SYNTHESIS OF HETEROCYCLIC CHIRAL DIAMINES
AND USE OF DIAMINE-BASED CHIRAL GUANIDINES
TO DETERMINE ENANTIOPURITY OF AMINO ACIDS
by
Leo Mui
A thesis submitted in conformity with the requirements
for the degree of Master of Science,
Graduate Department of Chemistry
University of Toronto
© Copyright by Leo Mui 2010
ii
Synthesis of Heterocyclic Chiral Diamines and Use of Diamine-Based Chiral
Guanidines to Determine Enantiopurity of Amino Acids
Leo Mui
Master of Science
Graduate Department of Chemistry
University of Toronto
2010
ABSTRACT
The chiral vicinal diamine moiety is “privileged” and is widely found in catalysts and bio-
active compounds. A series of seven chiral vicinal diamines with heterocyclic substituents
have been synthesized with great enantiospecificity via the resonance assisted hydrogen
bond driven diaza-Cope rearrangement reaction using 1,2-bis(2-hydroxyphenyl)-1,2-
diaminoethane and heterocyclic aldehydes as starting materials.
This thesis will also discuss the development of a new guanidine-based chiral shift rea-
gent for determining the enantiopurity and the absolute configuration of α-amino acids by
proton nuclear magnetic resonance (1H NMR) spectroscopy. The chiral shift reagent is easi-
ly synthesized from the commercially-available 1,2-diphenyl-1,2-diaminoethane. This
method is advantageous over many previously described procedures for determining amino
acid enantiopurity as it does not require prior derivatization of the analyte.
iii
Dedicated to my parents.
I love and appreciate you two more than I can ever express.
iv
Acknowledgements
Although this Master’s thesis has only my name on the cover, the work contained within it would
not be possible without the support of a large number of individuals.
First of all, I would like to thank Professor Jik Chin for being a fantastic research supervisor,
mentor, and friend these past three years. The amount of knowledge I gained from working with
him since the summer before I started my fourth year of undergrad is immense. I really appreciate
the great ideas he thought of and I loved our discussions about chemistry and business at lunch. He
got me excited about a new projects, and he picked me up when I felt down when things did not
turn out well. Prof. Chin was always there when I need to go talk to him about whatever and I deep-
ly appreciate that.
Thanks also go out to all members of the Chin Research Group past and present for being com-
panions in and outside the lab: Soon Mog So, Ewa Golas, Eun Jee Koh, Quanyi Zhao, Cindy Yen, Yen
Nguyen, Ali Rizvi, Rida Mourtada, Elisângela Vinhato, Sandy Hong, and Hae-Jo Kim. A very special
thanks goes to Hyunwoo Kim for all that he has taught me, the chemistry discussions we had, and
all the patience he has shown to me and all my questions!
I would like to acknowledge Dr. Andy Dicks, Vangelis Aktoudianakis, Prof. Robert Batey and
Chris Smith for their guidance and mentorship during my first forays into chemical research.
I would also like to thank Prof. Ronald Kluger for reading my thesis manuscript. I have really en-
joyed getting to know him and hearing all his interesting stories!
Many other people in the Department of Chemistry have helped me complete my work, and I
would like to acknowledge and thank them here:
• Anna Liza Villavelez and Denise Ing for helping me sorting out all the paperwork and bu-
reaucracy that comes with being a grad student;
• Tim Burrow, Darcy Burns, Adina Golombek, and Rose Balazs for helping me sort out all
the problems I had with NMRs;
• Ken Greaves, Donald Foy, Melvyn De Souza, and Jim for helping me get what I needed
and for making trips to the Chem Stores so much fun;
• Patricia Meindl for helping me locate obscure journals and references;
• Mike Dymarski and Linda Scott for helping out with all the building-related problems
v
I would also like to acknowledge my many friends (too many to list here) who have supported
me through graduate school. Thanks for the coffee, thanks for coming out to pub nights, thanks for
the serious chats, thanks for the fun ones, thanks for playing softball…thanks for being friends. In
particular, my friendships with Bryan Li, Anne Motwani, Eric “Ricco” Woroshow, Ben Lau, Rida
Mourtada, Jenny “#2” Wong, Mike “Chudzy” Chudzinski, Scott “Scoot” McAuley, and Michelle Nagy
have been especially special for me. Bryan, Anne, Ben, Rida, Michelle: you need to get nicknames.
Eric: thanks for reading through this thesis looking for typos and stylistic and grammatical prob-
lems. Scott: this is proof that we were once friends, before we started Lunanos.
As I conclude, I would like to thank my family for their unquestioning support through the years.
Mom, Dad, Tim, and Grandma—they have all helped me so much during the course of my studies. I
do not know how I would have completed this degree without them all being there beside me. I am
very lucky to have the family that I do.
Leo Mui
September 29, 2010
vi
Table of Contents
Abstract .................................................................................................................................................................................. ii
Dedication ............................................................................................................................................................................ iii
Acknowledgements .......................................................................................................................................................... iv
Table of Contents ............................................................................................................................................................... vi
List of Tables ..................................................................................................................................................................... viii
List of Figures...................................................................................................................................................................... ix
List of Schemes ..................................................................................................................................................................... x
List of Abbreviations ....................................................................................................................................................... xii
CHAPTER 1 Chiral Vicinal Diamines .......................................................................................................................... 1
1.1. Applications in Catalysis ................................................................................................................................... 1
1.2. Applications in Medicinal Chemistry ........................................................................................................... 4
1.3. Historical Overview of Diamine Syntheses ................................................................................................ 4
1.4. Research Objectives ............................................................................................................................................ 6
1.5. Summary .................................................................................................................................................................. 6
1.6. References ............................................................................................................................................................... 8
CHAPTER 2 Synthesis of Heterocyclic Chiral Vicinal Diamines ............................................................... 10
2.1 The Diaza-Cope Rearrangement .................................................................................................................. 10
2.2 Heterocyclic Diamines...................................................................................................................................... 16
2.3 Experimental and Results ............................................................................................................................... 26
2.4 Conclusions and Future Directions ............................................................................................................. 32
2.5 Spectra .................................................................................................................................................................... 32
2.6 References ............................................................................................................................................................. 40
vii
CHAPTER 3 Chiral Cyclic Guanidine for Determining Enantiopurity and Absolute
Configuration of Amino Acids ...................................................................................................................................... 42
3.1 Methods of Determining Enantiopurity and Absolute Configuration of Amino Acids .......... 42
3.2 Development of Cyclic Guanidine Chiral Shift Reagent for Amino Acids .................................... 46
3.3 Results..................................................................................................................................................................... 54
3.4 Experimental ........................................................................................................................................................ 58
3.5 Conclusions and Future Directions ............................................................................................................. 60
3.6 Spectra .................................................................................................................................................................... 60
3.7 References ............................................................................................................................................................. 68
Appendix 1 ......................................................................................................................................................................... 70
Appendix 2 ......................................................................................................................................................................... 71
viii
List of Tables
Table 2-1. Synthesized heterocyclic diamines and respective isolated yields ............................................. 21
Table 3-1. Chemical shifts of the diagnostic peaks for the D- and L- forms of six amino acids in CDCl3
with 7% DMSO-d6 v/v using the of chiral shift reagent 1 and salicylaldehyde 13 ..................................... 56
Table 3-2. 1H NMR-measured ratios of the L- and D- forms of Val at seven known ratios and
associated absolute and relative errors of the method ......................................................................................... 57
ix
List of Figures
Figure 1-1. Graphical summary of Chapter 1 ............................................................................................................... 7
Figure 2-1. Partial 1H NMR spectra of the reaction of (R,R)-3a and 4a in DMSO-d6 (70 mM) at 1 h, 2h,
and 7 h; of isolated (R,R)-5’a in DMSO-d6 .................................................................................................................... 20
Figure 2-2. Partial 1H NMR spectra for diimines 13d, f, and g ............................................................................ 23
Figure 2-3. Partial 1H NMR spectra for diimines 15a–c, and e ............................................................................ 24
Figure 3-1. Partial 1H NMR spectrum of a mixture of 1, 1·L-Val, and 1·D-Val in CDCl3. ............................ 50
Figure 3-2. Partial 1H NMR spectrum of the diastereomers (S,S)-1·13·D-Phe and (S,S)-1·13·L-Phe in
CDCl3 (50 mM) ........................................................................................................................................................................ 52
Figure 3-3. Partial 1H NMR spectra of L-Met, L-Phe, L-Thr, L-Ala, D-Ser, and L-Asn 1 h after 1, 13, and
amino acid were mixed in CDCl3 (50 mM) .................................................................................................................. 53
Figure 3-4. Partial 1H NMR spectra of the diagnostic peaks of 1·13·DL-Phe (racemate used) .............. 54
Figure 3-5. Partial 1H NMR spectra of the diagnostic peak of 1·13·amino acid complex ........................ 55
Figure 3-6. Partial 1H NMR spectra of the diagnostic peak of 1·13·D-Val 1·13·L-Val complex at
various known D : L molar ratios ..................................................................................................................................... 56
x
List of Schemes
Scheme 1-1. Noyori's asymmetric transfer hydrogenation of ketones. ............................................................ 2
Scheme 1-2. Fu's Suzuki arylation of chloroamides using an aryl vicinal diamine as a ligand for the
nickel catalyzed reaction ...................................................................................................................................................... 2
Scheme 1-3. Ding's diamine-based phosphoramidite as a ligand for a rhodium-catalyzed
hydrogenation ........................................................................................................................................................................... 2
Scheme 1-4. Monotosylated vicinal diamine used as bifunctional organocatalyst for enantioselective
Michael additions .................................................................................................................................................................... 3
Scheme 1-5. Highly stereoselective synthesis of warfarin catalyzed by diamine 6 ..................................... 3
Scheme 1-6 . Examples of biologically-active compounds containing the vicinal diamine moiety ....... 4
Scheme 1-7. Two methods to synthesize 11 developed by Corey and colleagues ....................................... 5
Scheme 1-8. Two routes to vicinal diamines developed by Petersen et al ...................................................... 5
Scheme 1-9. SmI2 mediated homocoupling of sulfinyl imines to produce enantiopure chiral vicinal
diamines ...................................................................................................................................................................................... 6
Scheme 2-1. General scheme of the diaza-Cope rearrangement reaction ..................................................... 10
Scheme 2-2. The mechanism of the Fischer indole synthesis involves a [3,3]-sigmatropic
rearrangement as the key step ........................................................................................................................................ 10
Scheme 2-3. The diaza-Cope rearrangement reaction studied by Vögtle and Goldschmitt ................... 11
Scheme 2-4. Resonance structures demonstrate how π-delocalization enhances hydrogen bond
strength by causing a separation of charge ................................................................................................................ 12
Scheme 2-5. Resonance assisted hydrogen bonding in the Watson-Crick base pairing of adenine and
thymine ...................................................................................................................................................................................... 12
Scheme 2-7. Ground-state conformations of meso and chiral diimines .......................................................... 13
Scheme 2-6. Transition-state geometries of meso and chiral diimines undergoing DCR ........................ 13
Scheme 2-8. General synthetic scheme of a variety of chiral vicinal aryl diamines using the diaza-
Cope rearrangement ............................................................................................................................................................ 14
xi
Scheme 2-9. Key intermediates involved in the synthesis of alkyl diamines using the diaza-Core
rearrangement reaction ...................................................................................................................................................... 15
Scheme 2-10. General synthetic scheme for heterocyclic chiral vicinal diamines 6a–g .......................... 17
Scheme 2-11. Intermediates formed in the reaction of (R,R)-3a and 4a to produce (R,R)-5’a .............. 20
Scheme 2-12. Chiral diamines 6 react with enantiopure aldehyde R-12 to give two diastereomers. 22
Scheme 2-13. Structure and molecular weight of 6e·2HCl and 6e·4HCl ........................................................ 25
Scheme 3-1. The imine formed between 2 and L-Ala exist predominantly as the P helix in aprotic
solvents ...................................................................................................................................................................................... 43
Scheme 3-2. The attachment of azo groups to 2 amplifies the CD signals for receptor 3 ....................... 43
Scheme 3-3. The bioinspired amino acid receptor 9 binds stereoselectively to amino acids by imine
formation controlled by hydrogen bonding ............................................................................................................... 48
Scheme 3-4. The synthetic scheme to reach cyclic guanidine 1 from (S,S)-10 in one transformation
step followed by a basic workup ..................................................................................................................................... 49
Scheme 3-5. The hydrogen bond between receptor 1’s guanidinium group and an amino acid’s (AA)
carboxylate group is charged and is especially strong .......................................................................................... 49
Scheme 3-6. Interactions of the ternary complex (S,S)-1·13·L-Phe. The proton associated with 1H
NMR diagnostic peak is outlined and bolded ............................................................................................................. 51
xii
List of Abbreviations
Note: This list does not include common SI units, atomic symbols, amino acid abbreviations, or abbre-
viations used in standard IUPAC nomenclature.
9-BBN 9-borabicyclo(3.3.1)nonane
AA amino acid
Ar aryl or aromatic
B3LYP Becke, three-parameter, Lee-Yang-Par (exchange functional)
Bn benzyl
Bu butyl
CAS chrome azurol S
Cbz carboxybenzyl
CD circular dichroism
cod 1,5-cyclooctadiene
CSR chiral shift reagent
DCR diaza-Cope rearrangement
δ chemical shift
Δ change in
DFT density functional theory
DMSO dimethyl sulfoxide
DMSO-d6 trideuterio(trideuteriomethylsulfinyl)methane (deuterated DMSO)
dpen 1,2-diphenyl-1,2-diaminoethane
ε molar extinction coefficient
ee enantiomeric excess
e.g. exempli gratia
equiv equivalents
xiii
et al. et alii or et aliæ
Et3N triethylamine
GC gas chromatography
HMPA hexamethylphosphoramide
HPLC high-performance liquid chromatography
HRMS high resolution mass spectra
ie. id est
λ wavelength
LC liquid chromatography
Me methyl
MeOH methanol
MSG monosodium glutamate
NMR nuclear magnetic resonance
Ph phenyl
ppm parts per million
RAHB resonance-assisted hydrogen bond
rt room temperature
THF tetrahydrofuran
TLC thin-layer chromatography
Ts toluenesulfonyl (tosyl)
TS transition state (activated complex)
UV-vis ultraviolet-visible
ver. Version
v/v volume/volume (ratio)
1
CHAPTER 1
Chiral Vicinal Diamines
Not all molecules are created equal. Chiral vicinal diamine moieties are privileged structures in
asymmetric catalysis because of their utility and enantioselectivity for a variety of reactions.1 The
diamine substructure can be found in a wide range of organometallic catalysts and organocatalysts
to provide asymmetry for reactions such as hydrogenations, oxidations, hydrolyses, ring-openings,
and carbon-carbon bond-formations.2 Outside of the glassware, vicinal diamines appear in the
structure of important antibacterial, anticancer, antidepressant, antiviral,
and antihypertensive pharmaceuticals and drug candidates.2 Until recent
developments by Chin and colleagues, it was difficult to access a wide li-
brary of different enantiopure chiral vicinal diamines because of the lack
of a synthetically simplistic and general method to make these privileged
structures. This introductory chapter will provide a brief background on
the use of chiral vicinal diamines in catalysis and pharmaceuticals, and will also overview some key
methods that have been used to make these molecules.3
1.1. Applications in Catalysis
In nature, organisms have evolved efficient enzymes to conduct asymmetric catalysis of biomol-
ecules—that is, reactions in enzymes could result in only a single enantiomer of the products. In
the chemistry laboratory, one of the most active areas of research is in finding methods to make
enantiopure compounds by “enzyme-like” enantioselective methods rather than classical methods
that rely on resolution of enantiomers after synthesis.1 In that endeavour, diamine-based asym-
metric catalysts have been used in many reactions. Several interesting examples will be highlight-
ed here, but many more examples can be found in recent reviews and the ref-
erences therein.2, 4-8
1.1.1. As Ligands for Organometallic Catalysts
The vicinal diamine moiety can be found in Noyori’s transfer hydrogena-
tion catalyst (1), which uses formic acid or isopropanol as a source of hydro-
gen to reduce ketones asymmetrically with an 80% conversion and 94% ee
(Scheme 1-1).9 Further improvements were made by Mioskowski et al., who
NH2TsHN
Ru
Cl
1
2
showed that the replacement of the tosyl (Ts) group on the amine with a more electron-
withdrawing perfluorobutylsulfonate group increases the conversion to 100% with a >99% ee in
less than half the reaction time than the original reaction.10 This example underscores the im-
portance of having an easy-to-access library of diamines as replacing the phenyl groups in 1 with
more electron-withdrawing aryl groups could also have been used in the tuning of the catalyst.
