7# NW'T 4KFz x *7T R Experiment VIII Modeling Molecular Structure pre-lab assignment Reading: Before coming to your discussion section, read: 1. Sections 8.3 ->8.7 in Olmstead and Williams. 2. The remainder of this experiment in this manual. Pre-lab assignment to be handed in: Prepare written responses to the following questions and submit your work to your instructor at the beginning of your discussion period: 1. Which of the atoms in the molecules to be modeled in Part I can exist with expanded octets? What criteria do you use in deciding? 2. Use Figure 5 to predict the dihedral angle for H2O2. 3. The hydrogen atoms in H2O2 are partially positively charged. Will the hydrogens want to be as close as possible or as far apart as possible? What dihedral angle makes the hydrogens come as close as possible? What dihedral angle makes them as far apart as possible? Introduction Chemists often use models to help them to represent and understand the behavior of molecules, which are too small to be seen. One example of models is the "ball and stick" type wooden or plastic structures often used to represent molecules. Such representations give us some sense of the way atoms are arrayed in molecules and of the molecule's three-dimensional structure. However, if a model is to be really useful it must be more than an artistic representation. It must serve as an aid to understanding a structure or phenomenon. It must be a tool for predicting properties and/or behavior. It must help us go beyond a simple appreciation of a static object and give us some sense the object's potential. In this experiment, we will deal with two types of models. In Part I we will work with some traditional "ball and stick" molecular models and in Part II we will explore the computer programs QUANTA/CHARMm that model the conformational energy of molecules. In both parts of the experiment, we try to emphasize the value of the model as a source of information on molecular behavior or as a facilitator for predicting such behavior. The exercises described in this experiment are designed to be somewhat open-ended. Redo things that are initially confusing or build new molecules and try to predict their shapes, polarity, etc. Look at some more complex structures with QUANTA. First predict the molecule's behavior and then see what the program tells you. Such "play" is an ideal way to build your scientific intuition. Devise your own theories (working models), test them, modify them and test them again. Part I. The Geometrical Structure of Molecules In class we are currently working with Lewis Structure representations for molecules and the VSEPR theory for predicting molecular shape and polarity. This model building exercise is designed to give you a more concrete and three dimensional view of these representations to help you to get the most information possible out of these simple models for molecules. Procedure Using models it is relatively easy to see both geometry and polarity , as well as to deduce Lewis structures. In this exercise, you will assemble models for a sizable number of common chemical species and interpret them in terms of geometry, polarity and possible isomerism. You may work in pairs. The models you will use consist of wooden balls (atoms), pegs (single bonds) and springs (used for multiple bonds). The balls represent the nuclei and inner core electrons. The pegs and springs represent valence shell bonding or nonbonding (lone pair) electron pairs. Most of the heavy atoms in this exercise (C, N, O, F, S, Cl, Sb and I) obey the octet rule. In their bonded structures, such atoms have four electron pairs around a central core and are represented in the model set as black balls with four holes. When these atoms participate in a multiple bond, more than one of their electron pairs (holes) are connected to the same atom. Springs are required for these bonds because of the angles between the holes on the same atom. A few of the structures will require "expanded octet" atoms. Atoms with an expanded octet can accommodate more than eight electrons. Atoms in the third and higher periods of the periodic table in the transition metal or representative groups (IIIA-VIIA) can have expanded octets. Initially we will assume that all heavy atoms obey the octet rule, and in our procedure we will discover the atoms that require "expanded octets". A. Constructing Lewis Structures In assembling molecular models of the kind we are considering, it is usually desirable to use a systematic approach. The method suggested below parallels the method for writing Lewis structures that we have discussed in class. We will illustrate the recommended method by developing a model for formaldehyde, CH2O. 1. Determine the total number of valence electrons and select the appropriate number of pegs. Recall that for the "main group elements," (of the Periodic Table) which we will be using (H, C, N, O, F, S, Cl, Sb and I), the number of valence electrons on an atom is equal to the group number. If the structure to be constructed has one or more electric charges (+or -), add 1 electron to the total for each negative charge and subtract 1 electron for each positive charge. Each peg in our models represents 2 electrons, so once you have determined the total number of electrons, count the number of pegs to be used in your structure. For formaldehyde each H contributes 1 valence electron, the C contributes 4 valence electrons and the O contributes 6 valence electrons to the overall structure. This yields a total of (2 + 4 + 6) or 12 valence electrons. Therefore, our structure will require 6 pegs. 2. Select the appropriate wooden balls and deduce a skeleton structure for the molecule. Each hydrogen atom in our models is represented by a yellow ball with 1 hole (accommodating 1 electron pair) and, initially, each "heavy atom" will be represented by a black ball having 4 holes (accommodating 4 electron pairs, an "octet"). Thus select one ball of the appropriate type for each atom in your molecular formula. The skeleton structure is the sequence of attachments (which atoms are bonded to which) within the molecule. Usually this can be discerned by the order in which the atoms are presented in the molecular formula. Often the first atom in the formula will be the central atom with those following all being attached to it. For more complex structures (e.g. ethyl alcohol in Section C.1.c, below) the skeleton structure will sometimes be suggested by subdivisions in the molecular formula (CH3CH2OH ethyl alcohol). Often there will be several possible and/or reasonable skeleton structures for a given molecular formula. Such molecular formulas are said to have several possible structural isomers (see B.5, below). Note that if your skeleton structure requires more than four atoms to be attached to a central atom, that central atom will require an "expanded octet". In such cases, replace the black ball (4 holes) with an appropriate expanded octet ball (5 holes, light blue or 6 holes, white) and proceed to Step3. For formaldehyde we will require 2 yellow balls (2 H's) and 2 black balls (1 C and 1 O). We can choose a skeleton structure having C (black ball with 4 holes) as the central atom with the H's (yellow balls with 1 hole) and the O (black ball with 4 holes) attached to it. 3. Assemble a single bonded structure for the molecule. Simply connect the adjacent atoms in your skeleton structure by inserting each end of a wooden peg into holes in adjacent atoms. If there are no empty holes in any of the atoms and no unused pegs at this point the structure is complete, proceed to Part B. If there are empty holes or extra pegs, proceed to step 4. For formaldehyde, we will require three pegs and our single bonded structure will have three of the holes in the C occupied by pegs attached to the O and each H. Note that in this case there is 1 empty hole in the C, there are 3 empty holes in the O and there are 3 unused pegs. So we proceed to Step 4. 