An unnatural amino acid that mimics a tripeptide β-strand and forms β-sheetlike hydrogen-bonded dimers

Article abstract of DOI:10.1021/ja001142w

Unnatural amino acid 2 (5-HO₂CCONH-2-MeO-C₆H₃-CONHNH₂) duplicates the hydrogen-bonding functionality of one edge of a tripeptide β-strand. It is composed of hydrazine, 5-amino-2-methoxybenzoic acid, and oxalic a

Full text of DOI:10.1021/ja001142w

7654
J. Am. Chem. Soc. 2000, 122, 7654-7661
An Unnatural Amino Acid that Mimics a Tripeptide â-Strand and
Forms â-Sheetlike Hydrogen-Bonded Dimers
James S. Nowick,* De Michael Chung, Kalyani Maitra, Santanu Maitra,
Kimberly D. Stigers, and Ye Sun
Contribution from the Department of Chemistry UniVersity of California, IrVine
IrVine, California 92697-2025
ReceiVed March 31, 2000
Abstract: Unnatural amino acid 2 (5-HO2CCONH-2-MeO-C6H3-CONHNH2) duplicates the hydrogen-bonding
functionality of one edge of a tripeptide â-strand. It is composed of hydrazine, 5-amino-2-methoxybenzoic
acid, and oxalic acid groups and is designated by the three-letter abbreviation “Hao” to reflect these three
components. The 2,7-di-tert-butylfluorenylmethyloxycarbonyl (Fmoc*)- and tert-butyloxycarbonyl (Boc)-
protected derivatives of Hao are prepared efficiently and in high yield by the condensation of suitably protected
derivatives of hydrazine, 5-amino-2-methoxybenzoic acid, and oxalic acid. Fmoc*-Hao and Boc-Hao behave
like typical Fmoc- and Boc-protected amino acids and can be incorporated into peptides by standard solid-
and solution-phase peptide synthesis techniques using carbodiimide coupling agents. Hao-containing peptide
9 (i-PrCO-Phe-Hao-Val-NHBu) forms a â-sheetlike hydrogen-bonded dimer in CDCl3 and CD3OD-CDCl3
solutions. Peptides containing Hao and natural amino acids display hydrogen-bonding surfaces that are
complementary to the hydrogen-bonding edges of protein â-sheets.
Introduction
Recognition between exposed edges of â-sheets is an
important mode of protein-protein interaction.1 These â-sheet
interactions between proteins are, for example, critical in the
binding of neuronal nitric oxide synthase inhibitory protein to
neuronal nitric oxide synthase,2 PDZ domains to membrane
receptor and ion channel proteins,3 and Ras oncoproteins to the
Raf kinase (Figure 1).4 Interactions between â-sheet edges are
also widely involved in protein dimerization and in peptide and
protein aggregation.
The edges of protein â-sheets provide alternating arrays of
hydrogen-bond donors and acceptors, with the pattern: donor-
acceptor, donor-acceptor, donor-acceptor, etc. In â-sheet
interactions between proteins, these edges hydrogen bond
together. Chemical decoys that duplicate the hydrogen-bonding
edges of protein â-sheets hold promise as inhibitors of protein-
protein interactions. Although this promise has not yet been
achieved, it has been pursued by several researchers within the
past decade. Michne and Schroeder created a bicyclic compound
that mimics the hydrogen-bonding pattern of one edge of a
peptide â-strand to block a putative â-sheet interaction between
lymphocyte function-associated antigen-1 (LFA-1) and inter-
cellular adhesion molecule-1 (ICAM-1).6 Rebek, Pallai, and co-
workers have developed bi- and tricyclic â-strand mimics to
(1) Maitra, S.; Nowick, J. S. â-Sheet Interactions Between Proteins. In
The Amide Linkage: Structural Significance in Chemistry, Biochemistry,
and Materials Science; Greenberg, A., Breneman C. M., Liebman, J. F.,
Eds.; Wiley: New York, 2000; Chapter 15.
Figure 1. Molscript5 diagram of the complex between the Ras-binding
domain of the c-Raf1 kinase (upper) and the Ras analogue, Rap1A
(lower). (PDB reference 1gua.4)
(2) Liang, J.; Jaffrey, S. R.; Guo, W.; Snyder, S. H.; Clardy, J. Nat.
Struct. Biol. 1999, 6, 735-740.
(3) Doyle, D. A.; Lee, A.; Lewis, J.; Kim, E.; Sheng, M.; MacKinnon,
R. Cell 1996, 85, 1067-1076.
inhibit a postulated â-sheet interaction between gp120 and the
CD4 receptor.7 Related studies by Schrader and Kirsten have
focused on the development of peptidomimetic compounds that
mimic the hydrogen-bonding pattern of one edge of a peptide
â-strand and bind peptides through â-sheet interactions.8
(4) Nassar, N.; Horn, G.; Herrmann, C.; Scherrer, A.; McCormick, F.;
Wittinghofer, A. Nature, 1995, 375, 554-560.
(5) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946-950.
(6) Michne, W. F.; Schroeder, J. D. Int. J. Pept. Protein Res. 1996, 47,
2-8.
10.1021/ja001142w CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/28/2000

The Peptidomimetic Unnatural Amino Acid, Hao
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7655
Both hydrogen bonding and other noncovalent interactions
contribute to â-sheet interactions between proteins; the hydrogen-
bonding interactions between the edges of protein â-sheets are
generally accompanied by additional polar and hydrophobic
interactions between the amino acid side chains. Contacts with
other structures, such as helices, can also be present. Col-
lectively, these important interactions provide strength and
specificity to the protein-protein interactions. A variety of
systems that mimic and block these interactions have been
developed.9-12
Scheme 1
Compounds that mimic the structures and hydrogen-bonding
patterns of protein â-sheets but that have not specifically been
targeted toward binding proteins have also been reported.13 In
pioneering studies during the late 1980s, Kemp and co-workers
described a 2,8-diaminoepindolidione molecular template that
mimics the hydrogen-bonding functionality of one edge of a
peptide â-strand and have coupled this â-strand mimic to
peptides to generate intramolecularly hydrogen-bonded â-shee-
tlike structures.14 Our own research group has subsequently
developed â-strand mimics based on aminoaromatic derivatives
and has combined these â-strand mimics with urea-based turn
units and peptide strands to create a variety of â-sheetlike
structures, which we have termed artificial â-sheets.13,15,16
Recently, we reported that combination of a 5-amino-2-
methoxybenzoic acid hydrazide â-strand mimic with an oxala-
mide linker and tripeptides generates artificial â-sheets that form
well-defined â-sheet dimers (Scheme 1).17 Intrigued by the
prospect of developing smaller and simpler systems that interact
through â-sheet formation, we realized that the 5-amino-2-
methoxybenzoic acid hydrazide and oxalamide groups within
Scheme 2
these molecules could be viewed collectively as an unnatural
amino acid that duplicates the hydrogen-bonding functionality
of one edge of a tripeptide â-strand. The amino acid, 2,
comprises hydrazine, 5-amino-2-methoxybenzoic acid, and
oxalic acid groups. We have designated this amino acid by the
three-letter abbreviation “Hao” to reflect these three components.
Scheme 2 illustrates its structure and shows its relationship to
a tripeptide.
