Solid phase syntheses of oligoureas

Article abstract of DOI:10.1021/ja9631256

Isocyanates 7 were formed from monoprotected diamines 3 or 6, which in turn can be easily prepared from commercially available N-BOC- or N-FMOC-protected amino acid derivatives. Isocyanates 7, formed in situ, could be coupled directly to a solid support f

Full text of DOI:10.1021/ja9631256

1556
J. Am. Chem. Soc. 1997, 119, 1556-1564
Solid Phase Syntheses of Oligoureas
Kevin Burgess,* Javier Ibarzo, D. Scott Linthicum, David H. Russell,
Hunwoo Shin, Aroonsiri Shitangkoon, Reiko Totani, and Alex J. Zhang
Contribution from the Department of Chemistry, Texas A & M UniVersity,
College Station, Texas 77843-3255
ReceiVed September 5, 1996X
Abstract: Isocyanates 7 were formed from monoprotected diamines 3 or 6, which in turn can be easily prepared
from commercially available N-BOC- or N-FMOC-protected amino acid derivatives. Isocyanates 7, formed in situ,
could be coupled directly to a solid support functionalized with amine groups or to amino acids anchored on resins
using CH2Cl2 as solvent and an 11 h coupling time at 25 °C. Such couplings afforded peptidomimetics with an
N-phthaloyl group at the N-terminus. The optimal conditions identified for removal of the N-phthaloyl group were
to use 60% hydrazine in DMF for 1-3 h. Several sequences of amino acids coupled to ureas (“peptidic ureas”) and
of sequential urea units (“oligoureas”) were prepared via solid phase syntheses and isolated by HPLC. Partition
coefficients were measured for two of these peptidomimetics, and their water solubilities were found to be similar
to the corresponding peptides. A small library of 160 analogues of the YGGFL-amide sequence was prepared via
Houghten’s tea bag methodology. This library was tested for binding to the anti-â-endorphin monoclonal antibody.
Overall, this paper describes methodology for solid phase syntheses of oligourea derivatives with side chains
corresponding to some of the protein amino acids. The chemistry involved is ideal for high-throughput syntheses
and screening operations. The products can be expected to have an interesting range of pharmacological properties
and enhanced proteolytic stabilities relative to the corresponding peptides.
Introduction
on a support thereby facilitating separations. Research on solid
phase syntheses of small molecules is proliferating as a
consequence.13-15 There is also considerable interest in the
formation of supported oligomers with a defined sequence.16,17
Well-designed oligomers can be formed on a support by
repeating the same types of coupling reactions. They can have
chemically diverse side chains, but they need not have pro-
teolytically labile amide bonds.
Much of the combinatorial chemistry1-6 now practiced in
pharmaceutical companies around the world originated from
research involving solid phase peptide syntheses.7,8 This was
not a coincidence. Peptide chemistry was one of the few areas
for which techniques in solid phase syntheses were sufficiently
well developed to permit experiments like those conceived by
Furka,9 Houghten,10 Lam,11 and others.12 Paradoxically, how-
ever, peptides are not ideal candidates for many avenues of
pharmaceutical development. Their major attribute is that they
can present an infinite number of pharmacological profiles by
forming permutations of acidic, basic, hydrophobic, hydrophilic,
and aromatic side chains. The main reason peptides are not
used more extensively as pharmaceuticals is that their bioavail-
abilities are not high, mainly due to proteolytic degradation.
The focus of combinatorial research is therefore moving away
from peptides toward other molecular types that can be prepared
At the present time there are several approaches to the
formation of novel oligomers of defined sequence. “Peptoids”
[poly(N-alkylglycines)]18-21 and per-N-methylated peptides22
illustrate the concept of using unusual polyamide structures. As
the initial stages of the work reported in this manuscript were
in progress, a paper by Schultz and co-workers appeared
describing oligocarbamates prepared and tested on a plate with
small photochemically addressable zones.23 Since then there
has been other research in this area24 (including the preliminary
communication of the work reported in this paper).25 Somewhat
later, Janda and co-workers christened azapeptides26,27 contain-
X Abstract published in AdVance ACS Abstracts, February 1, 1997.
(1) Pinilla, C.; Appel, J.; Blondelle, S.; Dooley, C.; Dorner, B.; Eichler,
J.; Ostresh, J.; Houghten, R. A. Biopolymers 1995, 37, 221.
(2) Carell, T.; Wintner, E. A.; Sutherland, A. J.; Rebek, J.; Dunayevskiy,
Y. M.; Vouros, P. Chem. Biol. 1995, 2, 171.
(13) Thompson, L. A.; Ellman, J. A. Chem. ReV. 1996, 96, 555.
(14) Ellman, J. A. Acc. Chem. Res. 1996, 29, 132.
(3) Gordon, E. M.; Gallop, M. A.; Patel, D. V. Acc. Chem. Res. 1996,
29, 144.
(4) Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon,
E. M. J. Med. Chem. 1994, 37, 1233.
(15) DeWitt, S. H.; Czarnik, A. W. Acc. Chem. Res. 1996, 29, 114.
(16) Liskamp, R. M. J. Angew. Chem., Int. Ed. Engl. 1994, 33, 633.
(17) Gennari, C.; Salom, B.; Potenza, D.; Williams, A. Angew. Chem.,
Int. Ed. Engl. 1994, 33, 2067.
(5) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop,
M. A. J. Med. Chem. 1994, 37, 1385.
(6) Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J.; Steele,
J. Tetrahedron 1995, 51, 8135.
(7) Bodanszky, M. Principles of Peptide Synthesis; Springer-Verlag:
Berlin, 1984.
(18) Simon, R. J.; Kania, R. S.; Zuckermann, 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.
(19) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J.
Am. Chem. Soc. 1992, 114, 10646.
(8) Bodanszky, M. The Practice of Peptide Synthesis; Springer-Verlag:
Berlin, 1984.
(9) Furka, A.; Sevestyen, F.; Asgedom, M.; Dibo, G. Int. J. Peptide Res.
1991, 37, 487.
(10) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley,
C. T.; Cuervo, J. H. Nature 1991, 354, 84.
(11) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski,
W. M.; Knapp, R. J. Nature 1991, 354, 82.
(20) Kessler, H. Angew. Chem., Int. Ed. Engl. 1993, 32, 543.
(21) Kruijtzer, J. A. W.; Liskamp, R. M. J. Tetrahedron Lett. 1995, 36,
6969.
(22) Ostresh, J. M.; Husar, G. M.; Blondelle, S. E.; Dorner, B.; Weber,
P. A.; Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11138.
(23) Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S.
P. A.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G. Science
1993, 261, 1303.
(12) Fodor, S. P. A.; Rava, R. P.; Huang, X. C.; Pease, A. C.; Holmes,
C. P.; Adams, C. L. Nature 1993, 364, 555.
(24) Moran, E. J.; Wilson, T. E.; Cho, C. Y.; Cherry, S. R.; Schultz, P.
G. Biopolymers 1995, 37, 213.
S0002-7863(96)03125-3 CCC: $14.00 © 1997 American Chemical Society

Solid Phase Syntheses of Oligoureas
J. Am. Chem. Soc., Vol. 119, No. 7, 1997 1557
ing several consecutive urea bonds as “azatides” and described
an elegant method for preparation of these materials on poly-
(ethylene glycol) as a liquid phase support.28
Scheme 1. Preparation of Monoprotected Diamines from
N-BOC-Protected Amino Acids
Other recent studies complement our current research in other
ways, with respect to either formation of isocyanates or coupling
of isocyanates to amines in supported reactions. The synthetic
basis of Nowick’s studies on molecular scaffolds29,30 involves
urea bond formation by reaction of amines with isocyanates in
solution. Those researchers have developed pertinent ways to
produce isocyanates from amino acid derived materials in the
course of these studies.31,32 Phosgene or triphosgene can be
used for such reactions.33 Meanwhile Hutchins and Chapman
have been exploring solid phase methods to prepare small
molecules containing urea bonds. To achieve this goal, sup-
ported amines were reacted with bis(4-nitrophenyl)carbonate or
(4-nitrophenyl)chlorocarbonate to give anchored (4-nitrophenyl)-
carbamates, which were then coupled with other amines in
solution.34,35 Since that work, others have used urea bond
formation in syntheses of small molecules on a solid phase
wherein that particular step was not the focus.36,37
The objective of the work reported here was to study the
potential of solid phase syntheses of oligoureas.25 Oligourea
backbones of the type outlined here have the potential to support
a selection of chemically diverse side chains on a framework
that is unlikely to be easily hydrolyzed by proteases. Prepara-
tions of starting materials are described, and details of the
coupling steps used in the solid phase syntheses are given.
Solubility properties of some of the products have been
elucidated using partition coefficients. Finally, the expertise
accumulated in the course of this work was then used to prepare
a 160-member pilot library for a test screen using a monoclonal
antibody.
Scheme 2. Preparation of a Monoprotected Diamine from
FMOC-Tyr(tBu)
Results and Discussion
Preparation of the Monomers. Monoprotected diamines
were required for the syntheses of oligoureas and peptide/
oligourea chimeras. Scheme 1 gives the route followed to obtain
these starting materials from amino acids which do not have
reactive side-chain functional groups. This sequence was
outlined in the communication of this work,25 and experimental
details are given in the appropriate section of this paper.
