Creating diversity by site-selective peptide modification: A customizable unit affords amino acids with high optical purity

Article abstract of DOI:10.1021/ol402800a

The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes, but still is a difficult synthetic challenge. Herein natural hydroxyproline is introduced as a convertible unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, and to achieve the mild, efficient, and site-selective modification of peptides.

Full text of DOI:10.1021/ol402800a

ORGANIC
LETTERS
2013
Vol. 15, No. 22
5778–5781
Creating Diversity by Site-Selective Peptide
Modification: A Customizable Unit Affords
Amino Acids with High Optical Purity
Ivan Romero-Estudillo and Alicia Boto*
´ ´
Instituto de Productos Naturales y Agrobiologıa del CSIC, Avda. Astrofısico Fco.
ꢀ
Sanchez, 3, 38206-La Laguna, Tenerife, Spain
alicia@ipna.csic.es
Received September 28, 2013
ABSTRACT
The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery
processes, but still is a difficult synthetic challenge. Herein natural hydroxyproline is introduced as a “convertible” unit for the production of a variety
of optically pure amino acids, including expensive N-alkyl amino acids, and to achieve the mild, efficient, and site-selective modification of peptides.
The modification of peptides has elicited much interest,
in order to provide new peptide drugs,1 probes for mole-
cular imaging,2 and peptide catalysts for green chemistry.3
During the discovery process of bioactive or catalytic
peptides, libraries of analogues of a lead compound are
prepared. Their activities are studied next, in order to
determine structureꢀactivity relationships and then design
new generations of modified peptides.
In the traditional approach to obtain these peptides,
each compound is synthesized de novo; in each case, a
modification with respect to the parent peptide is intro-
duced.1ꢀ3 This serial procedure is costly in time and
materials. When the peptides are particularly difficult to
obtain (e.g., long or difficult sequences, macrocycles) or
when only part of the peptide requires modification, a site-
selective transformation would be preferable.4,5 In this
alternative strategy, a single (or a few) starting peptide is
used to generate the other library members, by selective
conversion of a “customizable” (or “tunable”) residue.
This strategy has recently attracted much attention in
academia and industry, and customizable units such as
glycine, dehydroalanine, 4-azidoproline, allylglycine, cy-
steine, and aziridine carboxylic acids have been reported6ꢀ8
and used for the selective modification of small peptides.7,8
(5) (a) For the “Tag-and-Modify” approach to protein modification,
see: Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res.
2011, 44, 730ꢀ741. (b) See also: Takaoka, Y.; Ojida, A.; Hamachi, I.
Angew. Chem., Int. Ed. 2013, 52, 4088–4106. (c) Dıaz-Rodrıguez, A.;
ꢀ
Davis, B. G. Curr. Opin. Chem. Biol. 2011, 15, 211–219. (d) Basle, E.;
(1) (a) Sewald, N.; Jakubke, H. D. Peptides: Chemistry and Biology;
Wiley-VCH: Weinheim, 2002. (b) Handbook of Biologically Active Pep-
tides; Kastin, A. J., Ed.; Academic Press: San Diego, 2006. (c) Acc. Chem.
Res. 2008, 41, no. 10, Special Issue on Peptidomimetics.
Joubert, N.; Pucheault, M. Chem. Biol. 2010, 17, 213–227. (e) Rabuka,
D. Curr. Opin. Chem. Biol. 2010, 14, 790–796. (f) Ostresh, J. M.; Husar,
G. M.; Blondelle, S. E.; Dorner, B.; Webert, P. A.; Houghten, R. A.
€
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11138–11142.
(2) Lee, S.; Xie, J.; Chen, X. Chem. Rev. 2010, 110, 3087–3111 and
references cited therein.
(6) (a) Seebach, D.; Bech, A. K.; Studer, A. Modern Synthetic
Methods, Vol. 7; Ernst, B., Leumann, C., Eds.; VCH: Weinheim, 1995.
(b) Datta, S.; Kazmaier, U. Org. Biomol. Chem. 2011, 9, 872–880. (c)
Deska, J.; Kazmaier, U. Chem.;Eur. J. 2007, 13, 6204–6211. (d)
Erdmann, R. S.; Wennemers, H. J. Am. Chem. Soc. 2010, 132, 13957–
13959. (e) Franz, N.; Menin, L.; Klok, H. A. Org. Biomol. Chem. 2009, 7,
(3) (a) Freund, M.; Tsogoeva, S. B. Peptides for Asymmetric Cata-
lysis. In Catalytic Methods in Asymmetric Synthesis; Gruttadauria, M.,
Giacalone, F., Eds.; John Wiley and Sons: Hoboken, NJ, USA, 2011. (b)
Roelfes, G. ChemCatChem 2011, 3, 647–648. (c) Colby-Davie, E. A.;
Mennen, S. M.; Xu, Y.; Miller, S. J. Chem. Rev. 2007, 107, 5759–5812.
(4) (a) Ebran, J. P.; Jensen, C. M.; Johannesen, S. A.; Karaffa, J.;
Lindsay, K. B.; Taaning, R.; Skrydstrup, T. Org. Biomol. Chem. 2006, 4,
3553–3564. (b) Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol.
2006, 10, 253–262. (c) Qi, D.; Tann, C. M.; Distefano, M. D. Chem. Rev.
2001, 101, 3081–3112.
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5207–5218. (f) Galonic, D. P.; Ide, N. D.; van der Donk, W. A.; Gin,
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D. Y. J. Am. Chem. Soc. 2005, 127, 7359–7369. (g) Galonic, D. P.;
van der Donk, W.; Gin, D. Y. J. Am. Chem. Soc. 2004, 126, 12712–
12713. (h) Dyer, F. B.; Park, C. M.; Joseph, R.; Garner, P. J. Am. Chem.
Soc. 2011, 133, 20033–20035. (i) Ricci, M.; Blakskjær, P.; Skrydstrup, T.
J. Am. Chem. Soc. 2000, 122, 1241–12421.
r
10.1021/ol402800a
Published on Web 10/30/2013
2013 American Chemical Society

