Dicyclopropylmethyl peptide backbone protectant

Article abstract of DOI:10.1021/ol901310q

Image Presented The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an

Full text of DOI:10.1021/ol901310q

ORGANIC
LETTERS
Dicyclopropylmethyl Peptide Backbone Protectant†
Louis A. Carpino,*,‡ Khaled Nasr,‡ Adel Ali Abdel-Maksoud,#,‡
Ayman El-Faham,‡,∇ Dumitru Ionescu,‡,O Peter Henklein,§ Holger Wenschuh,|
Michael Beyermann,⊥ Eberhard Krause,⊥ and Michael Bienert⊥
2009
Vol. 11, No. 16
3718-3721
Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003,
Institute of Biochemistry, Humboldt UniVersity, Monbijoustr. 2, 10117 Berlin, Germany,
Jerini AG, InValidenstr. 130, 10115 Berlin, Germany, and Forschungsinstitut fu¨r
Molelulare Pharmakologie, Robert-Ro¨ssle-Str. 10, 13125 Berlin, Germany
carpino@chem.umass.edu
Received June 11, 2009
ABSTRACT
The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino
acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide
synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion
peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the
assembly of sequences containing the sensitive Asp-Gly unit.
We describe a new backbone protectant for the synthesis of
so-called “difficult peptides”, namely, those peptides for
which the presence of specific sequences of amino acids,
often hydrophobic amino acids, lead to slow deblocking and
incomplete coupling processes. Several approaches to over-
come these problems have been devised. These include the
use of more potent coupling reagents,1 pseudoproline inser-
tions,2 the depsipeptide technique,3 and the use of backbone
protection.4 Recently, dicyclopropylmethyl (Dcpm) and
dimethylcyclopropylmethyl (Dmcp) groups have been de-
scribed as amide protectants for Asn, Gln, and the C-terminal
position of linear peptide amides.5 In view of their utility as
amide protectants, consideration has now been given to their
possible use as peptide bond protectants. Of these two alkyl
residues, the Dcpm group was chosen due to its lesser steric
requirements and the fact that the appropriate N-substituted
amino acids could be synthesized via standard reductive
alkylation techniques. Finally, deblocking of the Dcpm
residue from an internal (tertiary amide) position is expected
to be enhanced and perhaps as rapid as the removal of the
† Abbreviations used: ACN ) acetonitrile; BSA ) bistrimethylsilylac-
etamide; Bsmoc ) benzothiophenesulfone-2-methyloxycarbonyl; Dcpm )
dicyclopropylmethyl; DIC ) diisopropylcarbodiimide; Dmcp ) dimethyl-
cyclopropylmethyl; DODT ) 3,6-dioxa-1,8-octanedithiol; Fmoc ) 9-fluo-
renemethyloxycarbonyl; N-HATU ) 1-[bis(dimethylamino)methylene]-1-
H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide; N-HBTU
) 1-[bis(dimethylamino)methylene]-1-H-benzotriazolium hexafluorophos-
phate 3-oxide; Hmb ) 2-hydroxy-4-methoxybenzyl; HOAt ) 1-hydroxy-
7-azabenzotriazole; Phth ) o-phthaloyl; TFA ) trifluoroacetic acid; TFFH
) tetramethyl fluoroformamidinium hexafluorophosphate.
‡ University of Massachusetts.
(1) (a) El-Agnaf, O. M. A.; Goodwin, H.; Sheridan, J. M.; Frears, E. R.;
Austen, B. M. Protein Pept. Lett. 2000, 7, 1. (b) Milton, S. C. F.; Milton,
R. C. L.; Kates, S. A.; Glabe, C. Lett. Pept. Sci. 1999, 6, 151.
(2) Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wuhr, T. Pept.
Res. 1995, 8, 145.
§ Humboldt University.
| Jerini AG.
⊥ Forschungsinstitut fu¨r Molelulare Pharmakologie.
# On leave of absence from the Protein Research Department, Genetic
Engineering Institute, Mubarak City for Scientific Research, Borg El-Arab,
Alexandria 21934, Egypt.
(3) (a) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Chem.
Commun. 2004, 124. (b) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez,
J.; Dos Santos, S.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.; Saucejde,
L.; Tuchscherer, G. Angew. Chem., Int. Ed. 2004, 43, 4172. (c) Carpino,
L. A.; Krause, E.; Sferdean, C. D.; Schu¨mann, M.; Fabian, H.; Bienert,
M.; Beyermann, M. Tetrahedron Lett. 2004, 45, 7519.
∇ On leave from the Department of Chemistry, Faculty of Science,
Alexandria University, Alexandria, Egypt.
O On leave from the Department of Chemistry, University of Bucharest,
70346 Bucharest, Romania.
10.1021/ol901310q CCC: $40.75
Published on Web 07/16/2009
 2009 American Chemical Society

