Proc. Natl. Acad. Sci. USAVol. 92, pp. 1208-1212, February 1995Chemistry

Hydrogen bonding in water using synthetic receptors(molecular recognition/adenine receptors/entropy-enthalpy compensation/hydrophobic interaction)

YOKO KATO, M. MORGAN CONN, AND JULIUS REBEK, JR.Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Julius Rebek, Jr., October 21, 1994

ABSTRACT Four water-soluble adenine receptors weresynthesized to study the influence ofhydrophobic interactionsand hydrogen bonding on molecular recognition in aqueoussolution. Association constants were measured in aqueoussolution at five temperatures from 3-27°C (pH 6, 51 mM ionicstrength). For the mono(imide) receptors, AH was -5.8kcal/mol (carbazole) and -9.2 kcal/mol (naphthalene). Theentropy of association for these was -13 cal mol-l K-1 (car-bazole) and -26 cal mol-l K-1 (naphthalene). The carbazolebis(imide) receptor showed a binding enthalpy of -7.4kcal/mol and entropy of -18 cal mol-l K-1. From this thefree energy at 298 K of a single hydrogen bond is estimated tobe only 0.2 kcal/mol. The enthalpy of a single hydrogen bondin this solvent-exposed system is estimated to be, at most, 0.8kcal/mol, indicating that enthalpy just compensates for theunfavorable entropy in this system. These values reflectstronger hydrophobic interactions with the more polarizablenaphthalene, as well as enthalpy-entropy compensation ef-fects.

Complementarity of size, shape, and chemical surface pro-vides much of the driving force for molecular-recognitionphenomena; yet allocating the binding affinities to individualintermolecular forces is difficult. For example, site-directedmutagenesis has been used to obtain hydrogen-bonding con-tributions in the context of enzyme-substrate and enzyme-transition state complementarity (1-4). The peptide-bindingvancomycins have likewise been used (5, 6) and have recentlyyielded additional insights concerning the contributions ofentropic effects (7). Water-soluble receptors for nucleic acidcomponents have given a measure of ionic interactions (8-11)and now permit a direct assessment of hydrogen bonding inwater. We report here our preliminary results in that context.The structures involve Kemp's triacid derivatives that result

in molecules with inwardly directed functionality. These mol-ecules were outfitted with imides and stacking surfaces forcomplementarity to the purine nucleus of adenines (Scheme1). Peripheral hydroxyl groups impart water solubility. Asoluble carbazole mono(imide) derivative 1 and bis(imide) 2were prepared for comparison with the naphthyl mono(imide)3 and bis(imide) 4 (Scheme 2). The synthesis follows well-trodden paths (11, 12) (Fig. 1). Receptor 1 binds adenines ina manner similar to receptor 3 but provides a different

Scheme 1

3 4

Scheme 2: Water-soluble receptors

hydrophobic platform. The arrangement of imides in receptor2 is known to chelate adenines in organic solvents throughsimultaneous Watson-Crick and Hoogsteen base-pairingmodes with high binding affinities (Ka > 105 M-1), dominatedby hydrogen bonding (13). In contrast, hydrophobic effectscontribute most of the affinity of related imide receptors foradenine in water (11, 12) and overwhelm the hydrogen-bond-ing component.

Titrations of 9-ethyladenine with the receptors were done atfive different temperatures from 3-27°C at pH 6 (cacodylatebuffer) and ionic strength of 51 mM (NaCI), monitoring theimide proton shift as described (11). Van't Hoff (1/T vs. In K)analysis was used to determine enthalpic and entropic contri-butions to binding. Comparing these thermodynamic param-eters gives the details of interactions involved in binding9-ethyladenine, particularly hydrogen bonding, in a solvent-exposed environment.

MATERIALS AND METHODSGeneral. Air/water-sensitive reactions were done in flame-

dried glassware under argon. Unless otherwise stated, allcommercially available reagents were used without furtherpurification. 'H NMR spectra were obtained on Bruker AC-250, Varian XL-300, Varian UN-300, and Varian VXR-500spectrometers. All chemical shift values are reported in ppmdownfield from tetramethylsilane for organic solvents, or2,2-dimethyl-2-silapentane-5-sulfonate for H20/2H20. Spec-tra taken in CDCl3 were referenced to either tetramethylsilane(0.00) or residual CHCl3 (7.26). Spectra taken in dimethylsulfoxide (DMSO)-d6 were referenced to residual solvent

Abbreviations: DMF, N,N-dimethylformamide; DMSO, dimethyl sul-foxide; FAB, fast atom bombardment; HRMS, high-resolution massspectrometry; THF, tetrahydrofuran.

