Metal Carboxylate Salts As an Avenue to Protecting Group Free Peptide Couplings

Metal Carboxylate Salts as an Avenue to Protecting Group Free Peptide Couplings

Isaac Rakofsky

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Chemistry University of Toronto

Metal Carboxylate Salts as an Avenue to Protecting Group Free Peptide Couplings

Isaac Rakofsky

Master of Science

Department Of Chemistry University of Toronto

2018 Abstract Peptide coupling has had a long history, beginning its story in 1882 using the silver salt of glycine, continuing to solid phase peptide synthesis and the introduction of protecting groups.

However protecting groups create a lot of needless waste. This thesis explores potential work in the area regarding the use of metal carboxylates as means to get around the need for these groups, as well as taking things further to do more than a single coupling couplings in one pot in solution phase chemistry. Despite variable success throughout, making a one-pot tripeptide in good yield and low epimerization was indeed achieved, with the help of these metal carboxylates and ONp esters.

Acknowledgments

I’d like to thank, first and foremost, Dr. Robert Batey for accepting me into his lab, and allowing me to the time to become a fully-fledged chemist under his guidance. Thank you to Anna Liza whose patience I tried many times, your administrative work was always appreciated. Thank you Darcy, Jack, Shawn and the whole NMR team over the years for helping me analyze my work and getting me the machines that I needed. Thank you to Dr. Sophie Rousseaux and all of the above, without who I would have never been able to get this thesis out before the deadline. All your kindness is appreciated.

I’d like to thank Marvin Morales who handled all of my training and familiarized me with standard organic lab protocols. Next I’d like to thank PJ. Roest who helped coach and to get my chemistry up to speed as well as suggesting ideas for experiments over the course of the degree, to varying degrees of success. I’d also like to thank Anika Tarasewicz who also helped me massively with chemistry, and also sat through my stress rants, especially in these last few weeks of my degree. Maja Chojnacka, always eager to help me out as well, and great company when wanting to get sushi for lunch or being there when needing to unwind. Honourable mention to the rest of the Batey group for being a great bunch of people for more than just chemistry.

Finally I’d like to thank my mom, dad and my brother Joseph who is my best friend, for helping me get to this point, and without them I probably would have never even gotten here. Joseph, I super miss you and I’m always looking forward to the next time we’re able to get together (living across the country makes it difficult). Success isn’t made by only one individual and it takes a village to raise a person.

P.S. Shout out to Auntie Edie, Auntie Rachel and Uncle Sheldon P.P.S. Shout out to all my Montreal friends

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Table of Contents

Acknowledgements.………………………………………………….…………….……………. iii

List of Tables.………………………………………………………...………………………….. vi

List of Figures.………………..…………………………………………………………………. xii

List of Schemes.……….………..……………………………………………………………… xiii

List of Appendices.….……………...…………………………………………………………….. x

List of Abbreviations.………………………………………...………………………………….. xi

Chapter 1 Introduction: History and Classic Methods of Amide Bond Formation………………1

1.1 Brief History of the Peptide Bond …………………………...……………………….1

1.2 Epimerization of Amino Acids….…………………………………………………….3

1.3 Common Method of C-terminus Activation ……...………………………………….5

1.3.1 Acid Chlorides ...……..……………………………………………………..6

1.3.2 Carbonyl diimidazole (CDI)……………………………………….………..7

1.3.3 Anhydrides…………………………………………………………………..8

1.3.4 Activated Esters……………………………………………………………..8

1.4 Direct Coupling using Ru …………………………………………………………...10

1.5 Overall Summary…………………………………………..………………………...11

1.6 References……...…………………………………………..………………………...11

Chapter 2 Calcium Carboxylate Salts and Their Use in Peptide Synthesis…………...……….. 14

2.1 The Utility of Carboxylate salts in Amide Bond Formation ....……………………...14

2.2 Results and Discussion….…………………………………………………………... 22

2.3 Conclusion………………………………… ……...………………………………... 28

2.4 References …………………………………….…...………………………………... 30

Chapter 3 Activated Esters and One-Pot Solution Phase Tripeptide Synthesis………………... 31

3.1 Protecting-Group Free Couplings of Amino Acids ....……………………….……... 31

3.2 Results and Discussion….…………………………………………………………... 34

3.3 Conclusion………………………………… ……...………………………………... 52

3.4 References …………………………………….…...………………………………... 54

Chapter 4 Copper-Lysine Complexes and Their Derivatizations………………………..…….. 55

4.1 The Utility of Carboxylate salts in Amide Bond Formation ....……………………...55

4.2 Results and Discussion….…………………………………………………………... 57

4.3 Conclusion………………………………… ……...………………………………... 62

4.4 References …………………………………….…...………………………………... 63

Chapter 5 Copper-Lysine Complexes and Their Derivatizations………………………..…….. 64

5.1 General Experimental……………………………………...... ……………………... 64

5.2 Results and Discussion….…………………………………………………………... 65

5.3 References………………………………… ……...………………………………... 93

List of Tables Table 2.1 Comparing modified Goodreid methods A and B for chosen model substrate .…….. 24

Table 2.2 Comparing the reactivity of the calcium and TBA salts of benzoic acid 2.16b and….25

Table 2.3 Comparing the reactivity of the calcium and TBA salts of Boc-Ala-OH 2.18a and ... 26

Table 2.4 Reaction of the calcium salt of Boc-Phe-OH 2.20a with select amines ….…………. 27

Table 3.1 Optimization of different carboxylate salts of leucine with different activating….…. 35

Table 3.2 Use of base and the free amino acid instead of amino acid salts …...…….…………. 37

Table 3.3 Altering conditions for tripeptide formation …..………………………….…………. 39

Table 3.4 Anthranilic acid as the middle “tripeptide” piece …..…………………….…………. 41

Table 3.5 Alternate methods for the formation of the lithium salt 3.14a and the effect on ……. 44

Table 3.6 Alternate methods for the formation of the metal salt 3.14b-e and the effect on ...…. 46

Table 3.7 Effect of using Leu salts instead of Phe salts on epimerization…………………….... 47

Table 3.8 Various reaction conditions for tripeptide Cbz-Phe-Phe-Ile-OMe 3.15 formation…...49

Table 3.9 Optimization of one pot-tripeptide conditions for formation of 3.15……..…………. 50

Table 3.10 Preliminary scope of tripeptides …...……………………………………………...... 51

Table 4.1 Decomplexation reactions ..………………………………………………………...... 58

Table 4.2 Improving formylation conditions for copper complex 4.9………………………...... 61

List of Figures

Figure 1.1 Various Amino Acids and the pKa of their α-protons ..……………………………… 4

Figure 1.2 Assorted activating groups used for created the activated ester …...………………… 9

Figure 1.3 Mechanism of activation and coupling using [Ru] and ethoxyacetylene ….……….. 11

Figure 2.1 Select carboxylate salts reacted with amines by Batey and colleagues …………….. 15

Figure 2.2 H-NMR in CDCl3 of reaction shown in scheme 2.9 at 1.0 and 2.0 h …...………….. 22

Figure 3.1 Other potential activated acids for future testing …..………………………………..37

Figure 3.2 VT experiment displaying the methyl ester peak of non-coalescing peaks at 25 °C.. 42

Figure 4.1 Original proposed complex of copper-glycine …….……………………………….. 56

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List of Schemes Scheme 1.1 Formation of protected dipeptide via silver(I) salt of glycine …..……………….…. 1

Scheme 1.2 Formation of Gly-Gly from diketopiperazine ...……....…………………………….. 1

Scheme 1.3 First example of the use of acid chlorides for peptide bond formation …………….. 2

Scheme 1.4 Method of generating peptide chains ……………………………………………….. 3

Scheme 1.5 Oxazolone formation causing epimerization of the -stereocentre in peptide…..….. 5

Scheme 1.6 Mechanism of Acid Chloride Formation from SOCl2 and (COCl)2……………..….. 6

Scheme 1.7 Epimerization of the -stereocentre via ketene formation………………………….. 7

Scheme 1.8 Mechanism of CDI activation and amine addition ...... …………………………….. 7

Scheme 1.9 Formation of carbonic mixed anhydride 1.28 through ethyl chloroformate 1.27..…..8

Scheme 1.10 Mechanism of activation by HOBt & HOAt ………………………….…………. 10

Scheme 1.11 Reaction conditions for amide bond formation using ethoxyacetylene as a..……..10

Scheme 2.1 General method for the direct formation of amide bonds from metal carboxylates.. 15

Scheme 2.2 Formation of Cbz-Phg-Val-OBzl 2.8…...……………………………….………….16

Scheme 2.3 TiCl4 based method used by Liguori Group for amide bond formation ..…………. 16

Scheme 2.4 Proposed mechanism by Liguori group for TiCl4 activated amidation ...…………. 17

Scheme 2.5 General scheme for carboxylate salt formation and coupling by Kodomari group .. 18

Scheme 2.6 General procedure for the formation of the isobutyl anhydrides of the amino……..19

Scheme 2.7 Unintended reactivation of 2nd amino acid during 2nd step………..…….…………. 20

Scheme 2.8 The two methods used to generate calcium salts from the corresponding free……. 21

Scheme 2.9 Initial testing on coupling time required for reaction of the simple calcium salt….. 22

Scheme 2.10 General method for generating lithium salts published by Batey and coworkers... 23

Scheme 2.11 Using the Goodreid methods A and B on substrate 2.16a to verify yields for….... 23

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Scheme 2.12 First attempts to utilize a one-pot approach for tripeptide synthesis using an……. 28

Scheme 3.1 Activation using p-NPCF and coupling of free acid in one pot ….…….…………..31

Scheme 3.2 Formation of benzotriazole esters and subsequent coupling to free amines ……….32

Scheme 3.3 One-pot activation and coupling of a free amino acid (AA2) using a mixed ethyl ...33

Scheme 3.4 In situ ONp ester activation of benzoic acid using EDC and subsequent coupling...38

Scheme 3.5 Coupling of two amino acids and one acid activation in a one-pot fashion ………. 39

Scheme 3.6 Two sequential in-situ ONp ester activations and couplings in one-pot ….………. 40

Scheme 3.7 Possible explanation of low yields from Table 3.4 …….……………….…………. 42

Scheme 4.1 Synthesis of Selective Monomethylated Lysine ….…………………….…………. 56

Scheme 4.2 Formation of selective ε-Cbz copper lysine complex …….…………….…………. 57

Scheme 4.3 Reduction of lysine ……………….…………………………………….…………. 59

Scheme 4.4 Attempts at obtaining the dimethylated ε-nitrogen product …...……….…………. 60

List of Appendicies Appendix 1H and 13C NMR Spectra……………………………………………………....…..... 94

Abbreviations

°C degrees Celsius AA amino acid Ala alanine Alloc allyloxycarbonyl appt apparent At 7-azabenzotriazol Boc tert-Butoxycarbonyl br broad Bzl benzyl Bt benzotriazol Cbz carboxybenzyl CDI carbonyldiimidizol DCC N,N dicyloheyxlcarbodiimide DCM dichloromethane DIC N,N′-diisopropyl carbodiimide DIPEA N,N-diisopropyl ethylamine DMAP 4-dimethylaminopyridine DMF N,N-Dimethyl formamide DMSO dimethyl sulfoxide EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide Eoc ethoxycarbonyl equiv equivalent Et Ethyl Fmoc fluorenylmethyloxycarbonyl g gram Gly glycine h hour HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate HBTU N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate HMDS hexamethyl disilizane HOAt 1-Hydroxy-7-azabenzotriazole HOBt hydroxybenzotriazole HOSu N-hydroxysuccinimide IBCF isobutyl chloroformate Ile isoleucine L litre Leu leucine Lys lysine M molar Me methyl min minutes mL millilitre

xi mmol millimoles NHS N-Hydroxysuccinimide NMM N-methylmorpholine n/a not available NMR nuclear magnetic resonance OMe methyl ester ONp p-nitrophenol ester OSu N-Hydroxysuccinimide ester o/n over night PFP Pentafluorophenol PG protecting group PNP p-nitrophenol Phe phenylalanine Phg phenylglycine Piv pivalic pKa power of the acid dissociation constant p-NPCF p-nitrophenyl chloroformate ppm parts per million PPTS pyridinium p-toluenesulfonate Pro proline quant. quantitative rt. room Temperature SM starting Material SUMO small ubiquitin-like modifier TBA tetrabutyl ammonium TDBTU N,N,N',N'-Tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3- yl)uranium tetrafluoroborate TEA triethylamine THF tetrahydrofuran TLC thin-layer chromatography TMS trimethylsilyl/trimethylsilane Tr trityl μL microlitre Val valine VT variable temperature

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Chapter 1 Introduction: History and Classic Methods of Amide Bond Formation

1.1 Brief History of the Peptide Bond The first reported formation of a peptide bond was published in 1882, during the attempted protection of glycine using the benzoyl protecting group. The glycine silver salt 1.1 was used to form the benzoyl protected dipeptide 1.3 when reacted with benzoyl chloride 1.2 (Scheme 1.1). This work was completed by Theodore Curtius during his PhD under Hermann Kolbe.1, 2

Scheme 1.1 Formation of protected dipeptide via silver(I) salt of glycine

In 1901, Emile Fischer and Earnest Fourneau reported the synthesis of the first unprotected dipeptide, H-Gly-Gly-OH 1.7, from the hydrolysis of diketopiperazine 1.6 formed from the glycine ethyl ester (Scheme 1.2).3 The following year at the 14th meeting in Germany for German scientists and physicians, Fisher and Fourneau coined the term “peptide” which we use today.4

Scheme 1.2 Formation of Gly-Gly from diketopiperazine

The next major innovation in peptide chemistry, a procedure that still sees use, is the formation of acyl chlorides as an activator, developed by Fischer in 19055 (Scheme 1.3). In 1907, Fischer published a paper showing the successful generation of an eighteen-residue sequence consisting primarily of glycine residues with a few leucine residues. 6

Scheme 1.3 First example of the use of acid chlorides for peptide bond formation

The next major stepping stone was published in 1932 by Zervas and Bergmann, the Cbz temporary “protecting group”, removable by hydrogenation, allows for the uninhibited growth of peptide chains in the N to C direction7. The Boc group was introduced 25 years later by McKay and Albertson as an acid labile group able to be used orthogonally to Cbz, and the Fmoc group was published as a base labile group by Carpino and Han a number years later in 1970.8,9 Despite being revolutionary at the time of their discovery, protecting groups are always preferred to not be used in cases that are deemed unnecessary. In both solution and solid phase, several deprotection steps are still required using extra time and extra solvent, potentially lowering the overall yield and occasionally running the risk of side reactions, despite attempts to prevent the formation of additional products.10,11

That all being said however, the innovation that occurred in 1963 would change how peptides were built, namely the use of solid phase peptide synthesis introduced by R. B. Merrifield.12 Solid phase peptide synthesis differs from solution phase synthesis, in that one of the initial “protecting groups” acts as a tether to a solid bead or resin, as opposed to being free floating in solution. At this point solid phase and solution phase chemistry have a similar approach to generating peptide chains (Scheme 1.4). Beginning with an O-protected amino acid, an N- 3 protected residue is added to the mixture. They are coupled together using one of several C- terminal activators. This is followed by N-terminus deprotection. The process repeats until the desired chain length is obtained, at which point one final deprotection is used to release the free acid as well as free any protected sidechains.

Scheme 1.4 Method of generating peptide chains

The advantage that solid phase introduces here is that solution phase syntheses often require isolation after each coupling whereas solid phase allows for the building of long chains with only a single isolation after a final cleavage from the resin. No innovation in the field has matched the level of solid phase peptide synthesis.

1.2 Epimerization of Amino Acids The α-protons of amino acids are particularly acidic due to the electron withdrawing nature of the nearby carbonyl as well as the nearby amine, and side chain at the tertiary carbon. The pKa of the α-proton tends to range between 16 and 17, with the phenylalanine α-proton being one of the more acidic ones at 16.2, making it a good example to use when testing new conditions.13 Notably, phenylglycine is one exception with an α-proton acidity of 14.9, meaning it could easily 13 racemize in water, which has a pKa of 15.7. Organic bases in the form of alkyl tertiary amines + are often chosen for deprotonation of amino acids, requiring at least a pKa of 9 for the RNH3 , the zwitterionic form of amino acids. The conjugate acids of alkyl tertiary amines usually have pKa values of around 12, low enough that they should not cause racemization problems for the α- proton of amino acids, but high enough that no issues with deprotonation of the ammonium are 4

present. Notably, a Lewis acid would be able to lower the pKa of the α-proton by pulling electron density away from the C-H bond.

Figure 1.1 Various Amino Acids and the pKa of their α-protons

An issue that often occurs when synthesizing peptide chains longer than two residues, and is often the reason for their isolation in solution phase, is the problem of centre epimerization (Scheme 1.5). The process begins by activating the C-terminus using one of many activating groups, such as acyl chlorides or different activated esters 1.13. Following this, the nitrogen from the terminal amino acid will push electrons into the amide bond 1.13, resulting in nucleophilic attack into the activated terminal acid affording oxazolone 1.14. From here even mildly basic conditions can deprotonate the hydrogen at the α-carbon, forming the anionic species 1.15, as the

α-hydrogen has a pKa of approximately 9, low enough for the typically used organic bases to deprotonate it.14 At this point, protonation can occur from either one of the faces of 1.15 leading to epimerization of that stereocentre, resulting in diasteriomers 1.17 and 1.18. This issue is much less of a problem if the peptide is built in the C to N direction as the peptide cannot cyclize on itself since there is no terminal activated acid.15

Scheme 1.5 Oxazolone formation causing epimerization of the -stereocentre in peptide coupling

Despite this however, a disadvantage is that the N-terminal of the incoming residue must be protected to prevent it from attacking its own activated ester, preventing protecting group free couplings.

1.3 Common Method of C-terminus Activation Over the years, several methods of C-terminus activation have been used. No one method has been deemed best above all others and couplings can often be very system dependant. The factors contributing to the success of a coupling method can range from the solvent chosen, the presence of sterically hindered or racemization prone residues and formation of potential side products.15 Overall the general aim is to increase yields, decrease racemization, improve ease of final purification, decrease the cost of reagents, and to decrease reaction time whenever possible. Several of the more common options are outlined below, including their advantages and disadvantages.

1.3.1 Acid Chlorides Acid chlorides were one of the earliest invented activated acylating agents, as previously described. These compounds are commercially available, and there are also numerous methods for their formation. Several reagents exist for forming the acid chloride from the carboxylic acid. 16 17 18 19 Some of the more common reagents include SOCl2 1.19, (COCl)2 1.21, PCl3 and POCl3.

The mechanisms of acid chloride formation for SOCl2 and (COCl)2, are very similar, in that both begin with nucleophilic attack of the acid onto the activating reagent, followed by chloride ion loss, which then attacks into the intermediates 1.20 or 1.22, generating HCl, and SO2 or, CO2 and CO respectively (Scheme 1.6). Catalytic DMF may also be used to activate these species in a process that proceeds through an iminium ion.

Scheme 1.6 Mechanism of Acid Chloride Formation from SOCl2 and (COCl)2

Despite their ease of creation, acid chlorides carry with them several limitations. For one, base is often required to neutralize the HCl that is formed out of the reaction after coupling of the two amino acids, which is a particularly concern when using the acid labile Boc PG. Further concerns involve hydrolysis of the acid chloride, racemization, and side reactions.15 Racemization occurs through ketene formation. Beginning with the deprotonation of the α- proton, the ketene is formed resulting in stereochemical scrambling after nucleophilic attack by the amine (Scheme 1.7).20

Scheme 1.7 Epimerization of the -stereocentre via ketene formation.

1.3.2 Carbonyl diimidazole (CDI) CDI allows for the one-pot activation and coupling of two amino acids using only one reagent (Scheme 1.8).21 The lone pair of electrons on CDI 1.15 initially deprotonates the acid.15 This is followed by nucleophilic attack of the acid onto the carbonyl group of the protonated CDI, with subsequent loss of imidazole. The imidazole then attacks back onto the newly formed anhydride forming the acylimidazole species 1.24 along with another equivalent of imidazole and CO2. Finally, the second amine or amino acid partner is added and the dipeptide 1.26 is formed. Notably, the CDI acts as a base as well, and additional base is not required. CDI has been used on both large and small scale production, is fairly inexpensive, and is available on a large scale. One of the major downfalls of this reagent is that aromatic amines react sluggishly to form the intermediate 1.24. 22 For standard peptide synthesis this issue is not a problem.

