- Altera Corporation - University Program April 2014 1. QUARTUS II INTRODUCTION USING VERILOG DESIGNS For Quartus II 13.1 2Background Computer Aided Design (CAD) software makes it easy to implement a desired logic circuit by using a programmable logic device, such as a field-programmable gate array (FPGA) chip. A typical FPGA CAD flow is.
- Page 1 University Program's web site. An easy way to begin working with the DE1-SoC Computer and the Nios II processor is to make use of a utility called the Altera Monitor Program.
This link keeps changing, but the old software is still around the website. There is also a simulator add-on available for Quartus version 11.0 that works like the old Quartus simulator. It is available in the University program section at Altera's website. Altera's University Program cores can be downloaded from their website, however to save others the 1.5 GB download the SD core and associated HAL for Quartus 10.1 are available here. With the above files saved in the project directory, SOPC automatically added the core to the tree view on the left (otherwise the core could be imported via the.
Quartus II Tutorial IntroductionAltera Quartus II is available forWindows and Linux. The instructions here are from version 11.0, withsome updates for versions 12.0, 12.1 and 13.0.
This is accomplished by selecting 'File - New - University Program VWF'. Test vectors created with this tool can be used in simulation of your circuits by running the ModelSim-Altera simulation tool. The simulator can be started from within the Waveform Editor, or by using the Altera Nativelink flow.
I try to keep it up todate.On my YouTube channnel, I have a.Using Quartus II. Simulation Note: Since version 11.1 of Quartus II, the QSimsimulator has been automatically included with Quartus II, forWindows and Linux. For simple simulations, it is easy to use.Following are instructions for simulations using either one.(using QSim) (using ModelSim).In this tutorial, we will showyou how you capture the schematic design for the automatic door opener circuitusing Altera Quartus II software.The ProblemWe are designing a circuit for anautomatic door like those you see at supermarkets.
The door shouldopen only when a person is detected walking through or when aperson presses a switch (such as the wheelchair button) to havethe door open. If you're using a version of Quartus II lower than13.0, use the.Simulation using QSim for version 13.0 Note: In version 13.0 of Quartus II, QSim can beopeneddirectly from within QuartusII, however it only works with Cyclone devices.
If you'vealready chosen a non-Cyclone device, switch to a Cyclone device to dothesimulation. Simulation using QSim for versions 11 and 12 Note: QSim can't be opened automatically from within QuartusII. You can invoke it by typingquartussh -qsimat a command prompt. (Run this in the directory where you find theQuartus II executable file.).
Open the Altera U.P.
Full text of 'UNIVERSITY OFIILINOIS LIBRARYAT U ^.NA-CHAMPAIGNDigitized by the Internet Archivein 2012 with funding fromUniversity of Illinois Urbana-ChampaignTOPICSI Semester 1979-80X-— ^Generation and Synthetic Utilityof Carbanions Stabilized byDivalent SulfurPeter BeckerrThe Design, Synthesis, and Biology ofIntercalating AgentsDavid W. RobertsonJ HERTZBERG - NEW METHOD, INC. EAST VANDALIA ROAD, JACKSONVILLE, ILL. 62650Q, -TITUE NO.B.ACCOUNT NO.:NRTK«LOT AND TICKET NO.132257982.PT ♦ 1Q.
547.IG»L 5^- R' I2 T. C=SCH 2 LiLl y +1 R = Ph Y32 R = Me - AReaction of a-lithiosulfides with a variety of electrophiles givesthe expected products in good yield; j2.j. 2^ with carbon dioxide gives2-methylthioacetic acid. Methylenation of ketones 10 has been performedwith 1 and 1 along with higher homologs has been used in the synthesis ofepoxides. 11 Homologation of trialkyl boranes 12 has been effected with 2^,as well as the synthesis of terminal alkynes from carboxylic acids.
