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Aggregation of acridine orange: Crystal structure of acridine orange tetrachlorozincate 2C17H19N3·2HCl·ZnCl2·CH3COOH
BIOIIVORGANK
CHEMISTRY
6,2944
(1976)
29
Aggregation of A&dine Orange: Crystal Structure of Acridine Orange Tetrachlorozincate ~C,,H,,N,-~HCI-ZUC~~-CH~COOH S. K. OBENDORF,? JENNY PICKWORTH HELEN M. BERMAN, and H. L. CARRELL
GLUSKER,*
*
PAUL R. HANSEN,
The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania I91 I I
ABSTRACT The crystal structure of the biological stain, “acridine orange,” has been determined. This compound, when crystallized from ethanol, is shown to be a zinc chloride double salt of acridine orange, containing, in addition, acetic acid of crystallization. These additional components are residuals from the method of acridine orange. This complex, 2 aaidine of preparation orange-2HCl.ZnCl, lCH,COOH, (2C, -,HX ,N, -2HC1-ZnCl,-CH,COOH) crystallizes in the monoclinic space group P2,, a = 9.965 (2), b = 21 SO7 (6), c = 9.645 (2) A, p = 113.98” (2), V = 1888.7 (8) A’, FW = 800.0, Z = 2, D, = I.41 g-cni3, Dabs = 1.43 (9) g.cni3 _Threedimensional diffraction data were collected with CuKa radiation, and the structure refined to R = 0.065 for 1885 observed reflections. In the crystal structure hydrogen bonds are formed,
INTRODUCTION Acridine orange is used as a biological stain for nucleic acids and polysaccharides. It also shows mutagenic and antibacterial activity_ The interaction of acridine derivatives, such as acridine orange, proflavine and 9-aminoacridine, with DNA has been studied extensively. There appear to be two main types of binding. One type, at low acridine concentrations, involves intercalation [l-3] _ The acridine derivative becomes insetted between the base
*From the Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111. This research was supported by grants CA-10925, CA-06927 and RR-05539 from the National Institutes of Health, U.S. Public Health Service, AG370 from the NationaI Science Foundation, and by an appropriation from the Commonwealth of Pennsylvania. j-Present address: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853. -&Towhom all correspondence and reprint requests should be addressed. 0 American Elsevier Publishing Company, Inc_, 1976
S_ K. OBENDORF,
30
et al.
vti the protonated nitrogen atom of the central rings of two a&dine orange cations, to two chloride ions in a Zncl, =- tetrahedral grouping_ These two acridine orange moIecules are stacked in parallel pIanes, approximately 3.4 A apart, with the long axes of the ring systems inclined at 26.S” to each other. Thus an apparent dime&z&ion of the acridine.orange is facilitated by the anions present, resulting in the complex studied. The two -N(CH,), groups of
each acridime orange molecule are not protonated in this crystalline form. The mode of molecular packing found here may be relevant to models for the external stacking of acridine orange around a DNA molecule_ The importance of
removing
any
zinc
salt from
acridine
orange
preparations
prior
to
aggregation studies is stressed.
pairs of DNA (a hydrophobic interaction perpendicular to the plane of the a&dine ring system), with an appropriate conformational adjustment of the nucleic acid to accommodate this additional (3-4 A thick) molecule. The other mode of interaction, occurring at high dye concentrations, is via external stacking (a hydrophilic interaction, probably in the plane of the acridine ring system)_ The central nitrogen atom of the a&dine ring system, which is protonated at neutral pH can hydrogen bond to a hydrogen bond acceptor (for an ionized phosphate group)_ This is the predominant mode of example, interaction at high concentrations of acridine derivatives, and may be important in their action as antibacterial agents, since they are active as bacteriostats only at pH vahres at which the central nitrogen atom is protonated [4, 51. EXPERIMENTAL
SECTION
The source of the compound studied was the commercial product, “_4cridihe Orange, Basic Orange 14, Bioiogical Stain” from Matheson, Coleman and Bell_ Crystals were grown from ethanol as needles from which a small single crystal was cut, size O-10 X 0.27 X 0,11 mm_ Cell data are given in Table 1. Intensity data were collected on an automated four circle diffractometer with monochromatized CuKa radiation with the 6-28 scan technique to a maximum sine/A = 0.61 cm-’ _ A total of 3495 reflections were scanned of which 1885 were above the threshhold vahre of 3oQ (where o(l) was determined from counting statistics)_ There was no intensity loss with time during the data collection_ Values of o(F) for observed data were calculated from the formula o(F) = (F/2) [ ]02(r)l12 J + ?j2 ] s where 6 is a measured instrumental uncertainty, 0.0207, determined from the variation of the intensities of periodically monitored check reflections‘. An X-ray absorption correction (,$CuKcr) = 36.3 cm-‘) was applied when the total contents of the crysta1 had been found. The density of the crystal, measured in dichioromethane and tetrachloromethylene, showed a huge experimental error, presumably because there were several components in the crystals with different sohrbilities in chlorinated hydrocarbons_ The structure was solved, with difficulty, by a combination of Patterson, Fourier and direct methods (using the program MULTAN [6]).The unexpected presence of zinc in the crystals was revealed in Fourier maps as a peak approximately 2.2 A from each of four cholride ions. Therefore analyses of
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
31
TABLE I Cell Data Formula: 2Cr 7Hr s N, l2HCl* ZnClz Formula weight: 800.0 Cell dimensions: a = 9.965 (2) A b = 21.507 (6) 9.645 (2) ;I 113.98 (2)O v= 1888.7 A3 Space group 1 Density:
P2r z=
calculated observed
lCHs COOH
2 1.41 g-cm3 1.43 (9) g- cmW3
crystals from the same batch were made. A spectroscopic assay of acridine orange in a solution of crystals (3.6 X 1f16 M acridine orange), complemented by an atomic absorption spectroscopic zinc determination (2.16 X lo* M Znl [7], showed that there were two a&dine orange molecules per zinc atom in the crystals, as found from the crystal structure. The zinc chloride present in these crystals had been used as a cataIyst in the preparation of acridine orange [5J and was obviously not removed in the subsequent recrystallization. The structure (consisting of acridine orange hydrochloride and zinc chloride) was refined, first by the interpretation of electron density maps, and then by full-matrix least-squares methods using UCLALS4 [83_ The weights during refmement were l/(02 (F)), with zero weight for those reflections below the threshhold values. The quantity minimized in the least squares calculation was The atomic scattering factors used were: Zn, International Co[llFol-IFc1112. Tables, Volume III [9] ; for Cl, 0, N, C, those of Cromer and Mann [lo] ; and for H, those of Stewart et al. [ 113. The anomalous dispersion corrections for Zn and Cl were those of Cromer and Liberman 1121. The refinement stopped at an R value of approximately a.1 O_ A difference map revealed four high peaks which were interpreted as acetic acid of also residual from the method of preparation_ The high crystallization, temperature factors of these atoms indicate that the acetic acid molecule is only loosely bound in the crystal lattice. Further refinement with isotropic temperature factors for the acetic acid atoms and ail hydrogen atoms included in their calculated positions gave the final R value of 0.065. Final parameters are listed in Table 2. A table of calculated and observed structure factors is available.’ Illustrations and molecular geometry calculations were done via the CRYSNET system [ 13 ] _ ‘Tables of FoFc values may be obtained from the senior author or the E&tcrial office of Bioinorganic Chemistry.
32
S_ K_ OBENDORF,
et aL
DISCUSSION MolecuIar Description The formula of the salt studied is:
There are two acridine orange cations, designated “A” and “B”, within each asymmetric unit of the crystal_ Coordinates are listed in Table 2. Estimated standard deviations for carbon, nitrogen, oxygen, and particularly hydrogen atom positions are high owin g to the presence of the heavier zinc and four chIoride ions in the structure. Therefore none of the differences in bond lengths between cations “A- and “B- (and between the two haIves of each cation) are considered significant and averaged distances and angles are shown in Fig. 1. The C-N bonds to the -N(CH& groups range from 130 to 1.39 (2) 8, indicative of much double bond character, as found in 9aminoacridine (C-N = 1.321 (3) A [141. The cations of both “A” and “B” are monoionized (pK, = 10.45, pKb = -3-15) with the proton located on the nitrogen atom of the central ring (N( 1 OA), N( 1 OB))_ These protons are involved in hydrogen bond formation to chloride ions in the ZnCb2- anion. One ZnC14 2- anion (at I-x, y-‘55, -2) forms hydrogen bonds to one of each “A” (at x, y. z) and ‘73” (at x, y, z-l) acridine orange cation_
H
H
H
H
H
H
H
FIG_ l_ Averaged angles and distances in acridine orange cations. Estimated standard deviationsare approximately0.02 A and 1”.