The corresponding protected diamine derived from tyrosine
was required for the work that is described here, in addition to
those derivatives shown in Scheme 1. Masking of the phenolic
hydroxyl functionality was assumed to be necessary if this
compound was to be used in solid phase synthesis. The ideal
type of side-chain protecting group would be one that can be
removed under the conditions employed for cleavage of the
peptidomimetics from the resin. Scheme 2 shows the route used
to obtain the tert-butyl- and phthaloyl-protected phenolic
diamine 5, which meets this requirement.
Experimentally, both Schemes 1 and 2 are convenient and
begin with commercially available starting materials. Mono-
protected diamines are accessible through BOC-protected amino
acids (Scheme 1) whereas FMOC-Tyr(t-Bu) provides the
requisite acid labile side-chain protecting group required for the
synthesis of compound 6 (Scheme 2). For long-term storage
of starting materials in this project, the differentially protected
diamines 2 and 5 were chosen; the selective N-deprotection and
isocyanate formation steps to give 7 were performed im-
mediately prior to the coupling reactions, as indicated below.
Alternatively, the starting materials could be stored as hydro-
chloride salts of the amines after removal of the BOC group
from compounds 2.
(25) Burgess, K.; Linthicum, D. S.; Shin, H. Angew. Chem., Int. Ed.
Engl. 1995, 34, 907.
(26) Hess, H.-J.; Moreland, W. T.; Laubach, G. D. J. Am. Chem. Soc.
1963, 85, 4040.
(27) Gante, J. Synthesis 1989, 405.
(28) Han, H.; Janda, K. D. J. Am. Chem. Soc. 1996, 118, 2539.
(29) Nowick, J. S.; Powell, N. A.; Martinez, E. J.; Smith, E. M.; Noronha,
G. J. Org. Chem. 1992, 57, 3763.
(30) Nowick, J. S.; Abdi, M.; Bellamo, K. A.; Love, J. A.; Martinez, E.
J.; Noroha, G.; Smith, E. M.; Ziller, J. W. J. Am. Chem. Soc. 1995, 117,
89.
(31) Nowick, J. S.; Powell, N. A.; Nguyen, T. M.; Noronha, G. J. Org.
Chem. 1992, 57, 7364.
(32) Nowick, J. S.; Holmes, D. L.; Noronha, G.; Smith, E. M.; Nguyen,
T. M.; Huang, S.-L. J. Org. Chem. 1996, 61, 3929.
(33) Majer, P.; Randad, R. S. J. Org. Chem. 1994, 59, 1937.
(34) Hutchins, S. M.; Chapman, K. T. Tetrahedron Lett. 1994, 35, 4055.
(35) Hutchins, S. M.; Chapman, K. T. Tetrahedron Lett. 1995, 36, 2583.
(36) Dressman, B. A.; Spangle, L. A.; Kaldor, S. W. Tetrahedron Lett.
1996, 37, 937.
As the work reported here was approaching the final stages,
Nowick and co-workers reported a method for forming very
(37) Buckman, B. O.; Mohan, R. Tetrahedron Lett. 1996, 37, 4439.

1558 J. Am. Chem. Soc., Vol. 119, No. 7, 1997
Burgess et al.
and prior to the coupling reactions was shown to be unnecessary,
so the crude reaction mixtures were used. After a specific
coupling time with isocyanate 7a, a tea bag was removed from
the reaction mixture and the contents were treated with hydrazine
in DMF for 12 h and then cleaved from the resin by treatment
with TFA overnight. A series of experiments performed in this
way indicated that the yield of the corresponding urea was
optimal after approximately 11 h coupling time at room
temperature; the product yields were suboptimal after 22 h and
diminished further after 40 h.
Figure 1. Variation of yield in the phthaloyl deprotection step as a
function of reagent, solvent, and reaction time.
similar isocyanates but using aqueous conditions.31 We have
briefly tested the same conditions with our amine starting
materials and find that method to be equally suitable and perhaps
even more convenient.
Racemization in the formation of the monoprotected diamine
products 3 and/or in couplings of the corresponding isocyanates
was considered to be unlikely given the nature of the transfor-
mations involved. Nevertheless, we thought it prudent to check,
at least in one case. The isocyanate derived from phenylalanine
7a was therefore reacted with optically pure 1-phenylethylamine
and with a racemic sample of the same amine, in two separate
experiments. Only one of the two diastereomeric ureas 8 formed
in the second reaction could be detected in the first.
Figure 1 depicts some data concerning the various reagents
and reaction times examined for the deprotection step. A 60%
hydrazine in DMF for 1-3 h, gave the optimal yield of product.
Use of extended reaction times, other reagents (e.g., methyl-
hydrazine and methylamine), and other solvents each gave less
of the desired material. Use of the tetrachlorophthaloyl protect-
ing group39,40 in place of phthaloyl was briefly examined (data
not shown), but it was evident that this change afforded no
advantage.
Syntheses of Oligoureas and Peptidic Oligoureas. Figure
2 shows some of the products that were made via solid phase
syntheses, cleaved from the resin, and isolated in the yields
indicated after HPLC purification. A solid supported amine
intermediate is generated in each cycle; consequently, a positive
ninhydrin test41 was used to test for the presence of this group
after the deprotection steps, and evidence for complete coupling
was derived from a negative ninhydrin test. The overall yields
are in the 9-46% range, after four or five cycles of coupling
and deprotection. Each compound was prepared only once, so
these yields depicted are from initial synthetic attempts and
might be improved with practice. The HPLC trace of the crude
reaction product from the preparation of CH Yu‚CH Gu‚CH Gu‚
Conditions for Coupling and Deprotection. Attempts were
made to optimize the conditions for coupling of the isocyanates
7 to solid supported amines. To do this it was desirable to
identify an appropriate internal standard. 4-Methylbenzene-
sulfonamide was chosen because its maximum absorbance at
220 nm is close to the typical λmax for the oligourea compounds
(210 nm). Thus a batch of Rink’s amide resin was treated with
4-methylbenzenesulfonyl chloride such that approximately 20%
of the reactive amine groups were converted to sulfonamides.
The remainder of the reactive amines were coupled with FMOC-
Phe. FMOC deprotection then gave a batch of resin with a
convenient internal standard for HPLC analyses. Typically,
portions of the resin were packaged in “tea bags” 38 so that
aliquots could be conveniently removed and analyzed at
intervals.
2
2
2
CH Fu‚CH Lu-amide is typical; this is shown in the Supporting
2
2
Information.
Figure 2 also illustrates the abbreviations that we use to depict
the sequence. For example, a CH Fu indicates a urea moiety
2
The first issue to be addressed in the optimization process
was the coupling step. Preparation of the isocyanates used in
this research were performed in dichloromethane, and this
solvent was also selected for the coupling reactions. Removal
of excess phosgene and solvent after the isocyanate formation
arranged in such a way that if the peptidomimetic is arranged
with the “N-terminus” on the left, then the methylene group
(39) Debenham, J. S.; Fraser-Reid, B. J. Org. Chem. 1996, 61, 432.
(40) Stangier, P.; Hindsgaul, O. SYNLETT 1996, 179.
(41) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
(38) Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5131.

Solid Phase Syntheses of Oligoureas
J. Am. Chem. Soc., Vol. 119, No. 7, 1997 1559
Figure 2. Some oligoureas prepared on a solid phase and isolated by HPLC.
Table 1. Partition Coefficients for Two Peptides and the
we found that proton assignments were possible using much
the same techniques as are employed for peptides and proteins.42
DQF-COSY spectra43 facilitated assignment of most of the
proton signals; several illustrative spectra of this kind are given
in the Supporting Information. There were, however, breaks
in connectivities caused by the urea bonds. Consequently, more
complete assignments required use of ROESY spectra44 to define
NH protons which are associated with the same urea linkage.
An illustrative ROESY spectra of one product is also given in
the Supporting Information. The only remaining ambiguities
arise from overlapping CH2 resonances of the backbone. On
the basis of these observations, we predict that conformational
assignments of oligoureas in solution are possible.
Corresponding Oligoureasa
[pH 7.4 buffer]
P )
[octanol]
sequence
partition coefficient P
FFRF-amide
CH Fu‚CH Fu‚CH Ru‚CH Fu-amide
0.77
1.50
3.12
2.18
2
2
2
2
YGGFL-amide
CH Yu‚CH Gu‚CH Gu‚CH Fu‚CH Lu-amide
2
2
2
2
2
a Measured for the peptide/peptidomimetic partitioned between pH
7.4 phosphate buffer and 1-octanol; see the Experimental Section for
details.
Synthesis of a Library of Leu-Enkephalin Analogues. A
160-membered pilot library of peptidic oligoureas was prepared
to illustrate that more ambitious screening projects would be
possible using this methodology. The library was designed with
YGGFL-amide as a basis; this peptide is shown in the top left
of Figure 3. The plate was arranged in such that, progressing
down and to the right, more D-amino acids and more ureas units
(from both L- and D-amino acids) were incorporated.
precedes the side chain substituent derivative of L-phenylalanine
(F).
