohols as a function
of n and time at pH 7.5 at room temperature

Equilibrium constants for the oxidation of
N,N,N-triethylaminoalcohols as a function
of n and time at pH 8.0 at room temperature

Equilibrium constants for the oxidation of
N,N,N-triethylaminoalcohols as a function

of n and time at pH 8.8 at room temperature .

Equilibrium constants for the oxidation of
N,N,N-triethylaminoalcohols as a function
of n and time at pH 10.0 at room temperature

Apparatus for the preparation of chloro-
hydrins from diols . . . . .

ix

Page

57

58

59

6O

. 61

62

69

INTRODUCTION

Horse liver alcohol dehydrogenase (HLADH) (E.C.1.1.1.1.)
is a nicotinamide adenine dinucleotide (NAD+/NADH)-dependent
enzyme that catalyzes the reversible oxidoreduction reaction
of a variety of primary and secondary aliphatic and cyclic
alcohols, ketones and aldehydes, and some steroids. The enzyme
has been extensively studied since its discovery in the late
nineteenth century. Several reviews on its structure, proper-
ties, and mechanism of reaction have been published.1"4

The overall reaction is represented by equation 1.

HLAQH'

RlR CHOH + NAD+ f —+ R R2c=o + NADH + H+ (eq. 1)

2 1

The equilibrium of the reaction overwhelmingly favors the
reduction of the carbonyl compounds. A typical equilibrium
constant for the oxidation of primary alcohols is about

8.0 x 10"12 M.

Although the mechanisms of the catalytic reaction has
been extensively investigated by many researchers through
the use of various kinetic methods,5"9 it is not completely
agreed upon. At the present time, the mechanistic sequence
generally accepted by most is a modified Theorell-Chance

10

ordered Bi-Bi mechanism.3’ This is shown in Figure 1.

E--Zn—-H O

2

NAD+

NAD+--E--Zn~-H20
Isomerization H+
E--2n--0H'

NAD+

RCHZOH -\

‘-9uzo
W

E--Zn-- OCHZR

HAD+

V
E--Zn--O=CHR
NADH

K"O=GI*I1’I
/

NADH

Ismmerization

 

\ .

NADH—-E--2n.--H2
NADH
Eu» mam-320

Figure 1. - Modified Theorell-Chance mechanism
of HLADH

The structure of the enzyme has been thoroughly worked
out. X-ray crystallography of the enzyme has recently been

0
extended to 2.4 A resolution.11’ 12

The active enzyme has a
molecular weight of 80,000 and is a.dimer of identical subunits.
Each subunit is further divided into a coenzyme binding site
and a substrate binding site, and firmly binds a "catalytic"
and a "structural" zinc atom. The catalytic zinc atom is situ-
ated at the bottom of a predominantly hydrophobic substrate
binding pocket some 20 X from the surface of the enzyme. It

is bonded to the enzyme through three bonds with the sulfhydryl
and imidazole groups of cysteine 46, Histidine 67, and Cysteine
174. The fourth position of the coordination sphere of zinc

is occupied by either a water molecule or hydroxide depending
on the pH of the solution.

The structure of the reduced coenzyme (NADH)13is shown

in Figure 2.

33151)

 

Figure 2. - Structure of Reduced Nicotinamide
Adenine Dinucleotide

4
The stereochemistry of the hydrogens on the C-4 position of
of the nicotinamide ring is labeled.

Alcohol dehydrogenases are divided into type A and type
B enzymes depending on which hydrogen atom on the C-4 position
of the nicotinamide ring of NADH is transferred to the sub-
strate. The A enzymes transfer the ro-R hydrogen and the B
enzymes the ro-S hydrogen. HLADH belongs to the A enzyme
category. Furthermore, HLADH is not only stereospecific with
respect to the coenzyme, but also with respect to the substrate.
It transfers the C-4 hydrogen of the coenzyme exclusively or
predominantly onto the re-face of the carbonyl group of the.
carbonyl compound; conversely, it oxidizes exclusively or pre-
dominantly secondary alcohols having the S-configuration. This
stereospecificity with the substrate and coenzyme is illustrated
in Figure 3.

At the present time, there is no definitive picture on
the spatial orientation between the substrate and the coenzyme
or on the role of the catalytic zinc atom in the reaction and
its interaction with the substrate in the active site pocket.

It is very important to have a detailed knowledge of the
spatial arrangement between the coenzyme and the substrate in
the ternary enzyme complex in order to understand the reaction
mechanism of alcohol dehydrogenases. Even though there have
been several models proposed on this subject, none has been

unambiguously confirmed or unanimously accepted.

 

 

0
/ coma s L
l + ,
‘\\+ ,’
T HA 0H
2
+ S-conr180
(NAD )
Z = ribose-diphosphate-ribose-adenine
Figure 3. - Stereospecificity of hydrogen transfer

between substrate and coenzyme

I
Some researchers consider steric effects the dominating factor

in controlling and determining the orientation. Others pro-
pose that hydrophobic and hydrophilic interactions play an
important part.

Prelog suggested a model shown in Figure 414 to explain
the stereochemistry of alcohols obtained from the reduction

of cyclic ketones with a type B enzyme from Curvularia falcata.

 

Figure 4. - Prelog's model

In his model, steric interactions would be minimized
with the smaller group, '5', of the carbonyl compound placed
on the side of the carbamido group of NADH, if the carbonyl
group of the ketone was pointed down towards the pyridinium
nitrogen. However, when this orientation was applied to the
A type enzymes, it predicted products having the opposite
configuration (R-configuration) from those obtained experi-

15

mentally (S-configuration). With this observation and

the results of his group's work on some type A alcohol dehy-

drogenases,16'18

Karabatsos proposed that the carbamido group
played no role in determining the stereochemistry of the pro-
duct. He suggested the arrangement between the substrate and
coenzyme shown in Figure 5,16 where the larger group, 'L',

not the smaller group, '5', of the carbonyl compounds was put

 

Figure 5. - Karabatsos' model

on the carbamido side of the nicotinamide ring. The model
still maintained the carbonyl group pointing down towards the
pyridinium nitrogen. With this spatial orientation, the pro-
duct with the S-configuration was expected. In addition, his
group17’ 18 had also found that hydrophobic and hydrophilic
interactions might be more important than steric interactions
in determining the spatial arrangement of substrate-coenzyme.
Their findings are summarized in Table 1.

The results indicated that for nonpolar aliphatic alkyl
substituents on the carbonyl compound, the predominant product
was the S-enantiomer as predicted by the Karabatsos' model.
However, when the substituent group was sufficiently hydrophilic,
the product configuration could be completely reversed as evi-

denced from the result of the oxidation of hydroxyacetone by

glycerol dehydrogenase (Gly-DH).

 

 

 

 

 

Table 1. — Effects of hydrophobic and hydrophilic
groups on product stereospecificity
+ ADH e. +
R1R2C=0 + NADH + H < RIRZCHOH + NAD
R1 R2 Enzyme % R-config. % S-config.
CH3- CH3CH2- HLADH, Gly-DH 33-36 64-67
CH3- ClCHz- HLADH 48 52

Both Karabatsos' and Prelog's models had the carbonyl
group of the carbonyl compound pointed down towards the pyri-

19 However,

dinium nitrogen as initially suggested by Kosower.
there is no direct evidence against having the carbonyl group
oriented in some other direction. Indeed, the models proposed

by Graves, Clark, and Ringold,20 21

and by Cerinka and Hub,
Figure 6, had the carbonyl group pointed away from the pyri-
dinium nitrogen or towards the carbamido group. These alter-
native arrangements could change the entire spatial orienta-
tion of the substituent groups of the carbonyl compound with
respect to the coenzyme.

Therefore, it is clear that unless the spatial orienta-
tion of the carbonyl group with respect to the coenzyme is
determined, the arguments of the importance of steric, hydro-

phobic, and hydrophilic control of product stereospecificity

are meaningless.

