Synthesis of Chiral Spin-Labeled Amino Acids

Article abstract of DOI:10.1021/acs.orglett.9b04216

Spin-labeled amino acids (SLAAs) are often used to determine intermolecular distances and conformations in proteins via double electron-electron resonance. Currently available SLAAs can be difficult to incorporate selectively and have little resemblance to natural side chains in proteins. Enantioselective synthesis of three spin-labeled l-amino acids is described, starting from readily available 2,2,6,6-tetramethyl-4-piperidinone. These SLAAs better replicate canonical residues in proteins and aim for biological incorporation via genetic incorporation or solid-phase peptide synthesis.

Full text of DOI:10.1021/acs.orglett.9b04216

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Chiral Spin-Labeled Amino Acids
Wayne Vuong,† Fabricio Mosquera-Guagua,† Randy Sanichar,† Tyler R. McDonald,† Oliver P. Ernst,‡
Lei Wang,§ and John C. Vederas*,†
†Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
‡Departments of Biochemistry & Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8
§Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: Spin-labeled amino acids (SLAAs) are often used to determine intermolecular distances and conformations in
proteins via double electron−electron resonance. Currently available SLAAs can be difficult to incorporate selectively and have
little resemblance to natural side chains in proteins. Enantioselective synthesis of three spin-labeled L-amino acids is described,
starting from readily available 2,2,6,6-tetramethyl-4-piperidinone. These SLAAs better replicate canonical residues in proteins
and aim for biological incorporation via genetic incorporation or solid-phase peptide synthesis.
pulsed electron paramagnetic resonance (EPR) techni-
que known as double electron−electron resonance
A
(DEER)1−3 can measure interspin distances of electrons
ranging from 15 to >100 Å and is a highly effective tool to
study protein conformations.4−6 It usually depends on
incorporation of two spin labels that have a stable unpaired
electron,1,2,6−8 often as a modified amino acid residue.6,7,9 The
distances between these two spin-labels can be used to
triangulate conformational changes in proteins stepwise by
site-directed spin-labeling of various topological sites.10
Another approach comprises a single spin-labeled protein
residue from which ligand binding can be observed.11,12 Using
spin-labeled ligands is already well established in EPR
spectroscopy, and additional information about number of
binding sites and affinity can be gained.13 A common motif is a
fully α-substituted nitroxide radical akin to TEMPO (2,2,6,6-
tetramethylpiperidin-1-yl-oxy).7,14 Such moieties can be
attached by selective modification of existing amino acid
residues (e.g., alkylation or disulfide bridge formation to
cysteine sulfhydryl by 1 or 2, respectively) (Figure 1).
However, specific chemical modification of complex proteins
having many similar residues can be difficult to achieve. This
approach also generates extended flexible side chains on the
protein that may have varied conformations. Alternatively,
direct incorporation into the protein backbone is especially
attractive. This requires either solid phase peptide synthesis
(SPPS) (e.g., 3 or 4)7,15 or genetic incorporation (e.g., 5).16
Unnatural amino acids can be site-specifically incorporated by
expansion of the genetic code with utilization of specifically
evolved tRNA/aminoacyl-tRNA synthetase pairs.17−19 How-
ever, currently available amino acids such as 2,2,6,6-
tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid
(TOAC) (3) or the β-amino acid (4) (Figure 1), lack
Figure 1. Structures of spin-labels for site-specific incorporation into
proteins.
resemblance to canonical amino acids, making the generation
of synthetases and the genetic approach more difficult. In
addition, TOAC is α,α-disubstituted and prone to changing
the natural protein conformation.7
In the current study, we describe the synthesis of three chiral
spin-labeled α-amino acids 6−8 (Figure 2) whose core