Dmcp residue from the terminal amide position. This
expectation follows from the curious fact recorded in the
literature6 that secondary amide, N-t-butylbenzamide 1, is
stable toward TFA, whereas the analogous tertiary amide
N-t-butyl-N-methylbenzamide 2 loses the t-butyl residue upon
treatment with the same acid. It has long been recognized
that N-t-butyl amides such as 1 differ from the corresponding
oxygen analogues (t-butyl esters) in being stable toward
acidic deblocking.7 The sensitivity of 2 could perhaps be
explained on the basis of its being a tertiary alkyl-based
tertiary amide which might be subject to more facile N-
relative to O-protonation with subsequent ready loss of the
t-butyl cation in TFA.8
described imine N-n-propyl dicyclopropyl-ketimine.13 Re-
duction and benzoylation gave the corresponding tertiary
amide which was tested for its stability toward TFA under
conditions normally used for the final deprotection step
during Fmoc/t-Bu-based solid phase peptide assembly. As
expected, the Dcpm residue was readily removed with the
formation of N-(n-propyl)benzamide.
A general method for the synthesis of N-Dcpm amino acids
was then developed. A key intermediate 3, obtainable by
treatment of the commercially available, or easily synthesized
dicyclopropyl ketone, with ammonia in the presence of
titanium tetrachloride, has been described in the patent
literature.14 Reaction of 3 with an amino acid ester gave
imine 4 (Scheme 1) which upon reduction with sodium
In the present work, the high acid sensitivity of the internal
Dcpm residue was verified, thus allowing for its use as a
backbone protectant. Regarding the susceptibility of an
N-Dcpm-substituted amino acid or amino acid ester to
coupling reactions, it is well-known that coupling to N-
methyl amino acids is difficult9 and coupling to N-Dcpm
amino acids is expected to be even less facile. However, the
steric requirements of the Dcpm group may be unique since
in its coupling chemistry the Dcpm-bearing model amino
acid R,R-dicyclopropylgycine resembles its simple R,R-
dimethyl analogue, H-Aib-OH, more than its extremely
hindered analogue, R,R-diisopropylglycine.10 Since H-Aib-
OH is an easily handled amino acid for peptide incorporation,
similar effects are to be expected for the N-Dcpm residue.10
Indeed, as shown in the present work, for the relatively
unhindered amino acids glycine and alanine, coupling to the
N-Dcpm derivatives is readily achievable. On the other hand,
for the more hindered amino acids such as valine, isoleucine,
and threonine, special acylating techniques may be required.
The currently most commonly used backbone protectant,
the Hmb group,4 differs from the Dcpm residue in being
acylated via prior reaction at the o-hydroxyl group followed
by an O f N acyl shift.11
Scheme 1
cyanoborohydride or triacetoxyborohydride in the presence
of acetic acid gave the amino acid derivative 5 which in the
case of the benzyl ester (5, R′ ) Bn) underwent standard
catalytic hydrogenolysis to give the free amino acid 6
(Scheme 2).
Scheme 2
Prior to preparation of appropriate amino acid derivatives,
a model experiment was carried out with the previously
For relatively unhindered amino acids (e.g., 6, R ) H,
Me) the amino function is readily attacked by an appropriate
acylating agent. Thus, the Fmoc15 and Bsmoc16 derivatives
7 (R′′ ) Fm, Bsm) are readily obtained via the corresponding
chloroformates using the Bolin technique.17 Analogously,
treatment with Fmoc-Gly-Cl18 or Fmoc-Gly-F19 yields the
corresponding protected dipeptide 8 (R ) H, Me).
(4) (a) Johnson, T.; Quibell, M.; Owen, D.; Sheppard, R. C. J. Chem.
Soc., Chem. Commun. 1993, 369. (b) Johnson, T.; Quibell, M. Tetrahedron
Lett. 1994, 35, 463. (c) Johnson, T.; Quibell, M.; Sheppard, R. C. J. Pept.
Sci. 1995, 1, 11. (d) Ede, N. J.; Ang, K. H.; James, I. W.; Bray, A. W.
Tetrahedron Lett. 1996, 37, 9097.
(5) 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.; Ionescu, D.; 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.
(6) Catt, J. D.; Matier, W. D. J. Org. Chem. 1974, 39, 566.
(7) Callahan, F. M.; Anderson, G. W.; Paul, R.; Zimmerman, J. E. J. Am.
Chem. Soc. 1963, 85, 201.
(8) Compare: Guiheneuf, G.; Abboud, J.-L. M.; Lachkar, A. Can.
J. Chem. 1988, 66, 1032.
(9) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97, 2243.
(10) (a) Yamada, T.; Sano, A.; Yanagihara, R.; Miyazawa, T. Peptide
Chemistry 1996, Proc. of the 34th Japanese Peptide Symposium; Kitada,
C., Ed.; Protein Research Foundation, Osaka, 1997; p 357. (b) Yamada T.;
Gohda, S.; Sano, A.; Yanagihara, R.; Miyazawa, T.; Kuwata, S. Peptides
1996, Proc. of the 24th European Peptide Symposium; Ramage, R., Epton,
R., Eds.; p 927.
It was demonstrated that in the synthesis of 5 (R ) Me,
R′ ) Bn) via 3 no significant loss of configuration
(12) Miranda, L. P.; Meutermans, W. D. F.; Smythe, M. L.; Alewood,
P. F. J. Org. Chem. 2000, 65, 5460.
(11) To make it more suitable for use with hindered amino acids,
Miranda and Alewood and co-workers12 have modified the Hmb residue
by the introduction of a nitro group into the benzylic ring. While this change
has the desired effect, the resulting protectant cannot be removed by acid-
catalyzed deblocking.
(13) Pocar, D.; Stradi, R.; Trimarco, P. Tetrahedron 1975, 31, 2427.
(14) Kurono, M.; Kondo, Y.; Matsumoto, Y.; Sawai, K. U.S.P. 5,110,945
( 1992).[Chem. Abstr. 1992, 117, 111466].
(15) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404.
Org. Lett., Vol. 11, No. 16, 2009
3719

