Azatides: Solution and liquid phase syntheses of a new peptidomimetic

Article abstract of DOI:10.1021/ja9535470

An efficient method has been developed for the solution and liquid phase syntheses of a biopolymer mimetic consisting of 'α-aza-amino acids' linked in a repetitive manner to form what we term an azatide oligomer. To construct this biopolymer mimetic, thre

Full text of DOI:10.1021/ja9535470

VOLUME 118, NUMBER 11
MARCH 20, 1996
© Copyright 1996 by the
American Chemical Society
Azatides: Solution and Liquid Phase Syntheses of a New
Peptidomimetic
Hyunsoo Han and Kim D. Janda*
Contribution from the Departments of Molecular Biology and Chemistry, The Scripps Research
Institute, 10666 N. Torrey Pines Road, La Jolla, California 92037
ReceiVed October 23, 1995X
Abstract: An efficient method has been developed for the solution and liquid phase syntheses of a biopolymer
mimetic consisting of “R-aza-amino acids” linked in a repetitive manner to form what we term an azatide oligomer.
To construct this biopolymer mimetic, three stages of research were pursued as follows: (1) development of general
synthetic procedures that allowed the synthesis of a wide variety of Boc-protected aza-amino acid monomers, (2)
optimization of solution phase procedures for the coupling of aza-amino acids in a repetitive manner, and (3) design
and synthesis of a linker that would support azatide synthesis using a liquid phase synthetic format. The successful
completion of these three phases of research demonstrates that oligoazatides can now be rapidly assembled on a
homogeneous polymeric support. The long term prospectus of this new biopolymer is the exploration of peptide
structure as well as a potential source of new peptidomimetic libraries.
Introduction
side chain manipulations, amino acid extensions,4 deletions,5
substitutions,1a,b and, most recently, backbone modifications.6
It is this latter development that has been exploited for the
synthesis of biomimetic polymeric structures. Such progress
has been fueled by the suggestion that peptidomimetics may
provide novel scaffolds for the generation of macromolecules
with new properties of both biological and chemical interest.6
(4) (a) Freidinger, R. M.; Veber, D. F.; Perlow, D. S.; Brooks, J. R.;
Saperstein, R. Science 1980, 210, 656. (b) Paruszewski, R. Rocz. Chem.
1973, 47, 735. (c) Stachowiak, K.; Khosla, M. C.; Plucinska, K.; Khairallah,
P. A.; Bumpus, F. M. J. Med. Chem. 1979, 22, 1128.
(5) (a) Rivier, J.; Monahan, M.; Vale, W.; Grant, G.; Amoss, M.;
Blackwell, R.; Guillemin, R.; Burgus, R. Chemia 1972, 26, 303. (b)
Sarantakis, D.; McKinley, W.; Jaunakais, I. Clin. Endocrinol. 1976, 5, 2755.
(6) (a) Hagihara, M.; Anthony, N. J.; Stout, T. J.; Clardy, J.; Schreiber,
S. J. J. Am. Chem. Soc. 1992, 114, 6568. (b) Simon, R. J.; Kania, R. S.;
Zuckerman, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.;
Wang, L.; Rosenberg, S.; Marlowe, C. K.; Spellmeyer, D. C.; Tan, R.;
Frankel, A. D.; Santi, D. V.; Cohen, F. E.; Bartlett, P. A. Proc. natl. Acad.
Sci. U.S.A. 1992, 89, 9367. (c) Smith, A. B., III; 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. (d) Cho, C. Y.;
Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S. P. A.; Adams, C. L.;
Sundaaram, A.; Jacobs, J. W.; Schultz, P. G. Science 1993, 261, 1303. (e)
Liskamp, R. M. J. Angew. Chem., Int. Ed. Engl. 1994, 33, 633. (f) Burgess,
K.; Linthicum, D. S.; Shin, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 907.
Peptidomimetics have become immensely important for both
organic and medicinal chemists.1 Synthetic interest in these
surrogate peptide structures has been driven by the pharmaceuti-
cal industry’s needs for molecules with improved pharmaco-
kinetic properties.2 Biophysical studies on these pseudopeptides
has allowed elucidation of the functional role of the peptide
backbone,3 and with an ever-increasing level of synthetic
sophistication, the degree of peptide mimicry within a pepti-
domimetric can be tailored to a chemist’s needs. Indeed, the
alteration of peptides to peptidomimetics has included peptide
* To whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, March 1, 1996.
(1) (a) Spatola, A. F. In Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York,
1983; pp 267-357. (b) Sherman, D. B.; Spatola, A. F. J. Am. Chem. Soc.
1990, 112, 433. (c) Hirschmann, R. Angew. Chem., Int. Ed. Engl. 1990,
29, 1278. (d) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699.
(2) Hodgson, J. Bio/Technology 1993, 11, 683.