13Trost has formed cyclobutanones 14 and cyclopentenes 15 from adducts ofphenylthiocyclopropyl lithium with ketones.Allyl and benzyl sulfides are more readily metallated, and even thedianions of allyl and benzyl thiol 16 have been reported. Trialkyl boranecomplexes 17 and complexation with heteroatoms 18 have been used to directa-alkylation of ambident allyl anion J3. However, the copper reagent givespredominantly y- alky la t ion. 19 Reported syntheses of terpenes, 20 jasmonoids 21and prostaglandin F 2 22 employed species J3. Cecropia juvenile hormoneshave been made via dihydrothiapyrans.
23 Alkylation followed by sigmatropicrearrangement has proven useful. 24Metallated vinyl sulfides serve as acyl anion equivalents and havebeen prepared by addition of organometallic reagents to acetylenic sulfidesand thioketenes and transmetallation.
25 Direct proton abstraction is theormost widely used route and there have been a number of recent examples. DMetallation of 1,3-dien-l-yl sulfides has also been reported. 27 Higherhomologs have been metallated in the a-position.
28Derivatives of thiols such as thioimidates, dithiocarbonates and thio-esters have been deprotonated on the sulfur-bearing carbon and are thoughtto be dipole stabilized 4. 29 Mono and dithiocarbamates also have beendeprotonated.
30 The above compounds are generally limited to the methyland allyl cases; however, a-lithioisopropyl 2,4,6-triisopropylthio-benzoate has been formed in good yield. Gillman and F. Soc, 71, 4062 (1949).2. Chen., 31, 4097 (1966)3. Peterson, ibid., 32, 1717 (1967).4. Bryson, Tetrahedron Lett., 1961 (1977).5.
Mendoza and D. Chem., 44, 1352 (1979).6. Screttas and M. Micha-Screttas, ibid., 44, 713 (1979); T.
Daniewski and R. Weisenfeld, Tetrahedron Lett., 4665 (1978).7. Damonc, and A. Krief, ibid., 1617 (1975);D. Meyer, and A. Chem., 846 (1977).8.
Peterson, Organomet. Rev., A 2, 295 (1972).9. Pichat and J. Beaucourt, J. Com., 10, 103 (1974).10.
Kurata, Tetrahedron Lett., 737 (1972);T. Shono, et al., ibid., 2807 (1978); R. Sowerby and R. Soc, 94, 4758 (1972).11. Shanklin, et al., J.
Soc, 95, 3429 (1973); W. H.Pirkle and P. Chem., 43, 3803 (1978); W.
Altera University Program Qsime Online
H.Pirkle and P. Rinaldi, ibid., 44, 1025 (1979).12. Nigishi, et al., J. Chem., 40, 814 (1975). Yokomatsu, and S.
Shibuya, ibid., 43, 4366 (1978).14. Trost, et al., J. Soc, 99, 3080,3088 (1977); B. M.Trost and J.
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Chem., 43, 2938 (1978); B. Vladchick, Synthesis, 821 (1978).15. Soc, 98, 248 (1976).16.
Seebach and K.-H. Geiss, Angew.
Chem., 86^, 202 (1974); M.Pohmakotr, K.-H. Geiss, and D.
Seebach, Chem. Ber., 112, 1420 (1979).17. Yatagai, and K. Soc, Chem.Commun., 157 (1979).18. Hayashi, and T.
Mukaiyama, Chem. Lett., 259 (1972).19. Oshima, et al., Bull. J., 48, 1567 (1975).20. Chem., 43,4915 (1978); M.
Matsuki, and S. Ito, Tetrahedron Lett.,1121 (1976); J. Biellmann and J. Ducep, ibid., 3707 (1969); E.
EVan Tamelen, et al., J. Soc, 94, 8228 (1972).21. Biellmann and D. Schirlin, Syn.
Coram., 8, 409 (1978).22. Noyori, Tetrahedron Lett., 311 (1970).23. Sotter and R. Soc, 95, 4444 (1973).24. Thiaclaisen, L. Brandsma, and H.
Verkraijsse, Rec Trav. Chim.Pays-Bas, 9^, 319 (1974); K. Yamamoto, and H.
Soc, 95, 4446 (1973). Evans, ibid.,100, 2242 (1978).25. GrSbel and D. Seebach, Synthesis, 357 (1977) and referencescited in section 5.1.26. Cookson and P. Commun., 990(1976) and 821,822 (1978).27.