Despite these advances, the site-selective modification of
peptides remains difficult,4 particularly when several units
of the “tunable” residue are present in the peptide. The
introduction of serine (or threonine) residues as customiz-
able units8 represented considerable progress: the serine
residues with free hydroxy groups were transformed, while
protected serine units remained unchanged (e.g., conver-
sion 1f3, Scheme 1). Orthogonal protecting groups allow
further residue differentiation.
Scheme 1. Introduction of Hyp as “Customizable” Unit (Right)
Presents Important Advantages over Use of Ser (Left)
In most of the reported procedures,8 the serine (or
threonine) lateral chain was completely removed and
replaced by other chains using scissionꢀaddition pro-
cesses. However, the addition step (either of nucleophiles
or electrophiles) usually took place with low stereoselec-
tivity, providing low diastereomeric excesses.
Therefore, the ultimate goal is the development of new
“customizable” units which could be transformed selec-
tively, even if several tunable units were present, and which
could be converted into unnatural chains with high stereo-
selectivity. In addition, the customizable units should
come from commercial, readily available amino acids,
preferably natural ones. This paper reports the use of
natural L-hydroxyproline derivatives as customizable units
in tandem radical scissionꢀoxidation processes and the
feasibility of this approach for the site-selective peptide
modification (conversion 4f6, Scheme 1).
The regioselectivity of the scission was aninitial concern.
Although the fragmentation could take place via the
C4ꢀC5 or the C3ꢀC4 bond, it was expected that the
first case would be favored,9 since the resulting primary
C-radical would be stabilized by the adjacent nitrogen
and then readily oxidized to an acyliminium ion 5.10 The
scission of the C3ꢀC4 bond would yield a nonstabilized
primary C-radical, which would be disfavored.
The initial studies of the transformation of hydroxypro-
line derivatives under oxidative radical fragmentation
conditions were performed with methyl carbamate 7
(Scheme 2).7f
Scheme 2. Study of the Conversion of Hydroxyproline
Derivatives into N-Alkyl 4-Oxohomoalanine Derivatives
On treatment with (diacetoxyiodo)benzene and iodine,
and under irradiation with visible light, the N-alkyl 4-oxo-
L-homoalanine derivative 8 was obtained in good yield;
(7) (a) For other interesting approaches, see: Saavedra, C. J.; Boto,
ꢀ
A.; Hernandez, R.; Miranda, J. I.; Aizpurua, J. M. J. Org. Chem. 2012,
ꢀ
77, 5907–5913. (b) Saavedra, C. J.; Boto, A.; Hernandez, R. Org. Lett.
no C3ꢀC4 scission products were detected. In order to
check that no epimerization took place in this step, the
(ꢀ)-menthyl carbamate 9 underwent the reaction, affording
exclusively compound 10.
2012, 14, 3542–3545. (c) Uhlig, N.; Li, C.-J. Org. Lett. 2012, 14, 3000–
3003. (d) Monfregola, L.; Leone, M.; Calce, E.; De Luca, S. Org. Lett.
2012, 14, 1664–1667. (e) Saavedra, C. J.; Boto, A.; Hernandez, R. Org.
ꢀ
ꢀ
Biomol. Chem. 2012, 10, 4448–4461. (f) Saavedra, C.; Hernandez, R.;
Boto, A.; Alvarez, E. J. Org. Chem. 2009, 74, 4655–4665. (g) Chapman,
C. J.; Hargrave, J. D.; Bish, G.; Frost, C. G. Tetrahedron 2008, 64, 9528–
9539. (h) Lin, Y. A.; Chalker, J. M.; Floyd, N.; Bernardes, G. J. L.;
Davis, B. G. J. Am. Chem. Soc. 2008, 130, 9642–9643. (i) Wan, Q.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 9248–9252 and
references cited therein.
(8) (a) Ricci, M.; Madariaga, L.; Skrydstrup, T. Angew. Chem., Int.
Ed. 2000, 39, 242–246. (b) Dialer, H.; Steglich, W.; Beck, W. Tetrahedron
2001, 57, 4855–4861. (c) Saavedra, C. J.; Boto, A.; Hernandez, R. Org.
Lett. 2012, 14, 3788–3791.
(9) (a) Boto, A.; Hernandez, D.; Hernandez, R.; Montoya, A.;
Suarez, E. Eur. J. Org. Chem. 2007, 325–334 and references cited therein.
(10) Boto, A.; Romero-Estudillo, I. Org. Lett. 2011, 13, 3426–
3429.
(11) Jamieson, A. G.; Boutard, N.; Sabatino, D.; Lubell, W. D.
Chem. Biol. Drug Des. 2013, 81, 148–165.
A prerequisite for a high-quality “customizable unit” is
that it can be transformed into a diversity of amino acids
while keeping the stereochemical integrity. Thus, the
transformation of compounds 8 and 9 into other unnatural
amino acids, by transformation of the aldehyde and
N,O-acetal “handles”, was studied next (Scheme 3). For
instance, the reductive amination of compound 8 with a
chiral amine followed by intramolecular lactamization
afforded compounds (10R)-11 or (10S)-11 in high optical
purity; only traces of a minor diastereomer coming from
epimerization could be detected (dr g 96:4).11
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Org. Lett., Vol. 15, No. 22, 2013
5779

