Solid-phase synthesis of artificial β-sheets

Article abstract of DOI:10.1021/ja9710971

This paper describes the solid-phase syntheses of artificial β-sheets 1-4, which mimic the structure and hydrogen-bonding patterns of protein β-sheets. In these compounds, molecular templates induce β-sheet structures in attached peptide strands. The temp

Full text of DOI:10.1021/ja9710971

J. Am. Chem. Soc. 1997, 119, 7665-7669
7665
Solid-Phase Synthesis of Artificial â-Sheets
Darren L. Holmes, Eric M. Smith, and James S. Nowick*
Contribution from the Department of Chemistry, UniVersity of California, IrVine,
IrVine, California 92697-2025
ReceiVed April 7, 1997X
Abstract: This paper describes the solid-phase syntheses of artificial â-sheets 1-4, which mimic the structure and
hydrogen-bonding patterns of protein â-sheets. In these compounds, molecular templates induce â-sheet structures
in attached peptide strands. The templates consist of di- and triurea derivatives, which hold peptide and peptidomimetic
strands in proximity, and â-strand mimics, which hydrogen bond to the peptide strands. The syntheses involve
constructing the “lower” peptide strand on Merrifield resin, attaching the di- or triamine portions of the di- or triurea
templates, connecting the “upper” peptide and peptidomimetic strands, and cleaving the resulting artificial â-sheets
from the resin. The artificial â-sheets were prepared in 8-13 steps from leucine Merrifield in 33-67% overall
yield.
Compounds that mimic the structure and hydrogen-bonding
patterns of protein â-sheets are of interest as drug candidates
and as model systems with which to study protein structure and
stability.1 We have termed these compounds artificial â-sheets,
and we are synthesizing and studying artificial â-sheets of
increasing size and complexity with the goals of learning about
â-sheet structure and developing biologically active peptido-
mimetic compounds. Our artificial â-sheets consist of an
oligourea molecular scaffold and attached peptide and pepti-
domimetic strands. The molecular scaffold holds the strands
in proximity, promoting the formation of a hydrogen-bonded
â-sheet structure. Some of the structures incorporate a rigid
â-strand mimic, which hydrogen bonds to the adjacent peptide
strands and further stabilizes the â-sheet structure. The fol-
lowing cartoons illustrate these structures.
We recently reported solution-phase syntheses and structural
studies of artificial â-sheets 1-3.1b,2 In artificial â-sheet 1, a
diurea template links two peptide strands and induces an
intramolecularly hydrogen-bonded parallel â-sheet conformation.2a
Artificial â-sheet 2 mimics an antiparallel â-sheet structure and
incorporates both a diurea template and a â-strand mimic.2b In
artificial â-sheet 3, the â-strand mimic and peptide strands are
report efficient procedures for the solid-phase syntheses of these
compounds using Merrifield resin.
elongated.2c Artificial â-sheet 4 contains a triurea template, a
â-strand mimic, and two peptide strands.3 In this paper, we
X Abstract published in AdVance ACS Abstracts, August 1, 1997.
(1) (a) Schneider, J. P.; Kelly, J. W. Chem. ReV. 1995, 95, 2169. (b)
Nowick, J. S.; Smith, E. M.; Pairish, M. Chem. Soc. ReV. 1996, 25, 401.
(c) Service, R. F. Science 1996, 274, 34.
(2) (a) Nowick, J. S.; Smith, E. M.; Noronha, G. J. Org. Chem. 1995,
60, 7386. (b) Nowick, J. S.; Holmes, D. L.; Mackin, G.; Noronha, G.;
Shaka, A. J.; Smith, E. M. J. Am. Chem. Soc. 1996, 118, 2764. (c) Nowick,
J. S.; Pairish, M.; Lee, I. Q.; Holmes, D. L.; Ziller, J. W. J. Am. Chem.
Soc. 1997, 119, 5413.
Results
Artificial â-sheets 1-4 were synthesized by constructing the
“lower” peptide strand on Merrifield resin, attaching the di- or
(3) Structural studies of artificial â-sheet 4 will be described in a
forthcoming paper. Studies of a closely related artificial â-sheet comprising
a triurea template, a â-strand mimic, and two peptide strands are described
in ref 1b.
S0002-7863(97)01097-4 CCC: $14.00 © 1997 American Chemical Society

