Tetrahedrontitters, Vol.38, No. 36, pp.6323.6326, 199701997 ElsevierScienceLtd

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Fluorescence Sensing of a Ribonucleoside 5’-Triphosphate

Steven Patterson, Bradley D. Smith*, and Richard E. Taylor*

Departmentof Chemistryand Biochemistry,Universityof Notre Dame,NotreDame,Indiana46556,USA

Abstract: Conjugation of a fluorescent srrccharidesensor to poly(allylamine) produces a sensingcompound with selectivity for riborrucleoside5’-triphosphate. An associationconstant (log Ka) of 5.6was determined for uridine 5’-triphosphatein pH 7.3 phosphatesolution (15 mM, 298 K), whereas logKa waaless than 2 for uridine,uridine5’-monophosphate,andundine 5’-diphosphate.01997 Elsevier Science Ltd.

A primaryaimin abioticsupramolectdarchemistryis to inventmoleculardeviceswithspeeiticfunctionssuchas recognitionandcatalysis. Themajorityofcasesreportedin the literaturedescribea compoundthathaspromisingattributesbut there is a need to convertthe lead candidateinto a second-generationversionwithimprovedbindingaffinityand/orbindingselectivity. At presentthereare few waysto addressthis problem.]Wehaveinitiateda programtodevelopgeneralmethodsof improvingreceptorbindingthroughthe incorporationof a tunableseeondaryrecognitiondomain. Asa startingpoint,we havechosento exploremethodsthatexpandupon the binding characteristicsof boronicacids which are knownto form covalent complexeswith diol-contairtingmoleculessuchas carbohydrates.2~3

We have modifiedthe carbohydratereceptor,1, inventedby Shinkaiand coworkers.z.gThis receptordesignwas chosenfor a numberof reasons. It bindsneutralmonosaccharidesin aqueousmethanolsolutionwitha selectivityfor fructose(Ka= 103M-1).2Compound1 is fluorescentandis thoughtto signalbindingviamodulationof a photoinducedelectrontransferquenchingmechanism.zAlthoughfluorescenceprovideslimitedinformationabout the molecular details of binding, it is nonethelessa straightforwardway of measuringassociation.Tbisis an importanttechnicalpointwithsugarreceptorsbecausemostcarbohydrateslackaneasilyobservablechromophore.

T

1; ; ;B(OH)2

ti

1 R=H

# 1> 2 R= 4-C#14C02H

T\\\1, , ,N

@m1;o c

3t.la+0-

In this report we describethe versatilederivative2, whichcontainsa benzoicacid linker groupthatallowsconjugationto a rangeof secondmybindingfunctionality.As a demonstrationof its versatility,we haveattached 2 to a cationic polyamineand produceda fluorescentsensorwith greatly enhancedaffinity for aribonucleoside5’-triphosphate.Nucleotidesareimportantbiologicalcompoundsandthereis a needto developfluorescentnucleotidesensors.sTo date,onlya fewattemptshavebeenreported.6.7

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Compound2 was preparedby the sequenceshownin Scheme 1. The secondaryammonium5 wastreatedwiththe knownorgartoboron62to provide7,8whichwaseasilyhydrolysizedto 2 withaqueouslithiumhydroxide.A controlversion,3, whichdoesnotcontaina boronicacidwaspreparedby a similarsequence.

Scheme 1

+tfH3 Br -

P ao :1 —

CHSO 4

w<+

NH2 Cl-

&0 1; ,

CH30

x00‘B’ Br

U*1; ~

b

T\\\ 4/ / / :.0

“dN 1;do 1;(a) 9-anthraldehyde,CH30H, aieves; NaBH4; HCI; (71 %); (b) 6, K2C03; (4B %).

Initially, the bindingabilitiesof the free receptors,1 and2, were determinedin aqueoussolution(pH7.3, 15rnMphosphatebuffer)by fluorescencetitrationmethods. Compound2 (logKa = 4.6) binds fructosemore tightly than parent 1 (logKa = 3.7),9 but the fluorescenceenhancementat sensorsaturationis smaller(two-foldfluorescenceincreasefor 2 comparedto a three-foldenhancementfor 1). Compounds1 and2 wererdsoassayed for associationwith nucleosidesand nucelotides. In all cases there was little or no change inreceptorfluorescencesuggestingveryweakbinding. Toenhancenucleotideaffinity,receptor2 wasattachedtopoly(allylamine)PA (MW 50,000- 65,000) via an amidelinkage.lo A loadinglevelof107. was employed(i.e., the ratio of sensor 2 to allylamine monomer was 0.1). A control polymer was also prepared byfunctionalizing10% of PA withfluorophore3.

