Indian Journal of Chemistry Vol. 438, September 2004, pp. 1944-1949

Synthesis and fluorescence properties of 3-benzoxa- and thiazol-2-ylquinoline-5 or 7 -maleimides

J S Nair & K N Rajasekharan*

Department of Chemistry, University of Kerala, Kariavattom, Trivandrum 695 581 India

Email: rajkn 51 @sancharnet.in

Received 24 April 2003; accepted (revised) I March 2004

Synthesis of four new 3-benzoxa/thiazol-2-ylquinolinemaleimides and their fluorescence properties are described . Among these, the 3-benzothiazol-2-ylquinoline-5-maleimide shows very good enhancement in fluorescence upon thiol addition and thus would serve as a sensitive thiol detection and quantitation reagent. Simi larly, 3-benzoxazol-2-y lquinoline-7-maleimide forms a fluorescent conjugate with a model protein BSA that has good emission properties, both in the native and in the denatured state, as shown by fluorescence spectroscopy and SDS-PAGE experiments, thus ind icating that 3-benzoxazol-2-ylquinoline-7-maleimide would be useful as a protein label that gives highly fluorescent protein bioconjugates.

IPC: Int.Cl7 C 07 D 215/00

Fluorolabels that are bioconjugatable to proteins find extensive application in various areas of bio­sciences.t·2 For these protein labelling reactions, the fluoroprobe should contain a reactive linker group.3

Maleimide group is a well established linker group which reacts readily with thiol groups in proteins to form an adduct.4 Quinoline is one of the smaller sized fluorophores and forms highly fluorescent deri­vatives.5 This ability has been exploited in the detection of amino acids in li ving cells even to a zeptomole level.5 Apart from its compact size, quinol ine as a fluorophore has other advantages such as being fluorescent in polar media and favourable solubility properties conferred by the presence of the heteroatom in the aromatic ring. These and other desirable emission properties of aminoquinolines have led to their use in the preparation of sensitive fluoro­genic enzyme substrates.6

·7 However, substituted

aminoquinolines have not been used in the design of fluoroprobes . We now report the synthesis of 5- and 7- amino-3-hetarylquinolines and their corresponding 5- and 7-maleimido derivatives as novel thiol-reactive fluoroprobes along with an evaluation of their emission properties and suitability as fluoroprobes.

Results and Discussion Maleimides are accessib le in a two step reaction

from the respective amines through the intermediate maleamic ac ids. 8 The required 7-amino-3-(2-hetaryl)-

quinoline in turn could be obtained by Combes quinoline synthesis9 using substituted malon­dialdehydes.10 The Vil smeier-Haack formylation of 2-methylbenzoxazole 1 and 2-methylbenzothiazole 2 would afford 10 the required 2-(benzoxazol-2-yl)·· malondialdehyde 3 and 2-(benzothiazol-2-yl)malon·· dialdehyde 4. We now find that the Combes reaction of 2-(benzoxazol-2-yl)malondialdehyde 3 with m­phenylenediamine affords the hitherto unreported 5·· amino-3-benzoxazol-2-ylquinoline S (Scheme I) in addition to the 7-amino-3-benzoxazol-2-ylquinoline 7 as observed by Naik et a/. 10 Some 7-acetamido-3-benzoxazol-2-ylquinoline 7a was also obtained from the reaction, but isolable amounts of the 5-acetamido-3-benzoxazol-2-ylquinoline Sa was not obtained; thi s derivative Sa was prepared later by acety lating S. The structure of the compound S was identified on the basis of its mass and 1H NMR spectra. The compound showed a M+ ion peak at m/z 261 which was the base peak of the EI mass spectrum. In its 1H NMR spectrum, the compound showed two downfield singlets at o 9.68 and 9.05 which could be assigned to H-2 and H-4 in the pyridine moiety . The benzoxazole moiety gave rise to two sets of two-hydrogen multiplets at 07.38··7.45 and 7.60-7 .64. Another upfi eld multiplet due to one hydrogen at 06.86 - 6.92 indicated a hydrogen ortho to an amino group. The two hydrogens of the NH2 group showed a rather broad signal at o 4.47. Two other two-hydrogen

