NXO building blocks for backbone modification of peptides and preparation of pseudopeptides

Article abstract of DOI:10.1021/jo902518r

The design and synthesis of novel building blocks for peptide modification, termed NXO, is reported. We describe the utility of these building blocks to prepare new pseudo- and oligopeptides and demonstrate the efficient assembly of modified tripeptides using both conventional liquid phase peptide synthesis and solid supported synthesis. Pertaining to the study of NXO in peptide mimicry, the structure of NXO-incorporating tripeptide β-strand mimics was investigated in the N^LAlaO incorporating β-sheet model compound 13. Evidenced by spectroscopy and computation, 13 selectively adopts a β-structure in chloroform and characteristically samples the NXO-modified backbone site in the core β-sheet region. ROESY (HNMR) and molecular dynamics data suggest that when disrupting the cross-strand hydrogen-bonding pattern by switching the solvent from CDCl₃ to d₃-MeOH, the main conformation with peptide and NXO-peptide backbones similar to that in root mean square deviation of corresponding backbone atoms (rmsd) is preserved.

Full text of DOI:10.1021/jo902518r

pubs.acs.org/joc
NXO Building Blocks for Backbone Modification of Peptides and
Preparation of Pseudopeptides
Constantin Rabong, Ulrich Jordis,* and Jaywant B. Phopase*
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria
jaywant.phopase@dcu.ie; ujordis@pop.tuwien.ac.at
Received November 26, 2009
The design and synthesis of novel building blocks for peptide modification, termed “NXO”, is
reported. We describe the utility of these building blocks to prepare new pseudo- and oligopeptides
and demonstrate the efficient assembly of modified tripeptides using both conventional liquid phase
peptide synthesis and solid supported synthesis. Pertaining to the study of NXO in peptide mimicry,
the structure of NXO-incorporating tripeptide β-strand mimics was investigated in the NLAlaO
incorporating β-sheet model compound 13. Evidenced by spectroscopy and computation, 13
selectively adopts a β-structure in chloroform and characteristically samples the NXO-modified
backbone site in the core β-sheet region. ROESY (HNMR) and molecular dynamics data suggest that
when disrupting the cross-strand hydrogen-bonding pattern by switching the solvent from CDCl3 to
d3-MeOH, the main conformation with peptide and NXO-peptide backbones similar to that in root
mean square deviation of corresponding backbone atoms (rmsd) is preserved.
Introduction
at any one of the repeating elements (NH, CHR, and CO)
or a combined (CONH) unit of the peptide. Synthetically,
modification at the R-carbon in the peptide backbone
remains the most challenging task. Only a few examples of
backbone modification at the R-carbon in peptides exist.4
The most common manipulation involving the R-carbon
atom of peptides is the inversion of stereochemistry to
yield D-amino acids.5 Azapeptides2a,4a,d and oxalo-retro
Though naturally occurring and synthetic peptides are pro-
mising leads for drug discovery, their therapeutical applica-
tion has been limited.1 This arises from their rapid metabolism,
poor bioavailability, and short duration of action. Many
peptide analogues have been synthesized to date through the
modification of not only the amino acid side chain, but also the
backbone structure.2,3 The general strategy involved in peptide
backbone (-NH-CHR-CO)n modification is the change
(3) (a) Han, H.; Janda, K. D. J. Am. Chem. Soc. 1996, 118, 2539–2544. (b)
Hagihara, M.; Anthony, N. J.; Stout, T. J.; Clardy, J.; Schreiber, S. J. J. Am.
Chem. Soc. 1992, 114, 6568–6570. (c) Simon, R. J..; et al. Proc. Natl. Acad.
Sci. U.S.A. 1992, 89, 9367–9371. (d) Smith, A. B.; Keenan, T. P.; Holcomb,
R. C.; Sprengeler, P. A.; Guzman, M. C.; Wood, J. L.; Carroll, P. J.;
Hirschmann, R. J. Am. Chem. Soc. 1992, 114, 10672–10674. (e) Cho, C.
Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S. P.; Adams, C. L.;
Sundaaram, A.; Jacobs, J. W.; Schultz, P. G. Science 1993, 261, 1303–1305.
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McCluskey, J.; Rossjohn, J. Nat. Rev. Drug Discovery 2007, 6, 404–414. (c)
Perez, J. J.; Corcho, F.; Llorens, O. Curr. Med. Chem. 2002, 9, 2209–2229. (d)
Bursavich, M. G.; Rich, D. H. J. Med. Chem. 2002, 45, 541–558. (e) Dougherty,
J. M.; Probst, D. A.; Robinson, R. E.; Moore, J. D.; Klein, T. A.; Snelgrove, K. A.;
Hanson, P. R. Tetrahedron 2000, 56, 9781–9790. (f) Hruby, V. J.; Balse, P. M.
Curr. Med. Chem. 2000, 7, 945–970. (g) Steer, D. L.; Lew, R. A.; Perlmutter, P.;
Smith, A. I.; Aguilar, M.-I. Curr. Med. Chem. 2002, 9, 811–822.
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F. Org. Biomol. Chem. 2009, 7, 128–134. (b) Cardillo, G.; Gentilucci, L.;
Tolomelli, A. Mini Rev. Med. Chem. 2006, 6, 293–304. (c) Melendez, R. E.;
Lubell, W. D. J. Am. Chem. Soc. 2004, 126, 6759-6764 and references cited
therein. (d) Marshall, G. R. Tetrahedron 1993, 49, 3547–3558. (e) Han, H.; Yoon,
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2492 J. Org. Chem. 2010, 75, 2492–2500
Published on Web 03/18/2010
DOI: 10.1021/jo902518r
r
2010 American Chemical Society

Rabong et al.
JOCArticle
NXO constitutes a short-range, dipole-organized motif.
Its “N-” and “O”-retrons have an inherent bias toward
antiparallel dipole configuration.7 By incorporating such
features into an NXO-modified backbone, they should lend
themselves naturally to β-structure mimicry. It is instructive
to note that NXO-modified peptides are β-sheet-tripeptide
mimics that comprise the oxalo-retro modification and
azapeptide modification on either ends. While doing so,
the R-carbon atoms of the amino acids at both ends of the
tripeptide are converted from sp3-hybridized, chiral centers
to sp2 centers that impose additional rigidity and extended
conjugation to the peptide strand (Figure 2).