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Proc. Natl. Acad Sci. USA 92 (1995) 1209

R

COOMe

CU(NO3)2-2.5H2O, ACO Sa FAc2O, 30 °C, 2h 5b F

H2, 10% Pd-C Kc FTHF, RT (quant)

N

cI~g

Bn Bn6 OBn

py, reflux4h (67%)

.-

RI=H

RI=NH2

NaOH (aq)RT (89%)

0 OBn R

HN$OBn HP

0< 0HOBn \NH 1.(COCI)2, DMF, THF NH

RT, 1 h (quant)

K 2. PrNH2, THFLCOOR RT (75%)

EtOH,THF(7a R-MeH

7b R-H HBr(g), HC02H (8 R-OBn0OC,5min(84%) .1 R OH

H2N NH2

'COOMe

NH2 6, py (25%)

12

H2N NH2 6, py (54%)

14

HBr(9), HCO2H13 0 OC, 5 min (quant)

HO_-

HBr(g), HCO2H HO-1S 0 OC, 5 min (88%)

FIG. 1. Synthesis scheme. py, Pyridine; THF, tetrahydrofuran; RT, room temperature; quant, quantitative; DMF, N,N-dimethylformamide;Pd-C, palladium on activated carbon; (g), gaseous, (aq), aqueous.

(2.50). Fourier transform IR spectra were taken on a Perkin-Elmer IR spectrometer. Flash chromatography was done byusing silica gel 60 (EM Science; 230-400 mesh).

Synthesis. N-[(Methoxycarbonyl)methyl]-3-nitrocarbazole(5b). Alkyl carbazole 5a (13) (2.20 g, 9.194 mmol) was addedto a solution of Cu(NO3)242.5 H20 (4.6 mmol, 1 equivalent) inAc2O (10 ml) and AcOH (15 ml) at 32°C. Additional AcOH (5ml) was added; the mixture was stirred for 5 hr and then pouredinto water (300 ml). Filtration and drying gave 2.485 g (95%)of the mononitro product as yellow solid, which was usedwithout further purification: 1H NMR (300 MHz, acetone-d6)8 9.114 (d, 1H,J = 2.4 Hz),8.401 (dd, 1H,J= 8.4,0.6 Hz),8.366(dd, 1H, J = 9.0, 2.4 Hz), 7.738 (d, 1H, J = 9.0 Hz), 7.660 (d,1H, J = 8.1 Hz), 7.589 (dd, 1H, J = 7.6, 1.1 Hz), 7.385 (dd,1H, 7.3, 1.5 Hz), 5.431 (s, 2H), 3.727 (s, 3H).

N-[(Methoxycarbonyl)methyl)]-3-aminocarbazole (5c). Ni-trocarbazole 5b (478 mg, 1.681 mmol) dissolved in THF (50ml) was hydrogenated at balloon pressure for 1.6 hr with 10%(wt/wt) palladium on activated carbon [218 mg, 46% (wt/wt)].The suspension was filtered through Celite and concentratedin vacuo. The solid was used immediately without furtherpurification.

N-[(Methoxycarbonyl)methyl]-3- (([cis,cis-2, 4-dioxo-1, 5, 7-tris(benzyloxymethyl)-3-azabicyclo[3.3. 1]non- 7-ylJcarbonylJ-

amino)carbazole (7a). Acid chloride 6 (11) (1.036 g, 1.799mmol) was refluxed with aminocarbazole 5c (1.5 equivalent) inpyridine to yield 953 mg (67%) of the monoimide 7a aftergravity chromatography (silica gel; 10-15% EtOAc/CHCl3),which was used without further purification: 1H NMR (500MHz, DMSO-d6) 8 10.670 (s, 1H), 9.357 (s, 1H), 8.067 (d, 1H,J = 7.1 Hz), 7.5-7.2 (m, 21H), 5.331 (s, 2H), 4.511 (s, 2H), 4.506(s, 2H), 4.476 (s, 2H), 3.772 (d, 2H, J = 9.0 Hz), 3.650 (s, 3H),3.458 (s, 2H), 3.404 (d, 2H, J = 9.0 Hz), 2.545 (d, 2H, J = 14.4Hz), 2.338 (d, 1H, J = 12.5 Hz), 1.608 (d, 2H, J = 13.7 Hz),1.596 (d, 1H, J = 12.9 Hz).

