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J . Org. Chem. 1995,60, 2391-2395 2391A New Method for the Synthesis of Heptamethine Cyanine Dyes:Synthesis of New Near-Infrared Fluorescent Labels

Narasimhachari Narayanan*gt and Gabor PatonayDepartment of Chemistry, GeorgiaState University,Atlanta, Georgia30303

Received J anuar y 3, 1995@

A new uncatalyzed synthesis of heptamethine cyanine dyes is described. The reaction involvesheating amixture of N-alkyl-substituted quaternary salts derived from 2,3,3-trimethylindole or2, 3, 34r i methyl benzi ndol eand 2- chl or o- l - f or myl - 3- ( hydr oxymet hyl ene) cycl ohex- l - eneo reflux ina mixture of 1-butanol and benzene (7:3) as solvent. No catalyst is used, and water is removed asan azeotrope. The resulting chloro compounds possess strong absorption and fluorescence propertiesin the near-infrared region and are convertedtodyes bearing reactive functionalities such as hydroxyand isothiocyanate groups useful as fluorescent tags for nucleic acids and proteins. Severalsymmetric and nonsymmetric dyes have been synthesized in high yields.Introduction

Near infrared (near-IR) dyes are importantassensitiz-ers in several appli~ationsl-~uch as silver halidephotography, laser dyes, pleochroic dyes for LC displays,xerography, cosmetic ingredients, and dyes for polymers.Polymethine cyanine dyes are increasingly used asfluorescent tags inDNA ~equencing,~,~mmunoassay,6-6and flow ~ytometry.~-ll he advantagesof the near-IRdyes in bioanalytical methods12J3 nclude their strongspectral properties in the longer wavelength region(700-1000 nm) with minimal background from biomoleculesand high sensitivity.

Cyanine dyes have traditionally been synthesized bya condensation reaction between a heterocyclic basecontaining an activated methyl group and an unsaturatedbisaldehyde or its equivalent, usually as Schiff base inthe presence of a catalyst. Sodium acetate has mostfrequently14-16 been used as a catalyst. In addition toethanol, solvents such as acetic acid andor acetic anhy-dride have also been commonly used as in the synthesis+Present address: LI-COR, Inc., 4421 Superior Street, Lincoln, NE@Abstractpublished inAdvance AC S Abstracts, March 20, 1995.(1)Zollinger, H. Color Chemistry;VCH: Weinheim, 1991.(2) Fabian, J .;Nakazumi, H.; Matsuoka, M. Chem.Rev. 1992, 92,1197.(3) Warning, D. R., Hallas, G., Eds. The Chemistry and Applicationsof Dyes; Plenum: New York, 1990.(4 ) Middendorf, L. R.; Bruce, J . C.; Bruce, R. C.; Eckles, R. D.; Grone,D. L.; Roemer, S.C.; Sloniker, G. D.; Steffens, D. L.; Sutter, S. L.;Brumbaugh, J .A,; Patonay, G. Electrophoresis 1992,13,487.(5)Brumbaugh, J .A.; Middendorf, L . R.; Grone, D. L.; Ruth, J .L.Proc. Natl. Acad. Sci. U S A . 1988,85, 5610.(6) Bhattacharyya, A. Indian J .Biochem. Biophys. 1986,23, 171.(7) Chen, E. Y.; Kuang, W.; Lee, A. L. Methods 1991,3,3.(8)Williams, R.J .;Lipowska, M.; Patonay,G.;Strekowski, L.Anal.(9) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S.R.; Lewis, C. J .;( I O) Ernst, L. A.; Gupta, R. K.; Mujumdar, R. B.;Waggoner, A. S.(11)Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S.R.; Waggoner, A.(12) Boyer, A. E.; Lipowska, M.; Zen, J.-M.; Patonay, G.; Tsang,V.(13)Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 63, 321A.(14)Makin, S. M.; Boiko, L. I.; Shavrygina, 0.A. Zh. Org. Khim.(15)Slominski, Yu. L.; Radchenko, I. D.; Popov, S.V.; Tolmachev,( 16)Sosnovskii, G. M.; Lugovskii, A. P.; Tischenko, I. G.Zh. Org.

68504.