Full details of the library preparation are given in the
experimental section, but the salient details are summarized here.
Rink’s amide resin was packaged into tea bags. Amide bonds
were formed via coupling FMOC-protected amino acids acti-
vated using PyBOP/HOBt, and the urea bonds were formed by
direct reactions of isocyanates as described above. Deprotec-
tions of the amino acids were performed using piperidine in
the usual way,45 and the ureas were deprotected using hydrazine
as outlined above. Finally, the peptides and peptidomimetics
were cleaved from the resin using a TFA, phenol, water,
ethanedithiol, thioanisole “cocktail”. The scavengers in that
mixture were included to protect the phenolic side chains from
Relative Solubilities of Oligoureas and the Corresponding
Peptides. Aqueous buffer/octanol partition coefficients were
measured for two peptides and for the corresponding oligourea
sequence (Table 1). In one case the oligourea was more
hydrophilic, and in the other it was less. Thus the relative
hydrophilicity of peptides and oligoureas can be comparable,
but the solubilities of both classes of compounds will be
influenced by factors other than molecular composition, includ-
ing association of molecules and folding into relatively long
lived conformations in solution. Solubility factors are sequence
dependent, but so far, we have not prepared an oligourea that
is markedly insoluble in water. Concerns that oligoureas might
be very insoluble due to intermolecular hydrogen binding were
thereby alleviated, at least for these sequences of four to five
residues.
(42) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986.
(43) Piantini, U.; Sorensen, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982,
104, 6800.
(44) Bothner-By, A. A.; Stephen, R. L.; Lee, J.; Warren, C. D.; Jeanloz,
R. W. J. Am. Chem. Soc. 1984, 106, 811.
(45) Atherton, E.; Sheppard, R. C. Solid Phase Peptide Synthesis, A
Practical Approach; IRL Press: Oxford, U.K., 1989.
Assignment of 1H NMR Resonances for the Peptidodomi-
metics. The one-dimensional proton NMR spectra of these
molecules are complicated due to overlapping peaks. However,

1560 J. Am. Chem. Soc., Vol. 119, No. 7, 1997
Burgess et al.
Table 2. IC50 Values for Binding of Peptides/Peptidomimetics to
the anti-â-endorphin Antibody in a Competitive Radioimmunoassay
entry
sequence
IC50 (µM)
1
2
3
4
5
6
7
YGGFL-OH
0.0794
0.295
108
50.1
25.1
63.1
79.4
YGGFL-NH2
YGCH GuFL-NH2
2
YCH GuGFL-NH2
2
YCH GuGF(D-L)-NH2
2
YCH GuGFCH Lu-NH2
2
2
YCH GuFL-NH2
2
cleavage cocktail under vacuum, the peptide/peptidomimetic
residues in small vials were dissolved in 2:1 H2O:MeCN. The
hydrophobic impurities were then extracted via treatment with
diethyl ether. Evaporation of the aqueous portions (speed vac)
gave colorless residues. Analyses of these via HPLC (215 nm)
showed one major peak in almost every case. MALDI analyses
showed a peak corresponding to the molecular ion for all the
samples.
Screening a Library of Leu-enkephalin Analogues in a
Competitive Radioimmunoassay. A monoclonal antibody
(mAb) selective for the YGGF sequence was used to screen
the pilot library shown in Figure 3. The sequence YGGFL-
OH is known to be a good substrate for this mAb, but the mAb
had less affinity for the peptides/peptidomimetics prepared here,
all of which had carboxamide residues at the C-terminus. This
was not a concern in this project, however, because the overall
objective of the screening exercise was to probe the effect of
systematically introducing more urea linkages on the binding
efficiency, i.e. the change in binding affinity on progressing
from top/left to bottom/right in Figure 3. The absolute values
of the binding efficiency were really of little consequence in
this work.
Peptides/peptidomimetics that had 70% or more of the binding
affinity of YGGFL-amide with respect to the anti-â-endorphin
antibody are shown in Figure 3. These data indicated that five
peptidic-oligoureas were relatively tight binders to the mAb.
To confirm this, these five compounds were resynthesized and
purified by HPLC for determination of IC50 values. These data
are given in Table 2. It emerges that, relative to YGGFL-amide,
the IC50 values are decreased by approximately 2 orders of
magnitude when a urea bond is incorporated at the G2 and/or
the L5 position. The mAb has less affinity for substrates with
other amide-for-urea substitutions. Substitution of G2 and G3
with a single urea entity was also explored in one case. Table
2, entry 7, shows that the peptide YCH GuFL-NH2 bound with
2
approximately the same affinity as the analogues comprised of
five residues.
Substitution of amide bonds with the urea fragments used in
this work elongates the backbone of the peptidomimetics by
several atoms. This study shows that incorporation of urea units
does not necessarily negate all the binding efficiency, but it
was reduced in the limited number of compounds screened. This
is not a surprising observation. The changes effected in this
library were systematic, but they were not designed to logically
enhance any particular activity.
Preparation of Monomers For Preparation of Other
Oligoureas. The monomer building blocks used to prepare the
library described above were derived from amino acids without
side-chain functionalities (Scheme 1) except for the tyrosine
derivative 6. Preparations of other monomers will be necessary
if the methodology described in this paper is to be expanded to
other situations. In preliminary work toward that goal, the
monomer units corresponding to Asp and Lys were also
prepared as shown in Scheme 3. These two syntheses demon-
Figure 3. Screening of a peptidomimetic library with the anti-â-
endorphin antibody. X ) L-amino acid; x ) D-amino acid; X ) an
urea entity formed from L-amino acid; x ) an urea entity formed from
a D-amino acid.
the tert-butyl cations liberated from the Tyr-derived residues.
After filtration (effectively performed by agitating the tea bags
which contained only the spent resin) and removal of the

Solid Phase Syntheses of Oligoureas
J. Am. Chem. Soc., Vol. 119, No. 7, 1997 1561
Scheme 3
Our optimism regarding the potential of this group of
compounds is shared by others in the field. Very recently,
approximately 1 year after we communicated our preliminary
results,25 another group has reported syntheses of oligourea
compounds on a solid phase.50,51 Their approach is slightly
different. They used (4-nitrophenyl)carbamates instead of
isocyanates as the “activiated” coupling partners (although these
carbamates possibly react via isocyanates formed in situ).
Moreover, they used azides rather than phthaloyl functionality
to mask the “N-terminal” nitrogen. It appears that approach
works well for coupling several simple monomer units but does
not offer any very significant advantage over the route we
originally communicated and elaborated here. In unpublished
work, we have designed synthetic routes for preparation of
appropriately side-chain protected monomers corresponding to
nearly all the protein amino acids. The latter results will be
reported in due course.
Experimental Section
General Synthetic Procedures. Melting points were uncorrected.
High-field NMR spectra were recorded on a Varian XLAA 200 (1H at
200 MHz, 13C at 50 MHz), a Varian Unity+ 300, a Varian XLR 300,
a Varian XL-400, or a Varian Unity+ 500 machine. 1H chemical shifts
are reported in δ relative to CHCl3 (7.24 ppm) as internal standard,
and 13C chemical shifts are reported in ppm relative to CDCl3 (77.0
ppm) unless otherwise specified. Multiplicities in 1H NMR are reported
as (br) broad, (s) singlet, (d) doublet, (t) triplet, (q) quartet, and (m)
multiplet. IR spectra were recorded on a Mattson RS/1. Centrifuge
evaporation system is Jouan RC 10.22 system. Thin layer chroma-
tography was performed on silica gel 60 F254 plates from Whatman.
Flash chromatography was performed on SP silica gel 60 (230-600
mesh ASTM). Other chemicals were purchased from commercial
suppliers and used as received. The alcohols 1 and 4 were formed via
the reduction process outlined in Schemes 1 and 2 or, for most of this
work, purchased directly from Advanced ChemTech Chemical Co.
(S)-N2-tert-Butoxycarbonyl-3-phenyl-N1-phthaloyl-1,2-diamino-
propane (2a). Diethyl azodicarboxylate (3.2 mL, 16.4 mmol, 1.0
equiv) was added over 10 min to an ice-cold mixture of N-BOC
phenylglycinol 1a (2.91 g, 16.4 mmol, 1.0 equiv), PPh3 (5.25 g, 20.0
mmol, 1.2 equiv), and phthalimide (2.70 g, 18.4 mmol, 1.1 equiv) in
80 mL of THF. The reaction mixture was stirred for 3 h, the THF
was evaporated under vacuum, and the residue was purified via flash
chromatography (4.5:1 hexane:EtOAc eluant) to afford 3.73 g (73%)
strate production of synthons with acidic side chains protected
as tert-butyl esters and with basic side chains protected as tert-
butoxycarbonyl groups. In summary, simple monomers can be
prepared from BOC-protected amino acids (Scheme 1), and we
anticipate that most of the side-chain functionalized monomers
corresponding to the protein amino acids will be available from
FMOC-protected amino acids (Schemes 2 and 3).
