MIDA borylated Isocyanides Enable Efficient Access to Bioactive Boropeptides and Other Derivatives

by

Adam Daniel Zajdlik

A thesis submitted in conformity with the requirements for the degree of Masters of Science

School of Graduate Studies Department of Chemistry

University of Toronto

© Copyright by Adam Daniel Zajdlik 2017

ii

MIDA borylated Isocyanides Enable Efficient Access to Bioactive Boropeptides and Other Derivatives

Adam Daniel Zajdlik

Masters of Science

School of Graduate Studies

Department of Chemistry University of Toronto

2017

Abstract

MIDA-containing alpha-boryl isocyanides are isolable molecules which allow one-step

access to boroalkyl-functionalized heterocycles as well as biologically active boropeptides

through a multicomponent approach. Among these derivatives are 6-boromorpholinones,

novel borocycles with nanomolar IC50 values for 20S proteasome inhibition. MIDA = N-

methyliminodiacetyl.

iii

Table of Contents Table of Contents ................................................................................................................ iii

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

List of Figures .................................................................................................................... vii

List of Schemes ................................................................................................................. viii

List of Appendices .............................................................................................................. ix

ix

Introduction .......................................................................................................................... 1

Chapter 1 Boryl Isocyanides ................................................................................................ 2

1.1 Synthesis and Scope ................................................................................................. 2

1.2 Functionalization of the isocyanide ......................................................................... 4

1.2.1 Ugi 4-Component Reaction ......................................................................... 6

1.3 Passerini 3-Component Reaction ............................................................................. 8

1.4 Boromorpholinones ................................................................................................ 10

1.4.1 pKa Determination ..................................................................................... 11

1.4.2 Proteasome Inhibition Assays .................................................................... 11

1.5 ClpP Substrate ........................................................................................................ 13

1.5.1 n-Octyl substituted boryl isocyanide ......................................................... 13

1.5.2 Attempted ring-closure conditions ............................................................. 14

1.6 Computational analysis of BMN ring-opening ...................................................... 16

1.6.1 Thermodynamic Equilibria ........................................................................ 16

1.6.2 Kinetic Profiling ......................................................................................... 18

1.7 Activity-based profiling (Ben Cravatt, Scripps) .................................................... 20

1.8 Chiral Variants ....................................................................................................... 22

Chapter 2 ............................................................................................................................ 27

Experimental ................................................................................................................. 27

2.1 General Information ............................................................................................... 27

iv

2.2 Synthesis of α-boryl isocyanides ........................................................................... 28

2.3 Deuterium Exchange Experiments ........................................................................ 29

2.4 Attempted α-deprotonations of α-boryl isocyanides .............................................. 29

2.4.1 Accidental MIDA tetradeuteration ............................................................ 29

2.5 Other functionalizations of α-boryl isocyanides .................................................... 30

2.5.1 Preparation of α-boryl tetrazole ................................................................. 30

2.5.2 Preparation of α-boryl isothiocyanate ........................................................ 30

2.5.3 Preparation of α-boryl thioureas ................................................................ 31

Passerini 3-Component Reactions ................................................................................ 32

3.1 General preparation of N-(MIDA boromethyl)-α-acyloxyamides ......................... 32

3.2 Determination of P3CR Product Relative Stereochemistry ................................... 32

3.3 Deprotection of P3CR Products / Discovery of Boromorpholinones .................... 33

3.3.1 Assay yield and calibration curve .............................................................. 33

3.4 VT NMR experiments ............................................................................................ 34

3.5 Calibration Curve ................................................................................................... 34

3.6 NMR Yield............................................................................................................. 35

Ugi 4-Component Reactions ......................................................................................... 36

4.1 Proline Ugi Reaction .............................................................................................. 36

4.2 Preparation of MIDA-Velcade® ............................................................................. 36

MIDA-boropeptides ...................................................................................................... 38

5.1 Preparation ............................................................................................................. 38

5.2 Preparation, purification and characterization ....................................................... 39

5.3 Determination of boropeptide yield using 1H NMR and external standard ........... 39

Boromorpholinones ....................................................................................................... 41

6.1 General preparation of 6-boromorpholin-3-ones ................................................... 41

6.2 pKa determination .................................................................................................. 41

6.3 Assay yield and calibration curve .......................................................................... 42

v

6.4 20S proteasome inhibition assays .......................................................................... 43

6.4.1 MIDA-bortezomib (AZ-4-285A) ............................................................... 43

6.4.2 boromorpholinone (AZ-4-180) .................................................................. 44

References .......................................................................................................................... 46

Appendices ......................................................................................................................... 49

vi

List of Tables Table 1. Preparation of alpha-boryl isocyanides ........................................................................ 2

Table 2. Preparation of protected boropeptides using α-boryl isocyanide 2a ............................ 7

Table 3. P3CRs involving α-boryl isocyanides ......................................................................... 9

Table 5. Reaction condition screening for preparation of enantiopure boryl aldehydes ......... 25

vii

List of Figures Figure 1. The borylamide motif ................................................................................................ 1

Figure 2. pKa determination of 11ad. ....................................................................................... 11

Figure 3. In vitro cytosolic chymotrypsin-like 20S proteasome inhibition by 11ad ............... 12

Figure 4. potency of AZ-1 vs. potential ClpP inhibitor in 20S protease inhibition ................. 14

List of Schemes Scheme 1. Functionalization of MIDA α-boryl isocyanide 2a. ................................................. 5

Scheme 2. U4CRs involving α-boryl isocyanide 2a. ................................................................. 6

Scheme 3. Deprotection/Condensation of P3CR product 10ad. .............................................. 10

Scheme 4. Preparation of precursor to potential BMN ClpP inhibitor .................................... 13

Scheme 5. Attempted promoted ring closure to form BMN .................................................... 14

Scheme 6. Computational modeling of AZ-1 hydrolysis ....................................................... 16

Scheme 7. Equilibrium between AZ-1 and trans-amide bond containing boronic acid .......... 17

Scheme 8. Proposed pathway for amide bond rotation of AZ-1 hydrolysis products ............. 17

Scheme 9. Kinetic profiling of BMN hydrolysis ..................................................................... 19

Scheme 10. Preparation of N-carboxymethyl proline .............................................................. 22

Scheme 11. Preparation of cyclohexyl N-(COMP) boryl aldehyde ........................................ 24

Scheme 12.Oxidation and Curtius rearrangement of COMP boryl aldehydes ........................ 26

ix

List of Appendices

Appendix A. Compounds send to Cravatt lab, Scripps, La Jolla ............................................. 49

Appendix B. Attempted U4CRs using simple and OTBDMS aziridine aldehyde dimers ...... 52

Appendix C. Serendipitous discovery of α-boryl carbamoyl azide ......................................... 53

Appendix D. Hetero-Diels-Alder reaction of α-boryl aldehydes ............................................. 55

Introduction Boronic acids and their derivatives are useful as synthetic building

blocks as well as biologically active targets of synthesis. Both the

biological activity and chemical reactivity of boronic acids stem from

boron’s Lewis acidity. While useful in a broad range of applications,

boron’s propensity to undergo reactions with Lewis bases becomes problematic for functional

group compatibility during synthesis.[1] Reagents that streamline installation of a carbon-boron

bond in stereochemically complex, heteroatom-rich environments, are expected to find

application not only as starting materials but also as valuable endpoints of synthesis.

The borylamide motif (Figure 1) is commonly found in the structures of biologically active

boropeptides.[2} Efforts in our lab have been focused on the amphoteric aziridine aldehyde- and

isocyanide-driven macrocyclizations of linear peptides and peptidomietics.[3] This

methodology is enabled by the development of functionally dense, heteroatom-rich

environments where a kinetic barrier prevents two otherwise reactive functional groups from

prematurely reacting with each other.[4] Here we expand the scope of this methodology to

include amphoteric boron-containing building blocks for use in multi-component preparation

of boropeptides and their derivatives

BHO

NH

OOH

Figure 1. The

borylamide motif

2

Chapter 1 Boryl Isocyanides

1.1 Synthesis and Scope

As part of our efforts to develop amphoteric boron-transfer reagents, we considered an

isocyanide/boron combination. Isocyanides are 1,1-amphoteric molecules that enable

heterocycle synthesis[5] and participate in multi-component reactions (MCRs) such as Ugi and

Passerini processes.[6] Herein, we outline the preparation and utility of bench-stable α-boryl

isocyanides. We demonstrate the application of these novel derivatives in heterocycle synthesis

as well as in MCRs to generate biologically active boropeptides. As part of our study, we

demonstrate a two-step synthesis of both diastereomers of Velcade®, an FDA approved, boron-

containing proteasome inhibitor used to treat multiple myeloma.[7] Furthermore, our

documented discovery of a novel

class of boron-containing heterocycles, with biological activity comparable to Velcade®,

will be useful in efforts to prepare libraries.[a,b, d, f, g, l, n, o, p]

BO OMeN

OO

NCOR

HSiCl3, Et3N

CH2Cl2, 0°C to 23°C BO OMeN

OO

NCR

1 2

Table 1. Preparation of alpha-boryl isocyanides

Starting Material R Product Yield [%][b]

____________________________________________________

1a isobutyl 2a 75

1b cyclohexyl 2b 31

1c phenyl 2c 30

[a]Thereactionswerecarriedoutusingα-borylisocyanate(1.0equiv.),

trichlorosilane(1.6equiv.)andtriethylamine(3.6equiv.)inanhydrous

CH2Cl2at0°Cfor30minutesfollowedby6hoursat23°C.[b]Yieldsof

isolatedproductsaftersilicagelchromatography.