4. Add lone pairs. If you have additional pegs, place them in the empty holes. If all of the holes are filled and all of the pegs used at this point, the structure is complete2; proceed to Part B. If there are empty holes or extra pegs, proceed to step 5. For formaldehyde, we can fill 3 of the 4 empty holes. Thus, we are left with 1 empty hole on either the C or the O and we proceed to Step 5. 5. Add Multiple bonds and/or expand octets. a. If you have empty holes, your molecule is said to be unsaturated since there are not sufficient electrons to fill the octet of all atoms separately. Thus, some atoms will need to share more than one electron pair with one or more of their neighbors. A double bond can be created by using a lone pair of electrons on one atom to help fill an empty hole on an adjacent atom (using 2 lone pairs from a single atom to fill 2 empty holes on a single adjacent atom produces a triple bond). To locate the most appropriate sites for multiple bonds, arrange the lone pairs in your single bonded structure so that each atom with an empty hole is adjacent to at least 1 atom with at least 1 lone pair. To complete the multiple bonds, we require more flexibility in bonding than is available with wooden pegs, so we will use springs to form the double and triple bonds. At each site where there is an empty hole and an adjacent lone pair, replace both the lone pair peg and the peg representing the bond between the adjacent atoms with springs. This will provide the structural flexibility to allow you to create the multiple bond by inserting the free end of the lone pair spring into the empty hole on the adjacent atom. Complete octets of all atoms (fill all empty holes) by creating multiple bonds. Usually there will be more than one arrangement of multiple bonds that will fill all of the holes. At this point in our exercise, any one of the possible multiply bonded structures is sufficient. Your structure should now be complete; proceed to Part B. For formaldehyde we can use a structure with an empty hole on C and 3 lone pairs on O or one with an empty hole on O and 1 lone pair on C. In either case we replace the cO bond peg and one lone pair with springs and then form a double bond between C and O by connecting them with the two springs. This completes the structure for formaldehyde since all holes are filled and all pegs and springs are used. b. If you have additional pegs. Your molecule has more electrons than can be accommodated in octets on all heavy atoms and the octet of one or more of the atoms in the structure will need to be "expanded". Atoms from the third row of the Periodic Table or below can accommodate more than 8 valence electrons; thus they can have "expanded octets". Select the atoms in your structure which can have expanded octets (see prelab assignment), and replace them in the structure with either a light blue (5 holes 10 electrons) or a white (6 holes 12 electrons) ball depending on the number of extra lone pairs of electrons they will hold in your final structure. Fill the extra holes with your unused pegs and your structure should be complete; proceed to Part B. B. Interpretation of Molecular Models 1. Draw the Lewis Dot Formula for your Model. Simply write down the atoms, bonds, and lone pairs. This exercise should help you gain a sense of the three dimensional structure represented by Lewis dot formulas. Ultimately, it will be very useful for you to use the two dimensional Lewis formulas to visualize three dimensional structures without the aid of "ball and stick" models. Lewis structures are much quicker and easier to produce and when you can "see them in three dimensions" they will be very powerful reasoning tools for you to use in predicting the shape and properties of molecules. For formaldehyde the Lewis dot formula is: :O: C / \ H H 2. Examine the Molecule's Molecular Geometry and describe the general overall shape of the molecule. Do the outer most atoms form a recognizable solid form (e.g. a cube, a pyramid, a tetrahedron, a bipyramid, an octahedron, etc.)? Is the molecule flat (planar) or are the atoms all in a line (linear)? If the molecule is planar, does it have an easily recognizable regular shape (e.g. a triangle, a square, a pentagon, etc.)? Formaldehyde is flat (all of its atoms lie in one plane) with its peripheral atoms forming the vertices of a triangle. Such a structure is called trigonal planar. 3. Predict the Molecule's Polarity. Polar bonds are formed when two atoms with different electronegativity bond. The more electronegative atom will get more than its fair share of electrons, and be the negative end of the bond. The less electronegative atom will have some electron density pulled away, so it will be the positive end of the dipole. The total dipole moment of the molecule is the vector sum of the bond dipoles. For example, both CO2 and SO2 have polar bonds. However, the bond dipoles in carbon dioxide oppose each other and cancel out, since CO2 is a linear molecule. In SO2 the bond dipoles don't cancel, therefore making the whole molecule polar.  Note that the difference in electronegativity between C, at 2.5, and H, at 2.1, is not sufficient to give a significant bond dipole. The difference between C, at 2.5, and O, at 3.5, is markedly polar. Does your molecule contain polar bonds? If so, which end of each polar bond is more positive and which more negative? From the shape noted in 2, above, predict whether the molecule as a whole should be polar. If you believe it to be polar, indicate the direction of the molecule's dipole moment on the Lewis Dot Formula you drew in 1, above. 4. Examine the Molecule's Potential for Resonance. If your molecule has multiple bonds, resonance is possible. If it is possible to arrange the multiple bonds in more than one way and still maintain the same skeleton structure then each of the possible structures is a resonance structure. In such cases, the actual structure of the molecule is best represented by an average of all of the resonance structures weighted such that the most reasonable resonance structures (those with no isolated formal charges on atoms) contribute more to the average structure than do the less reasonable ones (those containing atoms with formal charge). As we will see in later class discussions, the existence of resonance structures for a molecule can be an indication that it is more stable than we would predict from any one of its individual resonance structures. There is only one place for the double bond in formaldehyde. Thus, it has no stable resonance structures. 