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ABSTRACT
EXCITED STATES OF INDOLE AND AZAINDOLES

By

Li Lillian Yang

Excited state characteristics of indole and various aza-substituted
indoles, 2-, 3-, 4-, 5-, and 7-azaindoles, are studied. A comparative
study of the absorption spectra in the gas phase and in solution as well
as emission spectra at room and cryogenic temperatures has been performed.
Both fluorescence and phosphorescence spectra and triplet state lifetimes
are investigated. Various environments (solvents) are used and spectral
shifts are interpreted in terms of various interactions, i.e., dispersion,
dipole-induced dipole, dipole-dipole interactions and hydrogen bonding.
These shifts are correlated with charge density changes at the aza and
pyrrolic nitrogens. These charge densities are calculated using Pariser-
Parr-Pople method. From the absorption and emission studies, excited
state dipole moments and pK's are obtained.

The knowledge of the absorption and dissipation of excitation energy
by these molecules is important in radiation biophysics and photobiology,
and in using these molecules as fluorescent probes to probe changes in
their natural environment. Information gained regarding the chemistry of
the excited states of these molecules is valuable in further understanding

of photoinduced proton transfer phenomenon.

EXCITED STATES OF INDOLE AND AZAINDOLES

By

Li Lillian Yang

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Biophysics

1974

c3500)

(7‘53

To my parents

ii

ACKNOWLEDGEMENTS

I would like to express my thanks to my research adviser, Prof. M.
Ashraf El-Bayoumi, for his assistance with the work herein reported.
Dr. El-Bayoumi has contributed greatly to the development of my scientific
understanding. The constructive criticism and advice of the thesis com-
mittee members, Dr. E. McGroarty, Dr. A. Haug, and Dr. C. H. Suelter are
also greatly appreciated. Special thanks go to my colleague and best
friend, Dr. Ph. Avouris, for numerous discussions and encouragement.

This research was supported by the College of Osteopathic Medicine,
with the cooperation of the Biophysics Department, Michigan State Univer-

sity, East Lansing, Michigan.

iii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
CHAPTER 1 GENERAL INTRODUCTION
CHAPTER 2 EXPERIMENTAL
(A) Compounds Studied
(B) Purification of Solvents
(C) Spectral Measurements
CHAPTER 3 VAPOR SPECTRA OF INDOLE AND AZAINDOLES
Introduction
Results and Discussions
Indole

Azaindoles

CHAPTER 4 ABSORPTION SPECTRAL SHIFTS
-CORRELATION WITH EXCITED STATES CHARGE DENSITIES

Introduction

Results and Discussions
Indole
Azaindoles

CHAPTER 5 EMISSION SPECTRA OF INDOLE AND AZAINDOLES

Introduction

Results and Discussions
Solvent Effects on Luminescence Spectra
Excited State Dipole Moment Studies
Excited State pK Studies

Indole
7-Azaindole

BIBLIOGRAPHY

iv

vi

13
13
15

25
25
34
34
4O
53
53
57
57
62
63
63
65

73

Table

Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

1Lb(____) and 1La(") Absorption Band Energies of
Indole and Azaindoles in the Vapor Phase.

Experimental (Vapor) and Calculated Electronic
State Energies of Indole and Azaindoles

Absorption Spectral Shifts for Indole in Different
MGdia.

Contributions of Dipole-Dipole Interaction and
Hydrogen Bonding Interactions to the Observed
Absorption Spectral Shifts for Indole.

Absorption Spectral Shifts for Azaindoles in
Different Media.

Contributions of Dipole-Dipole Interaction and
Hydrogen Bonding Interactions to the Observed
Absorption Spectral Shifts for Azaindoles.

Fluorescence (F) and Phosphorescence (P) Spectra of
Indole and Azaindoles in Different Media.

Lowest Singlet Excited State Dipole Moments of
Indole and Azaindoles.

Lowest Singlet Excited State pK's of Indole.and
Azaindoles.

21

22

35

39

41

42

S8

64

71

Figure

Figure

Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure

Figure

Figure

Figure

Figure

Figure
Figure
Figure
Figure

Figure

10.

ll.

12.

13.

14.

15.

16.

LIST OF FIGURES

Experimentally Studied Compounds.

Ground and Excited State Potential Energy Curves

and Vibronic Trans
Schematic Absorpti
Indole Vapor Absor
2-Azaindole Vapor
3-Azaindole Vapor
4-Azaindole Vapor
S-Azaindole Vapor
7-Azaindole Vapor

Calculated Charge

itions.

on Band in a Vibronic Spectrum.
ption Spectrum.

Absorption Spectrum.
Absorption Spectrum.
Absorption Spectrum.
Absorption Spectrum.
Absorption Spectrum.

Densities for Indole.

Illustrating Change of Solvation After Excitation

or Emission.
Demonstration of
Levels of a Polar
with a Non-polar

Solvent Shifts du

Hydrogen Bondings
(i) diethyl ether

Calculated Charge
Calculated Charge
Calculated Charge
Calculated Charge

Schematic Thermoc
of Excited-State

Solvent Strain Effects on Energy
Solute in a Polar Solvent Compared
Solvent and Vapor. ..

e to Hydrogen Bonding.

Between Indole and Solvents
(ii) ethanol (iii) water.

Densities for 3-Azaindole.
Densities for 4-Azaindole.
Densities for 5-Azaindole.
Densities for 7-Azaindole.

hemical Diagram for Determination
pKa Values.

vi

10
10
14
16
17
18
19
20

23

28

30

31

37
44
46
48

50

56

Figure

Figure
Figure

Figure

20.

21.

22.

23.

vii
Emission Spectra of Indole in Different Media at
Room Temperature.
Emission Spectrum of 2-Azaindole in BM? at 77°K.
Fluorimetric Titration Curve of Indole (25°C).

Fluorimetric Titration Curve of 7-Azaindole.

6O

61

66

68

CHAPTER 1

GENERAL INTRODUCTION

Indole is a unique and important molecule to both biochemists and
biophysicists. It is the chromophore of tryptophan, an amino acid found
in most proteins. Among the three amino acids that absorb light in the
ultraviolet region, tryptophan has a maximum extinction coefficient of 700
(at 280 nm) which is five times that of tyrosine (at 278 nm) and fifteen
times that of Phenylalanine (at 260 nm). Thus, tryptophan accounts for
most of the direct energy deposition from UV irradiation in protein.

Indole is also the chromophore of many other important biological sub-
stances like: serotonin (a transmitter substance found at chemical synapses
between neurons), indole acetic acid (a plant growth hormone), and lysergic
acid (LSD), bufotenin and psilocybin (hallucinogenic drugs).

Purine an important biological molecule is an aza-substituted indole
(3,5,7-triazaindole). Purine is the chromophore for'adenine and guanine,
two of the bases found in DNA and RNA. Purine is also the chromophore of
ATP and NAD coenzymes.

Azaindoles are of interest as possible metabolite antagonists of purine
and of physiologically active indoles such as serotonin, tryptophan, and
NN-diehyl-lysergamide. Competition has been demonstrated between 7-aza-
indole and indoles in bacteria, viruses, fungi, and protozoa. 7-Azatryp-
tophan has been incorporated into bacterial protein in the place of tryp-
tophan by a tryptophan-requiring mutant of §;_ggli, but growth soon ceasedl.

l

2
T2 bacteriophages behave somewhat similarlyl, 7-azaindole inhibits the

. . . . 2
conver31on of indole into tryptophan 1n the mould Neurospgra crassa ,

 

and 7-azatryptophan prevents the uptake of tryptophan by the protozoon
Tetrahymenagpyriformis? These compounds should be of considerable inter-
est in other systems, particularly those in which tryptophan synthesis is
carried out, for it should be possible there to determine more exactly
the point of interference.