N-(Carboxymethyl)-3-([[cis,cis-2, 4-dioxo-1, 5, 7-tris(ben-zyloxymethyl)-3-azabicyclo[3.3. 1]non-7-yl]carbonylJamino)-carbazole (7b). Imide 7a (953 mg, 1.20 mmol) was hydrolyzedwith 1 M NaOH (2 equivalents) in THF (3.6 ml) and EtOH (2ml) at room temperature for 6.5 hr. Organic solvents wereevaporated, and the residue was suspended in water (10 ml).After acidification with 10% HCl, the solid was filtered anddried under vacuum to give 831 mg (89%) of white powder,which was used without further purification: high-resolutionmass spectroscopy (HRMS) electron impact calculated forC47H45N308, 779.3207; found, 779.3210.

N-[[(Propylamino)carbonyl]methyll-3-([[cis,cis-2,4-dioxo-1,5, 7-tris(benzyloxymethyl)-3-azabicyclo[3. 3. 1]non- 7-yl]-

6, py(51%)

3

4

Chemistry: Kato et aL

Proc. Natl. Acad Sci USA 92 (1995)

carbonyljamino)carbazole (8). Acid 7b (102 mg, 0.131 mmol)was dissolved in anhydrous THF (4 ml) under argon. CatalyticDMF (<1 gl) was added, followed by oxalyl chloride (80,1,7.0equivalents). The reaction was stirred at room temperature for45 min, and solvent was removed in vacuo. The residue wasredissolved in anhydrous THF on ice under argon. Pro-pylamine (150 ,ul, 13.9 equivalents) was added. The reactionwas warmed to room temperature and stirred for 53 hr. Solventwas evaporated, then taken up in chloroform (50 ml), washedwith 1 M HCl (50 ml) and brine (25 ml), and dried overNa2SO4. Evaporation of solvent followed by chromatography(silica gel; 30-50% EtOAc/CH2Cl2) yielded 7 (80 mg, 75%) asa white powder: IR (KBr) 3356, 1701, 1533, 1493, 1465, 1363,1323, 1201, 1099, 746, 698 cm-1; 1H NMR (500 MHz, DMSO-d6) 8 10.660 (s, 1H), 9.348 (s, 1H), 8.233 (br t, 1H), 8.063 (s,4H), 7.487 (d, 2H, J = 8.5 Hz), 7.41-7.25 (m, 18H), 7.185 (t,1H,J = 7.3 Hz), 4.969 (s, 2H), 4.511 (s, 4H), 4.505 (s, 2H), 4.476(s, 2H), 3.772 (d, 2H, J = 9.0 Hz), 3.461 (s, 2H), 3.403 (d, 2H,J = 8.8 Hz), 3.042 (q, 2H, J = 6.1 Hz), 2.545 (d, 2H, J = 13.9Hz), 2.346 (d, 1H, J = 12.5 Hz), 1.606 (d, 3H, J = 13.7 Hz),1.426 (q, 2H, J = 7.3 Hz), 0.840 (t, 3H, J = 7.3 Hz); HRMS [fastatom bombardment (FAB) in 3-nitrobenzyl alcohol) calcu-lated for C5oH53N407 (M+H), 821.3914; found, 821.3914.