TABLE
2
Atomic Parametew Y
zn2* cl-( 1) Cl_(2) S(3) Cr(4) C(lA) C(2A) C(3A)
C(4A) C(sA) C(a) C(7A)
WA) C@A) N( 10A) C(llA) C(12A) C( 13A) C( 14A) N( 15A) C(l5A) C( 15’A) N(16A) C(l6A) C( 16’A) C(lB) C(2B) C(3B) C(4B) C(5B) C(6B) C(7B) C(8B) C(9B) N(lOB) C(lIB) C(l2B) C( 13B) C( 14B) N(l5B) C(15B) C( 1 S’B) N( 16B) C( 16B) C( 16’B)
0.7009( O-8906( O-7691( 0.5329( 0.5928(
2) 5) 5) 5) 6)
O-7541(18) O-8213(16) O-7906( 17) O-6734(16) 0.3!25( 19) O-2456( 18) 0.2757(19) 0.3771(21) 0.5735( 19) O-4996( 12) O-6095( 18) O-461-1( 18) O-6406( 17) O-4273( 19) O-8653( 13) O-9786( 17) O-8419(21) 0.1459(13) O-0676(26) 0. I 176(20) O-7444( 15) O-7682( 15) O-6993( 16) 0.5860(15) O-3287( 14) O-2959( 16) O-3803( 17) O-4760(16) 0.6123(16) 0.46X6(1 I) O-5607( 1.5) 0.5037( 15) O-6388(15) O-4303( 15) O-7258( 13) O-6567(20) O-8330(16) O-1825(13) 0.0909( 16) O-1529(19)
O-4839( 1) O-4183( 3) O-5756( 2) O-4439( 3) 0.5055< 3) O-2405( 8) O-1941( 8) O-1303( 8) 0.1158( 8) O-1721( 9) O-2209( 11) O-2851( 9) 0.3014(10) O-2726( 8) O-1494( 6) O-1634( 9) O-2565( 8) O-2262( 8) O-1944( 8) O.OSSl( 6) 0_1020(10) 0.0199(10) 0.2033( 8) O-2493( 15) 0.1379(14) O-1809( 9) O-1220( 9) 0.0711( 8) 0.0889( 7) O-2357( 7) O-2965( 8) O-3491( 8) O-3338( 7) 0.2593( 8) O-1642( 6) O-1482( 8) O-2750( 7) O-1987( 7) O-2243( 8) O-0131( 6) -0.0400( 10) -O-0032( 10) O-3088( 6) O-2598( 8) O-3729( 9)
z O-0774( 0.1231(
3) 7)
0.2059( 6) O-1.543( 6) .0_1766( 6) O-0716(21) 0.0313(19) 0.0430(19) 0.0858(20) O-2724(21) 0.3 194(22) 0.3 113(22) 0.2642(23) 0.1705(21) 0_1771(14) 0.1294(21) 0.2193(21) O-1226( 19) O-2257( 19) O.OOVO( 16) .O.OS 16(20) O-0286(28) 0.3785( 16) O-4245(28) 0.3991(25) 0_5591(19) O-5376(19) O-5735( 19) O-6304( 18) O-8002( 19) O-8240( 18) O-8003( 19) O-7387(18) O-6466( 19) 0.7 1 l6( 14) O-6496( 18) O-7053( 17) O-6239( 18) O-7364( 18) 0.5544( 16) 0.5879(22) O-4899( 19) O-8679( 16) O-8842(22) O-8964(24) 33
S:K. OBENDORF,
34
Bll ZI?’ cl-( l_) cl-(z) a-(3) a-(4) C
C(9B) N( IOB) C(llB) C(12B) C( 13B) C( 14B) N(i5B) C( 1 SB) C(15’B) N( 16B)
4_4q 1) 6.15( 2) 6.36( 1) 5.03( 1) 12.13( 3) 7.29( 7) 6.10( 6) 6.94( 6) 2.31( 6) 3.99( 8) 2.56( 6) 3_95( 7) 8.55( 9) 4.85( 9) 5.39( 5) l-41( 7) 6_69( 6) 3.86( 7) 4_25( 7) S-23( 5) 2.02( 6) 9.36( 9) 4_70( 5) 12.37( 12) 5.58( 8) 3.22( 6) 2_89( 5) 4.56( 6) 5.53( 6) 2.58( 5) 3.43( 5) 2.22( 6) 5.46( 6) 6.60( 6) 2.37( 4) 4.7q 5) 3_91( 6) 5.56( 6) 3_09( 5) S-28( 5) 12.38( 8) S-15( 6) 5.28( 5)
B22
3.20( I) 5.51( 2) 4.4q 2) 5.90( 2) 4.62( 2) 3_6f( 8) 4_81( 8) 2.94( 8) 3.66( 8) 6.25( IO) 9_53( 13) 6.46( IO) 4_42( 9) 3.96( 8) 2.92( 5) 4.66( 8) 3_07( 8) 3.55( 7) -4.09( 8) 3_46( 6) 3.88( 13) 5.66( 11) 8.21( 9) 16.76(21) 12.54(18) 5.86( 9) 8_55( 10) 5.73( 8) 3.83( 7) 4.18( 7) 4.31( 7) 3.64( 8) 3.14( 7) 4.66( 7) 4.27( 5) 4.44( 9) 3.31( 7) 2.94( 6) 4_OL( 7) 5_98( 7) 7.16(11) 9.56( 12) 3.96( 6)
B 33 4.86( 1) 11.34( 3) 7.70( 2) 7.67( 2) 5.42( 2) 5.18( 9) 4.42( 7) 3.97( 7) 3_75( 8) 1_25( 9) 3.24( 9) 4_94( 9) 5.04(11) S-15( 9) 1.97( 5) 4.42( 8) 3.24( 8) 4.08( 7) 2.42( 7) 4.36( 6) 2.8q 8) 11.18(12) 3.80( 6) 6.72( 9) 4.14( 11) 3.85( 8) 4.68( 8) 3.29( 7) 4.10( 7) 3.64( 8) 3.18( 7) 3.93( 9) 3.43( 7) 5_02( 8) 3.94( 5) 3.93( 7) 4.58( 7) 4.53( 7) 2.58( 7) 6_5q 7) S-88( 9) 7.23( 8) 7.13( 7)
et aL
B12
O.ll( l-87( -O-63( -l-15( O-83( -l-17( -l-61( 0.32( 0.27( O-14( O-57( 0.70( 1.39( -0.47( -0.79( 0.38( 0.14( -O-13( 0.54(
J313
1) 2) 1) I) 2) 6) 6) 7) 6) 8) 8) 7) 8) 7) 4) 7) 6) 6) 7)
0.04( 5) -O-16( 8) 3.29( 9) -O-43( 6) O.OO( 15) -0.54(1 I) - 1.92( 6) -1.06( 7) -O-08( 7) -O-95( 5) -O.Ol( 5) 0.21( 5) -0.99( 6) -O-58( 6) -l.OO( 6) -O-52( 4) -0.99( 6) 0.33( 5) -l-17( 5) -0.98( 5) 0.22( 5) - l-84( 9) O.S7( 7) -0.18( 5)
2.6q 1) 4.20( 3) 4_41( 2) 3_46( 2) 4.33( 3) 3.53( 9) 3.27( 7) 334( 8) 1.18( 8) -O-35( 9) 1.21( 8) 1_50( 9) 3.72(10) 1.95( 9) 0.73( 6) 0.61( 8) 1.80( 8) l-08( 8) 0.99( 8) 1.57( 6) l-03( 8) 7_37( 14) l-25( 6) 7_14(14) 2.12(11) O-42( 7) l.OO( 7) l-43( 6) 2.99( 8) O-81( 7) 0.33( 7) -l-71( 7) 1.16( 7) 2.24( 8) l-27( 5) 1.72( 7) l-20( 7) 2.37( 7) -O-25( 6) 3.07( 7) 6.34(2 I) 3.09( 9) 3.29( 7)
B23
-O-06( 1) 0.24( 2) -l-79( 2) -O-19( 2) -O-08( 2) O.lO( 7) 0.07( 7) 1.02( 7) 0.38( 7) 0.31( 9) -0.10(10) -l-64( 8) 0.32( 10) O.ll( 8) 0.77( 5) O-76( 8) O-11( 7) -0.03( 6) O-08( 7) O.ll( 6) O-08( 10) l-38(11) -l-33( 7) - 1.07( 14) -0.17(13) O-25( 8) O-63( 8) l-62( 8) O-44( 7) 0.41( 7) 0.30( 7) -O-16( 7) 1.40( 6) 1.35( 7) O.OS( 5) 0.44( 7) 1.59( 6) -0.81( 6) 0.28( 6) 0.60( 6) -O-83( 9) -I.79(10) -0.80( 6)
ACRIDINE ORANGE TETRACHLOROZINCATE
C(16B) C( 16’B)
3.97( 6) 7.28( 8)
3.72( 7) 4.72( 8) x
0(1X)
0(2X) C(3X) C(4X) H(IA) H(2A) ‘H(4A) H(5A) H(7A) H(SA) H(9A) H( 1OA) H(lSA1) H( 15A2) H( lSA3) H( 15’Al) H( 15’A2) H( lS’A3) H( 16Al) H( 16A2) H(16A3) H(16’Al) H( 16’A2) H( 16’A3) H(W H(2B) H(4B) H(5B) U(7B) H(SB) H(9B) H( 10B) H(15Bl) H( 15X32) H(15B3) H(lS’B1) H(15’B2) H( 15’B3) H(16Bl) H( 16B2)
0.851( 2) O-814( 2) 0.802( 3) 0.784( 3) O-783( 15) 0.896( 15) O-639( 15) O-285(25) O-220(15) 0.398(15) O-604(15) O-471(15) l-074(15) O-943( 15) O-993(15) O-742( 15) 0_857(15) O-920( 15) -0.024(15) O-127(15) O-041( 15) 0.031(15) 0.203( 15) O.OSS(15) 0.799(15) 0.84q15) 0.528( 15) O-280( 15) O-368(15) O-531(15) O-668(15) 0.41q15) O-637( 15) O-562(15) O-723( 15) 0.81 l(15) O-933(15) 0.838(15) 0.034( 15) 0.019(15)
5_76( 9) 11.75(12)
- 1.77( 6) -O-69( 7) Y
O-371( I) O-471( 1) 0.410( 2) 0.399( 1) O-285( 9) 0.205( 9) 0.072( 9) O-127( 9) O-318( 9) O-347( 9) O-317( 9) 0.105( 9) O.OSl( 9) O_OSS( 9) O-148( 9) O-012( 9) -O-006( 9) O-012( 9) 0.230( 9) O-264( 9) 0.28r;( 9) 0.124( 9) O.l?l( 9) O-132( 9) O-214( 9) O-113( 9) O-056( 9) O-200( 9) O-393( 9) O-369( 9) O-293( 9) O-130( 9) -O-031( 9) -O-049( 9) -O-077( 9) 0.024( 9) O-007( 9) -O-047( 9) O-276( 9) 0.245( 9)
35
STRUCTURE
l-73( 9) 5.17(10)
-1.23( -4.03(
8) 9)
t
B
O-487( 2) 0.578( 2) 0.553( 3) 0.692( 3) 0.066( 18) -0.009( 18) O-084( 18) O-271( 18) O-341(18) O-259( 18) O-171(18) O-177(18) 0.01 l(18) -0.160(18) - 0.046( 18) 0.025( 18) -0.050(18) O-132(18) O-424( 18) 0.531(18) O-354( 18) 0.308( 18) 0.413(18) O-486( 18) 0.531(18) O-493( 18) 0.654( 18) O-829( 18) O-528( 18) O-716(18) O-623( 18) O-740( 18) O-680(18) O-500( 18) O-609( 18) O-399( 18) O-568( 18) O-457( 18) O-941( 18) 0.782( 18)
17.4( 5) 18.4( 5) 15.2( 8) 13.8( 8) 4.2( 10) 3.9(10) 3.7(10) 4.9( 10) 4.5( 10) 5.0(10) 4.3( 10) 3.3( 10) 4.5( 10) 4.5( 10) 4.5( 10) 5.5( 10) 5.5( 10) 5.5( 10) 7_6( 10) 7_6(10) 7.6( 10) 6.8( 10) 6.8( 10) 6.8( 10) 4.5( 10) 5.2(10) 3.6( 10) 3.7(10) 4.6( 10) 4.1(10) 4.3(10) 3.7( 10) 7.1(10) 7.1(10) 7.1(10) 6.0( 10) 6.0( 10) 6.0( 10) 5.0(10) 5.0(10)
36
S. K. OBENDORF,
et ai.