Any advanced scaffold must aim to meet the requirements
of a peptidomimetic in terms of being a “peptide foldamer”:8
diversifiable, highly designable, efficiently prepared, and
selectively adopting an in silico predictable secondary struc-
ture. NXO, its features endowed within each monomer,
unites the “beta-oid” template with the strand increment at
the repeating unit level. Featuring the backbone polarization
pattern of R-amino acids (Figure 2), it is amenable for use in
mixed peptides and as a biopolymer in its own right.
FIGURE 1. X = any amino acid, R1-R4 = H, alkyl, aryl, protect-
ing group.
peptides6 are further examples of R-carbon modification;
although studies toward biologically active analogues of
these have had significant success,2a,6d their synthetic ex-
ploitation remained underexplored.2,4a,d,6
In this article, we describe the design of novel building
blocks and their use in peptide synthesis and as tripeptide β-
strand mimics to prepare β-sheet-like compounds.
Synthesis of Orthogonally Protected NXO Building Blocks.
Our first goal was to prepare orthogonally protected NXO
building blocks 3 starting from different amino acids that can
be incorporated into peptide synthesis from either the C- or
N-terminal after selective deprotection. As numerous Boc,
Fmoc, Z-protected substituted hydrazines,9 as well as a
variety of different oxalic acid diesters 2, are synthetically
available,10 a large variety of orthogonally protected NXO
building blocks can be prepared.
We developed a robust one-pot methodology to prepare
NXO building blocks starting from commercially available
amino acids (Scheme 1). The amino acid 1 is reacted with the
appropriate oxalic acid diester 2, prepared in near-quantita-
tive yields by reacting corresponding alkyl oxalyl chlorides
with 4-nitrophenol in pyridine-CH2Cl2, in DMF at 50 °C for
1.5 h. Further treatment of the resulting reaction mixture
with an appropriately protected hydrazine at room tempera-
ture affords the desired NXO building blocks. The advan-
tage of this method is that the amino acids do not require any
protection at the amino group, thus decreasing the number
of synthetic steps.11 This method allowed for the efficient
preparation of the building blocks presented in Table 1 in
good to excellent yields. The chiral purity of compounds 3c
and 3f was analyzed by HPLC, using a Chiral Technologies
Chiral Cel OD-H column, eluting with 90% n-heptane/0.1%
diethylamine/9.9% isopropanol as mobile phase. The mini-
mum detection limit of the other enantiomer was established
to be <0.04%; therefore, the chiral purity of the NXO
building blocks exceeded 99.96% in both cases.
Results and Discussion
We envisioned modifying amino acids to give new kinds of
pseudopeptides with reverse functionalities on either end by
incorporating an oxalic acid functionality at the amine end and
a hydrazine functionality at the carboxylic acid end. Thus, the
amino group in the parent amino acid is converted to the oxala-
mide (and therefore becomes the C-terminus of the pseudo-
peptide) and the acid is converted to the azapeptide (thus being
converted to the N-terminus); however, the amino acid core of
the parent amino acid remains unchanged (Figure 1). We
designated this new class of pseudopeptides by a three-letter
abbreviation as “NXO”, where “N” represents the hydrazide
part, “O” represents the oxalamide part, and “X” is the three-
letter abbreviation of the parent amino acid (see Table 1).
(6) (a) Ranganathan, D.; Saini, S. J. Am. Chem. Soc. 1991, 113, 1042–
1044. (b) Ranganathan, D. Pure Appl. Chem. 1996, 68, 671–674. (c) Ranga-
nathan, D.; Vaish, N. K.; Shah, K. J. Am. Chem. Soc. 1994, 116, 6545–6557.
(d) Murahashi, S-I.; Mitani, A.; Kitao, K. Tetrahedron 2000, 41, 10245-1049
and references cited therein.
(7) (a) Guenther, R.; Hofmann, H.-J. J. Am. Chem. Soc. 2001, 123, 247–
255. (b) Thormann, M.; Hofmann, H.-J. THEOCHEM 1999, 469, 63-76 and
references cited therein; see also refs 4d and 6b. These references contain several
important points which guided our design considerations employing substituted
hydrazines and oxalates (at the peptidic C- and N-ends, respectively) to replace amides
as well as stereogenic R-amino-carbons in the peptide backbone: Introduced nitrogens
were shown to be substantially pyramidal and hence potentially stereogenic (ref 7a) and
to engage heavily in hydrogen bonding to neighboring carbonyl oxygen (C5 interactions
and also, to a lesser extent, C8 interactions). In general, azapeptides were considered to
be a poor choice when extended strand mimics are sought after and described as
intrinsically biased for helix/turn formation (ref 7b; see also 8b). However, they possess
a high propensity for turn mimicry as they sample all known β-turn types within the
configuration space also adopted by amino acids. Aza-amino acids at the i þ 2 position
favor a type II turn, whereas at the i þ 1 site, a type I turn is preferred. On the other hand,
dicarbonylhydrazides were shown to have a propensity for an N-N bond orientation
with perpendicular lone pairs (ref 7a) but there is also experimental evidence that when
a dicarbonyl-substituted aza-linkage is allowed to engage in hydrogen bonding,
planarity can be enforced. Ab initio methods employing correlation-polarization basis
sets with explicitly treated aqueous solvation on formylated azaglycine gave the relevant
backbone dihedrals exclusively in the core βand Rregion (Φ≈(-70)° and (-90)° and
ψ ≈ þ165° and (-25)° for βand R, respectively). Moreover, stereogenicity was highly
pronounced in the β region but an overtly planarized nitrogen is observed in the R
region. Most importantly, the authors concluded that side chains on the azapeptide
nitrogens had almost no influence on the backbone torsion profile established before.