In view of their importance, it is not surprising to see that the
spectroscopy of indole has attracted great attention. Our spectral
study of indole and various aza~substituted indoles, 2-, 3-, 4-, 5-, and
7-azaindole, include detailed studies of the solution absorption and emission
spectra of these molecules at room and cryogenic temperatures. Both f1Uo-
rescence spectra and phosphorescence spectra and lifetimes are studied.
Various environment (solvents) are used and spectral shifts are inter-
preted in terms of various interactions, i.e., dispersion, dipole-induced
dipole interaction, dipole-dipole interaction and hydrogen bonding inter-
action. These shifts are correlated with charge density changes at the
aza and pyrrolic nitrogens. These charge densities are calculated by
using Pariser-Parr-Pople method. The results of.this.investigation should
be helpful in the area of radiation biophysics because of the knowledge
we gained on the way these molecules dissipate the light energy they
absorb and the effect of the environment on these processes. An under-
standing of the spectroscopy and the effect of the environment on the
emissive properties of molecules like indole (tryptophan) will improve
their use as intrinsic fluorescent probes in proteins for the study of
processes like denaturation, dimerization, etc. Gas phase absorption

spectra of these molecules have also been studied. The analysis of these

spectra is helpful in identifying electronic transitions and in studying

the effect of aza-nitrogen substitution on the spectra.

CHAPTER 2

EXPERIMENTAL

(A) Compounds Studied
Molecules studied here are: indole, 2-, 3-, 4-, 5-, and 7-azaindoles

(Figure l). The means of purification are described below for each mole-

cule.

(1) Indole: purchased from Calbiochem was recrystallized once from an

alcohol-water mixture then vacuum sublimed slowly (one day).

(2) 2-Azaindole (Benzpyrazole): purchased from Aldrich was recrystallized

two times from water.

(3) 3-Azaindole (Benzimidazole): purchased from Aldrich was recrystallized

in water then vacuum sublimed slowly.

(4) 4-Azaindole: 4-azaindole oxalate salt was kindly supplied by Dr. Nachod
of Sterling-Winthrop Research Institute. The salt was
liberated by NaZCO3, then the free 4lazaindole was extracted
by ether, and was further purified by sublimation and re-
crystallization.

(5) S-Azaindole: kindly supplied by Dr. Nachod of Sterling-Winthrop Research
Institute, was vacuum sublimed slowly.

(6) 7-Azaindole: purchased from Aldrich was recrystallized from cyclohexane
three times.

(B) Purification of Solvents

(1) water: double distilled water was used.

4

Indole

3-Azaindole

/

Z.

—_—-——

\ N
H

S-Azaindole

\

\2/

I2

2-Azaindole

§

\
/

\\\\ N
H
4-Azaindole

/\ \

/

N
N H

7-Azaindole

Figure 1. Experimentally Studied Compounds

(2) Ethanol: 200 proof ethanol was fractionally distilled. When the UV
absorption spectrum in 10 cm cell did not show any benzene
absorption, the ethanol was considered pure. Ethanol was
kept refluxing and freshly distilled when needed.

(3) Diethyl Ether: Anhydrous ether was refluxed over Na ribbons, then

distilled in a 1 meter column.

(4) 3-Methylpentane (3MP): A modified method of Potta'was used. Phillips
Pure Grade 3MP was mixed with 50:50 sulfuric
acid and nitric acid, stir overnight. Repeat
by only using concentrated sulfuric acid until
reddish color is gone. Neutralization by
stirring with diluted Na2C03 for one hour was
followed by stirring with distilled water and
stored over CaClz. The solvent was then refluxed
over Na ribbon and distilled through 1 meter
column.

(C) Spectral Measurements

(1) Absorption Spectra: all UV absorption spectra were taken on a Cary

15 spectrophotometer with a resolution of about

1 A. A 10 cm absorption cell with quartz window
was used for vapor spectra. This modified cell
was wrapped with nichrome wire which was secured
with silicone cement. Vacuum stopcock was attached
to the only opening of the absorption cell to
provide a 10”6 Torr when connected to the vacuum
line. For solution absorption spectra, 1 cm path

length cells were used in all cases. A matched cell

(2) Emission Spectra:

were used as reference.
emission spectra were determined using an Aminco-
Keirs spectrophotophosphorimeter equipped with a
high pressure xenon arc lamp and IP21 phototube.
Some of the samples were degassed prior to their
use in luminescence studies. This was done by
freezing the sample with liquid nitrogen, evacuation
above the sample to a pressure of about 10"6 Torr,
allowing the sample to thaw, then refreezing and
continuing this freeze-thaw cycle until the vacuum
line ionization gauge did not quiver when the stop-

cock was opened after a freeze.

CHAPTER 3

VAPOR SPECTRA OF INDOLE AND AZAINDOLES

Vapor spectra of indole, 2-, 3-, 4-, 5-, and 7-azaindoles were mea-
sured. Our purpose is to study the effect of aza-substitution at differ-
ent positions on the energy and intensities of the low energy absorption
bands. These energy changes will be correlated with charge densities
changes as a result of excitation, the latter are calculated using Pari-
ser-Parr-Pople method. Moreover this correlation study is helpful in
identifying electronic transitions, particularly in cases where absorption
bands greatly overlap.

Introduction

 

Describing the molecule, with N atomic nuclei, in terms of a Born-
Oppenheimer state function which separates the electronic and nuclear
motion, one writes:

w(q.<2) = we(q.Q)wv(Q) (1)
with the vibrational state function WV, depending only on the nuclear
corrdinates, Q, whereas the electronic state function We, depends on
electronic coordinate, q, and the nuclear coordinates, Q, the indepen-
dence of the electronic and nuclear motions implies that the electronic
energy Ee and vibrational energy Ev will simply add to give the total
energy of a Vibronic state:

E = Ee + Ev (2)

Each of the 3N-6 vibrations in a polyatomic molecule may be described in

terms of displacements along normal coordinates of the molecule. The
vibrational energy states can be described approximately by a simple one-
dimensional harmonic oscillator expression that is used for diatomic
molecules, so the nth vibrational mode
Evn= (v + 35)hvn vn = O,1,2,...... (3)
th

v is the vibration quantum number for n‘

n vibrational mode, vn is its

fundamental frequency. If the vibrational modes are independent of each

others, the total energy of the molecule can be expressed as

3N—6
E = Ee + z (vn + $2)th (4)
n = 1
When a transition occurs between two Vibronic levels, the change in
energy 3N-6 3N-6
AB = Ee + Z (vm'+%)h‘h' - Z (vn"+%)hvn" (5)
m = l n = l
and in terms of wave numbers
__ 3N-6 3N-6
AE =v + Z Z (VmI-Jm' - vn'k‘fn" (6)
m = l n = 1
with vibrational frequencies in the ground (3") and excited (3”) electronic
states, and the symbol 360 being used to denote the transition between the
zero vibrational level of one electronic state and the zero level of the
other, i.e., when all the vibrational quantum numbers are zero.

The population distribution of each of the vibrational level is
characterized by Boltzmann factor e-E/kT. At room temperature kT= 210 cm-
and since the energy differences between vibrational states in organic
molecules vary from two to twenty times this energy, the majority of
molecules will be those for which vn" = 0. Therefore most of the absorp-

tion intensity will result from vn" = 0 molecules undergoing to the

various excited vibronic states characterized by vm' (Figure 2 and 3).

10

  
 

One Excited State

"‘I
V

l.
T

   

' vvvvv

AAAA-

Ground State

Energy

C!

l
T

 

 

 

Internuclear Distance

Figure 2. Ground and Excited State Potential Energy Curves
and Vibronic Transitions.

0l+0ll

Absorption

 

 

 

Wavenumber (cm-1)

Figure 3. Schematic Absorption Band in a Vibronic Spectrum.