expensive dehydrohomoglutamic acid 15. The reduction
of alkenyl chains could then afford unusual alkyl chains.
The derivatization of the amino acid lateral chain can
be accompanied by transformation of the N,O-acetal
function, by saponification, reduction, allylation, or alkyla-
tion.13 For instance, by reduction of the acetal, com-
pound 15 is converted into the N-methyl dehydrohomo-
glutamate 16 (Scheme 4). The N-alkylated amino acids
usually reach high market values, since the introduction
of N-methyl amino acids into peptides can affect their con-
formation and activity. Despite this, not many N-methyl
amino acids are commercially available. The present method
affords an efficient route to these compounds.
Scheme 3. Study of the Conversion of N-Alkyl 4-Oxohomoalanine
8 into Other Amino Acids
Scheme 4. Synthesis of High-Profit N-Methyl Amino Acid 16
Once the conversion of the hydroxyproline unit into
new, optically pure residues had been studied, its applica-
tion for site-selective peptide modification was explored.
For instance, the tripeptide 17 (Scheme 5) afforded the
aldehyde derivative 18 in good yields. In a similar way,
the dipeptide 19 underwent scission to give the aldehyde
derivative 20, which was converted into the leucine-
dehydrohomoglutamate derivative 21; no other isomers
were isolated.
Finally, the tetrapeptide 22 (Scheme 6) which contains
two tunable residues (a threonine and a hydroxyproline
units) underwent cleavage of the unit with the free hydroxy
group, while the other tunable residue, whose lateral
chain was protected, remained unchanged. The resulting
tetrapeptide 23 was reduced to the lactone derivative 24.
By careful control of the conditions, the N,O-acetal was
removed, releasing the NH group.
The scission of hydroxyproline units in internal positions
inthe peptide (for instance, conversion25f26, Scheme7) is
particularly interesting for conformationꢀactivity studies.
Such cleavage would decrease the system rigidity, produ-
cing conformational or even secondary structure changes
which could be used to modulate the biological or catalytic
activity. Besides,byremoval oftheN,O-acetal groups in the
scission products, new H-bonds could be formed, allowing
other conformational changes.
Whena secondaryamine (proline benzyl ester) was used,
the 4-aminohomoalanine derivative 12 was isolated (80%
yield). The reductive amination also gave good results
using amino acids with primary amino groups. For in-
stance, the aldehyde 8 was converted into the dipeptide
13 on treatment with a threonine derivative, followed by
reduction with inexpensive methanolic NaBH4. The pre-
paration of dipeptides such as 12 and 13 would be valuable
to synthesize branched peptides.
The reductive amination could also allow the prepara-
tion of medical probes.12 For instance, complexes of crown
ethers or cyclenes with different metals have been used in
molecular imaging to detect tumors.2,11 The ready trans-
formation of aldehyde 9 (Scheme 3) into the 15-aza-crown-5
derivative 14 in excellent yield and optical purity provides a
valuable probe component.
The aldehyde can also be transformed into alkenyl chains,
as shown by treatment of aldehyde 8 (Scheme 4) under
HornerꢀWadsworthꢀEmmons conditions to give the
(13) (a) For modification of both handles, see: Kesselring, D.;
Maurer, K.; Moeller, K. D. J. Am. Chem. Soc. 2008, 130, 11290–
11291. (b) Meffre, P. Amino Acids 1999, 16, 251–272. (c) Rush, J.;
Bertozzi, C. R. Org. Lett. 2006, 8, 131–134 and references cited therein.
(d) Substituted Hyp derivatives would provide new functionalized
handles: Sharma, R.; Lubell, W. D. J. Org. Chem. 1996, 61, 202–209.
(12) Bartholoma, M.;Valliant, J.;Maresca, K. P.;Babich, J.;Zubieta, J.
Chem. Commun. 2009, 493–512.
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Org. Lett., Vol. 15, No. 22, 2013