NAIR eta/.: SYNTHESIS AND FLUORESCENCE PROPERTIES OF 3-BENZOXATHIAZOL-2-YLQUINOLINE MALEIMIDES 1945

~N}-Me~1 ~X DMF

I X=O 2X=S

cc N

SX=O 6X=S

I Maleic + anhydride

Q_; N

7X=0 8X=S

Maleic I anhydride +

cc NHCOCH=CHCOOH

N

9 X=O 10 X= S

NHCOCH=CHCOOH

II X=O 12 X= S

~Q' Q X 0 N 0 - N,;

N~ ~ ~

13 X= 0 14 X= S ISX=O

16 X= S

Scheme I

multiplets were seen at 87.79-7 .85 and 7.65-7.68 due to 7-H and 8-H, respectively . These signals are compatible with a 5-amino-3-benzoxazol-2-ylquino­line structure 5 for the compound. In contrast, the compound 7 in its 1H NMR spectrum showed a singlet due to one hydrogen and two doublets due to the other two hydrogens in the benzene ring of quinoline. The 1H NMR data of 7a was overall similar to that of 7, except for an additional singlet due to the acetyl group. In a reaction starting from

2-(benzothiazol-2-yl)malondialdehyde, we have now isolated the 5-amino-3-benzothiazol-2-ylquinoline 6, the isomeric 7-amino-3-benzothiazol-2-ylquinoline 10

8 and 7-acetamido-3-benzothiazol-2-ylquinoline Sa; the acetyl derivative 5-acetamido-3-benzothiazol-2-ylquinoline 6a was prepared separately .

The aminoquinolines so obtained were then reacted with maleic anhydride to prepare the maleamic acid derivatives 9-12. These were then cyclised to 3-benzoxa/thiazol-2-ylquinolinemaleimides 13-16. The

1946 INDIAN J. CHEM., SEC B, SEPTEMBER 2004

1H NMR spectra of all these maleimides showed a characteristic singlet in the region o 6.98-7.10 due to the two maleimide ring hydrogens. EI mass spectrum of 13 and 15 showed moderate and weak M+ ions at mlz 341 while the EI mass spectrum of 14 and 16 each exhibited M+ ions at m/z 357 that were the base peaks in the respecti ve mass spectrum.

The sy nthetic part of the work described herein show that in the Combes reaction leading to the ami nes 5-8, the Friedel-Crafts acy lat ion step lead ing to heterocycl isation occurs at the para-position of the free amino group in m-phenylenediamine. This provides the regia-isomers 7 and 8 approximately in a 12:1 ratio, on average, in preference to the isomeric 5 and 6 formed by the reaction at the ortho-pos ition. These amines 5-8 gave in the next step the maleamic ac id derivati ves in 77-89% yield, with no observable preferences between the oxa and thi a analogs, but the maleimide formation proceeded better in the case of the sulphur analogs 14 and 16.

The fluorescence spectra of 7-amino-3-hetaryl­quinolines and those of their acetyl and maleimide derivatives have now been examined. A so lution of quinoline sulphate served as the reference and all emiss ion intensities were normali sed with respect to it. It is now observed that in methanol , the 7-amino-3-benzazol-2-ylquinolines 7 and 8 are more flu orescent th an their 5-amino analogs 5 and 6. Further, the thia analogs 6 and 8 are more flu orescent than the oxa derivatives 5 and 7. In summary, the 7-amino-3-benzothi azo l-2-ylquinoline 8 is fo und to be the most fluorescent amine among the four aminoquinolines now studied (Table I).

We have also determined the emission property of

8 in J: J MeOH-phosphate buffer (0.1 M, pH 8.0). It has been found that the emission maximum is red­shifted to 470 nm but the aqueous environment quenches the fluorescence since the relative emission intensity decreased (Table 1) . In short, our study showed that compound 8 would be useful as a fluorescent hetary l amine that could be used in the preparation of fluorogenic enzyme substrates which could be used to detect and quantify enzy matic amide bond hydrolys is. It is hi ghly fluorescent with a!:::,.").._ of 50 nm between the lcmax of the amine and its amide in aqueous buffer so lutions. Further, the compound 8 can be selectively excited at a wavelength of 391 nm at which it has a substanti al extinction coefficient of absorption. Further, at thi s wavelength of excita ti on , its amide form Sa does not absorb.