Chem. 1993,65,601.Waggoner, A. S.Bioconjugate Chem. 1993,4, 105.Cytometry 1989, 10, 3.S.Cytometry 1989, 10, 11.C. W.Anal. Lett. 1992,25, 415.1977,13,2440.A. I. Zh. Org. Khim. 1983,19,2134.Khim. 1983,19,2143.

0022- 3263/ 95/ 1960- 2391$09. 00/ 0

Scheme12 1 + P

ButanollBenzeneNo catalyst

7(X, =OPhR)

of heptamethine pyrylium dyes.l7 Although this proce-dure is simple and straight forward, it suffers fromseveral disadvantages. The purification of the productis very difficult because of the side products due toaniline; the use of a catalyst interferes with the purityof the product, as often noticed in NMR and IR, andwarrants repeated purification; the reaction is generallyfaster and cannot be employed for the synthesis ofnonsymmetric dyes in onepot; scaling up of the reactionproducts to larger gram quantities leads to severaladditional problems resulting in poor quality and yield.Results and Discussion

In this paper, we describe a new and simple methodfor the uncatalyzed synthesis of heptamethine cyaninedyes which are potential precursors for making function-alized near-infrared labels. This approach involves heat-ing a mixture of a quaternary salt of a heterocyclic basecontaining an activated methyl group 1 (2 equiv) and2- chl oro- l - f ormyl - 3- (hydroxymethyl ene)cycl ohex-l - ene21,an unsaturated bisaldehydel7J8 derived from cyclohex-anone(1equiv) to reflux in a mixture of 1-butanol and(17) Reynolds, G. A.; Drexhage, K. H. J . Org. C hem. 1977,42, 885.(18)Organic Syntheses; Wiley: New York, 1973; Collect.Vol. 5,p215.

0 1995 American Chemical Society

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2392 J . Org. Chem., Vol.60,No. 8, 1995 Narayanan and PatonayTable1. Synthesisof SymmetricChl oro Dyes 4 fromQuaternary Salts1

absyield maxproduct Z Y Ri(X) t(h) (%) (nmPQuaternary Salts 1Y

l a Hl b Hl c Hi d OCHsl e OCH,I f H%I H

ZCMe2CMe2CMe2CMe2CMe20S

OH Pa, X:!=CI2 4 X2 H

Bisaldehydes 2benzene (7:3) as solvent, without using any catalyst(Scheme1).The water formed during the reaction isremoved as an azeotrope by a Dean-Stark condenser.The reactions generally require 3-12 h for completion.The resulting product is generally pure after simplefiltration of the dye from the crude reaction mixturefollowed by washings with diethyl ether. In some casesfurther purification was not necessary, as revealed byproton NMR, while in other cases a flash chromato-graphic filtration would be sufficient for better purity. Awide range of 2-methy-1-alkyl quaternary saltsofvariousindole 1 and benzoindole derivatives 3 undergo thisreaction in a facile manner to form the correspondingsymmetric dyes (Tables1and 2)inhigh yields. It seemed

Quaternaty Salts 3z R2(W

3a CMe2 (CHz)4S033b CMe2 CHzC Hd )3c CMe2 (CH&NHn.HBr(Br)3d CMe2 Phthalimidobutyl(Br)30 0 CH2CHdI)3f S CHzCHdI)

initially that the chlorine in the bisaldehyde 2a wasimportant for making the aldehydic protons acidic andreactive in the absence of a catalyst. However, thesmooth reaction of the bisaldehyde2b19(which lacks theC1)with quaternary salt3b to form the dye5f (Table 2)

6proved that the C'I was not needed for the bisaldehyde

(19) Makin, S. M.; Boiko, I. I.; Berezhnaya,M. I.; Boiko, T. N.Zh.Org.Khim. 1977,10,24. See also ref 14.

