the conformational characteristics of congo red, evans blue and trypan blue

Computers and Chemistry 24 (2000) 429–450

The conformational characteristics of Congo red, Evans blueand Trypan blue�

M. Skowronek a, I. Roterman a,*, L. Konieczny b, B. Stopa b, J. Rybarska b,B. Piekarska b, A. Gorecki c, M. Krol a

a Department of Biostatistics and Medical Informatics, Collegium Medicum, Jagiellonian Uni6ersity, Kopernika 17,Medical Informatics, 31-501 Krakow, Poland

b Institute of Biochemistry, Collegium Medicum, Jagiellonian Uni6ersity, Kopernika 7, 31-034 Krakow, Polandc Stanisl*aw Staszic Uni6ersity of Mining and Metallurgy Mickiewicza 30, Poland

Received 23 April 1999; accepted 10 November 1999

Abstract

The structures of the closely related bis–azo dyes Evans blue, Trypan blue and Congo red, which appeared to havedifferent self-assembly properties and correspondingly different abilities to form complexes with amyloids and someother proteins, were compared in this work. Ab initio and semi-empirical methods were used to find the optimalstructures and partial charge distributions of the dyes. The optimal structures were searched using different widelyused programs. The structures of Congo red and evans blue were found to be planar, except for the torsion on thecentral diphenyl bond connecting the two halves of the dye. Both symmetrical parts of the molecules appeared veryclose to planarity. However, Trypan blue exhibits non planarity on the di-azo bonds, as well as on the central bondbetween the symmetrical parts of the dye. In a consequence, the non planarity of this molecule is higher than in thecase of its isomer, Evans blue and co ngo red as well. The extra rotation around the azo bonds extorted by the closeproximity of the sulfonic groups may be the direct cause of its poor self-assembling and complexation propertiesversus Evans blue. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Congo red; Evans blue; Trypan blue

www.elsevier.com/locate/compchem

1. Introduction

The strong self-assembly tendency of bis–azo dyes inwater solution has been known for years (Jelinek,1970), but not until present has this property beenidentified with the ligation capability (Stopa et al.,1997). Interest in understanding Congo red complexa-tion properties is growing, since it is commonly used asa specific reagent for amyloid proteins (Caughey and

Race, 1992; Burgevin et al., 1994; Pollack et al., 1995;Sadler et al., 1995; Piekarska et al., 1996; Stopa et al.,1997; Demaimay et al., 1998; Stopa et al., 1998; Zhen etal., 1999). However, many other non amyloid proteins,especially those rich in bstructure, have also been ob-served to bind these dyes when destabilized proteinmolecules become available for penetration (Rybarskaet al., 1991; Kaszuba et al., 1993; Roterman et al., 1993;Konieczny et al., 1997; Stopa et al., 1997; Roterman etal., 1998; Stopa et al., 1998). Since a correlation hasbeen found between binding to protein and the self-as-sembling tendency, the search for the structuralparameters of bis–azo dyes, that influence their mutualinteraction between molecules, is becoming a key prob-

� Workshop on New Trends in Computational Methods forLarge Molecular Systems, Szklarska Poreba, Poland, 1–6 July1999, Edited by W. Andrzej Sokalski and Morris Krauss.

* Corresponding author.

0097-8485/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.

PII: S 0097 -8485 (99 )00089 -3

M. Skowronek et al. / Computers & Chemistry 24 (2000) 429–450430

Table 1

Congo red geometric parameters as found using HF/3-21G*1, Weiner ’91, cff91, cvff, esff and topology data base

Weiner ’91 CF91 CVFF ESFFAtom Bond HF/3-21G*

1 C2 H 1.07 1.08 1.09 1.08 1.082 1

1.39 1.38 1.393 C 1.403 1 1.381.08 1.09 1.081.07 1.084 H 4 3

1.395 3 1.39 1.41 1.405 C1.36 1.46 1.436 N 1.426 5 1.421.24 1.28 1.231.25 1.257 67 N

1.408 7 1.37 1.45 1.43 1.418 C1.39 1.41 1.399 C 1.409 8 1.401.08 1.09 1.081.07 1.0810 910 H

1.3511 9 1.39 1.39 1.38 1.4011 C1.74 1.75 1.7612 S 1.7612 11 1.721.44 1.54 1.591.43 1.5913 1213 O

1.4314 12 1.44 1.54 1.59 1.4314 O1.58 1.61 1.6815 O 1.5815 12 1.580.99 0.96 0.960.97 0.9616 H 16 15

1.4317 11 1.57 1.43 1.39 1.4117 C1.41 1.42 1.4018 C 1.4118 17 1.411.08 1.08 1.081.07 1.0819 1819 H

1.3620 18 1.40 1.38 1.39 1.3920 C1.08 1.0821 H 1.0821 20 1.081.071.40 1.41 1.381.40 1.3922 C 22 20

1.0723 22 1.08 1.08 1.08 1.0823 H1.40 1.43 1.4124 C 1.4124 22 1.361.08 1.08 1.081.07 1.0825 2425 H

1.4126 27 1.42 1.43 1.43 1.4126 C1.41 1.42 1.4327 C 1.4027 8 1.401.37 1.43 1.361.33 1.4528 N 28 27

1.0029 28 1.00 1.00 1.03 1.0129 H1.00 1.00 1.0230 H 1.0130 28 1.001.40 1.44 1.411.38 1.4131 531 C

1.0732 31 1.08 1.09 1.08 1.0832 H1.31 1.3833 C 1.3933 31 1.401.381.08 1.09 1.081.07 1.0834 3334 H

1.3835 31 1.40 1.45 1.42 1.4035 C1.50 1.45 1.4236 C 1.4136 35 1.491.38 1.45 1.421.40 1.4237 3637 C

1.0738 37 1.08 1.09 1.08 1.0838 H1.39 1.3839 C 1.4039 37 1.401.381.08 1.09 1.081.07 1.0840 H 40 39

1.3841 39 1.39 1.42 1.40 1.4041 C1.36 1.4542 N 1.4342 41 1.421.421.24 1.28 1.22 1.2543 N 1.2543 42

M. Skowronek et al. / Computers & Chemistry 24 (2000) 429–450 431

Table 1 (Continued)

HF/3-21G* Weiner ’91 CF91Bond CVFFAtom ESFF

1.40 1.37 1.40 1.4344 C 1.4044 431.40 1.41 1.4045 44 1.4345 C 1.411.33 1.37 1.4346 N 1.4646 45 1.451.00 1.00 1.0047 46 1.0347 H 1.011.00 1.00 1.00 1.0248 H 1.0148 461.45 1.42 1.4349 45 1.4249 C 1.411.41 1.41 1.4050 C 1.4150 49 1.401.07 1.08 1.0951 50 1.0851 H 1.081.36 1.40 1.3852 C 1.3952 50 1.401.07 1.08 1.0953 52 1.0853 H 1.081.40 1.401 1.4054 C 1.3854 52 1.391.07 1.08 1.0955 54 1.0855 H 1.081.3656 C 1.4056 54 1.38 1.39 1.391.07 1.08 1.0957 56 1.0857 H 1.081.41 1.42 1.4258 C 1.4058 56 1.411.43 1.42 1.4459 58 1.4059 C 1.411.72 1.74 1.7560 S 1.7660 59 1.761.43 1.44 1.5461 60 1.5961 O 1.431.43 1.44 1.5462 O 1.5962 60 1.431.42 1.58 1.6263 60 1.6863 O 1.58

64 6364 H 0.97 1.00 0.96 0.95 0.961.40 1.40 1.4065 44 1.4065 C 1.401.07 1.08 1.0866 H 1.0866 65 1.081.40 1.40 1.4167 41 1.4067 C 1.40

68 6768 H 1.07 1.08 1.09 1.08 1.081.39 1.40 1.4369 36 1.4069 C 1.41

70 6970 H 1.07 1.08 1.09 1.08 1.08

HF/3-21 G* Weiner ‘91 CFF91 CVFFAtom ESFFAngle

1 C2 H

119.65 118.22 119.313 1 2 116.943 C 118.31120.89 118.774 H 118.364 3 1 119.22 119.64120.04 120.11 121.865 3 1 120.455 C 120.79123.93 122.41 145.826 N 100.406 5 3 145.10115.80 123.98 151.277 6 5 153.267 N 148.00120.54 128.858 C 124.328 7 6 121.35 121.61113.55 114.51 151.739 8 7 153.749 C 147.90117.10 117.86 145.8210 H 100.4010 9 8 145.10122.84 122.70 124.3211 9 8 121.3511 C 121.61116.73 117.65 120.6512 S 117.5312 11 9 115.97108.94 106.44 112.0413 12 11 113.0313 O 112.33112.68 105.60 115.0814 O 110.8314 12 11 111.96