(3) (a) Marshall, G. R. In Chemical Recognition in Biological Systems;
Creighton, A. M., Turner, S., Eds.; The Chemical Society: London, 1982;
p 278. (b) Farmer, P. S. In Drug Design; Ariens, E. J., Ed.; Academic
Press: New York, 1980; p 121.
0002-7863/96/1518-2539$12.00/0 © 1996 American Chemical Society

2540 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Han and Janda
Scheme 1. Preparation of Boc-Protected Alkylhydrazine
Monomers
Scheme 2. Routes for Solution Phase Diazatide Synthesis
Figure 1. Comparison of a peptide, azapeptide, and azatide.
The most common manipulation involving the R-carbon atom
of peptides is the inversion of stereochemistry to yield D-amino
acids.1a The importance of this substitution in affording
compounds with improved biological potencies, altered con-
formational properties,7 and increased resistance to enzymatic
degradation has been widely recognized and exploited.8 Re-
placements of the R-hydrogen of the common amino acids by
a methyl group or any other substituent (NH2CRR′CO2H) are
both further examples of R-alkyl modification.1a Azapeptides,
however, are peptides in which one (or more) of the R-carbon(s)
has been replaced by a trivalent nitrogen atom (Figure 1).9 This
transformation results in a loss of asymmetry associated with
the R-carbon and yields a structure that can be considered
intermediate in configuration between D- and L-amino acids.1a,10
Interest in this R-carbon replacement unit stems from its ability
to provide resistance to enzymatic cleavage and its capacity to
act as a selective inhibitor of cysteine11 and serine proteases.12
While the synthesis of azapeptides has been reported,1d,9,13 the
synthesis of a “pure azapeptide”, or what we will term an
“azatide”, has yet to be accomplished.1d,14 Herein, we report
how monomeric “R-aza-amino acids” can be coupled in a linear,
stepwise, chain-lengthening fashion to construct azatides by
either solution phase or liquid phase synthetic methodologies.
Results and Discussion
For the synthesis of oligoazatides, an alphabet of suitably
protected aza-amino acid constituents needed to be prepared.
The tactic that we took was to synthesize de noVo Boc-protected
alkylhydrazine monomers substituted with a variety of functional
groups. Two principal routes are used in their syntheses
(Scheme 1): (1) reduction of Boc-protected hydrazones,13b
derived from the reaction of Boc-carbazate with either an
aldehyde or a ketone (eq 1), and (2) alkylation of hydrazine
with an alkyl halide followed by Boc-protection of the resulting
alkylhydrazine (eq 2).15 The outgrowth of these methods is the
transient protection of either the “amino- or carboxy-terminal”
functionality of the aza-amino acid and an ability to create a
unique alphabet of R-aza-amino acid R groups.
(7) Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.;
Galligan, J. J.; Burks, T. F. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5871.
(8) Dooley, C. T.; Chung, N. N.; Wilkes, B. C.; Schiller, P. W.; Bidlack,
J. M.; Pasternak, G. W.; Houghten, R. N. Science 1994, 266, 2019.
(9) Gante, J. Synthesis 1989, 405 and references cited therein.
(10) Aubry, A.; Marraud, M. Biopolymers 1989, 28, 109.
To convert these Boc-protected aza-amino acids into acylating
agents that would allow stepwise chain lengthening, the hydra-
zine portion of the molecule had to be activated (Scheme 2).
Activation of this moiety is a challenging problem since the
Boc-alkylhydrazines are poorer nucleophiles than simple amines
or amino acids. Consequently, we required a highly activated
carbonyl synthon that would allow facile coupling of two Boc-
protected aza-amino acids to form the azatide linkage. Fur-
thermore, this coupling reaction had to be controllable, such
that symmetrical dimer formation could be minimized. Our
initial attempts to couple two aza-amino acids together using
p-nitrophenyl chloroformate, carbonyldiimidazole, bis(2,4-dini-
trophenyl) carbonate, or trichloromethyl chloroformate were
(11) Magrath, J.; Abeles, R. H. J. Med. Chem. 1992, 35, 4279.
(12) (a) Elmore, D. T.; Smyth, J. J. Biochem. J. 1968, 107, 103. (b)
Barker, S. A.; Gray, C. J.; Ireson, J. C.; Parker, R. C. Biochem. J. 1974,
139, 555. (c) Gray, C. J.; Al-Dulaimi, K.; Khoujah, A. M.; Parker, R. C.
Tetrahedron 1977, 33, 837. (d) Gupton, B. F.; Carroll, D. L.; Tuhy, P.
M.; Kam, C.-M.; Powers, J. C. J. Biol. Chem. 1984, 259, 4279. (e) Powers,
J. C.; Boone, R.; Carroll, D. L.; Gupton, B. F.; Kam, C.-M.; Nishino, N.;
Sakamoto, M.; Tuhy, P. M. J. Biol. Chem. 1984, 259, 4288.