3

At the outset, we were aware of the known propensity of tricoordinate boron to react with

isocyanides.[8] Moreover, rapid decomposition upon exposure to air has limited synthetic

applicability of boron-containing isocyanides.[9] To circumvent this undesired reactivity, we

focussed our search on fragments with tetra coordinate boron. In N-methyliminodiacetyl

(MIDA) boronates, an intramolecular coordinative stabilization of boron’s empty p-orbital

effectively masks its Lewis acidity.[10] The tetracoordinate boronate fragment is tolerant to a

range of functional group transformations, allowing access to various borylated derivatives.

These include α-boryl aldehydes, [11] which contain a carbon-boron bond adjacent to an

electrophilic aldehyde, and α-boryl isocyanates 1, a group of reagents that are now readily

available from a protocol recently reported by our lab among others.[12]

1.2 Functionalization of the isocyanide

The structural integrity of α-boryl isocyanates 1 in various functionalizations prompted us to

attempt a trichlorosilane-mediated deoxygenation,[13] which gratifyingly afforded the

corresponding isocyanides 2 as solid materials stable to column chromatography (Scheme 1). To

explore the properties of these compounds we first investigated the possibility of α-proton removal

given the documented utility of α-metalated isocyanides.[5] During attempted α-deprotonation

using weak bases (10 equiv. of Et3N or 15 equiv. of NaHCO3) there was no observable decrease

in the 1H NMR integration of the α-proton signal. Under strongly basic conditions (1 equiv. of

potassium tert-butoxide) followed by exposure to deuterated solvents (MeCN-d3) a similar lack of

�-deprotonation was observed. Interestingly, tetradeuteration of the MIDA moiety was observed

5

in both the 1H NMR and HRMS spectra. These results showcase the dual nature of the MIDA

moiety as a protective group for both the carbon-boron bond and the α-proton.[14]

The protected nature of the α-proton led us to focus our efforts on a variety of isocyanide

functionalizations (Scheme 1). Selenium-catalyzed sulfurization gave rise to α-boryl

isothiocyanate 3a in moderate yield.[15] Reaction of isothiocyanate 3a with various amines afforded

the corresponding thioureas 4aa – 4ab in quantitative yield.[16] Reaction of the isocyanide 2a with

in situ-generated hydrazoic acid yielded the α-boryl tetrazole 5a in moderate yield.[17]

Scheme 1. Functionalization of MIDA α-boryl isocyanide 2a. [a] α-Boryl isocyanide 2a (1.0 equiv.), sulfur (1.2 equiv.), selenium (5.0 mol %)

and triethylamine (2.4 equiv.) in THF at 80°C for 15 min. [b] α-Boryl

isothiocyanate 3a (1.0 equiv.) and amine (2.0 equiv.) in THF at 23°C. [c] α-Boryl

isocyanide 2a (1.0 equiv.), HCl (2.0 mol%) and TMSN3 (1.5 equiv.) in TFE/Et2O

at 60°C for 6.5 h. [d] Yields of isolated products after silica gel chromatography.

B(MIDA)

NC

B(MIDA)

NN

NN

B(MIDA)

NCS3a (77%)[d]

B(MIDA)

NH

S NH

R

4aa, R = ethyl (79%)[d]

4ab, R = tert-butyl (90%)[d]

2a5a (78%)[d]

[a] [c]

[b]

BOO

MeN

OOR

= R-B(MIDA)

1.2.1 Ugi 4-Component Reaction

Successful retention of the carbon-boron bond during functionalization of the isocyanide moiety

prompted us to investigate more challenging transformations. Isocyanide 2a participated in an Ugi

4-component reaction (U4CR) with 2-pyrazinyl carboxylic acid, phenylacetaldehyde and

ammonia to afford the MIDA-Velcade® analogue 7a in 55% isolated yield (Scheme 2). We then

attempted to generalize this reaction by employing amino acids as both the acid and amine

component. When isocyanide 2a was reacted with L-proline and isobutyraldehyde the

borodipeptide 8a was obtained in 7% isolated yield (Scheme 2. U4CRs involving α-boryl

isocyanide 2a.).[18] In a number of other reactions, we have found that methanol solvolyzes the

MIDA moiety yielding decomposition products. We attributed the low yield of 8a to the

nucleophilicity of the solvent. The reaction was attempted using trifluoroethanol (TFE) as the

solvent given its decreased nucleophilicity relative to methanol; however, the desired product was

not obtained.[5 a]

Scheme 2. U4CRs involving α-boryl isocyanide 2a.

B(MIDA)

NC

HNCOOH

O

MeOH-40°C (3 h) to 23°C (15 h)

(MIDA)B

NH

2a 8a (7%)

B(MIDA)

NC

2a

+

+ PhO

N

N COOH

NH3

TFE, 23°C, 12 h

(MIDA)B

NH

7a (55%)

+

O

N COOMe

O HN

ON

N

Ph

7

We therefore shifted our focus to U4CRs without solvent participation using N-protected amino

acids and peptides as the carboxylic acid component and ammonia as the amine. The reactions

proceeded at room temperature to give diastereomeric mixtures of the desired protected

boropeptides 9 in moderate to excellent yields (Table 2). In most cases, diastereomers were

separable by preparative reverse-phase high-performance liquid chromatography (HPLC). For

larger peptides separation became more difficult due to considerable peak overlap.

Table 2. Preparation of protected boropeptides using α-boryl

NC

B(MIDA)

2a

(Boc-N)-Peptide-COOHR O + NH3

TFE, 23°C

HN

ONH

R

Peptide(N-Boc)

O

9

B(MIDA)

Entry Peptide/AA[b] R Product Time (d) Yield (%)[c]

__________________________________________________________

1 G benzyl 9a 7 69

2 G isopropyl 9b 6 79

3 F Isopropyl 9c 10 74

4 V benzyl 9d 6 57

5 GG benzyl 9e 6 92

6 FA benzyl 9f 6 91

7 PLF benzyl 9g 7 63

8 PGLF benzyl 9h 12 76

[a]Thereactionswerecarriedoutusingα-borylisocyanide2a(1.0equiv.),aldehyde(1.0equiv.),ammonia(1.5equiv.,7NsolutioninMeOH)andpeptide(1.0equiv.)inTFEat23°C.[b]BocprotectedatN-terminus;writtenfromN-toC-terminususingstandard1-letteraminoacidabbreviations.[c]Yieldofdiastereomericproductsdeterminedbycomparisonof1HNMRintegrationwith3,4,5-triiodobenzoicacidasaninternalstandard.

1.3 Passerini 3-Component Reaction

The successful participation of isocyanide 2a in U4CRs led us to explore its applicability

in Passerini 3-component reactions (P3CRs).[5a] We found this reaction to proceed smoothly

with a variety of aldehydes affording minimal by-products. Generally, the P3CR products 9

could be isolated in an acceptably pure form with simple aqueous workup. The two

diastereomers could be separated by flash column chromatography on silica gel in the vast

majority of cases. The reaction proceeded with moderate to excellent yields (Table 3). The rate

of the reaction could be increased at elevated temperature and pressure (100°C, µwave, 25

min); however, this occurred at the expense of selectivity for the desired product.

Dichloromethane was found to be an ideal solvent as protic solvents did not yield the desired

product and the isocyanide was not soluble in diethyl ether or tetrahydrofuran.

9

We then attempted to remove MIDA from the P3CR product 10ad under standard

aqueous basic conditions to afford the corresponding free boronic acids.[ 18c] We found that

under the deprotection conditions, hydrolysis of the ester also occurred followed by

condensation of the resulting free hydroxyl with the newly formed boronic acid yielding

disubstituted 6-boromorpholinone 11ad as an air-stable white solid (Scheme 3.

Deprotection/Condensation of P3CR product 10ad.Scheme 3). The relative stereochemistry of borocycle

11ad was determined by computational modelling of the starting material. Computational

prediction (MPW1PW91, 6-311G(2d, p)) of several 1H NMR chemical shift differences

B(MIDA)

NCR1

CH2Cl2, 23°C

(MIDA)B

NH

R1

2

10

R2 OPh COOH

OR2

O

OPh

Table 3. P3CRs involving α-boryl isocyanides.[a]

Entry R1 R2 Product Time (d) Yield [%][b]

_____________________________________________________

1 isobutyl phenyl 10aa 7 81

2[c] isobutyl isopropyl 10ab 4 68

3 isobutyl 4-F-phenyl 10ac 4 60

4 isobutyl benzyl 10ad 4 56

5 isobutyl 3-pyradinyl 10ae 2 79

6 isobutyl 4-Me-phenyl 10af 4 50

7 isobutyl 2-Br-phenyl 10ag 7 93

8 cyclohexyl phenyl 10ba 7 53

9 cyclohexyl isopropyl 10bb 7 46

10

between the two diastereomers of P3CR product 10ad correlated well with experimental

observations allowing an inference of the relative stereochemistry.

1.4 Boromorpholinones We attempted similar deprotection/condensations with a number of other P3CR products;

however the borocyclic products (identified by LRMS and crude 1H NMR) could not be

obtained in sufficient purity for characterization. Attempted purification of these compounds

by acidic/basic extraction, recrystallization and chromatography (silica, neutralized silica,

alumina (basic/neutral) ) were all unsuccessful. We are confident that techniques commonly

used for the purification of unprotected boropeptides (such as centrifugal partition liquid

chromatography) could be successfully employed to isolated these boromorpholinone adducts.

Scheme 3. Deprotection/Condensation of P3CR product 10ad.

BO OMeN

OO

NH

O

PhO

OPh

NaOHTHF/H2O

23°C, 10 min.

B

NH

OHO Ph

O

10ad

11ad (79% yield)

11

1.4.1 pKa Determination

The unique structure of boromorpholinone

11ad led us to investigate its behaviour in

aqueous solution. To determine the pKa of the

boronate moiety, the 11B NMR spectrum was

taken at various pHs in buffered aqueous

methanol (Figure 2).[19] A sharp decrease in the

signal at 19.3 ppm was observed at a pH of 9.0

accompanied by a sharp increase in a signal at

2.9 ppm. ESI mass spectra of these samples

identified compound 11ad as the primary

component in the lower pH samples while

pentadetuerated boronate 12ad was identified in the samples at higher pH. From this data we

approximated the pKa of the boronate moiety as 9.0.