99.63 104.65 111.9515 12 11 110.2115 O 103.6816 H 16 15 12 115.03 111.52 104.06 106.32 91.82

119.62 118.11 117.7417 11 9 121.5017 C 119.64122.74 120.72 121.6818 C 120.5818 17 11 121.54119.03 123.03 97.1219 18 17 96.4419 H 97.61120.98 120.27 121.6920 C 120.5820 18 17 121.54119.65 119.74 147.7621 20 18 146.0621 H 147.36120.64 120.46 120.4622 C 120.6222 20 18 119.85120.15 119.64 146.3523 22 20 146.6523 H 145.61

24 C 24 22 20 119.50 120.37 120.56 123.43 120.44118.20 116.52 146.7125 24 22 147.4025 H 146.06118.73 119.96 119.2426 C 119.2726 17 11 120.44126.27 125.64 118.5527 8 7 119.6227 C 119.11

28 N 28 27 8 120.69 121.08 119.36 120.10 118.24122.60 122.40 111.5129 28 27 115.0829 H 110.34


Table 1 (Continued)

Bond HF/3-21G* Weiner ’91 CF91 CVFF ESFFAtom

121.46 112.88 117.72 112.4230 H 30 28 27 117.24118.83 121.17 121.68119.46 121.5031 5 331 C120.12 117.90 118.9432 H 119.4332 31 5 118.91120.24 116.28 116.61120.51 117.2533 C 33 31 5118.38 119.56 121.34 120.1834 H 34 33 31 119.85120.38 121.08 121.62119.47 121.4435 C 35 1 3120.89 121.16 121.6836 C 121.4236 35 1 120.64120.90 106.82 151.28120.69 140.1537 C 37 36 35120.26 138.44 134.6838 H 136.0338 3736 119.69121.26 121.16 121.68120.45 121.4239 37 3639 C119.64 117.94 118.9940 H 119.5440 39 37 120.58120.29 121.63 120.55120.51 120.9141 C 41 39 37

42 N 118.8042 41 39 145.85 105.23 100.58116.61123.96 151.52 154.60115.80 150.0243 42 4143 N128.82 119.75 119.3844 C 120.0144 43 42 120.55125.70 121.68 123.03126.27 120.2145 C 45 44 43121.00 120.22 120.2146 N 120.2546 45 44 120.69123.44 113,93 114.17122.60 110.7047 46 4547 H122.03 114.90 115.1248 H 112.4548 46 45 117.24118.42 119.75 119.38118.03 120.2149 C 49 45 44

120.6050 49 45 120.03 121.68 123.02 120.2250 C122.83 122.40 121.42120.30 122.7151 H 51 50 49120.43 121.78 121.2452 C 121.3652 50 49 121.51120.01 120.26 119.87120.35 120.2053 H 53 52 50

119.5054 52 50 120.31 119.87 120.34 119.6154 C119.83 119.78 119.65119.71 120.0755 54 5255 H

120.6456 54 52 120.42 120.03 120.60 119.8856 C116.57 117.01 117.14 117.2057 H 57 56 54 119.97120.81 121.87 120.56120.58 121.4958 C 58 49 45120.06 122.40 121.4259 C 122.7159 58 49 118.73124.15 123.34 121.10123.64 123.1760 S 60 59 58106.38 108.07 111.9261 O 111.9961 60 59 108.95105.53 116.34 113.79112.66 111.2962 60 5962 O104.66 114.27 111.3663 O 105.5963 60 59 99.63111.64 103.78 104.77115.06 91.5064 H 64 63 60114.37 119.07 119.62 118.8365 C 65 44 43 113.55117.92 145.62 145.37117.09 145.6466 H 66 65 44118.75 117.98 119.1167 C 118.3267 41 39 119.45121.04 119.76 120.30119.06 119.9868 H 68 67 41120.82 121.86 120.43 120.7069 C 69 36 35 120.64120.50 119.29 116.94119.47 118.4170 69 3670 H

Weiner ‘91 CFF91 CVFFAtom ESFFDihedral HF/3-2 1 G*

2 H3 C

−2.09 −0.72 1.50−0.82 −0.684 3 1 24 H179.28 179.82 179.915 C 179.845 3 1 2 179.52

−179.83 145.82 100.40180.00 145.106 N 6 5 3 1−0.50 −5.54 0.287 N −6.067 6 5 3 −0.92179.53 −179.58 −179.80179.91 −179.818 7 6 58 C178.26 176.00 178.319 C 176.439 8 7 6 179.37

0.40 1.00 1.10−0.52 0.2110 H 10 9 8 711 C 180.00 −179.56 178.22 −179.14 177.9914 9 8 7

178.06 −179.63 178.72178.38 −179.6612 S 12 11 9 87.13 −2.21 −3.0713 O −0.3013 12 11 9 5.53

120.97 122.34 121.29133.91 129.4014 O 14 12 11 915 O −113.66 −120.02 −115.18 −120.86 −118.4915 12 11 9

−127.15 123.60 61.60 176.2316 H −171.2216 15 12 11


Table 1 (Continued)

HF/3-21G* Weiner ’91 CF91Bond CVFFAtom ESFF

−1.25 −2.44 −0.34 −0.06417 C 0.3017 11 9 8−178.32 179.48 178.5518 17 11 9 −179.8118 C 179.87

3.17 1.29 1.1719 H 0.5119 18 17 11 0.08178.00 178.78 −178.8020 18 17 11 −179.8420 C −179.77

21 H −179.8121 20 18 17 −179.38 −178.73 179.91 −179.87−0.50 −0.51 0.1929 20 18 17 0.0322 C 0.27

0.18 0.08 0.3923 H 0.1523 22 20 18 0.56179.50 179.67 −178.0424 22 20 23 −179.9324 C −178.96

25 24 22 2025 H 179.50 179.94 179.41 179.77 179.880.20 0.07 0.6726 17 11 9 0.0926 C 0.53

27 8 9 1127 C 0.00 −0.52 0.64 0.08 0.030.18 −2.09 0.42 0.0628 N 0.0528 27 8 7

−179.14 −0.69 −19.5429 28 27 8 −0.4729 H −13.03179.23 179.11 −144.5930 H −179.3030 28 27 8 −136.06179.91 179.93 180.0031 5 6 7 179.8731 C 179.52179.63 179.59 −179.6232 H 179.4032 31 5 3 180.00

−179.54 −179.35 179.9333 31 5 3 180.0033 C 179.80178.75 177.32 −179.0234 H 177.9734 33 31 5 −178.97

0.28 2.43 −0.2035 1 3 5 0.3935 C −0.1036 35 1 336 C 179.37 177.81 168.41 171.15 160.01

48.35 36.24 32.8437 36 35 1 25.9037 C 26.191.14 2.50 −0.4238 H 0.8338 37 36 35 −0.10

178.77 177.58 179.0739 37 36 35 178.5639 C 179.3340 39 37 3640 H −179.54 −179.65 179.14 −178.06 179.26

0.53 0.34 0.9141 39 37 36 0.9641 C 0.9242 41 39 3742 N −179.92 −179.80 160.01 −159.42 −170.31

43 N 179.7343 42 41 39 179.81 177.73 179.94 178.93−179.08 179.92 179.0344 43 42 41 −179.6644 C 179.78

0.00 −0.31 2.2145 C 0.0345 44 43 42 1.68−0.14 0.13 −2.2846 45 44 43 0.0646 N −1.07179.41 179.80 −159.7047 H −159.4247 46 45 44 −170.31−0.41 −0.77 26.5648 46 45 44 25.4048 H 47.47179.79 −179.93 179.8149 C 179.9349 45 44 43 179.70179.87 179.38 117.8050 49 45 44 179.9450 C 178.32

51 50 49 4551 H −0.80 0.07 0.20 −0.71 0.72−179.91 179.94 179.8152 50 49 45 −179.8552 C 178.02

179.89 −179.76 170.4553 H 179.8553 52 50 49 179.220.51 0.72 −0.2054 52 50 49 −0.6554 C −0.07

55 54 52 5055 H −178.84 −179.24 179.86 179.80 179.770.51 0.38 0.1656 54 52 50 0.0356 C −0.06

57 56 54 5257 H −177.67 −179.17 179.22 179.67 179.5258 C 58 49 45 44 0.73 −0.89 −0.92 −0.92 −0.85

1.46 0.13 1.2959 58 49 45 0.1659 C 0.35177.23 178.21 178.6360 S 179.5260 59 58 49 179.03

5.68 5.87 178.0961 60 59 58 177.4461 O 162.5762 60 59 5862 C −133.88 −120.91 −58.85 −57.76 −84.88

113.65 119.90 49.3863 60 59 58 58.2063 O 50.6464 63 60 5964 H 171.83 127.18 −129.28 −178.98 −177.08

−0.63 −0.8065 C 0.1665 44 45 49 −0.72 0.370.50 0.34 0.9666 65 44 43 −0.3466 H 0.22

−9.92 −0.95 −8.2167 C −8.9667 41 42 43 −0.92179.52 179.48 179.7668 67 41 39 −179.5868 H 179.83

69 C 69 36 35 1 179.95 −179.03 178.71 179.87 179.661.36 2.72 0.7170 69 36 35 −1.8170 H 1.18


lem (Stopa et al., 1997). The present study approachesthis problem theoretically, by comparing the structuresof selected bis–azo dye molecules, which are related in

their molecular architecture but differ in ligation prop-erties. These are Congo red, Evans blue and Trypanblue. Of special interest are Evans Blue and Trypan

Figure 1. Differences between structural parameters (bond length, angle, dihedral) as calculated according to HF/3-21G* andWeiner ‘91. (a) Congo red (bonds, angle, dihedrals numbers as in Table 1); (b) Evans blue (bonds, angles, dihedrals numbers as inTable 2).


