'A^C MM *Äi - DTICdole, but indole -3-acetic acid is an order of magnitude more effi- cient than tryptophol. The brightest indoles are all ^-substituted. Since 2-substi- tution reduces

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DISCLAIMER NOTICE

THIS DOCUMENT IS THE BEST

QUALITY AVAILABLE.

COPY FURNISHED CONTAINED

A SIGNIFICANT NUMBER OF

PAGES WHICH DO NOT

REPRODUCE LEGIBLY.

MRB3009Q3

Technical Report No. 5

'he

Office of Naval Research and

Advanced Research Projects Agency ARPA Order No. 299, Amend. 6

Contract Nonr 4511(OO) Task NR ."556-46^

CHEMILUMINESCENT SYSTEMS

Monsanto Research Corporation Boston Laboratory

Everett, Massachusetts 02149 Tel: 617-389-0480

30 September 1965

Reproduction In whole or In part Is permitted for any purpose of the United States Government.

• MONSANTO RKMANCH CORPOIC.TiON •

*mnmtmMmm

ABSTRAGT

r

>5axlmum brightness and efficiency of the chemllumlnescent autoxjdatlon of Indoles In basic ..'olutlon in polar anrotlc sol- vents is found for the 5- and ö-substltuted skatoles. 2,3-Di- methylindole-5-c8rboxylic acid yields greater peak brightness than akatole at the standard 5 x 10'^M concentration in DMSO, although with lower efficiency than skatole. The fluorescence spectrum of a basic solution of orthoacetamidoacetophenone, in DMSO, matches the chemilumlnescence emission spectrum of 2,3-dimethyllndole In peak wavelength and contour.

1 11

I • MOMSANTO «KMAWCH COIWOMATtON 0 wu. ■ im—i in, n mwmk.m ''a

TABLE OF CONTENTS

I. INTRODUCTION

II. CHEMILUMINESCEKCE OP INDOLE DERIVATIVES . . .

A. MONOSUBSTITUTED ALKYL INDOLES

B. INDOLES WITH OTHER SUBSTITUENTS IN THE BENZENE MOIETY . .

C. INDOLES WITH OTHER SUBSTITUENTS IN THE PYROLE MOIETi .....

III. INVESTiaATION OP INDOLE CHEMILUMINESCENCE . .

A. SOLVENT AND CONCENTRATION EFFECTS ....

B. THE MECHANISM OP INDOLE OXIDATION ....

1. General Background 2. Discussion ...

C. CHEMILUMINESCENCE AND FLUORESCENCE SPECTRA OP THE INDOLES .....

D. CHEMILUMINESCENCE DECAY KINETICS

IV. HETEROGENEOUS CATALYSIS ...........

A. INTOODUCTION ...............

B. EXPERIMENTAL

V. INSTRUMENTATION

A. INTRODUCTION ......

B. EXPERIMENTAL

VI. FUTURE WORK .....

VII. REFERENCES . .

APPENDIX I

APPENDIX II - ORGANIC SYNTHESIS . .

Page

1

2

2

6

16

16

21

21 21

23

30

34

34

34

37

37

37

40

41

43

48

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• MOM9ANTO »«.«*>»€* Cnn^OHATiO* •

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LIST OP TABLES

i ^e

Chemilumlnrscence of Monosubatituted Alkyl- Indoles In DMSO . . - 3

Chemilumlnescence of Indoles with Substituted Benzene Moiety 4

Indoies with Substltuents on the Pyrole Moiety. 7

Tabulation of Brighter Chemllumlnescent Indolc Derivatives -- In Order of Peak Gross Brightness , 14

Tabulation of Brighter Chemllumlnescent Indole Derivatives -- In Order of Plgure-of-Merit . t 15

Observed Emission Decay Initial Half-Lives . , 19

Chemilumlnescence Parameters of some Indoles in HPT Solvent 20

Uncorrected Chemilumlnescence and Fluorescence Spectra of Indoles ,,.... 24

Fluorescence Spectra of Possible Indole Oxidation Products 28

Decay Kinetics of Indole Chemilumlnescence . . 31

Chemllumlnescent Reaction en Silica Qel Adsorbent 35

iv

• MONSANTO KESCAftCH CORPOHATION

LIST OF FIGURES

Figure . No. Page

1 Concentration Dependence of Peak Brightness . . 17

2 Concentration Dependence of Flgure-of-Merit . . 18

3 S^-Dlsiabstltuted Indole Oxidation Products . . 22

4 Chemlluminescence Spectrum of Skatole and Fluorescence Spectra of Skatole and its Oxidation Products ... 26

5 Comparison of the Fluorescence Spectrum of the Anion of o-Acetamido acetophenone to the Chemlluminescence Spectrum of 2,5-Dlmethyllndoie 29

6 Kinetic Plot of the Chemlluminescence Decay Curve of 3-Ethylindole . 32

7 Kinetic Plot of the Chemlluminescence Decay Curve of 2,3-Dimethylindole 33

8 Chemlluminescence Spectra of Lucigenin in Ethanol Solution and on Silica Qel 36

9 Relative Sensitivity of Spectrometer vs Wavelength 39

« MONfeANTO WCStARCH COflPOftATION •

. I. INTRODUCTION

The objective of thla research Is the discovery of bright chemiluminescent reactions suitable for developrr^nt into practi-

! cal field Systeme. The previous reports in this series have out- lined the results of a survey of fhe chemiluminescent autoxidative

- reactions of a number of different functional classes of organic compounds in basic solution in polar aprotic solvents.

In this report the major emphasis is en the chemlluminescence of substituted indoles. A survey has been made of the influence of substituents on the chemiluminescence parameters for commercially available compounds. A few structurally significant indoles nave been synthesized. VPC analyses have been confined to determina- tions of related compound purities.