5. Examine the Molecule's Potential for Structural Isomerism. If it is possible to arrange the atoms of your molecule in another skeleton structure that is different from the one chosen in Part A.2., then its molecular formula does not describe a unique molecule. Two or more structures that have the same molecular formula but different skeleton structures (sequences of attachment of atoms) are called structural isomers. Note, if there is only one possible structure, there are no isomers. If your molecule has structural isomers, build a model of at least one of them and compare it with your original model. It is possible to arrange the atoms of formaldehyde with the O as the central atom and the C and 2 H's as the peripheral atoms. Build a model for this structure. This second isomer is not a stable molecule probably because the central O has a formal charge of +2 and the C has a formal charge of -2. Molecules with such separated formal charges are not usually very stable. C. Molecules to Explore 1. Simple Structures a. Methane Construct a model of methane (CH4). Place the model on your desk top and note the symmetry of this saturated tetrahedral molecule. Carry out the additional structural analyses described in Part B above. b. Dichloromethane Convert your methane molecule to dichloromethane (CH2Cl2) by replacing two hydrogen atoms with chlorine atoms. Do the additional structural analyses described in Part B above. Draw the Lewis dot structures for all of the structural isomers of CH2Cl2 that you think may exist. Build structures of all of your isomers and compare them to be sure that they are not superimposable upon each other. Actually, how many structural isomers are there with the molecular formula CH2Cl2? c. Ethyl alcohol (ethanol, CH3CH2OH) and dimethyl ether (CH3OCH3) Construct models for ethyl alcohol (ethanol) and dimethyl ether. These two compounds are structural isomers, but they have very different properties. Ethanol is very soluble in water, while dimethyl ether is not. The boiling point of ethanol is much higher (78.5C) than dimethyl ether (-24C). These differences are caused by the presence of the O-H bond, which allows hydrogen bonding to be an important contribution to the forces between molecules in ethanol. 2. Additional Molecules Build a model for each of the following molecular formulas and then analyze your model according to the procedure listed in Part B above. Note that some of the species listed are ions; be sure to add or remove the appropriate number of electrons when calculating the total number of electrons in these structures. Molecular Formulas H2O NH3 H2O2 C2H4 C3H4 NH4+ SO2 SF4 ClOS(-,2) SO42- SbF5 IA(-,3) Part II. Conformational Analysis The shape of a molecule is determined by the bond lengths and angles between its atoms. The bond length between a pair of bonded atoms, A-B, is primarily determined by the hybridization of the two atoms. The bond angle of three directly bonded atoms, A-B-C, is determined primarily by the hybridization of the central atom, B. However, these lengths and angles are not enough to predict the shape of complex molecules. Rotation angles around bonds are also necessary to specify the conformation of a molecule. As you have discovered or will discover in Part I, singly bonded atoms can rotate around their bonding axis. This rotation of single bonds usually requires only small amounts of energy. Thus, at room temperature many singly bonded atoms rotate freely. Single bond rotations play an important role in determining molecular shapes. The amount of rotation around a bond determines the conformation of the bond. The quantity which specifies the conformation of a bond is the dihedral angle. The dihedral angle is the angle between the substituent atoms attached to the bonded atoms being considered. For example, in hydrogen peroxide two possible conformations of the OO bond are shown in Figure1. In this case, the dihedral angle of the OO bond is the angle between the hydrogen atoms attached to each oxygen atom. Some dihedral angles are energetically more favorable than others. We only consider those bonds whose rotation will change the relative positions of atoms in the molecule (e.g. the OO bond but not the HO bonds of H2O2).  Figure 1: Dihedral angles for H2O2. The goal of this exercise is to find the lowest energy conformation of hydrogen peroxide. The most important factors that effect the energy of the molecule as the dihedral angle changes are the dihedral, Van der Waal's, and electrostatic energies. Dihedral Energy: Two possible conformations of ethane are shown in Figure 2. The eclipsed  Figure 2. Eclipsed and staggered ethane.conformer is higher in energy than the staggered form. The increase in dihedral energy of the eclipsed form is caused by the repulsion of the electrons in the C-H bonds on different ends of the molecule. In the staggered form, the bonds are further apart thus reducing the electron-electron repulsion between the bonds. A plot of the dihedral energy of ethane is shown in Figure 3. The energy penalty of having eclipsed bonds rather than staggered bonds is seen to be 2.7 kcal/mol (11.3 kJ/mol). The energy curve has three minima because the three atoms attached to each end of the molecule are the same. Therefore, the conformations with f = 0, 120, and 240 are all identical eclipsed conformations. The conformations with f = 60, 180, and 300 are all identical with staggered, low energy conformations. Locate these energies in Figure 3.  Figure 3. Dihedral energy in ethane. In the structures all hydrogens are equivalent, however one particular hydrogen on the front of the molecule and one on the back are shown with a dot so that you can follow the change in the dihedral angle over a full 360. Van der Waal's Energy: Van der Waal's interactions between pairs of atoms influence the energy of a conformation. Van der Waals interactions are used in conjunction with dihedral energies to predict the lowest energy conformation. Van der Waal's interactions are often the most important factors in determining the overall molecular conformation (shape). Such interactions are extremely important in determining the three-dimensional structure of many biomolecules, especially proteins. You will also learn that Van der Waals interactions also act between molecules and are responsible for the liquefaction of non-polar substances like O2 and N2. A plot of the Van der Waal's energy as a function of distance between two hydrogen atoms is shown in Figure 4. When two atoms are far apart, an attraction is felt. When two atoms are very close together, a strong repulsion is present. Attractions lower the energy of a molecule, while repulsions raise the energy of a molecule. A measure of the size of an atom is its Van der Waal's radius. The distance that gives the lowest, most favorable, energy of interaction between two atoms is the sum of their Van der Waals radii. The lowest point on the curve in Figure 4 is this point. Interactions of two atoms separated by more than this minimum energy distance are governed by attractive forces. At distances smaller than the minimum energy distance, the atoms repel each other. Calculate the Van der Waal's radius of a hydrogen atom from Figure 4. The Van der Waal's interaction is commonly modeled by the Lennard-Jones equation. We often use the terms Van der Waals and Lennard-Jones interchangeably. Figure 4 is calculated using the Lennard-Jones equation. Our molecular modeling program uses the term Lennard-Jones in its print out.  