 

1'sg ONHZ.
L
Figure 6. -
Graves model Cervinka and Hub model
. . . . 22 23
It was with th1s 1n m1nd that Miedema, May, and

Kroon24 set out to synthesize some coenzyme analogs by attach-

ing one end of the substrate molecule onto the carbamido group

of NAD+. These coenzyme analogs are shown in Figure 7.

I NHPICHZ)n-CH(OK)R
\\| R 8 H, 033
Z n 8 2 to 6

Figure 7. - Structure of NAD+ analogs

10

By reducing the number of possible orientations that each

face of the substrate could have with respect to the coenzyme,
and by determining the configuration of the coenzyme analogs
that reacted under the enzymatic conditions, it would be possi-
ble to deduce the orientation of the carbonyl group in the
complex. The possible arrangements are illustrated in Figure
8. However, the results would not be definitive unless the
reaction is strictly intramolecular. Experiments on the co-
enzyme analogs with added NAD+ in the enzyme system indicated.

otherwise.24

An increase in reactivity in the system with
the coenzyme analogs and NAD+ over that of the coenzyme ana-

logs alone acting as both the coenzyme and the substrate led

 

 

 

 

*R-conf18 c

Figure 8. - Possible spatial arrangements of
the NAD+ analogs

11

to the conclusion that both intra- and inter-molecular reac-
tions could be possible. Therefore, the question of the
actual orientation of the substrate and the coenzyme remained
unanswered.

The role of the catalytic zinc atom in the active site
is not clearly understood. From the X-ray structure deter-
mination of HLADH, the inference may be drawn that the only
groups in the enzyme that might be responsible for binding
and polarizing the reactive part of the substrate are the
catalytic zinc atom, the zinc bound water molecule or hydro-
xide ion, the hydroxyl group of Serine 48, and indirectly, the
imidazole group of Histidine 51 through its hydrogen bonding
to Serine 48. The active site of HLADH as observed from X-ray

studies is shown in Figure 9.

Coenzyme fi/J__Thr 178
‘::::§l—M fi\\\\b"0ye 17h

320

Hydrophobic Poem/gar I48 8} 6
His 7
7‘ (3 His 51

 

Figure 9. - Active site of HLADH as observed
from X-ray studies

Two mechanisms involving the catalytic zinc atom have

been suggested. The first was based on an electrophilic

catalysis3’ 25a

mediated by the zinc atom.

In this mechanism,

alcohol was bonded directly to zinc as the negatively charged

alcoholate ion replacing the hydroxide ion on zinc. The other

mechanism was a general acid-base catalysis

25a; b with the

hydroxide ion remaining on the zinc atom and the neutral alco-

hol attached as a fifth ligand to zinc. The two mechanistic

arrangements are depicted in Figure 10.

   

Coenzyme
+
l/jL—Thr 178
-'_0
\
Cys 17h
R2“" ‘j’m on 11.6
E

R1
Alco plate I!

/1\Ser I48 a
;& His 67
ff His 51

Electrophilic catalysis

Figure 10.

Coenzyme

\N+
*\\ />-Thr 178

0....
0y: 17h

   
   

R -—c 0-—
Aloohol 2 >0“. Cya 116
R1 HT“ $0
Ill?
Hydroxide H
ion "TT’T; I
His 67

I Acid-base catalysis

- Mechanistic arrangements in the

ternary complex of HLADH

13

The importance of the deep hydrophobic substrate binding
pocket of the enzyme is well established. The hydrophobic
interaction between the substrate and the enzyme is essential
in facilitating substrate binding and catalytic reaction.

Brooks and Shore26

showed that while ethanol reacts readily
with HLADH, methanol shows little or no activity at all. It
is difficult to explain the relative reaction rates of metha-

27 that for

nol and ethanol of 1:1400. It has been proposed
alcoholic substrates the proper molecular orientation for
hydride transfer at an optimum rate could be obtained by the
substrate binding to two sites, a hydroxyl binding site and a
hydrophobic binding site. Thus, methanol, with only one carbon
atom in the molecule, apparently is not large enough to bind
at both sites at the same time. On the other hand, ethanol
and the higher homologs are able to bind at both sites. Con-
sequently, the affinity for methanol by the enzyme is drasti-
cally reduced. This is reflected in the observation that
50 mM ethanol, as compared to 1 M for methanol, is required
for saturation. Furthermore, increased alkyl chain length in
the alcohol enhances this affinity of the enzyme-coenzyme
binary complex for the substrates, since only 1.5 m! l-propanol
is required for saturation and it reacts four to five times
faster than ethanol.

This thesis describes the synthesis of the quaternary
ammonium halides of primary aminoalcohols shown in Figure 11

and their reactivity with HLADH at six pHs at room temperature.

These compounds of variable alkyl chain length with a large

14

+ .-
RBN -(CH2)n-CH20H X
R = CH3, CH3032
n = 1-9
X = 01, I
Figure 11. - Structure of quaternary ammonium halides

of primary aminoalcohols

bulky group at one end and a hydroxyl group at the other
could serve as probes into the effective length and size of
the hydrophobic substrate binding pocket. They also could
serve as a preliminary study of the viability of this kind of
compound as possible substrates for the enzyme. If they are
successful as substrates, the procedure could be extended to
similar compounds with secondary aminoalcohols or carbonyl
groups and could further be extended to the study of other
enzyme systems.

In recent years, both the catalytic and the structural
zinc atoms of the enzyme have been completely or selectively

28, 29 3O

replaced by either cobalt or cadmium. Bobsein and

Myers31

recently published the Cadmium-113 NMR spectra of the
totally cadmium-substituted HLADH. With the compounds studied
here, if the bulky group is too large to enter the cleft
leading into the active site region of the enzyme, then only
those molecules with an alkyl chain long enough to reach into

the catalytic site would react. It would then seem probable

15

that by using Cadmium NMR spectroscopy with these compounds
and cadmium-substituted HLADH, the two catalytic mechanisms
involving zinc and the role of zinc in the catalytic reaction

could be further clarified.

RESULTS AND DISCUSSION

Preparations of the quaternary

ammonium salts of aminoalcohols

0f paramount importance for the studies described in this
thesis was the availability of various aminoalcohols to con-
vert to the desired quaternary ammonium salts. Since only a
few of the desired N,N-dialkylaminoalcohols are commercially
available, and since separation of the various mong-, Elf, and
tri-alkylated aminoalcohols is difficult and tedious, and
since not all of the aminoalcohols are readily available
(only up to six carbons long are available commercially),
direct alkylation of the aminoalcohols is not practical and
suitable preparative methods had to be devised. Before pro-
ceeding with a discussion of the kinetic studies, the prepa-
ration and identification of the aminoalcohols and their qua-
ternary ammonium salts will be described.

The quaternary ammonium salts were prepared according
to the general reaction routes shown in equation 2 for the
iodides and in equation 3 for the chlorides. The starting
point for the iodides varied depending on the availability
of the starting materials (diols or N,N-dialkylaminoalcohols).

16

17

H0-(CH2)n-OH + HCl —————-9 H0-(CH2)n-Cl

H0-(CH2)n-Cl + RZNH———_> RZN-(CH2)n-OH (eq. 2)
+ -

RZN-(CH2)n-0H + RI-—-————+ R3N -(CH2)n-0H I

R

CH3. CH3CH2

4--1O

I'I

, + - (eq. 3)
H0-(CH2)n-Cl + R3N-——————+ R3N —(CH2)n-0H Cl
R = CH3
n = 6-——10

With the exception of the butamethylene chlorohydrin,
all of the other chlorohydrins not purchased were prepared
from the corresponding diols. Butamethylene chlorohydrin was
prepared from dry, gaseous hydrogen chloride and tetrahydro-
furan primarily because these reagents are inexpensive and
readily available.