(13) (a) Bentley, P. H.; Morley, J. S. J. Chem. Soc. C 1966, 60. (b)
Dutta, A. S.; Morley, J. S. J. Chem. Soc., Perkin Trans. 1 1975, 1712. (c)
Furr, B. J. A.; Giles, M. B. J. Chem. Soc., Perkin Trans. 1 1979, 379. (d)
Quibell, M.; Turnell, W. G.; Johnson, T. J. Chem. Soc., Perkin Trans. 1
1993, 2843.
(14) The earliest attempts to make pure azatides can be dated to Gante
and co-workers: (a) Gante, J. Chem. Ber. 1965, 98, 3340. (b) Gante, J.;
Krug, M.; Lauterbach, G.; Weitzel, R. Proc. Am. Pept. Symp. 13th 1993,
1994, 299. But the methodology that was reported does not allow azatide
stepwise chain lengthening in a repetitive manner of anything but hydrazine
units.
(15) 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.

Solution and Liquid Phase Syntheses of Azatides
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2541
Table 1. Prepration of Diazatides Starting from
1-R′-Hydrazinecarboxylic acid, 1,1-Dimethylethyl Ester
the compound to be synthesized. The essence of this technology
is that it avoids a number of difficulties found in solid phase
synthesis and preserves the positive aspects of solution phase
synthesis. We have demonstrated the advantages of using liquid
phase synthesis through the construction of both peptide and
small molecule combinatorial libraries.19
A leucine-enkephalin peptide sequence (YGGFL) was
chosen as the first azatide mimetic to be synthesized. This
pentamer was selected as the N-terminal sequence within this
unit (YGGF) is common to most natural opioid peptides.20 The
successful diazatide-coupling procedure described in Scheme
compd
R1
R2
R3
R4
yield (%)
5
6
7
8
9
10
11
H
methyl
H
H
H
H
H
H
H
methyl
methyl
methyl
isobutyl
isopropyl
H
H
H
H
H
H
H
H
methyl
methyl
benzyl
isobutyl
isobutyl
isopropyl
92
91
90
85
84
82
84
2 implies N-to-C-terminal construction of the azatide.
A
p-substituted benzyl ester spacer unit that would accommodate
directional synthesis on MeO-PEG and withstand the rigors of
Boc-chemistry was designed (12; Scheme 3). It was reasoned
that 12 attached to MeO-PEG would be stable against acidolysis
due to the presence of the p-benzoate substituent, and the
oligoazatide could be liberated by catalytic hydrogenation
generating a free amino group. Thus methyl p-(hydroxymethyl)-
benzoate was O-protected as the tert-butyl ether by treatment
with isobutylene and acid. Subsequent hydrolysis of the methyl
ester with lithium hydroxide provided 12. Linker 12 was
coupled to MeO-PEG with the aid of DCC/DMAP, and upon
deprotection with trifluoroacetic acid (TFA) gave the MeO-PEG-
benzyl-OH (13) support ready for azatide synthesis. Synthesis
of the azatide pentamer YaGaGaFaLa was accomplished in a
repetitive stepwise fashion as shown in Scheme 3. Because of
the unique physical properties of the MeO-PEG homopolymer,
each coupling/deprotection reaction could be purified by
precipitation of the modified homopolymer. Furthermore MeO-
PEG allows reaction progress to be conveniently monitored by
either proton-NMR spectroscopy or the Kaiser ninhydrin test.21
On the basis of our linker strategy, the pentamer and the benzyl
protecting group of aza-tyrosine could be liberated in a single
step using catalytic hydrogenation to give the Boc-protected
pentamer (overall yield: 56.7% from 13). This compound was
converted to the desired Leu-enkephalin azatide by treatment
with trifluoroacetic acid (Scheme 3).
unsuccessful, as they suffered from complicated side reactions,
poor reaction yields, and/or prolonged reaction time. We
reasoned that these results were due to either insufficient leaving
ability of p-nitrophenol and imidazole or steric hindrance of
the o-nitro group in the case of bis(2,4-dinitrophenyl) carbonate.
To overcome these problems, we opted to use bis(pentafluo-
rophenyl) carbonate (1) as the carbonyl activation element.16
Our decision to use this reagent was based on three factors.
First, the pentafluorophenol functionality is a powerful electron-
withdrawing group, while the fluoro substituents minimize steric
problems. Second, the bis(pentafluorophenyl) carbonate can be
readily prepared from phosgene and a sodium pentafluorophe-
nolate solution. Third, the compound is a highly crystalline
solid which is easy to handle.