A principal result of this study has been the idontificaticn of some classes of substituents and substitutional positions which promote chemiluminescence brightness and efficiency in the autoxi- dation of indole and the identification of a group of indoles with chemiluminescence peak brightness or efficiency of the order of skatole. The compound 2,j5-Cimethylindole-5-carboxylic acid has been synthesized and has been founo to possess greater peak emis- sion brightness than skatole at the standard 5 x 10"3M conc3ntra- tion.

For several of the better indoles, brightness and efficiency parameters have been obtained as a function of concentration and solvent. The chemiluminescence and fluorescence spectra have been determined for additional indoles and the fluorescence spectra of some possible reaction products have been determined. The fluores- cence spectrum of one peak reaction product has been shown to match the chemiluminescence emission spectrum of 2,3-dimethyllndole in peak wavelength and contour.

Preliminary experiments are reported on chemiluminescence spectral shifts observed in the oxidation of lucigenin adsorbed on silica.

i • MONSANTO ««SfAltCH COft^ONATtON •

*

II. CKEMILUMIN^SCENCE OP INDOLE DERIVATIVES

A • MONOSUBSTITUTED ALKYL IND.QLES

To obtain a rational basts for the structural optimization of the chemilumlneBcence of the indoles, we have compared the gross peak brightness of the seven monomethyl compounds and of 3-ethyl- indole. The results obtained are summarized in Table 1. VPC anal- yses of the compounds were performed with emphasis upon determina- tion of the skatole content. Gorrectiont were applied for the skatole impurity assuming additivlly of the peak brlghtnebs at the observed peak. Compared to irdole, the 5- and 6-methyi compounds reveal large flgures-of-merit (PMJ, about an order of magnitude below that of skatole. These large FM values are due largely, how- ever, to long emission decay times rather than to nigh brightness. The skatole impurity content of T-methylindole was not determined. Both the internal evidence (decay time and brightness) and the be- havior of polysubstituted indoles (see following) lead us to sus- pect skatole contamination at the 0.3-0.5^ level. Thus, again compared to indole, the 4- and 7-substitutions have only t? very modest effect on the chemllumlnescence. Both 1- and 2-8ubstitution> on the other hand, depress the chemllumlnescence markedly. The l-^havior of the N-methyl compound is, of course, of special interest since the very low brightness implies that the probable initial step in the oxidation is proton removal, in analogy to the luminol reac- tion (ref. l).

It is clear from these results that the 3,5- and 3»6-dimethyl- indoles and 3,5i6-trimethylindole should be investigated. To ob- tain a standard skatole sample the commercial u'K) material was purified by zone refining (a^O zone passes). The apparent 5^ in- crease in efficiency over the stock skatole is not considered to be significant at present since wide variations have been observed in previous comparisons made at long Intervals (see concentration dependence section following).

B. INDOLES WITH OTHER SUBSTITUENTS IN THE BENZENE MOIETY

With the exception of T-azaindole, all the compounds listed in Table 2 are 5- and 6-substituted. In large part, this reflects the availability of the materials. We have attempted to determine the chemllumlnescence of 4- and 7-benzyloxyindole,but to date have found such extreme variability that the results are not Interpret- able.

The outstanding indoles in this group are the 5-cyano and 5- carboxyl and the 6-benzyloxy and 6-methoxy compounds, all of which are characterized by long decay half-lives rather than by high brightness. The 5-halogen8 show Increasing efficiencies with de- creasing electronegativity. 5-Bromoindole indeed possesses the long decay time of the more efficient members of this class but

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CH^ILÜMINESCKNCE OF INDOLfcS WITO SUBSTriWRD BOß!SIE MOIETV'

Compound &nd Structure

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Pe«k 0» Current

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• MOW»AKTO IIC9CAACH COHPOHATIC*» *

r Table 2

(Continued)

Compound and Structur«

Time to Peak Ot Current 08 Peak

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1 I

• MONSANTO NeUANOH COftPOSATION •

at much lower brightness. The 6-nltrolndole compound Is found to be the least efficient of the Indolea, as may be expected from tue known poor fluorescence efficiency of nltro-substltuted compounds (ref. 2).

C. INDOLES WITH OTHER 5UBSTITUENTS IN THE PYROLE MOIETY

Substitution at the 2-posltlon of an indole derivative reduces the chemllumlnescence efficiency markedly in all compounds Investi- gated.*

A relatively large number of ^-substituted Indoles, particu- larly ska Die derivatives of structure,

CH2R

have been Investigated. T^e results are presented 1 Table 3 and are summarized in order of peak brightness in Table 4a and in order of figure-of-merit in Table 4B. The data generally support the conclusions derived from the monomethyl indole study.

Although quantitative analysis of substituent effects is not feasible, since most compounds of this group are of unkno/n purity, the qualitative effects are consistent for the brighter species. Thus, 5- and 6-substitutlon by methyl» metnoxy and benzyloxy radi- cals Increases the chemllumlnescence efficiency of the lndole-3 acetic acid, as does 5-carboxyl substitution. Similar effects are observed in tryptophan On the other hand, 5-substituti^n ^ amino or hydroxyl groups leads to marked reduction in efficiency.

The effects of ^-substitution are both extremely marked and complex. Thus tryptophan Is far less efficient than 3-ethyl in- dole, but indole -3-acetic acid is an order of magnitude more effi- cient than tryptophol.

The brightest indoles are all ^-substituted. Since 2-substi- tution reduces both efficiency and brightness, an obvious candidate for a brighter and more efficient indole is 3-niethylindole~5-car- boxylic acid. A thorough investigation of the 3,5,6-indoles as a class is clearly desirable.

A number of related heterocyclic compounds have been investi- gated with no outstanding candidates found. T'iese "esults are given in Appendix I, together with the results for additional com- pounds Included in the chemllumlnescence survey.

