An improved, scalable synthesis of bis-amino acids

Article abstract of DOI:10.1016/j.tetlet.2016.09.032

trans-4-Hydroxy-L-proline derived bis-amino acids are chiral, cyclic building blocks that display two alpha-amino acids that are differentiated from each other with protecting groups. They are assembled into spiroligomers—rigid, shape-programmable spirocyclic oligomers that are both stereochemically and functionally diverse. The synthesis presented here focuses on recent improvements that allow for a convenient, large-scale synthesis of twelve stereochemically pure bis-amino acids from inexpensive trans-4-hydroxy-L-proline. The bis-amino acids differ in stereochemistry as well as the amine protecting group, one of which (para-nitrobenzyl carbamate) has not been previously incorporated into bis-amino acids.

Full text of DOI:10.1016/j.tetlet.2016.09.032

Accepted Manuscript
An Improved, Scalable Synthesis of bis-Amino Acids
Jae Eun Cheong, Conrad T. Pfeiffer, J.D. Northrup, Matthew F.L. Parker,
Christian E. Schafmeister
PII:
S0040-4039(16)31190-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.09.032
TETL 48102
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 July 2016
31 August 2016
7 September 2016
Please cite this article as: Cheong, J.E., Pfeiffer, C.T., Northrup, J.D., Parker, M.F.L., Schafmeister, C.E., An
Improved, Scalable Synthesis of bis-Amino Acids, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/
j.tetlet.2016.09.032
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An Improved, Scalable Synthesis of bis-Amino Acids
Jae Eun Cheong§‡, Conrad T. Pfeiffer§‡, JD Northrup§‡, Matthew F. L. Parker†, Christian E. Schafmeister§*
§Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122,
United States of America
†Department of Radiology & Biomedical Imaging, University of California, San Francisco
‡Author’s contributed equally
*Corresponding author. Tel.: +1 2152047128; e-mail: meister@temple.edu
Abstract
Trans-4-hydroxy-L-proline derived bis-amino acids are chiral, cyclic building blocks that display two alpha-
amino acids that are differentiated from each other with protecting groups. They are assembled into
spiroligomers – rigid, shape-programmable spirocyclic oligomers that are both stereochemically and
functionally diverse. The synthesis presented here focuses on recent improvements that allow for a
convenient, large-scale synthesis of twelve stereochemically pure bis-amino acids from inexpensive
trans-4-hydroxy-L-proline. The bis-amino acids differ in stereochemistry as well as the amine protecting
group, one of which (para-nitrobenzyl carbamate) has not been previously incorporated into bis-amino
acids.
Keywords:
Spiroligomer; Bis-amino acids; Amino acids; Peptidomimetics; Diastereomers
1. Introduction
Proteins act as scaffolds to position multiple chemically active groups in three-dimensional space, either
inward in the case of enzymes, or outward to interact with other proteins. The fields of peptidomimetics
and foldamers seek to mimic the functions of proteins using unnatural building blocks that create specific
three-dimensional structures. 1-5 Common challenges shared by all foldamers is predicting and controlling
2,5-7
folding into specific three-dimensional structures.
To this end, we have developed spiroligomers,8
oligomers composed of trans-4-hydroxy-L-proline derived bis-amino acids9-11 connected through pairs of

amide bonds to form ladder molecules containing linkages formed by diketopiperazines (DKP) rather than
single amide bonds. By forming spirocyclic DKPs, the spiroligomer backbone has no rotatable bonds;
therefore, they are inherently rigid molecules with complex and predictable three-dimensional shapes.
Functionalization is possible at specific positions on this backbone, which altogether allows for a pre-
organized, stereochemically and functionally diverse oligomer that has been utilized for a variety of
applications including demonstration of a transesterification catalyst,12 a proline-aldol catalyst,13 an
aromatic Claisen rearrangement catalyst,14 a spiroligomer that binds the protein MDM2,15 the formation of
supramolecular metal binding complexes,16 the first donor-bridge-acceptor molecule that demonstrated
acceleration of electron transfer in water,17 a metal binding mechanical molecular actuator,18 and
molecular rulers.19
2. Results and discussion
The goal of this work is to scale up the synthesis of trans-4-hydroxy-L-proline derived bis-amino acids to
make this chemistry more accessible to others, to lower the environmental impact of the synthesis, and to
access larger quantities of bis-amino acids in less time. The syntheses presented here demonstrate
recent advancements that allow for the large-scale synthesis (~200-250 grams theoretical yield of
hydantoin products) of twelve different bis-amino acids, some of which have been previously reported by
our group but now have much improved syntheses.8-10,20 Increasing the concentration of the reactions and
workup procedures, while increasing the scale of the synthesis and purifications, has led to a reduction in
the overall volume of organic solvents needed throughout the synthetic effort. Additionally, we have
reduced the equivalents of carcinogenic chromium trioxide and toxic potassium cyanide used during
synthesis. As described by our group,20,21 the overall shape of any spiroligomer is controlled by the
stereochemistry of each bis-amino acid subunit, making it important to have access to all stereoisomers.
Furthermore, having synthetic access to different, orthogonal protected versions of the bis-amino acids
allows more flexibility in the choice of chemistry used to assemble them into functional spiroligomer
nanostructures. (e. g. solution phase versus solid phase.)