N-t[(Propylamino)carbonyl]methylj-3-([[cis,cis-2,4-dioxo-1,5, 7-tris (hydroxymethyl)-3-azabicyclo[3. 3. 1]non- 7-yl]-carbonyljamino)carbazole (1). Benzylated receptor 8 (72 mg,87.7 ,umol) was dissolved in HCOOH (4.6 ml) and stirred onice for 40 min. HBr (gaseous) was bubbled through the solutionfor 9 min, followed by Ar (gaseous) for 2 hr. Solvent wasremoved in vacuo. The waxy solid was dissolved in MeOH (1ml) and precipitated by addition of ether (5 ml). The productwas filtered and dried under vacuum to give 1 (40.5 mg, 84%)as a white solid: IR (KBr) 3326 (br), 1693, 1540, 1492, 1466,1432, 1325, 1204, 1056, 804, 749 cm-1; 1H NMR (300 MHz,DMSO-d6) 8 10.445 (s, 1H), 9.200 (s, 1H), 8.245 (br t, 1H),8.115 (s, 1H), 8.075 (d, 1H, J = 8.1 Hz), 7.486 (d, 1H, J = 8.1Hz), 7.398 (m, 3H), 7.177 (t, 1H, J = 7.5 Hz), 4.969 (s, 2H),3.770 (d, 2H,J = 10.5 Hz), 3.389 (s, 2H), 3.318 (d, 2H,J = 10.2Hz), 3.040 (q, 2H,J = 6.5 Hz), 2.375 (d, 2H,J = 14.1 Hz), 2.205(d, 1H, J = 12.6 Hz), 1.487 (d, 2H, J = 14.7 Hz), 1.425 (q, 2H,J = 6.9 Hz), 1.328 (d, 1H, J = 12.9 Hz), 0.839 (t, 3H, J = 7.5Hz); HRMS (FAB in 3-nitrobenzyl alcohol) calculated forC29H35N407 (M+H), 551.2506; found, 551.2510.

N-[(Methoxycarbonyl)methyl]-3, 6-bis(t[cis,cis-2, 4-dioxo-1, 5, 7-tris (benzyloxymethyl) -3-azabicyclo[3. 3. lJnon- 7-yl]carbonylJamino)carbazole (lOa). Diamine 9 (13) (264 mg,0.981 mmol) and acid chloride 6 (11) (1.354 g, 2.35 mmol) weredissolved in pyridine (60 ml) and stirred overnight under argonat room temperature. The deep red solution was concentrated.The resulting solid was taken up in CH2Cl2 (150 ml) andwashed with 1 M HCI (200 ml) and brine (100 ml), dried overNa2SO4, filtered, and concentrated to give a red solid that wasflash chromatographed [silica gel; 35, 40% (vol/vol) ethylace-tate/CHC13] to give 402 mg of 10a (30% yield) as a pale yellowsolid that was used without further purification: IR (KBr)3431, 3212, 2923, 2860, 1703, 1699, 1467, 1453, 1281, 1196,1101, 738, 698 cm-1; 1H NMR (300 MHz, DMSO-d6) 8 10.641(s, 2H), 9.322 (s, 2H), 8.120 (s, 2H), 7.44-7.24 (m, 34H), 5.312(s, 2H), 4.518 (s, 8H), 4.480 (s, 4H), 3.789 (d, 4H,I = 8.64 Hz),3.745 (s, 3H), 3.46-3.38 (m, 8H), 2.574 (d, 4H, J = 14.2 Hz),2.348 (d, 2H, J = 12.9 Hz), 1.64-1.58 (m, 6H).

N-(Carboxymethyl)-3,6-bis(t[cis,cis-2,4-dioxo-1,5, 7-tris(ben-zyloxymethyl)-3-azabicyclo[3.3.lJnon-7-yl]carbonyllamino)-carbazole (lOb). Methylester 10a (671 mg, 0.498 mmol) washydrolyzed in 1 M NaOH (4 ml), THF (16 ml), and EtOH (25ml) with stirring at room temperature for 30 min. The reactionmixture was cooled to 0°C, and 10% HCl was added until pH3. Organic solvents were removed in vacuo, and the resultingaqueous suspension was extracted by CH2Cl2, dried overMgSO4, filtered, and concentrated to yield crude 10b (quan-

titative) which was used without further purification: 1HNMR(300 MHz, DMSO-d6) 810.64 (s, 2H), 9.32 (s, 2H), 8.07 (s, 2H),7.42-7.26 (m, 34H), 4.72 (br s, 2H), 4.52 (s, 8H), 4.48 (s, 4H),3.79 (d, 4H, J = 8.9 Hz), 3.46 (s, 4H), 3.42 (d, 4H, J = 9.4 Hz),2.57 (d, 4H, J = 13.8 Hz), 2.35 (d, 2H, J = 12.9 Hz), 1.62 (d,4H, J = 14.2 Hz), 1.59 (d, 2H, J = 12.9 Hz).

