Methyl calcein blue and other analogues of calcein blue

METHYL CALCEIN BLUE AND OTHER ANALOGUES OF CALCEIN BLUE GERALDINEM. HUITINK and HARVEYDIEHL Department of Chemistry. lowa State University Ames. Iowa, U.S.A. 50010 (Receiaed 11Drc~~whrr

1973.Accepted

25 Fh-uary

1974)

Summary-4-Metbylumbeliiferone-8-methylenesarcosine (Methyl Calcein Blue) and four related metaliofluorochromi~ ~ndj~torsderjved from umbeliiferone. ~methy~umbeili~erone, and Cmethylesculetin by condensation with formaldehyde and iminodiacetic acid or glycine have been synthesized, the structures established. the absorbance and fluorescence measured as functions of pH, and the reactions with copper(H) and calcium studied with attention to the effects on fluorescence. All of the compounds display a maximum fluorescence at pH about 9. The fluorescence of each is quenched by copper(H). The calcium derivatives of the compounds derived from the umbeliiferones and iminodia~tic acid fluoresce at high pH but those from the umbel~irerones and glycine or sarcosine do not. At high pH. 4-methylesculetin and the amino-acids derived from it do not fluoresce either alone or in the prescence of calcium.

In an earlier paper’ we described the properties of ~methylumbelliferone-8-methyleneiminodiacetic acid, a compound earlier given the common name Calcein Blue by Wilkins.2 Wilkins3 was also the first to prepare Methyl Calcein Blue, a metallofluorochromic indicator synthesized by the condensation of 4_methylumbelliferone, formaldehyde and sarcosine (N-methylglycine). As with Calcein Blue, the work reported by Wilkins on Methyl Calcein Blue was limited to explaining some advantages of the compound as an indicator, and to reporting its use as an indicator in the determination of aluminium, nickel and manganese by titration with hydroxyethylethylenediaminetriacetic acid (HEDTA). We have now examined Methyl Calcein Blue in some detail; it proves to be 4-methylumbelliferone-8-meth~lene~rcosine ’ 1*5H,O (structure IX below}. We have also examined four closely related compounds: umbelliferone-8-methyleneim~nodia~tic acid (V), umbelliferone-8-methyleneglycine . Q5H20 (VII), 4-methylumbelliferone-8-methyleneglycine (VIII), and 4-methylesculetin-8-methyleneiminodiacetic acid.Q5H,O (X).

I RI = H. R, = H (Umbeliiferone) II III IV V VI WI VIII IX X

R, R, R, R, R, R, R, RI R3

= = = = = = = = =

CH,. R2 H. R, = CH3. R* H. R2 = CH,. R2 H, R2 = CH,. R2 CH,. R2 CH3. R,

= H (4-Methylumbelliferone) H (Esculetin) = H (4-Methylesculetin) -CH,N(CH,CO,H)> = -CH,N(CH2COZH)2 (Cakein Blue) -CHZNHCH,CO,H = -CH2NHCH2C0,H = - CH,N(CH,)CH,C02H (Methyl Calcein Blue) = -CH2NICH2C02H)I 1193

GERALDINE M. HCITINKand HARVEIIDIEHL

1194

The investigation of these compounds followed essentially the same course as that of Calcein Blue and the calculations and reasoning follow the pattern presented in the earlier paper.’ Results are given in the following section, and the summary and discussion are confined to the differences found between these compounds and Calcein Blue. These differences are significant inasmuch as the nature of the chelating group in some of the compounds has been considerably altered. A more detailed treatment of this work and a compilation of the graphs presenting the potentiometric. absorbance. and fluorescence data will be found in the thesis of G. M. Huitink.4

EXPERIMENTAL Synthesis

and intermediate

compounds

The Mannich condensation was used in the synthesis of compounds V-X, the molar proportions of reactants found best being hydroxycoumarin 1.0. amino-acid 15. and formaldehyde 15. The solvents and procedures given below for the individual compounds are the optimum found in the course of numerous preparations. The chemicals used were commercial materials without further purification: disodium iminodiacetate monohydrate, Geigy Chemical Company; glycine. Eastman Organic Chemicals; sarcosine. Mann Research Laboratories, Inc. The formaldehyde used was a 37% aqueous solution. All water used was distilled and then demineralized by passage through Amberlite MB-l.

