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presented by
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SYNTHESIS AND CHARACTERIZATION OF SOME
BENZO-CRYPTANDS AND OF SOME

LANTHANIDE CRYPTATES

BY

A. Rashid Kausar

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1978

‘l’ /‘/

ABSTRACT

SYNTHESIS AND CHARACTERIZATION OF SOME
BENZO-CRYPTANDS AND OF SOME

LANTHANIDE CRYPTATES.

By
A. RASHID KAUSAR

The first part of the dissertation describes the im-
proved methods for preparing cryptand chemical precursors.
Most of the improvements involved new routes to the start-
ing materials or a more judicious selection of solvents
and reaction temperatures. For the first time, detailed
and complete methods for the synthesis of some benzo-cryp-

tands (g, 31 and EQ) are provided.

F‘é’} ..
"$0

52,

Y=H,23=2=l L5.)
8N02I2BN023231 (ll)
=NH2,28NH2:2:I (2g)

A. Rashid Kausar

The benzo-cryptands are analyzed by their infrared, ultra-
violet and mass spectra and elemental analysis. Their
proton-nmr spectra reveal that the coupling patterns for
the methylene protons in these closely related ligands are
not at all alike. They all show different sets of signals
in the methylene region between 2.5-4.5 ppm.

Chemical procedures outlined in the second part of
this research work encapsulate the tripositive lanthanide
metal ions inside the cryptand cavity in order to produce
a new series of cryptate complexes. Most success in the
synthesis of cryptate complexes was achieved by employing
a dehydrating agent and by use of dry acetonitrile as sol-
vent.

The hydrated lanthanide nitrate salts were used to
provide solid samples of cryptate complexes. In the nitrate
complexes, infrared spectra show, that an anion is almost
certainly bonded to the metal ion in alkyl-cryptate com-
plexes, whereas in benzo-cryptate, all the nitrate anions
are bonded to lanthanide metal ion. The shift of (C-0)

1 also afforded an evi-

bands to lower energy by 40-60 cm-
dence for the complexation of lanthanide metal ions by
cryptands (37;).

Ultraviolet spectra of 28:2:1 (é) ligand and of its
complexes are markedly different. Upon encryptation, a

sizeable decrease in extinction coefficient and some

band shifting is measured.

A. Rashid Kausar

In studies to date, the most useful method of charac-
terization has been proton nmr spectroscopy. Spectra of
free and complexed benzo-cryptands show noticeable dif-

3+

ferences, even for the diamagnetic La complexes. The

lH-nmr spectrum of the diamagnetic [La(ZB:2:l)]3+ provides
strong evidence that the metal ion indeed resides in the
central cavity of the molecule. The four aromatic protons
in the free 28:2:1 ligand are recorded as an A2B2 doublet
whereas in the complex, these are found as an AA'BB'
symmetric multiplets, with a net downfield chemical shift.
The paramagnetic lanthanide cryptates show induced para-
magnetic shifts, in both directions, in the proton nmr
spectrum of the ligand. These induced shifts are also
indicative of a complex where the metal ion resides in the
intramolecular cavity of the ligand.

The last part of the thesis details the experiments
in which a lanthanide cryptate is used as a labelling
reagent. After diazotization, the metal cryptate is
coupled with a protein or an enzyme. The generation of a
visible color and an increased absorption in the azo region
(330 nm) provided an evidence of covalent attachment through
and azo linkage. The presence of metal ion in the labelled
protein is determined by encapsulating a radioactive lan-

thanide metal ion in the cryptate which is coupled with

protein after diazotization in aqueous solution.

TO

.... my parents, especially my father, who

wants me to go always for the best;

.... my teachers, some here; some there, whose

many valuable advices helped me to

acquire the goal;

.... my brothers, who really helped me get

insight of the reality of the life;

I dedicate this piece of the research.

ii

ACKNOWLEDGMENTS

The author wishes to express his gratitude to Dr.
Otto A. Gansow for his guidance throughout the course
of this investigation. Thanks are also extended to Dr.
Michael W. Rathke whose doors were always open with en-
couragement. Dr. L. Sousa, whose friendship and under-
standing will never be forgotten, I owe many thanks.
The financial assistance from the Department of Chem-
istry, Michigan State University is gratefully acknowledged.
Finally, thanks to the past and present members of
"OG's Group" for making my stay in the 0.8. a pleasant

and memorable experience.

iii

TABLE OF CONTENTS

Chapter

LIST or TABLES . . . . . . . . . . . .
LIST OF'FIGURES. . . . . . . . . . . .

CHAPTER I GENERAL SURVEY OF THE
SUBJECT O I O O O O O O O O

1.1. The Cryptands and the Cryptates.

1.2. Lanthanide Ions Coordination
Chemistry. . . . . . . . . .

CHAPTER II SYNTHESIS OF CRYPTANDS AND
THEIR CHEMICAL PRECURSORS.

2.1. Outline of Synthetic Methods
2.2. Experimental . . . . . . . .

2.3. Results and Discussion . . .

2.3.1. Cryptands and Their Pre-

cursor Preparations.

2.3.2. Characterization of
Cryptands. . . . . .

CHAPTER III PREPARATION AND CHARACTERIZATION

OF SOME LANTHANIDE CRYPTATES.

3.1. Introduction . . . . . . . .
3.2. Experimental . . . . . . . .

3.3. Results and Discussion . . .

3.3.1. Formation of Cryptates

3.3.2. Spectroscopic Studies.

3.3.3. Electrochemical Studies.

3.3.4. Analytical Measurements.

iv

Page

vi

vii

13
13
16
28

28

35

41
41
43
46
46
49
71

75

Chapter Page

CHAPTER IV DIAZO COUPLING OF LANTHANIDE
BENZO-CRYPTATES TO PROTEINS. . . . . . 77

4.1. Introduction . . . . . . . . . . . . . . 77
4.2. Experimental . . . . . . . . . . . . . . 81
4.3. Results and Discussion . . . . . . . . . 86

REFERENCES 0 O O O O O O O O O O O O O O O O O O O 9 6

Table

LIST OF TABLES

Page
Dissociation Rates and Stability
Constants Data of the Cryptates . . . . . 6
Absorption Wavelengths for
[Ln(23:2:l)]3+ Cryptates in
Acetonitrile and Water. . . . . . . . . . 53

Assignment of Bands due to

Nitrate Ion in Alkyl Cryptates. . . . . . 59
Vibrational Modes for sz

Nitrates in Ln(2B:2:1) (N03)3. . . . . . 65
Analytical Data on Some

Lanthanide Cryptates. . . . . . . . . . . 76

vi

Figure

10

LIST OF FIGURES

1H-NMR spectrum of [Pr(2:2)](NO3)3. . .

1H-NMR spectra of cryptands 2B:2:l,

2BN02:2:1 and 2BNHZ

Ultraviolet spectra of [Ln(28:2:l)]3+

:2:1 0 O O O O O O O

cryptates (a) in acetonitrile and
(b) in water. . . . . . . . . . . . . . .

Infrared spectra of (a) [Ln(2:2:l)]3+
cryptates and (b) [Ln(2:2:2)]3+
cryptates . . . . . . . . . . . . . .
Infrared spectra of [Ln(2B:2:1)]3+
cryptates . . . . . . . . . . . . . . .

1H-NMR spectra of cryptand 23:2:1

and its lanthanum complex in CD3CN. .

1H-NMR spectrum of a deuterated

acetonitrile solution of [Pr(2B:2:l)]3+ .

1H-NMR spectrum of a deuterated aceto-

:2:1)]3+ .
2
(aq):

nitrile solution of [Pr(ZBNH
Cyclic voltammograms of Eu3+
[Eu(2B:2:1)]3+ and [Eu(ZBNH :2:1)]3+

2
cryptates O O O I O O O O O O O O O O O O

Ultraviolet spectra of an Azo-tyrosine, an

Azo-histidine and an Azo-Nuclease . . . .

vii

Page

11

38

51

56

61

66

69

72

74

80

Figure Page

11 1H-NMR spectrum of a deuterated aceto-

nitrile solution of 31. . . . . . . . . . 88
12 Ultraviolet spectra of Bovine Serum

Albumin and labelled Azo-B.S.A. . . . . . 91
13 Ultraviolet spectra of Ribonuclease-A

and labelled Azo-Ribonuclease-A . . . . . 93
14 Plot of ultraviolet absorption (at

330 and 276 nm) and radioactivity
(cpm) vs tube numbers carrying the
labelled and unlabelled Ribonuclease—

A O O O O O C O O O O O O O O O O I O O O 94

viii

CHAPTER 1

GENERAL SURVEY OF THE SUBJECT

The chemistry of synthetic macrocycles has been ex-
plored extensively only in the last decade. Simmons and
Park1 reported the synthesis of some macrobicyclic diamines
(l) in 1968. In the preceding year, Pedersen2 had published
his fascinating discovery of a broad range of macrocyclic
polyethers (2) now called the "crowns". The preparation
of macrobicyclic polyether diamines (3,4) by Lehn and co-
workers3 in 1969 combined chemical features of two closely
related classes of molecules together in the form of "the

cryptands".

Each of the macrocycles (4:4) has the principal struc-
tural feature of a cavity in the center of the molecule
which may serve the purpose of holding a cation.

The background information on cryptands and cryp-
tates, and lanthanide ions coordination chemistry are
provided in the following sections. This may familiarize
the reader with both of these rather different areas of

chemistry.

1.1. The Cryptands and the Cryptates

 

The polyoxadiaza-macrocycles (cryptands 2,4) differ
from the crowns in their bicyclic nature. All of the
crowns are two dimensional monocycles of topology (A).

The addition of another strand to monocycle (A) would lead
to a bicycle of topology (B) which has a three dimensional

cavity:

Because these bicycles have an inside and an outside,
pyramidal inversion at the nitrogen atoms can lead to

different conformers. The bicycles can be in the exo-exo

form (where both lone pairs of the nitrogen atoms are
directed away from the cavity), the exo-endo form (where
one lone pair is directed towards the cavity and the
other away from it), and the endo-endo form (where both

lone pairs are directed towards the molecular cavity).

I/ICT—‘13\\\ l/lcy"13\) ((4)-‘CT\\
""'"""N =N N

N N N
‘0—07 ‘— §O~0 ’ (to 0/)
o C—
0—0 \0 0—0
axe-m oxo-ondo ondo-cndo

Cryptands of the type (3) or (4) are also called
[2]-cryptands by Lehn where [2] denotes their bicyclic
nature. A nomenclature for these molecules was also
devised by Lehn.3 The molecule (3), for example, is
denoted as 2:2:1 cryptand according to the number of oxygen
atoms present in each of the three strands of the ligand.
The replacement of an ethylenedioxy unit by a catechol
group in (2) would lead to a new cryptand (g) which is
denoted 2B:2:l.

Coordination compounds formed by these macrobi-
cyclic cryptands (éfé) can be referred to as [2]-cryptates.
Various [2]-cryptates have been synthesized with a variety

of metal cations including the alkali and alkaline earth

I-‘o
"00%

bad

cations,4 Cu2+, Ni2+, C02+, Zn2+, Pb+, Ag+, Tl+, Hg

0*

2+

and Cd2+.5 The cryptates of tripositive metal ions have,
of course, been prepared and characterized only in our
laboratory.

The stability constants (Ks) for the cryptates are

the measure of the strength of complexation in solu-

tion:
L +Mn+ slisL M s
[]S[ m]._[ Mls+m
[L]S = solvated ligand, cryptand
[Mn+-mS] = n-fold charged cation - solvated
[UM]:+ = solvated cryptate

Some of the factors which affect the formation and
thermodynamic stabilities of these ion-macrocycle com-

plexes include:

i) type and number of hetero atoms in the ring;

ii) relative size of the ion and the molecular cavity;

iii) electrical charge of the ion; and

iv) solvent.

 

Results of stability measurements for alkali and

alkaline earth cryptates with ligands (3) and (4) were

reported by Lehn and Sauvage6 in 1971. From their data,

they concluded that these cryptands Show a great cation
selectivity. The maximum stability for a given cation
depends to a great extent on the nature and shape of
the ligand. It was also noticed that changing the
solvent from water to methanol markedly increased the
thermodynamic stabilities of the inclusion complexes.
The following table includes the data on stability con-
stants of some [2]-cryptates in water.6 An important
information that can be extracted from their kinetic
investigations is that dissociation rates of alkaline
earth cryptates are between 102 - 106 slower than those

of the alkali metals.7

The cryptands (3) and (4) usually form 1:1 complexes

8 X-ray crystal

with alkali and alkaline earth cations.
structure of some of these [ZJ-cryptates have been per-
formed by Weiss.9 The metal ion was shown to reside in

a central location of the molecular cavity. In all the

[2]-cryptates studied by x-ray, the bicyclic systems are

in the endo-endo form because in this conformer all the

heteroatoms may participate in the complexation.

