PROGRESS TOWARD A NOVEL ORTHO-FUNCTIONALIZED CYCLIC PHENYLALANINE DIPEPTIDE BARTHOLOMEW WAYNE NEFF, B.S. A DISSERTATION CHEMISTRY

Transcription

1 PROGRESS TOWARD A NOVEL ORTHO-FUNCTIONALIZED CYCLIC PHENYLALANINE DIPEPTIDE by BARTHOLOMEW WAYNE NEFF, B.S. A DISSERTATION IN CHEMISTRY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved December, 199 /

2 ^^ ACKNOWLEDGMENTS Cb^* ^ I would like to thank the Robert A. Welch Foundation and the National Institutes of Health for their continuedfinancialsupport for this research. I would also like to thank the faculty, staff, and graduate students in the Department of Chemistry and Biochemistry for their knowledge, support, and kindness throughout my years at Texas Tech. I thank Professor Robert D. Walkup for sharing his incredible synthetic expertise and Professor John N. Marx for introducing me to the world of Nuclear Magnetic Resonance. Lastly, I would like to express my sincerest gratitude to Dr. David M. Bimey. I thank him for his patience, trust, and encouragement. He is a great chemist and professor, but an even better man. I consider it an honor to call him my mentor. Most of all, I would like to thank Wendi. She makes the impossible possible. As You Wish 11

3 TABLE OF CONTENTS ACKNOWLEDGEMENTS ABSTRACT LIST OF FIGURES ii iv v CHAPTER L INTRODUCTION 1 Substance P 1 Cyclic Peptides 7 n. SYNTHETIC CONSIDERATIONS 9 Cyclization Methods 9 Protecting Groups 11 Retrosynthetic Analysis 12 in. SYNTHETIC METHODS 14 The Boc-Fmoc-Allyl Ester SEM approach 14 The Boc-Sultam MPM approach 18 The Boc-Fmoc-/-Butyl Ester MPM approach 21 IV. CONCLUSIONS 26 V. EXPERIMENTAL METHODS 28 REFERENCES 43 APPENDIX A. SUPPLEMENTARY EXPERIMENTAL METHODS 45 APPENDIXB. PROTECTING GROUP SUMMARY

4 ABSTRACT Substance P, a neuropeptide found in various mammalian tissues, is involved a variety of physiological processes including the regulation of blood pressure, smooth muscle contraction, and cell growth. Assays have shown that the phenylalanine residues (Phe7 and Phe8) of Substance P are particularly important for receptor binding. Modeling studies, based on NMR data and P-methyl substitiution, seem to indicate a preferred conformation for the phenyl sidechains. This preferred conformation for these sidechains can be approximated, without backbone distortion, through the use of a sidechain ortho 0-CH2-O-CH2-CH2-O-CH2-0 linkage between the two residues. We report progress toward the synthesis of an ortho-linked, cyclic phenylalanine dipeptide [cyclo(boc-phe-phe-o-t-bu)] for incorporation into a Substance P analog as described above. We also report the development of a synthetic method, based on the camphorsultam-derived synthesis of phenylalanine, for the formation phenylalanines containing novel sidechain functionality. These ortho-functionalized phenylalanines are then modified with the appropriate protecting groups, coupled to form the acyclic dipeptide, and cyclized via a crown ether-like SN2 coupling with ethylene glycol. IV

5 LIST OF FIGURES 1. Spacefillingmodel of the lowest energy conformation of Substance P 3 2. Examples of typical ortho linkages incorporated into models of Substance P 5 3. Backbone structure overlay of cyclic Substance P analog with Substance P 6 4. Design of a cyclic Substance P analog 7 5 Retrosynthetic analysis of the cyclic phenylalanine dipeptide 13

6 CHAPTER I INTRODUCTION Substance P Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), an undecapeptide found in various mammalian tissues, has been found to play a role in various physiological processes among which are the ability to lower blood pressure, stimulate smooth muscle contraction^ and cytokine release,^ and promote cell growth. ^'^ It is one of five known mammalian neuropeptides which belong to the tachykinin family, each of which share a common carboxy terminal sequence corresponding to residues 7 and 9-11 of Substance P.^ The receptors for these neurokinins exist as three subtypes: NKl, NK2, and NK3. Although each of the tachykinins will bind to all three receptors, each shows a particular affinity for one individual receptor. For Substance P, physiological activity results from stimulation of the NKl Receptor. Because of the apparent role which Substance P plays in physiological processes relevant to various pathological conditions, it is important to understand the structural requirements for biological activity. The goal of such research is the development of potent Substance P agonists or antagonists in the hopes of eventually arriving at potent chemotherapies which target the NKl receptor. Assays of various Substance P analogs have indicated that the six amino acid residues at the carboxy terminus of the peptide are required for most of Substance P's binding to its receptor, and that the two phenylalanine residues at positions 7 and 8 are particularly important in the binding process.^

7 Furthermore, previous work by Bimey et al., utilizing P-methylphenylalanine substitution at residues 7 and 8 of Substance P indicate that even this subtle conformational restraint on the niolecule can have dramatic effects on its ability to bind to the NKl receptor. In the course of the previous research with p-methylphenylalanine substitution, the ^H Nuclear Magnetic Resonance (NMR) and molecular modeling data, reported by Hicks et al.,^ were used to begin a molecular modeling study of Substance P and the P-methyl analogs. Figure 1 shows a CPK model of the lowest energy conformation of Substance P based on these data. Limited energy minimizations of these structures show large changes in the preferred molecular orientation for the phenyl sidechains due to the steric impact of the P-methyl substituent, presumably the origin of the dramatic changes in binding affinity seen in biological assays.^ Assuming that the low energy conformation in the above study is indeed the biologically active conformation of Substance P, the activities of the P- methyl analogs can be rationalized in terms of a conformational bias for or against the sidechain arrangement depicted in the NMR and modeling data. The implications of the previous statement are the foundation of the current research. If the two phenyl sidechains of residues 7 and 8 of Substance P can be tied together with an appropriate spacer, then an analog of Substance P, having the appropriate sidechain arrangement for biological activity, would result. If, as expected, this bridge between the sidechains forces the peptide to spend more time in the biologically active conformation, then an increase in binding affinity should occur.

8 Figure I: Space filling model of the lowest energy conformation of Substance P.

9 There is a distinct advantage to this approach of enhancing biological activity. Conformational changes in the peptide induced by steric bulk are not limited to the sidechains themselves. Backbone distortion can, and does, result in some cases which may negate or enhance effects due to changes in sidechain position. It is then impossible to deduce which specific changes were responsible for the observed variations in biological activity. On the other hand, the inclusion of a tether between two sidechains need not add any significant steric bulk. Actually, in this case, the tether is specifically designed so as to mimic the low energy conformation. Its function is simply to reduce the number of undesirable, higher energy conformers. With this goal in mind, a number of possible connections at the ortho positions of the two sidechains were modeled in the hope offindinga suitable bridge which closely approximated the low energy conformation of Substance P itself (see Figure 2). Linkage models varied in terms of length,fianctionality,and difficulty of incorporation. The best overlap between Substance P and a cyclic analog occurs when the bridge between the two phenyl groups consists of an 0-CH2-O-CH2-CH2-O-CH2-0 linkage. Figure 2f shows a line drawing of this linkage between the two phenyl sidechains, while Figure 3 shows an overiay of the tethered Substance P analog with Substance P.