Lundin and Fu recently reported an asymmetric Suzuki arylation of racemic α-chloroamides to
give enantioenriched α-arylamides using one of two vicinal diamines (2a and 2b) as a ligand.11 It
was found that the installation of CF3 groups on the phenyl groups of 2a give an increase of ee from
85 to 92%, and an increase in yield from 74 to 89% (Scheme 1-2).11 Further improvements on both
the yield and ee may be possible by using differently substituted aryl diamines.
Vicinal diamines were also used as synthetic precursors for monodentate phosphoramidite lig-
ands (3) in rhodium-catalyzed asymmetric hydrogenation of acrylates in this reaction carried out
by Ding and co-workers (Scheme 1-3).12, 13
Scheme 1-1. Noyori's asymmetric transfer hydrogenation of ketones.
Scheme 1-2. Fu's Suzuki arylation of
chloroamides using an aryl vicinal di-
amine as a ligand for the nickel cata-
lyzed reaction.
Scheme 1-3. Ding's diamine-based phosphoramidite as a ligand for a rhodium-catalyzed hydrogenation.
3
1.1.2. As Organocatalysts
Research into organocatalysis—catalysts that are purely organic and are metal-free—has been
growing exponentially since the turn of the century.14 MacMillan is one of the
leaders in this field with his development of imidazolidinone catalyst 4, which
also contains the vicinal diamine motif. Catalyst 4 has been used for a variety
of reactions such as indol alkylation15, Michael additions 16, 17, aldol condensa-
tions18, allylation and arylation19, and cascade reactions20.
Wang et al. recently reported very enantioselective (up to 98% ee) Michael additions of acetone
to a wide range of nitrostyrenes using the bifunctional catalyst 5 originally developed by Noyori6
for transfer hydrogenation (Scheme 1-4).21 The monotosylation of aryl vicinal diamine are relative-
ly simple, so a variety of monotosyl bifunctional catalysts may be developed in the near future.
Unmodified diamines can also be used as organocatalysts—for example, Chin et al. used (R,R)-
1,2-bis(2-methylphenyl)-1,2-diaminoethane (6) to catalyze the stereoselective synthesis of warfa-
rin from 4-hydroxycoumarin and trans-4-phenyl-3-buten-2-one in 99% yield and 92% ee (Scheme
1-5).22
O
OH
O
Ph
O
+
O
OH
O
OPh
6
acetic acid
NH2H2N
6
Scheme 1-5. Highly stereoselective synthesis of warfarin catalyzed by diamine 6.
Scheme 1-4. Monotosylated vicinal diamine used as bifunctional organo-
catalyst for enantioselective Michael additions.
4
1.2. Applications in Medicinal Chemistry
The vicinal diamine moiety is found in many bioactive molecules that are used in the pharma-
ceutical industry. Due to the recent outbreaks of the influenza virus, much attention has been
brought to the antiviral drug Tamiflu® (oseltamivir, 7) by both the media and the medical com-
munity.2, 23 Another well-known drug containing the chiral vicinal diamine motif is Eloxatin® (ox-
aliplatin, 8), which is used in colorectal cancer chemotherapy.2 Many β-lactam antibacterials, like
the penicillins (9) and cephalosporin, also contain diamines in their structure.2 Nutlin-3 (10) is an-
ticancer drug candidate that is based on an imidazoline formed from a meso diaryl vicinal diamine.2
1.3. Historical Overview of Diamine Syntheses
Due to the presence of the chiral vicinal diamine moiety in many different catalysts and phar-
maceuticals, many methods of vicinal diamine synthesis have been developed through the years.
However, many of these methods are of limited scope, have low yields, or are not stereoselective
and require tedious resolution.2, 23
The synthesis of one of the most commonly used diamines, 1,2-diphenyl-1,2-diaminoethane
(dpen, 11), can be done through the hydrolysis of isoamarine prepared from benzaldehyde and
ammonia24 or from reductive amination of benzil and cyclohexanone followed by the reduction of
the intermediate imidazole. The racemic dpen can then be resolved using tartaric acid to give en-
antiopure dpen.25
O
OO
O
HN
NH2
H2N
NH2
Pt
O
OO
O
7 8
N
S
O
H
COOH
HN
O
R
9 NHN
O
O
N
N
O
O
Cl
Cl
10
Scheme 1-6 . Examples of bio-
logically-active compounds
containing the vicinal diamine
moiety.
5
Scheme 1-7. Two methods to synthesize 11 developed by Corey and colleagues.
Pedersen et al. developed a method to use NbCl4(THF)2 to promote homocoupling of nitriles and
TMS imines to yield racemic diamines.26 The authors were able to make a variety of diamines with
good yield and varying diastereomeric excess, including four diamines with heterocyclic substitu-
ents.26 Kise and Ueda published syntheses that use Zn-TiCl4 to reductively couple aryl oximes and
azines, but their methods were plagued by very poor diastereomeric excess, producing a large
amount of the meso along with the chiral diamine.27
The resolution of racemic diamines are generally used by forming a diastereotopic salt with en-
antiopure L-tartaric acid, but the process can be tedious and cause great loss of yield, and unless
the goal is to obtain both enantiomers, the even the maximum yield is only 50%.2 Employing an
enantioselective synthesis of vicinal diamines would alleviate these problems. An enantioselective
method was developed by Xu et al. by using a samarium iodide homocoupling of N-tert-
butanesulfinyl imines followed by an acidic work up to give chiral vicinal diamines with high ee’s
but a large variation in yields (Scheme 1-9).28
Scheme 1-8. Two routes to
vicinal diamines developed
by Petersen et al.
6
Scheme 1-9. SmI2 mediated homocoupling of sulfinyl imines to produce enantiopure chiral vicinal
diamines.
An important disadvantage of the above method is the requirement of a stoichiometric amount
of samarium. An asymmetric catalytic method is to use Sharpless asymmetric dihydroxylation of
stilbenes to give cis-vicinal diols, which can be transformed into the required vicinal diamines. Un-
fortunately, this procedure involves a hazardous step which may be difficult to scale up, as the di-
ols are converted into diazides using sodium azide.29
Chin and colleagues have developed a facile method to reach chiral vicinal diamines with aryl,
alkyl, or mixed aryl-alkyl substituents with excellent ee’s in two steps using the diaza-Cope rear-
rangement. The developments are detailed in Chapter 2.
1.4. Research Objectives
Two major research objectives related to chiral vicinal diamines have been achieved by the au-
thor and are described in this thesis. First, seven chiral vicinal diamines with heterocyclic substitu-
ents have been synthesized using the diaza-Cope rearrangement method leading to very good
yields and excellent enantioselectivity (Chapter 2). Second, a vicinal diamine-based chemical shift
reagent for determining the enantiopurity of free, nonderivatized amino acids was developed
(Chapter 3).
1.5. Summary
A graphical summary of this chapter can be found on the following page (Figure 1-1).
7
Fig
ure
1-1
. S
um
ma
ry o
f th
e v
ari
ou
s m
eth
od
s
of
chir
al
vic
ina
l d
iam
ine
sy
nth
esi
s a
nd
th
e
ma
ny
ap
pli
cati
on
s o
f ch
ira
l v
icin
al
dia
min
es
in c
he
mis
try
an
d b
iolo
gy
. T
he
re
sea
rch
ob
jec-
tiv
es
of
this
th
esi
s (w
he
re R
is
a h
ete
rocy
cle
)
are
hig
hli
gh
ted
in
bo
xe
s.
8
1.6. References
(1) Yoon, T. P.; Jacobsen, E. N. Science 2003, 299, 1691-1693.
(2) Kim, H.; So, S. M.; Chin, J.; Kim, B. M. Aldrichim. Acta. 2008, 41, 77-88.
(3) Kim, H. Hydrogen Bond-Directed Stereospecific Interactions in A) General Synthesis of Chiral
Vicinal Diamines and B) Generation of Helical Chirality with Amino Acids, University of Toron-
to, Toronto, 2009.
(4) Trost, B. M.; Crawley, M. L. Chem. Rev. 2003, 103, 2921-2944.
(5) Trost, B. M.; Machacek, M. R.; Aponick, A. Acc. Chem. Res. 2006, 39, 747-760.
(6) Noyori, R. Adv. Synth. Catal. 2003, 345, 15-32.
(7) Denmark, S. E.; Stavenger, R. A. Acc. Chem. Res. 2000, 33, 432-440.
(8) Hems, W. P.; Groarke, M.; Zanotti-Gerosa, A.; Grasa, G. A. Acc. Chem. Res. 2007, 40, 1340-1347.
(9) Noyori, R.; Hashiguchi, S. Acc. Chem. Res. 1997, 30, 97-102.
(10) Mohar, B.; Valleix, A.; Desmurs, J.; Felemez, M.; Wagner, A.; Mioskowski, A. Chem. Commun.
2001, 2572-2573.
(11) Lundin, P. M.; Fu, G. C. J. Am. Chem. Soc. 2010, 132, 11027-11029.
(12) Liu, Y.; Ding, K. J. Am. Chem. Soc. 2005, 127, 10488-10489.
(13) Liu, Y.; Sandoval, C. A.; Yamaguchi, Y.; Zhang, X.; Wang, Z.; Kato, K.; Ding, K. J. Am. Chem. Soc.
2006, 128, 14212-14213.
(14) List, B. Chem. Rev. 2007, 107, 5413-5415.
(15) Austin, J. F.; MacMillan, D. W. C. J. Am. Chem. Soc. 2002, 124, 1172-1173.
(16) Paras, N. A.; MacMillan, D. W. C. J. Am. Chem. Soc. 2002, 124, 7894-7895.
(17) Borths, C. J.; Carrera, D. E.; MacMillan, D. W. C. Tetrahedron 2009, 65, 6746-6753.
(18) Mangion, I. K.; Northrup, A. B.; MacMillan, D. W. C. Angew. Chem. Int. Ed. 2004, 43, 6722-6724.
(19) Beeson, T. D.; Mastracchio, A.; Hong, J.; Ashton, K.; MacMillan, D. W. C. Science 2007, 316, 582-
585.
(20) Simmons, B.; Walji, A. M.; MacMillan, D. W. C. Angew. Chem. Int. Ed. 2009, 48, 4349-4353.
9
(21) Peng, L.; Xu, X.; Wang, L.; Huang, J.; Bai, J.; Huang, Q.; Wang, L. Eur. J. Org. Chem. 2010, 1849-
1853.
(22) Kim, H.; Yen, C.; Preston, P.; Chin, J. Org. Lett. 2006, 8, 5239-5242.
(23) Kotti, S. R. S. Saibabu; Timmons, C.; Li, G. Chem. Biol. Drug Des. 2006, 67, 101-114.
(24) Corey, E. J.; Kühnle, F. N. M. Tet. Lett. 1997, 38, 8631-8634.
(25) Corey, E. J.; Lee, D.; Sarshar, S. Tet. Asym. 1995, 6, 3-6.
(26) Roskamp, E. J.; Pedersen, S. F. J. Am. Chem. Soc. 1987, 109, 3152-3154.
(27) Kise, N.; Ueda, N. Tet. Lett. 2001, 42, 2365-2368.
(28) Zhong, Y.; Izumi, K.; Xu, M.; Lin, G. Org. Lett. 2004, 6, 4747-4750.
(29) Hilgraf, R.; Pfaltz, A. Adv. Synth. Catal. 2005, 347, 61-77.
10
CHAPTER 2
Synthesis of Heterocyclic Chiral Vicinal Diamines
This chapter describes the enantioselective synthesis of seven chiral vicinal diamines that con-
tain heterocyclic substituents. The syntheses were carried out using the diaza-Cope rearrangement
reaction, using enantiopure 1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane and the appropriate het-
erocyclic aldehyde as the starting materials. This method can be used to produce a wide variety of
heterocyclic diamines with very good yields from micro- to multi-gram scales.
2.1 The Diaza-Cope Rearrangement
2.1.1 History and Physical Organic Chemistry of the Diaza-Cope Rearrangement and
Resonance-Assistance Hydrogen Bonds
The diaza-Cope rearrangement (DCR), as the name aptly implies, is an analogue of the more
well-known Cope rearrangement,1 where two carbon atoms are replaced by nitrogens. In this
chapter, DCR will exclusively signify the thermal [3,3]-sigmatropic reaction involving the 1,2-
diiminoethane moiety—where the carbons in the 2- and
6- positions of the Cope rearrangement are replaced by
nitrogens (Scheme 2-1). Nitrogens substituted in other
positions are known as well. For example, the currently
accepted mechanism of the Fischer indole synthesis in-
volves a version of the DCR where nitrogens are positioned the 3- and 4-positions so that an
enamine (2) rearranges into a diimine (2’) (Scheme 2-2).2
NH
NH2
O+
NH
NNH
NH-H2O
[3,3]
NHNHN
H
2
2'
H+
Scheme 2-2. The mechanism
of the Fischer indole synthesis
involves a [3,3]-sigmatropic
rearrangement as the key
step.
Scheme 2-1. General scheme of the
diaza-Cope rearrangement reaction.