Everhardus, H. Eeuwhorst, and L. Commun., 801 (1977); R. Grafing and L. Brandsma, Synthesis,578 (1978); R. Everhardus, R. Grafing and L.
Brandsma, Rec Trav.Chim. Pays-Bas, 97, 69 (1978).28.
Muthukrishman and M. Schlosser, Helv. Acta, 59, 13 (1976);J.
Chem., 44, 303 (1979).29. Rev., 78, 275 (1978) and referencescited in section IV.30. Sakurai, and T. Lett., 1483 (1977);T. Mimura, and A. Ari-Izumi, Tetrahedron Lett., 2425 (1977)and references cited therein.31.
Becker, unpublished results.-3-THE DESIGN, SYNTHESIS, AND BIOLOGY OF DNA INTERCALATING AGENTSReported by David W. RobertsonSeptember 27, 1979Intercalation is the noncovalent insertion of planar aromatic mole-cules between two successive base pairs of double-helical DNA.
SinceLerman's classic delineation of intercalation 1 over two decades ago, manyantitumor agents, mutagens, carcinogens, and teratogens have been found toexert their effects through intercalative binding to DNA. Because of thebiological and clinical importance of intercalation, molecular biologists,oncologists, and chemists have intensively investigated the nature of thisphenomenon. In this paper the evidence adduced for intercalation will besurveyed, and the design, synthesis, and biological applications of DNAintercalating agents will be examined.Structural Requirements for Intercalation. Ethidium bromide (1), aphenanthridinium trypanocide, and proflavin (2), a powerful frameshift mutagen,are archetypical intercalators (Figure 1). Both inhibit DNA and RNA synthesis 2and are two of the more widely studied intercalators. As these two examplesindicate, molecules which intercalate are planar aromatic molecules; largedeviations from molecular planarity in the form of bulky substituents such asin 2,7-di-t-butylproflavin 3 prevent intercalation. In addition to the endo-cyclic nitrogens which most high-affinity intercalators possess, many inter-calators also contain exocyclic amine groups.
These, if positioned properly,result in enhanced binding affinity. ^Figure 19 10 12H 2 N3 'NH 2proflavin (2)ethidium bromide (1)Methods by Which Intercalation and the Resultant DNA Deformations areStudied. In the normal DNA duplex, the adjacent parallel base pairs are invan der Waals contact with one another. A planar molecule is accommodated inthe helix by a local unwinding of the deoxyribose backbone; this separatesthe base pairs sufficiently to allow the insertion of the intercalator whilestill maintaining the integrity of the interbase Watson-Crick hydrogenbonding.
The DNA-intercalator complex is stabilized by many physicalprocesses; hydrophobic interactions, electronic interactions between the -rr-clouds of the intercalator and DNA bases, and hydrogen bonding have all beenshown to contribute to the stabilization of the complex. Because intercala-tive interactions perturb the physical and chemical characteristics of boththe DNA and the intercalator, a number of physicochemical probes can be usedto examine the interaction of an intercalator with DNA.Fluorescence and Visible Spectroscopy. Upon intercalation of ethidiumbromide into DNA, hypochromic and bathochromic shifts are produced in thevisible spectrum of ethidium. 5 When the intercalator is treated with-4-increasing concentrations of DNA, the absorbance spectrum of the drug shiftsprogressively towards a limit which represents the spectrum of the drug whenfully intercalated (Figure 2).
The curves pass through a well-definedisosbestic point, indicating that two forms of the drug, free and bound, arecontributing to the absorbance.Figure 2^V 1 1freefully complexed400 450 500 550 600Wavelength (nm)From a combination of the visible spectrum and a Scatchard plot, theassociation constant and number of binding sites in the DNA can be estimated.For ethidium bromide the approximate association constant is 5 x 10 M, withup to one ethidium per two base pairs being bound. The nonlinearity of theScatchard plots 5 and many other lines of evidence indicate that two types ofbinding are possible. After the high-affinity intercalative sites areexhausted, more positively charged ethidium molecules can interact with thenegatively charged phosphates of the DNA. At a ratio of one drug per nucleo-tide residue, an electrostatically neutral complex precipitates. 5When ethidium is complexed with DNA there is a dramatic increase in thefluorescence of the intercalator 6 (Figure 3). The most plausible explanationfor the fluorescence enhancement is the immersion of the ethidium into ahydrophobic region of the nucleic acid upon intercalation.