10 (a) (b) \ ^ (c) (d) O O ^l^ (e) (f) ^^^ Figure 2: Examples of typical ortho linkages incorporated into models of Substance P. Rather than focus on the entire eleven residues of Substance P, the focus of this research is the synthesis of a cyclic phenylalanine dipeptide with a linker as shown in Figure 4. For later incorporation into Substance P, the dipeptide must be synthesized using orthogonal amine and acid protecting groups. This allows deprotection of the amine and coupling of the first six residues followed by acid deprotection and coupling to the final three residues of Substance P. The resulting cyclic Substance P analog can then be studied in biological assays.

11 Figure 3: Backbone structure overlay of cyclic Substance P analog with Substance P

12 (a) NHR" (b) Boc-Phe-Phe-O/Bu 1) amine deprotection 2) DCC, residues 1-6 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-O/Bu 1) acid deprotection 2) DCC, residues 9-11 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Figure 4: Design of a cyclic Substance P analog. The figure shows (a) the ortho-cyclic phenylalanine dipeptide, and (b) its incorporation into Substance P. Cyclic Peptides In the literature, it appears that these types of linkage between residue sidechains have not been previously utilized. More commonly, sidechain-to-sidechain cyclization occurs via cysteine or related disulfide bridges, examples of which are the antitumor 7

13 depsipeptide FR-901,228^^ and the 5 opioid agonist c[d-pen^d-pen^]enkephalin (DPDPE).^^ Otherwise, the cyclization typically makes use of other reactivity inherent in the residue sidechain, specifically tyrosine in the case of the macrocyclic norstatine-based inhibitors of HIV protease.^^ This last case is an atypical example of cyclization for the purpose of conformational restramt. For the most part, experimenters rely on the incorporation of steric bulk into the peptide to achieve a measure of conformational restraint. Examples of this are the aforementioned P-methylphenylalanine, 2'-6'-dimethyl- P-methyltyrosines (TMT),^^ and P-(trimethylsilyl)alanine.^^ This research appears to be thefirstto synthesize a sidechain-to-sidechain cyclic peptide to reduce conformational flexibility. Moreover, the attempt to accomplish this task by synthesizing the necessary fimctionality into the otherwise natural phenylalanine residues appears to be a departure fi-om the standard approach to the study of peptide conformation. 8

14 CHAPTER II SYNTHETIC CONSIDERATIONS Cyclization Methods Without question, the most important aspect of the synthesis is the cyclization step itself We envisioned two pathways by which cyclization could occur: synthesis of the two tethered amino acids and ring closure by peptide bond formation (scheme la), and synthesis of the acyclic dipeptide and insertion of the tether via substitution of a dinucleophile to form two bonds in a reaction similar to that of crown ether synthesis (scheme lb). Both of these methods are highly sensitive to reagent concentrations and care must be taken so as to avoid polymerization and 2+2 adducts. The synthesis of the cyclic dipeptide could be designed to incorporate either a one or two centered cyclization step. Either choice givesrise to its own set of benefits and limitations. The one-centered cyclization would occur via afinalamide bond formation between the two phenylalanine residues. Prior to cyclization, this method necessitates using four orthogonal and base stable groups for protection of the amino and acid moieties of both residues, followed by an attachment of the ortho bridge to one, then the other amino acid. The benefit of this method lies in the cyclization method itself The onecentered reaction contains only one mode of reaction, namely the attack of an amine on an activated ester via dicyclohexylcarbodiimide (DCC) chemistry. The only possible side products arisefi"ompolymerization and the non-reacting amino and acid moieties can be

15 (a) HO O NH NHR" DCC RO NHR" (b) -RO NHR" o- o- \ / NHR" 23 R'=rBu R" = Boc Scheme 1 protected with standard protecting groups used commonly in reactions involving peptide bond formation. Alternatively, the two-centered cyclization gives rise to the possibility of multiple products including the 2+2 adduct or several acyclic 2+1 products. Additionally, the more harsh SN2 conditions made necessary by the ether nature of the ortho bridge may be incompatible with the reactive, albeit protected, functionality elsewhere in the molecule. Even with the difficulties posed by the two-centered reaction, the decision was made to follow this pathway because it required fewer protecting groups and because the inclusion of the linkage late in the synthesis gives rise to the possible incorporation of a number of different linkages, resulting in a modest library of cyclic dipeptides, with minimal modification of the overall synthesis. 10

16 Protecting Groups Because of the peptide nature of thefinalproduct and the use of solid phase peptide synthesis conditions to arrive at thefinalsubstance P analog, the most obvious choices of protecting groups for the amino and acid moieties of the individual amino acids and the resulting dipeptide are those consistently used in industrial peptide synthesis. These have been developed with a keen eye toward orthogonality so that a selective deprotection of one or more groups may be achieved. The most common acid protecting groups are the /-butyl and allyl ester, which are acid and transition metal labile, respectively. For the amino groups, the /-butyl carbamate (Boc), 9-fluorenylmethyl carbamate (Fmoc), and the benzyl carbamate (Cbz) are most extensively used and are removed by dilute acid, secondary amines, and catalytic hydrogenation, respectively. However, the conditions used for the removal of the Cbz group are also capable of cleaving benzyl ethers and could therefore destroy the ortho linkage in the dipeptide. For this reason, the Cbz group was eliminatedfi-omfurther consideration. As mentioned earlier, the nature of the cyclic product requires the synthesis of unnatural amino acids having functionality at the ortho position of the phenyl sidechains. The most obvious choice for this, based on the nature of the eventual linkage, is an hydroxymethyl. Alcohols are easily modified to an appropriate leaving group and the benzylic nature should facilitate the nucleophilic substitution of an ethylene glycol unit to complete the cyclization. This hydroxymethyl is present early in the synthesis and must therefore be protected with a suitable group which orthogonal to those amino and acid protecting groups which are introduced later. It must also be possible to remove the 11