“Who if“ wno‘r‘
' .
r.

Table 1. Dissociation Rates and Stability Constants
Data of the Cryptates.

 

 

Cavity Cation

 

Ligand Size Cation Radius kd/a-l log KS
in A

4 1.4 % Na+ 1.12 27 3.9
4 K+ 1.44 7.5 5.4
4 Rb+ 1.58 38 4.35
4 Ca2+ 1.18 0.21 4.4
4 Sr2+ 1.32 7.5x1o'5 8.0
4 Ba2+ 1.49 1.7x1o“5 9.5
3 1.15 i Na+ 14.5 5.3
3 Ca2+ 6.6xlO-4 6.9
3 Sr2+ 1.5x10"4 7.3

‘m -r. .- rm waif—Tr—
I

 

 

 

[Rb(2:2:2)]+

1.2. Lanthanide Ions Coordination Chemistry

Lanthanum and the 14 elements which follow it in

the periodic table comprise a series of chemically simi-
lar elements. The common oxidation number for these
rare earths is +3. That is also by far the most thermo-
dynamically stable oxidation state. As the series is
traversed, electron filling takes place in the inner

4f orbitals rather than in 5d. Because the valence 4f
electrons are shielded from the environment, their con-
tribution to the chemistry of these elements is minimal.
Unlike d-orbitals of d-block transition elements, f-
orbitals of the lanthanides do not exhibit large crystal

field effects upon complexation.

 

 

All the rare earth cations are strongly electro-
positive and are considered to be "class A" cations.lo
Hence, the lanthanides show ionic bonding and form stable
complexes with hard anions. The lanthanide complexes
extensively studied so far are formed from anionic ligands
like carboxylates, acetates and B-diketonates. The ex-
treme insolubility of their hydroxides in basic pH pre-
vents complex formation in water except with strongest
multidentate ligands like EDTA. In other words, coor-
dinated water is so strong a ligand, that all others

must compete with it for binding sites.

3+

The observation that Ln —»N interactions were much

weaker than Ln3+

'— O in aqueous media led to the thought
that no complexes with a nitrogen donor ligand could
be prepared. However, the use of non-aqueous solvents
recently helped to produce a series of new lanthanide
coordination compounds. Complexes of the lanthanides
have been successfully prepared with ethylenediamine
and similar ligands.11
Among the d-transition elements, geometries of com-
plexes and the coordination numbers are mostly determined
by d-orbital participation in bonding. In contrast, the
absence of f-orbital interactions among lanthanides re-
sults in a wide variety of coordination geometries and

in coordination numbers ranging from 6 - 12.12 An ex-

ample of a ligand effect on the structure of a

 

lanthanide complex can be seen from the work of Hoard

13'14 They determined through x-raY

and coworkers.
studies that in the following two complexes, protonation
weakens the bonding to an arm of the EDTA ligand, and

another water molecule is accommodated.

 

OH20

[La(EDTA)(H20)3]-l [La(HEDTA"H2°)430

It is seen that it is the ligand which influences the co-
ordination sphere as well as the geometry of the coordin-
ation complex rather than the electronic structure of
the metal ion.

There are very few reports of macrocyclic complexes
with lanthanide metal ions as compared to those of the
alkali and alkaline earth metal cations. G. DePaoli and

15

coworkers reported the formation of lanthanide crown

complexes of dibenzo-lB-crown-6 and benzo-lS-crown-S.

10

In another extensive study with the same crowns, King and

16 published results contrary to what DePaoli had

3+

Heckley
reported. King and Heckley found that the post Sm
lanthanides do not yield pure 1:1 complexes with any of
the crowns studied whereas DePaoli and coworkers had

reported that they obtained analytically pure complexes

3+ ions also. However, the 1H-NMR results

with post Sm
presented by King and Heckley are not convincing because
they do not find paramagnetic shifts in the ligand spec-
trum due to the paramagnetic lanthanide ions. If a

3+, was in the cavity

paramagnetic metal ion, such as Eu
of the crown ligand, then induced chemical shifts for
the ligand protons should have been observed.

Another paper recently published by G. Duyckaerts
and coworkers17 deals with the lanthanide complexes of
a diaza-lB-crown-6, commonly known as 2:2. They were
able to isolate all the lanthanide macrocycle complexes
in analytically pure form. In the 1H-NMR spectra of the

3+ 3+, they did observe

ligand in complexes with Pr and Eu
large induced paramagnetic shifts as shown below in
Figure 1. No studies of lathanide cryptate formation
have been reported other than the present work.

The [Zj-cryptands are known to possess, in some of
their conformations, a spheroidal cavity which may pre-
sent oxygen and nitrogen binding sites to a encapsulated

metal ion. Hence, if the spherical tripositive lanthanide

metal ions whose ionic radii vary in the range 0.85-1.15 A

ll

 

 

 

 

 

 

 

 

k‘f-‘“rn :thquf”-

-20 ~10 0 IO 20
Figure 1. NMR spectrum of a deuterated nitromethane
solution of Pr(NO3)3-2:2. A similar spectrum
was obtained for Eu(NO3)3 with CH2 peaks at
-10.85. -6.54. -5.29. -l.39. +9.30 and +ll.28
ppm from TMS. The NMR spectrum of pure macro-
cycle exhibits peaks at -3.60, -3.50 and -2.85

ppm 0

could be encapsulated inside the cryptand cavity, they
might produce a series of systematically varied, sub-
stitutionally inert, yet thermodynamically stable
cryptate complexes. Moreover, the cryptand may be func-
tionalized and the entire cryptand (or cryptate) could
then be chemically attached to a large macromolecule or
a solid support. In summary, these cryptates could find
use in nmr, synthesis, and medicine.

The first part of this research work describes the
improved methods for preparing cryptand chemical pre-

cursors and several modifications in the literature

' “I”

 

‘lw'i .
‘.

12

syntheses of the cryptands. Next, the chemical pro-
cedures outlined in the second part, encapsulate the tri-
positive lanthanide metal ions firmly inside the cryp-
tand cavity. These cryptates are characterized by their
infrared, ultraviolet, and proton nmr spectra. Finally,
the experiments described in the third part, deal with

covalently attaching the cryptates to a protein.

NP“ 2qu n- (“CI—A.“ a “up

CHAPTER 2
SYNTHESIS OF CRYPTANDS AND THEIR CHEMICAL PRECURSORS

2.1. Outline of Synthetic Methods

4 published details of syntheses

Lehn and coworkers
of alkyl cryptands in 1973. These preparations require
the high dilution condensation of diamines with diacid
chlorides to yield cyclic diamides and are similar to

18

those described by Stetter and Marx and by Simmons and

19

Park. The first condensation yields the monocycle

which is again condensed with a molecule of diacid
chloride to obtain the bicyclic cryptand of desired size

and structure.

”mono/“N... . "\

.9.
4.

C 0 CI

03.4% _8_
l

C)

liAlH4 "N,“ n f'\

T, K/o\/l

13

14

d
d

Jul/9H9 I

8
§3

- S
K.

i)HC|,l’I"UX

PO ’\\ ii)Dowexl-X8
N\/\°\/\N <—— (OHHOM
L’OLJOV/ '

.3.
4, 7,13,19,22 - pontooxa-
LID-diam -bicyclic-

(8=5=8) tricosane or
cryptand 232:)

 

 

In the synthesis of benzo cryptands, such as 2B:2:l
(3), the second cyclization involves the reaction of
monocycle (3) with the diacid chloride derived from

catechol. Details of synthetic procedures are present-

ed in the following section of the thesis.

T. v». F T *-'\-i£'\-_r“.. a. ~cfi?‘ '32P.—

15

2. I"\o
” e W

WSW L674”

 

 

0'
15.
13' l
i)BH3/'I’HF
ii) HCI , reflux
iii)Dowoxl-X8 (OH)
/’\c{'\o,\\ L
N\V/hfi1~/fi\~ -<———-
oJ

Cryptand 28:23]

 

Some cryptands are commercially available from E.
Merck and Co., Parish Chemical Co. and PCR, Inc. at
rather high prices. The benzo cryptands (3, 2_7 and a)
are not commercially available and were synthesized in
our laboratory. Since several of the syntheses employed
by Lehn were found more laborious than required, alter-
nate and improved synthetic routes were devised for

the synthesis of the cryptands.

16

2.2. Experimental
(a) General

Most reactions were performed under dry nitrogen
atmosphere unless water was used as solvent. Toluene
used in high dilution condensations was purified by
distillation over sodium metal just prior to use. All
other solvents were reagent grade and were used without
purification.

The diglycolic acid and 4-nitrocatechol used were
purchased from Aldrich Chemical Company. The resins,
Dowex l-x8 (Cl form) 50-100 mesh and Dowex SOW-x8 (H+
form) 100-200 mesh, were purchased from J. T. Baker Chemi-
cal Company and were used without any purification.

Melting points were determined in air employing a
Thomas-Hoover apparatus using a capillary tube and are
uncorrected. Ultraviolet spectra were taken by using a
Cary-l7 spectrophotometer. Infrared spectra were re-
corded with a Perkin-Elmer Model 457 spectrophotometer.
Proton NMR spectra were obtained using a Bruker WH-180
or a Varian T-60 spectrometer. Mass spectra were obtained
by our departmental technician who used a Hitachi RMU-6
operating at 70 eV.

Elemental analyses were performed by Spang Laboratories
of Eagle Harbor, Michigan or Galbraith Laboratories of

Knoxville, Tennessee.

ni‘ua u . n

 

w‘oa‘

17

(b) Preparative Procedures

Triethyleneglycol ditosylate or 2-ditosylate (1;)-
To an erlenmeyer flask equipped with a stir bar was added
400 ml of pyridine and 75 grams (0.5 mole) of triethylene
glycol. After cooling to 5-10 °C in an ice bath, 95.33
grams (0.5 mole) of p-toluenesulfonyl chloride was next i
added to the reaction flask in two portions over a period
of 20 minutes. Then another 95.33 grams (0.5 mole) of p-

toluenesulfonyl chloride was added in the same way. Near

 

the completion of this addition, a slurry formed. Stirr-
ing was continued for another one and one-half hour after
the addition had been completed. Next the slurry was
dumped into a solid ice and water mixture whereupon the
ditosylate solidified and pyridine hydrochloride salt
dissolved. After filtration, triethyleneglycol ditosylate
was recrystalized from ethanol (yield: 178 g, 78%, mp

78 9c): 18 NMR (CDC13)62.4(6H, singlet), 3.43-3.62 (88,
singlet and triplet), 4.1 (4H, triplet) and 7.2-7.65 (4H,

two doublets, J = 8.0 Hz); mass spectrum m/e 458.

l,8-diazido-3,6-dioxaoctane or 2-diazide (33) -

 

NOTE: All organic azides are known to be explosive.
Although this particular diazide can be distilled, it is
advisable to use it immediately after preparation and
without further purification.

The 2-ditosylate, 45.8 gms (0.1 m), was placed in a

18

round bottom flask and 100 ml ethanol and 50 ml of water
added. To this slurry, 15 gms of NaN3 was next added.
The mixture dissolved upon heating to 60 °C after one-
half hour and was refluxed for 8-10 hours. The solvent
was rotory evaporated whereupon a paste was obtained which
was extracted with ether. The ether was rotory evaporated,
yielding the crude Z-diazide as a yellow oil (16 gms,
80%, bp 83 °c at 0.3 mm): IR (neat) 1890 cm'1 due to
asymmetrical azide stretchings; 1H NMR (CDC13) 63.3
(4H, triplet) and 3.55 (8H, singlet and triplet); mass
spectrum m/e 178.

Caution: Upon one occasion, the original azide reac-
tion slurry, was let to go to dryness resulting in a

violent explosion.

l,8-diamino-3,6-dioxaoctane or 2-diamine (3) -

A three neck flask equipped with a mechanical
stirrer, a reflux condenser, and a dropping funnel was
charged with 5 gms of LiAlH4. The flask was evacuated,
filled with dry nitrogen and attached to a bubbler to
monitor the flow of N2. To this was slowly added 300
ml dry THF (predistilled over LiA1H4) through dropping
funnel while the flask was kept cold (5-10 °C) in an
ice bath. Next, 10 gms of 2-diazide in 30 ml dry THF
was added to the slurry of LiA1H4 in THF over a period
of one hour. After the addition was complete, the slurry

was refluxed for 10 hours. Excess hydride was destroyed

19

by dropwise addition of an aqueous 5% NaOH solution un-
til the evolution of hydrogen gas ceased. The slurry
was filtered to remove hydroxide salts. Solvent from
the filtrate was rotory evaporated and a yellow oil
obtained. The crude reaction product was distilled to
give colorless 2-diamine (6.1 gm, 82%, bp 78 °C/0.2 mm):
1H NMR (CDC13) 61.38 (4H, broad singlet), 2.85 (4H, trip-
let), 3.48-3.60 (8H, singlet and triplet); mass spectrum

m/e 148.

