EXPERIMENT 5: DIPEPTIDE RESEARCH PROJECT

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1 EXPERIMENT 5: DIPEPTIDE RESEARCH PROJECT Pre-Lab Questions: None. 64

2 I. Background Information DIPEPTIDE RESEARCH PROJECT Methods developed by organic chemists for the synthesis of biopolymers have had extraordinary impact in the biological sciences and technology. Methods for the synthesis of proteins and nucleic acids (both DNA and RNA) are reasonably well developed. Considerable progress has been made in recent years on the synthesis of complex polysaccharides (carbohydrates). But room for improvement remains in all of these areas. There are several challenges associated with the development of methods for the synthesis of biopolymers. The syntheses must be efficient. In other words, they must yield the desired product in high yield. The syntheses must place the individual monomers in precisely the correct order in the biopolymer. For example, there are six ways in which three distinct monomeric units can be ordered in an asymmetric linear sequence (abc, acb, bac, bca, cab, cba). Usually, the goal is to make just one of these in the synthesis process. And it is critical that the structural elements of the monomers not be inadvertently altered in the course of the synthesis. The dipeptide research project addresses this last challenge: assuring the structural integrity of monomers as they are added to a growing polymer chain. Specifically, you will study the stereochemical integrity of N-protected amino acids during the peptide bond forming reaction that is a critical recurring step in the chemical synthesis of polypeptides. For a more thorough description of the chemical synthesis of polypeptides, you should consult your CHEM 337 textbook. A key step in these syntheses is the following: Prot = Protecting Group 65

3 In this reaction, an amino acid bearing a protected amino group (1) is coupled to a second amino acid bearing a protected carboxylic acid group (2). Typically, each of the amino acids bears at least one asymmetric carbon atom (starred). In the desired product (3), the stereochemistry at each of these stereocenters is unaltered, as shown above. Early on in the development of methods to achieve this coupling, it was discovered that the stereochemistry at the starred carbon in the N-protected partner 1 can be altered under certain circumstances. It was found that, under some circumstances, in addition to the desired product 3 in the above coupling reaction, an undesired by-product 4 was also formed. Because the synthesis of polypeptides involves many such coupling reactions, even a very small degree of loss of stereochemical integrity in each coupling reaction poses a very serious problem. Imagine, for example, the synthesis of an undecapeptide (11-mer), requiring ten sequential coupling steps. If each coupling is accompanied by 10% inversion of the stereocenter in the N- protected coupling partner, it is a simple mathematical exercise to demonstrate that the desired undecapeptide, with the original monomer stereochemistry intact in the product at all stereocenters, will constitute a minority of a complex mixture of diastereoisomers. For this reason, considerable effort has been invested in attempting to understand the mechanistic origin of this undesired isomerization reaction, with the expectation that this understanding will improve our ability to suppress it. To make a long story short, there is considerable evidence that the loss of stereochemical integrity of the N-protected component is a consequence of activation of the carboxyl group, and includes participation of a carbonyl group oxygen common to N- protecting groups, as shown in Scheme I. Activation of the carboxylic acid group in 1 by a dehydrating agent converts the hydroxyl group into a leaving group X (see 5). If amine 2 is able to capture intermediate 5 directly, desired dipeptide 3 can result. Unfortunately, the data suggest that activated intermediate 5 is able to undergo a rapid intramolecular cyclization to afford intermediate 6. Again, if intermediate 6 is captured directly by amine 2, the desired dipeptide 3 can be produced. It appears, though, that intermediate 6 is sufficiently acidic that under some conditions it undergoes loss of the proton on the stereocenter to afford the achiral enolate 7. Enolate 7 can be reprotonated to form 8, the enantiomer of 6. Reaction of amine 2 with 8 can then afford undesired dipeptide 4. 66

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5 If the chemistry depicted in Scheme I is responsible for the undesired production of 4, then we can imagine avenues to suppress this undesired reaction. For example, we could attempt to modify reaction conditions so as to suppress the rate of which intermediate 6 is deprotonated, because this is the key step in loss of the stereochemical integrity of 1. Alternatively, we could seek reaction conditions that accelerate the rate at which amine 2 captures 5 or 6, as these lead, presumably irreversibly, to the desired dipeptide 3. The experimental variables we can modify include the structure of the N-protecting group, the dehydrating agent, the solvent, the acidity of the reaction medium, and the reaction temperature. Another variable is the inclusion (or lack thereof) of additional nucleophiles capable of intercepting 5 or 6 prior to their progression to 7, to create a new intermediate capable of progressing to desired dipeptide 3. II. Your Goal This research project will proceed in two distinct phases: In Phase I, the goal of your team of four students is to quantify unequivocally the degree, if any, of loss of stereochemical integrity during a coupling reaction for two different N-protecting groups under one set of reaction conditions. Your team of four students will choose, from the lists below, a single set of coupling conditions you wish to study. Or, you may choose some other set of conditions not on the list if approved by the course instructor and we are able to obtain the necessary chemicals. The two N-protecting groups you will compare are N-acetyl (N- Ac, CH 3 CO-) and N-carbobenzyloxy (N-Cbz, C 6 H 5 CH 2 OCO-). It is our goal to complete Phase I within the first four of the six class sessions devoted to this study. All groups will then share their experimental data from their Phase I experiments with one another. Your final report will need to summarize the findings of all groups, and your own group s Phase II data (see below). In Phase II, it is not at this time known exactly what we will choose to do. We will consider the data from Phase I, in deciding what to do in Phase II. It may be that Phase II time will be needed just to complete the Phase I goals. Or it may be that we choose to repeat some or all Phase I experiments to confirm their accuracy. Or it may be that on the basis of the Phase I results we wish to test some new reaction conditions to improve the reaction outcome. At or near the end of Phase I we will need to make our choice. In your final report, you will want to be sure to indicate which coupling methods are best at producing the desired dipeptide. You may wish to suggest a mechanistic explanation for the findings. Note that your grade will NOT be influenced by the degree to which your particular coupling reaction produces the desired dipeptide rather than its undesired isomer. Indeed we will gain the most insights if some coupling methods can be shown to yield a very high degree of loss of stereochemical integrity. 68

