Functionalized analogues of an unnatural amino acid that mimics a tripeptide β-strand

Article abstract of DOI:10.1021/ol8021897

(Chemical Equation Presented) This paper introduces polar and hydrophobic variants of the unnatural amino acid Hao, which mimics the hydrogen-bonding functionality of one edge of a β-strand. In these variants, the methyl side chain of Hao is replaced with

Full text of DOI:10.1021/ol8021897

ORGANIC
LETTERS
2008
Vol. 10, No. 22
5293-5296
Functionalized Analogues of an
Unnatural Amino Acid that Mimics a
Tripeptide ꢀ-Strand
Tatyana V. Khasanova, Omid Khakshoor, and James S. Nowick*
Department of Chemistry, UniVersity of California, IrVine,
IrVine, California 92697-2025
jsnowick@uci.edu
Received September 18, 2008
ABSTRACT
This paper introduces polar and hydrophobic variants of the unnatural amino acid Hao, which mimics the hydrogen-bonding functionality of
one edge of a ꢀ-strand. In these variants, the methyl side chain of Hao is replaced with acidic, basic, and hydrophobic groups. These modifications
can impart improved solubility and additional side-chain interactions to peptides containing Hao.
Peptidomimetic templates that mimic or induce helix, turn,
or ꢀ-sheet structures are useful for studying and controlling
the conformations and interactions of peptides and proteins.1
Our research group previously introduced the unnatural
amino acid Hao as a tripeptide ꢀ-strand mimic that forms
hydrogen bonds from only one edge (Figure 1).2 We have
developed Hao-containing peptides that fold to form ꢀ-sheet
structure, dimerize through edge-to-edge ꢀ-sheet interaction,
and antagonize ꢀ-sheet aggregation.3 Other research groups
have investigated Hao and related structures in peptidomi-
metic compounds and hydrogen-bonded assemblies.4,5
The original unnatural amino acid Hao provides the
hydrogen-bonding functionality of the peptide main chain
but lacks side-chain functionality. In this paper, we introduce
variants of Hao with acidic, basic, and hydrophobic side
chains: HaoK, HaoD, HaoF, and HaoL (Figure 1). We have
developed these variants to address specific problems with
solubility and folding of Hao-containing peptides that we
(3) (a) Nowick, J. S.; Lam, K. S.; Khasanova, T. V.; Kemnitzer, W. E.;
Maitra, S.; Mee, H. T.; Liu, R. J. Am. Chem. Soc. 2002, 124, 4972–4973.
(b) Nowick, J. S.; Chung, D. M. Angew. Chem., Int. Ed. Engl. 2003, 42,
1765–1768. (c) Chung, D. M.; Nowick, J. S. J. Am. Chem. Soc. 2004, 126,
3062–3063. (d) Chung, D. M.; Dou, Y.; Baldi, P.; Nowick, J. S. J. Am.
Chem. Soc. 2004, 126, 9998–9999. (e) Woods, R. J.; Brower, J. O.;
Castellanos, E.; Hashemzadeh, M.; Khakshoor, O.; Russo, W. A.; Nowick,
J. S. J. Am. Chem. Soc. 2007, 129, 2548–2558. (f) Khakshoor, O.; Demeler,
B.; Nowick, J. S. J. Am. Chem. Soc. 2007, 129, 5558–5569.
(4) For a few examples, see: (a) Yu, H.; Daura, X.; van Gunsteren, W. F.
Proteins: Struct., Funct., Bioinf. 2004, 54, 116–127. (b) Bonauer, C.; Zabel,
M.; Ko¨nig, B. Org. Lett. 2004, 6, 1349–1352. (c) MiltSchitzky, S.;
Mitchlova, V.; Stadlbauer, S.; Koenig, B. Heterocycles 2006, 67, 135–160.
(d) Yang, Y.; Yan, H.-J.; Chen, C.-F.; Wan, L.-J. Org. Lett. 2007, 9, 4991–
4994.
(1) For a few reviews, see: (a) Nowick, J. S. Acc. Chem. Res. 2008,
ASAP Article, DOI: 10.1021/ar800064f. (b) Hanessian, S.; Auzzas, L. Acc.
Chem. Res. 2008, ASAP Article, DOI: 10.1021/ar8000052. (c) Patgiri, A.;
Jochim, A. L.; Arora, P. S. Acc. Chem. Res. 2008, ASAP Article, DOI:
10.1021/ar700264k. (d) Sakai, N.; Mareda, J.; Matile, S. Acc. Chem. Res.
2008, ASAP Article, DOI: 10.1021/ar700229r. (e) Horne, W. S.; Gellman,
S. H. Acc. Chem. Res. 2008, ASAP Article, DOI: 10.1021/ar800009n. (f)
Seebach, D.; Gardiner, J. Acc. Chem. Res. 2008, ASAP Article, DOI:
10.1021/ar700263g. (g) Robinson, J. A. Acc. Chem. Res. 2008, ASAP
Article, DOI: 10.1021/ar700259k.
(5) (a) Li, Z.-T.; Hou, J.-L.; Li, C. Acc. Chem. Res. 2008, ASAP Article,
DOI: 10.1021/ar700219m. (b) Gong, B. Acc. Chem. Res. 2008, ASAP
Article, DOI: 10.1021/ar700266f.
(2) Nowick, J. S.; Chung, D. M.; Maitra, K.; Maitra, S.; Stigers, K. D.;
Sun, Y. J. Am. Chem. Soc. 2000, 122, 7654–7661.
10.1021/ol8021897 CCC: $40.75
Published on Web 10/21/2008
2008 American Chemical Society