11

There is a fairly strong absorption progression approximately equally

spaced, beginning with 0m' + O" and continue to higher energies vm' + 0"

which are separated by the energy of excited state fundamental 35'. The
concentration of other vibration level (v"> 0) increases exponentially
with temperature, the intensity of absorption bands resulting from
molecules intially in other vibrational levels will also increase, these
are called ”hot bands”. These bands characterized as vm' t vn” can be
easily identified by their temperature dependence. For indole and aza-
indoles with 3N-6 equals 42 and 39 respectively, therefore, one expects
to see 42 or 39 progressions of excited state frequencies. It is also
possible for combination frequencies to appear in which two vibrations
are simultaneously excited during the transition. Considering the fine‘
structure of each member in a progression, for instance the first member,
if having only one quantum of vibrational energy (vn" = 1, all others
vn" = O) undergoes a transition to the excited electronic state with no

vibrations excited (all vm' = 0), this first member will be constructed

with 42 or 39 fine structures. But from the transition probability:

P « {mfg-8'(q,Q‘)§I(Q)we"(q.<’2‘>dquow",m.(Q)w;;n..(Q) er}2 (7)

with M(Q) as transition moment operator, and Born-Oppenheimer wave func-
tion for ground (W”) and excited (W') states. The first integral, elec-
tronic transition moment integral, determines the transition probability
between electronic states We' and We". The second integral is called

the Franck—Condom overlap integral which determined the probability of
the transition occured between particular vibrations characterized by

vm' and vn". It is found diat the Franck-Condon factors for may of these

transitions vanish identically or are very small, so that not all possible

transitions between vibronic states will be observed. But for the

12

non-vanishing Franck—Condon integrals the magnitude determines the rela-
tive intensities of the vibronic bands which do appear. The assignment of
transitions in the absorption spectra of nitrogen heterocyclic molecules
is usually accomplished by comparing to spectra to those corresponding
aromatic hydrocarbons. The perturbation on the energy level of an aromatic
hydrocarbon caused by the substitution of a nitrogen atom into the ring

is usually very small. Spectra of various polyacenes are similar and have
been classified by Platt5’6, they contain a symmetry~forbidden transition
with long wavelength, low intensity, and allowed transition bands, with
higher energy. The long-wavelength band is a long-axis polarized 1Lb
transition in the Platt5 notation; slightly higher in energy is a short-
axis polarized lL transition. There are situations where the 1Lb and.

a

1La levels cross, e.g., naphthacene.

Substituting of an azanitrogen into the ring of an aromatic hydro-
carbon causes small shifts in the energy levels. These shifts may be to
longer or shorter wavelengths depending on the molecules, usually the
shifts are small enough that the order of the energy level remains un-
changed. This substitution destroys the symmetry property of the 1Lb
band, causes the transition to be less forbidden, in other words, sub-
stitution of nitrogen, intensifies the 1Lb bands. Also N* + n transition7
is introduced as a result of aza-substitution. An "* + n transition
arises from a non-bonding 0- hybridized orbital to an anti-bonding fl *
orbital. An “* + n transition is generally lower in intensity than

"* * " transitions, and usually appears at longer wavelength than the
lowest n* + n transition. But when the molecule size increase the

n*.+ n band of aza-aromatic molecule shift to the red faster than

n*‘+ n, in this case the n* + n transition is hidden under n* + n

13

bands. The magnitude of the shifts also depends on the direction of
polarization of a given transition and the position of the substitution.
The energy of an electronic transition is more greatly affected by substi-
tution along the axis of polarization, thus the 1Lb is more greatly affected
by substitution in the 8-positions, and 1La by substitution in the o -
positions.
Results and Discussions

Vapor spectrum of indole has been published by Hollas8. He assigned
the 1LbO-O transition at 35233.2 cm-l. The 1Lb region of the 3-azaindole
(benzimidazole) vapor spectrum has been published by Gordon and Yangg.
Indole, 2-, 3-, and 7-azaindole vapor Spectra have been taken earlier in
our lab by Richard Wagnerlo. These spectra were repeated in addition the
vapor spectra of 4-, and 5-azaindoles have been measured for the first time.
Our purpose here is to compare these spectra and to investigate the effect
of aza-substitution on energies and intensities of transitions. These
energy changes will be correlated with charge densities calculated by
using Pariser-Parr-Pople method.
Indole

Observation of the vapor spectrum (Figure 4) of indole indicates one
region by sharp vibrational structure centered at 284 nm, and the other
with diffuse vibrational structure centered at 260 nm. Using Platt no-

5 l

l l
tation , they correspond to 1Lb + A and L + A transition bands res-
a

pectively. Hollas did not study the lLa-+ 1A transition. Our observation

1 as the beginning of a sequence,

-1 .
another sequence starts at 278.2 nm or 720 cm above the first sequence.

shows the 1LbO-O at 283.85 nm or 35229.8 cm-

Using this 720 cm”1 as a fundamental and goes to the blue, one can observe

further sequences beginning at 272.2 nm and 267.3 nm. In addition one

.Esuuomam cowumuomn< uoam> mHowcH .d muswwm

A55 maozmumiz

 

l4

 

omN owN 0am 0am omm oqu omu cam
_ . _ . . . n . _
my
Ga
8
0
w
... _ I
fl : 1“ N
m_ a a I
a” a m
z a a w
5 _ 3 ,. I I
._ r .?4

w. «I f m

i

_

 

15

can observe two diffuse peaks at 253.8 nm and 259.8 nm with an energy
separation of about 900 cm'1 which presumablly belong to the 1La transi-
tion.

Azaindoles

 

Vapor spectrum of 2-azaindole shown in Figure 5 exhibits two elec-
tronic transition bands: one is characterized by sharp vibrational features
centered at 285 nm, the other with diffuse vibrational structure centered
at 245 nm. These two bands are quite well separated. The 1LbO-O transi-
tion is assigned at 290.3 nm or 34447.1 cm-l. Other vibrational sequences
start at 284.2 nm (35186.4 cm‘l), 279.2 nm (35816 cm'l) and 273.7 nm
(36536.3 cm-l) with an average separation of 700 cm-1. The diffuse band
shows two peaks at 252.2 nm (39651 cmfl) and 245.2 nm (40783 cm-l) with
1100 cm"1 separation.

All other azaindoles, 3-, 4-, 5-, and 7-, are analysed in the same
way, and both their vapor absorption spectra (Figure 5-9) and the corres-
ponding peaks (Table 1) from experimental data as well as Pariser-Parr-

10 are shown in Table 2. From Wagner's10 calculation,

Pople calculations
charge density in the 1La and lLb states are significantly different in
indole (Figure 10). Considering the electronegativity, nitrogen is higher
than carbon. Aza-substitutions of those places whose charge density in-
crease show red shifts in the transition band. The charge density for

the 2-, 4-, 5-, and 7eazaindoles increased in the lLb state, the corres-
ponding transitions are red shifted. This is also true for the 1La state
for 4-, and 7-azaindoles. Also there is a charge density decrease at

both 1

L and 1Lb state of 3 position of indole, the vapor spectrum shows

a

blue shifted 1La and 1Lb bands. The charge density interpretation of
vapor spectra agree qualitatively with the data. For further quantitative

analysis, charge density becomes an unsatisfactory approximation.

l6

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Figure 10.

10a.

10b.

23

Calculated Charge Densities for Indole

Numbers at each atomic position denote n -electron
charge densities in electron units for ground state
(top number), 1La state (middle number). and 1Lb
state (bottom number).

Numbers at each atomic position denote n -electron
charge density differences from ground state (in
electron units) for 1L state (top number), and
state (bottom number) aith plus sign indicating an
increase in electron density and minus sign indi-
cating a decrease in electron density.