Shown in Scheme 2 are two solution phase routes to
diazatides. In the first case, carbamate 2 is utilized for the
coupling reaction. Thus, a Boc-protected aza-amino acid is
added dropwise to 1 granting activation of the 1-R′-hydrazine-
carboxylic acid, 1,1-dimethylethyl ester. The activated complex
formed, 2, is not isolated but instead immediately reacted Via
the addition of a second Boc-protected alkylhydrazine to
complete the diazatide coupling. This coupling procedure
provides diazatides in good yield with few side reactions in an
acceptable reaction time. Results using this coupling method
are summarized in Table 1. From this table, it is evident that
the coupling process seems to be quite general, as both simple
Glya-Glya (superscript a refers to an aza-amino acid linkage)
and sterically demanding (Vala-Vala) azatides can be synthesized
in <1 h. The latter result is extremely important as it dictates
whether the stepwise coupling of aza-amino acids is feasible.
Whereas coupling through activated 1-R′-hydrazinecarboxylic
acid, 1,1-dimethylethyl ester was successful, the coupling of
activated 2-R′-hydrazinecarboxylic acid, 1,1-dimethylethyl ester
was not (Scheme 2). From these findings, we surmise that the
activated complex is not carbamate 2 but rather the isocyanate
3.17
Tandem mass spectrometry,22 when coupled with any soft
ionization method, has emerged as an important tool for the
elucidation of sequences of peptides and nucleotides. We used
this technique for the sequence determination of our azatide.
Thus, the Leu-enkephalin azatide was subjected to ESI-tandem
mass spectrum analysis. In the acidic matrix employed for ESI
experiments, Leu-enkephalin azatide would exist as a (M + H)+
ion with a proton located on the R-nitrogen atom of Leua (i.e.,
the most basic residue). In the gas phase, (M + H)+ ions
undergo proton transfer to other basic sites to allow charge
delocalization. For an azatide, proton trnasfer would prefer-
entially occur on the more basic tertiary amide nitrogens over
the secondary amide nitrogens. The protonation of a tertiary
amide nitrogen causes bond cleavage between the R-nitrogen
and carbonyl carbon to generate X- and A-type fragments
(Figure 2). When there is no preferential protonation between
two secondary amide nitrogens such as in the urea linkage
involving the Glya residues, cleavage is possible on either side
The techniques described above allow R-azatide chain build-
ing to be performed in an iterative manner. To prepare a small
well-defined R-azatide, we chose to use polymer-supported
liquid phase synthesis.18 Liquid phase synthesis uses a soluble
linear homopolymer [poly(ethylene glycol) monomethyl ether
(MeO-PEG)] which serves as a terminal protecting group for
(18) Geckeler, K. E. In AdVances in Polymer Science; Abe, A., et al.,
Eds.; Springer-Verlag: Berlin, 1995; Vol. 121, p 31 and references cited
therein.
(16) Efimov, V. A.; Kalinkina, A. L.; Chakhmakhcheva, O. G. Nucleic
Acids Res. 1993, 21, 5337.
(17) We hypothesized this to be the case based on findings reported by
Abeles (see ref 11). For an activated 2-R′-hydrazinecarboxylic acid, 1,1-
dimethylethyl ester, the intermediate is untenable because of the carbamate’s
substitution pattern.
(19) Han, H.; Wolfe, M. M.; Brenner, S.; Janda, K. D. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 6419.
(20) Meo, T.; Gansch, C.; Inan, R.; Hollt, V.; Weber, E.; Herz, A.;
Riethmuller, G. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4084.
(21) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1979, 34, 595.
(22) (a) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.;
Hauser, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233. (b) Biemann,
K. Methods Enzymol. 1990, 193, 455.

2542 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Han and Janda
Scheme 3. MeO-PEG-Supported Leu-Enkephalin Azatide
Synthesis
Figure 2. Fragmentation patterns of (M + 1)+ ion of peptides and
azatides.
The azatide oligomer sequence synthesized (Tyra-Glya-Glya-
Phea-Leua) provided a chance to assess any biological activity
that this azatide biopolymer sequence may possess (Vide supra).
Monoclonal antibody 3-E7 was raised by Meo et al. against
the antigen â-endorphin and, like the δ-opioid receptor,
recognized the N-terminal portion of the protein.20 The antibody
also binds tightly to [Leu5]enkephalin [Tyr-Gly-Gly-Phe-Leu]
(Kd ) 7.1 nM) and a variety of related opioid peptides.24
A
competition ELISA method was used to investigate if the Tyra-
Glya-Glya-Phea-Leua sequence could bind to IgG 3-E7.19 At 1
mM the azatide pentamer showed no propensity to compete with
the natural peptide for 3-E7. While this result at first glance
appears to be disappointing, it was not completely unexpected.
The bound conformation(s) of enkephalin has been studied
extensively for over the past 15 years.25 While the exact
bioactive conformation of this peptide remains shrouded, it is
thought that the active form of this peptide resides in some sort
of a â-turn,26 this being based on X-ray crystallographic data
which showed that the glycine residues at the second and third
position of enkephalin force a type I′ 4 f 1 â-turn.27 Although
we have yet to obtain an X-ray structure on 16, physiochemical
data do exist on diacylhydrazines.28 Simple unsubstituted diacyl
hydrazines (i.e., glycine azatides) contain a dihedral (φ) angle
of ca. -175°, while N-substituted ones (i.e., all other azatides)
possess a dihedral (φ) angle of ca. -110°. Taken as a whole,
these data suggest that 16 should adopt a more extended
conformation within the critical glycine region. In essence then
this azatide oligomer would have difficulty in achieving the
orientation displayed by the antigenic determinant (Tyr-Gly-
Gly-Phe-Leu) that elicited IgG 3-E7, the outcome being that
16 is nonligand for 3-E7.