7666 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
Holmes et al.
triamine portions of the di- or triurea templates, connecting the
“upper” peptide and peptidomimetic strands, and cleaving the
resulting artificial â-sheets from the resin. The compounds were
prepared in 0.4-0.6 g batches starting with 1.5 g (1.2 mmol)
of tert-butoxycarbonyl (Boc)-leucine Merrifield resin. The
syntheses involve 8-13 steps and proceeded in 33-67% overall
yield.
Scheme 1
Artificial â-Sheet 1. Artificial â-sheet 1 was prepared by
constructing the lower dipeptide on the solid support, adding
the diamine portion of the diurea template as carbamoyl chloride
7, and connecting the upper dipeptide strand as the correspond-
ing peptide isocyanate. Carbamoyl chloride 7 was prepared
from N-phenylethylenediamine (5), as shown in eq 1. Treatment
of N-phenylethylenediamine with 1.0 equiv of di-tert-butyl
dicarbonate afforded Boc-protected amine 6. Amine 6 was
converted to carbamoyl chloride 7 using modified Schotten-
Baumann conditions.4 These conditions involve addition of a
solution of phosgene in toluene to a solution of amine 6 in a
stirred, ice-cooled, biphasic mixture of methylene chloride and
saturated aqueous sodium bicarbonate solution. This procedure
is exceptionally convenient; the carbamoyl chloride is generated
in near quantitative yield and good purity and is used without
further purification. The top peptide strand was introduced as
a peptide isocyanate, valylalanine methyl ester isocyanate.
12-15 h. Under these reaction conditions, the Michael addition
proceeds to completion cleanly, with little addition of a second
equivalent of acrylonitrile.6 Reaction of amine 11 with valyl-
alanine methyl ester isocyanate, followed by aminolysis with
methylamine, affords artificial â-sheet 1. The aminolysis step
cleaves both the alanine methyl ester group and the leucine
linkage to the resin, generating both methyl amide groups of 1.
Chromatographic purification afforded artificial â-sheet 1 in
67% overall yield.
Artificial â-Sheet 2. Artificial â-sheet 2 contains a â-strand
mimic in place of the valylalanine peptide strand that is present
in 1 and was prepared in an analogous fashion. The â-strand
mimic was introduced as isocyanate 15, which was prepared
from 5-nitro-2-methoxybenzoic acid2c,7 (12), as shown in eq 2.
Peptide isocyanates are readily prepared by treatment of peptide
hydrochloride salts with a solution of phosgene in toluene, using
similar modified Schotten-Baumann conditions.4 Peptide iso-
cyanates react cleanly and in high yield with amines to form
ureas, making these isocyanates particularly attractive building
blocks for the construction of peptide derivatives and peptido-
mimetic compounds.4
Scheme 1 illustrates the synthesis of artificial â-sheet 1. Boc-
leucine Merrifield resin (8) was homologated to the correspond-
ing Boc-phenylalanylleucine dipeptide (9) by standard solid-
phase peptide synthesis techniques.5 The Boc protective group
was removed by treatment with trifluoroacetic acid (TFA), the
free amino group was then liberated by treatment with triethyl-
amine (TEA), and the amino group was coupled with carbamoyl
chloride 7 in the presence of TEA. Because carbamoyl chlorides
are less reactive than most activated carboxylic acid derivatives,
the reaction of carbamoyl chloride 7 with resin-bound peptides
requires prolonged (e.g., 20 h) reaction times at ambient
temperature or heating (e.g., 50 °C for 2 h). The resulting
intermediate (10) was treated with TFA to remove the Boc
protective group and TEA to liberate the amino group. Michael
addition of the amino group to acrylonitrile proceeds smoothly
to afford intermediate 11 when a 3:1:1 mixture of tetrahydro-
furan (THF), methanol, and acrylonitrile is used as solvent. This
solvent swells the resin and gives complete reaction within ca.
(4) Nowick, J. S.; Holmes, D. L.; Noronha, G.; Smith, E. M.; Nguyen,
T. M.; Huang, S.-L. J. Org. Chem. 1996, 61, 3929.
(5) Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis, 2nd ed.;
Pierce Chemical Company: Rockford, IL, 1984.