Conclusions
We suspect that oligoureas may have unrealized potential with
regard to developing leads for pharmaceutical development. The
reactions described in this paper facilitate formation of isocy-
anate monomers from amino acids and the formation of
oligoureas from these via solid phase reactions. This chemistry
is amenable to formation of supported libraries via split
syntheses and to solution phase libraries. We envisage that the
techniques could be adapted for automated syntheses providing
one compound per well or for generation of mixtures and
deconvolution via the iterative screening10,46 or positional scan47
approaches most closely associated with Houghten. For target-
oriented applications, attempts would be made to match the
steric demands of the urea group with the intended receptor
and small biased libraries could be formed. Alternatively, the
“alphabet” of isocyanate monomers could be expanded to
facilitate random screening of larger libraries. In general,
proteolytic stabilities are likely to be increased when amide
bonds are substituted with ureas. Moreover, pharmaceutically
interesting ureas have been proved to be efficacious,48,49 and it
seems reasonable that other bioactive compounds in this class
would be discovered if screening efforts in this area were
intensified. Consequently, we have considerable optimism
regarding the potential of this category of compounds in
biomedical applications.
1
of the product as a white powder: mp 155-157 °C; H NMR (400
MHz, CDCl3) δ 7.83 (dd, J ) 3, 5 Hz, 2H), 7.69 (dd, J ) 3, 5 Hz,
2H), 7.33-7.22 (m, 5H), 4.64 (d, J ) 8 Hz, 1H), 4.32 (br s, 1H), 4.28-
3.65 (m, 2H), 2.95-2.83 (m, 2H), 1.22 (s, 9H); 13C NMR (50 MHz,
CDCl3) δ 168.33, 155.38, 137.16, 133.81, 132.35, 129.26, 128.59,
126.70, 123.26, 79.41, 50.85, 41.93, 39.45, 28.18; IR (KBr) 3367, 2979,
2939, 1772, 1720, 1677, 1641, 1510, 1431 cm-1; FAB-HRMS for
C22H25N2O4 [MH] calcd m/z 381.1814, found 381.1818.
N1-tert-Butoxycarbonyl-N2-phthaloyl-1,2-diaminoethane (2b). This
compound was prepared using N-BOC glycinol 1b (10.1 g, 62.7 mmol)
in essentially the same procedure as outlined for the conversion of 1a
into 2a (Vide supra). The product 2b was formed as a white powder
(17.1 g, 94%): mp 136-138 °C; 1H NMR (400 MHz, CDCl3) δ 7.86-
7.83 (m, 2H), 7.73-7.70 (m, 2H), 4.82 (br s, 1H), 3.83 (t, J ) 6 Hz,
2H), 3.43 (q, J ) 6 Hz, 2H), 1.33 (s, 9H); 13C NMR (50 MHz, CDCl3)
δ 168.31, 155.86, 133.87, 132.30, 123.26, 79.50, 39.83, 38.16, 28.27;
IR (KBr) 3394, 2971, 2944, 1772, 1703, 1519, 1434 cm-1; FAB-HRMS
for C15H19N2O4 [MH] calcd m/z 291.1344, found m/z 291.1367.
(S)-N2-tert-Butoxycarbonyl-4-methyl-N1-phthaloyl-1,2-diamino-
pentane (2c). This compound was prepared using N-BOC leucinol
1c (0.9 g, 4.4 mmol) in essentially the same procedure as outlined for
the conversion of 1a into 2a (Vide supra). The product 2c was formed
as a white powder (1.3 g, 86%): mp 124-129 °C; 1H NMR (300 MHz,
(46) Blake, J.; Litzi-Davis, L. Bioconjugate Chem. 1992, 3, 510.
(47) Pinilla, C.; Appel, J. R.; Blanc, P.; Houghten, R. A. Biotechniques
1992, 13, 901.
(48) Getman, D. P.; DeCrescenzo, G. A.; Heintz, R. M.; Reed, K. L.;
Talley, J. J.; Bryant, M. L.; Clare, M.; Houseman, K. A.; Marr, J. J.; Mueller,
R. A.; Vazquez, M. L.; Shieh, H.-S.; Stallings, W. C.; Stegeman, R. A. J.
Med. Chem. 1993, 36, 288.
(49) Lam, P. Y. S.; Jadhav, P. K.; Eyermann, C. J.; Hodge, C. N.; Ru,
Y.; Bacheler, L. T.; Meek, J. L.; Otto, M. J.; Rayner, M. M.; Wong, Y. N.;
Chang, C.-H.; Weber, P. C.; Jackson, D. A.; Sharpe, T. R.; Erickson-
Viitanen, S. Science 1994, 263, 380.
(50) Kim, J.-M.; Bi, Y.; Paikoff, S. J.; Schultz, P. G. Tetrahedron Lett.
1996, 37, 5305.
(51) Kim, J.-M.; Wilson, T. E.; Norman, T. C.; Schultz, P. G. Tetrahedron
Lett. 1996, 37, 5309.

1562 J. Am. Chem. Soc., Vol. 119, No. 7, 1997
Burgess et al.
acetone-d6) δ 7.86 (m, 4H), 5.81 (m, 1H), 4.11 (m, 1H), 3.66 (m, 2H),
1.56 (m, 1H), 1.39 (m, 1H), 1.20 (s, 9H), 0.99 (d, J ) 7 Hz, 3H), 0.94
(d, J ) 7 Hz, 3H); 13C NMR (75 MHz, acetone-d6) δ 164.90, 157.35,
130.76, 129.33, 119.63, 74.48, 44.50, 39.63, 37.88, 24.40, 21.62, 19.42,
chromatography (2.3:1 hexane:EtOAc eluant) to afford 2.4 g (96%) of
the product as a white powder: mp 132-134 °C; 1H NMR (300 MHz,
acetone-d6) δ 7.85 (d, J ) 7 Hz, 2H), 7.81 (m, 4H), 7.65-7.55 (m,
2H), 7.44-7.30 (m, 4H), 7.27 (d, J ) 8 Hz, 2H), 6.91 (d, J ) 8 Hz,
2H), 6.53 (m, 1H), 4.33 (m, 1H), 4.12-4.01 (m, 2H), 3.89 (m, 2H),
3.04 (dd, J ) 6, 14 Hz, 1H), 2.94 (dd, J ) 9, 14 Hz, 1H), 1.26 (s, 9H);
13C NMR (75 MHz, acetone-d6) δ 164.98, 152.85, 151.00, 141.22,
139.02, 130.90, 130.04, 129.15, 124.46, 123.93, 122.30, 122.14, 120.70,
119.76, 116.73, 74.42, 63.01, 48.93, 43.96, 38.62, 34.65; IR (KBr) 3377,
2981, 2964, 2930, 1776, 1709, 1700, 1434 cm-1; FAB-HRMS for
C36H34N2O5Na [MNa] calcd m/z 597.2365, found m/z 597.2374.
(S)-3-[(4-tert-Butyloxy)benzyl]-N1-phthaloyl-1,2-diaminopro-
pane (6). Diethylamine (0.36 mL, 3.5 mmol, 10 equiv) was added to
the solution of 0.2 g of compound 5 (0.35 mmol) in 5 mL of THF.
The mixture was stirred at 25 °C for 12 h, the THF was evaporated
under vacuum, and the residue was purified via flash chromatography
(1:4 hexane:EtOAc eluant) to afford 0.07 g (56%) of the product as an
oil: 1H NMR (200 MHz, DMSO-d6) δ 7.81 (s, 4H), 7.03 (d, J ) 9 Hz,
2H), 6.81 (d, J ) 9 Hz, 2H), 4.06 (m, 1H), 3.73 (dd, J ) 5, 7 Hz, 2H),
2.89 (dd, J ) 4, 13 Hz, 1H), 2.61 (dd, J ) 9, 13 Hz, 1H), 1.25 (s, 9H);
13C NMR (50 MHz, DMSO-d6) δ 168.87, 154.43, 134.71, 132.77,
130.41, 124.30, 123.42, 109.65, 78.00, 60.49, 43.65, 40.18; IR (neat)
3228, 2976, 2931, 1714, 1646, 1435 cm-1; FAB-HRMS for C22H25N2O3
[MH] calcd m/z 353.1865, found m/z 353.1862.
18.17; IR (KBr) 3248, 2953, 2870, 1770, 1708, 1685, 1529, 1431 cm-1
;
FAB-HRMS for C19H27N2O4 [MH] calcd m/z 347.1971, found m/z
347.1977.
(S)-N2-tert-Butoxycarbonyl-1-[(4-methoxy-2,3,5-trimethylben-
zenesulfonyl)guanidino]-N1-phthaloyl-1,2-diaminopentane (2d). This
compound was prepared using N-BOC argininol 1d (4.1 g, 8.7 mmol)
in essentially the same procedure as outlined for the conversion of 1a
into 2a (Vide supra). The product 2d was formed as an oil (3.5 g,
1
65%); H NMR (300 MHz, CDCl3) δ 7.68-7.61 (m, 2H), 7.50-7.47
(m, 2H), 6.51 (s, 1H), 6.46 (br s, 2H), 4.80 (br s, 1H), 4.41 (m, 1H),
3.80 (s, 3H), 3.54 (m, 2H), 3.17 (m, 2H), 2.70 (s, 3H), 2.63 (s, 3H),
2.11 (s, 3H), 1.51-1.37 (m, 4H), 1.18 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 165.38, 155.09, 153.46, 135.42, 133.37, 130.85, 129.61,
128.22, 121.47, 120.15, 108.52, 76.18, 73.69, 52.29, 46.39, 39.16, 37.63,
26.90, 24.97, 22.81, 21.11; IR (neat) 3340, 2978, 1772, 1712, 1439,
1367 cm-1; FAB-HRMS for C29H39N5O7SNa [MNa] calcd m/z 624.2468,
found m/z 642.2478.
