Efficient introduction of protected guanidines in boc solid phase peptide synthesis.

Article abstract of DOI:10.1021/ol016139b

[reaction: see text] Reaction of primary amines with pyrazole 1 results in rapid and efficient guanidinylation, either in solution or on solid phase. The reaction affords sulfonamide-protected products required for BOC solid phase peptide synthesis (SPPS)

Full text of DOI:10.1021/ol016139b

ORGANIC
LETTERS
2001
Vol. 3, No. 15
2341-2344
Efficient Introduction of Protected
Guanidines in BOC Solid Phase Peptide
Synthesis
Yongda Zhang and Alan J. Kennan*
Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523
kennan@lamar.colostate.edu
Received May 18, 2001
ABSTRACT
Reaction of primary amines with pyrazole 1 results in rapid and efficient guanidinylation, either in solution or on solid phase. The reaction
affords sulfonamide-protected products required for BOC solid phase peptide synthesis (SPPS) in a single step under mild conditions.
Incorporation of orthogonally protected side chain amines permits the synthesis of peptides containing arginine analogues, one of which
could not be prepared by coupling of preformed amino acids.
The widespread occurrence of guanidine groups in natural
products has inspired considerable development of synthetic
methodology.1 In particular, a number of elegant methods
for on-resin functionalization have been disclosed, though
most involve more than one step.2 Here we report a new
reagent (1), capable of guanidinylating primary amines with
impressive speed and efficiency. Reaction with side chain
amines during peptide synthesis allows one-step introduction
of protected guanidines compatible with BOC methodology.
The synthesis of 1 and its application in both solution and
solid phase contexts is described.
In the course of peptide-based molecular recognition
studies, we sought to prepare sequences containing chain-
shortened or -elongated arginine analogues. Given the
standard use of N-tosyl-protected arginine in BOC synthesis,
initial efforts targeted N-tosyl monomers. Although the
corresponding amino acids proved simple to prepare, their
use in peptide synthesis was impractical.3 Thus, an alternative
strategy of on-resin functionalization was pursued. Incorpo-
ration of amino acids bearing FMOC-protected side chain
amines, followed by sequential treatment with piperidine and
a guanidinylating agent, was expected to afford the desired
peptides.
(1) (a) Moroni, M.; Koksch, B.; Osipov, S. N.; Crucianelli, M.; Frigerio,
M.; Bravo, P.; Burger, K. J. Org. Chem. 2001, 66, 130-133. (b) Brewer,
M.; Rich, D. H. Org. Lett. 2001, 3, 945-948. (c) Katritzky, A. R.; Rogovoy,
B. V.; Chassaing, C.; Vvedensky, V. J. Org. Chem. 2000, 65, 8080-8082.
(d) Barawkar, D. A.; Kwok, Y.; Bruice, T. W.; Bruice, T. C. J. Am. Chem.
Soc. 2000, 122, 5244-5250. (e) Linton, B. R.; Carr, A. J.; Orner, B. P.;
Hamilton, A. D. J. Org. Chem. 2000, 65, 1566-1568. (f) Schneider, S. E.;
O’Neil, S. N.; Anslyn, E. V. J. Am. Chem. Soc. 2000, 122, 542-543. (g)
Baker, T. J.; Luedtke, N. W.; Tor, Y.; Goodman, M. J. Org. Chem. 2000,
65, 9054-9058. (h) Luedtke, N. W.; Baker, T. J.; Goodman, M.; Tor, Y.
J. Am. Chem. Soc. 2000, 122, 12035-12036. (i) Feichtinger, K.; Sings, H.
L.; Baker, T. J.; Matthews, K.; Goodman, M. J. Org. Chem. 1998, 63,
8432-8439. (j) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J.
Org. Chem. 1998, 63, 3804-3805.
(2) (a) Burgess, K.; Chen, J. In Solid-Phase Organic Synthesis; Burgess,
K., Ed.; John Wiley & Sons: New York, 2000; pp 1-23. (b) Gomez, L.;
Gellibert, F.; Wagner, A.; Mioskowski, C. Chem.sEur. J. 2000, 6, 4016-
4020. (c) Dahmen, S.; Braese, S. Org. Lett. 2000, 2, 3563-3565. (d) Zapf,
C. W.; Creighton, C. J.; Tomioka, M.; Goodman, M. Org. Lett. 2001, 3,
1133-1136. (e) Ghosh, A. K.; Hol, W. G. J.; Fan, E. J. Org. Chem. 2001,
66, 2161-2164. (f) Mamai, A.; Madalengoitia, J. S. Org. Lett. 2001, 3,
561-564.
An appropriate reagent was suggested by reports that
pyrazole 2 produces bis-Boc protected guanidines from
(3) For example, use of N-Boc-R-amino-γ-N-tosylguanidinobutyric acid
gave little or no desired product even using extended coupling times and
excess reagents.
10.1021/ol016139b CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/27/2001