4a4b4c4d4e4f4g4h

CMe2 HCMe2 HCMez HCMe2 OCH3CMe2 OCH3CMez HO HS H

a Measured in MeOH. *Represents crude yield and convertedto 4f.Table2. Synthesis of SymmetricChl oro Dyes 5 fromQuaternary Salts3

absyield maxproduct R z W t(h) (%I (nmIb5a (CHd4SOdH) 8 82 8225b CzHdI) 6 95 8165c (CH2)3NHyHBr(Br) 7 95e 8205d CzHdI) 8 92 8215e (CH2)3NCS(Br) 6 86 8225f CzHdIY 8 88 7835g CzHdI) 2 75 7485h CzHdI) 8 85 830

a Z =CMez except 5g where Z =0 and 5h where Z = S.Measured in MeOH. Represents crude yield and converted to5e. C1=H.tobe reactivetoundergothisreaction, especially withouta catalyst. In contrast to the Schiff base method, thereaction of l a and 2a in refluxing ethanol with sodiumacetate as catalyst did not form any product over threedays, suggesting that the reaction involves an equilibri-um step.An important featureof the current method is that theslower rate of the reaction allows oneto prepare non-

symmetric dyes derived from two different heterocyclesin a single pot in a fairly good yield. The syntheses ofthese nonsymmetric dyes become important when changesin the spectral and physical properties of the dyes aredesired for specific application and compatibility withinstrumentation. These changes can be incorporated byappropriately modifying the structural designof the dyes.Reports in the literature concerning the syntheses ofnonsymmetric dyes reveal that they always involve twoHowever, they do not limit the synthesis tothe formation of pure nonsymmetric dye alone. Sym-metric dyes have always been the byproducts. The newmethod provides an easy procedure for the synthesis ofnonsymmetric dyes. For instance, the chloro dye6a isobtained by first heating a mixture of 3c and2a (1:l)oreflux in butanol-benzene (7:3) with removal of waterfor 2h (Scheme 2). The second quaternary saltof l a isthen added and refuxing resumed for 12 h (Table 3). Thenonsymmetric dye6a is the major product, although thesymmetric dyes4a and5c have also been isolated whichare easily separable by chromatography on silica gel.

~(20) For reviews on the synthesis of nonsymmetric cyanine dyes,see: Ficken,G. E. Cyanine Dyes. InTheChemistry ofsynthetic Dyes;Venkataraman, I C, Ed., Academc Press: New York, 1071;pp 212-334.(21) Hammer, F. M The Cyanine Dyes and Related Compounds;Wiley: New York, 1964.(22) Venkataraman,K. TheChemistryof Synthetic Dyes;AcademcPress: New York, 1952, pp 1143.

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Synthesis of Heptamethine Cyanine DyesScheme2

J .Org. Chem., Vol.60, No. 8, 1995 2393Table4. Conversion of Symmetric Chloro Dyes4 toFunctional Dyes 7a

- H20 , Reflux nBuOH-Benzene

6a (R1=C4H8S03)(X2=CI; X=NH2.HBr)6b (Rl=CzH5)

6e (Rq=CdHeS03)6f (Ri=CzH5)(X2sCI; X-NCS )cs . Na2C03,CSCh n CHC13, rt

R4PhOH?OM e H(X2'=OPh%; X=NCS)Table3. Synthesisof Nonsymmetric Chloro Dyes 6 fromQuaternary Salts1and3

absyield maxDroduct RdX) R t(h) (%) (nWa796795804801804800

a Measured in MeOH. b Represents crude yield and convertedto6eand6f,respectively.The dye7a with a reactive isothiocyante group waslabeledtoaDNA oligomer and usedinourearlier studieson DNA sequencing application^^ However, a majorconcern was to eliminate the cleavage of the enol etherfrom the chromophore which resulted in the failure ofdetection by fluorescence and responsible for the appear-ance of smearing in the sequence ladder which renderedthe sequence nonreadable. A n alternative strategy forattaching the linker arm such as the one through thenitrogen of the heterocycle was thought of, for exampleby the use of phthalimidobutyl group. Attempts madeto deprotectZ4 he phthalimide group on the dyes6c and6d by either acid-catalyzed (refluxing in concentratedHC1) or base-catalyzed hydrolysis (methyl hydrazine)were not successful to furnish the amine in good yield.However, a new approach avoiding the protective groupsaltogether was found to be effective by the use ofhydrobromide salt of the propylamine by itself.25 Nofurther protection was required in this case and theprocedure proved to be highly efficient. The dyes in the( 23)Shealy,D. ; ipowska, M.; Lipowski, J .; Narayanan, N.; Sutter,( 24)Green, T. W.; Wuts, P. G. M. Protective groups in organic(25)3-Bromopropylamine hydrobromide was used for preparing

S.;Strekowski, L.; Patonay, G.Anal. Chem. 1996, 67,247.synthesis; J ohn Wiley and Sons: New York, 1991.quaternary salt of l , l , 2- tr i methyl - l H- benz(e)i ndol e.

abst yield maxproduct Y R Rl(X) (mid (%I (nm)b7a H7c H7d H70 H7f H7g H7h Ha Z =CMez.