17 hydroxymethyl protection selectively in the presence of those same amino and acid protecting groups. This protecting group, as well as those mentioned above will be discussed in more detail in the section entitled "Synthetic Methods", below. It should be noted that the choice of appropriate protecting groups is central to this synthesis. For the most part, this synthesis makes use of well known and studied reactions to achieve the target compound, rather than more elaborate or difficult types of chemistry. Therefore, the ability of the chosen protectmg groups to withstand the given reaction conditions, rather than the reactions themselves, will be seen to dictate the success or failure of this research. Retrosynthetic Analysis Thefirstmtermediate in a retrosynthetic analysis (Figure 5) of the cyclic dipeptide 23 could be considered to be the acyclic precursor (22) to thefinalproduct. This dipeptide is formed in turn by DCC coupling of two of the same basic ortho-functionalized phenylalanine residues, one as the protected acid free amine 19, the other as the free acid protected amine 12b. Both of these are synthesized through different pathways from the same amino acid precursor 5b (which willfi^omnow on be referred to as the "common intermediate"), which is in turn made up of three parts: a glycine equivalent to which the sidechain is introduced, a chiral auxiliary to dictate the stereochemistry of the alkylation, and an alkylating agent 4b which will become the functionalized sidechain. This initial portion of the synthesis, namely the formation of the amino acid precursor, has been 12

18 widely used for the synthesis of a number of natural and unnatural amino acids, yet its use for sidechainfiinctionalizedamino acids remains unreported 17 NHR" 'RO NHR" 22 OR RO HO OR / Br- O- V 4b Figure 5. Retrosynthetic analysis of the cyclic phenylalanine dipeptide. 13

19 CHAPTER III SYNTHETIC METHODS Though many aspects of this synthesis changed greatly as the research progressed, three major pathways evolved. Each of these differ primarily in the identity of protecting groups used. The Boc-Fmoc-Allyl Ester SEM Approach Initially, the 2-trimethylsilylethoxymethyl (SEM) group was seen as an appropriate protecting group for the sidechain hydroxyl function. It is stable to the conditions necessary for liberation of the amine and acid functions in the common intermediate, as well as the Lewis acid catalyzed transesterification to the allyl ester. Like most silyl protecting groups, it is typically removed under mild conditions with afluoridesource such as tetra-«-butyl ammoniumfluoride (TBAF). The SEM group also shows good orthogonality to the Boc, Fmoc, and allyl ester protecting groups. The synthesis began with the formation of the sultam derived N- (diphenylmethylene) glycinate equivalent 3 (scheme 2) by the method of Josien, Martin, and Chassaing^"^ with one modification. The methyl N-(diphenylmethylene)glycinate 2 was reportedly purified by distillation. In this case, however, the product was crystallized directly from the crude oil in good yields. 14

20 XN PhMgBr NH 1 J NH3CI MeO-^ MeO \ ^ THF I CH2CI2 MejAl Toluene 1) nbuli, TllF 2) Br 5a R= SEM 5b R= MPM OR 4a R= SEM 4b R=MPM Scheme 2 The alkylating agent is prepared from the commercially available 1,2-benzene dimethanol by bromination of one hydroxyl group to form bromomethylbenzyl alcohol (4) followed by protection of the other with SEM chloride to form 5a. Initial attempts to form the bromomethylbenzyl alcohol by the method of Cowell and Stille^^ via reaction with bromomethylenedimethylammonium bromide^^ proved difficult and gave disappointing yields. A dramatic improvement in both ease and yield was achieved by reaction of the benzene dimethanol with triphenylphosphine (PPhs) and carbon tetrabromide (CBr4), as shown in scheme 3. A statistical transformation with yields of the monobromide in the range of 50% were expected but yields in the range of 70-80% were consistently observed, possibly due to the steric bulk of the ylide complex. The alkylation 15

21 of 3 with the SEM protected benzyl bromide 4a to form the common intermediate 5a was then carried out, again by the method described by Chassaing and coworkers (scheme 2, R= SEM). OH Br Br CBr4, PPh3 SEMCl, Et3N o: CH2CI2 CH2CI2 OH OH OSEM Scheme 3 Due to the ortho functionality at the sidechain, it was thought best to avoid the free amino acid intermediate so as to limit the rather harsh purification conditions (ion exchange chromatography) and possible water solubility. Fortunately, Oppolzer and Lienard^^ describe methods by which both protected amino acids may be synthesized without going through the free amino acid. The common intermediate was cleaved first to the amine 7a (not shown), then protected as either the Fmoc or Boc sultam derived phenylalanine, compounds 8a and 9a, respectively (scheme 4, sultam chiral auxiliary denoted as "Nc"). The sultam of the Boc derivative can then be removed with basic hydrolysis to give Boc-Phe(OSEM)-OH (12a) directly. Alternatively, the transformation of the Fmoc derivative to H2N-Phe(0SEM)-allyl ester requires an additional step. The sultam of the Fmoc derivative 8a isfirsttransesterified by the method of Oppolzer and Lienard^^ to give the allyl ester 10, then the Fmoc is removed with piperidine to give the free amino ester 11. 4a 16

22 Nc = O ^ "^-«---NHFmoc j 1) INHCl,^^-^ THF OR 8a 2) FmocCl, Na7Co3 (aq) '^^^^ 5a,5b OR 1) INHCl THF 1 2) B0C2O THF NHBoc n.^^-/oh Ti(OEt)4/THF LiOH, H2O2 THF 2) piperdine THF O.0-^^NH2 DCC HOBT THF HO NHBoc OR 11 NHBoc Once the amino-allyl ester 11 and Boc-acid 12a have been made, the two are coupled using DCC chemistry to form dipeptide 13. The SEM protection was to be removed followed by mesylation of the two ortho hydroxymethyl groups, and the peptide cyclized using the dianion of ethylene glycol. Unfortunately, at this point, the first major failure of a protecting group was observed. Deprotection of the SEM group was not 17

23 afforded by either TBAF or cesium fluoride. Attempts to use trifluoroacetic acid (TFA) resulted in not only deprotection of the SEM groups, but also cleavage of the Boc and allyl ester protection and the peptide bond itself (presumably to small amounts of water in the TFA). In fact, the Boc group was used specifically for its acid lability and the best case scenario for the use of TFA would require a subsequent reprotection of the amine functionality. NHBoc TFA - Multiple Cleavage Products OSEM OSEM 13 Scheme 5 The Boc-Sultam MPM Approach After the failure of the SEM group, the search began for a more appropriate protection for the ortho functionality. Through model studies, the 4- methoxyphenylmethyl (MPM) group was shown to withstand the varied conditions of the synthesis. Also, its oxidative removal with dichlorodicyanoquinone (DDQ) showed no adverse affects on any of the other protection in the molecule. MPM protection of the bromomethylbenzyl alcohol is achieved through the addition of o-methoxyphenylmethyl-2,2,2-trichloroacetimidate, prepared fi-om methoxyphenylmethanol and trichloroacetonitrile, with an acid catalyst, to form 4b (scheme 6). The synthetic route to 5b (scheme 2) and the MPM protected intermediates 18

