An expedient synthesis of peptidyl N-alkylamides by "hOPE" strategy

Article abstract of DOI:10.1016/j.cclet.2012.03.006

We have developed an expedient approach, "HOPE" (hybrid orthogonal protocol with ease) strategy for the synthesis of peptidyl N-alkylamides. This new strategy was characterized by following points: incorporating Boc and Fmoc protocols together on Merrifie

Full text of DOI:10.1016/j.cclet.2012.03.006

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 565–568
www.elsevier.com/locate/cclet
An expedient synthesis of peptidyl N-alkylamides
by ‘‘HOPE’’ strategy
*
Hao Lin, Xiao Xiao Yang, De Xin Wang
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation Beijing 100050, China
Received 22 December 2011
Available online 18 April 2012
Abstract
We have developed an expedient approach, ‘‘HOPE’’ (hybrid orthogonal protocol with ease) strategy for the synthesis of
peptidyl N-alkylamides. This new strategy was characterized by following points: incorporating Boc and Fmoc protocols together
on Merrifield resin, removal of SPG (side-chain protecting groups) without the damage of linker structure on the resin, and the
ammonolysis of linker as the last step could achieve the introducing N-alkylamide structure into C-terminal and releasing product
from resin-support simultaneously. In present work, eight peptidylamides with different alkylsubstitution at C-terminal were
conveniently synthesized by HOPE strategy.
# 2012 De Xin Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: ‘‘HOPE’’ strategy; Solid-phase peptide synthesis; Peptidyl N-alkylamide
Many C-terminal modified peptides are used as active pharmaceutical ingredients (APIs), such as leuprolide [1],
DAGO [2], octreotide [3], and some of them have been applied in clinic for years. Also some C-terminally mutant
peptides are found in nature and have potential interest as therapeutic agents [4]. Usually, it is difficult to prepare C-
terminal modified peptide by conventional solid-phase peptide synthesis (SPPS), because the final products released
from resin always have a COOH or CONH2 structure at C-terminal. To our knowledge, some labs have been engaged
in the synthesis of peptide N-alkyl amides, and the general ideas they focused were as follows: (1) total liquid-phase
synthesis [5]; (2) Boc protocol on Merrifield resin followed by ammonolysis and HF deprotection [6]. (3) Fmoc
protocol on aryl hydrazide resin followed by ammonolysis and TFA deprotection [7]; (4) Fmoc protocol on 2-Cl-Trt
resin followed by liquid-phase coupling of C-terminal carboxylic acid (liberated from Trt resin by 5% TFA-DCM)
with N-alkyl amine [8,13]; (5) Fmoc protocol on N-alkyl linker resin followed by photolysis or TFA cleavage [9–11].
It is obvious that some protocols related in references are probably not suitable for large scale production by
following points: (A) SPG (side-chain protecting group) deprotection by HF treatment in Boc protocol; (B) additional
liquid-phase coupling of protected peptide C-terminal carboxylic acid with N-alkyl amines; or (C) special linker resins
with limited N-alkyl substitution were used instead of case B. Hence, there is a need to develop a more practicable
approach to the on-resin synthesis of C-terminal modified peptides. We envisaged that the key points in this new
* Corresponding author.
E-mail address: wangdx@imm.ac.cn (D.X. Wang).
1001-8417/$ – see front matter # 2012 De Xin Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2012.03.006

566
H. Lin et al. / Chinese Chemical Letters 23 (2012) 565–568
Scheme 1. Retrosynthetic route of peptidyl N-alkylamide.
Scheme 2. Synthesis of peptidyl N-alkylamide.