11
The first detailed studies of DCR were reported by Vögtle and Goldschmitt in the1970s.3-5 They
made a series of meso-diimines meso-5 and meso-5’ by the condensation of meso-1,2-bis(2-
hydroxyphenyl)-1,2-diaminoethane (meso-hpen, meso-3) with two equivalents of aryl aldehyde 4
(Scheme 2-3).5 They found that, at 80°C, the rearrangement heavily favoured the rearranged
diimine meso-5’, speculating that the equilibrium was controlled by a “Salicyl-Effekt” (“salicyl ef-
fect”) that catalyzed the reaction by the formation energetically-favourable internal hydrogen
bonds.5 From a synthetic point of view, this finding was important, as a series of meso-1,2-diamines
were readily accessed by acid hydrolysis of the rearranged diimines (meso-5’).
Vögtle and Goldschmitt noticed early on that the same rearrangement reaction with meso-1,2-
diphenyl-1,2-diaminoethane (meso-dpen) was less favoured,3 but besides remarking on the “salicyl
effect”, they did not elucidate the physical basis of how the ortho-hydroxyl groups of meso-5 and
meso-5’ affect the equilibrium. At first glance, it may be difficult to explain the thermodynamic dif-
ference between meso-5 and meso-5’ as they are very similar in structure and both species can
form an internal hydrogen bond between the imino nitrogen and phenolic hydrogen. That “first
glance” lasted for thirty years before Chin et al. reported that the DCR in question was controlled
by resonance-assisted hydrogen bonds (RAHBs).
A brief side discussion on RAHBs would be appropriate here. “Resonance-assisted hydrogen
bonding” was a term coined by Gilli and colleagues6, 7 to describe unusually short, strong hydrogen
bonds that are assisted by π-delocalization. Early studies examined homonuclear O–H···O RAHBs
in the enol form of acetoacetone,6, 7 and a more recent study examined heteronuclear N–H···O
RAHB systems.8 Although heteronuclear RAHBs are weaker and less symmetric compared to their
homonuclear counterparts, it is nonetheless still stronger than regular hydrogen bonds.
Scheme 2-3. The diaza-Cope rearrangement reaction studied by Vögtle and Goldschmitt.
12
The DCR studied here involves a heteronuclear O–H···N RAHB system (Scheme 2-4). It is im-
portant to understand that the hydrogen in this case remains covalently bonded to oxygen—RAHB
does not involve a proton transfer, nor is the hydrogen equally shared between the two heteroa-
toms. The strength of RAHBs is derived from the formation of a partially positive hydrogen bond
donor (oxygen in this case) and a partially negative acceptor (nitrogen in this case) through π-
delocalization. Electron donation to the imine nitrogen reduces its electronegativity and thus in-
creases the energy of its lone pair to makes it a better hydrogen
bond acceptor. Electron withdrawal from oxygen increases the hy-
drogen’s electronegativity and makes it a better hydrogen bond do-
nor. Even though it took until the late 1980s for RAHBs to be named
and described, it is neither uncommon nor rare: a close examination
of Watson-Crick base pairing (Scheme 2-5) and α-helixes show that
their hydrogen bonds are also resonance-assisted.6
By now, it should be clear why the rearrangement of meso- 5 to
meso-5’ in Scheme 2-3 favours the product— meso-5’ has two salicyl
imine moieties that form two internal RAHBs while meso-5 can only form two regular hydrogen
bonds. X-ray crystallography studies show that, in meso-5’ (R = o-OMe), the C=N imine bond is
longer and the C–O phenolic and the N···H hydrogen bonds are shorter than the corresponding
bonds in meso-5 (R = o-OMe),, as expected if π-delocalization was occurring in meso-5’ (R = o-
OMe).9 Furthermore, 1H NMR in DMSO-d6 show the phenolic proton of the rearranged diimine me-
so-5’ (R = o-OMe) resonating extremely downfield at 13.4 ppm, indicating a much stronger hydro-
gen bond formation compared to the initial diimine meso-5 (R = o-OMe), whose phenolic proton
resonates at 10.0 ppm.9 Having experimental evidence of RAHB formation in this diimine system,
Chin et al. calculated [DFT B3LYP/6-31++G(d,p)] that the rearranged diimine was 6.62 kcal/mol
more stable than the initial diimine, resulting in an equilibrium of the forward rearrangement
equalling 1.4 × 105.9
Scheme 2-4. Resonance
structures demonstrate
how π-delocalization (light
dashes) enhances hydrogen
bond (heavy dashes)
strength by causing a sepa-
ration of charge.
Scheme 2-5. Resonance
assisted hydrogen bonding
in the Watson-Crick base
pairing of adenine and thymine.
13
Vögtle and Goldschmitt found that DCR with meso diimines go through a boat-like transition
state while chiral diimines go through a chair-like transition state, having observed that the rear-
rangement of meso diimines always gives meso products, and chiral initial diimines always lead to
chiral rearranged diimines (Scheme 2-7). In addition, as aryl groups in the chair-like transition
state prefer to occupy the pseudo-equatorial positions, DCR with chiral diimines proceed with very
high enantiospecificity resulting in
an inversion of stereochemistry.10
In the ground state, meso-5, me-
so-5’, and (R,R)-5’ have been shown
by crystallography to be in the stag-
gered conformation (Scheme 2-6).10
It can be seen the transition state of
the rearrangement of meso-5 and me-
so-5’ would require an eclipsed con-
formation to bring the imine nitrogens closer together, while (R,R)-5’ is already preorganized for
rearrangement.10 The result of
this geometry is that the entropic
barrier for rearrangement of chi-
ral-5 (R = o-OMe) is 5.2 kcal/mol
lower than meso-5 (R = o-OMe) at
25°C.10 So, even though the en-
thalpic barrier for the chiral
diimine is slightly higher than the
meso diimine, the rate constant for
the DCR of the chiral diimine was
found to be 30 times greater than that of the meso diimine at 50°C.10
It should be noted that, while the formation of RAHBs is a major “push” factor in favouring the
rearranged diimine in DCR when an ortho-hydroxy group is present, Chin and colleagues have
studied the effect of other weak forces on the rearrangement extensively. Steric strain,11 conjuga-
tion,12 aromatic ring electronics, and the oxyanion effect are all factors that affect the thermody-
namics and kinetics of DCR. Manuscripts of the electronic effect and the oxyanion effect, as well as
an account of this chemical research are currently in preparation for publication.
Scheme 2-7. Transition-state geometries of meso and chi-
ral diimines undergoing DCR.
Scheme 2-6. Ground-state conformations of meso and chiral diimines.
14
2.1.2 Using the Diaza-Cope Rearrangement in Diamine Synthesis
F
F
F
F
F
NO2OMeO
F
Cl
CF3
CF3 CF3F3C
CN
Cl
HN
O
OMeNMe2 OH
OMe
OMeMeO
OMe
Ar =
NH2
NH2
OH
OH
N
N
OH
OH
Ar
Ar
[3,3]
(R,R)-3 or(S,S)-3
(R,R)-5 or(S,S)-5
N
N
OH
OH
Ar
Ar
(S,S)-5' or(R,R)-5'
H+, H2ONH3Cl
NH3Cl
Ar
Ar
(S,S)-6 or(R,R)-6
2.5 equiv.
ArCHO 4
"Mother Diamine" "Daughter Diamine"
H2OOH
OHC
Scheme 2-8. General synthetic scheme of a variety of chiral vicinal aryl diamines using the diaza-
Cope rearrangement.
The RAHB effect on the DCR of 5 to 5’ can be exploited in the synthesis of chiral vicinal diamines
6. The condensation of 3 with 4 in DMSO or ethanol results in diimine 5’ exclusively in a very ste-
reospecific manner (Scheme 2-8).13 As the rearrangement goes through a chair-like transition state
where all substituents prefer to occupy a pseudo-equatorial position, an apparent inversion of ste-
reochemistry occurs (ie. (R,R)diimines rearrange to make (S,S) diimines), generally with >99%
ee.13 The formation of the opposite enantiomer or the meso-diimine would require one or more of
the aryl groups to be placed in the energetically unfavourable pseudo-axial position.10
15
The crude rearranged diimine 5’ can be subjected to acid hydrolysis conditions to yield the hy-
drochloride salt of a new diamine (6) and two equivalents of salicylaldehyde (Scheme 2-8). Purifi-
cation of diimine 5’ is generally not needed as DFT calculations [B3LYP/6-31G(d)] have shown that
the equilibrium constant for the rearrangement of diimine 5 is 9.3 × 104 at 25°C when Ar = C6H5.
Because of the variety of aromatic aldehydes (4) available commercially, the generality of this syn-
thetic process was tested with a large library of aryl substituents (Scheme 2-8), all yielding enanti-
opure (>99% ee) diamines in moderate to high yields (70–90%).14 This diamine synthesis is con-
ducted at ambient or slightly elevated temperatures, and has been shown to be effective from an
NMR scale (10 mg of 3) to a large preparative scale (15 g of 3).
NH2
NH2
OH
OH
N
N
OH
OH
(R,R)-3 (R,R)-9
N
N
OH
OH
(S,S)-9'
O
2.5 equiv
7 HN
N
OH
O
(R,R,R,R)-8
Scheme 2-9. Key intermediates involved in the synthesis of alkyl diamines using the diaza-Core
rearrangement reaction.
The synthesis of diamines with alkyl groups as opposed to aromatic groups using a modification
of the above procedure has been reported. When 3 is mixed with two or more equivalents of an
alkyl aldehyde like isobutyraldehyde (7) at room temperature, the only isolated product is the im-
idazolidine-dihydro-1,3-oxazine 8 (Scheme 2-9).12 The equilibrium between fused ring 8 and
diimine 9 greatly favour the former compound in the case of alkyl diamines, probably because the
stability brought on by conjugation between the imine and an aromatic ring is not available.12 In
addition, the barrier (activation energy) of DCR to 9’ is higher, possibly because of greater steric
bulk at the transition state.15 The best synthetic method to overcome this barrier is to conduct the
DCR in refluxing toluene (111°C) with a Dean-Stark trap to remove water, which despite the
harsher conditions still gives a variety of alkyl diamines in good yields and >99% ee.12
Colloquially, (R,R)- and (S,S)-3 have been affectionately termed the “mother diamine”, as it pro-
duces “daughter diamines” 6 that share half its features and half the features of the aldehyde 4.
However, in light of the preference towards gender-neutral language in scientific writing, these
terms will not be used further in this thesis.
16
N
NH2
N+
O
O-SO
N
Zantac® (ranitidine)
S
O
O
NH N
H
N
Imigran® (sumatriptan)
O
NH
N
HN
Doralese® (indoramin)
2.2 Heterocyclic Diamines
2.2.1 Possible Applications in Chemistry and Biology
One class of chiral vicinal diamines that is missing from the above discussion are ones that con-
tain heterocycles. Heterocyclic amines are important structures in biology.
One of the most popularly used pharmaceuticals with a heterocyclic amine
is Plavix® (clopidogrel), which is used to reduce blood clots in patients.16
Some of the more well-
known biological compounds
with this moiety are the monoamine neurotransmit-
ters histamine and serotonin, which are enzymati-
cally converted from histidine and tryptophan, re-
spectively.17 The heterocyclic diamines described
here can prove to be interesting because they can be seen as dimers of these biologically-active
moieties.
Histamine is not only a neurotransmitter but also acts to control gastric acid secretion by acting
on the H2 histamine receptor.17 Pharmaceuticals that act as H2 receptor antagonists are used to
treat gastroesophageal acid reflux, ulcers, and other symptoms of acid hypersecretion.18 An exam-
ple of a popular drug that treats this symptom is Zantac® (ranitidine), which is often preferred
because of its relatively mild side-effects.17 Serotonin is responsible for the regulation of a wide
variety of biological process, from digestion to cardiovascular function to modulation of mood.19
The related melatonin (O-methylated and N-acetylated serotonin) is another ubiquitous signalling
molecule and drug as it is involved in regulation of blood pressure, immune function, and sleep cy-
cles.20 An example of a drug that acts as serotonin agonist is Imigran® (sumatriptan), which is a
prescription medication for severe migraine headaches.17 While adrenaline does not contain a het-
erocycle, a drug that acts as an adrenergic receptor antagonist to treat enlarged prostate glands is
Doralese® (indoramin).17
17
The heterocyclic diamines reported here can also be of great utility for chemists looking to
“tune” the function of their organometallic catalysts or organocatalysts. For example, heterocycles
can provide strategically placed hydrogen bonding sites to stabilize a particular transition state
structure, or can act as Brønsted acid and bases in a reaction. Now that these heterocyclic diamines
are easily accessible, it is hoped that both chemists and biologists would start to realize the poten-
tial functions for these structures in their research area.
2.2.2 Synthesis
The two main solvents that are used for the DCR process are ethanol and DMSO. Ethanol is gen-
erally the preferred solvent for a number of reasons. Generally, both the starting diamine 3 and the
rearranged diimine 5’ are not very soluble at room temperature; however, most aromatic alde-
hydes and the reaction intermediates (see below) are quite soluble. In effect, upon the addition of
two or more equivalents of the soluble aldehyde to a suspension of 3 in ethanol, the mixture be-
comes homogeneous as the reaction proceeds before the product 5 precipitates out upon comple-
tion. Not only can this method easily produce pure 5’ as a solid, there is no need to monitor the re-
action by TLC or NMR as the reaction progress can be determined by visual inspection. In addition,
Ar =
NH2
NH2
OH
OH
(R,R)-3 or(S,S)-3
N
N
OH
OH
Ar
Ar
(R,R)-5' or(S,S)-5'
NH3Cl
NH3Cl
Ar
Ar
(R,R)-6 or(S,S)-6
2.5 equiv.ArCHO 4
DMSO
c. HCl(aq)
THF
O O SBr
N
S
HN
N
TsN HN
HN
O
O
6a 6b 6c 6d
6e 6f 6g
Scheme 2-10. General synthetic scheme for heterocyclic chiral vicinal diamines 6a–g.
18
ethanol would be the better solvent for large scale syntheses due to its lower toxicity, lower envi-
ronmental impact, and lower costs.21, 22
Initially, ethanol was used as the solvent for the synthesis of heterocyclic diimines, but a num-
ber of complications arose. In some cases, the addition of the aldehyde did not turn the reaction
mixture homogeneous. In others, the rearranged diimine 5’ was isolated as a thick gum as a com-
plex mixture of the aldehyde and reaction intermediates. In another case, the only recovered solid
was a small amount of initial diimine 5. In addition, as some heterocyclic diimines 5’ were more
soluble in ethanol than corresponding phenyl-based diimines, isolated yields were sometimes
dramatically lowered when ethanol was employed. The greater solubility is possibly related to fa-
vourable solvent-solute interactions as ethanol can form hydrogen bonds with both donors and
acceptors on the heterocycle.