Apparatus

and procedures

Electrophoresis studies were done with a Model R. Series D. Beckman Paper Electrophoresis Cell (Durrum Type). NMR spectra were obtained with a Varian Associates A-60 Nuclear Magnetic Resonance Spectrometer. The spectra of III, V, VII, VIII, and IX were obtained in a deuterium oxide-potassium carbonate solvent. those of IV and X in D,-dimethylsulphoxide. Tetramethylsilane and Tier’s salt were used as standards. in D,-dimethylsulphoxide and Da0 respectively. Measurements of pH were made with a Corning Model IO pH-meter equipped with a Beckman No. 40495 high alkalinity glass electrode and a Beckman asbestos fibre S.C.E. Potentiometric titrations were carried out in @IM potassium chloride medium with O*IM sodium hydroxide made up in DIM potassium chloride. Solubility measurements (of compounds V and X)as a function of pH were made by shaking buffers with excess of compound for 12 hr. An appropriate volume of the filtrate was then adjusted to a specific pH (I@0 for V. 4.0 for X) diluted to specific volume with @IM potassium chloride. and the absorbance measured (at 367 nm for V. 342 nm for X), and the concentration calculated from calibration curves. The intrinsic solubility. Si, was obtained by linear extrapolation to I/[H+] = 0 of plots of S vs. l/[H+] and the acid dissociation constant was obtained5 from a plot of pK = pH - log[(g/s ) - I]. For both V and X the plots were linear and both parallel and close to that for Calcein Blue.’

Absorption

spectra

The absorption spectra of I, II, V, VII. VIII and IX were obtained at intervals of 05 pH over the pH range 1.5-13.0. Solutions were prepared by mixing 375 ~1 of a 3.1 I x 10e3M stock solution of the compounds. 0.25 ml of O*OlM EDTA and 10 ml of buffer. The solutions were then diluted to 25 ml with O.IM potassium chloride. Absorption spectra were obtained with a Cary Model I5 Recording Spectrophotometer. The pH was checked after the absorbance measurement. Additional absorbance measurements were made on solutions containing the acidic form and the basic form of the compounds and on solutions of pH numerically equal to the estimated pK, + 0.0. 0.2. 0.4 and 0.6. the measurements being made on a Beckman DU spectrophotometer. Values for the acid dissociation constants were obtained from the results by using the equation pK, = pH -lo&A - AHA)/(Ar( - A), which is simply derived from the mathematical equation defining K,, the terms A. A HA. and AA being the absorbances of the solutions of pH = pK, + x, and of solutions in which the substances were entirely in the acid or salt forms. The absorption spectra of IV and X at pH 4 and 9 were obtained for buffered solutions. Solutions containing I ml of 3.1 I x IOe3M stock solution of the compounds and 0.75 ml of @lM EDTA in 75 ml of O.IM potassium chloride were titrated spectrophotometrically with @2M potassium hydroxide. Values for the acid dissociation constants were obtained from graphs of absorbance vs. pH; absorbances obtained from buffered solutions of IV and X could not be used because. of serious chemical reaction of the compounds with the buffer components.