Fluorescent reagents that show en hancement in emiss ion upon reaction with thiol groups find va lu able use in va ri ous field s of biosc iences. It was Kanaoka 11 who pioneered the use of fluorescen t maleimides in the study of protein thiols. Maleim ides bearing a fluorogenic moiety should have a very low flu orescence before bioconjugation. Upon reac ti on with a thiol group in a biomolecule such as a protein , a large increase in fluorescence emiss ion in tensity should occur thereby this increased signal may be used as a qu anti tat ion tool. Among the currentl y used fl uorescent maleimides, 7-dimethylamino-4-methyl­coumarin-3-maleimide (DACM) 12

•13 is the most

prominent one. Its thiol adduct emit at around 457 nm when exc ited at 385 nm in ethanol. The remarkab ly long wavelength emiss ion of compound 6 at 571 nm prompted us to develop maleimide probes based on the four new aminohetarylq uinolines 5-8. The

Table I- Em iss ion properties" of aminobenzazoly lqu inolines in methanol.

Compd Quinoline derivatives Acx "-em REI

5 5-Amino-3-benzoxazol-2-yl 406 558 39

7 7 -Ami no-3-benzoxazol-2-y I 387 444 374

Sa 5-Acetamido-3-benzoxazol-2-yl 325 468 19

7a 7-Aceta mido-3-benzoxazol-2-y I 307 395 295

6 5-Amino-3-benzothiazol-2-yl 404 571 33

8 7-Amino-3-benzothiazol-2-yl 391 449(470)b 524

6a 5-Acetamido-3-benzothiazol-2-yl 335 449 106

Sa 7 -Acetamido-3-benzothiazol-2-y I 358 406(420) 346

• Referen ce used was quinine sulphate in O. IM H2S04, to which arbitrary relative e missio n intensity (REI) of I 00 has been assigned. The solution was exc ited at 332 nm. All solutions used had an optical density of 0 .150 ± 0.005 at the respective Acx. b Value in parenthesis are Acrn in I : I MeOH-phosphate buffer O.IM, pH 8.0.

---------------------

NAIR eta/.: SYNTHESIS AND FLUORESCENCE PROPERTIES OF 3-BENZOXATHIAZOL-2-YLQUINOLINE MALEIMIDES 1947

Table II- Emission properties before and after reaction with DIT

Compd" /..ex (nm) >..\;;~~ nm) Rei. Emission (xI 06

) Enhancement b .c. d

13 320 457 0.018

13 + DIT 320 467 1.17 65

IS 333 405 0 .029

IS+ DIT 333 408 3.5 120

14 333 458 0.006

14 + DIT 333 469 1.2 200

16 332 417 0.04

16 + DIT 332 417 2.2 55

"Sol vent MeOH bA fter add iti on of 2 ~LL dithiothreitol (DIT) to the cuvette (4 mL) <Expressed relative to the emiss ion in the absence of DIT dEx pressed re lative to quinine su lphate (0.1 M H2S04 , OD = 0.150) excited at 332 nm

(Relative em ission RE = 0.89xl06)

3-benzothiazol-2-ylquinoline-5-maleimide 14 ex hibits very low fluorescence (Table II) , but upon the addition of dithiothreitol (OTT), a 200-fold increase

in emission occurs with a >..\;;~~ of 469 nm and this

>..\;,·~~~ places the maleimide 14 alongside with the

currently used ones, OACM A.\~;~~ of the thiol adduct,

457 nm in ethanol) and N-[4'-(7-diethylamino-4-

methylcoumarin-3-yl)phenyl]mal eimide (CPM; >..\~',',~~

of the thiol adduct, 465 nm in MeOH). The data from these experiments (Table II) have thus revealed th at 14 would serve as a sensit ive thiol detec tion and quantitation reagent because of the large increase in the emi ssion signal that resulted upon reac tion with a thiol compound.