This reduces therate of excited state-solvent proton transfer, and increases the fluorescencelifetime by a factor of 12. 7Figure 31000Fluorescenceintensity 500300 400 500 600A (nm)NMR Spectroscopy. Numerous X H- and 31 P-NMR experiments have been con-ducted on mixtures of complementary oligonucleotides and intercalators. 8 10When 9-aminoacridine in D 2 is titrated with increasing concentrations ofdGpC, the nonexchangeable protons experience a linear upfield shift untila 1:2 acridine-dinucleotide stoichiometry is reached 11 (Figure A).
Thissuggests the formation of a minihelix with the acridine sandwiched betwef n-5-Figure 459i ri i— r—— r—60si-2^6.1.o'I'a-. 0.5 ppm (H-l, H-10) relative to the corresponding protons in uncomplexedethidium. 12 Calculated values for the upfield shifts based upon the atomicdiamagnetic anisotropy and ring current contributions compare favorably withthe experimentally determined values.Circular Dichroism and X-Ray Studies. When ethidium is mixed with7—9double-helical DNA, circular dichroism is induced in the nonchiral ethidium. RVarious dichroism studies have indicated that the plane of the inter calator isapproximately parallel to the planes of the adjacent base pairs.Complexes of intercalators and complementary oligonucleotides can some-times be crystallized, allowing the direct visualization of inter calativebinding in a miniature double helix. 13 All the X-ray studies confirm thepostulates which were based on spectroscopic data.Hydrodynamics.
Intercalation, because of the necessary local unwindingof the helix, results in a lengthening and stiffening of the DNA molecule.This causes the intercalated DNA to become more rod-like than its pristinecounterpart; as a consequence the intrinsic viscosity of DNA solutions isincreased. 1 Because the additional length due to an intercalator is approxi-mately the same as that of an additional base pair and most intercalatorsare of less molecular weight than a base pair, the average molecular weightper unit length of the DNA decreases; this results in a decrease of thesedimentation coefficient. 1A unique method of studying intercalation is the interaction of theintercalating agent with covalently closed, supercoiled DNA. These DNAsdisplay large changes in their supercoiling as a result of local intercalativehelix unwinding. ' 5 As increasing concentrations of intercalator are added,the right-handed supercoiling decreases until open circles are obtained.Additional intercalating agent results in more unwinding until torsionalstrains produce left-handed supercoils (Figure 5). The progress of the titra-Figure 5-^right-handedleft-handed-6-tion is most conveniently monitored by changes in the sedimentation coeffi-cient of the DNA (Figure 6).
All known intercalating agents remove andreverse the supercoils of closed circular duplex DNA.Figure 6right-handedsupercoilsleft-handedsupercoilsopen circles0.02 0.06 0.1drug molecules bound per nucleotideIntercalative Photoaf f inity Labelling Reagents. Intercalators such asethidium bromide have been widely used to probe numerous biological structuressuch as tRNA, 5S-RNA, and chromatin; they have also been of great utility instudies of biological processes such as DNA replication and transcription,and the mechanism of frameshift mutagenesis. 16 One of the major disadvantagesof using noncovalently attached intercalators in the study of biologicalstructures and processes is that during the time of a biological or physicalassay the intercalators can move from site to site. 17 In in vivo studies,cell fractionation techniques can rearrange concentration gradients and drugdistributions among subcellular compartments. 18 This problem can be circum-vented by the use of intercalating agents which can be covalently attachedto the DNA or juxtaposed proteins. Photoaf f inity labelling is a techniquewhich permits this covalent attachment.