Figure 4: Van der Waal's interactions between two hydrogen atoms in a molecule, such as ethane. The curve is calculated from the Lennard-Jones potential. Electrostatic Energy: Atoms in molecules have partial electric charges, which are caused by the polarity of the bond to the atom. For example, the hydrogens in H2O and H2O2 are slightly positive, because the O-H bond is polar. The O-H bond is polar because oxygen is more electronegative than hydrogen. Like-partial charges repel and opposite-partial charges attract. These attractions and repulsions are called electrostatic interactions. Electrostatic interactions have an effect on the conformations of molecules. In H2O2 (Figure 1) for example, the two positively charged hydrogens will try to avoid each other. With a 0 dihedral the atoms are close together and electrostatic repulsion will be at maximum. As the dihedral angle increases, the H atoms are further apart, decreasing the repulsions, and therefore lowering the energy. The conformational energy of a bond is the sum of the dihedral energy plus the Van der Waals energy plus the electrostatic energy. The lowest energy conformation is the angle that gives the smallest total energy. In other words, bonds find a compromise among competing forces to determine the lowest energy conformation. The goal of this experiment is to use a computer model to determine the lowest energy conformation of hydrogen peroxide, H2O2. The process is called molecular modeling. The computer makes small changes in the position of every atom and calculates the energy after every move. The move is kept if the energy is lowered, otherwise the atom is returned to its original position. This process is repeated many times until an overall energy minimum is reached. One full cycle, where each atom is moved once, is called a minimization step. Hundreds of steps may be necessary to find a reasonable structure for the molecule. In summary the conformational energy of a molecule is determined by the: 1. Dihedral energy: The electrons in bonds on the opposite atoms of a bond repell each other. 2. Van der Waal's energy: At large distances atoms are attracted to each other, but at very short distances, atoms repell each other. 3. Electrostatic energy: Like charges repell and opposite charges attract. 4. The conformational energy = Dihedral + Van der Waal's + Electrostatic Procedure The dihedral energy for H2O2 is different than ethane's, because of the presence of non-bonding orbitals. The dihedral energy plot that CHARMm uses for H2O2 is shown in Figure 5. Use Figure 5 to predict the dihedral angle in H2O2. The Van der Waal's energy for H2O2, Figure 6, is also different than ethane's, because of the polarity of the bond. Find the Van der Waal's radius of a H in an O-H bond from Figure 6. Figure 5. Dihedral energy in H2O2.  Figure 6. Van der Waal's interaction between two hydrogens, both of which are attached to oxygen. We will use the "QUANTA" and "CHARMm" programs on our Silicon Graphics workstations, to produce our model of H2O2 for this experiment. An outline of the procedure is: A. Input a rough structure for H2O2 using the ChemNote application. B. Compare the 0 and 180 conformational energies using CHARMm. B. Minimize the structure (i.e. find the lowest energy conformation) using CHARMm. C. Measure the dihedral angle and H atom distances using the Geometry palette in QUANTA. The specifics of the procedure follow: A. Input a rough structure for H2O2 . Using the following procedure you will construct a computer model of the hydrogen peroxide molecule. 1. Pull down the Applications menu, slide right on "Builders," and choose "2D Sketcher." This process opens the ChemNote application. 2. Click on the single bond icon in the bonds palette. The single bond is the horizontal line in the middle left hand portion of the screen. 3. Position the cursor near the middle of the screen. Hold down the left mouse button and drag the bond a short ways to the right. The default atom type is carbon, therefore the single bond is assumed to have a carbon atom at each end. We must now change these carbons to oxygens. 4. Click on the oxygen icon in the atoms palette in the upper left portion of the screen. 5. Click the left button on the mouse with the cursor over each end of the single bond. ChemNote is picky about where you click, if an oxygen atom doesn't appear at the end of the bond, try moving the mouse and clicking the left mouse button again. Usually clicking on the bond near the end of the bond works well. The molecule should now appear as below: HOOH If your molecule does not look like that above, pull down the File menu and choose "New", answer "No" to "Save changes first?", and start over. 6. Next save your molecule as a file and return to QUANTA by pulling down the File menu and choosing "Return to Molecular Modeling." The system responds by asking if you wish to "Save changes first?" Click on "Yes." The File Librarian dialog is displayed. First change to the small_molecules/ folder by clicking on Small_molecules in the file list. If you can't see the small_molecules/ entry, use the scroll bar to the right of the file list by clicking in the dark blue area beneath the red scroll bar. Now type in a file name for your structure. File names should not contain any spaces or punctuation. For example, H2O2 followed by your initials would be a good file name, but remember not to use spaces. 7. QUANTA then calculates the sum of the partial charges on the atoms in your molecule. The sum should be zero, but is often not because the program just uses a table of approximate values for each atom. The actual desired charge on the molecule is entered in the blue edit field; for H2O2 the total charge should be zero. Any difference between the calculated charge and the desired charge is smoothed over the atoms in the molecule. Choose the "CT, CH1E,CH2E, CH3E, C5R, C6R, C5RE, C6RE, and HA types" smoothing option and choose OK. (The smoothing you chose is over all carbon atoms and non polar hydrogens - of which H2O2 has none.) 8. If a molecule was in the QUANTA window when you started, you will be asked how you want to display your molecule. Choose "Use the new molecule ____ only." B. Find the energy of the 0 and 180 dihedral angle structures: The following procedure will allow you to compare the energy of two conformations of the molecule. 1. Reorient the molecule so that you can see all the atoms by holding down the center mouse button and moving the cursor within the molecule window. The molecule will rotate as you move the cursor around the screen. 2. To determine the contribution to the total energy from the dihedral, Lennard-Jones, and electrostatic energies, click on the "CHARMm energy" option in the Modeling palette. The total energy is listed on the top right portion of the screen. The detailed results are printed in the QUANTA Textport window at the bottom of the screen. Click on the scroll bar, which is at the left side of the Textport window to see the top two lines that list the bond and angle energies. Record the various contributions to the energy in the second column of the table below. The units are kcal/mol. The bond energy term is the energy caused by strain of the bond lengths from their normal values, and the angle term is the energy caused by strain of the A-B-C type bond angles from their normal values. Contribution0 dihedral (kcal/mol)180 dihedral (kcal/mol)minimized (kcal/mol)difference: 180-minimizedconformer favored bond energyangle energydihedral energyLennard-JonesElectrostatictotal 3. To convert the dihedral angle to 180, select "Torsions..." from the Modeling palette. The "Select Torsions" palette will appear. Pick the first atom defining the torsion by clicking on one of the oxygens. A green atom label should appear each time you select an atom; click again if the labels do not appear. Pick the second atom defining the torsion by clicking on the second oxygen atom. Select "Finish" in the torsions palette. The Dials palette will now change to show only one dial, that for torsion 1. If the dials palette isn't completely visible, click on the border of its window (notice that the cursor changes to a >| symbol when you are on the border of a window). Click on the "torsion 1" dial until the dihedral angle is near 180 (-180 is the same as 180). Click on "Save Changes" in the Modeling Palette. You