(S)-3-Phenyl-N1-phthaloyl-1,2-diaminopropane hydrochloride (3a).
A solution of compound 1a (1.04 g, 2.7 mmol) in 10 mL of 6 M HClaq
(60 mmol, 22 equiv) and 10 mL of THF was stirred at 25 °C for 12 h.
A white powder precipitated. This was collected and washed with
diethyl ether to afford 0.72 g (84%) of the product as a white powder:
Procedure for Formation of Isocyanates in Situ. The conversion
of amine 3a into isocyanate 7a is typical of the isocyanate formation
procedure used throughout this work. Compound 7a was prepared by
mixing 95 mg of 3a (0.3 mmol), triethylamine (0.34 mL, 2.4 mmol, 8
equiv), and 0.2 mL of a 2 M phosgene solution (0.39 mmol, 1.3 equiv)
in 5 mL of CH2Cl2 at 0 °C. The mixture was stirred for 2 h, then
added to the solid supported amine.
1
mp 263-264 °C; H NMR (400 MHz, DMSO-d6) δ 8.31 (br s, 2H),
7.86-7.81 (m, 4H), 7.32 (d, J ) 4 Hz, 4H), 7.22 (m, 1H), 3.82 (dd, J
) 9, 14 Hz, 1H), 3.73 (m, 1H), 3.63 (dd, J ) 4, 14 Hz, 1H), 3.15 (dd,
J ) 5, 14 Hz, 1H), 2.86 (dd, J ) 9, 14 Hz, 1H);13C NMR (50 MHz,
DMSO-d6) δ 167.62, 135.75, 133.87, 131.62, 128.67, 128.20, 126.43,
122.57, 50.14, 38.94, 36.26; IR (KBr) 3468, 3053, 3024, 1773, 1710,
1600, 1434 cm-1; FAB-HRMS for C17H17N2O2 [MNa] calcd m/z
281.1290, found m/z 281.1293.
Test for Racemization of 7a. The isocyanate 7a (prepared in situ
as described immediately above) was added to a solution of racemic
2-phenylethylamine (0.013 mL, 0.1 mmol, 0.33 equiv) in 0.5 mL of
CH2Cl2. The reaction mixture was stirred for 12 h at 25 °C, the solvent
was evaporated under vacuum, and the residue was purified by
preparative RP-HPLC (Vydac C18 column, 22 mm × 25 cm, 10 µm)
with a linear gradient obtained by mixing solvent A (0.1% TFA in
water) and solvent B (0.1% TFA in acetonitrile) and programmed to
increase from 50 to 65% B over 35 min with a flow rate of 5 mL
min-1. The peak with a retention time of 27.3 min was collected and
lyophilized to afford 6 mg (14%) of a white powder: This material
was a near 1:1 mixture of the two diastereoisomers of compound 8:
1H-NMR (300 MHz, acetone-d6) δ 7.84-7.79 (m, 4H), 7.32-7.19 (m,
10H), 5.86 (m, 1H), 5.31 (m, 1H), 4.68 (m, 1H), 4.50 (m, 1H), 3.73
N2-Phthaloyl-1,2-diaminoethane (3b). This compound was pre-
pared using 2b (17.1 g, 59 mmol) in essentially the same procedure as
outlined for the conversion of 2a into 3a (Vide supra). The product
3b was formed as a white powder (10.9 g, 81%): mp 268-270 °C; 1H
NMR (400 MHz, DMSO-d6) δ 8.35 (br s, 3H), 7.88-7.82 (m, 4H),
3.86 (t, J ) 6 Hz, 2H), 3.06 (m, 2H); 13C NMR (50 MHz, DMSO-d6)
δ 167.98, 134.38, 132.12, 123.07, 37.30, 35.45; IR (KBr) 3475, 3011,
2909, 2886, 1779, 1709, 1432 cm-1; FAB-HRMS for C10H10N2O2 [MH]
calcd m/z 191.0821, found m/z 191.0837.
(S)-4-Methyl-N1-phthaloyl-1,2-diaminopentane (3c). This com-
pound was prepared using 2c (1.45 g, 4.2 mmol) in essentially the same
procedure as outlined for the conversion of 2a into 3a (Vide supra).
The product 3c was formed as a white powder (0.87 g, 74%): mp
3
(m, 2H), 2.93 (m, 2H), 1.30 (d, J ) 7 Hz, /2H), 1.18 (d, J ) 7 Hz,
3/2H); FAB+ for C26H26N3O3 [MH] calcd m/z 427, found m/z 427. When
this experiment was repeated using 7a and optically pure (S)-2-
phenylethylamine, only one diastereomer of 8 was observed. This
material was isolated as a white powder: 1H-NMR (300 MHz, acetone-
d6) δ 7.83 (m, 4H), 7.27-7.15 (m, 10H), 5.88 (m, 1H), 5.34 (m, 1H),
4.68 (m, 1H), 4.50 (m, 1H), 3.70 (m, 2H), 2.89 (m, 2H), 1.14 (d, J )
7 Hz, 3H); FAB+ for C26H26N3O3 [MH] calcd m/z 427, found m/z 427.
CH FuCH FuCH RuCH Fu-NH2 (9). The general procedure for forma-
1
252-255 °C; H NMR (300 MHz, DMSO-d6) δ 8.25 (br s, 2H), 7.89
(m, 4H), 3.76 (m, 2H), 3.43 (m, 1H), 1.86 (m, 1H), 1.52 (m, 2H), 0.95
(d, J ) 6 Hz, 1H), 0.91 (d, J ) 6 Hz, 1H); 13C NMR (75 MHz, DMSO-
d6) δ 165.62, 131.76, 129.32, 120.45, 45.39, 41.63, 36.46, 21.18, 19.97,
19.14; IR (KBr) 3477, 3010, 2873, 1776, 1716, 1608, 1429 cm-1; FAB-
HRMS for C14H19N2O2 [MH] calcd m/z 247.1446, found m/z 247.1465.
(S)-1-[(4-Methoxy-2,3,5-trimethylbenzenesulfonyl)guanidino]-N1-
phthaloyl-1,2-diaminopentane (3d). This compound was prepared
using 2d (0.11 g, 0.18 mmol) in essentially the same procedure as
outlined for the conversion of 2a into 3a (Vide supra). The product
3d was formed as a oil (0.05 g, 53%): 1H NMR (300 MHz, CD3OD)
δ 7.91 (m, 4H), 6.63 (s, 1H), 3.76 (s, 3H), 3.52 (m, 1H), 3.30 (m, 2H),
3.26 (m, 2H), 2.67 (s, 3H), 2.61 (s, 3H), 2.09 (s, 3H), 1.69 (m, 2H),
1.29 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 165.46, 155.33, 153.63,
134.91, 133.40, 131.20, 130.35, 128.83, 121.17, 119.99, 108.29, 66.12,
51.55, 47.92, 35.54, 27.69, 25.07, 24.33, 21.24, 19.81; IR (neat) 3367,
3070, 2953, 2926, 2854, 1716, 1458, 1378 cm-1; FAB-HRMS for
C24H32N5O5S [MH] calcd m/z 502.2124, found m/z 502.2110.
2
2
2
2
tion of oligoureas via stepwise coupling of phthaloyl-protected isocy-
anates to Rink’s amide resin was as follows. Manual oligourea
synthesis was carried out in a 30 mL vessel fitted with a course glass
frit and agitated using a manual wrist action shaker (Burrel, Model
75). All reactions were carried out at 25 °C. Phthaloyl deprotection
was performed by shaking the resin with a 60% solution of 98%
hydrazine hydrate in DMF (5 mL, 12 h). DMF (5 × 1 min, ca. 10
mL) and CH2Cl2 (5 × 1 min, ca. 10 mL) washing cycles were used
after each coupling step and each deprotection step. The isocyanate
7a was prepared by mixing 76 mg of 3a (0.24 mmol), 0.27 mL
triethylamine (1.92 mmol, 8 equiv), and 0.16 mL of a 2 M phosgene
solution (0.32 mmol, 1.3 equiv) in 10 mL of CH2Cl2 at 0 °C. The
mixture was stirred for 2 h, then used for the coupling reaction without
further purification. Rink’s amide resin (0.064 g of 0.62 mmol g-1
capacity) was first swelled in DMF (ca 10 mL) for 2 h, then reacted
with the solution of 7a (0.24 mmol, 3 equiv in 10 mL of CH2Cl2). The
mixture was then shaken for 5 h and subjected to one washing cycle.