H( i6333)
O_i52(15)
0.224(
H(I6’Bl) H( I6%2) H( 16’B3)
0.124(15) O-242( 15) 0.069( 15)
0.374( 9) O-399( 9) O-390( 9)
9)
0.941(18)
5.0(10)
0.985(18) 0.920( 18) O-807( 18)
5.6( 10) 5.6(10) 5.6(10)
uPositionalparametersare givenas fractionsof cell edges Anisotropictemperaturefactors are expressedas exp [-%(hz a*‘B, I + k* b*’ Bz 1 + Q*c*‘B, 3 + 2hka*b*B, 2 + 2hPa*c*!3, 3 + 2kQb*c?B, 3)J and isotropictemperaturefactorsas exp (-B sit?~/h*) with B valuesgiven in A* _ The standarddeviationsfor each parameter,determinedfrom the invertedfull matrix, and given in parentheses,apply to the last specified digits. Hydrogenatom positionsare calculated,and, in the cese of methyl groups,give the best fit to a difference map_ Values for esd’s for hydrogen atoms are based on averageisotropic refmement results.Temperaturefactors for hydrogenatoms are based on those of the atom to which-theyare attached_
(a)
FIG. 2. Acridine orange-zincchloride complex. Space-f3ug diagramsof the complex. The stippled areas are representativeof the van der Waals radii of atoms. (a) View onto the cl(z)-Zn*+-CI(4) pIane; (b) View along the planesof the aaidine orangecations.
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
37
TABLE 3 (a)
(b)
Angles between rings Definitions AI AII AH1 BI BII BHI
attached contains attached attached contains attached
Angles between planes AIrAII lSO AII:AIII l-l0 ALAI11 0.6O
BIB11 BII:BIII BI:BIII
Equations in A with respect Molecule A 3.36177 x - 1.25393 y -2.70144x -20.68579~ 898342 x - 5.75 124 y
to N( 15A) N( 10A) to N(16A) to N( 15B) N( 1OB) to N( 16B)
0.8O 1.6” 2.3O
to fractional coordinates x, y, and z + 6.95756 z - 2.74442 = X, + 1.39447 z + 5.51491 = YA - 6.53252
z - 2.44724
= ZA
Molecule B 3.65805 x - 1.2911Oy + 6-74123 z - 6-27654 = XB -6.35191x-15.97997~ i-4-296602 +3.83416 =YB 6.75079 x - 14.33613 y - 5.39638 z + 3.08480 = Zn (c)
Angles between principal axes of of moIecuIes A and B 1.8O Angle between 26.5O Angle between 26.5” Angle between
best planes through three rings molecular planes 3-4 A apart intermediate axes of molecules long axes of molecules
The three carbon atoms attached to each nitrogen atom in the -N(CHs)z groups, i-e_, attached to N( 1 SA), N( 16A), N( 15B), and N( 16B) are coplanar with the nitrogen atom Therefore these nitrogen atoms do not have an additional proton on them since this should cause a tetrahedral arrangement to occur. (In a crystalline form of proflavine hydrochloride studied in this laboratory one amino group had an additional proton attached to it in a tetrahedral arrangement [ 151.) It is, however, probable that the positive charge on the a&dine orange cation is distributed among the three nitrogen atoms (N(LO), N( 15), N( 16)). The -N(CH& groups are slightly tilted with respect to the plane of the rest of the ring system These tilts are 5.5O, 5.3O, 2.1°, and 6.9” for the groups including N( 15A), N( 16A), N( 15B), and N( 16B), respectively_ The zmc chloride-acridine orange complex is illustrated in Fig. 2. As shown iu Table 3 and Fig. 2b, each cation is approximately planar, the maximum
38
S. K_ OBENDOBF,
et al_
deviation between rings being 2-3&_ This planarity seems important for intercalation in DNA as shown by the crystal study of the ApU: Paminoacridine complex [ 161 and the f-iodo UpA: ethidium complex [ 171_ Molecular Packing The overlap of the ring systems of acrldine orange lying in parallel planes approximately 3-45 A apart, is shown in Fig. 3a and b. The cations stack throughout the crystal (Fig. 3b). The long axes of the acridine orange cations are not parallel but inclined at 26.S” to each other- In the crystal structures of proflavine hydrochloride crystals [ 1S] , 9-aminoacridine hydrochloride [ 141 and acridine [ 18, 191, with no complex anions present, the ring systems lie in paraBe planes approximately 3.4 A apart but with their long axes parallel. This rotation of approximately 3Op in the angle between the long axes of the ring systems is probably caused by the presence of the ZnCl~*- complex anion. In proflavine hemisulpbate [ 20,211 a similar situation occurs with the long axes of the cations Inclined at 150’ [= 18Q30°) to each other. A comparison of the stacking in the ZnC&‘- and the SOe2- complexes is shown in Fig_ 3a vs. c. The sobent of crystallization, presumed to be acetic acid, fits in a pocket between the chloride ions, the methyl groups, and the more hydrophobic regions of the a&dine orange molecules. The temperature factors for atoms in this group are high indicating considerable disorder_ Zinc Interactions One of the principal points of interest in this crystal structure is the interaction of the tetrachlorozincate anions with the aromatic acridine orange cations. A&dine orange has been shown to dimerize in solution [22, 231 and the possible participation of anions in this dimerization was noted by Lamm and Neville 1241. As shown in Table 4 and Fig. 2 the ZnCb2- anion forms hydrogen bonds to two acridine orange cations (“A” and “B”)_ Thus effectively a complex has been formed which involves a dimer of a&dine orange. McGinnety [ 25 ] found that in a ZnCb*- anion, in the absence of any “crystal forces” a value of 2.294 A would be expected for the Zn*“. - --Cl- distance. Deviations from this distance, and from true tetrahedral symmetry, are given in Table 4. Taylor and McCall [26-281 studied some zinc chloride complexes of nucleic acid bases They found zinc ions coordinated to ring nitrogen atoms of the bases and, via coordinated chloride ions or water molecules, to keto or amino groups on the bases. Stacking of Acridine Orange in the Presence of Anions In the ZnC1,*- anion the Cl---*Cl distances are approximately 3.7 A_ This distance is similar to the thickness of the aromatic ring system of acridine orange_ In this crystal structure the cations pack in parallel planes approximately 3.45 A apart with no differentiation in distance apart of the pairs of cations that complex to ZnCb *-.
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
39
FIG. 3. Comparisons of stacking in acridie orange tetrachlorozincate (this work) and proflavine hemisutphate [20, 211. Views onto the molecular planes. The proflavine cation bas been numbered to conform with the orientation for acridme orange. Molecule A is darkened. In the proflavine salt 0 denotes oxygen of sulphate, W denotes water, both hydrogen bonded to -NH groups. (a) Acridiie orange tetrachlorozincate. 26.5” between rings; (b) Acridine orange tetrachlorozincate. Stacking through the crystal; (c) Proflavine hemisulphate. 150” between rings.