24 in scheme 4 is then identical to that of the SEM derivatives until the MPM deprotection of the dipeptide, with one major improvement. The previous alkylation step (3a to 5a) involved a difficuh purification of the product viaflashchromatography. With MPM protection (3b to 5b), however, the alkylation product 5b is crystauine and can simply be filteredfi-omthe reaction mixture. OH CBr4 PPhj /^^v^j^ NH MPMO-^CCl3 Br OH CH2CI2 \^::=^'^ OT-T ptsoh CH2CI2 OMPM 4b Scheme 6 In the process of bringing up the necessary starting materials, it was noticed that the synthesis of the Fmoc-allyl ester derivative might be unnecessary. In the course of model studies for MPM protection, it was shown that the sultam remained intact during MPM deprotection. If the suham could be used as a "protecting group" for the acid functionality, then the free amine 7b could be used as outlined in scheme 7, rather than taking the four steps to the MPM protected amino-allyl ester derivative 11 indicated in scheme 4. Boc-Phe(OMPM)-OH (12b)and H2N-Phe(0MPM)-Sultam (7b) were coupled to form the MPM protected dipeptide 14. Deprotection of the MPM group, to form the diol 15, followed by mesylation of the ortho hydroxy Sanctions, giving 16, made possible an 19

25 O"^^ OMPM 1) INHCl THF NHBoc ' -. ^ 2) B0C2O THF 6b INHCl THF LiOH, H2O2 THF NH, NHBoc DCC HOBT 7b OMPM NHBoc OMPM OMPM 14 Scheme 7 attempt at the cyclization. After several attempts, it became clear that the sultam was not stable to the nucleophilic conditions of the cyclization and that an intramolecular esterification was likely occurring between one of the side chains and the previously sultam-protected acid moiety (scheme 8). Unfortunately, only very small quantities of this compound could be isolated and, as a result, it was not flilly characterized. However, it was possible to determine by NMR that the sultam had been cleaved. The NMR spectra 20

26 also showed that an ethylene glycol unit had been incorporated into the molecule. Cyclization to form the lactone, rather than the desired product, was surmised by the complex splitting of one benzylic CH2 and the simplified signal of the other, as compared to the pseudo-symmetrical signals for these four hydrogens in the starting material. NHBoc NHBoc DDQ, ph 4 Buffer CH2a2 OMPM OMPM MsoO CH'2Cl2 NHBoc O o.n-^""^.--nhboc KO^^ ^OK THF Scheme 8 The Boc-Fmoc-/-Butyl Ester MPM Approach At this point, a great deal of time and effort had been devoted to the synthesis and the failure of the sultam to survive the cyclization begged the question of whether this method of cyclization was indeed more favorable than the more linear, one-centered route. Since the synthetic methodology had been worked out up to the cyclization itself, it 21

27 seemed logical to make one last attempt at the two-centered cyclization. But, rather than continue with the allyl ester strategy, it was decided to opt for the /-butyl ester, one of the least reactive esters in the presence of nucleophiles. Again, the synthesis remained virtually identical to that of the two previous pathways, with the exception that H2N-Phe(0MPM)-0-/-Bu (19) was formed from the common intermediate 5b, as shown in scheme 9. This was accomplished by again cleaving the imine and protection of the amine 7b with Fmoc to give 8b. The sultam was the removed to form the free acid 17 which was then protected as the /-butyl ester, compound 18 (It should be noted that several methods were tried for the /-butyl protection on both the Fmoc-acid and the free amino acid with little or no success. It was not until the method of Jackson and coworkers^^ was used, which makes use of an O- /-butyl-2,2,2-trichloroacetimidate in a similar manner to MPM protection, that an effective transformation was achieved). After Fmoc deprotection of 18 with piperidine, the free amine 19 was coupled with the free acid 12b to give Boc-Phe(OMPM)-Phe(OMPM)-0-/- Bu (20). DDQ-mediated MPM deprotection of this dipeptide, followed by mesylation via methanesulfonic anhydride (MS2O) gave compounds 21 and 22, respectively (scheme 10). 22

28 OMPM NHFmoc NHBoc 1) INHCl THF 1) INHCl THF 8b OMPM 2) FmocCl, Na2C03 (aq) THF 5b 2) B0C2O THF 1) LiOH, H2O2, THF 2) NH t-buo-^^cclg LiOH, H2O2 THF 3) piperidine t-buo NH. DCC HOBT NHBoc OMPM t-buo NHBoc 19 OMPM OMPM 20 Scheme 9 23

29 t-buo NHBoc t-buo NHBoc DDQ, ph 4 Buffer CH2CI2 OMPM OMPM MS2O CHoCl 2'-i2 r-buo O o,n-l^-^^ NHBoc Scheme 10 The cyclization was carried out using Boc-Phe(OMS)-Phe(OMS)-0-/-Bu, compound 22, with ethylene glycol and potassium hydride in tetrahydrofuran (THF) at a concentration of 6mM (high dilution). No reaction occurred within 24 hours (as monitored by thin layer chromatography), presumably due to limited solubility of the alkoxide, and addition of 5ml/7-dioxane resulted in no noticeable change within an additional 24 hours. At this time, an additional 0.5 equivalents of ethylene glycol that had been premixed with lithium diisopropylamide (LDA) in/?-dioxane was added to the solution. Preliminary NMR data indicate the presence of products which are thought to correspond to the 1+1 (compounds 23 and 24), 2+1 (25), and 2+2 adducts (26), as well as other compounds which remain unidentified at this time (scheme 11). 24

30 t-buo NHBoc 23 t-buo O o.n-^^^-^-nhboc t-buo NHBoc 24 t-buo NHBoc t-buo Scheme 11 25

31 CHAPTER IV CONCLUSIONS As of the writing of this dissertation, the yields of the cyclized Boc-/-Butyl dipeptide remain too low to provide an adequate means of arriving at the desired cyclic dipeptide. Repeated attempts to optimize reaction conditions have failed to increase product yields. However, as noted in the section entitled "Cyclization Methods," a synthesis involving an attachment of the linkage prior to amide bond formation remains a viable route to the desired end product. While the completion of the synthesis in this manner lies outside the scope of the current research, some initial work has been completed toward this alternate pathway for the benefit of future researchers. Experimental methods and NMR data for these new intermediates are included in Appendix A. In the future, after sufficient quantities of the cyclized dipeptide have been made, it will be possible to submit the material for incorporation into Substance P. From previous statements concerning the need for orthogonal protection at the amine and acid functionalities of the dipeptide, it might appear that the use of the Boc and /-butyl ester is not appropriate for the needs of peptide synthesis. Fortunately, there is literature precedent for the selective removal of the Boc protecting group. Gibson, Bergmeier, and Rapoport^^ have shown that the N-Boc group can be removed in the presence of the /- butyl ester through the use of a 1 M solution of HCl in ethyl acetate. This degree of orthogonality should prove sufficient in this case. If, however, the methods for coupling 26