Summary
of the carbonyl group. Conversely, Y- and B-type fragmenta-
tions of peptides result from protonation of amide nitrogens and
hence cleavage of amide bonds (Figure 2). This prediction was
manifested in the collision-induced dissociation (CAD) pattern
of Leu-enkephalin azatide 16 shown in Figure 3. The MS-MS
of the (M + H+) ion at m/z 517 produced daughter peaks at
m/z 403, 255, 197 (A-type), 321, and 263 (X-type), and MS-
MS-MS on m/z 403 (M - Leua + H+) gave granddaughter
peaks at m/z 255, 197, 139 (A-type), 207, 149 (X-type), 239,
197, and 123 (Y-type). Peaks at m/z 297, 149, 107, and 91
represent A-type fragments involving cleavage of side chain of
Tyra. Mass difference between homologous A-type ions cor-
responds to elements -CONHNR-. Predicted m/z values for A2-
A5 fragments were obtained by sequentially adding the incre-
mental masses of Glya, Glya, Phea, and Leua to that for A1 at
m/z 139. A similar arguement can be made for X- and Y-type
fragments, confirming the Tyra-Glya-Glya-Phea-Leua sequence
of Leu-enkephalin azatide.
In conclusion, we have developed solution and liquid phase
methodologies for the stepwise synthesis of azatides. In spite
of our inability to show biological activity for azatide 16, we
believe that azatides will be of considerable interest as an
accessible new material with potential for novel biological
properties. Furthermore, the structural and pharmacological
(23) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
(24) Cwirla, S. E.; Peters, E. A.; Barrett, R.; Dower, W. J. Proc. Natl.
Acad. Sci. U.S.A. 1990, 87, 6378.
(25) Garner, B.; Nakanishi, H.; Kahn, M. Tetrahedron 1993, 49, 3433.
(26) (a) Bradbury, A. F.; Smyth, D. G.; Snell, C. R. Nature 1976, 260,
165. (b) Lowe, G. H.; Bart, S. K. Proc. Natl. Acad. Sci. U.S.A. 1978, 75,
7. (c) Manavalan, P.; Momany, F. A. Int. J. Pept. Protein Res. 1981, 18,
256.
(27) (a) Smith, G. D.; Griffin, J. F. Science 1978, 199, 1214. (b) Ishida,
T.; Kenmotsu, M.; Mino, Y.; Inoue, M.; Fujiwara, T.; Tomita, K.; Kimura,
T.; Sakakibara, S. Biochem. J. 1984, 218, 677.
(28) (a) Olivato, P. R.; Guerrero, S. A. J. Chem. Soc., Perkin Trans. II
1983, 1053. (b) Graybill, T. L.; Ross, J. T.; Gauvin, B. R.; Gregory, J. S.;
Harris, A. L.; Ator, M. A.; Rinker, J. M.; Dolle, R. E. Bioorg. Med. Chem.
Lett. 1992, 2, 1375.

Solution and Liquid Phase Syntheses of Azatides
J. Am. Chem. Soc., Vol. 118, No. 11, 1996 2543
Figure 3. CAD spectra of m/z 517 (M + 1)+ and 403 peaks for compound 16.
a solution of 2-N-Boc-alkylhydrazine (1 equiv) and DMAP (1 equiv)
in methylene chloride (2 mL) was added. The resulting mixture was
stirred for 30 min at room temperature. Removal of solvent and flash
chromatography23 gave the desired unsymmetrical diazatide.
Boc-Glya-Glya-Boc: 1H-NMR (300 MHz, CDCl3) δ 1.35 (s, 18H),
7.33 (br s, 2H), 7.77 (s, 2H); 13C-NMR (75 MHz, CDCl3) δ 27.9, 81.3,
156.2, 157.1; HRMS (FAB) calcd for C11H22N4O5‚Cs+ 423.0645, found
423.0655.
Boc-Alaa-Alaa-Boc (symmetrical): 1H-NMR (300 MHz, CDCl3)
δ 1.42 (s, 18H), 3.09 (s, 6H), 7.43 (s, 2H); 13C-NMR (75 MHz, CDCl3)
δ 28.1, 38.3, 81.5, 156.1, 157.0; HRMS (FAB) calcd for C13H26N4O5‚-
Cs+ 451.0958, found 451.0976.