The use of the polar aprotic DMSO proved to be a good general solvent for the synthesis of all
the heterocyclic diimines (5’a–g) described here (Scheme 2-10). In this synthesis, enantiopure di-
amine 3 was fully dissolved in DMSO at room temperature to make a 0.3 M solution, then 2.5
equivalents of aldehyde 4 was added either as a weighed solid (4b, e, f, g) or as a liquid added by a
volumetric syringe (4a, c, d) in one portion. The homogeneous reaction mixture was allowed to
stir at room temperature until only the diimine 5’ and excess aldehyde 4 is detected by 1H NMR. If
the reaction was not complete after approximately 15 h, it was heated at 60°C for 1.5 h before cool-
ing to room temperature prior to isolation. The room temperature reaction mixture was then very
slowly added to five volumes of vigorously stirring brine that had been cooled to -15°C to cause the
precipitation of diimine 5’ as a solid. The solid should be allowed to stir in brine for approximately
1 h to ensure the formation of a good powder. If the DMSO solution was added too quickly, if the
brine was not stirring fast enough, or if room temperature brine was used, the diimine would
clump together to form either a thick gum or a sticky solid that heavily reduced yield when filtered.
1H NMR analysis showed that this is caused by the incorporation of a large amount of aldehyde 4
into the product, and thus could be prevented by the slow addition of the reaction mixture. After
the successful collection of solid 5’ by vacuum filtration, the DMSO-brine mixture was washed
away with at least 10 volumes of distilled water. The diimines were generally allowed to dry under
vacuum for 24 h before proceeding to hydrolysis.
Upon the addition of the first equivalent of aldehyde 4 to diamine 3 in DMSO, an intermediate
imidazolidine 11 can be detected by 1H NMR, and it exists in equilibrium with monoimine 10,
which attacks another equivalent of aldehyde to form 5 which then rearranges to form 5’. Howev-
er, 10 and 5 are short-lived species, as 1H NMR spectra taken over time only shows 11 converting
19
to 5’. Imidazolidines 10 can be easily identified by an AB quadruplet 4.4 ppm (C*-H) and a sharp
singlet at approximately 5.3 ppm (NH-CHAr-NH), and diimines 5’ can be identified by sharp sin-
glets at approximately 5.3 ppm (C*-H), 8.5 ppm (CH=N), and a broad singlet at approximately 13.0
ppm (RAHB O-H). A representative example for the reaction of (R,R)-3 with 4a to give (R,R)-5’ai in
70 mM DMSO-d6 at room temperature can be seen in Scheme 2-11and Figure 2-1.
Upon drying, diimines 5’ were dissolved in THF (0.1 M) and concentrated hydrochloric acid was
added to the solution at room temperature. Usually, the product diamines 6 precipitate out of the
solution from immediately to up to 60 minutes upon the addition of the acid. The solid was allowed
to stir in the acidic solution for an additional 30 minutes to ensure the formation of a fine powder.
The solid was then collected by vacuum filtration, and the precipitate is washed thoroughly with
THF and allowed to dry. Most of the diamines were analytically pure by 1H NMR after the washing;
however, 6c and 6d needed to be purified by recrystallization from a methanol, water, and THF
system. The diamines were obtained at good to excellent overall yields (68–90%, Table 2-1) with
excellent enantiopurity (>99%).
i An important point about nomenclature and Cahn-Ingold-Prelog (CIP) rules when dealing with heterocyclic dia-
mines must be made here. At the stereogenic centre of diamines with phenyl or alkyl groups, CIP rules assign the
groups attached to the carbon in decreasing priority as follows: -NH2 > -CHArNH2 > -Ar > -H. When both the start-
ing and synthesized diamines have the same priority order, the R and S nomenclature reflect the inversion in ste-
reochemistry (ie. (S,S) diimines rearrange to (R,R) diimines). However, the presence of a heteroatom (N, O, S) in
the 2-position of the heterocycle gives it the highest priority. Thus in these cases, the priority rules around the ste-
reogenic centre is: -Ar > -NH2 > -CHArNH2 > -H. Because the starting diamine 3 and diamines 6a–e are named us-
ing different priority orders, in these cases (S,S) diimines rearrange to (S,S) diimine even though an inversion of
the sense of stereochemistry still occurs. Diamines 6f−g have a carbon atom in the 2-position so it follows the for-
mer priority rankings. In reviewing literature on these diamines, extra care is recommended to ensure that no er-
rors in nomenclature have been made by the authors.
20
Figure 2-1. Partial 1H NMR spectra of the reaction of (R,R)-3a and 4a in DMSO-d6 (70 mM) at 1 h,
2h, and 7 h; of isolated (R,R)-5’a in DMSO-d6.
Scheme 2-11. Intermediates
formed in the reaction of
(R,R)-3a and 4a to produce (R,R)-5’a.
21
Table 2-1. Synthesized heterocyclic diamines and respective isolated yields.
Product Overall
Yield (%) Product
Overall
Yield (%)
6a
90
6e
72
6b
77
6f
82
6c
84
6g
68
6d
83a
a – Yield assuming 6d is the dihydrochloride salt
2.2.3 Determination of Enantiopurity
The enantiopurity of the product diamines were determined by 1H NMR using either (1R)-
myrtenal (12) as the chiral derivatizing agent in a method developed described by Yen23, or by us-
ing (1R)-2,2’-dihydroxy-(1,1’-binaphthalene)-2-carboxaldehyde (14).23,24
22
Scheme 2-12. Chiral diamines 6 react with enantiopure aldehyde R-12 to give two diastereomers.
The protons of the two diastereomers have different chemical shifts on 1H NMR. The enantiopurity
of 6 can be determined by integration ratio of the peaks belonging to (R,R,R)- and (S,S,R)-13.
(1R)-(−)-Myrtenal is a commercially-available chiral terpenoid aldehyde that has been used
previously by Mangeney et al. and Dufrasne et al. to determine the enantiopurity of amines. Alde-
hyde 14 was an intermediate in the synthesis of a chiral shift reagent developed by Chin et al. to
determine the enantiopurity of amino acids (see Chapter 3).ii
Although the enantiomeric diamine salts (R,R)-6 and (S,S)-6 have identical 1H NMR spectra, they
become the diastereomers (R,R,R)-13 and (S,S,R)-13 respectively upon addition
to an enantiopure sample of R-12 (Scheme 2-12), and the same occurs when the
diamines add to R-14 to give the diastereomers (R,R,R)-15 and (S,S,R)-15. Dia-
stereomers have different physical properties, and also have different 1H NMR
spectra. Successful chiral shift reagents, like R-12 and R-14, produce diastere-
omers whose spectra are different enough to have clear baseline separation of at
least one of the proton peaks. By integrating both sets of peaks, the molar ratio of
the two diastereomers in the sample can be determined. Further discussion on chiral shift rea-
gents, particularly in the context of determination of the enantiopurity of amino acids, can be found
in Chapter 3.
The partial 1H NMR spectra of the diagnostic peaks in the diastereomeric diimines 13 (Figure
2-2) and 15 (Figure 2-3) show that both enantiomers of the seven diamines synthesized were pro-
duced with >99% ee. Different diamines were analyzed under different conditions—see section
2.3.3 for details on the method for each diamine.
ii The synthesis of 14 can be found in the supplementary information of reference 24.
23
Figure 2-2. Partial 1H NMR spectra for diimines 13d, f, and g. The enantiopurities of diamines 6d,
6f, and 6g were determined using R-12 to form the diastereomeric diimines 13. The 1H NMR of
13d and 13f were obtained in CDCl3, and the 1H NMR of 13g was in DMSO-d6. The upper trace of each set of spectra was obtained by starting with a mixture of (R,R)-6 and (S,S)-6.
24
2.2.4 Determination of Protonation State
Diamines 6d, e, and g contain nitrogen atoms in their heterocycles that could be protonated
during the acidic hydrolysis step in addition to the two amine groups. It is difficult to determine
whether these diamines exist as the dihydrochloride (if heterocycle was not protonated) or tetra-
hydrochloride (if heterocycle was protonated) salt simply by looking at their 1H NMR spectra, as
the N-H protons are exchangeable and often come out as a broad peak, reducing the accuracy of
integration. Mass spectrometry also leads to inconclusive results as it is difficult to tell the protona-
Figure 2-3. Partial 1H NMR spectra for diimines 15a–c, and e. The enantiopurities of diamines 6a–
c, and 6e were determined using R-14 to form the diastereomeric diimines 15. The 1H NMR of
15a–c were obtained in CDCl3, and the 1H NMR of 13e was in DMSO-d6. The upper trace of each set
of spectra was obtained by starting with a mixture of (R,R)-6 and (S,S)-6.
25
tion state of the substrate prior to ionization. To that end, Chin and co-workers have developed an
ingenious method to determine whether a diamine salt is di- or tetraprotonated without having to
resort to gravimetric analysis or acid-base titration.
First, the molecular weight of both the dihydrochloride (MW2) and tetrahydrochloride (MW4)
salt of a diamine is calculated. Using the molecular weight of the dihydrochloride salt, the mass of
0.05 mmol (n2) of the diprotonated compound (m2) is calculated. Then, the number of millimoles of
the tetrahydrochloride salt (n4) that mass m2 represents is calculated. Mass m2 of the diamine 6 is
weighed out, placed in 0.7 mL DMSO-d6, and to that is added 5n2 moles (0.25 mmol) of triethyla-
mine to fully neutralize and fully solubilize 6. After neutralization, 3n2 moles (0.15 mmol) of salic-
ylaldehyde is added to form diimine 5’. A 1H NMR spectrum is obtained of this mixture, and the in-
tegration ratio between diimine 5’ and salicylaldehyde is determined. If the molar ratio between
5’ and salicylaldehyde is 1:1, then the diamine is the dihydrochloride salt, as n2 moles of 6 reacts
with 2n2 moles of salicylaldehyde, leaving n2 moles excess. However, if the diamine is the tetrahy-
drochloride salt, the molar ratio would be1:������
��
, as only 2n4 moles of salicylaldehyde reacts,
leaving 3n2−2n4 moles excess.
The diamine salt 6e can be used as an example of
how its protonation state was determined (Scheme
2-13). First, 13.3 mg of 6e (n2 = 0.05 mmol, n4 = 0.04
mmol) is added to 0.7 mL DMSO-d6, followed by the
addition of 35 μL triethylamine (0.25 mmol) and 16 μL
salicylaldehyde (0.15 mmol). After 1 h, the 1H NMR
spectrum was obtained and it was determined that
the integration ratio of the diimine peaks and the sa-
licylaldehyde peaks was 2 :1, thus indicating a 1 : 1
molar ratio. Therefore, 6e exists as the dihydrochloride salt.
Unfortunately, the protonation state of diamine 6d was not able to be obtained as it appeared to
decompose under these conditions. However, for the purpose of yield calculations, it was assumed
to exist as the dihydrochloride salt as thiazoles are generally less basic than imidazoles.
Scheme 2-13. Structure and molecular
weight of 6e·2HCl and 6e·4HCl.
26
2.3 Experimental and Results
2.3.1 General Information
The following compounds were obtained commercially and were used without further purifica-
tion or drying: dimethylsulfoxide (DMSO), furfural (2-furaldehyde), 5-bromo-2-furaldehyde, 2-
thiophenecarboxaldehyde, 2-thiazolecarboxaldehyde, 4-imidazolecarboxaldehyde, 1-(p-
toluenesulfonyl)-3-indolecarboxaldehyde, 5-formyluracil, sodium chloride, tetrahydrofuran (THF),
concentrated (37%) hydrochloric acid, methanol, triethylamine, (1R)-myrtenal, DMSO-d6, D2O, and
CDCl3 (0.05% v/v TMS). The following compounds were synthesized by the Chin research group:
(R,R)- and (S,S)-1,2-bis(2-hydroxyphenyl)ethane-1,2-diamine, and (1R)-2,2’-dihydroxy-(1,1’-
binaphthalene)-2-carboxaldehyde. All reactions were held under an ambient atmosphere. Spec-
trometers used to record 1H and 13H nuclear magnetic resonance (NMR) spectra were the Varian
Mercury 400, the Varian NMR System 400, and the Bruker Avance III (400) at The Centre for Spec-
troscopic Investigation of Complex Organic Molecules and Polymers in the Department of Chemis-
try at the University of Toronto. High resolution mass spectra (HRMS) were recorded at the Ad-
vanced Instrumentation for Molecular Structure facility in the Department of Chemistry at the Uni-
versity of Toronto.
2.3.2 General Procedures
Diaza-Cope Rearrangement. To a stirring solution of 250 mg (1.02 mmol) of (R,R)- or (S,S)-1,2-
bis(2-hydroxyphenyl)ethane-1,2-diamine [(R,R)-or (S,S)-3] in 10.2 mL DMSO (100 mM) was added
3.06 mmol (3 equiv) of the appropriate aryl aldehyde in one portion. The reaction was allowed to
stir at room temperature for 15 h or until complete by NMR (DMSO-d6). The reaction mixture was
then poured slowly into a beaker with vigorously stirring -15°C brine (50 mL) to cause the precipi-
tation of the rearranged diimine (5’a–g). The diimine suspension in brine was allowed to stir for
30 min., or until it had warmed to room temperature, before the solid diimine was collected by
vacuum filtration. The diimine was washed with at least 125 mL distilled water and allowed to dry
under vacuum. The diimine was not isolated or purified and was used directly for the next step.
Hydrolysis. To a stirring solution of 1.00 mmol diimine (5’a–g) from the diaza-Cope rearrange-
ment in 10 mL THF (100 mM) is added 300 μL 37% aqueous hydrochloric acid solution. The reac-
tion was allowed to stir at room temperature for 1 h or until the product diamine salt (6a–g) had
completely precipitated out as a fine powder. The diamine was collected by vacuum filtration,
washed with 20 mL THF, and allowed to dry under vacuum for 2 h. If required, the diamine can be
recrystallized from methanol/water/THF.
27
Proof of Protonation State. Into a small glass vial the diamine salts 6d, e, and g (0.05 mmol of the
presumed dihydrochloride salt, see section 2.2.4 for explanation) were dissolved in 0.70 mL
DMSO-d6, followed by the addition of 35 μL triethylamine (0.25 mmol) and 16 μL salicylaldehyde
(0.15 mmol). The solution was allowed to stir for 1 h at room temperature before the 1H NMR spec-
trum was obtained.
2.3.3 Specific Procedures and Results
1,2-bis(furan-2-yl)ethane-1,2-diamine dihydrochloride (6a)
6a was obtained as an off-white crystalline solid with a 90% overall yield and was analytically pure
without recrystallization. 6a is a known compound and has been previously prepared.25-27
1H NMR (400 MHz, DMSO-d6, ppm): δ = 9.17 (br s, 6H, NH), 7.68 (dd, J = 1.8, 0.8 Hz, 2H, ArH), 6.51
(dd, J = 3.4, 0.8 Hz, 2H, ArH), 6.44 (dd, J = 3.4, 1.8 Hz, 2H, ArH), 5.09 (s, 2H, C*H).