32 the dipeptide tofragmentsof Substance P prohibit the use of these two groups in conjunction with each other, the Boc protection can be removed and replaced with an Fmoc or other suitable group. This research has made significant progress toward a cyclic ortho-functionalized phenylalanine dipeptide which appears to be the first attempt to create a sidechain-tosidechain cyclized peptide. In the course of the research, several improvements have been made in existing methodologies, particularly for the mild and efficient synthesis of bromomethylbenzyl alcohol and for the purification of the methyl N-(diphenylmethylene) glycinate. Upon successful synthesis of sufficient quantities of this dipeptide (~ mg), the cyclic Substance P analog can be made and studied. It is expected, by reasoning that the lowest energy conformers are likely those involved in biological activity, that this analog will display increased affinity for the NKl receptor. In addition, in the process of developing the cyclic dipeptide, this research has resulted in the synthesis of several sidechain-functionalized phenylalanine derivatives which mayfindapplication in other areas of study. This also serves as a good method for the synthesis of other sidechain functionalized amino acids thereby increasing the number of options available to those investigate peptides through the use of conformational restraint. 27

33 CHAPTER V EXPERIMENTAL METHODS All reagents and compounds whose syntheses are not specifically listed are commercially available and were used as provided. Reaction solvents were distilled for purity under conditions appropriate for each solvent. Flash column chromatography (FC) was carried out using silica gel, mesh, 60 angstrom, BET surface area ~500m /g, pore volume ~0.75cmVg. ^H and ^^C NMR samples were analyzed on Bruker AF-200 (200 MHz) and/or Bruker AF-300 (300 MHz) spectrometers. Sultam derived N-(diphenybiethylene)glycinate (3): Prepared (including intermediate compounds 1 and 2) by the method of Josien, Martin, and Chassaing, ^'^ with the following modification: evaporation of the solvent from the workup of the final step gives a viscous yellow oil which crystallizes upon standing for hours. Faster crystallization can be achieved (~5 hours) by the addition of seed crystals to the crude oil. o-bromomethylbenzyl Alcohol (6): To a stirred solution of 5.Og 1,2-benzenedimethanol (36.2mmol) in 150ml methylene chloride at 0 C was added 9.97g triphenylphosphine (PPhj) (38.0mmol) followed by 12.61g CBr4 (38.0mmol). Upon complete addition, the solution was allowed to warm to room temperature and stirred 12 hours. The solution was washed several times with brine, dried over MgS04, and concentrated on a rotary evaporator. FC with 8:2 hexanes:ethyl acetate gave 5.58g (76.6%) of a white crystalline solid: ^H NMR (CDCI3) (s, IH), 4.62 (s, 2H),

34 (s, 2H), (m, 4H); '^C NMR (CDCI3) , 62.72, , , , , , (Trimethylsilvl)ethoxvmethyl o-bromomethylbenzyl Ether (4a): To a stirred solution of 2.68g 6 (13.33mmol) in 100ml methylene chloride at 0 C under N2 was added 8.58g tetra-«-butylammonium bromide (26.66mmol) followed by 3.25ml diisopropyl ethyl amine (7Pr2NEt) (18.66mmol). After 10 minutes, 2.59ml SEM chloride (14.66mmol) was added via syringe and the solution was allowed to stir 2 hours. The solution was then washed several times with brine, dried over MgS04, and the solvent was removed using a rotary evaporator. FC in 8:2 hexanes:ethyl acetate gave 4.03g (91.4%) of the clear colorless liquid: ^H NMR (CDCI3) (s, 9H), 0.96 (m, 2H), 3.65 (m, 2H), 4.70 (s, 2H), 4.74 (s, 2H), 4.75 (s, 2H), 7.34 (m, 4H); ''C NMR (CDCI3) , 18.07, 43.55, 65.37, 66.67, 94.15, , , , , , /7-Methoxybenzyl o-bromomethylbenzyl Ether (4b): To a stirred suspension of 0.16g NaH as a 60% dispersion in mineral oil (3.89mmol NaH) in 50ml dry tetrahydrofuran (THF) at room temperature under Ar was added 4.84ml/7-methoxybenzyl alcohol (38.9mmol) via syringe. After 30 minutes, 3.90ml trichloroacetonitrile (38.9mmol) was added slowly by syringe and the solution was allowed to stir for an additional 2 hours. The solution was then washed several times with brine, dried over MgS04, and concentrated on a rotary evaporator. The resulting red oil (crude O- methoxyphenylmethyl 2,2,2-trichloroacetimidate) was added to a stirred solution of 5.58g 6 (27.8mmol) in 50ml methylene chloride with stirring at room temperature under Ar. A 29

35 catalytic amount of p-toluenesulfonic acid monohydrate (~ 2mmol) was added and the solution was allowed to stir 20 hours. The solution was washed once with a saturated aqueous NaHCOs solution, once with brine, dried over MgS04, and concentrated on a rotary evaporator. Separation of the trichloroacetamide by-product by trituration of the residue in methylene chloride and hexanes followed by FC in 95:5 hexanes:ethyl acetate gave 7.89g (88.6%) of the clear colorless oil: ^H NMR (CDCI3) ( s, 3H), 4.55 (s, 2H), 4.63 (s, 2H), 4.69 (s, 2H), 6.93 (d, 2H), 7.32 (m, 6H); ^^C NMR not available. Sultam Derived N-(diphenylmethylene) Ortho-SEM Protected Phenylalanine Equivalent (5a) (Conmion Intermediate): The preparation for this compound is identical to that of 5b except for purification. After workup and solvent evaporation, FC with a gradient elution from 9:1 to 7:3 hexanes:ethyl acetate gives the puffy white solid in 56% yield: ^H NMR (CDCI3) (s, 9H), 0.68 (s, 3H), 0.83 (s, 3H), 0.92 (m, 2H), 1.26 (m, 3H), 1.76 (m, 3H), 1.98 (m, IH), 3.09 (dd, IH, J= 8,13 Hz), 3.29 (s, 2H), 3.36 (dd, IH, J= 6,13 Hz), 3.58 (m, 2H), 3.85 (m, IH), 4.39 (s, 2H), 4.61 (s, 2H), 5.04 (dd, IH, J= 6,8 Hz), 6.81 (d, 2H), 7.05 (d, 2H), 7.38 (m, 8H), 7.52 (d, 2H); ''C NMR (CDCI3) , 17.95, 19.68, 20.38, 26.31, 32.63, 37.11, 38.04, 44.40, 47.47, 48.14, 53.00, 64.93, 64.98, 66.33, 93.83, , , , , , , , , , , , , , , , , , , Sultam Derived N-(diphenylmethylene) Ortho-MPM Protected Phenylalanine Equivalent (5b) (Common Intermediate): To a stirred solution of 8.44g of the glycine equivalent 3 (17.4mmol) in 100ml dry THF at -78 C under Ar was added 9.5ml 30