occurred at the alanine residue as shown by conversion
to 7 (R ) Me, R′′ ) Fm). Coupling of the Fmoc-protected
acid to proline atached to a Rink amide resin by means
of N-HATU/DIEA in DMF gave the corresponding
dipeptide resin which was Dcpm-deblocked and cleaved
from the resin by treatment with TFA to give Fmoc-Ala-
Pro-NH220 which was contaminated by only 0.95% of the
DL-isomer showing that no more than this amount of
contamination could have been present in the sample 5
used or could have been formed during the coupling
process. Further work will be required to determine the
exact source of the contamination, but the amount is small
enough not to impinge upon the present work.
The methodology described was also applied to the
synthesis of the valine derivatives 5 (R ) iPr, R′ ) Bn) and
6 (R ) iPr); however, this more hindered amino acid could
not be acylated via Fmoc-Cl or Bsmoc-Cl to give 7 (R )
iPr, R′′ ) Fm or Bsm). Similarly, the coupling of a simple
protected amino acid (e.g., Z-Phe-OH) to 5 (R ) iPr, R′ )
Bn) did not succeed with a variety of standard coupling
reagents (e.g., N-HBTU,21,22 N-HATU,22 TFFH,23 etc.).
However, with the more potent acylating agent Phth-Phe-
Cl, coupling in the presence of BSA24 gave the dipeptide in
56-62% yield. Details will be provided in a subsequent
publication.
for all subsequent coupling steps. The remainder of the
synthesis was carried out in the normal manner, but the only
material obtained following workup and removal of the
peptide from the resin was the N-Dcpm-pentaalanine deriva-
tive (Figure 2, Supporting Information) showing that the
system shut down following introduction of the Dcpm-Ala
unit.
However, since previous studies had suggested that Bsmoc
amino acids were more reactive than their Fmoc
counterparts,3c it was simply necessary to substitute Bsmoc-
Ala-OH for the Fmoc analogue in this synthesis, and
remarkably, about 50% of the desired decaalanine product
was obtained. The 11-mer is accompanied by about 50% of
the N-Dcpm-substituted deletion sequence which had been
the sole product formed in the all Fmoc case (Figure 3,
Supporting Information). If HOAt/DIC was used in place
of N-HATU and coupling continued for a longer time, the
reaction could be pushed toward completion (Figure 8,
Supporting Information).
A sequence which is expected to be subject to difficulties
due to the presence of the base-sensitive Asp-Gly unit26 is
dodecapeptide 9, which on attempted synthesis under stan-
dard conditions via N-HBTU gave only the aminosuccin-
imide cyclization product. With Fmoc-(Dcpm)-Gly-OH
substituted for Fmoc-Gly-OH under the same conditions
As a first sequence in which to examine the utility of the
Dcpm residue as a backbone protectant, the difficult de-
caalanine sequence built onto arginine was examined.25
Previously this system has been shown to be subject to the
effects of aggregation leading to both deblocking and
coupling deficiencies. An automated synthesis of the de-
caalanine sequence using 4 equiv of Fmoc amino acid with
coupling via N-HATU gave material contaminated by several
des-Ala units as well as undeblocked Fmoc-containing
segments (Figure 1, Supporting Information). An attempt to
improve the synthesis was made by substituting Fmoc(D-
cpm)-Ala-OH for Fmoc-Ala-OH to introduce the fifth alanine
unit and at the same time reduce the effect of aggregation
(N-HBTU), the result was still unsatisfactory (only 17% of
9), but if N-HATU was substituted for N-HBTU, the desired
dodecapeptide amide was obtained in a yield of 91% along
with only 8% of the des-Asp deletion peptide (see Figure
9a,b, Supporting Information).
A classical “difficult sequence” was then examined,