1.046
0.962
1.093

1.012
1.175
0.970

-0.084
+0.047

+0.163
-0.042

 

 

0.988
1.133 1.194
1.156 1.003
1.135
1.096
1.175
1.101
1.118 a
1.264 1.480
1.232 1.210
1.382
Figure 10a
+0.145 -0.l91
+0.168 -O. 059
+0.079
+0.005

 

 

/\
V\N
+0 146 H

+0.114 -0.270
-0.098

Figure 10b

CHAPTER 4
ABSORPTION SPECTRAL SHIFTS

-CORRELATION WITH EXCITED STATES CHARGE DENSITIES

Introduction

 

Vapor phase absorption spectra under reduced pressures represent
spectra of essentially isolated molecules; high-resolution spectra show
vibrational and rotational structures. The disappearance of rotational
structure in vapor spectra due to high pressure and pressure broadening
are manifestations of intermolecular interactions. In solution, the
solute molecules are in contact or solvated by solvent molecules, this
eliminates all rotational structures, and may cause blurring of vibration
structure. At liquid nitrogen temperature (77°K), vibrational structure
may become well resolved again.

Shifts due to solvent effects are often the result of several in-
dividual effects which may reinforce or cancel one another. They arise
due to differences in interactions with the medium in the different elec-
tronic states. Interpretation should be related to the 0-0 band of an
absorption or fluorescence spectrum; but it is often difficult to locate
to 0~0 band in solution spectra. Therefore, shifts are usually referred
to the maxima, which are not exactly affected the same way as 0-0 band.

Bayliss and McRae11

pointed out that most solvent effects can be
explained in terms of four main factors: (1) momentary transition dipole

during optical absorption process (2) difference in the permanent dipole

25

26

moment between the ground and excited states of the solute (3) of Franck-
Condon effects and (4) the dipole moment of the solvent. Solvent effects
depend on various intermolecular interactions, such as dispersion forces,
dipole-induced dipole, dipole-dipole, and hydrogen bonding. Dispersion
forces are always operative in all solutions, whenever the solute and
solvent are polar or not. This effect occurs when the transition dipole
of the solute induces a momentary polarization in the solvent and is thus
present in all solutions. Dipole-induced dipole and dipole-dipole inter-
actions produce shifts in absorption spectra which may be to higher or
lower energies. For non-polar solutes in non-polar or polar solvents,
only dispersion forces are operative, which produced a moderate polari-
zation red shift12’13. For polar solutes in nonjpolar solvents, dipole?
induced dipole interactions take place, which produced either a red or

a blue shift, depending on whether the excited state dipole moment of

the solute increases or decreases. For_polar solutes in polar solvents,
dipole-dipole interactions can also cause a red or blue shift depending
on whether the excited state dipole moment increases or decreases. In
this case dipole-dipole interaction is the dominant cause for spectral
shifts. -

Franck-Condon principle plays a major role in solvent effects on both
absorption and emission spectra. A solute molecule is surrounded by solvent
molecules in equilibrium in solution. This equilibrium of ground state
depends on (1) packing factor, which depend on geometry of the solvent
and solute and (2) orientation factor, which depends on the mutual orien-
tation interaction if the solute and solvent are polar or can form hydrogen

bonding. The geometry, charge density and dipole moment of solute my be

different in the excited state; therefore, the equilibrium configuration

27

of the solvent cage will also be different in the excited state. However,
according to the Franck-Condom principle an optical transition occurs in
a time (IO-lssec.) that is short compared with the period of nuclear
motions. Therefore, the solvent configuration around the excited solute
molecule, after this vertical transition, does not correspond to the
equilibrium excited configuration, but to a conformation geometrically
identical of the solvated ground state, i.e., a Franck-Condon state
configuration. The orientation energy of this configuration is higher
than that of the excited state equilibrium configuration, which can be
reached by solvent relaxation of the system. The time required for geo-
metrical rearrangement of solute is around 10’13sec. and solvent reorien-

tation around 10'llsec. Since the lifetime of an excited singlet state

is of the order of 10-9

sec., there is plenty of time for excited state
equilibrium to be reached before deactivation occurs if the solvent is

not viscous. Also the ground state configuration after fluorescence is
not the equilibrium ground state configuration but a state of strain

whose energy is higher thantfluu:of the ground state euqilibrium configu- .
ration (Figure 11).

The non-equilibrium configuration experiences two types of solvent

strain upon solute moleculesll. (l) packing strain and (2) orientation

strain14. Packing strain results from an actual change in geometric
size of the molecule in the excited and ground states. Generally the
percentage change of the size of organic molecules is small and this
strain can be neglected except in specific instances. The orientation
strain results from the non-equilibrium orientation of the solvent cage

around the solute molecule in its excited state and includes dipole-

polarization and dipole-dipole interactions. Orientation strain is

28

Unstable (Franck-Condon)
Excited State
Configuration

Q 0 Stable Excited State

C:;::::::%:) .\\ Configuration
“00

ll ‘*

QCZDQ
0 O

hV

OO
OCZDO
Q 0 /’00

hv

 

/

I
(::::::) Unstable Ground State
0 Q Conf igurat ion

Stable Ground State
Configuration

Figure 11. Illustrating Change of Solvation After Excitation or Emission
The large and small ovals representing solute and solvating
solvent molecules are purely diagrammatic and are intended
to represent a higher degree of solvation in the stable
configuration of the excited state.

29
particularly important when solute and solvent are both polar and when
the permanent dipole moment changes upon excitation. Modification of
the strength of any hydrogen bonding would also give importance to this
type of strain. If the dipole moment decreases in the excited state, the
absorption undergoes a blue shift due to orientation strain, if the dipole
moment increases in the excited state, this will cause a red shift. The
fluorescence spectrum undergoes a red shift in both cases (Figure 12).
Several studies have been made of the effect of hydrogen bonding on solvent
shifts, that was first discussed by Kasha7. He pointed out that absorption
bands corresponding the n * + n transition blue shift in hydrogen bonding
media. Later Brealey and Kasha15 demonstrated that hydrogen bonding is
the main influence in the n *‘+ n blue shift phenomenon in hydroxylic
solvents. However, Pimentel16 pointed out the dipole-induced dipole and
dipole-dipole interaction produce small solvent shifts compared with those
to hydrogen bonding. Pimentel discussed the influence of hydrogen bonding
formation on electronic transition in terms of the Franck-Condom principle.
In the case when hydrogen bonding is stronger in the ground state than the
excited state (Figure 13a), the hydrogen bonding energy Wé<Wé, and the
excitation energy implied by Franck-Condon principle-is labeled w. But
in the case when the hydrogen bonding is weaker in the ground state (Figure
13b), then We >Wg.

Solvent shifts due to hydrogen bonding can be formalized as follows:

va-vo=Ava=wg-we+we (8)
.- = A = .. .-
vf v0 vf wg we wg (9)
When hydrogen bonding is stronger in the ground state Wé< Wé, so Ava> 0,

i.e., a blue-shift which exceeds W8 - Wé by we will be observed in absorp-

tion. Similarly the shift observed in emission, will be less than WE ~ Wé

30

Franck-Condon

Excited State
Vapor ,F‘ir‘—\ Equilibrium
.‘ Excited State ,’ \\ Excited State

\ I \
F—u—

 

I

 

 

}‘ ku—

 

 

;_;4___
\\ I Franck-Condom
\L_, _-_mxa._ I Ground State
u___l

\
*—

 

\

 

 

 

 

Ground State Equilibrium

Ground State

(non-polar solvent) (polar solvent)

 

 

(i)
Vapor
-_-_—?r__\\ Excited State Franck-Condom
\ .
\_._........ ~~ Exalted State
I“ ‘ “‘~‘ Equilibrium
‘ Excited State
——‘—\_._L_-- - x
‘T‘ ’ Franck-Condom
Ground State Equilibrium Ground State
Ground State
(non-polar solvent) (polar solvent)

(ii)

Figure 12. Demonstration of solvent strain effects on energy levels of
a polar solute in a polar solvent compared with a non-polar
solvent and vapor. (i) Solute dipole moment decreases in the
excited state; (ii) solute dipole moment increases in the
excited state.-

Figure

13.