Figure 2.
Fmoc* derivatives of Hao variants.8
and protecting groups of the side chains. The syntheses of
1a-1d begin with ethyl or allyl 5-nitrosalicylate and involve
alkylation of the phenol group to introduce the side chains,
conversion of the ester group to the hydrazide, and conver-
sion of the nitro group to the oxamic acid.9
Figure 1. Tripeptide ꢀ-strand, Hao ꢀ-strand mimic, and Hao
variants.
Alkylation of ethyl 5-nitrosalicylate with Boc-protected
3-amino-1-bromopropane,10 tert-butyl bromoacetate, benzyl
bromide, or isopentyl bromide gives ethers 2a-2d (Scheme
1). Alkylation to form 2a and 2c proceed smoothly at
70-100 °C. For 2b, the temperature must be kept below 50
°C to minimize undesired reactions. For 2d, sodium iodide
is added to increase the rate of alkylation. Saponification of
the ethyl ester groups of 2a-2d is sluggish at room
temperature but occurs in 2-5 h upon heating at reflux in
aqueous THF (Scheme 1). Carboxylic acids 3a, 3c, and 3d
have encountered in our own research, and we anticipate
that these variants and types of side chains will be useful to
others.6,7
The amino acid Hao contains a methoxy group that imparts
rigidity through intramolecular hydrogen bonding and blocks
unwanted intermolecular hydrogen-bonding interactions. To
provide additional functionality, we have now replaced the
methyl group with aminopropyl, carboxymethyl, benzyl, and
isopentyl groups, which resemble the side chains of lysine,
aspartic acid, phenylalanine, and leucine, respectively. The
polar side chains offer the promise of enhanced water
solubility and electrostatic interactions, while the hydropho-
bic side chains offer the possibility of enhanced hydrophobic
interactions. To allow use in standard Fmoc-based solid-
phase peptide synthesis, we have prepared the Fmoc*
derivatives Fmoc*-HaoK(Boc)-OH (1a), Fmoc*-HaoD(t-Bu)-OH
(1b), Fmoc*-HaoF-OH (1c), and Fmoc*-HaoL-OH (1d)
(Figure 2).8
Scheme 1. Introduction of the Side Chains
The syntheses of Hao analogues 1a-1d are similar to the
synthesis of Fmoc*-Hao-OH that we reported previously but
require an alkylation reaction to introduce the different side
chains and tactical changes to tolerate the functional groups
(6) (a) Park, S. B.; Ho, T. H.; Reedy, B. M.; Connolly, M. D.; Standaert,
R. F. Org. Lett. 2003, 5, 2437–2440. (b) Kase¨mi, E.; Zhuang, W.; Ju¨rgen,
P. R.; Fischer, K.; Schmidt, M.; Colussi, M.; Keul, H.; Yi, D.; Co¨lfen, H.;
Schlu¨ter, A. D. J. Am. Chem. Soc. 2006, 128, 5091–5099. (c) Alam, M. A.;
Kim, Y.-S.; Ogawa, S.; Tsuda, A.; Ishii, N.; Aida, T. Angew. Chem., Int.
Ed. 2008, 47, 2070–2073. (d) Li, M.; Yamato, K.; Ferguson, J. S.; Singarapu,
K. K.; Szyperski, T.; Gong, B. J. Am. Chem. Soc. 2008, 130, 491–500
(7) Kang, S. W.; Gothard, C. M.; Maitra, S.; Atia-tul-Wahab; Nowick,
J. S. J. Am. Chem. Soc. 2007, 129, 1486–1487
(8) Fmoc* is the 2,7-di-tert-butyl derivative of the 9-fluorenylmethoxy-
carbonyl (Fmoc) group. The tert-butyl groups enhance solubility in organic
solvents: Stigers, K. D.; Koutroulis, M. R.; Chung, D. M.; Nowick, J. S. J.
Org. Chem. 2000, 65, 3858–3860.
.
are readily isolated by neutralization with strongly acidic ion-
exchange resin (Amberlite IR-120) and removal of THF.
Competing reactions during the hydrolysis of 2b are a
problem. To circumvent this problem, we selected the
orthogonal allyl protecting group and have used allyl
.
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Org. Lett., Vol. 10, No. 22, 2008