namely, the prion peptide (106-126) 10 which was previously
examined by Jobling et al.27 Using Fmoc chemistry with
(16) Carpino, L. A.; Ismail, M.; Truran, G. A.; Mansour, E. M. E.;
Iguchi, S.; Ionescu, D.; El-Faham, A.; Riemer, C.; Warrass, R. J. Org. Chem.
1999, 64, 4324.
(17) Bolin, R. R.; Sytwu, I.; Humiec, F.; Meienhofer, J. Int. J. Pept.
Protein Res. 1989, 33, 353.
N-HBTU coupling and introducing the Hmb residue at
positions 114 and 119, these workers obtained a low yield
of the desired peptide (7.3%) along with a number of deletion
sequences. With Fmoc (Dcpm)-Gly-OH introduced at the
same two positions and using N-HATU for coupling using
normal Fmoc amino acids except for positions 113 and 118
which were introduced by Bsmoc-Ala-OH, a relatively clean
sample of the desired peptide was obtained (crude yield 41%,
purity 89%) (see Figure 10a,b, Supporting Information). The
(18) Carpino, L. A.; Cohen, B. J.; Stephens, K. E., Jr.; Sadat-Aalaee,
S. Y.; Tien, J.-H.; Langridge, J. Org. Chem. 1986, 51, 3732.
(19) Carpino, L. A.; Sadat-Aalaee, D.; Chao, H. G.; DeSelms, R. H.
J. Am. Chem. Soc. 1990, 112, 9651.
(20) Authentic samples of the two diastereomeric dipeptides were
prepared by normal solution techniques and shown to be base line separated
on a C-18 HPLC column (see Supporting Information). For a prior report
on the Ala/Pro dipeptide amide without experimental data, see: Li, J.; Wilk,
E.; Wilk, S. Arch. Biochem. Biophys. 1995, 323, 148.
(21) Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Zioudrou, C.
Synthesis 1984, 572
.
(22) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang,
C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mu¨gge, C.; Wenschuh,
H.; Klose, J.; Beyermann, M.; Bienert, M. Angew. Chem., Int. Ed. 2002,
(25) Compare: (a) Larsen, D.; Holm, A. Int. J. Pept. Protein Res. 1994,
43, 1. (b) Kates, S. A.; Sole, N. A.; Beyermann, M.; Barany, G.; Albericio,
F. Peptide Res. 1996, 9, 106.
41, 441
.
(23) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995, 117, 5401.
(24) Compare: (a) Carpino, L. A.; Beyermann, M.; Wenschuh, H.;
Bienert, M. Acc. Chem. Res. 1996, 29, 268. (b) Wenschuh, H.; Beyermann,
M.; Winter, R.; Bienert, M.; Ionescu, D.; Carpino, L. A. Tetrahedron Lett.
1996, 37, 5483. (c) Carpino, L. A.; Ionescu, D.; El-Faham, A.; Henklein,
P.; Wenschuh, H.; Bienert, M.; Beyermann, M. Tetrahedron Lett. 1998,
39, 241.
(26) For a series of papers dealing with this general problem, see: (a)
Mergler, M.; Dick, F.; Sax, B.; Weiler, P.; Vorherr, T. J. Pept. Sci. 2003,
9, 36. (b) Mergler, M.; Dick, F.; Sax, B.; Sta¨helin, C.; Vorherr, T. J. Pept.
Sci. 2003, 9, 518. (c) Mergler, M.; Dick, F. J. Pept. Sci. 2005, 11, 650.
(27) Jobling, M. F.; Barrow, C. J.; White, A. R.; Masters, C. L.; Collins,
S. J.; Cappai, R. Lett. Pept. Sci. 1999, 6, 129.
3720
Org. Lett., Vol. 11, No. 16, 2009

only major byproduct was a small amount of the methionine
sulfoxide derivative.
Acknowledgment. We are indebted to the National
Science Foundation (NSF CHE-9003192) and the National
Institutes of Health (GM-09706) for support of the work
in Amherst.
In conclusion, it has been shown that the N-dicyclopro-
pylmethyl residue can be used as an amide backbone
protectant in the case of relatively unhindered amino acids.
N-Dcpm amino acids can be readily synthesized via treatment
of dicyclopropyl ketone imine hydrochloride with an amino
acid ester followed by sodium cyanoborohydride or sodium
triacetoxyborohydride reduction.
Supporting Information Available: Experimental pro-
cedures and compound characterization data. This material
isavailablefreeofchargeviatheInternetathttp://pubs.acs.org.
OL901310Q
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