An Efficient Synthesis of a Probe for Protein Function: 2,3-Diaminopropionic Acid with Orthogonal Protecting Groups

Article abstract of DOI:10.1021/ol0361599

(Matrix presented) An efficient and cost-effective synthesis of N(α)-Boc₂-N(β)-Cbz-2,3-diaminopropionic acid is reported. The synthesis starts from commercially available N(α)-Boc-Asp(OBn)-OH and employs a Curtius rearrangement to establish the β-nitrogen. Proper protection of the α-nitrogen is essential for the success of the Curtius rearrangement.

Full text of DOI:10.1021/ol0361599

ORGANIC
LETTERS
2004
Vol. 6, No. 2
213-215
An Efficient Synthesis of a Probe for
Protein Function: 2,3-Diaminopropionic
Acid with Orthogonal Protecting Groups
Ethan A. Englund, Hosahudya N. Gopi, and Daniel H. Appella*
Department of Chemistry, Northwestern UniVersity, EVanston, Illinois 60208
dappella@chem.northwestern.edu
Received November 4, 2003
ABSTRACT
An efficient and cost-effective synthesis of N(r)-Boc2-N(â)-Cbz-2,3-diaminopropionic acid is reported. The synthesis starts from commercially
available N(r)-Boc-Asp(OBn)-OH and employs a Curtius rearrangement to establish the â-nitrogen. Proper protection of the r-nitrogen is
essential for the success of the Curtius rearrangement.
Incorporation of 2,3-diaminopropionic acid (Dap, Figure 1)
has frequently been used to probe many aspects of peptide
and protein structure. For example, substituting Dap for
substitution can also be used to probe the importance of salt
bridge formation.3
In our work, we are exploring the effects of Dap
substitution in RNA-binding peptides. Many RNA-binding
proteins and peptides rely on highly cationic amino acid
sequences in order to achieve high binding affinity.4 There-
fore, such RNA-binding sequences always contain large
numbers of lysine and arginine residues. To design effective
RNA-binding drugs, molecules must be designed so that high
binding affinity and selectivity to an RNA target can be
achieved. The highly charged nature of lysine and arginine,
combined with their extended side chains, can contribute to
nonspecific binding, which greatly reduces selectivity. Mov-
ing the charge closer to the peptide backbone should reduce
the propensity of the cationic charge to interact with random
anionic species. Confining the charge associated with lysine
(and arginine) to positions that are closer to the polypeptide
backbone could impact the secondary structure of the peptide
and the binding interaction between polypeptides and RNA.
NMR studies indicate that not all charged residues in RNA-
binding polypeptides are directly coordinated to the RNA
fold.5 We theorize that incorporation of Dap (and guanylated
Dap) into peptides with known RNA binding targets could
Figure 1.
lysine has been used to demonstrate that side chain length
affects the stability of R helix formation in simple polypep-
tides.1 Lysine-associated hydrophobic interactions, arising
from interactions with the methylenes of lysine, have also
been investigated using Dap, in combination with N-
methylated analogues of Dap.2 In addition, lysine/Dap
(1) (a) Groebke, K.; Renold, P.; Tsang, K. Y.; Allen, T. J.; McClure, K.
F.; Kemp, D. S. Proc. Natl. Acad. Sci. 1996, 93, 4025. (b) Padmanabhan,
S.; York, E. J.; Stewart, J. M.; Baldwin, R. L. J. Mol. Biol. 1996, 257, 726.
(2) Kretsinger, J. K.; Schneider, J. P. J. Am. Chem. Soc. 2003, 125, 7907.
(3) (a) Marqusee, S.; Baldwin, R. L. Proc. Natl. Acad. Sci. 1987, 84,
8898. (b) Merutka, G.; Stellwagen, E. Biochemistry 1991, 30, 1591.
(4) Draper, D. E. J. Mol. Biol. 1999, 293, 255.
10.1021/ol0361599 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/25/2003