Analogues of Cal&n Blue

1195

Fluorescence excitation and emission spectra of I. II, V, VII, VIII and IX were obtained with an AmincoBowman Spectrofluorometer and Mosely XY Recorder. A fiuoromctcr, G. K. Turner Associates. Model 10. equipped with a flow-through cell. was used to monitor Ruorometric titrations. Stock solutions. 3.1 I x IOe3M. of the various compounds were prepared, a little potassium hydroxide being added to facilitate dissolution. Volumes of 15 111of I, II, V, VII, VIII and IX were mixed with 0.25 ml of 0.01M EDTA and 10 ml of huffcr solution and diluted to 25 ml with @lM potassium chloride. The fluorescence exatation and emission spectra were then obtained and the pH again measured. The pH range 1.5- 130 was covered at intervals of 05 pH. Plots of relative fluorescence I‘S.pH were made and acid dissociation constants taken as the pH at the point of inflection of these curves. The effect of copper on the fluorescence of V, VII. VIII and IX over the pH range 35-10~5 was studied by essentially the same procedure hut with omission ofthe EDTA and addition of 15 ~1 of 3.1 I x 10s3M copper(H). Similarly the effect of calcium was determined over the pH range 7G 13.0. The relative fluorescence of IV and X was obtained at pH 2+ I I.0 by fluorometric titration of solutions containing 7 111ofstock soiution and 0.75 ml of 0.01 M EDTA in 75 ml ofO.lM potassium chloride with @2M potassium hydroxide. Acid dissociation constants were ohtaincd from the titration results. The effects of copper(H) were investigated in the same way. omittin the EDTA and adding 7 ~1 of 3.1 I x 10e3M copper(H).

~I??~~~~~~~(~~~IZ~, (I) was prepared by the procedure of Or~uf~j~~~uc~~oI~s:~m.p. 234 235’. literature value 227128 The preparation of 4-r~1zrh~~irrrr~hrl/~~~~~o~7~~ (II) was reported earlier. ’ Esculetin (III) was prepared by the method of Amiard and Allias:.‘mp. 26;. 271 . literature value 270 4-Af~,rll~~ls~~~letir, (IV) was prepared by the method of Orc/trllic S.WI/WWS:”m.p. 279-28 I . literature value 272 274”. Urlrh~ll~~ronP-8-rtlc~thylenri,ninoditrcrric acid (\‘) was prepared with the same procedure and ratios of reactants as for VI (Culcrin Blue).’ Yellow crystals were obtained. which were unchanged on heating to 300;. The neutralization equivalent found was 308.3. and analysis gave C 54%““. I-l 4.4”,. N 4.4:“: C,,H,,NO, (m.w. 307.2) requires C 54,730;. H 4.27%. N 4.56%. t.‘,rhrllik~,loric-S-~1t~,r~f~~~,~7~~~~~~~~11~ (VII) was prepared with the same procedure and ratios of reactants as for IX. hut no spongy material formed and the product precipitated immediately. Yellow crystals were obtained. which were unchanged on heating to 300”‘.The equivalent weight round by neutralization was 256.5. The water content found by the Karl Fischer method was 0.41 mole per mole of VII. Analysis gave C 55.5”/, H 46%, N 5.7”,: C, :H 1, NO,. jH,O (m.w. 258.2) requires C 55571,. H 4.69:; N 5.43%. 4-Methylurnhe[Iiferone-8-methp ~~,~j~~~~~.~,j~f~~ (VIII) was prepared in the same way as VII. Yellow crystals. unchanged on heating to 3w’ were obtained. The equivalent weight round was 264.9. Analysis gave C 59.1:;, H S%;;, N 547.6: CtsH,3NOs (m.w. 263.2) requires C 59.30”,. H 4984,. N 5.329,. MPI/IJ/ Co/cc+ B/W (4-methylumbelliferone-8-methylenesarcosine. IX) was prepared as follows. A mixture of 0.12 mole (2144 go of 4-methylumbelliferose. 0.18 mole (16.03g) of sarcosine and 0.18 mole (17.76 ml) of 370, the dropuise addition of dilute hydrochloric acid (1 + 5). A precipitate formed which was filtered off and washed with demineralized water and acetone. The product was slurried three times with acetone and then air-dried. Yello\h- crystals. unchanged at temperatures up to 300’ were obtained. The neutralization equivalent found ~3s 303.6. Analysis gate C SW’,,, H 60”,,. N 4.4”,,: C,,H, $N05. llHzO (m.w. 304.3) requires (‘ 5526”,.. H 596“,,. N 4W’,,. 4-111‘~rh~~l~~.stt,l~,rirl~lI~,rR~~/rrlrirltirrodiacrri~ ucid (X) was prepared in the same way as for VI. Fine grey crystals UL’~L’ obtained. unchanged on heating to 300.. The neutralization equivalent found was 348.8. Analysis gave C S 1.7”,,. H 3,7”.. N +O”,: C, 5H, ,NOs fH,O (m.w. 346.3) requires C 52.027;. H 4*66x, N 4.06%.