Figure 2. Torsional potential as a function of the dihedral angle between two benzene rings in the Congo red molecule calculatedaccording to the HF/3-21G* and Weiner ‘91 methods. (a) Congo red; (b) Evans blue.

blue molecules, two structural isomers presentingsignificantly different complexation capabilities(Rybarska et al., 1991). While Congo red and Evansblue are easily engaged in complexation with proteins,the ligation property of Trypan blue is poor. Theseobserved differences seem promising, in the search foran explanation of the complexation mechanism ofCongo red and other bis–azo dyes.

2. Materials and methods

2.1. Programs and computers

The quantum mechanical calculations aimed at theoptimal structural geometry, torsional potential, elec-trostatic potential, dipole moments and partial chargedistributions of the dyes, were performed with Gaussian

94 ver. A1 (Frisch et al., 1995) using an HP-ConvexExemplar SPP-1600/XA-32. The molecular mechanicscalculations, aimed at energetic optimization of themonomer structure of the dyes, were done using AM-BER 4.1 (Pearlman et al., 1995) with a Convex 3210computer. All of these calculations were done at theCYFRONET academic computer center in Krakow.

The optimal structure and partial charge distribu-tions of the dye molecules were also calculated, usingthe Insight II program, applying the cvff, cff91 and esffforce fields. All calculations using Insight II were doneon an octane work station (Silicon Graphics) (Hagler etal., 1979, 1985; Maple et al., 1990; Insight II, 1995).

2.2. Geometry and partial charges distribution

The first step in modeling the supramolecular system,was to define the optimal geometry for the trans and cis


Figure 3. Partial charge distribution for the trans+ conformation as calculated using the Mulliken, ChelpG, Merz and Kollman andResp methods. (a1) Congo red-Mulliken-31G*, ChelpG/6-31G*, Merz–Kollman/6-31G* and Resp/6-31G* methods; (a2) Congo redcis− Resp single-conformational versus trans+ Resp single-conformational versus cis and trans+ Resp multi-conformational;(b)-Evans blue-Resp single and multiconformational; (c)-Trypan blue-Resp single-conformational


Fig. 3. (Continued)

isomers of the dye monomer molecules. These calcula-tions used the 3-21G* (HF/3-21G*) Hartree–Fockquantum-mechanical model (Binkley et al., 1980; Gor-don et al., 1982; Pietro et al., 1982).

The stationary points on the potential energy sur-faces were found for all analyzed molecules. The tor-sional potential for Congo red and Evans blue wasadditionally calculated on the basis of HF/3-21G* for

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450438Table 2

Congo red partial charge distribution as found using the different methods (listed in the column headings)

MK trans ChelpG trans Mulliken trans CFF91 trans CVFF trans ESFF transAtom RESP cisRESP trans RESP cis-trans

−0.2171 C −0.127−0.146 −0.100 −0.055−0.140 −0.143 −0.144 −0.1900.218 0.127 0.100 0.1060.1330.1452 H 0.1510.1440.142

−0.042–0.065 −0.196 −0.127 −0.100 −0.002–0.071 −0.068 −0.1353 C0.108 0.068 0.244 0.127 0.100 0.1070.0864 H 0.090 0.087

0.271 0.199 0.199 −0.1040.1650.057 0.2245 C 0.058 0.055−0.1070.001 −0.430 −0.199 −0.199 −0.106−0.007 −0.003 −0.0516 N−0.163−0.147 −0.306 −0.199 −0.199 −0.106−0.142 −0.144 −0.2267 N

0.178 0.199 0.199 0.0090.0538 C 0.2440.0870.0940.080−0.1329 C −0.127−0.085 −0.100 0.077−0.101 −0.094 −0.223 −0.067

0.293 0.127 0.100 0.1070.1600.14110 H 0.1900.1470.151−0.278−0.041 −0.395 0.073 0.028 0.146−0.080 −0.061 −0.08611 C

1.655 0.608 0.655 0.62212 S 0.942 0.971 0.957 1.117 1.301−0.651 −0.302 −0.339 −0.412−0.602−0.50713 O −0.556−0.510−0.513

−0.634−0.527 −0.682 −0.302 −0.339 −0.412−0.533 −0.531 −0.57614 O−0.689−0.628 −0.785 −0.501 −0.356 −0.480−0.629 −0.628 −0.65115 O

0.500 0.424 0.350 0.4320.5030.513 0.50616 H 0.514 0.5130.2330.005 0.079 0.000 0.000 −0.0240.030 0.019 0.04717 C

−0.234−0.165 −0.235 −0.127 −0.100 −0.055−0.141 −0.151 −0.22318 C0.275 0.127 0.100 0.1060.16719 H 0.1810.1580.1540.163

−0.056 −0.006 −0.191 −0.127 −0.100 −0.10620 C −0.114−0.092 −0.1040.218 0.127 0.100 0.1060.1010.145 0.13621 H 0.142 0.147

−0.178−0.156 −0.216 −0.127 −0.100 −0.106−0.146 −0.151 −0.17622 C0.212 0.127 0.100 0.10623 H 0.145 0.144 0.145 0.150 0.126

−0.178 −0.127 −0.100 −0.055−0.061−0.16524 C −0.165−0.173−0.1800.0900.129 0.198 0.127 0.100 0.1060.134 0.132 0.13225 H

−0.1320.078 −0.056 0.000 0.000 0.0110.075 0.076 0.07626 C0.424 0.083 0.105 −0.2090.4040.145 0.14327 C 0.146 0.146

−0.833−0.640 −0.987 −0.580 −0.665 −0.391−3.646 −0.642 −0.72028 N0.4210.378 0.404 0.249 0.280 0.2640.378 0.378 0.40329 H

0.471 0.249 0.280 0.2640.31130 H 0.2740.2420.2460.240−0.204−0.221 −0.201 −0.127 −0.100 −0.002−0.211 −0.214 −0.34931 C

0.210 0.127 0.100 0.1070.1280.14932 H 0.1790.1470.144−0.095−0.075 −0.219 −0.127 −0.100 −0.055−0.074 −0.076 −0.02433 C

0.217 0.127 0.100 0.10634 H 0.123 0.121 0.121 0.118 0.1000.010 0.000 0.000 −0.1020.060−0.00835 C −0.028−0.011−0.013

0.040−0.002 0.010 0.000 0.000 −0.102−0.004 −0.004 0.00936 C−0.055 −0.094 −0.218 −0.127 −0.100 −0.05537 C −0.084 −0.089 −0.088

0.217 0.127 0.100 0.1060.1050.1250.12438 H 0.126 0.125−0.222−0.225 −0.201 −0.127 −0.100 −0.002−0.206 −0.214 −0.32139 C

0.179 0.134 0.210 0.127 0.100 0.10740 H 0.155 0.146 0.150

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Table 2 (Continued)

MK trans ChelpG trans Mulliken trans CFF91 trans CVFF trans ESFF transRESP cisAtom RESP cis-transRESP trans

0.167 0.191 0.271 0.199 0.199 −0.10441 C 0.0470.050 0.048−0.430 −0.199 −0.199 −0.106−0.10242 N −0.0060.008−0.0030.021

−0.225 −0.170 −0.306 −0.199 −0.199 −0.10643 N −0.140−0.159 −0.1480.178 0.199 0.199 0.00970.0540.19644 C 0.080 0.076 0.0780.424 0.000 0.122 −0.20945 C 0.165 0.178 0.171 0.188 0.393

−0.987 −0.497 −0.402 −0.391−0.80946 N −0.70−0.645−0.666−0.6240.402 0.418 0.404 0.249 0.140 0.26447 H 0.3790.373 0.376

0.471 0.249 0.140 0.2640.29648 H 0.2480.2350.2510.219−0.056 0.000 0.000 0.01149 C 0.062 0.064 0.063 0.038 −0.134−0.177 −0.127 −0.100 −0.055−0.05850 C −0.151−0.167−0.169−0.165