provide important benefits such as decreased nonspecific
binding. Incorporation of nonnatural residues also discour-
ages decomposition of peptides in vivo.6 Given the high cost
of Dap, and our desire to incorporate this residue into
multiple positions within polypeptides, we have developed
an efficient and economical route to a protected form of Dap.
Extensive studies using Dap can be limited due to the cost
associated with purchasing this compound. Although com-
mercially available, the typical price for Dap (with orthogonal
protecting groups) is about $35/mmol.7 This cost can be
prohibitive when using an excess of monomer in the solid-
phase synthesis of a polypeptide containing multiple Dap
residues. Furthermore, only the L-enantiomer is available
commercially.
encountered were the constant need to exchange protecting
groups, the high cost of bis-[trifluoroacetoxy]-iodo benzene
(∼$0.95/mmol11), and the requirement to use an excess (at
least 2 equiv) of this reagent. Therefore, we began to develop
a more direct and less costly route to Dap.
We felt that a Curtius rearrangement of an aspartic acid
derivative would afford an isocyanate that could be trapped
with benzyl alcohol to directly afford a derivative of Dap
with orthogonal protecting groups suitable for solid-phase
peptide synthesis. Starting from Boc-Asp-OBn (1), a Curtius
1
rearrangement proceeded to give a product with H NMR
consistent with the expected isocyanate intermediate 2
(Figure 3). To our surprise, multiple efforts to trap the
The most common synthesis of Dap involves a Hoffman
rearrangement of protected asparagine using a trivalent iodine
reagent, most often bis-[trifluoroacetoxy]-iodo benzene
(Figure 2). When this reagent is used, the trifluoroacetic acid
Figure 3. Cyclization with a single Boc-protecting group during
Curtius rearrangement.
Figure 2. Hoffman rearrangement with bis-[trifluoroacetoxy]-iodo
benzene.12
supposed isocyante failed. Additional 13C NMR, IR, and mass
spectrometry data confirmed that cyclic urea 3 was present
instead. In the 1H NMR of 3, the urea proton (H*) appeared
in the same region (5.3-5.6 ppm in CDCl3) as most
carbamate protons, which was initially deceptive. Also, there
was no discernible change in the couplings or chemical shifts
of the R or â protons to indicate urea versus isocyanate
formation. Deprotection of the Boc group from 3 provided
the unprotected urea, which was also fully characterized. A
number of studies have shown that carbamate-protected
amines can trap isocyanates via intramolecular cyclization.13
In our case, the cyclization to form a five-membered ring
urea was especially efficient. We were unable to intercept
the isocyanate with other nucleophiles.
We were able to prevent the intramolecular trapping of
the isocyanate after the Curtius rearrangement by dually
protecting the reactive nitrogen.14 Starting from protected
aspartic acid 4, which is commercially available, the car-
boxylic acid was first converted to methyl ester 5, followed
by introduction of a second Boc group13a to give 6 (Scheme
formed in solution8 is believed to catalyze the hydrolysis of
the isocyanate intermediate to an amine, reducing urea
formation from reaction of amine with remaining isocyanate.9
However, the acid generated can also remove a Boc
protecting group. As shown in Figure 2, Boc-protected
asparagine produced significantly lower yields than Cbz-
protected asparagine when subjected to the same conditions
for the Hoffman rearrangement. Fmoc-protected asparagine
had poor solubility under the same reaction conditions, which
accounts for the failure of the Hoffman rearrangement when
using this form of protected asparagine. If Cbz protection is
used, which is optimal, then an R-amine protecting group
must be converted to a Boc or Fmoc group in order to be
compatible with solid-phase peptide synthesis.10 Because we
required access to large amounts of Boc-protected Dap, the
Hoffman-based synthesis of Dap quickly became prohibitive
to the progress of our research. The main problems we
(5) Battiste, J. L.; Mao, H.; Rao, N. S.; Tan, R.; Mahandiram, D. R.;
Kay, L. E.; Frankel, A. D.; Williamson, J. R. Science 1996, 273, 1547.
(6) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1669.
(7) Advanced ChemTech, Louisville, KY.
(8) Boutin, R. H.; Loundon, G. M. J. Org. Chem. 1984, 49, 4277.
(9) Radhakrishna, A. S.; Parham, M. E.; Riggs, R. M.; Loundon, G. M.
J. Org. Chem. 1979, 44, 1746.
(11) Aldrich, Milwaukee, WI.
(12) Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 4, 266.
(13) (a) Theon, J. C.; Morales-Ramos, A. I.; Lipton, M. A. Org. Lett.
2002, 4, 4455. (b) Salituro, F. G.; Agarwal, N.; Hofmann, T.; Rich, D. H.
J. Med. Chem. 1987, 30, 286. (c) Bodanszky, M.; Ondetti, M. A. Peptide
Synthesis; Interscience Pubpishers: New York, 1966; Chapter 5.
(14) Roberts, J. L.; Chan, C. Tetrahedron Lett. 2002, 43, 7679.
(10) Arttamangkul, S.; Murray, T. F.; DeLander, G. E.; Aldrich, J. V. J.
Med. Chem. 1995, 38, 2410.
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Org. Lett., Vol. 6, No. 2, 2004