For protein labelling experiments, the 3-benzo­xazol-2-ylquinoline-7 -maleimide 15 appeared as the best fluorol abel among the four we have now prepared as its OTT adduc t was the most emi ss ive (Table II) . In such labelling, any of the unreacted or hydrolysed male imide reagent can be removed by dialysis or chromatography, thus avoiding any background fluorescence due to any excess fluoro­label. Experiments based on conjugating these four maleimides 13-16 with bov ine serum albumin (BSA), selec ted as a reference protein , and the exa mination of the fluo rescence of the protein bands after sodium dodecyl sulph ate-po lyacry lamide ge l e lec trophores is (SDS-PAGE) experiments, revea led that the bio­conjugate of 15 with BSA is the most fluorescent band on the gel among the fou r examined now. Thi s showed that compound 15 would be useful as a new,

reactive protein label that gives highly fluorescent bands on PAGE gels. We also observed that among the four maleimide-BSA conjugates now prepared, that of BSA-15 was the most fluorescent in the nati ve form as well.

Experimental Section

Reagents and so lvents were from Merck Indi a and Fluka. The instruments used were Bruker WM 400 and AC 300F and Varian EM 390 NMR and JEOL-0 300 ElMS spectrometers, Jasco FP750 and SPEX Fl uorolog F 112X spectrofluorimeters and Biorad Gel Scanner. Melting points are uncorrected. Elementa l analyses were done at CORJ, Lucknow, Indi a.

Preparation of aminobenzazol-2-ylquinolines. General procedure. A mi xture of 2-benzoxa/thiazo l-2-ylmalondialdehyde, 111-phenylenediamine, p­toluenesulphonic acid (1 mmole each) and glac ial aceti c acid (25 mL) was ref! uxed fo r 6 hr, cooled and poured into ice water and neutrali zed with aqueous sod ium hydroxide ( 10%) so lution . The crude so lid was filtered, dried, and purifi ed by column chromato­graphy using neutral alumina.

5-Amino-3-benzoxazol-2-ylquinoline 5. It was prepared fro m 2-benzoxazol-2-ylmalondialdehyde and m-phenylened iamine and was obtained (6%) as the first co mponent which e luted in benzene during co lumn chromatographi c purifi ca tion, m. p. 228°C; 1H NMR (400 MHz, CDCI 3): o 4.47 (b, 2H, Nf-1 2),

6.86-6.92 (m, IH, 6-H) , 7.38-7.45 (m, 21-l , 5'- f-l , 6'- f-I ); 7.60-7.64 (m, 2H, 4' -H , 7'-f-1), 7.65-7 .68 (m, Il-l , 8-f-1 ),7.79-7.85 (m, I f-1 , 7-f-I ), 9.05 (s, I H, 4- 1-1 ), 9.68 (s,

1948 INDI AN J. CHEM., SEC B, SEPTEMBER 2004

11-1 , 2-H); ElMS: m/z(%) 262(37), 26 1(M+, 100), 260(10), 245(12) , 244(44), 234(5), 169(46), 168(12), 149( 12), 143(18), 142(40), 141( 14), 1 16(22), 1 15(25), I 14( 17), 89( 12), 64(5 1 ), 63(75).

7-Amino-3-benzoxazol-2-ylquinoline 7. 1t was obtained (65 %) as the second eluted component in CHCI 3, m. p 243°C; 11-1 NMR (400MHz, DMSO-r/6) :

86.3 (s, 21-1, Nl-1 2) , 7.00 (s, 1 H, 8-H), 7.09 (del, 1=8 Hz, 2Hz, lH , 6-H), 7.36-7.45 (m, 21-1 , 5'-H, 6'-H), 7.78 (m, 2H, 4'-1-1, 7'-H), 7.85 (d, 1=81-I z, 1 H , 5-1--1), 8.79 (d, 1=21-l z, IH , 4- l-1), 9.31 (d, 1=21-l z, 11-1 , 2-H); ElMS : m/z(%) 262(15), 261(M+, 100), 260(15), 234(5), 169( 10), 167( 14), 149(43), 142( 18), 11 6(7), 11 5(6), 91(5), 71( 12), 63(34), 57(26), 44(26).