will next be asked how you want to name the file for the changed structure. Choose "Overwrite ___" for the "Saving option" and click OK. Then select "CHARMm Energy" from the Modeling palette. Record the various contributions to the energy in the third, 180 column of the table. Which conformation is most stable? Which energy contribution differs the most? Add up each term to verify that the total steric energy is the sum of the listed terms. C. Minimize the structure by adjusting the bond lengths and angles. The following procedure will produce a computer model of hydrogen peroxide with correct bond lengths and angles. This model should be similar to the model you produced or will produce in Part I. 1. We need to make sure the default minimization settings are chosen. You need only do this step once. If you minimize other molecules after H2O2 you can skip to the next step. First pull down the CHARMm menu, slide right on "CHARMm Mode", and choose "PSF." Next pull down the CHARMm menu and choose "Minimization Options;" a dialog box will appear. Choose the "Conjugate Gradient" method, which is the most accurate method. Also, make sure the following values are entered in the edit fields: Number of Minimization Steps 50 Coordinate update frequency 5 Energy gradient tolerance 0.0001 Energy value tolerance 0 Initial step size 0.02 Step value tolerance 0 Click on OK when you are finished. 2. Click on the "CHARMm Minimization" option in the Modeling palette. 3. As the calculation proceeds the results are printed in the window at the bottom of the screen and the total energy is printed at the top right of the main window. The program does 50 minimization steps each time you click on the "CHARMm Minimization" option. However, usually more than 50 iterations are required to find the minimum energy. Repeatedly click on the "CHARMm Minimization" option until the energy no longer changes. The structure changes as the minimization proceeds so that you can visually follow the course of the minimization. 4. Then select "CHARMm Energy..." from the Modeling palette. Record the various contributions to the energy in the fourth, minimized column of the table. Add up each term to verify that the total steric energy is the sum of the listed terms. Calculate the difference in energy between the 180 and minimized sturctures for each contribution in the 5th column. In the 6th column record which conformer is favored by each contribution. Finally, from the difference column, decide which contribution dominates the conformational preference. In the minimized structure, you will notice a contribution from the bond and angle terms. The bond energy term is the energy caused by strain of the bond lengths from their normal values, and the angle term is the energy caused by strain of the A-B-C type bond angles from their normal values. The bond lengths and angles in the final minimized structure deviate from their normal values, because in so doing the other energy terms can be even lower. 5. Click on "Save Changes" in the Modeling palette to save the minimized structure. You will next be asked how you want to name the file for the minimized structure. Choose "Overwrite ___," and click OK. C. Measure the dihedral angle , H<> H atom distance, and charges. Using the following procedure you will be able to rotate the molecule in space to get a better perspective on the bond rotations, you will also be able to determine the dihedral angle and distance between the hydrogen atoms. As you choose options, notice that instructions for what to do next are printed at the bottom line of the molecule window in the light grey bar. 1. While holding down the middle mouse button, reorient the molecule so that you can clearly see all four atoms. 2. Click on the title bar (very top of the window) of the Geometry palette to make the palette fully visible 3. Make sure "Show distance monitors," and "Show dihedral monitors" are highlighted in blue. If they are not select them by clicking. 4. Next choose the "Distance" option and then click on the two hydrogen atoms. The distance between the two atoms should be printed on the screen. Record this value in your notebook. How does the H-H distance compare with the minimum in Figure 6? Are the Van der Waal's forces between the hydrogen atoms attractive or repulsive at this distance in Figure 6? 5. Choose the "Dihedral angle" option and then click on the four atoms in the sequence H-O-O-H. The dihedral angle will be printed on the screen. You can reorient the molecule, by holding down the center mouse button, to see the dihedral angle better, if necessary. Record this value. 6. To finish up, click on "Clear ID" and then click on the border of the Modeling palette to bring that window back to the top. 7. Pull down the Draw menu, slide right on "Label Atoms," and select "Atomic Charge." Record the charges on the atoms. To remove the atom charges from the screen, pull down the Draw menu, slide right on "Label Atoms," and select "Off." CONCLUSION The minimum energy structure is a compromise among all the energy terms. What angle do you predict using the dihedral energy alone (Figure 5)? What angle do you predict using the electrostatic interaction alone: that is, did you expect attractions or repulsions between the hydrogen atoms and based on that what dihedral angle do you predict? How is the compromise struck between the dihedral and electrostatic terms? For H2O2 the Van der Waals interaction is not very important because the hydrogen atoms are so far apart where the attractive forces are small. The experimental dihedral angle is 93. How close did you come in your model? Building Structural Complexity Hydrogen peroxide is a very simple molecule, since it only has one dihedral angle. The true importance and utility of conformational analysis is evident in studies of much more complicated molecules, especially large biomolecules like proteins and nucleic acids. In determining the conformation of large molecules all the dihedral angles must be varied to find the minimum energy conformation. Such studies can take hours of computer time. Hydrogen peroxide is a good starting point for understanding these more complex systems. Dihedral energies, Van der Waals energies, and electrostatic interactions work the same for each dihedral angle in a protein or nucleic acid as they do for H2O2. Chemistry gains much of its beauty and utility as we study larger and larger molecules. At first sight, a large molecule such as a protein can appear bewildering. How do we deal with the complexity, and what questions are important to ask? Chemists often look at large molecules as being made up of small molecules. Nature is very conservative, the same interactions that act in small molecules are active in the very largest molecules. Therefore, mentally breaking down large systems into their component small molecules is an important part of understanding large systems. The bond angles and lengths we find in small molecules are good predictors of the bond angles and lengths in large systems. The dihedral angle study we just completed for H2O2 is a good example of how we might begin to build up the conformations of large molecules. Figure 3 shows what we can expect from C-H bonds. We just need to work through large molecules on a bond-by-bond basis. Proteins are composed of many amino acids joined by peptide bonds; that is, proteins are polymers made from amino acid monomers. Three of the 20 naturally occuring amino acids are shown in Figure 7. Also shown is the peptide bond between to glycines. The peptide bond is formed when the O=C-OH on one amino acid reacts with the H2N- on the next amino acid, forming an O=C-NH- bond.  