Dipeptide surrogates containing asparagine-derived tetrahydropyrimidinones: Preparation, structure, and use in solid phase synthesis

Full text of DOI:10.1021/ja9639271

J. Am. Chem. Soc. 1997, 119, 4305-4306
4305
wide range of amino acid side chains (Table 1), including
D-amino acids. Proline can also be employed in this reaction,
although in more modest yield.11
Dipeptide Surrogates Containing
Asparagine-Derived Tetrahydropyrimidinones:
Preparation, Structure, and Use in Solid Phase
Synthesis
Compounds 5 are sufficiently (and orthogonally) protected
to function in peptide synthesis schemes. Standard treatment
with trifluoroacetic acid (TFA) affords corresponding free acids
6 in excellent yield (Table 1). Isolation of free amines 7 after
selective removal of the Fmoc group proved somewhat more
difficult. Standard treatment with piperidine in DMF results
in complete deprotection. Unfortunately, the product amines
exhibit enough water solubility to make isolation of pure
compounds difficult. Success was achieved with certain of the
derivatives through use of Me2NH as deprotection agent. Under
these conditions, excess base is removed by evaporation to leave
the desired material contaminated with fluorene residue, which
is easily removed by filtration. However, both 5b and 5d
afforded variable amounts of a largely insoluble material, which
has yet to be identified, upon attempted isolation of the desired
free amine. While direct acetamide formation proved advan-
tagous in most cases, the yield of 7b could not be brought to
an acceptable level. Fortunately, these problems were not
evident in a solid phase peptide synthesis (SPPS) deprotection
sequence. For example, compound 6b was attached to a solid
support and deprotected with piperidine/DMF under standard
conditions. Acetamide formation and isolation of the product
after cleavage from the resin occurs in 77% yield, as opposed
to 40% for the solution phase synthesis. Finally, the aminal
functionality can be opened with dilute aqueous acid, which
also liberates the free asparagine carboxylic acid functionality
to afford 8. Each of the compounds 8a-g is identical with the
dipeptide formed by conventional methods.
The structure obtained from a single crystal X-ray analysis
of compound 6b is depicted in Figure 112 and has similarity to
related compounds studied both in this laboratory13 and oth-
ers.14,15 In particular, the peptide linkage is maintained in the
equatorial plane to allow maximum amide resonance. This
comes at the expense of the C2 aryl group, which is stationed
as a flagpole substituent on the six-membered ring in a boat
conformation. The two flagpole bonds are nearly parallel, with
a dihedral angle of only 1.7°, and these substituents are held in
close proximity (2.71 Å). The key torsion angle around the
amino acid residue (C(Ala)-N1-C6-CO2H) is 73.2°, some-
what larger than that found in proline (approximately 65°).
Intermolecular hydrogen bonds between the free asparagine acid
functionality and the adjacent lactam system afford linear arrays
of molecules in the solid state. Adjacent linear sequences are
held together by edge-to-face π-stacking forces between the
fluorene fragments of the Fmoc protection group (see figures
in Supporting Information).
Joseph P. Konopelski,*,1a Lubov K. Filonova,1a and
Marilyn M. Olmstead1b,2
Department of Chemistry and Biochemistry
UniVersity of California, Santa Cruz, California 95064
Department of Chemistry, UniVersity of California
DaVis, California 95616
ReceiVed NoVember 13, 1996
The amino acid asparagine is the focal point for varied studies
of polypeptide and protein structure and function. Asparagine
acts as an efficient C-terminal R-helix cap3 and serves as the
linkage point for the oligosaccharide unit of N-linked glyco-
peptides.4 These ubiquitous cell-surface biomolecules function
as recognition elements in cell-cell interactions and are
implicated in many biological functions and disease states.
However, asparagine-containing polypeptides and proteins
require special considerations upon attempted laboratory syn-
thesis. Use of the amino acid with an unprotected side-chain
amide can lead, by dehydration, to nitrile and/or succinimide
derivatives and, even if the synthesis is successful, general
solubility difficulties.5 An array of protective groups for the
primary amide, including trityl and others5 and, more recently,
dimethylcyclopropylmethyl,6 have been promoted to alleviate,
or at least reduce, these difficulties.