This paper reports our studies of the amino acid Hao: the
preparation of its N-protected derivatives, the use of these
derivatives in peptide synthesis, and the remarkable propensity
of a Hao-containing peptide to form a â-sheetlike dimer.
(7) (a) Roberts, J. C.; Pallai, P. V.; Rebek, J., Jr. Tetrahedron Lett. 1995,
36, 691-694. (b) Boumendjel, A.; Roberts, J. C.; Hu, E.; Pallai, P. V.;
Rebek, J., Jr. J. Org. Chem. 1996, 61, 4434-4438.
(8) (a) Schrader, T.; Kirsten, C. Chem. Commun. 1996, 2089-2090. (b)
Kirsten, C. N.; Schrader, T. H. J. Am. Chem. Soc. 1997, 119, 12061-12068.
(9) (a) Smith, A. B., III; Guzman, M. C.; Sprengeler, P. A.; Keenan, T.
P., Holcomb, R. C.; Wood, J. L.; Carroll, P. J.; Hirschmann, R. J. Am.
Chem. Soc. 1994, 116, 9947-9962. (b) Smith, A. B., III; Hirschmann, R.;
Pasternak, A.; Guzman, M. C.; Yokoyama, A.; Sprengeler, P. A.; Darke,
P. L.; Emini, E. A.; Schleif, W. A. J. Am. Chem. Soc. 1995, 117, 11113-
11123. (c) Smith, A. B., III; Benowitz, A. B.; Sprengeler, P. A.; Barbosa,
J.; Guzman, M. C.; Hirschmann, R.; Schweiger, E. J.; Bolin, D. R.; Nagy,
Z.; Campbell, R. M.; Cox, D. C.; Olson, G. L. J. Am. Chem. Soc. 1999,
121, 9286-9298.
(10) (a) Abbenante, G.; March, D. R.; Bergman, D. A.; Hunt, P. A.;
Garnham, B.; Dancer, R. J.; Martin, J. L.; Fairlie, D. P. J. Am. Chem. Soc.
1995, 117, 10220-10226. (b) March, D. R.; Abbenante, G.; Bergman, D.
A.; Brinkworth, R. I.; Wickramasinghe, W.; Begun, J.; Martin, J. L.; Fairlie,
D. P. J. Am. Chem. Soc. 1996, 118, 3375-3379. (c) Reid, R. C.; March,
D. R.; Dooley, M. J.; Bergman, D. A.; Abbenante, G.; Fairlie, D. P. J. Am.
Chem. Soc. 1996, 118, 8511-8517.
Results
Hao is readily prepared as its 2,7-di-tert-butylfluorenylm-
ethyloxycarbonyl protected derivative (8) by the condensation
of suitably protected derivatives of hydrazine, 5-amino-2-
methoxybenzoic acid, and oxalic acid, as shown in Scheme 3.
The 2,7-di-tert-butylfluorenylmethyloxycarbonyl (Fmoc*) group
is used in place of the popular fluorenylmethyloxycarbonyl
(Fmoc) group to improve the solubility of Hao and its precursors
in organic solvents.18 Reaction of 2,7-di-tert-butylfluorenylm-
ethyloxycarbonyl chloride (Fmoc*-Cl) with anhydrous hydrazine
affords Fmoc*-hydrazine (3). Coupling with 2-methoxy-5-
nitrobenzoyl chloride (4)16c gives hydrazide 5. Reduction of the
nitro group generates amine 6. Condensation with ethyl oxalyl
chloride gives amide 7. Hydrolysis of the ethyl ester group with
NaOH, followed by passage of the reaction mixture through
acidic ion exchage resin, yields Fmoc*-Hao (8). When this
procedure was performed on a multigram scale, a 75% yield
for the conversion of Fmoc*-Cl to Fmoc*-Hao was obtained.
The Boc-protected derivative of Hao (Boc-Hao) was prepared
in a similar fashion from Boc-hydrazine.
(11) Janetka, J. W.; Raman, P.; Satyshur, K.; Flentke, G. R.; Rich, D.
H. J. Am. Chem. Soc. 1997, 119, 441-442.
(12) Zutshi, R.; Franciskovich, J.; Shultz, M.; Schweitzer, B.; Bishop,
P.; Wilson, M.; Chmielewski, J. J. Am. Chem. Soc. 1997, 119, 4841-4845.
(13) Nowick, J. S.; Smith, E. M.; Pairish, M. Chem. Soc. ReV. 1996, 25,
401-415.
(14) (a) Kemp, D. S.; Bowen, B. R. Tetrahedron Lett. 1988, 29, 5077-
5080. (b) Kemp, D. S.; Bowen, B. R. Tetrahedron Lett. 1988, 29, 5081-
5082. (c) Kemp, D. S.; Bowen, B. R.; Muendel, C. C. J. Org. Chem. 1990,
55, 4650-4657.
(15) Nowick, J. S. Acc. Chem. Res. 1999, 32, 287-296.
(16) (a) Nowick, J. S.; Smith, E. M.; Noronha, G. J. Org. Chem. 1995,
60, 7386-7387. (b) Nowick, J. S.; Holmes, D. L.; Mackin, G.; Noronha,
G.; Shaka, A. J.; Smith, E. M. J. Am. Chem. Soc. 1996, 118, 2764-2765.
(c) Nowick, J. S.; Pairish, M.; Lee, I. Q.; Holmes, D. L.; Ziller, J. W. J.
Am. Chem. Soc. 1997, 119, 5413-5424. (d) Holmes, D. L.; Smith, E. M.;
Nowick, J. S. J. Am. Chem. Soc. 1997, 119, 7665-7669. (e) Smith, E. M.;
Holmes, D. L.; Shaka, A. J.; Nowick, J. S. J. Org. Chem. 1997, 62, 7906-
7907. (f) Tsai, J. H.; Waldman, A. S.; Nowick, J. S. Bioorg. Med. Chem.
1999, 7, 29-38. (g) Junquera, E.; Nowick, J. S. J. Org. Chem. 1999, 64,
2527-2531.
(17) Nowick, J. S.; Tsai, J. H.; Bui, Q.-C. D.; Maitra, S. J. Am. Chem.
Soc. 1999, 121, 8409-8410.
(18) Stigers, K. D.; Koutroulis, M. R.; Chung, D. M.; Nowick, J. S. J.
Org. Chem. 2000, 65, 3858-3860.

7656 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Nowick et al.
Scheme 3
Scheme 4
standard coupling reagents, but proceeded smoothly using the
symmetrical anhydride in CH2Cl2-DMF (8:2).20 The Hao and
Phe residues were introduced by double coupling with DCC
and HOBt; the isobutryic acid residue was introduced by single
coupling under similar conditions.23,24 Cleavage from the resin
with TFA then afforded tripeptide 9. Scheme 4 illustrates this
synthesis.
When “indole resin” recently became commercially available,
we tried using it to prepare tripeptide 9.25 This resin bears an
indole-3-carboxaldehyde linker, which allows the butylamine
portion of 9 to be introduced by reductive amination. Subsequent
introduction of the amino acid residues with DCC and HOBt
proceeded smoothly. The product produced by this route proved
slightly more pure than that generated on the PEG-PS resin with
the PAL linker. For this reason, and because of the absence of
the need of the symmetrical anhydride, we consider this linker
superior and have adopted it for subsequent solid-phase
syntheses of related N-alkylamides.