H. Lin et al. / Chinese Chemical Letters 23 (2012) 565–568
567
approach should be: (1) the incorporation of Fmoc and Boc protocol on Merrifield resin together; (2) keeping the
benzyl ester linker integrity during SPG deprotection; (3) Fmoc protocol must be applied for the sake of all SPGs
should be removed without the damage to resin linker; (4) ammonolysis as the multi-functional step for introducing N-
alkyl amide structure and liberating product from resin simultaneously. We would like to represent this new approach
in brief terms of HOPE strategy based on the feature: hybrid orthogonal protocol with ease.
In present work, the synthesis of eight peptidyl amides with different N-alkyl substitution was disposed based on the
following retrosynthetic analysis (Scheme 1).
For HOPE strategy, Merrifield resin was our prior choice of solid support, because of the benzylester linkage was
stable to TFA in the course of eliminating a-N-Boc group and SPGs related in Fmoc protocol, and easy to be
ammonolized by amine to release the targeted product: peptidyl N-alkylamide.
A crucial point in HOPE strategy is all SPGs must be removed by TFA condition without the damage to benzylester
linker on the resin. Therefore, Fmoc protocol would be qualified and be disposed only after Boc protocol. In other words,
some residues with chemically inert side-chain, such as Ala, Gly, Ile, Leu, Phe, Pro and so on, could be assembled before
the performance of Fmoc Protocol by Boc protocol if they are located in the region of C-terminal (Scheme 2).
In present synthesis, some non-proteinogenic residues, such as Aca (6-aminocaproic acid) and b-Ala were
introduced for products 1, 2 and 4–8, acting as a form of structure modification. Different amines were used in
ammonolysis aimed at enhancing C-terminal structural diversity. In order to understand the different yields of
ammonolysis with different blocking amines, compounds 5–8 were designed, they had the same sequence but different
alkyl substitutions of the C-terminal. The overall yield of products was calculated by integrating the yield of sequential
assembly on solid-support based on the resin-weight increment and the yield of ammonolysis (Table 1).
From Table 1, it is evident that the yields of crude products are dependent mainly on the yields of ammonolysis. The
yields of product 1–4, 7 and 8 are quite reasonable. Moderate yields and low purity of peptides 5 and 6 were obtained
and indicated the effect of stereohindrance from isopropyl and dimethyl groups on N atom was conspicuous.
For comparing HOPE strategy with conventional strategy, peptide 2 and 4 were also prepared by standard Fmoc
protocol [12], the synthetic route of compound 4 was presented (Scheme 3).
Table 1
The yields, purity and ESI-MS analysis of compound 1–8.
Compound C-terminal Yields of Yields of
structure sequential ammonolysis yields of
assembly crude products separation
Overall
Purity before Purity after Overall yields of Calculated ESI m/z obsd.
HPLC
HPLC
final products
molecular
weight
separation
1
2
3
4
5
6
7
8
CONHMe 87.9%
CONHMe 95.5%
95.1%
96.2%
96.0%
93.6%
50.6%
71.5%
93.5%
95.8%
83.6%
91.9%
76.0%
85.5%
40.7%
57.5%
89.5%
93.0%
85.0%
94.2%
78.5%
70.2%
65.7%
55.7%
76.7%
81.8%
97.65%
98.72%
96.64%
95.46%
94.73%
93.25%
94.61%
95.87%
62.9%
74.5%
51.7%
47.8%
21.3%
27.8%
57.3%
64.6%
877.49
1415.75
1100.63
598.45
878.4981 MH+
1416.7555 MH+
1101.6226 MH+
599.4687 MH+
1015.5132 MH+
1001.4449 MH+
1015.5157 MH+
1001.5009 MH+
CONHEt
CONHPr
79.2%
91.1%
CONHi-Pr 80.5%
CONHMe2 80.5%
1014.52
1000.51
1014.52
1000.51
CONHPr
CONHEt
95.7%
97.0%
Table 2
Benefit comparison between HOPE and Fmoc protocol in the synthesis of products 2 and 4.
Protocol
Resin
Fmoc protocol
Cl-Trt-
HOPE protocol
Merrifield
Liquid-phase reaction
Coupling steps
2
0
2
4
2
4
2
4
11
10
9
8
Yields of amine introduced to C-terminal
86.3%
85.1%
81.9%
71.6%
96.2%
93.6%
91.9%
85.5%
Total yields of crude product

568
H. Lin et al. / Chinese Chemical Letters 23 (2012) 565–568
Scheme 3. Synthesis of peptidyl N-alkylamide compound 4 by Fmoc strategy.
It was obvious that two extra-steps, coupling with propylamine and deprotection with TFA in solution phase, were
indispensable in conventional Fmoc strategy. Compared with HOPE strategy, standard Fmoc protocol was more
tedious and lower yield (Table 2).
In summary, we have developed HOPE strategy for solid-phase synthesis of peptidyl N-alkylamide. This new
strategy was mainly characterized by the incorporation of Fmoc/Boc chemistry on Merrifield resin, removal of SPGs
without the damage of linkage and the ammonolysis of linker at the last step to achieve both the assembling of C-
terminal N-alkylamide and releasing final product from resin support simultaneously. In view of being more feasible
and more economical, HOPE strategy would be a reasonable way to synthesize C-terminal modified peptides,
especially peptidyl N-alkylamides.
References
[1] P. Chrisp, E.M. Sorkin, Drugs & Aging 1 (1991) 487.
[2] J. Kowalski, Neuropeptides 32 (1998) 301.
[3] S.W. Lamberts, N. Engl. J. Med. 334 (1996) 246.
[4] M.F. Songster, G. Baramy, Methods Enzymol. 289 (1997) 126.
[5] D.H. Coy, A.V. Schally, Ann. Clin. Res. 10 (1978) 139.
[6] John J. Nestor Jr., B. Vickey, US Patent, US4690916.
[7] Y.H. Woo, A.R. Mitchell, J.A. Camarero, Int. J. Pept. Res. Ther. 13 (2007) 181.
[8] H.Y. Xu, Q. Zhu, Chinese Patent, CN101195653A.
[9] A. Ajayaghosh, V.N.R. Pillai, J. Org. Chem. 55 (1990) 2826.
[10] R. Ramage, S.L. Irving, C. Mclnnes, Tetrahedron Lett. 34 (1993) 6599.
[11] S. Mourtas, C. Katakalou, A. Nicolettou, et al. Tetrahedron Lett. 44 (2003) 179.
´ ´
[12] J. Bodi, H. Su¨li-Vargha, K. Ludanyi, et al. Tetrahedron Lett. 38 (1997) 3293.
[13] K.G. Estep, C.E. Neipp, L.M. Stephens Stramiello, et al. J. Org. Chem. 63 (1998) 5300.