13a.

13b.

Solvent Shifts due to Hydrogen Bonding.

.1

Hydrogen Bonding Is Stronger in the Ground State

Hydrogen Bonding Is Stronger in the Excited State

32

  

A "'"""""""""""’ ""'""""""T-

yv.

e1

3!

_I

 

H/J

 

WI

 

 

J—I———-—

 

 

 

 

summzm

._wg

9
t"

 

 

 

W
.....___...._..:L._._ ....

-..i

‘J

W

 

 

 

 

V

 

 

 

 

 

RLA B) y

33

by W8 and will be either a red or a blue shift depending on the specific
case. When hydrogen bonding is weaker in the ground state Wg< Wé, both
emission and absorption spectra will show a red shift. The well charac-
terized hydrogen bonds have energies in the range of 1-7 kcal/mole (350-
2500 cm-l). According to the above discussion, a blue shift in absorption
may exceed the ground state hydrogen bonding energy, hence, the expected

blue shift occurs in the range of 350-2500 cm-1 or larger than 2500 cm

But a red shift in absorption should never exceeds Wé, i.e., should not be
larger than 2500 cm'l. For n * + n transition in hydrogen bonding media,
a blue shift in absorption is usually observed. This is due to the de-
crease in charge density around the lone pair atom as a result of lone
pair promotion. Thus, the hydrogen bond is always stronger in the ground
state.

So a red shift in absorption spectrum indicated that the hydrogen
bonding is stronger in the excited state16. This may indicate an increase
in acidity or basisity depending on the functional group of the chromo-
phore involved in hydrogen bonding.

As discussed above, when both solvent and solute are polar, electronic
excitation to the Franck-Condon state is followed by a relaxation process,
such that the solvent molecules rearrange themselves to the excited-equi-
librium state. This kind of relaxation happens very fast at room tempera-
ture. But at very low temperature, the solvent forms rigid glass, and the
solvent molecules are inhibited to relaxation process before emission occurs.
In this case fluorescence occurs from a non-equilibrium Franck-Condon state
or an intermediate state. Since Franck—Condon state is always higher in
energy than the equilibrium excited state, fluorescence at liquid nitrogen

temperature is blue shifted with respect to that in solution at room

temperature.

34

Results and Discussions

The absorption spectra of indole and azaindoles were run in different
media (3-methylpentane, diethyl ether, ethanol, water and dichloromethane).
The purpose is to interpret spectral shifts in terms of the various possible
interactions. These include: (1) dipole-dipole effects which reflect
changes in the permanent dipole moment as a result of excitation (2) po-
larization shifts (3) hydrogen bonding (a) hydrogen bonding involving the
pyrrolic hydrogen with the oxygen of ether, alcohol or water (b) hydrogen
bonding involving the aza-nitrogen with the proton of alcohol or water (c)
hydrogen bonding involving the pyrrolic nitrogen with the proton of alcohol
or water. We analyzed the data by using 3-methylpentane (3MP) as a refer-
ence and the results are now discussed for each individual case.
Indole

Spectral shifts of indole in different media are shown in Table 3.
In spite of the red shift observed in ether and ethanol solutions, a blue

shift is observed in water. In hydrocarbon the shifts observed relative

 

to vapor are attributed to general polarization red shifts as well as
dipole-induced dipole interaction. The dipole moment of indole in the
ground state is 2.3D and in the excited state 7.3D. Both types of inter-
actions (i.e., dispersion and solute dipole-induced dipole interaction)
should cause a red shift (Figure 12) compared with vapor spectra (Table 2).
Our results show a red shift of '510 cm-1 and r897 cm'1 for 1Lb and 1La
respectively.

In gghgg, besides the dispersion and dipole-induced dipole interaction,
dipole-dipole interaction is taking place, because ether has a dipole moment

17

of 1.3D In addition spectral shifts due to hydrogen bonding interaction

between the pyrrolic hydrogen and ether lone pair oxygen must be taken into

35

mmm+

«mo:

Now:

New:

omen

quwm

emmmm

oomom

Nmmom

waom

monm

Au-a.yw

memH

w.mmN
com
Hum
m.HmN
m.HmN
m.m©N

Agave

.chmz ucwuwmmHn 5H oHowcH sow mumHLm Hmnuowam coHumuomn¢

Hom+

HNH:
HNH-
Ho u

NNH+

A soypq
HI

A

mmmmm
mqwqm
Nqum
mmmqm
anqm

women

58y»

Ono H

m.mwN
an
wwm
wwN
m.mwN
owm

Asses

Homm>

92m
NHono
omum
moum

owe

ESvaE

.m mHan

36

consideration. These effects should cause a further red shift if the
pyrrolic hydrogen is more acidic in the excited state and because the
excited state dipole moment is larger than the ground state dipole moment.
Indeed, an additional red shift of ~121 cm-1 and -762 cm-1 for 1Lb and 1La
respectively, are observed due to change from 3MP to ether. In ethanol
gpd water, an additional hydrogen bond can be formed involving the n -
electron on the pyrrolic nitrogen and the lone pair oxygen of the solvent
(Figure 14). If the nitrogen is less basic in the excited state, a blue
shift will be expected. In alcohol the red shift is less than the observed
in ether. Moreover a blue shift is observed in water. These results could
only be interpreted in terms of a hydrogen bond involving the pyrrolic
nitrogen which becomes weaker in the excited state. This indicated that-
the charge density on that nitrogen decreases upon excitation to 1Lb or
1L states.
3

Also for quantitative study of each of the shifts, absorption spectrum

of indole in dichloromethane was taken (Table 3). Dichloromethane has a
17

dipole moment of 1.5D but can not form hydrogen bonds with indole.

Spectral shifts in going from 3MP to CH2C12 can be useful in obtaining

shifts that are solely due to dipole-dipole interaction Avd d° Using
Onsager's19 formula
Avd-d = Zuo( 111 - u0){(n2'1)/2n2+l) - (D-1)/(2D+1)}/a3hc (10)

where “o and U1 represent the dipole moment of the solute molecule in the
ground and excited state, a is an effective cavity radius appropriate to
the solvent, D is the dielectric constant of the solvent, n is the re-
fractive index of solvent, one can therefore estimate shifts due to this
effect in other polar solvent. The red shift due to dipole-dipole inter-

- 1 ' .
action in ether calculated by equation 10 is ~92 cm 1 for Lb band, which

37

A

0H
«4
"—4

.umuma Haaav Hoameum_aaav “meow assumes Hay

mucm>Hom mam oHowcH awosumm

Anne

vflmwwu

 

mwGHwaom cwwonwhm

um um

/

2 1...... :Q:\

- /

.aa magmas

38

indicates that a red shift of about -30cm-1 is due to hydrogen bonding
effects for lLb band. Similarly for the 1La band, dipole-dipole red
shift is -530 cm”1 and the hydrogen bonding red shift is ~232 cm-l.
From IR datal8, one may calculate the relative strengths of hydrogen
bonding involving the oxygen lone pair of ethanol and water. Assuming the
spectral shifts due to hydrogen bonding are proportional to the relative
strength of hydrogen bonding in the ground state, one may estimate spectral
shifts due to hydrogen bonding €::N-H°---O) in ethanol and water (Table 4).
Charge density calculations using Pariser—Parr—Pople method was per~
formed10 to test these findings. The charge densities of indole in the
ground, 1Lb and 1La states are shown in Figure 10a, and the charge density
difference is shown in Figure 10b with plus sign indicating an increase in
electron density and minus sign indicating a decrease in electron density.
From Figure 10b, we noticed that charge density decreases at the pyrrolic
nitrogen such decrease is more pronounced in the 1La state compared to
1Lb state. This implies an increase in the acidity of the pyrrolic hydrogen
as a result of excitation to 1Lb and particularly to 1La state which will
give rise to a red shift of the 1Lb and 1La bands. The magnitude of that
shifts will be greater for 1La band. The decrease in charge density at the
pyrrolic nitrogen means that hydrogen bonding of the solvent proton with
the pyrrolic nitrogen n ~electron charge density is stronger in the ground
state. This should cause a blue shift, specially the 1La band. Since the
acidity of water is about 100 times stronger than ethanol, we expect a much
larger blue shift in water than ethanol due to this hydrogen bonding inter-
action. This explains the smaller magnitude of the red shift in ethanol
and the change in sign of the shift (blue) observed in water.