To evaluate the synthetic and structural properties of Hao,
we used these Hao derivatives to prepare the tripeptide i-PrCO-
Phe-Hao-Val-NHBu (9) by several different methods. We chose
this compound as a simple, nonfunctionalized peptide derivative
that would be suitable for 1H NMR studies in chloroform
solution. Initially, we had prepared the corresponding methyl-
amide, i-PrCO-Phe-Hao-Val-NHMe. However, the limited (low
millimolar) solubility of this compound made its study by NMR
inconvenient. For this reason, we performed all subsequent
studies on the more soluble butylamide.
To evaluate the behavior of Hao in solution-phase syntheses,
we synthesized peptide 9 in solution by sequential coupling of
Val-NHBu, Boc-Hao, Boc-Phe, and isobutyric acid using
1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide hydrochloride
(EDC) and HOBt in a mixture of THF and DMF. This synthesis
also proceeded smoothly, although the limited solubility of the
Hao-containing intermediates necessitated using DMF as a
cosolvent. Collectively, these studies show that Fmoc*-Hao and
Boc-Hao behave like regular amino acids in both solid-phase
and solution-phase peptide syntheses with carbodiimide coupling
agents.26
We first prepared tripeptide 9 manually on poly(ethylene
glycol)-polystyrene (PEG-PS) resin with a tris(alkoxy)benzyl-
amide (PAL) linker19 using a modified version of the backbone
amide linker (BAL) strategy described by Barany, Albericio,
and co-workers.20 In our modification, we introduced the butyl
group by converting the PAL amino group to its o-nitrobenze-
nesulfonamide by treatment with o-nitrobenzenesulfonyl chlo-
ride (NsCl), alkylation with n-butyl iodide and 1,3,4,6,7,8-
hexahydro-1-methyl-2H-pyrimido[1,2-a]pyrimidine (MTBD),
and removal of the nitrobenzenesulfonamide group with mer-
captoethanol and DBU.21,22 As described by Barany, Albericio,
and co-workers, coupling of Fmoc-valine to the resulting
sterically hindered (Bu)PAL-PEG-PS proved difficult using
To evaluate the effect of Hao upon peptide structure, we
studied tripeptide 9 by 1H NMR spectroscopy. 1H NMR
chemical shift, NOE, and dilution titration studies indicate that
Hao derivative 9 forms a remarkable â-sheetlike hydrogen-
(19) (a) Albericio, F.; Barany, G. Int. J. Pept. Protein Res. 1987, 30,
206-216. (b) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.;
Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem. 1990, 55, 3730-
3743.
(20) (a) Jensen, K. J.; Alsina, J.; Songster, M. F.; Va´gner, J.; Albericio,
F.; Barany, G. J. Am. Chem. Soc. 1998, 120, 5441-5452. (b) Alsina, J.;
Jensen, K. J.; Albericio, F.; Barany, G. Chem. Eur. J. 1999, 5, 2787-
2795.
(21) (a) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301-
2302. (b) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690-
2691.
(22) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36,
6373-6374.
(23) Interestingly, we obtained better coupling of Fmoc*-Hao using DCC
and HOBt than using HATU or HATU and HOAt, which are often better
peptide coupling agents. For descriptions of HATU and HOAt, see: Carpino,
L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
(24) Our experiences suggest that the coupling of Hao is slightly less
efficient than that of ordinary amino acids. Double coupling was performed
in each step in the synthesis to ensure complete reaction, although it was
not clear that it was required.
(25) Estep, K. G.; Neipp, C. E.; Stephens Stramiello, L. M.; Adam, M.
D.; Allen, M. P.; Robinson, S.; Roskamp, E. J. J. Org. Chem. 1998, 63,
5300-5301.

The Peptidomimetic Unnatural Amino Acid, Hao
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7657
Scheme 5
the Val γ (methyl) protons and the Phe R, â, and δ protons.
Scheme 5 illustrates these NOEs with arrows. These long-range
NOEs cannot easily be explained by intramolecular contacts,
but are wholly consistent with the dimeric structure.
Intrastrand NOE data also provide evidence that 9 adopts a
â-strandlike conformation. Notably, the aromatic amino proton
(Hd) exhibits a strong NOE with the aromatic H6 proton but
exhibits no NOE with the aromatic H4 proton. Similarly, the
Val R-proton exhibits a stronger interresidue NOE with buty-
lamide proton Hf and a weaker NOE with the Val NH proton
(He). When a 300 ms mixing time is used in the Tr-ROESY
experiment, the Phe R-proton exhibits weak inter- and intraresi-
due NOEs with both the Hao NH proton Hb and the Phe NH
proton (Ha). The weakness of the interresidue NOE appears to
result from an unusually short transverse relaxation time (and
associated T1F) for the Hao NH proton Hb. The short relaxation
time of this proton, evidenced by the broadness of its peak in
the 1H NMR spectrum, should result in loss of phase coherence
during the mixing period of the Tr-ROESY experiment and
generate an anomalously weak NOE. Consistent with this
explanation, the Phe R-proton gives a relatively strong inter-
residue NOE with the Hao NH proton Hb and little or no
intraresidue NOE with the Phe NH proton (Ha) when a shorter
(100 ms) mixing time is used. Coupling constant data provide
bonded dimer in CDCl3 solution. Scheme 5 illustrates the
structure of this dimer.
In this structure, NH protons Hb, Hc, Hd, and Hf are hydrogen-
1
bonded, while NH protons Ha and He are not. In the H NMR
spectrum in CDCl3 solution, hydrogen-bonded NH protons
typically appear about 2 ppm downfield of non-hydrogen-
bonded NH protons. Peptide amides generally appear at ∼6 ppm
when not hydrogen-bonded and at ∼8 ppm when hydrogen-
bonded.13 In Hao derivative 9, the chemical shift of the (non-
hydrogen-bonded) phenyalanine NHa group is 6.35 ppm in 7
mM CDCl3 solution at 295 K, while that of the (hydrogen-
bonded) valine butylamide NHf group is 7.93 ppm. The three
NH protons of Hao, Hb, Hc, and Hd appear at 11.74, 11.18, and
10.71 ppm, respectively. These values are comparable to those
of the corresponding hydrogen-bonded protons in artificial
â-sheets 1a and b (11.3, 11.2, and 10.7 ppm, respectively).17
The remaining NH proton (He) appears at 8.41 ppm. This proton
belongs to an oxalamide system and appears significantly
downfield of that of the NH proton of N,N′-dibutyloxalamide
in dilute CDCl3 solution (7.44 ppm, 7 mM), conditions at which
it is not hydrogen-bonded. The downfield shifting of this proton
relative to the dibutyloxalamide control appears too small to
arise from a typical hydrogen bond, and may instead result from
magnetic anisotropy associated with the valine carbonyl group
and Hao aromatic ring.