Scheme 1.8 Mechanism of CDI activation and amine addition 8

1.3.3 Anhydrides Anhydride formation is a third commonly used method of C-terminal activation. While forming the symmetrical anhydride by using DCC or other carbodiimides, results in 50% waste of the initial amino acid, the formation of mixed anhydrides as activators results in no such loss. A common difficulty in mixed anhydride formation is the regioselectivity. The incoming amino acid has the potential to attack into either one of the two carbonyls at the formed anhydride, potentially resulting in unwanted side-product. Using Piv-Cl to form the Piv mixed anhydride overrides this however, as the t-butyl group carries enough steric bulk which makes attacking into the wrong carbonyl highly unlikely.23 Alternatively, forming the carbonic anyhydride 1.28 from reagents like isopropyl or ethyl chloroformate 1.27 also achieves this selectivity due to the additional resonance provided by the carbonate, resulting the carbonyl of the carbonate being a worse electrophile (Scheme 1.9).24,25

Scheme 1.9 Formation of carbonic mixed anhydride 1.28 through ethyl chloroformate 1.27

1.3.4 Activated Esters Activated esters are one of the broader options of carboxylic acid activators and as such only a few of the more common categories of which will be discussed. The theme of activated esters is the use of an electron poor activated ester, with a reactive acyl group and a good leaving group. Activated esters have a tendency to react under mild conditions, react cleanly, and with reduced racemization when compared to other methods.15

Some of the more common activating groups include PNP 1.29,26 PFP 1.30,27 HOSu 1.31,28 HOBt29 1.32, and HOAt 1.33 (Figure 1.2).29 PNP and PFP esters are more easily isolated compared to other more reactive options, often using DCC or EDC to pre-activate the acid. They 9 are storable for some time at lower temperatures and have become commercially available over the years. PNP and N-hydroxy succinimide esters offer the option of not requiring a C-terminal protecting group on the incoming acid due to being worse electrophiles.30 HOBt and HOAt derived esters are also usually activated by using a carbodiimide reagent, often offering lower racemization than even other alternatives.15

Figure 1.2 Assorted activating groups used for created the activated ester

The development of HBTU and HATU, allowed for in-situ activation of the esters without the need for adding additional reagents such as EDC and DCC, allowing for a one-pot method achieving easy activation and coupling. The mechanism of this is described below (Scheme 1.10). Beginning with deprotonation of the acid in question, the free acid then acts as a nucleophile to attack onto the iminium ion group of the coupling reagent 1.34 or 1.35, leading to loss of the deprotonated forms of 1.34 or 1.35 respectively. This group then attacks the intermediate 1.36 forming the more stable activated ester 1.37 or 1.38, producing tetramethyl urea as a side product. Finally the second amino acid is added and attacks the activated carbonyl group leading to formation of the dipeptide (or longer peptide).

Scheme 1.10 Mechanism of activation by HOBt & HOAt

1.4 Direct Coupling using Ru Recently, new methods have been developed allowing for the direct coupling of carboxylic acids and amines under catalytic conditions.31,32,33 One such method uses acetylene and ethoxyacetylene as coupling reagents by making use of a ruthenium catalyst. The scheme using the more reactive ethoxyacetylene is displayed below (Scheme 1.11). The scope of the acid includes cyclic, aryl, heteroaryl, alkyl and amino acid groups. The amine scope included amino acids, alkyl, benzyl and cyclic compounds. The amide is formed in poor (13%) to excellent (99%) yields.33

Scheme 1.11 Reaction conditions for amide bond formation using ethoxyacetylene as a coupling reagent 11

Figure 1.3 Mechanism of activation and coupling using [Ru] and ethoxyacetylene The proposed mechanism demonstrates the coordination of the alkyne and carboxylate to the ruthenium followed by addition of the acid to generate ethoxyacetylene 1.39. Following protonolysis the activated species 1.40 was afforded, and finally coupling occurred.

1.5 Overall Summary

Peptide coupling has had a long history, beginning its story in 1882 using the silver salt of glycine. Over the years many innovations such as solid phase peptide synthesis and the introduction of protecting groups have allowed the formation of easily formed peptide chains of 30-40 residues, and has been intrinsic to the pharmaceutical industry as well as to biotechnology.34 In spite of this, relatively little effort has been made to reduce the waste, and lower the step counts by removing the once revolutionary protecting groups from the process. The lack of protecting groups would mean less steps for the deprotection of the amino acids, cheaper starting reagents, less solvent waste, and an overall more efficient method of achieving the same product. This thesis explores potential work in that area. 12

1.6 References

1 Jaradat, D. M. M. Springer Nature. 2017, Advance online publication https://doi.org/10.1007/s00726-017-2516-0. 2 Curtius, T. J Prakt Chemie. 1882, 26, 145–208. 3 Fischer, E.; Fourneau, E. Ber. Dtsch. Chem. Ges. 1901, 34, 2868–2879. 4 Suresh, B. VV. Resonance. 2001, 6, 68–75. 5 Fischer, E. Ber. Dtsch. Chem. Ges. 1905, 38, 605–619. 6 Fischer, E. Ber. Dtsch. Chem. Ges. 1907, 40, 1754–1767. 7 Bergmann, M.; Zervas, L. Ber. Dtsch. Chem. Ges. 1932, 65, 1192–1201. 8 McKay, F.C.; Albertson, N. F. J. Am. Chem. Soc. 1957, 79, 4686–4690. 9 Carpino, L.A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748–5749. 10 King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Peptide Protein Res. 1990, 36, 255–266. 11 Wade, J. D.; Mathieu, M. N.; Macris, M.; Tregear, G. W. Let. In Pep. Sci. 2000, 7, 107–112 12 Merrifeld R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154. 13 Stroud, E. D.; Fife, D. J.; Smith, G. G. J. Org. Chem. 1983, 48, 627–628. 14 Metrano, A. J.; Miller, S. J. J. Org. Chem. 2014, 79, 1542–1554. 15 Montalbetti, C. A. G. N.; Falque, V. Tetrahedron. 2005, 61, 10827–10852. 16 Chu, W.; Tu, Z.; Mcelveen, E.; Xu, J.; Taylor, M.; Luedtke, R. R.; Mach, R. H. Bioorg. Med. Chem. 2005, 13, 77–87. 17 Adamas, R.; Ulrich, L. H. J. Am. Chem. Soc. 1920, 42, 599–611. 18 Pearson, A. J.; Roush, W. R.; Handbook of Reagents for organic Synthesis: Activating Agents and Protecting Groups; Eds.; Wiley, New York, 1999, p333. 19 Klosa, J. J.; Prakt. Chem. 1962, 19, 45–55. 20 Luknitskii, F. I.; Vovsi, B. A. Usp. Khim. 1969, 38, 1072–1088. 21 Paul, R.; Anderson, W. J. Am. Chem. Soc. 1960, 82, 4596–4600. 22 Woodman, E. K.; Chaffey, J. G. K.; Hopes, P. A.; Jose, D. R. J. Gilday, J. P. Org. Proc. Res. Dev. 2009, 13, 106–113. 23 Wittenberger, S. J.; Mclaughlin, M. A. Tetrahedron Lett. 1999, 40, 7175–7178. 24 Chu, W.; Tu, Z.; McElveen, E,; Xu, J.; Taylor, M,; Luedtke, R. R.; Mach, R. H. Bioorg. Med. Chem. 2005, 13, 77–87. 13

25 Benoiton, N. L.; Lee, Y. Chen, F. M. F. Int. J. Peptide Protein Res. 1988, 31, 577-580. 26 Gangwar, G. M.; Pauletti, T.; Siahaan, J.; Stella, V. J.; Borchardt, R. T. J. Org. Chem. 1997, 62, 1356–1362. 27 Kisfaludy, L.; Schon, I; Szirtes, T.; Nyeki, O.; Low, M. Tetrahedron Lett. 1974, 19, 1785– 1786. 28 Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. Soc. 1964, 85, 1839–1842. 29 Li, P;, Xu, J. Tetrahedron Lett. 2000, 41, 721–724. 30 Hashimoto, C.; Takeguchi, K.; Kodomari, M. Synlett. 2011, 1427−1430. 31 Lundberg, H.; Tinnis, F.; Selander, N.; Adolfsson, H. Chem. Soc. Rev. 2014, 43, 2714−2742. 32 Sbatini, M. T.; Boulton, L. T.; Sheppard, T. D. Sci. Adv. 2017, 3, E17028 33 Krause, T.; Baader, S.; Erb, B.; Gooßen, L. J. Nat. Commun. 2016, 7, 11732 34 Palomo, J.M. RSC Adv. 2014, 4, 32658−32672 14

Chapter 2 Calcium Carboxylate Salts and Their Use in Peptide Synthesis

2.1 The Utility of Carboxylate salts in Amide Bond Formation Metal carboxylates are a largely unexplored area of chemistry in regards to their use in peptide synthesis and amide bond formation, especially in reactions of free carboxylic acids, acyl chlorides and other activating acylating reagents.1 Despite their underutilization they do have the potential to offer several advantages, including the possibility for direct coupling, acting as a pseudo-protecting group or even increasing the solubility of the corresponding acid all of which could potentially be combined to develop a new method for synthesizing peptides.1 The limitations of current methods of peptide generation include very wasteful solution phase chemistry that require isolation after each coupling or solid phase peptide synthesis that, while offering ways to make long chains reliably, cannot be done well on larger scale should large quantities of products be necessary without the proper equipment. Furthermore, both typically require the use of protecting groups that subsequently require deprotection, further adding to the waste and step counts. With the limitations of these current techniques in mind, the following methods were considered while attempting to come up with alternatives.

In addition to the above, other uses of metal carboxylates include the in-situ formation of acid chlorides for the synthesis of amide bonds,1 and similarly coupling amines and alkali metal dye carboxylates using DIC and HOBt with tertiary amines and PPTS.2 However, given that neither of these methods are suitable when the formed acid chloride is unstable or when having an acid sensitive system, the Batey group set out to come up with an alternative. Furthermore these salts would be bench stable unlike other extremely electrophilic activated esters or acid chlorides which have a tendency to decompose over time. The group’s solution was to develop the first general method to directly couple metal carboxylate salts with amines via a one-pot protocol that would circumvent the need for the formation of an acyl chloride (Scheme 2.1).1

Scheme 2.1 General method for the direct formation of amide bonds from metal carboxylates by Batey and colleagues

It had been shown in the work done by Goodreid1, a previous lab member, that the lithium salts of many substrates ranging from alkyl, alkenyl, aryl and heteroaryl carboxylic acids, as well as amino acids, were able to be formed easily from the corresponding acids when stirred with lithium hydroxide in a mixture of water and acetonitrile for 10 min and followed up with lyophilization for isolation (Scheme 2.2). Shown below are a select few salts that were formed. (Figure 2.1). Of particular note was that substrate 2.5 was tolerant of the conditions. The use of sodium or potassium in-lieu of lithium salts did not seem to negatively affect the reaction.

Figure 2.1 Select carboxylate salts reacted with amines by Batey and colleagues

Following isolation, the salts were able to be activated with HBTU utilizing no additional base (except for cases where an HCl salt of amino acids are used), and were successfully coupled with primary and secondary amines after 1 to 2 h stir times. This base free approach is particularly 16 advantageous when applied to more sensitive amide couplings, as shown in the specific example provided below using the amino acid phenylglycine which is particularly sensitive to racemization due to acidity of the proton at the α-carbon.1,3 Most notably 2.8 was formed with only 5% racemization when ran at room temperature and 3% when ran from 0-10 °C with 97% and 96% yields respectively (Scheme 2.2). The full scope included several different C-protected amino acids as the amine source. These substrates gave yields ranging from 61% up to quantitative with minimal racemization in the final products.1

Scheme 2.2 Formation of Cbz-Phg-Val-OBzl 2.8

A recent publication by the Liguori group in September 2017 describes a general procedure for 4 the direct condensation of amines and carboxylic acids in the presence of TiCl4. The reaction itself was carried out over a period of 2 h at 85 °C using pyridine as the solvent, which acted as a base and also neutralized any hydrochloric acid that develops during the reaction (Scheme 2.3).

Scheme 2.3 TiCl4 based method used by Liguori Group for amide bond formation

The reaction scope includes simple primary, secondary and aryl amines including butylamine, the bulkier diethylamine, and aniline respectively while the carboxylic acids were aromatic, benzylic, vinyl or simple aliphatic ones. Noteworthy is that none of the substrates tested were chiral molecules and their proposed mechanism suggested that after deprotonation by pyridine, the TiCl3 carboxylate salt forms, at which point either the amine can add in directly, with 17 pyridine acting as an activator, or the chloride anion adds in generating the electrophilic acyl chloride (Scheme 2.4). Given the harsh conditions of this reaction as well as the high temperature used, it is undoubtedly inappropriate for peptide bond formation, despite the reaction’s interesting conditions.

Scheme 2.4 Proposed mechanism by Liguori group for TiCl4 activated amidation

A second use, and an interesting consequence of the reduced nucleophilicity, of the carboxylate functionality with these metal salts is that it has allowed carboxylate salts of amino acids to be used as pseudo-protecting groups in peptide coupling.5 In 2011, the Kodomari group published a paper describing the synthesis of dipeptides starting with an N-protected –ONp or –ONSu activated ester and coupling it to a calcium carboxylate salt of a free amino acid.6

Scheme 2.5 General scheme for carboxylate salt formation and coupling by Kodomari group

Beginning with the free amino acid, the metal salts were generated after being stirred with the corresponding alkali earth metal hydroxides (Scheme 2.5). However, the magnesium and barium salts gave yields of around 30% after being reacted with the activated esters, and calcium hydroxide became the standard for this method to give the calcium salts. Alkali metals were not tried as it was thought that having two AA units per cation (as for Ca2+) would increase the hydrophobicity and therefore allow the reaction to occur in organic solvents rather than in water. After drying, the amino acid salt was mixed with either the PNP or NHS activated ester of alanine in DMF, giving roughly a 90% yield of the final dipeptide after 5 h of stirring, and near 100% yield by HPLC, prior to isolation, after 24 h.

The advantages the authors of this method detailed included the reduced nucleophilicity of the carboxylic acid thereby preventing unwanted side-products despite the lack of C-terminal protection as well as its ease for formation and removal. This lack of an actual protecting group leads to greener chemistry due a higher atom economy as well as the fact that the calcium cation is easily removable after the reaction by a simple wash with citric acid as opposed to a C- terminal protections that need an additional step to remove. Furthermore, the extra step can lead to substrate loss or can cause unwanted side reactions.5 The salts are easily accessible, easy to work with, and are bench stable.

Another use of these salt complexes has been described in the work by A. Gorassini and colleagues published in 2013 which makes use of the hydrophobicity of the tetrabutyl ammonium salt.7 Other older examples, albeit less elegant, of these salts also exist.8-9

Scheme 2.6 General procedure for the formation of the isobutyl anhydrides of the amino acids and their subsequent coupling, developed by the Gorassini group

For Grassini’s procedure, first an N-protected amino acid was stirred with NMM (N- methylmorpholine) and IBCF (isobutylchloroformate) in DMF to afford the mixed carbonic isobutyl anhydride, at which point the NMM·HCl salt that formed was filtered off. A free amine in a 55% solution of aqueous TBA-OH (tetrabutylammonium hydroxide) in DMSO was added to the mother liquor, stirred for 30 min affording the dipeptide after purification and recrystallization (Scheme 2.6). The reason for the addition of the TBA-OH was to increase the hydrophobicity and solubility of the amino acids in organic solvents. They had previously found that under aqueous conditions, the water would hydrolyze the mixed isobutyl anhydride and thus were required to switch to a dry solvent. The yields ranged from 69% to 95% covering aliphatic, aromatic, polar and negatively charged side-chains. During initial screenings Gorassini and coworkers had attempted to reduce the amount of water needed to dissolve the free phenylalanine by adding NaOH instead, and interestingly the result was that after addition to the anhydride, sodium phenylalaninate precipitated out of the THF, to which the group claimed that the lower concentration in the THF as well as the presence of a strong base caused a transfer of the anhydride from unreacted 2.9 to activate the newly formed 2.12, resulting in the undesired tripeptide 2.13 in initially a 42% yield (Scheme 2.7).

Scheme 2.7 Unintended reactivation of 2nd amino acid during 2nd step

Finally, despite all of the group’s success, when trying to use the method to form a tripeptide from the activated dipeptide, they ended up with a 9% epimerization of the middle amino acid of the final tripeptide, which they had postulated had gone through the classic mode of amino acid epimerization of activated dipeptides as mentioned in the introduction (Scheme 1.5). At the time of publication the group was investigating a way to remedy this problem but offered no insight regarding a solution. It was shown that different salts could have a serious impact on solubility and reactivity as well as to be aware that racemization could well become an issue.

Protecting groups require additional steps to add and remove thus creating unnecessary waste. Furthermore, excessive C-terminus deprotections can result in loss of product and increase the likelihood of unexpected side reactions.6 Also if direct activation of said salt would be possible, it would be conceivable to aim for a larger peptide than one formed from just two amino acids. 21

Given the procedures by the Batey and Kodomari groups for the use of the carboxylate salts, specifically the calcium carboxylate salts, we postulated that it would be possible to first use the calcium salt of an amino acid to selectively amidate a PNP activated ester, and then to follow it up with the activation of the calcium salt with HBTU for a second amidation reaction either with a second calcium salt or a classically C-terminal protected amino acid thereby establishing a one pot procedure to make a peptide chain longer than merely two amino acids. The first goal therefore was to investigate the viability of the direct activation of calcium salts via HBTU followed by amide bond formation, as had been done with the alkali metal salts.

2.2 Results and Discussion The first thing that had to be accomplished was to make a calcium salt, a simple procedure based on work by the Kodomari group which involved the stirring of a free carboxylic acid in deionized water with 1.1 equiv of the metal hydroxide for 30 min at a rather high concentration (Scheme 2.8, Method 1).6 The first salt synthesized was the simple calcium acetate 2.14.

Scheme 2.8 The two methods used to generate calcium salts from the corresponding free-acids

For convenience, the drying procedure from the Kodomari group was changed from the obtained solid being washed with dry THF and diethyl ether after being ground, and dried over SiO2, to the solvent simply being removed under reduced pressure. It was first necessary to test, using the simplest of substrates, 2.14, if the coupling time of the Goodreid method1 which utilized at a standard 1 h stir after the addition of the amine in question, needed to be increased for the calcium salt when compared to the alkali metal salts (Scheme 2.9). 22

The Goodreid method was a method developed by the previous group member Dr. Jordan Goodreid as a means of coupling alkali metal carboxylates with amines to form amide bonds.1

Scheme 2.9 Initial testing on coupling time required for reaction of the simple calcium salt 2.14

The reaction was followed by 1H NMR of the crude reaction mixture based on the disappearance of the singlet of the calcium acetate at 1.87ppm, and the appearance downfield at 2.05 ppm of the singlet of 2.15. Interestingly, the salt was still present in roughly 10% after 1 h, but had disappeared nearly completely after 2 h (Figure 2.2).

1.0 h 2.0 h

Figure 2.2 H-NMR in CDCl3 of reaction shown in scheme 2.9 at 1.0 and 2.0 h

Next it was necessary to pick what would be used as a model substrate to compare the yields of different salts. Benzoic acid was chosen for its simplicity, yet still providing some steric bulk. It would then be coupled to the leucine methyl ester. As the 2013 paper published by Goodreid and 23

Batey hadn’t covered the substrate benzoic acid 1.4, it was necessary to first test the yield of this reaction by forming lithium benzoate 2.16a using the original protocol. Producing the lithium salt involved stirring the metal hydroxide in a 3:2 mixture of acetonitrile to water followed by cooling and lyophilization (Scheme 2.10).

Scheme 2.10 General method for generating lithium salts published by Batey and coworkers

With carboxylate 2.16a in hand, both Goodreid methods A and B to produce the amide bond were evaluated.1 The primary difference between the methods is that Method A adds all reagents into the flame-dried vial at once, while Method B allows for a 1 h stir time between the lithium salt and HBTU in DMF prior to the syringe addition of a DMF solution containing the amine to be coupled and DIPEA (in the case of the amine being an HCl salt). Both methods then require stirring for 1 h after all of the reagents are added to the vial.

Scheme 2.11 Using the Goodreid methods A and B on substrate 2.16a to verify yields for comparative purposes

Giving the very respectable 98% and 99% yields for Methods A and B respectively of product 2.17a, we next tested if there would be a difference in yield between the methods when using a 24 different salt. Additionally, we considered it unusual that in the general procedure for the calcium salt formation when following the published work of Kodomari, that an excess of calcium hydroxide was added. Although this would cause the reaction to be more likely to go to completion, it also meant that some of the mass would be unreacted calcium hydroxide, and it was unknown whether or not the additional base would negatively affect any subsequent reaction as none of the workup steps were likely to remove the unreacted base. Furthermore the high concentration of the reaction, although very convenient, would not be suitable for less water soluble compounds. With these two issues in mind Calcium Salt Method 2 was created, which used 1/5th the concentration and only 1.0 equiv of the calcium hydroxide instead of the usual 1.1 equiv (Scheme 2.8). Using this procedure the calcium salts were obtained in quantitative yields. The next step was to test model substrate 2.16b created by both calcium salt methods along with both Goodreid methods of amide bond formation to see what the optimal conditions would be for formation of 2.17a (Table 2.1).

Table 2.1 Comparing modified Goodreid1 methods A and B for chosen model substrate

Entry Goodreid Method Calcium Salt Method %Yield (Scheme 2.9) 1 A 1 88 2 B 1 82 3 A 2 96 4 B 2 90

The isolated yields of 2.17a were obtained by column chromatography using an eluant of 1:3 ethyl acetate to hexanes. Subsequently, using the altered Method 2 for formation of the calcium salt 2.16b led to improved final yields by 8% when compared with the approaches using the original Method 1 for formation of 2.16b. Moreover, adding all of the reagents to the vial at once using the Goodreid Method A resulted in an unanticipated small increase in yield over Method 25

B. As such when those two methods were combined, the best yield for 2.16b of 96% was comparable to that obtained using the lithium salt (Table 2.1, Entry 3).