Boc-Alaa-Alaa-Boc: 1H-NMR (300 MHz, CDCl3) δ 1.43 (s, 9H),
1.45 (s, 9H), 3.08 (s, 3H), 3.09 (s, 3H), 6.45 (br s, 1H), 7.05 and 7.62
(br s, 1H); 13C-NMR (75 MHz, CDCl3) δ 28.2, 28.3, 37.9, 81.2, 81.4,
155.4, 156.6, 156.8; HRMS (FAB) calcd for C11H22N4O5‚Cs+ 451.0958,
found 451.0965.
properties of these azatides may provide important leads for
the drug industry, and biophysical studies of these polymers
could enhance our understanding of receptor-ligand interac-
tions. Combinatorial library construction of this new biomimetic
polymer may provide a means to fabricate global peptidomi-
metic libraries. Future studies will address these possibilities.
Experimental Section
General. Methylene chloride and methanol were dried over CaH2
and powdered magnesium, respectively. Poly(ethylene glycol) mono-
methyl ether (MeO-PEG, MW ) 5000) was purchased from Aldrich
and dried over P2O5 under vacuum before use. All other solvents and
chemicals were obtained from commercial sources and used without
further purification, unless otherwise stated. NMR spectra were
obtained on a Bruker AM-300 spectrometer. Boc-protected methyl-
hydrazines, isopropylhydrazines, isobutylhydrazines, benzylhydrazines,
(p-O-benzylhydroxybenzyl)hydrazines,13b,15 and pentafluorophenyl car-
bonate16 were prepared according to literature procedures.
General Solution Phase Diazatide-Coupling Procedure. To a
stirred solution of pentafluorophenyl carbonate (50.0 mg, 13.0 mmol)
in methylene chloride (5 mL) over a period of 20 min was added a
solution of 1-N-Boc-alkylhydrazine (1 equiv) and DMAP (1 equiv) in
methylene chloride (2 mL) dropwise. Upon completion of the addition,
Boc-Alaa-Phea-Boc: 1H-NMR (300 MHz, CDCl3) δ 1.37 (s, 9H),
1.41 (s, 9H), 3.1 (s, 3H), 4.50 (br s, 2H), 6.10 and 6.59 (br s, 1H), 7.22
(m, 5H), 7.37 and 7.55 (br s, 1H); 13C-NMR (75 MHz, CDCl3) δ 27.8,
28.2, 38.5, 54.3, 81.5, 81.6, 127.3, 128.3, 128.6, 137.3, 155.5, 156.4,
156.5; HRMS (FAB) calcd for C19H30N4O5‚Cs+ 527.1271, found
527.1289.
Boc-Alaa-Leua-Boc: 1H-NMR (300 MHz, CDCl3) δ 0.90 (d, J ) 7

2544 J. Am. Chem. Soc., Vol. 118, No. 11, 1996
Han and Janda
Hz, 6H), 1.40 (s, 9H), 1.45 (s, 9H), 1.84 (m, 1H), 3.07 (s, 3H), 3.35
(br s, 2H), 6.30 and 6.56 (br s, 1H), 7.20 and 7.36 (br s, 1H); 13C-
NMR (75 MHz, CDCl3) δ 19.9, 26.3, 28.1, 28.3, 38.0, 55.7, 81.1, 82.0,
154.5, 156.1, 157.6; HRMS (FAB) calcd for C16H32N4O5‚Cs+ 493.1427,
found 493.1447.
The polymer 14 was dissolved in TFA/methylene chloride and stirred
for 30 min to remove the Boc-group. Precipitation with ether and a
separate wash with absolute ethanol and diethyl ether followed by
drying over P2O5 under vacuum gave the trifluoroacetate salt of (p-O-
benzylhydroxybenzyl)Tyra-O-benzyl-PEG-OMe. This salt was dis-
solved in methylene chloride and neutralized with diisopropylethylamine
(DIPEA; 1 equiv). To the resulting mixture were added the pentafluo-
rophenyl carbamate of Boc-carbazate (5 equiv) and DMAP (5 equiv).
The reaction mixture was stirred for 4 h. Precipitation with diethyl
ether followed by washing with absolute ethanol and diethyl ether and
then drying over P2O5 under vacuum gave the product Boc-Glya-(O-
benzyl)-Tyra-O-benzyl-PEG-OMe. Repetition of this cycle of depro-
tection, neutralization, and coupling with Glya, Phea, and Leua produced
the Leu-enkephalin azatide 15 (137 mg, 62.4% from 13): 1H-NMR
(300 MHz, CD3OD) δ 0.87 (6H), 1.42 (9H), 1.91 (1H), 4.43 (2H),
4.96 (2H), 5.12 (2H), 6.83 (2H), 7.13 (2H), 7.37 (7H), 7.95 (2H). The
multiplicity of peaks is not described due to the peak broadening.