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At least two different types ofintercalative photoaf f inity labels have been developed: the furocoumarins(Figure 7) and azide analogs of ethidium and acridine.Figure 7psoralin (3)angelicin (A)The furocoumarins are a group of naturally occurring and syntheticcompounds which manifest interesting photobiological effects such as skinThey have been used therapeutically since antiquity; thesensitization,19fruit and seed extracts used by the ancient Egyptians to treat vitiligocontained furocoumarins. 20 A more recent medicinal application is thephotochemo therapy of psoriasis and other skin diseases19The mechanism by which furocoumarins exert their photobiological effectsis photoreaction with DNA; a linear relationship normally exists between the-7-biological effects and the extent of photoreaction.
2 1 In the absence ofelectromagnetic excitation there is no disruption of cellular processes.Furocoumarins intercalate into DNA and form cyclobutane adducts with thepyrimidine bases in DNA when the complex is irradiated with 365 nm light.^The linear furocoumarins, the psoralens (Figure 7), are capable of formingeither monofunctional or difunctional adducts with DNA. The difunctionaladducts are due to the reaction of both the 3,4 and 4', 5. double bonds withpyrimidine bases in each strand of duplex DNA. This crosslinks the DNAstrands and prevents them from becoming separated even under conditionswhich normally denature DNA. The crosslinking has been demonstrated bydenaturation-renaturation kinetics 22 and by electron microscope studies. 23Cole has shown that the inhibition of certain bacterial functions by psoralinis approximately equal to the rate of formation of a single psoralen cross-link per DNA molecule. 24Numerous derivatives of psoralen have been prepared in order to optimizetheir DNA interaction characteristics.
Hearst and Rapoport reported 2 - 5 that4'-aminomethyl-4,5 l,8-trimethylpsoralen is the best derivative for photo-reaction with DNA. It can bind to DNA to the extent of one drug moleculeper five base pairs with a 65% photoattachment efficiency.The angular furocoumarins, the angelicins (Figure 7), form only mono-functional adducts and have a low ability to produce photosensitized effects.^The angelicins, however, may be the furocoumarins of choice if inhibition ofDNA or RNA synthesis is the only desired biological effect. Crosslinking isa very severe type of damage to a cell's genome and often leads to undesirableresults.9-Azidoacridine, another type of intercalative photoaffinity label, wasprepared to help elucidate the mechanism of 9-aminoacridine ' s frameshiftmutagenicity. 26 Surprisingly, 9-azidoacridine is not a frameshift mutagen;it is a base pair substitution mutagen with or without photolytic activa-tion. Placing the azide moiety at other positions on 9-aminoacridine wouldperhaps yield a photoaffinity label which still maintains the frameshiftmutagenicity of the parent compound.A more suitable photoaffinity label for the frameshift mutagen studiesis the fluorescent intercalator 8-azidoethidium bromide. 7 18 27 Structure-activity relationships of various phenanthridinium analogs of ethidiumbromide indicated that the 8-amino group could be modified with littlechange in its interaction with DNA.
4 This was found to be true with 8-azidoethidium bromide. In the dark, 8-azidoethidium bromide and ethidium bromideare competitive inhibitors in DNA binding studies and cause similar pertur-bations in the hydrodynamic properties of native DNA. The azide analogbinds strongly to DNA (K a =2-3xl0 5 M 1 ), and can be photoattached to DNAwith efficiencies approaching 75%. 27 Unlike 9-azidoacridine, 8-azidoethidiumbromide behaves as a frameshift mutagen both before and after covalentattachment to DNA.Antibiotic Intercalators. Many naturally occurring antibiotics havebeen shown to exert their effects through intercalative binding to DNA.The actinomycins, 28 ' 29 quinomycins, 30 and triostlns 31 all intercalate intoDNA with very high affinity. The two most heavily studied intercalatorsare actinomycin D (5, Figure 8) and echinomycin (60, a member of thequinomycin antibiotic family.
EVan Tamelen, et al., J. Soc, 94, 8228 (1972).21. Biellmann and D. Schirlin, Syn.
Coram., 8, 409 (1978).22. Noyori, Tetrahedron Lett., 311 (1970).23. Sotter and R. Soc, 95, 4444 (1973).24. Thiaclaisen, L. Brandsma, and H.
Verkraijsse, Rec Trav. Chim.Pays-Bas, 9^, 319 (1974); K. Yamamoto, and H.