RESULTS

The results for the investigation of the absorption and fluorescence spectra are summarized in Table 1. and the NMR data and their interpretation are given in Table 2. The absorption spectra of IV at various pH values resembled those of I and II but a second maximum was present (Fig. 1). The fluorescence spectra of IX are shown in Fig. 2. Unlike that of II. but resembling that of Calcein Blue (Ref. 1, Fig. 5). the fluorescence decreased at high pH. the maximum occurring at pH 8.85 (see Fig. 3).

1196

GERALDINEM. HUTINK and HARVEY DIEHL

Wavelength,

nm

Fig. 1. Absorption spectra of 4-methylesculetin (compound IV) at (1) pH 4; (2) pH 9.

Table 1. Spectrophotometric

data

Compound

pH

Absorption* E. *in,,, nm ~l.~no/e-‘.cm-’

IJmbelliferone*t

4 10

325 365

1.49 x IO4 1.93 x lo4

CMethylesculetin

4 9 4 10

340 360 325 367

1.16 x 1.46 x 1.49 X 1.61 x

lo4 lo4 IO4 IO4

9

4 10

322 369

1.53 x lo4 1.90 x IO4

4 9

4 IO

322 365

I.52 x IO4 1.85 x IO4

4 9

4 10

321 360

1.58 x IO4 1.85 x IO*

4 9

4 9

342 360

1.26 x lo4 1.50 x IO4

9

Umbelliferone-lmethyleneiminodiacetic acid’t Umbelliferone-8methyleneglycine CMethylumbelliferone-8-methyleneglycine*t Methyl Calcein*t Blue CMethylesculetinmcthyicnciminodiacetic acid

pH 4 10

4 9

Fluorescence Excitation L,,,. Emission A,, nm nm 330 370

460 460

370 shoulder 460 at 300 nm) 328 455 370 455 (fluorescence maximal at pH 9.1) 330 455 370 455 (fluorescence maximal at pH 8.5) 328 450 366 450 (fluorescence maximal at pH 8.5) 328 448 365 448 (fluorescence maximal at pH 8.85) 369 459

* Absorption spectra and variation ofabsorbance with pH similar to those 4-methylumbelliferone and Calcein Blue (Ref. 1, Figs. 2 and 3). t Fluorescence spectra and variation of fluorescence with pH similar to those of Calcein Blue and 4-methylumbelliferone (Ref. I. Figs 4 and 5).

Table 2. NMR data Compound

6. ppln Pattern

III

IV

V

VII

VIII

IX

X

6.08 6.74 7.02 3.19 4.42 5.94 768 6.63 7.29

3.54 4.05 5.82 7.53 6.52 7.f1

2.14 3.52 4.07 5.72

207 2.72 3.67 4.15 5.67

253 3.05 4.56 6.10

AB AB

AB

t AB i

I

Peaks

Proton integration

Doublet Doublet Singlet Singlet

1 1 I I

Singlet Singlet Singlet Singlet Singlet Doublet Doublet Doublet

1 I

J

Coupling

)

9

Singlet Singlet Doublet Doublet Doublet Doublet

2 2

Singlet Singlet Singlet Shlglet Doublet Doublet

3 2 2

Singlet Singlet Singlet Singlet Singlet Doublet Doublet

3

Singlet Singlet Singlet Singlet

3 4 2 1

Singlet

1

1 9

9

ortho

1

1 1

orthu

1

1 9

absent in IV by analogy with II

(-N--CHz--Ar) 3 4 6 5

CH, at 4 (-N-CH,-CO;) (-N-CH2--Ar) 3 6 5

)z

confirms location of mcthylcnciminodidcetic acid at 8 )

confirms location of methyleneglycine at 8

absence of peak at 6.16ppm in II and or-rho protons confirm location of methyleneglycine at 8