Solid-Phase Synthesis of Artificial â-Sheets
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7667
Scheme 2
Scheme 3
Carboxylic acid 12 was converted to the corresponding methyl-
amide (13) by successive treatment with thionyl chloride and
methylamine. Reduction of the nitro group of 13 afforded
N-methyl-5-amino-2-methoxybenzamide, which darkened rap-
idly in air and was stored and handled as its amine hydrochloride
salt (14). Amine hydrochloride 14 was converted to the
corresponding isocyanate 15 using modified Schotten-Baumann
conditions; chloroform was used as a solvent, instead of
methylene chloride, to minimize the formation of emulsions.
Treatment of resin-bound amine 11 with isocyanate 15,
followed by aminolysis with methylamine, generated artificial
â-sheet 2 (Scheme 2). Chromatography of the crude aminolysis
product afforded pure 2 in 63% yield from Boc-leucine
Merrifield resin.
Artificial â-Sheet 3. Artificial â-sheet 3 comprises a peptide
strand and a â-strand mimic attached to a diurea molecular
scaffold. The peptide strand and â-strand mimic of 3 are longer
than those of 2: the peptide is a tripeptide instead of a dipeptide,
and the â-strand mimic is composed of two aromatic rings rather
than one. The synthesis of 3 is similar to that of 2, with the
exception that the â-strand mimic is assembled from two halves
on the solid support. The first half is introduced as isocyanate
18, which was prepared as shown in eq 3. Carboxylic acid 12
converted to the Boc-phenylalanylisoleucylleucine tripeptide 19
by standard solid-phase techniques. The Boc group was
removed by treatment with TFA, the free amino group was then
liberated by treatment with TEA, and the amino group was
coupled with carbamoyl chloride 7 to form intermediate 20. This
intermediate was converted to cyanoethylamine 21 by removal
of the Boc group with TFA, liberation of the free amino group
with TEA, and Michael addition to acrylonitrile. The first half
of the â-strand mimic was then introduced by reaction of the
amino group of 21 with isocyanate 18. Removal of the tert-
butyl ester protective group with TFA and treatment with
isobutyl chloroformate afforded mixed anhydride 22. The
second half of the â-strand mimic was introduced by coupling
mixed anhydride 22 with hydrazine 23.2c Artificial â-sheet 3
was liberated from the resin by aminolysis with methylamine.
Column chromatography, followed by preparative reverse-phase
HPLC, afforded pure 3 in 33% overall yield.
was converted to tert-butyl ester 16 by successive treatment
with thionyl chloride and potassium tert-butoxide. Reduction
of the nitro group generated amine 17, which was converted to
the isocyanate using modified Schotten-Baumann conditions.
The isocyanate was used in the preparation of artificial â-sheet
3, as shown in Scheme 3. Boc-leucine Merrifield resin (8) was
(6) Nowick, J. S.; Mahrus, S.; Smith, E. M.; Ziller, J. W. J. Am. Chem.
Soc. 1996, 118, 1066.
(7) de Paulis, T.; Janowsky, A.; Kessler, R. M.; Clanton, J. A.; Smith,
H. E. J. Med. Chem. 1988, 31, 2027.

7668 J. Am. Chem. Soc., Vol. 119, No. 33, 1997
Holmes et al.
Scheme 4
27 was prepared from diamine 24,6 as shown in eq 4. Diamine
24 was converted to Nps-protected monoamine 26 by treatment
with N-(2-nitrophenylsufenyl)saccharin.9 The reaction proceeds
at the “middle” nitrogen with high yield and selectivity, because
this nitrogen is aliphatic and is thus much more basic and
nucleophilic than the “lower” nitrogen, which is aromatic.
Monoamine 26 was then converted to carbamoyl chloride 27
using the modified Schotten-Baumann conditions. This com-
pound is not stable and is used immediately after preparation,
without further purification.
To evaluate carbamoyl chloride 27 as building block for the
triurea molecular scaffold, we used it to prepare triurea 32, a
compound that we had previously prepared by solution-phase
techniques.6 Triurea 32 was prepared on Merrifield resin, as
shown in Scheme 4. Boc-phenylalanine Merrifield resin (28)
was deprotected by treatment with TFA and TEA and was
coupled with carbamoyl chloride 27 to give monourea 29.
Removal of the Nps protective group by treatment with
ammonium thiocyanate and 2-methylindole,10 followed by
treatment with valine methyl ester isocyanate,4,11 generated
diurea 30. The Boc protective group was then removed by
treatment with TFA, and the free amine was liberated by
treatment with TEA and subjected to acrylonitrile to form
cyanoethylamine 31. The third urea group was introduced by
treatment with isoleucine methyl ester isocyanate,4,11 and the
triurea was liberated from the resin by transesterification with
methanol and triethylamine in dimethylformamide (DMF).12
Column chromatography, followed by preparative reverse-phase
HPLC, afforded pure triurea 32 in 52% overall yield.
Artificial â-Sheet 4. Artificial â-sheet 4 was prepared on
Merrifield resin using carbamoyl chloride 27 to form the triurea
molecular scaffold and isocyanate 35 to introduce the â-strand
mimic. Isocyanate 35 was prepared from 5-nitro-2-methoxy-
benzoic acid (12), as shown in eq 5. Acid 12 was converted to
the corresponding acid chloride by treatment with thionyl
chloride and then coupled with isobutyric hydrazide to form
intermediate 33. Reduction of the nitro group to form amine
34, followed by treatment with phosgene using modified
Schotten-Baumann conditions, afforded the isocyanate.
Triurea 32. In the solid-phase syntheses of artificial â-sheets
1-3, the diurea molecular scaffold is introduced as a derivative
of ethylenediamine (7), in which one of the amino groups is
protected as a Boc carbamate and the other is activated as a
carbamoyl chloride. Because artificial â-sheet 4 is based upon
a triurea molecular scaffold, a means of constructing the triurea
molecular scaffold was required. We initially investigated a
variety of SN2 alkylation and reductive alkylation strategies for
constructing the requisite diethylenetriamine portion of the
molecular scaffold on the solid support. These alkylation
reactions proved insufficiently clean to allow an efficient solid-
phase synthesis.
(9) Romani, S.; Bovermann, G.; Moroder, L.; Wu¨nsch, E. Synthesis 1985,
512.
(10) (a) Wu¨nsch, E.; Spangenberg, R. Chem. Ber. 1972, 105, 740. (b)
Lu¨scher, I. F.; Schneider, C. H. HelV. Chim. Acta 1983, 66, 602.
(11) Nowick, J. S.; Powell, N. A.; Nguyen, T. M.; Noronha, G. J. Org.
Chem. 1992, 57, 7364.
For this reason, we ultimately settled upon introduction of
the triurea molecular scaffold as a derivative of diethylenetri-
amine (27), in which two of the amino groups are protected
with Boc and o-nitrophenylsufenyl8 (Nps) groups. Derivative
(8) Zervas, L.; Borovas, D.; Gazis, E. J. Am. Chem. Soc. 1963, 85, 3660.
(12) Reference 5, p 91.