Soc, 95, 4446 (1973). Evans, ibid.,100, 2242 (1978).25. GrSbel and D. Seebach, Synthesis, 357 (1977) and referencescited in section 5.1.26. Cookson and P. Commun., 990(1976) and 821,822 (1978).27.
Everhardus, H. Eeuwhorst, and L. Commun., 801 (1977); R. Grafing and L. Brandsma, Synthesis,578 (1978); R. Everhardus, R. Grafing and L.
Brandsma, Rec Trav.Chim. Pays-Bas, 97, 69 (1978).28.
Muthukrishman and M. Schlosser, Helv. Acta, 59, 13 (1976);J.
Chem., 44, 303 (1979).29. Rev., 78, 275 (1978) and referencescited in section IV.30. Sakurai, and T. Lett., 1483 (1977);T. Mimura, and A. Ari-Izumi, Tetrahedron Lett., 2425 (1977)and references cited therein.31.
Becker, unpublished results.-3-THE DESIGN, SYNTHESIS, AND BIOLOGY OF DNA INTERCALATING AGENTSReported by David W. RobertsonSeptember 27, 1979Intercalation is the noncovalent insertion of planar aromatic mole-cules between two successive base pairs of double-helical DNA.
SinceLerman's classic delineation of intercalation 1 over two decades ago, manyantitumor agents, mutagens, carcinogens, and teratogens have been found toexert their effects through intercalative binding to DNA. Because of thebiological and clinical importance of intercalation, molecular biologists,oncologists, and chemists have intensively investigated the nature of thisphenomenon. In this paper the evidence adduced for intercalation will besurveyed, and the design, synthesis, and biological applications of DNAintercalating agents will be examined.Structural Requirements for Intercalation. Ethidium bromide (1), aphenanthridinium trypanocide, and proflavin (2), a powerful frameshift mutagen,are archetypical intercalators (Figure 1). Both inhibit DNA and RNA synthesis 2and are two of the more widely studied intercalators. As these two examplesindicate, molecules which intercalate are planar aromatic molecules; largedeviations from molecular planarity in the form of bulky substituents such asin 2,7-di-t-butylproflavin 3 prevent intercalation. In addition to the endo-cyclic nitrogens which most high-affinity intercalators possess, many inter-calators also contain exocyclic amine groups.
These, if positioned properly,result in enhanced binding affinity. ^Figure 19 10 12H 2 N3 'NH 2proflavin (2)ethidium bromide (1)Methods by Which Intercalation and the Resultant DNA Deformations areStudied. In the normal DNA duplex, the adjacent parallel base pairs are invan der Waals contact with one another. A planar molecule is accommodated inthe helix by a local unwinding of the deoxyribose backbone; this separatesthe base pairs sufficiently to allow the insertion of the intercalator whilestill maintaining the integrity of the interbase Watson-Crick hydrogenbonding.
The DNA-intercalator complex is stabilized by many physicalprocesses; hydrophobic interactions, electronic interactions between the -rr-clouds of the intercalator and DNA bases, and hydrogen bonding have all beenshown to contribute to the stabilization of the complex. Because intercala-tive interactions perturb the physical and chemical characteristics of boththe DNA and the intercalator, a number of physicochemical probes can be usedto examine the interaction of an intercalator with DNA.Fluorescence and Visible Spectroscopy. Upon intercalation of ethidiumbromide into DNA, hypochromic and bathochromic shifts are produced in thevisible spectrum of ethidium. 5 When the intercalator is treated with-4-increasing concentrations of DNA, the absorbance spectrum of the drug shiftsprogressively towards a limit which represents the spectrum of the drug whenfully intercalated (Figure 2).