* A valid comparison cannot be made for the very weak N-methyl vs 1,2-dimethylindole. since impjrity effects are clearly predominant,

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Table k&

liBUIATION OF BRIOHTER CHEMILUMINESvENT INDOLE DERIVATIVES IN ORDER OP PEAK GROSS BRIGHTNESS

Compound l/lp

1. 2,3-Dlmethyllndole-5- l^ carboxyllc acid

2. 5-Methyllndole 84

3. 2^-Dimethyllndole 60

4. 5-Methoxylndole-3-acetlc acid 45

5. 5-Eenzyloxylridole-3-acetic acid 41

6. 5-Methylindole-3-acetlc acid 25

7. 3-Ethyllndole 20

8. 2,5-Dlmethyl"3-propyl, tech. 17*

9. 2,3,7-Trlmethylindoie 14

10 Indole-3-acetlc acid 9

* Crude sample.

14

• MONSANTO »SSCARCH COft^OltATIOM •

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Table ^b

TABULATION OF nHiüHTER CHEMILUMINESCENT INDOLE DERIVATIVES IN ORDER OF FIGURE-OF-MERIT

Compound FM x 10~3

1. 3-Methyllrviole 26

2. 5-Ethyllndole 13

3. 2,3-Dlfnethylindole-5-carboxyllc acid 6

4. 5-Benzyloxyindüle~3-acetic acid 6

5. 5-Methoxyindole-3-acetic acid 4.5

6. 6-Benzyloxyindüle* 4.4

7. Ethyl-2,3-dimethylirdüle-5- 4.2 carboxylate

8. Indole-5-carbüxylic acid* 3.8

9. 2,3-^iJTiethylindole 3.6

10. 6-Methoxyindole* 3.2

Compounds with average emission decay times of the order of one hour. With the exception of indole- 5-carboxylir acid, individual decay times are highly irreproducible.

15

• MONSANTO MCSKAMCH COftPOHi "'ON •

III. INVESTIGATION OP INDGLE CHEMILUMINESCENCE

A. 30LVENT AND CONCENTRATION EFFECTS

We previously reported on the chemllumlnescence peak bright- ness of skatole as a function of concentration in DMSO and DMP (ref. >). These data have been extended and re-evaluated. Ihr. concentration dependence of the peak brightness is ^iven in Fig- ure 1 for skatole in the above solvents and in HPT vhcxamethyl- phosphorictriamide), and for two additional indoles in DMF. These data are presented as the figure-of-merit/mole in Figure 2. This parameter is approximately related to the quantum efficiency of the reaction assuming that the emission spectrum and the order of reaction are constant, which is roughly true in the low concentra- tion regions (see following sections). The optimum concentration for the brighter systems is clearly about 10"2M.

The emission decay times have been observed to be highly irreproducible and apparently unpredictable functions of concen- tration for a given indole. For skatole in HPT we find the emis- sion decay half-life to be a decreasing function of concer -ratlonj it is an increasing function in DMF and in DMSO a function with a maximum (Table 5). Both lndole-> acetic acid and 2,j5-dimethylin- dole also show decay time maxima in DMP. However, since the ob- served maximum dev*. tion of a single value is of the order of the total Increment observed, we do not consider the results cefini- tlvo. As may be seen from Table 5 the regular family of curves in Figure 2 in part reflects the relatively small increment in tijd over the concentration range studied. Only for skatole in DMP is the maximum ratio as great as a factor of two.

As pointed out by Lee and Seliger (ref. 4), the observed rates of chemiluminescent reaction are subject to large variations presumably as a result of trace Impurity catalysis, although the integrated emission, at least for luminol, is constant. We have attempted to incorporate a first order correction to this effect by use of the figure-of-merit, but propose in future photometry to measure the integrated emission directly. Greater piecision could also be obtained by determination of the half-lives from kinetic analysis where simple rate law behavior is observed.

We have examined the chemiluminescence parameters of some representative indoles in HPT with the results given in Table 6. The very large variation observed in the figure-of-merit ratio between HPT and DMSO, for different indolea, althougn difficMlt to understand, suggests that further work in HPT is desirable to confirm and extend the above results.

16

• MONSANTO RCSCARCH COWORATION •

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D 2,3-Dlmethylindole In DMF

O Skatole in DMSO

A Skatole in HPT

O Indole-3-acetic acid in DMP

i io-3 lO'2

Concentration, moles/liter

10 -i

;

i I

Figure 1. Concentration Dependence of Peak Brightness of Several Indoles

I ■

• MON«*.yTO »ESEA«CH CCm^OWATlON •

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Concentration, moles/liter

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18

• MONSANTO ReSCAKCH CORPORATiON •

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•

Table 5

OBSERVED EMISSION DECAY INITIAL HALF-LIVES

Compound

Skatole

Skatoie

Skatole

2,3"Dimethyllndole

Indole-3-acetlo acid

Solvent

1/2 at Low Cone. seconds

«5 x ID"1 *

M

ti/2 at High Cone., seconds

«*7 x 1C~2M

DMSO 240 200

HPT 925 765

DMF 240 (10-3M)

450

DMF 30 35

DMF 640 540

ti^j Maximum Observed, seconds_

235

(at 10"2M)

40

900

19

I • MONSANTO ftKSEARCH COM^ORATIO^ «

-^ "■—i^WW.iM» »IIIW^——

Tabl« 6

CHEMILUMINESCBHCE PARAJ«T8R3 OF SOME INDOUBS B« K?T SOLVSKT

Indolt Conotntr*tlon: 5 x lO'*M BAD« ConcwTitratlon: 0.1N

SQaaaunfl tafl StPiftbm

Tint tc t^» tjf Peak 0» Current 0, Peak, 0« Peak,

I/Io etcottdH »econd»

Of L~Tryptoph«n

^V-I-CH.-CH-COOH

2.2

0.15

^: 60

Figure-of• Merit,

eeconde

152

Figure-of-Merit in DM30

n^re-oMHerll In HPT

0.1'

300 i^liO 216 0.5

HH.