Two other things needed to be tested before moving on to the activated ester portion of the planned reaction. The first would be the viability of other substrates and the second would be considering the possibility of making use of the TBA salt given that they should be highly soluble in organic solvents as had been shown in previous work.10 Using the same altered method used for generating the calcium salts, TBA salts 2.16c and 2.18b were made in quantitative yields, however removing the water by evaporation was extremely arduous, even after several washes with dichloromethane. Even then, the salt of the amino acid was obtained as a thick oil, making it necessary to re-dissolve the salt in DMF to be able to add it to the reactions in an accurately measurable amount. Using Goodreid Method A, and salts 2.16b-c and 2.18a-b with H-Leu-OMe·HCl, morpholine and pyrrolidine, the products 2.17a-c and 2.19a-c were formed (Table 2.2 and Table 2.3). Hünig’s base (DIPEA) was only added when it was necessary to remove the proton generated from the HCl salt of the C-terminal protected amino acids. The stirring times were increased to 2 h based on the findings shown in Figure 2.2. Table 2.2 Comparing the reactivity of the calcium and TBA salts of benzoic acid 2.16b and 2.16c, and their reaction with select amines

Entry Reactant Counter ion Amine Use of %Yield Product DIPEA (1.1 equiv) 1 2.16b Ca2+ H-Leu-OMe · Yes 96 2.17a HCl 2 2.16b Ca2+ Morpholinea No 90 2.17b 3 2.16b Ca2+ Pyrrolidine No 80 2.17c + 4 2.16c Bu4N H-Leu-OMe · Yes 90 2.17a HCl + a 5 2.16c Bu4N Morpholine No 83 2.17b + 6 2.16c Bu4N Pyrrolidine No 75 2.17c a 1.5 equiv. of amine was used based on Goodried’s protocol1 26

Table 2.3 Comparing the reactivity of the calcium and TBA salts of Boc-Ala-OH 2.18a and 2.18b, and their reaction with select amines

Entry Reactant Counter Amine Use of %Yield Product ion DIPEA 1 2.18a Ca2+ H-Leu-OMe · HCl Yes 99 2.19a 2 2.18a Ca2+ Morpholinea No 97 2.19b 3 2.18a Ca2+ Pyrrolidine No 83 2.19c + 4 2.18b Bu4N H-Leu-OMe · HCl Yes 88 2.19a + a 5 2.18b Bu4N Morpholine No 93 2.19b + 6 2.18b Bu4N Pyrrolidine No 76 2.19c a 1.5 equiv. of amine was used based on Goodried’s protocol1

The products were isolated using column chromatography. The pyrrolidine products were particularly troublesome and each needed to be columned a second time using 4% MeOH in dichloromethane. Otherwise columns were run around 25-40% ethyl acetate in hexanes as eluant. Calcium benzoate and alaninate gave yields ranging from 83% to 99% in the couplings, while the TBA salts produced yields ranging from 75% to 93% depending on the substrate, showing that on top of being more difficult to work with, the TBA salts also showed inferior reactivity under this set of conditions despite fully dissolving in the solutions, unlike their calcium salt counterparts.

Given the excellent yields of 80% to 99%, it was necessary to test a few bulkier substrates to confirm the viability of the calcium salts. 2.20 was chosen as the goal for the salt to be viable for amino acid coupling, and as Phe is one of the bulkier hydrophobic options it was a logical choice (Table 2.4). Indeed coupling of 2.20 with various coupling partners was successful, resulting in a 75% yield with the bulky, rigid proline and above 90% yield with the other tested substrates.

Table 2.4 Reaction of the calcium salt of Boc-Phe-OH 2.20a with select amines

Entry Amine Use of DIPEA %Yield Product # (1.1 equiv.) 1 H-Val-OMe · HCl Yes 90 2.21a 2 Morpholine No 93 2.21b 3 H-Pro-OMe · HCl Yes 75 2.21c a 1.5 equiv. of amine were used based on Goodried’s protocol1

Given the successful results thus far it was hypothesised that the Goodreid Method could be extended to a tripeptide sequence using a one-pot approach, thereby achieving two activations and two couplings with only one isolation required (Scheme 2.12). The reasoning behind this was that the calcium salt was claimed to have been able to act as a “protecting group” for the carboxylic group thereby preventing cross reactivity that would normally be seen with free amino acids. Generating the free lithium, calcium and TBA salts of leucine posed no problems and gave quantitative yields of 2.22a, 2.22b and 2.22c (See chapter 3) respectively. Phe was chosen as the amino again simply due to the fact that compounds containing it stained better on TLCs. In order to minimize the activation of the second amino acid by HBTU prior to the first coupling, salt 2.20 was stirred with the coupling reagent for 1 h first, as per Goodreid Method B, and a smaller excess was added compared to that used previously (as for Scheme 2.11). After the addition of the Leu salt, the lithium version of which was also tested, the reaction was further stirred for 2.5 hours, the additional time given to ensure the reaction went to completion. Finally the last step was achieved using Goodreid Method A, with all of the additional reagents added at once, and stirred for an additional 2 hours.

Scheme 2.12 First attempts to utilize a one-pot approach for tripeptide synthesis using an N- protected amino acid 2.20

While the results were disappointing, there was potential for improvement. Using the calcium salt as the 2nd amino acid resulted in a 48% yield of tripeptide 2.23 whereas the lithium salt resulted in a 40% yield. The TLC showed the presence of at least 6 separate products, most of which went unidentified but did include the dipeptide of compound 2.20 coupled to the alanine methyl ester forming Boc-Phe-Ala-OMe, indicating that the initial coupling had not gone to completion. Although it was not realized until later, what were initially thought to be rotomers were in fact diastereomers that resulted from partial epimerization of the 2nd amino acid (discussed in detail in Chapter 3).

2.3 Conclusion It has been successfully demonstrated that the calcium salt could be effectively substituted for the lithium salt using the Goodreid Method of amide coupling in excellent yields using several different carboxylic acids as well as several different amines, including the formation of several dipeptides. Furthermore the conditions for making the calcium salts have been optimized to account for less polar acids and reduced the use of excess base. When the calcium salts were compared with the TBA salt, which had been shown to be used specifically to increase the 29 solubility of amino acids in organic solvents,10 the calcium salt demonstrated improved results under this set of conditions. However preliminary results when attempting to make a tripeptide using a one-pot approach met with only partial success. The next step therefore would be to conduct a scope study where a free acid is coupled to a pre-activated ester, including the HOBt ester, to test if that was causing part of the problem, as well as to see if HOBt was an appropriate activating group when using a free amino acid rather than a C-terminal protected one. This would also show if the calcium was actually failing to act as a protecting group as had been originally suggested or if perhaps other salts would be able to work in its place. The results of these studies are described in Chapter 3.

2.4 References

1 Goodreid, J. D.; Duspara, P. A.; Bosch, C.; Batey, R. A. J. Org. Chem. 2013, 79, 943−954. 2 Ficht, S.; Rö glin, L.; Ziehe, M.; Breyer, D.; Seitz, O. Synlett. 2004, 2525−2528. 3 Liang, C.; Behnam, M. A. M.; Sundermann, T. R.; Klein, C. D. Tetrahedron Lett. 2017, 58, 2325−2329. 4 Leggio, A.; Bagalà, J.; Belsito, E. L.; Comandè, A.; Greco, M.; Liguori, A. Chemistry Central Journal. 2017, 11, 87−87. 5 Bodansky, M.; Klausner, S. Y.; Ondetti, A. M. Peptide Synthesis, 2nd ed.; John Wiley & Sons: New York, 1976; 49−50. 6 Hashimoto, C.; Takeguchi, K.; Kodomari, M. Synlett. 2011, 1427−1430. 7 Verardo, G.; Gorassini, A. J. Pept. Sci. 2013, 19, 315–324. 8 Lansbury, T. P., Jr.; Hendrix, C. J.; Coffman, I. A. Tetrahedron Lett. 1989, 30, 4915−4918. 9 Chen, S.-T.; Wang, K.-T. J. Chem. Soc., Chem. Commun. 1990, 1045−1047. 10 Verardo, G.; Gorassini, A. J. Pept. Sci. 2013, 19, 315–324. 31

Chapter 3 Activated Esters and One-Pot Solution Phase Tripeptide Synthesis

3.1 Protecting-Group Free Couplings of Amino Acids Protecting groups in peptide coupling are often required to prevent undesired side reactions or the formation of unwanted bonds.1 However, additional steps are required for their protection and deprotection often reducing the overall product yield. In addition, the extra time required to perform the additional steps, can lead to increased epimerization and/or cause unwanted side reactions under certain conditions.2,3 In light of this, since the early 2000’s, many methods have been developed to create dipeptides or add on one additional residue to a chain, most often using an N-protected terminus, activating the acid in some way, and using an unprotected C-terminal free acid as the nucleophile.

Besides the two methods already discussed in Chapter 2, both of which make use of carboxylate salt formation, (Scheme 2.5, Scheme 2.6),2,3 many other methods have also been established. Early on, azides4 and N-hydroxysuccinimide5 esters were used in couplings between N-protected amino acids and a second free amino acid. In 2002 Keillor and colleagues published a method which makes use of the unique reactivity of PNP, taking the reaction conditions a step further than what had already been established by doing both the activation and coupling in one pot rather than isolating the ONp ester (Scheme 3.1).6

Scheme 3.1 Activation using p-NPCF and coupling of free acid in one pot

The aforementioned group used p-NPCF, base, and DMAP (0.1 equiv) as a way to activate the ester, followed by the addition of a free amino acid in acetonitrile and water, obtaining yields of various dipeptides and tripeptides between 15% and 98%. The tripeptides were made from the isolated dipeptide using this method. Although useful, the procedure isn’t without its problems. Firstly, the method requires the use of 4 equivalents of the 2nd amino acid and base, as well as being conducted in an aqueous environment. Furthermore p-NPCF is fairly expensive, being around 150$ for 25g as of 2018.7 Nonetheless, this study demonstrated an alternate method to form the ONp ester in good yield and showed that racemization using this method was not a problem.

A second method published in 2005 used a previously established method of forming benzotriazole esters, and used these isolated intermediates to form dipeptides (Scheme 3.2).8

Scheme 3.2 Formation of benzotriazole esters and subsequent coupling to free amines

With yields ranging between 72% and 98% yield for the formation of the benzotriazole esters, then 68% to 90% for the subsequent dipeptide formation, this method offers the advantages of using cheaper reagents and shorter reaction times than Keillor’s PNP method described above. Furthermore only 1.0 equiv of the amino acid is used in the coupling step. However, it still uses an aqueous environment and the product yields are only for the coupling step, whereas the previous paper had managed to do both the activation and coupling steps in one pot. Subsequently, this group would go on to extend their method to the formation of tripeptides but using the dipeptide benzotriazole as the starting material.9 The procedure was changed to lower the reaction temperature to -10 °C during the formation of the benzotriazole (Bt) ester as well as for the coupling to help prevent epimerization of the middle amino acid, which they claimed was 33 a problem at room temperature. None the less it still required four separate reactions and four separate isolations to form the free C-terminus tripeptide.

Another mixed anhydride that has been shown to be effective at coupling free amino acids is the ethyl anhydride, that was initially published in 2011 by the Imai group (Scheme 3.3).10

Scheme 3.3 One-pot activation and coupling of a free amino acid (AA2) using a mixed ethyl anhydride

Unlike the previous synthesis there was no isolation step after activation. Furthermore of all the methods discussed this one was achieved with the shortest reaction time. The method was tolerant to most -amino acid residues, although it was not tolerant to histidine due to its nucleophilic nitrogen.

Given the above there were several options to evaluate as to which activated esters would be tested for the reaction with the carboxylic salt. The results from Chapter 2 revealed that using the calcium salts in lieu of the alkali metal salts had no negative effect on the yields. It was suspected that based on the paper by Kodomari2 that PNP esters would be a strong contender and as such were chosen as one of the test substrates. Also, the isopropyl mixed anhydride would have some potential based on the paper by Gorassini.3 More standard activating groups were also chosen since they are often found in either standard activating group formation or solid phase peptide synthesis, acyl chlorides and the N-hydoxybenzotriazoles esters respectively were chosen as well. Benzotriazole esters was not chosen as substrates due to their being significantly less common as well as not having been established as a carboxylate coupling partner.

Curiously, despite the fact that several examples of PG group free coupling had been previously established, isolation was required after every step coupling which hindered the effectiveness of 34 these aforementioned methods. To overcome these challenges, our goal was to develop a one pot method for tripeptide formation that would not require several isolations, and that could potentially form the basis of methods to create longer chains of peptides in solution phase in good yield and without excessive side products.

3.2 Results and Discussion The standard method we used was to dissolve the activated acid in DMF in a flame dried vial, the same solvent used by both Kodomari and Goodreid.2,11 The free amino acid would then be added, and stirred for 24 h. The product yield was then determined by NMR by comparing a premeasured amount 1,4 dioxane to the alpha-proton of leucine at 4.47 ppm in DMSO in product 3.3, following citric acid and aqueous washes.

Table 3.1 Optimization of different carboxylate salts of leucine with different activating groups on 3.1 or 3.3 (full Table next page)

Entry Compound Compound Lithium Calcium TBA Salt Product number Salt % salt % % Yield # Yield Yield (2.22c)c (2.22a)c (2.22b)c 1 3.1a 94 80 95 3.2

2 3.1b 31 45 40 3.2

3 3.1c 11 13 52 3.2

4 3.1d 10 60 13 3.2

5 3.1e 40 18 72 3.2

6 3.1fa 68 50 40 3.2

7 3.1gb 14 11 1 3.2

8 3.3 15 33 25 3.4

a Generated in situ using HATU b Generated in situ using EDC c 1H NMR yields using 1,4-dioxane as an internal standard

Table 3.1 reveal some interesting results. The PNP activated ester 3.1a gave the best outcomes across the board, with only the reaction with the calcium salt showing a slightly lower percentage yield for 3.1a. More importantly however was the –ONp ester was the only activating group 36 tested in Table 3.1 whose NMR showed the presence of a single proton in the α-proton region. This indicated the presence of only a single product for this set of reactions. All other activating groups showed at least three α-protons, indicating multiple products. All other activating groups showed at least 3 α-protons, indicating multiple products. After this result, the effectiveness of the activating group coupled to the acid salt seemed highly dependent on the counter ion. For example, although the isobutyl anhydride was indeed fairly effective when using the TBA salt (only one other alpha proton at 3.97 ppm was observed), when running the reaction with the lithium or calcium salts there were as many as 4 to 5 other products present based on the number of α-protons, also explaining the low yields (Table 3.1, Entry 5). Conversely, the Piv-mixed anhydride gave just as many products when using the lithium or TBA salts, but only a second alpha proton at 4.25 ppm was apparent when using the calcium salt, also explaining the higher yield of 3.2 (Table 3.1, Entry 4). Notably EDC didn’t give side products but also gave poor product yields (Table 3.1, Entry 7). Both N-hydroxyl benzotrizole esters gave product 3.2 as the primary one, but NMR showed that several other products were formed based on the number of alpha protons present, even using the lithium salt with HATU which gave the highest yield of 3.2 under these conditions. (Table 3.1, Entry 6) The acyl chlorides gave some of the poorest yields of 3.2 with the exception of the acid chloride and the TBA salt which again gave fewer side products (Table 3.1 Entry 3). Surprisingly though, despite the poor yield of the calcium and lithium salts with the acid chloride, when using the lithium salt, many products were produced in a similar ratio. Meanwhile, the calcium salt gave more of the product with the α-proton with the peak at 4.25 ppm (Table 3.1 Entry 3). Notably the peak at 4.25 ppm was often the largest secondary peak in the reactions from table 3.1, and in some cases the largest peak, although its exact source was never investigated. These results also explain the low yield of the attempt to form tripeptides when using only HBTU (Scheme 2.12), as many products were being formed despite the supposed protecting properties of the calcium salt claimed by the Kodomari group (Scheme 2.12).2

Overall this scope study showed that the ratio of products, and which product is the primary one, is extremely dependant on the salt used for a particular activating group and that further investigation was not warranted at the time. This data set also demonstrates that stating that calcium can act as a general protecting group is likely false. Rather, the ONp ester is simply 37 selective for the amine to act as nucleophile over the acid, thereby generating minimal side products, especially since it has been shown several times that the ONp ester can react selectively with the amine (a more recent example uses in situ activation of the ester followed by a single coupling).6 One other substrate that might be interesting to explore in future studies is OSu ester 3.1h and the the benzotriazole ester 3.1i, simply due to their established success in the coupling of free amino acids demonstrated by the Kodomari and Katrizky groups respectively (Figure 3.1). 2,12

Figure 3.1 Other potential activated acids for future testing

With our results indicating that the most complete reactions under all conditions tested were achieved using –ONp esters, they were chosen to move forward with further studies. The lithium salts were chosen over the TBA salts as they were all solids, unlike most of the TBA salts which were mostly oils and harder to work with. Furthermore, the lithium salt had a higher yield when compared to the calcium salt (Table 3.1 Entry 1). It was also imperative that the reaction go to completion to avoid side products as the end goal was to do a second coupling without isolation. Next, the effectiveness of the salt was compared to standard conditions in which DIPEA was used, using the same general method as above (Table 3.2). Table 3.2 Use of base and the free amino acid instead of amino acid salts

Entry Equivalents of DIPEA % Yield of 3.2a 1 0.0 Trace 2 1.1 31 3 2.0 47 4 5.0 70 a 1H NMR yields using 1,4-dioxane as an internal standard 38

As it turned out, utilizing even 5 equiv of base still did not match the yield obtained when using the carboxylates, even when compared to the calcium salts (Table 3.2). As such, the metal carboxylates were used in the reactions described below.

As the goal of this project was to make longer peptides, it was imperative to attempt in situ activation, followed by a coupling (Scheme 3.4). The method that the Batey lab had used previously to form ONp esters,13 and had been used to make every ONp ester in this project, was attempted here, and followed up in a one pot fashion with the lithium salt (Scheme 3.4).

Scheme 3.4 In situ ONp ester activation of benzoic acid using EDC and subsequent coupling with 2.22a

Disappointingly the yield of 3.2 was a mere 20% by NMR even after 72 h of stirring, and further optimization was not attempted.

The next step was to form two amide bonds in one pot by combining the direct HBTU activation of a metal salt with the coupling of a free amino acid with the activated ester (Scheme 3.5). First the ONp ester was dissolved in DMF in a flame dried vial and stirred for 24 h, at which point HBTU was added to the mixture. In a separate flame dried vial the free acid and base (used to neutralize the HCl salt), was stirred in DMF, then added by syringe and stirred for 2 additional hours. This was successfully achieved in 80% yield affording product 3.5 using benzoic acid as the first pseudo amino acid.

Scheme 3.5 Coupling of two amino acids and one acid activation in a one-pot fashion

Following this, tripeptide 3.7 was successfully synthesized using the same method (Table 3.3). The initial yield of 3.7 was found to be inadequate, and as such the reaction was run again using the sodium salt with doubling the volume of DMF solvent. The volume of solvent was increased, due to the poor solubility of the lithium salt which would stick on the walls of the vial. Despite not dissolving well, simply having the vial fuller helped, as the yield increased to 82% (Table 3.3, Entry 3). The sodium salt saw no change over the lithium salt. From this point every reaction used 2 mLs of solvent in steps 1 and 2 respectively.

Table 3.3 Altering conditions for tripeptide formation

Entry Counter Ion M+ Leucine Salt # Volume Of Solvent X Isolated (mL) %Yield of 3.7 1 Li+ 2.22a 1 73 2 Na+ 3.8 1 74 3 Li+ 2.22a 2 82

Following the success of the formation of the tripeptide 3.7 it was attempted to form the ONp activated ester in-situ. In-situ reactivation using PNP would mean that one would be able to continue to use unprotected C-terminal amino acids due to the selectivity of the PNP ester, as previously discussed. The method develop by Keillor and coworkers was tried in combination

6 with an amino acid salt rather than adding a free amino acid in an H2O/MeCN solution. As well, it was attempted to do this a second time in the same pot without needing to isolate the dipeptide unlike in the original protocol.6 Furthermore a second set of conditions using oxalylchloride and DIPEA was attempted (Scheme 3.6). Unfortunately, although both methods gave better results than previous attempts, the product yields were still considered to be unacceptable.

Scheme 3.6 Two sequential in-situ ONp ester activations and couplings in one-pot

We next investigated the possibility of using anthrinilic acid in the sequence, as the anthranilic motif is found in many peptide-like sequences as well as in potential drugs and anti-cancer agents.14 As such, anthranilic acid and its lithium salt were tested using this method (Table 3.4).

Table 3.4 Anthranilic acid as the middle “tripeptide” piece

Entry DIPEA, Step Li Salt of Acid (1.0 % Conversion of % Conversion of % Yield 1 equiv) 3.6b after 24 h 3.6b after 48h of 3.10 (1.1 equiv) (step 1) (step 1) 1 No No n/a 20 n/a 2 Yes No 74 80 17 3 No Yes 90 95 37 4 Yes Yes n/a 98 37

To minimize the amount of activated ester present under the previous conditions, it was required to stir for 48 hours for step 1 instead of the usual 24 hours, resulting in 95% conversion of starting material (Table 3.4, Entry 3). When compared to the using the free acid with DIPEA rather than the lithium salt, there was 4x the amount of starting material 3.6 used and only half the yield of 3.10 was obtained as compared with the lithium salt method (Table 3.4, entry 2). Despite this however, the yield of 3.10 was only 37%. One possibility discussed based on the typical racemization mechanism for activated dipeptides,15 was that after HBTU activation giving 3.12, the oxygen from the amide bond could attack in resulting in the cyclized product 3.13(Scheme 3.7). However, no spectroscopic data was collected on any other products from this reaction as it had been discussed after the fact and further investigation was not perused.