In general, the yields of the chlorohydrins were reasona-
bly good (about 45 to 75%) except for pentamethylene chloro-
hydrin (about 20%). For this reaction to be successful, it
is important to remove the chlorohydrin from the reaction
vessel by continuous extraction with a suitable solvent to
minimize the formation of such by-products as the dichlorides.
The yields of the chlorohydrins were increased considerably

(from 10--20% to about 50--60%) by this method over those

18

32 without the continuous removal

obtained from other methods
of the chlorohydrins. Those methods gave lower yields of the
desired product and considerably larger amounts of the
dichlorides.

The low.yield of pentamethylene chlorohydrin is probably
due to the instability of this compound when heated at high
temperature (140--145°) for an extended period of time (over-
night). In fact, this compound decomposed slightly on standing
for about a month at room temperature and required redistilla-
tion prior to its use.

The identity of the quaternary ammonium salts was ascer-
tained by qualitative chemical analysis (see experimental),
by proton NMR, and by mass spectroscopy with chemical ioniza-
tion in methane gas (CI-MS). Figures 12 and 13 show a couple
of the CI-MS spectra of the quaternary ammonium salts. None
of the mass spectra obtained gave the ion peak of the parent
compound. Instead, the ion peak of the dealkylhalogenated
quarternary ammonium salt (N,N-dialkylaminoalcohol) and a
much more intense m + 1 ion peak were observed (m is the mass
of the N,N-dialkylaminoalcohol). A typical mass spectral
pattern for chemical ionization with methane gas33’ 34 was
observed in all cases. Some of the ion peaks of m + 1,

m + 15, m + 29, and m + 41, of variable intensity, due to the
interactions of H+, CH3+, CZHS+ ions from methane with the
parent compound respectively, were observed in the spectra.

The ion corresponding to the dehydration product of the m + 1

ion was also observed in the spectra indicating the presence

19

of a hydroxyl group. With all the iodides studied, the methyl
iodide peak (m/e = 142) and that of its protonated ion (m/e =
143) appeared in every spectrum. The 143 peak was always more
intense than the 142 peak. In contrast to the iodides, the
chlorides gave no peak for the methyl chloride (m/e = 50) or

for its protonated ion (m/e = 51). The CI-MS spectrum of
N,N,N-trimethyl-10-hydroxydecyl ammonium chloride is shown in
Figure 12. The dealkylhalogenated ion of mass 201 has 8% of

the intensity of the m + 1 ion (m/3 = 202), whose intensity is
100%. The m + 15 ion (m/e = 216) and the m + 29 ion (m/e = 230)
have relative intensities of 4% and 17% respectively. The m/e =
164 ion is the dehydration product of the m + 1 ion. Figure

13 shows the mass spectrum of N,N,N-triethyl-10-hydroxydecyl
ammonium iodide. In the spectrum appear the m/e = 229 ion

(4% intensity) that corresponds to the dealkylhalogenated qua-
ternary ammonium salt, the m + 1 ion (m/e = 230, 53% intensity),
and the m + 29 ion (m/e = 258, 7% intensity). In addition, the
methyl iodide ion (m/e = 156, 9% intensity) and its protonated
ion (m/e = 157, 100% intensity) are observed. Although no

good explanation for not observing the methyl chloride ion

or its protonated ion in the mass spectrum can be offered,
nevertheless, the mass spectra are consistent with the struc-

tures of the quaternary ammonium salts.

20

mcwcopgu Eavcossm PXuowzxocuzgnofi

 

 

 

 

 

-Pzgumswcatz.z.z do szcuuoam mums cowaa~rcow paupsmgu 1 .NH ocamwd
QNN can am. am. av. an. on. on a» u\:
.. Pan—pppr-pF —P~.-p-P Fr-..chthp-uo.F-p-bonppPpr..p-nlnnhpp puppEup-pE-nnpp—p-n
— a: ‘ ___
Bu 3 3
no on .
2K .8. .
raga
ahoa m.o ”onzuaonm
ooow ”onauaaoaaoa
endgame «mum acaaoaom .
mg 8.8.

 

 

21

Own
r.

 

 

 

 

muwuo_ Eapcossa phomczxocvxgtofi
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. .J ¥t
8%. no

mo .
loan

haoa.m.o .ouzuuohm .

ooom ”oasnanoasoe

escapes .uam_a:¢aeaom .

toga.

22

The proton NMR spectra of the N,N,N-trimethyl- and the
N,N,N-triethylalkyl ammonium salts were taken at 250 MHz,
with deuterium oxide as the solvent and spectral grade acetone
as the internal standard (6 = 2.05). They are consistent with
and support the structures of the salts. Some of these
spectra are shown in Figures 14 to 17. In the methyl series
(Figures 14 and 15), the methyl group and the methylene group
of the quaternary nitrogen appear as a singlet (6 = 2.9) and
as a multiplet (6 = 3.1 to 3.4 for n = 10 to n = 2), respect-
ively. The methylene group next to the oxygen appears as a_
triplet (except for n = 2, which is a multiplet) at 6 = 3.4
to 3.9 for n = 10 to n = 2. All other methylene groups in
the carbon chain appear as multiplets in the range 6 = 1.1 to
1.9. Understandably, no hydroxyl hydrogen was observed with
water as the solvent. In the ethyl series (Figures 16 and
17), except for the hydrogens of the ethyl group, all other
groups of hydrogens have chemical shifts and splitting pat-
terns that are similar with those of their counterparts in
the methyl series. The multiplicity and chemical shifts of
the methyl (triplet at 6 = 1.1) and the methylene (quartet
at 6 = 3.1 to 3.2) of the ethyl group are typical of such

hydrogens in similar compounds.

23

assumcmasmu Eco; um Amo.m u 0v mcopmum ucmucmum
.mcgmucw ;a_3 vaxo sawcmaamw cw mvpuow Eavcossm
Pagoosxocos;-~-_»;ooswco-z.2.2 co azcbooam mzz cooocm NI: 9mm - (.ofi oa=n_u
we. _. N‘. m :

jd.dd._.%fi_l__4__-1d_diqq_4-d.qu4fi-___-_d4__4

.4 . 1 14(fi1111

 

 

 

 

24

w

«caumcmasmu soon we Amotm u cv mcoumoo uguucmum
panacea? sup: mcpxo Ezycouamu :? mcpcopzu Ezpcosao
_»uwu»xoccactofitPagumswcutz.z.z mo Escuuoam mzz copes; sz omm

o .. N m
.4 4 4 4 4 4 4 4 4,44 4,414 4 41444144 414 4 4.4 4 4 414 d

J

 

 

 

 

 

.mfi manor;

4 4 4 4 4 414 4.

25

mcsumcmaemu Eco; an Amo.~ n my mcoumum ucmucaum
yuccaucw spy: mupxo Engmuamu cw oupuo. sarcossm
_»;oosxocoss-~-Pagooacu-z.z.z Lo sacoooam «=2 coooca sz omm -

w a... .. . . . r

Jq~—_—44-d‘-__——1444d4q4.44—4fi4d-444——d—————

 

1111/4

 

 

N

 

mull

 

 

 

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.0“ ooszE

:

26

&

«causewasmu Eco; um Amo.m u my mcououm ucmucmum
chcmucp saw: mupxo E:_cmu:mu =4 ouwuow Ez—cossm
—»umuxxocvxguoaastumwsutz.z.z we sasuomam mzz couoca sz emu . .nfi ogaawu

o _. N m

 

4444441444d44qddiddddfiddqudfiqqud—qJ-Jd

 

 

ll;

 

 

 

 

 

 

 

 

27

Initial rate studies of the

 

quaternary ammonium salts of aminoalcohols

The initial rates of the aminoalcohols measured at
340 nm at the six pHs studied are shown in Figures 18 and
19, and in Tables 2 and 3. The relative rates at each pH
for both the methyl and the ethyl series of aminoalcohols
are given in Tables 4 and 5 respectively.