N-[[(Propylamino)carbonyl]methylJ-3, 6-bis([[cis,cis-2, 4-dioxo-1,5, 7-tris(benzyloxymethyl)-3-azabicyclo[3.3. 1]non-7-yl]carbonylJamino)carbazole (11). Acid 10b (388 mg, 0.291mmol) was dissolved in THF (8.8 ml). A catalytic amount (1drop) of DMF and then oxalylchloride (177 gl, 7 equivalents)were added, and the mixture was stirred at room temperaturefor 1 hr. This mixture was concentrated in vacuo, filled with Arand cooled in an ice bath. The solids were redissolved in THF(8.8 ml), and propylamine (335 pul, 14 equivalents) was added,and then the mixture was stirred for 24 hr. Solvent wasevaporated. The residue was taken up in CHCl3 (80 ml),washed with 1 M HCl (100 ml) and brine (80 ml), then driedover Na2SO4, filtered, and concentrated. The resulting residuewas flash chromatographed (silica gel; 2 and 5% MeOH/CHCl3) to yield the desired product (307 mg, 76%): IR (KBr)3328, 2923, 2853, 1701, 1534, 1493, 1453, 1200, 1099, 737, 697cm-1; 1H NMR (300 MHz, DMSO-d6) 6 10.645 (s, 2H), 9.324(s, 2H), 8.222 (t, 1H,J = 5.68 Hz), 8.107 (s, 2H), 7.46-7.21 (m,34H), 4.950 (s, 2H), 4.56-4.45 (m, 12H), 3.786 (d, 4H, J = 8.96Hz), 3.462 (s, 4H), 3.416 (d, 4H, J = 9.12 Hz), 3.044 (dt, 2H,J = 5.87, 6.28 Hz), 2.573 (d, 4H, J = 13.85 Hz), 2.346 (d, 2H,J = 12.04 Hz), 1.64-1.58 (m, 6H), 1.432 (q, 2H, J = 7.10 Hz),0.850 (t, 3H,J = 7.42 Hz); HRMS (FAB in glycerol) calculatedC83H87N6013 (M+H), 1375.6331; found, 1375.6326.

N-t[(Propylamino)carbonylJmethylJ-3, 6-bis([[cis,cis-2,4-dioxo-1, 5, 7-tris(hydroxymethyl)-3-azabicyclo[3.3. 1]non- 7-yl]carbonyl)amino)carbazole (2). The benzyl-protected cleft 11(158 mg, 0.115 mmol) was dissolved in formic acid (10 ml) andplaced in an ice bath, HBr (gaseous) was bubbled for 3 min.The yellow solution was concentrated in vacuo to give orangesolids, which were triturated in MeOH/Et2O, 5:1 (6 ml)overnight. The solids were separated and washed with ether togive receptor 2 as off-white solids (100 mg, quantitative): IR(KBr) 3381, 2933, 2873, 1692, 1544, 1492, 1472, 1201, 1058cm-1; 1H NMR (300 MHz, DMSO-d6) 8 10.414 (s, 2H), 9.156(s, 2H), 8.231 (t, 1H, J = 5.54 Hz), 8.176 (s, 2H), 7.454 (d, 2H,J = 8.8 Hz), 7.371 (d, 2H,I = 8.8 Hz), 4.940 (s, 2H), 4.10-3.30(m, 18H), 3.045 (td, 2H, J = 6.88, 6.03 Hz), 0.852 (t, 3H, J =7.33 Hz); HRMS (FAB in glycerol) calculated C41H51N6013(M+H), 835.3514; found, 835.3520.

2-(t[cis,cis-2,4-Dioxo-1,5, 7-tris(benzyloxymethyl)-3-azabicy-clo[3.3.1]non-7-yl]carbonyljamino)naphthalene (13). Acidchloride 6 (11) (181 mg, 0.31 mmol) was coupled to ,B-naph-thylamine (68 mg, 0.48 mmol) in pyridine (5 ml) with catalyticdimethylaminopyridine. The dark red solution was stirredovernight, and the solvent was removed in vacuo. The resultingoil was dissolved in CH2Cl2 (15 ml) and washed with 2 M HCl(2 ml). Upon concentration, the product was flash chromato-graphed (silica gel; hexanes/ethyl acetate, 5:2) to give 53 mg(25%) of product, as a pale pink solid: mp 75-82°C; IR (KBr)3100, 2800, 1690, 1680 cm-1; 1H NMR (250 MHz, CDCl3) 88.02 (d, 1H,J = 1.8 Hz), 7.93 (br s, 1H), 7.77-7.71 (m, 2H), 7.62(br s, 1H), 7.47-7.15 (m, 19 H), 4.59 (d, 2H, J = 12.1 Hz), 4.53(d, 2H,J = 12.1 Hz), 4.49 (s, 2H), 3.80 (d, 2H,J = 9.2 Hz), 3.55(d, 2H, J = 9.2 Hz), 3.46 (s, 2H), 2.55 (d, 2H, J = 14.1 Hz), 2.45(d, 1H, J = 13.9 Hz), 1.78 (d, 1H, J = 13.4 Hz), 1.64, (d, 2H,J = 14.2 Hz); HRMS (electron impact) calculated forC43H42N206, 682.3042; found, 682.3040.