Scheme 3.7 Possible explanation of low yields from Table 3.4 The first tripeptide, Boc-Phe-Leu-Ala-OMe 3.7 had been analyzed using HPLC to confirm that there was only a single diastereomer as only one peak was present. However it was much to our chagrin to see that the 1H NMR of Cbz-Phe-Phe-Ile-OMe 3.15, showed two distinct peaks at 3.65ppm and 3.63 in DMSO. In response, a run of this tripeptide was done on the HPLC, which showed two very close but distinct peaks with the same mass. At this point we were still unsure whether the extra peak in the 1H NMR was due to rotomers or diastereomers as proline is known to be rigid, and the two Phe side chains were quite bulky. Until now many doubles of peaks had been assumed to be rotomers and ignored, so a variable temperature NMR experiment was performed to determine if the peaks would coalesce thereby indicating whether rotomers over diastereomers were present, since the latter would have 1H NMR peaks that would not coalesce at higher temperatures (Figure 3.2).

25 °C 80 °C Figure 3.2 VT experiment displaying the methyl ester peak of non-coalescing peaks at 25 °C compared to 80 °C of Cbz-Phe-Phe-Ile-OMe 3.15 43

As can be observed, the peaks in question did not coalesce to a single peak, thus confirming that diastereomers were present (Figure 3.2). When subjecting two other tripeptides to the same level of scrutiny they too did not stand up to the test and what we had thought to be rotomers turned out to be diastereomers. Even the initially evaluated sequence of 3.7 had peaks that did not coalesce using VT NMR despite showing only one peak in the normal phase HPLC analysis. While two peaks were visible on the HPLC of other tripeptides, as the ratio was eventually decreased, the peaks became less discernable, showing that HPLC was not an ideal method to determine the purity of these compounds. Thus began the arduous task of going back and attempting to prevent the epimerization of the tripeptide.

Our next step was to determine which of the three pieces of the tripeptide was causing the problem and, as there were only two peaks, meaning two products, only one of the one amino acid residues had likely epimerized. Racemic LD-Phe-O-Li+ was tested first, and used in the place of the L lithium salt. The amino acid salt was found to be the cause of the problem as ratio between the peaks at 3.63 ppm and 3.65 ppm had become 1:1, expected from a racemic mixture since these had been identified as indicative of the two diastereomers (Figure 3.2). Also no new peaks appeared in the 1H NMR thereby indicating that a new diastereomer was not introduced. It was therefore hypothesized that epimerization could be occurring during the reaction itself or during the preparation of the salt. Notably, although the work by Batey and co-workers had shown that making lithium salts on N-protected amino acids had no associated racemization problems, free amino acids had not been tested.11 As such the first thing attempted was to change the conditions for the formation of the lithium salt (Table 3.5).

Table 3.5 Alternate methods for the formation of the lithium salt 3.14a and the effect on epimerization in the formation of 3.15

Entry Salt Formation Method % Yield of % Epimerizationa 3.15 1 AA (1.0 equiv), LiOH (1.0 equiv), 93 35 3.0:2.0 MeCN:H2O, rt, 30 min 2 N/Ab 70 40 3 AA (1.0 equiv), LiOH (1.0 equiv), 91 34 c 3.0:2.0 MeCN:H2O, rt, 30 min d 4 AA (1.0 equiv), Li2CO3, (1.0 equiv), Crude Only 25 3.0:2.0 MeCN:H2O, rt, 30 min 5 AA (1.0 equiv), LiOH (1.0 equiv), 3.0:2.0 Crude Onlyd 29 MeCN:H2O, 0 °C, 30 min 6 AA (1.0 equiv), TMSOLi (1.0 equiv), 77 27 MeCN, rt, 30 min, filter d 7 AA (1.0 equiv), LiBF4 (1.0 equiv), Crude Only 17 DIPEA (1.05 equiv), MeCN, rt, 30 min, filter 8 AA (1.0 equiv), LiClO4 (1.0 equiv), 80 13 DIPEA (1.05 equiv), MeCN, rt, 30 min, filter 9 AA (1.0 equiv), LiClO4 (1.0 equiv), 77 12 DIPEA (3.0 equiv), MeCN, rt, 30 min, filter 10 1) AA (1.0 equiv), DIPEA (3.0 equiv), 70 12 MeCN, rt, 30 min, 2) LiClO4, 30 min, filter a Determined by comparing the –OMe singlets at 3.63 ppm and 3.65 ppm in DMSO at rt. b H-Phe-OH (1.0 equiv) and DIPEA (3.0 equiv) were used instead of a salt, as control experiment c Solvent evaporated by lyophilization instead of under reduced pressure d Only a crude 1H NMR was taken, the product was not isolated and no internal standard was used as tracking the yield was not the main goal of these experiments

The first set of changes done were entries 3-5 on the table above. The largest decrease of centre racemization was from changing the LiOH for Li2CO3 (Table 3.5, Entry 4). This was most likely - 2- due to the OH anion being a stronger base than the CO3 anion. Seeing as this significantly decreased the epimerization, the lithium source was the focus of further changes rather than the temperature or the evaporation method. Although several crude reaction mixturess were obtained over the course of this scope study, it quickly became apparent that product isolation was required. This was because as the secondary peaks became smaller in the subsequent NMRs, a pure product was required for accurate assessment. Remarkably, when a control reaction run was tested using only DIPEA and Phe, the epimerization was even worse than under initial conditions (Table 3.4, Entry 2). When compared with later results this was crucial since it showed that, despite not functioning as a protecting group, the preformation of the salt would be essential for this reaction to function as intended. Working towards even gentler conditions, eventually pre-stirring the amino acid with DIPEA combined with using lithium perchlorate as the lithium carboxylate source allowed the easy filtration of the formed salt after 30 min resulted in the lowest epimerization so far at 12%, down from the originally obtained 35% epimerization (Table 3.5, Entries 9, 10). Using weaker bases than DIPEA was attempted, as it was considered that the lithium counterion might be acting as a Lewis acid making the α-proton particularly acidic. Unfortunately, attempts to use an excess of 2,6 lutidine and even dimethylamine weren’t strong enough to deprotonate the free amine completely, thus resulting in no salt formation. This low level of epimerization was not satisfactory however, given how perfected peptide couplings have relatively low epimerization amounts, and further investigation was required. Therefore, the next step was to try the calcium salts again, despite lower yields in the initial trials (Table 3.1, Entry 1). Other salts were tried as well (Table 3.6).

Table 3.6 Alternate methods for the formation of the metal salt 3.14b-e and the effect on epimerization in the formation of 3.15

Entry Salt # M+ Salt Formation Method % % Yield Epimerizationa of 3.15 2+ 1 3.14b Ca AA (1.0 equiv), Ca(OH)2 (1.0 equiv) 63 11 H2O, rt, 10 min 2+ 2 3.14b Ca AA (1.0 equiv), CaCO3 (1.0 equiv) 38 6 H2O, rt, 2 h 2+ 3 3.14b Ca AA (1.0 equiv), CaCO3 (1.0 equiv) 35 9 H2O, rt, o/n 4 3.14b Ca2+ 1)AA (1.0 equiv), DIPEA (3.0 equiv), 70 12 MeOH, rt, 30 min b 2) Ca(ClO4)2· 4 H2O (1.0 equiv), 30 min + 5 3.14c Bu4N AA (1.0 equiv), TBA-OH (1.0 equiv), 66 22 H2O, rt, 30 min 6 3.14d Na+ 1)AA (1.0 equiv), DIPEA (3.0 equiv), 70 11 MeOH, rt, 30 min b 2) NaBF4 (1.0 equiv), 30 min 7 3.14e K+ 1)AA (1.0 equiv), DIPEA (3.0 equiv), 26 10 MeOH, rt, 30 min b 2) KBF4, 30 min a Determined by comparison of the –OMe singlets at 3.63 ppm and 3.65 ppm in DMSO b After evaporation under reduced pressure, redissolution in MeCN and filtration

Forming the salt using calcium hydroxide had given a promising lead, giving the lowest epimerization yet at 11% despite the lower product yield of 63% (Table 3.6, Entry 1). While the use of calcium carbonate to form calcium salts had appeared to have more potential initially, the yields using the salts were very low and the masses of the formed salts were giving over 100% yield. It was hypothesized that despite salts formed from Li2CO3 were functioning as intended, that the CaCO3 was causing a problem due to the fact that Ca(CO3H)2, formed after the first deprotonation of the amino acid, could be a worse base than both CaCO3 and LiCO3H, causing 47 the formation of the salt to stop at the midway point, which would also explain why the yields are about half the amount when compared to the calcium salt formed from calcium hydroxide (Table 3.6, Entries 1, 3). Other salts were tried as well, including the TBA, Na+ and K+ salts 3.14c-e, but these caused additional epimerization, gave similar results to those already obtained or were too insoluble, respectively. At this point a wall was hit, and it seemed as though the epimerization would not go below 10%. However, we were unsure since when calcium perchlorate was tried, it gave very similar epimerization rates when compared to the lithium perchlorate, despite the fact that lithium cation is much more Lewis acidic than calcium and thus should presumably have resulted in lower epimerization amounts. The next step was to see if perhaps there was simply a problem with the Phe residue, as there had been a precedent for it to be more easily epimerized (Table 3.7). 16

Table 3.7 Effect of using Leu salts instead of Phe salts on epimerization

Entry Salt # M+ Salt Formation Method % Yield % of 3.16 Epimerizationa 1 2.22a Li+ 1) AA (1.0 equiv), DIPEA (3.0 equiv), 33c 15 MeCN, rt, 30 min, 2) LiClO4 (1.0 equiv), 30 min, filter 2+ 2 2.22b Ca AA (1.0 equiv), Ca(OH)2 (1.0 equiv) 78 9 H2O, rt, 10 min 2+ 3 2.22b Ca AA (1.0 equiv), CaCO3 (1.0 equiv) 22 12 H2O, rt, o/n 4 3.8 Na+ 1)AA (1.0 equiv), DIPEA (3.0 equiv), 40c 14 MeOH, rt, 30 min b 2) NaBF4 (1.0 equiv), 30 min a Determined by comparison of the –OMe singlets at 3.61 ppm and 3.63 ppm in DMSO b After evaporation under reduced pressure, redissolution in MeCN and filtration c Significant amounts of the tetrapeptide Boc-Phe-Leu-Leu-Ile-OMe were isolated following column chromatography

Disappointingly, the epimerization of 3.16 was roughly the same as that of 3.15 (c.f. Table 3.6 Entry 10, Table 3.7). However these results did demonstrate that using alkali metals as salts could result in the formation of the undesired tetrapeptide in significant yield. Although the reasoning was never explored in detail, Gorassini and coworkers had observed a similar situation when forming the sodium salt using sodium hydroxide but coupling it with the isopropyl mixed anhydride of an amino acid.3 They had claimed that it was due to the insolubility of the salt over the TBA salt that resulted in the transfer the isopropyl anhydride. However, the calcium salt was found to be more insoluble than the lithium salt so it is not clear as to whether this reasoning is correct. The results also demonstrated that perhaps the lithium salts, even made under mild conditions, would not be appropriate coupling partners.

At this point, it was attempted to change the reaction conditions rather than the salt, as no improvement had been made to reduce the level of epimerization beyond 10%. On initial screening many different conditions were attempted (Table 3.8). As adding extra equivalents of DIPEA had previously caused epimerization, using a weaker base and less base was attempted. Furthermore reducing the reaction time in step one was trialed as well. Coupling reagents, such as HATU and TDBTU, known to be better at reducing epimerization were tried, as well as adding extra equivalents of HBTU. All these failed and showed no improvement to the baseline epimerization rate. Finally, adding two equivalents of HOBt prior to the addition of HBTU and adding the methyl ester of the AA before the addition of HBTU were surprisingly both found to cut the epimerization in half (Table 3.8, Entries 5,10). Addition of DMAP prior to the HBTU made the ratio worse.

Table 3.8 Various reaction conditions for tripeptide Cbz-Phe-Phe-Ile-OMe 3.15 formation and the effect on epimerization levels

Entry Change to Protocolb % Yield of % 3.15 Epimerizationa 1 - 63 11 2 DIPEA (1.0 equiv) in step 2 59 14 3 pyridine (1.1 equiv) instead of DIPEA 28 19 4 15 h stir for step 1 63 12 5 Addition of HOBt (2.0 equiv) prior to HBTU 70 5 6 Addition of DMAP (0.2 equiv) prior to HBTU 77 18 7 H-Phe-OH + pyridine (3 equiv.), 24 12 no metal salt, in step 1 8 HBTU (3.3 equiv) 73 12 9 methyl ester (3.0 equiv), DIPEA (3.3 equiv) 77 12 10 Add methyl ester solution prior to HBTU 63 6 (Reverse) 11 HATU instead of HBTU 70 12 12 TDBTU instead of HBTU 77 12 a Determined by comparison of the –OMe singlets at 3.63 ppm and 3.65 ppm in DMSO b Calcium salt made from Ca(OH)2

After much testing, further reducing the epimerization and trying to improve the yield of the reaction was attempted (Table 3.9) (“Reverse” refers to adding the final AA prior to HBTU as Table 3.8, Entry 10). Overall, it seemed that along with reversing the order of addition, more equivalents of HOBt decreased the epimerization, while adding additional equivalents of the last amino acid further increased the yield of 3.15 up to 77% (Table 3.8, Entries 5, 9, 10.) Pre- dissolving the HBTU and adding it dropwise also lowered the epimerization when compared to adding it all at once (Table 3.9, Entry 7). However, it was not found to have enough of an impact compared to simply reversing the order of addition to justify its further use. In the end, using 3.0 equivalents of HOBt and 2.0 equivalents of the amino acid was determined to be the appropriate conditions for this tripeptide formation (Table 3.9, Entry 14).

Table 3.9 Optimization of one pot-tripeptide conditions for formation of 3.15

Entry Change to Protocolb % Yield of Epimerizationa 3.15 1 - 63 11 2 Addition of HOBt (1.0 equiv) prior to HBTU 77 8 3 Addition of HOBt (4.0 equiv) prior to HBTU 73 3 4 Addition of HOAt (2.0 equiv) prior to HATU, 77 5 HATU used instead of HBTU 5 methyl ester (3.0 equiv), DIPEA (3.3 equiv), 80 4 reverse 6 Addition of HOBt (2.0 equiv) prior to HBTU, 72 4 reverse 7 Dissolve HBTU in DMF and add dropwise, 77 5 reverse 8 Addition of HOBt (1.0 equiv) prior to HBTU, 77 5 reverse 9 Addition of HOBt (2.0 equiv) prior to HBTU, 84 3 methyl ester (3.0 equiv), DIPEA (3.3 equiv), reverse 10 Addition of HOBt (1.0 equiv) prior to HBTU, 80 5 methyl ester (3.0 equiv), DIPEA (3.3 equiv), reverse 11 Addition of HOBt (4.0 equiv) prior to HBTU, 70 2 reverse 12 Addition of HOBt (2.0 equiv) prior to HBTU, 77 3 methyl ester (2.0 equiv), DIPEA (2.2 equiv), reverse 13 Addition of HOBt (3.0 equiv) prior to HBTU 80 2 methyl ester (3.0 equiv) , DIPEA (3.3 equiv), reverse 14 Addition of HOBt (3.0 equiv) prior to HBTU, 80 2 methyl ester (2.0 equiv), DIPEA (2.2 equiv), reverse a Determined by comparison of the –OMe singlets at 3.63 ppm and 3.65 ppm in DMSO b Calcium salt made from Ca(OH)2

Using these conditions (Table 3.9, entry 14) a small reaction scope study was performed.

Table 3.10 Preliminary scope of tripeptides

Entry AA1 AA2 AA3 % % Product Structures Epimeri- Yield zation 1 Cbz- Phe Ile- 2 82 3.15 Phe OMe (3.6b)

2 Boc- Phe Ile- 3 80 3.17 Phe OMe (3.6a)

3 Fmoc- Phe Ile- <1 60 3.18 Phe OMe (3.6c)

The scope demonstrated that all three of the most common protecting groups are tolerant of the conditions, albeit with a reduced yield for the Fmoc amino acids due to low solubility. Epimerization was not an issue for any of these compounds, with it being slightly worse when using the Boc protecting group, but still kept at acceptable levels.

3.3 Conclusion Overall, a one pot solution phase tripeptide synthesis has been successfully achieved, with one activation and two couplings in a single vessel in good yield and low epimerization. This is the first example of forming a tripeptide in one pot using a protecting group free method with minimal racemization and in good yield. The results that led up to the point where making the preliminary scope was possible were also fairly intriguing. They showed the utility of HOBt to reduce centre racemization under these conditions (Table 3.8 Entry 5). The results also demonstrated that using DIPEA is not appropriate when forming tripeptides in a one pot manner when using HBTU (Table 3.5 Entry 2). One more interesting result demonstrated effects using diverse salts have on different activating groups during amide couplings (Table 3.1). We have also shown the advantages of using pre-prepared amino acid salts over the traditional free acid and base method, despite the salts not truly acting as a protecting group for the carboxylic acid as had been anticipated.

Future work would be to, firstly, expand the scope and try some of the amino acids that would be more likely to cause problems such as arginine. Also, it would be interesting to study the relationships between different salts and what makes certain ones have a higher affinity to specific substrates, such as the why TBA salt gives better yields for the carbonic isopropyl mixed anhydride when compared to the calcium and lithium salts. Alternatively, coupling together pieces larger than one residue, possibly coupling three dipeptides to make a hexamer would be an interesting experiment. Another direction to continue would be to see if a set of conditions could be found such that peptide chains longer than three residues could be formed, testing other activating groups known to be effective for free amino acid coupling like benzotriazol or ONSu. HBTU, although effective for a final coupling step, would not be useful if needing to couple a second unprotected amino acid. For this, ideally one would use the free activator in solution along with another reagent to activate the acid prior to the addition of the unprotected acid, followed by using the free activator in solution to reactivate subsequent residues. As such, a repeatable in situ activation would be required; which was not developed over the scope of this project. It will also have to be done with a very mild activating reagent, such as one of the ones previously mentioned to prevent multiple product formation, and could result in a very efficient, atom economic, nearly protecting group free method of solution phase peptide formation. 53

Furthermore the finalized optimized conditions might also improve the yields of the anthranillic acid reactions if the problem had turned out to be HBTU related as the epimerization partly had been. (Table 3.4) 54

3.4 References

1 Isidro-Llobet, A.; Alvarez, M. Albericio, F. Chem. Rev. 2009, 109, 2455−2504 2 Hashimoto, C.; Takeguchi, K.; Kodomari, M. Synlett. 2011, 1427−1430. 3 Verardo, G.; Gorassini, A. J. Pept. Sci. 2013, 19, 315–324. 4 Bodanszky, M.; Klausner, Y. S.; Ondetti, M. A. Peptide Synthesis. John Wiley & Sons: Newyork, 1976. 5 Anderson, G. W.; Zimmerman, J.E.; Callahan, F. M. J. Am. Chem. Soc. 1964, 86, 1839. 6 Gagnon, P.; Huang, X.; Therrien, Eric.; Keillor, J.W. Tetrahedron. Lett. 2002, 7717−7719. 7 CAS #: 7693-46-1, Sigmaaldrich.com 8 Katritzky, A. R.; Angrish, P.; Hur, D. Suiuki, K. Synthesis. 2005, 3, 397−402. 9 Katritzky, A. R.; Angrish, P.; Hur, D. Suiuki, K. Synthesis. 2006, 3, 411−424. 10 Ezawa, T.; Jung, S.; Kawashima, Y.; Noguchi, T.; Imai, N. Tetrahedron: Asymmetry. 2017, 75−83. 11 Goodreid, J. D.; Duspara, P. A.; Bosch, C.; Batey, R. A. J. Org. Chem. 2013, 79, 943−954. 12 Katritzky, A. R.; Wang, M.; Yang, H.; Zhang, S.; Akhmedov, N. G. Arkivoc. 2002, 8, 134. 13 MRM, exp-062 (personal communication, June 7th, 2016) 14 Congui, C.; Cocco, M. T.; Lilliu, V.; Onnis, V.; J. Med. Chem. 2005, 48, 8245−8252. 15 Montalbetti, C. A. G. N.; Falque, V. Tetrahedron. 2005, 61, 10827–10852. 16 Stroud, E. D.; Fife, D. J.; Smith, G. G. J. Org. Chem. 1983, 48, 627–628. 55

Chapter 4 Copper-Lysine Complexes and Their Derivatizations

4.1 Lysine, Derivative Function, and Methods of Derivatization The amino acid lysine and its numerous modifications are intrinsic to epigenetic effects for the purposes of gene expression in regards to histones.1 The ε-amino group is able to undergo mono- , di- and trimethylation as well as acylation, SUMOylation and ubiquitinylation.2 Acylation has largely been associated with gene activation while SUMOylation with gene repression.2 The effect of methylation on the other hand is highly dependent on the specific residue; for example the trimethylation of the ε-amino group of lysine 4 of the H3 histone fragment acts as an activator while mono methylated lysine is enriched at enhancer sequences. Conversely, with lysine 9 of the H3 histone fragment monomethylation is associated with gene suppression whereas di- and trimethylated species act as gene activators.3 Furthermore a link has been shown to exist between a mutation in a protein which acts as a demethylase for the H3K4me3 residue, and brain development deficiencies and autism.4 Also, in Huntington’s disease the same residue has been shown to have an over occurrence of trimethylation, most likely resulting in neuronal dysfunction.5 As peptide therapeutics gain popularity, understandably so due to their high efficacy, selectivity and relative safety, with 140 in testing as of 2014, a demand for ease of access is on the rise.6 As such these modified lysine residues make ideal targets for mimicking given their functionality. In addition, their use as probes for chemical biology studies is also of interest. Unfortunately, buying the modified residues is extremely expensive when compared to the free lysine, and current methods for selective derivatization require several steps. This is partly due to issues of regioselectivity. For example, the following is a method developed in 1991 for the specific monomethylation of the ε-amino group (Scheme 4.1).7

Scheme 4.1 Synthesis of Selective Monomethylated Lysine

Lysine derivative 4.5 was synthesized over 4 steps and in 50% overall yield. Despite being able to be scaled up efficiently, it was postulated that a simple methylation should be able to be done more concisely than in four steps with a process that requires several days. Similarly, achieving protection regioselectively is usually very difficult and either requires several additional reagents over several steps or the use of enzymes to gain the desired specificity.8

Reacting copper ions with amino acids to from stable AA-copper complexes have been a part amino acid chemistry since at least the early 1900s, the structure of which was first proposed in 1904 by Ley (Figure 4.1).9

Figure 4.1 Original proposed complex of copper-glycine.

