Solid phase synthesis of aspartyl peptide aldehydes

Article abstract of DOI:10.1016/S0040-4039(00)01615-4

An efficient method for the solid phase synthesis of aspartyl aldehyde peptides has been developed. It uses a low cost synthetic process for the preparation of Fmoc-protected Weinreb amide linker. This procedure covers several tetrapeptide aspartyl aldehydes as well as biotinylated tetrapeptide aspartyl aldehydes. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01615-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8857–8860
Solid phase synthesis of aspartyl peptide aldehydes
Xiao-He Tong* and Anita Hong
AnaSpec Inc., 2149 O’Toole Ave. cF, San Jose, CA 95131, USA
Received 3 August 2000; accepted 18 September 2000
Abstract
An efficient method for the solid phase synthesis of aspartyl aldehyde peptides has been developed. It
uses a low cost synthetic process for the preparation of Fmoc-protected Weinreb amide linker. This
procedure covers several tetrapeptide aspartyl aldehydes as well as biotinylated tetrapeptide aspartyl
aldehydes. © 2000 Elsevier Science Ltd. All rights reserved.
Substrate-based inhibitor designs have led to the synthesis of some tetrapeptide aspartyl
aldehydes. These aldehydes have been found to be inhibitors of caspases.1–5 Thus it is desirable
to develop a simple and efficient solid phase synthetic method of producing aspartyl peptide
aldehydes.
Two methods for the solution synthesis of aspartyl peptide aldehydes have been described in
the literature. The first method, the aspartyl aldehyde moiety was protected at the corresponding
O-benzylacylal, which can be coupled, and hydrogenolyzed to afford the desired compound.3
Another general strategy afforded the racemization-free peptidic aspartyl aldehydes by using a
semicarbazone derivative.4 There are several studies concerning the solid phase synthesis of
peptide aldehydes that have been published.6–14 However, only one study has been concerned
with aspartyl peptide aldehyde.13
In this paper we describe a convenient synthetic method of tetrapeptide aspartyl aldehydes
from their corresponding Weinreb amides resin by using lithium aluminium hydride. Based on
Fehrentz’s work6,7 a low cost synthetic route for preparation of Fmoc-protected Weinreb amide
linker has been developed as shown in Scheme 1.
* Corresponding author. Tel: 4085260554; fax: 4084525059; e-mail: xhtong@yahoo.com
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01615-4

8858
Scheme 1. Synthesis of the Weinreb amide linker
Methoxy-amine hydrochloride was reacted with low cost t-butyl acrylate in the presence of
DIEA, the resulting alkylated methoxy-amine 1 was protected with Fmoc group using FmocꢀCl
in the presence of Na2CO3 to obtain compound 2. After deprotection of the t-butyl group by
TFA/DCM (1:1), the solid Fmoc protected Weinreb amide linker 3 was obtained in three simple
steps with good yields.
Linker 3 was coupled to the MBHA resin with PyBOP to yield Fmoc protected Weinreb
MBHA resin. Peptide synthesis was performed using Fmoc strategy (Scheme 2). Following
removal of the Fmoc group with 20% piperidine in DMF, FmocꢀAsp(OtBu)ꢀOH was coupled
to the resin using HATU/DIEA activation. Other couplings were performed with a fourfold
excess of activated amino acid using an activation solution of Fmocꢀamino
acid:PyBOP:HOBt:DIEA (1:1:1:2). Upon completion of the chain assembly, the peptide–resin
was treated with TFA/scavenger to remove the side chain protecting groups, then the peptide
was reduced and cleaved from the resin with LiAlH4 (8–10 equiv.) in THF at −20°C to obtain
the peptide aldehyde15 (Table 1 and Fig. 1). It is important to remove side chain protecting
groups before the peptide is cleaved from solid phase, since the esters of aspartyl and glutamyl
are easily reduced to the hydroxyl groups with LiAlH4, even at −20°C. On the other hand, the
carboxylic groups are more stable, and they can not easily be reduced at the same conditions.
Scheme 2. SPPS of the tetrapeptide aspartyl aldehydes

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Table 1
Result of synthesis
Aldehyde peptide
% Yielda
Retention time, minb
Ac-Leu-Glu-His-Asp-H
Ac-Val-Glu-Ile-Asp-H
Ac-Asp-Gln-Met-Asp-H
Ac-Ile-Glu-Thr-Asp-H
Biotin-Asp-Glu-Val-Asp-H
Biotin-Glu-Ser-Met-Asp-H
25
20
25
25
20
15
9.7
11.1
7.9
9.0
7.8
9.1
a Yield of purified peptides based on the substitution of the resin.
b Retention time on RP HPLC, 1 ml/min, 0–60% B in 20 min. A: 0.1% TFA in H2O, and B: 0.09% TFA in CH3CN.
Figure 1. HPLC of the crude AcꢀIleꢀGluꢀThrꢀAspꢀH
For biotinylated tetrapeptide aspartyl aldehydes, more synthesis steps were needed in the
solution phase synthesis.5 Our method can directly and effectively synthesize these kinds of
compounds.
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15. The peptidyl-resin were suspended in anhydrous THF and placed in a cooling bath at −20°C. LiAlH4 (1 M in
THF; 8–10 mol equiv.) were slowly added and the reaction was stirred for 45 min. The reaction was then
hydrolyzed with 1 M potassium hydrogenosulfate solution. The resulting mixture was filtered in order to
eliminate the resin, which was washed twice with 1 M potassium hydrogenosulfate solution. The liquid phases
were gathered and concentrated under reduced pressure at room temperature to evaporate most of the THF. The
crude peptide aldehyde solution was checked by reversed phase HPLC of solutions A (0.1% TFA in H2O) and
B (0.09% TFA in CH3CN) as the solvent system (crude purity 60–85%) in a gradient mode from 100% (A) to 60%
(B) in 20 min and then immediately purified with preparative RP HPLC (pure compound yield after lyophiliza-
tion: 15–25%). We found aspartyl peptide aldehydes to be very unstable and one of the reasons why pure yields
were not very good. A study7 showed that purification of the peptide aldehyde by chromatography (silica gel or
reversed phase) induced a loss of the optical integrity of the C-terminal residue.
.