36 w-butyllithium (2.0M in hexanes, 19.0mmol). After 30 minutes, a solution of 6.1 Ig 4b in 10ml THF was added slowly by syringe and the solution was allowed to warm to room temperature and stir for 12 hours. The solution was washed several times with brine, dried over MgS04, and concentrated on a rotary evaporator. The residue was taken up in diethyl ether and triturated with hexanes to precipitate 8.85g (75.1%) of a fine white powder: ^HNMR (CDCI3) (s, 3H), 0.84 (s, 3H), 1.31 (m, 2H), 1.79 (m, 4H), 1.98 (m, IH), 3.06 (dd, IH, J= 8,13 Hz), 3.30 (s, 2H), 3.32 (dd, IH, J= 6,13 Hz), 3.79 (s, 3H), 3.88 (m, IH), 4.32 (m, 4H), 5.05 (dd, IH, J= 6,8 Hz), 6.85 (d, 4H), 7.25 (m, 12H), 7.61 (m, 2H); [a]d (c = g/ml, CHCI3). H2N-Phe(0SEM)-Sultam (7a): To a stirred solution of 1.12g 6a (1.63mmol) in 25ml THF at room temperature was added 3.0ml IM aqueous HCl (3.00mmol). After stirring for 2 hours, the solution was washed tv^ce with a saturated NaHCOs (aq) solution, dried over MgS04, and concentrated on a rotary evaporator. FC with a gradient elution from 8:2 to 1:1 hexanes:ethyl acetate gave 0.82g (96.5%) of the clear, colorless oil. NMR spectra were not taken for this compound. It was used in crude form for subsequent amine protection. H2N-Phe(0MPM)-Sultam (7b): To a stirred solution of 1.OOg 6b (1.47mmol) in 25ml THF at room temperature was added 3.0ml IM aqueous HCl (3.00mmol). After stirring for 2 hours, the solution was washed twice with a saturated NaHCOs (aq) solution, dried over MgS04, and concentrated on a rotary evaporator. FC with a gradient elution from 8:2 to 1:1 hexanes:ethyl acetate gave 0.75g (98.7%) of the clear, coloriess oil: ^H NMR (CDCI3) (s, 3H), 0.85 (s, 3H), 1.27 (m, 2H), 1.61 (broad singlet, 2H), 31

37 1.73 (m, 3H), 1.80 (m, 2H), 1.94 (m, IH), 2.92 (dd, IH, J= 8, 13 Hz), 3.08 (dd, IH, J= 8, 13 Hz), 3.35 (d, IH), 3.39 (d, IH), 3.76 (s, 3H), 4.29 (dd, IH, J= 8, 8 Hz), 4.48 (s, 2H), 4.55 (d, IH), 4.61 (d, IH), 6.84 (d, 2H), 7.23 (m, 6H); ^'C NMR not available; [ajd (c = g/ml, CHCI3). Fmoc-Phe(OSEM)-Suham (8a): To a stirred solution of 0.96g 6a (1.40mmol) in 50ml THF at room temperature was added 3.0ml IM aqueous HCl (3.00mmol). After stirring for 2 hours, the solution was washed twice with a saturated NaHC03 (aq) solution, dried over MgS04, and concentrated on a rotary evaporator. The residue was then dissolved in 25ml THF and stirred at room temperature under Ar. To this was added 15ml of a 10% Na2C03 (aq) solution (loml/mmol) followed by 0.44g 9-fluorenylmethyl chloroformate (Fmoc-Cl) (1.70mmol) and the solution was allowed to stir six hours. The solution was washed twice with brine, dried over MgS04, and concentrated using a rotary evaporator. FC in 85:15 hexanes:ethyl acetate gave 0.95g (85.6%) of a clear colorless oil: *HNMR (CDCI3) (s, 9H), 0.89 (m, 2H), 0.96 (s, 3H), 1.12 (s, 3H), 1.32 (m, 3H), 1.87 (m, 2H), 2.09 (m, 2H), 2.94 (t, IH, J= 12 Hz), 3.32 (t, IH, J= 9 Hz), 3.51 (s, 2H), 3.64 (m, 2H), 3.95 (s, IH), 4.19 (m, 3H), 4.51 (d, IH), 4.75 (m, 3H), 5.02 (m, IH), 6.71 (s, IH), 7.30(m, 8H), 7.50(d, IH), 7.57 (d, IH), 7.70 (d, 2H); ''CNMR5-1.39, 14.11, 18.14, 19.86, 20.85, 22.64, 26.37, 31.57, 32.78, 33.79, 38.33, 44.62, 47.07, 47.78, 48.78, 52.92, 56.63, 65.06, 65.76, 66.67, 67.54, 93.85, , , , , , , , , , , , , , , , Fmoc-Phe(OMPM)-Sultam (8b): To a stirred solution of I.OOg 6b (1.47mmol) in 25ml THF at room temperature was added 3.0ml IM aqueous HCl (3.00mmol). After 32

38 stirring for 2 hours, the solution was washed twice with a saturated NaHC03 (aq) solution, dried over MgS04, and concentrated on a rotary evaporator. The residue was then dissolved in 25ml THF and stirred at room temperature under Ar. To this was added 15ml of a 10% Na2C03 (aq) solution (loml/mmol) followed by 0.46g Fmoc-Cl (1.76mmol) and the solution was allowed to stir one hour. The solution was washed twice with brine, dried over MgS04, and concentrated on a rotary evaporator. FC with 8:2 followed by 7:3 hexanes: ethyl acetate gave 1.09g (>99%) of the clear coloriess oil: ^H NMR (CDCI3) (s, 3H), 1.12 (s,3h), 1.39 (m,2h), 1.89 (m, 3H), 2.14 (d, 2H), 2.85 (t, IH, J= 12 Hz), 3.26 (dd, IH, J= 4,13 Hz), 3.52 (s, 2H), 3.73 (s, 3H), 3.96 (m, IH), 4.17 (m, 4H), 4.60 (m, 3H), 5.00 (m, IH), 6.85 (d, 2H), 6.98 (d, IH), 7.39 (m, 14H); ^^C NMR (CDCI3) , 20.89, 32.81, 33.58, 38.36, 44.67, 47.04, 47.79, 48.79, 52.94, 55.17, 56.75, 65.08, 66.64, 69.78, 72.35, , , , , , , , , , , , , , , , , , , , : [a]d (c = g/ml, CHCI3). Boc-Phe(OSEM)-Sultam (9a): To a stirred solution of 2.92g 6a (4.25mmol) in 50ml THF at room temperature was added 8.00ml IM aqueous HCl (8.00mmol). After stirring for 5 hours, the solution was washed twice with a saturated NaHCOs (aq) solution, dried over MgS04, and concentrated on a rotary evaporator. The residue was then dissolved in 25ml dry THF and stirred at room temperature under Ar. After syringe addition of 1.17ml di-/er/-butyl dicarboxylate (B0C2O) (5.18mmol), the solution was allowed to stir 4 hours. The solution was washed twice with brine, dried over MgS04, and concentrated on a rotary evaporator. FC with 8:2 followed by 7:3 hexanes:ethyl 33