Studies on oxalo-retro bispeptides (see refs 6a-c) had shown that a peptide-isosteric
MeO2C-Leu-HNCO2CNH-Leu-CO2Me unit is mainly in a bicyclo[3.3.0] motif, that is,
extensive next-neighbor hydrogen bonding occurs, imposing strong planarizing bias
toward an extended conformation.
(8) (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173–180. (b) Hill, D. J.;
Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101,
3893–4012.
(9) (a) Dutta, A. S.; Morley, J. S. J. Chem. Soc., Perkin Trans. 1 1975, 712–
1720. (b) Quibell, M.; Turnell, W. G.; Johnson, T. J. Chem. Soc., Perkin
Trans. 1 1993, 2843–2849. (c) Bentley, P. H.; Morley, J. S. J. Chem. Soc.,
Perkin Trans. 1 1966, 60–64. (d) Biel, J. H.; Drukker, A. E.; Mitchell, T. F.;
Sprengeler, E. P.; Nuhfer, P. A.; Conway, A. C.; Horita, A. J. Am. Chem.
Soc. 1959, 81, 2805–2813. (e) Rutjes, F.-P. J. T.; Teerhuis, N. M.; Hiemstra,
H.; Speckkamp, W. N. Tetrahedron 1993, 49, 8605–8628.
(10) (a) Vashchenko, Y. N.; Moshchitskii, S. D.; Kirsanov, A. V. Zh.
Obshch. Khim. 1962, 32, 3765–3768. (b) Chakraborty, K.; Devakumar, C.
J. Agric. Food Chem. 2006, 54, 1868–1873.
(11) Some of these building blocks have been prepared by us in a more
classical manner before, see: PCT Int. Appl. 2007, WO2007095980.
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TABLE 1. Orthogonally Protected NXO Building Blocks
entry
R2
R4
R3, R1
X
NXO-name
[R]20D (c 0.5, CH3OH)
isolated yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Boc
Fmoc
Fmoc
Fmoc
Fmoc
Cbz
Me
Me
But
But
But
But
Me
Me
But
Me
Me
But
But
Me
Me
But
Me
But
But
But
Me
But
But
But
But
H, H
H, H
H, H
H, Me
Me, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Trp
Trp
Trp
Boc-NPheO-OMe (3a)
Fmoc-NPheO-OMe (3b)
Fmoc-NPheO-OBut (3c)
Fmoc-N(Me)PheO-OBut (3d)
Fmoc-(Me)NPheO-OBut (3e)
Cbz-NPheO-OBut (3f)
-28.6
-19.4
-18.0a
þ26.4
-17.4
-23.0
24.6
-37.2
-37.2
-37.4
-10.2a
-12.4
-10.4
-9.0
-43.4
-35.2
-43.8
-68.6
-70.8
-31.4
-47.6
-6.2
92
61
94
83
87
89
94
85
83
78
88
84
94
94
75
88
88
98
99
93
97
81
78
67
82
Cbz
Cbz-NPheO-OMe (3g)
Fmoc
Fmoc
Cbz
Fmoc
Fmoc
Cbz
Cbz
Boc
Fmoc
Cbz
Fmoc
Cbz
Fmoc
Cbz
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc-NTrpO-OMe (3h)
Fmoc-NTrpO-OBut (3i)
Cbz-NTrpO-OMe (3j)
Tyr(2Br-Z)OH
Tyr(2Br-Z)OH
Tyr(2Br-Z)OH
Tyr(2Br-Z)OH
Leu
Fmoc-NTyr (2Br-Z) O-OMe (3k)
Fmoc-NTyr (2Br-Z) O-OBut (3l)
Cbz-NTyr (2Br-Z) O-OBut (3m)
Cbz-NTyr (2Br-Z) O-OMe (3n)
Boc-NLeuO-OMe (3o)
Leu
Leu
Pro
Pro
Ala
Ala
Fmoc-NLeuO-OBut (3p)
Fmoc-NLeuO-OMe (3q)
Fmoc-NProO-OBut (3r)
Cbz-NProO-OBut (3s)
Fmoc-NAlaO-OBut (3t)
Cbz-NAlaO-OMe (3u)
Ser(Bn)-OH
Glu(OBn)OH
Thr(Bn)OH
Val
Fmoc-NSer(Bn)O-OBut (3v)
Fmoc-NGlu(OBn)O-OBut 3w)
Fmoc-NThr(Bn)O-OBut (3x)
Fmoc-NValO-OBut (3y)
-22.8
-8.0
-31.6
aOptical rotation was measured in DMF.
synthesis methods. The Boc protective group was removed
by treatment with TFA-CH2Cl2 (1:1) and the free amino
group was then liberated by treatment with diisopropylethy-
lamine (DIPEA). The resulting amine was coupled to Boc-L-
Ala with DIC, HOBt to give 11. The Boc group of 11 was
removed and the liberated amine was coupled to Boc-L-
phenylalanine by employing similar reaction conditions as
above. Finally, peptide 5 was isolated as the sodium salt from
the resin by hydrolysis with sodium methoxide. The sodium
salt was neutralized with 1 N HCl and the structure of
pseudopeptide 5 (43% overall yield with respect to the resin
loading) was verified with MALDI-TOF.
Preparation of an NXO-Tripeptide β-Strand Mimic. The
NXO-modified peptide backbone can adopt extended con-
formations as in a tripeptide β-strand by virtue of the
conformational restraints and extended conjugation im-
posed by NXO’s azapeptide “N” and oxalo-retro peptide
“O”-motifs. Thus, we anticipated that, by exchanging both
termini of a given amino acid to an oxalamide and azapep-
tide, respectively, sufficient strand planarization bias12 could
be introduced to maintain a strand-extended alignment
capable of multiple intramolecular hydrogen bonding.
To evaluate the structural properties of NXO acting as a
tripeptideβ-strand mimic, weimplemented an NXO derivative
to prepare the β-sheet model compound 13 (see Scheme 4). 13
was designed by using a proven model system reported by
Nowick and co-workers that consists of an oligourea molec-
ular scaffold, β-strand mimic, and peptide strand.13 There, it
FIGURE 2. The donor-acceptor pattern of a tripeptide vs NXO
peptide.
