TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 8693–8696
Covalent capture purification of polypeptides after SPPS via a
linker removable under very mild conditions
Jean Vizzavona,† Matteo Villain‡ and Keith Rose*
Department of Medicinal Chemistry, University Medical Center, Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland
Received 14 June 2002; revised 24 September 2002; accepted 25 September 2002
Abstract—The covalent purification of polypeptides possessing an N-terminal cysteine or threonine residue via formation of a
thiazolidine or oxazolidine with an aldehyde-functionalized-resin has been successfully demonstrated. To extend the applicability
of this approach to any possible N-terminal residue, a special linker derived from (S)-4-amino-2-hydroxy-butyric acid was
incorporated into peptidyl-resin. This linker represents the connecting point between the capture unit (cysteine) useful for the
isolation of the desired polypeptide and the desired N-terminus. The target polypeptide was recovered by periodate oxidation,
which cleaved the covalent bond between the linker and the last residue of polypeptide under very mild conditions. © 2002
Elsevier Science Ltd. All rights reserved.
After solid-phase peptide synthesis (SPPS), whether the
Boc or Fmoc strategy is used, the purification step is a
major problem to obtain the desired peptide with a
satisfactory yield, completely devoid of the accumula-
tion of truncated or deleted chains generated on the
resin.
cifically captured from
a
complex mixture by
chemoselective reaction or affinity chromatography. A
variety of handles have already been reported in the
literature, including ones used in covalent1 or in
affinity-type purification of the desired peptide.2–8 How-
ever, none of these methods can be acceptable for a
fully deprotected peptide due to the harsh conditions
required for handle removal (cyanogen bromide, pipe-
ridine, high pH). Therefore, their use has been limited.
Many groups1–6 have sought to functionalize the N-ter-
minal residue of full-length peptides with a removable
purification handle as a final step in SPPS. After cleav-
age from the resin, these handled-peptides can be spe-
As reported recently in the literature,9 covalent purifica-
tion of polypeptides possessing either N-terminal cys-
teine or threonine residue can be successfully applied
after SPPS to isolate the desired compound via the
specific formation of a thiazolidine or oxazolidine
ring.10
Abbreviations: HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3,tetramethyl-
uronium hexafluorophosphate; BOP, 2-(1H-benzotriazole-1-yl)-oxy-
tris(dimethylamino)phosphonium hexafluorophosphate; HOAt, 1-
hydroxy-7-aza-benzotriazole; DIEA, diisopropylethylamine; DCCI,
dicyclohexylcarbodiimide; NaBH3CN, sodium cyanoborhohydride;
BrBzl, bromide benzyl; AcOH, acetic acid; Boc2O, di-tert-butyl dicar-
bonate; Boc, tert-butyloxycarbonyl; LiAlH4, lithium aluminum
hydride; ZCl, benzyloxycarbonylchloride; HCl·HN(OCH3)CH3, N,O-
dimethylhydroxylamine hydrochloride; NaIO4, sodium metaperio-
date; TFA, trifluoroacetic acid; HF, liquid hydrogen fluoride; DCM,
dichloromethane; DMF, dimethylformamide; THF, tetrahydrofuran;
MeOH, methanol; SPy, S-pyridine; TCEP, tris(2-carboxyethyl) phos-
phine hydrochloride; CH3CN, acetonitrile; HPLC, reversed-phase
high performance liquid chromatography; rt, room temperature.
Keywords: covalent capture; purification; removable linker; periodate
oxidation; large polypeptide.
Here, we present an extension of this approach using a
special linker, the 4-amino-2-hydroxy-butyryl group
(Ahb) linked to the last residue of a growing peptide
chain. As shown in Fig. 1, we synthesized an appropri-
ate Ahb derivative (4) before coupling it into peptidyl-
resin,
starting
from
commercially
available
(S)-4-amino-2-hydroxy-butyric acid (1). After Boc
deprotection, we coupled a cysteine residue to the free
amine. This generated a peptide possessing all the char-
acteristics necessary for covalent capture purification.
* Corresponding author. Present adress: GeneProt Inc., CH-1217
One great advantage of using the Ahb group in protein
purification is demonstrated by the removal conditions:
this group can be efficiently and completely removed by
periodate oxidation (NaIO4), a procedure known not to
† Present adress: RMF Dictagene S.A., En Marin, CH-1014 Lau-
sanne, Switzerland.
‡ Present adress: GeneProt Inc., CH-1217 Meyrin/Geneva, Switzer-
land.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02110-X