39 acetate gave 2.38g (89.9%) of the clear, colorless oil: ^H NMR (CDCI3) (s, 9H), 0.92 (m, 5H), 1.08 (d, 3H), 1.30 (s, 9H), 1.86 (m, 5H), 2.05 (d, 2H), 2.86 (t, IH, J= 13 Hz), 3.24 (dd, IH, J= 4,13 Hz), 3.38 (m, IH), 3.48 (s, 2H), 3.64 (m, 2H), 3.89 (s, IH), 4.50 (d, IH), 4.72, (m, 3H), 4.92 (m, IH), 7.24 (m, 3H), 7.51 (m, IH). Boc-Phe(OMPM)-Sultam (9b): To a stirred solution of 1.50g 6b (2.06mmol) in 25ml THF at room temperature was added 4.00ml IM aqueous HCl (4.00mmol). After stirring for 2 hours, the solution was washed twice with a saturated NaHCOs (aq) solution, dried over MgS04, and concentrated on a rotary evaporator. The residue was then dissolved in 25ml dry THF and stirred at room temperature under Ar. After syringe addition of 0.70ml B0C2O (2.88mmol), the solution was allowed to stir 4 hours. The solution was washed tv^ce with brine, dried over MgS04, and concentrated on a rotary evaporator. FC with 8:2 followed by 7:3 hexanes:ethyl acetate gave 1.36g (99.4%) of the clear, colorless oil: 'H NMR (CDCI3) (s, 3H), 1.12 (s, 3H), 1.28 (s, 9H), 1.31 (m, 2H), 1.87 (m, 3H), 2.10 (d, 2H), 2.81 (t, IH, J= 12 Hz), 3.20 (dd, IH, J= 4,13 Hz), 3.50 (s, 2H), 3.79 (s, 3H), 3.92 (m, IH), 4.30 (d, IH), 4.57 (q, 3H), 4.94 (m, IH), 6.22 (d, IH), 6.89 (d, 2H), 7.35 (m, 6H); ^^C NMR (CDCI3) , 20.82, 26.24, 28.13, 32.66, 38.23, 44.55, 47.59, 48.58, 52.77, 55.11, 55.89, 64.88, 69.69, 72.04, 78.92, , , , , , , , , , , , , ; [a]d (c = g/ml, CHCI3). Fmoc-Phe(OSEM)-Allyl Ester (10): In a 50ml high pressure tube equipped with a stir bar and a teflon screw cap was placed 1.43g 8a (1.92mmol) dissolved in 20ml THF and 20ml allyl alcohol and the tube was purged with Ar. After addition of 2.0ml titanium 34

40 ethoxide (Ti(0Et)4), the cap was tightly fitted and the solution was stirred at 130 C for 24 hours. The solution was then washed with dilute acid followed with brine, dried over MgS04, and the solvent was removed on a rotary evaporator. Removal of the majority of the remaining allyl alcohol was accomplished under vacuum. FC in 85:15 hexanes:ethyl acetate gave 0.89g (84.7%) of the clear coloriess oil: *H NMR (CDCI3) (s, 9H), 0.97 (m, 2H), 3.08 (dd, IH, J= 10,14 Hz), 3.25 (dd, IH, J= 5,14 Hz), 3.65 (m, 2H), 4.21 (m, 3H), 4.65 (m, 7H), 5.22 (m, 2H), 5.87 (m, IH), 6.39 (d, IH), 7.30 (m, 8H), 7.51 (m, 2H), 7.72 (d, 2H); ^^C NMR (CDCI3) , 18.26,29.84,34.08,47.21,55.61,65.64, 65.79, 66.81, 67.58, 93.98, , , , , , , , , , , , , , , , , , , , H7N-Phe(0SEM)-Allyl Ester (11): To a stirred solution of 0.35g 10 (0.81mmol) in 25nil THF under Ar was added 0.09ml piperidine (0.89mmol) via syringe. After 4 hours, the solution was washed with brine, dried over MgS04, and the solvent removed on a rotary evaporator. FC in 8:2 hexanes:ethyl acetate gave 0.14g (62.9%) of the clear coloriess oil: ^H NMR (CDCI3) (s, 9H), 0.93 (m, 2H), 1.46 (s, 2H, broad), 2.84 (dd, IH, J= 9,14 Hz), 3.16 (dd, IH, J= 5,14 Hz), 3.63 (m, 2H), 3.74 (dd, IH, J= 5,9 Hz), 4.57 (d, 2H), 4.63 (d, 2H), 4.71 (s, 2H), 5.22 (m, 2H), 5.83 (m, IH), 7.22 (m, 3H), 7.34 (m, IH); ^'C NMR (CDCI3) , 18.02, 37.79, 55.62, 65.22, 65.46, 67.15, 94.00, , , , , , , , , ; [ajo (c = g/ml, CHCI3). 35

41 Boc-Phe(OSEM)-OH (12a): To a stirred solution of 0.30g 9a (0.48mmol) in 40ml THF and 10ml H2O (4:1 THF:H20) was added 0.05g lithium hydroxide (~2mmol) and 2.0ml of a 30% H2O2 (aq) solution. After 2 hours, the solution was washed once with IM aqueous HCl, once with brine, dried over MgS04, and concentrated on a rotary evaporator. FC in chloroform gave 0.20g (quant.) of the clear colorless oil: ^H NMR (CDCI3) (s, 9H), 0.95 (m, 2H), 1.33 (s, 9H), 2.98 (m, IH), 3.12 (m, IH), 3.64 (m, 2H), 4.43 (m, IH), 4.60 (dd, 2H), 4.73 (s, 2H), 5.21 (d, IH), 7.24 (m, 4H); ^'C NMR (CDCI3) , 18.04, 28.23, 34.37, 48.96, 54.87, 65.44, 67.19, 93.76, , , , , , , , ; [ajo (c = g/ml, CHCI3). Boc-Phe(OMPM)-OH (12b): To a stirred solution of 1.52g 9b (2.48mmol) in 40ml THF and 10ml H2O (4:1 THF:H20) was added 0.24g lithium hydroxide (9.92mmol) and 1.12ml of a 30% H2O2 (aq) solution. After 2 hours, the solution was washed once with IM aqueous HCl, once with brine, dried over MgS04, and concentrated on a rotary evaporator. FC in chloroform gave 1. log (98.1%) of the clear colorless oil: 'H NMR (CDCI3) (s, 9H), 3.04 (m, IH), 3.23 (m, IH), 3.77 (s, 3H), 4.47 (m, 5H), 6.23 (s, IH), 6.86 (d, 2H), 7.28 (m, 6H), 9.5 (s, IH, broad); ^'C NMR not available; [ah (c = g/ml, CHCI3). Boc-Phe(OSEM)-Phe(OSEM)-Allvl Ester (13): To a stirred solution of 0.27g of the amine 11 (0.74mmol), 0.31g of the acid 12a (0.74mmol), O.llg 1-hydroxybenzotriazole (HOBT) (0.81mmol), and 0.14ml /Pr2NEt (0.81mmol) at room temperature under Ar was added 0.17g dicyclohexylcarbodiimide (DCC) (0.81mmol) and the solution was allowed to stir overnight. The solution was fihered to remove the 36