S. K. OBENDORF,
40
et al.
TABLE 4 Molecular Packing (a)
Hydrogen bondsa D-H----.A
H . . . ..A
N( 1OA)‘-H( 1OA)“. Cl(4) N(lOB)‘-H(1OB)’ ---=C1(2) l
(b)
l
D-H-*.=-A 180° 161°
2.231( 2.36
l
3322
ZnC!4 ‘- compIex distance A zncq 1) CK2) Cl(3) CY4)
2.254 2.281 2.259 2.288
* - 0.040 -0.013 - 0.035 - 0.006
*Variation from 2.294 A (McGinnety)
C1( I)**= +X(2) CKf) ----Cl(3) Ci(I)---•l(4) C&2) ----C1(3) C1(2)* - lCl(4) CK3) ---X1(4) l
tVariation (c)
distance A
Angle at Zn2+
3.786 3.738 3.702 3.586 3.702 3.721
113.2 111.8 109.2 104.3 108.2 109.9
ii-3.7 +2.3 -0.3 -5.2 -1.3 +0_4
from 109.5”
ZnCtr *= - - - acridine orange A
B
Zn2+ Zn2+
CX2) C](4)
C N( 10B)’ N( IOA)”
A-B
B-C
A-C
ABC
2.28111 2.288
3.322A 3.227
4.948A 4.323
122.9O 101.9O
‘l-x yrty I-t ‘Cl-x IA+v -2 .. @Hydrogen atoms are in calculated positions_ The structure of proflavine hemisulphate [20,21 J shows hydrogen bonding of the sulphate groups to proflavine molecules. The S-0 distance is 1.40 5 so that O-O distances are approximately 2.40 A within one anion. This distance is not sufficient to span the 3.4 h distance between stacked proflavine molecuIes and therefore the proflavine molecules that are found to stack in the crystal are hydrogen-bonded to different sulphate groups.
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
41
Blears and Danyluk [29] report a greatly increased amount of stacking in a&dine orange on adding sodium chloride_ This may be due to complex formation with, for example, hydrated sodium ions or with chloride ions. In acid soiution disaggregation occurs, presumably due to the formation of non planar ,C& -NHqH groups (cf_ the nonstacking of -&Ha groups in proflavine I1 51). The l&R results of Blears and Danyluk [29] are consistent with the stacking of the ring systems in planes 3.4 A apart. They found that, in solution, protons on C(4) and C(5) are oriented more nearly over the centers of adjacent rings than are the other ring protons. In the crystal structure reported here, the protons on C( 1B) and C(5B) lie on either side of the center of cation “A” (see Fig. 3a).
Model for Binding of Acridine Orange to DNA The structure of acridine orange tetrachlorozincate is a model of the situation that may occur when acridine molecules bind externally to a DNA molecule_ Therefore, as shown in Fig. 4, the stacking found in the acridine orange tetrachlorozincate structure, with an angle of 26.5* between the long axes of the cations “A” and “B,” was continued so that a helical arrangement was generated_ This model contained between 13 and 14 cations per revolution_ The central nitrogen atoms of the rings (N( 10)) were approximately 6 A from the helical axis. Hypothetical water molecules, hydrogen bonded to these nitrogen atoms and in the plane of the acridine ring system, Iay 4 A from the helical axis. Thus it is possible for acridine orange molecules to hydrogen bond (vin water or
FIG. 4. HeiicaI arrangementof acridine orange cations obtained by continuing the stacking obtained by complexation with Z&l, *-_
42
S_ K_ OBENDORF,
et aL
an anion) to the reactive groups (C=O or C-NH*) on the bases of DNA (in the major groove)_ Since the helix generated for acridine orange (Fig. 4) has a higher rise per 360° turn than B-DNA (46 a vs. 34 A), additional intercalation of acridine orange, with local unwinding of DNA, may be necessary to f&hate this type of “external binding” of acridine orange. It should, however, be pointed out that Iocal stacking of acridine orange can occur when the cations lie in a variety of orientations with respect to the helix axis in the wide groove of DNA {2,301_ It is likely that acridine orange also binds to the external phosphate groups of DNA It was shown by Neidle and Jones 1203 that one sulphate group (and hence, by arralogy, one phosphate group) cannot promote stacking of acridine orange cations hydrogen bonded to it. In addition, acridiue orange molecules, hydrogen bonded to adjacent phosphate groups on the phosphodiester backbone of DNA, would bareIy overIap if they were oriented parallel to the bases. More overlap is possible if they are oriented perpendicular to the bases. However, it is possible that, if a suitable metal ion is bound to a phosphate group, then acridine orange cations may stack if they hydrogen bond to water molecules coordinated to the metal. From size considerations suitable hydrated cations that could form hydrogen bonds to acridines and also pro-mote stacking include those of Mg’*, Pt*+, Co”“, Mn*‘, and possibIy Cat+ and Na’_ These hydrated ions would, however, be positively charged and the complex formed might be less stable than that with a compIex anion (such as ZnCL *-)_ Thus there are several possible situations in the interaction of acridine orange with DINA that could be envisaged to cause stacking: ( 1) Acridme orange cations could be directly hydrogen bonded to phosphate groups on DNA, If the acridine orange molecules stack, they must be hydrogen bonded to diffprenr phosphate groups- This stacking, owing to the arrangement of phosphate groups on the phosphodiester backbone of DNA, is minir-nal, as mentioned earlier. acridine orange flhosphate DNA ~phosphate-acridine orange (2) Acridme orange cations could form hydrogen bonds to hydrated cations bound to the phosphate groups on DNA. In this case only one phosphate group wouId be invoIved in the stacking of two acridine orange molecules. /Ha DNA-phoghate-metal
O-acridine
orange
\ H2 O-acridiue
orange
(3) Acridme orange_cations could form hydrogen bonds to water molecules that are directly hydrogen bonded to the bases of DNA (in the major groove)_ , DNA,
I-&0 - acridine orange H2 0 -
acridine orange
ACRIDINE
ORANGE TETRACHLOROZINCATE
STRUCTURE
43
This is consistent
with our results as extrapolated in Fig. 4 in which the acridine orange cations are assumed to be oriented parallel to the bases of DNA, but it is
also consistent with a model that involves a&dine molecules inclined, in the major goove, at various angles to the helix axis. It should be noted that DNA and RNA polymerases contain zinc in stoichiometric amounts and this metal appears to be required for catalysis [7, 3 1,321. The presence of zinc as a contaminant of a&dine orange has faciliated the stacking of aromatic rings and this fact may be biologically relevant to the role of zinc in polymerases and in the unwinding and rewinding of DNA [ 33]_ We conclude that it is important to consider the effects of compiex anions, when studying the aggregation of acridine orange. Zinc chloride, often present because it is used as a catalyst in the preparation, is not readily removed by recrystallization, and causes dimerization of acridiue orange.
We thank Dr. AL Sutin for the compound, Dr. N. C. Seeman for helpful discussions and for providing data OR the 9aminoacridine: ApU complex, Drs. Bernard Poiesz for the zinc analysis, and Miss Roberta Talacki and Mr. Harvey Gilmartin for technical assistance_-
REFERENCES
. 1. L. S. Lerman,J. MoL Biol. 3,18-30 (1961). 2. L. S. Lerman,Proc_ Nat_ Acad. Sci. USA 49,94-102 (1963). 3. L. S. Lennan,J. Cell Camp. PhysioL 64, (suppl. 1) 1-18 (1964). 4. A. Albert and R. J. GoIdacre,Natire 161,95 (1948)5. A. Albert, The Acridines (2nd ed.) Edward Arnold, London (1966). 6. G. Gem-mm,P. Main, and M. Mi.WooJfson,Acta Cryst. A27,368-376 (1971). 7. B. 3. Poiesz, G. Seal, and L. A. Loeb,&oc Nat. Acad. Sci. USA 71,4892-4896 (1974). 8_ P. K. Gantzel, R. A. Sparks, R. E_ Long, and K. N. Trueblood, UCLALS 4 Program in Fortran N (1969). 9_ international Tables for X-ray Crystallography, Vol. III, Kynoch Press, Birmingham (1962), pp_ 201-207. 10. D. T. Cramer and J. hiann,Acta Cryst. A24,321-324 (1968). 11. R. F. Stewart, E. R- Davidson, and W. T. Simpson, 3_ Chem. Phys. 42, 3175-3187 (1965). 12. D. T. Cramer and D. Liberman,J. Chem Phys. 53,1891-1898 (19,70). 13. H. J. Bernstein, L. C- Andrews, H_ M. Berman, F. C. Bern&in, G. H. Campbell, H. L. CarrelJ,H. B. Chiang, W. C. Hamilton, D. D. Jones, D. Klunk, T. F. Koenle, E. F. Meyer, C. N. Morimoto, S- S. Sevian. R. K. Stodola, hf. M. Strongson, and T. V. Willoughby, Cry&et-a network of intelligentremote graphics tenninals_SecondAnnuaJ AEC ScientificComputer Information Exchange Meetings,Proceedmgsof the Technical Program, 148-158 (1974). 14. R. Talacki, 8. L. Carrell, and J. P. Glu&er,Acta Cry& B30,1044-1047 (1974). 15. S. K. Obendorf, H. L. Carrell, and J. P. Glnsker, Acta Cryst. B30 1408-1411 (1974). 16. N. C. Seeman,R- 0. Day, and A. Rich,Nature 253,324-326 (1975). 17. C-C. Tsai, S. C. Jam, and H. hi. Sobell, Proc. Nat. Acad. Sci USA 72,628-632 (1975). 18. D. C. Phillips,Acfa Crysf. 9,237-250 (1956). 19. D. C. Phillips,F. R. Ahmed, and W. H. Bames,Actu Cr>st_ 13,365377 (1960). 20. S. Neidle andT. A- Jones,Nature 253,284-285 (1974).