Scheme 1
Scheme 2
In conclusion, we have developed a convenient synthesis
of orthogonally protected Dap. On the basis of the com-
mercial prices of the reagents and the yields of this synthesis,
using the Curtius rearrangement to access orthogonally
protected Dap proves to be half as costly as using the
Hoffman-based procedures and less time-consuming. It
should be noted that this synthesis is optimal for accessing
Dap for use in solid-phase peptide synthesis using the Boc
group to protect the R nitrogen. For solid-phase peptide
synthesis using the Fmoc strategy, the Dap monomer requires
an Fmoc group on the R nitrogen and a Boc group on the â
nitrogen. This arrangement of protecting groups could
certainly be made via our synthetic route using the following
series of protecting group manipulations on 10: deprotection
of the Boc groups followed by Fmoc protection and then
removal of the Cbz protecting group followed by Boc
protection. However, the Hoffman-based approach would
provide a shorter route to R-Fmoc, â-Boc Dap since less
protecting group manipulation would be necessary. Further
studies in our group will be directed toward incorporating
this amino acid into RNA binding peptides in order to probe
the effects on binding affinities.
1). The benzyl ester was then removed via hydrogenolysis
under pressure (50 psig). Next, the Curtius rearrangement
was performed by first converting the acid 7 to a mixed
anhydride, followed by conversion to the corresponding acyl
azide, and then refluxing in toluene for 2 h. These conditions
clearly formed isocyanate 8, which has been characterized
by 1H NMR, 13C NMR, and IR. Once isolated, the isocyanate
was trapped with benzyl alcohol, using CuCl as a Lewis acid,
to form the Cbz-protected amine 9.15 Hydrolysis of the
methyl ester then gave protected Dap 10.
A minor side product that we noticed in this synthetic route
results from loss of one Boc group of the diprotected
nitrogen. Partial monodeprotection most likely occurs during
acidic workup after the ester hydrolysis. This byproduct is
1
easily identified in the H NMR spectra (see Supporting
Information for data) and present in quantities of less than
1
10% relative to the main product (estimated by H NMR).
We have used 10 directly in solid-phase peptide synthesis
without complications in the deprotection or coupling steps
associated with peptide synthesis. The partially deprotected
byproduct is not detrimental to the conditions of solid-phase
peptide synthesis.
To confirm the enantiomeric purity of 9, we prepared 13
and compared the optical rotation to the identical compound
prepared from commercial Dap. Conversion of 9 to 13 is
shown in Scheme 2. Boc-Dap(Fmoc)-OH (from Advanced
Chemtech) was methylated to give 13. The optical rotations
of both compounds were within experimental error of each
other, confirming that racemization does not occur in our
synthesis of Dap.
Acknowledgment. We gratefully acknowledge financial
support from Northwestern University’s Weinberg College
of Arts and Sciences, Department of Chemistry, and VP of
Research.
Supporting Information Available: Experimental pro-
cedures and 1H NMR characterization for compounds 4-10
and 13, 13C NMR characterization available for compounds
4-9, and IR for 8. This material is available free of charge
via the Internet at http://pubs.acs.org.
(15) Duggan, M. E.; Imagire, J. S. Synthesis 1989, 131.
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