CH,at 4 (-N-CH,) (-N-CH,-CO;) (-N-CHZ--Ar) 3

: 2

1 1 1

3 4 5 x

(--N-CH2---CO;

1

1 1

Notes

3 8 5 (--N-CH,-CO, (-N-CHZ---Ar) 3 4 6 5>

f 4 2

I 1 1 1

Position

1 9

6 5

CHs at 4 (-N-CH,-CO;)t (--N-CH,-Ar) 3 5

absence of peak at 6.16 ppm in II and orrho protons confirm location of methyie~~r~osine at X

olefinic proton* aromatic proton*

* Only one aromatic proton is present and there is no direct evidence enabling a particular assignment to the 5 - or to the 8 - position. The position of the two peaks coincided with those of IV (6.08 ppm for position 3 -. 7-02 ppm for position 5 - f and lie close to those of II, 111and VI, so that the probable assignment is 601 to position 3 - and 7.00 to position 5-. The methyleneiminodiacetic acid group thus occupies position 8. 1197

1198

GERALDINE

M.

HUITINK

and HARVEYDIEHL

Table 3. Acid dissociation constants of Methyl Calcein Blue and related compounds

Compound

Value found

Constant

Umbelliferone (I)

Method

Group involved

PK,

7.83 768

Absorbance Fluorescence

Phenol

PK,

7.82 7.78

Absorbance Fluorescence

Phenol

4-Mcthylesculetin (IV)

PK,

769 7.40 7.35 11.72

Potentiometric Absorbance Fluorescence Fluorescence

First Phenol

Umbelliferone-8-methyleneiminodiacetic acid (V)

PK, PKZ

PK,

2.94 6.90 6.95 6.90 11.18

Solubility Potentiometric Absorbance Fluorescence Fluorescence

PKI

6.65

Potentiometric Absorbance Fluorescence Fluorescence

4-Methylumbelliferone

(II)

PK,

Umbeiliferone-8-methyleneglycine (VII)

4-Methylumbelliferone-8methyleneglycine (VIII)

6.79

6.70 11.70

PK, PK,

6.85

PKZ

12.12

Potentiometric Absorbance Fluorescence Fluorescence

PK,

6.78 6.71 6.60 12.98

Potentiometric Absorbance Fluorescence Fluorescence

PK,

3.03 6.22 6.35 668 11.38

Solubility Potentiometric Absorbance Fluorescence Fluorescence

6.88

6.85 4-Methylumbelliferone-8methylene sarcosine (IX) (Methyl Cal&n Blue) CMethylesculetin-8methyleneiminodiacetic acid (X)

PKZ

PK,

350

400

Wavelength,

450

Second Phenol

Carboxyl Phenol

Ammonium Phenol

Ammonium Phenol

Ammonium Phenol

Ammonium Carboxyl Phenol

Ammonium

500

nm

Fig. 2. Fluorescence excitation and emission spectra of Methyl Calcein Blue (compound IX). (I) Excitation, pH 4, emission monochromator set at 448 nm. (2) Emission, pH 4. excitation monochromator set at 328 nm. (3) Excitation, pH 9, emission monochromator set at 448 nm. (4) Emission, pH 9, excitation monochromator set at 365 nm.

1199

Analogues of Cal&n Blue

PH

Fig. 3. Vaktion of fluorescence of Methyl Cal&n Blue (compound IX) with pH. Emission monochromator set at 448 nm for all curves. (I) Excitation monochromator set at 328 nm. (2) Excitation monochromator set at 365 nm. (3) In the presence of excess of calcium. excitation monochromator set at 365 nm. (4) In the presence of copper(H), excitation monochromator set at 328 nm for pH < 6 and at 365 nm for pH > 6.