The chemical shifts of the amino acid R-protons of 9 are also
consistent with the dimeric â-sheetlike structure shown in
Scheme 5. Protein R-protons generally resonate downfield of
those in random coils by several tenths of a ppm in â-sheets.27
In 9, the Phe and Val R-protons appear at 5.38 and 4.62 ppm
respectively, substantially downfield of the corresponding
random coil values (4.66 and 3.95 ppm). Also consistent with
the dimeric structure, the chemical shift of the Phe R-proton in
9 is comparable to the limiting chemical shift of the Phe
R-proton in the dimer of artificial â-sheet 1a (5.29 ppm).17
Interstrand NOES are a hallmark of â-sheets.28 1H NMR
transverse-ROESY (Tr-ROESY)29 studies in CDCl3 solution (7
mM, 30 °C) show that Hao derivative 9 exhibits strong NOEs
between the Phe and Val R-protons and weaker NOEs between
3
further evidence for a â-strandlike conformation, with JHNR
values of 8.4 Hz (Phe) and 9.6 Hz (Val).13,30
Peptide derivative 9 is too strongly dimerized in pure CDCl3
to allow its dimerization constant to be accurately determined
1
by H NMR dilution titration studies. The NH groups Ha, Hb,
and Hc of 9 exhibit very little concentration dependence in
chemical shift (0.05-0.07 ppm from 0.16 to 2.6 mM) in CDCl3
solution and show saturation at higher concentrations. These
data indicate that 9 is virtually completely dimerized at NMR
accessible concentrations. Analysis of this very limited titration
data set reveals a dimerization constant of ∼106 M-1. This value
is dramatically larger than that of the tripeptide i-PrCO-Phe-
Leu-Val-NHBu, which was prepared as a control and found to
have a dimerization constant of 100-200 M-1 by fitting
dimerization isotherms to the NH and R-proton chemical shifts
at varying concentrations. Several pentapeptides were also
1
prepared as controls but were found to be too insoluble for H
NMR titration studies.
Addition of the competitive solvent CD3OD to the CDCl3
weakens the dimerization of 9, allowing a dimerization constant
1
to be determined by H NMR dilution titration studies. Thus,
dilution titration studies, in which the chemical shifts of the
R-protons were monitored as a function of concentration and a
dimerization isotherm was fitted to the shift data, reveal a
dimerization constant (Kdim) of 900 M-1 in 10% CD3OD-
CDCl3.31-33 Figure 2 illustrates these data and the fitted
isotherms. In this solvent system, the control tripeptide i-PrCO-
(26) Hao can also be constructed during the convergent synthesis of larger
molecules that contain it. Thus, we have also prepared 9 convergently, by
forming its oxalic acid amide bond using EDC (i-PrCO-Phe-NHNH-
COArNH2 + HO2CCO-Val-NHBu f 9). In addition, we have found that
simple esters of Hao and its derivatives are exceptionally reactive, allowing
their facile direct coupling with simple amines, even in dilute solution (e.g.,
i-PrCO-Phe-Hao-OEt + RNH2 f i-PrCO-Phe-Hao-NHR). This exceptional
reactivity of Hao ester derivatives also allows their facile base-promoted
hydrolysis and transesterification.
(30) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986; pp 166-168.
(31) The titration data were analyzed by nonlinear least-squares fitting
1/2
the following equation to the data: δobs δfree ((1 + 8Kdim[9]tot
)
-1)/
)
4Kdim[9]tot + δbound (4Kdim[9]tot - (1 + 8Kdim[9]tot)
1/2 +1)/4Kdim[9]tot, where
δobs ) observed chemical shift, δfree ) chemical shift of 9, δbound ) chemical
shift of dimer, [9]tot ) total concentration of 9 in solution, and Kdim ) the
equilibrium constant for formation of the dimer complex. The quantities
(27) (a) Wishart, D. S.; Sykes, B. D.; Richards, F. M. J. Mol. Biol. 1991,
222, 311-333. (b) Wishart, D. S.; Sykes, B. D.; Richards, F. M.
Biochemistry 1992, 31, 1647-1651.
δ
free, δbound, and Kdim were allowed to vary during the fitting procedure, the
δ
obs was measured during the titration, and the quantity [9]tot was calculated
from the volumes and concentrations of the solutions used in the titration.
(32) The value of Kdim (900 M-1) was estimated to be accurate to within
(100 on the basis of multiple independent titrations. For the Val R-proton,
δfree and δbound were calculated to be 4.11 and 4.58 ppm, respectively; for
the Phe R-proton, δfree and δbound were calculated to be 4.83 and 5.26 ppm,
respectively.
(28) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986; pp 125-129.
(29) (a) Hwang, T.-L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114, 3157-
3159. (b) Hwang, T.-L.; Shaka, A. J. J. Magn. Reson., Ser. B 1993, 102,
155-165.

7658 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Nowick et al.
Figure 2. 1H NMR chemical shift of R-protons of 9 as a function of
concentration in CDCl3 solution at 295 K. The curves are dimerization
isotherms that best fit the data points.
Scheme 6
Figure 3. Molecular model of Ac-Ala-Hao-Ala-NHMe docked to the
â-sheet edge of the Ras analogue, Rap1A. Modeling was performed
using MacroModel V6.5 with the AMBER* force field and the figure
was rendered using Molscript.5
difference is that the dimerization interface of peptide derivative
9 is longer and has six intermolecular hydrogen bonds, while
the examples described above have four. More significantly, 9
differs from these other systems in that its key structural unit is
an unnatural amino acid that can be combined with natural
amino acids to give hybrid peptides that are complementary to
protein â-sheets.
This unnatural amino acid, Hao, imparts a â-sheetlike
conformation to peptides that contain it and facilitates their
dimerization through â-sheet interactions. In hybrid peptide 9,
Hao provides a â-strandlike edge with an alternating array of
hydrogen-bond donors and acceptors that is preorganized to
dimerize. Unlike peptides composed solely of natural amino
acids, Hao-containing peptides do not readily form higher
oligomers, because the aromatic ring of Hao blocks the other
edge of the peptide strand.
Artificial â-sheets 1 can also be viewed as hybrid peptides,
consisting of the tripeptide mimic Hao, the turn forming
dipeptide replacement, -N(Ph)CH2CH2N(CH2CH2CN)CO-,
and a tripeptide region; written as a peptide, the structure of 1a
is i-PrCO-Hao-N(Ph)CH2CH2N(CH2CH2CN)CO-Phe-Ile-Leu-
NHMe. In artificial â-sheets 1, Hao serves as a template that
organizes the tripeptide region into a â-sheet that can dimerize.
Thus Hao imparts both structure (conformation and folding) and
function (dimerization) to peptides that contain it.
Phe-Leu-Val-NHBu self-associates too weakly to allow accurate
determination of its dimerization constant by 1H NMR titration.
Analysis of the limited data sets available from 1H NMR dilution
titration revealed a dimerization constant of ∼5 M-1
.
Attempts to crystallize 9 to determine its solid-state structure
by X-ray crystallography proved unsuccessful. A smaller
homologue i-PrCO-Hao-NHMe (10) did crystallize but did not
form â-sheetlike hydrogen-bonded dimers in the solid state.