0.198 0.127 0.100 0.10651 H 0.134 0.138 0.136 0.132 0.082−0.216 −0.127 −0.100 −0.106−0.174−0.19852 C −0.174 −0.170 −0.172−0.213 0.127 0.100 0.10653 H 0.152 0.150 0.151 0.158 0.126−0.191 −0.127 −0.100 −0.106−0.01654 C −0.058−0.095−0.088−0.100

0.137 0.104 0.218 0.127 0.100 0.1060.14255 H 0.1430.145−0.235 −0.127 −0.100 −0.055−0.231−0.138 −0.19956 C −0.127 −0.150

0.1670.145 0.275 0.127 0.100 0.1060.157 0.151 0.16957 H0.2040.008 0.079 0.000 0.000 −0.0240.003 0.006 0.06958 C

−0.394 0.073 0.028 0.146−0.29059 C −0.109−0.041−0.030−0.0511.3010.931 1.655 0.608 0.655 0.6220.935 0.934 1.10860 S

−0.682 −0.302 −0.339 −0.412−0.634−0.57261 O −0.522 −0.525 −0.524−0.652 −0.302 −0.339 −0.41262 O −0.505 −0.507 −0.506 −0.556 −0.602−0.784 −0.501 −0.356 −0.480−0.68863 O −0.641−0.619−0.623−0.616

0.502 0.502 0.500 0.424 0.350 0.43264 H 0.509 0.511 0.510−0.132 −0.127 −0.100 0.077−0.05765 C −0.200−0.105−0.108−0.098

0.293 0.127 0.100 0.10766 H 0.152 0.152 0.153 0.193 0.156−0.195 −0.127 −0.100 −0.002−0.08767 C −0.114−0.070−0.072−0.067

68 H 0.2440.085 0.127 0.100 0.1070.086 0.085 0.104 0.809−0.217 −0.127 −0.100 −0.055−0.145−0.12569 C −0.133 −0.119 −0.150

70 H 0.2180.139 0.127 0.100 0.1060.135 0.137 0.148 0.120

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Evans blue partial charge distribution and geometric parameters as found according to HF/3-21G* and Weiner ‘91 methods

MQ Angle (°) MM MQ Dihedral (°) MM MQCharge (ecu)Atom Bond (A, ) MM

1 C −0.13822 C 1.081.072 10.1460

1.39 3 1 2 119.97 118.483 C −0.1124 3 1 1.38120.76 118.75 4 3 1 2 −0.81 −2.144 3 11.084 H 0.1099 4 3 1.07120.48 120.24 5 3 1 2 178.79 177.355 C −0.0093 5 3 1.39 1.39 5 3 1122.46 121.77 6 5 3 1 179.97 179.786 5 36 N 1.361.426 50.1383

1.24 7 6 5 117.07 124.71 7 6 5 3 −0.03 0.61−0.20357 N 7 6 1.25119.00 126.96 8 7 6 5 −179.97 −178.578 7 68 C 1.371.408 70.8411

9 8 7 115.68 115.66 9 8 7 6 −179.669 C−170.83 −0.1288 9 8 1.40 1.39-90117.46 119.60 10 9 8 7 −0.611 −5.7310 9 810 H 1.081.0710 90.1563121.58 121.06 11 9 8 7 179.8811 C 172.70−0.1891 11 9 1.35 1.38 11 9 8120.21 116.26 12 11 9 8 177.37 −172.6312 11 91.0812 H 0.1757 12 11 1.06120.35 120.50 13 11 9 8 −0.44 0.5313 C −0.0157 13 11 1.41 1.41 13 11 9121.53 119.65 14 13 11 9 −179.05 −178.4714 13 1114 C 1.421.4314 13−0.0581

15 S 123.700.9646 124.37 15 14 13 11 2.27 5.7615 14 1.72 1.74 15 14 13112.75 105.40 16 15 14 13 −42.53 −59.5416 15 141.43 1.4316 O −0.5269 15 15

17 15 14−0.5155 108.60 106.32 17 15 14 13 −176.45 179.3317 15 1.43 1.4417 O18 15 14−0.6242 99.51 104.74 18 15 141 3 70.04 59.4718 15 1.58 1.4318 O

115.20 111.51 19 18 15 14 −174.93 −128.5719 18 1519 H 1.000.9719 180.512620 14 13−0.0567 119.95 118.17 20 14 13 11 −179.33 −177.0720 14 1.36 1.4020 C

119.25 119.12 21 20 14 13 179.61 178.7021 20 141.0821 H 0.1924 2l 20 1.07122.18 122.65 22 20 14 13 0.03 −1.8022 C −0.0553 22 20 1.39 1.40 22 20 14116.04 116.15 23 22 20 14 −176.80 −174.2523 22 2023 S 1.751.7123 220.9392

24 23 22−0.5054 110.15 105.94 24 23 22 20 −20.53 −28.6024 23 1.43 1.4424 O110.75 106.35 25 23 22 20 −154.40 −149.8825 23 2225 O 1.441.4425 23−0.5234100.46 104.01 26 23 22 20 92.73 90.0526 O −0.6181 36 23 1.58 1.43 26 23 22115.37 111.42 27 26 23 22 −169.90 −122.9027 26 2327 H 1.000.9727 260.5137121.38 119.66 28 22 20 14 0.93 −0.5828 C 0.1196 28 22 1.41 1.41 28 22 20121.70 119.68 29 28 22 20 177.24 −176.5729 28 221.3629 N −0.5534 29 28 1.32119.61 122.02 30 29 28 22 −7.23 −4.7430 H 0.3604 30 29 1.00 1.00 30 29 28118.85 120.41 31 29 28 22 178.53 179.9731 29 2831 H 0.3000 1.001.0031 29

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Table 3 (Continued)

MQ Angle (°) MM MQ Dihedral (°) MM MQBond (A, )Atom MMCharge (ecu)

119.42 119.7732 C 32 13 11 90.0824 1.41 2.3032 13 1.42 1.42 32 13 11124.83 124.53 33 8 7 6 0.22 6.7833 8 70.152433 C 1.421.3833 8

34 33 8−0.3469 119.40 118.62 34 33 8 7 0.04 12.9734 33 1.35 1.3734 O110.18 113.6235 H 35 34 33 80.1897 −0.26 23.7635 34 0.99 0.96 35 34 33120.29 118.95 36 5 3 1 −0.05 0.0336 5 31.40 1.4036 C 0.0071 36 5

37 36 5−0.1568 121.03 120.35 37 36 5 3 179.59 179.5737 36 1.52 1.5237 C110.42 111.00 38 37 36 5 −179.75 177.5338 H 0.0667 38 37 1.08 1.09 38 37 36110.90 109.74 39 37 36 5 60.45 57.6739 37 360.066739 H 1.091.0839 37

40 37 360.0667 110.911 109.70 40 37 36 5 −59.97 −62.4040 37 1.08 1.0940 H41 C 118.55−0.1525 119.77 41 36 5 3 −0.03 0.2841 36 1.38 1.39 41 36 5

119.13 119.08 42 41 36 5 178.83 177.0842 41 360.144042 H 1.081.0742 4143 41 360.0492 121.62 120.30 43 41 36 5 0.04 −0.3743 41 1.40 1.4043 C

120.43 120.78 44 43 41 36 179.97 179.8244 C 0.0054 44 43 1.49 1.49 44 43 41120.43 120.84 45 44 43 41 49.42 38.1145 44 430.152745 C 1.381.3945 44

46 45 440.1445 119.24 119.41 46 45 44 43 1.19 2.5246 45 1.07 1.0846 H47 C 121.620.0068 121.51 47 45 44 43 −179.97 −179.8447 45 1.38 1.40 47 45 44

120.41 119.92 48 47 45 44 −179.62 −179.8148 47 451.51 1.5248 C −0.1635 48 4749 48 470.0679 110.42 111.01 49 48 45 45 0.10 1.5049 48 1.08 1.0949 H50 48 470.0679 110.88 109.66 50 48 47 45 −119.67 −118.5250 48 1.08 1.0950 H

110.94 109.70 51 48 47 45 −119.91 121.3751 48 4751 H 1.091.0851 480.06791.39 52 47 45 118.55 119.76 52 47 45 44 0.03 −0.2752 4752 C 1.39−0.0060

117.25 119.28 53 52 47 45 179.97 179.9753 52 471.42 1.6853 N 0.1370 53 5254 53 52−0.2081 117.08 124.68 54 53 52 47 179.80 −179.8454 53 1.25 1.2454 N

110.00 126.97 55 54 53 52 179.97 179.4255 C 0.0921 55 54 1.40 1.37 55 54 53124.82 124.57 56 55 54 53 −0.18 −6.0956 55 540.139156 C 1.421.3856 55