primary amines.4 Also encouraging was an analogue’s ability
To probe the reactivity of 1 toward amines, solution
couplings with a handful of simple substrates were attempted
(Figure 2). Reactions with simple primary amines were rapid
to functionalize an ornithine side chain on solid phase,
although the guanidiniylation demanded somewhat elevated
temperatures and extended reaction times.5 More problemati-
cally, either analogue would require that guanidinylation
immediately precede cleavage from the resin, since removal
of the R-amino Boc group would also deprotect any newly
formed guanidine. In the context of longer sequences, this
would require orthogonal side chain protecting groups that
could withstand repeated coupling iterations. A simpler
approach involving side chain deprotection and functional-
ization before further synthesis would permit the use of
protecting groups that need not be removed until final
cleavage from the resin. Thus, an ideal reagent would
efficiently generate BOC-compatible protected guanidines
from side chain amines. In keeping with the sulfonamide
strategy, we examined N-tosyl-protected 1 and discovered
that it was an extremely efficient means of accomplishing
this objective (Figure 1).
Figure 2. Solution phase reactions. Reaction times for guanidi-
nylation of each substrate with 1.1 equiv of 1 in THF at room
temperature. Reaction yields were quantitative, except for substrates
marked n.r., which gave no product after 1 day.
and quantitative upon mixing with 1.1 equiv of 1 in THF at
room temperature.7 Aniline and tert-butylamine were also
reasonable substrates, though considerably more sluggish.
Further reduction in nucleophilicity (e.g., p-nitroaniline)
suppressed the reaction entirely. Piperidine was similarly
unreactive, confirming that residual base from side chain
deprotections would not compete for 1.
With the efficacy of 1 in solution demonstrated, attention
turned to its use in peptide synthesis. Target sequences were
selected to address a variety of issues (Figure 3). Guanidi-
Figure 1. General guanidinylation reaction.
The required reagent can be prepared on a gram scale from
commercially available pyrazole 3 via a two-step sequence
(Scheme 1).6 The mono Boc derivative of 3 (Boc2O, iPr2-
Scheme 1. Reagent Preparation
Figure 3. Test sequences for on-resin guanidinylation, presented
using one-letter codes.11 Unusual side chains are depicted explicitly.
Peptides 5Fm and 7Fm were prepared only in resin-bound form.
NEt/CH2Cl2, 90%) produced 1 upon reaction with TsCl
(NaH/THF 46%).
(4) (a) Drake, B.; Patek, M.; Lebl, M. Synthesis 1994, 579-582. (b)
Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett. 1993, 34,
3389-3392.
nylation conditions were tested by preparation of a short
monoguanidine peptide (4), which could not be prepared by
standard methods. Thus, fragment 4a, terminating in an
R-BOC-γ-FMOC-protected diaminobutyric acid, was syn-
(5) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org. Chem. 1992,
57, 2497-2502.
(6) See Supporting Information for experimental details.
2342
Org. Lett., Vol. 3, No. 15, 2001