The results shown indicate that there is a general ‘
pattern in the initial velocities of the substrates. With
a few exceptions, the initial rate reaches a maximum with
the 7-carbon alkyl chain aminoalcohols in both series. The
higher homologs in the same series taper off in reactivity
while the shorter alkyl chain aminoalcohols show less reac-
tivity or, in some cases, do not react at all at the sub-
strate concentration used.

The effect of hydrophobic binding to enzymatic activity
is vividly illustrated here. It has long been recognized
that hydrophobic binding plays an important part in enzyme

35

catalysis. With HLDAH, the activity of primary aliphatic

alcohols increases as the chain length increases. Kroon,24
in his work with the aminoalcohols CH3CH0H(CH2)nNH2, where
n = 3,4,5, and 6, as substrates for HLADH, observed a rela-
tive reactivity rate of 1.0/1.5/2.0/34 respectively. The

present work also shows a similar trend in relative activity.

The sudden surge in reactivity in going from the 6-carbon

alkyl chain aminoalcohol to the 7-carbon chain aminoalcohol

28

No.04
mN.o

No.OH
mH.o

No.OH
wH.o

moo.ou
H~H.o

eoo.on
fimo.o

moo.ou
mmo.o

OH

HodH no.04 mo.o4 mo.o« ooo.o4 coc.on hoo.cw
om.o m¢.o Nu.o mm.o who.c mmo.o ono.c
No.04 no.0“ mo.o« Ho.oh moo.o4 moo.c4 o~c.c«
m~.o m~.o sm.c NH.o mmc.o mHo.c mec.o
No.o4 Ho.OH No.on 940.0“ moo.OH moc.c« moo.o4
mH.o Hm.o mm.o nmo.o evo.o emo.o eec.c
moo.ou o~o.o4 o~o.on coo.o« moo.0H
mNH.o ew~.o m¢~.c mmo.o moo.o 111 111
moo.o« Hoo.ou coo.on Noo.o4
Hmo.o m~o.o «mo.o moo.o 111 1-1 111
moo.ow moo.ow moo.ou Noo.om
cmo.o omo.o emo.o moo.o 1-- 111 111
m m m m m e m

.=Ps\oE== .o.o .e: cam on wood; .oaoucu

c N mmmauacmaemu Eco; no mza me um
rx :o A zuv+z A :uv mo copuouwxo as» com moons powuwcfi .

mo:.ow
eeo.o

mac.c«
m~o.o

coo.c4
Nmo.o

.N m—nmp

o.o~

m.w

m.m

o.m

m.o

:a

29

00.04
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.=_s\o4

-x =o=4N=uv+zmAN=

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n

:3 .o.o .2: oem so

0

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oco.qh
~40.c

m

mmuok

~0.04
no.0

40.04
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040.04 000.04
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~00.04 m00.04
040.0 c00.0

e m

4owupc4

mmzumcmasmu Eco; an ng xpm an
:00 mo :o44o04xo as» com moans 4m444c4

000.04
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40.04
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000.04
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--- m.4
--- 0.4
--- m.o
N "C IQ
1 .m mPQMF

30

004

004

004

004

004

004

04

404

00

00

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N4

004

o
-x zocfim

0m4

4m4

N44

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um

40

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180+:

04m

404

“M4

NM4

me

m04

a tom: 04

N44

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u c :44: :a come um
:80 co =o.pmo_xo oco coc mount o>4oa4o¢ -

0N

0N

mm

N4

04

em

em

mm

m4 0.04

04 0.0

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31

004

004

004

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me“ mmfi
Nofi mm“
so mo4
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84 AN
mm ---
a m
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-x 10:0 :80 +2

4 :0

004

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m: new: 04

00

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00

mm

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00

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32

0.8 r

 

n
1\

O

o

”I
I

.0
:r
j

0

o

w
I

002 '-

001 -

 

 

 

Figure 18. - Initial rates for the oxidation
of (CH3)CN+(CH2)nOH X“ as a function
of pH and n at 340 nm at room
temperature

v3honm, 00D. unit/Mina

 

33

 

 

 

 

0.8.
I
0.7-
J ,,
0.6r
_2§_.
0.5? ‘ 6.5
£3 7.0
0 7.5
CD 8.0
o,h- I 8.8
D 10.0
0.3- T
O
o 2 J.
. I o |
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I
m
I . bN/A/
O _ .qfl-o ‘/;
—F_2I |3r Fhi T5! '6' j7' l8! '9' w'10‘
a
Figure 19. - Initial rates for the oxidation of

(CH3CH2)3N+(CH2)n0H x‘ as a function
of pH and n at 340 nm at room
temperature

34

observed in this work and from 5-carbon to 6-carbon observed

in Kroon's work can be explained by the best "fit" phenomenon.

Since the active site of the enzyme is situated at the bot-

tom of a deep hydrophobic substrate binding pocket, the sub-

strate that would best fit into this pocket is expected to

be kinetically favored to bind to the enzyme. It would pro-

duce the most favorable combined hydrophobic, hydrophilic,

and steric interactions with the enzyme, and would require

the lowest activation energy in the rate determining step of

the catalytic sequence. It would seem that in the reaction

of HLADH with aminoalcohols, the 7-carbon chain aminoalcohols

are the most favored substrates at most of the pHs studied.
The effect of pH on the reactivity of the substrates

with HLADH is shown in Figures 18 and 19. The reactivity

of all substrates increases as the pH increases. At pH

values of 7.5 or lower, not all substrates react. Indeed,

as the pH decreases, the reactivity of the short alkyl chain

aminoalcohols decreases. At the lowest pH used, 6.5, only

the 9-carbon and the 10-carbon alkyl chain aminoalcohols in

the ethyl series show some activity. This is perhaps an

indication that the enzyme undergoes a conformational change

at the hydrophobic substrate binding pocket as the pH changes.

The substrate binding pocket becomes more constricted at

lower pHs, and consequently, the enzyme becomes more selective

with respect to the size and length of the substrate. Probably,

the bulky group attached at one end of a molecule is too

large to fit into the cleft at low pH and must remain outside

35
of it. Thus, only those substrates with an alkyl chain long
enough to reach the active site will be able to react. This
could be an important factor in designing NMR experiments to
determine the role of zinc in the catalytic process. If the
metal did play a role in the binding of the sdbstrate at the
active site, then at low pH and with a totally cadmium-sub-
stituted HLADH, only these substrates that showed activity at
that pH would have any appreciable effect on the Cadmium NMR
spectrum. 0n the other hand, if the metal did not interact
with the substrate directly, none of the substrates, either
active or inactive at that pH, would have much effect on the
Cadmium NMR spectrum.

The quaternary ammonium salts of the aminoalcohols used
in this study have thus proven to be useful probes of the
hydrophobic substrate binding pocket of HLADH in terms of the
depth of the cleft at the bottom of which is the active site.
Their use as probes to other alcohol dehydrogenase enzyme
systems whose structure is not known is something that is
recommended for future studies.

The N,N,N-trimethyl and the N,N,N-triethyl derivatives
of the aminoalcohols showed similar reactivities where acti-
vities were observed. The enzyme reacted with all of the
substrates, in both series, at pHs 8.0 and above. It seemed
desirable to test aminoalcohols with even bulkier groups
attached at the amino group to see if the enzyme would show
selectivity among substrates at higher pH. Attempts to pre-

pare the propyl series of aminoalcohols were unsuccessful,

36

as only the 2-carbon alkyl chain compound was successfully
synthesized. Further work in this area is required in order
to probe the substrate binding pocket at high pHs.
Equilibrium studies of thegguaternary

ammonium salts of aminoalcohols with

HLADH

The timed study of the equilibrium constants of the
aminoalcohols at six pHs at room temperature are listed in
Tables 6 and 7 for the methyl and ethyl series, respectively.
These are also shown graphically as a function of pH and
alkyl chain length at 36 hours in Figures 20 and 21, and as
a function of time and alkyl chain length at each pH in
Figures 22 to 33. The absorbance of the reduced nicotinamide
adenine dinucleotide at 340 nm was recorded every twelve hours
and the equilibrium constants were calculated by using equa-
tion 4 (see experimental). The equilibrium constants shown
are the average of six determinations for each substrate at
each pH.