Solid-Phase Synthesis of Artificial â-Sheets
J. Am. Chem. Soc., Vol. 119, No. 33, 1997 7669
Scheme 5
Artificial â-sheet 4 was prepared as shown in Scheme 5. The
Boc-phenylalanylleucine dipeptide resin (9, from Scheme 1) was
deprotected and coupled with carbamoyl chloride 27 to afford
monourea 36. The Nps group was removed,10 and the liberated
amino group was coupled with valylalanine methyl ester
isocyanate4 to form diurea 37. Removal of the Boc protective
group and Michael addition of the primary amino group to
acrylonitrile afforded cyanoethylamine 38. Reaction with
isocyanate 35, followed by aminolysis of with methylamine,
generated artificial â-sheet 4. Column chromatography, fol-
lowed by preparative reverse-phase HPLC, afforded pure
artificial â-sheet 4 in 40% overall yield.
Discussion and Conclusions
The solid-phase syntheses of artificial â-sheets 1-4 and
triurea 32 are efficient, allowing the assembly of each of these
complex molecules in a few days to a week. These syntheses
encompass a variety of solid-phase synthetic reactions, including
peptide coupling, the reaction of carbamoyl chlorides and amines
to form ureas, the reaction of isocyanates and amines to form
ureas, Michael addition, the removal of three different types of
protective groups, and aminolysis and transesterification reac-
tions. Although the syntheses require 8-13 steps, they can be
performed rapidly because excess reagents are used to drive
the reactions to completion, byproducts and excess reagents are
washed away, and purification of resin-bound intermediates is
not required. The yields are good, averaging 91-95% per step.
We have also synthesized all of these compounds by solution-
phase techniques.2,6,13 The solution-phase syntheses involve
forming the same bonds as the solid-phase syntheses, albeit in
a slightly more convergent fashion. The overall yields of the
two types of syntheses are comparable; the solution-phase
syntheses give slightly lower average yields per step, but require
fewer steps in the longest linear sequence. The main advantage
of the solid-phase technique is convenience. Because interme-
diates need not be isolated, purified, and characterized, it is
possible to carry out reactions more rapidly. For this reason,
solid-phase synthesis is now our preferred method for the
preparation of artificial â-sheets. We have recently applied these
methods to the multiple parallel solid-phase synthesis of a library
of 16 artificial â-sheets, which we are using to study the â-sheet-
forming propensities of different amino acids. The solid-phase
methods have facilitated this endeavor, and we will publish this
study in a forthcoming paper.
Group, the Upjohn Company, and Hoffman-La Roche Inc.
D.L.H. and E.M.S. thank the National Institute of Aging for
support in the form of a training grant (National Research
Service Award AG00096-12). J.S.N. thanks the following
agencies for support in the form of awards: the Camille and
Henry Dreyfus Foundation (New Faculty Award, Teacher-
Scholar Award), the National Science Foundation (Young
Investigator Award, Presidential Faculty Fellowship), the Arnold
and Mabel Beckman Foundation (Young Investigator Award),
and the Alfred P. Sloan Foundation (Alfred P. Sloan Research
Fellowship).
Supporting Information Available: Experimental proce-
1
dures; H NMR spectra and HPLC traces of artificial â-sheets
1-4 (24 pages). See any current masthead page for ordering
and Internet access instructions.
Acknowledgment. This work was supported by the National
Institutes of Health Grant GM-49076, Zeneca Pharmaceuticals
(13) Nowick, J.; Smith, E. M. Unpublished results.
JA9710971