Tyra-Glya-Glya-Phea-Leua-Boc. Compound 15 (137 mg, 23.7 µmol)
was hydrogenated with 10% Pd/C (100 mg) in methanol (5 mL) under
a balloon containing 1 atm of hydrogen for 4 h. All volatiles were
removed in Vacuo, and the residue was extracted with absolute ethanol.
This ethanol solution was concentrated and purified by preparative thin
layer chromatography. The desired material was observed as a single
band at Rf ) 0.4 (13.25 mg, 90.7%; TLC solvent, methylene chloride:
methanol, 9:1): 1H-NMR (300 MHz, CD3OD) δ 0.93 (d, J ) 7 Hz,
6H), 1.43 (s, 9H), 1.47 (s, 9H), 1.95 (m, 1H), 3.27 (br s, 2H), 4.17 and
5.19 (br s, 2H), 4.50 (br s, 2H), 6.76 (d, J ) 6.7 Hz, 2H), 7.12 (d, J )
6.7 Hz, 2H), 7.33 (m, 5H); MS m/z (ESI, positive) 639 (M + Na)+,
617 (M + 1)+.
Boc-Leua-Leua-Boc: 1H-NMR (300 MHz, CDCl3) δ 0.88 (d, J )
7 Hz, 6H), 0.90 (d, J ) 7 Hz, 6H), 1.42 (s, 9H), 1.46 (s, 9H), 1.86 (m,
2H), 3.27 (br s, 4H), 6.33 and 6.57 (br s, 1H), 7.11 and 7.23 (br s,
1H); 13C-NMR (75 MHz, CDCl3) δ 20.4, 20.4, 27.6, 27.8, 28.5, 28.6,
56.7, 59.5, 81.8, 82.3, 156.3, 156.5, 158.1; HRMS (FAB) calcd for
C19H38N4O5‚Cs+ 535.1897, found 535.1881.
Boc-Vala-Vala-Boc: mp 101-102 °C; 1H-NMR (300 MHz, CDCl3)
δ 0.09 (br s, 12H), 1.40 (s, 9H), 1.45 (s, 9H), 4.32 (br s, 1H), 4.61 (m,
2H), 6.27 (br s, 1H), 6.79 (br s, 1H); 13C-NMR (75 MHz, CDCl3) δ
19.3, 19.8, 28.0, 28.3, 48.4, 48.6, 81.0, 81.6, 157.1, 157.4, 157.9; HRMS
(FAB) calcd for C17H34N4O5‚Cs+ 507.1584, found 507.1599.
Linker Preparation: Methyl p-O-tert-Butyl(hydroxymethyl)ben-
zoate. Isobutylene was liquidified in a sealed bottle at -78 °C. A
solution of sulfuric acid (0.5 mL) and methyl p-(hydroxymethyl)-
benzoate (2.00 g, 12.0 mmol) in dry ethyl ether (20 mL) was added to
the isobutylene solution (8 mL) at -78 °C and stirred overnight at
room temperature. The resulting mixture was cooled to 4 °C; then
ice-cooled water was added. The ether layer was dried over magnesium
sulfate and evaporated to give the desired product as a white solid (2.59
g, 96.8%): mp 34-36 °C; 1H-NMR (300 MHz, CDCl3) δ 1.29 (s, 9H),
3.89 (s, 3H), 4.49 (s, 2H), 7.40 (d, J ) 6.7 Hz, 2H), 7.98 (d, J ) 6.7
Hz, 2H); 13C-NMR (75 MHz, CDCl3) δ 27.5, 51.8, 63.4, 73.6, 126.8,
128.7, 129.5; HRMS (FAB) calcd for C13H18O5‚Cs+ 355.0310, found
355.0323.
p-O-tert-Butyl(hydroxymethyl)benzoic Acid (12). Methyl p-O-
tert-butyl(hydroxymethyl)benzoate (2.02 g, 9.10 mmol) was dissolved
in a 0.8 M LiOH solution in methanol and H2O (34 mL; methanol:
H2O, 3:1). The reaction mixture was stirred until the starting material
disappeared as judged by TLC (methylene chloride:ethyl ether, 9:1).
The reaction mixture was acidified by the addition of 1 N HCl and
extracted with methylene chloride. The methylene chloride layer was
dried over magnesium sulfate and then evaporated to give the desired
Tyra-Glya-Glya-Phea-Leua‚2CF3COOH (16). Tyra-Glya-Glya-Phea-
Leua-Boc (13.25 mg, 21.5 µmol) was dissolved in TFA/methylene
chloride (5 mL) and stirred for 30 min. All volatiles were removed in
Vacuo to give the desired product as a white hygroscopic solid (16.0
mg, 100%): 1H-NMR (300 MHz, CD3OD) δ 1.05 (d, J ) 6.7 Hz,
6H), 2.09 (m, 1H), 3.07 (br s, 2H), 4.22 and 5.26 (br s, 2H), 4.65 (br
s, 2H), 6.77 (d, J ) 6.8 Hz, 2H), 7.17 (d, J ) 6.8 Hz, 2H), 7.35 (m,
5H); MS m/z (ESI, positive) 539 (M + Na)+, 517 (M + 1)+.