7b OCH3 NCS (CH2)4S03(Na) 15 90 772NCS (CHMOdNa) 30 94 795(CHhOH (CH2)4SOdNa) 20 90 76820 92 769CS CzHdI)(CHd30H CzHdI) 20 90 763(CH2)20H CZH6(I) 25 90 766N(CHZCOZM~)~zHdI) 25 85 770H (CH2)3NCS(Br) 20 80 776Measured in MeOH.

Table5. Conversion of Nonsymmetric Chloro Dyes6 toFunctional Dyes8abst yield maxproduct Ri R4 (min) (%) (nmIa

8a (CH2)4S03(Na) H 25 81 7888b (CH&S03(Na) OCH3 30 80 7908c CzHdI) H 25 82 784a Measured in MeOH.

form of hydrobromide salt (dyes4c,5c,6a,and6b)wereused in the subsequent step. They were purified afterconvertingtothe corresponding isothiocyanate derivative(compounds4f, 5e,6e,and6f respectively). Thismethodserves dual advantages of not only simplifying thereaction procedure butalsomakesitfaster by eliminatingprotection and deprotection steps.The vinylic chlorine on the cyclohexane bridgehead ofthe dye is reactive and can be replaced by avariety ofnucleophiles.26 The chloro compound4a in general canbe converted to7a,by using 4-N-tert-butoxycarbonyl (t-BOO-protected phenolz4 and sodium hydride in DMF.The protective group is removed by trimethylsilyl chlo-ridez7 TMSC) n the subsequent step followed by conver-sion of the amino group to isothiocyanate derivative. Wenow simplified this lengthy procedure by avoiding theprotection by t-BOC and deprotection by TMSC steps.Thus, when the 4- i sot hi ocyanatophen~ ~~as directlyused with the chloro dyes (4,5, and6) for this purposean efficient conversion to the 4-isothiocyantophenolderivative of the dyes was obtained (Tables 4 and5).Afacile reaction of4bwith other nucleophiles from phenolssuch as HOPhCHZCHzOH, HOPh(CH2)30H, HOPhN-(CHzCOzCH3)z and sodium hydride also formed thesubstitution products(7f, e,and7g, espectively) in highyields (Table4) .The functionalized dyes have been designed to meetthe needs of our current research such as solubility,absorption, and emission wavelength suitable for instru-mentation, for example for use with diode laser whichexcites at 780 nm. A complete study on the spectralproperties of these dyes and applications in DNA se-quencing and protein conjugation for studies in immu-noassay methods are under investigation. The resultswill be published elsewhere.

ConclusionsIn summary we have developeda new, simple, andefficient procedure forafacile synthesis of heptamethine(26)Strekowski, L.; Lipowska, M.; Patonay, G.J .Org. Chem. 1992,57. 4578.. ( 27) ipowska, M.; Patonay, G.; Strekowski, L. Synth. Commun.( 28)See Experimental Section for preparation.1993, 23, 087.

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2394 J . Org. Chem., Vol.60,No. 8, 1995 Narayanan and PatonayPreparation of Symmetric Chloro Dyes 4and5. Rep-resentative Procedure for 4a: Quaternary salt la (1.18g,4 mmol) and 0.345 g (2 mmol) of bisaldehyde2awere dissolvedin 150mL of a mixture of 1-butanol and benzene (7:3) in aflask equipped with a Dean-Stark trap. The mixture washeated at reflux with constant stirring and the water formedwas collected in the trap. The progress of the reaction wasmonitored by the Vis-near-IR spectrometry. The product hadasharp peak at782 nm. After3h, the reaction was cooledtort, and the solvents were removed in vacuo. The residue waswashed with ether to give the product 99%. The purity of the