Synthesis of Pseudopeptides 4 and 5. In an effort to demon-
strate the applicability of NXO building blocks, we utilized
them to prepare pseudopeptides via liquid (4, Scheme 2) and
solid phase (5, Scheme 3) protocols. Both pseudopeptides 4
and 5 were chosen as model peptides in order to demonstrate
the ease of integrating the NXO building blocks in the
synthesis of oligopeptides by using routine chemical manip-
ulations. Pseudopeptide 4 was prepared from Boc-NPheO-
OMe (3a) (Scheme 2).
The Boc protective group was removed by treatment with
TFA-CH2Cl2 (1:1), and the free amine was liberated by
treatment with triethylamine (TEA). The amino group was
then coupled to Boc-L-alanine with DCC/HOBt to give 6.
The methyl ester was hydrolyzed by using LiOH in methanol
to afford acid 7, which after coupling to 8 with EDCI/HOBt
affords compound 4 (73% overall yield).
(12) Archer, E. A.; Gong, H.; Krische, M. J. Tetrahedron 2001, 57, 1139–
1159.
(13) (a) Nowick, J. S.; Tsai, J. H.; Bui, Q.-C. D.; Maitra, S. J. Am. Chem.
Soc. 1999, 121, 8409–8410. (b) Nowick, J. S. Acc. Chem. Res. 1999, 32, 287–
296. (c) Nowick, J. S.; Lam, K. S.; Khasanova, T. V.; Kemnitzer, W. E.;
Maitra, S.; Mee, H. T.; Liu, R. J. Am. Chem. Soc. 2000, 124, 4972–4973. (d)
Nowick, J. S.; Chung, D. M.; Maitra, K.; Maitra, S.; Stigers, K. D.; Sun, Y. J.
Am. Chem. Soc. 2000, 122, 7654–7661. (e) Nowick, J. S. Org. Biomol. Chem.
2006, 4, 3869–3885. (f) Stigers, K. D.; Sun, Y. J. Am. Chem. Soc. 2000, 122,
7654–7661. (g) Nowick, J. S. Org. Biomol. Chem. 2006, 4, 3869–3885.
Scheme 3 illustrates the solid phase synthesis of pseudo-
peptide 5. Resin bound NXO peptide 10 was constructed on
hydroxymethyl polystyrene resin by standard solid phase
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SCHEME 1. One-Pot Protocol To Prepare Orthogonally Protected NXO Building Blocks from R-Amino Acids
SCHEME 2. Solution-Phase Synthesis of Pseudopeptide 4
SCHEME 3. Solid-Phase Synthesis of Pseudopeptide 5
was shown that “hybrid” mimics where natural amino acids, in
conjunction with conformationally restricted aromatic assem-
bly templates, can selectively adopt well-defined secondary
structure motifs.14 Therefore, we aimed to test the foldability
of an NXO-modified β-strand mimic in an oligo-urea turn unit
(termed “molecular scaffold”13) in the context of what Nowick
and co-workers have described as an “artificial β-sheet.”15
To assess the performance of NXO in sheet-mimicry, we
evaluated the same sequence of amino acids (Phe-Ile-Leu-
NHMe) at corresponding16 backbone positions. Knowing
that Ala displays a rather insignificant intrinsic β-sheet
propensity17 we have, hence, incorporated an NLAlaO unit
into the lower strand section.
The synthesis of β-sheet-mimic 13 started from diamine 14.
Compound 14 was converted into urea 15 by reaction with
phosgene to generate a carbamyl chloride group at the aniline
nitrogen, followed by reaction with Boc-carbazate in dichlor-
omethane. Subsequent deprotection of the Boc-protected hy-
drazide with TFA-CH2Cl2 (1:1) followed by liberation of the
free amino group by treatment with DIPEA gave the amine
that afforded, after coupling with compound 18, compound 16
in 90% yield. Deprotection of Fmoc with 10% piperidine in
DMF and further reacting the reaction mixture with acryloni-
trile gave compound 17 in 50% yield. Compound 17 was con-
verted into NXO-modified artificial β-sheet 13 by reaction with
isocyanate 19. Purification of the crude residue by column chro-
matography afforded 68% of 13.
(14) Nowick, J. S. Acc. Chem. Res. 2008, 41, 1319-1330 and references
cited therein.
(15) Nowick, J. S.; Smith, E. M.; Noronha, G. J. Org. Chem. 1995, 60,
7386–7387.
(16) (a) Nowick, J. S.; Holmes, D. L.; Mackin, G.; Noronha, G. A.;
Shaka, J.; Smith, E. M. J. Am. Chem. Soc. 1996, 118, 2764–2765. (b) Nowick,
J. S.; Pairish, M.; Lee, I. Q.; Holmes, D.; Ziller, J. W. J. Am. Chem. Soc. 1997,
119, 5413–5424.
(17) (a) Smith, C. K.; Withka, J. M.; Regan, L. Biochemistry 1994, 33,
5510–5517. (b) Smith, C. K.; Regan, L. Acc. Chem. Res. 1997, 30, 153–161.
(c) Merkel, J. S.; Sturtevant, J. M.; Regan, L. Structure 1999, 7, 1333–1343.
(d) Hughes, R. M.; Waters, M. L. Curr. Op. Struct. Biol. 2006, 16, 514–524.
Conformation Analysis of 13. Part 1: ROESY NMR
Studies. Extensive attempts to obtain single crystals of 13
suitable for X-ray diffraction analysis (XRD) have failed to
date. Thus, secondary structure was investigated by solution
NMR. Analysis of 13 in CDCl3 at 290 K with 250 ms mixing
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SCHEME 4. Synthesis of NXO-Incorporating β-Sheet Mimic 13
time at a concentration of 5 mM suggests population of the
sample with a major conformer amounting to g94.8% based
on averaged signal integration of H1, N-H28, and N-H32.
The most compelling among the data are the NOEs between
the lower strand substituent methyl and the phenylalanine
benzyl-CH2, H31TH16, and between the upper strand iso-
leucine side chain and the R-amino proton, H30TH10 and
H30TH12 (Figure 3; see also the Supporting Information).