^-Cyenoindole

NC

(8.2*1.8)10-» 30*30 '♦SO ± 290 3.0*1.6 (2.8 * 1.5/10 "

TG? Inrtole~5-carboxyllc Acli H.6 x 10'' H6

HC.C.

180 8.3 2.1 x 10

Indole-2-carboxyllc Acid

On ^^IPCOOH I1

5-Methoxylndole

8.« *0 -c 356 •;.5 x 10

6.U * 0.2 40*10 39 2^5 * J* (6.* * 0.9)10"*

CH,0

^

1,2-Dlmethyllndole 8 x 10"4 240 560 0.3 10

• Datum corrected for 0.1% skatole Impurityi 99.71* pure by VPC analyeie.

20

• MOM8ANTO «WMAMCH COHPOIIATION «

tv*1 -" -*"g^:^—WO—HBWJi

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B. THE MECHANISM OF INDOLE OXIDATION

1. Qeneral Background

The identii'lcatlon ol the light-emitting species In the In- doie chemlluminescence reaction would greatly aid attempts to op- timize the system. A number of references relating to the oxida- tion of 2,3-di8ub3tituted indoles have appeared in the literature during the jDast fifteen years. The work of Robertson (ref, 5-7.) and Witkop (ref. 8 and 9; is especially pertinent.

While none of the literature work relates to reactions In dimethylsulfoxide solution, the reaction products that were iden- tified give clues to the types of reactions that might take place in our chemiluminescent system. A summary of this literature work is given below in Figure J>.

2. Discussion

From data available in the literature, several compounds emerge as candidates for the chemiluminescent species in indole oxidation reactions.

The excited state of o-acetamidoacetophenone (H) must be con- sidered as the prime candidate for the light emitting species in the 2,3-dimethylindole (F) oxidation reaction. The fluorescence emission of (Hj in basic dlmethylsulfoxide solution corresponds closely to the chemlluminescence emission of (P) (see Section III.C). This type of intermediate might explain the difference in light emission between compounds (A) and (F). While (F) forms a dicarbonyl derivative (K) which is analogous to (H), (K) reacts to produce compound (C) (ref. 7, 8); (H) apparently does not react further (ref. 7). In addition, the anion of (H), Icompound (L)I has a structure that is very similar to (M), the anion which is postulated to be the emitting species in the lophine chemllumines- cence reaction (ref. 10).

O-Formamldoacetophenone, the skatole intermediate analogous to (H), is currently being prepared as a further test for this type of intermediate.

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Tetrahydrocarbazolyl hydroperoxide (B) and Its 5-carbomeLhoxy der vatlve give off flashes of blue and green light when thermally decompcsed. This Indicates that the energy available f-om perox- ide decomposition is of the proper magnitude to be detected as visual chemiluminescence. ^he fact that tetrahydrocarbszoie (AJ is not chemilumlnescent under our reaction conditions may be due to the facile formation of nonfluorescent products such as (c) in solution.

Finally, the )-lndoxyls (D a.id J) should be considered as r possible chemilumlnescent Intermediates. This type of compound

is known to be formed from 2,3-disubstituted indoles under basic autoxidation conditions. These compounds are noted for their in-

^ tense green fluorescence in solution (ref. 5-7 ■• Since the in- doles chemilumlnesce in the green region of the visible spectrum,

i the excited state of (J ) is a possibility for the chemilumlnescent species in the oxidation of 2,3-dimethylindole (F).

While it is tempting to postulate detailed reaction mechanisms to explain the observed substituent effects, we feel that this speculation should be deferred until most of the proposed interme- diates are examined experimentally. Compounds G, I, arid J ai-e currently being synthesized to test the above mentione 1 hypotheses.

C. CHEMILUMINESCENCE AND FLU,.tSSCSNCS SPECTRA OF THE INDOLES

The indoles for which chemiluminescence spectra have been determined are given in Table 7. The emission spectra peak in the green at about 500 nm. The fluorescence peak wavelengths of the parent indoles are in the near ultraviolet at about 350 nm. Addi- tion of base produces a rather large shift to the red of the order of 50 nm (with the exception of 7-methyllndole). In all examples the resulting anion fluorescence peak is 50-100 nm below the chem- iluminescence peak wavelength.

The fluorescence spectra of oxidized solutic is of skatole and 2,3-dlmethylindole (ref. 3) possess emission bands which corres- pond to the chemiluminescence emission spectra in shape and peak wavelength (Figure 4) indicating that for these compounds the emittor species is reasonably stable to further oxidation. The uncorrected fluorescence spectrum of base-free skatole in DMSO peaks at 356 with a shoulder at 370 nm. The corrected spectrum (relative phc'-ons/cm"1 ) peaks at 350 nm and eliminates the shoulder. During the course of the oxidation of skatole, the anion fluores- cence at ca. k20 nm is observed to diminish in intensity, and the intensity at ca. ^90 nm increases.

, The overall oxidation reaction may be formulated as shown below, where several react tori steps are required for the produc- tion of the Intermediate symbolized as X.

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P (dark reaction)

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•^ F (radlationleos de-excitation processes )

The fluorescence emission spectra of several possible oxida- tion products of the indoles have been obtained in an attempt to identify the emitting species. The observed emission peaks are given in Table 8. One of the products isolated from the aqueous persulfate oxidation of indole ms anthranillc acid (ref. 11). Tie fluorescence of the anion oS anthranillc acic in DMSC-0.1M t- bütc^ide peaks at a wavelength 20 nm to the blue of the; Indole chemllumlnescence- The fluorescence spectrum of the oxidized solution does not reveal the 471 nm band characteristic of this anion, ruling out this species.