Solvent effects on the absorption spectrum of indole lead to the

39

oom+

owHu

mooHu

NNm+

07H:

0 m

mmm+

mom-

NHmn

HNH+

mmHu

mOum

NMN:

0mm:

0 um

, .oHowGH How mumHnm Hmuuownm aoHumuome< wo>kmmno
meu ou chHuomuoucH wcchom cowonw%m cam COHuomuowaH-mHomHnumHomHa mo meHuanHucoo

9......3A

maoaae-maoaae

a5.5V;

Oooooooom-IZ\

/
afloaae-maouae

mGOHuomuoucH

mumum

.8 magma

40

following conclusions: (1) The permanent dipole moment of the ground (1A),
1Lb and 1La state follows the order 1La >1Lb> 1A. (2) The charge density
at the pyrrolic nitrogen as calculated by Pariser-Parr-Pople method
decreases in the excited states, which cause blue shifts due to hydrogen
bonding involving the proton of the solvent particularly water, this is
especially true for 1La state. The calculated charge density change
(Figure 10b) for the 1La and 1Lb states has a ratio of 2.721, and the

blue shifts of 1La and lLb bands that are attributed to hydrogen bonding
with the pyrrolic n ~electron (H-N°°'°H) have a ratio of 2.8:1 in ethanol
and 2.3:1 in water. This is a good agreement and is not surprising inview
of the fact that the energy of the hydrogen bond involving the pyrrolic
nitrogen reflects directly the n-charge density at that site. (3) The.
decrease in charge density at the pyrrolic nitrogen due to excitation
causes the pyrrolic hydrogen to be more acidic in the excited states par-
ticularly the 1L3. This causes red shifts but quantitative correlation

is not expected since the acidity of the hydrogen does not reflect quanti-
tative changes in charge density at the pyrrolic nitrogen.

Azaindoles

 

Absorption spectra of 2-, 3-, 4-, 5-, and 7-azaindoles have been taken
in water, ethanol, ether, dichloromethane and 3-methylpentane. Spectral
shift data are presented in Table 5 and are interpreted in a similar way
as indole but also taking into consideration the fact the azanitrogen
forms hydrogen bonds with protic solvents (Table 6).

Spectral red shifts in hydrocarbon with respect to the vapor reflect
an increase in the excited state permanent dipole moment particularly
in the 1L state. After attributing spectral shifts to the various inter-

a

actions (Table 6), we conclude that in the case of 2-azaindole there is a

41

mmHH+

HNH-
HNH-
Hem:
Hem:
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sea.
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42

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0mm:

wmm+
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mHN+
mo 1
ommu

H8 +
m8 -
mom-

mOum

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mom:

mm u
mum:

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msu cu mcoHuomuwucH wchcom cowouw%m wow aOHuomuoucH oHomHQnoHoaHQ mo wGOHuDBHuucoo .o mHan

possibility that the charge density on the azanitrogen increases, leading
1 \ v

to a stronger hydrogen bond of the typeéyNzoo-H in the excited state.

(The Pariser-Parerople calculation for 2-azaindole is not available

because of the adjacent nitrogens). This causes a red shift and will

therefore reduce the blue shift associated with hydrogen bonding with

the pyrrolic nitrogen (HuN----H). For 3—azaindole, the hydrogen bonding

 

effect caused quite a blue shift which indicated that the n ~electron
charge density for both pyridinic and pyrrolic nitrogen decreassed in the
excited state. This is consistent with the charge density calculated by
Pariser—Parr—Pople method (Figure 15). The reason for smaller dipole-
dipole interaction in the case of 3-azaindole compared to other azaindoles

reflects the smaller changes in the permanent dipole moment upon excita-

 

tion. In 4-azaindole only lLa state is available because the 1LB band is
completely submerged. Hydrogen bonding effect involving the pyrrolic
nitrogen is smaller compared with indole. This can be explained as an
increase in shifts due to hydrogen bonding with the azanitrogen which
reduces the blue shift caused by interaction with the pyrrolic nitrogen.
This is also consistent with charge density calculation (Figure 16). Same
arguement has been applied to 5:, and 7-azaindole spectral shifts which
are also consistent with charge density calculation (Figure 17 and 18).
In the case of 7-azaindole, hydrogen bonding is fully dominated by the spec-
tral red shift due to hydrogen bonding involving the azanitrogen.

Solvent effects on the absorption spectra of azaindoles lead to the
following conclusions: (1) The 1La and 1Lb state permanent dipole moment
are larger than the ground state permanent dipole moment, with 1La larger

than the lLb state. (2) The charge density at the pyrrolic nitrogen

decreases in the excited state which gives rise to a blue shift in’hydrogen

Figure 15.

15a.

15b.

Calculated Charge Densities for 3-Azaindole.

Numbers at each atomic position denote n -electron
charge densities in electron units for ground state
(top number), 1Lb state (middle number), and L

a
state (bottom number).

Numbers at each atomic position denote n -electron
charge density defferences from ground state (in
electron units) for 1Lb (top number), and 1La state
(bottom number) with plus sign indicating an increase
in electron density and minus sign indicating a de-
crease in electron density.

H‘C>H‘

H

.047
.932
.113

.014
.140
.005

 

-0.115

"00 0861

 

 

+0.12
-0.009

45

0.988
1.098 1.248
1.176 1.181

1.090

N

 

1.062
1.079
1.134
1.121 1.472
1.223 1.351
1.278 1.233
Figure 15a
+0.1oo
+0.188 -0.067
-0.158
no
‘+o.017
+0.072

 

\ N
+0 102 “:3

+0.157 -0.121
-00239

Figure 15b

Figure 16.

16a.

16b.

46

Calculated Charge Densities for 4-Azaindole.

Numbers at each atomic position denote n -electron
charge densities in electron units for ground state
(top number), lLb state (middle number), and 1La
state (bottom number).

Numbers at each atomic position denote n -electron
charge density differences from ground state (in
electron units) for lLb state (top number), and L
state (bottom number) with plus sign indicating an3
increase in electron density and minus sign indicating
a decrease in electron density.

1.004
0.952
1.038

1.015
1.160
0.976

-0.052
+0.034

+0.145
-0.039

47

1.063
1.228 1.189
1.230 0.987

1.121

/ n\\/_

 

 

 

1.094
1.175
1.098
lh§---

1.105 1.481

1.281 1.188

1.249 1.384

Figure 16a

+0.165

+0.167 -0.202
+0.081
+0.004

 

 

\N
+0. 176 EN:

+0.144 -0.293
-0.097

Figure 16b

Figure 17.

17a.

17b.

Calculated Charge Densities for 5-Azaindole.

Numbers at each atomic position denote n -electron
charge densities in electron units for ground state
(top number), 1 state (middle number), and La
state (bottom number).

Numbers at each atomic position denote n -electron
charge density differences from ground state (in
electron units) for state (top number), and L
state (bottom number) with plus sign indicating an8
increase in electron density and minus sign indicating
a decrease in electron density.