57 56 55−0.3524 119.40 118.60 57 56 55 54 −0.04 −13.0557 56 1.35 1.3757 O58 57 560.1992 110.19 113.57 58 57 56 55 0.24 −23. 9358 57 0.99 0.9658 H

120.65 119.57 59 56 55 54 179.80 170.8459 56 551.42 1.4359 C 0.0799 59 5660 59 560.1354 121.16 121.38 60 59 56 55 179.26 −176.3660 59 1.46 1.4360 C61 60 59−0.5648 121.25 120.94 61 60 59 56 −2.45 3.7161 60 1.32 1.3661 N

118.85 120.33 62 61 60 59 2.28 −1.0562 616062 H 1.001.0062 610.303563 61 600.3617 119.62 122.01 63 61 60 59 −171.95 −176.2263 61 1.00 1.0063 H

117.05 119.30 64 60 59 56 178.32 −177.3664 60 59−0.472964 C 1.411.4164 6065 64 600.9306 122.54 123.95 65 64 60 59 −175.56 −175.3865 64 1.71 1.7465 S66 65 64−0.5214 110.76 106.45 66 65 64 60 −27.97 −36.3866 65 1.44 1.4466 O

110.14 105.87 67 65 64 60 −161.83 −157.7067 65 6467 O 1.441.4367 650.5039100.45 103.99 68 65 64 60 84.90 83.7168 O −0.6166 68 65 1.58 1.43 68 65 64115.36 111.40 69 68 65 64 170.20 122.7669 68 650.97 0.9969 H −0.5136 69 68

70 64 60−0.0854 121.37 119.65 70 64 60 59 2.00 −2.7070 64 1.39 1.4070 C71 70 640.1998 118.56 118.56 71 70 64 60 178.67 −178.9871 70 1.07 1.0871 H

122.18 122.65 72 70 64 60 −0.93 0.7772 70 6472 C −0.0368 1.401.3672 70

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Table 3 (Continued)

MQ Angle (°) MM MQ Dihedral (°) MM MQBond (A, )Atom MMCharge (ecu)

1.74 73 72 70 116.35 117.30 73 72 70 64 −178.53 −175.7173 7273 S 1.720.9531108.60 106.33 74 73 72 70 −5.13 −2.6374 73 7274 O 1.441.4374 73−0.5118

1.43 75 73 72 112.75 105.47 75 73 72 70 −139.04 −123.8075 7375 O 1.43−0.525599.51 104.71 76 73 72 70 108.40 117.1876 73 721.4376 O −0.6259 76 73 1.58

115.18 111.56 77 76 73 72 175.09 128.6177 H 0.5148 77 76 0.97 0.99 77 76 73118.50 118.24 78 59 56 55 −0.21 3.9378 59 5678 C 1.421.4278 59−0.0129

−0.2152 79 78 S9 119.42 119.75 79 78 79 76 1.28 0.5779 78 1.4179 C 1.41119.41 123.16 80 79 78 79 176.40 176.2880 79 7880 H 1.081.0680 790.1853120.42 120.51 81 79 78 59 −1.41 −2.3281 C −0.1128 81 79 1.35 1.38 81 79 78120.95 119.34 82 81 79 78 179.91 −178.9882 81 7982 H 1.071.0782 81−0.1528120.28 118.95 83 52 47 45 0.03 0.0583 C −0.1073 83 82 1.39 1.40 83 52 47118.76 121.02 84 83 52 47 179.50 179.4384 83 S21.0884 H 0.1080 84 83 1.08120.48 120.81 85 83 52 47 −0.06 0.1085 C −0.1451 85 83 1.37 1.38 8S 83 52119.98 120.30 86 85 83 52 178.76 177.2486 85 8386 H 0.1482 86 85 1.081.07

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Table 4

Trypan blue partial charge distribution and geometric parameters as found using HF/3-21G* and Weiner ‘91 methods

Weiner ‘91 HF/3-21G*Atom Dihedral (°)Charge (ecu) Weiner ’91 HF/3-21G*Bond (A, ) Weiner ’91 HF/3-21G* Angle (°)

−0.11931 C2 H 0.1388 2 1 1.08 1.07

118.16 119.843 1 21.383 C −0.1552 3 1 1.38118.40 120.72 4 3 1 2 −3.19 0.454 H 0.1495 4 3 1.08 1.07 4 3 1120.16 120.02 5 3 1 2 177.29 178.153 15 C 1.39 51.395 3−0.0020

1.42 6 5 3 121.76 121.00 6 5 3 1 179.80 −179.110.01386 N 6 5 1.42123.20 115.31 7 6 5 3 2.93 −24.616 57 N 1.25 71.257 6−0.0300129.10 120.56 8 7 5 6 −175.37 175.468 C −0.0466 8 7 1.40 1.40 8 7 6116.72 114.92 9 8 7 6 −171.18 −174.638 79 C 1.42 91.419 8−0.1610

9 81.0540 120.79 119.81 10 9 8 7 −4.54 4.6610 9 1.74 1.75 1010 5104.51 109.76 11 10 9 8 −53.83 54.5811 10 9−0.530411 O 1.431.4311 10

12 10 9−0.5013 105.44 107.36 12 10 9 8 −174.53 −172.6312 11 1.43 1.4212 O103.6213 O 102.22−0.5325 13 10 9 8 65.75 −59.9213 12 1.43 1.56 13 10 9116.02 114.15 14 13 10 9 −0.14 73.0414 13 100.9714 H 0.4070 14 13 1.00120.16 121.00 15 9 8 7 172.65 −178.1615 C −0.0624 I5 14 1.40 1.34 1S 9 8119.64 120.18 16 15 9 8 −178.40 179.9016 15 916 H 1.071.0816 150.1704120.00 120.29 17 15 9 8 1.90 −0.2017 C −0.0543 17 16 1.40 1.43 17 15 9117.77 119.46 18 17 15 9 −179.58 179.6018 17 151.4018 C −0.1639 18 17 1.40121.82 120.51 19 18 17 15 1.92 0.7719 H 0.1723 19 18 1.08 1.07 19 18 17119.41 118.57 20 18 17 15 −178.36 179.9720 18 1720 C 1.361.4020 19−0.0138

21 20 181.0514 120.80 119.01 21 20 18 17 −176.00 179.4021 20 1.73 1.7421 S104.03 110.00 22 21 20 18 100.83 19.9322 21 2022 O 1.431.4322 21−0.5400105.66 109.55 23 21 20 18 −20.42 154.1623 O −0.5260 23 22 1.43 1.43 23 21 20104.33 98.64 24 21 20 18 −139.38 −92.4824 21 2024 O 1.581.4324 23−0.6430

25 24 210.5200 109.90 115.67 25 24 21 20 123.22 163.932S 24 1.00 0.9725 H120.12 122.41 26 20 18 17 −2.52 −1.4426 20 1826 2526 C −0.1905 1.381.40

27 26 200.1740 117.07 119.12 27 26 20 18 −176.00 −177.4827 26 1.08 1.0727 H

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Table 4 (Continued)

HF/3-21G* Angle (°) Weiner ‘91 HF/3-21G* Dihedral (°) Weiner ’91 HF/3-21G*Bond (A, )Atom Weiner ’91Charge (ecu)

121.31 121.2428 C 28 26 20 180.2062 2.80 1.5328 27 1.40 1.38 28 26 20117.25 119.67 29 28 26 20 179.51 179.7329 28 26−0.823829 N 1.351.3529 28

30 29 280.4043 123.11 119.82 30 29 28 26 0.36 0.1830 29 1.00 1.0030 H1.00 31 29 28 118.22 119.97 31 29 28 26 −179.90 179.7231 H 31 300.3617 1.00

121.11 119.96 32 17 15 9 1.11 −0.87632 17 151.41 1.4132 C 0.1113 32 3133 8 70.2240 123.13 124.93 33 8 7 6 6.40 5.7933 32 1.42 1.3733 C34 33 8−0.3927 119.84 120.11 34 33 8 7 10.74 −1.5234 33 1.37 1.3534 O

113.34 111.10 35 34 33 8 22.67 −3.1035 34 3335 H 1.000.9635 340.2504119.00 120.8036 C 36 5 3 10.0540 0.62 −1.3836 3S 1.40 1.40 36 5 3120.30 120.93 37 36 5 3 179.40 −179.0437 36 5−0.186537 C 1.511.5237 36

38 37 360.0717 111.01 110.62 38 37 36 5 176.72 −178.1538 37 1.09 1.0838 H109.67 110.44 39 37 36 5 56.80 62.2439 H 0.0717 39 38 1.09 1.08 39 37 36109.51 111.14 40 37 36 5 −63.23 −58.0740 37 360.071740 H 1.081.0940 39