6 III. Procedure 1. Your instructor will assign you to a group of four students. 2. Your group should select a single coupling reagent and set of reaction conditions to use in all Phase I reactions. 3. Your group should decide what data you will need unequivocally to quantify the stereochemical outcome of couplings with both N-acetyl and N-Cbz protecting groups. It is required that you discuss your plan with your TA. 4. Your group should decide how to divide the labor to achieve the Phase I goal. Again, it is required that you discuss your proposed division of labor with your TA. 5. After you have collected your data, your group should prepare a concise Phase I summary (including copies of the relevant spectra) of your findings to be shared with your peers. Limit the context of this report to a description of what coupling conditions you studied, and the results you obtained (yield, ratio of products). You should not in this Phase I report speculate as to why you obtained whatever were your results. In other words, report for example that the ratio was 10:1, and how you determined that ratio, not why you believe these particular reaction conditions led to that ratio. 6. As we near the end of Phase I, we will use the Tuesday lectures to discuss results and choose our Phase II experiments. 7. At the end of Phase II, every student will turn in his or her own final report on the entire project. [Note: It is okay, if you so choose, to work on the final report with one or more other students or even to turn in a jointly prepared report, but please so indicate on your report.] Your report should emphasize the results of your own group, but in the discussion section attempt to place your group s results in the context of the Phase I results of the entire class. List of Available Protected Amino Acids NOTE: All of these protected amino acids are expensive. For that reason, as well as to reduce the volume of waste, we ask that you run couplings on a 0.5 mmol or smaller scale, or about 100 mg per coupling per amino acid for the heavier compounds. N-Acetyl-L-alanine N-Acetyl-DL-alanine 69

7 N-Cbz-L-Alanine N-Cbz-DL-Alanine L-Phenylalanine methyl ester HCl DL-Phenylalanine methyl ester HCl List of Coupling Methods/Conditions Solvent: Typically CH 2 Cl 2. One group could try DMF (dimethylformamide), but it will be difficult to separate from the product. Try for about 0.5 M concentration in each amino acid (~1 mmol/2 ml). Temperature: Typically 25 C. Dehydrating Agent: Typically DCC, dicyclohexylcarbodiimide. You should only need a slight excess. Other coupling agents include: o EDAC (1-ethyl-3-(3-diethylaminopropyl)carbodiimide) which yields a water soluble by-product to simplify purification 70

8 o PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) Additives: Triethylamine. You will need to neutralize the HCl present in the methyl esters with one equivalent of a base. Triethylamine can be used. If you think it would be interesting to add more, try it! o 4-DMAP (4-dimethylaminopyridine) Another base. Possible substitute for Et 3 N. o HOBt (1-hydroxybenzotriazole). The hydroxyl group of HOBt is a powerful nucleophile. It can intercept an activated carboxyl group, to give an intermediate still reactive enough to react with a primary amine. Usually added stoichiometically (not catalytically). Order of Addition: Typically all chemicals except the dehydrating agent are combined in a solvent in which they are soluble at the desired temperature, and then the dehydrating agent is added to initiate the reaction. An alternative plan would be to withhold the C-protected amino acid (the nucleophile amine) during the activation step. This would prolong the lifetime of intermediates such as 5 and 6 in Scheme I. To achieve this, you would want to mix all chemicals except the dehydrating agent and C-protected amino acid, then add the dehydrating agent, and finally (after some time for the activation step to occur) add the C-protected amino acid. You can probably find many examples of coupling procedures in the chemical literature if you wish to do so. Remember: In this project it is our goal to study the impact of the N-protecting group and coupling conditions on the stereochemical outcome of the reaction. If all groups manage to suppress the isomerization step, we won t learn very much. You may wish to follow the coupling reaction by TLC. To work-up your reaction mixture recognize that the dipeptides will be soluble in organic solvents, whereas most of the residual starting materials and some by-products will be soluble in one or more of water, aqueous sodium bicarbonate, or 0.1 M aqueous hydrochloric acid. You are strongly encouraged to characterize your crude product. If necessary to interpret the data, you could further purify by column chromatography, but this will take time. 71