Although the complex has somewhat fallen out of use since, it has still found utility in such chemistry as the regioselective protection of lysine using the Fmoc and Alloc groups.10 Given that the procedure for the Alloc protection was one pot and very straightforward, it was hoped that from that monomethylation would be possible as well as other derivatizations.

4.2 Results and Discussion Based on the procedure by Lajoie, product 4.6 was produced (Scheme 4.2).10

Scheme 4.2 Formation of selective ε-Cbz copper lysine complex

HCl·Lysine, was dissolved in deionized water, mixed with 1.0 eq. of copper carbonate and refluxed for 45 min. It was important to maintain the temperature at around 95 °C as when it became hotter than this, the deep blue solution would turn a muddy brown, subsequent reactions would fail and the NMRs of the reactions would show nothing of value. Following a hot filtration, the solution was cooled to 0 °C, the pH set to 9 using Na2CO3 and benzyl chloroformate added over 1 h to give quantitative yields of 4.6. A disadvantage of the copper complexes that was quickly established was that NMR analysis provided no usual data, since the unpaired electron of the Cu(II) creates a strong local magnetic field, and at the concentrations present in the samples, the 1H NMR peaks were broadened to the point of it seeming as though no lysine was present in the NMR sample.11 This would mean that decomplexation would be required for every copper complex synthesized. Several known conditions were evaluated to test for decomplexation of 4.6 as well as one non-literature method (Table 4.1).

Table 4.1 Decomplexation reactions

Entry Decomplexation Conditions %Yield 10 1 Thioacetamide (2.0 equiv), H2O, 50 °C, 2.5 h n/a 12 2 Na2S (1.5 equiv), H2O, rt, 20 min 48 3 (COOH)2 (1.03 equiv), Dilute HCl (aq, 0.01M), 0 80 °C, 2 h13 14 4 EDTA (1.5 equiv), H2O, Reflux, 5 h <70 5 conc. HCl, MeCN, rt, 10 min 45

When attempting to decomplex using thioacetamide, a hydrophobic layer formed at the top of the flask, resulting in a mixture of products and only partial decomplexation (Table 4.1, Entry 1). Although Lajoie had obtained near quantitative yield using this method on the same substrate on a larger scale, the reaction was not reattempted, since the results obtained were not positive and thioacetamide is fairly toxic. Similarly the source material for oxalic acid decomplexation had reported a 74% yield, only a small amount of a pale blue solid was isolated which was not the desired product (Table 4.1, Entry 3). The best result was obtained using EDTA, although after recrystallization, 1H NMR analysis showed a small amount of unidentified impurities (Table 4.1 Entry 4). Notably every set of conditions used HCl to form the lysine salt at the end of the reaction. As such reaction in non-polar conditions was attempted to establish whether product 4.7 could be isolated without any residual copper. As it turned out the copper was able to complex with the acetonitrile, leading to a very simple set of conditions to decomplex requiring no heating, no toxic additives, and a very short stir time with only a straightforward work-up of washing with MeCN being required (Table 4.1 Entry 5). Unfortunately, for this particular substrate, the yield could not be increased. Adding any water to the solution would result in no precipitate, and using HCl in 1,4-dioxane instead of an aqueous solution saw no change in yield and using hexanes as a solvent resulted in incomplete decomplexation. Nonetheless, it was still useful for decomplexation of small amounts of products to simply to obtain an NMR.

The next attempt to selectively reduce the newly formed carbamate 4.7 had mixed results (Scheme 4.3).

Scheme 4.3 Reduction of lysine

Positively, it turned out that using conditions appropriate for reducing a carbamate15 also decomplexed the copper species 4.6. Unfortunately, the yields of 4.8 obtained from both Path A and Path B were greatly over inflated at 163% and 205% respectively after work up. This might possibly be due to residual copper present as it would not show up in the NMR. However, given that the NMRs only showed product peaks, the material was pushed forward to the next step, despite clearly requiring further purification. Furthermore attempts to oxidize the alcohol functionality of the product 4.8 from both Path A and B using the Jones reagent only led to starting material recovery.

Also, attempted was one-pot dimethylation using reductive amination or a methylating agent (Scheme 4.4). The reductive amination was done in a two-step one-pot fashion, forming the lysine copper complex 4.9, then adding formaldehyde and sodium cyanoborohydride to the hot- filtered solution, targeting the dimethylated product. To our dismay, even these milder reductive

60 conditions decomplexed the copper-lysine, affording tetramethylated product 4.10 in 67% crude yield. Further purification was not attempted. Again with the goal of doing a one-pot dimethylation, iodomethane and DBU were added to the solution following a hot filtration of the copper-lysine complex. Even after overnight stirring, decomplexation using conc. HCl resulted no observable product 4.10 by NMR.

Scheme 4.4 Attempts at obtaining the dimethylated ε-nitrogen product

Finally, several conditions for the formylation of ε-nitrogens of 4.9 using N-formylsaccharin were attempted (Table 4.1). This reagent was chosen due to its reported success in 2011 by Cossy and coworkers as a cheap, chemoselective formylating reagent that can be used under aqueous conditions.16 Small amounts of product 4.12 were, in each instance, subjected to acidic conditions to afford the analyzable product 4.11. Initial attempts resulted in very little conversion to 4.12, even when raising the temperature to 80 °C and stirring overnight (Table 4.1 Entry 2). It was noticed however that adding the N-formyl saccharin to the flask changed the solution of the

61 copper lysine complex from dark blue to a lighter blue. The pH of the solution had shifted towards being more acidic, and so K2CO3 was added to the solution regularly to assure that the pH remained around 9 (Table 4.2 Entries 5-7). Furthermore, the reaction was conducted at 0 °C variable time. Conversion was 87% by NMR after 2 h of stirring, further reaction progress was slow. Cleavage using concentrated HCl revealed that the ε-nitrogen had been successfully formylated, but that free saccharin remained.

Table 4.2 Improving formylation conditions for copper complex 4.9

Entry Conditions % Conversion to 4.12 1 4.9, N-Formylsaccharin (2.5 equiv), H2O, rt, o/n 20 2 4.9, N-Formylsaccharin (2.5 equiv), H2O, 80 °C, o/n 10 3 4.9, N-Formylsaccharin (2.0 equiv), H2O, 0 °C, 2 h 15 4 4.9, N-Formylsaccharin (2.0 equiv), H2O, 0 °C, 3 h 20 5 4.9, N-Formylsaccharin (2.0 equiv), K2CO3, H2O, 0 °C, 2 h 87 6 4.9, N-Formylsaccharin (2.0 equiv), K2CO3, H2O, 0 °C, 3 h 90 7 4.9, N-Formylsaccharin (2.0 equiv), K2CO3, H2O, 0 °C, 15 h 95

Regrettably, despite the success, we were unable to separate the formylated lysine from the saccharin. All solvents tried either dissolved both the product and the saccharin, or neither, both before and after decomplexation. Also, column chromatography of the uncomplexed lysine resulted required such polar conditions to elute the amino acid from the column that the silica would have dissolved, creating the same problem. Reverse phase silica chromatography was not attempted. Adding extra protecting groups to allow the lysine to be less polar would have negated the whole point of trying to reduce the step count by using these complexes and therefore was not attempted

4.3 Conclusion Overall, minor successes were made over the course of these experiments. One success was using HCl in MeCN to quickly decomplex the lysine and filter the product that had not been previously reported, and a second was the findings that standard reducing conditions also took the complex apart. Nonetheless, insufficient progress had been made towards making and isolating any of the lysine derivatives in short sequence. The complex was hard to work with as it mostly needed to be used in an aqueous environment due to its high polarity. Also, it was sensitive to environments in which the pH was lower than 8. The complex itself could not be analyzed by NMR and therefore needed to be decomplexed after each reaction to determine its success. Purification of most of the subsequent compounds required recrystallization if the impurities couldn’t be removed utilizing more non-polar solvents. At this stage further work on the project has been suspended.

4.4 References

1 Kang, T. J.; Yuzawa, S.; Suga, H. Chem. & Biol. 2008, 15, 1166−1174. 2 S.L. Berger. Nature 2007, 447, 407-412. 3 Akbarian, S.; Huang, H. S. Biological Psychiatry 2009, 65, 198−203 4 Iwase, S.; Lan, F.; Bayliss, P.; de la Torre-Ubieta, L.; Huarte, M.; Qi, H.H.; Whetstien, J. R.; Bonni, A.; Roberts, T. M.; Shi, Y. Cell 2008, 128, 1077−1088. 5 Ryu, H.; Lee, J.; Hagerty, S. W.; Soh, B. Y.; McAlpin, S. E.; Cormier, K. A. Smith, K. M.; Ferrante, R. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19176−19181. 6 Fosgerau, K.; Hoffmann, T.; Drug Disc. Today 2015, 20, 122−128. 7 Belyaev, A. A.; Krasko E. V.; Synthesis. 1991, 417−419. 8 Su, J.; Sheng, X.; Li, S.; Sun, T.; Liu, G.; Hao, A.; Org. Biomol. Chem. 2012, 10, 9319−9324. 9 Ley, H. Z.; Elektrochem. 1904, 10, 954−954. 10 Crivici, A.; Lajoie, G. Synth. Comm. 1993, 23, 49−53. 11 Reid, R. S.; Attaelmannan, M.A. J. Inorg. Biochem. 1998, 69, 59−65. 12 Nowshuddin, S.; Reddy, R. Tetrahedron Lett. 2006, 47, 5159−5161. 13 Liu, Y.; Jia, G.; Ling, X.; Lan, N. Zheng, Y.; Li, S.; Zhang, L.; Liu, L.; Zhang, R.; Xue, Y.; Can. J. Chem. 2012, 90, 557−559. 14 Kuwata, S.; Watanabe, H.; Bull. Chem. Soc. Jpn. 1965, 38, 676−677. 15 Rahman, O.; Kilhberg, T.; Langstrom, B. J. Chem. Soc. Perkin Trans. 1. 2002, 23, 2699−2703. 16 Cochet, T.; Bellosta, V.; Greiner, A.; Roche, D.; Cossy, J. Synlett. 2011, 13, 1920−1922

Chapter 5 Experimental

5.1 General Experimental 1D 1H and 13C spectra for new compounds can be found in the Appendix. Tetrahydrofuran was freshly distilled from sodium/benzophenone under nitrogen. Anhydrous N,N-dimethylformamide was obtained as  99.9% pure and stored under nitrogen over 4 Å molecular sieves. All other solvents were ACS grade or better from commercial suppliers and used as received. All amino acid materials and coupling reagents were purchased from Aapptec. All other reagents were purchased from Aldrich, VWR or other commercial suppliers and used as received. Flash chromatography on silica gel (60 Å 230-400 mesh, obtained from Silicycle) was performed with reagent grade solvents. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 pre-coated plates and visualized with a UV lamp and either KMNO4, bromocresol green or cerium ammonium molybdate. IR Spectra for all compounds were taken using a Shimadzu FTIR-8400S. Mass spectra were obtained by the University of Toronto Advanced Instrumentation for Molecular Structure (AIMS) mass spectrometry facility; high- resolution mass spectra (HRMS) were recorded on ABI/Sciex QStar mass spectrometer (ESI) for positive mode and 6538 UHD Accurate-Mass Q-TOF LC/MS using Mass Hunter Qual (ESI) for negative mode. All 1D (1H and 13C) NMR spectra were obtained on Varian Mercury 400MHz, Bruker 400 MHz, Agilent 600 MHz and Agilent 700 MHz spectrometers as solutions in deuterated solvents. Chemical shifts are reported in δ ppm values. 1H and 13C chemical shifts were internally referenced to the residual proton resonance in CDCl3, CD3OD, D2O and DMSO- 13 d6, except in cases where D2O had to be used in C spectra due to solubility issues. Peak multiplicities are designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; dq, doublet of quartets; td, triplet of doublets; qqd, quartet of quartet of doublets; J, coupling constant in Hz and rounded to the nearest 0.5 Hz.

5.2 Procedures and Characterizations

Method 1 of calcium salt formation (Scheme 2.8) Acetic acid (1.20 g, 20.0 mmol) or benzoic acid 1.4 (2.44 g, 20.0 mmol) was loaded into a round bottom flask, and stirred at rt and either dissolved or suspended in H2O (100 mL, 200 M).

Ca(OH)2 (815 mg, 11.0 mmol) was then added to the solution and stirred for an additional 30 min. The solvent was then evaporated under reduced pressure and dried in vacuo overnight. This afforded either a white dense or fluffy powder of products 2.14 or 2.16b in quantitative yields, respectively.1

Calcium acetate (2.14)

Isolated as dense white powder, (1580 mg, quant): IR (solid) νmax 3240, -1 1 13 1536, 1446, 1415 cm ; H NMR (400 MHz, D2O) δ 1.86 (3H, s); C NMR

(101 MHz, D2O) δ 181.62, 23.26.

Method 2 of calcium salt formation (Scheme 2.8) and general reaction conditions for calcium salts made from a carboxylic acids and Ca(OH)2

Carboxylic acid (4.0 mmol) was loaded into a round bottom flask, dissolved H2O (100 mL, 40

M) and stirred at rt. Ca(OH)2 (148 mg, 2.0 mmol) was then added to the solution and stirred for an additional 30 min. The solvent was then evaporated under reduced pressure and dried in vacuo overnight. This afforded the products as white powders in quantitative yields. The amount of solvent and starting material was sometimes doubled without any effect on purity or isolated yield.

Calcium benzoate (2.16b)

Isolated as fluffy white powder, (560 mg, quant): IR (solid) νmax 3293, 3058, 1595, 1538, 1435, 1385 cm-1; 1H NMR (400 MHz, 13 D2O) δ 7.89 (2H, d, J = 8.0 Hz), 7.60-7.45 (3H, m); C NMR - (101 MHz, D2O) δ 175.80, 135.98, 131.33, 128.81, 128.30; HRMS (ESI ) m/z calcd for C7H5O2 [M – Ca]-: 121.0295, found 121.0298.

Calcium Boc-L-alaninate (2.18a) Isolated as a white powder, (830 mg, quant): IR

(solid) νmax 3276, 2978, 1569, 1416, 1365, 1163 -1 1 cm ; H NMR (400 MHz, DMSO-d6) δ 6.25 (1H d, J = 6.5 Hz), 3.75 – 3.65 (1H, m), 1.37 (9H, s), 1.17 (3H, d, J = 7.0 Hz); 13C NMR (101 MHz, - DMSO-d6) δ 176.53, 153.49, 76.39, 49.53, 27.24, 18.41; HRMS (ESI ) m/z calcd for C8H14NO4 [M – Ca]-: 188.0928, found 188.0928.

Calcium (tert-butoxycarbonyl)-L-phenylalaninate (2.20) Isolated as a white powder, (1120 mg, quant): IR

(solid) νmax 3324, 2978, 1675, 1574 ,1411, 1165 -1 1 cm ; H NMR (399 MHz, DMSO-d6) δ 7.26 – 7.08 (5H, m), 6.16 (1H, d, J = 6.0 Hz), 4.02 – 3.89 (1H, m), 3.10 (1H, dd, J = 13.5, 5.0 Hz), 2.91 (1H, 13 dd, J = 13.5, 7.0 Hz)., 1.32 (9H, s). C NMR (101 MHz, DMSO-d6) δ 154.66, 145.42, 138.99, 129.43, 127.68, 125.64, 77.36, 55.99, 37.67, 28.20, 27.83; HRMS (ESI-) m/z calcd for - C14H18NO4 [M – Ca] : 264.1241, found 264.1244.

Calcium L-leucinate 2.22b

Isolated as a white powder, (1200 mg, quant): IR (solid) νmax -1 1 3283, 2954, 2870, 1571, 1413 cm ; H NMR (400 MHz, D2O) δ 3.39 (1H, dd, J = 8.5, 5.5 Hz), 1.61 – 1.48 (1H, m), 1.58 – 1.38 (2H, m), 0.89 (3H, d, J = 6.5 Hz,). 0.87 (3H, d, J = 6.5 Hz,). 13C - NMR (101 MHz, D2O) δ 181.51, 54.14, 42.69, 24.31, 22.32, 21.14; HRMS (ESI ) m/z calcd for - C6H12NO2 [M – Ca] : 130.0874, found 130.0875.

Calcium L-phenylaninate (3.14b)

Isolated as a white powder, (735 mg, quant): IR (solid) νmax 3340, -1 1 3028, 1556, 1495, 1408 cm ; H NMR (400 MHz, D2O) δ 7.57 – 7.23 (m, 5H), 3.67 (1H, dd, J = 7.5, 5.5 Hz), 3.07 (1H, dd, J = 14.0, 5.5 Hz), 2.91 (1H, dd, J = 14.0, 7.5 Hz).13C NMR (101

MHz, D2O) δ 178.59, 136.84, 129.39, 128.82, 127.11, 56.79, - - 38.73; HRMS (ESI ) m/z calcd for C9H10NO2 [M – Ca] : 164.0717, found 164.0718.

General Method of synthesizing lithium salts from carboxylic acids and LiOH·H2O 2 (Scheme 2.10, Table 3.5) or Li2CO3 (Table 3.5) Carboxylic acid (1.0 or 8.0 mmol) was loaded into flask, and suspended in a 3:2 solution of

MeCN:H2O (5 or 25 mL). LiOH·H2O (42 mg, 1.0 mmol or 336 mg, 8.0 mmol) or Li2CO3 (37 mg, 0.5 mmol) was then then added to the solution and stirred until completely dissolved (~ 10- 30 min). For Scheme 2.10 and Table 3, Entry 3, the reaction solution was then transferred to a lyophilization flask, cooled to -78 °C using a cold bath of dry ice and acetone, and left to lyophilize for a day on the lyophilizer to afford the pure product and white fluffy solids 2.16a or 3.14a in quantitative yields. In instances other than Scheme 2.10 and Table 3, Entry 3, the solvent was then evaporated under reduced pressure and dried in vacuo overnight affording a white or tanned powder with no loss in either yield or purity compared to that obtained with lyophilization.

Lithium benzoate (2.16a)

Isolated as a loose, fluffy, white powder, (1010 mg, quant): IR (solid) νmax 3058, 1600, 1557, -1 1 1403 cm ; H NMR (400 MHz, DMSO-d6) δ 7.88 – 7.75 (2H, m), 7.26 – 7.14 13 (3H, m); C NMR (101 MHz, DMSO-d6) δ 170.12, 139.58, 129.20, 129.03, - - 127.15.; HRMS (ESI ) m/z calcd for C7H5O2 [M – Li] : 121.0295, found 121.0295.

Lithium anthranilate (3.9)

Isolated as a tan powder, (1144g, quant): IR (solid) νmax 3385, 3271, 1611, -1 1 1585, 1520, 1395 cm ; H NMR (400 MHz, DMSO-d6) 7.82 (1H, dd, J = 8.0, 2.0 Hz), 6.99 (1H, ddd, J = 8.0, 7.0, 2.0 Hz), 6.67 (2H, s), 6.55 (1H, dd, J = 8.0, 13 1.0 Hz), 6.39 (1H, ddd, J = 8.0, 7.0, 1.0 Hz). C NMR (101 MHz, DMSO-d6) δ - 173.05, 150.28, 132.04, 130.29, 119.34, 115.34, 113.77; HRMS (ESI ) m/z calcd for C7H6NO2 [M – Li]-: 136.0404, found 136.0402.

General Method of synthesizing TBA salts from carboxylic acids and TBA-OH

Carboxylic acid (4.0 mmol) was loaded into a round bottom flask, and stirred at rt in H2O (100 mL). 40% TBA-OHaq (2.69 mL, 4.0 mmol) was then added to the solution and stirred for an additional 30 min. The solvent was then evaporated under reduced pressure and dried in vacuo over a week. No further purification was necessary. This afforded a translucent semi-solid or yellow oil depending on the product. The yellow oils were then made into a solution using DMF. Post-reaction NMR analysis showed full conversion from starting material and purity of the product. Exact yields were not determined as products were not fully dry even after 7 days in vacuo, but the crude products could be used in this state.

Tetrabutyl ammonium benzoate (2.16c)

Isolated as a translucent semi-solid: IR (solid) νmax 3241, 2957, 2874, 1638, -1 1 1596, 1558, 1485, 1358 cm ; H NMR (400 MHz, DMSO-d6) δ 7.87 – 7.79 (2H, m), 7.24 (dd, J = 5.0, 2.0 Hz, 3H), 3.22 – 3.11 (8H, m), 1.56 (8H, tt, J = 13 8.0, 8.0 Hz), 1.35 – 1.25 (8H, m), 0.92 (12H, t, J = 7.5 Hz). C NMR (101 MHz, DMSO-d6) δ 168.14, 141.11, 128.87, 128.14, 126.83, 57.52, 23.07, 19.18, 13.45; HRMS (ESI-) m/z calcd for - C7H5O2 [M – Bu4N] : 121.0295, found 121.0298.