The across-strand NOE between the tert-butyloxalamido
group and the terminal methylamide, H34TH1, provides
further strong evidence for the observed conformation to be
in an intramolecularly strand-paired arrangement.18 The
presence of a weak NOE between the alanine methyl group
and the terminal methylamide, H31TH1, seems to reflect
a curvature previously observed in β-sheet models at the
C-end section.19 Further, the vicinal coupling constant of
the “X”-R-amino-proton with its neighboring amide-NH,
3JH30THN32, was found to be 8.5 Hz.
(18) (a) Nowick, J. S.; Smith, E. M.; Ziller, J. W.; Shaka, A. J. Tetrahedron
2002, 58, 727–739. (b) Chung, D. M.; Dou, Y.; Baldi, P.; Nowick, J. S. J. Am.
Chem. Soc. 2005, 127, 9998–9999. see also ref 13d.
(19) Several NOEs account for o- and m-aniline-protons H25 and H26 to
be close to the strand section C30-C28, suggesting the phenyl ring to be
orthogonal to the lower strand amide backbone. H26 showed a medium
NOE to the leucine-region C6-C7 multiplet, consistent with the previously
invoked antiparallel edge-to-edge dimer formation in artificial β-sheet
mimics, where a strand of one molecule binds the other strand of a second
molecule in a 180° rotated fashion. ( Nowick, J. S. Org. Biomol. Chem. 2006,
4, 3869-3885. On dimeric aggregation in particular: Nowick, J. S., Tsai, J.
H.; Bui, Q.-C. D.; Maitra, S. J. Am. Chem. Soc. 1999, 121, 8409-8410. On
self aggregation in β-sheets: Osterman, D. G.; Kaiser, E. T. J. Cell. Biochem.
1985, 29, 57-72. ). By contrast, the minor conformation gave rise to a strong
NOE H16TH14 which was weak in the main conformer; H29TH30 was seen
but no H29TH31. Moreover, a strong crosspeak H21TH5/H4 in the minor
conformer infers an alternative dimer pattern formation;homodimer or
same-strand aggregation;to be present here ( Nowick, J. S. Org. Biomol.
Chem. 2006, 4, 3869-3885 and references cited therein).
FIGURE 3. ROESY interactions in 13. Spectra were recorded with
a 250 ms mixing time at 290 K in CDCl3 and d3-MeOH. Full and
dotted arrows represent the observed conformational key NOE
enhancements in CDCl3 and d3-MeOH, respectively. Numbering is
as used throughout the text. Notice that an intermolecular interac-
tion is shown at the top of the figure; see also ref 19.
ROESY in d3-MeOH exhibited some markedly different
crosspeaks. Again, the spectra showed almost exclusively a
single signal set to be populated in solution with the minor
set amounting to <4.7% by averaged signal integration.
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FIGURE 4. The minimum-energy conformer of 13 generated from stochastic searching with OPLS-AA parameters and also the central motif
in rmsd-space during a 100 ns, 290 K MD simulation (see also the Supporting Information). Dotted lines indicate hydrogen bonding.
Weak-to-medium NOEs of the aniline-protons with the cyano-
ethylene side chain protons H22 and a prominent medium-
to-strong NOE between the phenylalanine para-proton and the
tert-butyloxalamide, H19TH34, suggested a conformation
with an alternating turn region to be present. Moreover, the
crosspeaks H9TH10, H10TH14, and H31TH32 were weaker
in comparison to spectra recorded in CDCl3 and there was a
previously unobserved very weak interaction H3TH11/H13.
In particular, TOCSY in d3-MeOH gave insight into the
hydrogen bonding behavior of the, now well-resolved, N-H
signals (except for the N-hydrazide protons, N-H28 and 29,
which exchanged under these conditions). All amide N-H
signals were shifted downfield, some up to 1.2 ppm relative to
measurements in CDCl3, indicating strong augmentation of
backbone hydrogen bonding in the more polar solvent (see
Table 2, Supporting Information).
Conformation Analysis of 13. Part 2: Computational Ana-
lysis. Locating the Ensemble Minimum. To verify and substan-
tiate the picture derived from NMR analysis, we performed
exhaustive (>105 conformations generated) stochastic confor-
mational searching around a loosely constrained, OPLS-AA20-
derived input geometry satisfying the above-mentioned (see
Figure 3 and the Supporting Information) conformationally
diagnostic NOE requirements. An extended structure (Figure 4),
across-strand associated via two “chelating” hydrogen bonds
was detected as the energy-lowest conformer by 0.8 kcal.21 This
conformer was then freed of all restraints and subjected to
molecular dynamics (MD), using the OPLS-AA parameter set
and GB/SA22 as an implicit solvation model.
strand paired, extended sheet-like conformations within the
sampled time span of 100 ns. The turn diaminoethane unit re-
mains in an arrangement displaying N-C23-C24-N antiperi-
planar (ap) during the entire simulation. At these temperatures,
the hydrazide and in particular the oxalamide modifications are
favored in a narrow torsional region (ap) þ160° and -160°,
respectively (see the Supporting Information).
Several indicative upper-to-lower strand hydrogen bonding
interactions are present, notably O27arHN20 (sampled 94%
of the simulation time with an average lifetime of 31.5 ps),
O29arH28 (52.5%, 2.6 ps), O8arH29 (22.2%, 7.5 ps),
O8arH32 (19.2%, 9 ps), O14arH29 (14.8%, 2 ps), and
1
O8arH33 (12.7%, 2.8 ps). Moreover, from 600 MHz H
NMR in CDCl3, we had observed 3JH30THN32 to give a value
of 8.5 Hz.23 Since the dihedral corresponding to this torsion was
adequately reproduced in the MD simulation (the dihedral
displaying a maximum at -148° and thus satisfying the unpar-
ameterized Karplus equation24) we can, therefore, assess Φ
(C32a-N32-C30-C29a) and Ψ (N32-C30-C29a-N29) in
the NLAlaO-modified strand. Φand Ψ display maxima at -82°
and þ79°, respectively, thus sampling NLAlaO in the core β-
sheet-region of the Ramachandran plot.25,26
(23) (a) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992,
31, 1647–1651. (b) Yu, H.; Daura, X.; van Gunsteren, W. F. Proteins 2004, 54,
116-127; see also: (c) Daura, X. Theor. Chem. Acc. 2006, 116, 297-306 and
references cited therein. (d) Santivieri, C. M.; Jimenez, M. A.; Rico, M.; van
Gunsteren, W. F.; Daura, X. J. Peptide Sci. 2004, 10, 546–565.