Dissociutiorz cotrstnrm

Spectrophotometric titration (Fig. 4), fluorescence data (Fig. 51, potentiometric titration and solubility data were used as appropriate for obtaining the acid dissociation constants, which are summarized in Table 3. The solubility data are given in Table 4. In the potentiometric titrations. V gave two breaks in the curve, one at a = 1.0 and pH 5.12. the second at u = 2-Oand pH 9.34 (a = number of equivalents of base added per equivalent of acid). The pH at N = 05 was 4.30. but this was not a good value for pK,, the solubility of V being so low that the sample dissolved completely only as the first break was being approached. The pH at a = l-5 gave a good value (690) for pKz. VII gave a single break at 0 = 1.0. pH = 8.45. The pH at a = 0.5 (6.65) indicated that the proton of the phenol group was neutralized and that the acetic acid group formed a zwitter-ion that was not titrated. \*I[11and IX behaved similarly to VII and X behaved similarly to V. Copper(I1) quenched the fluorescence of VII, VIII and IX over the pH’ range 7-10.5, and that of IV (Fig. 5). V (pH 4-10.5), VI (Ref. 1, Fig. 1) and X (similar to IV) over a wider range of pH. Two breaks occurred in the curve for the ~uorimetric titration of X with copper(H) at pH 9.1. indicating the addition of first one and then a second ion of copper. Table 4. Solubility data. 3.26 3.84 3.96 4.06 22.66 65.66 84.80 94.33 7.20 3.34 I.58 0.52 * S. = Intrinsic solubiiit!.

3.76 3.55

406 4.41 5.63 12.94

1200

GERALDINE

3

M.

HWINK

5

and

7

HARVEY DIEHL

9

II

13

Fig. 4. Absorbance of 4-methylesculetin as a function of pH at (1) 340 nm; (2) 360 nm.

In the prescence of calcium at high pH, V fluoresces intensely, in this respect resembling VI (Ref. 1, Fig. 5).Titration of V with alkali in the presence of a tenfold amount of calcium yielded a curve closely resembling that for the same titration of VI (Ref. 1, Fig. I). The formation constant calculated’ from the curves was KC.,,- = l-5 x 10’. In the presence of calcium at high pH, VII and VIII do not guoresce, behaving similarly to IX (Fig. 3). Unlike V and VI, X does not fluoresce in the presence of calcium at high pH. The parent material IV (4-methylesculetin) differs from the umbelliferones I and II in not fluorescing at high pH, and this behaviour is carried over to the methyleneiminodiacetic acid derivative (X).

PH

Fig. 5. Fluorescence of Cmethylesculetin as a function of pH, (1) alone; (2) in the presence of copper(H). Excitation monochromator set at 370 run, emission monochromator set at 460 nm.

1201

Anaiopues of Calcein Blue DISCUSSION

The acid dissociation constants of the various compounds studied are summarized in Table 3. Among the methyleneimino acid compounds, only the three derived from iminodiacetic acid, V, VI and X, have a titratable carboxyl group, and each of these only one. All six compounds V-X are therefore zwitter-ions, a carboxyl group proton having been transferred to a nitrogen atom. The remaining free carboxyl group in each of the compounds V, VI and X is unusually strong for a carboxyl group, pK I = 2,94, 2.97 and 3.03, respectively. owing to the presence of the positive charge on the neighbouring ammonium ion. This effect is exerted also on the neighbouring phenolic groups, the phenolic groups of V-X each being an order of magnitude more acidic than those of the parent phenols I, II and IV. As is almost always the case, the ultraviolet absorption and the fluorescence excitation spectra of each of these compounds practically superimpose. Although UmbelIiferone and 4-methylumbelliferone fluoresce at high pH, the methyleneimino acid derivatives of them do not. For this the explanation offered earlier’ is offered again, that neutralization of the ammonium ion reduces the rigid, hydrogen-bonded structure attached to the aromatic nucleus and by allowing unrestricted rotation about the bonds in the methyleneimino acid group provides a non-radiative mechanism for the dispersion of the energy of the excited state. The fluorescence of the two compounds derived from iminodiacetic acid and the umbelliferones, that is V and VI, is restored at high pH by calcium and this is attributed, as before,’ to the formation of the tight, non-rotating, multidentate chelate structure about the calcium atom, involving the phenoI and both acetic acid groups. The fluorescence of those compounds lacking a second acetic acid group (VII, VIII and IX) is not restored at high pH by calcium. Unlike the umbelliferones, Cmethylesculetin does not fluoresce at high pH; this is attributed to opening of the pyrone ring, the one phenohc group having been neutralized below pH 9, the pH at which maximum fluorescence occurs. The fluorescence of the iminodiacetic acid derivative. X. is not restored by calcium at high pH as happens with V and VI; presumably again this is because of ring opening. The properties of the iminodiacetic acid derivatives of umbelliferone and of 4-methylumbelliferone are so alike that only the latter, Calcein Blue, need be considered for practical use as a metallofluorochromic indicator because not only is umbelliferone dificult to prepare (4-methy~umbel~iferone being relatively easy) but umbel~iferone and its derivatives decompose in alkaline solutions far more rapidly than do 4-methylumbelliferone and Calcein Blue.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