41 36 5−0.1621 119.79 118.29 41 36 5 3 −0.04 1.7041 40 1.40 1.3841 C42 41360.1422 118.90 119.18 42 41 36 5 177.10 178.1242 41 1.08 1.0742 H

121.53 121.61 43 41 36 5 0.35 −0.8743 41 361.40 1.4043 C 0.0018 43 4244 43 410.0008 120.89 120.29 44 43 41 36 179.70 179.7144 43 1.50 1.4944 C45 44 430.1646 120.85 120.31 45 44 43 41 48.89 36.2645 44 1.38 1.3945 C

119.57 119.35 46 45 44 43 2.25 1.1146 45 4446 H 1.071.0846 450.14021.38 47 45 44 121.51 121.48 47 45 44 43 179.85 179.9747 4547 C 1.400.0454

119.91 120.58 48 47 45 44 179.83 179.8148 47 451.52 1.5148 C −0.1850 48 4749 48 470.0720 110.98 110.44 49 48 47 45 −0.86 1.4049 48 1.09 1.0849 H

109.67 111.01 50 48 47 45 −120.83 121.4050 H 0.0720 50 48 1.09 1.08 50 48 47109.70 110.74 51 48 47 45 119.02 −118.2551 48 470.072051 H 1.081.0951 48

52 47 450.0161 119.75 118.38 52 47 45 44 −1.04 −0.4052 47 1.39 1.3952 C53 52 470.0850 119.30 117.65 53 52 47 45 −178.80 179.5853 52 1.36 1.4253 N

122.99 115.57 54 53 52 47 176.38 168.2054 53 521.24 1.2454 N −0.0943 54 5355 54 530.0229 129.15 120.48 55 54 53 52 175.26 176.9355 54 1.37 1.4055 C56 55 540.2225 122.94 125.15 56 55 54 53 −6.03 −0.2556 55 1.42 1.3756 C

119.88 119.88 57 56 55 54 −11.30 1.1257 56 5557 O 1.351.3757 560.344658 57 560.1850 113.78 110.85 58 57 56 55 −24.37 −0.7658 57 0.96 1.0058 H

119.30 120.68 59 56 55 54 170.82 −178.6259 56 550.075359 C 1.431.4259 5660 59 560.2033 122.38 122.66 60 59 56 55 −177.04 −179.5960 59 1.42 1.4360 C61 60 590.8016 123.37 122.39 61 60 59 56 0.67 0.4461 60 1.35 1.3561 N

118.80 119.88 62 61 60 59 −1.04 −0.2262 61 6062 H 1.001.0062 610.3472122.58 119.87 63 61 60 59 179.18 179.5063 H 0.4010 63 61 1.00 1.00 63 61 60119.41 117.70 64 60 59 56 −179.84 −179.9064 60 591.40 1.3864 C −0.1930 64 60

65 64 600.1693 121.73 119.65 65 64 60 59 179.80 −178.4265 64 1.08 1.0765 H66 64 60−0.0157 121.33 121.20 66 64 60 59 −0.79 0.5066 64 1.40 1.3866 C

118.85 118.63 67 66 64 60 177.76 179.3267 66 6467 S 1.0348 1.741.7367 66

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Table 4 (Continued)

HF/3-21G* Angle (°) Weiner ‘91 HF/3-21G* Dihedral (°) Weiner ’91 HF/3-21G*Bond (A, )Atom Weiner ’91Charge (ecu)

104.41 110.1268 O 68 67 66 64−0.5331 44.23 25.9868 67 1.44 1.43 68 67 66105.77 109.63 69 67 66 64 165.54 160.2069 67 66−0.525869 O 1.431.4469 67

70 67 66−0.6520 104.02 98.43 70 67 66 64 −75.46 −86.6070 67 1.44 1.5870 O0.97 71 70 67 110.55 115.72 71 70 67 66 −123.73 173.3271 H 71 700.5270 1.00

120.10 122.44 72 66 64 60 1.68 −1.4572 66 641.40 1.3672 C −0.1076 72 6673 72 660.1584 119.05 120.84 73 72 66 64 178.92 177.8573 72 1.08 1.0773 H74 72 66−0.0905 119.40 118.58 74 72 66 64 −1.61 1.4074 72 1.41 1.4074 C

117.82 119.30 75 74 72 66 179.87 179.7375 74 7275 C 1.431.4075 74−0.0645120.30 118.84 76 75 74 7276 H −0.710.1550 0.5776 75 1.08 1.07 76 75 74120.09 120.28 77 75 74 72 179.59 179.7477 75 74−0.116577 C 1.341.4077 75

78 77 751.0001 119.00 122.23 78 77 75 74 −178.97 −177.6178 77 1.74 1.7578 S104.75 110.01 79 78 77 75 −130.84 104.8079 O −0.4864 79 78 1.44 1.42 79 78 77105.32 111.72 80 78 77 75 −10.15 −119.4280 78 77−0.549480 O 1.431.4480 78

81 78 77−0.5657 103.92 98.77 81 78 77 75 109.41 −8.6881 78 1.43 1.5881 O82 H 115.780.4809 114.40 82 81 78 77 −0.91 −126.6082 81 1.00 0.97 82 81 78

118.98 120.79 83 52 47 45 1.53 0.8783 52 471.40 1.3983 C −0.2261 83 8284 83 820.1882 121.42 119.02 84 83 52 47 176.93 178.2284 83 1.08 1.0784 H

85 C 120.17−0.0732 120.00 85 83 52 47 −0.89 −0.7385 83 1.40 1.38 85 83 82118.40 119.80 86 85 83 52 177.00 178.5686 85 8386 8586 H 0 1301 1.071.08


Table 5Energetic characteristics of Congo red, Evans blue and Trypan blue monomers as found using HF/3-21G* and Weiner ‘91 methodsand discover force-field (in italics)a

Energy (kcal/mol) CR cis−CR trans+ EB trans+ EB cis− TB trans+

−2781.677 −4246.195HF/3-21G* (Hartree) −4246.195−2781.677 −4246.160−21.512Weiner’91 total −21.314 −44.502 −44.485 −90.514

321.474321.9235.183 50.816Weiner’91internal 50.84135.060 44.111

130.084131.8820.000Weiner’91 hbond 0.000 3.464 3.463 −4.321

**24.705 52.946Weiner’91 vdW 52.93124.765 −102.542

202.834201.267−81.202 −151.729Weiner’91 electrostat −151.731−81.343 −32.293−11.714−11.757

Dipole moment (D)10.445 3.5134.610 7.647HF/3-21G* 4.476

4.904Weiner ‘91 11.095 3.657 7.496 4.396

a Discover force-field does not distinguish the h-bond component.

Table 6

CR MQTransition (kcal/mol) CR MM EB MQ EB MM

trans+–trans− 4.531 6.483 4.838 7.8586.015 4.5874.273 7.384trans−–trans+

cis+–cis− 4.355 6.076 4.536 7.4196.510 4.8064.600 7.895cis−–cis−

3.041 1.098trans+–cis+ 2.7911.2432.389 0.8720.866 2.299cis+–trans+

2.594 0.847 2.316trans−–cis− 0.9982.843 1.123 2.7751.123cis−–trans−

the rotation on the bond placed between two benzenerings in the benzidine fragment.

The partial charge distributions for all moleculeswere calculated, using the RESP method in the AM-BER 4.1 program (Cornell et al., 1993; Cieplak et al.,1995). This method searches for the partial charges,that represent the best fit to the quantum-mechanicalelectrostatic potential around the molecule. The targetelectrostatic potential is the one defined on surfacelayers for distances which are 1.4, 1.6, 1.8 and 2.0multiples of the van der Waals radii, which have beenfound to best represent the intermolecular interactions(Bondi, 1964). The electrostatic potential values in eachgrid point, were calculated according to the Hartree–Fock wave function (631G (d)) (Krishnan et al., 1980;Frisch et al., 1984) in the Gaussian 94 program. Multi-conformational ( cis and trans isomers) (Bayly et al.,1993) 1° fit was applied to the Congo red molecule(Cornell et al., 1993). One-conformational (trans) 2° fit

was applied to the Evans blue calculation, aimed at thesymmetrical distribution of the partial charges on thehydrogen atoms in the methyl groups. The Trypan bluepartial charge distribution, was found after one-confor-mational (trans) 1° fitting.

Other fitting procedures were used to calculate thepartial charge distribution for the trans conformationof Congo red to point out the influence of and depen-dence on, the method used: Mulliken (Mulliken, 1955),ChelpG (Breneman and Wiberg, 1990) or Merz–Koll-man (HF/6-31G (d) (Besler et al., 1990)). The results ofthe last two methods agreed with those of the electro-static potential fit procedure.

The partial charge distributions obtained using theRESP procedure (Bayly et al., 1993) were taken forfurther calculation of the supramolecular system andmolecular dynamics simulation (Skowronek et al.,1999). This was done because of the need for compati-bility with AMBER 4.1 force-field parameterization.