Diacidchloride of diglycolic acid (1) or l-diacid

chloride: - To a stirred mixture of 125 gms of PC15 in

 

400 ml chloroform at room temperature under nitrogen at-
mosphere, was added 40.2 gms of diglycolic acid in small
portions (2-3 gms). After a short while, a slurry formed.
Stirring and addition were continued at room temperature
for 5-6 hours. During this time the slurry dissolved.
Solvent was removed by using a rotory evaporator and an
oil was obtained. Upon distillation under vacuum (1.05
mm). the Pool3 was distilled first at 25 °c, next the
diacid chloride was collected as a colorless oil (bp =

1

37-38 °C): H NMR (CDC13) 64.52 (4H, singlet); mass

spectrum m/e 170.

20

1,7,13-Trioxa-4,lO-didza-cyclgpentadecane (3) gg

cryptand 2:1

 

(a) Cyclization to form 5,9-Dioxo-l,7,13-trioxa-4,10-

diazacyclopentadecane (3): - The reaction of 2-diamine
(3) and l-diacid chloride (1) was carried out under high
dilution conditions and at low temperature (5-10 °C)
to minimize formation of polymers.

To a S-litre three neck flask equipped with a mechani-
cal stirrer and 2-dropping funnel, all kept under a nitro-
gen atmosphere, 1.5 9 of dry toluene was added. The vessel
was then cooled by an ice bath. The 2-diamine, 14.5 gm
in 500 ml of toluene, and diacid chloride, 17.1 gms in 500
ml of toluene, were added separately to the two dropping
funnels attached to the flask. The temperature of the
toluene in the flask was lowered to 5-10 °C by the ice
and was kept cold throughout the reaction by addition of
more ice to the bath. Dropwise, simultaneous, addition
was started while stirring. The addition was completed
in 7 hours. The resulting mixture was stirred overnight
and then filtered. The amine hydrochloride salt was
removed by filtration and the solvent was stripped to
dryness on the rotory evaporator. A white solid was
obtained in this way which was eluted with 50/50 CHC13-
C6H6 from a column (18" long, 1" diameter) packed with 50
grams of alumina. After removal of solvent by a rotory

evaporator, the unreduced monocycle (3) was obtained as

21

a white solid (mp 149-150 °c, yield 60%): 1

H NMR (CdCl3)
63.60-3.67 (singlet and multiplet), 4.08 (singlet) and

7.15 (singlet); mass spec m/e 246.

(b) Reduction to form monocycle 2:1 (3) -

A 1-1 three neck flask was equipped with a mechanical
stirrer, a dropping funnel, and a reflux condenser. The
flask was evacuated and kept under a nitrogen atmosphere.

Then, 5 gms of LiAlH4 and 200 ml of dry THF was added

 

to the flask which was cooled in an ice bath (5-10 °C). E
To this cold slurry, 5 gms of monocycle (3) was added

as a solid in portions exceeding not more than 3/4 gm.
After 8 hours of reflux, excess LiAlH4 was destroyed by
dropwise addition of 5% NaOH solutions. The hydroxide
salt was filtered and the filtrate was concentrated on

a rotory evaporator. This yielded a white solid which was
recrystalized from petroleum ether (mp 89-90 °C, yield
85%): 1H NMR.(CDC13) 51.9 (2H, singlet), 2.75 (8H, trip-
let) and 3.60 (12H, singlet and triplet); mass spec m/e
218.

Bis(l,2-ethyl acetoxy)benzene (31)

30.3 gms of ethylbromoacetate was slowly added to a
stirred mixture of 27 grams of anhydrous ch03 in 500
ml acetone at room temperature. Next, 7 gms of cathecol
in 100 ml of acetone was added drOpwise over a period of

one-half hour. After addition, the mixture was refluxed

22

overnight. The salts formed were removed by filtration
and the solvent was rotory evaporated leaving an oil which

1H NMR

was distilled (bp 145 °C/0.l mm, yield 82%):
(CDC13) 61.4 (6H, triplet), 4.32 (4H, quartet), 4.85
(4H, singlet) and 6.98 (4H, singlet); mass spec m/e 282.
Anal. Calcd for C14Hl806: C, 59.59; H, 6.38. Found:

C, 59.36; H, 6.46.

Bis-(l,2-ethylacetoxy)-4-nitrobenzene (33)

The procedure described for preparation of (31) was
employed. After the rotory evaporation step, a solid was
obtained which was recrystalized from ethanol to yield
light yellow crystals of (33) (mp 74—76 °C, yield 88%):
1:1 NMR (00913) 51.45 (611, triplet), 4.30 (4H, quartet).
4.8 (4H, singlet), 6.92 (1H, doublet), 7.7 (1H, doublet)
and 7.82 (1H, quartet); mass spec m/e 327.

Anal. Calcd for C14H17N08: C, 51.37; H, 5.19. Found:

C, 51.12; H, 5.11.

Hydrolysis of (31) to form 1,2-Bis-(oxyacetic acid)-

 

benzene (33)

5 gm of Bis-(1,2-ethylacetoxy) benzene (31) was re-
fluxed in water with 0.5 gm of Dowex 50W-x8 (H+ form).
The ethanol formed upon hydrolysis of diester (31) was
distilled as the azeotrope in order to drive the reaction
to completion, thus forming the diacid (33). After 8

hours of reflux, the mixture was filtered to remove the

23

resin. Upon cooling the diacid solidified and was re-

crystalized from hot water (mp 181-182 °C, yield 78%):

1H NMR (d6-DMSO) 64.80 (4H, singlet), 6.98 (4H, singlet);
mass spec. m/e 226:
Analysis: Calcd. for C10H1006: C, 53.10; H, 4.42.

Found: C, 50.87; H, 3.98.

Hydrolysis of (33) to form 1,2-Bis-(oxyacetic acid)-

 

 

4-nitrobenzene (33)

 

The same procedure was used as described for the prep-
aration of (33). Upon recrystalization from hot water,
a light yellow solid was obtained (mp 173-176 °C, yield
85%): 1H NMR (d6-DMSO) 64.88 (4H, singlet), 6.95 (1H,
doublet), 7.55 (1H, doublet) and 7.75 (1H, quartet);
mass spec m/e 271:
Analysis. Calcd. for C10H9N08: C, 44.28; H, 3.32.
Found: C, 41.43; H, 3.82. (Note: Calcd. with 1 mole

of water of crystalization: C, 41.52; H, 3.80).

1,2-Bis-(oxyacetyl chloride)benzene (33)

To a 1 2 3 neck flask equipped with a dropping funnel,
a reflux condenser, and a mechanical stirrer, 10 gms of
diacid (33) was added. The flask was evacuated and kept
under nitrogen atmosphere. Then 40 ml of freshly dis-
tilled thionyl chloride was added dropwise. The suspension
was heated slowly and then to reflux. The solid goes into

solution after one hour. After 5 hours of reflux, the

24

excess thionyl chloride was evaporated using a water
aspirator. A solid was obtained in this way which was
recrystalized from petroleum ether/acetone to yield white
crystals of diacid chloride (13) (mp 50.5-51.5 °C, yield
90%); 1H NMR (CDC13) 64.99 (4H, singlet), 6.95 (4H, singlet);
mass spec. m/e 269.

Anal. Calc. for ClOH 04C12: C, 45.63; H, 3.04; Found:

8
c, 45.81; H, 3.20.

l,2-Bis(oxyacetyl chloride)-4-nitrobenzene (13)

 

The procedure applied was the same as that used for
the preparation of (33). Recrystalization from petroleum
ether/acetone yielded a light yellow solid (mp 76-78°C,

yield 95%): 1

H NMR (CDC13) 65.15 (4H, singlet), 6.98
(1H, doublet), 7.70 (1H, doublet) and 7.88 (1H, quartet);
mass spec m/e 308.

Anal. Calcd. for C10H7NO6C12: C, 38.96; H, 2.27. Found:

C, 38.90; H, 2.32.

Preparation of 4,7,13,18,21:pentaoxa-5,6-benzo-l,10-

diazabicyclo[8.5.8]tricosane or 2B:2:1 (3)

 

(a) Cyclization to form 2,9-dioxo-4,7,l3,18,21-
pentaoxa-5,6-benzo l,10-diazabicyclo[8.5.8]tricosane

(14).

A 3-2 three neck flask equipped with a mechanical

stirrer and two dropping funnels was evacuated and refilled

25

with nitrogen. Then 800 ml of dry toluene was added.

The flask was then cooled by an ice bath. Next 5 gms of
monocycle 2:1 (3) and 6 gms of triethyl amine were mixed
together in a separate flask and adjusted to 250 ml
volume by adding toluene. Separately, 6.02 gms of diacid
chloride (33) was dissolved to 250 ml volume with toluene.
These two solutions were added to the two dropping funnels.
Then stirring was begun and the toluene cooled to 5-10 °C
by an ice bath. Then the solutions from dropping funnels
were added drOpwise simultaneously to cold toluene in the
flask. The addition was completed in 5 hours. After the
addition, stirring was continued for another 12 hours.

The triethyl amine hydrochloride salt formed was then
filtered and the supernatant liquid rotory evaporated.
Upon evaporation a paste was obtained which was dissolved
in 50 m1 of 50/50 CHCl3/benzene and passed through a
column (18" long, 1" diameter) packed with 30 gms of
alumina. Following evaporation of solvent by rotory
evaporation, a fluffy solid of (34) was obtained which was
used as such in the following reduction procedure (mp

154-157 °C, yield 86%).

(b) Diborane Reduction of (34)

Six gms of (34) were dissolved in 25 ml of THF in a
l 2 three neck flask. Then 30 m1 of 1M diborane in THF

solution was added dropwise to the flask. The resulting

26

solution was refluxed for 8 hours. The excess diborane
was destroyed by dropwise addition of water. Upon re-
moval of solvent by rotory evaporation, the diborane salt

of 2B:2:1 (13) was obtained as a white solid.

(c) HCl Salt Formation

 

The diborane salt of 2B:2:1 (13) obtained in the pre-
vious step was dissolved in 50 m1 of 6N HCl and was re-
fluxed for 8 hours. The water was rotory evaporated to

yield the dihydrochloride salt of 2B:2:1 (13).

(d) Cryptand 28:2:1 (é)

 

A column 18" long and l" in diameter was packed with
Dowex l-x8 ion exchange resin (5H form). The first resin
commercially obtained was in the chloride ion form and
was treated with KOH to obtain the hydroxide form prior
to this reaction. The dihydrochloride salt (13) was
dissolved in 50 ml of water and passed through the column.
The column was washed with water until there was no basic
reaction of effluent to litmus. The water was rotary
evaporated to yield a white solid of 2B:2:1 (3). Upon
recrystalization from methanol, white crystals of 23:2:1

were obtained (mp 74-75 °C, yield 78%): 1

H NMR (CDBCN)
62.46-3.06 (12H, multiplets), 3.44-4.18 (16H, multiplets)

and 6.90 (4H, singlet); mass spec m/e 380; U.V. (CH3CN)

27

Amax at 274 nm.
Anal. Calcd. for C20H32N205: C, 63.16; H, 8.42. Found:

C, 62.99; H, 8.60.

Preparation of 4,Zyl3y18L21:pentaoxa-5,6,4-(nitro)
benzo 1,10-diazabicycloE8,5,8]tricosaneLcryptand

ZBNOZ:2:1 (31)

(a) Cyclization to form 2,9-dioxo-4L7Ll3,18,21-
pentaoxa-S,6,4-nitrobenzo 1,10 diaza bicyclo 8LS,8

tricosane (11)

The same procedure was used as for the preparation
of (34). The diacid chloride used in this reaction was
(13). A light yellow fluffy solid of (11) was obtained in

this case which was reduced further as follows.

(b) Diborane Reduction and Formation of 28n0-:2:1
5‘ z

(2_7_)

The same procedures were used as described in the
synthetic scheme for 2B:2:1. Upon recrystalization from
methanol, yellow crystals of ZBN02:2:1 (11) were obtained
(mp 125 °c, yield 83%): 1H NMR (CD3cn) 62.44-3.14 (12H,
multiplets) 3.24-4.24 (16H, multiplets), 6.98 (1H, doublet),
7.70 (1H, doublet) and 7.85 (1H, quartet); mass spec

m/e 425; U.V. (CH3CN) Ama at 238, 300 and 338 nm.

x
Anal. Calcd. for C20H31N3O7: C, 56.47; H, 7.29. Found:

28

C, 56.53; H, 7.40.