Herein we report our initial results on a novel class of
protected asparagine building blocks. On one level, these cyclic
surrogates function as protected, organic-soluble asparagine
residues for peptide synthesis. However, as opposed to more
conventional protective groups, the tetrahydropyrimidines em-
ployed herein are of known absolute configuration and confor-
mation. Such topological certainty allows for their incorporation
into rationally designed polypeptides as stereochemically defined
peptidomimetics.7 Finally, the heterocycle is easily transformed
to the natural amino acid within the polypeptide framework.8
This allows for both the direct comparison between the
conformationally restricted and native polypeptide and the
observation of the changes that occur in the process.
Imine 3 is obtained in nearly quantitative yield from 4-chloro-
3-nitrobenzaldehyde (4) and commercially available asparagine
tert-butyl ester, in accord with the results of Seebach and co-
workers.9 Although no desired product came from the use of
more conventional amino acid activated carboxyl residues
(including p-nitrophenyl ester, anhydride, acyl fluoride, acyl
imidazolide), treatment of 3 with Fmoc-protected amino acid
chlorides10 in anhydrous benzene with pyridine as base led to
desired heterocycle 5. Yields of 5 vary from 58-66% for a
(11) The Pro-Asn dipeptide residue is found in a number of important
biomolecules, including the circumsporozoite surface protein of the malaria
parasite Plasmidium falciparum. See: (a) Bisang, C.; Weber, C.; Inglis, J.;
Schiffer, C. A.; van Gunsteren, W. F.; Jelesarov, I.; Bosshard, H. R.;
Robinson, J. A. J. Am. Chem. Soc. 1995, 117, 7904-15.
(12) Crystals of 6b are orthorhombic, a ) 7.654(2), b ) 12.190(3), c )
32.529(6) Å, space group P212121, Z ) 4, r ) 1.337 g/cm3 for C29H27-
ClN4O9. A total of 2934 independent reflections were measured with nickel-
monochromated Cu KR radiation at 130(2) K on a Siemens P4 diffracto-
meter in the θ range of 2.72 to 56.06°. The structure was solved by using
direct methods and refined to a final R value of 4.01%. The primary program
used was SHELXTL, version 5.03, 1994, by G.M. Sheldrick.
(13) (a) Konopelski, J. P.; Chu, K. S.; Negrete, G. R. J. Org. Chem.
1991, 56, 1355-6. (b) Chu, K. S.; Negrete, G. R.; Konopelski, J. P.; Lakner,
F. J.; Woo, N.-T.; Olmstead, M. M. J. Am. Chem. Soc. 1992, 114, 1800-
1812.
(1) (a) University of California, Santa Cruz. (b) University of California,
Davis.
(2) Author to whom correspondence concerning X-ray crystallography
should be addressed.
(3) Zhou, H. X.; Lyu, P. C.; Wemmer, D. E.; Kallenbach, N. R. J. Am.
Chem. Soc. 1994, 116, 1139-40.
(4) Dwek, R. A. Chem. ReV. 1996, 96, 683-720.
(5) Sieber, P.; Riniker, B. Tetrahedron Lett. 1991, 32, 739-42.
(6) Carpino, L. A.; Chao, H.-G.; Ghassemi, S.; Mansour, E. M. E.;
Riemer, C.; Warrass, R.; Sadat-Aalaee, D.; Truran, G. A.; Imazumi, H.;
El-Faham, A.; Imazumi, H.; Ismail, M.; Kowaleski, T. L.; Han, C. H.;
Wenschuh, H.; Beyermann, M.; Bienert, M.; Shroff, H.; Albericio, F.; Triolo,
S. A.; Sole, N. A.; Kates, S. A. J. Org. Chem. 1995, 60, 7718-9.
(7) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699-1720.
(8) Pseudo-prolines of serine, threonine (oxazolidine), and cysteine
(thiazolidine) have been described as reversible protecting groups. See:
Wohr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.; Mutter,
M. J. Am. Chem. Soc. 1996, 118, 9218-27.
(14) Seebach, D.; Lamatsch, B.; Amstutz, R.; Beck, A. K.; Dobler, M.;
Egli, M.; Fitzi, R.; Gautschi, M.; Herrado´n, B.; Hidber, P. C.; Irwin, J. J.;
Locher, R.; Maestro, M.; Maetzke, T.; Mourin˜o, A.; Pfammatter, E.; Plattner,
D. A.; Schickli, C.; Schweizer, W. B.; Seiler, P.; Stucky, G.; petter, W.;
Escalant, J.; Juaristi, E.; Quintana, D.; Miravitlles, C.; Molins, E. HelV.
Chim. Acta 1992, 75, 913-34.
(9) Juaristi, E.; Quintana, D.; Lamatsch, B.; Seebach, D. J. Org. Chem.