5-nitrosalicylate to prepare acid 3b. Alkylation of allyl
5-nitrosalicylate with tert-butyl bromoacetate to give ether
4 followed by catalytic deprotection with Pd(PPh3)4 affords
3b (Scheme 2).
Scheme 4. Introduction of the Oxamic Acid Group
Scheme 2. Introduction of the D(t-Bu) Side Chain
Coupling of acids 3a-3d with Fmoc*-hydrazine2 gives
the Fmoc*-protected hydrazides 5a-5d (Scheme 3). Acids
3a and 3b require nonacidic coupling conditions (EDC,
HOAt)11 to avoid loss of the acid-labile protecting groups,
whereas acids 3c and 3d can be coupled as the acid chlorides,
as in the original synthesis of Fmoc*-Hao-OH.2 The former
conditions are more convenient and higher-yielding and
should also be suitable for 3c and 3d (and Fmoc*-Hao-OH)
if desired.
chloride followed by saponification of the resulting oxamate
esters 7a-7d gives Hao analogues 1a-1d. The saponifica-
tion occurs rapidly, because the oxamate ester groups are
especially electrophilic. The resulting oxamate salts are
readily converted to the acids with strongly acidic ion-
exchange resin. Although the oxamic acid group (RNH-
Scheme 3. Introduction of the Fmoc*-Hydrazide Group
Introduction of the oxamic acid group by reduction of the
nitro group, acylation of the resulting aniline group, and
hydrolysis of the resulting oxamate ester affords the desired
Fmoc*-protected Hao variants (Scheme 4). Reduction of the
nitro group of Fmoc*-protected hydrazides 5a-5d gives
anilines 6a-6d. Although catalytic hydrogenation of hy-
drazides 5a, 5b, and 5d with Pd/C is efficient, it causes the
removal of the labile benzyl side chain of hydrazide 5c. To
selectively reduce the nitro group of hydrazide 5c, we used
SnCl2.12 Acylation of anilines 6a-6d with ethyl oxalyl
Figure 3
peptides 8-10.
.
Hao-, HaoK-, and HaoD-containing macrocyclic ꢀ-sheet
Org. Lett., Vol. 10, No. 22, 2008
5295

COCO2H) is more acidic than a regular carboxylic acid, the
Boc and tert-butyl ester groups of 1a and 1b appear to be
stable under typical storage and handling conditions.
useful tools for studying and controlling the conformations
and interactions of peptides and proteins. We will report
applications and studies of peptides containing these polar
and hydrophobic variants of Hao in due course.
The functionalized Hao building blocks 1a-1d are readily
prepared in gram quantities and permit the creation of Hao-
containing peptides with modified properties. The Fmoc*-
HaoX-OH building blocks 1a-1d can be coupled in solid-
phase peptide synthesis using either DIC and HOAt or HCTU
and 2,4,6-collidine in DMF as coupling reagents.11,13-15 We
have used building blocks 1a and 1b to prepare HaoK- and
HaoD-containing macrocyclic ꢀ-sheet peptides 9 and 10 as
analogues of macrocyclic ꢀ-sheet peptides 8, which we had
reported previously (Figure 3).3f Macrocyclic ꢀ-sheet pep-
tides 8 form tetramers through ꢀ-sheet interactions in water
and hold promise as inhibitors of ꢀ-amyloid aggregation or
as ligands to bind protein ꢀ-sheets. Our efforts to study these
biologically relevant properties have been hampered by
precipitation of peptides 8 with anionic buffers and proteins.
HaoD and HaoK analogues 9 and 10 have shown improved
solubility and have allowed us to proceed with our studies.
These functionalized Hao building blocks increase the
arsenal of ꢀ-sheet peptidomimetic templates and should be
Acknowledgment. We thank the National Institutes of
Health for grant support (GM-49076).
Supporting Information Available: Experimental pro-
cedures and spectroscopic and analytical data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL8021897
(11) Abbreviations: EDC, N-ethyl-N′-(3-dimethylaminopropyl)carbodi-
imide hydrochloride; HOAt, 1-hydroxy-7-azabenzotriazole; DIC, N,N′-
diisopropylcarbodiimide; HCTU, O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (ref 13).
(12) Bellamy, F. D.; Ou, K. Tetrahedron Lett. 1984, 25, 839–842.
(13) Although HCTU is commonly thought of as the uranium isomer,
the actual structure of this compound is likely the guanidinium isomer. See:
(a) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang, C.;
Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mu¨gge, C.; Wenschuh,
H.; Klose, J.; Beyermann, M.; Bienert, M. Angew. Chem., Int. Ed. 2002,
41, 441–445. (b) Sabatino, G.; Mulinacci, B.; Alcaro, M. C.; Chelli, M.;
Rovero, P.; Papini, A. M. Lett. Pept. Sci. 2002, 9, 119–123.
(9) The ethyl and allyl esters of 5-nitrosalicylic acid can be easily
prepared from 5-nitrosalicylic acid by Fischer esterification.
(10) Pearson, D. A.; Lister-James, J.; McBride, W. J.; Wilson, D. M.;
Martel, L. J.; Civitello, E. R.; Dean, R. T. J. Med. Chem. 1996, 39, 1372–
1382.
(14) Coupling of Hao and its variants in the presence of stronger bases,
such as N,N-diisopropylethylamine, appears to result in decomposition of
the oxamate group.
(15) For the use of 2,4,6-collidine as a base in peptide synthesis, see:
Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695–698.
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