Preparation of 4,7,13,18L21-pentaoxa-5L§,4-(amino)
benzo 1,10 diazabicycqu8yS,81tricosane, cryptand

ZBNH2:2:1 (28).

 

One hundred mg of 2BN02:2:1 (11) was dissolved in 10
m1 of dry ethylacetate, and 30 mg of 10% Pd/C obtained
from ROC/RlC Chemical Co. was added to the solution.
This mixture was hydrogenated at room temperature and
atmospheric pressure for 24 hours. The catalyst was
removed by filtration and the solvent was rotary evap-

1H NMR

orated to yield 85 mg of light brown paste:
(CD3CN) 62.35-3.30 (14H, multiplets), 3.35-4.26 (16H,
multiplets), 6.10 (1H, quartet), 6.26 (1H, doublet) and
6.62 (1H, doublet); mass spec m/e 395.

Anal. Calcd. for C N30 : C, 58.11; H, 8.47. Found:

20H33 5

C, 58.27; H, 8.20.
2.3. Results and Discussion

2.3.1. Cryptands and Their Precursor Preparations

4 have published the complete

Lehn and coworkers
descriptions of preparative methods used to synthesize
the cryptands 3-4. The high dilution condensations of

diacid chlorides with diamines are the most important

29

reactions in the cryptand syntheses. The starting ma-
terials, diamines or acid chlorides, are not commercially
available. The preparative procedures reported were found
to be more laborious than required and so were replaced

by more efficient and simple methods and are discussed
below.

The synthesis of the diamine (3) published by Lehn
employed Gabriel's pthalimide method to produce the hydro-
chloride salt of (3). This salt can be liquid-liquid
extracted for 2-3 days to obtain the pure diamine.

Phthalimide synthesis of 1,8-diamino-3,6-dioxaoctane

(3) (after Lehn4)

CQ\
2' '+
ClCHZCHZOCHZCHZOCHZCH2C1 + ,/h R
CO
l,2-Bis(2-chloroethoxy) Potassium phthalimide
ethane ‘
0 0
g H
____,DMF \VCH CH OCH CH OCH CH N/C
C,/ 2 2 2 2 2 2 \‘C

H H
O O

Triethylene glycol diphthalimide

-—--—9HINCH CH OCH CH OCH CH NH + ,

1,8-Diamino-3,6-dioxaoctane Phthalhydrazide

30

comi
HCl - + + -

NaOH CCOXa
em, NH 2CH 2CH ZOCH ZCHZOCH 2CH 2NH2 +

2-3 days COONa

l,8-Diamino-3,6-dioxaoctane (3)

This laborious method has been replaced by a simple

and efficient three step preparation:

0 on o 015 O N‘
E 21:0 C 2""1'N3 E 3
0 0" o 015 0 N3
\__J \__J
_l_§_ 16
O NH2
mm, C
.______,
THF 0 NH2
\..J
.6.

One could also use the dichloride instead of ditosylate
in the above scheme but that requires a longer time for
nucleophilic substitution by the azide ion.

In the synthesis of cryptand 2B:2:1 or 2BN02:2:1,
the monocycle (3) was obtained by condensing the 2-diamine
(3) with l-acid chloride (1). The acid chloride used was
obtained by reaction of the parent diacid with PCl5 in

chloroform. This preparation of the diacid chloride is

much simpler and more efficient than the procedure

31

published by Lehn. In the high dilution condensation,
toluene was used instead of benzene which gave a com-
parable yield of monocycle (3). The use of toluene en-
abled the reaction to be performed at a colder tempera-

ture.

qunor-V—‘nwz Ono
6

'3: '—_—__*’ th H
C °C)
03.4% _3_
l

[HUHh HN/-\C(_\Mr_\

V K/OV

The second cyclization in the preparation of cryptands
required the reaction of the monocycle (3) and benzo-2-
diacid chloride (33) or 4-nitrobenzo-2-diacid chloride
(13). The diacid chlorides were prepared in the follow-
ing way. The treatment of catechol or 4-nitro catechol
with ethyl bromoacetate in acetone/K2C03 went smoothly
to give the benzo diestre (31) or 4-nitrobenzodiestre
(33) which was then hydrolyzed to obtain the diacid (33)

or (13), respectively.

32

on ocuzcozu
+ 2 BVCH2C02C' _____.)
Y OH y ocuzcow

Y: H or N02 Earl—8

 

00-12(00).) Dowex SOW-XB
o <-——-( (H+)/ H 20
Y OCH2COOH

‘9or2

Reaction of the diacid with thionyl chloride provided the

desired diacid chloride.

0
o ocnzcoon 5°02 00423-0
--———a
Y ocnzcooa y 00,2 - c _ C,
3
Y=flt33
g N02: 2‘

The second condensation proceeded in high yield as

presented in the following reaction scheme, to yield the

benzo cryptands 23:2:1 (3) or ZBNO :2:1 (11). Toluene was

2
employed as a solvent and triethylamine was added to con-

sume the HCl evolved during reaction:

33

 

oh ' of‘
cum/A oflmcu :6NHCI H38:N/:\/\O\o/’\\\N:Bl-{3
o o

64 92
Y
‘25—".29- __:_3_or_fi_

 

Dowex I-XB
(6H)

 

l

/—\

o o
[/‘ofi Y=H,2B:2:l(§)
o

a EU =NOz:23N02‘2“ (Z?)

0 :2:1 ligand can also be obtained by direct
2

nitration of 2B:2:1 with HNO3/H0Ac in chloroform. The

The 2BN

reaction went smoothly to give the hydronitrate salt which

34

was passed through a column packed with Dowex l-x8 (OH).

:2:l was obtained after recrystalization from

Pure ZBN 2

O
methanol.

/’\of—\D /_\
N\/\o\/\~ + HNO3/H0Ac -—__. F0 0
OJ °3NH‘N\/\°\/\N:HNO3

be o\,/

(E?

5 N03

Dovgcx l-X8
(0H)

 

)

r‘oflo

N\/‘o\/‘N

Lie OJ
{3.5

N02

Catalytic hydrogenation was used to reduce the

2B :2:1 cryptand to obtain 2B :2:1. Ethylacetate was
NO2 NH2

successfully used as solvent. Hydrogenation with Pd/10% C
at room temperature and pressure was completed in 24 hours.

A brown paste was obtained which proved to be 23 :2:1,

NH
2
as confirmed by 1H-NMR, mass spectral data and elemental

analyses.

35

 

{ESQ emu/:23 3233

\VO OJ EtOAc \Vo OJ
@NOZ @8012
_2_Z ZEN-{2‘2” (Zfl)

2.3.2. Characterization of Cryptands

Those precursors already reported by other workers
were routinely checked for purity by spectral charac-
terization. Those precursors prepared for the first time
were characterized by their lH-NMR, U.V., and mass spec-
trum and by elemental analysis. Spectral results for the
cryptands will now be discussed.

The 2B:2:1 cryptand is a white solid whereas ZBNO :2:1
2

is a yellow solid, and ZBNH :2:1 a brown viscous paste.
2

Ono’w
V 04(31):

Y

Y=H,2B=2=l (5)
=N02,2BN02:2 :1 (:12)
=NH2 ,ZBNH232H (23)

36

One would expect that the proton NMR spectrum of these
three closely related ligands should be similar in the
region of - O — CH2 and — N-—CH2, but different in the
aromatic region. However, their proton NMR spectra
reveal that the coupling patterns for the methylene protons
in all three ligands are not at all alike. They all show
different sets of signals in the methylene region between
2.5-4.00 ppm. The four aromatic protons in 2B:2:1 ap-
peared as almost a singlet whereas three completely
resolved signals were obtained for the three aromatic
protons in 2BN02:2:1 and 2BNH2:2:1 cryptands.
2B:2:1 (5)

Infrared spectroscopy confirmed the presence of an
ether linkage by a strong and broad band centered at
1132 cm-1. The aromatic C—H bending vibrational band
denoted the presence of ortho di-substitution on the
benzene ring by a strong band centered at 740 cm-1.
The ultraviolet spectrum was taken in both acetonitrile
and water solvents and compared with a model compound,
1,2-dimethoxybenzene, which shows a broad absorption
at Amax 275 nm characteristic of catechol derivatives.
The 2B:2:1 cryptand showed a Amax at 274 nm in aceto-
nitrile and at 272 nm in water.

The proton nmr spectrum provided further evidence

for the structure. The 180 MHz spectrum shown in

37

Figure 2 contain separate complex multiplets for both
the N—CH2 and O—CH2 protons. The twelve methylene
protons adjacent to nitrogens appeared between 2.46-
3.06 ppm whereas the methylene protons next to oxygens
are found between 3.44-4.18 ppm. The O—CH2 protons of the
benzo strand were located downfield relative to the
aliphatic strand O-CH2 protons. Hence the four protons
of the ggz-O—benzene ring are split into a complex
multiplet between 3.9-4.18 ppm while the remaining O—CH2
protons give a complex pattern between 3.44-3.77 ppm.
The signal due to the four remaining aromatic protons

were centered at 6.90 ppm.

 

 

ZBNOZ:2:1 (27) and 2BNH2:2:1 (28)

Spectral results from these two new cryptands were
compared wherever possible with those obtained from the
model compounds l,2-diethoxy-4-nitrobenzene or 1,2-
diethoxy-4-aminobenzene.

The U.V. spectrum of 1,2 diethoxy-4-nitrobenzene in
ethanol shows two absorptions with Amax at 241 and 338

nm. The absorptions found for 2B 2:1 in CH CN showed

No‘

2 3

Xmax at 238 and 340 nm.

The 1H-NMR spectrum in the aromatic region for
2BN02:2:1 and 4-nitro 1,2-diethoxy benzene and for
ZBNH2:2:1 and 4-amino 1,2 diethoxy benzene are similar.
All have 3 aromatic protons which are chemically and

38

 

 

 

 

 

 

 

 

 

 

 

 

“3:33: ""2 T2 mm: m’
I"z“ “x
0435-
"f’goifiz:
Hx
HA
HM
' n
10 (£17 £6 ‘; :5 2;
ppm
l &
°2'bél: ZBNOZ: 2 :| Aliphatic O-CHz-
5x 03'
a- N'CHZ-
”x "A
" 1|,
Dalw_,flflk3_w__wfi_
l ' 1 ‘ lefl l 1 . l l 1
3.0 70 3.0 If so ‘0 3'0 10
(ppm)
28 :2: | Aliphatic o-cuz-
u

365?

cog—g:

4212-1
____JW WlL

 

 

 

L1.” 1
°° 1° 10” no

 

(ppm)

Figure 2. 1H-NMR spectra of cryptands 28:2:1, ZBNO :2:1
2

and ZBNH2:2:1.

39

magnetically nonequivalent. As shown below, depending

upon whether Y is —N02 or —NH2, the proton M will be the

>'
1:
)<
an?”

 

one most affected and proton X least affected by the
shielding or deshielding effects of the benzene ring

substituents.

Chemical Shifts in ppm

HA .HM HX

Y = N02 6.98 (doublet) 7.85 (quartet) 7.7 (doublet)

I<
II

NH2 6.62 (doublet) 6.10 (quartet) 6.25 (doublet)

The coupling constants JAM and JMX in both cryptands were
9 Hz and 3 Hz, respectively. Similar to that is found
for 2B:2:1, the methylene protons attached to oxygens
in the substituted benzo cryptands divide into two groups.

In ZBN :2:1 the O—CH2 protons were found in the region

0
2
3.24-4.24 ppm whereas in ZBNH :2:1 they fall between
2

3.35-4.26 ppm. The methylene protons adjacent to nitrogen,

40

i.e., N-CH2 were found between 2.44-3.14 ppm in ZBNO :2:1
2
and between 2.35-3.30 in the case of ZBNH :2:1. Spectral

2
assignments are as shown in Figure 2.

CHAPTER III

PREPARATION AND CHARACTERIZATION OF SOME

LANTHANIDE CRYPTATES

3.1. Introduction

 

A11 rare earth elements exhibit the thermodynamically
stable +3 oxidation state. All trivalent lanthanides are
formed by loss of f electrons except for lanthanum and

1 valence

lutetium which are formed by loss of their 32d
electrons. Since the f-orbitals are effectively shielded
by the xenon core electrons, their unavailability almost
excludesauurinteraction in bond formation. This is in con-
trast to d-block transition elements where d-orbitals of
metals participate in metal-ligand covalent bond formation,
whereas cation-ligand interactions in lanthanides are pri-
marily electrostatic. Therefore, the chemical properties
of the lanthanide cations are similar to the group IA,

IIA and IIIA cations rather than to the d-transition
metals. These lanthanide cations are considered to be
"class A" or "hard" cations. This idea is supported by
the tendency of these metals to form complexes with "hard"
anions or neutral molecules containing oxygen(s) or a
combination of oxygen(s) and nitrogen(s).