1.4.2 Proteasome Inhibition Assays

Given the known propensity of boropeptide analogues to inhibit members of the 20S

proteasome[2n, q] we decided to investigate the interaction of 6-boromorpholinone 11ad with

various classes of this protease family. We found that borocycle 11ad inhibited the

chymotrypsin-like members of the 20S proteasome with an IC50 of 76 nM (compared to

Velcade® in the same assay; IC50 = 0.3 nM). Figure 3 illustrates this activity with the results

obtained in the same studies for Velcade®, the current benchmark for proteasome inhibition. A

similar correlation was observed for the caspase-like and trypsin-like members of the 20S

proteasome. Both 11ad and Velcade® exhibited much weaker inhibition of the caspase-like

NH

BHO O Ph

O ND

BDO

ODPh

OODODpKa ~ 9.0

11ad 12ad

Figure 2. pKa determination of 11ad.

12

enzymes (IC50s of 2.0 µM and 3.2 nM respectively) and no inhibition of the trypsin-like

members. The syn-diastereomer of 11ad was prepared from anti-10ad and exhibited inhibition

of the chymotrypsin-like enzymes with an IC50 roughly 20 times greater than that of the anti-

isomer (52 nM vs. 2.9 nM in the same assay).

While the boromorpholinone inhibitors showed weaker inhibition than Velcade®, we are

confident that structural optimization has the potential to significantly improve these results.

The pyrazine side chain in Velcade® does not exhibit a defined interaction with the active site

binding pockets within the 20S proteasome causing a lack of selectivity and therefore a range

of undesired side effects.[20]

Figure 3. In vitro cytosolic chymotrypsin-like 20S proteasome inhibition by 11ad.[a] [a] Results were obtained in duplicate and averaged. Errors shown indicate 1 standard error of mean.

1.5 ClpP Substrate

1.5.1 n-Octyl substituted boryl isocyanide

The Schimmer lab prepared an

inhibitor of ClpP with the

structure shown below (A).

They noted that this inhibitor was unstable under biological conditions due to the propensity of the

β-lactam to undergo hydrolysis. We hypothesized that utilizing the boromorpholinone core in place

of the β-lactam core with the same side chains would impart added stability under biological

conditions while maintaining the selectivity for ClpP over other serine proteases. To test this

hypothesis we set out to prepare the boromorpholinone analogue shown below (B) via the

procedure outlined above.

Scheme 4. Preparation of precursor to potential BMN ClpP inhibitor

BO OMeN

OO

n-Oct NH

O

O

OPh

NBO OMeN

OO

n-Bu NCO

BO OMeN

OO

n-Oct NC

HSiCl3, Et3N

DCM, 0°C to 23°C4h, 60% crude

N

O

Ph COOH

DCM, 23°C, 3h39% isolated

B

NH

O

n-Octyl O

NHOO

O

N

A B

14

1.5.2 Attempted ring-closure conditions

The deprotection/cyclization of the P3CR product was attempted under standard aqueous basic

conditions to yield a mixture of free boronic acid and cyclized product. The reaction was repeated

with addition of 4Å molecular sieves and acetic anhydride to promote ring closure (Scheme 5).

Over several hours the ratio (ion count, ESI MS) between the cyclized product and the free boronic

acid increased in favor of the cyclized product. After filtration and lyophilization, the 1H NMR

indicated a mixture of cyclized and open-chain products. A reliable set of conditions to affor

selective ring closure without the formation of impurities remains underway.

Scheme 5. Attempted promoted ring closure to form BMN

An ideal ClpP inhibitor would be selective

and would therefore not inhibit members of

the 20S proteasome. Given the relative

expense of the ClpP assays we first

screened ClpP-1 against the 20S

proteasome. Using a technique analogous

to that used for AZ-1, the IC50 value for the

BO OMeN

OO

n-Oct NH

O

O

OPh

N

1) NaOHTHF/H2O, rt

2) Ac2O, 4Å ms23°C, o/n

B

NH

O

n-Oct O

HO NB

NH

OH

n-Oct O

AcO NOH

B

NH

OHO

O

AZ-1IC50 ~ 20 nM

B

NH

OHO

On-Oct

Ph N

ClpP-1IC50 ~ 1 µM

Figure 4. potency of AZ-1 vs. potential ClpP

inhibitor in 20S protease inhibition

15

crude reaction mixture of about 1 µM for the CT-L enzymes (compare to 20nM for AZ-1) (Figure

4). Efforts to test the inhibitor in ClpP inhibition assays to see if there is a therapeutic window

between ClpP and 20S protease inhibition remain underway.

1.6 Computational analysis of BMN ring-opening

While the β-lactone ClpP inhibitor used by the Schimmer lab is selective for ClpP, its beta-lactone

core is unstable in vivo and undergoes rapid hydrolysis, resulting in a total loss of activity. Given

that the AZ-1 has a pKa of about 9 in aqueous buffered methanol, there is good reason to believe

that the BMN core would remain hydrolytically stable in vivo. To test this hypothesis

computational modelling of BMN ring hydrolysis and condensation processes were carried out.

1.6.1 Thermodynamic Equilibria

We first addressed the thermodynamic nature of the hydrolysis equilibrium. Using

Gaussian ’09 as a computational engine (geometry optimization at B3LYP 6-31G+(d) and single-

point energy calculation at B3LYP 6-311G++(d,p)) (Scheme 6).[21] The results indicate that

hydrolysis to the free boronic acid with a cis-amide bond is energetically disfavored. Note that all

energies noted below are in kJ/mol.

Scheme 6. Computational modeling of AZ-1 hydrolysis

Though the results indicate that AZ-1 remains unhydrolyzed in aqueous medium, the

B

NH

O

O

PhHOB

NH

O

OPh

HOHO H

H2O∆H = 160.3

+

17

possibility of alternate lower energy pathways and intermediates remained unaddressed. For

example, a hydrolysed intermediate with a trans-amide might exhibit enhanced thermodynamic

stability. Computational modeling of this equilibrium (Scheme 7) suggests that the trans-amide

containing hydrolysis product is in fact more stable than AZ-1.

Scheme 7. Equilibrium between AZ-1 and trans-amide bond containing boronic acid

Scheme 8. Proposed pathway for amide bond rotation of AZ-1 hydrolysis products

B

NH OH

O PhHOHO

B

NH

O

O

PhHOH2O

∆H = –151.7+

B

NH

O

O

PhHOB

NH

O

OPh

HOHO H

B

NH OH

O PhHOHO

8.8 kcal/mol

BNH

O

OPh

HO

HO HO HH

H2O3.3 kcal/mol

Global Minimum

+

B

NH

O

O

Ph

HOHO H

HO

H

0.2 kcal/mol

8.6 kcal/mol

134.7 kcal/mol

B

NOH

O PhHO

H2O +

12.1 kcal/mol

18

Yet experimental evidence indicates that this AZ-1 is the predominant species so we sought an

explanation for this seemingly erroneous result. To probe a potential solvent effect, a handful of

the most reasonable potential configurations were modelled with a discrete water molecule

included (Scheme 8). The results suggest that the global minimum energy species is the cyclic

BMN coordinated at boron to a single molecule of water. Worth note is relatively large difference

in energy between the 5 and 6 membered cycles (Scheme 8).

1.6.2 Kinetic Profiling

The kinetic feasibility of hydrolysis was also investigated by modeling several potential transition

states. Also modeled were the N-methylated derivatives to probe the feasibility of reactivity fine

tuning via N-substitution. The results are summarized in Scheme 9. The results indicate that the

inclusion of a water molecule lowers the activation energy considerably. the activation energy is

high for both AZ-1 and the N-Me derivative. This suggests that the reaction is governed by

thermodynamics and supports our observation that the cyclic BMN is the predominant species in

solution. Also worth note is the stabilizing effect of N-methylation towards hydrolysis.

19

Scheme 9. Kinetic profiling of BMN hydrolysis

B

NH

O

O

R

R

OB

NH

O

O

R

R

HOH

HHO

H

HO

B

NH

O

O

R

R

O B

NH

O

O

R

R

HOH

HHOH

HOHO H

O HH

Ea = 24.0

Ea = 19.2

∆E = 3.3 kcal/mol

Ea = 5.2

Ea = 4.9

B

N

O

O

R

R

OB

N

O

O

R

R

HOH

HHO

H

HO

B

N

O

O

R

R

O B

N

O

O

R

R

HOH

HHOH

HOHO H

O HH

Ea = 26.0

Ea = 20.4

∆E = 7.7 kcal/mol

Ea = 10.8

Ea = 3.2

CH3 CH3

CH3CH3

B

NH

O

O

R

R

OB

NH

O

O

R

R

H3COH3C

HHO

H

HO

B

NH

O

O

R

R

O B

NH

O

O

R

R

H3COH3C

HHOH

HOHO H

O HH

Ea = 24.3

Ea = 16.5

∆E = -0.5 kcal/mol

Ea = 4.7

Ea = 5.3

B

NH

O

O

R

R

OB

NH

O

O

R

R

HOH

HHO

CH3

H3CO

B

NH

O

O

R

R

O B

NH

O

O

R

R

HOH

HHOH3C

H3COHO H

O HH

Ea = 6.1

Ea = -0.3

∆E = 0.5 kcal/mol

Ea = 5.8

Ea = 5.3

1.7 Activity-based profiling (Ben Cravatt, Scripps)

In collaboration with the Cravatt lab, a small library of boron containing compounds (see Appendix

A) were sent to Scripps for activity-based profiling. Positive results were obtained for 3

compounds: either diastereomer of the boromorpholinone and the boryl tetrazole. Both

diastereomers of the boromorpholinone inhibited ADEP (an acyl amino acid releasing enzyme) in

cells from mouse spleen, liver and testes. The boryl tetrazole inhibited an unidentified serine

hydrolase with a molecular weight of approximately 26 kDa. Ecouraged by these results, we sent

a second small library for analysis (Appendix A).