(24) (a) Karplus, M.; Anderson, D. H. J. Chem. Phys. 1959, 30, 6–10. (b)
Karplus, M. J. Chem. Phys. 1959, 30, 11–15. (c) Karplus, M. J. Am. Chem.
Soc. 1963, 85, 2870–2871.
(25) Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V. J. Mol.
Biol. 1963, 7, 95–99.
Molecular Dynamics Trajectory Analyses, CDCl3. At 290
and 300 K in CDCl3, 13 presents a trajectory of strand-
(26) While the NLAlaO Φ, ψ torsions in 13 appear to be at the lower end
for what is expected for the central residue of a β-strand ( Avbelj, F.; Moult, J.
Biochemistry 1995, 34, 755-764. ), there is evidence that many Ala-based
proteins exhibit a maximum of Φ that is shifted around -90°. Φ around -80°
was a minimum configuration as computed by ab initio methods in a two-
stranded Ala dipeptide model ( Shamovsky, I. L.; Ross, G. M.; Riopelle, R. J.
J Phys. Chem. B 2000, 104, 11296-11307 and references cited therein; see
also: Smith, L. J.; Bolin, K. A.; Schwalbe, H.; MacArthur, M. W.; Thornton,
J. A.; Dobson, C. M.; J. Mol. Biol. 1996, 255, 494-506).
(20) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem. Soc.
1996, 117, 11225–11236.
(21) for instructive overviews on hydrogen bonding see: Schneider, H.-J.
Angew. Chem., Int. Ed. 2009, 48, 3924–3977. Steiner, T. Angew. Chem., Int.
Ed. 2002, 41, 48–76.
(22) Wojciechowski, M.; Lesyng, B. J. Phys. Chem. B 2004, 108, 18368–
18376.
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ꢀ
˚
FIGURE 5. 13, Percentage-weighted rmsd[A]-histogram of the 100 ns MD runs at 290 K. Solid and dotted lines indicate CDCl3 (ε = 4.8) and
MeOH (ε = 32.5) trajectories, respectively; RMSDs sampled at e25% atom, 50% atom, and 75% atom: 4.37, 4.87, 5.23 and 4.55, 4.95, 5.42 for
CDCl3 and MeOH, respectively.
Molecular Dynamics Trajectory Analyses, MeOH. It has
been recognized that shielding of the peptide backbone
dipoles by polar solvent molecules is a major contributor
to solvation free energy. This in turn has a decisive influence
on backbone conformation in proteins.27 Upon switching
from an aprotic, nonpolar solvent to one of relatively higher
dielectric permittivity that is capable of hydrogen bonding,
the polar solvent will compete with the solute for hydrogen-
bonding interactions and introduce an increased desolvation
free-energy penalty at the polar backbone. Then, as often the
decisive influence that stabilizes the folded conformations is
intrastrand hydrogen bonding,28 the ensemble of conforma-
tional states in the solute-solvent system will experience a
reorganization.
With β-sheet mimic 13 in MeOH, fewer stabilizing
strand-strand hydrogen bonds were observed compared
to CDCl3. The diaminoethane turn-linker, displayed exclu-
sively (ap) in CDCl3, is sampled partially (sc) similar to prior
observation in competitive solvents.29 In this configuration,
the linker can place the backbone carbonyl dipoles in an
orientation with increased probability for favorable hydro-
philic interaction,27b the pronounced increase in backbone
solvation being mirrored in the observed downfield shifting
of corresponding amino protons (see Table 2, Supporting
Information). Strikingly, the lack in cross-strand hydrogen
bonding interactions is unreflected in the backbone rmsd
(Figures 5 and 6) that is still centered around rmsd 4.9, in the
same region as the two ensemble maxima in the CDCl3
simulation. In the ROESY experiments, measurements in
both solvents showed a single but different major conforma-
tion. The observed H22TH25 and H19TH34 interactions
are well-reproduced by molecular dynamics and subset
analysis explains why no NOE across the strand-ends was
observed in the more polar solvent: In MeOH, the NXO-
mimic is still vastly sampled in a folded conformation with
both strands extended and aligned, however, with relatively
diminished across-strand hydrogen bonding. An increase in
rotational flexibility, in particular the pronounced fraying of
strand ends and less well-defined side chain rotamer prefer-
ences in the more polar solvent, is mirrored in overall
decreased subset lifetimes (see Figure 5 and rmsd histograms
in Figures 5 and 6 in the Supporting Information). Also, a
minor fraction of conformation space represents the un-
folded molecule.
Conclusion
Oligopeptides usually display a highly dynamic solution
behavior, populating multiple low-energy conformations.30
When one sets about a task related to this field of research,
the structural plurality has to be defined. It is known that
β-sheets in proteins exhibit potential energy surfaces with
multiple energy-minima.31 Yet, biological action is exerted
via chemical entities appended to the secondary structure
element; the arising tertiary structure then plays the decisive
role in directing molecular recognition, e.g., receptor-ligand
(27) (a) Avbelj, F. J. Mol. Biol. 2000, 300, 1335–1359. (b) Baldwin, R. L.
J. Mol. Biol. 2007, 371, 283-301 and references cited therein.
(28) Prabhu, N.; Sharp, K. Chem. Rev. 2006, 106, 1616-1623 and
references cited therein.
(29) Junquera, E.; Nowick, J. S. J. Org. Chem. 1999, 64, 2527–2531. See
also ref 22.
(30) Dill, K. A. Biochemistry 1990, 29, 7133-55; see also ref 27b.
(31) (a) Lacroix, E.; Kortemme, T.; Lopez de la Paz, M.; Serrano, L. Curr. Op.