Tetrabutyl ammonium L-alaninate (2.18b)

Isolated as a thick yellow oil: IR (solid) νmax 3542, 3248, 2958, 2875, -1 1 1638, 1597, 1558, 1488, 1358 cm ; H NMR (400 MHz, DMSO-d6) δ 6.01 (1H, d, J = 4.5 Hz), 3.37 – 3.25 (1H, m), 3.22– 3.18 (8H, m), 1.57 (8H, tt, J = 8.0, 8.0 Hz), 1.35 (9H, s), 1.35 – 1.25 (8H, m), 1.10 (3H, d, J = 7.0 Hz), 0.93 (12H, t, 69

13 J = 7.5 Hz). C NMR (101 MHz, DMSO-d6) δ 173.46, 154.46, 77.08, 57.51, 50.29, 40.15, - - 28.19, 23.06, 19.18, 13.44; HRMS (ESI ) m/z calcd for C7H14NO4 [M – Bu4N] : 188.0928, found 188.0930.

Tetrabutyl ammonium L-leucinate (2.22c) 1 Isolated as a thick yellow oil: H NMR (400 MHz, DMSO-d6) δ 3.22 – 3.14 (8H, m), 2.89 – 2.69 (1H, m) 1.75 – 1.60 (1H, m), 1.57 (8H, tt, J = 8.0, 8.0 Hz), 1.43 – 1.36 (1H, m), 1.35 – 1.25 (8H, m), 1.17 – 1.01 (1H, m), 0.93 (12H, t, J = 7.5 Hz), 0.84 (3H, d, J = 6.5 Hz), 0.80 (3H, d, J = 6.5 Hz).

Tetrabutyl ammonium L- phenylalaninate (3.14c)

Isolated as a thick, brown semi-soild: IR (solid) νmax 2959, 2874, 1597, -1 1 1492, 1371 cm . H NMR (400 MHz, Methanol-d4) δ 7.32 – 7.27 (4H, m), 7.25 – 7.18 (1H, m), 3.48 (1H, dd, J = 8.5, 4.5 Hz), 3.28 – 3.19 (8H, m), 3.13 (1H, dd, J = 13.5, 4.5 Hz), 2.78 (1H, dd, J = 13.5, 8.5 Hz), 1.79 – 1.55 (8H, m), 1.50 – 1.39 (8H, m), 1.03 (12H, t, J = 7.5 Hz); 13C NMR (101 MHz, MeOD) δ 180.31, 139.98, 130.50, 129.43, 127.40, 59.43, 58.78, 42.33, 24.75, 20.68, 13.98. HRMS (ESI-) m/z calcd - for C9H10NO2 [M – Bu4N] : 164.0717, found 164.0717.

Goodreid Method B (Scheme 2.9) for amide bond formation2

To a flame dried flask under N2, 2.14 (40 mg, 0.25 mmol) was loaded and stirred in DMF (2 mL). HBTU (209 mg, 0.55 mmol) was then added to the reaction flask and stirred for 2 h. Benzylamine (54 mg, 0.50 mmol) was then added to the vessel and stirred for an 1-2 additional hours. Reaction progress was monitored by NMR and was found to give full conversion to product 2.15 after 2 h of stir time.

Goodreid Method B (Scheme 2.11, Table 2.1) for amide bond formation, coupling with an amino acid HCl salt to form product 2.17a

To a flame dried flask under N2, metal carboxylate 2.16a or 2.16b (64 mg, 0.5 mmol or 71 mg, 0.25 mmol) was loaded and stirred in DMF (6.66 mL). HBTU (209, 0.55 mmol) was then added to the reaction flask and stirred for 1 h. In a separate flame dried flask under N2, H-Leu- 70

OMe·HCl (91 mg, 0.5 mmol) was stirred in DMF (6.66 mL), and DIPEA (71 mg, 0.55 mmol) was added and stirred for ~10 min. The amino acid and base solution was then transferred via syringe into the primary reaction mixture and stirred or an additional 1-2 h (1 h for Li+ salt and 2 h for the Ca2+ salt). The solution was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (3.0:1.0 hexanes:EtOAc) afforded a white glassy product 2.17a in yields ranging from 82% to 99% depending on the starting material.

Methyl benzoyl-L-leucinate (2.17a)

Isolated as a white solid, (123 mg, 99% yield): Rf = 0.60 (33% 1 EtOAc:67% hexanes); H NMR (400 MHz, DMSO-d6) δ 8.72 (1H, d, J = 7.5 Hz), 7.94 – 7.82 (2H, m), 7.60 – 7.53 (1H, m), 7.50 – 7.43 (2H, m), 4.50 (1H, ddd, J = 10.5, 7.5, 4.5 Hz), 3.64 (s, 3H), 1.88 – 1.73 (1H, m), 1.73 – 1.62 (1H, m), 1.63 – 1.51 (1H, m), 0.92 (3H, d, J = 6.5 Hz), 0.88 (3H, d, J = 6.5 Hz).; 13C

NMR (101 MHz, DMSO-d6) δ 173.08, 166.53, 133.71, 131.43, 128.23, 127.44, 51.84, 50.90, 24.45, 22.83, 21.15. Other spectroscopic data in accordance with previously obtained results.3,4

Goodreid Method A (Scheme 2.11, Table 2.1, 2.2, 2.3, 2.4) for amide bond formation2 + + To a flame dried flask under N2, carboxylate salt (1.0 equiv of Li or Bu4N salts or 0.5 equiv of Ca2+ salt) was loaded and stirred in DMF (4.3 mL). Amine (1.0 equiv of most amines or 1.5 equiv of morpholine) was then added to the stirred solution. DIPEA (71 mg, 0.55 mmol, 1.1 equiv) was added immediately afterward if the amine was the HCl salt of an amino acid. Finally, HBTU (209 mg, 0.55 mmol) was then added to the reaction flask and stirred for 1-2 h. (1 h for + + 2+ Li or Bu4N salts and 2 h for the Ca salt). The solution was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (conditions being product dependant) afforded the amides as white glassy solids. The product yields ranged from 75% to 99% depending on the starting material and product afforded.

Morpholino(phenyl)methanone (2.17b) Using Goodreid Method A. Column conditions: 40% EtOAc:60% hexanes.

Isolated as a yellow oil, (as high as 86 mg, 90% yield): Rf = 0.35 (33% 1 EtOAc:67% hexanes); H NMR (400 MHz, DMSO-d6) δ 7.51 – 7.41 (3H, m), 13 7.43 – 7.36 (2H, m), 3.75 – 3.45 (6H, m), 3.45 – 3.20 (2H, m); C NMR (101 MHz, DMSO-d6) δ 169.04, 135.58, 129.56, 128.41, 126.98, 66.07, 59.75, 42.15. Other spectroscopic data in accordance with previously obtained results.5,6

Phenyl(pyrrolidin-1-yl)methanone (2.17c) Using Goodreid Method A. Gradient of 20% to 100% EtOAc/hexanes. Isolated

as a brown oil, (as high as 70 mg, 80% yield): ): Rf = 0.45 (30% EtOAc:70% 1 Hexanes); H NMR (399 MHz, DMSO-d6) δ 7.53 – 7.47 (2H, m), 7.46 – 7.39 (3H, m), 3.46 (2H, dd, J = 7.0, 7.0 Hz), 3.35 (2H, dd, J = 6.5, 6.5 Hz), 1.97 – 1.71 (4H, m); 13C

NMR (176 MHz, DMSO-d6) δ 168.26, 137.28, 129.67, 128.20, 126.99, 48.93, 45.88, 25.96, 23.93. Other spectroscopic data in accordance with previously obtained results.5,6

Methyl (tert-butoxycarbonyl)-L-alanyl-L-leucinate (2.19a) Using Goodreid Method A. Column conditions: 20% EtOAc:80% hexanes. Isolated as a clear, colourless oil, (as high as 156 mg,

99% yield): Rf = 0.30 (20% EtOAc:80% hexanes); IR (solid) νmax 3293, 2952, 1749, 1674, 1525 cm-1; 1H NMR (400 MHz, DMSO- d6) δ 8.07 (1H, d, J = 7.5 Hz), 6.88 (1H, d, J = 7.5 Hz), 4.28 (1H, ddd, J = 10.0, 8.0, 5.0 Hz), 3.99 (1H, dq, J = 7.5, 7.0 Hz), 3.60 (3H, s), 1.72 – 1.57 (1H, m), 1.61 – 1.50 (1H, m), 1.53 – 1.41 (1H, m), 1.37 (9H, s), 1.15 (3H, d, J = 7.0 Hz), 0.88 (3H, d, J = 6.5 Hz), 0.83 (3H, d, J = 6.5 Hz); 13 C NMR (101 MHz, DMSO-d6) δ 172.88, 172.87, 154.94, 77.94, 51.77, 50.07, 49.38, 39.84, 28.15, 24.08, 22.77, 21.26, 17.96. Other spectroscopic data in accordance with previously obtained results.7,8

72 tert-Butyl (S)-(1-morpholino-1-oxopropan-2-yl)carbamate (2.19b) Using Goodreid Method A. Column conditions: Gradient of 30% to 50% EtOAc/DCM. Isolated as a pale yellow oil, (as high as 125 mg,

97% yield): Rf = 0.40 (30% EtOAc:70% DCM); R (solid) νmax 3312, -1 1 2977, 2931, 2858, 1704, 1639, 1441 cm ; H NMR (400 MHz, CDCl3) 5.49 (1H, d, J = 7.5 Hz), 4.62 (1H, dq, J = 7.5, 7.0 Hz ), 3.65– 3.7 (5H, m), 3.63 – 3.51 (2H, m), 3.52 – 3.41 (1H, m), 1.44 13 (9H, s), 1.30 (3H, d, J = 7.0 Hz); C NMR (101 MHz, CDCl3) δ 171.47, 155.22, 79.80, 66.95, + + 66.73, 46.08, 42.55, 28.52, 19.47. HRMS (ESI ) m/z calcd for C12H23N2O4 [M + H] : 259.16578, found 259.16512. tert-Butyl (S)-(1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (2.19c) Using Goodreid Method A. Column conditions: 40% EtOAc:60%

hexanes. Isolated as a yellow oil, (as high as 100 mg, 83% yield): Rf =

0.25 (35% EtOAc:65% hexanes); IR (solid) νmax 3299, 2975, 2879, -1 1 1704, 1635, 1447 cm ; H NMR (400 MHz, DMSO-d6) δ 6.89 (1H, d, J = 7.5 Hz), 4.20 (1H, dq, J = 7.5, 7.0 Hz), 3.45 – 3.37 (2H, m), 3.36 – 3.21 (2H, m), 1.93 – 1.82 (2H, m), 1.75 (2H, m), 13 1.36 (9H, s), 1.13 (3H, d, J = 7.0 Hz); C NMR (101 MHz, DMSO-d6) δ 170.61, 154.89, 77.82, + + 47.55, 45.59, 28.19, 25.72, 23.61, 17.11. HRMS (ESI ) m/z calcd for C12H23N2O3 [M + H] : 243.17087, found 243.17034.

Methyl (tert-butoxycarbonyl)-L-phenylalanyl-L-valinate (2.21a) Using Goodreid Method A. Column conditions: 20% EtOAc:80%

hexanes. Isolated as a white solid, (190 mg, 90% yield): Rf = 0.40 1 (33% EtOAc:67% hexanes); H NMR (400 MHz, CDCl3) δ 7.33 – 7.24 (3H, m), 7.24 – 7.19 (3H, m), 6.33 (1H, d, J = 8.0 Hz), 4.46 (1H, dd, J = 8.5, 5.0 Hz), 4.38 – 4.29 (1H, m), 3.69 (3H, s), 3.08 (2H, d, J = 7.0 Hz), 2.10 (1H, qqd, J = 7.0, 7.0, 5.0 Hz), 1.42 (9H, s), 0.87 (3H, d, J = 7.0 Hz), 0.80 (3H, d, J = 7.0 Hz); 13 C NMR (176 MHz, CDCl3) δ 171.88, 171.23, 155.56, 136.76, 129.48, 128.78, 127.04, 80.33, 57.37, 52.19, 31.41, 28.39, 18.94, 17.89, 17.88. Other spectroscopic data in accordance with previously obtained results.9

73 tert-Butyl (S)-(1-morpholino-1-oxo-3-phenylpropan-2-yl)carbamate (2.21b)

Using Goodreid Method A. Column conditions: 30% EtOAc:70%

hexanes. Isolated as a pale yellow oil, (155 mg, 93% yield): Rf = 0.35

(30% EtOAc:70% hexanes); IR (solid) νmax 3299, 2975, 2027, 2858, -1 1 1705, 1634, 1495, 1437 cm ; H NMR (400 MHz, CDCl3) δ 7.34 – 7.25 (3H, m), 7.24 – 7.16 (2H, m), 5.40 (1H, d, J = 9.0 Hz), 4.79 (1H, ddd, J = 9.0, 5.5, 5.5 Hz), 3.66 – 3.51 (2H, m), 3.52 – 3.36 (3H, m), 3.34 – 3.22 (1H, m), 3.03 (1H, dd, J = 13.0, 5.5 Hz), 13 2.98 – 2.83 (3H, m), 1.43 (9H, s); C NMR (176 MHz, CDCl3) δ 170.42, 155.17, 136.48, 129.69, 128.73, 127.24, 79.96, 66.61, 66.21, 50.92, 46.12, 42.36, 40.65, 28.50. Other spectroscopic data in accordance with previously obtained results.10

Methyl (tert-butoxycarbonyl)-L-phenylalanyl-L-prolinate (2.21c)

Using Goodreid Method A. Column conditions: Gradient of 20% to 50% EtOAc/hexanes. Isolated as a darker yellow oil, (141 mg, 75% 1 yield): Rf = 0.35 (30% EtOAc:70% hexanes); H NMR (400 MHz,

CDCl3) δ 7.32 – 7.27 (3H, m), 7.25 – 7.18 (2H, m), 5.24 (1H, d, J = 9.5 Hz), 4.65 (1H, ddd, J = 9.5, 7.0, 6.5 Hz), 4.54 – 4.45 (1H, m), 3.75 (3H, s), 3.66 – 3.55 (1H, m), 3.22 – 3.12 (1H, s), 3.09 (1H, dd, J = 13.5, 7.0 Hz), 2.91 (1H, dd, J = 13.5, 6.5 Hz), 2.26 – 2.10 (1H, m), 2.00 – 1.82 (3H, m), 1.38 (9H, s); 13C NMR (176

MHz, CDCl3) δ 172.43, 170.78, 155.28, 136.50, 129.84, 128.45, 126.87, 79.74, 59.04, 53.37, + + 52.31, 46.95, 39.30, 29.14, 28.44, 25.01; HRMS (ESI ) m/z calcd for C20H29N2O5 [M + H] : 377.20765, found 377.20675. Other spectroscopic data in accordance with previously obtained results.11

Formation of Boc-Phe-Leu-Ala-OMe in one-pot using HBTU activation (Scheme 2.12)

To a flame dried vial under N2, metal carboxylate 2.20 (71 mg, 0.125 mmol) was added to DMF (1 mL). HBTU (101 mg, 0.265 mmol) was then added to the vial and the resulting solution was stirred for 1 h. Carboxylate salt 2.22a or 2.22b (34 mg, 0.25 mmol or 38 mg, 0.125 mmol) was then added to the vial, and stirred for an additional 2.5 h. In a separate flame dried vial under N2 74

H-Leu-OMe·HCl (35 mg, 0.25 mmol) was stirred in DMF (1 mL) and DIPEA (36 mg, 0.275 mmol) was added and stirred for ~10 min. Additional HBTU (101 mg, 0.265 mmol) was added to the primary reaction vessel. The amino acid and DIPEA solution was then transferred via syringe into the primary reaction mixture and stirred or an additional 2 h. The solution was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (1.0:1.0 hexanes:EtOAc,) afforded a colourless, clear, crystalline product 2.23 in 48% yield using the calcium salt or 40% yield using the lithium salt, and resulting in 25% and 46% racemization of the leucine residue respectively as determined by NMR.

Formation of 4-nitrophenyl benzoate 3.1a from benzoyl-Cl

To a flame dried flask under N2 were loaded 4-nitrophenol (668 mg, 4.8 mmol), DMF (5 mL) and DIPEA (775 mg, 6.0 mmol). Benzoyl-Cl (562 mg, 4.0 mmol) was then added by syringe and the resulting solution was stirred o/n. The contents of the flask was then poured into a separatory funnel and H2O (15 mL) and EtOAc (15 mL) were added and the funnel shaken. EtOAc was then added until all precipitate was dissolved and the aqueous layer was drained. The organic layer was then washed with H2O (15 mL x 2), 1 M HCl (10 mL x 2), 2 M NaOH (10 mL x 5), and brine (10 mL x 3). The organic layer was then dried with Na2SO4 and filtered. The resulting solution was then evaporated under reduced pressure to afford pure product 3.1a (590 mg) in 61% yield as a very pale yellow solid.

4-Nitrophenyl benzoate (3.1a) Isolated as an off white solid, (590 mg, 61% yield): 1H NMR (399

MHz, CDCl3) δ 8.38 – 8.29 (1H, m), 8.25 – 8.17 (2H, m), 7.74 – 7.64 (1H, m), 7.61 – 7.50 (2H, m), 7.47 – 7.38 (2H, m). Spectroscopic data in accordance to previously reported data.12

Formation of –OBt ester 3.1b from benzoyl-Cl

To a flame dried flask under N2 were loaded HOBt (735 mg, 4.8 mmol), DMF (5 mL) and DIPEA (775 mg, 6.0 mmol). Benzoyl-Cl (562 mg, 4.0 mmol) was then added by syringe and the resulting solution was stirred o/n. The contents of the flask was then poured into a separatory 75

funnel and H2O (15 mL) and EtOAc (15 mL) were added and the funnel shaken. EtOAc (~30 mL) was then added until all precipitate was dissolved and the aqueous layer was drained. The organic layer was then washed with H2O (15 mL x 2), 1 M HCl (10 mL x 2), 2 M NaOH (10 mL x 5), and brine (10 mL x 3). The organic layer was then dried with Na2SO4 and filtered. The resulting solution was then evaporated under reduced pressure. The product was then loaded onto a silica column and quickly eluted (17:3 hexanes:EtOAc) to avoid decomposition, affording the white solid product 3.1b (607 mg) in 63% yield.

1H-Benzo[d][1,2,3]triazol-1-yl benzoate (3.1b)

Isolated as white solid, (607 mg, 63% yield): 1H NMR (400 MHz,

CDCl3) δ 8.34 – 8.26 (2H, m), 8.15 – 8.08 (1H, m), 7.83 – 7.76 (1H, m), 7.67 – 7.60 (2H, m), 7.59 – 7.53 (1H, m), 7.51 – 7.41 (2H, m). Spectroscopic data in accordance to previously reported data.13

Formation of mixed anhydrides 3.1d-e from benzoic acid 1.4

To a flame dried flask under N2, benzoic acid 1.4 (366 mg, 3.0 mmol) was loaded. Enough distilled THF (~3 mL) was then added to the vessel until the acid was completely dissolved. Pre- dried triethylamine (304 mg, 3.0 mmol) was then added to the flask by syringe, and the solution was then submerged in an ice bath and cooled to 0 °C. Piv-Cl ((362 mg, 3.0 mmol) or isobutyl chloroformate (410 mg, 3.0 mmol) was then added to the cooled solution dropwise over 5 min. The solution was stirred and monitored by TLC until the disappearance of starting material was apparent (~1.5 h). The precipitate salt was filtered off into a flame dried flask and the solvent was evaporated under reduced pressure affording products 3.1d or 3.1e. The flask was then placed in the fridge immediately after a small sample was taken for NMR analysis. Quantitative yields of the thick pale yellow oils were obtained.

Benzoic pivalic anhydride (3.1d) Isolated as a pale yellow oil, (5% starting material remaining): 1H NMR

(400 MHz, CDCl3) δ 8.05 (2H d, J = 7.0 Hz), 7.64 (1H, t, J = 7.5 Hz), 7.49 (2H, app t, J = 7.5 Hz), 1.37 (9H, s).

Benzoic (isobutyl carbonic) anhydride (3.1e) Isolated as a pale yellow oil, (10% starting material remaining): 1H

NMR (400 MHz, CDCl3) δ 8.12 – 8.05 (2H, m), 7.65 (1H, tt, J = 7.0, 1.5 Hz), 7.50 (2H, app t, J = 7.5 Hz,), 4.14 (2H, d, J = 6.5 Hz), 2.19 – 2.04 (1H, m), 1.01 (6H, d, J = 6.5 Hz).

General method for –ONp ester formation To a flask charged with PNP (668 mg, 4.8 mmol) and an N-protected amino acid (4.0 mmol, 1.0 equiv), EtOAc (40 mL) was added and the mixture stirred until dissolution occurred. EDC (920 mg, 4.8 mmol) was then added and stirred o/n. The contents of the flask was then poured into a separatory funnel and H2O (10 mL) was added and the funnel shaken then the aqueous layer was removed. The organic layer was then washed with H2O (10 mL x 2), 1 M HCl (10 mL x 2), 2M

NaOH (10 mL x 5), and brine (10 mL x 2). The organic layer was then dried with Na2SO4 and filtered. The solvent was then evaporated under reduced pressure and the product dried in vacuo o/n, affording a yellow solid or occasionally, a yellow oil. In some cases further purification was required. In such instances the resulting solid was either recrystallized from a mixture of hexanes and EtOAc to afford a white solid or, if very insoluble, washed with EtOAc, and Et2O. Yields ranged from 59% to 75%.