Figure 7. Three common amino acids. On the bottom a peptide bond is formed between two glycines. Each of the bonds in the amino acids can be analyzed as we have done with H2O2. Figure 3 shows what we can expect for the C-H bonds in glycine, alanine, and serine. In the CH141/ folder on the lab computers you will find the structures of some more complex molecules. Choose one of the following projects concerning these molecules. The purpose of the projects is foremost to have some fun learning about molecular modeling and mechanics. The other purpose is to begin to appreciate how complex molecules are built from small molecule building blocks. Project 1: Tri-metaphosphate (Inorganic Chemistry) The structure of the tri-metaphosphate ion is one example of how large molecules are built from small molecule building blocks, Figure 8. Tri-metaphosphate is made from three phosphates in a cyclic structure. Tri-metaphosphate is used for corrosion protection in some municipal water supplies. It complexes strongly with Ca+2, and the question we ask here is why is the complex so stable? Why does tri-metaphosphate like to interact with Ca+2.  Figure 8. Tri-metaphosphate ion. We will first build the structure of phosphoric acid, H3PO4, to determine the basic structure of the phosphate backbone. Then we will look at the complex that forms from tri-metaphosphate ions and Ca+2. We will minimize the tri-metaphosphate- Ca+2 complex and then use solid models with Van der Waals spheres to visualize the complex with Ca+2. Procedure 1. Pull down the Applications menu, slide right on "Builders," and choose "2D Sketcher." This process opens the ChemNote application. 2. Click on the single bond icon in the bonds palette. 3. Position the cursor near the middle of the screen. Hold down the left mouse button and drag the bond a short ways to the right. Now return to the original starting point and drag a second bond to the left. Return to the original starting point a drag a bond upward. Now click on the double bond icon in the bonds palette. Position the cursor at the original starting point and drag a double bond downward. The structure should appear as below:  4. Click on the oxygen icon in the atoms palette in the upper left portion of the screen. 5. Click the left button on the mouse with the cursor over the outer ends of the single bonds. Then also click on the outer end of the double bond. 6. Click on the phosphorus icon in the atoms palette in the upper left portion of the screen. Then click on the central atom in your structure. The molecule should now appear as below:  If your molecule does not look like that above, pull down the File menu and choose "New", answer No to "Save changes first?", and start over. Next save your molecule as a file and return to QUANTA by pulling down the File menu and choosing "Return to Molecular Modeling." The system responds by asking if you wish to "Save changes first." Click on "Yes." The File Librarian dialog is displayed. First change to the small_molecules/ folder by clicking on small_molecules/ in the file list. If you can't see the small_molecules/ entry, use the scroll bar to the right of the file list by clicking in the dark blue area beneath the light blue scroll bar. Now type in a file name for your structure. File names should not contain any spaces or punctuation. For example, H3PO4 followed by your initials would be a good file name, but remember not to use spaces. 7. QUANTA then calculates the sum of the partial charges on the atoms in your molecule. The sum should be zero, but is often not because the program just uses a table of approximate values for each atom. The actual desired charge on the molecule is entered in the blue edit field, for H3PO4 the total charge should be zero. Any difference between the calculated charge and the desired charge is smoothed over the atoms in the molecule. Choose the "CT, CH1E, CH2E, CH3E, C5R, C6R, C5RE, C6RE, and HA types" smoothing option and choose OK. (The smoothing you chose is over all carbon atoms and non polar hydrogens - of which H3PO4 has none.) 8. You will then be asked "Which molecule do you want to use?" Choose "Use the new molecule ____ only." Then click "OK." 9. In QUANTA rotate the structure of H3PO4 to familiarize yourself with the molecule. 10. If the doubly bonded O doesn't have two lines running to it, indicating a double bond, pull down the Draw menu, slide right on "Bond Style" and choose "Multi-Vector Bonds." 11. Switch to the Geometry palette by clicking on its border. Turn off "Distance" and "Dihedral" by clicking on them, if they are highlighted. Measure the O-P=O bond angle by clicking on "Bond Angle.," and then clicking on an the O, P and O atoms in turn, which are bonded to each other. Make sure one of the O atoms is the doubly bonded O atom. The angle should be displayed. Record this value. 12. Next we will study the complex. Pull down the File menu and choose "Open." In the File Librarian, click on the CH141/ folder. In this folder click on the file "Ca_trimetaphosphate." At the bottom of the dialog box click on "Replace." Finally click on "Open." 13. In the molecule window should appear the tri-metaphosphate anion (-3 charge) and spaced away from the ring a Ca2+ ion. You won't be able to see the Ca2+ ion at first; use the middle mouse button to rotate the complex around so that the Ca2+ ion is to the side of the ring. Measure the O-P=O bond angle as you did in step 11. Make sure the singly bonded O is not inthe ring, rather choose an O that points toward the Ca2+ ion. Record this value. ARe the values in phosphoric acid and trimetaphosphate ion similar? 14. Click on "CHARMm Minimization" repeatedly in the Modeling palette. The complex should form. Did the bond angle change much when the complex formed? Does the ring structure need to change to accomidate the Ca2+ ion? Click on "Save Changes" in the Modeling palette. This time make sure to choose "Save to a New Filename." Then click "OK." In the File Librarian, type in a new file name (remember: no spaces or punctuation). 15. To help visualize the interactions in the complex we will display a space filling model of the structure. Pull down the Draw menu, slide right on "Solid Models," and choose "Van der Waal's." A new object will be displayed showing each atom as a sphere with the radius of the sphere equal to the atom's Van der Waal's radius. You can reorient the structure in the normal way -- hold down the center mouse button and move the cursor in the molecule window. From this space filling model and the change in bond angle upon complex formation, you should be able to appreciate why the complex is so strong. When you have finished, click on the "No" box underneath the "Delete" column in the "Object Managment" window to remove the solid object. You should also try other options including "Ball and Stick" models in "Solid Models" and a "Raster Models." "Ray Trace" gives the best quality, but rotations aren't possible in ray trace mode. Click a mouse button to exit "Raster Models" or "Ray Trace" mode. Project 2: Methyl Alcohol and Alcohol Dehydrogenase (Organic and Biochemistry) The structure of methyl alcohol, CH3OH, is a good model for any alcohol no matter how complex. In this project you will build a model of methyl alcohol and measure the H-C-O-H dihedral angle. You will compare the model value with your prediction based on Figure 3. Some alcohols like methyl alcohol are toxic, so cells have developed a mechanism to convert alcohols into other substances. Other alcohols are necessary for the proper functioning of cells, like the amino acid serine. Once again biochemical pathways are necessary to produce and degrade these alcohols. Enzymes are the catalysts that increase the rate of biochemical reactions. Enzymes are usually proteins, which are polymers composed of amino acids. Serine is one of the amino acids found in proteins. You will observe a QUANTA model of the amino acid serine. One of the enzymes that is used to convert alcohols into other substances is alcohol dehydrogenase. You will also observe the structure of this important enzyme. Starting with methyl alcohol helps to understand the amino acid serine, and serine is one of the amino acids in alcohol dehydrogenase. Our goal of this project is to show you how very large molecules can be mentally broken down into smaller molecules, so that the large molecules become easier to understand. Procedure 1. Predict the dihedral angle in methyl alcohol. The methyl group will behave the same as in ethane, so Figure 3 will be helpful. 