2.6.3 Cell permeability (Ahmed Aman, OICR)

Bortezomib 0 5.2 Low >10 High 28 MIDAbortezomib 0 5.2 Low >10 High 50 MIDAbortezomib* 0 4.1 Low >10 High 33 BoromorpholinoneAZ1 7.7 9.5 Moderate 1.2 Negative 55 Digoxin(P-gpcontrol) 0.1 11.2 Low >10 High 73 Atenelol(negcontrol) 0.3 0.3 Low 1.1 N/A 93 Metoprolol(Poscontrol) 13.8 10.5 High 0.8 Negative 70 *monitoringforBortezomib

21

The similarity in potency and selectivity of 6-boromorpholinone AZ-1 and MIDA-

bortezomib 7a compared to bortezomib led us to question whether their structural differences

might give rise to differences in cell permeability. We subjected each of these compounds to a

Caco-2 screening assay[22] and found that AZ-1 exhibited greatly improved cell permeability

compared to both MIDA-bortezomib 7a and bortezomib. The latter two yielded similar cellular

permeability (see Supporting Information). In a control experiment, we found that in the absence

of cells and cell lysates, MIDA-bortezomib is partially hydrolyzed to the free boronic acid when

incubated at 37°C in buffered aqueous solution (see Supporting Information). We therefore

attribute the results of both the 20S proteasome inhibition and Caco-2 assays for this compound to

its buffer-mediated hydrolysis forming the active species during the assay.

22

1.8 Chiral Variants

Our boron methodology thus far has lacked a straightforward means of preparing chiral variants

of our reagents. Use of the pinene-derived MIDA ligand (PIDA) was met with inconsistent and

limited success. We therefore set out to develop other chiral MIDA derivatives for the preparation

of enantioenriched boron-containing building blocks. We previously hypothesized that the

instability of PIDA-boryl aldehydes and isocyanates was due to the bulk of the chiral N-alkyl

substituent. Thus a derivative might gain stability by shifting the steric bulk away from nitrogen.

We opted to derive chirality from readily available amino-acid starting materials. Proline seemed

to be an ideal retrosynthetic target as its chirality is conformationally locked and its nitrogen is

already disubstituted, which allowed a one step alkylation from proline to the N-alkylated

derivative using bromoacetic acid.[xxiii] (Scheme 10).

Scheme 10. Preparation of N-carboxymethyl proline

NH

O

OH +O

OHBrNaOH (pH 12)

H2O, 0°C to rt, 4h

N

O

OH

N-COMP

O

OH

23

We then attempted to ligate N-COMP to commercially available vinyl boronic acids using the

standard procedure for MIDA boronates. Initially, trans-2-cyclohexylvinylboronic acid was used,

however overlap of the pyrrolidine and cyclohexyl methylene groups made analysis of the

epoxidized product difficult (Scheme 11). At first glance, the reaction appeared to product a single

diastereomer by 1H NMR; however, upon rearrangement to the aldehyde, the relative integrations

of the two aldehyde H (crude 1H NMR) signals indicated a 34:66 diastereomeric ratio. The spots

for the two diastereomers do not separate on TLC. I therefore prepared the p-tolyl substrate to get

a better understanding of the stereoselectivity of the epoxidation reaction (Scheme 3). Under

standard conditions the epoxidation produced a mixture of epoxide diastereomers; however upon

work up, complete conversion to the aldehyde occurred with a diastereomeric ratio of 9:1.

Lowering the temperature to –30°C resulted in only one observable diastereomer by 1H NMR. It

is possible that during the epoxidation work-up, Brønsted acid catalyzed rearrangement occurs

due due to the presence of the carboxylic acid byproduct of the epoxidizing agent. This

rearrangement would be stereospecific due to the nature of the epoxide-opened intermediate. This

would explain the difference in stereoselectivity between the reaction and work-up steps.

24

Scheme 11. Preparation of cyclohexyl N-(COMP) boryl aldehyde

With proof-of-concept in hand we chose the n-Bu compound for its relatively low steric

size and relatively high conformational freedom. These traits usually rank compounds low in terms

of stereoselectivity and tailoring a method to suit their needs results in improved horizontal

scalability. Oxiranyl-COMP boronates were prepared analogously to their MIDA cousins. The

reactions were monitored by 1H NMR which may have provided inaccurate results for low

temperature data points as the reaction may have gone to completion during the time between

transfer from the reaction vessel and quenching. Better yields and selectivities were observed using

THF as the solvent. Table 4. Reaction condition screening for preparation of enantiopure boryl

aldehydes summarizes the results and highlights the importance of reaction time. Longer reaction

times under identical conditions result in progressive stereodegradation, which supports the

hypothesis of a byproduct-catalyzed epimerization,

Cy B(OH)2

COOH

N COOHtoluene, DMSO

130°C, o/nB O

O

N

OO

Cy

mCPBADCM, 0°C to 23°C

1hB O

O

N

OO

CyO

90% isolatedappears to be >95:5 dr

DCM, -30°C to 0°C1h

BO O

NOO

O

97% cruded.r. 34:66

BF3·Et2O

25

Table 4. Reaction condition screening for preparation of enantiopure boryl aldehydes

Entry Solvent[a] Temp (°C) Time Conversion d.r.[b]

1 DCM –40 to 23 5 min[c] 100%[c] 70:30

2 DCM –30[d] to 23 1 h 100% 75:25

3 DCM –78 to –30 30 min 100% 65:35

4 Et2O[e] –100 to 23 6 h 100% 70:30

5 THF -100 to 23 1 h 15% >95:5

6 THF -100 to 23

3 h @ 23°C ~ 70% >95:5

o/n @ 23°C 100% 85:15

[a] All solvents were distilled anhydrously prior to use, all reactions were run at 0.02 M

[b] Determined by integration of 1H NMR aldehyde signals

[c] 100% conversion (1H NMR) after 5 min (temperature –25°C)

[d] BF3·Et2O added over 1 h as 0.2 M solution in DCM

[e] SM not soluble, 0% conversion until reaction mixture sonicated at 23°C

B OO

N

OOn-Bu

OB OO

N

OOn-Bu

mCPBADCM, 0°C to 23°C

o/n1 2

BO ON

OO

n-BuO

BF3·Et2O

Conditions

26

Scheme 12.Oxidation and Curtius rearrangement of COMP boryl aldehydes

BO ON

OO

OnBu

BO ON

OO

COOHnBu

BO ON

OO

NCOnBu

NaClO2, NaH2PO4cyclohexenetBuOH, H2O23°C, 2.5 h

97%, with retention

DPPA, Et3NMeCN, 50°C, 1h

67%, with retention

(9:1 dr) (9:1 dr) (9:1 dr)

27

Chapter 2

Experimental

2.1 General Information

NMR spectra were recorded at r.t. on Bruker Advance III 400 and Varian Mercury 400

instruments. 13C NMR spectra were registered with broad-band decoupling. Recorded shifts for

protons are reported in parts per million (δ scale) downfield from tetramethylsilane and are

referenced to residual protium in the NMR solvents (CDCl3: δ 7.26, DMSO-d6: 2.54 or MeCN-d3:

δ 1.94, centre line). Chemical shifts for carbon resonances are reported in parts per million (δ scale)

downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent

(CDCl3: δ 77.16, DMSO-d6: 40.45 or MeCN-d3: δ 1.32, centre line). 11B NMR was recorded at r.t.

on a Bruker Advance III 400 MHz spectrometer and referenced to an external standard of

BF3·Et2O. Data are represented as follows: chemical shift δ in ppm, multiplicity (s singlet, d

doublet, t triplet, q quartet, m multiplet, br broad), coupling constant J in Hz and integration.

Flash column chromatography was carried out using Silicycle 230-400 mesh silica gel. Thin-layer

chromatography (TLC) was performed on Macherey Nagel pre-coated glass backed TLC plates

(SIL G/UV254, 0.25 mm) and visualized using a UV lamp (254 nm) or KMnO4 or Curcumin stain

in case of no UV activity.

Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum 100 instrument equipped with a

single-reflection diamond/ZnSe ATR accessory.

Anhydrous solvents were obtained by distillation under nitrogen prior to use. Dichloromethane

was purified using an MBraun Solvent Purification System. All other solvents were of reagent

grade quality. All reagents, catalysts and ligands were purchased from Combi-Blocks Inc., Sigma-

Aldrich, Strem-Chemical Company, VWR International and used as received unless otherwise

noted.

It should be noted that for compounds that were prepared as part of routes that were ultimately

unsuccessful or for use by other group members/supervisors, full characterization and/or

28

experimental details may not be provided below. Furthermore, intermediates used directly in

subsequent reactions without purification are characterized by crude 1H NMR only.

2.2 Synthesis of α-boryl isocyanides

To a flame-dried flask flushed with Ar was added freshly distilled DCM (10 mL) which

was cooled to 0°C. Trichlorosilane (4.86 mmol, 0.5 mL, 1.3 eq.) was added followed by dropwise

addition of triethylamine (11.22 mmol, 0.82 mL, 3.0 eq.). Cyclohexyl substituted α-boryl

isocyanate (3.74 mmol, 1.1 g, 1.0 eq.) was added and the resulting suspension was stirred

vigorously at 0°C for 30 minutes. The resulting solution was allowed to warm to rt. Stirring was

continued until starting material was completely consumed as indicated by TLC or crude 1H NMR.