The peracetic acid-hydrogen peroxide oxidation of skatole and tryptophan yields as reaction products 3-methyloxindole and i-oxytryptophan, respectively (ref. 12). Tne fluorescence peak for oxlndole anion is, however found approxima.ely 100 nm to the blue relative to the chemllumlnescence peak of Indole.

The oxidative ring cleavage product of sketole is ortho for- mamido f.cetophenone (see following section). We have determined the fluorescence spectrum of the related compound, ortho amino acetophone. Both neutral and basic solutions of this compound peak at ^5^ nnu In the basic solution a shoulder appears at ap- proximately 510 nm. At present, it is not clear whether or not this spectrum is produced by the original species. The fluores- cence spectra of the anion will be obtained as a function of con- centration to resolve the problem and that of its formyl deriva- tive.

A possxble oxidation product of 2-methyllndole is N-acetyl anthranillc acid. However, the peak of the fluorescence spectrum of the anion of this acid is at 448 nm or to the short wavelength side of all the chemllumlnescence spectra of the Indoles.

The oxidative ring cleavage product of 2,3-dimethylindole is orthoacetamidoacetophenone. We find that the fluorescence of the basic solution of this compound in DMSO peaks at a wavelength of 5l6 nm. There is excellent agreement between the contour of the fluorescence peak and the chemiluminedcence emission from 2,3- dinethy1indole (Figure 5)« The Inference, of course, is that the chemllumlnescence results from the formation of the acetophenone product in the excited state.

27

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O üpectrum of ?,3- dlmethyllndol«, b x ICr'M'.O.lM base

i»30 ^jOC ^20 S^O b

It Is of interest to note that 3~fcthyllndole, unlike skatole, does not show a stable oxidation product fluorescence. However, for reasons which are not known,, the skatole product fluorescence was not noted until the oxidation was repeated with the zone- refined material.

D. GHEMILUMIKESCSNCE DECAY KINETICS

We have examined in a preliminary way the kinetics of some ehe iiiluminetcence decays as determined from gross brightness photom- eter tracings. In general, the recordings do not exceed one half- life in duration ard any conclusions drawn must be taken as tenta- tive since it is recognized that unless apparent reaction order Is maintained over several half-lives trivial fluctuations can easily lead to Improper identification.

Random selection and analysis of a dozen decay curves gave the results shown in Table 9. The decay is found to be linear in three examples,, exponential in six, and corresponds to no simple analytical function in the remaining three. Th-:> skatole results are of particular Interest since they would Imply a change of reaction order with concentration if confirmed. This result is not at all surprising for the postulated consecutive and parallel reactions that must occur In the indole oxidation (ref. Ij5).

The peak emission decay (initial) half-lives listed in t^e last column are derived from the fitted functions and are, there- fore, more reliable averages than the values given in tables of chemlluminescence parameters. They are, however, generally smaller values than the "experimental" results. T.n part this results from the fact that the "kinetic" times are true decays from established steady state concentrations by the nature of the fitting processj that is, simple exponential decay does not occur from the observed peak of the brightness curve.

Figures 6 and 7 give examples of the exponential and linear decay curves observed. The plotted points are evenly spaced val- ues read off the photometer tracings.

30

• MONSANTO ftKMAftCH COftPOffATION •

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TablÄ 9

DECAY KINETICS OF INDOLE CHEMIL'JMDJESCENCE

C9mpow^ Cone., mol-r Run No.

Decay Function

Rate Constant

ty«, seconds

Indole-J-acetlc add

5 x lO-3 £-21-4 - Ioe-kt 2,9 x 10"3 240

L-U'yptophan 5 x lO'3 !,-26-1,2 . Ioe-kt (l.9±0.02)l0-98ec"1, 443^4

2,7-DlJs»thyiir!dole 5 x lO"3 7-22-1,2 • loe (l,83iO.07)l0-«8ec-1, 38*1

3-gthyllndcle ? x 10'5 8-4-1,2 _ -kt »loe (].66±0.9)10"38ec"1. 41/^22

5-Methylindole 6.6 > 10-a 3-5-5 , -kt •■ I^ 2.9 x \0-3aec-i 240

3-HethyUndole 2 x 10-a 3-^-1 u loe"^ 2.0 x 10-3sec': >2

3-Methyllr:dole lO"» 3-5-2 Indeterminate - -

^-Methyllndol« 2.2 x lO"3 3-3 I - -lokt 1.1 z 10"« amp/eec 3-0

2,3-Dtniethyllnd. 1? 'j x K 3 4-30 1 - -lokt 5.1 x lO"2 a-mp/aec yc

2,3-Dlinethyllndc In DKF

1« 1.8$ x 1C-3 '-ib-2 I - -lokt 1.05 x ID-1 amp/se'?

5-Benzyloxy-indole- 5 x 10~3 3-acetic acid

l-£f

2.3,7-Trlmethy;- Indole

5 x lO"3

Indeterml i&te

6-13-1,2,3 ;.ide»errnlnate

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IV. HETEROGENEOUS CATALYSIS

A. INTRODUCTION

Several preliminary experiments have been performed to initi- ate the investigation of heterogeneous catalysis in chemilumines- cent reactions.

B. EXPERIMENTAL

A simple, qualitative procedure was used to measure chemi- luminescence emission resulting from the oxidation of luminol and lucigenin catalyzed by silica gel G. Sheets of silica gel were prepared by coating the gel on glass slides. The slides were dipped in a water-silica gel slurry and were air-dried at room temperature. Methanol solution of the organic compound was sprayed from an atomizer onto the silica gel and dried. The base and oxi- dant, if any, were sprayed independently on the dry slides.