7-Acetamido-3-benzoxazol-2-ylquinoline 7a. This compound was obtained as the third component to be eluted out from the co lumn in 30: I chloroform­methanol (60%), m. p. >300°C; 1H MR (400MI-I z, DMSO-c/6) : 8 2. 14(s, 31-1), 7.40-7 .56 (m, 21--1), 7.80 (cl, J=4l--l z, li--1 ), 7.84 (t, J=4Hz, I H ), 8.1 3 (d, 1=31-l z, I H), 8.50 (s, I H), 9.52 (s, I 1-1 ), I 0.47 (s, 1 1-1 ); ElMS: m/z(%) 304 (2 1 ), 303 (M +, 95), 262 ( 4 1 ), 261 ( I 00), 260 (33), 235 ( 10), 234 ( 16), 142 ( 17), 116 (9), 115 (8), 114 (8), 63 (20), 44 (48).

5-Amino-3-benzothiazol-2-ylquinoline 6. It was obtained from 2-benzothiazol-2-ylmalonclialclehyde and was obta ined (5%) as th e first component to be el uted in benzene during co lumn chromatography, m. p. 245°C; 11-1 NMR (400 MH z, CDCI3): 84.57 (b, 21-1, N l-1 2), 6.90 (d, 1=81-l z, I H, 5-f-1) , 7.56 (t, J=6Hz, 11--1 , 7-1--1 ), 7.62 (d, 1=61-lz, I f-1, 8-1-1 ), 7.60 (m, 21-1, 5'-H, 6'-1--1 ), 7.98 (cl, 1=81--l z, 1H, 4'-1-1), 8. 12 (cl , J=8Hz, 11--1 , 7'­H), 8.92 (d, 1=2£-l z, IH, 4-1-l ), 9.52 (cl, 1=21--l z, 1 H, 2-H); ElMS: m/z (%) 279( 18), 278(57) , 277(M +, 1 00), 276(30), 26 1 ( I I ), 245( 11 ), 244(54 ), 143(9), 1 42(28), 138(25), 136(28), 135(14) , 125( I 5), I 09( 18), 71 (22), 69(23), 57(38).

7-Amino-3-bcnzothiazol-2-ylquinoline 8. During the above purifi cati on, thi s compound eluted (69%) as the second component in CHCI 3 ; m. p. 252-54°C; 1 HNMR (400MHz, DMSO-c/6) : 84.30 (b, 2 1--1 , Nl-fz) , 7.06 (del , 1=21--l z, J=9.5H z, I 1-1 , 6-H) , 7.28 (d, 1=21-lz, Il-l , 8-1--1 ), 7.42 (t, J=6Hz, 11--1, 5'-f-1 ), 7.53 (t, 1=81-I z, 11-1 , 6'-l--1), 7.76 (cl , 1=9.51--!z, 11-1 , 5-1--1), 7.95 (d, 1=81-l z, I H, 4'-H), 8. 10 (d, 1=81-lz, 11--1 , 7'- 1--1 ), 8.64 (d, 1=21-l z, IH, 4- 1--1 ), 9.44 (cl, 1=21-lz, I f--1 , 2-1-1); ElMS: m/z (%) 278(20), 277(M +, 100), 276(30), 25 1( 10), 142( 13), 138( 11 ).

7 -Acctamido-3-bcnzothiazol-2-ylq uinoline Sa. This was obtained (6%) as the third compound to be eluted in a 30: I chloroform-methanol, m. p. 303°C;

EIMS: m/z (%) 320(12), 319(M+, 49), 278(22), 277(M-42, 100), 251(11), 222(11), 105( 12), 91 ( 13), 71( 19).

Preparation of 5-acetamido-3-benzoxa/thiazol-2-ylquinolines Sa and 6a . The acetyl deriva ti ves Sa and 6a of 5-amino-3-hetarylquinolines 5 and 6 were prepared by heating the respective am ines ( I mmole) w ith acetic anhydride (2 mL) at 95 °C for 30 min. The mixture was then poured into cold water and stirred well. The solid was filtered , washed with water and dried. It was purified by recrystal l isation from benzene.

5-Acctamido-3-benzoxazol-2-ylquinoline Sa : yield 68%, m. p. 266°C; 1 I-1 NM R (90 MI--I z, CDCI 3) :

8 2.42 (s, 31-1) , 7.48 (t, I H ), 7.56 (t, 1 1-J ), 7.75-7 .85 (m, 21-1), 8.00-8.10 (m, 2H), 8. 14 (d, !H), 8.94 (s, 11-1 ), 9.52 (s, 1 H); ElMS: 111/z(%) 3 19(M +, 29), 277(35), 244(6), 125(12), Ill (24), 99(26), 97(49), 85(67), 83(53 ), 7 I (79), 58( 1 00).