Instead of adopting a â-strandlike conformation that could form
â-sheetlike dimers (Scheme 6, conformer A), this compound
adopted a conformation in which rotation about the Ar-NH
bond of the Hao unit occurred (Scheme 6, conformer B). These
results provide a reminder that although Hao is more confor-
mationally constrained than a peptide, it can adopt a conforma-
tion that is not â-strandlike as well as one that is.34
Discussion
Within the past couple of years, considerable interest in
developing systems that dimerize strongly has emerged. Leading
examples have been developed in the laboratories of Meijer,35
Zimmerman,36 and Gong.37 Peptide derivative 9 is similar to
these systems in that it dimerizes with comparable strength. One
Hybrid peptides containing Hao display hydrogen-bonding
surfaces that are complementary to the hydrogen-bonding edges
of protein â-sheets. Figure 3 illustrates this complementarity
through a model of Hao-containing peptide Ac-Ala-Hao-Ala-
NHMe docked to the â-sheet edge of the Ras analogue, Rap1A.
The edge of this simple tripeptide provides an alternating pattern
of hydrogen-bond donors and acceptors that matches that of
the protein. An intriguing application of Hao would be to
incorporate it into peptides that bind to the edges of protein
â-sheets. Such peptides offer the appealing promise of blocking
â-sheet interactions between proteins. To achieve this promise,
we will have to learn how to make Hao-containing peptides
(33) Interestingly, peptide derivative 9 shows a strong propensity to form
dimers in the electrospray mass spectrum, and the relative intensities of
the dimer and monomer peaks correlate with the concentration of 9
introduced into the spectrometer.
(34) Although these X-ray crystallographic studies show that 10 adopts
this alternative conformation in the solid state, the 1H NMR Tr-ROESY
studies establish that 9 does not adopt this alternative conformation in its
dimer in CDCl3 solution.
(35) Beijer, F. H.; Kooijman, H.; Spek, A. L.; Sijbesma, R. P.; Meijer,
E. W. Angew. Chem., Int. Ed. 1998, 37, 75-78. (b) Beijer, F. H.; Sijbesma,
R. P.; Kooijman, H.; Spek, A. L.; Meijer, E. W. J. Am. Chem. Soc. 1998,
120, 6761-6769.
(36) Corbin, P. S.; Zimmerman, S. C. J. Am. Chem. Soc. 1998, 120,
9710-9711.

The Peptidomimetic Unnatural Amino Acid, Hao
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7659
and saturated aqueous NaCl (150 mL), dried over MgSO4, filtered, and
concentrated to yield 11.70 g of hydrazide 5 (99%) as a white fluffy
solid. An analytical sample was purified by column chromatography
on silica gel: mp 126-127 °C; IR (CHCl3) 3410, 1749, 1682, 1616
cm-1; 1H NMR (500 MHz, CDCl3) δ 9.42 (br s, 1 H), 9.08 (br s, 1 H),
8.38 (dd, J ) 9.1, 3.0 Hz, 1 H), 7.60 (br s, 4 H), 7.41 (d, J ) 6.7 Hz,
2 H), 7.22 (br s, 1 H), 7.12 (d, J ) 9.2 Hz, 1 H), 4.51 (d, J ) 7.0 Hz,
2 H), 4.22 (t, J ) 6.8 Hz, 1 H), 4.11 (br s, 3 H), 1.37 (s, 18 H); 13C
NMR (125 MHz, CDCl3) δ 162.2, 161.6, 156.0, 150.0, 143.5, 142.1,
138.7, 128.9, 128.8, 124.9, 121.9, 120.2, 119.2, 112.0, 68.6, 57.3, 47.0,
34.9, 31.6; HRMS (LSIMS) m/z for C31H35N3O6 [M]+ calcd 545.2526,
found 545.2530. Anal. Calcd for C31H35N3O6: C, 68.24; H, 6.47; N,
7.70. Found: C, 68.13; H, 6.47; N, 7.75.
Amine 6. A 500-mL, three-necked, round-bottomed flask equipped
with a stopper, a septum, a magnetic stirring bar, and a three-way
stopcock connected to a vacuum line and to a balloon filled with
hydrogen was charged with hydrazide 5 (11.70 g, 21.44 mmol), 10%
Pd/C (1.20 g), CH3OH (100 mL) and THF (200 mL). The flask was
evacuated and filled with hydrogen (3×), and the reaction mixture was
allowed to stir under hydrogen. After 2 h, the flask was evacuated and
opened to air, the suspension was filtered through Celite, and the Celite
bed was rinsed thoroughly with ethyl acetate (150 mL). The filtrate
was evaporated to dryness to obtain 11.01 g of an off-white solid.
Purification by column chromatography (gradient elution with EtOAc:
hexanes, 1:20-3:1, on a 7.5 cm d × 19 cm h column of silica gel)
yielded 8.60 g (76% from Fmoc*-Cl) of amine 6 as a white fluffy
solid: mp 108-121 °C; IR (CHCl3) 3392, 1736, 1668, 1624 cm-1; 1H
NMR (500 MHz, CDCl3) δ 9.72 (br s, 1 H), 7.64-7.62 (m, 4 H), 7.55
(dd, J ) 2.5, 0.8 Hz, 1 H), 7.41 (d, J ) 7.4 Hz, 2 H), 7.18 (br s, 1 H),
6.85-6.81 (m, 2 H), 4.48 (d, J ) 7.3 Hz, 2 H), 4.23 (t, J ) 7.3 Hz, 1
H), 3.92 (s, 3 H), 3.56 (br s, 2 H), 1.36 (s, 18 H); 13C NMR (125 MHz,
CDCl3) δ 164.5, 156.2, 150.8, 149.9, 143.7, 140.7, 138.7, 124.8, 122.0,
120.2, 119.6, 119.2, 118.4, 113.0, 68.5, 56.6, 47.1, 34.9, 31.6; HRMS
(LSIMS) m/z for C31H37N3O4 [M]+ calcd 515.2784, found 515.2799.
Anal. Calcd for C31H37N3O4: C, 72.21; H, 7.23; N, 8.15. Found: C,
72.04; H, 7.30; N, 8.15.
Amide 7. To an ice-cooled solution of amine 6 (8.60 g, 16.7 mmol)
and pyridine (1.60 mL, 19.8 mmol) in CH2Cl2 (150 mL), was added
ethyl oxalyl chloride (2.10 mL, 18.8 mmol) in drops over 2 min. After
15 min, the solution was transferred to a separatory funnel with CH2-
Cl2 (50 mL), washed with H2O (150 mL) and saturated aqueous NaCl
solution (150 mL), dried over MgSO4, filtered, and concentrated to
yield 10.17 g (99%) of amide 7 as a white solid: mp 222-225 °C; IR
(CHCl3) 3390, 1732, 1710, 1672 cm-1; 1H NMR (400 MHz, CDCl3) δ
9.74 (br s, 1 H), 9.43 (br s, 1 H), 8.37 (d, J ) 8.8 Hz, 1 H), 8.26 (d,
J ) 2.8 Hz, 1 H), 7.63-7.60 (m, 5 H), 7.41-7.39 (m, 2 H), 7.05 (d,
J ) 9.2 Hz, 1 H), 4.48 (d, J ) 7.2 Hz, 2 H), 4.41 (q, J ) 7.2 Hz, 2 H),
4.22 (t, J ) 7.0 Hz, 1 H), 4.01 (s, 3 H), 1.40 (t, J ) 7.4 Hz, 3 H), 1.35
(s, 18 H); 13C NMR (125 MHz, CDCl3) δ 163.5, 161.1, 156.0, 154.7,
154.2, 149.9, 143.6, 138.6, 131.0, 125.5, 124.8, 124.2, 121.9, 119.4,
119.2, 112.2, 68.4, 63.7, 56.5, 47.0, 34.8, 31.7, 13.9; HRMS (LSIMS)
m/z for C35H41N3O7 [M]+ calcd 615.2944, found 615.2953. Anal. Calcd
for C35H41N3O7: C, 68.27; H, 6.71; N, 6.82. Found: C, 68.53; H, 6.57;
N, 6.62.
that selectively recognize other molecules in preference to
dimerizing and we will have to achieve this recognition in
aqueous solution. Studies directed toward these goals are
underway in our laboratories and will be reported in due course.