Struct. Biol. 1999, 9, 487-493 and references cited therein. (b) Street, A. G.;
Mayo, S. L. AS 1999, 96, 9074-9076.
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FIGURE 6. 13, Backbone-rmsd superposition of the two single most abundantly sampled motifs of the 100 ns MD runs at 290 K. Blue stick
and red line renderings represent CDCl3 (ε = 4.8) and MeOH (ε = 32.5) trajectories, respectively. Hydrogens omitted for clarity.
interactions. To obtain tight interactions, backbone-modify-
ing peptidomimetic scaffolds must aim at reproducing the
spatial arrangements adopted by the lining functionalities as
closely as possible while at the same time maintaining
straightforward synthetic diversification. Then, for our pur-
poses, we require strand-extended ensembles of folds with
the strands in parallel alignment before and after the turn,
sampling a satisfactorily small rmsd section of conforma-
tional space. In general, foldability is thought of as an
enthalpic gain that must be realized via weak interactions,
eventually overcompensating the entropic penalty for intras-
trand ordering.32 β-sheets in proteins are characteristically
stabilized by cross-strand hydrogen bonds; this may be
favorably so because they preferentially occur in a hydro-
phobic pocket of the protein where the solvent cannot
effectively compete in the donor-acceptor interaction.27a
On the contrary, a small oligomer peptidomimetic like 13 will
likely always experience full solvent exposure. In a compet-
ing environment such as a polar solvent, the resulting
reversibility of association and thus kinetic lability of folds33
are decisive impediments toward successful hydrogen bond-
ing directed self-assembly without relying on any support
provided by additional conformation-fixing strand incre-
ments (templates), covalent cross-linking, and the like.
NMR studies of NXO-based β-mimics verify the presence
of substantial amounts of folded conformers in solution.
Pertinent cross-strand NOEs in the mimics resemble those in
the closest analogues known in the literature and are well-
reproduced by molecular dynamics simulations in implicit
solvent. Strongly (ap) constrained “N” hydrazide and “O”
oxalamide backbone dihedrals rigidify the potential energy
surface in the extended NLAlaO-tripeptide, which, in turn,
restrains the “X” amino acid orientation in the octapeptide
β-sheet mimic 13 to characteristic Φ, Ψ values. When 13 is
subjected to a competitive solvent, the turn linker gives way
and thus ensemble homogeneity is decreased. Still, the core
conformer populates the same rmsd space. These studies of
an NXO-modified β-mimic encourage further endeavors
toward NXO-peptidomimetics that fold to predictable sec-
ondary structure in aqueous solution.34
Experimental Section
Preparation of Orthogonally Protected NXO Building Blocks
3. General Procedure. The mixture of amino acid 1 and alkyl-4-
nitrophenyloxalate (1.1 equiv) 2 in dry DMF was stirred at 50 °C
until the solution had become clear (1.5-2.0 h). The reaction
mixture was allowed to cool to room temperature. Subse-
quently, HOBt (1.05 equiv), DCC (1.05 equiv), and the corre-
sponding protected hydrazine (1.0 equiv) were added and the
mixture was stirred at room temperature under argon for 5 h.
DMF was evaporated under reduced pressure on a rotary
evaporator; the residue obtained was dissolved in ethyl acetate
and precipitated dicyclohexyl urea was removed by filtration.
The ethyl acetate solution was washed with 1 N HCl and 10%
NaHCO3 followed by brine and dried over Na2SO4. After
evaporation of solvent, the crude compound was purified by
flash column chromatography to give the required compound 3
as a solid.
Preparation of Pseudopeptides 4. Trifluoroacetic acid (TFA,
1.5 equiv) was added slowly with stirring at 0 °C to a solution of
3 in CH2Cl2. The reaction mixture was stirred under argon at
room temperature for an additional 30 min. Solvent was eva-
porated and the crude residue was dried well under high
vacuum. The material obtained was dissolved in CH2Cl2 and
neutralized by triethylamine (TEA) (2 equiv). In another flask,
Boc-L-amino acid (1 equiv), EDCI HCl (1 equiv), and HOBt (1
3
equiv) were dissolved in CH2Cl2. TEA (1 equiv) was added to
this mixture and the reaction mixture was stirred for 15 min at
room temperature under argon. Then, the first prepared solu-
tion was added to this mixture and stirred overnight at room
temperature. The solvent was removed by rotary evaporation
(32) Jager, M.; Deechongkit, S.; Koepf, E. K.; Nguyen,J.; Gao, J.;
Powers, E. T.; Gruebele, M.; Kelly, J. W. J. Pept. Sci. 2008, 90, 751-758
and references cited therein; see also refs 8b and 27b.
(34) NXO building blocks are offered by Senn Chemicals AG, CH-8157
Dielsdorf, www.sennchem.com, who have obtained an exclusive license from
the Vienna University of Technology.
(33) Baldwin, R. L. J. Biol. Chem. 2003, 278, 17581–17588.
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and the residue obtained was dissolved in ethyl acetate. The
ethyl acetate solution was washed with 1 N HCl, 10% NaHCO3,
and brine, dried (Na2SO4), and concentrated to give 6 as a
colorless solid.
A solution of LiOH (12 equiv) in a minimum amount of water
was added dropwise to a solution of 6 in MeOH. The reaction
mixture was further stirred for 1 h. MeOH was then evaporated
and the residue obtained was dissolved in water, washed with
ether, acidified with 1 N HCl, and extracted with ethyl acetate.
The combined ethyl acetate layers were washed with brine, dried
(Na2SO4), and concentrated. The crude compound was tritu-
rated with diethyl ether and filtered to give 7 as a colorless solid.
mixture was stirred overnight at room temperature under argon.
The solvent was evaporated and the crude compound was
purified by column chromatography to give 333 mg (50%) of
product as a white solid; mp 62-63 °C.