The PA-2 conjugatewas titratedwith fructose,uridineand variousuridine5’-phosphatederivativesinaqueoussolution (pH 7.3, 15 mM phosphatebuffer). The resultingchangesin fluorescenceare showninFigure 1. Titrationof PA-2 with fructoseshowedweakenedaffinity(logKa = 2.7)comparedto boronicacidreceptor2 alone. It is possiblethis is due to aggregationof the hydrophobicsensinggroups. However,thefluorescencespectrumfor PA-2in aqueousphosphatesolutionshowsno excimeremission,indicatingthat thesensinggroupsare dispersedalongthe PA chain. Therefore,it appearsthat the cationicPA and its solvationshellof phosphateionsstericallyencumberfructosebindingto thesensingboronicacidgroups.

1.30-

1.20-

I/lo1.10-

1.004

0.900-0.20

solutecone (mM)

Figure 1. Change in fluorescence emission (kex 389 nm, l.em 423 nm) for PA-2 upontitration with: O 5’-UT#-; ■ thctose; A 2’-deoxy-5’-UT#-,+ 5’-UMP2-,and x 5’-UDP3-,in phosphatebuffer solution(PH 7.3, 15mM), T = 298 K.

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Titrationof PA-2 with uridine,5’-UMP2-and 5’-UDP3-producednegligiblechangesin fluorescence.However,5’-UTP4-was foundto bind very strongly(logKa = 5.6).9 Althoughthe changein fluorescenceissmall,the highassociationconstantfor 5’-UT~-is particularlyimpressivesincebindingoccursin thepresenceof competingphosphatebuffer. The associationconstantis comparableto the best aftlnities observedwithLehn’sbis(intercrdand)andtris-acridinecryptand.bIn addition,PA-2exhibitsa bindingselectivitythat is quitedifferentto Lehn’sreceptors. The lack of responseto 2’-deoxy-5’-UTP,a compoundincapableof chelatingwith the boronicacid, is evidencein favorof the bindingmodeshownin Scheme2. Furtherevidencethat theboronic acid is crucial for sensingis the observationthat in phosphatebuffer the control polymer PA-3 isunresponsiveto 5’-UTP’f_or anyof thecompoundsdescribedin Figure1.

PA-2 (10 % loading)w+

N.DHO? ‘ \..-=

Anth-

&

:1a a @“ a

Scheme 2

UTP&

-2 H20

P

weak fluorescence enhancedfluorescence

Competitive titration experimentsprovided a further demonstrationof the sensing selectivity fornucleoside triphosphate. Titration of PA-2 with 5’-UTP4-in the presence of excess 5’-UMP2-(1 mM)produceda titrationcurve that was essentiallyidenticalto that seen in Figure 1. The high selectivityfor 5’-UTP4-over5’-UDP3-and5’-UMP2-is rationalizedin termsof a competitionbetweenthe analyteandphosphatebufferfor the cationicpolymerPA-2. It appearsthat onlywhenthe analytechargereaches-4 doesbindingtothe cationic PA-2 become highly favorable. When the titrations are repeated in HEPES (N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonicacid)bufferall of the nucleotidesinducea modestfluorescentresponsein the order5’-UTP4-> 5’-UDP3-> 5’-UMP2-> fructose. In this case, the anionicanalytescan morereadilydisplacethezwitterionicHEPESfromthecationicPA-2chainandgainaccessto the sensinggroups. Asimilar result was very recently reported by Anslyn.l1 In HEPESbuffer, the control polymerPA-3 alsoshowedweak fluorescentresponsesto the differentnucleotides,suggestingthat in HEPES there are moresignalingpathwaysavailableto PA-2thantheboron-mediatedmechanismshownin Scheme2.

The fluorescenceenhancementfor PA-2 saturatedwith 5’-UT@-is lower than that observedwithfructose. This suggests that the anthracenefluorescenceis partially quenchedby the pyrimidinebase, inagreement with the known fluorescence quencing properties of nucleobases.b,lz Thus the observedfluorescenceincrease in phosphatebuffer is a compromisebetween the enhancementdue to boronic acidchelation,andquenchingdueto interactionbetweenthesnthraceneandnucleobase.

In summary,conjugationof a fluorescentdiol-sensorto a cationicpolymerchain producesa sensingcompoundwith selectivityfor ribonucleoside5’-triphosphate.Thus,we havedemonstratedan applicationof ageneralconceptwheretheintroductionof secondaryinteractionenhancestheaftlnityandselectivityinherentto amolecularreceptor. Studiesin progressare attemptingto furtherimprovethesepropertiesthroughthe use ofmorehighlyfictionalized secondruyrecognitiondomains.