The curves pass through a well-definedisosbestic point, indicating that two forms of the drug, free and bound, arecontributing to the absorbance.Figure 2^V 1 1freefully complexed400 450 500 550 600Wavelength (nm)From a combination of the visible spectrum and a Scatchard plot, theassociation constant and number of binding sites in the DNA can be estimated.For ethidium bromide the approximate association constant is 5 x 10 M, withup to one ethidium per two base pairs being bound. The nonlinearity of theScatchard plots 5 and many other lines of evidence indicate that two types ofbinding are possible. After the high-affinity intercalative sites areexhausted, more positively charged ethidium molecules can interact with thenegatively charged phosphates of the DNA. At a ratio of one drug per nucleo-tide residue, an electrostatically neutral complex precipitates. 5When ethidium is complexed with DNA there is a dramatic increase in thefluorescence of the intercalator 6 (Figure 3). The most plausible explanationfor the fluorescence enhancement is the immersion of the ethidium into ahydrophobic region of the nucleic acid upon intercalation.
This reduces therate of excited state-solvent proton transfer, and increases the fluorescencelifetime by a factor of 12. 7Figure 31000Fluorescenceintensity 500300 400 500 600A (nm)NMR Spectroscopy. Numerous X H- and 31 P-NMR experiments have been con-ducted on mixtures of complementary oligonucleotides and intercalators. 8 10When 9-aminoacridine in D 2 is titrated with increasing concentrations ofdGpC, the nonexchangeable protons experience a linear upfield shift untila 1:2 acridine-dinucleotide stoichiometry is reached 11 (Figure A).
Thissuggests the formation of a minihelix with the acridine sandwiched betwef n-5-Figure 459i ri i— r—— r—60si-2^6.1.o'I'a-. 0.5 ppm (H-l, H-10) relative to the corresponding protons in uncomplexedethidium. 12 Calculated values for the upfield shifts based upon the atomicdiamagnetic anisotropy and ring current contributions compare favorably withthe experimentally determined values.Circular Dichroism and X-Ray Studies. When ethidium is mixed with7—9double-helical DNA, circular dichroism is induced in the nonchiral ethidium. RVarious dichroism studies have indicated that the plane of the inter calator isapproximately parallel to the planes of the adjacent base pairs.Complexes of intercalators and complementary oligonucleotides can some-times be crystallized, allowing the direct visualization of inter calativebinding in a miniature double helix. 13 All the X-ray studies confirm thepostulates which were based on spectroscopic data.Hydrodynamics.
Intercalation, because of the necessary local unwindingof the helix, results in a lengthening and stiffening of the DNA molecule.This causes the intercalated DNA to become more rod-like than its pristinecounterpart; as a consequence the intrinsic viscosity of DNA solutions isincreased. 1 Because the additional length due to an intercalator is approxi-mately the same as that of an additional base pair and most intercalatorsare of less molecular weight than a base pair, the average molecular weightper unit length of the DNA decreases; this results in a decrease of thesedimentation coefficient. 1A unique method of studying intercalation is the interaction of theintercalating agent with covalently closed, supercoiled DNA. These DNAsdisplay large changes in their supercoiling as a result of local intercalativehelix unwinding. ' 5 As increasing concentrations of intercalator are added,the right-handed supercoiling decreases until open circles are obtained.Additional intercalating agent results in more unwinding until torsionalstrains produce left-handed supercoils (Figure 5). The progress of the titra-Figure 5-^right-handedleft-handed-6-tion is most conveniently monitored by changes in the sedimentation coeffi-cient of the DNA (Figure 6).
All known intercalating agents remove andreverse the supercoils of closed circular duplex DNA.Figure 6right-handedsupercoilsleft-handedsupercoilsopen circles0.02 0.06 0.1drug molecules bound per nucleotideIntercalative Photoaf f inity Labelling Reagents. Intercalators such asethidium bromide have been widely used to probe numerous biological structuressuch as tRNA, 5S-RNA, and chromatin; they have also been of great utility instudies of biological processes such as DNA replication and transcription,and the mechanism of frameshift mutagenesis. 16 One of the major disadvantagesof using noncovalently attached intercalators in the study of biologicalstructures and processes is that during the time of a biological or physicalassay the intercalators can move from site to site. 17 In in vivo studies,cell fractionation techniques can rearrange concentration gradients and drugdistributions among subcellular compartments. 18 This problem can be circum-vented by the use of intercalating agents which can be covalently attachedto the DNA or juxtaposed proteins. Photoaf f inity labelling is a techniquewhich permits this covalent attachment.