Conclusions
In summary, the unnatural amino acid Hao is a tripeptide
â-strand mimic that reproduces the hydrogen-bonding pattern
of one edge of a peptide â-strand. Fmoc*-Hao and Boc-Hao
behave like typical Fmoc- and Boc-protected amino acids and
can be incorporated into peptides by standard peptide synthesis
techniques. The resulting peptide-peptidomimetic hybrid com-
pounds form â-sheetlike dimers. We anticipate that hybrid
peptides containing Hao may serve as antagonists to block
â-sheet interactions between proteins. We will pursue this idea
in future studies.
Experimental Section
General. Commercial grade reagents and solvents were used without
further purification. CH2Cl2 and THF were dried prior to use by passage
through anhydrous Al2O3 as described by Grubbs and co-workers;38
DMF was dried in an analogous fashion by percolation through 3 Å
molecular sieves.39 PS-PEG-PAL-Fmoc resin was obtained from PE
Biosystems. High-resolution mass spectra were obtained by liquid
secondary-ion ionization (LSI) of samples in a m-nitrobenzyl alcohol
matrix bombarded with Cs+ ions at 25 kV (instrumental variation σ )
2 mmu). All solution-phase reactions were performed with magnetic
stirring; moisture-sensitive solution-phase reactions were carried out
in flame- or oven-dried glassware under nitrogen. Solid-phase reactions
were performed with mechanical shaking and were, where appropriate,
monitored by qualitative ninhydrin tests.40 Reaction mixtures and
product solutions were concentrated by rotary evaporation; where
appropriate, the residue was further dried using a vacuum pump.
Fmoc*-hydrazine (3). A chilled (0 °C) solution of Fmoc*-Cl18
(18.72 g, 50.47 mmol) in ether (150 mL) was added to a stirred, ice-
cooled solution of anhydrous hydrazine (6.40 mL, 202 mmol) in ether
(100 mL) over 2 min.41 The ice-bath was removed, and the solution
was stirred for 3 h. The reaction mixture was transferred to a separatory
funnel with ether (50 mL) and washed with water (3 × 150 mL)
followed by saturated aqueous NaCl (150 mL). The organic layer was
dried over MgSO4, filtered, and concentrated to afford 18.40 g (100%)
of Fmoc*-hydrazine (3) as a white foamy solid: mp 152-154 °C; IR
(CHCl3) 3450, 3352, 1726, 1632 cm-1; 1H NMR (500 MHz, CDCl3) δ
7.63 (d, J ) 8.0 Hz, 2 H), 7.57 (br s, 2 H), 7.41 (dd, J ) 8.0, 1.6 Hz,
2 H), 6.11 (br s, 1 H), 4.43 (d, J ) 7.1 Hz, 2 H), 4.17 (t, J ) 6.8 Hz,
1 H), 3.78 (br s, 2 H), 1.37 (s, 18 H); 13C NMR (125 MHz, CDCl3) δ
158.7, 149.9, 143.7, 138.7, 124.8, 121.8, 119.2, 67.8, 47.2, 34.9, 31.6;
HRMS (LSIMS) m/z for C23H30N2O2 [M]+ calcd 366.2307, found
366.2293. Anal. Calcd for C23H30N2O2: C, 75.38; H, 8.25; N, 7.64.
Found: C, 75.53; H, 7.96; N, 7.59.
Hydrazide 5. 2-Methoxy-5-nitrobenzoic acid16c (4.33 g, 22.0 mmol)
was stirred with oxalyl chloride (5.80 mL, 66.0 mmol) and DMF (10
µL) in THF (50 mL) for 1 h. The solvent was removed, and a solution
of the resulting acid chloride in CH2Cl2 (50 mL) was added over ∼2
min to a stirred, ice-cold solution of Fmoc*-hydrazine (3) and pyridine
(1.87 mL, 23.1 mmol) in CH2Cl2 (80 mL). The ice-bath was removed
and the resulting solution was stirred for 30 min. The reaction mixture
was washed with H2O (150 mL), saturated aqueous NaHCO3 (150 mL),
Fmoc*-Hao (8). To a solution of 7 (10.17 g, 16.52 mmol) in THF:
H2O (300 mL, 4:1) was added 1.00 M NaOH solution (16.55 mL, 16.55
mmol) in a single portion. After 30 min, the solution was passed through
a column of Amberlite IR-120(plus) ion-exchange resin (4 cm d × 15
cm h, 100 mL, 1.9 mmol/mL) and concentrated to yield 9.62 g (99%)
of Fmoc*-Hao (8) as a light tan solid: mp 168-183 °C; IR (KBr)
1
3700-2400 (br), 3377, 1738, 1693 cm-1; H NMR (500 MHz, CD3-
SOCD3) δ 14.10 (br s, 1 H), 10.81 (s, 1 H), 9.85 (s, 1 H), 9.60 (s, 1
H), 8.17 (d, J ) 2.5 Hz, 1 H), 7.88 (dd, J ) 9.0, 2.6 Hz, 1 H), 7.77 (s,
2 H), 7.75 (d, J ) 8.1 Hz, 2 H), 7.45 (d, J ) 8.0 Hz, 2 H), 7.17 (d, J
) 9.1 Hz, 1 H), 4.35 (d, J ) 7.3 Hz, 2 H), 4.24 (t, J ) 7.2 Hz, 1 H),
3.87 (s, 3 H), 1.37 (s, 18 H); 13C NMR (125 MHz, CD3SOCD3) δ
164.9, 162.1, 156.6, 156.2, 153.7, 149.5, 143.8, 138.1, 130.8, 124.6,
124.6, 122.6, 122.1, 121.7, 119.3, 112.3, 66.8, 56.1, 46.6, 34.7, 31.4;
HRMS (LSIMS) m/z for C33H37N3O7 [M]+ calcd 587.2631, found
587.2631. Anal. Calcd for C33H37N3O7: C, 67.45; H, 6.35; N, 7.15.
Found: C, 67.67; H, 6.23; N, 6.90.
(37) Gong, B.; Yan, Y.; Zeng, H.; Skrzypczak-Jankunn, E.; Kim, Y. W.;
Zhu, J.; Ickes, H. J. Am. Chem. Soc. 1999, 121, 5607-5608.
(38) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.;
Timmers, F. J. Organometallics 1996, 15, 1518-1520.
(39) Burfield, D. R.; Smithers, R. H. J. Org. Chem. 1978, 43, 3966-
3968.