The phthaloyl protecting group was removed, and then the resin was
(S)-N2-[(9-Fluorenylmethoxy)carbonyl]-3-[(4-tert-butyloxy)ben-
zyl]-N1-phthaloyl-1,2-diaminopropane (5). Diethyl azodicarboxylate
(1.1 mL, 6.5 mmol, 1.5 equiv) was added over 10 min to an ice-cold
mixture of N-FMOC tyrosinol 4 (0.96 g, 4.4 mmol, 1.0 equiv), PPh3
(1.71 g, 6.5 mmol, 1.5 equiv), and phthalimide (1.71 g, 6.5 mmol, 1.5
equiv) in 50 mL of THF. The solution was stirred for 3 h, the THF
was evaporated under vacuum, and the residue was purified via flash

Solid Phase Syntheses of Oligoureas
J. Am. Chem. Soc., Vol. 119, No. 7, 1997 1563
washed, as described immediately above. Three cycles of deprotection
and coupling were then performed in the same manner. After removal
of the final phthaloyl protecting group, the resin was washed thoroughly
with DMF then CH2Cl2.
(m, 5H), 6.98 (d, J ) 8 Hz, 2H), 6.67 (d, J ) 8 Hz, 2H), 6.18 (br s,
1H), 6.13 (m, 1H), 6.11 (m, 1H), 6.07 (m, 1H), 6.05 (m, 1H), 6.02 (m,
1H), 5.99 (m, 1H), 5.97 (m, 1H), 5.86 (m, 1H), 5.60 (m, 2H), 3.97 (m,
1H), 3.81 (m, 1H), 3.64 (m, 1H), 3.29 (m, 2H), 3.27 (m, 2H), 3.19 (m,
1H), 3.14 (m, 1H), 3.09 (m, 2H), 3.04 (m, 2H), 2.84 (m, 2H), 2.73 (m,
1H), 2.60 (m, 2H), 2.58 (m, 2H), 1.59 (m, 1H), 1.13 (m, 1H), 0.86 (d,
J ) 6 Hz, 3H), 0.81 (d, J ) 6 Hz, 3H); 13C NMR (75 Hz, DMSO-d6)
δ 158.98, 158.54, 158.35, 158.09, 156.87, 138.92, 138.73, 129.97,
129.16, 128.11, 127.74, 125.90, 115.13, 60.35, 51.10, 49.13, 47.48,
44.88, 43.54, 41.78, 38.46, 37.43, 21.90, 14.56; IR (KBr) 3374, 2956,
2933, 1684, 1653, 1558, 1437 cm-1; MALDI+ for C33H54N11O6 [MH]
calcd m/z 700.8684, found m/z 700.4268.
Cleavage of the peptide from the resin was effected using the
following procedure. The resin was completely dried under high
vacuum, and then a phenol/1,2-ethanedithiol/thioanisole/water/TFA
mixture (5:2.5:1:5:81.5, v/v/v/v/v, respectively, 15 mL) was added to
the resin in a flask. The reaction mixture was agitated at 25 °C for 9
h. The resin was filtered and washed with TFA (5 mL), and the filtrate
was evaporated to ca. 2 mL. Deionized water (20 mL) was added,
and the resulting aqueous solution was washed with Et2O (2 × 5 mL)
and lyophilized. The crude peptidomimetic was further purified by
preparative RP-HPLC (Vydac C18 column, 22 mm × 25 cm, 10 µm)
with a linear gradient obtained by mixing solvent A (0.1% TFA in
water) and solvent B (0.1% TFA in acetonitrile). The gradient was
programmed to increase from 5 to 60% B over 37 min with a flow
rate of 5 mL min-1. The peak with a retention time of 34.3 min was
collected and lyophilized to afford 5 mg (9%) of the product as a white
powder: 1H-NMR (400 MHz, DMSO-d6) δ 7.87 (br s, 2H), 7.62 (t, J
) 5 Hz, 1H), 7.30-7.10 (m, 10H), 6.34 (d, J ) 9 Hz, 1H), 6.19-6.03
(m, 10H), 5.70 (bs, 2H), 4.14 (m, 1H), 3.99 (m, 1H), 3.89 (m, 1H),
3.54 (m, 1H), 3.51-3.40 (m, 2H), 3.31-3.26 (m, 4H), 3.10-3.00 (m,
3H), 2.79-2.25 (m, 7H), 1.49-1.15 (m, 2H); FAB-HRMS for
C37H55N12O4 [MH] calcd m/z 731.4469, found m/z 731.4468.
CH GuCH FuCH FuCH AuA-NH2 (12). The crude peptide was pre-
2
2
2
2
pared using a protocol very similar to that used for the oligourea 9
described above, except that the quantities were different. Rink’s amide
resin (0.31 g of 0.62 mmol g-1 capacity) was used. The isocyanate
for each coupling step were prepared from 3 (0.58 mmol, 3 equiv),
triethylamine (0.65 mL, 24 equiv), and 0.29 mL of a 2 M phosgene
solution (0.58 mmol, 3 equiv) in 10 mL of CH2Cl2 at 0 °C. The mixture
used to cleave the peptidomimetic for the resin was phenol/1,2-
ethanedithiol/thioanisole/water/ TFA (3:1:2:2:40, w/v/v/v/v, respec-
tively); 12 mL of this was applied at 25 °C for 9 h. The crude peptide
was further purified using the same column and solvent system. The
gradient was programmed to increase from 20 to 80% B over 35 min
with a flow rate of 5 mL min-1. The peak with a retention time of
21.6 min was collected and lyophilized to afford 46.6 mg (46.3%) of
YGCH GuCH FuL-NH2 (10). The crude peptide was prepared using
2
2
1
a protocol very similar to that used for the oligourea 9 described above,
except that the quantities were different. Rink’s amide resin (0.46 g
of 0.53 mmol g-1 capacity) was used. The isocyanates were prepared
by mixing of 3 (0.73 mmol, 3 equiv), triethylamine (0.8 mL, 24 equiv),
and 2 M phosgene solution (0.37 mL, 0.73 mmol, 3.9 equiv) in CH2-
Cl2 (10 mL) at 0 °C. Cleavage of the peptide from the resin was
agitated in a mixture of phenol/1,2-ethanedithiol/thioanisole/water/TFA
(3:1:2:2:40, w/v/v/v/v, respectively, 12 mL) at 25 °C for 9 h. The
crude peptide was further purified by preparative RP-HPLC (Vydac
C18 column, 22 mm × 25 cm, 10 µm) with a linear gradient obtained
by mixing solvent A (0.1% TFA in water) and solvent B (0.1% TFA
in acetonitrile). The gradient was programmed to increase from 20 to
80% B over 35 min with a flow rate of 5 mL min-1. The peak with
a retention time of 20.1 min was collected and lyophilized to afford
25 mg (17%) of the product as a white hygroscopic solid: mp 125-
127 °C; 1H NMR (400 MHz, DMSO-d6): δ 9.37 (s, 1H), 8.71 (t, J )
6 Hz, 1H), 8.07 (br d, J ) 2 Hz, 1H), 7.96 (m, 1H), 7.28-7.24 (m,
3H), 7.19 (d, J ) 7 Hz, 3H), 7.04 (d, J ) 9 Hz, 2H), 6.91 (s, 1H), 6.70
(d, J ) 9 Hz, 2H), 6.12 (s, 1H), 6.05-6.01 (m, 2H), 5.95 (d, J ) 8 Hz,
1H), 4.05 (m, 1H), 3.97 (m, 1H), 3.79 (dd, J ) 6, 17 Hz, 1H), 3.73-
3.69 (m, 2H), 3.10-2.92 (m, 7H), 2.82 (dd, J ) 8, 14 Hz, 1H), 2.70
(dd, J ) 6, 14 Hz, 1H), 2.59 (dd, J ) 7, 14 Hz, 1H), 1.60-1.51 (m,
1H), 1.42-1.28 (m, 3H), 0.87 (d, J ) 4 Hz, 3H), 0.85 (d, J ) 4 Hz,
3H); 13C NMR (50 MHz, DMSO-d6): δ 165.37, 156.08, 155.99, 147.16,
143.90, 140.72, 137.59, 134.11, 132.19, 131.43, 129.81, 129.33, 127.56,
127.02, 125.35, 125.13, 120.09, 114.68, 66.30, 65.11, 46.74, 41.96,
the product as a white hygroscopic solid: mp 125-127 °C; H NMR
(400 MHz, DMSO-d6): δ 7.78 (br s, 3H), 7.40 (s, 1H), 7.29-7.23 (m,
4H), 7.20-7.16 (m, 6H), 7.00 (s, 1H), 6.28 (m, 2H), 6.27-6.08 (m,
4H), 6.04 (m, 2H), 4.09 (m, 1H), 3.93 (m, 1H), 3.84 (m, 1H), 3.58 (m,
1H), 3.44 (m, 2H), 3.28 (m, 2H), 3.07 (m, 2H), 2.89 (m, 1H), 2.67 (m,
4H), 2.54 (m, 3H), 1.19 (d, J ) 7 Hz, 3H), 0.91 (d, J ) 7 Hz, 3H); 13
C
NMR (50 MHz, DMSO-d6): δ 175.17, 159.07, 158.73, 158.39, 157.53,
138.77, 129.09, 128.17, 125.99, 50.54, 49.91, 48.49, 45.83, 45.25, 44.54,
44.32, 37.01, 19.76, 18.70; IR (KBr) 3450, 2921, 1644, 1567, 1562,
1505, 1498, 1458 cm-1; MALDI+ for C30H47N10O5 [MH] calcd m/z
626.37, found m/z 626.82, 649.20 [MNa], 664.99 [MK].