The chemiluminescence of the base catalyzed peroxidation of lucigenin was observed (Table 10). The blue-green emission ap- peared upon spraying of the final reaction component, ''he silica gel was wet during the course of the chemiluminescence, permitting the components (except for the solid substrate) to diffuse toward each other in the liquid phase. The decay of the light intensity was measured photometrically. The time required for the Intensity to diminish to one h\lf of its value was on the order of t Tee minutes. The decay does not obey simple first order, second order or nonintegral order kinetics.

The chemiluminescence spectrum of the lucigenin oxidation in homogeneous ethanol solution is a broad, structured band between 420 nm and 490 nm (Figure 8). The emission from silica gel Is shifted to the red, giving a broad band peaking at 505 nm. Simi- lar emission spectra are obtained with either ammonia or sodium hydroxide. The oxidation of luminol by potassium tertiary butox- ide and oxygen does not lead to chemiluminescence in the system investigated. This quenching of chemiluminescence upon adsorption on silica has also been observed for the indoles. Quenching of the fluorescence of the parent compound upon absorption also occurs This is, presumably, the key factor in the quenching of the chemi- luminescence.

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Table 10

CHEMILUMIN2SCENT REACTION ON SILICA GEL ADSORBENT

Organic Compound

Lumlnol

Base

Luclgenln 2M NaOH

Luc igenin 4M NaOH

0.05 to 0.1M K+tBuO", DMSO solution

Qxidant

10% H^ü2 in et'nanol

10% HaOg in ethanol

0:

Observation

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V. INSTRUMENTATION

A, INTRODUCTION

The determination of the absolute fluorescer«e spectra, quan- tum yields, and brightness of chemilumlnescent reactions requires calibration of the spectral sensitivity of the spectrometer. We have performed a relative calibration of the spectral sensitivity of our spectrometer in the near ultraviolet and visible spectrum by comparison to fluorescence standards.

B. EXPERIMENTAL

The fluorescence standards were selected from the literature (ref. Ik„ 15) to cover the spectral range between 320 nm and 660 nm. A low-pressure mercury lamp was used as the excitation source, oriented at a 90° angle relative to the spectrometer.

The conditions under which the fluorescence standards were used is given below:

^l) 5.0 x 10"4M Naphthalene in ethanoi; M.C.B., recrystallized from ethanol, mp 79-80oCj spectral range 32k to 570 nm (ref. 14); 254 nm excitation.

(2) 4.3 x 10"6M Anthracene in ethanol; Eastman Kodak blue-violet fluorescent grade, used without further purification, spec- tral range 370 to 400 nm (ref. 14); 254 nm excitation.

(3) lxlO"4M Quinine Sulfate in IN sulfuric acid*, M.C.B.. mp 2290C; (The sulfate was used without further purification.) spectral range 400 to 500 nm (ref. 15); 3130 and 3330 A excitation*,

(4) 1 x 10"4M N,N-Dimethyl-m-nitroaniline in 30^ by volume fluo- rescent grade benzene and 70^ by volume of M.C.B. spectro- grade heptane**, spectral range 500 to 660 nm (ref. 15); 3130 nm and 3330 nm excitation.

The emission spectrometer is a Mod 1700 Spex Czerny-Turner scanning spectrometer, equipped with a 1200-groove/mm grating

The literature mp is 235«2 C. The lower melting point is at- tributed to the presence of water of recrystallization, c.f. mp of quinine sulfate ^HgO is 2030C.

** M.C.B. spectrograde hexane contained some trace amount of fluorescent material and was not used. Llppert reports the spectrum in this solvent but the spectrum will be similar in heptane.

37

• MONSANTO fttMANCN CONPOAATION t

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blazed at 3000 A, and an E.M.I. 9558Q photomultlpller. The emit- ters are chosen so that the wavelength of each spectrum is over- lapped by the adjacent spectra, permitting a continuous relative spectral sensitivity callbrt-tion. The sensitivity of the instru- ment is obtained by taking the ratio of the observed photocurrent, measured in microamperes, at a given wavelength and bandwidth, to the true relative spectral distribution of the emitter in units photons/unit frequency Interval, To obtain the relative sensltl- tivlty, the ratio at 376 nm was defined as unity. The sensitivi- ties at the wavelengths where the anthracene intensity crosses raphthalene and quinine sulfate were calculated, and the relative sensitivity continued as a smooth function through the wavelengths of these emitters.

The relative sensitivity function obtained is shown in Fig- ure 9- The curve is characterized by two maxima, at 376 nm and at ^1 nm. The spectral range covered by each compound is desig- nated in the legend.

The wavelength of the exciting source was chosen as to be near the maximum of the absorption band. This selection snould reduce the effect of traces of iluorescent Impurities, The con- centrations were chosen to minimize the effects of self absorp- tion. The gross features of the sensitivity function resembles the reported quantum efficiency curve of the photomultlpller in the blue and that reported by Parker for a similar system (ref. 16),

38

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VI. FUTURE WORK

Synthesis of a number of probable Intermediates in the oxida- tion -»f 3-methyi and 2,3-dimethyllrjdoies will be carried out, and ^he proper 'ies of these compounds investigated. The hydroperoxides and other reactive intermediates will be examined as possiblt- can- didates for chemlluminescent reactants of high brightness and efficiency.

Synthesis of 3-methyllndole-5~carboxylic acid, and possibly other 5»6-substltuted indoles, will be undertaken in the continu- ing effort to obtain highly efficient chemilumlnescence compounds. Research on optimum conditions for chemilumlnescence of the brighter indoles will be continued, with emphasis upon the effect of senai- tizers, solvents, oxldants, and base strength.