(40) Stewart, J. M.; Young, J. D. Solid-Phase Peptide Synthesis; Pierce
Chemical Company: Rockford, Illinois, 1984; pp 105-106.
(41) Addition of the Fmoc*-Cl to the NH2NH2 is essential; addition of
NH2NH2 to the Fmoc*-Cl resulted in substantially diminished yields of
Fmoc*-NHNH2.

7660 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Nowick et al.
Boc-Hao. To an ice-cooled solution of pure 5-NH2-2-MeO-C6H3-
CONHNHBoc16f (3.91 g, 13.9 mmol) and Et3N (2.30 mL, 16.7 mmol)
in CH2Cl2 (75 mL) was added ethyl oxalyl chloride (1.70 mL, 15.3
mmol) in drops over 1 min. After 15 min, the solution was transferred
to a separatory funnel with CH2Cl2 (125 mL), washed with H2O (100
mL) and saturated aqueous NaCl (100 mL), dried over MgSO4 and the
solvent was removed to yield 5.25 g (99%) of 5-EtO2CCONH-2-MeO-
C6H3-CONHNHBoc as a white solid: mp 184-185 °C; IR (CHCl3)
3388, 1714, 1674 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.62-9.78 (m,
2 H), 8.37 (dd, J ) 9.0, 2.8 Hz, 1 H), 8.33 (d, J ) 2.8 Hz, 1 H), 7.47
(br s, 1 H), 7.02 (d, J ) 9.1 Hz, 1 H), 4.44 (q, J ) 7.2 Hz, 2 H), 4.00
(s, 3 H), 1.47 (s, 9 H), 1.44 (t, J ) 7.2 Hz, 3 H); 13C NMR (125 MHz,
CDCl3) δ 163.2, 161.1, 155.1, 154.7, 154.2, 130.9, 125.3, 124.3, 119.5,
112.1, 81.7, 63.7, 56.5, 28.2, 14.0; HRMS (LSIMS) m/z for C17H23N3O7
[M]+ calcd 381.1536, found 381.1533. Anal. Calcd for C17H23N3O7:
C, 53.54; H, 6.08; N, 11.02. Found: C, 53.19; H, 5.76; N, 10.81.
To a solution of 5-EtO2CCONH-2-MeO-C6H3-CONHNHBoc (2.45
g, 6.42 mmol) in THF:H2O (120 mL, 4:1), was added 1.00 M NaOH
solution (6.50 mL, 6.50 mmol) in a single portion. After 30 min, the
solution was passed through a column of Amberlite IR-120(plus) ion-
exchange resin (2.5 cm d × 10 cm h, 1.9 mmol/mL) and concentrated
to yield 2.24 g (99%) of Boc-Hao as a white solid: mp 218-219 °C;
with three 10 mL-portions of 20% piperidine in DMF (10 min per
treatment, draining between treatments), washed with DMF (3 × 10
mL), CH2Cl2 (3 × 10 mL), methanol (3 × 10 mL), CH2Cl2 and
methanol (alternately, 3×, with 10 mL-portions of each solvent), CH2-
Cl2 (2 × 10 mL), ether (2 × 10 mL) and dried under a stream of dry
nitrogen to give PS-PEG-PAL(Bu)-Val.
Coupling of Hao. The PS-PEG-PAL(Bu)-Val resin was shaken with
DMF (10 mL), Fmoc*-Hao (8, 440 mg, 0.750 mmol), DCC (154 mg,
0.748 mmol), and HOBt‚H2O (101 mg, 0.66 mmol) for 2 h. The solution
was then drained, and the coupling treatment of resin was repeated for
an additional 2 h. The solution was drained, and the resin was washed
with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL), methanol (3 × 10 mL),
CH2Cl2 and methanol (alternately, 3×, with 10 mL-portions of each
solvent), CH2Cl2 (2 × 10 mL), and ether (2 × 10 mL) and dried under
a stream of dry nitrogen. The resin was shaken with three 10 mL-
portions of 20% piperidine in DMF (10 min per treatment, draining
between treatments), washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10
mL), methanol (3 × 10 mL), CH2Cl2 and methanol (alternately, 3×,
with 10 mL-portions of each solvent), CH2Cl2 (2 × 10 mL), ether (2
× 10 mL) and dried under a stream of dry nitrogen to give PS-PEG-
PAL(Bu)-Val-Hao.
Coupling of Phe. The PS-PEG-PAL(Bu)-Val-Hao resin was shaken
with DMF (10 mL), Fmoc-Phe (281 mg, 0.725 mmol), DCC (150 mg,
0.725 mmol), and HOBt‚H2O (98 mg, 0.64 mmol) for 2 h. The solution
was then drained, and the coupling treatment of resin was repeated for
an additional 3 h. The solution was drained, and the resin was washed
with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL), methanol (3 × 10 mL),
CH2Cl2 and methanol (alternately, 3×, with 10 mL-portions of each
solvent), CH2Cl2 (2 × 10 mL), ether (2 × 10 mL) and dried under a
stream of dry nitrogen. The resin was shaken with three 10 mL-portions
of 20% piperidine in DMF (10 min per treatment, draining between
treatments), washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL),
methanol (3 × 10 mL), CH2Cl2 and methanol (alternately, 3×, with
10 mL-portions of each solvent), CH2Cl2 (2 × 10 mL), and ether (2 ×
10 mL), and dried under a stream of dry nitrogen to give PS-PEG-
PAL(Bu)-Val-Hao-Phe.
1
IR (KBr) 3600-3400, 3336, 3273, 1720, 1691, 1649 cm-1; H NMR
(500 MHz, CD3SOCD3) δ 10.75 (s, 1 H), 9.64 (s, 1 H), 8.93 (s, 1 H),
8.14 (d, J ) 1.5 Hz, 1 H), 7.83 (dd, J ) 8.8, 1.8 Hz, 1 H), 7.13 (d, J
) 9.0 Hz, 1 H), 3.84 (s, 3 H), 1.41 (s, 9 H); 13C NMR (125 MHz,
CDCl3) δ 164.8, 162.1, 156.6, 155.2, 153.8, 130.7, 124.6, 122.7, 121.7,
112.3, 79.1, 56.0, 28.1; HRMS (LSIMS) m/z for C15H19N3O7 [M]+ calcd
353.1223, found 353.1230. Anal. Calcd for C15H19N3O7: C, 50.99; H,
5.42; N, 11.89. Found: C, 50.86; H, 5.39; N, 11.82.
Solid-Phase Synthesis of i-PrCO-Phe-Hao-Val-NHBu (9) on
PEG-PS-PAL Resin. Alkylation of the Resin. PS-PEG-PAL-Fmoc
resin (1.071 g, 0.175 mmol/g loading, 0.187 mmol) was shaken with
three 10 mL-portions of 20% piperidine in DMF (10 min per treatment,
draining between treatments), washed with DMF (3 × 10 mL), CH2-
Cl2 (3 × 10 mL), methanol (3 × 10 mL), CH2Cl2 and methanol
(alternately, 3×, with 10 mL-portions of each solvent), CH2Cl2 (2 ×
10 mL), ether (2 × 10 mL), and dried under a stream of dry nitrogen.