Initially, the equilibrium constants were calculated
after a reaction time of 30 minutes to an hour. However, it
soon became apparent that this reaction time was not adequate,
as many of the reactions had not reached equilibrium. The
reaction time was then extended to three days. Measurements
were made at twelve hour intervals up to three days, and then

a final measurement was taken at the end of the seventh day.

37

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38

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46

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47

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48

04

04

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04 x

ON
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49

1300f
11000
900-
700-
500-
300-

1 00 f
80‘?-

Mx1013

eq’

’40?

20 ”

 

 

 

Figure 20. - Equilibrium constants for the oxidation
of N,N,N-trimethy1aminoaicoho1s as a function
of pH and n at 36 hours at room temperature

1500

1300

1100

900

700

500

300

M 2:10"3

0 100
O

50'
no
30
20

10

Figure 21.

 

 

50

c: I C) . c> v

 

 

12

36

h8
6O

72

 

00000000

 

- Equiiibrium constants for the oxidation
of N,N,N-trimethylaminoaicohols as a function
of time and n at pH 6.5 at room temperature

51

Mx1013

  

   

 

 

 

 

 

1300 F
r
1100 —
900 -
700 ‘
500 —
300 -
100‘:
80"-
l I--
?r
0
a
60— T1me(Hour)
A 12
_ A 21., .......
o 36 ___
ho — O #8 --—
I 60 -------
- c1 72 ---_
20 -
o4.
llllllllllllllllll
2 3 4 5 6 7 8 9 10
n
Figure 22. - Equiiibrium constants for the oxidation

of N,N,N-trimethyiaminoaicohols as a function
of time and n at pH 7.0 at room temperature

 

52

 

 

 

1300—
11004 47’ 0::
If. /’_7 '\
_ (Jr,
900’ /,/§ /§“§
.4 I" //
7oo— .-7-/§--§..--§ ----- I}.
500- _———A
_ ‘.__A/‘
300-
m .-
fib
v- 100fi'
K
’1' 9o-
0'
go
soi-
'T Time (Hour)
50*— A 12
'A an
ho- . 36 —__
(3 he 44....
30.. I 60 -------
a 72 -.m.um
20*-
10L
Ob?
“LI I I I l I l I I I I I I I I I I I
2 3 h 5 6 7 8 9 10
n
Figure 23. - Equiiibrium constants for the oxidation

of N,N,N-trimethy]aminoaicohois as a function
of time and n at pH 7.5 at room temperature

Figure 24.

700—

SOOF

300r

1004-

20

I

 

53

 

DIOCDD

(0*“2f’

o

’@
’

I.’

fl.’
’ /
I

cufl!

 

- Equiiibrium constants for the oxidation of
N,N,N-trimethy1aminoaicohois as a function
of time and n at pH 8.0 at room temperature

54

200 F

100 +— 1... x: ..... A ..... A

 

 

 

Figure 25. - Equiiibrium constants for the oxidation of
N,N,N-trimethyiaminoaicohois as a function
of time and n at pH 8.8 at room temperature

55

 

 

 

 

 

 

23 4
21 l
\ 1 - I
.), . _
l. |
1.9. -- ' I '. - ‘
A .A- 4A-
17
JL.
15
13
2? 11
<3
” 9
:l 5
Ir
0
"‘ 3
1 Time (Hour)
A 12
0.8 A 24 .......
I 36 ——-
0.6 0 “8 "'"'.
I 60 - ------
o.“ D 72 -.-.-.-.
0.2
O
1 I I l l l T f I I I Ti] l l 1 l I
2 3 h 5 6 7 8 9 10
n
Figure 26. - Equilibrium constants for the oxidation of

N,N,N-trimethyiaminoaicohois as a function
of time and n at pH 10.0 at room temperature

56

 

   

1000r-
800 —
600 r
hOO -
200 4
100 :“r
r- #H
tn '4. 6.5
<3 A .
'- 80 4 7 0
K . ' 705
:I o 8.0
‘8‘ - I 808
>4 D 10.0
6O'F-
1+0 L
20 ~ ' .
E+-=~
r f
./ -
' -/ 40"
o 4. mama-2&3"

 

 

Figure 27. - Equilibrium constants for the oxidation of
N,N,N-triethyiaminoaicohols as a function
of pH and n at 36 hours at room temperature

1100

I —|

900

'—

700

l

500“

300-

,1
100’4

M 1:1013

Keq, __
I

60-

no

 

57

Time (Hour)
12

 

 

c: I (3 I t» v
;r
a:
|
I

 

 

 

Figure 28.

- Equiiibrium constants for the oxidation of
N,N,N-triethyiaminoaicohois as a function
of time and n at pH 6.5 at room temperature

58

 

 

 

 

 

 

r
1100 -
900 —
700 -
500-
300 " Time (Hour)
A 12
"0 o 36
I; 904- 0 43
E" I 60
.0. 80- D 72
0
x:
70-
4?
50b
30-
/
:— ./
/
1°" ‘31 V0:
V3 .,fii
OI- ‘A ..._.. (’L‘/
I I T I I I I l I I I I I I I I I r
2 3 LI 5 6 7 8 9 10
n
Figure 29. - Equilibrium constants for the oxidation of

N,N,N-triethyiaminoaicohols as a function
of time and n at pH 7.0 at room temperature

59

 

 

 

 

1200-
Iooo~ £4 ...... §
_ ‘ 1,? C“;
o"? ‘
800’ ' I40
. 'iZ// ‘\Q
05/
600" f?’ _A..
_ ./ ,6 “6
“OOI- -fl§i‘ §\\\\
fl;/ ‘
200
100,
m 1
«13 90
x
I" 70
5453‘ so
30
Time (Hour)
10 ‘ 2
4|: A an .......
I 36 ---
3 o hB ---$
.. I 60 --------
2 D 72 .......
1
0
fl I I I I I I I I I I I l I I I I
2 3 ‘4 5 6 7 8 9 10
n
Figure 30. - Equiiibrium constants for the oxidation of

N,N,N-triethy1aminoa1cohois as a function
of time and n at pH 7.5 at room temperature

700

600

500

1.00

300

200

100

I

22

\

M x 1013

18

eq’

14

10.

\\]

Tilllllllllll

 

60

0.0.9)

  

flme(mmr)

 

 

Figure 31

- Equiiibrium constants for the oxidation of
N,N,N-triethyiaminoaicohois as a function of
time and n at pH 8.0 at room temperature

210

190

150

130

110

90

7O

13
Keq’ g x 10

50

30
15

11

\\

TIII lllllll

1 j l

T

“T

 

DIOODD

 

Time (Hour)
12

61

  
 
 
 
 
  

 

24 .......
36 ———
43 __4_.
60 -------
72 m-_,4

 

Figure 32.

- Equiiibrium constants for the oxidation of
N,N,N-triethyiaminoaicohois as a function
of time and n at pH 8.8 at room temperature

62

  

 

 

 

23 F
21 0
19 —
17 —
15 -
13 —
«V
g 11 -
K _
El 9 r
‘8‘—
>47?
1.
5 _
4
3 F
— Time (Hour)
1 1, A 12
I A an
0.6: .35---
: 0 he _.4-.
0.2 7 I 60 ......
0.1 1: a 72 ...... -
o...
]Ill|lllllllllllll
2 3 1+ 5 6 7 8 910
a
Figure 33. - Equiiibrium constants for the oxidation of

N,N,N-triethyiaminoaicohois as a function
of time and n at pH 10.0 at room temperature

63

The results showed that most of the substrates had reached
equilibrium within the three day period; and by the end of
the third day, the equilibrium constant had remained rela-
tively constant or already had started decreasing, thus
indicating that some degradation had begun to take place.
Both series of the salts of the aminoalcohols showed
significant changes in the equilibrium constant both as a
function of alkyl chain length and of pH. The equilibrium
constant of the N,N,N-trimethyl series increased as the
alkyl chain of the aminoalcohol increased from C-Z to C-7
and remained relatively constant up to C-IO, while that of
the N,N.N-triethyl series increased from C-2 to C-8 and
' remained constant up to C-10. The equilibrium constants
reached a maximum between pH 7.0 and 7.5 and then decreased
as the pH was increased further.