44
S. K. OBENDORF,
et al.
21. A_ Jones and S. NeidIe.Actn Cry&_ B31,1324-1333 (1975)_ 22_ V_ Zanker,Z. Physik Chem 199,225258 (1952). 23_ I)_ H_ Turner, G_ W_ Flynn. S. K. Lundberg, L. D. FalIer, and N. Sutin, Nurure 239, 215-217 (1972). 24_ M. E_ Lamm and D. M. Neville, Jr., J. Phys Chem 69,3872-3877 2% J. A_ McGinnety,Ino~ them 13,1057-1061 (1974)_ 26. M. R. Taylor, Acra Ckyxt. B29,884-890 (1973)_
(1965).
27_ M. J. McCaII and M. R. Taylor, Biochirn Siophys Acfa 390,137-139 (1975). 28_ M. R. TayIor and M. J. McCaU,Acta Cryst. B31, S42 (1975). 29. D_ J_ Blear-s and S. S. Danyluk, J_ Amer. Chenz Ser. 89,21-26 (1967). 30. C_ Houssier and E. Fredericq, Biochim. Biophys Acta 120.434447 (1966). 31_ C_ F_ Springate, A_ S. Mihivan. R. Abramson, J. L. Engle, and L. A. Loeb, /. Biol. Chem 248,5987-5993 (1973). 32. L. A. Loeb, The Enzymes, VoL X (3rd Edition), Academic press, inc_, New York, San Francisco, London (1974). pp_ 173-209. 33_ Y. A_ Shin and G. L. Eichhom, Biochemistry 7,1026-1032 (1968). Received I7September
1975; revised-24 October 1973
CHEMISTRY
6,2944
(1976)
29
Aggregation of A&dine Orange: Crystal Structure of Acridine Orange Tetrachlorozincate ~C,,H,,N,-~HCI-ZUC~~-CH~COOH S. K. OBENDORF,? JENNY PICKWORTH HELEN M. BERMAN, and H. L. CARRELL
GLUSKER,*
*
PAUL R. HANSEN,
The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania I91 I I
ABSTRACT The crystal structure of the biological stain, “acridine orange,” has been determined. This compound, when crystallized from ethanol, is shown to be a zinc chloride double salt of acridine orange, containing, in addition, acetic acid of crystallization. These additional components are residuals from the method of acridine orange. This complex, 2 aaidine of preparation orange-2HCl.ZnCl, lCH,COOH, (2C, -,HX ,N, -2HC1-ZnCl,-CH,COOH) crystallizes in the monoclinic space group P2,, a = 9.965 (2), b = 21 SO7 (6), c = 9.645 (2) A, p = 113.98” (2), V = 1888.7 (8) A’, FW = 800.0, Z = 2, D, = I.41 g-cni3, Dabs = 1.43 (9) g.cni3 _Threedimensional diffraction data were collected with CuKa radiation, and the structure refined to R = 0.065 for 1885 observed reflections. In the crystal structure hydrogen bonds are formed,
INTRODUCTION Acridine orange is used as a biological stain for nucleic acids and polysaccharides. It also shows mutagenic and antibacterial activity_ The interaction of acridine derivatives, such as acridine orange, proflavine and 9-aminoacridine, with DNA has been studied extensively. There appear to be two main types of binding. One type, at low acridine concentrations, involves intercalation [l-3] _ The acridine derivative becomes insetted between the base
*From the Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111. This research was supported by grants CA-10925, CA-06927 and RR-05539 from the National Institutes of Health, U.S. Public Health Service, AG370 from the NationaI Science Foundation, and by an appropriation from the Commonwealth of Pennsylvania. j-Present address: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853. -&Towhom all correspondence and reprint requests should be addressed. 0 American Elsevier Publishing Company, Inc_, 1976
S_ K. OBENDORF,
30
et al.
vti the protonated nitrogen atom of the central rings of two a&dine orange cations, to two chloride ions in a Zncl, =- tetrahedral grouping_ These two acridine orange moIecules are stacked in parallel pIanes, approximately 3.4 A apart, with the long axes of the ring systems inclined at 26.S” to each other. Thus an apparent dime&z&ion of the acridine.orange is facilitated by the anions present, resulting in the complex studied. The two -N(CH,), groups of
each acridime orange molecule are not protonated in this crystalline form. The mode of molecular packing found here may be relevant to models for the external stacking of acridine orange around a DNA molecule_ The importance of
removing
any
zinc
salt from
acridine
orange
preparations
prior
to
aggregation studies is stressed.
pairs of DNA (a hydrophobic interaction perpendicular to the plane of the a&dine ring system), with an appropriate conformational adjustment of the nucleic acid to accommodate this additional (3-4 A thick) molecule. The other mode of interaction, occurring at high dye concentrations, is via external stacking (a hydrophilic interaction, probably in the plane of the acridine ring system)_ The central nitrogen atom of the a&dine ring system, which is protonated at neutral pH can hydrogen bond to a hydrogen bond acceptor (for an ionized phosphate group)_ This is the predominant mode of example, interaction at high concentrations of acridine derivatives, and may be important in their action as antibacterial agents, since they are active as bacteriostats only at pH vahres at which the central nitrogen atom is protonated [4, 51. EXPERIMENTAL
SECTION
The source of the compound studied was the commercial product, “_4cridihe Orange, Basic Orange 14, Bioiogical Stain” from Matheson, Coleman and Bell_ Crystals were grown from ethanol as needles from which a small single crystal was cut, size O-10 X 0.27 X 0,11 mm_ Cell data are given in Table 1. Intensity data were collected on an automated four circle diffractometer with monochromatized CuKa radiation with the 6-28 scan technique to a maximum sine/A = 0.61 cm-’ _ A total of 3495 reflections were scanned of which 1885 were above the threshhold vahre of 3oQ (where o(l) was determined from counting statistics)_ There was no intensity loss with time during the data collection_ Values of o(F) for observed data were calculated from the formula o(F) = (F/2) [ ]02(r)l12 J + ?j2 ] s where 6 is a measured instrumental uncertainty, 0.0207, determined from the variation of the intensities of periodically monitored check reflections‘. An X-ray absorption correction (,$CuKcr) = 36.3 cm-‘) was applied when the total contents of the crysta1 had been found. The density of the crystal, measured in dichioromethane and tetrachloromethylene, showed a huge experimental error, presumably because there were several components in the crystals with different sohrbilities in chlorinated hydrocarbons_ The structure was solved, with difficulty, by a combination of Patterson, Fourier and direct methods (using the program MULTAN [6]).The unexpected presence of zinc in the crystals was revealed in Fourier maps as a peak approximately 2.2 A from each of four cholride ions. Therefore analyses of
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
31
TABLE I Cell Data Formula: 2Cr 7Hr s N, l2HCl* ZnClz Formula weight: 800.0 Cell dimensions: a = 9.965 (2) A b = 21.507 (6) 9.645 (2) ;I 113.98 (2)O v= 1888.7 A3 Space group 1 Density:
P2r z=
calculated observed
lCHs COOH
2 1.41 g-cm3 1.43 (9) g- cmW3
crystals from the same batch were made. A spectroscopic assay of acridine orange in a solution of crystals (3.6 X 1f16 M acridine orange), complemented by an atomic absorption spectroscopic zinc determination (2.16 X lo* M Znl [7], showed that there were two a&dine orange molecules per zinc atom in the crystals, as found from the crystal structure. The zinc chloride present in these crystals had been used as a cataIyst in the preparation of acridine orange [5J and was obviously not removed in the subsequent recrystallization. The structure (consisting of acridine orange hydrochloride and zinc chloride) was refined, first by the interpretation of electron density maps, and then by full-matrix least-squares methods using UCLALS4 [83_ The weights during refmement were l/(02 (F)), with zero weight for those reflections below the threshhold values. The quantity minimized in the least squares calculation was The atomic scattering factors used were: Zn, International Co[llFol-IFc1112. Tables, Volume III [9] ; for Cl, 0, N, C, those of Cromer and Mann [lo] ; and for H, those of Stewart et al. [ 113. The anomalous dispersion corrections for Zn and Cl were those of Cromer and Liberman 1121. The refinement stopped at an R value of approximately a.1 O_ A difference map revealed four high peaks which were interpreted as acetic acid of also residual from the method of preparation_ The high crystallization, temperature factors of these atoms indicate that the acetic acid molecule is only loosely bound in the crystal lattice. Further refinement with isotropic temperature factors for the acetic acid atoms and ail hydrogen atoms included in their calculated positions gave the final R value of 0.065. Final parameters are listed in Table 2. A table of calculated and observed structure factors is available.’ Illustrations and molecular geometry calculations were done via the CRYSNET system [ 13 ] _ ‘Tables of FoFc values may be obtained from the senior author or the E&tcrial office of Bioinorganic Chemistry.