The reaction solution was cooled to 0°C and satd aq. NaHCO3 (10 mL) was added very slowly.

The resulting suspension was stirred at 0°C until bubbling ceased at which point it was allowed to

warm to rt. The layers were separated and the aqueous layer was washed with EtOAc. The

combined organic layers were washed with satd aq. NaHCO3/H2O (50/50) and satd aq.

NaHCO3/brine (50/50), dried over Na2SO4, filtered and concentrated. The crude residue was

purified by flash column chromatography on neutralized silica (slurry prepared in hexanes/Et3N

95:5, eluent; EtOAc/Et3N 95:5 → EtOAc/MeCN/Et3N 70:25:5) to yield the desired product.[xxiv]

Additiona of several extra equivalents of triethylamine once the reaction reaches completion prior

to addition of satd NaHCO3(aq.) ensures that the pH remains above 7. Performing the quench

under a heavy stream of N2 facilitates removal of Et3NHCl formed via hydrolysis of HSiCl3 which

is otherwise challenging to remove during extraction. Addition of small amounts (~2-3 vol%) of

Et3N to all organic phases during extraction improves product recovery by marginalizing

hydroloysis of the isocyanide.

R

B(MIDA)

NCO

HSiCl3, Et3N

DCM, 0°C -> rt5 - 24h R

B(MIDA)

NC30 - 75%

3 examples

29

2.3 Deuterium Exchange Experiments A 5 mg sample of the isocyanide (AZ-2-96A1) was dissolved or suspended in 0.5 mL of solvent.

Base was added and the tube was shaken for 30 seconds. The 1H NMR spectrum was measured

within 10 minutes.

Base Solvent Result

Et3N (10 Eq.)* D2O

No visible decrease in α-proton integration Et3N (10 Eq.) D3COD

NaHCO3 (15 Eq.) D2O *MIDA Removed 1H NMR Spectra obtained for these experiments are included below.

2.4 Attempted α-deprotonations of α-boryl isocyanides

2.4.1 Accidental MIDA tetradeuteration

A suspension of the isocyanide (10 mg, 0.040 mmol) in anhydrous DCM (0.1 mL) was cooled to

-78°C in a flame-dried flask under Ar. To the mixture was added KOtBu (5.2 mg, 0.044 mmol)

and the resulting solution was stirred at -78°C for 30 minutes. To the solution was added MeI (2.49

µL, 0.40 mL) and the reaction mixture was stirred at -78°C for 30 minutes and at r.t. for 1.5 h after

1) KOtBu, DCM-78°C, 30min2) MeI-78°C (30min) -> rt (1.5h)

B

NCO

HN

OOOB

NCO

HN

OOO

D D D DHH H H

30

which the solvent had evaporated. To the resulting residue was added MeCN-d3 and 1H NMR was

taken.

2.5 Other functionalizations of α-boryl isocyanides

2.5.1 Preparation of α-boryl tetrazole

To a solution of the isocyanide (50 mg, 0.20 mmol) in TFE (0.5 mL) was added HCl (400 µL, 4.0

µmol, 2.0 mol%) and TMSN3 (40 µL, 0.30 mmol). The vial was sealed and heated to 60°C with

stirring for 2.5h at which point TLC indicated that the reaction had gone to completion. The solvent

was removed and the crude product was purified via flash column chromatography using

EtOAc/MeCN 1.0 → 8:2 as an eluent. The purified product was isolated as an off-white solid (46

mg, 78%).

2.5.2 Preparation of α-boryl isothiocyanate

Elemental sulfur (15 mg, 0.48 mmol) and selenium (2.0 mg, 0.020 mmol, 5 mol%) were suspended

in THF (0.8 mL) and Et3N (134 µL, 0.96 mmol) was added. The isocyanide (100 mg, 0.40 mmoL)

was added, the vial was sealed and the resulting suspension was stirred at 80°C for 15 min at which

point TLC indicated that the reaction had gone to completion. The solution was allowed to cool to

r.t., filtered (rinsing with EtOAc) and concentrated. The crude product was purified via flash

column chromatography using hexanes/EtOAc (6:4 → 1:0) and was isolated as an off-white solid

B(MIDA)

NC

TMSN3HCl (cat.)

Et2O/MeOH60°C, 6.5h

B(MIDA)

NHC N

NN

B(MIDA)

NC

S, Se (cat.)

THF, 80°, 15 min

B(MIDA)

N CS

31

(88 mg, 77%).

2.5.3 Preparation of α-boryl thioureas

To a solution of the isothiocyanate (45 mg, 0.158 mmol) in THF (0.5 mL) was added amine (0.32

mmol) and the resulting solution was stirred at r.t. until TLC or LRMS indicated that the reaction

had gone to completion. The solvent was removed and the crude product was purified via flash

column chromatography using a gradient of hexanes/EtOAc (1:0 → 0:1) as the eluent. purified

products were isolated as off-white solids. When 3,5-bis(trifluoromethyl)aniline was used as the

amine, only starting material remained after 2 days of heating at 50°C. Carrying out the reacoin

with dry solvents and reagents (triethylamine and THF) under inert atmostphere results in greater

selectivity for the desired isothiocyanate over byproducts. Excess sulfer and selenium can be

filtered to provide nearly quantitative yields of the desired product in acceptable purity.

B(MIDA)

N CS RNH2

THF, rt9-24 h

B(MIDA)

NH

S NH

R

32

Passerini 3-Component Reactions

3.1 General preparation of N-(MIDA boromethyl)-α-acyloxyamides

To a 10 mL vial charged with α-boryl isocyanide (0.20 mmol), phenyl acetic acid (27 mg, 0.20

mmol) and aldehyde (0.20 mmol) was added DCM (0.5 mL) and the vial was sealed. The resulting

suspension was stirred magnetically at r.t. until the reaction was complete as indicated by TLC.

The crude product mixture was pipetted directly onto silica gel (4 g, as a slurry in hexanes) and

the product diastereomers were eluted using a hexanes/EtOAc gradient (1:0 → 0:1). In some cases,

EtOAc/MeCN (8:2) was required for complete elution of the product. In most cases, TLC indicated

complete separation of product diastereomers after column; however, 1H NMR indicated trace

amounts of either diastereomer in each sample. Diastereomeric ratios were determined using 1H

NMR proton integrations of the N-Me signal. It should be noted that reactions utilizing

nicotinaldehyde were carried out in the dark until TLC indicated full consumption of starting

material. For cases in which the relative stereochemistry has not yet been assigned, the

placeholders “A” and “B” are used and represent the diastereomers which eluted first and second

from the column, respectively. The 1H NMR spectra for these diastereomers have been arbitrarily

labelled with either one. This will be remedied when their relative stereochemistries have been

elucidated.

3.2 Determination of P3CR Product Relative Stereochemistry

Using GaussView 5.0.9 as a computational engine, the structures of AZ-4-188 and AZ-4-

189 were built (both the syn and anti diastereomers of each) and the optimization + frequencies

calculation was carried out using the following parameters: Theory: DFT, Basis Set: B3LYP, 6-

Ph COOH

DCM, 2-7dNH

R' OO

O

OPh

R'

BO OMeN

OO

RB

O OMeNOO

NCRNH

OO

OPh

R'

BO OMeN

OO

R

+

33

31G(d), additional keyword: “scrf=(solvent=acetonitrile) geom=connectivity”. Using the

optimized structures from these calculations, the NMR calculation was carried out using the giao

method and the following computational parameters: Theory: DFT, Basis Set: RMPW1PW91, 6-

311G(2d,p), additional keyword: “scrf=(solvent=acetonitrile) guess=input geom=connectivity”

The isotropic Eigenvalues for the chemical shifts of the protons of interest were recorded in Table

1 (Supporting Information) along with their scaled values and the corresponding experimental

values. The computational isotropic Eigenvalues were subject to scaling factors developed by

Tantillo, et al. (slope: -1.0823, intercept 31.8486). The trends are visualized as follows: when

comparing analogous protons between product diastereomers, the more upfield proton is

highlighted in red while the more downfield proton is highlighted in blue.

3.3 Deprotection of P3CR Products / Discovery of Boromorpholinones

See Boromorpholinones.

3.3.1 Assay yield and calibration curve

AZ-4-160 (3.46 mg) was dissolved in DMSO (136.2 µL) to make a 50 mM stock solution.

DMBA (11.66 mg) was dissolved in DMSO (782 µL) to make a 50 mM stock solution. To 998 µL

of MeCN was added 2 µL of the 50 µL AZ-4-160 stock (100 µM). This solution was subject to

dilution into an appropriate amount of MeCN with10 µL of 50 mM DMBA stock to a final volume

of 1 mL. Each solution was subject to reverse-phase LCMS (5 µL injection, gradient of

MeCN/H2O 5:95 to 95:5 (plus 0.1% formic acid)) with two single-ion monitoring channels (one

for DMBA (m/z = 150.2) and one for AZ-4-160 (m/z = 509.2)). The integration of the P3CR

product peak (relative to the DMBA standard) was plotted against the concentration and a linear

fit applied. Response = 0.1554[AZ-4-160]; R2 = 0.9402.

34

To a solution of phenylacetaldehyde (79 µL, 0.67 mmol, 1.0 equiv.) and phenylacetic acid

(92 mg, 0.67 mmol, 1.0 equiv,) in MeCN (1.7 mL) was added isobutyl α-boryl isocyanide (170

mg, 0.67 mmol, 1.0 equiv.) and the reaction mixture was stirred under microwave conditions

(100°C) for 25 min at which point TLC indicated complete consumption of the isocyanide starting

material. The level of solvent did not decrease in the microwave vial. 2 µL of the reaction mixture

was added to DMBA stock (2 µL) and MeCN (978 µL) and the resulting solution was subject to

the same LCMS conditions used to construct the calibration curve (above). Interpolation of the

calibrated response for the reaction mixture indicated a 9.0% yield of the desired product. The

remaining reaction mixture was concentrated and subject to flash column chromatography

(hexanes/EtOAc 8:2 to 0:1) to afford the desired diastereomeric products (272 mg, 79%). 1H NMR

indicated only a small amount of impurities present in the purified products.