13C NMR (100 MHz, D2O, ppm): δ = 145.3, 142.7, 112.7, 111.1 (4 aromatic carbons), 49.1 (1 aliphat-
ic carbon).
Enantiopurity was confirmed using (1R)-2,2’-dihydroxy-(1,1’-binaphthalene)-2-carboxaldehyde
(14) as the chiral shift reagent. To 1.1 mg of enantiopure 6a (0.004 mmol) dissolved in 0.40 mL
methanol was added 1.7 μL triethylamine (0.012 mmol, 3.0 equiv), followed by 3.8 mg of 14 (0.012
mmol, 3.0 equiv). The solution was allowed to stir overnight, and when the methanol was removed
by evaporation. The solid diimine 15a was re-dissolved in 0.60 mL CDCl3 to give the following di-
agnostic peaks by 1H NMR: (S,S)-15a δ = 5.09 ppm; (R,R)-15a δ = 5.13 ppm.
1,2-bis(5-bromofuran-2-yl)ethane-1,2-diamine dihydrochloride (6b)
6b was obtained as a white crystalline solid with a 77% overall yield and was analytically pure
without recrystallization.
28
1H NMR (400 MHz, DMSO-d6, ppm): δ = 8.96 (br s, 6H, NH), 6.64 (d, J = 3.4 Hz, 2H, ArH), 6.61 (d, J =
3.4 Hz, 2H, ArH), 4.99 (s, 2H, C*H).
13C NMR (100 MHz, DMSO-d6, ppm): δ = 147.4, 122.9, 114.8, 112.9 (4 aromatic carbons), 49.1 (1
aliphatic carbon).
HRMS (ESI) calculated for C10H10Br2N2O2 [M+H]+: 348.9181, found: 348.9187.
Enantiopurity was confirmed using (1R)-2,2’-dihydroxy-(1,1’-binaphthalene)-2-carboxaldehyde
(14) as the chiral shift reagent. To 1.7 mg of enantiopure 6b (0.004 mmol) dissolved in 0.40 mL
methanol was added 1.7 μL triethylamine (0.012 mmol, 3.0 equiv), followed by 3.8 mg of 14 (0.012
mmol, 3.0 equiv). The solution was allowed to stir overnight, and when the methanol was removed
by evaporation. The solid diimine 15b was re-dissolved in 0.60 mL CDCl3 to give the following di-
agnostic peaks by 1H NMR: (S,S)-15b δ = 5.00 ppm; (R,R)-15b δ = 5.04 ppm.
1,2-bis(thiophen-2-yl)ethane-1,2-diamine dihydrochloride (6c)
6c was obtained as a white crystalline solid with an 84% overall yield and was analytically pure
after recrystallization. 6c is a known compound and has been previously prepared.25
1H NMR (400 MHz, DMSO-d6, ppm): δ = 9.20 (br s, 6H, NH), 7.55 (dd, J = 5.1, 1.1 Hz, 2H, ArH), 7.28
(dd, J = 3.6, 1.1 Hz, 2H, ArH), 7.01 (dd, J = 5.1, 3.6 Hz, 2H, ArH), 5.27 (s, 2H, C*H).
13C NMR (100 MHz, DMSO-d6, ppm): δ = 134.4, 129.2, 128.0, 127.1 (4 aromatic carbons), 52.5 (1
aliphatic carbon).
Enantiopurity was confirmed using (1R)-2,2’-dihydroxy-(1,1’-binaphthalene)-2-carboxaldehyde
(14) as the chiral shift reagent. To 1.2 mg of enantiopure 6c (0.004 mmol) dissolved in 0.40 mL
methanol was added 1.7 μL triethylamine (0.012 mmol, 3.0 equiv), followed by 3.8 mg of 14 (0.012
mmol, 3.0 equiv). The solution was allowed to stir overnight, and when the methanol was removed
by evaporation. The solid diimine 15c was re-dissolved in 0.60 mL CDCl3 to give the following di-
agnostic peaks by 1H NMR: (S,S)-15c δ = 5.02 ppm; (R,R)-15c δ = 5.07 ppm.
29
1,2-bis(thiazol-2-yl)ethane-1,2-diamine hydrochloride (6d)
The diaza-Cope rearrangement to make 5’d required an additional 1.5 h of heating at 60°C after 15
h room temperature stirring to reach completion. After hydrolysis and filtration, 6d should be
washed with 10 mL cold methanol after the THF washing. 6d was obtained as an off-white crystal-
line solid with an 83% overall yield and was analytically pure after recrystallization.
1H NMR (400 MHz, DMSO-d6, ppm): δ = 8.88 (br s, 6H, NH), 7.88 (d, J = 3.2 Hz, 2H, ArH), 7.80 (d, J =
3.2 Hz, 2H, ArH), 5.42 (s, 2H, C*H).
13C NMR (100 MHz, DMSO-d6, ppm): δ = 159.6, 143.6, 123.7 (3 aromatic carbons), 52.9 (1 aliphatic
carbon).
HRMS (ESI) calculated for C8H10N4S2 [M+H]+: 227.0419, found: 227.0418.
Enantiopurity was confirmed using (1R)-(−)-myrtenal (12) as the chiral shift reagent. To 7.5 mg of
enantiopure 6d (0.025 mmol) dissolved in 0.50 mL methanol was added 10.5 μL triethylamine
(0.075 mmol, 3.0 equiv), followed by 11.4 μL of 12 (0.075 mmol, 3.0 equiv). The solution was al-
lowed to stir overnight, and when the methanol was removed by evaporation. The solid diimine
13d was re-dissolved in 0.60 mL CDCl3 to give the following diagnostic peaks by 1H NMR: (S,S)-13d
δ = 5.04 ppm; (R,R)-13d δ = 5.00 ppm.
1,2-bis(imidazol-4-yl)ethane-1,2-diamine dihydrochloride (6e)
NH2
NH2
HN
N
HN
N� 2 HCl
The hydrolysis of 5’e was conducted at 50°C due to poor solubility of the diimine in THF. 6e was
obtained as a light yellow crystalline solid with a 72% overall yield and was analytically pure with-
out recrystallization.
30
1H NMR (400 MHz, DMSO-d6, ppm): δ = 8.80 (s, 2H, ArH), 7.74 (s, 2H, ArH), 5.30 (s, 2H, C*H).
13C NMR (100 MHz, D2O, ppm): δ = 136.5, 125.1, 120.6 (3 aromatic carbons), 48.0 (1 aliphatic car-
bon).
HRMS (ESI) calculated for C8H13N6 [M+H]+: 193.1196, found: 193.1200.
Enantiopurity was confirmed using (1R)-2,2’-dihydroxy-(1,1’-binaphthalene)-2-carboxaldehyde
(14) as the chiral shift reagent. To 1.1 mg of enantiopure 6e (0.004 mmol) dissolved in 0.60 mL
DMSO-d6 was added 1.7 μL triethylamine (0.012 mmol, 3.0 equiv), followed by 3.8 mg of 14 (0.012
mmol, 3.0 equiv). The solution was mixed well and after 1 h the solution gave the following diag-
nostic peaks by 1H NMR: (S,S)-15e δ = 5.26 ppm; (R,R)-15e δ = 5.23 ppm.
1,2-bis(N-p-toluenesulfonyl-indol-3-yl)ethane-1,2-diamine dihydrochloride (6f)
The diaza-Cope rearrangement to make 5’f required an additional 1.5 h of heating at 60°C after 15
h room temperature stirring to reach completion. 6f was obtained as white crystalline solid with
an 82% overall yield and was analytically pure without recrystallization.
1H NMR (400 MHz, DMSO-d6, ppm): δ = 9.13 (br s, 2H, NH), 8.23 (s, 2H, ArH), 7.94 (d, J = 7.6 Hz, 2H,
ArH), 7.67 (d, J = 8.2 Hz, 2H, ArH), 7.42 – 7.22 (m, 8H, ArH), 7.03 (d, J = 8.2 Hz, 4H, ArH), 5.61 (s,
2H, C*H), 2.22 (s, 6H, CH3).
13C NMR (100 MHz, DMSO-d6, ppm): δ = 145.3, 133.6, 133.0, 130.0, 129.1, 126.7, 126.3, 125.2,
123.5, 119.7, 115.2, 112.8 (12 aromatic carbons), 48.5, 21.0 (2 aliphatic carbons).
HRMS (ESI) calculated for C32H30N4 O4S2 [M+H]+: 599.1781, found: 599.1802.
Enantiopurity was confirmed using (1R)-(−)-myrtenal (12) as the chiral shift reagent. To 16.8 mg
of enantiopure 6f (0.025 mmol) dissolved in 0.50 mL methanol was added 10.5 μL triethylamine
(0.075 mmol, 3.0 equiv), followed by 11.4 μL of 12 (0.075 mmol, 3.0 equiv). The solution was al-
lowed to stir overnight, and when the methanol was removed by evaporation. The solid diimine
31
13f was re-dissolved in 0.60 mL CDCl3 to give the following diagnostic peaks by 1H NMR: (R,R)-13f
δ = 4.91 ppm; (S,S)-13f δ = 4.86 ppm.
1,2-bis(uracil-5-yl)ethane-1,2-diamine dihydrochloride (6g)
The diaza-Cope rearrangement to make 5’g required an additional 1.5 h of heating at 60°C after 15
h room temperature stirring to reach completion. The hydrolysis of 5’g was conducted at 50°C due
to poor solubility of the diimine in THF. 6g was obtained as white crystalline solid with a 68%
overall yield and was analytically pure without recrystallization.
1H NMR (400 MHz, DMSO-d6, ppm): δ = 11.45 (s, 2H, C(O)NHC(O)), 11.30 (d, J = 6.0 Hz, 2H,
C(O)NHCH), 8.70 (br s, 6H, NH), 7.66 (d, J = 6.0 Hz, ArH), 4.70 (s, 2H, C*H).
13C NMR (100 MHz, D2O, ppm): δ = 164.7, 152.2, 144.8, 104.5 (4 aromatic carbons), 52.2 (1 aliphat-
ic carbon).
HRMS (ESI) calculated for C10H13N6O4 [M+H]+: 281.0992, found: 281.0991.
Enantiopurity was confirmed using (1R)-(−)-myrtenal (12) as the chiral shift reagent. To 8.8 mg of
enantiopure 6g (0.025 mmol) dissolved in 0.65 mL DMSO-d6 was added 10.5 μL triethylamine
(0.075 mmol, 3.0 equiv), followed by 11.4 μL of 12 (0.075 mmol, 3.0 equiv). The solution was
mixed well and after 1 h the solution gave the following diagnostic peaks by 1H NMR: (R,R)-13g δ =
7.29 ppm; (S,S)-13g δ = 7.35 ppm.
32
2.4 Conclusions and Future Directions
In this work, both enantiomers of seven heterocyclic chiral vicinal diamines were synthesized
using the diaza-Cope rearrangement method. Enantiopure (>99% ee) diamines, five of which are
novel compounds, were prepared at good to excellent overall yields (68–90%). This facile method
avoids the use of metal catalysts, inert atmospheres, harsh conditions, or any need for column
chromatography. This protocol has shown to be effective from milligram to gram scales, making
the large-scale production of these diamines comparatively simple.
Because of the versatility of the chiral vicinal diamine moiety, there are a number of directions
this development can lead to in the near future. As one example, diamine 6g contains two uracil
moieties which can be employed in both the in vitro and in vivo molecular recognition of biomole-
cules with the adenine moiety such as ATP and NAD(H). As discussed earlier, the heterocyclic
amine moiety is found in a variety of neurotransmitters and pharmaceuticals. The availability of
these diamines now may lead to the development of new useful drugs that targets these receptors.
In catalysis, a well-placed hydrogen donating or accepting group in an organocatalyst or as a
ligand for organometallic catalysts can provide extra stability towards a particular transition state
structure to increase reaction rate and selectivity. Nitrogen heterocycles like imidazole and thia-
zole (moieties in 6d and 6e respectively) can also be exploited for acid/base properties as activity
of a catalyst or drug can be modulated by the protonation and deprotonation, or cyclization or
opening of the heterocycle.
Heterocyclic diamines have the advantage of having hydrogen bond acceptors, hydrogen bond
donors, or both, in their structures. This feature can make these diamines very useful as part of
molecular receptors, catalytic ligands, and organocatalysts. Because heterocycles are pervasive in
natural biomolecule and synthetic pharmaceuticals, these heterocyclic diamines can play a key role
in the discovery and synthesis of new drugs. Adding heterocycles to the library of diamines pro-
duced by Chin and colleagues will allow researchers to better “tune” their receptors, catalysts, or
drugs to make them more effective.
2.5 Spectra
All proton (1H) and carbon (13C) NMR spectra were processed using MestReNova (ver. 6.1.1-
6384) from Mestrelab Research S.L. The proton in DMSO-d5 has appears as a quintet at 2.50 ppm in
DMSO-d6. The proton in H2O and HDO appear as a broad singlet at approximately 3.4 ppm in
DMSO-d6. The carbon of DMSO-d6 appears as a septet at 39.52 ppm.
33
1,2-bis(furan-2-yl)ethane-1,2-diamine dihydrochloride (6a)
34
1,2-bis(5-bromofuran-2-yl)ethane-1,2-diamine dihydrochloride (6b)
012345678910ppm (1H)
0102030405060708090100110120130140150160170ppm (13C)
35
1,2-bis(thiophen-2-yl)ethane-1,2-diamine dihydrochloride (6c)
012345678910ppm (1H)
36
1,2-bis(thiazol-2-yl)ethane-1,2-diamine hydrochloride (6d)
012345678910ppm (1H)
0102030405060708090100110120130140150160170ppm (13C)
37
1,2-bis(imidazol-4-yl)ethane-1,2-diamine dihydrochloride (6e)
012345678910ppm (1H)
0102030405060708090100110120130140150160170ppm (13C)
38
1,2-bis(N-p-toluenesulfonyl-indol-3-yl)ethane-1,2-diamine dihydrochloride (6f)
012345678910ppm (1H)
0102030405060708090100110120130140150160170ppm (13C)
39
1,2-bis(uracil-5-yl)ethane-1,2-diamine dihydrochloride (6g)
0123456789101112ppm (1H)
0102030405060708090100110120130140150160170180ppm (13C)
40
2.6 References
(1) Cope, A. C.; Hardy, E. M. J. Am. Chem. Soc. 1940, 62, 441-444.
(2) Clayden, J.; Greeves, N.; Warren, S.; Wothers, P. Organic Chemistry; Oxford University Press:
New York, 2006 .
(3) Vögtle, F.; Goldschmitt, E. Angew. Chem. Int. Ed. Engl. 1973, 12, 767-768.
(4) Vögtle, F.; Goldschmitt, E. Angew. Chem. Int. Ed. Engl. 1974, 13, 480-482.
(5) Vögtle, F.; Goldschmitt, E. Chem. Ber. 1976, 109, 1-40.
(6) Gilli, G.; Bellucci, F.; Ferretti, V.; Bertolasi, V. J. Am. Chem. Soc. 1989, 111, 1023-1028.