Scheme 1: Reaction scheme detailing the synthesis of all four stereoisomers of hydantoins. Compound
12 is the enantiomer of 5, and compound 11 the enantiomer of 6. (a) Cbz-Cl, NaHCO3, 1:10
dioxane/water (b) Jones reagent, acetone, 0 oC to rt; (c) Isobutylene, H2SO4 (cat.), CH2Cl2, 0 oC to rt; (d)
(NH4)2CO3, KCN, 1:1 EtOH/water, sealed tube; (e) (i) AcOH/Ac2O, Reflux; (ii) 2M HCl, reflux
The synthesis begins with the Cbz protection of the pyrrolidine nitrogen of trans-4-hydroxy-L-proline (1)
employing Schotten-Baumann conditions,22 as shown in Scheme 1. The Schotten-Baumann conditions
allow the pyrrolidine protection in a single step at room temperature. The next step of the synthesis
oxidizes the secondary alcohol, 2, to the corresponding ketone, 3, via Jones oxidation. We have
attempted many other oxidation methods for this step; however, only a method using trichloroisocyanuric
acid (TCCA/TEMPO)23 provided comparable yields. Unfortunately, this reaction also produced cyanuric
acid as a side product that could not be removed without extensive chromatography, which led to lower
yields in subsequent reactions. Since the other oxidation methods were fruitless, we reduced the number
of equivalents of carcinogenic chromium trioxide used during the reaction to 38% of what was previous
reported (from 8 equivalents to 3), and compensated for this reduction by increasing the reaction time.
The next step of the synthesis is the tert-butyl protection of 3 to yield ester 4.9 A Bucherer-Bergs
reaction24 is used to form the diastereomeric hydantoins 5 and 6 in a sealed, round bottom flask at room
temperature. In this synthesis, the number of equivalents of toxic potassium cyanide is reduced to 70% of
what we had previously reported (from 1.5 equivalents to 1.05).
The diastereomers formed from the Bucherer-Bergs reaction, 5 and 6, were obtained in a combined yield
of 63% (5:1 ratio of ss:sr) over four reactions after purification by normal phase flash chromatography with
a 0-10% IPA in DCM gradient which was carried out in multiple batches due to volume limits of our

automated flash chromatography system. By switching to an IPA/DCM solvent system, we have improved
the separation of an impurity that elutes just prior to the major product peak. Also of note, it is possible to
isolate and recycle the DCM and IPA by extractive distillation25, which would be financially and
environmentally responsible, should the scale of the purification be further increased in the future.
Scheme 2. Reaction scheme detailing the specific protecting group conversion, and hydrolysis of the
hydantoin. Compounds shown in orange are the enantiomers of those shown in black, and those shown
in red are the enantiomers of those shown in blue. (a) 5 wt % Pd/C, H2 (g), THF, Boc2O; (b) (i) 5 wt %
Pd/C, H2 (g), THF; (ii) Ar(g), pNZ-Cl, DIPEA; (c) (i) (Boc)2O, DMAP, THF, rt (ii) 2 M KOH, 0 oC
After separation of the hydantoin diastereomers, the Cbz group attached to the pyrrolidine nitrogen may
be converted to a Boc protecting group (Scheme 2, a) or a p-nitrobenzyloxycarbonyl protecting group
(pNZ, Scheme 2, b.)22,26 Synthetic access to multiple protecting groups is advantageous when
assembling bis-amino acids into spiroligomers, since one protecting group may be more suitable
depending on the implemented synthetic method (e. g. solution versus solid phase synthesis.) Once the
desired pyrrolidine nitrogen protecting group is installed, each diastereomer is simultaneously Boc
protected at both of the imide and amide nitrogens of the hydantoin, followed by the hydrolysis of the
hydantoin under mild conditions by addition of 2 M KOH. This hydrolysis reveals the second amino acid at
what was originally the position of the alcohol of trans-4-hydroxy-L-proline. Each bis-amino acid is
isolated by precipitation via neutralization of the basic solution, with yields that range from 21.5% for 27 to
91.7% for 25, with the average being 61% from the Cbz-protected hydantion 5, 6, 11, or 12. They are
then dried and stored at room temperature in a desiccator until needed. The synthetic steps outlined have

also been applied to cis-4-hydroxy-D-proline, which was obtained by epimerizing the C-2 acid of trans-4-
hydroxy-L-proline using a published method.27,28
3. Conclusion
We have greatly improved and increased the scale of the synthesis of proline-based bis-amino acid
monomers. This synthesis is not only divergent, with all twelve bis-amino acids stemming from a common
trans-4-hydroxy-L-proline feedstock, but it is also more convenient and environmentally friendly than our
previous work. Three different protected forms of the bis-amino acid (pNZ, Cbz, and Boc) are described,
which increase the versatility of these building blocks for different solid-phase and solution phase
syntheses of oligomers.
Acknowledgements
This material is based on work supported in part by the National Science Foundation under Grant No.
1300231 and the NIH/NIGMS GM067866.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at XXXXX
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Graphical Abstract

Highlights




Synthesis of spiroligomer precursors (bis-amino acids) scaled up five-fold.
Divergent synthesis leads to twelve stereochemically pure bis-amino acids.
Incorporated the orthogonal protecting group para-nitrobenzyl.
More environmentally friendly, reducing chromium and cyanide required.