As a class, the complexes of lanthanides show very

41

42

significant differences in their properties and chemical
bonding comparedtxad-transition elements. Non-directional,
electrostatic bonding is the rule rather than directional
covalent bonding. The coordination numbers of 6 through

12 have been observed for the lanthanide complexeleb.

The stereochemistry of the complexes is thus influenced

by the topology of the ligand rather than by crystal field
effects.

The extreme insolubility of lanthanide hydroxides
prevents complex formation in water except with the strongest
multidendate ligands like EDTA. Even effective poly-
nitrogen chelates like ethylenediamine and triethylene-
tetramine form complexes of appreciable stability only

10a

in non-aqueous media. The use of non-aqueous solvents

thus helped to successfully prepare a series of lanthanide

coordination compounds with ethylenediamine and similar

ligands.lla-e

The macrocyclic complexes, crowns and cryptates, of

alkali and alkaline earth cations have been formed and

20,21

characterized during the last decade. Cyclic poly-

ethers, dibenzo-lB-crown-G, benzo-ls-crown-S, and 18-crown-

6, are reported to form complexes with tripositive lanthanide

16,22

metal cations. F. A. Hart and coworkers recently

reported an x-ray structure of [Ln(syn-di-(cis-cyclohexyl)-

l8-crown-6)(No3)3]. The metal ion was shown to reside

23

inside the molecular cavity. In a paper published by

 

43

G. Duyckaerts and coworkers,17

the preparation and spectro-
scopic prOperties of complexes of diaza-lS-crown-6 with all
the lanthanide metal ions was reported. It was established
that the crown ether adopts a non-planar conformation in the
lanthanide complexes isolated. The studies of lanthanide
[Zl-cryptate formation have been reported only in a recent .

paper from our laboratory.24

The research work presented
here describes the preparation and characterization of

lanthanide [2]-cryptates and elaboratescnlour previous

 

publication.

3.2. Experimental

General: All reactions were performed under strictly
anhydrous conditions. Acetonitrile was distilled over P205
just prior to use. Filtration was performed using high
vacuum techniques. Filtrates were kept under a nitrogen
atmosphere even though the systems appear to be stable to

air, although diliquesent.

Materials: Hydrated lanthanide nitrate salts were

 

purchased from Ventron Corporation, Beverly, MA and Research
Organic and Inorganic Corporation, Belleville, NJ and were
used without further purification. The cryptands were
synthesized in our laboratory and kept away from light

and air. Spectral grade acetonitrile was purchased from

Burdick & Jackson, Inc. of Muskegon, MI.

44

Spectroscopic and Analytical Studies: All proton NMR
spectra were determined on a Bruker WH-180 spectrometer
at prob temperature in d3-acetonitri1e using TMS as an
internal standard. Infrared spectra were recorded by using
a Perkin-Elmer Model 457 spectrometre with KBr plates and
nujol (for 650-1600 cm'l region) and hexaflarochlorobutadiene

1 region) mulls. Ultraviolet spectra of

(for 1600-3500 cm“
acetonitrile solutions of cryptates were taken by using a

Cary-l7 spectrophotometer. The cyclic voltammograms of
3+

 

aqueous solutions of Eu and Yb3+-benzo cryptates were
recorded by Professor M. Weaver's research group on a PAR
174 electroanalyzer. Melting and decomposition temperatures
were determined in capillaries in air and are uncorrected.
Elemental analyses were performed by Spang Laboratories

of Eagle Harbor, MI or Galbraith Laboratories of Knoxville,

TN.

(a) Preparation of Ln-cryptates (N03)3 (Ln=32, 23, 13),

(Scheme 1)

0.2 mm of cryptand (3-3,2_7_,13) and 0.2 mm of hydrated
lanthanide nitrate were taken together in a 100 m1 round
bottom side arm flask. The flask was evacuated, filled
with dry nitrogen gas and placed in a preheated (60-70°C)
oil bath. Next, 25 ml of dry acetonitrile was added to the

flask and the stirring was started. After 2 hours of

reflux, the solution was filtered using high vacuum.

45

To the filtrate, ethyl ether was added dropwise until
cloudy. The solution was refrigerated under nitrogen
atmosphere where the crystals of lanthanide cryptates

grew. The solids were filtered, washed with cold ether

and dried for 2-3 hours using high vacuum. The yield of
the cryptate solids were in the range of 60-70%. The spec-
tral and analytical data of cryptates are presented and

discussed in the following sections of this chapter.

(b) Preparation of Ln-cryptates (N03)3 (Ln = 33, 33, 33,

99.: 11:12: 21: 92: Eli: 213: Q and £43.): (Scheme 2)~

In this procedure, the hydrated metal nitrates were
dehydrated in situ prior to complexation. A solution of
0.2 mm of hydrated metal nitrate, 2 ml of triethylortho-
formate and 25 ml of dry acetonitrile was stirred and re-
fluxed for 5 hours. A solution of 0.2 mm of benzo-cryp-
tands (_5_,_2_7_,13) in 5 m1 of dry acetonitrile was added to the
dehydrated metal salt solution. The stirring and refluxing
was continued for another 4 hours (for 33 through 33), 16
hours (for 33, 3y) and 25 hours (for 32 through 33). After
cooling, the solution was filtered, and to the filtrate
was added ethyl ether dropwise to cloudiness. The mixture
was refrigerated from which the solid cryptate salts
precipitated. The solid was filtered and dried for 4-5

hours using high vacuum. NOTE: In the case of Yb, the

46

cryptate salt precipitates after 25 hours. This solid
was filtered, washed with ether and dried. The spectral
and analytical data for the cryptates are discussed and

described in following sections of this chapter.

3.3. Results and Discussion fl

3.3.1. Formation of Salt Complexes (Cryptates)

 

It is important to note that the cryptands (§f§,27, 8)

“A *‘ -‘-_' **—‘
t' .
C

are tertiary amines, pK m 10.5, pK m 7.5. The lanthanides
tend to precipitate as hydroxide in the presence of any
water. Only by using the non-aqueous medium can one suc-
cessfully prepare the lanthanide cryptates. Even a very
slight amount of hydration water in the reaction medium
hampered the whole cryptate synthesis especially with
smaller lanthanides.

The [Ln(cryptand 27§1_Z!_§) (N03)3](Ln = g3, 93, gr)
are successfully prepared using anhydrous acetonitrile
solvent. It is observed that formation of lanthanide
cryptate with cryptand 2:2:2 (g) is more facile than
with other cryptands studied. This is probably due to

its larger cavity size.

47
Scheme 1:
[M(H20)6](NO3)3 + Cryptand (§,-§,31,_§)
anhydrous CH3CN/reflux

M(cryptand) (N03)3 + 6H20

M=£e:22:££

 

Cryptands y

Ci)
0 °)
[$3

3 a = l
_ / ‘0‘ ‘o’\
i a — 2
Nx/‘°\/‘N
é. Y=H \ o U
_2_Z Y=N02 Q
Efi y = NH

The precipitate formation and longer reflux time with
heavier lanthanides prompted us to use a dehydration pro-
cess in order to strip the water molecules from the first
coordination sphere. The precipitates do not form in ap—
preciable amounts when the metal salts are dehydrated prior

to addition of cryptand. Most of the success obtained, so

48

far, is by using the following scheme 2 of dehydration.

Scheme 2

dehydrating dry CN3CN
[Ln(H20)n](NO3)3 + agent

A

reflux7' [Ln(CH3CN)nJ(NO3)3

CH CN
[Ln(CHBCN)n](NO3)3 + cryptand (5,27,28) 3

[Ln(cryptand)](N03)3

Ln = Nd through Lu

The dehydrating agent used is triethyl or trimethyl ortho-

formate which removes water as shown below:

OR CH3CN
H - C\-—OR + [M(H20)n](N03)3———) [M(CH3CN)n](NO3)3 +
0R

E?
ROH + H - C - OR

The following is the list of the various lanthanide cryptates

prepared to date in pure form by this worker.

 

V“... ‘. "A u: iflhfi'.
.I

49

i) [Ln(2:2:2)](NO3)3 Ln = La, Ce, Pr
(ii) [Ln(2:2:l)](N03)3 Ln = La, Ce, Pr
(iii) [Ln(ZB:2:1)](N03)3 Ln = La, Ce, Pr

(iv) [Ln(ZBNozz2:l)](NO3)3 Ln = La, Ce, Eu

(v) [Ln(ZBNH2:2:l)](NO3)3 Ln = Pr, Eu

Although these cryptates appear to be relatively stable to

 

air and moisture, they are however, best stored under nitro- :

gen gas. '4

3.3.2. Spectrosc0pic Studies

 

(a) Ultraviolet Spectra

The aliphatic cryptands (3,4) have no significant ab-
sorptions in the u.v.-visible region. However, the cryp-
tands (§.gz,g§) are derivatives of catechols and so show
the absorptions characteristic of catechol ethers.

The ultraviolet spectra of cryptand 2B:2:1 (g) and its
lanthanide cryptates are quite different. The catechal
ethers in organic solvents show a single absorption peak
in the region of 275 nm, the cryptand 28:2:1 show a
Amax at 274 nm in acetonitrile and at 272 nm in water.
Upon encryptation, a fairly large decrease in extinction
coefficient and a second peak about 5-8 nm to the longer

wavelength side of the major peak are observed.

The effects of encryptation of a metal ion on the

50

ultraviolet spectrum of cryptand 28:2:1 are shown in

Figure 3a and 3b. As can be seen from Figure 3a-b, the
changes in the shape of spectra among the several com-
plexes are slight and complex formation is suggested

by a hypsochromic shift and a decrease in absorbance.

This also is seen for the complexes of benzo-crowns pre-
pared with a variety of metal cations.2 The following table
lists the absorption wavelengths for the cryptates prepared
using 28:2:1 as a ligand. The shape of the u.v. spectrum
has been used for qualitative detection of complexing in
benzo-crown ether complexes. However, since the new peak

is not very well separated from main absorption peak, no
conclusions can be drawn as to its origin except to say

that the metal ion perturbs slightly the degenerate

n-w* transition of a benzene ring in the metal complexes
resulting in a slight change in the degeneracy of the transi-

tion.

(b) Infrared Spectra:

Infrared spectroscopy was used more extensively for
characterization of the complexes. It provided several
important types of informations. First, the presence of
water or any solvent molecule could be seen, also, absorp-

tions due to NO3 ion vibrations, in the solid complexes

were easily identified in the infrared spectra. Second,

the coordination of cryptands to metal ions resulted in a

 

51

Figure 3. Ultraviolet spectra of [Ln(ZB:2:l)]3+
cryptates (a) in acetonitrile and (b) in

water.

ABSORBANCE

ABSORBANCE

 

 

52

 

 

1- 2824 +
2-[La(za=2=l)]3
3- [Eu(2a=2= 0]”

  

\x

L l

l l l I l \ ____,,_ , _
220 230 240 250 260 270 280 $0 0' IO 320

WAVELENGTH (nm)
3b

 

l- 23=2=I
2- [Fr (23:24)?’
3- [Eu (28:2: 0]”
4- [La (23 =2: 013+

 

 

  
    

2
\

l l l l I \—

220 230 240 250 260 270 230 290 300 3:0 320

WAVELENGTH (nm)
38

 

 

53

Table 2. Absorption Wavelengths for [Ln(28:2:1)](NO3)3
Cryptates in Acetonitrile and H20.