1991, 56, 2553-7.
(10) Review: Carpino, L. A. Acc. Chem. Res. 1996, 29, 268-74.
(15) Beaulieu, F.; Arora, J.; Veith, U.; Taylor, N. J.; Chapell, B. J.;
Snieckus, V. J. Am. Chem. Soc. 1996, 118, 8727-8.
S0002-7863(96)03927-3 CCC: $14.00 © 1997 American Chemical Society

4306 J. Am. Chem. Soc., Vol. 119, No. 18, 1997
Communications to the Editor
Scheme 1a
Scheme 2
a (a) 4, (MeO)3CH, 98%; (b) Fmoc-Xxx-Cl, C5H5N, 58-66%.
Table 1. Yields of Free Acid 6, Amine 7, and Native Dipeptide 8
from 5
Scheme 3
1 Prepared by treatment with Me2NH; isolated as the corresponding
acetamide by direct treatment with Ac2O. 2 Prepared by binding cor-
responding acid 6 to NovaSyn PR-500 resin and treating with piperidine/
DMF. Cleavage from resin results in carboxamide, isolated as the
corresponding acetamide derivative by direct treatment with Ac2O.
a (a) Diisopropylcarbodiimide, HOBt; (b) 30% piperidine, DMF; (c)
Fmoc-Xxx-OH, HATU, DIEA, DMF; (d) 95% TFA; (e) 0.25 M HCl,
THF.
conventional manner in all respects. The solubility of 12 in
organic solvents is poor.
These dipeptide surrogates offer attractive scaffolds from
which to explore issues of polypeptide synthesis and structure.
Incorporation of these building blocks into model polypeptides
and the elaboration of their influence on local conformations
are the subject of ongoing studies in these laboratories and will
be reported in due course.
Acknowledgment. This paper is dedicated to Professor Amos B.
Smith, III, recipient of the ACS Award for Creative Work in Synthetic
Organic Chemistry, in recognition of his many contributions. Partial
support of this work by the American Cancer Society (DHP-79) is
gratefully acknowledged. Grants for the purchase of NMR equipment
from NSF (BIR-94-19409) and the Elsa U. Pardee Foundation are
gratefully acknowledged. We wish to express our sincere thanks to
Dr. Nicolay Kulikov (Affymax Research Institute) for many helpful
discussions. In addition, we thank both our collaborators in the research
group of Professor G. Cardillo (Bologna) for many helpful discussions
and NATO for funding this collaboration in the form of a Collaborative
Research Grant (CRG 950049).
Figure 1. Structure of 6b. Only hydrogens on heteroatoms are shown.
Compounds 5a-g display the full range of cis-trans amide
isomer populations, with 5c and 5g appearing as single isomers,
while 5f, derived from L-tyrosine benzyl ether, affords two sets
of 1H and 13C NMR (CDCl3) resonances in almost equal
amounts. Experiments with 5f in DMSO-d6 over the range from
room temperature to 100 °C suggest that this cis-trans
isomerization around the dipeptide bond has a relatively low
barrier, with a coalescence temperature of 70 °C.16
Supporting Information Available: Experimental procedures and
1H NMR spectra for key compounds and X-ray data for 6b (62 pages).
See any current masthead page for ordering and Internet access
instructions.
JA9639271
It was of interest to determine if these dipeptides could be
employed in the SPPS of polypeptides, particularly with the
heterocycle at the C-terminal position.17 To this end, the
previously described 6b-resin adduct was deprotected with
piperidine/DMF. Resulting free amine 9 was coupled with
Fmoc-Val-OH to give the desired tripeptide, followed by
deprotection and coupling with Fmoc-Gly-OH in similar manner
to afford 10. Cleavage from the resin with TFA gave expected
tetrapeptide 11 as the Fmoc-protected primary amide in 82%
yield from 6b. Compound 11, which is readily soluble in
common organic solvents, was treated with mild aqueous acid
to afford tetrapeptide 12, identical with material made in the
(16) By comparison, Snieckus and co-workers15 have studied amide re-
stricted rotation in compound i, and report overall coalescence at 100 °C.
(17) Asparagine has been a troublesome amino acid when directly linked
to the resin. See: (a) Albericio, F.; Van Abel, R.; Barany, G. Int. J. Peptide
Protein Res. 1990, 35, 284-6. (b) Carey, R. I.; Huang, H.; Wadsworth, J.
L.; Burrell, C. S. Int. J. Peptide Protein Res. 1996, 47, 209-13.