Scheme 5. Selective Modification of the Hyp Residue in Peptides
Scheme 7. Selective Scission of Internal Hyp Residues, and
Generation of Cationic Residues for Peptide Antimicrobials
Scheme 6. Selective Converison of Only One Customizable Unit
In summary, the use of natural and commercial hydro-
xyproline as a “customizable” unit allows the produc-
tion of a diversity of unnatural amino acids with high
optical purity, including expensive N-alkyl amino acids,
many R-alkylglycines, components of medical probes, and
branched peptides. The initial one-pot scissionꢀoxidation
process generates a residue with two reactive handles
which can be manipulated independently.
This “tunable” unit can also be used for the mild,
efficient, and site-selective modification of peptides, gen-
erating different derivatives from a single parent peptide.
Unlike other site-selective strategies, it is possible to dis-
criminate between several “tunable” units. The applica-
tions of this method to create diversity in the search
for antimicrobial peptides and for conformation/activity
studies will be reported in due course.
Acknowledgment. This work was supported by Plan
Nacional de IþD (CTQ2009-07109), MICINN/MINECO,
Spain), and European Regional Development (FEDER)
Funds. I.R.E. (Spanish Research Council) thanks CSIC for
a JAE predoctoral contract. We thank Prof. W. Lubell for
valuable suggestions for expanding this work.
The functionalization of the scission products would be
useful to create libraries of bioactive compounds. For
instance, the generation of 4-amino homoalanines could
be interesting for the development of antibiotic peptides,1a,
b
where a balance of nonpolar and cationic residues is
necessary for pharmacological utility.14 The conversion of
26 into 27 in very good yield highlights the feasibility of this
approach. The chosen morpholino-containing unit can be
found in commercial antimicrobials such as Cobicistat.15
Supporting Information Available. Experimental details,
and 1H and 13C NMR for all new products. This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) Savage, P. B. Eur. J. Med. Chem. 2002, 759–768.
(15) De Clercq, E. Med. Chem. Rev. 2013, 33, 1278–1303.
The authors declare no competing financial interest.
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