2- (t[cis,cis-2, 4-Dioxo-1, 5, 7-tris(hydroxymethyl) -3-aza-bicyclo[3.3.1]non-7-yl]carbonyl)amino)naphthalene (3). Thebenzyl-protected host 13 (53 mg, 0.08 mmol) was dissolved informic acid (10 ml) at 0°C. HBr (gaseous) was bubbled throughthe solution with stirring for 10 min. The solvent was removedunder reduced pressure to yield host 3 as a very pale pink solid

1210 Chemistry: Kato et al.

Proc. Natt Acad Sci. USA 92 (1995) 1211

(32 mg, quantitative), which was precipitated with methanoland CH2Cl2: mp 289-290°C; IR (KBr) 3600-3100, 2800, 1690,1680 cm-; 1H NMR (500 MHz, DMSO-d6) 8 10.36 (br s, 1H),9.32 (br s, 1H), 8.06 (s, 1H), 7.82-7.76 (m, 3H), 7.57 (dd, 1H,J = 1.9, 8.7 Hz), 7.45-7.38 (m, 2H), 3.73 (d, 2H, J = 7.0 Hz),3.38 (s, 2H), 3.31 (d, 2H,J = 7.0 Hz,), 2.37 (d, 2H,J = 13.8 Hz),2.18 (d, 1H, J = 13.0 Hz), 1.46 (d, 2H, J = 14.1 Hz), 1.32 (d,1H, J = 13.0 Hz); HRMS (electron impact) calculated forC22H24N206, 412.1634; found, 412.1632.

2, 7-Bis[([[cis,cis-2,4-dioxo-1,5, 7-tris(benzyloxy)methyl]-3-azabicyclo[3.3. 1]non-7-yljcarbonyl)amino]naphthalene (15).Acid chloride 6 (11) (496 mg, 0.862 mmol) and 2,7-diamino-naphthalene (5.8 mg, 0.372 mmol) were refluxed for 2 hr in 15ml of pyridine under Ar. The solvent was removed in vacuo.

The resulting black residue was dissolved in CH2Cl2 (50 ml),washed with 1 M HCI (75 ml) and brine (50 ml). The organiccompounds were dried over MgSO4, filtered, and dried to givea reddish-brown residue that was flash chromatographed(silica gel; 20% EtOAc/CH2Cl2) to give 249.6 mg (54%) oforange solids: mp 86-90°C; IR (KBr) 3200, 3000, 1690, 1680cm-1; 1H NMR (250 MHz, acetone-d6) 8 9.56 (s, 2H), 8.76 (s,2H), 8.00 (d, 2H, J = 1.5 Hz), 7.67 (d, 2H, J = 8.8 Hz), 7.40(dd, 2H, J = 2.0, 9.0 Hz), 7.39-7.25 (m, 30H), 4.60-4.54 (m,6H), 3.88 (d, 4H, J = 9.0 Hz), 3.54 (s, 4H), 3.52 (d, 4H, J = 7.2Hz), 2.69 (d, 4H, J = 13.8 Hz), 2.53 (d, 2H, J = 13.0 Hz), 1.77(d, 6H, J = 13.5 Hz).