Scheme 2. Example of Peptide Guanidinylation
thesized by SPPS (Scheme 2).8 Following FMOC removal,
quantitative guanidinylation of the side chain amine was best
achieved using 2 equiv of 1 in 3:1 THF:DMF at room
temperature for 1 h.9 Completion of the synthesis and
cleavage from the resin with anhydrous HF afforded the
desired peptide 4. An analogous homoarginine peptide (5)
was prepared using the same methods, confirming the ability
to functionalize side chains of different length.10
Although crude products were judged reasonably clean by
reverse-phase HPLC, verification was desired that observed
side products were not due to inefficient guanidinylation.
Thus, peptide 5Fm was prepared, in which the functional-
ization site of 5 remains FMOC-protected. The full-length
peptide was then deprotected, and half the resulting resin
was reacted with 1 under standard conditions. Individual
cleavage of both resin samples afforded samples of 5 and
5Lys, whose side chain is not guanidinylated. Since the two
peptides differ only in whether they were guanidinylated, a
comparison of crude HPLC traces should reflect the reaction
efficiency. An overlay of the two traces demonstrates that
indeed all significant byproducts are present in both samples
and are thus attributable to errors in peptide synthesis rather
than guanidinylation (Figure 4).
Figure 4. Reaction efficiency. Crude HPLC chromatograms of
peptides 5 and 5Lys. Both were prepared from a common synthesis
and differ only in whether the deprotected amine side chain was
guanidinylated (5, dotted line) or not (5Lys, solid line) before
cleavage from the resin.
Thus, longer sequences containing two (6), four (7), and eight
(8) such groups were examined (Figure 3). It was anticipated
Once the basic application of 1 in peptide synthesis had
been demonstrated, multiple substitutions were investigated.
Since R-amino-γ-guanidinobutyric acid residues could not
be installed by coupling functionalized monomers, they were
selected as a challenging test case for the new methodology.
(7) Sample procedure: A mixture of 1 (21.8 mg, 0.06 mmol) and amine
(0.055 mmol) in 0.5 mL of THF was stirred at room temperature for 5
min. Water and ethyl acetate were added, and the organic layer was washed
with 0.5 M citric acid, water, and brine and dried over Na2SO4. After
removal of solvent in vacuo, the residue was chromatographed (SiO2; 3:1
ethyl acetate:hexane) to afford pure product.
(8) Peptides were synthesized according to standard Boc protocols,
employing the in situ neutralization procedure developed by Kent: Schno-
elzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H. Int. J. Pept.
Protein Res. 1992, 40, 180-193.
(9) Functionalization of all resin-bound amines was verified by the Kaiser
test. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595-598.
Figure 5. Sequential vs global guanidinylation. Crude HPLC
chromatograms of peptide 7 synthesized by either: FMOC removal
and guanidinylation immediately after incorporation of R-amino-
butyric acid (solid line) or simultaneous deprotection and guanidi-
nylation of all four amines in the fully synthesized peptide (dotted
line).
(10) The identity of each peptide was confirmed by electrospray mass
spectrometry of a sample purified by reverse-phase HPLC. See Supporting
Information.
(11) One-letter abbreviations: A ) alanine, E ) glutamic acid, K )
lysine, L ) leucine, N ) aspartic acid, Q ) glutamine, X ) R-aminobutyric
acid.
Org. Lett., Vol. 3, No. 15, 2001
2343

that the ability to introduce robustly protected guanidines
should be particularly useful in these contexts. Indeed, all
three peptides were easily prepared using 1. Each guanidine
was introduced in the same manner as above, by side chain
deprotection and functionalization prior to chain elongation.
Crude HPLC analysis confirmed synthetic efficiency.
To further demonstrate the benefits of sequential guanidi-
nylation, peptide 7 was also prepared according to a global
functionalization strategy. Thus, 7Fm, which retains all four
FMOC groups, was synthesized. The completed peptide was
then exposed to piperidine in DMF to liberate the amine side
chains. Simultaneous functionalization of all four positions
proceeded without incident, and after cleavage from the resin
the crude material was compared to that prepared above via
the sequential approach.
As expected, the globally functionalized material suffered
a considerable degradation in purity. Presumably this is due
to undesired FMOC removal during repeated exposure to
coupling and deprotection conditions. An overlay of crude
HPLC traces (Figure 5) reveals an increase in side products
(from 18 to 22 min) and a dramatic reduction of the desired
product (22 min).
The investigations reported above clearly demonstrate the
value of 1 as an efficient reagent for the preparation of
guanidine-containing peptides. The reagent can be prepared
on large scale from inexpensive commercial materials. It
permits one-step installation of N-tosyl guanidines under mild
conditions and affords products compatible with subsequent
exposure to repeated coupling cycles. In particular, the
method has been validated in the context of several sequences
that could not be prepared by simple coupling of prefunc-
tionalized monomers. This chemistry should prove extremely
useful in the construction of peptides containing a wide
variety of guanidinylated side chains.
Acknowledgment. We thank Colorado State University
and the Petroleum Research Fund, administered by the
American Chemical Society, for financial support.
Supporting Information Available: Experimental details
for the preparation of 1, along with characterization data for
solution and peptide products. This material is available free
of charge via the Internet at http://www.acs.org.
OL016139B
2344
Org. Lett., Vol. 3, No. 15, 2001