42 dicyclohexyl urea by-product, washed with brine, dried over MgS04, and concentrated on a rotary evaporator. FC in 8:2 hexanes:ethyl acetate gave 0.55g (96.5%) of the clear coloriess oil: ^H NMR (CDCI3) (s, 18H), 0.95 (m, 4H), 1.29 (s, 9H), 2.80 (m, IH), 3.08 (m, 3H), 3.63 (m, 4H), 4.27 (m, IH), (m, IIH), 5.20 (m, 2H), 5.75 (m, IH), 7.00 (m, IH), 7.21 (m, 9H); *'C NMR (CDCI3) , 14.00, 18.07, 18.10, 24.65, 25.41, 28.18, 34.67, 34.88, 53.10, 55.79, 61.19, 65.48, 65.78, 66.94, 67.48, 79.51, 93.34, 94.18, , , , , , , , , , , , , , , , Boc-Phe(OMPM)-Phe(OMPM)-Sultam(14): To a stirred solution of 1.14g of the amine 7b (2.22mmol), 0.6lg of the acid 12b (1.47mmol), 0.22g HOBT (1.62mmol), and 0.31ml 7Pr2NEt (1.76mmol) at room temperature under Ar was added 0.33g DCC (1.62mmol) and the solution was allowed to stir overnight. The solution was filtered to remove the dicyclohexyl urea by-product, washed with brine, dried over MgS04, and concentrated on a rotary evaporator. FC with gradient elution from 8:2 to 1:1 hexanes:ethyl acetate gave 1.13g (84.3%) of the clear colorless oil: ^H NMR (CDCI3) (s, 3H), 0.96 (s, 3H), 1.23 (s, IIH), 1.81 (m, 3H), 2.03 (m, 2H), 2.78 (m, 2H), 3.04 (m, 2H), 3.43 (m, 2H), 3.76 (s, 6H), 4.48 (m, 9H), 5.20 (m, IH), 5.63 (m, IH), 6.85 (m, 4H), 7.25 (m, 12H); ^'C NMR (CDCI3) ,20.83,26.29,28.16,32.83,35.15, 38.31, 44.70, 47.65, 48.61, 52.73, 54.11, 55.21, 69.81, 70.08, 71.80, 72.55, 79.24, , , , , , , , , , , , , , , , , , , ; [ajo (c = g/ml, CHCL3). 37

43 Boc-Phe(OH)-Phe(OH)-Sultam (15): To a stirred solution of 1.13g dipeptide 14 (1.24mmol) in 40ml methylene chloride under Ar was added 40ml ph 4 potassium hydrogen phthalate (KHP) buffer and 0.84g dichlorodicyanoquinone (DDQ) (3.72mmol). After 5 hours, 50ml ethyl acetate was added and the solution was washed five times with 20ml aliquots of saturated aqueous NaHC03 to remove unreacted DDQ. The solution was dried over MgS04 and concentrated on a rotary evaporator. FC with gradient elution from 8:2 to 1:1 hexanes:ethyl acetate gave 0.65g (78.3%) the clear viscous oil: ^H NMR (CDCI3) (s, 3H), 1.13 (s, 12H), 1.34 (m, 2H), 1.85 (m, 3H), 2.08 (m, 2H), 2.76 (m, IH), 2.96 (m, IH), 3.22 (m, 2H), 3.52 (s, 2H), 3.89 (s, IH), 4.33 (m, IH), 4.60 (m, 6H), 5.05 (m, IH), 5.58 (d, IH), 7.22 (m, 8H), 7.55 (d, IH). Boc-Phe(OMS)-Phe(OMS)-Sultam(16): To a stirred solution of 0.65g of the dipeptide diol 15 (0.97mmol) in 25ml distilled methylene chloride at 0 C under Ar was added 0.51ml /Pr2NEt (2.91mmol) followed by 0.37g methanesulfonic anhydride (MS2O) (2.13mmol) and the solution was allowed to stir for 2 hours. The solution was washed with brine, dried over MgS04, and concentrated on a rotary evaporator. FC in 1:1 hexanes:ethyl acetate gave 0.12g (48.0%) of the clear viscous oil: ^H NMR (CDCI3) (s, 3H), 0.90 (s, 3H), 1.29 (s, IIH), 1.88 (m, 5H), (m, loh), 3.43 (s, 2H), 3.75 (m, IH), 4.30 (m, IH), 4.35 (m, 4H), 5.12 (d, IH), 6.57 (d, IH), 7.29 (m. 8H). Fmoc-Phe(OMPM)-OH (17): To a stirred solution of 1.09g 8b (1.48mmol) in 40ml THF and 10ml H2O (4:1 THF:H20) was added 0.05g LiOH (2.22mmol) and 0.25ml of a 30% H2O2 (aq) solution. After 2 hours, the solution was washed once with IM aqueous HCl, once with brine, dried over MgS04, and concentrated on a rotary 38

44 evaporator. FC in chloroform gave 0.76g (95.0%) of the clear coloriess oil: ^H NMR (CDCI3) (dd, IH, J= 11,13 Hz), 3.30 (dd, IH, J= 4,13 Hz), 3.70 (s, 3H), 4.09 (t, IH), 4.30 (m, 3H), 4.60 (m, 4H), 6.89 (d, 2H), 7.00 (d, IH), 7.36 (m, 12 H), 7.72 (d, 2H), (s, IH, broad); '^C NMR (CDCI3) , 46.83, 54.95, 55.49, 66.59, 69,84, 72.27, , , , , , , , , , , , , , , , , , , , , , ; [a]d (c = g/ml, CHCI3). Fmoc-Phe(OMPM)-0-/-Bu (18): The following procedure is derived from the method for /-butyl ester synthesis developed by Armstrong et al^^. To a stirred solution of l.olg 17 (1.88mmol) dissolved in methylene chloride (2ml/mmol) at room temperature was added a solution of 0.82g 0-/-butyl 2,2,2-trichloroacetimidate in cyclohexane (2ml/nmiol). The trichloroacetamide by-product began to precipitate within one hour. After 5 hours, ~0.5g solid NaHCOs was added and the solution was stirred for an additional 30 minutes. The solution was then filtered through glass wool and the solvent removed on a rotary evaporator. FC in 8:2 hexanes:ethyl acetate gave 0.83g (74.4%) of the clear colorless oil: ^H NMR (CDCL3) (s, 9H), 2.96 (dd, IH, J= 8.8, 13.8 Hz), 3.14 (dd, IH, J= 5.4, 13.8 Hz), 3.75 (s, 3H), 4.10 (dd, IH, J= 5.4, 8.8 Hz), 4.23 (d, 2H), 4.34 (d, 2H), 4.58 (m, 3H), 6.44 (d, IH), 6.36 (d, 2H), 7.29 (m, loh), 7.47 (t, 2H), 7.70 (d, 2H); '^C NMR (CDCI3) , 34.18, 47.20, 55.33, 56.22, 66.78, 70.12, 72.47, 81.96, , , , , , , , , , , , , , , , , , ; [a]d (c = g/ml, CHCI3). 39