4-Nitrophenyl (tert-butoxycarbonyl)-L-phenylalaninate (3.6a) Used General method for -ONp ester formation. Isolated as a crystalline, off white solid, (1000 mg, 65% yield): IR (solid) -1 1 νmax 3375, 1762, 1689, 1523 cm ; H NMR (400 MHz,

DMSO-d6) δ 8.32 (2H, d, J = 16.0 Hz), 7.67 (1H, d, J = 7.0 Hz), 7.39 – 7.21 (7H, m), 4.44 (1H, ddd, J = 9.5, 7.0, 7.0 Hz), 3.16 (1H, dd, J = 13.0, 7.0 Hz), 13 3.08 (1H, dd, J = 13.0, 9.5 Hz), 1.38 (9H, s); C NMR (101 MHz, DMSO-d6) δ 170.47, 155.55, 77

155.19, 145.11, 137.08, 129.25, 128.31, 126.64, 125.42, 122.80, 78.70, 55.55, 36.07, 28.08; + + HRMS (ESI ) m/z calcd for C20H22N2O6 [M + H] : 387.15561, found 387.15555.

4-Nitrophenyl ((benzyloxy)carbonyl)-L-phenylalaninate (3.6b) Used General method for -ONp ester formation. Isolated as a fluffy, white, crystalline solid, (1260 mg, 75% - yield): IR (solid) νmax 3357, 3029, 1759, 1694, 1524 cm 1 1 ; H NMR (400 MHz, DMSO-d6) δ 8.30 (2H, d, J = 9.0 Hz), 8.13 (1H, d, J = 7.0 Hz), 7.38 – 7.22 (12H, m), 5.05 (2H, s), 4.58 (1H, ddd, J = 9.5, 7.0, 6.0 Hz 3.21 (1H, dd, J = 13.5, 6.0 Hz), 3.09 (1H, dd, J = 13 13.5, 9.5 Hz); C NMR (101 MHz, DMSO-d6) δ 170.15, 156.07, 155.03, 145.14, 136.89, 136.77, 129.24, 128.32, 128.31, 127.83, 127.65, 126.70, 125.36, 122.80, 65.65, 55.74, 36.14; + + HRMS (ESI ) m/z calcd for C23H21N2O6 [M + H] : 421.13996, found 421.14103.

4-Nitrophenyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalaninate (3.6c) Used General method for -ONp ester formation. Isolated as a white solid, (1220 mg, 60% yield): IR 1 (solid) νmax 3326, 1764, 1696, 1524. H NMR (400

MHz, DMSO-d6) δ 8.31 (2H, d, J = 9.0 Hz), 8.16 (1H, d, J = 7.5 Hz), 7.89 (2H, d, J = 7.5 Hz), 7.66 (2H, d, J = 7.0 Hz), 7.40 (2H, app t, J = 7.5 Hz), 7.36 – 7.22 (9H, m), 4.54 (1H, ddd, J = 9.5, 7.0, 6.0 Hz), 4.42 – 4.27 (2H, m), 4.21 (1H, t, J = 7.0 Hz), 13 3.21 (1H, dd, J = 13.5, 6.0 Hz), 3.10 (1H, dd, J = 13.5, 9.5 Hz) ; C NMR (101 MHz, DMSO-d6) δ 170.15, 156.00, 155.05, 145.14, 143.63, 140.71, 136.98, 129.24, 128.31, 127.60, 127.01, 126.67, 125.36, 125.08, 122.82, 120.08, 65.67, 55.66, 46.59, 36.10; HRMS (ESI+) m/z calcd for + C30H25N2O6 [M + H] : 509.17126, found 509.17052.

General procedure for formation of product 3.2 via activated ester and carboxylate salt of amino acid coupling (Table 3.1 except Entries 6 and 7,)

To a flame dried vial under N2, loaded with an activated acid 3.1a-e or 3.3 (0.25 mmol, 1.0 equiv), DMF (1 mL) was added and the mixture stirred until dissolution occurred. A leucine salt 78

2.22a-c (0.275 mmol, 1.1 equiv) was then added to the solution and the mixture was stirred for 24 h. The contents of the vial were then transferred to a separatory funnel and dissolved in

EtOAc (20 mL). This was then washed with a 5% citric acid solution (2 mL x 4), H2O (2 mL x

3) and brine (2 mL x 2). The organic layer was then dried with Na2SO4, filtered and evaporated under reduced pressure. The crude product was used to obtain NMR yields by comparing the α- proton at 4.40 ppm to a premeasured amount of 1,4 dioxane for product 3.2. Product 3.4 was compared to the α-proton at 4.22 ppm. Yields ranged from 10% to 96%.

Formation of product 3.2 via in-situ activated ester and carboxylate salt of amino acid coupling (Table 3.1 / Entries 6 and 7)

To a flame dried vial under N2, loaded with benzoic acid 1.4 (31 mg, 0.25 mmol), DMF (1 mL) was added and the mixture stirred until dissolution occurred. At this point, either HATU (95 mg, 0.25 mmol) was added and stirred for 1 h, or EDC·HCl (48 mg, 0.25 mmol) was added and stirred o/n. Leucine salt 2.22 (103 mg, 0.275 mmol) was then added to the solution and the mixture was stirred for an additional 24 h. The contents of the vial were then transferred to a separatory funnel and dissolved in EtOAc (20 mL). This was then washed with a 5% citric acid solution (2 mL x 4), H2O (2 mL x 3) and brine (2 mL x 2). The organic layer was then dried with

Na2SO4, filtered and evaporated under reduced pressure. The crude mixture was used to obtain NMR yields by comparing the α-proton at 4.4 ppm to a premeasured amount of 1,4 dioxane. Yields ranged from 1% to 68%.

Procedure for the formation of product 3.2 through free amino acid and base formation with –ONp esters (Table 3.2)

To a flame dried vial under N2, loaded with –ONp ester 3.1a (61 mg, 0.25 mmol), DMF (1 mL) was added and the mixture stirred until dissolution occurred. Leucine (36 mg, 0.275 mmol) was then added to the solution, followed by DIPEA (0 – 162 mg, 0 – 1.25 mmol), and the soution stirred for 24 h. The contents of the vial were then transferred to a separatory funnel and dissolved in EtOAc (20 mL). This was then washed with a 5% citric acid solution (2 mL x 4),

H2O (2 mL x 3), and brine (2 mL x 2). The organic layer was then dried with Na2SO4, filtered and evaporated under reduced pressure. The crude mixture was used to obtain NMR yields by 79 comparing the α-proton at 4.40 ppm to a premeasured amount of 1,4 dioxane for product 3.2 to afford yields of trace amounts to 70%.

Procedure for the formation of product 3.2 through in-situ –ONp activation with EDC (Scheme 3.4)

To a flame dried vial under N2, loaded with benzoic acid 1.4 (31 mg, 0.25 mmol), DMF (2 mL) was added and the mixture stirred until dissolution occurred. para-Nitrophenol (42 mg, 0.3 mmol) and EDC·HCl. (58 mg, 0.3 mmol) were then added to the vial, and the mixture was stirred for 2 h. 2.22a (37 mg, 0.275 mmol) was then added the mixture and stirred for a further 72 h, during the course of which the reaction was tracked by TLC. The contents of the vial were then transferred to a separatory funnel and dissolved in EtOAc (20 mL). This was then washed with a 5% citric acid solution (2 mL x 4), H2O (2 mL x 3) and brine (2 mL x 2). The organic layer was then dried with Na2SO4, filtered and evaporated under reduced pressure. The crude mixture was used to obtain NMR yields by comparing the α-proton at 4.40 ppm to a premeasured amount of 1,4 dioxane for product 3.2 affording an NMR yield of 20%.

Initial conditions for formation of the one-pot two couplings and one activation product 3.5 (Scheme 3.5)

To a flame dried vial under N2, loaded with –ONp ester 3.1a (61 mg, 0.25 mmol), DMF (1 mL) was added and the mixture stirred until dissolution occurred. Leucine salt 2.22a (34 mg, 0.25 mmol) was then added to the vial and its contents stirred for 24 h. In a separate flame dried vial under N2 charged with H-Ala-OMe·HCl (35 mg, 0.25 mmol), DMF (1 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. HBTU (104 mg, 0.275 mmol) was then added to the initial vial, followed by the amino acid and base solution via syringe and the resulting mixture was stirred for an additional 2 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded product 3.5 in 80% yield.

Methyl benzoyl-LD-leucyl-L-alaninate (3.5)

Isolated as a glassy, translucent solid, (64 mg, 80% yield): Rf =

0.30 (30% EtOAc:70% Hexanes); IR (solid) νmax 3268, 3070, -1 1 2955 1750, 1630, 1537 cm ; H NMR (400 MHz, DMSO-d6) δ 8.42 – 8.36 (2H, m), 7.95 – 7.86 (2H, m), 7.60 – 7.41 (3H, m), 4.63 – 4.52 (1H, m), 4.27 (1H, m), 3.63* (1.4H, s) / 3.62 (1.6H, s), 1.75 – 1.62 (2H, m) 1.58 – 1.46 (1H, m) 1.32 (1.6H, d, J = 7.5 Hz) / 1.28* (1.4H, d, J = 7.5 Hz), 0.99 – 0.82 (6H, m); 13C

NMR (101 MHz, DMSO-d6) major diastereomer: δ 172.96, 172.24, 166.21, 134.15, 131.19, 128.11, 127.48, 51.77, 51.35, 47.55, 40.35, 24.36, 23.09, 21.44, 16.81; minor diastereomer: δ 172.92, 172.15, 166.09, 134.12, 131.22, 128.14, 51.80, 51.43, 40.65, 24.42, 23.03, 21.38; HRMS + + (ESI ) m/z calcd for C17H25N3O4 [M + H] : 321.18143, found 321.18085; 46% epimerization asterisk (*) denotes minor diastereomer

Improved conditions for formation of a tripeptide in one-pot product 3.7 (Table 3.3)

To a flame dried vial under N2, loaded with –ONp ester 3.6a (97 mg, 0.25 mmol), DMF (1-2 mL) was added and the mixture stirred until dissolution occurred. Leucine salt 2.22a or 3.8 (34 mg, 0.25 mmol or 38 mg, 0.25 mmol) was then added to the vial and its contents stirred for 24 h.

In a separate flame dried vial under N2 charged with H-Ala-OMe·HCl (35 mg, 0.25 mmol), DMF (1-2 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. HBTU (104 mg, 0.275 mmol) was then added to the initial vial, followed by the amino acid and base solution via syringe and the resulting mixture was stirred for an additional 2 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded product 3.7 in 73% to 82% yield.

Methyl (tert-butoxycarbonyl)-L-phenylalanyl-LD-leucyl-L-alaninate (3.7) Isolated as a glassy, translucent solid, (85 mg, 73% yield): -1 IR (solid) νmax 3312, 2955, 1753, 1691, 1643, 1527 cm ; 1 H NMR (400 MHz, DMSO-d6) δ 8.38 (0.8H, d, J = 7.0 Hz) / 8.15* (0.2H, d, J = 7.0 Hz), 8.07* (0.2H, d, J = 8.5 Hz) / 7.87 (0.8H, d, J = 8.5 Hz), 7.23 – 7.22 (4H, m), 7.22– 7.15 (1H m), 7.02* (0.8H, d, J = 8.5 Hz) / 6.91 (0.2H, d, J = 8.5 Hz), 4.42 – 4.34 (1H, m), 81

4.30 – 4.21 (1H, m), 4.20 – 4.12 (1H, m), 3.62* (0.6H, s) / 3.60 (2.4H, s), 2.95 (1H, dd, J = 14.0, 5.0 Hz), 2.71 (1H, dd, J = 13.5, 10.5 Hz), 1.70 – 1.60 (1H, m), 1.45 (1.6 H, appt t, J = 7.5 Hz) / 1.38* (0.4H, appt t, J = 7.5 Hz), 1.33 – 1.21 (12H, m), 0.90 (2.4H, d, J = 6.5 Hz) / 0.79* (0.6H, d, J = 6.5 Hz) 0.74 (2.4H, d, J = 6.5 Hz) / 0.68* (0.6H, d, J = 6.5 Hz); 13C NMR (101 MHz,

DMSO-d6, major diastereomer) δ 172.81, 171.80, 171.26, 155.17, 138.14, 129.15, 127.94, 126.09, 78.04, 55.66, 51.78, 50.46, 47.47, 41.28, 37.21, 28.06, 23.90, 23.04, 21.73, 16.76; 13C

NMR (101 MHz, DMSO-d6, minor diastereomer) δ 172.75, 171.76, 137.68, 129.19, 126.14, 78.10, 55.99, 47.54, 41.01, 37.46, 27.76, 27.75, 23.84, 23.09, 21.35, 17.06; HRMS (ESI+) m/z + calcd for C24H38N3O6 [M + H] : 464.27606, found 464.27696. 21% epimerization. Asterisk (*) denotes minor diastereomer

Two sequential in-situ ONp ester activations and couplings in one-pot using p-NPCF (Scheme 3.6)

To a flame dried vial under N2, Boc-Phe-OH (66 mg, 0.25 mmol) was dissolved in DMF (2 mL). TEA (25 mg, 0.25 mmol) was then added and the solution was cooled to 0 °C. To the cooled mixture p-NPCF (50 mg, 0.25 mmol) was added dropwise over 5 min, at which point DMAP (3 mg, 0.025 mmol) was added as well, and the resulting mixture was stirred for 50 min. The vial was allowed to warm to room temperature, and leucine salt 2.22a (34 mg, 0.25 mmol) was then added and the mixture stirred for an additional 24 h. The solution was then again cooled to 0 °C and additional triethylamine (25 mg, 0.25 mmol) was added followed by additional and p-NPCF (50 mg, 0.25 mmol) added dropwise over 5 min, and stirred for 50 min. In a separate flame dried vial under N2 charged with H-Ala-OMe·HCl (35 mg, 0.25 mmol), DMF (2 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. The amino acid and base solution was added to the primary reaction mixture via syringe and was stirred for an additional 24 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded product 3.7 in 25% yield.

Two sequential in-situ ONp ester activations and couplings in one-pot using oxalyl chloride and PNP (Scheme 3.6)

To a flame dried vial under N2, Boc-Phe-OH (66 mg, 0.25 mmol) was dissolved in DMF (2 mL). para-Nitrophenol (45 mg, 0.325 mmol) was then added to the reaction mixture along with DIPEA (68 mg, 0.525 mmol) and the solution was cooled to 0 °C. Oxalyl chloride (33 mg, 0.26 mmol) was then added dropwise over 5 min, and the solution was stirred for 30 min while warming to room temperature. Leucine salt 2.22a (34 mg, 0.25 mmol) was then added and the mixture stirred for an additional 24 h. Additional DIPEA (68 mg, 0.525 mmol) was then added to the reaction mixture which was then cooled back down to 0 °C and oxalyl chloride (33 mg, 0.26 mmol) was added dropwise via syringe over 5 min and allowed to warm to room temperature. In a separate flame dried vial under N2 charged with H-Ala-OMe·HCl (35 mg, 0.25 mmol), DMF (2 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. The amino acid and base solution was added to the primary reaction mixture via syringe and was the mixture stirred for an additional 24 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded product 3.7 in 10% yield.

Conditions for formation of the one-pot two couplings and one activation product 3.10 (Table 3.4)

To a flame dried vial under N2, loaded with –ONp ester 3.6b (105 mg, 0.25 mmol), DMF (2 mL) was added and the mixture stirred until dissolution occurred. Lithium anthranilate 3.9 (36 mg, 0.25 mmol) or anthranilic acid (34 mg, 0.25 mmol) was then added to the vial, as well as DIPIA (0 or 36 mg, 0 or 0.275 mmol) and its contents stirred for 48 and tracked by NMR. In a separate flame dried vial under N2 charged with H-Ile-OMe·HCl (46 mg, 0.25 mmol), DMF (2 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. HBTU (104 mg, 0.275 mmol) was then added to the initial vial, followed by the amino acid and base solution via syringe and the resulting mixture was stirred for an additional 6 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column (except Table 3.4 Entry 1). Flash chromatography (30% EtOAc, 70% Hexanes) afforded pure product 3.10 in 17% to 37% yield.

Methyl (2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)benzoyl)-L- isoleucinate (3.10) Column conditions: 30% EtOAc:70% hexanes. Yellow

oil, (as high as 50 mg, 37% yield): Rf = 0.4 (30%

EtOAc:70% hexanes); IR (solid) νmax 3250, 2963,1719, 1644, 1586, 1511, 1445 cm-1 ; 1H NMR (400 MHz,

DMSO-d6) δ 11.46 (1H, s), 8.90 (1H, d, J = 7.5 Hz), 8.45 (1H, d, J = 8.0 Hz), 7.98 (1H, d, J = 8.0 Hz), 7.84 (1H, d, J = 8.0 Hz), 7.54 (1H, t, J = 8.5 Hz), 7.39 – 7.15 (11H, m), 4.98 (2H, s), 4.43 (1H, t, J = 7.5 Hz), 4.29 – 4.20 (1H, m), 3.66 (3H, s), 3.23 (1H, dd, J = 14.0, 4.0 Hz), 2.88 (1H, dd, J = 14.0, 11.0 Hz), 2.02 –1.91 (1H, m), 1.57 – 1.37 (1H, m), 1.24 (1H, appt dt, J = 13.5, 8.0 Hz), 13 0.98 – 0.78 (6H, m); C NMR (101 MHz, DMSO-d6) δ 171.76, 170.33, 168.41, 156.06, 138.25, 138.11, 136.76, 132.09, 129.02, 128.80, 128.22, 128.15, 127.67, 127.45, 126.31, 122.82, 120.85, 120.31, 65.60, 57.99, 57.05, 51.72, 36.57, 35.64, 25.19, 15.45, 10.87; HRMS (ESI+) m/z calcd + for C31H36N3O6 [M + H] : 546.26041, found 546.26016.

General method of tripeptide formation (Table 3.5, 3.6, 3.7)

To a flame dried vial under N2, loaded with –ONp ester 3.6b (105 mg, 0.25 mmol), DMF (2 mL) was added and the mixture stirred until dissolution occurred. A Phe salt 3.14a,c-e or a Leu salt 2.22a, 3.8, or Phe salt 3.14b or Leu salt 2.22b (0.25 mmol, 1.0 equiv or 0.125 mmol, 0.5 equiv), (methods of preparation differ), was then added to the vial and its contents stirred for 24 h. In a separate flame dried vial under N2 charged with H-Ile-OMe·HCl (46 mg, 0.25 mmol), DMF (2 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. HBTU (104 mg, 0.275 mmol) was then added to the initial vial, followed by the amino acid and base solution via syringe and the resulting mixture was stirred for an additional 2 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded pure product 3.15 in 22% to 93% yield and with 9% to 35% epimerization as measured by 1H NMR through integration of the OMe peaks at 3.63 ppm and 3.65 ppm or 3.61 ppm and 3.63 ppm (3H).

General method of tripeptide formation (Table 3.5, Entry 2)

To a flame dried vial under N2, loaded with –ONp ester 3.6b (105 mg, 0.25 mmol), DMF (2 mL) was added and the mixture stirred until dissolution occurred. Phenyl alanine (41 mg, 0.25 mmol) was then added to the vial along with DIPEA (97 mg, 3.0 equiv) and its contents stirred for 24 h.

In a separate flame dried vial under N2 charged with H-Ile-OMe·HCl (46 mg, 0.25 mmol), DMF (2 mL), was added. HBTU (104 mg, 0.275 mmol) was then added to the initial vial, followed by the amino acid solution via syringe and the resulting mixture was stirred for an additional 2 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded product 3.15 in 70% yield and with 40% epimerization as measured by 1H NMR through integration of the OMe peaks at 3.63 ppm and 3.65 ppm (3H).

Method of synthesizing lithium salt from H-Phe-OH and LiOH·H2O at 0 °C (Table 3.5 Entry 5) H-Phe-OH (165 mg, 1.0 mmol) was loaded into a flask, and suspended in a 3:2 solution of

MeCN:H2O (5 mL). The solution was submerged into an ice bath and the temperature lowered to

0 °C. LiOH·H2O (42 mg, 1.0 mmol) was then added to the solution and the mixture stirred for 30 min while the temperature was maintained at 0 °C. The solvent was then evaporated under reduced pressure and dried in vacuo overnight affording white powder 3.14a in quantitative yield.

Method of synthesizing lithium salt from H-Phe-OH and TMSOLi (Table 3.5 Entry 6) H-Phe-OH (165 mg, 1.0 mmol) was loaded into a flask, and suspended in MeCN (5 mL). TMSOLi (96 mg, 1.0 equiv) was then added to the solution and the mixture was stirred for 30 min. The Phe salt was then filtered off to afford pure white powder 3.14a in 98% yield.

Preliminary Method of synthesizing lithium salt from H-Phe-OH and LiClO4 or LiBF4 (Table 3.5 Entries 7, 8, 9)

H-Phe-OH (165 mg, 1.0 mmol) was loaded into a flask, and suspended in MeCN (5 mL). LiClO4 or LiBF4 (106 mg, 1.0 mmol or 94mg, 1.0 mmol) was then added to the solution followed by 85

DIPEA (136 mg, 1.05mmol or 387 mg, 3.0 mmol) and stirred for 30 min. The Phe salt was then filtered off to afford pure white powder 3.14a in 83% to 90% yield.

+ - + - General Method of synthesizing metal salts from amino acids and M ClO4 or M BF4 (Table 3.5 Entry 10, Table 3.6 Entries 4, 6, 7 , Table 3.7 Entries 1, 4) Free AA (1.0 mmol, 1.0 equiv) was loaded into a flask, and suspended in MeCN (5 mL) or dissolved in MeOH (80 mL). DIPEA (387 mg, 3.0 equiv) was then added and the solution stirred + - + - for 30 min. M ClO4 or M BF4 (1.0 mmol, 1.0 equiv) was then added to the solution and the mixture was stirred for an additional 30 min. If MeOH was used, the solvent was evaporated under a stream of air, and the residue was redissolved in MeCN (5 mL). In both cases, the MeCN suspension was then filtered off to afford a white or tan powder in 85% to quantitative yields.