32
S_ K_ OBENDORF,
et aL
DISCUSSION MolecuIar Description The formula of the salt studied is:
There are two acridine orange cations, designated “A” and “B”, within each asymmetric unit of the crystal_ Coordinates are listed in Table 2. Estimated standard deviations for carbon, nitrogen, oxygen, and particularly hydrogen atom positions are high owin g to the presence of the heavier zinc and four chIoride ions in the structure. Therefore none of the differences in bond lengths between cations “A- and “B- (and between the two haIves of each cation) are considered significant and averaged distances and angles are shown in Fig. 1. The C-N bonds to the -N(CH& groups range from 130 to 1.39 (2) 8, indicative of much double bond character, as found in 9aminoacridine (C-N = 1.321 (3) A [141. The cations of both “A” and “B” are monoionized (pK, = 10.45, pKb = -3-15) with the proton located on the nitrogen atom of the central ring (N( 1 OA), N( 1 OB))_ These protons are involved in hydrogen bond formation to chloride ions in the ZnCb2- anion. One ZnC14 2- anion (at I-x, y-‘55, -2) forms hydrogen bonds to one of each “A” (at x, y. z) and ‘73” (at x, y, z-l) acridine orange cation_
H
H
H
H
H
H
H
FIG_ l_ Averaged angles and distances in acridine orange cations. Estimated standard deviationsare approximately0.02 A and 1”.
TABLE
2
Atomic Parametew Y
zn2* cl-( 1) Cl_(2) S(3) Cr(4) C(lA) C(2A) C(3A)
C(4A) C(sA) C(a) C(7A)
WA) C@A) N( 10A) C(llA) C(12A) C( 13A) C( 14A) N( 15A) C(l5A) C( 15’A) N(16A) C(l6A) C( 16’A) C(lB) C(2B) C(3B) C(4B) C(5B) C(6B) C(7B) C(8B) C(9B) N(lOB) C(lIB) C(l2B) C( 13B) C( 14B) N(l5B) C(15B) C( 1 S’B) N( 16B) C( 16B) C( 16’B)
0.7009( O-8906( O-7691( 0.5329( 0.5928(
2) 5) 5) 5) 6)
O-7541(18) O-8213(16) O-7906( 17) O-6734(16) 0.3!25( 19) O-2456( 18) 0.2757(19) 0.3771(21) 0.5735( 19) O-4996( 12) O-6095( 18) O-461-1( 18) O-6406( 17) O-4273( 19) O-8653( 13) O-9786( 17) O-8419(21) 0.1459(13) O-0676(26) 0. I 176(20) O-7444( 15) O-7682( 15) O-6993( 16) 0.5860(15) O-3287( 14) O-2959( 16) O-3803( 17) O-4760(16) 0.6123(16) 0.46X6(1 I) O-5607( 1.5) 0.5037( 15) O-6388(15) O-4303( 15) O-7258( 13) O-6567(20) O-8330(16) O-1825(13) 0.0909( 16) O-1529(19)
O-4839( 1) O-4183( 3) O-5756( 2) O-4439( 3) 0.5055< 3) O-2405( 8) O-1941( 8) O-1303( 8) 0.1158( 8) O-1721( 9) O-2209( 11) O-2851( 9) 0.3014(10) O-2726( 8) O-1494( 6) O-1634( 9) O-2565( 8) O-2262( 8) O-1944( 8) O.OSSl( 6) 0_1020(10) 0.0199(10) 0.2033( 8) O-2493( 15) 0.1379(14) O-1809( 9) O-1220( 9) 0.0711( 8) 0.0889( 7) O-2357( 7) O-2965( 8) O-3491( 8) O-3338( 7) 0.2593( 8) O-1642( 6) O-1482( 8) O-2750( 7) O-1987( 7) O-2243( 8) O-0131( 6) -0.0400( 10) -O-0032( 10) O-3088( 6) O-2598( 8) O-3729( 9)
z O-0774( 0.1231(
3) 7)
0.2059( 6) O-1.543( 6) .0_1766( 6) O-0716(21) 0.0313(19) 0.0430(19) 0.0858(20) O-2724(21) 0.3 194(22) 0.3 113(22) 0.2642(23) 0.1705(21) 0_1771(14) 0.1294(21) 0.2193(21) O-1226( 19) O-2257( 19) O.OOVO( 16) .O.OS 16(20) O-0286(28) 0.3785( 16) O-4245(28) 0.3991(25) 0_5591(19) O-5376(19) O-5735( 19) O-6304( 18) O-8002( 19) O-8240( 18) O-8003( 19) O-7387(18) O-6466( 19) 0.7 1 l6( 14) O-6496( 18) O-7053( 17) O-6239( 18) O-7364( 18) 0.5544( 16) 0.5879(22) O-4899( 19) O-8679( 16) O-8842(22) O-8964(24) 33
S:K. OBENDORF,
34
Bll ZI?’ cl-( l_) cl-(z) a-(3) a-(4) C
C(9B) N( IOB) C(llB) C(12B) C( 13B) C( 14B) N(i5B) C( 1 SB) C(15’B) N( 16B)
4_4q 1) 6.15( 2) 6.36( 1) 5.03( 1) 12.13( 3) 7.29( 7) 6.10( 6) 6.94( 6) 2.31( 6) 3.99( 8) 2.56( 6) 3_95( 7) 8.55( 9) 4.85( 9) 5.39( 5) l-41( 7) 6_69( 6) 3.86( 7) 4_25( 7) S-23( 5) 2.02( 6) 9.36( 9) 4_70( 5) 12.37( 12) 5.58( 8) 3.22( 6) 2_89( 5) 4.56( 6) 5.53( 6) 2.58( 5) 3.43( 5) 2.22( 6) 5.46( 6) 6.60( 6) 2.37( 4) 4.7q 5) 3_91( 6) 5.56( 6) 3_09( 5) S-28( 5) 12.38( 8) S-15( 6) 5.28( 5)
B22
3.20( I) 5.51( 2) 4.4q 2) 5.90( 2) 4.62( 2) 3_6f( 8) 4_81( 8) 2.94( 8) 3.66( 8) 6.25( IO) 9_53( 13) 6.46( IO) 4_42( 9) 3.96( 8) 2.92( 5) 4.66( 8) 3_07( 8) 3.55( 7) -4.09( 8) 3_46( 6) 3.88( 13) 5.66( 11) 8.21( 9) 16.76(21) 12.54(18) 5.86( 9) 8_55( 10) 5.73( 8) 3.83( 7) 4.18( 7) 4.31( 7) 3.64( 8) 3.14( 7) 4.66( 7) 4.27( 5) 4.44( 9) 3.31( 7) 2.94( 6) 4_OL( 7) 5_98( 7) 7.16(11) 9.56( 12) 3.96( 6)
B 33 4.86( 1) 11.34( 3) 7.70( 2) 7.67( 2) 5.42( 2) 5.18( 9) 4.42( 7) 3.97( 7) 3_75( 8) 1_25( 9) 3.24( 9) 4_94( 9) 5.04(11) S-15( 9) 1.97( 5) 4.42( 8) 3.24( 8) 4.08( 7) 2.42( 7) 4.36( 6) 2.8q 8) 11.18(12) 3.80( 6) 6.72( 9) 4.14( 11) 3.85( 8) 4.68( 8) 3.29( 7) 4.10( 7) 3.64( 8) 3.18( 7) 3.93( 9) 3.43( 7) 5_02( 8) 3.94( 5) 3.93( 7) 4.58( 7) 4.53( 7) 2.58( 7) 6_5q 7) S-88( 9) 7.23( 8) 7.13( 7)
et aL
B12
O.ll( l-87( -O-63( -l-15( O-83( -l-17( -l-61( 0.32( 0.27( O-14( O-57( 0.70( 1.39( -0.47( -0.79( 0.38( 0.14( -O-13( 0.54(
J313
1) 2) 1) I) 2) 6) 6) 7) 6) 8) 8) 7) 8) 7) 4) 7) 6) 6) 7)
0.04( 5) -O-16( 8) 3.29( 9) -O-43( 6) O.OO( 15) -0.54(1 I) - 1.92( 6) -1.06( 7) -O-08( 7) -O-95( 5) -O.Ol( 5) 0.21( 5) -0.99( 6) -O-58( 6) -l.OO( 6) -O-52( 4) -0.99( 6) 0.33( 5) -l-17( 5) -0.98( 5) 0.22( 5) - l-84( 9) O.S7( 7) -0.18( 5)
2.6q 1) 4.20( 3) 4_41( 2) 3_46( 2) 4.33( 3) 3.53( 9) 3.27( 7) 334( 8) 1.18( 8) -O-35( 9) 1.21( 8) 1_50( 9) 3.72(10) 1.95( 9) 0.73( 6) 0.61( 8) 1.80( 8) l-08( 8) 0.99( 8) 1.57( 6) l-03( 8) 7_37( 14) l-25( 6) 7_14(14) 2.12(11) O-42( 7) l.OO( 7) l-43( 6) 2.99( 8) O-81( 7) 0.33( 7) -l-71( 7) 1.16( 7) 2.24( 8) l-27( 5) 1.72( 7) l-20( 7) 2.37( 7) -O-25( 6) 3.07( 7) 6.34(2 I) 3.09( 9) 3.29( 7)
B23
-O-06( 1) 0.24( 2) -l-79( 2) -O-19( 2) -O-08( 2) O.lO( 7) 0.07( 7) 1.02( 7) 0.38( 7) 0.31( 9) -0.10(10) -l-64( 8) 0.32( 10) O.ll( 8) 0.77( 5) O-76( 8) O-11( 7) -0.03( 6) O-08( 7) O.ll( 6) O-08( 10) l-38(11) -l-33( 7) - 1.07( 14) -0.17(13) O-25( 8) O-63( 8) l-62( 8) O-44( 7) 0.41( 7) 0.30( 7) -O-16( 7) 1.40( 6) 1.35( 7) O.OS( 5) 0.44( 7) 1.59( 6) -0.81( 6) 0.28( 6) 0.60( 6) -O-83( 9) -I.79(10) -0.80( 6)
ACRIDINE ORANGE TETRACHLOROZINCATE
C(16B) C( 16’B)
3.97( 6) 7.28( 8)
3.72( 7) 4.72( 8) x
0(1X)
0(2X) C(3X) C(4X) H(IA) H(2A) ‘H(4A) H(5A) H(7A) H(SA) H(9A) H( 1OA) H(lSA1) H( 15A2) H( lSA3) H( 15’Al) H( 15’A2) H( lS’A3) H( 16Al) H( 16A2) H(16A3) H(16’Al) H( 16’A2) H( 16’A3) H(W H(2B) H(4B) H(5B) U(7B) H(SB) H(9B) H( 10B) H(15Bl) H( 15X32) H(15B3) H(lS’B1) H(15’B2) H( 15’B3) H(16Bl) H( 16B2)
0.851( 2) O-814( 2) 0.802( 3) 0.784( 3) O-783( 15) 0.896( 15) O-639( 15) O-285(25) O-220(15) 0.398(15) O-604(15) O-471(15) l-074(15) O-943( 15) O-993(15) O-742( 15) 0_857(15) O-920( 15) -0.024(15) O-127(15) O-041( 15) 0.031(15) 0.203( 15) O.OSS(15) 0.799(15) 0.84q15) 0.528( 15) O-280( 15) O-368(15) O-531(15) O-668(15) 0.41q15) O-637( 15) O-562(15) O-723( 15) 0.81 l(15) O-933(15) 0.838(15) 0.034( 15) 0.019(15)
5_76( 9) 11.75(12)
- 1.77( 6) -O-69( 7) Y
O-371( I) O-471( 1) 0.410( 2) 0.399( 1) O-285( 9) 0.205( 9) 0.072( 9) O-127( 9) O-318( 9) O-347( 9) O-317( 9) 0.105( 9) O.OSl( 9) O_OSS( 9) O-148( 9) O-012( 9) -O-006( 9) O-012( 9) 0.230( 9) O-264( 9) 0.28r;( 9) 0.124( 9) O.l?l( 9) O-132( 9) O-214( 9) O-113( 9) O-056( 9) O-200( 9) O-393( 9) O-369( 9) O-293( 9) O-130( 9) -O-031( 9) -O-049( 9) -O-077( 9) 0.024( 9) O-007( 9) -O-047( 9) O-276( 9) 0.245( 9)