The resin was then shaken with CH2Cl2 (10 mL), collidine (149 µL,
1.13 mmol), and 2-nitrobenzenesulfonyl chloride (125 mg, 0.565 mmol)
for 3 h.22 The solution was drained, and the resin was washed with
CH2Cl2 (3 × 10 mL), methanol (3 × 10 mL), CH2Cl2 and methanol
(alternately, 3×, with 10 mL-portions of each solvent), CH2Cl2 (2 ×
10 mL), ether (2 × 10 mL) and dried under a stream of dry nitrogen.
The resin was then shaken with DMF (10 mL); 1,3,4,6,7,8-hexahydro-
1-methyl-2H-pyrimido[1,2-a]pyrimidine (MTBD, 108 µL, 0.752 mmol)
and 1-iodobutane (215 µL, 1.89 mmol) for 8 h.21 The solution was
drained, and the resin was washed with DMF (3 × 10 mL), CH2Cl2 (3
× 10 mL), methanol (3 × 10 mL), CH2Cl2 and methanol (alternately,
3×, with 10 mL-portions of each solvent), CH2Cl2 (2 × 10 mL), ether
(2 × 10 mL) and dried under a stream of dry nitrogen. The resin was
then shaken with DMF (10 mL), DBU (140 µL, 0.939 mmol), and
â-mercaptoethanol (130 µL, 1.86 mmol) for 3 h. The solution was
drained, and this treatment was repeated to ensure complete deprotec-
tion. The resin was washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10
mL), methanol (3 × 10 mL), CH2Cl2 and methanol (alternately, 3×,
with 10 mL-portions of each solvent), CH2Cl2 (2 × 10 mL), ether (2
× 10 mL) and dried under a stream of dry nitrogen, to give PS-PEG-
PAL(Bu).
Coupling of Val. DCC (0.393 g, 1.90 mmol) was added to a solution
of 1.27 g of Fmoc-Val (3.74 mmol) in dichloromethane (47 mL). After
10 min, the resulting white suspension was filtered through a glass
frit, the filtrate was concentrated, and the residue (Fmoc-Val anhydride)
was suspended in 8:2 CH2Cl2:DMF (20 mL). The PS-PEG-PAL(Bu)
resin was shaken with half (10 mL) of the suspension for 2 h, the
solution was drained, and the resin was shaken with the other half (10
mL) of the suspension for 2 h.20 The solution was drained, and the
resin was washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL),
methanol (3 × 10 mL), CH2Cl2 and methanol (alternately, 3×, with
10 mL-portions of each solvent), CH2Cl2 (2 × 10 mL), and ether (2 ×
10 mL), and dried under a stream of dry nitrogen. The resin was shaken
Coupling of Isobutyric Acid. The resin was shaken with DMF (10
mL), isobutyric acid (67 µL, 0.72 mmol), DCC (150 mg, 0.73 mmol),
and HOBt‚H2O (100 mg, 0.65 mmol) for 1.5 h. The solution was
drained, and the resin was washed with DMF (3 × 10 mL), CH2Cl2 (3
× 10 mL), methanol (3 × 10 mL), CH2Cl2 and methanol (alternately,
3×, with 10 mL-portions of each solvent), CH2Cl2 (2 × 10 mL), and
ether (2 × 10 mL), and dried under a stream of dry nitrogen to give
PS-PEG-PAL(Bu)-Val-Hao-Phe-CO-i-Pr.
Cleavage and Purification. The PS-PEG-PAL(Bu)-Val-Hao-Phe-
CO-i-Pr resin (0.969 g) was allowed to stand with 10% TFA in CH2-
Cl2 (47 mL) for 1 h without agitation. The solution was filtered and
concentrated, and the resultant oil was dissolved in CH2Cl2 (100 mL)
and washed with saturated aqueous K2CO3 (100 mL). The organic layer
was removed, dried over MgSO4, filtered, and concentrated. The
resultant solid (71 mg) was dissolved in hot MeOH (3 mL) and allowed
to precipitate. The precipitate was collected in two crops by filtration
and dried under vacuum to give 46 mg (39% yield, 46% corrected for
resin losses) of i-PrCO-Phe-Hao-Val-NHBu (9) as a white solid: mp
152 °C dec; IR (KBr) 3442, 1641; 1H NMR (CDCl3, 400 MHz) δ 11.73
(br s, 1 H), 11.18 (d, J ) 7.8 Hz, 1 H), 10.70 (s, 1 H), 8.65 (d, J ) 2.5
Hz, 1 H), 8.49 (dd, J ) 9.0, 2.5 Hz, 1 H), 8.41 (d, J ) 9.4 Hz, 1 H),
7.91 (br s, 1 H), 7.18-7.25 (m, 3 H), 7.12 (d, J ) 6.6 Hz, 2 H), 7.04
(d, J ) 9.2 Hz, 1 H), 6.34 (d, J ) 7.2 Hz, 1 H), 5.38 (appar. quartet,
J ) 6.8 Hz, 1 H), 4.61 (appar. t, J ) 8.5 Hz, 1 H), 4.04 (s, 3 H), 3.35
(appar. sextet, J ) 6.8 Hz, 1 H), 3.23 (dd, J ) 13.7, 6.3 Hz, 1 H),
3.08-3.16 (m, 2 H), 2.40 (appar. septet, J ) 6.9 Hz, 1 H), 2.17 (septet,
J ) 7.0 Hz, 1 H), 1.51 (appar. pentet, J ) 7.3 Hz, 2 H), 1.35 (sextet,
J ) 7.4 Hz, 2 H), 1.14 (d, J ) 6.9 Hz, 3 H), 1.13 (d, J ) 6.8 Hz, 3 H),
1.06 (d, J ) 6.6 Hz, 3 H), 1.05 (d, J ) 6.7 Hz, 3 H), 0.90 (t, J ) 7.3
Hz, 3 H); 13C NMR (125 MHz, CDCl3) δ 176.7, 169.8, 165.1, 159.8,
158.8, 157.8, 154.2, 135.5, 131.6, 129.5, 128.4, 127.1, 126.4, 124.5,
119.1, 111.8, 59.2, 56.6, 51.4, 39.9, 39.2, 35.5, 31.9, 31.6, 20.2, 19.7,
19.2, 19.0, 13.8; HRMS (SIMS) m/z for C32H44N6O7Na [M + Na]+
Calcd 647.3169, found 647.3195. A sample of 9, prepared by solid-
phase synthesis on indole resin,25 was subjected to elemental analysis:

The Peptidomimetic Unnatural Amino Acid, Hao
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7661
Anal. Calcd for C32H44N6O7: C, 61.52; H, 7.10; N, 13.45. Found: C,
61.39; H, 7.12; N, 13.28.
Award), the Alfred P. Sloan Foundation (Alfred P. Sloan
Research Fellowship), and the American Chemical Society
(Arthur C. Cope Scholar Award). The authors offer special
thanks to Professor A. J. Shaka, for his insightful comments on
the Tr-ROESY experiment.
Acknowledgment. We thank the NIH and NSF for grant
support (GM-49076 and CHE-9813105). J.S.N. thanks the
following agencies for support in the form of awards: the
Camille and Henry Dreyfus Foundation (Teacher-Scholar
JA001142W