Lithium L-leucinate (2.22a) Using Method of synthesizing metal salts from amino acids and perchlorates or tetrafluoroborates. Isolated as a white powder (115 mg 85% yield); IR (solid) -1 1 νmax 2956, 2869, 2913, 1575, 1508,1406 cm ; H NMR (400 MHz, D2O) δ 3.67 – 3.58 (1H, m), 1.76 – 1.52 (3H, m), 0.91 (3H, d, J = 5.0 Hz). 0.89 (3H, d, J = 13 - 5.0 Hz) C NMR (176 MHz, D2O) δ 176.70, 53.51, 40.33, 24.11, 21.99, 20.87. HRMS (ESI ) m/z - calcd for C6H12NO2 [M – Li] : 130.0873, found 130.0874.

Sodium L-leucinate (3.8) Using Method of synthesizing metal salts from amino acids and perchlorates or tetrafluoroborates. Isolated as a tan powder (150 mg, 98% yield); IR (solid) -1 1 νmax 2957, 2614, 1578, 1511, 1406 cm ; H NMR (400 MHz, D2O) δ 3.77 – 3.68 (1H, m), 1.83 – 1.62 (3H, m), 0.98 (3H, d, J = 5.0 Hz). 0.97 (3H, d, J = 13 - 5.0 Hz). C NMR (101 MHz, D2O) δ 175.83, 53.48, 39.95, 24.17, 22.03, 20.90. HRMS (ESI ) - m/z calcd for C6H12NO2 [M – Na] : 130.0873, found 130.0874.

Lithium L- Phenylalaninate (3.14a) Using Method of synthesizing metal salts from amino acids and perchlorates or tetrafluoroborates.Isolated as a fine white powder (171 mg, quant); IR (solid) -1 1 νmax 1594, 1554, 1423 cm ; H NMR (400 MHz, D2O) δ 7.32 – 7.12 (5H, m), 3.56 (1H, dd, J = 7.5, 5.5 Hz), 2.98 (1H, dd, J = 14.0, 5.5 Hz), 2.83 (1H, dd, J = 13 14.0, 7.5 Hz). C NMR (101 MHz, D2O) δ 178.87, 136.96, 129.39, 128.79, 127.06, 56.84, 38.91. - - HRMS (ESI ) m/z calcd for C9H10NO2 [M – Li] : 164.0717, found 164.0718.

Sodium L-phenylalaninate (3.14d) Using Method of synthesizing metal salts from amino acids and perchlorates or tetrafluoroborates. Isolated as a fine white powder (150 mg, 98% yield): IR -1 1 (solid) νmax 3029, 2394, 1557, 1493, 1409, 1306 cm ; H NMR (400 MHz,

Methanol-d4) δ 7.37 – 7.26 (4H, m), 7.28 – 7.19 (1H, m), 3.65 (1H, dd, J = 8.5, 4.5 Hz), 3.24 (1H, dd, J = 14.0, 4.5 Hz), 2.90 (1H, dd, J = 14.0, 8.5 Hz); 13C NMR (176 - MHz, Methanol-d4) δ 177.62, 137.79, 129.01, 128.14, 126.24, 57.06, 39.89. HRMS (ESI ) m/z - calcd for C9H10NO2 [M – Na] : 164.0717, found 164.0717.

Potassium L-phenylalaninate (3.14e) Using Method of synthesizing metal salts from amino acids and perchlorates or tetrafluoroborates. Isolated as a white flakey powder (202 mg, quant): IR (solid) -1 1 νmax 3031, 2422, 2130, 1558, 1494, 1409, 1306 cm ; H NMR (400 MHz, D2O) δ 7.44 – 7.26 (5H, m), 3.96 (1H, dd, J = 7.9, 5.2 Hz), 3.25 (1H, dd, J = 14.5, 5.2 13 Hz), 3.09 (1H, dd, J = 14.5, 8.0 Hz). C NMR (101 MHz, D2O) δ 173.87, 135.09, 129.36, - - 129.11, 127.70, 56.03, 36.34. HRMS (ESI ) m/z calcd for C9H10NO2 [M – K] : 164.0717, found 164.0715.

Calcium salt formation from amino acid and CaCO3 (Table 3.6 Entries 2, 3 Table 3.7 Entry 3)

Free amino acid (2.0 mmol, 1.0 equiv) was loaded into a flask, and dissolved in H2O (50 mL).

CaCO3 (100 mg, 1.0 mmol) was then added to the solution and the resulting mixture stirred for 87 either 2 h or o/n. The solvent was then evaporated under reduced pressure and dried in vacuo overnight. This afforded a white powder in yields exceeding 100% (See Chapter 3 for details).

General method of tripeptide formation (Table 3.8, 3.9)

To a flame dried vial under N2, loaded with –ONp ester 3.6b (105 mg, 0.25 mmol), DMF (2 mL) was added and the mixture stirred until dissolution occurred. Phe salt 3.14b (46 mg, 0.125 mmol) was then added to the vial and its contents stirred for 24 h. In a separate flame dried vial under

N2 charged with H-Ile-OMe·HCl (46 mg, 0.25 mmol), DMF (2 mL), followed by DIPEA (36 mg, 0.275 mmol) were added. HBTU (104 mg, 0.275 mmol) was then added to the initial vial, followed by the amino acid and base solution via syringe and the resulting mixture was stirred for an additional 2 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography (30% EtOAc, 70% Hexanes) afforded pure product 3.15 in 28% to 80% yield and with 2% to 18% epimerization as measured by 1H NMR through integration of the OMe peaks at 3.63 ppm and 3.65 ppm (3H). Changes to procedure as reported in Table 3.8 and Table 3.9. (Chapter 3)

Optimized general method of tripeptide formation (Table 3.10)

To a flame dried vial under N2, loaded with –ONp ester AA1 (0.25 mmol, 1.0 equiv), DMF (2 mL) was added and the mixture stirred until dissolution occurred. The calcium salt of AA2 (0.25 mmol, 1.0 equiv) was then added to the vial and its contents stirred for 24 h. In a separate flame dried vial under N2 charged with AA3·HCl (0.50 mmol, 2.0 equiv), DMF (2 mL), followed by DIPEA (71 mg, 0.550 mmol) were added. HOBt (115 mg, 0.75 mmol) was then added into the initial vial, followed by the syringe addition of the AA3 solution. Finally, HBTU (104 mg, 0.275 mmol) was then added to the vial, and the resulting mixture was stirred for an additional 2 h. The mixture was then concentrated down to a thick residue under a stream of air o/n, and then loaded directly onto a silica column. Flash chromatography afforded pure product 3.15, 3.17, 3.18 in 60% to 80% yield and 1% to 3% epimerization as measured by 1H NMR through integration of the OMe at peaks 3.63 ppm and 3.65 ppm (3H).

Methyl ((benzyloxy)carbonyl)-L-phenylalanyl-L-phenylalanyl-L-isoleucinate (3.15) Using Optimized Method. Column conditions: 30% EtOAc:70% hexanes. Isolated as a white solid, (114 mg,

82% yield): Rf = 0.35 (30% EtOAc:70% hexanes); IR -1 1 (solid) νmax 3276, 2963, 1737, 1693, 1643, 1533 cm ; H

NMR (400 MHz, DMSO-d6) δ 8.27 (1H, d, J = 8.0 Hz), 8.10 (1H, d, J = 8.5 Hz), 7.42 (1H, d, J = 9.0 Hz), 7.37 – 7.14 (15H, m), 4.92 (s, 2H), 4.70 – 4.63 (1H, m), 4.24 (2H, s), 3.62 (3H, s), 3.02 (1H, dd, J = 14.0, 5.0 Hz), 2.94 – 2.77 (2H, m), 2.65 (1H, dd, J = 14.0, 10.5 Hz), 1.86 – 1.72 (1H, m), 1.47 – 1.36 (1H, m), 1.47 – 1.36 (1H, m), 0.86 13 (3H, d, J = 5.5 Hz) 0.83 (3H, d, J = 5.5 Hz); C NMR (101 MHz, DMSO-d6) δ 171.67, 171.24, 171.12, 155.63, 137.97, 137.42, 136.95, 129.24, 129.10, 128.23, 127.95, 127.95, 127.61, 127.33, 126.22, 126.15, 65.15, 56.37, 56.06, 53.36, 51.62, 37.53, 37.43, 36.29, 24.73, 15.35, 11.09. + + HRMS (ESI ) m/z calcd for C33H40N3O6 [M + H] : 574.29171, found 574.29147.

Methyl (tert-butoxycarbonyl)-L-phenylalanyl-L-phenylalanyl-L-isoleucinate (3.17) Using Optimized Method. Column conditions: Gradient of 20% to 40% EtOAc/Hexanes. Isolated as a white solid,

(108 mg, 80% yield): Rf = 0.30 (35% EtOAc: 65%

hexanes); IR (solid) νmax 3274, 2966, 1742, 1688, 1644, 1 1519 H NMR (400 MHz, DMSO-d6) δ 8.31 (1H, d, J = 4.5 Hz), 7.94 (1H, d, J = 8.0 Hz), 7.39 – 7.08 (10H, m), 6.87 (1H, d, J = 10.0 Hz), 4.78 – 4.56 (1H, m), 4.22 (1H, appt t, J = 7.5 Hz), 4.16 – 4.03 (1H, m), 3.62 (3H, s), 3.00 (1H, dd, J = 14.0, 5.0 Hz), 2.88 – 2.76 (2H, m), 2.69 – 2.57 (1H, m), 1.85 – 1.72 (1H, m), 1.47 – 1.35 (1H, m), 1.27 (9H, s), 1.11 (1H, s), 0.86 (3H, d, J = 6.0 Hz) 0.82 (3H, d, J = 6.0 Hz); 13C NMR (101 MHz,

DMSO-d6) δ 171.65, 171.28, 171.10, 154.97, 138.02, 137.36, 129.30, 129.07, 127.93, 127.92, 126.21, 126.08, 78.06, 56.35, 55.84, 53.16, 51.62, 37.73, 37.48, 36.30, 28.06, 24.73, 15.36, + + 11.08. HRMS (ESI ) m/z calcd for C30H42N3O6 [M + H] : 540.30736, found 540.30835.

Methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanyl-L-phenylalanyl-L- isoleucinate (3.18) Using Optimized Method. Column conditions: 30% EtOAc:70% hexanes. Isolated as a white

solid, (100 mg, 60% yield): Rf = 0.30 (35%

EtOAc: 65% Hexanes); IR (solid) νmax 3275, 2959, 1737, 1691, 1644, 1535 cm-1; 1H NMR

(400 MHz, DMSO-d6) δ 8.29 (1H, d, J = 8.0 Hz), 8.14 (1H, d, J = 8.0 Hz), 7.89 (2H, d, J = 7.5 Hz), 7.63 (2H, t, J = 8.0 Hz), 7.55 (1H, d, J = 9.0 Hz), 7.45 – 7.37 (2H, m), 7.38 – 7.12 (12H, m), 4.71 – 4.63 (1H, m), 4.35 – 4.05 (5H, m), 3.64 (3H, s), 3.05 (1H, dd, J = 14.0, 5.0 Hz), 3.00 – 2.80 (2H, m), 2.72 (1H, dd, J = 14.0, 11.0 Hz), 1.85 –1.71 (1H, m), 1.48 –1.33 (1H, m), 1.27– 13 1.11 (1H, m), 0.84 (3H, d, 7.0 Hz), 0.82, (3H, d, 7.0Hz); C NMR (101 MHz, DMSO-d6) δ 171.68, 171.29, 171.14, 155.59, 143.72, 143.67, 140.62, 140.60, 138.03, 137.42, 129.22, 129.14, 127.95, 127.57, 127.02, 126.21, 126.16, 125.29, 125.19, 120.04, 65.62, 56.37, 56.04, 53.38, + 51.62, 46.51, 37.51, 37.42, 36.28, 24.72, 15.34, 11.09. HRMS (ESI ) m/z calcd for C40H44N3O6 [M + H]+: 662.32301, found 662.32228

Formation of Lysine Copper Complex

A stirred solution of Lysine·HCl (365 mg, 2.0 mmol) in H2O (20 mL) was heated to reflux.

CuCO3 (619mg, 2.8 mmol) was then added and the mixture was refluxed for 45 min. The hot suspension was filtered to give a clear dark blue solution which was transferred to a lyophilization flask and was cooled to -78 °C using a cold bath of dry ice and acetone, and left to lyophilize for a day on the lyophilizer to afford the product 4.8.

Formation of Copper Complex 4.6 (Scheme 4.2)

A stirred solution of Lysine·HCl (25.57 g, 140 mmol) in H2O (250 mL) was heated to reflux.

CuCO3 (32.06 g, 145 mmol) was then added and the mixture was refluxed for 45 min. The hot suspension was filtered to give a clear dark blue solution. The resulting solution was then cooled to 0 °C and the pH was set to 9 using Na2CO3. Benzyl chloroformate (34.97 g, 205 mmol) was then added dropwise over one hour and the resulting suspension was allowed to warm to room temperature and stirred for an additional 12 h. Finally, the solid pale blue product was filtered off 90 affording quantitative yield.15 Copper complexes are paramagnetic and as such NMR analysis cannot be performed. They are also not stable under mass spec. conditions.

Formation of 4.7 from Copper Complex 4.6 using sodium sulphide (Table 4.1 Entry 2)

A suspension is formed from copper complex 4.6 (620 mg, 1.0 mmol) in H2O (15 mL). Sodium sulphide (117 mg, 1.5 mmol) was then added to the mixture, and stirred for 30 min. The solid was then filtered off to afford a clear solution which was then neutralized with dilute HCl. The solution was cooled and the white precipitate was filtered off, affording product 4.7 (270 mg, 48%) yield.16

Formation of N6-((benzyloxy)carbonyl)-L-lysine 4.7 from Copper Complex 4.6 using EDTA (Table 4.1 Entry 4)

To a solution EDTA (438 mg, 1.5 mmol) in H2O (15 mL) was added copper complex 4.6 (620 mg, 1.0 mmol) and the mixture heated to reflux and stirred for 5 h. The white solid was then filtered off, and recrystallized from a 50% acetic acid solution, affording white crystalline solid 4.7 (440 mg, 70% yield).17 Spectroscopic data in accordance with previously obtained results.18

Formation of 4.7 from Copper Complex 4.6 using HCl (Table 4.1 Entry 4) To a suspension of copper complex 4.6 (910 mg, 1.5 mmol) in MeCN (35 mL) was added HCl (12 M, 625 μL) dropwise, and the mixture was stirred for 10 min. The solid was then filtered off, and washed with acetonitrile until almost colourless, affording the product, 4.7 as a pale orange solid in (378 mg, 45% yield).

N6-((benzyloxy)carbonyl)-L-lysine (4.7)

Isolated as pale orange solid (378 mg, 45% yield). 1H NMR (400 MHz,

DMSO-d6) δ 8.40 (3H, br s), 7.51 – 7.10 (5H, m), 5.00 (2H, s), 3.83 (1H, s), 2.98 (2H, br d, J = 6.0 Hz), 1.78 (2H, br s), 1.48 – 1.23 (4H, 13 m). C NMR (176 MHz, DMSO-d6) δ 170.82, 156.07, 137.25, 128.35, 127.73, 127.69, 65.12, 51.95, 39.91, 29.57, 28.83, 21.59.

Note: Some present copper cause broad peaks in 1H NMR spectra. Cannot determine multiplicity.

Product 4.8 from products 4.6 and 4.7 using lithium aluminium hydride (Scheme 4.3) Product 4.7 (63 mg, 0.2 mmol) or 4.6 (310 mg, 0.5 mmol) was stirred in a solution of THF (4.2 mL or 21.0 mL). 1 M LiAlH4 in THF (800 μL, 0.8 mmol or 2.5 mL, 2.5 mmol) was then added to the solution and the mixture was refluxed o/n. Excess LiAlH4 was decomposed with H2O, and the precipitate was filtered off. The clear solution was partially evaporated under reduced pressure and then acidified to a pH of 2 using conc. HCl. Extraction using Et2O followed by evaporation under reduced pressure of the aqueous layer afforded the product reddish brown oil 4.8 in amounts significantly higher than expected for quantitative yields (95 mg, 163% yield, or 326mg, 205% yield), but nonetheless showing clean NMRs (See chapter 4).20

(S)-2-amino-6-(methylamino)hexan-1-ol (4.8)

1 Isolated as reddish brown oil (95 mg, 163% yield). H NMR (400 MHz, D2O) δ 3.55 (1H, dd, J = 12.5, 3.5 Hz), 3.41 – 3.30 (1H, m), 3.13 – 3.02 (1H, m), 2.84 – 2.73 (2H, m), 2.43 (3H, s), 1.55 – 1.33 (4H, m), 1.27 – 1.13 (2H, m).

Product 4.10 from lysine using reductive amidation (Scheme 4.4)

A stirred solution of Lysine·HCl (365 mg, 2.0 mmol) in H2O (10 mL) was heated to reflux.

CuCO3 (486 mg, 2.2 mmol) was then added and the mixture was refluxed for 1 h min. The hot suspension was filtered to give a clear dark blue solution. After being cooled to rt, 37% formaldehyde(aq) (606.3 mg, 10.0 mmol) was added to the solution, followed by NaBH3CN (314 mg, 5.0 mmol) and was stirred for 1.5 h. The brown mixture was then filtered to afford a clear yellow solution, which was then acidified with conc. HCl, and again the precipitate was filtered off, affording the crude and undesired product 4.10 as a brown solid (440 mg, 70% yield)

N2,N2,N6,N6-tetramethyllysine hydrochloride (4.10)

Isolated as brownish solid (440 mg, 70% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.27 (1H, dd, J = 9.0, 4.5 Hz), 2.92 – 2.81 (2H, m), 2.59 (12H, s), 1.82 – 1.63 (1H, m), 1.62 – 1.41 (3H, m), 1.21 – 1.05 (2H, m).

Sample procedure to form ε-formyl lysine (Table 4.2 Entry 6)

To a vial was loaded copper complex 4.9 (88 mg, 0.25 mmol) and H2O (1.25 mL), K2CO3 was added until the pH was adjusted to 9. The solution was then cooled to 0 °C and n-formyl saccharin (211 mg, 1.0 mmol) was added to the solution. The mixture was stirred for 2 h maintaining the temperature and adding additional K2CO3 to keep the pH at 9. The solvent was evaporated under reduced pressure to afford product 4.12 in 90% conversion based on small decomplexation gaining access to compounds 4.13 followed by NMR analysis. Purification of the product was never achieved due to the polar nature of both the formylating agent and the lysine complex, as well as being unable to be columned. Other procedures altered as per Table 4.2.

5.3 References

1 Hashimoto, C.; Takeguchi, K.; Kodomari, M. Synlett. 2011, 1427−1430. 2 Goodreid, J. D.; Duspara, P. A.; Bosch, C.; Batey, R. A. J. Org. Chem. 2013, 79, 943−954. 3 Karnik, A. V. ; Kamath, S. S. J. Org. Chem. 2007, 72, 7435−7438. 4 Reddy, K. R.; Maheswari, C. U.; Venkateshwar, M.; and Kantam M. L. Eur. J. Org. Chem. 2008, 3619–3622. 5 Ohshima, T.; Iwasaki, T.; Maegawa, Y.; Yoshiyama, A.; Mashima, K. J. Am. Chem. Soc. 2008, 130, 2944-2945. 6 Glynn, D.; Bernier, D.; Woodward, S. Tetrahedron Lett. 2008, 49, 5687–5688. 7 Kise, N.; Takaoka, S.; Yamauchi, M.; Ueda, N. Tetrahedron Lett. 2002, 43, 7297–7300. 8 Rella, M. R.; Williard, P. G.; J. Org. Chem. 2007, 72, 525−531. 9 Dhiman, R.S.; Opinska, L. G.; Kluger, R. Org. Biomol. Chem. 2009, 9, 5645−5647 10 Pradhan, T. K.; Krishnan, S.; Vasse, J.; Szymoniak, J. J. Am. Chem. Soc. 2011, 13, 1793−1795. 11 Selvakumar, S.; Sivasankaran, D.; Singh, V. K. Org. Biomol. Chem. 2009, 7, 3156–3162 12 Arisawa, M.; Igarashi, Y.; Kobayashi, H.; Yamada, T.; Bando, K.; Ichikawa, T.; Yamaguchi, M. Tetrahedron. 2011, 67, 7846−7859. 13 Fujisaki, F.; Marumi, O.; Sumotomo, K. Chem. Pharm. Bull. 2007, 55, 124−127. 15 Crivici, A.; Lajoie, G. Synth. Comm. 1993, 23, 49−53. 16 Nowshuddin, S.; Reddy, R. Tetrahedron Lett. 2006, 47, 5159−5161. 17 Kuwata, S.; Watanabe, H.; Bull. Chem. Soc. Jpn. 1965, 38, 676−677. 18 Casadio, Y. S.; Brown, D. H. Chirila, T. V.; Kraatz, H.; Baker, M. V. Biomacromolecules 2010, 11, 2949–2959. 20 Rahman, O.; Kilhberg, T.; Langstrom, B. J. Chem. Soc. Perkin Trans. 1. 2002, 23, 2699−2703. 94

Appendix 1H and 13C NMR Spectra

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