1
product as a white solid (1.72 g, 90.9%): mp 147-149 °C; H-NMR
Azatide 16 Competition ELISA for Anti-â-Endorphin Mono-
clonal Antibody. Each well of a Costar 96-well plate that was used
in the competition was initially coated with 25 µL of Tyr-Gly-Gly-
Phe-Leu-CO-NH-(CH2)2-NH-CO-(CH2)2-SS-BSA (5-20 mg/mL) in 60
mM sodium bicarbonate/30 mM sodium carbonate, pH 9.3, overnight.
The wells were washed 10 times with deionized water and blocked
with 100 µL of 3% BSA (all in PBS with 0.5% Tween) to prevent
nonspecific adsorption. After incubating for 30 min at 37 °C in a moist
chamber, the 3% BSA was then shaken out and 25 µL of 3% BSA and
25 µL of 16 (competing antigen) were added to the first well and serially
diluted across the plate; the same process was then continued in the
first well of the second row. Well 12 was used as the positive control.
The anti-â-endorphin antibody (diluted in 1% BSA/PBS with 0.5%
Tween) was added to each well (25 µL), and the plate was incubated
at 37 °C for 2 h. The plate was washed 20 times with deionized water,
25 µL of a 1:1000 dilution of goat anti-mouse IgG glucose oxidase
conjugate (Cappel) in 1% BSA was added to each well, and the plate
was incubated at 37 °C for 1 h. The plates were washed 20 times
with deionized water, and bound antibody was detected by the addition
of 50 µL of developing agent [0.6 mL of 20% glucose, 40 µL of 92
mM 2,2′-azinobis(3-ethylbenzothiazolinesulfonate), and 40 µL of 25
µM horseradish peroxidase in 5 mL of phosphate buffer, pH 6.0] to
each well. Thirty minutes later, the plates were read at 405 nm.
(300 MHz, CDCl3) δ 1.39 (s, 9H), 4.50 (s, 2H), 7.42 (d, J ) 6.8 Hz,
2H), 8.06 (d, J ) 6.8 Hz, 2H); 13C-NMR (75 MHz, CDCl3) δ 27.6,
63.6, 73.9, 127.0, 128.0, 130.3, 146.4, 171.8; HRMS (FAB) calcd for
C12H16O3‚Na+ 231.0997, found 231.0986.
Synthesis of MeO-PEG-Linker-YaGaGaFaLa: Attachment of p-O-
tert-Butyl(hydroxymethyl)benzoic Acid (12) to MeO-PEG: MeO-
PEG-benzyl-OH (13). Compound 12 (125 mg, 601 µmol), MeO-PEG
(1.00 g, 200 µmol), and DMAP (611 µg, 50.0 µmol) were dissolved in
methylene chloride (10 mL), and DCC (124 mg, 601 µmol) was added.
The resulting mixture was stirred for 12 h. The precipitated urea was
filtered through Celite. Diethyl ether was slowly added to the filtrate
in order to precipitate the polymer. The polymer precipitate was washed
with cold absolute ethanol and ether and dried over P2O5 under vacuum.
This solid was dissolved in trifluoroacetic acid, and the resulting solution
was stirred for 9 min at room temperature. The whole reaction mixture
was poured onto an ice-cold diethyl ether solution with vigorous stirring.
The precipitate was collected, washed with cold absolute ethanol and
diethyl ether, and dried over P2O5 under vacuum (935 mg, 91.1%):
1H-NMR (300 MHz, CDCl3) δ 4.45 (t, J ) 7 Hz, 2H), 4.71 (s, 2H),
7.41 (d, J ) 7 Hz, 2H), 7.98 (d, J ) 7 Hz, 2H).
Construction of (O-Benzyl)Tyra-Glya-Glya-Phea-Leua-Boc on 13.
A mixture of 13 (195 mg, 38.0 µmol), pentafluorophenyl carbamate
of Boc-(p-O-benzylhydroxybenzyl)hydrazine (102 mg, 5 equiv), and
DMAP (23.2 mg, 5 equiv) in methylene chloride (5 mL) was stirred
for 24 h at room temperature. Diethyl ether was slowly added to this
mixture to precipitate the polymer product 14. The polymer product
was washed with absolute ethanol and diethyl ether and dried over
P2O5 under vacuum: 1H-NMR (300 MHz, CDCl3) δ 1.40 (s, 9H), 4.45
(2H), 4.55 (2H), 5.00 (2H), 5.15 (2H), 6.55 (1H), 6.88 (2H), 7.15 (2H),
7.38 (7H), 8.00 (2H).
Acknowledgment. Financial support for this research from
the R. W. Johnson Pharmaceutical Research Institute, The
Scripps Research Institute, and the Alfred P. Sloan Foundation
is gratefully acknowledged.
JA9535470