3.4 VT NMR experiments

I collected the 1H NMR (400 MHz, MeCN-d3) spectra of AZ-5-282A at 25, 40, 50, 60, 70 and

80°C. See NMR section.

3.5 Calibration Curve

Using purified P3CR product anti-AZ-4-160 a series of solutions from 5 to 100 µM in MeCN

(each containing 500 µM N,N-dimethylbenzamide (DMBA)). Plotted below is the calibrated

LCMS response (product signal / DMBA signal) as a function of cocentration. Attempts to monitor

the P3CR reaction which produces AZ-4-160 resulted in assay yields which were paradoxically

low compared to the isolated yields after column. Boron-containing compounds in general behave

BO OMeN

OO

NC

BO OMeN

OO

NH

O

O

OPh

Ph

PhO

Ph COOH

MeCN (0.40 M)100°C (microwave), 25 min

35

poorly on reverse-phase LCMS in our hands. Calibrated NMR yield is therefore recommended and

a verified method is detailed below.

3.6 NMR Yield

3,4,5-triiodobenzoic acid was chosen as an internal standard for several reasons: a) its electron-

poor nature shifts the aromatic proton signal downfield (> 8ppm) making it less likely to overlap

with product signals; b) the symmetry and substitution pattern of the compound means it will only

have one aromatic signal and it will be a singlet (more accurate and consistent integration); c) its

acidic nature makes it easily removable from organic solvents by washing with satd NaHCO3(aq.);

Note that this compound in particular was chosen because it was immediately available. It is

recommended that , if other options exist, a less reactive species (such as the tri-chloro analog) be

used. By comparative integration of the external standard signal with the N-Boc t-Bu signal of the

boropeptide products the concentration of the product can be calculated.

36

Ugi 4-Component Reactions

4.1 Proline Ugi Reaction

To a solution of L-Proline (23 mg, 0.20 mmol) in MeOH (2 mL) at -40°C was added boryl

isocyanide (50 mg, 0.20 mmol) and isobutyraldehyde (18 µL, 0.20 mmol). The resulting mixture

was stirred for 4 hours at -40°C followed by 15 hours at r.t. until the reaction was complete as

indicated by TLC. The solvent was removed and the the crude was suspended in EtOAc and

washed with 5% aqueous NaHCO3. The aqueous layer was washed twice with EtOAc and the

combined organic layers were washed with brine, dried over Na2SO4 filtered and concentrated.

Attempted purification of the product on silica gel (EtOAc/MeCN/Et3N 16:4:1 →10:10:1) failed

as all spots were eluted in the first several fractions. Successful purification of the product was

achieved using preparative TLC with EtOAc/MeCN (1:1) as the eluent. The product was isolated

as a light orange oil (an inseparable 10:6:4:3 mixture of 4 diastereomers) (6.2 mg, 7%). This

procedure was adapted from one developed by I. Ugi et. al.

4.2 Preparation of MIDA-Velcade®

To a solution of the carboxylic acid (25 mg, 0.20 mmol) was added ammonia (28.3 µL,

0.20 mmol, 7.0 N solution in MeOH) and the resulting mixture was stirred at r.t. for 10 minutes.

B(MIDA)

NCHN

COOH

O

MeOH-40°C (3h) -> rt (15h)

B(MIDA)

NH

ONMeOOC

N

N COOH

Ph CHO

H3N

TFE, 23°C, 3dBO OMeN

OO

NC2a

BO OMeN

OO

NH

7a

O HN

Ph ON

N

37

The aldehyde (23.1 µL, 0.20 mmol) and the isocyanide (50 mg, 0.20 mmol) were added and the

resulting mixture was stirred at r.t. for 3 days at which point TLC indicated that the reaction had

gone to completion. The reaction mixture was concentrated and the crude product was purified via

flash column chromatography on silica gel (neutralized with hexanes/Et3N (95:5)) using

EtOAc/MeCN/Et3N (20:0:1 → 16:4:1 → 10:10:1 → 4:16:1). To afford the desired product as an

off-white solid (54 mg, 55%).

38

MIDA-boropeptides

5.1 Preparation (Jen Hickey:) Commercially available N-Boc protected amino acids were used as supplied.

Fully protected resin-bound tri- and tetra-peptides H-Pro-Leu-Phe-OH and H-Pro-Gly-Leu-Phe-

OH were synthesized via standard Fmoc solid-phase peptide chemistry using an automated peptide

synthesizer. Fmoc removal was achieved by treatment with 20% piperidine in NMP for 5 and 10

minutes with consecutive DMF and NMP washes after each addition. For all Fmoc amino acid

coupling, the resin was treated once with 4.5 eq. of Fmoc amino acids, 4.5 eq. of HCTU and 9 eq.

of DIPEA in NMP for 60 minutes. Once the peptide was synthesized, following Fmoc removal,

the resin was treated with 1:3, HFIP:DCM, twice for one hour each, to afford cleavage from the

solid support. The solvent was then removed, followed by trituration with tert-butyl methyl ether

to give the linear peptide. (Adam Zajdlik:) The peptide products were then Boc-protected along

with commercially available H-Phe-Ala-OH and H-Gly-Gly-OH according to the following

method:

A solution of the peptide (1.0 equiv.) in THF (0.75M) and NaOH(aq.) (1.0 M, 2.0 equiv.)

was cooled to 0°C and Boc2O was added slowly. The resulting mixture was stirred at 0°C for 30

minutes and was then allowed to warm to 23°C. Once TLC indicated that the reaction had gone to

completion, the solution was cooled to 0°C and acidified to pH ~ 2 with 1.0 M HCl(aq.). Et2O was

added and the layers were separated. The aqueous layer was washed twice with Et2O and the

combined organic layers were dried over MgSO4, filtered and concentrated to yield the crude

products as white solids. The products were identified with LRMS (ESI, positive) and crude 1H

NMR and used in the Ugi 4-component reaction without further purification.

H2N-Peptide-COOH

Boc2O, NaOH

THF/H2O0°C to 23°C

Boc-NH-Peptide-COOH

39

5.2 Preparation, purification and characterization

To a solution of the crude N-Boc protected peptide (1.0 equiv.) in TFE (0.50 M) was added

ammonia (1.5 equiv., 7.0 N solution in MeOH) and the resulting mixture was stirred at 23°C for

10 minutes. The aldehyde (1.0 equiv.) and α-boryl isocyanide 2a (1.0 equiv.) were added and the

resulting mixture was stirred at 23°C until TLC indicated that the reaction had gone to completion

(6-12 d). The reaction mixture was concentrated and a small sample (5-15 mg) was taken for crude 1H NMR using 750 µL of a stock solution of 3,4,5-triiodobenzoic acid (48.1 mM in DMSO-d6) as

the solvent. The yield was calculated by comparative integration of the Boc tert-butyl signal and

the aromatic protons of the internal standard. The crude product was purified via reverse-phase

preparative high-performance liquid chromatography on an Agilent ZORBAX SB-C18 column (5

µM mesh, 9.4 x 250 mm) using a H2O-MeCN (with added 0.1% formic acid) gradient to afford

the desired products. Diastereomers are classified by their order of elution in reverse-phase

chromatography (A eluting first being the most polar, B eluting second being less polar than A,

and so on for C and D if applicable.)

5.3 Determination of boropeptide yield using 1H NMR and external standard

After the preparation of crude boropeptide (above) was complete, the reaction mixture was

concentrated and weighed. A 5-15 mg portion of the crude product was weighed into an NMR tube

and 3,4,5-triiodobenzoic acid was added (0.48 µM in DMSO-d6, 750 µL). The 1H NMR spectrum

was taken within 30 minutes. The yield was calculated by comparative integration of the aromatic

signal of the standard and the tBu signal of the product N-Boc group. In some cases the leucine

(Boc-N)-Peptide-COOHR O + NH3

TFE, 23°C NH

OHN

R

(Boc-N)-Peptide

O

9

BO OMeN

OO

NC2a

BO OMeN

OO

40

fragment methyl groups were used as the tBu signal overlapped with other signals.

41

Boromorpholinones

6.1 General preparation of 6-boromorpholin-3-ones

To a small vial charged with P3CR product AZ-3-160B (50 mg, 0.10 mmol) was added THF (1.5

mL) and NaOH (1.0 M (aq.), 0.40 mL, 0.40 mmol) and the resulting mixture was magnetically

stirred at r.t. for 10 minutes at which point TLC indicated that the reaction had gone to completion.

To the reaction mixture was added 1.5 mL of pH 7 aqueous phosphate buffer and 1.5 mL Et2O.

The layers were separated and the aqueous layer was washed 3 times with THF/Et2O (1:1). The

combined organic layers were washed with brine, dried over Na2SO4 and filtered. 2 mL 1,4-

dioxane was added and the solution was concentrated via rotary evaporation without submersion

into a water bath to remove only the THF and Et2O. The 1,4-dioxane was lyophilized to afford the

desired product as a white powder in 79% crude yield.