49

 

 

0.948
1.119 1.193
1.106 0.975
’ 1.166
1.111
1.057 a
1.156 U
1.095
1.185
1.083
0.993
1.141
0.944 ~\\\\‘§ *~‘-~‘.D\g
1.120 c
1.273 1.478
1.199 1.163
1.419
Figure 17a
+0.17l
+0.158 -0.218
-0.027
-0.054 ,,r”””’§‘fi‘\“\\“
+0.039 pg
+0.09O
-0.012

 

 

+0.148
-0.049 V\ N
H

+0.153
+0.079 -0.315

Figure 17b

Figure 18.

18a.

18b.

50

Calculated Charge Densities for 7-Azaindole.

Numbers at each atomic position denote n -e1ectron
charge densities in electron units for ground state
(top number), 1Lb state (middle number), and La
state (bottom number).

Numbers at each atomic position denote n -e1ectron
charge density differences from ground state (in
electron units) for 1 state (top number), and La
state (bottom number) with plus sign indicating an
increase in electron density and minus sign indicating
a decrease in electron density.

 

51

 

 

0.978
1.180 1.198
1.150 0.951
1.186
1.050
1.022
1.036
1.095
1.192
1.077
0.973
1.128.
0°952 \ /\N
1.187 1.473
1.356 1.105
1.293 1.440
Figure 18a
+0.202
+0.172 -0.247
-0.012
-0.028
-0.014
.+0.097
-00018
+0.155

 

 

N/ N

H

+0.169 -0.368
+0.106 -0.033

Figure 18b

52

bonding solvents. (3) The pyrrolic hydrogen is more acidic in the excited
state than the ground state, this causes a red shift. (4) The charge den-
sity at the pyridinic nitrogen decreased in 3-azaindole but increase in
4-, 5-, and 7-azaindoles, which causes a blue and red shift respectively.

The shift due to this effect may be large enough to govern the observed

spectral shift.

CHAPTER 5

EMISSION SPECTRA OF INDOLE AND AZAINDOLES

Introduction

 

Our interest here is to study the solvent effect on emission spectra
and to obtain values for excited state dipole moment and excited state
pK's for indole and azaindoles. The emission characteristics of a mole-
cule in different solvents is governed by (1) the influence of the di-
electric properties of the solvent on the excited state of the solute
(2) hydrogen bonding between solvent and solute in the excited state
(3) solvent relaxation during the lifetime of the solute's excited
state.

Idealy, it would be expected that the 0-0 absorption and emission
bands would coincide since the energy change is identical. But this is
not usually the case. If the solvent and solute are both nonpolar, or
one is polar, the frequency shifts in 0-0 absorption‘and emission bands
are predicted to be the same or nearly the same. Therefore the 0-0 bands
should be coincide or nearly so (Figure 12). In these cases, dispersion
and dipole induced dipole terms are the principal contributing factors to
solvent-solute interaction and the orientation strain is negligible leading
to no or very little difference in energy between the Franck-Condom state
and the equilibrium state. Thus the state that is reached by absorption
and that from which emission originates are nearly the same°

When the solute and solvent are both polar,and there is negligible

53

change in the dipole moment as a result of excitation, the frequency

shift for fluorescence is the same as that for absorption, again the 0-0
bands in the absorption and emission processes should be the same or nearly
so.

If the dipole moment of the solute changes (in magnitude and/or
direction) upon excitation and the solvent is polar, reorientation may
occur before emission. However, if the solvent is rigid, relaxation times
are several order magnitude larger than the excited state lifetime, so
emission occurs before solvent rearrangement takes place. If however
the polar solvent is fluid, relaxation is much more rapid emission may
occur from the equilibrium excited state where dipole reorientation is
completed. Therefore, absorption will occur to the metastable Franck-
Condon state ( ”ng), emission will occur from the equilibrium state
( Pemiss)' The quantitative expression for the A; in absorption is dif-
ferent from that in emission and the 0-0 bands will not coincide. The
difference (Stokes shift) is:

-— _ 2 2 2 3

Ava—f =Vabs 'vemiss - 2(ue -ug) {(D-1)/(D+2) - (n -l)/(n +2)}/a hc (11)
+ 2{ (ae “ag>(3ug2‘5u.2+2ugu.>}{ (D-1)/(D+2) - (n2-1)/(n2+2>}2/a6hc

where pg and “e represent the dipole moment of the solute molecule in the

ground and excited state, a is an effective cavity radius appropriate to

the solvent, D is the dielectric constant of the solvent, n is the refrac-

tive index of solvent, and ag andue are the polarizabilities of the

solute molecule in the ground and excited states.

The second term originates from the dipole-induced dipole interaction,

in many cases can be consider as a second order interaction term and makes

a negligible contribution to the shift, and the equation is simplified as

55

follows:

Asa-f = vabs ' ”emiss = 2(ue -ug)2{(D-1)/(D+2) - (Hz-1)/(n2+2)}/a3hc (12)
The dipole moment difference in ground and excited state can be obtained
directly from the Stokes shift. An estimate of the excited state dipole
can be made from experimental absorption and emission shift data and the
known ground state dipole moment using equation 12.

Another interesting consideration is the effect of electronic excita~
tion on the pKa of certain compounds in the excited state compared with the
ground state. This is of special interest to us in relation to the pheno-

20’21’22 has dis-

menon of proton-transfer in the excited state. Weller
cussed methods of determing the excited singlet-state dissociation constant,
pK*, from spectroscopic data. The determination of ApK = (pK - pK*) of

this system is based on the energy diagram shown in Figure 19 and the ther-

modynamic quantities from the equilibria:

X-H-------Y : x". ..... .H+-Y (13)
,‘ ...J‘ +
X—H‘A.oooooY : X "coco-OH "Y (14)

From the diagram in Figure 19, we get:

AE+E*=AE'+Ed (15)

d
where AE and AE' are the energy changes for the transition from the
ground electronic state to the lowest-excited singlet state of the proton
donor and acceptor, respectively. The dissociation energies in the

ground and excited states, Ed and Ed*, can be written

Ed .. Ed* -_- (AG - TAS) - (110* - T115") (16)

Assuming AS = AS*, then

AG - AG* -RT(1nK - 1nK*) = E - E *

d d
AE - AE' (17)

56

 

 

 

 

 

 

 

X-koooooo OH+"Y
X H‘" Y E “V M
- . dl
\ .
1 AE' = hv'
AE = hv
II
— +
l’Ed X ' "H -Y
X-Ho o o o oY

Figure 19. Schematic Thermochemical Diagram for Determination of
Excited-State pKa Values.

57

Since logK = -pK,
pK - pK* = (AE - AE')/2.303RT (18)
where the energy changes AE and aE' can be estimated from the frequencies
measured at the maxima of absorption and fluorescence by means of
AE = hu = (huA + th)/2 (19)
AE' = hu'= (th' + th')/2 (20)

Results and Discussions

 

 

Solvent Effects on_Luminescence Spectra

Emission spectra of indole and 2-, 3-, 4-, 5-, and 7-azaindoles were
determined at room temperature and at 77°K in dilute solution of 3MP, ether,
ethanol, water and acidic and basic media (Table 7 and Figures 20-21). A
considerable red shift in room temperature fluorescence spectra is observed
in all compounds when the medium is changed from non-polar to polar solvent.
Such red shift is due to solvent relaxation during the excited state life-
time from the metastable Franck-Condom state to an equilibrium excited
state before fluorescence occurs. The low temperature fluorescence spectra
establishes clearly that the emission do not arise from the equilibrium
state but from essentially the Franck-Condon state, the shifts shown, cor-
respond to those observed in the absorption spectra. “The low temperature
phosphorescence data and lifetimes are also shown in Table 7. The charge
distributions and pK's of the triplet states are not expected to changed
dramatically as in the first excited singlet state, we expect therefore
that the triplet states to have properties closer to those of the ground
state. The energies of the phosphorescence peaks in different solvents
are close to each other, but the phosphorescence lifetimes are usually
longer in the polar solvents. In Table 7, we notice that for 7-aza-

indole in 3MP and ethanol, two luminescence bands are observed at

58

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62

room temperature. Both luminescence bands are fluorescence bands, the
long wavelength fluorescence (F2) is absent in both ether and water.
The broad, structureless fluorescence F2 band arises from a tautomer
which is formed due to excited state double proton transfer that occurs
in the 7-azaindole hydrogen bonded dimer (in 3MP) and the 7-azaindole-
alcohol complex (in ethanol).
Excited State Dipole Moment Studies
The methods for the determination of excited state dipole moments
can be combined into four groups: (1) The method of spectral shiftslB’M’lg’23
based on the study of the displacements of the frequencies of electronic
transitions and such shifts are Obtained from the analysis of the absorption
and luminescence spectra in different media. (2) The method of electrical

polarization of fluorescence24’25.