Weiner ‘91 force-field parameterization (Weineret al. 1984, 1986) extended by the parameters pre-sented in the paper, discussing Congo red’s inter-action with cellulose (Woodcock et al. 1995), weretaken for further calculations. Weiner ‘91 force-fieldwas used to relax the structure of all the discussedmonomers, by the vacuum energy minimization proce-

dure: Ten steps of steepest descent, followed bythe conjugend gradient method until the energy gradi-ent value was B0.001 kcal/mol. The structures ob-tained after this procedure were taken for furthercalculations.

The torsional potential for rotation on the centralinterbenzene rings bond, was calculated additionally for

Figure 4. Dipole orientation and optimal structures (a) Congo red trans+ and cis ; (b) Evans blue trans and cis ; (c)optimal structureof Trypan blue trans+.


Congo red and Evans blue using the sameparameterization.

The optimal structure and partial charge distributionwere also calculated, using the programs in the InsightII package. The standard parameterization of cvff wasused for calculation of the optimal structure of Congored.

3. Results and discussion

3.1. Structure of monomer Congo red, E6ans blue andTrypan blue

The optimal structures of Congo red, Evans blue andTrypan blue in monomer form were calculated, usingboth quantum mechanics (HF/3-21G*) and molecularmechanics (Weiner ‘91). Additionally, discover force-fields (cff91, cvff, esff) were used to calculate the ge-ometry and partial charge distribution of Congo red.The results are shown in Table 1. Fig. 1 shows thedifferences between the bond lengths, inter-bond anglesand dihedral angles obtained, using quantum mechan-ics and molecular mechanics calculation approaches.

The differences in bond lengths did not appear to be\0.04 A, , with the exception of bonds influenced by thesulfonic groups and di-azo bonds, which differed morethan 0.06 A, . The discrepancies between angles werelower than 4.0°, with the exception of :8° differencesfor di-azo bonds and :7° for sulfonic groups. Thedihedral angle differences were negligible, except forthose of the freely rotating H atoms in the OH groups.The differences found between the dihedral angles inthe di-azo groups were B15°.

The dihedral angle between two phenyl rings, mostlyaffects the planarity of the molecule. The torsionalpotential (Fig. 2) of this rotation was calculated indetail, to examine the possibility of cis− trans+ confor-mational changes. Two methods were used to evaluatethe energy function related to this rotation: quantummechanics (HF/3-21G*) and molecular mechanics(Weiner ‘91) (Fig. 2.). The energy profiles appearedsimilar for Congo red and Evans blue. The relativeenergy barriers were reached for the common values ofthe dihedral angles for both analyzed dyes. The minimaof torsional potential differed about 15° between theresults from the quantum mechanics and molecularmechanics methods and they ranged from 36 to 48°(cis) or 128–143° (trans) on an absolute scale.

3.2. Partial charge distribution on Congo red, E6ans blueand Trypan blue

The distributions of the partial charges on the atomsin all the analyzed molecules in the trans+ conforma-tion were calculated using the Weiner ‘91 and HF/3-

21G* methods. Some other methods were also used forthe same purpose: Mulliken, ChelpG, Merz–Kollmanand Resp (Bayly et al., 1993). Differences between theobtained results, using these programs, can be seen inFig. 3. Three Insight II procedures for partial chargesdetermination (cvff, cff91 and esif) (Insight II,1995)were used additionally to determine the partialcharges on Congo red (Table 2).

The results indicated that the symmetry of the partialcharge distributions, agreed with the symmetry of thedye molecules. All of the methods attributed the highestpositive charge to the sulfur atoms. The lowest negativecharge was found on the oxygen atoms of the sulfonicgroups and the nitrogen atoms of the amino groups.Low differences, between partial charge distributionswere obtained, using the Resp and Merz–Kollmanmethods, probably due to similarities in electrostaticfield grid construction. The most significant discrepan-cies were found between the results obtained with theResp and Mulliken methods (Fig. 3 a1).

The data obtained with the Resp procedure werefinally taken for further calculations, to guarantee com-patibility with Amber 4.1 force-field, which was thenused to simulate the molecular dynamics.

The Resp procedure was also adopted to calculatethe partial charge distributions for the cis and transconformations of the Congo red molecule (Fig. 3 a2).Very low differences (B0.04 ecu) were found betweenthe partial charges calculated for these twoconformations.

The optimal structures of Congo red, Evans blue andTrypan blue were calculated, using the Resp procedure.Multi-conformational 1° fit was applied to the Congored molecule, to unify the partial charge distributionsfor the trans and cis isomers (Table 2) (Bayly et al.,1993; Cornell et al., 1993). 2° fit (Cornell et al., 1993)was applied to the Evans blue molecule (Table 3). TheTrypan blue trans conformation partial charge distribu-tion, was found after single-conformational 2° fitting(Cornell et al., 1993) (Table 4).

Dipole moments were calculated for the cis− andtrans+ conformations of Congo red and Evans blueand the trans+ conformation of Trypan blue, to char-acterize the molecule (Tables 5 and 6). The twice-highervalues of the dipole moment for cis, most likely derivefrom the symmetry of the molecule and the non planar-ity of the diphenyl fragment. The dipole moment in thecis− conformation, was found to be parallel to theaveraged plane of the phenyl rings. For trans+ it isperpendicular to the averaged plane of the diphenylrings (Fig. 4). The dipole moment orientation is just theopposite in Evans blue: while it is oriented parallel tothe averaged diphenyl plane in the trans conformation,it is perpendicular in the cis conformation.

The characteristics of the monomer structures ofCongo red, Evans blue and Trypan blue are presentedin Table 5. The vdW components of Evans blue and


Trypan blue dyes were found to differ significantly,even though they are isomers. This apparentdiscrepancy is likely caused by the attractive 1-4 vdWinteraction between the sulfonic and di–azo groups,which happens to occur only in Trypan blue.

The final structures of Congo red, Evans blue andTrypan blue together with a schematic presentation oftheir dipole moment orientations, are seen in Fig. 4.

3.3. Comparison of the monomer structures of Congored, E6ans blue and Trypan blue

The structures of Congo red, Evans blue and Trypanblue molecules, obtained using both quantummechanics and molecular mechanics methods, arecharacterized by inverse symmetry for trans andtwo-fold symmetry for cis. Pseudo-symmetry appearedin Trypan blue, whose two symmetrical halves differedin the dihedral angles on the di-azo bonds.

The torsional angle between two phenyl rings in thetrans+ conformation of the dyes, which was found tobe 48.35° for quantum mechanical methods and 36.23°for molecular mechanics in a vacuum, agrees well withthe experimental data (42–45°) (Adrian, 1958; Schmidand Brosa, 1972) and with other quantum mechanicscalculations (ab initio 47.1°) (Penner, 1986; Bastianenand Samdal, 1985). This angle can be as low as 20–25°,as reported for soluted and molten forms of diphenyl(Schmid and Brosa, 1972) and even 0 for its crystalversion (Trotter 1961; Hargreaves and Rizvi, 1962). Theplanar form of diphenyl in the crystal, may be causedby intermolecular interaction. It could be the result ofdense packing in the crystal cell. A correspondingsituation was observed in Congo red crystal, where oneof the three molecules present in the cell is planar whilethe other two are rotated (25°) (Ojala et al., 1995). Thefinding of rather low energy barriers for the rotation onthe central bond, additionally confirms this phenomena.The relatively low energy barrier between trans and cissuggests that both may be expected to occur finally insolvent conditions.

The presence of polar groups (sulfonic and amino) isthe main reason for the high dipole moment. In Congored, the dipole moment is oriented co-planarly with theaveraged diphenyl plane of cis and perpendicularly inits trans version.

The particular structural attribute differentiatingTrypan blue from Congo red and Evans blue, basicallyconcerns the internal planarity of the symmetrical halfof the molecule. The displanarity (non planarity) ofTrypan blue is likely caused by strong attractive 1-4vdW interaction, between the sulfonic and di-azogroups. The dihedral angle on the di-azo bond is veryclose to 0° in Congo red and Evans blue, while inTrypan blue it reaches 5–6°. The specificity of thisinteraction is important in the molecular mechanics

approach. It influences the vdW component of Trypanblue (Table 5) and is probably also responsible for itsnon symmetry, while according to the calculations,both Congo red and Evans blue represent fullysymmetrical structures.

4. Conclusions

Congo red and two related isomeric dyes, Evans blueand Trypan blue, were selected for studies, intended tofind out why they differ in properties including self-as-sembly and interaction with proteins (Skowronek et al.,1998).