6.2 pKa determination To a solution of AZ-4-180 (25.4 mg, 0.097 mmol) in 4.1 mL of CD3OD was added 1.2 mL of

HEPES buffer (0.10 M in D2O). The pH was adjusted to 3.2 by addition of 2.0 M HClO4(aq.). A

250 µL aliquot was taken for 11B NMR. LRMS (ESI) showed this sample contained a mass

corresponding to the monodeuterated borocycle shown in Scheme 1. The pH was increased by ~

1 pH unit by addition of 2.0 M NaOH(aq.) until a pH of ~ 12 was reached. At each pH, a 250 µL

sample was taken for 11B NMR. LRMS (ESI) of the sample with pH 12.3 showed a mass

corresponding to the pentadeuterated boronate shown in Scheme 1. The 11B NMR spectra were

taken with a sweep width of 51000 Hz, 131000 data points, 90° pulse width, 1.2 second recycle

time, 10 Hz line broadening and a 2nd order polynomial fitting routine. This method was adapted

from a procedure developed by Eric Anslyn et al.

B(MIDA)

NH

O O

Ph

NaOHTHF/H2O10 min

NH

BHO O Ph

OO

Ph

+ Ph COOH

42

6.3 Assay yield and calibration curve

AZ-4-160 (3.46 mg) was dissolved in DMSO (136.2 µL) to make a 50 mM stock solution.

DMBA (11.66 mg) was dissolved in DMSO (782 µL) to make a 50 mM stock solution. To 998 µL

of MeCN was added 2 µL of the 50 µL AZ-4-160 stock (100 µM). This solution was subject to

dilution into an appropriate amount of MeCN with10 µL of 50 mM DMBA stock to a final volume

of 1 mL. Each solution was subject to reverse-phase LCMS (5 µL injection, gradient of

MeCN/H2O 5:95 to 95:5 (plus 0.1% formic acid)) with two single-ion monitoring channels (one

for DMBA (m/z = 150.2) and one for AZ-4-160 (m/z = 509.2)). The integration of the P3CR

product peak (relative to the DMBA standard) was plotted against the concentration and a linear

fit applied. Response = 0.1554[AZ-4-160]; R2 = 0.9402.

To a solution of phenylacetaldehyde (79 µL, 0.67 mmol, 1.0 equiv.) and phenylacetic acid

(92 mg, 0.67 mmol, 1.0 equiv,) in MeCN (1.7 mL) was added isobutyl α-boryl isocyanide (170

mg, 0.67 mmol, 1.0 equiv.) and the reaction mixture was stirred under microwave conditions

(100°C) for 25 min at which point TLC indicated complete consumption of the isocyanide starting

material. The level of solvent did not decrease in the microwave vial. 2 µL of the reaction mixture

was added to DMBA stock (2 µL) and MeCN (978 µL) and the resulting solution was subject to

the same LCMS conditions used to construct the calibration curve (above). Interpolation of the

calibrated response for the reaction mixture indicated a 9.0% yield of the desired product. The

remaining reaction mixture was concentrated and subject to flash column chromatography

(hexanes/EtOAc 8:2 to 0:1) to afford the desired diastereomeric products (272 mg, 79%). 1H NMR

indicated only a small amount of impurities present in the purified products.

BO OMeN

OO

NC

BO OMeN

OO

NH

O

O

OPh

Ph

PhO

Ph COOH

MeCN (0.40 M)100°C (microwave), 25 min

43

6.4 20S proteasome inhibition assays

6.4.1 MIDA-bortezomib (AZ-4-285A) Solutions of AZ-4-285 (each diastereomer) and bortezomib were prepared by serial dilution of 10

mM stocks in DMSO. To a feshly prepared sample of OCI-AML-2 human leukemia cells was

added 5 mL of freshly prepared lysis buffer containing 50 mM pH 7.5 HEPES buffer, 150 mM

NaCl, 1% Trition X-100 and 2 mM ATP. The cells were suspended by pipetting up and down

several times and were vortexed every 5 minutes for 30 minutes at 0°C. Each well of a 96 well-

plate was loaded with 87 µL of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCl

buffer, 150 mM NaCl and 2 mM ATP), 10 µL of cell lysate solution and 1 µL of each stock solution

of either AZ-4-285 or bortezomib (to final concentrations of 10 µM to 10 pM, in 1/10th dilution

increments). The resulting solutions were incubated at 37°C for 1h. To each well was added 2 µL

of 3.75 mM N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin in DMSO. The

fluorescence spectrum of each well was measured at 5 minute intervals over 30 minutes at 37°C

(using a Spectromax spectrometer by Molecular Devices, excitation: 360 nm; emission 460 nm).

The slope of the increase in fluorescence vs. time (converted to a percent of the slope for a blank

sample) was plotted against the inhibitor concentration (Figures 1-2). The IC50 of each of each

diastereomer of AZ-4-285 and bortezomib was calculated by applying a sigmoidal fit to each curve

shown in Figures 1-3 and interpolating to 50% enzyme activity. In each assay, rates were measure

in triplicate and averaged. Error bars represent 1 standard deviation. The IC50 values are tabulated

in Table 2.

44

6.4.2 boromorpholinone (AZ-4-180)

Solutions of AZ-4-180 and bortezomib were prepared by serial dilution of a 10 mM stock in

DMSO. To a feshly prepared sample of OCI-AML-2 human leukemia cells was added 5 mL of

freshly prepared lysis buffer containing 50 mM pH 7.5 HEPES buffer, 150 mM NaCl, 1% Trition

X-100 and 2 mM ATP. The cells were suspended by pipetting up and down several times and were

vortexed every 5 minutes for 30 minutes at 0°C. Each well of a 96 well-plate was loaded with 87

µL of freshly prepared assay buffer (containing 50 mM pH 7 Tris-HCl buffer, 150 mM NaCl and

2 mM ATP), 10 µL of cell lysate solution and 1 µL of each stock solution of either AZ-4-180 or

bortezomib (to final concentrations of 100 µM to 1 nM, in 1/10th dilution increments). The

resulting solutions were incubated at 37°C for 1h. To each well was added 2 µL of either 3.75 mM

N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin for the chymotrypsin-like assay, 3.75

mM t-butoxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin for the trypsin-like assay or 3.75

mM benzyloxycarbonyl-Leu-Leu-Glu-7-amino-4-methylcoumarin for the caspase-like assay (all

in DMSO). The fluorescence spectrum of each well was measured at 5 minute intervals over 30

minutes at 37°C (using a Spectromax spectrometer by Molecular Devices, excitation: 360 nm;

emission 460 nm). The slope of the increase in fluorescence vs. time (converted to a percent of the

slope for a blank sample) was plotted against the inhibitor concentration (Figures 1-3). The IC50

of each of AZ-4-180 and bortezomib was calculated by applying a sigmoidal fit to each curve

shown in Figures 1-3 and interpolating to 50% enzyme activity. In each assay, rates were measure

in duplicate and averaged. Error bars represent 1 standard deviation. Figure 4 shows a comparison

of the syn- and anti- isomers of AZ-4-180 in the chymotrypsin-like assay. It should be noted that

IC50 values obtained in the assays of syn- and anti-AZ-4-180 cannot be compared to those obtained

in the bortezomib comparison studies as different cell preparations were used. The IC50 values are

tabulated in Table 2. (in the figures below, 180 is referred to as 11ad).

Conclusion

45

The multi-component nature of our boropeptide preparation methodology facilitates diversity-

oriented synthesis by allowing addition of a second level of structural diversity in the same step as

boron integration. This facilitates the preparation of diverse inhibitor libraries and therefore

elucidation of a structure-activity relationship and optimization of a selective proteasome inhibitor.

In summary, we have demonstrated a straightforward deoxygenation of MIDA-protected α-

boryl isocyanates to yield the corresponding α-boryl isocyanides as bench-stable solids. The

isocyanide moiety participates in several functional group interconversions affording borylated

tetrazoles, isothiocyanates and thioureas. Isocyanide 2a also participates in several U4CRs

affording boropeptide derivatives including MIDA-Velcade®. As a component in P3CRs, the boryl

isocyanides gave rise to boryl acyloxyamide derivatives. One-pot deprotection and condensation

of the P3CR product 10ad gave access to a new class of boron-containing heterocycles (6-

boromorpholinones). These air-stable solids exhibited inhibition of chymotrypsin-like members of

the 20S proteasome with IC50s in the low nanomolar range. Computational analysis suggest that

these inhibitors are hydrolytically stable, a criterion required for clinical candidacy and one which

other inhibitors of this class often lack. They also exhibit improved cell permeability over the

current clinical standard. Preliminary efforts to develop an asymmetric route to these compounds

using a proline derived chiral auxiliary have yielded encouraging results. Given the step-economy

and experimental simplicity with which these inhibitors were obtained, we hope that this

methodology will be adopted for the efficient preparation of boropeptide libraries.