35
STRUCTURE
l-73( 9) 5.17(10)
-1.23( -4.03(
8) 9)
t
B
O-487( 2) 0.578( 2) 0.553( 3) 0.692( 3) 0.066( 18) -0.009( 18) O-084( 18) O-271( 18) O-341(18) O-259( 18) O-171(18) O-177(18) 0.01 l(18) -0.160(18) - 0.046( 18) 0.025( 18) -0.050(18) O-132(18) O-424( 18) 0.531(18) O-354( 18) 0.308( 18) 0.413(18) O-486( 18) 0.531(18) O-493( 18) 0.654( 18) O-829( 18) O-528( 18) O-716(18) O-623( 18) O-740( 18) O-680(18) O-500( 18) O-609( 18) O-399( 18) O-568( 18) O-457( 18) O-941( 18) 0.782( 18)
17.4( 5) 18.4( 5) 15.2( 8) 13.8( 8) 4.2( 10) 3.9(10) 3.7(10) 4.9( 10) 4.5( 10) 5.0(10) 4.3( 10) 3.3( 10) 4.5( 10) 4.5( 10) 4.5( 10) 5.5( 10) 5.5( 10) 5.5( 10) 7_6( 10) 7_6(10) 7.6( 10) 6.8( 10) 6.8( 10) 6.8( 10) 4.5( 10) 5.2(10) 3.6( 10) 3.7(10) 4.6( 10) 4.1(10) 4.3(10) 3.7( 10) 7.1(10) 7.1(10) 7.1(10) 6.0( 10) 6.0( 10) 6.0( 10) 5.0(10) 5.0(10)
36
S. K. OBENDORF,
et ai.
H( i6333)
O_i52(15)
0.224(
H(I6’Bl) H( I6%2) H( 16’B3)
0.124(15) O-242( 15) 0.069( 15)
0.374( 9) O-399( 9) O-390( 9)
9)
0.941(18)
5.0(10)
0.985(18) 0.920( 18) O-807( 18)
5.6( 10) 5.6(10) 5.6(10)
uPositionalparametersare givenas fractionsof cell edges Anisotropictemperaturefactors are expressedas exp [-%(hz a*‘B, I + k* b*’ Bz 1 + Q*c*‘B, 3 + 2hka*b*B, 2 + 2hPa*c*!3, 3 + 2kQb*c?B, 3)J and isotropictemperaturefactorsas exp (-B sit?~/h*) with B valuesgiven in A* _ The standarddeviationsfor each parameter,determinedfrom the invertedfull matrix, and given in parentheses,apply to the last specified digits. Hydrogenatom positionsare calculated,and, in the cese of methyl groups,give the best fit to a difference map_ Values for esd’s for hydrogen atoms are based on averageisotropic refmement results.Temperaturefactors for hydrogenatoms are based on those of the atom to which-theyare attached_
(a)
FIG. 2. Acridine orange-zincchloride complex. Space-f3ug diagramsof the complex. The stippled areas are representativeof the van der Waals radii of atoms. (a) View onto the cl(z)-Zn*+-CI(4) pIane; (b) View along the planesof the aaidine orangecations.
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
37
TABLE 3 (a)
(b)
Angles between rings Definitions AI AII AH1 BI BII BHI
attached contains attached attached contains attached
Angles between planes AIrAII lSO AII:AIII l-l0 ALAI11 0.6O
BIB11 BII:BIII BI:BIII
Equations in A with respect Molecule A 3.36177 x - 1.25393 y -2.70144x -20.68579~ 898342 x - 5.75 124 y
to N( 15A) N( 10A) to N(16A) to N( 15B) N( 1OB) to N( 16B)
0.8O 1.6” 2.3O
to fractional coordinates x, y, and z + 6.95756 z - 2.74442 = X, + 1.39447 z + 5.51491 = YA - 6.53252
z - 2.44724
= ZA
Molecule B 3.65805 x - 1.2911Oy + 6-74123 z - 6-27654 = XB -6.35191x-15.97997~ i-4-296602 +3.83416 =YB 6.75079 x - 14.33613 y - 5.39638 z + 3.08480 = Zn (c)
Angles between principal axes of of moIecuIes A and B 1.8O Angle between 26.5O Angle between 26.5” Angle between
best planes through three rings molecular planes 3-4 A apart intermediate axes of molecules long axes of molecules
The three carbon atoms attached to each nitrogen atom in the -N(CHs)z groups, i-e_, attached to N( 1 SA), N( 16A), N( 15B), and N( 16B) are coplanar with the nitrogen atom Therefore these nitrogen atoms do not have an additional proton on them since this should cause a tetrahedral arrangement to occur. (In a crystalline form of proflavine hydrochloride studied in this laboratory one amino group had an additional proton attached to it in a tetrahedral arrangement [ 151.) It is, however, probable that the positive charge on the a&dine orange cation is distributed among the three nitrogen atoms (N(LO), N( 15), N( 16)). The -N(CH& groups are slightly tilted with respect to the plane of the rest of the ring system These tilts are 5.5O, 5.3O, 2.1°, and 6.9” for the groups including N( 15A), N( 16A), N( 15B), and N( 16B), respectively_ The zmc chloride-acridine orange complex is illustrated in Fig. 2. As shown iu Table 3 and Fig. 2b, each cation is approximately planar, the maximum
38
S. K_ OBENDOBF,
et al_
deviation between rings being 2-3&_ This planarity seems important for intercalation in DNA as shown by the crystal study of the ApU: Paminoacridine complex [ 161 and the f-iodo UpA: ethidium complex [ 171_ Molecular Packing The overlap of the ring systems of acrldine orange lying in parallel planes approximately 3-45 A apart, is shown in Fig. 3a and b. The cations stack throughout the crystal (Fig. 3b). The long axes of the acridine orange cations are not parallel but inclined at 26.S” to each other- In the crystal structures of proflavine hydrochloride crystals [ 1S] , 9-aminoacridine hydrochloride [ 141 and acridine [ 18, 191, with no complex anions present, the ring systems lie in paraBe planes approximately 3.4 A apart but with their long axes parallel. This rotation of approximately 3Op in the angle between the long axes of the ring systems is probably caused by the presence of the ZnCl~*- complex anion. In proflavine hemisulpbate [ 20,211 a similar situation occurs with the long axes of the cations Inclined at 150’ [= 18Q30°) to each other. A comparison of the stacking in the ZnC&‘- and the SOe2- complexes is shown in Fig_ 3a vs. c. The sobent of crystallization, presumed to be acetic acid, fits in a pocket between the chloride ions, the methyl groups, and the more hydrophobic regions of the a&dine orange molecules. The temperature factors for atoms in this group are high indicating considerable disorder_ Zinc Interactions One of the principal points of interest in this crystal structure is the interaction of the tetrachlorozincate anions with the aromatic acridine orange cations. A&dine orange has been shown to dimerize in solution [22, 231 and the possible participation of anions in this dimerization was noted by Lamm and Neville 1241. As shown in Table 4 and Fig. 2 the ZnCb2- anion forms hydrogen bonds to two acridine orange cations (“A” and “B”)_ Thus effectively a complex has been formed which involves a dimer of a&dine orange. McGinnety [ 25 ] found that in a ZnCb*- anion, in the absence of any “crystal forces” a value of 2.294 A would be expected for the Zn*“. - --Cl- distance. Deviations from this distance, and from true tetrahedral symmetry, are given in Table 4. Taylor and McCall [26-281 studied some zinc chloride complexes of nucleic acid bases They found zinc ions coordinated to ring nitrogen atoms of the bases and, via coordinated chloride ions or water molecules, to keto or amino groups on the bases. Stacking of Acridine Orange in the Presence of Anions In the ZnC1,*- anion the Cl---*Cl distances are approximately 3.7 A_ This distance is similar to the thickness of the aromatic ring system of acridine orange_ In this crystal structure the cations pack in parallel planes approximately 3.45 A apart with no differentiation in distance apart of the pairs of cations that complex to ZnCb *-.