 

 

 

Ln Amax in CH3CN Amax in H20
La 268, 276 268, 276
Pr 270, 276 269, 275
Eu 267, 273 268, 273
Gd 272, 277 271, 277
Dy 272, 278 270, 276

Yb 273, 279 271, 278

_ 7‘ .‘Wa‘m
t: i r
‘ ‘. I

 

 

54

l in the absorp-

large shifts to lower energy by 40-60 cm-
tions due to C-O-C stretching vibrations. This large shift
was also helpful in detecting any free ligand present in the
complexes prepared. Similar shifts have been reported for
the lanthanide benzo-crown complexes.16
Infrared data have been shown to be useful in distin-
guishing between coordinated and non-coordinated nitrate

ion. The symmetry changes upon coordination to a metal ion

'. i\ ‘1'-

 

can be interpreted on the basis of group theory. The free gs

nitrate ion has the symmetry type, D h, whereas the coor-

3
2v point group. The D3h nitrate

group has three infrared allowed transitions (A5, 831 cm-l;

dinated nitrate has the C

E', 1390 cm-1; and E', 790 cm-l), whereas the coordinated

nitrate group (sz) has six infrared active band (A1,

1030 cm'l: 32, 810 cm'l; B1, 1480-1530 cm’l: A1, 1290 cm’l;

1 10a

A1' 740 cm- ; and B , 713 cm-l). Moreover, nitrate com-

1

plexes are known to give rise to combination frequencies
near 1750 and 2400 cm-1. The bands near 1750 cm-1 are mostly

well defined and often strong, whereas those near 2000-2400

cm 1 are weak, often broad and difficult to locate.
Ionic nitrate (D3h) exhibits one combination band in
1700-1800 cm-1 region whereas, in the coordinated nitrate

group, two combination bands are observed. Some research-

1

ers view 1700-1800 cm' region very important to differen-

tiate between ionic (D3h), mono-and bidendate (C2v) nitrate

groups.25’26

55

The assignments of the nitrate absorption bands in the
infrared spectra of lanthanide cryptates is based upon the
notations used by Faster and Hendricker. The same notations
have been used for the interpretation of the nitrate infra-
red data of lanthanide crown complexes by King and Heckley

and Duyckaerts and coworkers.16'17

(i) Infrared Spectra of [Ln(cryptand ; or 4)](NO3)3

Ln = 14.2.6312-

 

Figure 4a and 4b display infrared spectra of the ali-
phatic Ln(III) cryptates (Ln = EE'.EE' E£)° Infrared
spectra of the free macrocycles are also included for
comparison purposes.

The free cryptands (3 and 4) exhibit (C-O) bands at
1130 cm—1. Upon cryptate formation these absorptions are
found at lower frequencies as broad, unresolved bands.
The frequency of this (C-O-C) band in [Ln(cryptand 3)]
(N03)3 cryptates is 1072 cm-1 (Ln=La), 1075 cm-1 (Ln=Ce),
and 1072 cm-1 (Ln = Pr). In the cryptates formed by cryp-
tand 4, [Ln(2:2:2)](NO3)3,the (C-O) stretching bands are
recorded at 1087 cm'1 (Ln=La), 1085 cm”1 (Ln=Ce) and 1082
cm-1 (Ln = Pr). The shifting of this band towards lower
energy has been used as one of the diagnostic signs of
complex formation in cryptates and crowns.

The absence of any water band in the 3600-3200 cm-1

1

region and of any nitrile bands at 2200-2600 cm- regions

56

Figure 4. Infrared spectra of (a) [Ln(2:2:l)]3+

13+

cryptates and (b) [Ln(2:2:2) cryptates

57

 

[M220]

 

 

 

[Ln(2=2=l)]

I I I
I400 1200 1000 000 700

WAVENUMBER(CM")
4a

 

 

 

 

 

 
  

 

 

 

 

 

 

 

 

 

 

 

 

 

[Pr(2=2~2)]y

[08222)]?

[Lo(2=2-2)]3*

l

58

 

 

 

\\

 

 

 

 

I

 

B

Q

T?

 

2 :5

 

 

 

 

 

 

I400

I200

l000

WAVENUMBER (CM")
4b

800I 700

 

59

provide evidence that these cryptates are anhydrous and
free of solvent CH3CN.

The La, 93, and BE complexes derived from the cryptands
(3) and (4) show nitrate (NOS) absorptions corresponding
both to free (D3h) and coordinated (sz) nitrate groups.
The assignments of the nitrate bands recorded in Table 3
are based upon D3h and sz symmetry groups.
1)

Table 3. Assignment of Bands due to Nitrate ion (cm-

 

 

 

Assignment [Ln(2:2:1)](NO3)3 [Ln(2:2:2)](NO3)3
La Ce Pr La Ce Pr

v2 (03h) 1360 1360 1362 1380 1381 1380
v3 (sz) 818 813 815 832 835 835
v5 (sz) 738 735 735 752 760 758

 

 

(ii) Infrared Spectra of [Ln(2B:2:1)](NO3)3 Where
Ln = Lav P_r_v 3.31%! 22:921.: 119.: Ex: Er Ear

m. :2. and 22-

The infrared spectra of all lanthanide complexes show
marked changes compared to those of the pure cryptand
28:2:1 (5). The spectra obtained may be divided into three
types. Those of the EEFEE spectra are quite similar, but

different from the closely related spectra seen for gg-Qy,

60

which in turn are different from the spectra seen for Eg-
92° The infrared spectra within a given class, are in-
dependent of the lanthanide ion indicating that the metal-
cryptand interactions are essentially identical within the
experimental limitation of the infrared spectra.

No bands which could be attributed to water or any other
solvent, e.g., CH3CN are found in any of the spectra, es-
tablishing that the complexes are anhydrous and unsolvated.

The free cryptand (5) exhibit two different v(C-O)
bands at 1123 cm.1 (aliphatic Hzc-o-CHZ) and at 1253 cm-1-
(aliphatic-aromatic ether linkage; CHZ-O-benzene-CH).
These bands in all three classes of cryptates, are shifted
to lower frequencies by 50-60 cm'l. This lowering of energy
results from the metal-ether oxygen interaction upon cryp-
tate formation and is similar to that reported for all crown
and cryptate complexes. Assignments of the (C-0) absorp-
tions are presented in Figure 5.

The frequency of the C-H out-of-plane vibrations is
determined by the number of adjacent hydrogen atoms on
benzene ring. The ortho disubstituted benzene ring spectrum
shows these C-H deformation bands at 770-735 cm'l. The
C-H rocking modes of benzene ring in the free cryptand (5)
are recorded as a strong band centered at 738 cm-1.

Infrared bands due to nitrate groups in the three classes

of lanthanide (III) nitrate cryptate complexes indicates

the presence of only coordinated nitrate ligands. The

61

Figure 5. Infrared spectra of [Ln(2B:2:1)]3+

cryptates.

62

[Eu(2e=2zn]3’

[Pr(2£3=2:n]3+

    
 

[Lease-.0]?

 

 

 

23=2=l

 

 

I I I I
I600 I400 I200 I000 000 700

WAVENUMBER (CM")

 

63

 

[I.u(253=2=I)]3+

 

[Ho(2B=2=I)]3*

  
   
   
    

 

[0y(28=2 =I)]3*

 

 

 

 

 

[Gd (28:20)]?

 

 

 

 

I I I ' I
l700 I600 I400 I200 I000 000 700

WAVENUMBER (CM")

 

 

64

conclusion of nitrates all being coordinated to the metal
ion is based upon the fact that even if one nitrate of D3h
point group is present, along with coordinated nitrates,

1

there should be an absorption band around 1350 cm- plus

the band near 1300-1310 cm"1 due to coordinated nitrates.11a—e
This is also in agreement with the assignments of numerous
authors who have prepared various oxygen- and nitrogen-

11b-c,27

donor complexes of lanthanide nitrates. The assign-

ments of the nitrate bands recorded in Table 4 are based

upon the symmetry type sz point group (coordinated NO3

group).

(c) Proton-NMR Spectra:

Proton-nmr spectra were investigated only for the
lanthanide 28:2:1 complexes. Spectra of free and complexed
benzo-cryptands show noticeable differences. The spectrum
of diamagnetic lanthanum provides an evidence of a tight
complex made by encapsulation of metal ion by cryptand
28:2:1 (é). Spectra of some paramagnetic cryptates are

also recorded.
(i) Proton NMR spectra of Lanthanide 2B:2:1 Cryptates:

Proton nmr spectra of free cryptand (g) and its lan-
thanum (III) cryptate complex are presented in Figure 6.

Cryptate formation causes downfield shifts in all proton

65

Table 4. Vibrational Modes for C v Nitrates in Ln(2B:2:1)

 

 

 

 

 

2
(N03)3.

Ln v1 (A1) v4 (82) v2(Al) v3(Bl) v5(A1)
La 1470 vs 1315 vs 1055 s 813 m 771 m

Pr 1469 vs 1321 vs 1039 s 820 m 772 m

Nd 1460 vs,br 1315 vs 103985 818 m 755 m,br
Sm 1465 vs 1317 vs 1038 s 815 m 756 m,br
Eu 1469 vs 1318 vs 1023 s 817 m 751 w

Gd 1465 vs 1313 vs 1028 s 819 m 758 m, br
Tb 1469 vs 1315 vs,br 1028 s 818 m 755 m

Dy 1468 vs 1311 vs 1028 s 815 m 765 m

Ho 1463 vs 1318 vs 1028 s,br 812 m 745 m

Br 1460 vs 1322 vs 1031 s,br 813 m 742 m
Tm 1468 vs 1315 vs 1029 s,br 811 m 745 m

Yb 1475 vs,br 1318 vs,br 1020 811 m 760 m

Lu 1470 vs,br 1320 vs,br 1028 812 m 749 m

v = very, s = strong, br = broad, m = medium; and

w = weak.

66

[I.c:(2I3=2=I)]3+

 

 

 

 

 

I I It I I I L
7.5 7.0 W 5.0 4.0 3.0 2.5
[ppm]

 

 

 

.. v'o Ja/Ha

 

 

[ppm]

Figure 6. 1H-NMR spectra of cryptand 28:2:1 and its
lanthanum complex in CD3CN.

67

resonances. The proton nmr spectra of other diamagnetic
cation cryptates (alkali and alkaline earth), also exhibit
similar downfield shifts of the various methylene proton
resonances.28

The proton nmr spectrumcflfthe diamagnetic La (28:2:1)3+
provides strong evidence that the metal ion indeed resides
in the central cavity of the molecule. The four aromatic
protons in the free cryptand are recorded as an A282
doublet. Upon encryptation, the metal ion coordinates
with the oxygens attached to benzene ring and holds it
tight with a net result that the aromatic protons are
found as AA'BB' symmetric multiplets, centered at 7.15
ppm. This downfield shift in aromatic protons resonance
from 6.90 (in free cryptand) to 7.15 (in complex) is caused
by cryptate formation. The remaining methylene protons
are divided into two distinct groups. Both of these groups
are shifted downfield relative to their resonances in free
cryptand. The N-_C_2_§l2 protons are located between 2.58-3.62
ppm whereas the 0-932 protons are found in the range of
3.81-4.86 ppm. All of these methylene protons are split
into complex multiplets.

The proton nmr spectra of some paramagnetic lanthanide
cryptates are also recorded. They show induced paramag-
netic shifts in the proton nmr spectrum of the ligand.

The proton paramagnetic induced shifts are essentially

dipolar in origin.29'30

68

lH-nmr spectrum of Pr (2B:2:1)3+ is displayed in

The
Figure 7. The pure cryptand exhibits peaks only between
2.58-4.86 and at 6.90 ppm, whereas, the Pr (III) cryptate
exhibited peaks due to different protons between -23.4-21.4
ppm. There were a total of thirteen signals recorded,
ten of them were of single intensity and three of double -

intensity. One would expect sixteen peaks due to thirty-

two protons of the molecule, because the molecule has a I

 

plane of symmetry.

An attempt was made to assign some of the signals
observed in the spectrum. The more expanded and resolved
spectrum revealed that the signals #5, 6 and 9, of double
intensity, are singlet, singlet and a triplet, respectively.
Moreover, signals 7 and 10, of equal intensity, are very
much similar to each other and are distorted quartets.
The signals 7 and 10 are relatively sharp and located at
1.48 and -2.68 ppm. It is our opinion that these two
signals belong to the two sets of aromatic protons. It
is known that the coupling pattern of aromatic protons
are relatively less effected by the paramagnetism present

in the molecule.31

The presence of two singlets and one
triplet in the double intensity signals excludes the pos-
sibility of assignment to methylene protons of benzo

strand or the aliphatic-Z-strand. For these strands, all

three double intensity signals should be triplets. Probably

signal 5 and 6, located at 13.41 and 11.93 ppm, belong to

69

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7 9 IO
3 UL '2 I3
I L... 1 I
_J I 11 I I
300 -3.0 H -I6.6 -23.4
[ppm]
+ 5
[Pr(28321l)]3 6
4
A M
l w ' I L
22.0 ”.0
[ppm]
1

Figure 7. H-NMR spectrum of a deuterated acetonitrile

solution of [Pr(ZB:2:1)]3+.

70

four protons of 0-932 and four protons of N-_(_2_I_-I_2 of 1-
strand. The third double intensity signal (triplet),
found at -l.0 ppm, probably may be assigned to four protons
of an N-Qflz of a benzo-2-strand or an aliphatic-Z-strand.