1 36

It has been reported ’ that for most primary alipha-

tic alcohols the equilibrium constant is relatively constant

‘12 M. However, the re-

and has a value of about 8.0 x 10
sults obtained in this study are clearly inconsistent with

the values reported. On the other hand, they are consistent

24

with the results obtained by Kroon with secondary amino-

alcohols that showed that the equilibrium constant increased

with increasing chain length.' Furthermore, Pietruszko gt

37 observed the same trend with Z-enoic alcohols.

38-40

31
Theorell and coworkers found that the dissociation
constant of the binary complex of NAD+ and HLADH favored the

free oxidized coenzyme and enzyme at all pHs, whereas the'

64

dissociation constant of the binary complex of NADH and
HLADH favored the bound NADH at pHs lower than 9. In addi-
tion, all of the studies had indicated that the binary
complex was favored over the ternary complex with primary

41 Under these circumstances,

aliphatic alcohols and aldehydes.
the equilibrium constant measured for primary aliphatic
alcohols would in fact be a measure of the equilibrium be-
tween the free NAD+ and the bound NADH in the system and

would be independent of the structure of the substrate. Thus,
this would explain the relatively similar equilibrium constant
for primary aliphatic alcohols. Clearly, the data obtained

by this work cannot be a measure of the equilibrium between
the free NAD+ and the bound NADH. It is worth pointing out

that Hiner and Theorell,42 39

and Theorell and McKinley-McKee,
working with saturated straight chain fatty acids and amides
as inhibitors of HLADH, showed that the binding of the inhi-
bitor to the binary complex of coenzyme and enzyme was
greatly enhanced when the chain length was increased. In
fact, in a few cases, the dissociation constant favored the
ternary complex over the binary complex. Also the disso-
ciation constant of the ternary complex with primary ali-
phatic alcohols decreased as the chain length was increased,
although the binary complex was still favored in all cases
studied.

We suggest that hydrophobic interaction plays an import—
ant role in the binding of the substrate to the binary

complex and may affect the relative stability of the ternary

65

complex with respect to the binary complex. Conceivably,

in some cases, it could even make the ternary complex more
stable than the binary complex. The equilibrium constants
would then depend on the structure of the substrate and
might be a measure of the equilibrium between the ternary
complex and another identity. We suggest that as the length
of the carbon chain of the aminoalcohol derivative is in-
creased, the stability of the ternary complex increases.

The effect of pH on the equilibrium constant cannot be
explained satisfactorily. It would seem from equation 4
that at higher pH values, the equilibrium would favor the
oxidation of the alcohols and increase the equilibrium con-
stant. However, it is also known that at higher pH values,
NAD+ undergoes a side reaction to form a pseudobase,43’ 44
and at pH 10.0, the EE-dimer of HLADH, which is responsible
for the oxidoreduction of non-steroid substrates, is gra-
dually transformed to other isomers.45 These two factors
may explain the decrease in the equilibrium constant as the
pH is increased from 7.5 to 10.0.

In conclusion, this work has shown that: 1. The qua-
ternary ammonium salts of several aminoalcohols can be used
as substrates for HLADH. 2. The initial velocities and
equilibrium constants obtained at six pHs and at room tem-
perature are consistent with the premise that hydrophobic
interactions affect this enzymatic reaction significantly.
3. These substrates could be used as probes for the hydro-

phobic substrate binding pocket of the enzyme. The potential

66

use of these substrates, or structurally similar ones, to
elucidate the role of the catalytic zinc in this reaction,
or to study other alcohol dehydrogenases must await further

experimentation.

EXPERIMENTAL

Instrumentation

 

Proton NMR spectra were recorded either on a Varian
T-60 NMR spectrometer or a Bruker HP 250 NMR spectrometer.

Ultraviolet spectra for enzyme activity studies were
obtained with a Beckman DB-G spectrometer equipped with an
analog converter connected to a Sargent SR recorder.

The pH of the buffers was adjusted with an Instrumen-
tation Laboratory Model 245 pH meter.

Mass spectra were obtained with a Finnigan GC-MS
mass spectrometer. ~'

Melting points of the quaternary ammonium halides of
the aminoalcohols were determined with a Hoover capillary

melting point machine.

Materials

Crystalline horse liver alcohol dehydrogenase and the
oxidized form of nicotinamide adenine dinucleotide Grade III
were purchased from Sigma.

The compounds N,N-dimethyl-2-aminoethanol, N,N-dimethyl-
3-amino-1-propanol, N,N-diethyl-2-aminoethanol, N,N-diethyl-

3-amino-1—propanol, 6-chloro-1-hexanol, 1,5-pentanediol,

67

68

1,7-heptanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-

decanediol, and iodoethane were obtained from Aldrich.

Synthesis

 

A. Preparation of Chlorohydrins

I. 4-Chloro-1-butanol.46

Q + HCl ‘; ClCHZCHZCHZCHZOH

A 500 ml. three-necked round-bottomed flask contain-

 

ing 130 ml. (114 gm. or 1.58 moles) of tetrahydrofuran was
equipped with a reflux condenser, a thermometer dipping

into the liquid, and a bent glass tube arranged to introduce
gaseous hydrogen chloride near the bottom of the flask. The
condenser was connected to a soda lime tube to trap the
hydrogen chloride gas that might escape.

The tetrahydrofuran was heated to boiling in an oil
bath, and a slow stream of hydrogen chloride gas, produced
by dripping concentrated sulfuric acid onto a mixture of
sodium chloride and concentrated hydrochloric acid, was
bubbled into the liquid. The temperature increased slowly
at first, and then more rapidly. The reaction was stopped
when the temperature remained practically constant at about
1100 after six hours of heating. The liquid turned yellowish

brown at this point.

69

The liquid was cooled and the unreacted tetrahydro-
furan was removed by fractional distillation. The chloro-

hydrin was collected by vacuum distillation at 81-820/14mm
Hg.

11. Other chlorohydrins from the diols.47

H0-(CH2)n-OH + HCl-——-—-9’H0-(CH2)n-Cl + H20
n = 5, 7, 8, 9, 10

A special apparatus shown in Figure 34 was set up

for these preparations.

To condenser

 

 

 

 

 

  

Flask A'
(1000 m1.)
Flask B

(500 m1.)

 

 

 

 

 

Figure 34. - Apparatus for the preparation of
chlorohydrins from diols

70

About 0.89 mole of the diol was put in reaction
flask A together with 9.5 moles of concentrated hydrochloric
acid, 130 ml. of water, and 55 ml. of toluene.* Flask B
contained 350 ml. of toluene with boiling chips. Both flasks
were heated by oil baths, Flask A being kept at 950 and
Flask B at 140-1450. As the reaction proceeded, the product
chlorohydrin was continuously extracted and removed by
toluene flowing through the reaction mixture. After 12-24
hours of heating, the organic phase had turned brown and
the reaction was stopped. After the flasks were allowed to
cool, the organic phase in Flask A was forced out into
Flask B by adding water through a long stem glass funnel.
The aqueous phase was then extracted twice with 100 ml. por-
tions of toluene. Toluene was removed from the combined
organic mixture by either distillation or low pressure
rotary evaporation. The remaining liquid was fractionally
distilled at low pressure to obtain the chlorohydrin.
Table 8 gives the yield and distillation temperature and .

pressure of the chlorohydrins.