5-Acetamido-3-bcnzothiazol-2-ylquinoline 6a: yield 73 %, m. p. 266°C.

Synthesis of 3-hetarylquinolinemaleimides 13-16. 3-I-Ietarylquinoline a111 ines 5-8 (1 111mole) were treated w ith maleic anhydride (4 mmoles) in glacial acetic acid (8 mL) and stirred for I hr. The prec ipitated maleamic acids 9-12 (y ield 77-89%) were filtered, washed first w ith glac ial acet ic acid, then with diethyl ether and then dried. A mixtu re of maleamic ac ids 9-12 ( I mmole), acetic anhydride (10 mL), an hydrous sod ium acetate (50 mg), was heated to 120°C with st irring for I hr. The clear liquid was then coo led and poured dropwi se to ice-water with stirring. The so lid was filtered, washed w ith water and dried . It was purifi ed by elution w ith ch loroform from a co lumn of silica ge l to gi ve 3-benzoxa/thi azol-2-ylquino linemaleimides 13-16.

3-Bcnzoxazol-2-ylquinoline-5-maleimide 13: yield 58%; overall y ield from 5, 49%, 111. p. 220°C. Anal. Found: 1, 12. 19. Calc. for C20l--1 11 N30 3 (34 1.3) :

, 12.31 %; 1I--1 MR (90MI-l z, CDCI3) : 8 7.10 (s, 2H,CH=CI--I), 7.45 (m, 2H), 7.55(d, IH), 7.65 (d, 11-1), 7.82 (d, !H), 7.92 (t, lf-l), 8.35 (d, 11-1 ), 8.7 1 (s, 11-1), 9.80 (s, lH) ; ElMS: m/z(%) 34 1(M +, 74), 3 13(7), 297(16), 261(26), 191 (5), 149(8), 97(26), 85(36), 71(47), 57( 100), 44(48) .

3-Bcnzothiazol-2-ylquinolinc-5-maleimide 14: y ield 78 %, overa ll y ield from 6, 61 %, 111. p. 200°C. Anal. Found: N , 1 1.82. Calc. for C2o i-I 11 N30 2S (357.4): N, I 1.76 %; 1HNMR (400MHz, CDCh): 8 7.07 (s , 21-1 , HC=CI-1), 7.42 (t, 1=1 0.2Hz, I H), 7.5 I (d, 1=10.41--! z, 11-1), 7.55 (t, 1=10.41-lz, IH) , 7.88 (t, 1=5.6 1--! z, I 1-1), 7.95 (d, 1=10.61-lz, 1 H), 8.1 4 (cl,

NA IR e1 a/.: SYNTHES IS AND FLUORESCENCE PROPERTIES OF 3-BENZOXATHI AZOL-2-YLQUI NO LINE MALEIMID ES 1949

l=l0.5Hz, !H), 8.30 (d, 1=10.8Hz, 1H), 8.50 (d, 1=2.8Hz, 1H), 9.64 (d, 1=4Hz, 1H); ElMS: m/z(%) 358(12), 357(M+. 100), 330(41), 329(87), 328(45), 304(19), 303(28), 302(12), 275(16), 273(15), 261(28), 260( 12), 249( 12), 243( 11 ), 149(22), 140( 16), l 09(28), 82(18).

3-Benzoxazol-2-ylquinoline-7 -maleimide 15: yield 70%, overall yield from 7, 62%, m. p. 265°C. Anal. Found : C, 70 .1 9; H, 3.05; N, 12.46. Ca lc. for C2oHt1N30 3 (34 1.3): C, 70.38; H, 3.25; N, 12.31 %; 1HNMR (90 MHz, CDC13): o 6.98 (s, 2H, CH=CH), 7.40-7 .50 (m, 2H), 7.65 -7.80 (m, 2H), 7.88 (d, 1H), 8.08 (cl , 1H), 9.05 (s, 1H), 9.76(s, !H); ElMS: m/z(%) 341(M+, 4), 316(l0), 19 1( 10), 147(6), 97(10), 85(29), 83(42) , 71(42), 69(29), 58(100); FABMS; (NBA matrix): M+H+ 342.