A similar proton nmr spectrum of [Eu(28:2:1)]3+ is 5-
obtained. The whole spectrum is recorded between -15.0-18.5
ppm. Although the spectrum is very complex in the region

of CHDZCN, which was the solvent, still sixteen signals

 

PIC-DH.“ 'L‘_-. III—1 ~ .139.

of equal intensity are recorded. The peaks are found at
18.45, 17.14, 13.40, 10.12, 7.3, 6.93, 5.36, 4.62, -5.92,
-6.38, -8.64, -9.19, -9.56, -12.79, -14.17, and -15.18.
They all are of equal intensity which accounts for all

thirty-two protons of the molecule.

(ii) Proton NMR Spectra of Lanthanide 28 :2:1 Cryptates

NH2
The general features of the spectra obtained are similar

to those of cryptand 28:2:1. The proton nmr spectra of the

diamagnetic lanthanum complex causes downfield shift in

all the ligand protons of complex. Free cryptand, in

solution exhibits three aromatic protons at 6.10, 6.26,

and 6.62: whereas they are found at 6.23, 6.43, and 6.87

ppm, respectively in La(2BN :2:1)3+ in CD3CN. The N-ggz

H

2
methylene protons are recorded between 2.58-3.57 in the
complex spectrum and the O-Qfiz methylene protons are found
in the region 3.88-4.78 ppm. All of the methylene

proton signals are multiplets.

71

Praseodymium metal ion causes the paramagnetic shifts

in the proton nmr spectrum of Pr(ZBNH :2:1)3+ and are dis-

played in Figure 8. The peaks are logated at 19.80, 19.37,
15.44, 14.80, 14.34, 13.88, 13.68, 13.31, 12.99, 12.81,
12.63, 11.89, 11.38, -15.08, -16.14, -21.23, and -22.52 ppm. r“
The Eu(ZBNH2:2:1)3+ complex is not very soluble in CD3CN.
However, the proton nmr spectrum of a dilute solution ex-

hibits the induced shifts in the proton signals. The whole

spectrum is recorded between 25.17--16.87 ppm.

HI.

 

‘qc‘..‘| ' A:

The paramagnetic shifts obtained by the paramagnetic
cryptate complexes verify the metal ion residing in the
cavity of the cryptand. If the metal ion is not resided
in the cavity and just from an adduct, no appreciable
induced shifts in the ligand spectrum are observed.16
The proton nmr spectra reported for some lanthanide 313
complexes by Duyckaerts and coworkers, did exhibit the
induced paramagnetic shifts in methylene protons of the

ligand and is presented in Figure 1 (in Chapter I).

3.3.3. Electrochemical Studies

 

Within the lanthanide series, the variation of III/II
oxidation states is observed only for Eu3+/Eu2+, Yb3+/Yb2+
and perhaps Sm3+/Sm2+. The standard electrode potential
measured for these couples is -0.35, -1.15 and -1.55 volts,

respectively.32

72

 

WJ
I I I

-I508 -I6.I4 -2I.23 -22.52

 

 

[Pr(ZBNHé2=I)]3+

 

 

20 l0
[me1

1

Figure 8. H-NMR spectrum of a deuterated acetonitrile

solution of [Pr(2BNH :2:1)]3+.
2

73

The electrochemical studies reported were performed in
collaboration with Professor Weaver of our Department.

3+ has a marked effect on the

The encryptation of Eu
electrochemistry of this ion. The cyclic voltammograms
of Eu3+(aq)/Eu2+(aq) are electrochemically irreversible.
When the metal ion is encryptated, the oxidation-reduction
becomes electrochemically reversible. The cyclic voltam-
mogram serves as evidence that the metal ion resides in
the molecular cavity, resulting in reversible oxidation/
reduction.

Cyclic voltammograms of Eu(2B:2:1)3+/Eu(2B:2:l)2+

+
2 couple are re-

couple and Eu(ZBNH :2:l)/Eu(ZBNH :2:1)
2 2 2+

corded in Figure 9 along with the aqueous Eu3+/Eu couple.
The mean potential between the cathodic and anodic peaks in
Figure 9(a) -625 mV vs S.C.E. in 0.5 M NaClO4 is identified
to be the formal potential Ef for Eu3+(aq)/Eu2+(aq). In
contrast, Ef for Eu3+(28:2:1)/Eu2+(28:2:1) couple has been
determined to be -370 mV vs. S.C.E. in 0.5 M NaClO4. The
Ef for Eu3+(ZBNH2:2:1)/Eu2+(ZBNH2:2:1) couple measured is
-365 mV vs. S.C.E. in 0.5 M NaClO4. For a couple to be
electrochemical reversible, the separation of cathodic
and anodic peaks should be close to 57 mV at room tempera-
ture.33
The separation measured between the cathodic-anodic

peaks for the couples displayed in Figure 9 is close to the

value of making them electrochemically reversible.

74

 

 

 

 

 

(D _

'L' (2)

I .I
o —
-| .- .
3+ 0 0 2+
[Eu(zam-ézn] /[Eu(zaM52.I)]
I I . I I . I 1 .1 I
U) I
2 '— .—
(2)

I I" —I
O b ..
-l b Eu3+(oq)/Euz‘(oq)

' I I I l I -l I J_
'(IU'A) 2100 400 600 800 90C
-E (mv. vs. S.C.E.)
2 I...
(I)
'" (2)
, ..
O I—
4_ . 3+ _ . 2..
[Eu(2azzzl)] /[Eu(28:2:|)] "

 

Figure 9.

 

3+

Cyclic voltammograms of Eu (aq). [Eu(ZB:2:l)

+
and [Eu(ZBNH2:2:1)]3 cryptates.

13+

75

:2:1)3+ Show only
C’2
an extremely intense reductive peak. Probably, the electron

The cyclic voltammograms of Eu(ZBN

loss upon oxidation is taken up by -NO substituents on

2
benzene ring resulting in a chemical reaction so no oxida-
tive peak is observed.

Electrochemical studies of Yb3+(2B:2:1)/Yb2+(2B:2:1)
also gives similar results. The mean potential, Ef, for
this couple is measured. The Ef in 0.5 M NaClO4 solution
is determined to be -1100 mV vs. S.C.E.

These cyclic voltammogram measurements provide ac-
tually a proof that most likely, the metal ion is resided

in the intramolecular cavity making electrochemically

reversible couple of III/II oxidation states.

3.3.4. Analytical Measurements

The quantitative microanalyses were performed to compli-
ment the results obtained by the above mentioned methods.
The C, H analytical data of some lanthanide cryptates

of cryptand (3-5, 21, 8) are presented in Table 5.

76

 

 

 

Table 5. Analytical data on Some Ln (Cryptand 3—5, 21, __
(N03)3o
%C %H

Ln Cryptand Calc Found Calc Found
La 3 29.20 29.47 4.86 5.05
Ce 3 29.15 28.98 4.85 4.91
Pr 3 29.12 29.24 4.84 5.00
La 4 30.79 30.68 5.13 5.15
Ce 4 30.74 30.56 5.12 5.19
Pr 4 30.70 29.91 5.11 5.06
La 5 34.04 34.19 4.53 4.48
Ce 5 33.98 33.72 4.52 4.55
Pr 5 33.95 32.01 4.51 4.32
Ce 27 31.95 31.80 4.12 4.22
Eu 27 31.45 31.10 4.06 4.30
Pr 28 33.24 32.24 4.57 4.57
Eu 28 32.74 32.99 4.50 4.60

 

 

 

CHAPTER IV

DIAZO COUPLING OF LANTHANIDE BENZO CRYPTATES

TO PROTEINS

4.1. Introduction

The fact that cryptands may be functionalized during
or after their synthesis has not been extensively investi-
gated. The functional groups incorporated in cryptands
and cryptates could easily proyide a point of covalent
attachment, "a chemical hook", which could be used to bind
the cryptate to a macromolecule, for example a protein, an
enzyme or a steroidal hormone. Cryptand ZBNH2:2:1 (28)
is the first functionalized cryptand prepared which pro-
vides an amino group useful for attachment of a cryptate

on a macromolecule. The amino group of ZBN :2:1 may be

H
diazotized and coupled to a protein or an eniyme.

The coupling of proteins with diazonium compounds has
been studied extensively and has important applications
in immuno-chemistry, chemotherapy and other fields. Diazonium
compounds have been used to modify proteins, to study their
composition and structure, and to investigate their rela-

34-38 Diazonium compounds

tionship to functions of enzymes.
couple most smoothly with tyrosyl and histidyl residues

of proteins. Upon coupling with proteins, usually a visible

77

 

78

color is induced thus providing a way to analyze for the
attachment process.

There are a variety of diazonium reagents which have
been used to couple and modify proteins. Valle and Kagan39
observed that by coupling of Carboxypeptidase A with p-
azobenzenearsonate produced an absorption spectrum indica-
tive of arsanilazotyrosine. That was latter confirmed by
amino acid analysis and by measuring total protein bound
arsenic using Atomic Absorption Spectroscopy. Moreover,
they found that physicochemical properties of the deriva-
tive were essentially the same as those of native Carboxy-

h40 treated a variety of

peptidase-A. Valle and Faircloug
proteins with diazotized p-arsanilic acid to produce azo-
proteins. They found that both the azoproteins and the model
monoazotyrosyl derivatives exhibited absorption maxima
near 325 nm. Therefore they concluded that the bands
between 320-340 nm in the a20proteins arose from transi-
tions associated with the monoazotyrosyl chromophore in
proteins. This conclusion was strengthened by data ob-
tained with a poly-L-tyrosine polymer which contained azo-
tyrosyl chromophores and exhibited the expected absorption
maximum at 325 nm.

In another extensive study of affinity labelling of the
active site of staphylococcal Nuclease, P. Cuatrecasas

41 that this nuclease reacts stochiometrically at

reported
tyrosine-115 with the diazonium reagent (deoxythymidine-3'-

p-amino-phenylphosphate-5'-phosphate). Ultraviolet and

 

79

visible spectra of the nuclease and the model azo compounds
were compared. The spectra showed an absorption maximum
near 340 nm which suggested the presence of an azotyrosyl
moiety. The presence of an absorption maximum near 380

nm would be indicative of an azohistidyl moiety whereas

the absorptions at 330 and 420 nm would indicate the pres-

36’41 In another independent

42

ence of bis azotyrosyl moiety.
physicochemical study of azoinsulins done by W. Koltun,
he found an absorption maximum at 340 nm. It corresponds
closely to the absorption region of tyrosinemonoazo deriva-
tive whereas a shoulder at 380 nm corresponds to the ab-
sorption region of histidinemonoazo derivatives.

The covalent attachment of metal complexes to bio-
molecules has not been extensively explored. Attachment of
metal complexes is a promising new area of research because
metallic ions exhibit a wealth of spectroscopic and radio-
active pr0perties. The fact that EDTA is a strong chelating

43 to synthesize

agent inspired M. Sundberg and coworkers
an agent based on EDTA that can be used as a labelling re-
agent. A molecule which could be attached to biological
molecules and also complexed by metal ions might act as a
probe of the biological molecule due to the properties of
metal ions. These researchers used l-(p-aminophenyl) EDTA
(22) as a labelling reagent. After diazotization of amino

group they coupled to a variety of proteins. Upon covalently

attaching this reagent (32) to a human serum albumin for

 

80

 

 

 

 

 

 

 

 

 

“J
k)
2!
<1
CD
(I
0
GD
4 .
300 420 540
p.

 

ABSORBANCE

 

 

 

1 l l

 

Figure 10.

l
. 260 300 340 380 420 460 500 540 580

Mnm)

T

R- 'I’ ’ 9
- °'-P\ \P‘O‘O’NZ
I, a: &

0

Ultraviolet spectra of an Azo-tyrosine, an
Azo-histidine and an Azo-Nuclease.

81

1111n3+

example, radioactive ions were added in the pres-

3+

ence of a buffer. Thereafter the lllxn ions were com-

plexed by the chelating groups of the labelling reagent in

conjugated albumin.44

After diazotization and coupling of
(39) to human serum albumin and bovine fibrinogen, the pres-

ence of any azotyrosine in conjugated proteins was checked

‘ I -' ‘u‘ds‘!’

by viewing their absorption spectra. The presence of azo—

tyrosyl moieties was indeed indicated by an increased

 

absorption at 330 nm.45
HOOC- -v
- CH2\ /CH2" COO” L3"
N-C H - CHZ- N
HOOC-C ’ ‘
“2 © “'2' coon
NH. (22)

Preliminary accounts of diazotization and coupling of

cryptand ZBNH :2:1 (28) and some of its lanthanide cryptates
2 .__
are presented in this chapter.