(7) Bertolasi, V.; Gilli, P.; Ferretti, V.; Gilli, G. J. Am. Chem. Soc. 1991, 113, 4917-4925.
(8) Gilli, P.; Bertolasi, V.; Ferretti, V.; Gilli, G. J. Am. Chem. Soc. 2000, 122, 10405-10417.
(9) Chin, J.; Mancin, F.; Thavarajah, N.; Lee, D.; Lough, A.; Chung, D. S. J. Am. Chem. Soc. 2003, 125,
15276-15277.
(10) Kim, H.; Kim, H.; Alhakimi, G.; Jeong, E. J.; Thavarajah, N.; Studnicki, L.; Koprianiuk, A.; Lough,
A. J.; Suh, J.; Chin, J. J. Am. Chem. Soc. 2005, 127, 16370-16371.
(11) Kim, H.; Nguyen, Y.; Lough, A.; Chin, J. Angew. Chem. Int. Ed. 2008, 47, 8678-8681.
(12) Kim, H.; Staikova, M.; Lough, A.; Chin, J. Org. Lett. 2009, 11, 157-160.
(13) Kim, H.; Nguyen, Y.; Yen, C. P.; Chagal, L.; Lough, A. J.; Kim, B. M.; Chin, J. J. Am. Chem. Soc. 2008,
130, 12184-12191.
(14) Kim, H.; So, S. M.; Chin, J.; Kim, B. M. Aldrichim. Acta 2008, 41, 77-88.
(15) Kim, H. Hydrogen Bond-Directed Stereospecific Interactions in A) General Synthesis of Chiral
Vicinal Diamines and B) Generation of Helical Chirality with Amino Acids, University of Toron-
to, Toronto, 2009.
(16) Pereillo, J.; Maftouh, M.; Andrieu, A.; Uzabiaga, M.; Fedeli, O.; Savi, P.; Pascal, M.; Herbert, J.;
Maffrand, J.; Picard, C. Drug Metab. Disposition 2002, 30, 1288-1295.
(17) Joule, J. A.; Mills, K. Heterocyclic Chemistry, Fifth Edtion; Wiley: Chichester, 2010.
(18) Foti, C.; Cassano, N.; Panebianco, R.; Calogiuri, G. F.; Vena, G. A. Immunopharmacology and Im-
munotoxicology 2009, 31, 414-416.
(19) Berger, M.; Gray, J. A.; Roth, B. L. Annu. Rev. Med. 2009, 60, 355-366.
41
(20) Altun, A.; Ugur-Altun, B. Intl. J. Clin. Prac. 2007, 61, 835-845.
(21) Capello, C.; Fischer, U.; Hungerbühler, K. Green Chem. 2007, 9, 927-934.
(22) Alfonsi, K.; Colberg, J.; Dunn, P. J.; Fevig, T.; Jennings, S.; Johnson, T. A.; Kleine, H. P.; Knight, C.;
Nagy, M. A.; Perry, D. A.; Stefaniak, M. Green Chem. 2008, 10, 31-36.
(23) Yen, C. P. H. Highly Stereospecific Generation of Helical Chirality by Vicinal Diamine: Determi-
nation of Enantiomeric Purity, University of Toronto, Toronto, 2008.
(24) Chin, J.; Kim, D. C.; Kim, H.; Panosyan, F. P.; Kim, K. M. Org. Lett. 2004, 6, 2591-1593.
(25) Roskamp, E. J.; Pedersen, S. F. J. Am. Chem. Soc. 1987, 109, 3152-3154.
(26) Betschart, C.; Schmidt, B.; Seebach, D. Helv. Chim. Acta. 1988, 71, 1999-0201.
(27) Kise, N.; Ueda, N. Tet. Lett. 2001, 42, 2365-2368.
42
CHAPTER 3
Chiral Cyclic Guanidine for Determining Enantiopurity and
Absolute Configuration of Amino Acids
This chapter describes the development of a new cyclic guanidine 1 that is used to determine
the enantiopurity and the absolute configuration of α-amino acids using proton nuclear magnetic
resonance (1H NMR) spectroscopy. Representative examples of the twenty proteinogenic amino
acids were chosen to demonstrate the application of 1 as a chiral shift reagent (CSR) in a CDCl3-
DMSO-d6 solvent mixture. This method differs from many previously described procedures of de-
termining amino acid enantiopurity as it does not require prior chemical modification of the sam-
ple amino acid.
3.1 Methods of Determining Enantiopurity and Absolute Configuration of Amino
Acids
3.1.1 Circular Dichroism and Ultraviolet-Visible Spectroscopy
Circular dichroism (CD) and ultraviolet-visible (UV-vis) spectroscopy are both forms of absorp-
tion spectroscopy that can be used to determine the enantiopurity of amino acids. In CD spectros-
copy, right (clockwise) and left (counter-clockwise) circularly polarized light is passed through a
chiral environment (a solution of the amino acid sample). Chiral matter absorbs right circularly
polarized light (εR) to a different extent than left circularly polarized light (εL).1 The CD of a sample
at a particular wavelength (λ) is defined as the difference in absorption between the two forms of
light (CDλ ≡ Δε = εL − εR), which is often reported as the degree of ellipticity.1 Because the CD of en-
antiomers have the same amplitude but opposite signs for each wavelength, CD can be used to de-
termine the enantiopurity and possibly the absolute configuration of an amino acid sample.
43
Chin et al. have developed a universal sensor for amino acid enantiopurity based on CD and the
generation of helical chirality. Dihydroxybenzophenone (2) is not flat planar molecule—it exists as
a pair of enantiomers (P-2, M-2) with helical (twist) chirality. Bond rotation freely occurs in solu-
tion, and since the enantiomers have the same energy, a solution of 2 exists
as a racemic mixture. However, when a chiral amino acid is added and
imine formation occurs, the two helical forms become diastereomers and
one is strongly preferred due to steric effects and hydrogen bonding
(Scheme 3-1). Their addition of azo moieties to the commercially-available
dihydroxybenzophenone to give 3 amplified the CD absorption of the amino
acid-receptor complex. Unlike previous reports, they found that the CD pat-
terns for twelve proteinogenic amino acids were similar, and that the receptor interacted with the
amino acids with a high degree of stereospecificity. The enantiomeric excess of an amino acid
sample can then be determined by simply measuring the ratio of its CD and its UV-vis absorption at
a particular wavelength.
Other receptors for amino acids for CD analysis have been reported. For example Huang et al.
reported a chiral zinc-centred bisporphyrin tweezer that can be used to determine the absolute
configuration of amino acids by CD.2 However, unlike the above method that can use unmodified
amino acids, Huang’s method requires a two-step derivatization process.2 In a method developed
by Holmes et al., amino acids are reacted with 2-bromomethylquinoline and then complexed to a
copper (II) salt to create helical structures around the coordination sites.3 The chirality of the twist
is controlled by the chirality of the amino acid and the resulting CD spectra can be used to deter-
mine enantiomeric excess and absolute configuration.3
Anslyn et al. have recently developed a method to detect enantiopurity by using UV-vis absorp-
tion spectroscopy alone, without employing polarized light.4 A chiral receptor based on (S,S)- and
(R,R)- diaminocyclohexane is coordinated to two sites on a copper (II) centre, with a dye (CAS) oc-
cupying the two other coordination sites.5 The receptor-CAS complex absorbs at 602 nm (blue)
OOH OH
N NNN
3
Scheme 3-2. (below) The attachment of azo groups to 2 amplifies the CD signals for recep-tor 3.
Scheme 3-1. (left) The imine formed between 2 and L-Ala exist predominantly as the P helix in aprotic solvents
44
while free CAS absorbs at 429 nm (yellow).5 Both L- and D-amino acidsi can displace CAS from cop-
per, but D-amino acids were found to bind more tightly to the (S,S)-receptor.5 In this “indicator dis-
placement assay”, the ee of the added amino acid can be determined by a simple linear equation
from measuring absorbance at λmax of the receptor-CAS complex.4 A sample enriched in an L-amino
acid is expected to have a higher absorbance at that wavelength than a D-enriched sample since it
displaces less CAS from the (S,S)-complex. An advantage of this method is its use of free (nonderi-
vatized) amino acids, as the measurements are made in a buffered water-methanol solution, and it
works for at least 13 α-amino acids to an average error of 10.2%.5
3.1.2 Nuclear Magnetic Resonance Spectroscopy
Nuclei in enantiomers appear at identical chemical shifts in nuclear magnetic resonance spec-
troscopy. However, diastereomers formed from these enantiomers may show significant differ-
ences in chemical shifts, and these diastereomers can be formed using chiral shift reagents (CSRs).
CSRs are enantiopure reagents that either react to form covalent (or sometimes ionic) bonds with
the enantiomer to be analyzed, or bind to enantiomers by non-covalent means such as hydrogen
bonding.6
An example of a CSR for determining the ee and absolute configuration of an amino acid could
be found as early as the late 1960s.7 Pirkle and Beare used (R)-2,2,2-trifluorophenylethanol (4), a
derivative of Mosher’s acid,8 as a chiral NMR solvent for 14 proteinogenic amino
acids.7 The amino acids were converted to their methyl esters in order to solubil-
ize it in the chiral ethanol.7 The resolved peaks for 13 of the amino acids (Cys was
the exception) well separated enough to be analyzed despite the fact that the au-
thors were using a 100 MHz magnet.7 It should be noted that instead of calculating the area under
each peak by integration, the authors compared the heights of the peaks, as integration was a diffi-
cult task in 1969.7
CSRs do not have to be the NMR solvent (in fact most are not), it can be added in equimolar
quantities to the amino acid if they form a 1:1 complex. Malavašič et al. recent-
ly reported diketopiperazine 5 as a CSR for N-benzoylated amino acid methyl
esters in CDCl3.9 L-Alanine methyl ester was used as the enantiopure precur-
i The absolute configuration of all natural α-amino acids will be denoted using the more commonly used L- and D- convention as opposed to the Cahn-Ingold-Prelog designations of (S) and (R). For all natural α-amino acids, except for cysteine, L- is equivalent to S, and D- is equivalent to R. For cysteine, the reverse is true.
45
sor to 5, which was formed in three steps.9 This CSR does have a good scope, as out of the twelve
amino acids attempted, only His failed to resolve.9 Unfortunately, 5 is not a very practical CSR: it
involves multi-step syntheses for both the CSR and the amino acid derivatization, and it only re-
solves the diastereotopic peaks at sub-ambient temperatures (−20°C was required for good base-
line separation).9 Chiral crown ethers can also be used as a CSR for amino acids. Machida et al. used
(R)-18-crown-6-tetracarboxylic acid as a CSR for fourteen nonderivatized proteinogenic amino
acids in D2O, and found good resolution.10
A variety of CSRs for amino acids has been used over the years and can be separated into two
classes: organic and organometallic. Organometallic CSRs like cobalt porphyrins11, P*-chiral phos-
phapalladacycles (for 31P NMR)12, cerium (III)6 and samarium (III)13 propylenediaminetetraacetate,
and dysprosium (III) diethylenetriaminepentaacetate14 were common, but most suffered from
spectral line-broadening and most were not compatible with a wide range of free amino acids. Or-
ganic CSRs do not have this line-broadening problem, but most still require some sort of amino ac-
id derivatization. For example, Kaik et al.
reported a series of CSRs (two of which, 6
and 7, are shown here) derived from enan-
tiopure diaminocyclohexane.15 Only Ala,
Val, and Phe were analyzed, and were re-
quired to be either N-Boc protected (if to
be attached through the carboxyl group as an amide to the CSR), or were transformed into their
methyl esters (if to be attached through the amino group as an urea to the CSR).15 The 1H NMR
spectra were obtained in CDCl3 and yielded very well separated peaks for the diastereomers (from
0.11–0.40 ppm separation).15
(1R)-Myrtenal, used in Chapter 2 as a CSR for vicinal diamines, has also been used as a CSR for
amino acids.16 In CD3OD under basic conditions, an imine was formed between the amino acid and
myrtenal to give two diastereomers.16 Dufrasne et al. found that this method worked well with Phe
and Trp, but failed with Met.16 Chiral aldehyde 8, developed by Chin et al., was based on (R)-binol,
and is another CSR that uses imine formation to attach to an amino acid.17 The
major advantage of using 8 is that its salicylaldehyde moiety allows the formation
of a strong resonance-assisted hydrogen bond (RAHB, see Chapter 2) between the
–OH group on 8 and the imine nitrogen on the amino acid, allowing for rapid
imine formation.17 The RHAB proton appears as a singlet very downfield on 1H
NMR, at approximately 13.5 ppm, and is therefore very well removed from other
OH
CHO
OH
8
46
proton peaks.17 Good baseline resolution was found for the nine proteinogenic amino acids test-
ed.17 The major advantage of using 8 is that no prior derivatization of amino acids was required as
the imine was formed in situ in approximately 5 minutes.17
3.1.3 Liquid Chromatography and Gas Chromatography
One of the biggest disadvantages of using liquid chromatography (LC), and gas chromatography
(GC) to determine the enantiopurity of amino acids is that derivatization of the amino acid is al-
most always required. However, at a preparative scale, LC and GC methods can be useful to resolve
and isolate the two enantiomers.