3-Benzothiazol-2-ylquinoline-7 -maleimide 16: yield 75 %, overall yield from 8, 58%, m. p. 245°C. Anal. Found: C, 67.36; H, 3.18; N, 11.89. Calc. for C2oHt1N30 2S (357.4): C, 67.21; H, 3. 10; N, 11.76%; 1H NMR (90MHz, CDCl3): o 6.98 (s, 2H, HC=CH), 7.42-7.62 (m, 2H), 7.68-7.72 (d, 1H), 8.0 (d, 1H), 8.06 (d, 1H), 8. 17 (d, 1H), 8.38 (s, 1H), 9.70 (s, lH); ElMS: m/z(%) 358(23), 357(M+, 100), 356(10), 302(10), 277( 10), 160(23), 157(7), 153(12), 152(12), 151 (25), 144(13), 135(13), 124(10), 108(37) , 82(3 1), 69(63), 58(47), 45(36).

Protein modification and polyanylamide gel electrophoresis. For evaluation of fl uorolabels, bovine serum albumin (BSA) was used as a reference protein . A so lution of BSA (15 ~tM) in phosphate buffe r (0. 1M, pH 7.4) was treated with OTT (60 ~tM ).

After 30 min , a solution of maleimide 13-16 in DMSO was added to get a concentration of 300 ).!M. The labelling was al lowed to proceed for 30 min . Then it was treated with neat 2-mercaptoethanol to get a fina l concentration of 600 ~tM. The emi ssion intens ity of the maleimide-BSA conj ugates was then examined in the native state. The protein-BSA so lu tions were th en processed and separated by SDS-

PAGE. For thi s, the protei n was denatured by mi xing 50 ~LL of the labelled protein solution with 50 ~tL of denaturing solution contai ning Tris-HCL (O. l25M, pH 7.4), sod ium cloclecy l sulphate (4%), 2-mercaptoethanol ( l 0% ), glycero l (20%) and bromophenol blue (0.004%) . The gel was fixed in acetic acid-methanol mi xture for 15 min and was scanned by a CCD imager equipped Bio Rae! Gel Documentation system using a UV-Transillu min ator platform. The fluorescence data was co llected for 3 min and processed in a PC by the custom-software. This study showed that the BSA-[3-benzoxazol-2-ylquinoline-7-maleimicle] conjugate (BSA-15) was the most fluorescent among the four bioconjugates now examined in the nat ive state as well as on the gel.

Acknowledgement

The authors thank RSIC, Lucknow for NMR and MS spectra and also for the CHN analys is.

References

1 Gui lbault G G, Practical Fluorescence, 2"" ed n, (Ma rcel Dek­ker, New York), 1990, I.

2 Dewey T G, Biophysical and Biochemical Aspec/s of FluOI·es­cence Speclroscopy, (Ple num Press, New York), 1991 , I.

3 Haug land R P, Handbook of Fluorescen r Probes and Re­search Chemicals. 6'11 Ed n, (Molecular Probes lnc , Eugene), 1996, 1.

4 Gassman P G, Gilbert D P & Cole S M, J Org Chem, 42, 1977, 323 .

5 Liu J, Anal Chem, 68 , 1991 , 408. 6 Brynes P J, Bevilacqua P & Green A, Anal Biochem , 116,

1981' 408. 7 Brynes P J, Andrade P & Gorden D, Anal Biochem, 126,

1982, 447. 8 Cava M P, Deana A A, Muth K & Mitchell M J, Org Symh,

4 1, 1961, 93. 9 Combes, A, Bull Soc Chiln France, 49, 1888, 89.

10 Naik H A & Seshadri S, In dian J Chem, 158, 1977, 506. 1 1 Kanaoka Y, AngeiV Chem lnrl Edn , 66 , 1977, 137. 12 Mac hida M, Ushijima N, Machida M 1 & Kanaoka Y, Chem

?harm Bull, 23, 1975, 1385 . 13 Con·ie J E T, J Chem Soc Perkin Trans I, 1990, 2 1 5 I.