* Since nonamethylene glycol and decamethylene glycol are

quite soluble in toluene, ligroine (b.p. loo-110°) was

used as the extracting solvent instead of toluene.48

71

Table 8. - Yields and Boiling Points of the
Chlorohydrins ( H0-(CH2)n-Cl )

 

n b.p. OC/mm Hg % Yield
4 81-82/14 66.0
5 100-104/9 20.0
7 122-124/12 57.0
8 130-140/10 75.0
9 146-148/14 60.0
10 185-189/15 46.0

 

B. Preparation of N,N-dialkylaminoalkanols
I. 4-Dimethylamino-l-butanol.

H0-(CH2)4-Cl + (CH3)2NH-——————9'(CH3)2N-(CH2)4-OH + HC]

This aminoalcohol was prepared by a modified method

of Nakajima.49

An amount of 20 gm. of dimethylamine was
placed in a 250 ml. three-necked round-bottomed flask equipped
with a dry ice/acetone condenser, a separatory funnel, and

a magnetic stirrer. The flask was cooled to below 5° by

an ice-water bath. To the amine was added slowly 22 gm.

(0.2 mole) of butamethylene chlorohydrin through the sepa-
ratory funnel while the temperature was maintained at below
5°. After the addition was complete, the reaction vessel

was allowed to warm up to room temperature. This warming

allowed low boiling substance to evaporate. The remaining

72

liquid residue was vacuum distilled to obtain the product

4-dimethylamino-l-butanol, as a colorless viscous liquid.

11. S-Dimethylamino-l-pentanol.49

A mixture of 12.3 gm. (0.l mole) of pentamethylene
chlorohydrin and 25.0 gm. of a 40% aqueous solution of
dimethylamine was allowed to react in a 100 ml. round-
bottomed flask with a condenser and magnetic stirrer. The
reaction mixture was heated to 600 for about 4 hours until
it became homogeneous. The solution was then distilled
until the internal temperature reached 180°. A concentrated
solution of potassium hydroxide in methyl alcohol was added
to the cooled reaction mixture. The precipitated potassium
chloride was filtered off. After the addition of water,
ether was added to the biphasic mixture. The organic layer
was removed and saved. The aqueous layer was extracted
three times with 50 ml. portions of ether. The organic
layers were combined and dried with anhydrous magnesium
sulfate. After removal of the low boiling ether and methyl
alcohol by rotary evaporation, the remaining light brownish
liquid was distilled to obtain the product, 5—dimethylamino-

l-pentanol, as a colorless liquid.

73
III. N,N-Diethylaminoalkanols.

H0-(CH2)n-Cl + (CH3CH2)2NH‘--—)(CH3CH2)2N-(CH2)n-0H
n = 4 to 10 + HCl

The procedure used for the synthesis of these amino-

alcohols was the one used by N. w. Hartman.50

In a typical
experiment, 0.2 mole of diethylamine was refluxed in a 100
ml. round—bottomed flask equipped with dropping funnel, con-
denser and magnetic stirrer. To the refluxing diethylamine
was added dropwise 0.1 mole of the chlorohydrin. After the
addition was complete, the mixture was heated further for

8 to 12 hours. During this time, a white crystalline preci-
pitate began to form and the liquid turned brown. The heating
was stopped and the contents were allowed to cool. Addition
of 5.0 ml. of 16.4 0 sodium hydroxide to the cooled solution
caused the precipitation of sodium chloride. After 10 ml.
of water was added to dissolve the sodium chloride, 50 ml.
of ether was added to the mixture. The organic layer was
separated from the aqueous phase and the aqueous layer was
extracted three more times with 50 ml. portions of ether.
The combined ether extracts were dried over anhydrous potas-
sium carbonate by mechanical stirring until the turbidity
disappeared. The potassium carbonate was removed by fil-
tration, and the ether was removed by rotary evaporation.
The remaining brownish liquid was vacuum distilled to obtain

the desired product. Table 9 shows the boiling points and
percentage yields of the N,N-dialkylaminoalkanols prepared.

74

 

 

 

Table 9. - Boiling points and percentage yields
of N,N-Dialkylaminoalkanols
(CH3)2N-(CH2)n-0H
n . b.p. OC/mm Hg % Yield
4 55-57/2 45.5
5 62-63/3 48.0
n b.p. oC/mm Hg % Yield
4 92-95/4.8 33.6
5 114/14 55.0
6 72-74/0.04 70.0
7 146-148/7 72.0
8 140-141/6 68.0
9 135-137/1 65.5
10 133-134/4 63.0

 

Preparation of QuaternaryiAmmonium Halides

0f Aminoalcohols

I.

Quaternary ammonium chlorides.

. + -
H0-(CH2)n-Cl + (CH3)3N-———q9(CH3)3N -(CH2)n-OH C1

The following is a typical procedure for the prepara-

tion of quaternary ammonium chlorides.

75

An amount of 0.02 mole of chlorohydrin was allowed to
react with either a 25% aqueous solution of trimethylamine
(n = 6, 8) or with neat trimethylamine (n = 7, 9, 10) that
was produced by distilling the trimethylamine from a 25%
aqueous solution and condensing it with the aid of a dry
ice-acetone bath.

With the 25% aqueous solution of trimethylamine, the
reaction mixture was refluxed for about 24 hours. Low
boiling material was removed by rotary evaporation. The re-
maining liquid solidified on standing and was recrystallized
from ether-acetone solutions. The salt was dissolved in
acetone on a steam bath. To the acetone solution was added
ether until it became slightly cloudy. White crystalline
solid was obtained on cooling. . ,

With the neat trimethylamine, equimolar amounts of the
amine and chlorohydrin were allowed to react. The liquid
trimethylamine was placed in a 100 ml. round-bottomed flask
with a dry ice/acetone condenser and a dropping funnel.

The chlorohydrin was slowly added to the amine. After the
completion of the addition, the reaction mixture was allowed
to react for 6 hours at room temperature. Low boiling
material was removed by rotary evaporation and the resulting

white solid was recrystallized from ether—acetone solution.

II. Quaternary ammonium iodides.

+ -
(CH3)2N-(CH2)n-0H + CH3I-—————% (CH3)3N -(CH2)n-OH I

76
. + -

n = 2 to 10

The following is a typical procedure for the prepara-
tion of the quaternary ammonium iodides of various amino-
alcohols.

The N,N-dialkylaminoalc0hol (0.02 mole) was put in a
250 ml. round-bottomed flask equipped with condenser, dropping
funnel and magnetic stirrer. Anhydrous ether was then added
and the solution was heated to about 750 in an oil bath. An
equimolar amount (0.02 mole) of the alkyl iodide was added
dropwise to the solution. After the addition was complete,
the heating was maintained for another 2-3 hours. The reac-
tion mixture was then allowed to cool to room temperature.

Low boiling substances were removed by rotary evaporation.
The white solid that was formed on standing was recrystallized
from ether-acetone solution.

Table 10 gives the melting points and percentage yields
of the quaternary ammonium halides of the aminoalcohols, and

Tables 11 to 14 the proton NMR and mass spectral data.

Qualitative identification of

quaternary ammonium salts51 '

The test for quaternary ammonium salts was performed
as follows: a sample of the salt (0.1 gm) was dissolved
in water and excess concentrated nitric acid was added

followed by 1.0 ml of 0.1 N aqueous silver nitrate. A

77

white precipitate of silver halide was formed. Excess

amount of 1.0 N sodium hydroxide was added until the solution
was alkaline and the precipitate redissolved. The mixture
was extracted twice with ether and separated. Ether was
dried over anhydrous sodium sulfate and evaporated away.

A positive test for quaternary ammonium salts is indicated

when no residue remained after ether was removed.

78

Table 10. - Melting points and percentage yields of
quaternary ammonium halides of aminoalcohols

+ -
(R3N -(CH2)n-OH X )

 

 

 

R - CH3- n x m.p. °c % Yield
2 I 270-272 82
3 1 198-200 80
4 I 130-132 75
5 1 130-135 65
6 Cl 171-175 47
7 c1 201-205 40
8 Cl 195-200 38
9 Cl 218-223 41
10 Cl 195-202 31
R a CH3CH2- n x m.p. °c z Yie‘d
2 1 subl. 283 76
3 I 239-241 62
4 I 137-140 30
5 I 90-94 49
6 I ----* --
7 I 77-80 44.
8 I 58-62 40
9 I 70-72 38 \
10 I 85-90 39

 

*This was never isolated as a solid. It was a very viscous,
clear liquid.