G. M. H&ink, D. P. Poe and 11. Diehl, ~~~ff~~~.1974,21, to be published. D. H. Wilkins. ibid.. 1960.4, 182. Idrnl. Anal. Chirtl. Am. 1960. 23, 309. G. M. Huitink. Suhsritured Cmmarins as MerallqPuo~c7chror~~ic Indicators. shy. 1967. H. A. Krebs and J. C. Speakman. .f. Che~n. SOL 194.5.593. Organic Reacrions. 1953. VII. 20. G. Amiard and A. Allias. B& Sot. Chiin. F‘rar~cc,.1947. 512. Organic S~wrhes~s. Co//mire C’olurw 1. 1941. 360.

Ph.D.

Thsis.

Iowa State Univer-

1202

GEHALDINE M.

HUITINK and HARVEY DIEHL

Zwtmmenfasstmg4Methylumbelliferon-8-methylensarcosin (Methylcalceinblau) und vier damit verwandte, von Umbelliferon, 4-Methylumbelliferon und CMethylaesculetin durch Kondensation mit Formaldehyd und Iminodiessigsahre oder Glycin abgeleitete Metallguoreszenzfarbindikatoren wurden dargestellt und ihre Struktur festgelegt. Ihre Absorption und Fluoreszenz wurde in Abhlngigkeit vom pH gemessen und die Reakton mit Kupfer(I1) und Calcium im Hinblick auf Fluoreszenzeffekte studiert. Alle Verbindungen zeigen die starkste Fluoreszetu ungefahr bei pH 9. Bei alien wird die Fluoreszenz durch Kupfer(I1) geliischt. Die Calciumderivate der von den Umbelliferonen und Iminodiessigssiure abgeieiteten Verbindungen fluoreszieren bei hohem pH, die aus den Umbelliferonen und Glycin oder Sarcosin nicht. 4-Methylaesculetin und die davon abgeleiteten Aminos;iuren fluoreszieren bei hohem pH weder allein noch in Gegenwart von Calcium. R&urn&On a synthetid la 4-m&hylombeRiRrone-8-methyltnesarcosine (Bleu de Methylcalceine) ct quatre indicateurs metallofluorochromesderivees de l’ombelliferone. de la 4-methylombelliferone et de la Qmethylesculetine par condensation avec le formaldehyde et l’acide iminodiacetique ou la glycine, les structures ont et.6 etablies, I’absorption et la fluorescence mesuries en fonction du pH, et les reactions avec le cuivre(I1) et le calcium ont Cti Ctudites en portant I’attention sur les influences sur la fluorescence. TOW les compos6.s presentent une fluorescence maximale a un pH d’environ 9. La fluorescence de chacun d’eux est tteinte par le cuivre(I1). Les derives du calcium des composes derives des ombelliferones et de I’acide iminodiacetique sont fluorescents a pH Clevt, mais ceux provenant des ombelliferones et de la glycine ou de la sarcosine ne le sont pas. A pH eleve, la Cm&hylescuI&ine et les amino-acides qui en sont derives ne sont pas fluorescents. ni seuls ni en presence de calcium.