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Acknowledgments. This workwas supportedby a BayerPostdoctoralFellowshipand the NationalScienceFoundation(USA).

References and Notes

1. Gennari,C.; Nestler,H. P.; Piarulli,U.; Salem,B. LiebigsAnn.lRecueil 1997, 637-647.

2. (a) James, T. D.; Linnane, P.; Shinkai, S. .1. Chem. Soc., Chetn. Commun.1996, 281-288. (b) James,

T. D.; SamankumaraSandanayakeK. R. A.; Shinkai,S. Angew. Chem. Int. Ed. Engl. 1996,35, 1910-1922.

3. Smith,B. D. Supramol. Chem. 1996,7, 55-60.

4. To datetherehavebeenfewattemptsto alterthesensingpropertiesof 1 by syntheticmodification.TheRgroupin 1 has beenchangedfromhydrogento a secondboronicacid,2a crownether,zanda photochromicdiarylethene;Takeshita,M.; Uchida,K.; Irie, M. J. Chem. Soc., Chem. Commun.1996, 1807-1808.

5. Czarnik,A. W. Chemistry and Biology 1995,2,423-428.

6. (a) Cudic,P.; Zinic, M.; Tornisic,V.; Simeon,V.; Vigneron,J. P.; Lehn,J.-M.J. Chem. Soc., Chem.

Commun. 1995, 1073-1074. (b) Teulade-Fichou,M, -P.; Vigneron,J. -P.; Lehn, J. M. J. Chem. Soc.,

Perkin Trans. 21996, 2169-2174.

7. Takeuchi,M; Taguchi,M.; Shinmori,H.; Shinkai,S. BuZZ.Chem. Soc. Jpn. 1996,69, 2613-2618.

8. Characterizationdata for 7: mp 157-159“C; IH NMR(500MHz,CDC13)50.92 (s, 6H),3.37(s, 4H),

3.59 (s, 2H), 3.89 (s, 3H), 3.96 (s, 2H), 4.66 (s, 2H), 7.22 (app. t, J = 7.5 Hz, IH), 7.30-7.45(m, 8H),7.64 (d, J = 7.5 Hz, IH), 7.90 (d, J= 8 Hz, 2H), 7.94-7.96(m, 2H), 8.25-8.27(m, 2H), 8.35 (s, IH);13c NMR (75 MHz, CDC13)621.87, 31.42,50.77,51.98,58.63,58.70,71.74, 124.72,125.28,

125.35, 126.36, 127.39, 128.60,128.89,129.20,129.43,129.47,130.30,131.39,131.59, 134.04,143.95,145.70,167.14;MS (FAB+)mlz:557(M+, 12),366(13), 191(100);HRMS557.2788(calcdforC36H36BN04:557.2744).

9. AssociationconstantsweredeterminedbytheBenesi-Hildebrandmethodornordinearcurvefittingof theequationfor 1:1binding. Tsukube,H.;Furata,H.; Odani,A.;Takeda,Y.; Kudo,Y.; Inoue,Y.;Liu, Y.;Sakarnoto,H.; Kimura,K. in Comprehensive Supramolecular Chemist~, Atwood,J. L., Davies,J. E.D., MacNicol,D. D., Vogtle,F., Eds.;Pergamon,New York, 1996,vol. 8, Ch. 10.

10. Menger,F. M.; Eliseev,A. V.; Migulin,V. A. J. Org. Chem. 1995,60,6666-6667. Poly(allylarnine)hydrochloride(MW50,00- 65,000)wasconvertedto its freeamineformusinga ten-foldexcessof Dowexl-X8 resin. An aqueoussolutionof poly(allylamine)(0.5M of monomerunits)wastreatedwithsolutionsof 2 (0.1molarequivalentsof monomerunits)in dimethylforrnamideand l-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride(0.5molarequivalentsof monomerunits)in water. Thereactionwasstirredat roomtemperaturefor 14h beforepurificationbygelfiltration(SephadexG15)andremowdof thesolvent.The residue(IR 1620cm-l)wasredissolvedin methanol/waterto a makestocksolutionthatwas20 @f in sensorunit. A 200~L solutionof this stocksolutionwasdilutedto 3.0MLwithphosphatebuffer(15MM,pH 7.3)to givea finalsensorunitconcentrationof 1.3@f.

11. Metzger,A.; Lynch,V. M.; Anslyn,E. V.Angew. Chem. Int Ed. Engl. 1997,36, 862-865.

12. Lewis, F. D., Zhang, Y.; Letsinger,R. L. J. Am. Chem.Soc.,1997, 119,5451-5452.

(Received in USA 10June 1997;accepted 10July 1997)