2. The structure of methyl alcohol is easy to build. Just follow the instructions for building H2O2, except only change one of the carbons to an -OH. Leave the other end of the single bond alone. The structure will appear as below: OH Don't worry about the H atoms on the carbon, ChemNote will add them for you. Continue to follow the instructions for H2O2, including the "CHARMm Minimization" and the measurement of the dihedral angle. Does the dihedral angle agree with your predictions? 3. Now observe the structure for serine by pulling down the File menu and choosing "Open". In the File Librarian, click on the CH141/ folder. In this folder click on the file "serine" At the bottom of the dialog box click on "Replace." Finally, click on "Open." Reorient the molecule so that you can see it clearly, using the center mouse button and moving the cursor in the molecule screen. Switch to the Geometry palette by clicking on its border. Turn off "Distance" and "Angle" by clicking on them, if they are highlighted. Measure the H-C-O-H dihedral angle on the side chain by clicking on "Dihedral," and then clicking on an the H, C, O and H atoms in turn, which are bonded to each other. How does this dihedral angle agree with methyl alcohol? 4. Now observe the structure of alcohol dehydrogenase by pulling down the File menu and choosing "Open". In the File Librarian, click on the CH141/ folder. In this folder click on the file "6adh" At the bottom of the dialog box click on "Replace." Finally, click on "Open." Reorient the molecule so that you observe it from several different angles. Large molecules have a beauty that comes from their complexity and their utility. Why does nature choose such a complex structure for such a simple job? 5. To locate the serines in the enzyme, pull down the Draw menu, slide right on "Color atoms," and choose "Selection Tools." The Color Atoms palette will appear. Repeatedly click on "Next Color Numer" until *Color 8* in purple is displayed in the upper right hand corner of the QUANTA window. Choose "Residue type". A scroll list will be displayed of the common amino acids in proteins. Click on the "ser" entry and click on "OK." All the serines in the enzyme should then change color so that they will be easier to find. How many are there? (Answer this question with 1, 2, 3, or many.) Notice that the H atoms are not included in this structure. This is because the structure was determined by x-ray diffraction, which isn't sensitive to H atoms. 6. Choose "Finish" in the Color Atoms palette to return to the normal molecule window. 7. Pull down the Draw menu, slide right on "Color Atoms," and choose "Color by element." 8. A very common structural element in proteins is the alpha-helix. How many alpha-helical segments are there in alcohol dehydrogenase? To answer this question, it is easiest to construct a ribbon model of the enzyme. Do this in the following steps: pull down the Draw menu, slide right on "Solid models," and choose "Selection Tools." In the main QUANTA window, click on "Helix." In the Solid Display palette, click on "Protein Backbone." Click on "Finish" to return to the normal operating mode. Count the number of alpha-helical segments. 9. In the "Object Management" window in the lower right of the screen, click on the "Yes" box in the "Attached" column. The box should change to no. Then click on the border of the "Molecule Management" window to bring it forward. Click on the "Yes" box in the "Visible" column, to supress the drawing of the molecule. Count the number of alpha-helical segments. (Answer this question 1, 2, 3, several.) The flat regions with parallel ribbons shows the second common structural element in proteins: the beta pleated sheet structure. Project 3: Hydrogen Peroxide and Catalase (Inorganic and Biochemistry) Hydrogen peroxide is a common reactant and product in biochemical reactions. However, too much H2O2 can be toxic to cells, since H2O2 is such a strong oxidizing agent. Cells must be able to carefully regulate the concentrations of H2O2. Therefore, cells produce an enzyme, catalase, to destroy H2O2 : H2O2 > H2O + 1/2 O2 You will also remember this reaction as a lecture demonstration that evolves a cloud of steam when MnO2 is used as the catalyst. Enzymes are usually proteins, which are polymers composed of amino acids. However, many enzymes need additional help from other groups. Catalase contains a porphyrin ring that is complexed to an Fe3+ ion. Many other peroxidase enzymes, hemoglobin, and myoglobin, also contain a porphyrin ring that is complexed to an Fe3+ ion. The study of metal containing enzymes is an important part of Inorganic and Biochemistry. Why is the Fe3+ needed? In catalase, H2O2, which is a weak acid, dissociates yielding the hydroperoxide ion, HO2-: H2O2 > HO2- + H+ The hydroperoxide ion, HO2-, so produced binds directly to the Fe3+. In this project you will observe the structure of the porphyrin ring and then in catalase and cytochrome-c find the porphyrin ring and the associated Fe3+. Our goal of this project is to show you how very large molecules can be mentally broken down into smaller molecules, so that the large molecules become easier to understand. 1. Observe the structure for the porphyrin ring by pulling down the File menu and choosing "Open". In the File Librarian, click on the CH141/ folder. In this folder click on the file "porphyrin" At the bottom of the dialog box click on "Replace." Finally, click on "Open." Reorient the molecule so that you can see it clearly, using the middle mouse button and moving the cursor in the molecule screen. 