The absolute calibration of the spectrometer will be carrried out using Seliger's recent data for lumlnol as a secondary standard (ref. it). The photometers will be provided with integrating cir- cuits to permit total emission comparisons in an attempt to Improve reproduclbillty of the efficiency measurements.

k cell for convenient measurement of chemilumlnescence param- eters in heterogeneous reaction has been designed. The catalyst is incorporated in a porous Teflon membrane, permitting relatively free access of oxygen to the catalyst-reactant solution interface'. The emission can be observed through a window parallel to the mem- brane surface.

40

• MONftAHTO »CMtANCH COttPOftATION •

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VII. RFFERENCES

1. E. White In "Light and Life'1. W, D. McElroy and B. Glass (Editors i, Johns Hopki.is, Baltimore, 1961, p. 183.

See for example, W. west in "Chemical Applications of Spec- troscopy, Vol. IX. Technique of Organic Chemistry", A. Welssberger (Ed.), Interscience, New York, 1956, p. 707.

Tech, Reports no. 3 and k in this series.

J. Lee and H. H. Seliger, "Chemiluminescence and Fluorescence Quantum Yields of ...ILumin^1)..., J. Phys. Chem., in press.

R. J. S. Beer, L. McGrath and A. Robertson, J. Chem. Soc, 19^0, 2118, 3283. "~

R. J. S. Beer, 'Ti. Broadhurst and A. Robertson, J. Chem. Soc, 1^2, 4946. "

R. J. S. Beer, T. Donavanik and A. Robertson, J. Chem. Soc, l^i, ^139. ^

8. B. Witkop, J. Am. Chem. Soc, Ji» 1^28 (l950).

9, B. Witkop and A. Ek, J. Am. Chem. Soc. , £3, 5664 (l95l).

10. E. H. White and M. J. C. Harding, J. Am. Chem. Soc, 86, 5686 (1964). ■ ~ —

11. G. E. Philbrook, "Chemical and Enzymatic Studies on the Con- version of Chemical Energy to Light", Final Technical Report on Contract AP"APOSR-62-73; 44-63, University of Georgia, June 1964, AD 602798.

12. B. WitKop, Ann., ^8, 98 (1947).

13. See for example, R. Elderfield feditor) Heterocyclic Compounds, Vol. 3* John Wiley and Sons, 1952.

14. C. A. Parker, Anal. Chem., >4, 502 '1962).

15. S. Llppert, et al, Z. Analyt. Chom., JJO, 1 (1959).

16. C. A> Parker, "Advances in Photochemistry", Vol. 2, p. 305 et seq, Interscience Publishers, New York, 1954.

17. D. E. Vorkade and J. Lieste, Rec. Trav. Chim., 65, 912 (1946); CA., 41, 5876 (1947). "~"

41

• MONHANTO »rSEANCH COIIPOIIATiOM •

■1 1 ■■■«in ipi uimmm**m^*mfwt***m*m—mmgmam**L

18. E. Leete and L. Marlon, Jan. J. Chem., 21> 775 (1953).

19. D. E. Ames, R. E. Bowman, D. D. Evans, and W. A. Jones, J, Chem. Soc, 1984 (1956).

20. N. J. Leo'.mrd and S. N. Boyd, J. Qrg. Chem., 11, 405 (1946).

21. C. W. Huffman, J. Org. Chem., 22, 727 (1958).

42

• MOM»ANTO >K«eAI»CM CORPORATION •

f

APPENDIX I

CHEMILUKANESCENCE OP MISCSLLAHEOUS COMPOUNDS

Coapouod and Structure

B«n2Cfur«n

Op Benzlmldazole

OC^ 2-M«thylbenzlmldazole

Time to Peak 0« Current 0» Peak Ti/g of 0»

Ratio I/Io aec Peak sec

a) Heterocycllc Analogues of Ind^ne

6 x 10"8 15 55

7.6 x 10"4

1.3 x 10"*

50 120

18 72

Figure of Merit

2.1

9 x 10"8

9.^ x 10'

Vc H3

Benzoxazole

0- H

2-Metnylbenzoxazole

6 x 10'3

5 x 10'*

22

^C

72

^2

0.43

0.2

C-CHa 0^

Benzothlazole (2.6±0.6)10" 15 45 + 15 0.11

^H

2,-Methyl-Benzothlazole

oo- 2 x 10"3 12 12 2

43

4 x iO

*

• >r»f>*«0ANro l»«»tA«CM COM^OHATION •

—w WT"-" ■ " "»■^■w

APPENDIX I (Continued)

Time to

fompcntnd and Stricture

?,^-Dimcthylpyrrole

Peak 0« Current Ot Peak Ratio I/Io see

Ti/t of 0« Peak sec

58

Plgi're of Merit

b) Pyrrole Derivatives

1.2 x lO"8 6 0,62

H.cTf^CHa H

N-Methylpyrrol«

9 CHg 8 x 10'3 15 ?o 0.16

1,2,5-Trlmethy1pyrrole 10 15 l>.5 x 10'"

2,4-Dlmethyl-3-ethyl Pyrrolt

HaCAr^kcHa i K

;i.2 x '.0 60 30 0,29

5-Pheny1-2-pyrrole Proplonlc aeld

0? N^CHiCH.COOH 1,6 x 10' 0.14

c) Fluorene and Pluorenone Dcrivatlvea

2-Dliiiethylamlno fluorene

H NK

2-Ainlno fluorene

W H

(2.2±0.7)10 .a

^2

1,9 x 10'a ?6

44

5 + 1

\\

0,11

0.21

• MOMSAMYO l»t»«A>»CM CO*tPO«*riON •

I r

APPENDIX I (Contlnugd)