Syntheses of the Library of 160 Peptidomimetics (Figure 3).
Stepwise coupling of phthaloyl-protected isocyanates to Rink’s amide
resin was used as outlined above, except that the resin (16.6 mg of
0.62 mmol g-1 capacity) was packaged into approximately 1 cm2 “tea
bags” formed from a permeable membrane (McMaster-Carr, Atlanta,
GA: phone 404-346-7000). A 50 mL vessel was used and agitated
using a horizontal shaker (Eberbach 6000). All reactions were carried
out at 25 °C.
The following conditions were used for the formation of the urea
bonds. The isocyanate of each phthaloyl amino acid derivative was
prepared by mixing the appropriate amine (3 or 6) (3 equiv, relative to
the resin, throughout), triethylamine (24 equiv), and 2 M phosgene
solution (3.9 equiv) in 50 mL of CH2Cl2 at 0 °C. The Rink’s amide
resin in the tea bags was first swelled in DMF (ca. 50 mL) for 2 h,
then reacted with the isocyanate solution. The mixture was shaken
for 12 h, then washed (washing cycle as described in the preparation
of compound 9). The phthaloyl protecting group was removed using
a 12 h reaction time, and then the resin was washed again.
34.42; IR (KBr) 3380, 2934, 1671, 1559, 1517, 1455, 1438 cm-1
;
MALDI+ for C30H45N8O6 [MH] calcd m/z 613.3462, found m/z
613.3481.
CH YuCH GuCH GuCH FuCH Lu-NH2 (11). The crude peptide was
2
2
2
2
2
The conditions used for the formation of amide bonds were as
follows. FMOC amino acids were coupled using PyBOP/HOBt/NMM.
FMOC deprotection was performed by shaking the tea bags twice with
20% piperidine in DMF (50 mL, 30 min × 2). After removal of the
final protecting group, the resin was washed thoroughly with DMF.
Cleavage of the peptide from the resin (still in the tea bags) was
performed using individual vials containing a 1.5 mL of a mixture of
phenol/1,2-ethanedithiol/thioanisole/water/TFA (5:2.5:1:5:81.5, v/v/v/
v/v, respectively), agitated at 25 °C for 9 h. The solutions were
transferred to 2 mL centrifuge tubes; residual material in the bags was
collected and added to the centrifuge tubes by washing each bag with
0.5 mL of TFA. The solutions were evaporated to dryness on a vacuum
centrifuge. Purified water (1 mL) and acetonitrile (0.5 mL) was added
to each tube, and the resulting solutions were washed with Et2O (0.5
mL × 2) and lyophilized. The crude peptidomimetics were character-
ized by MALDI and HPLC.
prepared using a protocol very similar to that used for the oligourea 9
described above, except that the quantities were different. Rink’s amide
resin (0.161 g of 0.62 mmol g-1 capacity) was used. The isocyanates
for each coupling step were prepared by mixing 76 mg of compound
3 (0.6 mmol), triethylamine (0.67 mL, 4.8 mmol), and 0.39 mL of a 2
M phosgene solution (0.78 mmol) in 10 mL of CH2Cl2 at 0 °C. The
mixtures were stirred for 2 h, then used for the coupling reaction without
further purification. The mixture used to cleave the peptidomimetic
for the resin was phenol/1,2-ethanedithiol/thioanisole/water/TFA (5:
2.5:1:5:81.5, v/v/v/v/v, respectively, 5 mL); this was applied for 9 h at
25 °C. The crude peptide was further purified by preparative RP-HPLC
using the same column and solvent system programmed to increase
from 30 to 35% B over 25 min with a flow rate of 5 mL min-1. The
peak with a retention time of 19.3 min was collected and lyophilized
to afford 15 mg (21%) of the product as a white powder: 1H NMR
(500 MHz, DMSO-d6) δ 9.26 (br s, 1H), 7.80 (br s, 2H), 7.23-7.17

1564 J. Am. Chem. Soc., Vol. 119, No. 7, 1997
Burgess et al.
Preparation of Selected Compounds from the Library. Five
interesting peptidomimetics identified in the library screen, were
prepared again on a larger scale (0.1 mmol) using procedures very
similar to those already described for compounds 9-12. Throughout
the solvents A and B used in the HPLC purification were 0.1% TFA
in water and 0.1% TFA in acetonitrile, respectively. Only data for
characterization is given here.
Hz, 1H), 4.37 (m, 1H), 4.24 (m, 1H), 4.11 (t, J ) 6.1 Hz), 3.89 (d, J
) 6.1 Hz, 2H), 2.64 (m, 2H), 1.44 (s, 9H); 13C NMR (75 MHz, acetone-
d6) δ 169.40, 156.68, 145.26, 142.05, 135.10, 134.01, 128.50, 127.97,
126.14, 123.82, 120.77, 81.08, 67.13, 61.74, 48.76, 48.00, 39.16, 28.26;
IR (KBr) 3244, 3062, 3045, 2989, 1774, 1730, 1701, 1605, 1533, 1369
cm-1; [R]20D ) +11.33; FAB-HRMS for C31H30N2O6Na [MNa] calcd
m/z 549.2002, found m/z 549.2012.
tert-Butyl (S)-N4-Phthaloyl-3,4-diaminobutanate (16a). Diethyl-
amine (0.07 mL, 0.67 mmol, 10 equiv) was added to the solution of
0.036 g of compound 15a (0.068 mmol) in 1 mL of THF. The mixture
was stirred at 25 °C for 12 h, the THF was evaporated under vacuum,
and the residue was purified via flash chromatography (1:4 hexane:
EtOAc eluant) to afford 8 mg (39%) of the product as an oil. This
product can be used for the preparation of isocyanates without further
purification: 1H NMR (300 MHz, acetone-d6) δ 7.90-7.88 (m, 4H),
3.90 (m, 1H), 3.72 (dd, J ) 7, 14 Hz, 1H), 3.57 (dd, J ) 6, 14 Hz,
1H), 2.95 (br s, 2H), 2.28-1.89 (m, 2H), 1.44 (s, 9H); 13C NMR (75
MHz, acetone-d6) δ 172.97, 168.85, 135.14, 133.02, 123.82, 80.17,
58.07, 43.22, 32.50, 28.26; FAB+MS for C17H23N2O4 [MH] calcd m/z
305, found m/z 305.
YCH GuGFL-NH2. Gradient: 20 to 30% B over 47 min with a
2
flow rate of 5 mL min-1
. Retention time: 27.9 min. The product
was formed as a white powder (9 mg, 15%): 1H-NMR (500 MHz,
DMSO-d6) δ 9.36 (br s, 1H), 8.39 (m, 1H), 8.07 (br s, 2H), 8.06 (d, J
) 8 Hz, 1H), 8.01 (d, J ) 8 Hz, 1H), 7.26-7.16 (m, 5H), 7.10 (br s,
1H), 7.00 (d, J ) 8 Hz, 2H), 6.99 (br s, 1H), 6.71 (d, J ) 8 Hz, 2H),
6.25 (m, 1H), 6.19 (m, 1H), 4.49 (m, 1H), 4.19 (m, 1H), 3.79 (m, 1H),
3.65 (dd, J ) 5, 17 Hz, 1H), 3.49 (dd, J ) 5, 17 Hz, 1H), 3.14-2.91
(m, 6H), 2.82 (dd, J ) 8, 14 Hz, 1H), 2.78 (dd, J ) 10,14 Hz, 1H),
1.55 (m, 1H), 1.47 (m, 2H), 0.88 (d, J ) 7 Hz, 3H), 0.83 (d, J ) 7 Hz,
3H); IR (KBr) 3311, 3086, 2960, 1670, 1558, 1518, 1456 cm-1
;
MALDI+ for C29H42N7O6 [MH] calcd m/z 584.196, found m/z 584.388.
YCH GuGFl-NH2. Gradient: 22 to 29% B over 43 min with a flow
2
rate of 5 mL min-1
. Retention time: 34.8 min. The product was
(S)-N2-[(9-Fluorenylmethoxy)carbonyl]-N1-phthaloyl-N6-tert-bu-
tyloxycarbonyl-1,2,6-triaminohexane (15b). This compound was
prepared using 13a (2.87 g, 6.3 mmol) in essentially the same procedure
as outlined for the conversion of FMOC-Tyr(tBu)-OH into 5 (Vide
supra). The product 15b was purified via flash chromatography (3:2
hexane:EtOAc eluant) as a white solid (1.68g, 59%): mp 167-168
formed as a white powder (20 mg, 34%): 1H NMR (400 MHz, DMSO-
d6) δ 9.37 (br s, 1H), 8.41 (m, 1H), 8.18-8.05 (m, 4H), 7.27-7.16
(m, 6H), 7.01-6.98 (m, 3H), 6.71 (d, J ) 9 Hz, 2H), 6.28 (m, 1H),
6.17 (m, 1H), 4.51 (m, 1H), 4.11 (m, 1H), 3.78 (m, 1H), 3.63 (dd, J )
5, 17 Hz, 1H), 3.54 (dd, J ) 5, 17 Hz, 1H), 3.13-2.78 (m, 8H), 1.39
(m, 2H), 1.26 (m, 1H), 0.79(d, J ) 6 Hz, 3H), 0.73 (d, J ) 6 Hz, 3H);
IR (KBr) 3374, 3033, 2958, 1668, 1556, 1518, 1441 cm-1; MALDI+
for C29H42N7O6 [MH] calcd m/z 584.196, found m/z 584.554.