4.2. Experimental

Materials:

 

i-Amyl nitrite (CSHllONO) was purchased from Pflatz
and Bauer, Inc., Stamford, CT and was used as received.
Bovine Serum Albumin and Ribonuclease A were obtained from
Sigma Chemical Co., St. Louis, MO. The gel Sephadex G-25

was purchased from Pharmacia Fine Chemicals, Piscataway,

82

NJ; and the buffer PIPES was obtained from Calbiachem,
LaJolla, CA. All other chemicals were either reagent

grade or the best grade available.

Methods:

Diazotization of cryptand ZBNH2:2:1 was carried out only
in non-aqueous medium. The use of NaN02 as diazotization
reagent was avoided because of the fact that sodium cation
may form the complex with the cryptand. However, the diazo-
tization and coupling of lanthanide cryptates of ZBNH2:2:1
were done in aqueous solution using NaN02 as the diazotiz-
ing reagent.

Sephadex column was prepared to separate the proteins
from other small molecules. 10 gms of Sephadex G-25 was
weighed in a beaker and covered with water. The gel was
left soaked to swell overnight. The swollen gel was
packed on a 60 x 2.5 cm column to separate the proteins.
After each use, the gel was restored by washing five times
with water.

A 10‘2

M PIPES Piperazine-N,N'-bis(2-ethanesu1fonic
acid) buffer stock solution was made and pH was adjusted
to 6.50 by adding appropriate amounts of 0.1 M HCl. This
solution was used to elute the protein from Sephadex column.

The uv-visible measurements were made by using a Cary-l7

spectrophotometer. Radioactivity was measured by using a

Na(I) y-ray scintillation detector connected to an "Eber-

1ine" scalar-high voltage supply.

 

83

A. Preparation of diazo cryptand (30) and coupling to

 

N,N-dimethylani1ine:

 

Diazotization:

 

One hundred mg of cryptand 2BNH2:2:1 were placed in a r~
round bottom flask with a side arm. The flask was evacuat- 0
ed and kept under nitrogen atmosphere. 10 ml of THF was
added to the flask and the mixture was cooled (5-10°C)

with an ice bath. To this cold stirred mixture, 3 m1 of

 

i-amylnitrite was added dropwise. After 10 minutes a
white precipitate formed. The stirring was continued for

another 10 minutes while the mixture was kept cold.

Coupling:

Sixty mg of N,N-dimethyl aniline was dissolved in 5 ml
of THF. This solution was added dropwise to the above
diaztotized cold mixture over a period of 15 minutes. The
stirring was continued and the temperature was raised
slowly to room temperature. An orange-brown colored solu-
tion was formed during this period. After 2 hours the
stirring was stopped and the solvent was removed by rotory
evaporation, yielding (31), a dark orange-brown paste
(70 mg, 55%): u.v. (CHBCN) Imax at 290 and 375 nm; 1H-
NMR (CDBCN) 62.27-3.19 (18H of N-gflz and N-gia, 2 broad
singlet), 3.33-4.27 (16H, O-gflz), 6.18 (lHA,d), 6.7 (1HX.,d),
6.98 (lHM,d), 6.27 (2Hx.,d), and 7.33 (2HA.,d).

84

B. Diazotization of [La(2B :2:1)13+ and coupling to

 

N H
2
Bovine Serum Albumin:

 

Diazotization:

 

One mg (1.5 um) of [La(2B :2:l)](NO3)3 was dissolved

NH2 r~

in two ml of deionized water in a 50 m1 volumetric flask. .

The flask was placed in an ice bath and the solution cooled I

 

to 10-15°C. While stirring, 2 drops of concentrated HCl

3

and then 2 ml of 10- M NaNO2 stock solution were added

 

dropwise over a period of 10 minutes. After the addition was
complete, the stirring was continued for another 15 minutes.
Then stirring was stopped and the very light brown solution

(pH 3 l) was left in the ice bath.

Coupling to Bovine Serum Albumin

 

One hundred mg of protein was dissolved in 15 m1 of
water. This viscous solution was added slowly to the above
diazotized cold salt solution. The stirring was continued
for 1/2 hour at low temperature (10-15°C) and then one hour
at room temperature. During this time, the color of the
solution turned yellowish orange from very light brown. The
pH of the solution at this time was between 1-2. The yellow
colored solution of labelled B.S.A. with cryptate is analyzed
by its ultraviolet-visible spectrum; u.v. (H20) 1 at

max
270, 275, and 335 nm.

85

 

C. Diazotization of [Pr(2BNH2:2:l)1(NO3)3 and Coupling to

Ribonuclease-A.

 

Diazotization:

 

Ten mg of [Pr(2B :2:1)](NO ) was dissolved in 3 m1
NH2 3 3

 

of water in a 10 ml flask. The solution was cooled to 10- EE‘

15°C by an ice bath. While stirring, 2 m1 of 0.01 M NaNO2

stock solution and a drop of l M HCl was added to the flask.

The stirring at cold temperature was continued for another ';
y

25 minutes.

Coupling:

One hundred mg of Ribonuclease-A was dissolved in 5 m1
of water and the solution was cooled. To the cold protein
solution, diazotized salt solution was added dropwise. The
stirring was continued for 15 minutes at cold temperature
and another 2 hours at room temperature. A light yellow
colored solution was obtained which was analyzed for azo
linkage by taking u.v. spectrum of the yellow solution:
u.v. (H20) Amax at 276 and 330 nm.

NOTE: Same procedure was employed when radioactive

praseodymium cryptate was diazotized and coupled to Ribo-

nuclease-A.

86

4.3. Results and Discussion

The synthesis of a functionalized cryptand which on
one hand can encapsulate the metal ion inside its cavity
and on the other hand may interact, in some defined manner,
with biomolecules may in fact add new dimensions to the

44

field of bioinorganic chemistry. The cryptand 2BNH :2:1

2
and its lanthanide complexes may provide a series of sys-
tematically varied stable complexes which could be co-

46

valently attached to a macromolecule. The complete

details of the synthesis of cryptand ZBNH2:2:1 and some of
its lanthanide complexes have been described in Chapter II
and III of this thesis.

Diazotization and coupling of aromatic amines are
mostly carried out in aqueous solution using NaNO2 as

47 However, when diazotization has

diazotizing reagent.
to be carried out in non-aqueous solution, pentylnitrite
is employed usually as diazotizing agent and absolute
ethanol, benzene or THF may be used as a solvent.48'49

The reaction of aromatic diazonium ions with amino
acids or proteins possessing phenyl and imidazole rings
has long been known as a method of introducing an azo link-
age.50 The azo group is an intense chromophore that can
be attached to any molecule possessing the above reactive

functionalities.51

Exclusive substitution by one azo group
at the ortho position of the phenyl ring in tyrosine can

be achieved by the action of one equivalent of a diazotized

 

87

amine.52

 

z-z:z:

The aromatic amino group of cryptand ZBNH :2:1 or its
metal complexes may be modified through diazotization to
form covalent labelling reagents. The amino group of

ZBNH :2:1 is diazotized in non-aqueous media by using i-
2

amyl nitrite.

Of—‘E>

<55” 115°“
NH2 fi2
ZBNH2:2:I (2Q _3_Q

The diazotized species (29) is then coupled with N,N-
dimethyl aniline producing a highly colored molecule

(31). The proton nmr spectrum of this coupled dye is

displayed in Figure 11.

*1:

 

fl‘

88

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

H -1
HA: [—A‘. '7
(“*1
H w H
x x
IL" I olipho c O-CH2
] r——- —_
) N'CHE* ‘MCH31‘,
I .@E°°“"
‘0 L 0042
730 J IIII J l l l
- , 6 00 5.00 4 00 30° 2 00
[com]

Figure 11. lH-NMR spectrum of _3_]_._.

89

27)
o)
,I

 

‘03
0

i
oég
@I

:z=2:

n/
3:
(fl

 

|VEIK1IT£§W~ . "V. . . '

The lH-nmr spectrum of the coupled molecule (31) displays
the aliphatic methylene protons of either N-CH2 or o-CH2

as broad singlets. The N-CHZ protons of the cryptand moiety
and the six protons of N-CH3 of aniline fall between 52.27-
3.19. The O-CH2 protons are divided into two sets as is
seen in the cryptand itself. Four O-CH2 protons of benzo
strand are downfield relative to the aliphatic O-CH2
protons. All O-CH2 protons are displayed between 63.33-4.27.
The aromatic protons of the original cryptand are found as
an AMX set of three protons between 66.10-6.62. In the
coupled product, there are two different sets of aromatic
protons, an AMX set of three protons from the cryptand

moiety and four protons of an AéXé set from the aniline

90

moiety. Two doublets due to AéXé protons are found at

6.27 and 7.33 ppm whereas the three remaining aromatic
protons of AMX set fall between 6.13-7.19 ppm.

3+ (32) is carried

The diazotization of La(2BNH2:2:l)
out in aqueous solution. Sodium nitrite produces the
diazotized salt (33) which is then coupled to Bovine Serum ES:
Albumin. As the reaction of coupling proceeded, a yellowish -

orange coloration was induced into the solution.

 

roan row?

N .o\/‘N

Lo OJ \V0 0

(35.5 G}..-

nu, N20
(3 ) (3_3)

 

l‘\
— a m

91

The u.v.-visible spectra of Bovine Serum Albumin and the
modified protein are taken in aqueous solution. The protein
has only one absorption in ultraviolet region with Amax at
278 nm. The spectrum of the modified protein displays

three absorptions with A at 335, 275 and 270 nm. There

max
is no absorption above 500 nm. According to the litera-
ture, the absorption due to monoazotyrosyl residue in pro-
teins is found around 340 nm. It may be said with certainty
that the absorption found at 335 nm in the modified Bovine
Serum Albumin is due to the transition associated with
monoazo tyrosyl chromophore in protein. The absorption

spectra of protein and modified protein are displayed in

Figure 12.

A-B°S'A

B-FLaI2BN =20"

 

 

 

 

ABSORBANCE
7,2.

 

    

A

.I -

1__ l _L._.
240 300 400 500‘
WAVELENGTH (rim)

Figure 12. Ultraviolet spectra of Bovine Serum Albumin
and labelled Azo-B.S.A.

 

 

 

 

 

92

Coupling of Ribonuclease-A with [Pr(2B :2:1)J3+

NH2

cryptate also generates a visible colored solution. The
u.v.-visible spectra of native and labelled enzyme were
taken in aqueous solution. Ribonuclease-A has only one
absorption in ultraviolet region with Amax at 276 nm. The
spectrum of the labelled enzyme displays absorptions at
276-278 nm and 330 nm. The absorption spectra are shown
in Figure 13. The absorption at 330 nm in the labelled
enzyme spectrum is indicative of covalent attachment
through azo bond formation.

The question, whether the metal ion remains encapsulat-
ed in the cryptate cavity after the covalent attachment
on macromolecule, was answered by preparation of a radio-
active cryptate complex. The [Pr(2BNH2:2:1)]3+ was pre-
pared, diazotized and coupled with Ribonuclease-A.

The radioactive labelled enzyme was separated from
unlabelled enzyme, unreacted metal cryptate and other
smaller molecules by eluting from a column of a biogel,
Sephadex G-25. The biogel holds the molecules to a vary-
ing extent depending on their size and shape, hence mole-
cules are eluted in order of decreasing molecular size.

More than thirty fractions (each fraction m2 ml) were
collected and analyzed for their radioactivity and ab-
sorption in ultraviolet region. The Figure 14 displays
the results.

It is apparent from Figure 14 that the tube which

 

 

93

A . Ribonuclease - A

B . [Pr(28'=2=l)]3+

 

 

 

 

 

 

I.

I

g

m

o

2

:5;
UJ
o
<2, I:
00
(r
C)
U)
00
<1

\ A
B
I I J
230 250 300 330
WAVELENGTH (nm)
Figure 13. Ultraviolet spectra of Ribonuclease-A and

labelled Azo-Ribonuclease-A.

 

94

 

 

CPM
A330
/
6 _
10 Tab. N012 14 lb . 18 20 30
Figure 14. Plot of ultraviolet absorption (at 330 and

276 nm) and radioactivity (cpm) vs tube num-
bers carrying the labelled and unlabelled
Ribonuclease-A.

 

95

carries the highest radioactivity has the highest ab-
sorption at 330 nm (azo linkage) and at 276 nm (protein
moiety). Moreover, tubes which have only unlabelled
enzyme (absorption at 276 nm only) do not carry any
radioactivity. This indicates the absence of any kind
of interaction between protein and the metal ion.

It may be concluded with certainty that the metal
ion remains inside the cryptate cavity even after coupling
with protein. Biomolecules can therefore be labelled
this way through covalent attachment and the metal ion
inside the cryptate cavity can act as a probe for spectros-

copic purposes or as a radiotracer.

 

REFERENCES

 

10.

11.

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98

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