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
39
FIG. 3. Comparisons of stacking in acridie orange tetrachlorozincate (this work) and proflavine hemisutphate [20, 211. Views onto the molecular planes. The proflavine cation bas been numbered to conform with the orientation for acridme orange. Molecule A is darkened. In the proflavine salt 0 denotes oxygen of sulphate, W denotes water, both hydrogen bonded to -NH groups. (a) Acridiie orange tetrachlorozincate. 26.5” between rings; (b) Acridine orange tetrachlorozincate. Stacking through the crystal; (c) Proflavine hemisulphate. 150” between rings.
S. K. OBENDORF,
40
et al.
TABLE 4 Molecular Packing (a)
Hydrogen bondsa D-H----.A
H . . . ..A
N( 1OA)‘-H( 1OA)“. Cl(4) N(lOB)‘-H(1OB)’ ---=C1(2) l
(b)
l
D-H-*.=-A 180° 161°
2.231( 2.36
l
3322
ZnC!4 ‘- compIex distance A zncq 1) CK2) Cl(3) CY4)
2.254 2.281 2.259 2.288
* - 0.040 -0.013 - 0.035 - 0.006
*Variation from 2.294 A (McGinnety)
C1( I)**= +X(2) CKf) ----Cl(3) Ci(I)---•l(4) C&2) ----C1(3) C1(2)* - lCl(4) CK3) ---X1(4) l
tVariation (c)
distance A
Angle at Zn2+
3.786 3.738 3.702 3.586 3.702 3.721
113.2 111.8 109.2 104.3 108.2 109.9
ii-3.7 +2.3 -0.3 -5.2 -1.3 +0_4
from 109.5”
ZnCtr *= - - - acridine orange A
B
Zn2+ Zn2+
CX2) C](4)
C N( 10B)’ N( IOA)”
A-B
B-C
A-C
ABC
2.28111 2.288
3.322A 3.227
4.948A 4.323
122.9O 101.9O
‘l-x yrty I-t ‘Cl-x IA+v -2 .. @Hydrogen atoms are in calculated positions_ The structure of proflavine hemisulphate [20,21 J shows hydrogen bonding of the sulphate groups to proflavine molecules. The S-0 distance is 1.40 5 so that O-O distances are approximately 2.40 A within one anion. This distance is not sufficient to span the 3.4 h distance between stacked proflavine molecuIes and therefore the proflavine molecules that are found to stack in the crystal are hydrogen-bonded to different sulphate groups.
ACRIDINE ORANGE TETRACHLOROZINCATE
STRUCTURE
41
Blears and Danyluk [29] report a greatly increased amount of stacking in a&dine orange on adding sodium chloride_ This may be due to complex formation with, for example, hydrated sodium ions or with chloride ions. In acid soiution disaggregation occurs, presumably due to the formation of non planar ,C& -NHqH groups (cf_ the nonstacking of -&Ha groups in proflavine I1 51). The l&R results of Blears and Danyluk [29] are consistent with the stacking of the ring systems in planes 3.4 A apart. They found that, in solution, protons on C(4) and C(5) are oriented more nearly over the centers of adjacent rings than are the other ring protons. In the crystal structure reported here, the protons on C( 1B) and C(5B) lie on either side of the center of cation “A” (see Fig. 3a).
Model for Binding of Acridine Orange to DNA The structure of acridine orange tetrachlorozincate is a model of the situation that may occur when acridine molecules bind externally to a DNA molecule_ Therefore, as shown in Fig. 4, the stacking found in the acridine orange tetrachlorozincate structure, with an angle of 26.5* between the long axes of the cations “A” and “B,” was continued so that a helical arrangement was generated_ This model contained between 13 and 14 cations per revolution_ The central nitrogen atoms of the rings (N( 10)) were approximately 6 A from the helical axis. Hypothetical water molecules, hydrogen bonded to these nitrogen atoms and in the plane of the acridine ring system, Iay 4 A from the helical axis. Thus it is possible for acridine orange molecules to hydrogen bond (vin water or
FIG. 4. HeiicaI arrangementof acridine orange cations obtained by continuing the stacking obtained by complexation with Z&l, *-_
42
S_ K_ OBENDORF,
et aL
an anion) to the reactive groups (C=O or C-NH*) on the bases of DNA (in the major groove)_ Since the helix generated for acridine orange (Fig. 4) has a higher rise per 360° turn than B-DNA (46 a vs. 34 A), additional intercalation of acridine orange, with local unwinding of DNA, may be necessary to f&hate this type of “external binding” of acridine orange. It should, however, be pointed out that Iocal stacking of acridine orange can occur when the cations lie in a variety of orientations with respect to the helix axis in the wide groove of DNA {2,301_ It is likely that acridine orange also binds to the external phosphate groups of DNA It was shown by Neidle and Jones 1203 that one sulphate group (and hence, by arralogy, one phosphate group) cannot promote stacking of acridine orange cations hydrogen bonded to it. In addition, acridiue orange molecules, hydrogen bonded to adjacent phosphate groups on the phosphodiester backbone of DNA, would bareIy overIap if they were oriented parallel to the bases. More overlap is possible if they are oriented perpendicular to the bases. However, it is possible that, if a suitable metal ion is bound to a phosphate group, then acridine orange cations may stack if they hydrogen bond to water molecules coordinated to the metal. From size considerations suitable hydrated cations that could form hydrogen bonds to acridines and also pro-mote stacking include those of Mg’*, Pt*+, Co”“, Mn*‘, and possibIy Cat+ and Na’_ These hydrated ions would, however, be positively charged and the complex formed might be less stable than that with a compIex anion (such as ZnCL *-)_ Thus there are several possible situations in the interaction of acridine orange with DINA that could be envisaged to cause stacking: ( 1) Acridme orange cations could be directly hydrogen bonded to phosphate groups on DNA, If the acridine orange molecules stack, they must be hydrogen bonded to diffprenr phosphate groups- This stacking, owing to the arrangement of phosphate groups on the phosphodiester backbone of DNA, is minir-nal, as mentioned earlier. acridine orange flhosphate DNA ~phosphate-acridine orange (2) Acridme orange cations could form hydrogen bonds to hydrated cations bound to the phosphate groups on DNA. In this case only one phosphate group wouId be invoIved in the stacking of two acridine orange molecules. /Ha DNA-phoghate-metal
O-acridine
orange
\ H2 O-acridiue
orange
(3) Acridme orange_cations could form hydrogen bonds to water molecules that are directly hydrogen bonded to the bases of DNA (in the major groove)_ , DNA,
I-&0 - acridine orange H2 0 -
acridine orange
ACRIDINE
ORANGE TETRACHLOROZINCATE
STRUCTURE
43
This is consistent
with our results as extrapolated in Fig. 4 in which the acridine orange cations are assumed to be oriented parallel to the bases of DNA, but it is
also consistent with a model that involves a&dine molecules inclined, in the major goove, at various angles to the helix axis. It should be noted that DNA and RNA polymerases contain zinc in stoichiometric amounts and this metal appears to be required for catalysis [7, 3 1,321. The presence of zinc as a contaminant of a&dine orange has faciliated the stacking of aromatic rings and this fact may be biologically relevant to the role of zinc in polymerases and in the unwinding and rewinding of DNA [ 33]_ We conclude that it is important to consider the effects of compiex anions, when studying the aggregation of acridine orange. Zinc chloride, often present because it is used as a catalyst in the preparation, is not readily removed by recrystallization, and causes dimerization of acridiue orange.
We thank Dr. AL Sutin for the compound, Dr. N. C. Seeman for helpful discussions and for providing data OR the 9aminoacridine: ApU complex, Drs. Bernard Poiesz for the zinc analysis, and Miss Roberta Talacki and Mr. Harvey Gilmartin for technical assistance_-
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44
S. K. OBENDORF,
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