geometries more closely resemble the common amino acids
histidine, valine, and isoleucine/phenylalanine/tyrosine, re-
spectively.
Although chiral synthesis of α-amino acids is highly
developed,20,21 the spin-labeled targets 6−8 present some
special challenges. In addition to the nitroxyl radical broad-
ening conventional NMR signals, which makes direct structural
analysis more difficult, the tetrasubstitution of carbons
attached to nitrogen enables potential cation formation
under acidic conditions with subsequent undesired decom-
Received: November 24, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b04216
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Figure 2. Targeted chiral spin-labeled amino acids 6−8 for genetic or
synthetic incorporation into proteins.
position (e.g., elimination). The most readily available
commercial starting material, 2,2,6,6-tetramethyl-4-piperidi-
none, can be protected on nitrogen, but its N-acyl derivatives
have a strong tendency to lose the α-hydrogen next to the
carbonyl and eliminate the N-acyl moiety under basic
conditions. Hence relatively mild methods must be employed
during synthesis.
Our synthetic design for the target amino acids depends in
each case on attachment of the side chain to the α-carbon of a
glycinate complex to furnish the final products.
The reaction of 14 (synthesized from 2,2,6,6-tetramethyl-4-
piperidinone) (Scheme S1)22 with Belokon complex 1523 that
has been deprotonated with potassium tert-butoxide produces
16 (Scheme 1). Careful cleavage using a mixture of HCl and
Figure 3. Proposed mechanisms for degradation of the 6-membered
ring system in acidic and basic conditions.
elimination. To protect the nitrogen, a 2,2,2-trichloroethox-
ycarbonyl (Troc) group was chosen as it can be removed
under mild conditions (Zn) without direct attack at the
hindered N-acyl carbonyl. Unfortunately, such bulky electro-
philes fail to effectively alkylate the Belokon glycinate derived
from 15. Hence the strategy was altered to form the bond
between the α- and β-carbons in 7 and 8 by a more facile (less
hindered) nonstereoselective alkylation, followed by deracem-
ization using an imine ligand developed by Nian et al.27
The alternative strategy for synthesis of 7 and 8 commenced
with the generation of two protected electrophiles 19 and 22
(Scheme 2). This began with protection of nitrogen on 2,2,6,6-
tetramethyl-4-piperidinone with a 2,2,2-trichloroethoxycarbon-
yl (Troc) group. For production of 19, the ketone is subjected
to reduction by sodium borohydride to quantitatively generate
the secondary alcohol 18. Brosylation by 4-bromobenzene-
sulfonyl chloride then generates the desired electrophile 19. As
Scheme 1. Synthesis of Chiral Spin-Labeled Amino Acid 6
Scheme 2. Synthesis of 6-Membered Ring Electrophiles 19
and 22
MeOH gives 6 with an enantiomeric ratio of 90:10, as
demonstrated by Marfey’s assay (see the SI).24 This mixture
can be transformed to the enantiomerically pure S isomer
through catalytic destruction of the unwanted R isomer by D-
amino acid oxidase (see below).25,26
Production of amino acids 7 and 8 was initially attempted
via a similar strategy as for the production of 6. However,
direct alkylation onto the Belokon complex did not prove
productive with simple derivatives of 2,2,6,6-tetramethyl-4-
piperidinone. Exposure of this ring system to strongly acidic
conditions usually used for cleavage of the product complex
results in ring opening to form a tertiary cation that then
generates a variety of compounds including elimination
products (Figure 3). Although this can be somewhat mitigated
by N-acylation, subjecting such N-acyl derivatives with a small
leaving group (e.g., mesylate, bromide etc.) at the 4-position to
the basic conditions necessary for alkylation gives primarily
elimination.
It appeared that a large leaving group (e.g., brosylate) at the
4-position on the tetramethylpiperidine system would prefer to
be equatorial and could lock the conformation to hinder
B
DOI: 10.1021/acs.orglett.9b04216
Org. Lett. XXXX, XXX, XXX−XXX