It is becoming increasingly evident that Congo redand some other similar dyes which form chromonic,ribbon-like micellar mesophases in water solutions(Attwood et al., 1990) may interact with proteins assingle ligands, even while preserving their supramolecu-lar character in complexation (Attwood et al., 1990;Roterman et al. 1993; Stopa et al., 1997; Stopa et al.1998; Skowronek et al. 1998). The binding target ofthese dyes, is usually correlated with proteins rich in3b-conformation, particularly when these proteins be-come accessible to penetration by such a large ligand(Roterman et al. 1993; Piekarska et al., 1996; Stopa etal. 1997; Roterman et al. 1998; Skowronek et al. 1998;Stopa et al. 1998). Two isomeric dyes, Evans blue andTrypan blue, whose reactivity as protein ligands differssignificantly, were chosen for structural studies designto find the reason for those differences in behavior.Earlier experimental findings show that, while Evansblue dye molecules form relatively stable mixed micellarorganizations with Congo red and while both mayinteract with proteins in combined form, Trypan blue ispoorly engaged in such complexation (Kaszuba et al.,1993; Roterman et al. 1993). The observed difference inreactivity results most probably from the specific loca-tion of the sulfonic group in the Trypan blue molecule.The close proximity of the charged sulfonic group tothe di-azo group, polarizes them and introduces somenon planarity to this region of the molecule, as a resultof sub-sequenced extorted rotation. The resulting nonplanarity and sulfonic-group-derived charge located incentral region of the Trypan blue molecule disturbsselfassembling and inhibits its reactivity with proteinsas well. The findings support an earlier suggestion thatthe unique supramolecular organization represented byribbon-like micellar entities may give raise to the abilityof the ligands to bind to proteins rich in b-conforma-tion (Stopa et al. 1998).

The calculations in this work were done with variouswidely used programs, designed for molecular model-ing. Different approaches to quantum chemistry calcu-lations and different force-fields were used for the sameobject of analysis to find the reliable form of structure.


The obtained results seemed coherent, in despite of thedifferent parameterizations and program used.

Particularly suggestive, is the similarity between thedata obtained for supramolecular systems with the dis-cover and amber force-fields-programs with obviousdifferences in parameterization. This further argues forthe authenticty of the data.

Although the details of the mechanism of dye-proteincomplexation still elude a final explanation, there ismore and more support for the suggestion that thetightly associated ribbon-like micellar entities of bis–azo dyes represent the specific supramolecular organiza-tion responsible for ligation properties.

Independent studies concerning micelle formation bythe analyzed dyes will be published elsewhere, soon(Skowronek et al. 1999).

Acknowledgements

The authors are grateful to Magdalena Pogonowska,MD, of the ‘Medicus] Polish-American educationalfoundation, for obtaining professional literature for theInstitute of medical biochemistry, Collegium Medicum,Jagiellonian University in Krakow. Many thanks toProfessor Lucjan Piela of Warsaw University for criti-cal comments. Michael Jacobs helped edit themanuscrpt. Thanks to Ms. Anna Zaremba-Smietanskafor technical support. This work was supported byKBN POLAND (research grant 6P04A00211).

References

Adrian, F.J., 1958. J. Chem. Phys. 247, 606–616.Attwood, T.K., Lydon, J.E., Hall, C., Tiddy, G.I.T., 1990.

Liquid Cryst. 7, 657–668.Bastianen, O., Samdal, S., 1985. J. Mol. Struct. 128, 115–125.Bayly, C.I., Cieplak, P., Cornell, W.D., Kollman, P.A., 1993.

J. Phys. Chem. 97, 10269–10274.Besler, B.H., Merz, K.M., Kollman, P.A., 1990. J. Comp. Chem.

11, 431–437.Binkley, J.S., Pople, J.A., Hehre, W.J., 1980. J. Am. Chem. Soc.

102, 939–947.Bondi, A., 1964. J. Phys. Chem. 68, 441–451.Breneman, C.M., Wiberg, K.B., 1990. J. Comp. Chem. 11,

361–372.Burgevin, M.-C., Passat, M., Daniel, N., Capet, M., Doble, A.,

1994. Neurochemistry 5, 2429–2432.Caughey, B., Race, R.E., 1992. J. Neurochem. 59, 768–771.Cieplak, P., Cornell, W.D., Bayly, C.I., Kollman, P.A., 1995.

J. Comp. Chem. 16, 1357–1377.Cornell, W.D., Cieplak, P., Bayly, C.I., Kollman, P.A., 1993.

J. Am. Chem. Soc. 115, 9620–9631.Demaimay, R., Harper, J., Gordon, H., et al., 1998. J. Neu-

rochem. 71, 2534–2541.

Frisch, M.J., Trucks, G.W., Schlegel, H.B., 1995. GAUSSIAN94 (Revision A.1). GAUSSIAN Inc, Pittsburgh, PA.

Frisch, M.J., Binkley, S.J., Pople, J.A., 1984. J. Chem. Phys. 80,3265–3271.

Gordon, M.S., Binkey, J.S., Pople, J.A., Pietro, J.A., Hehre,W.J., 1982. J. Am. Chem. Soc. 104, 2797–2803.

Hagler, A.T., Osguthorpe, D.J., Dauber-Osguthorpe, P.,Hemple, J.C., 1985. Science 227, 1309–1315.

Hagler, A.T., Stern, P.S., Sharon, R., Becker, J.M., Naider, F.,1979. J. Am. Chem. Soc. 101, 6842–6852.

Hargreaves, E.D., Rizvi, H.S., 1962. Acta Cryst. 15, 365–372.Insight II, 1995. User Guide, October 1995, San Diego: Biosym/

MSI.Jelinek, Z.K., 1970. Particle size analysis. Wiley, New York, pp.

100–101.Kaszuba, J., Konieczny, L., Piekarska, B., Roterman, I., Ry-

barska, J., 1993. J. Physiol. Pharmacol. 44, 231–242.Konieczny, L., Piekarska, B., Rybarska, J., et al., 1997. Folia.

Histochem. Cytobiol. 35, 203–210.Krishnan, R., Frisch, M.J., Pople, J.A., 1980. J. Chem. Phys.

72, 650–657.Maple, J.R., Thacher, T.S., Dinur, U., Hagler, A.T., 1990.

Chem. Des. Autom. News 5, 5–10.Mulliken, R.S., 1955. J. Chem. Phys. 23, 1846–1883.Ojala, W.H., Ojala, C.R., Gleason, W.B., 1995. Antiviral Chem.

Chemother. 6, 25–33.Pearlman, D.A., Case, D.A., Caldwell, J.C., et al., 1995.

AMBER 4.1. University of California, San Francisco.Penner, G.H., 1986. J. Mol. Struct. 137, 191–193.Piekarska, B., Skowronek, M., Rybarska, J., et al., 1996.

Biochimie 78, 183–189.Pietro, J.A., Francl, M.M., Hehre, W.J., deFrees, D.J., Pople,

J.A., Binkley, J.S., 1982. J. Am. Chem. Soc. 104, 5039–5048.Pollack, S.J., Sadler, I.I.J., HawLin, S.R., Tailor, V.J., Sherman,

M.S., 1995. Neurisci. Lett. 197, 211–214.Roterman, I., No, K.T., Piekarska, B., et al., 1993. J. Physiol.

Pharmacol. 44, 213–232.Roterman, I., Rybarska, J., Konieczny, L., et al., 1998. Comp.

Chem. 22, 61–70.Rybarska, J., Konieczny, L., Roterman, I., Piekarska, B., 1991.

Arch. Immunol. Ther. Exp. 39, 317–327.Sadler, I.I.J., Smith, D.W., Shearman, M.S., et al., 1995. Neuro.

Rep. 7, 49–53.Schmid, E.D., Brosa, B., 1972. J. Chem. Phys. 56, 6267–6268.Skowronek, M., Stopa, B., Konieczny, L., et al., 1998. Biopoly-

mers 46, 267–281.Skowronek, M., Roterman, I., Konieczny, L., Stopa, B., Ry-

barska, J., Piekarska, B., 1999, J. Comput. Chem., in press.Stopa, B., Konieczny, L., Piekarska, B., et al., 1997. Biochimie

79, 23–26.Stopa, B., Gorny, M., Konieczny, L., et al., 1998. Biochimie 80,

963–968.Trotter, J., 1961. Acta Cryst. 14, 1135–1140.Weiner, S.J., Kollman, P.A., Case, D.A., et al., 1984. J. Am.

Chem. Soc. 106, 765–787.Weiner, S.J., Kollman, P.A., Nguyen, D.T., Case, D.A., 1986.

J. Comp. Chem. 7, 230–252.Woodcock, S., Henrissat, B., Sugiyama, J., 1995. Biopolymers

36, 201–210.Zhen, W., Han, H., Anguiano, M., Lemere, C.A., Cho, C.-G.,

Lansbury, P.T.J., 1999. Med. Chem. 42 (202), 2805–2815.

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the conformational characteristics of congo red, evans blue and trypan blue

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