46

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49

Appendices

Appendix A. Compounds send to Cravatt lab, Scripps, La Jolla

Shipment 1:

BO OMeN

OO

PhO

BO OMeN

OO

NN N

NB

O OMeNOO

NH

NH

SB

NH

O

O

PhHO B

NH

O

O

PhHO

BO OMeN

OO

NH

O HN

OPh

N

N BO OMeN

OO

NH

O HN

OPh

N

N B

NH

O HN

OPh

N

N

AZ-4-167 (3.85 mg)295.11 g/mol

AZ-Velcade (3.12 mg)384.24 g/mol

OHHO

AZ-4-225 (2.68 mg)261.13g/mol

AZ-Ph-BA (5.40 mg)275.07 g/mol

AZ-4-219 (3.45 mg)261.13 g/mol

AZ-4-235 (4.59 mg)357.28 g/mol

AZ-5-285A (3.49 mg)495.34 g/mol

AZ-5-285B (2.54 mg)495.34 g/mol

50

Shipment 2:

BO OMeN

OO

NPh CB

O OMeNOO

BO OMeN

OO

COOH

AZ-1-55 (6.42 mg)289.09 g/mol

AZ-1-10 (6.22 mg)294.11 g/mol

AZ-2-72 (9.14 mg)288.07 g/mol

AZ-1-57 (16.20 mg)255.08 g/mol

AZ-4-260 (4.76 mg)271.08 g/mol

OPh O

BO OMeN

OO

O

BO OMeN

OO

N C O

BO OMeN

OO

NH

BO OMeN

OO

COOH

PTC17-01 (3.60 mg)297.11 g/mol

ZH0615502 (15.76 mg)326.17 g/mol

AZ-4-234 (8.77 mg)329.22 g/mol

PTC50-01 (4.04 mg)243.07 g/mol

BO OMeN

OO

OH

BO OMeN

OO

NH

NH

S

O

O

BO OMeN

OO

COOH

PTC13-02 (4.77 mg)305.09 g/mol

Ph

ZH0700601 (4.37 mg)346.15 g/mol

BO OMeN

OO

NH

Ph O

O

ZH0616801(6.14 mg)389.26 g/mol

BO OMeN

OO

NH

Ph NH

O

ZH06167A01 (3.65 mg)389.26 g/mol

BO OMeN

OO

NH

Ph N

O

51

BOO

NMe

OO O

O

BOO

NMe

OO O

N

O

O

BOO

NMe

OO O

BOO

NMe

OO N

O

OBO

O

NMe

OO N

O OPh

O

O OPh

BOO

NMe

OO

NN

NN Cl

BOO

NMe

OO

NN

NN N(Boc)2 BO

O

NMe

OO S

N

S

BOO

NMe

OO S

N NN

N

Ph

BOO

NMe

OO O

O

BOO

NMe

OO N

NN

BOO

NMe

OO I

SA02360 (5 mg)

SA02353 (5 mg)

SA02316PB (5 mg)SA02388 (5 mg)

SA02371 (5 mg)

SA02377PA (5 mg)

SA02376 (5 mg)

SA02368 (5 mg)

SA02366 (5 mg)

SA02319 (5 mg)

SA02394 (5 mg)

SA02338 (5 mg)

BOO

NMe

OO OH

ZH07158 (5 mg)

Molecular Weight: 291.0644 Molecular Weight: 317.1016 Molecular Weight: 332.0732

Molecular Weight: 263.0543 Molecular Weight: 316.0738 Molecular Weight: 442.1839

Molecular Weight: 323.5001 Molecular Weight: 504.3014 Molecular Weight: 336.1943

Molecular Weight: 347.1574 Molecular Weight: 211.9711 Molecular Weight: 296.8554

Molecular Weight: 186.9583

52

Appendix B. Attempted U4CRs using simple and OTBDMS aziridine aldehyde dimers

To a solution of L-Phe (50 mg, 0.20 mmol, 1.0 equiv.) and TBDMSO dimer (prepared by Sean,

52 mg, 0.12 mmol, 0.6 equiv.) was added α-boryl isocyanide (50 mg, 0.20 mmol, 1.0 equiv.)

and the resulting solution was stirred at rt for 4 h at which point TLC indicated complete

consumption of the isocyanide. Attempted purification of the intermediate after this step

resulted in an inseparable mixture of diastereomers the 1H NMR of which could not be

interpreted. The reaction was repeated and thiobenzoic acid (118 µL, 1.0 mmol, 5.0 equiv.)

was added directly after the first step. The reaction mixture was stirred for 3 h until ESI MS

indicated complete consumption of the previous intermediate. RaNi (1.0 mL, slurry in H2O)

was added and the resulting mixture was stirred vigorously at rt overnight. ESI MS indicated

that the ring-opened intermediate was still present. A balloon of H2 gas was bubbled through

the reaction mixture and the resulting mixture was stirred at rt for 25 hours at which point ESI

MS indicated complete consumption of the previous intermediate. The resulting product was

purified by flash column chromatography using hexanes/EtOAc gradient. The isolated product

(22 mg, 21%) was characterized by various 1D and 2D NMR experiments.

NC

B(MIDA)

iBu

1) TBDMSO-dimer, L-PheTFE, 23°C, 4 h

2) Thiobenzoic acid, 3 h3) RaNi, o/n4) H2, 25 h

HNNH

O

Ph

NH2O

PGO

SPh

O

BO OMeN

OO

NC

O

NHN

OHL-Phe1)

TFE, 23°C, 7h

2) thiobenzoic acid, o/n

HNNH

O

Ph

NHO

BO O

MeN

OO

S

O

Ph

53

MIDA (1-((2R,3R,6S)-3-((benzoylthio)methyl)-6-benzyl-5-oxopiperazine-2-carboxamido)-3-methylbutyl)boronate (AZ-5-366)

To a solution of simple aziridine aldehyde dimer (34 mg, 0.24 mmol) and L-Phe (66 mg, 0.40

mmol) in 2,2,2-trifluoroethanol (1.2 mL) was added boryl isocyanide (100 mg, 0.40 mmol) and

the resulting suspension was stirred at 23°C for 7h at which point TLC indicated complete

consumption of the isocyanide starting material. Thiobenzoic acid (236 µL, 276 mg, 2.00 mmol)

was added and the resulting solution was stirred overnight at which point LCMS indicated that the

aziridine intermediate (m/z = 471) was consumed. The crude mixture was concentrated and taken

up into EtOAc and satd NaHCO3(aq.). The aqueous layer was washed twice with EtOAc and the

combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated.

The crude mixture was purified by flash column chromatography on silica gel eluting with

EtOAc/MeCN (1:0 to 6:4) to afford the desired diastereomeric products.

Appendix C. Serendipitous discovery of α-boryl carbamoyl azide

To a solution of the α-boryl carboxylic acid (750 mg, 2.58 mmol) in Et3N (313 mg, 0.43 mL, 3.09

mmol) and anhydrous MeCN (25 mL) was added diphenylphosphorinoazide (DPPA) (851 mg,

0.67 mL, 3.09 mmol) dropwise via a syringe at r.t. under N2 atmosphere. The resulting mixture

was heated to 50°C with magnetic stirring for 2 h at which point TLC indicated that the reaction

had gone to completion. The solvent was removed from the reaction mixture via rotary evaporation

and the resulting residue was purified via flash column chromatography (Hexanes/EtOAc 9:1 ➝

4:1 ➝ 1:1 ➝ EtOAc) to yield 125 mg (15%) of the desired product as a white solid.

Recrystallization was achieved by dissolving the product in a minimal amount of acetone and

layering the solution with hexanes in an NMR tube at rt.

To a solution of the azide (30 mg, 0.091 mmol) in dry THF (0.5 mL) was added CuI (1.0 mg, 4.6

Ph COOH

B(MIDA)DPPA, Et3N

MeCN, 50°C, 2h Ph NH

B(MIDA)

N3O

Ph NH

B(MIDA)

N3O

IPhEt3N, cat. CuI

THF, 2 wksPh NH

B(MIDA)

NO NN

PhI

54

µmol) and Et3N (18 mg, 25 µL, 0.181 mmol). The resulting suspension was stirred for 30 min at rt

and the alkyne (21 mg, 0.091 mmol) was added. After 2 weeks of stirring at room temperature,

TLC indicated that only starting material remained.

To a suspension of H2NOH·HCl (140 mg, 2.01 mmol) in MeOH (0.5 mL) was added Et3N (306

mg, 421 µL, 3.02 mmol) under N2 and the resulting mixture was stirred in a rt water bath for 10

minutes. DCM (4.5 mL) was added and the resulting solution was stirred for 10 minutes. The azide

(111 mg, 0.34 mmol) was added and the resulting solution was stirred for 1.5 hours until

consumption of starting material was observed on TLC. The crude mixture was concentrated,

loaded onto celite and purified by flash column chromatography on silica gel. No desired product

was obtained. 10 mg of a highly impure, unidentified compound was isolated but not characterized.

The azide (20 mg, 0.06 mmol) was dissolved in anhydrous THF (2 mL) and cooled to 0°C.

Hydrazine hydrate (18 mg, 18 µL, 0.362 mmol) was added slowly and the resulting solution was

allowed to warm to rt slowly. Stirring at rt was continued until consumption of starting material

was observed on TLC. The crude solution was concentrated and crude 1H NMR showed

decomposition.

The azide (17 mg, 0.051) was dissolved in THF (1.5 mL) and 1M aq. NaOH (0.15 mL, 0.15 mmol)

was added. The resulting solution was stirred for 20 minutes until consumption of starting material

Ph NH

B(MIDA)

N3O

H2NOH·HCl, Et3NMeOH/DCM, rt, 3h

Ph NH

B(MIDA)

NH

O OH

Ph NH

B(MIDA)

N3OTHF, 0°C -> rt, 3h

H2NNH2·H2O Ph NH

B(MIDA)

NH

O NH2

Ph NH

B(MIDA)

N3O

THF/H2O rt, 20 min

NaOHPh NH

B(OH)2

N3O

55

was observed on TLC. Aq. phosphate buffer (pH 7, 1.5 mL) was added and the layers were

separated. The aqueous layer was washed with THF/Et2O (1:1). The combined organic layers were

washed with brine, dried over Na2SO4, filtered and concentrated. 1H NMR showed a mixture of

products.

Appendix D. Hetero-Diels-Alder reaction of α-boryl aldehydes

In a flame-dried flask, the aldehyde (50 mg, 0.18 mmol) was dissolved in anhydrous solvent (see

Table 2) (0.5 mL) under N2. The resulting solution was cooled to -78°C if applicable and the Lewis

Acid was added (Table 2). The solution was stirred for 10 minutes at which point the diene was

added and the solution turned black almost immediately. Stirring was continued for 3 hours and

the mixture was then allowed to warm to room temperature (if it had previously been cooled).

Stirring was continued until the reaction was complete as indicated by TLC or crude NMR or until

the solvent had evaporated.

Ph

B(MIDA)O +

TMSO OMe

1) Lewis Acid Solvent2) Work Up

Ph

B(MIDA)O

O