Preparation of Compound 13. (all-S)-N-tert-Butyl-N0-(1-
{N0-[4-(1-(2-cyanoethyl)-3-{1-[2-methyl-1-(3-methyl-1-methylcar-
bamoylbutylcarbamoyl)butylcarbamoyl]-2-phenylethyl}ureido)-
2-phenylbutyryl]hydrazinocarbonyl}ethyl)oxalamide. The NH-
BOC derivative 24 (363 mg, 0.82 mmol) was deprotected with
5 mL of satd HCl in ethyl acetate at 0 °C for 60 min under Ar
atmosphere followed by evaporation, overnight drying. To this
crude HCl-salt were added 10 mL of CH2Cl2 and 10 mL of
saturated aqueous NaHCO3 and the biphasic mixture was
cooled to 0 °C in an ice bath. Stirring was stopped, the layers
were allowed to separate, and a 1.93 M solution of phosgene in
toluene (0.85 mL, 1.64 mmol) was added in a single portion via
syringe to the lower (organic) phase. Stirring was resumed
immediately, and the ice-cooled reaction mixture was stirred
vigorously for 10 min at 600 rpm. The layers were then allowed
to separate, the aqueous phase was extracted with CH2Cl2 (3 ꢀ 5
mL), and the combined organic layers were dried over Na2SO4,
filtered, and concentrated by rotary evaporation. The obtained
residue was dissolved in toluene (15 mL); hereto was added 17
(333 mg, 0.75 mmol). The reaction mixture was further refluxed
overnight, evaporated to dryness, diluted with ethyl acetate,
washed with 10% aq NaHCO3 and brine, dried over Na2SO4,
and evaporated to give crude compound, which was purified by
column chromatography to give (68%) the desired compound as
a white solid; mp 144-145 °C.
HOBt (1.05 equiv) and EDCI HCl (1.05 equiv) were added to
3
a solution of 7 in CH2Cl2; the mixture was stirred for an
additional 15 min. A solution of L-amino acid dimethylamide
(1.05 equiv) in CH2Cl2 was added followed by TEA (1.05 equiv)
then the mixture was stirred overnight at room temperature. The
solvent was removed by rotary evaporation and the residue
obtained was dissolved in ethyl acetate, washed with 1 N HCl,
10% NaHCO3, and brine, dried (Na2SO4), and concentrated to
give crude product, which was recrystallized from ethyl acetate
to give 4 as a colorless solid.
Preparation of NXO β-Sheet Mimic 13. Preparation of com-
pound 16: (S)-(4-{N0-[2-(tert-Butylaminooxalylamino)propio-
nyl]hydrazino}-4-oxo-3-phenylbutyl)carbamic Acid 9H-Fluoren-
9-ylmethyl Ester. To a solution of compound 15 (900 mg, 1.7
mmol) in CH2Cl2 (10 mL) was added TFA (10 mL) slowly with
stirring at 0 °C; the reaction mixture was further stirred under
argon at room temperature for 30 min (after overnight stirring
there is formation of byproduct so stirring only for 30 min is
crucial). Solvent was evaporated and the residue was dried well
under high vacuum and dissolved in CH2Cl2 (15 mL). In another
flask, compound 18 (377 mg, 1.74 mmol, prepared in three steps
and 67% overall yield from L-alanine methyl ester hydrochlo-
ride, see the Supporting Information), HOBt (247 mg, 1.83
mmol), and DCC (377 mg, 1.83 mmol) were dissolved in CH2Cl2
(15 mL) and the mixture was stirred for 15 min at room
temperature; hereto, a mixture of the above TFA salt of 15
and DIPEA (0.34 mL, 1.92 mmol) in CH2Cl2 (15 mL) were
added and the mixture was stirred overnight at room tempera-
ture. The precipitated dicyclohexyl urea (DCHU) was removed
by filtration and the filtrate was evaporated. Remaining DCHU
was removed by subsequent trituration with cold ethyl acetate
and filtration. The ethyl acetate solution was washed with 1 N
HCl (15 mL), 10% NaHCO3 (15 mL), and brine (15 mL), dried
(Na2SO4), and concentrated to give 1.1 g of crude compound,
which was purified by column chromatography to give 974 mg
(90%) of product as a white solid; mp 48-49 °C.
Computational Methods. Calculations were performed with
use of th eMolecular Operating Environment, Version 2007.09
(MOE, 1997-2007, Chemical Computing Group Inc.). Input
geometries were obtained from stochastic conformational
searching employing the OPLS-AA force field potential para-
meters and performed with soft distance constraints derived
from qualitatively assigned experimental NOE intensities. The
lowest energy conformers thus obtained were freed of all
restraints and subjected to molecular dynamics simulations in
an NVT ensemble. Solvents as in the NMR experiment were
treated implicitly by the generalized Born solvation model
setting the exterior dielectric to the value of ε = 4.8 or 32 D
for CDCl3 and d3-MeOH, respectively.
Acknowledgment. We thank Senn Chemicals AG for fi-
nancial support. Our gratitude to Prof. Oliver Zerbe and
Nadja Bross from the Institute of Organic Chemistry, Uni-
versity of Zurich, for the measurement of NMR spectra.
Preparation of compound 17: (S)-N-tert-Butyl-N0-(1-{N0-
[4-(2-cyanoethylamino)-2-phenylbutyryl]hydrazinocarbonyl}ethyl)-
oxalamide. A solution of compound 16 (915 mg, 1.49 mmol) in
10% piperidine in CH2Cl2 (15 mL) was stirred at room tem-
perature for 20 min. Solvent was evaporated and the reaction
mixture was dried well under high vacuum to remove the last
traces of piperidine. The residue thus obtained was crystallized
from ethyl acetate-pentane to give 575 mg (98%) of free amine.
The free amine was dissolved in methanol (15 mL); hereto was
added acrylonitrile (0.15 mL, 2.23 mmol) and the reaction
J.P. thanks the OAD-Osterreichischer Austauschdienst
(Austrian Exchange Service) for a doctoral grant. The
project was funded in part by the uni:invent PRIZE grant
(Z060414) of the Austria Wirtschaftsservice G.m.b.H. (aws).
€
€
Supporting Information Available: Experimental proce-
dures, characterization data for all new compounds, and spec-
tral and computational data. This material is available free of
charge via the Internet at http://pubs.acs.org.
2500 J. Org. Chem. Vol. 75, No. 8, 2010