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At least two different types ofintercalative photoaf f inity labels have been developed: the furocoumarins(Figure 7) and azide analogs of ethidium and acridine.Figure 7psoralin (3)angelicin (A)The furocoumarins are a group of naturally occurring and syntheticcompounds which manifest interesting photobiological effects such as skinThey have been used therapeutically since antiquity; thesensitization,19fruit and seed extracts used by the ancient Egyptians to treat vitiligocontained furocoumarins. 20 A more recent medicinal application is thephotochemo therapy of psoriasis and other skin diseases19The mechanism by which furocoumarins exert their photobiological effectsis photoreaction with DNA; a linear relationship normally exists between the-7-biological effects and the extent of photoreaction.
2 1 In the absence ofelectromagnetic excitation there is no disruption of cellular processes.Furocoumarins intercalate into DNA and form cyclobutane adducts with thepyrimidine bases in DNA when the complex is irradiated with 365 nm light.^The linear furocoumarins, the psoralens (Figure 7), are capable of formingeither monofunctional or difunctional adducts with DNA. The difunctionaladducts are due to the reaction of both the 3,4 and 4', 5. double bonds withpyrimidine bases in each strand of duplex DNA. This crosslinks the DNAstrands and prevents them from becoming separated even under conditionswhich normally denature DNA. The crosslinking has been demonstrated bydenaturation-renaturation kinetics 22 and by electron microscope studies. 23Cole has shown that the inhibition of certain bacterial functions by psoralinis approximately equal to the rate of formation of a single psoralen cross-link per DNA molecule. 24Numerous derivatives of psoralen have been prepared in order to optimizetheir DNA interaction characteristics.
Hearst and Rapoport reported 2 - 5 that4'-aminomethyl-4,5 l,8-trimethylpsoralen is the best derivative for photo-reaction with DNA. It can bind to DNA to the extent of one drug moleculeper five base pairs with a 65% photoattachment efficiency.The angular furocoumarins, the angelicins (Figure 7), form only mono-functional adducts and have a low ability to produce photosensitized effects.^The angelicins, however, may be the furocoumarins of choice if inhibition ofDNA or RNA synthesis is the only desired biological effect. Crosslinking isa very severe type of damage to a cell's genome and often leads to undesirableresults.9-Azidoacridine, another type of intercalative photoaffinity label, wasprepared to help elucidate the mechanism of 9-aminoacridine ' s frameshiftmutagenicity. 26 Surprisingly, 9-azidoacridine is not a frameshift mutagen;it is a base pair substitution mutagen with or without photolytic activa-tion. Placing the azide moiety at other positions on 9-aminoacridine wouldperhaps yield a photoaffinity label which still maintains the frameshiftmutagenicity of the parent compound.A more suitable photoaffinity label for the frameshift mutagen studiesis the fluorescent intercalator 8-azidoethidium bromide. 7 18 27 Structure-activity relationships of various phenanthridinium analogs of ethidiumbromide indicated that the 8-amino group could be modified with littlechange in its interaction with DNA.
4 This was found to be true with 8-azidoethidium bromide. In the dark, 8-azidoethidium bromide and ethidium bromideare competitive inhibitors in DNA binding studies and cause similar pertur-bations in the hydrodynamic properties of native DNA. The azide analogbinds strongly to DNA (K a =2-3xl0 5 M 1 ), and can be photoattached to DNAwith efficiencies approaching 75%. 27 Unlike 9-azidoacridine, 8-azidoethidiumbromide behaves as a frameshift mutagen both before and after covalentattachment to DNA.Antibiotic Intercalators. Many naturally occurring antibiotics havebeen shown to exert their effects through intercalative binding to DNA.The actinomycins, 28 ' 29 quinomycins, 30 and triostlns 31 all intercalate intoDNA with very high affinity. The two most heavily studied intercalatorsare actinomycin D (5, Figure 8) and echinomycin (60, a member of thequinomycin antibiotic family.
Both of these compounds differ from pre-viously mentioned intercalators in that they have peptide moieties, and-8-after incercalation of the chromophore, these peptides lie in the minorgroove of the DNA.
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