Here the degree of polarization of the
emission by solutions of the test substances in a powerful electric field
is measured, i.e., ultimately the anisotropy of the emission in the di-
rection of the field orienting the molecules is investigated. (3) The method
of electrical dichroism26’27. Here the dichroism of the absorption by
molecules in a solution placed in a powerful electric field is studied, i.e.,
the anisotropy of absorption in the direction of the orienting field is
considered. (4) The method based on the investigation of the splitting
of the fine rotational structure of the absorption bands of certain mole-
cules in the gas phase in a powerful electric field (the Stark effect,
which includes not only the excited electronic states but also the ground
state)28.

The method I used here is the spectral shifts method. The method is

23

based on the application of McRae13 and Ooshika formula to determine the

difference between shifts of absorption and fluorescence bands under the

63

conditions such that the orientation relaxation time of the solvent mole-
cules is much less than the lifetime of excited fluorescence state. The
following simple expression is then obtained:

._ 2 2 2 3

Ava-f = 2(pe - Hg) {(D-l)/(D+2) - (n -1)/(n +2)}/a hc (21)
as the first term of equation 11.

The solvent I used here for this study is a polar but non-hydrogen
bonding solvent CH2C12’ also assuming the cavity radius a in Onsager's19
theory of the reaction field to be 3 A for indole29 and azaindoles. No
hypotheses concerning the relative orientation of the moments ug and “e
but only the |Au|are considered. The Stokes shifts and the calculated
[Ap| are given in Table 8. The|Au|for indole and most of the azaindoles
are in the range of 5-6D, except 5-azaindole which showed a value of 9.1D
and 2~azaindole showed a value of 4.5D. Aza-substitution along the long

axis seems to perturb the system most.

Excited Stateng Studies

 

As we already pointed out in the introduction, Weller has shown that
electronic excitation may change drastically the acid-base properties of
a molecule.

If equilibrium is established during the excitedvstate lifetime, then
the pKa* and pr* can be determined in a way analogous to the spectrophoto-
metric determination of the ground state pK's by fluorometric titration.
For this reason we have studied the fluorescence intensity as a function
of pH (corrections were made for changes in the absorption spectra) for
indole and azaindoles. The results of indole and 7-azaindole are now
discussed, the data for other azaindoles are summarized in Table 9.

Indole

As we see, at both low and high pH, indole fluorescence is quenched.

64

Table 8. Lowest Singlet Excited State Dipole Moments of Indole and
Azaindoles.

Compound Stoke Shift (cm-1) A((D) ug(D) ue(D)
Indole 3666 5.0 2.3* 7.3
2-Azaindole 3109 4.5 1.8* 6.3
3-Azaindole 4531 5.5 4.0* 9.5
4-Azaindole 3944 5.2

5-Azaindole 12048 9.1

7-Azaindole 5924 6.4 3.6** 10.0

* Reference 17

** Reference 10

65

In the high pH region the quenching is ascribed to the ionization of the

pyrrolic hydrogen according to the reaction

\ _ ......
+ OH <-—-—-—- O + H20
N N1

H
From the midpoint of the titration curve (Figure 22), one get a pr*=12.3.
Exactly the same number was obtained by Donckt30. The excited state
pr* = 12.3 should be compared to the ground state pr = 16.9731, which
shows that indeed the pyrrolic hydrogen acidity has considerably increased
in the excited state.
In the low pH region the quenching is considered to be due to proto-

nation of the pyrrolic nitrogen according to the reaction

' \ + + —c \
H30 (""— +H20
N 2+
H l \H
In the same way one gets a pKa* = 1.8, in very good agreement with the

value pK * = 1.7 by Bridges and William332.
a

7-Azaindole

 

From the fluorometric titration of 7-azaindole (Figure 23), we get
a PKa* = 4.7. Besides the quenching upon increase of the concentration
of H3+0 and approximately below pH = 4, a new fluorescence band appears
with a maximum around 450 nm which is ascribed to the cation resulting
from the protonation of the N7 position. Since the cation is emitting it
provides us with an opportunity to test if euqilibrium is really established
in the excited state if a pKa* = 4.7 is really representing the true ther-
modynamic pKa* .

Following the method of Weller as derived in the introduction that

 

 

 

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Ill"! .1

Indole in water (25°C)

 

 

 

 

 

67

13

12

 

ll

10

 

 

 

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69

7- Azoindole in H2O
I

 

 

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13

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11

10

70

ApK = pK - pK* = (AE - AE')/2.303RT (22)
Using our data we find (25°C) that pKé - pKa* = 3.1. This result coupled

with Albert and Adler's33 ground state pKa = 4.59 gives us a thermodynamic
pKa* = 7.7. This shows that the rate of protonation is slow compared to
the deactivation rate of the excited state and so in the excited state

the ground state equilibrium is insignificantly perturbed.

The fluorimetric titration result for the pr* is 12.3, the same as
the one fOund for indole. Longworth et a1.34 gave a Apr = 7.5 for indole.
If we accept the Apr as being the same for 7-azaindole as for indole,
which appears reasonable, then the thermodynamic sz* = 9.5 to be compared
with the titrametric pr* = 12.3. This indicates that the ground state
equilibrium is perturbed significantly (pKB = 17) during the excited.
state lifetime but complete thermodynamic equilibrium is not attained.

A table showing the comparison of the titrametric pK's and thermo-
dynamic pK's is give in Table 9. In general the fluorimetric titration
need not give the true thermodynamic pK because it depends on the rate
with which equilibrium is attained. If the acid-base reaction is slow then
equilibrium may not be attained during the exicted state lifetime and
in this case the titrametric pK will be closer to the ground state pK.

For example compare the titrimetric and thermodynamic pKa* for 7-aza-
indole.

The pr* for all compounds studied is lower than 16.97, the ground
state pKB of indole. If we make the assumption that all these compounds
have approximately the same pKB as indole that means that the indolic
hydrogen becomes more acidic in the lowest excited state (lLb). This
fact is in accord with charge density calculations using Pariser-Parr-

l

Pople method where it is found that in the Lb state there is a reduction

71

Table 9. Lowest Singlet Excited State pK's of Indole and Azaindoles.

Compound

Indole

2-Azaindole
3-Azaindole
4-Azaindole
5-Azaindole

7-Azaindole

Compound

Indole

2-Azaindole
3-Azaindole
4-Azaindole
5-Azaindole

7-Azaindole

pK

5.33

6.94

K
pb

16.97

** Reference 34

pK*(titration) pK*(thermodynamic)
a a
1.8
2.4 5.6
5.5 8.5
4.5
4.7 7.7
Apl(1Lb) pK§(titration) pK§(thermodynamic)
-0.098 12.3
11.9 11.6
-0.239 11.9
-0.097 12.5
-0.059 11.3
-0.033 12.3 9.5**

72

(A01) of charge density compared to ground state in position 1. Since
Pariser-Parr-Pople method is n-electron method, no correlation can be made

with the protonation reaction (pK ) which involves the o -electron system.
a

BIBLIOGRAPHY

10.

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

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