crude product was satisfactory by proton NMR. However, thecrude was purified by column chromatography on silica gel,using CHCl3-methanol gradient, from 90% through 50%. Thefractions with absorption maximaat 782 nm were collected.Removal of solvent and drying under vacuum afforded purecrystalline material (1.35g, 93%). 'H NMR (DMSO-de): 61.67(s , 12H); 1.74-1.83 (m, 14H); 2.73(t,4H, J =5.6 Hz); 4.24(t,4H,J =6.8Hz);6.40(d,2H,J =14.0Hz);7.28(t,2H,J =7.6Hz); 7.43 (t, 2H, J =7.2 Hz); 7.5 (d, 2H, J = 7.6 Hz); 7.63 (d,2H, J =7.2 Hz); 8.28 (d, 2H, J =14.0 Hz). MS: 727(100).HRFAB: calcd 727.2642341; obsd, 727.262207; Am=0.00202mu). Elem. Anal. Calculated for c38H47ClN206S2. (Found):C, 59.10(59.34); H, 6.73(6.56); N, 3.63(3.61); C1 4.60(4.60);S,8.30(8.01).Conversion of Symmetric and Nonsymmetric ChloroDyes to the Phenoxy Derivative. RepresentativePro-cedure for 7a from 4a. Preparationof4-isothiocyanatophenol. To 4-aminophenol (5mmol) in amixture of CHC13(30 mL) and aqueous NazC03 (1g in 40 mL of water) wasadded thiophosgene (1.15 g, 0.76 mL, 10 mmol) with stirringatrt. After3h, the organic layer was washed and the solventwas removed. Purification of the crude residue on a silica gelcolumn (CHzC12 and petroleum ether 3:l) furnished the pureproduct in 65% yield. 'H NMR(DMSO-ds): 66.80 (d, 2H, J =8.8Hz); 7.28 (d, 2H, J =8.8Hz); 10.00(s,1H). IR: 2100.To a slurry of 65 mg of NaH (80% n mineral oil, washedwith hexane, 2.10 mmol) and 5 mL of anhydrous DMF, underNz, was addeda solution of 320 mg (2.15 mmol) of 4-isothio-cyanatophenol dissolved in 10 mL of DMF at0"C with stirring.After 30 min the reaction was allowed to warm to rt. Thesolution was added to the dye 4a (1.35 g, 1.86 mmol) in 50mL of DMF at rt with stirring under N2. The reaction wascomplete in15min. After 30 min the reaction was quenchedwith dry ice. DMF was removed in vacuo keeping the waterbath temperature below 40 "C. The residue was washed withether and dried. Chromatographic separation on a silica gelcolumn eluting with 10-50% methanol-CHCls gradient fur-nished the pure product. After solvent removal and vacuumdrying 1.44 g (90%) of pure dye was obtained. 'H NMR(DMSO-&): 6 1.32 (s,12H, CMe2); 1.76 (m, 8H); 1.96 (quintet,2H); 2.78(t,4H, J =7.2 Hz); 4.17(t,4H, J =7.2 Hz); 6.27 (d,2H, J =14 Hz); 7.21(t,4H, J =7.2 Hz); 7.28 (d, 2H, J =8.8Hz); 7.37 (d, 2H, J =8.0 Hz); 7.42 (t, 2H, J =8.0Hz); 7.52 (d,2H, J =8.0Hz); 7.75 (d, 2H, J =14.4 Hz). IR: 2096. MS:841(M+-Na). Elem. Anal. Calculated for C ~~H ~O N ~O ~2.5H20 (MW 908) (Found): C, 59.47 (59.56); H, 6.06 (6.13);N, 4.63 (4.63);S,10.57 (10.47); Na, 2.53 (2.55).Preparation of Nonsymmetric Chloro Dyes 6. Rep-resentative Procedure for 6a. To 0.428 g (1mmol) ofquaternary salt 3c and 0.173 g (1mmol) of bisaldehyde 2awas added 75 mL of1-butanol and benzene (7:3). The contentswere heated at reflux with stirring for 2 h after which theheating was discontinued and allowedto cool to rt. A 0.295 g(1mmol) portion of the quaternary salt la, dissolved as aslurry in 10 mL of the above solvent mixture, was added tothe reaction mixture. The heating was resumed at reflux for10 h with the removal of water. Solvents were distilledoff invacuo and the resulting residue was washed with 20 mL ofether. The crude material was filtered and dried to afford 850mg (99%).Conversion of Pr imary Amine Salt to I sothiocyanate.Representative procedure for 6e from 6a. A 640 mgportion of the crude dye6awas dissolved in 40 mL of CHC13.A solution of 600 mgofNazC03 in 20 mL ofwater was addedto the dye, and the heterogeneous mixture was stirred at rt.