2, 7-Bis([(cis,cis-2,4-dioxo-1, 5, 7-tris(hydroxymethyl)-3-azabicyclo[3.3.1]non-7-yl)carbonyl]aminojnaphthalene (4).The benzyl-protected host 14 (240 mg, 0.194 mmol) wasdissolved in formic acid (12 ml, 98%), cooled to 0°C, and HBr(gaseous) was bubbled for 15 min followed by argon for 10 min.Formic acid was removed under reduced pressure. The result-ing brown residue was triturated in diethyl ether overnight.The solids were triturated in ether and then dried to yield 119.8mg (88%) of pale purple powder. 1H NMR (300 MHz,DMSO-d6) 8 10.42 (s, 2H), 9.28 (s, 2H), 7.96 (s, 2H) 7.71 (d,2H, J = 9.0 Hz), 7.45 (d, 2H, J = 8.7 Hz), 4.40-3.90 (br s, 6H),3.77 (d, 4H, J = 10.5 Hz), 3.41 (s, 4H), 3.33 (d, 4H, J = 10.8Hz), 2.39 (d, 4H, J = 13.8 Hz), 2.20 (d, 2H, J = 13.8 Hz), 1.48(d, 4H, J = 13.2 Hz), 1.34 (d, 2H, J = 13.2 Hz); HRMS (FABin glycerol and methanol) calculated for C34H41N4012 (M+H),697.2721; found, 697.2716.

Titration. Each of the receptors was titrated with 9-ethyl-adenine at 3, 9, 15, 21, 27 ± 0.3°C as described (11). Dimer-ization constants for 9-ethyladenine were obtained at 3, 9, 15,21, 27 0.5°C from three protons (C2H, C8H, and ethyl CH2)and were averaged and incorporated into the calculation todetermine the binding constants. The dimerization at 10°C wasobtained through extrapolation of the five experimentallydetermined values.

Stoichiometry Determination. Stoichiometry of complexingfor receptors 2 and 4 with 9-ethyladenine was determined byusing the method of Job; a Job plot obtained for 4 with9-ethyladenine showed deviation from 1:1 binding due tocomplications from 2:1 (guest/host) binding (14). Elevensamples containing various ratios of receptor and 9-ethylad-enine in increments of 10% with a combined concentration of1 mM were prepared by using the same buffered solution as inthe titrations. NMR of these samples was taken at 10°C by

using the same procedure as in the titrations. The change inimide chemical shift was converted to concentration of com-plex in each sample, which was then plotted against the ratioof total receptor to total 9-ethyladenine concentrations.

RESULTS AND DISCUSSIONThe stoichiometry of the complex between 2 and 9-ethylad-enine was determined to be 1:1. The naphthalene-basedbis(imide) 4 showed a nonchelating 2:1 binding mode inaddition to 1:1 binding. Accordingly, this system was notfurther characterized. The titration results are summarized inTable 1.The values of association constants at 10°C when compared

with other water-soluble adenine receptors (ref. 11; temper-ature accurate to ±2°C; Kdimerization used here was determinedwith poor temperature control, ref. 12) show that the recog-nition elements within these receptors are focused on thepurine nucleus and involve a combination of hydrogen bondingand aryl stacking forces. Comparison of the results for thenaphthyl, fluorenyl (12), and carbazolyl surfaces indicates thatthe amide side chain of the latter is not involved in a directcontact with adenines.The AAH provided by the bis(imide) system is -1.6 kcal/

mol. This is the net difference between the strength of thenewly formed hydrogen bonds in the complex and in the bulksolvent, and the lost hydrogen bonds of the free receptor andpurine with the solvent (no net change in the number ofhydrogen bonds occurs). Because receptors 1 and 2 areexpected to have identical hydrophobic overlap with the boundadenine, the enhanced binding is primarily due to the twoadditional hydrogen bonds present in the complex between9-ethyladenine and 2. Thus, the enthalpy per hydrogen bondin water can be estimated to be 0.8 kcal/mol per hydrogenbond in the adenine receptor complex.The entropy difference seen in the bis(imide) system is -5

cal-mol-'K-1. The favorable liberation of bound water isopposed by the loss of entropy in fixing the amide rotors in oneof two equally probable positions (15) [convergent vs. diver-gent imides (16)] and fixing the purine nucleus into a moreconfined cleft. Statistically, restricting the amide rotor into oneof the two equally probable conformations is expected tocontribute -R ln(1/2) = 1.3 cal-mol-1K-1 to entropy ofbinding (17). When two imides chelate the adenine, hydrogenbonds and stacking are probably more effective in reducing thelow-frequency vibrations of 9-ethyladenine relative to thereceptor within the complex. The remaining -3.7 cal'mol-1 K-1 is expected to come from a tighter fit, resulting in2 rather than in the mono(imide) 1. Thus, AHjust compensatesfor the unfavorable AS and gives a small AG298 of 0.2 kcal/molfor a single hydrogen bond in this solvent-exposed system:

AG298 = [-1.6 - (298)(-3.7)]/2 = 0.2 kcal/mol.