2. How do you think the Fe3+ interacts with this ring? Answer this question by pulling down the Draw menu, sliding right on "Solid Models," and choosing "Van der Waal's." The Fe3+ is shown in grey. 3. Now observe the structure of catalase by pulling down the File menu and choosing "Open". In the File Librarian, click on the CH141/ folder. In this folder click on the file "8cat." At the bottom of the dialog box click on "Replace." Finally, click on "Open." Reorient the molecule so that you observe from several different angles. Large molecules have a beauty that comes from their complexity and their utility. Why does nature choose such a complex structure for such a simple job? Find the porphyrin ring. The position of the Fe3+ ion is shown grey. Where does the Fe3+ ion sit? 4. To see the porphyrin ring and the Fe3+ ion better, you may want to increase the size of the molecule on the screen. In the "Dials" box, which is in the lower right hand side of the screen, you will see six grey bars. Click on the right hand side of the "Scale" bar to increase the size of the molecule. Clicking on the left side of the "Scale" bar decreases the size. The further you click from the center of the bar, the bigger the movement. If you get lost and loose track of the molecule, click on "Reset" to return to the starting view. 5. The catalase enzyme is very large. The structure you see is only one half of the full enzyme; the other half is identical however. This structure also only has the backbone atoms. Side chain atoms and hydrogens are omitted, because their locations have not yet been determined. A smaller enzyme that also has a porphyrin ring is cytochrome c. Some cytochrome cs also use H2O2 in oxidation reactions. Open the cytochrome-c structure; it is also in the CH141/ folder under the name "1ccr." Locate the porphyrin ring. 6. A very common structural element in proteins is the alpha-helix. How many alpha-helical segments are there in cyctochrome-c? To answer this question, it is easiest to construct a ribbon model of the enzyme. Do this in the following steps: pull down the Draw menu, slide right on "Solid models," and choose "Selection Tools." In the QUANTA window, click on "Helix." In the Solid Display palette, click on "Protein Backbone." Click on "Finish" to return to the normal operating mode. Count the number of alpha-helical segments. (Dont try this with catalase, the computer will crash.) 9. Place a small amount of liver in a 3% solution of H2O2 and observe the results. What is the reaction that occurs? Report Part I. Prepare a chart listing all of the molecules and ions investigated and the results of your investigations from the Interpretation of Molecular Models section. Include Lewis Dot structures, Molecular Geometry, Molecular Polarity, Resonance Structures, and Structural Isomers. Part II. Answer the questions in the text above. These questions are repeated below for your convenience. H2O2 project: 1. Using Figure 4, find the Van der Waals radius of the hydrogen atom, when the H is in a C-H bond. Using Figure 6, find the Van der Waals radius of the hydrogen atom, when the H is in a C-H bond. 2. What dihedral angle do you predict using the dihedral energy alone (Figure 5)? 3. What dihedral angle do you predict using the electrostatic interaction alone: that is, did you expect attractions or repulsions between the hydrogen atoms and based on that what dihedral angle do you predict? 4. Reproduce your energy contribution table. Sum the energy terms from your CHARMm energy runs. Does the sum equal the total steric energy listed on the upper right-hand side of the screen? 5. Which structure, the 0 dihedral structure or the 180 dihedral structure, is more stable? Which energy contribution differs the most? 6. From the difference column in your table, which term, dihedral, Lennard-Jones, or electrostatic dominates the conformational preference of hydrogen peroxide? 7. From Figure 6, are the Van der Waal's forces between the hydrogen atoms attractive or repulsive at the minimum energy difference? 8. How is the compromise struck between the dihedral and electrostatic terms? To answer this question, compare your answers to questions 2 and 3 to the minimized value. Were the predictions too small or too large? Which prediction is the closest? 9. The experimental dihedral angle is 93. How close did you come in your model? For Project 1 on tri-metaphosphate: What is the O-P=O bond angle in phosphoric acid? What is the same angle in trimetaphosphate ion and in the Ca2+ complex? Comment on the size of the Ca2+ ion in relation to the distance between singly bonded O atoms in tri-metaphosphate ion. Why is the complex so strong? For Project 2 on methyl alcohol: Does the dihedral angle in methyl alcohol agree with your predictions based on Figure 3? Does the dihedral angle for the OH group in serine agree with the dihedral angle in methanol? How many serines are there in alcohol dehydrogenase? How many alpha-helical segments are there in alcohol dehydrogenase? For Project 3 on H2O2, catalase, and cytochrome-c: Where does the Fe3+ ion sit in catalase and cyctochrome-c? How many alpha-helical segments are there in cytochrome-c? What happens when you put liver in H2O2 solution? Write the equation for the reaction.  Developed from "The Geometrical Structure of Molecules: An Experiment using Molecular Models" from Chemistry 103 Laboratory Manual, S.F. Sontum ed, Department of Chemistry and Biochemistry, Middlebury College, Middlebury, Vermont, 1988, pp 75 - 91. 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xl}{xl P P}ie 1 1 S dv D sc CMT currentmatrix P lW sl 4.0 setmiterlimit np}b "q"" q"""""" " ":"" "9_"9_" c"GE"_?"HF "Et"I";"$"9"> "b"K "H"L"""""?m"?m"&q"MS"eM"NT "K"O"hz end %%EndProcSet 40 80 20 20 54 chemdict30 begin/hS x/sf x/lW x/bW x/cW x/sh T d 403 217 27 22 SPn/origstk x 65535 65535 65535 sBg 0 0 0 C np 3967 3448 M 3967 3472 l 3461 3764 l 3447 3760 l 3447 3748 l cp f np 4395 3697 M 4385 3715 l 3967 3472 l 3967 3448 l cp f np 3921 2970 M 3941 2970 l 3941 3480 l 3921 3480 l cp f np 3993 2970 M 4013 2970 l 4013 3480 l 3993 3480 l cp f np 4496 4250 M 4476 4250 l 4476 3870 l 4496 3870 l cp f np 3024 3527 M 3034 3509 l 3447 3748 l 3447 3760 l 3435 3765 l cp f np 3334 4223 M 3314 4217 l 3435 3765 l 3447 3760 l 3451 3787 l cp f np 3684 4150 M 3666 4160 l 3451 3787 l 3447 3760 l 3461 3764 l cp f np 5727 4071 M 5709 4081 l 5520 3744 l 5534 3728 l cp f np 6276 4186 M 6276 4206 l 5894 4207 l 5894 4187 l cp f np 5716 3254 M 5734 3262 l 5504 3723 l 5486 3715 l cp f np 5782 3288 M 5800 3296 l 5557 3779 l 5539 3771 l cp f np 1898 1133 M 1916 1147 l 1401 1444 l 1387 1440 l 1387 1428 l cp f np 2043 668 M 2063 674 l 1916 1147 l 1898 1133 l cp f np 1973 646 M 1993 652 l 1856 1094 l 1836 1088 l cp f np 964 1207 M 974 1189 l 1387 1428 l 1387 1440 l 1375 1445 l cp f np 1274 1903 M 1254 1897 l 1375 1445 l 1387 1440 l 1391 1467 l cp f np 1624 1830 M 1606 1840 l 1391 1467 l 1387 1440 l 1401 1444 l cp f np 2335 1389 M 2325 1407 l 1942 1185 l 1952 1167 l cp f np 2893 1466 M 2893 1486 l 2552 1487 l 2552 1467 l cp f np 4618 1183 M 4636 1197 l 4121 1494 l 4107 1490 l 4107 1478 l cp f np 4766 710 M 4786 716 l 4636 1197 l 4618 1183 l cp f np 4696 688 M 4716 694 l 4576 1144 l 4556 1138 l cp f np 3679 1255 M 3689 1237 l 4107 1478 l 4107 1490 l 4095 1495 l cp f np 3991 1963 M 3971 1957 l 4095 1495 l 4107 1490 l 4111 1517 l cp f np 4350 1890 M 4332 1900 l 4111 1517 l 4107 1490 l 4121 1494 l cp f np 5055 1439 M 5045 1457 l 4662 1235 l 4672 1217 l cp f np 5631 1516 M 5631 1536 l 5271 1537 l 5271 1517 l cp f np 4992 3457 M 5006 3461 l 5006 3473 l 4595 3710 l 4585 3692 l cp f np 5534 3728 M 5520 3744 l 5006 3473 l 5006 3461 l 5018 3456 l cp f np 4760 3056 M 4778 3046 l 5002 3434 l 5006 3461 l 4992 3457 l cp f np 5117 3005 M 5137 3011 l 5018 3456 l 5006 3461 l 5002 3434 l cp f np 7298 1253 M 7316 1267 l 6801 1564 l 6787 1560 l 6787 1548 l cp f np 7443 788 M 7463 794 l 7316 1267 l 7298 1253 l cp f np 7373 766 M 7393 772 l 7256 1214 l 7236 1208 l cp f np 6364 1327 M 6374 1309 l 6787 1548 l 6787 1560 l 6775 1565 l cp f np 6674 2023 M 6654 2017 l 6775 1565 l 6787 1560 l 6791 1587 l cp f np 7034 1967 M 7016 1977 l 6791 1587 l 6787 1560 l 6801 1564 l cp f np 7735 1509 M 7725 1527 l 7342 1305 l 7352 1287 l cp f np 8396 1586 M 8396 1606 l 7954 1607 l 7954 1587 l cp f np 7560 2070 M 7560 2090 l 7400 2090 l 7400 2070 l cp f gr count origstk sub{P}rp end chemsave restore ,  Helvetica .+N(O+KH(H +2 +(N+,H(H(O)H(O,Times ( glycylglycine (bO(=H +2 +(=(N+,H(fPH(MwO)H(!O(?H +2 +(?N+,H(hCH +3 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