1
°C; H NMR (300 MHz, DMSO-d6) δ 7.89-7.24 (m, 12H), 6.78 (m,
1H), 4.14 (m, 1H), 4.03 (m, 2H), 3.78 (m, 1H), 3.62 (m, 2H), 2.90 (m,
2H), 1.45-1.14 (m, 6H), 1.37 (s, 9H); 13C NMR (75 MHz, DMSO-d6)
δ 167.96, 156.03, 155.56, 144.01, 140.59, 134.20, 131.61, 128.68,
127.56, 126.97, 125.30, 120.04, 77.32, 74.20, 65.70, 50.00, 46.65, 36.70,
YCH GuGFCH2Lu-NH2. Gradient: 22 to 28.5% B over 40 min with
2
a flow rate of 5 mL min-1. Retention time: 30.9 min. The product
was isolated as a white powder (9 mg, 15%): 1H NMR (400 MHz,
DMSO-d6) δ 9.37 (br s, 1H), 8.40 (t, J ) 5 Hz, 1H), 8.06-8.01 (m,
4H), 7.23-7.16 (m, 5H), 7.00 (d, J ) 8 Hz, 2H), 6.70 (d, J ) 8 Hz,
2H), 6.25 (t, J ) 6 Hz, 1H), 6.14 (t, J ) 6 Hz, 1H), 5.79 (d, J ) 8 Hz,
1H), 5.45 (br s, 1H), 4.43 (m, 1H), 3.78 (m, 1H), 3.71 (dd, J ) 5, 16
Hz, 1H), 3.63 (m, 1H), 3.50 (dd, J ) 5, 16 Hz, 1H), 3.12-2.91 (m,
8H), 2.82 (dd, J ) 8, 14 Hz, 1H), 2.78 (dd, J ) 10, 14 Hz, 1H), 1.59
(m, 1H), 1.15 (m, 2H), 0.86 (d, J ) 7 Hz, 3H), 0.81 (d, J ) 7 Hz, 3H);
IR (KBr) 3374, 3093, 2958, 1670, 1558, 1518, 1438 cm-1; MALDI+
for C30H45N8O6 [MH] calcd m/z 613.743, found m/z 613.791.
31.04, 29.24, 28.26, 22.95; [R]20 ) +21.03; IR (KBr) 3350, 3329,
D
3049, 2978, 2943, 1776, 1709, 1689, 1529, 1446, 1438, 1400 cm-1
;
FAB-HRMS for C34H37N3O6Na [MNa] calcd m/z 606.2580, found m/z
606.2592.
N1-Phthaloyl-N6-tert-butyloxycarbonyl-1,2,6-triaminohexane (16b).
Diethylamine (0.32 mL, 3.1 mmol, 10 equiv) was added to the solution
of 0.20 g of compound 16a (0.34 mmol) in 3 mL of THF. The mixture
was stirred at 25 °C for 12 h, the THF was evaporated under vacuum,
and the residue was purified via flash chromatography (1:4 hexane:
EtOAc eluant) to afford 0.07 g (58%) of the product as an oil. This
product can be used for the preparation of isocyanates without further
purification: 1H NMR (300 MHz, acetone-d6) δ 7.90-7.88 (m, 9H),
5.97 (br s., 1H), 3.80 (m, 1H), 3.02 (m, 2H), 1.70-1.14 (m, 6H), 1.41
(s, 9H); 13C NMR (75 MHz, acetone-d6) δ 168.85. 156.66, 135.04,
133.01, 123.75, 78.28, 69.46,58.95, 40.96, 34.46, 31.89. 28.70, 24.13;
IR (neat) 3384, 2977, 2933, 2869, 2559, 2046, 1776, 1712, 1525, 1427,
1400 cm-1; FAB+MS for C19H28N3O4 [MH] calcd m/z 362, found m/z
362.
YGCH GuFL-NH2. Gradient: 20 to 40% B over 45 min with a
2
flow rate of 5 mL min-1
. Retention time: 29.1 min. The product
was isolated as a white powder (7 mg, 12%): 1H NMR (500 MHz,
DMSO-d6) δ 9.34 (br s, 1H), 8.17 (t, J ) 6 Hz, 1H), 8.00 (br s, 2H),
7.92 (d, J ) 8 Hz, 1H), 7.90 (d, J ) 4 Hz, 1H), 7.25-7.17 (m, 5H),
7.05 (d, J ) 9 Hz, 2H), 6.98 (br s, J ) 9 Hz, 2H), 6.69 (d, J ) 9 Hz,
2H), 6.20 (m, 1H), 6.10 (d, J ) 8 Hz, 1H), 4.35 (m, 1H), 4.21 (m,
1H), 3.96 (m, 1H), 3.89-3.62 (m, 2H), 3.02-2.93 (m, 6H), 2.79 (dd,
J ) 9, 14 Hz, 1H), 2.75 (dd, J ) 9, 14 Hz, 1H), 1.54 (m, 1H), 1.44
(m, 2H), 0.86 (d, J ) 7 Hz, 3H), 0.82 (d, J ) 7 Hz, 3H); IR (KBr)
3390, 3087, 2960, 1670, 1558, 1541, 1518, 1437 cm-1; MALDI+ for
C29H42N7O6 [MH] calcd m/z 584.196, found m/z 584.418.
Acknowledgment. We thank The National Institutes of
Health and The Robert A. Welch Foundation for direct support
for this research, K.B. thanks the NIH for a Research Career
Development Award and the Alfred P. Sloan Foundation for a
fellowship. NMR facilities were provided by the NSF via the
chemical instrumentation program. We also thank Chun-Yen
Ke, Carlos Martinez, and Destardi Moye for assistance with
the 2D NMR experiments. The MALDI mass spectrometry
research (financial support for A.J.Z.) is funded by the U.S.
Department of Energy, Division of Chemical Sciences, Office
of Basic Energy Sciences.
YCH GuFL-NH2. Gradient: 20 to 32% B over 40 min with a flow
2
rate of 5 mL min-1
. Retention time: 33.0 min. The product was
isolated as a white powder (6.4 mg, 20%): 1H NMR (400 MHz, DMSO-
d6) δ 9.48 (br s, 1H), 8.48 (br s, 1H), 8.16 (br s, 2H), 8.06 (br d, J )
9 Hz, 1H), 7.36-7.28 (m, 7H), 7.11 (d, J ) 9 Hz, 2H), 6.82 (d, J )
9 Hz, 2H), 6.31 (m, 1H), 6.24 (d, J ) 8 Hz, 1H), 4.48 (m, 1H), 4.33
(q, J ) 8 Hz, 1H), 3.84 (t, J ) 7 Hz, 1H), 3.62-3.56 (m, 1H), 3.53-
3.49 (m, 1H), 3.21-3.00 (m, 4H), 2.94-2.83 (m, 2H), 1.66 (m, 1H),
1.56 (m, 1H), 0.98 (d, J ) 6 Hz, 3H), 0.94 (d, J ) 6 Hz, 3H); IR
(KBr) 3406, 3089, 2958, 1670, 1558, 1518, 1439 cm-1; FAB-HRMS
for C27H39N6O5 [MH] calcd m/z 527.298, found m/z 527.297.
tert-Butyl (S)-N3-[(9-Fluorenylmethoxy)carbonyl]-N4-phthaloyl-
3,4-diaminobutanate (15a). This compound was prepared using 13a
(0.21 g, 0.53 mmol) in essentially the same procedure as outlined for
the conversion of FMOC-Tyr(tBu)-OH into 5 (Vide supra). The product
15a was purified via flash chromatography (3.3:1 hexane:EtOAc eluant)
as a white solid (0.20 g, 72%): mp 156-157.5 °C; H NMR (300
MHz, acetone-d6) δ 7.86-7.80 (m, 4H), 7.66 (d, J ) 7.2 Hz, 2H),
7.60 (d, J ) 7.5 Hz, 2H), 7.42 (m, 2H), 7.31 (m, 2H), 6.56 (d, J ) 9
Supporting Information Available: One- and two-dimen-
sional NMR data for a selection of the peptidomimetics prepared
in this work and an HPLC trace for the crude sample of
CH Yu‚CH Gu‚CH Gu‚CH Fu‚CH Lu-amide before HPLC purifica-
tion (19 pages). See any current masthead page for ordering
and Internet instructions.
2
2
2
2
2
1
JA9631256