To our knowledge other experimental values of hydrogen bondstrengths in water are free energy values (1-4, 18). In contrast,the estimated AG here is lower than the free energy valuesderived by Fersht and coworkers (1, 4) from site-directedmutagenesis studies. The lower value is attributed to therelatively exposed aqueous environment in which the present

Table 1. Association constants of the receptors with 9-ethyladenine and enthalpy and entropy of binding determined from Van't Hoff plots

Ka, M-1 (with 9-ethyladenine)* AH, AS, AG298,Receptor 30C 90C 10°C 150C 210C 270C kcal/mol cal/molFK kcal/mol

1 49±6 36±4 30±4 25±4 21±2 -5.8 -13 -1.92 79±6 62±3 45±4 35±2 28±2 -7.4 -18 -2.03 44±4 26±2 18±1 14±1 10±1 -9.2 -26 -1.5

*9-Ethyladenine dimerization constants of 13.9, 9.51, 8.8, 6.47, 4.60, 3.05 M-1 at 3, 9, 10, 15, 21, 270C, respectively, were incorporated into thecalculation; the uncertainties are at the 95% confidence level.

Chemistry: Kato et al.

Proc. NatL Acad Sci USA 92 (1995)

receptors operate vs. the more buried, less aqueous environ-ments at the active sites of the enzymes. Electrostatic effects,such as hydrogen bonds, are expected to be magnified in thelower dielectric of the enzyme interiors and have even beenproposed to be in the 10-20 kcal/mol range in special cases(19, 20). In the case at hand, the alternating hydrogen bonddonor-acceptor arrangement of the two new hydrogen bondsformed results in some unfavorable secondary interactions(21).The difference between receptors 1 and 3 lies solely in the

aromatic surfaces; the hydrogen bond component of thecomplexing is expected to be identical. The enthalpy differ-ence of -3.4 kcal/mol suggests that hydrophobic interaction isstronger with naphthalene than carbazole. This may be thereason for the 2:1 binding mode seen with receptor 4. As in thecomplexing studies with cyclophanes in various solvents bySmithrud and Diederich (22), the enthalpic component ofhydrophobic binding in water seems to be driven by theformation of stronger van der Waals interactions between thepolarizable aromatic surfaces, the loss of less favorable oneswith water (water has low molecular polarizability), and therelease of water from the reduced hydrogen-bonding environ-ment to the strongly hydrogen-bonding environment in thebulk solvent. The more polarizable character of the naphthylcompared to the inherently polarized carbazolyl surface ex-plains the more favorable AH for 3.The entropic difference between the complex of 1 and the

complex of 3 seems relatively large, -13 cal-mol-1K-1. Be-cause complex 3 showed stronger hydrophobic interactions, itappears that these favorable interactions result in a much moretightly bound and rigid complex than the corresponding car-bazole complex. Similar phenomena of more negative AHcorrelating to more negative AS have been seen with hydro-phobic effects in protein folding (23). Our observation of anenthalpy-entropy compensation effect suggests that even withsmall molecules, associations governed mostly by hydrophobiceffects in water will show this phenomena.

In summary, four water-soluble adenine receptors, mono-(imide) receptors 1 and 3, and bis(imide) 2 and 4 were syn-thesized. Association constants obtained at different temper-atures with 9-ethyladenine allowed determination of enthalpyand entropy of complexing. Comparison of the two carbazolereceptors 1 and 2 gives an estimate for the enthalpy of a singlehydrogen bond in aqueous solution of 0.8 kcal/mol. Compar-ison of the two mono(imide) receptors 1 and 3 showed thatnaphthalene has stronger hydrophobic interactions with

9-ethyladenine than carbazole. The entropy of binding isaccordingly less favorable for receptor 3, giving rise to overallsimilar free energies of binding for receptors 1 and 3 in thetemperature range studied.

We thank Prof. J. R. Williamson and Prof. J. Stubbe for helpfuldiscussions. M.M.C. thanks the Natural Science and EngineeringResearch Council (of Canada) for support in the form of a predoctoralfellowship. This work was supported by the National Institutes ofHealth.

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1212 Chemistry: Kato et aL