cyanine dye intermediates (dyeseries4,5, and6)whichcould be easily derivatized for applicationsin fluorescentlabelingof biologically important molecules. Thesalientfeatures associatedwith thenewmethodaresummarizedbelow. A fewer number of steps are involved in thesynthetic sequences. No catalyst isusedwhich resultsinenhanced puri ty andhighyields. Easy purif ication isaccomplished by simple chromatographic techniques. Theprocedure isexcellent for the synthesisof nonsymmetricdyes in onepot. Protection and deprotection steps arenot involved. The synthetic procedure is reproducibleand easy to scaleup for larger amounts for both sym-metric and non-symmetric dyes. Scaling up of thereactionhas been carried out on 5 g quantities for thefunctional dyes 7a and8a without any difficulty.A limitation tothismethod, however,was observed inthe reaction of quaternary salts of quinoline with 2a.1-Ethyl-2-methylquinoliniumodide gave only about 15%of the symmetric dye over a prolonged reaction timewhile, the 1- et hyl - 4- methyl qui nol i ni umodide did notundergo this reaction at all.

Experimental SectionGeneral. All starting materials for the dye synthesis wereobtained from Aldrich Chemical Co. except those for 1,1,2-trimethyl-VI-benzindole and2- methyl naphtho[2, 1- dl thi azol ewhich were obtained from Kodak. UV-vis-near-IR spectrawere measured in methanol. IR spectra were run as CHC13film on a KBr plate or as a KBr pellet. The IR absorptionfrequencies are reported in reciprocal centimeters. ProtonNMR spectra were run at400 MHz with TMS as an internalstandard. The chemical shifts are reported in ppm on6scale.Elemental analyses were performed at Atlantic Microlabs,Norcross, GA. Mass spectral measurements were made atGeorgia Institute of Technology. The reported yields are forpure and isolated compounds. On heating these compoundsundergo partial decomposition around 150 "C and then meltingis observed above 200 "C.Preparation of Quaternary Salts (1 and 3). Three

different methods were used depending upon the heterocyclicbase and the alkyl halides. Reactions were generally carriedout in 10 mmol scale.Method 1, Used for la, Id, and 3a. 1,4-Butanesultone7was mixed with the heterocyclic base in 3:l molar ratio inanhydrous 1,2-dichlorobenzene and heated for 12 hat120 "C.The pure salt was obtained by repeated crystallization fromacetone and methanol.Method 2, Used for lb, le-g, 3b, and 3e. Ethyl iodidewas mixed with the base ina5:l ratio in dry acetonitrile andheated at reflux for 15 h under N2. The salt was obtained asa solid which was filtered and crystallized from ether andacetone.Method 3, Used for IC, 3c, and 3d. RepresentativeProcedure for 3c. A mixture of 2.10 g ( 10mmol) of 2,3,3-trimethylbenzoindole and 2.30 g (10 mmol) of 3-bromopropyl-amine hydrobromide was heated in a pressure tube at 140 "C,in an oil bath with stirringfor 10 h. On cooling the melt, theproduct was a hard solid cake which was scraped off the tubeand washed withamixture of ether and CHCl3. The resultingslurry was filtered and dried and was used in the subsequentreactions without further purification. Yield: 4.20 g (93%).'H NMR(DMSO-ds):6 1.79(s, 6H); 2.97(s, 3H); 2.25 (quintet,2H); 3.17 (m, 2H); 4.71(t,2H,J =7.2 Hz); 7.74(t, H, =8.0Hz); 7.88 (t, lH, J =8.0 Hz); 7.99 (d, lH, J =8.0 Hz); 8.24 (d,lH, =8.0 Hz); 8.31 (d, lH, J =8.0Hz); 8.37 (d, lH, =8.0Hz).Preparation of Bisaldehydes. The bisaldehyde2awassynthesized by the reported procedure.17 lH NMR (DMSO-&): 6 1.58 (quintet, 2H, J =6.0 Hz); 2.37 (t,4H, J =6.0 Hz);10.85 (s, 1H). The bisaldehyde 2b was synthesized by amethod analogous to that reported.lQ

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