" Compound and Structure

'•-Methoxyfluorene

QCK3

OOT ■7

Tim« to Peak 0» Current 08 Peak Tx/t of 0» Figure of

Ratio I/IQ aec _r »k sec Merit

fe.5 ♦ O.J) 10"" 17 66 + 51* 1.8

2-Methylfluorep.e 8.4 x 10'8 ■c 10 0.84

3- Methoxyfluorenc

H XH

0.4 + 0.15 26 + 10 10 + " 4.2 _»• }.6

1-Plucrene-carboxyllc Aeld 6 x 10"*

H H,C00H

2«( 50 1.8

9.Pluorenone-l-carl3c;xyUc Acid

5.4 x 10'8

O COOH

4-Amlno-9-FlJorenone

NHa

00 1 1 x 10 12

1.52 "».S

24 26 x 10"

'

4-Methoxy-9-fluorenone OCH,

On©

2.2 x 10 -•j ol» ■■i . i 19

I 45

• MONSANTO ftCSCAHCH COKPORATIOK •

"WW '»■^—* . sc

APPENDIX I (Continued)

P«tk 0« Current Compound and Structure Ratio I/Io

2.Dlmtthyl«ffllno-9-nuor- 2.2 x 10"8 enone

Time to 0« Peak

■fc

17

Ti/t of 0» Figure of Peak sec Kerlt

SI

OrÄK(raj).

a-Pyrldoln

®;HK1@

0.53

d) Acylolnn and 1.2-dlketoneg

(2.^ + 0.3)10"Ä 31 ♦ 17 3+1 (e.t + 1.2 )10"e

e.e'-Dlmethyl^.S'^yridcln 3.5 x 10'1

If. CHs tfip

CHs

Barltwn BenMin-3,3'' Dlsulfonate

.««a ■Ba "^

■0.6 x 10"

i*0

■5 »10

7 x 10

«6 x 10"

2,2,-Pyridyl

&U^

l."» x 10' I? H.2 x 10'

e^'-Dlmethyl-Z^'-pyridyl 1.2 x 10**

Crt CHj

3.6 x TO'3

'6

I r r

UFEH5IX I (Continued)

Time to Peak Ct Current 0» Peak Ti/« of 0» Ftgure of

Compound and Structure S>tlo l/l-., a«e Peak BCC Merit

e) Compounda Containing sa Oleflnie Linkage

Sorblc Acid 1,0 x lO*5 10 SO CKaCl^CH-CH-CH-COOH

Tetracyanoethylene CN CN I ' C "C CN CN

K.8 x 10'a 10

39 x 10"?

48 x 10'3

Allyl benzene

K,C-CH-CHa-^

1.7 x 10 «10 .50 5.1 x 10

Tetracyanoqulno- dlmatfiane (TCNQJ

NO,

APPENDIX II

ORGANIC SYNTHESIS

1. 2.3-Dlmethvlindole-5-carboxyllc Acid

This indole was prepared in 30^ overall yield (three steps) according to the procedure of Verkade and Lieste (ref. 17),

a. 2-(p-Carbethoxyphenyl)amino-3-Dutanone (l)

Alkylation of ethyl p-aralnobenzoate with 3-bromo-2-butanone in aqueous alcohol with sodium bicarbonate as an acid scavenger provided I as a tan solid !46^) after charcoaling and recrystal- lization from diethyl ether-petroleum ether (bp 30-600c); mp 72.5- 73.50C.

b. Ethyl 2J3-Dimethylindüle-5-carboxylate (ll)

Reaction of I with the hydrochloride salt of ethyl p-amino- benzoate (prepared by treatment of the amino ester with anhydrous hydrogen chloride in absolute ethanol-diethyl ether; mp 206-208oC) yielded II as a light tan solid (70^) with mp 10g-llloC. Recrys- tallization from diethyl ether-petroleum ether (bp 30-60oC) raised the mp to 114-115.50C. No impurities were detected by VPC and NMR analysis.

c. 2..3-Dimethyl-5-carboxylic Acid

Saponificaticn of II with alcoholic potassium hydroxide, fol- lowed by acidification, provided the acid as a tan solid In 93% yield with mp 237-2390C. Charcoaling and recrystallization did not substantially chfnge the mp (mp 238-239«5CC).

2. 3-Ethylindole

This compound was prepared in 66% yield by the lithium alumi- num hydride reduction of 3-acetylindole according to the procedure of Leete and Marion (ref. 18). The crude product was purified by distillation (bp 83-840c/l3-i4 nm); the distillate solidified in the receiver to an off-white 19) 42eC, (ref. 19) 33-350C) with the structure.

3. O-Acetamidoacetophenone

solid The

mp 3>34gCj infrared

eported spectrum was

(ref- consistent

/

o-Acetamidoacetophenone, mp 72-740C (lit. mp 74-750C) was prepared in SCfi yield by the method of Leonard and Boyd (ref. 20). The formylation of o-aminoacetophenone is being attempted by means of a mixed anhydride acylation reaction (ref. 21).

48

• MONSANTO MESCA«CH COMPOMATION •

/?)) /^ß J> ^ J

MONSANTO RESEARCri CORPORATION BOSTON LABORATORY

Everett. Massachusetts 02149

ERRATA

Report No: MRB3009Q5 Report Title: Technical Report No. 5

"Chemiluminescent Systems" Report Period: 1 June - 1)L August 1965 Contract No: Nonr-4511(OO)

ARPA Order No. 299 Task 556-464

Report Date: ^0 September 1965

Please make corrections as noted below:

Page 1» para. 4, 1. 6: delete "peak"

Page 5, line 3, column 4: T^ is (6.4 ± 1.4 )l03

Page 9, line 1: formula for 5-hydroxytryptophan should be

H H 0 HO ^\ 1 I I!

NH 1

H

Page 19, line 1, column 5: tvz is 325> not 2;5.

Page 20, footnote: change 0,1^ to 0.3^

Page ?6: curve "d" should be labeled "e"j curve "e" should De labeled "d".

Page 51: lines 8, 9* 10, column 4: equations should read

I = lo - kt

not I = -lokt.