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indicated above, the bulky brosylate leaving group prefers an
equatorial position, thereby averting undesired elimination
upon exposure to the basic conditions of the subsequent
alkylation step.
the Boc protecting group affords amide 34 with high yield and
purity. Forgoing purification, 34 was then utilized as the
nucleophile in a subsequent amination step coupled to an in
situ Finkelstein reaction to furnish the target ligand 35
(Scheme 4).
The electrophile 22 was synthesized by transformation of 17
to olefin 20 via a Wittig reaction. Execution of this step must
be performed carefully as even slight increases in temperature
or improper addition of reagents lead to removal of the Troc
group or opening of the ring. Hydroboration−oxidation then
generates alcohol 21, which is then brosylated to produce 22.
Compounds 19 and 22 react with the enolate of 2328 to
form the desired racemic amino acid skeletons, which upon
mild treatment with hydroxylamine afford benzyl esters 24 and
25, respectively. Harsher imine hydrolysis attempts using acid
result in opening and degradation of the 6-membered ring. The
amino group is subsequently protected using trifluoroacetic
anhydride to obtain trifluoroacetamides 26 and 27.
Scheme 4. Synthesis of Kinetic Resolution Ligand 35
The Troc protecting group was then reductively cleaved
from compounds 26 and 27 using Zn dust suspended in acetic
acid to obtain free amines 28 and 29. The N-oxide radical was
then installed in good yield using m-chloroperoxybenzoic acid
(mCPBA) to furnish protected spin-labeled amino acids 30
and 31. Finally, removal of the trifluoroacetyl and benzyl
groups by gentle base hydrolysis yielded racemic free amino
acids 32 and 33, ready for deracemization by dynamic kinetic
resolution (DKR) (Scheme 3).27
DKR first requires the synthesis of ligand 35, developed by
Nian et al.27 This ligand was synthesized using an alternative
route, first by mesylation of Boc-protected L-proline. The
mesyl mixed anhydride intermediate then undergoes amide
bond formation following displacement by commercially
available 2-amino-4-chlorobenzophenone. Finally, removal of
The spin-labeled free amino acids 32 and 33 were then
complexed and kinetically resolved using ligand 35, providing
complexes 36 and 37, respectively, as bright red solids
(Scheme 5). At this point, it was found that the length of
Scheme 5. Kinetic Resolution of Amino Acids 32 and 33
Scheme 3. Synthesis of Racemic Amino Acids 32 and 33
time that the kinetic resolution step was allowed to proceed
affected both the enantiomeric ratio as well as yield of the
chirally enriched amino acid in the subsequent step. A kinetic
resolution period of 24 h appeared to be a good balance
between reaction yield and enantioselectivity, with longer
times prompting marginally better ratios at the cost of reduced
yields. Cleavage of complexes 36 and 37 was achieved by
transamination using hydroxylamine to afford enantiomerically
enriched amino acids 7 and 8 with L to D ratios of 90:10 and
78:22, respectively, based on Marfey’s assays.24
To improve enantiopurity, the amino acids 6, 7, and 8
obtained by cleavage from their respective complexes were
subjected to D-amino acid oxidase (DAAO). This catalytic
process selectively transforms the D-amino acids into α-keto
C
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acids, which can then be separated from the desired L-amino
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steric bulk. Fortunately, however, this was very effective for 6
and 8, which were then obtained enatiomerically pure (>99%)
based on Marfey’s assay.24
In conclusion, a facile and enantioselective synthesis of spin-
labeled amino acids 6−8 was achieved. Enantiopure 6 and 8
can be used directly for incorporation into proteins or peptides
by way of either SPPS or genetic incorporation methods.
Amino acid 7 (90:10 er) can be used directly for the latter but
may require further resolution of diastereomers for SPPS.
These amino acids are better mimics for replicating the steric
size and configuration of canonical amino acids in target
proteins. As spin-labels, they can allow better measurements of
distances in biological systems and more closely resemble side
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.9b04216.
(12) Reichenwallner, J.; Hauenschild, T.; Schmelzer, C. E. H.;
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Properties of Nitroxide Spin Labels for Use in Electron Paramagnetic
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Nitroxide Framework. Electron Paramagn. Reson. 2016, 25, 1−34.
(15) Brodrecht, M.; Herr, K.; Bothe, S.; de Oliveira, M.; Gutmann,
T.; Buntkowsky, G. Efficient Building Blocks for Solid-Phase Peptide
Synthesis of Spin Labeled Peptides for Electron Paramagnetic
Resonance and Dynamic Nuclear Polarization Applications. Chem-
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Experimental details and spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: john.vederas@ualberta.ca.
ORCID
Oliver P. Ernst: 0000-0002-8863-9444
Lei Wang: 0000-0002-5859-2526
John C. Vederas: 0000-0002-2996-0326
Notes
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Genetically Encoded Spin Label for Electron Paramagnetic Resonance
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the Genetic Code of Escherichia Coli. Science (Washington, DC, U. S.)
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(18) Wang, L.; Schultz, P. G. Expanding the Genetic Code. Angew.
Chem., Int. Ed. 2005, 44 (1), 34−66.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a Catalyst Grant from the
Canadian Institute For Advanced Research and by a Queen
́
Elizabeth II Graduate Scholarship (WV). We thank Bela Reiz
(University of Alberta) and Dr. Angelina Morales-Izquierdo
(University of Alberta) for their help in the acquisition and
interpretation of mass spectrometry data. We would also like to
thank Mark Miskolzie (University of Alberta) for his help in
the analysis of spectral data.
(19) Liu, C. C.; Schultz, P. G. Adding New Chemistries to the
Genetic Code. Annu. Rev. Biochem. 2010, 79 (1), 413−444.
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C(Sp3)-H Bond Functionalization of Glycine Derivatives. Youji
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