Takaya et al. were able to analyze the enantiomeric excess of wide variety of amino acids using
high-performance liquid chromatography (HPLC) by first derivatizing amino acid of interest into a
diastereomeric dipeptide by the addition of a second, enantiopure amino acid.18 More commonly,
though, chiral stationary phases are used instead. Many of these chiral columns are based on enan-
tiopure polysaccharides such as cellulose or cyclodextrin.19 Chiral stationary phases are also used
for GC: for example, Španik et al. uses cyclodextrin stationary phases to separate N-
trifluoroacetylated amino acid esters.20 One of the biggest advantages for using GC is the ability to
analyze the enantiopurity of a mixture many amino acids all at once, which would be very useful
for scientists studying complex matrices.21
3.2 Development of Cyclic Guanidine Chiral Shift Reagent for Amino Acids
3.2.1 Background
Amino acids, mostly known for their role as protein monomers, are an important class of bio-
molecules. Because of their main biological role, the twenty amino acids that are encoded by the
genetic code are known as the proteinogenic amino acids. All the proteinogenic amino acids have
the same backbone, with an amino group and the side chain (R) residing on the α-carbon with re-
spect to the carboxylate group. Amino acid side chains vary in form and function, including hydro-
phobic aliphatic hydrocarbons (e.g. Ala, Val), aromatic groups (e.g. Phe, Trp), uncharged polar
functionalities (e.g. Ser, Asn), acidic groups (e.g. Asp, Glu), and basic groups (e.g. Arg, His). This va-
riety plays a large part not only in dictating protein structure and function, but also gives amino
acids other important biological roles as biochemical precursors. For example, in vivo, Asp and Glu
are used to synthesize nucleotides22, Trp, Tyr, and Phe are precursors for monoamine neurotrans-
mitters like serotonin and epinephrine23, and Gly is employed in porphyrin biosynthesis23. Out of
47
the body, these amino acids are used as food additives like monosodium glutamate (MSG) and as-
partame (Asp-Phe methyl ester), and are valuable chiral building blocks for pharmaceuticals.23
In nature, the vast majority of amino acids have the L-configuration; however, D-Ala can be
found in bacterial cell walls as a defense mechanism against hydrolytic enzymes and antibiotics22,
24, and D-Ser was found as a neurotransmitter in mammal (rat) brains25, 26. Some L-Asp residues in
the tissue of elderly humans have been found to be capable of racemizing or epimerizing to form D-
Asp, which may affect function and cause diseases such as Alzheimer’s.27 Free D-amino acids and
their derivatives generally have very different metabolic routes than their more common enantio-
mers, and therefore may be useful as pharmaceuticals. Indeed, studies have been done to place D-
amino acid residues in antibiotic peptides in the fight against antibiotic resistance, D-Met has been
used to treat neuronal damage, and D-Phe has been used as an animal analgesic.28 D-amino acids
can also be good synthetic precursors for existing pharmaceuticals such as amoxicillin (D-Val),
seromycin (D-Ser), and tadalafil (D-Trp).28 The recent interest in D-amino acids has even spawned a
“D-Amino Acid Research Society” in Japan, which held an international conference on D-amino acid
research in 2009.29
Given the revelation of the utility of D-amino acids, it is no surprise that there
is also a rise in interest to produce an easy, efficient, and economical method of
producing D-amino acids from an L-amino acid feedstock. In nature, pyridoxal
(vitamin B6) is an important coenzyme for a range of enzymes, including amino
acids racemases.22, 30 Chin et al. have successfully produced an amino acid receptor 9 that pro-
motes the efficient conversion of L-amino acids into D-amino acids that was inspired by the salic-
ylaldehyde moiety of pyridoxal.31 Like the dihydroxybenzophenone-based enantiopurity sensors 2
and 3, resonance-assisted hydrogen bonds (RAHBs) drive the imine formation between aldehyde 9
and amino acids.31 The 1,3-diphenylurea moiety forms strong hydrogen bonds with the amino acid
carboxylate group to ensure high binding stereoselectivity (Scheme 3-3). The receptor also acted
as a CSR in DMSO-d6, so the epimerization reaction could be observed by 1H NMR.31 When the sys-
48
tem was near equilibrium, the authors integrated the peaks corresponding to 9·L-amino acid and
9·D-amino acid to find that out of thirteen proteinogenic amino acids, Ala had the smallest D:L ratio
(7:1) while Thr had the (20:1), with the other amino acids ranking more-or-less based on the steric
size of their side chains.31
O
O
9�L-Ala
N
NO
N O
O
H
H
H H3C H
9�D-Ala
O
O
N
NO
N O
O
H
H
H H CH3
H H
After the positive result, it was felt that more improvements can be made to construct an even
better method for the L- to D- conversion of amino acids. Some of the goals were to simplify the re-
ceptor to allow it to be synthesized in a small number of steps from cheap, readily commercially-
available starting materials, to be able to extract free amino acids from water into an organic sol-
vent like dichloromethane, to increase the speed of the conversion, to eliminate the need to add an
external base, and to obtain turnover for the L- to D- conversionii.
This chapter reports the completion of the first step towards this goal. A new chiral receptor
that acts as a 1H NMR CSR for free amino acids in CDCl3 (1) has been developed.
ii It should be noted that catalytic deracemization in an equilibrium system is forbidden by the second law of thermodynamics. Thus, to obtain turnover, the product D-amino acid must be removed from the system by a process such as enantioselective precipitation.
Scheme 3-3. The bioinspired amino acid receptor 9 binds stereoselectively to amino acids by imine formation controlled by hydrogen bonding. In a slightly basic solution, the proton on the α-carbon is rapidly protonated and deprotonated to cause the epimerization of the attached amino acid.
49
3.2.2 Synthesis of Chiral Shift Reagent
Scheme 3-4. The synthetic scheme to reach cyclic guanidine 1 from (S,S)-10 in one transformation step followed by a basic workup.
The amino acid CSR 1 is easily synthesized from the commercially-available (S,S)- 1,2-diphenyl-
1,2-diaminoethane (dpen, 10) and cyanogen bromide in dichloromethane, followed by treatment
with an aqueous base. This synthesis was a modification of a literature procedure.32 The formation
of the cyclic guanidinium bromide (1·HBr) in the first step is generally complete within 2 h. The
1·HBr salt is dissolved in water, and upon the addition of solid KOH tablets, 1 precipitates out im-
mediately and is extracted with dichloromethane. Generally, 1 collected
upon evaporating dichloromethane is analytically pure and no additional
purification is required. See section 3.4.2 for detailed experimental pro-
cedures.
When 1 is mixed with an amino acid, the guanidine is expected to form
a diastereomeric salt by deprotonating the carboxylic acid and establish-
ing a guanidinium-carboxylate complex. The interaction is estimated to
be particularly strong as it forms involves bidentate charged hydrogen
bonds (heavy dashes in Scheme 3-5) and favourable secondary hydrogen
bond interactions (light dashes in Scheme 3-5).33 The guanidinium-
carboxylate hydrogen bond here is expected to be even stronger than
the urea-carboxylate interaction in 9·AA as both the hydrogen bond ac-
ceptor and the donor are charged. The guanidinium-carboxylate interaction is seen in biology:
charged Arg residues are often sites for anion recognition in enzymes34, and artificial guanidines
can be used to bind to Asp and Glu residues on protein surfaces for treatment of diseases.35 In
chemistry, cyclic guanidines have previously been used for the recognition of amino acids and oth-
er carboxylates.36, 37
Scheme 3-5. The hydro-gen bond between recep-tor 1’s guanidinium group and an amino acid’s (AA) carboxylate group is charged and is especially strong.
50
3.2.3 Early Experiments
In one of the first successful extraction experiments, L-Val (0.375 mmol) and D-Val (0.125
mmol) dissolved in 1.0 mL D2O was stirred vigorously with 0.100 mmol of 1 dissolved in CDCl3.
The stirring was stopped and the two phases separated cleanly after approximately 10 minutes.
The CDCl3 NMR spectrum showed that approximately 75% of 1 (at 4.50 ppm) was bound to Val
(termed the “extraction efficiency”). The clearest peaks representing, L-Val and D-Val are from the
C*CH(CH3)2 of the side chain (at 1.95 and 1.70 ppm, Δδ = 0.25 ppm), which show a 0.75:0.25 ratio,
indicating that 1 does not bind stereoselectively. The other peaks corresponding to Val had over-
laps, so their integrations were not accurate.
While Met extracted well, with 85% extraction efficiency, other amino acids did not extract very
well: Ser showed no extraction, while the attempted extraction of His, Trp, Asn gave an inseparable
emulsion between the D2O and CDCl3 layers. Experiments with Ser, His, Trp, Asn used a 0.1 M solu-
tion of amino acid in hot D2O because of the amino acid’s lower solubility in water. Other amino
acids with even lower water solubilities were not used in this series of experiments. It is clear that
1 alone is not an effective enough receptor to be able to extract a large variety of amino acids into
chloroform.
3.2.4 Imine Formation with Substituted Salicylaldehydes
To attempt to better solubilize the amino acids, a series of substituted salicylaldehydes (11–13)
were added to form imines with the amino acids in situ, and all resulted in almost quantitative ex-
Figure 3-1. Partial 1H NMR spectrum of a mixture of 1, 1·L-Val, and 1·D-Val in CDCl3.
51
traction of the imino acid into the CDCl3
layer. First, 3,5-dinitrosalicylaldehyde
(11) was used because the two strongly
electron withdrawing nitro substituents,
along with the formation of a RAHB,
would result in rapid imine formation.
Although the imine formation with enantiopure L-Phe took place within 30 minutes, significant
racemization was detected within 2 h, lowering the enantiomeric excess to 0.69. Racemization was
unexpected, as receptor 9 required the addition of triethylamine to deprotonate the proton on the
α-carbon of the amino acid.31 The rapid racemization here is most probably due to a stabilization
of the anion on the α-carbon that develops after deprotonation by the electron withdrawing nitro
substituents, similar to the function of the pyridyl nitrogen in pyridoxal. Although a highly labile α-
proton is desirable for deracemization purposes, any racemization renders a CSR useless.
In an attempt to prevent racemization, an aldehyde with two electron donating substituents,
3,5-di-tert-butylsalicylaldehyde (12), was used in place of 11. However, the presence of the tert-
butyl groups greatly retarded the imine formation: after two hours of vigorously stirring, only 31%
of Phe extracted into the CDCl3 layer was in the imine form, and after 20 hours, imine formation
was only 79% complete. This slow imine formation was found for Val and Met as well, and no ex-
traction of Ser was observed even after 20 hours. Despite the lack of racemization with 12, imine
formation is much too slow for practical use as a CSR.
It was then thought that 5-chlorosalicylaldehyde (13), with one
mildly electron withdrawing group, represent a balance between the
need for rapid imine formation and the need to stop racemization. In
addition, at this point, the amino acid was no longer first dissolved in
D2O, as this represented an additional unnecessary step once it was
discovered that solid amino acids can go into a solution of 13 and 1.
Instead, free amino acid (0.120 mmol), aldehyde 13 (0.040 mmol),
and 1 (0.040 mmol) were weighed out (all three are solids) into a
glass vial together, and 0.80 mL CDCl3 was added to the solid mixture
and is vigorously stirred for 0.5–2 h. A serendipitous result was ob-
tained when DL-Phe was mixed with 13 and 1. The aromatic proton
at the 3 position of 13 (H-3, see Scheme 3-6) was pushed remarkably
upfield to 5.6–6.0 ppm (that proton on free 13 is at 6.9 ppm) when it
N
HN
NH
H
OOH
NO
Cl
H
H
Scheme 3-6. Interactions of the ternary complex (S,S)-1·13·L-Phe. The proton asso-ciated with 1H NMR diagnos-tic peak is outlined and bold-ed.
52
formed imines with D- and L-Phe, and the CSR 1 is able to induce excellent baseline resolution (Δδ
= 0.25 ppm) of the two diastereomers (Figure 3-2). It is not yet known why this remarkable shift
occurred. Thus, the use of salicylaldehyde 13 is useful because H-3 provides a diagnostic peak to
calculate ee for all amino acids, and because those peaks are well separated from others.
Figure 3-2. Partial 1H NMR spectrum of the diastereomers (S,S)-1·13·D-Phe and (S,S)-1·13·L-Phe in CDCl3 (50 mM) showing baseline separation of the proton at the 3-position (H-3) of salicylalde-hyde moiety in the imine formed from 13.
Unfortunately, when enantiopure amino acids were mixed with 13 and 1 in CDCl3 racemization
occurred. While Phe and Met had relatively slower racemization, Thr, Ala, Ser, and Asn showed
significant amount of racemization even after just 1 h of stirring (Figure 3-3).
3.2.5 Solvent Effect
Fortunately, it was discovered that racemization can be stopped by switching the solvent from
CDCl3 to DMSO-d6. However, the downfield shift of the diagnostic peak and its baseline separation
could not be observed from DMSO-d6 as the sole NMR solvent. Therefore, a mixed solvent system of
DMSO-d6 and CDCl3 was needed to obtain a well separated 1H NMR spectrum: DMSO-d6 was used to
dissolve the amino acid and form the imine·1 complex to prevent racemization, and the DMSO-d6
solution was diluted in CDCl3 to enhance peak separation (Figure 3-4). When 0.20 mL of a DMSO-d6
solution of 1·13·DL-Phe was diluted in 0.50 mL CDCl3, the peak resolution (Δδ)iii was just 0.02 ppm
and did not result in baseline separation (Figure 3-4a), the resolution increased as the DMSO-d6 :
CDCl3 ratio decreased, although the background noise increased due to the dilution. The best ratio
for the majority of amino acids tested, the one that balances resolution with noise minimization,
was 0.05 mL DMSO-d6 in 0.65 mL CDCl3 (7% DMSO-d6 v/v) (Figure 3-4c). Unfortunately, that
iii The peak resolution is measured by the distance between the midpoints of each of the two doublets of the diagnostic peaks in ppm.
53
means the analyte was present at low concentrations in the NMR solvent (approximately 7 mM).
To obtain less nosy 1H NMR spectra, each sample run at this concentration was subjected to 64 ac-
quisitions compared to the standard 8 as signal-to-noise ratio increases in proportion to the
square root of the number of acquisitions.38
Figure 3-3. Partial 1H NMR spectra of L-Met, L-Phe, L-Thr, L-Ala, D-Ser, and L-Asn 1 h after 1, 13, and amino acid were mixed in CDCl3 (50 mM) showing different amounts of racemization. In all six spec-tra, the more downfield doublet represents H-3 proton of the imine between 13 and the D-amino ac-id, while the more upfield doublet is from the imine between 13 and the L-amino acid. The normal-ized integration of each peak is indicated below the peak. Note: D-Ser was used to test racemization and watch for formation of L-Ser.
54
3.3 Results
3.3.1 Scope and Determination of Absolute Configuration of Amino Acids
Out of the seven amino acids tested (Ala, Asn, Met, Phe, Ser, Trp, and Val), five (Met, Phe, Ser,
Trp, and Val) showed baseline separation of its diagnostic peaks with no racemization detected
(Figure 3-5). For the two others, Ala did not dissolve well even after 5 h of vigorous stirring, and
Asn showed a small amount of racemization after 1 h of stirring (Figure 3-5). Three proteinogenic
amino acids were not compatible with this study and were not attempted: Cys showed intermolec-
ular attack of the imine by the side chain thiol group in early experiments, Gly is not chiral, and Pro,
having a secondary amine moiety, cannot form an imine.
As all of the amino acids shown in Table 3-1 follow the trend where the diagnostic peak associ-
ated with the D-amino acid is downfield of the peak associated with the L-amino acid, this method
can be used to determine the absolute configuration of amino acids.
Figure 3-4. Partial 1H NMR spectra of the diagnostic peaks of 1·13·DL-Phe (racemate used) in a) 0.20 mL DMSO-d6 and 0.50 mL CDCl3; b) 0.10 mL DMSO-d6 and 0.60 mL CDCl3; c) 0.05 mL DMSO-d6 and 0.65 mL CDCl3. The downfield peak is indicative of D-Phe, and the upfield peak is indicative of L-Phe.
55
Figure 3-5. Partial 1H NMR spectra of the diagnostic peak of 1·13·amino acid complex using the conditions outlined in section 3.4.3. Note that Asn undergoes a small amount of racemization dur-ing this process.
56
Table 3-1. Chemical shifts of the diagnostic peaks for the D- and L- forms of six amino acids in CDCl3 with 7% DMSO-d6 v/v using the of chiral shift reagent 1 and salicylaldehyde 13 by the condi-tions outlined in section 3.4.3.