79

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85

Activity of quaternarygammonium halides

of aminoalcohols with HLADH

Horse liver alcohol dehydrogenase was freshly prepared
daily for enzyme activity assays. Aqueous solutions of
NAD+ were kept frozen for no longer than three days. Hater
used for the preparation of solutions was deionized and
twice distilled. Buffer solutions were prepared as described

in Methods in Enzymology.52

The pH was adjusted with an
Instrumentation Laboratory pH meter. For pH 6.5, 7.0 and
7.5, 0.05 g sodium phosphate was used; for pH 8.0, 0.05 H
Tris.HCl was used; and for pH 8.8 and 10.0, 0.5 fl glycine/
NaOH was used.

The crystalline enzyme was reconstituted just prior to
use by dissolving 25 mg of the enzyme in 20 ml. of 0.05 5
sodium phosphate buffer, pH 7.0. All 15 m! stock solutions
of NAD+ were prepared by dissolving 0.1 gm of the coenzyme
in 10 ml. of water. Throughout the experiments 45 mfl sub-
strate stock solutions were used. Activity assay solutions
were made up of 0.1 ml NAD+ stock solution, 0.4 ml. of sub-
strate stock solution, 0.2 ml. of enzyme stock solution, and
the appropriate amount of buffer to bring the final volume
to 3.0 ml. The final concentrations of NAD+ and substrate
were 0.5 mg and 6.0 mfl respectively. The amount of enzyme
used for each assay was 0.5 unit or 0.25 mg.

The initial activity of the substrates with the enzyme

was measured by the increase in the UV absorbance at 340

nm due to the reduction of the pyridine ring of the nicotinamide

86

adenine dinucleotide. The increase of the absorbance for
the first three minutes was monitored on a Sargent SR re-
corder. Initial velocities of substrates were determined
from the best straight lines drawn as close to the initial
increase in absorbance as possible. At least six runs were
made for each substrate at each pH. Solutions minus the
enzyme were used as references (blanks) in all the runs.
All activity assays were performed at room temperature.

Equilibrium studies were run under the same conditions
as the initial velocity experiments. Ultraviolet absorbance
at 340 nm was measured at twelve hour intervals up to three
days and then at the seventh day. Triplicate samples were
run at the same time for each substrate at each pH at room
temperature. The entire experiment was again repeated. In
addition to the usual blank (solution minus enzyme), the
absorbance of a solution containing enzyme and coenzyme but
no substrate was also monitored. The absorbance was calcu-
lated to be that of the sample with enzyme, coenzyme and
substrate less that of the sample with only enzyme and
coenzyme.

The equilibrium constant for the oxidation of alcohols
by NAD+ and HLADH as represented in equation 1 was calcu-

lated by using equation 4,

2 +
Keq = + x (H 1- (eq. 4)
("AD )0 - X) (R1R2CHOH)0 ' X)

87

where (NAD+)0 is the initial concentration of the coenzyme;
(RIRZCHOH)0 the initial concentration of the alcohol; and

x the concentrations of NADH and R1R2C=0 at equilibrium.

The concentration of NADH was calculated from the absorbance
at 340 nm by using the reported extinction coefficient of

6.22 x 106 cmZ/mole.53

The proton concentration was fixed
by the pH of the buffer used. It was further assumed that

the extinction coefficient at 340 nm is independent of pH.

APPENDIX

APPENDIX

Nuclear Magnetic Resonance Studies
of Cadmium-lll-substituted

Horse Liver Alcohol Dehydrogenase

Cadmium-111 nmr studies of cadmium-substituted horse
liver alcohol dehydrogenase were initiated in 1977 in order
to elucidate the role of zinc in the enzymatic reaction and
to study changes at the active site of the reaction. The
cadmium-substituted enzyme was found to be 60% less active
than the native enzyme containing zinc.

The Cadmium-111 NMR spectra were recorded under various
conditions. The concentration of the totally cadmium-substi-
tuted enzyme ranged from a low 30 0M to a high of 580 u!
with or without 96% enriched cadmium-111 (purchased from
Oakridge Laboratory). The natural abundance of cadmium-111
is about 12%. High enzyme concentration, desirable for nmr
studies, was maintained by adding 1.0 M urea at the metal
exchange stage and during the subsequent removal of extraneous
cadmium ion from the system. The NMR spectra were usually
taken under the following conditions: 0.045 M sodium phos-
phate buffer, 1.0 M urea with 10% 020 as an internal lock,
pH 6.0; spectral width of 20,000 Hz; pulse anglé of 4 micro-
seconds (9 to 12°); no delay to a pulse delay of 0.2 seconds;

and the number of transients from 130,000 to 290,000. 1“
88

89

Unfortunately, under none of the conditions tried were we
able to observe any Cadmium NMR signals. After one and a
half years of work the project was abandoned.

In March, 1980, several months after this project was

31 published a communication

discontinued, Bobsein and Myers
on Cadmium-113 NMR spectra of totally cadmium-substituted
horse liver alcohol dehydrogenase. Their conditions were:
0.29 mM 90% enriched cadmium-113-substituted HLADH; 0.05 M
sodium phosphate buffer, pH 7.5 with 020 added; pulse angle,
70°; spectral width, 20,000 Hz; pulse delay of 3 seconds;
and 24,000 scans. The spectra showed two distinct signals,
one for each kind of cadmium.

NMR-wise, there is not much difference between cadmium-
113 and cadmium-111 (our probe for the Bruker 180 is just out
of the range for cadmium-113 NMR), although cadmium-113 is
about 10-to 15% more sensitive than cadmium-111. Therefore,
our failure to observe any nmr signal cannot be attributed
to differences between cadmium-111 and cadmium-113. The
most probable cause is an error in estimating the T1 of
the cadmium in the enzyme system from the T1 determined
from a solution of cadmium chloride and glycine at a ratio
of 1:4. The wrong estimate of T1 probably led to inadequate
delay time between pulses that in turn led to saturation.

This area of cadmium NMR spectroscopy of the enzyme
certainly merits further study since the ability to observe
the cadmium signals enables possible elucidation of the

role of the metal in the catalytic action of the enzyme.

90

Experimental

Cadmium-111 NMR spectra were taken with a Bruker NH
180 NMR spectrometer at 38.16 MHz equipped with a 20 mm
probe. The cadmium content, after metal exchange, in the
enzyme was determined by atomic absorption. Protein con-

54 with cry—

centration was determined by the Lowry method
stalline HLADH as the standard.
‘Metal exchange (cadmium for zinc) was accomplished by

d,30 the exchange was carried

a modification of Vallee's metho
out at 4° and the buffer solutions for dialysis were kept
under a continuous flow of argon. The following is a typical
experiment: A solution of crystalline HLADH in 0.045 M
sodium phosphate buffer, pH 7.0 was dialized against a liter
of the same buffer for 12 hours. The enzyme solution was
then dialyzed against a liter of 0.2 M sodium acetate buffer,
pH 5.5, with 0.1 mM cadmium chloride added* for 72 hours
with two changes of buffer. After the metal-exchange, extra-
neous cadmium was removed by dialyzing the enzyme solution
with a liter of 0.045 M sodium phosphate buffer, pH 6.0, for
24 hours with one change of buffer. The enzyme solution was

concentrated, whenever needed by ultrafiltration with an

Amicon ultrafiltration cell.

*at high concentrations of enzyme (0.5 mM or 40 mg/ml, or
higher), 1.0 M urea was added to the buffer solution to
stabilize the enzyme.

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10.

11.

12.

I

K.

K.

Co

I

I

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nd A.

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