Amino Acid δD (ppm)a δL (ppm)b Δδ (ppm)
Asnc 6.71 6.65 0.06
Met 6.71 6.58 0.13
Phe 6.58 6.43 0.15
Ser 6.67 6.62 0.05
Trp 6.57 6.39 0.18
Val 6.66 6.49 0.17
a – chemical shift of the diagnostic peak associated with the D-amino acid; b - chemical shift of the diagnostic peak associated with the L-amino acid; c – Asn showed some racemization before the spectra were taken, but still displayed baseline separation.
3.3.2 Determination of Amino Acid Enantiopurity
Figure 3-6. Partial 1H NMR spectra of the diagnostic peak of 1·13·D-Val 1·13·L-Val complex at var-ious known D : L molar ratios: a) 0.01 : 0.99; b) 0.05 : 0.95; c) 0.10 : 0.90; d) 0.20 : 0.80; e) 0.25 : 0.75; f) 0.40 : 0.60; g) 0.50 : 0.50. The measured integration of each peak is indicated below the peaks.
57
Table 3-2. 1H NMR-measured ratios of the L- and D- forms of Val at seven known ratios and associ-ated absolute and relative errors of the method.
En
try
Known Ratios Measured Ratios Error
D-Vala L-Vala eeb
(%) D-Valc L-Valc
eeb
(%)
Absoluted
(% ee)
Relativee
(%)
a 0.01 0.99 98 0.02 0.99 96 2 2.0
b 0.05 0.95 90 0.05 0.95 90 0 0.0
c 0.10 0.90 80 0.10 0.90 80 0 0.0
d 0.20 0.80 60 0.22 0.80 57 3 5.2
e 0.25 0.75 50 0.27 0.75 47 3 5.9
f 0.40 0.60 20 0.40 0.60 20 0 0.0
g 0.50 0.50 0.0 0.50 0.50 0.0 0 0.0
a – ratio of weight of solid amino acid added; b – enantiomeric excess (ee) = 100% × ([D-Val]−[L-Val])/([D-Val]+[L-Val]); c – ratio of 1H NMR integration from Figure 3-6; d – absolute error = |% eeknown−% eemeasured|; e – relative error = 100% × (|% eeknown−% eemeasured| / eeknown).
In order to determine if this method can be used to accurately determine the enantiomeric ex-
cess of a sample of amino acid, 1H NMR spectra were taken of Val at the following of L : D ratios
(with associated ee’s): a) 0.99 : 0.01 (98%), b) 0.95 : 0.05 (90%), c) 0.90 : 0.10 (80%), d) 0.80 : 0.20
(60%), e) 0.75 : 0.25 (50%), f) 0.60 : 0.40 (20%), and g) 0.50 : 0.50 (0%) (Figure 3-6). Solid L-Val
and D-Val were carefully weighed out at the prescribed ratios into a glass vial along with 1 and 13
before DMSO-d6 was added. After 1 h of vigorous stirring, a 1H NMR spectrum of the solution (7%
v/v solution in CDCl3) was obtained. The areas under the two diagnostic peaks were calculated by
integration, and from that the ee was calculated (Table 3-2). This experimental ee value was com-
pared with the known ee value for each ratio to give the absolute and relative error of each meas-
urement using the following formulae:
absolute error = |% eeknown−% eemeasured|
relative error = 100% × (|% eeknown−% eemeasured| / eeknown)
Because the highest relative error was 5.9% (Table 3-2, entry e), it can be concluded that this
chiral shift reagent can determine the enantiopurity of amino acids to an accuracy of approximate-
ly 6%.
58
3.4 Experimental
3.4.1 General Information
The following compounds were obtained commercially and were used without further purifica-
tion or drying: L-alanine, D-alanine, L-asparagine, D-asparagine, L-methionine, D-methionine, L-
phenylalanine, D-phenylalanine, L-serine, D-serine, L-tryptophan, D-tryptophan, L-valine, D-valine,
cyanogen bromide, dichloromethane, potassium hydroxide, anhydrous sodium sulfate, 5-
chlorosalicylaldehyde, DMSO-d6, and CDCl3 (0.05% v/v TMS). (S,S)-1,2-diphenyl-1,2-
diaminoethane was synthesized by the Chin research group. All reactions were held under an am-
bient atmosphere. Spectrometers used to record 1H nuclear magnetic resonance (NMR) spectra
were the Varian Mercury 400, the Varian NMR System 400, and the Bruker Avance III (400) at The
Centre for Spectroscopic Investigation of Complex Organic Molecules and Polymers in the Depart-
ment of Chemistry at the University of Toronto.
3.4.2 Synthesis of Chiral Shift Reagent
This synthesis was a modification of a procedure described by Lovick and Michael, reducing re-
action time from 48 h to 2 h and temperature from 82°C to room temperature, and eliminating the
requirement for purification by column chromatography.32
(S,S)-4,5-diphenylimidazolidin-2-imine hydrobromide (1·HBr)
Into a round-bottom flask was added 500 mg (2.355 mmol) of (S,S)-1,2-diphenyl-1,2-
diaminoethane (10) and 12 mL dichloromethane. In a separate vessel, 274 mg (2.591 mmol, 1.1
equiv) of cyanogen bromide was dissolved in 12 mL dichloromethane. Once solutions were cooled
to 0°C in an ice bath, the cyanogen bromide solution was added slowly to the stirring round-
bottom flask with the solution of 10. After ten minutes of stirring at 0°C, the flask was allowed to
warm to room temperature for an additional two hours of stirring. The dichloromethane was re-
moved by rotary evaporation to provide the 1·HBr salt in quantitative yield (751 mg) as a yellow
solid. The salt was carried forward for basification without purification.
1H NMR (400 MHz, CDCl3, ppm): δ = 7.38–7.19 (m, 10H, Ph-H), 4.64 (s, 2H, C*H), 3.44 (br s, 4H,
NH).
59
(S,S)-4,5-diphenylimidazolidin-2-imine (1)
The 1·HBr salt from the previous step (751 mg, 2.355 mmol) was fully dissolved in 25 mL H2O and
cooled in an ice bath before approximately 331 mg (5.888 mmol, 2.5 equiv) of potassium hydrox-
ide pellets were added. Upon the addition, a fine white suspension of 1 formed in the aqueous solu-
tion. The suspension was extracted into dichloromethane (3 × 25 mL), which was then dried thor-
oughly over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to give an-
alytically pure 1 in 97% yield (542 mg) as an off-white powder.
1H NMR (400 MHz, CDCl3, ppm): δ = 7.38–7.19 (m, 10H, Ph-H), 4.62 (s, 2H, C*H), 4.46 (br s, 3H,
NH).
3.4.3 General Procedure for Determining Enantiomeric Excess and Absolute Configu-
ration
Into a small glass vial was added the amino acid analyte (0.080 mmol), 9.5 mg of chiral shift rea-
gent 1 (0.040 mmol), and 6.3 mg of 5-chlorosalicylaldehyde 13 (0.040 mmol). To the solid mixture
was added 0.80 mL of DMSO-d6iv, and the mixture was allowed to stir vigorously for 1 h at room
temperature. After this time, 50 μL of the DMSO-d6 solution (avoiding the remaining solid amino
acid) was diluted in 0.65 mL CDCl3. To obtain the 1H NMR spectrum of the sample, the solvent lock
was set to CDCl3 (which provided a greater concentration of deuterons than DMSO-d6), and the
number of acquisitions was set to 64.
iv Non-deuterated DMSO could also be used here, as the large DMSO peak at 2.50 ppm is sufficiently far away from the diagnostic peaks.
60
3.5 Conclusions and Future Directions
In this work, a novel chiral shift reagent (CSR) for proteinogenic amino acids had been synthe-
sized and described. Cyclic guanidine 1 was synthesized from chiral diamine 10 in excellent yields
without needing purification. When 1 was used along with aldehyde 13, the enantiopurity of five
amino acids (Met, Phe, Ser, Trp, and Val) were easily determined by 1H NMR spectroscopy. The ad-
vantages of using this method were an easy synthesis of the CSR from commercially-available ma-
terials, and the ability to analyze free amino acids directly. The diagnosis peaks were well separat-
ed from others in the NMR spectra, and were found at similar locations for all amino acids, leading
to a facile and accurate (within 6% error) determination of the sample’s enantiomeric excess from
0% (50:50) to 96% (99:1). The diagnosis peaks also provided the absolute configuration of the
amino acid, as D-amino acids always appeared downfield to L-amino acids.
The advantage of having a CSR that is derived from chiral diamines is that this method could be
improved systematically by using the diaza-Cope rearrangement method to synthesize a variety of
diamine starting materials (see Chapter 2). Different aldehydes could substitute for 13 to eliminate
the problem of racemization for all amino acids and allow the use of higher concentrations of ana-
lyte in a single solvent system for NMR.
The development of this CSR represents the first step to the goal of developing an easy, efficient,
and economical method for the synthesis of a variety of D-amino acids and other unnatural amino
acids, which have great importance in the pharmaceutical industry.
3.6 Spectra
All proton (1H) NMR spectra were processed using MestReNova (ver. 6.1.1-6384) from Mestre-
lab Research S.L. The protons in tetramethylsilane (TMS) appears as a singlet at 0.00 ppm, DMSO-
d5 appears as a quintet at 2.58 ppm, CHCl3 appears as a singlet at 7.26 ppm.
61
(S,S)-4,5-diphenylimidazolidin-2-imine hydrobromide (1·HBr)
-0.50.00.51.01.52.02 53.03.54.04.55.05.56.06.57.07.58.08.5ppm (1H)
(S,S)-4,5-diphenylimidazolidin-2-imine (1)
-0.50.00.51.01.52.02 53.03.54.04.55.05.56.06.57.07.58.08.5ppm (1H)
62
1·13·D-Asn
1·13·L-Asn
63
1·13·D-Met
1·13·L-Met
64
1·13·D-Phe
1·13·L-Phe
65
1·13·D-Ser
1·13·L-Ser
66
1·13·D-Trp
1·13·L-Trp
67
1·13·D-Val
1·13·L-Val
68
3.7 References
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ed. Wiley-VCH: New York, 2000.
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(3) Holmes, A. E.; Zhan, S.; Canary, J. W. Chirality 2002, 14, 471-477.
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(5) Leung, D.; Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem. Soc. 2008, 130, 12318-12327.
(6) Wenzel, T. J.; Wilcox, J. D. Chirality 2003, 15, 256-270.
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(8) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543-2549.
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(14) Wenzel, T. J.; Miles, R. D.; Zomlefer, K.; Frederique, D. E.; Roan, M. A.; Troughton, J. S.; Pond, B. V.; Colby, A. L. Chirality 2000, 12, 30-37.
(15) Kaik, M.; Gajewy, J.; Grajewski, J.; Gawronski, J. Chirality 2008, 20, 301-306.
(16) Dufrasne, F.; Gelbcke, M.; Nève, J. Spectrochimica Acta Part A 2003, 59, 1239-1245.
(17) Chin, J.; Kim, D. C.; Kim, H.; Panosyan, F. P.; Kim, K. M. Org. Lett. 2004, 6, 2591-1593.
(18) Takaya, T.; Kishida, Y.; Sakakibara, S. J. Chromatogr. 1981, 215, 279-287.
(19) Okamoto, Y.; Yashima, E. Angew. Chem. Int. Ed. 1998, 37, 1020-1043.
(20) Španik, I.; Krupčik, J.; Skačáni, I.; Sandra, P.; Armstrong, D. W. J. Chromatogr. A. 2005, 1071, 59-66.
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(21) Schurig, V. J. Chromatogr. A 2001, 906, 275-299.
(22) Stryer, L. Biochemistry, 4th ed. W.H. Freeman: New York, 1995; .
(23) Voet, D.; Voet, J. G. Biochemistry, 2nd ed. Wiley: New York, 1995; .
(24) Friedman, M. Chem. Biodiversity 2010, 7, 1491-1530.
(25) Hashimoto, A.; Nishikawa, T.; Oka, T.; Takahashi, K. J. Neurochem. 1993, 60, 783-786.
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(27) Fujii, N. Biol. Pharm. Bull. 2005, 28, 1585-1589.
(28) Martínez-Rodríguez, S.; Martínez-Gómez, A. I.; Rodriguez-Vico, F.; Clemente-Jiménez, J. M.; Las Heras-Vazquez, F. J. Chem. Biodiversity 2010, 7, 1531-1548.
(29) D-Amino Acid Research Society DAA_HomePage. http://www.d-amino-acid.jp/ (accessed 09/20/ 2010).
(30) Shaw, J. P.; Petsko, G. A.; Ringe, D. Biochemistry 1997, 36, 1329-1342.
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70
APPENDIX 1
My Favourite Chemistry Things
Sung to the tune of “My Favorite Things” from the musical The Sound of Music.
Lyrics: Leo Mui
Music: Richard Rodgers
Easy quick workups and a column that cleanses
The smell of ether and a product that fluoresces
Brown Aldrich packages the courier brings
These are a few of my favourite things.
White powdery solids and high yields and ee's
Ground glass and valves brass and multiple degrees
Sealed vacuum chambers with rubber o-rings
These are a few of my favourite things.
Low-boiling solvents that leave with no traces
Girls in white lab coats and Erlenmeyer vases
Clean NMRs on a Friday in spring
These are a few of my favourite things.
When the flask breaks
When my hand stings
When I'm still a grad
I simply remember my favourite things,
And then I don't feel so bad!
Written on May 25, 2010, after a particularly trying day in the lab.
71
APPENDIX 2
Colophon
This thesis was typeset by the author in Microsoft Word 2010. The chemical structures and
schemes were created using Cambridgesoft’s ChemDraw Ultra 12.0, the NMR spectra were pro-
cessed in Mestrelab’s MestReNova (ver. 6.1.1), and additional processing of schemes and spectra
were done in Adobe Photoshop CS3. Literature citation and referencing was managed by Proquest’s
Refworks 2.0.
Cambria was chosen to be the font for the main text because of its compatibility with both the
print and electronic versions this thesis by offering more subtle serifs than full serif typefaces. De-
signed in 2004 by Jelle Bosma, Cambria is a very evenly-spaced typeface with high clarity that is
ideally suited for a document that is typeset with hyphenation and justified alignment. Cambria
also offers very elegant bold, italic, and small capital variations that flows very well with the main
text. Overall, the author chose Cambria for its superb readability, and its ability to be sharp, strong,
sturdy, and formal without the bland and cold sternness of a font like Times New Roman.
Arial was chosen to be used in chemical structures and schemes and to label spectra in this the-
sis. Arial’s progressive simplicity, smooth curves, and its “no-nonsense” clarity make it the ideal
font to be used to label atoms and structures. Despite the ubiquity of the font, and the unnecessary
hostility from proponents of Helvetica (of which the author also enjoys using), the author believed
that Arial, especially its bold variant, worked very well in this context, and formed a surprisingly
good pair with Cambria.
