C O M M U N I C A T I O N
Enzyme-cleavable linkers for peptide and glycopeptide synthesis
Beatrice A. Maltman, Mallesham Bejugam and Sabine L. Flitsch*
School of Chemistry, Edinburgh University, Kings Buildings, West Mains Road, Edinburgh,
UK EH9 3JJ. E-mail: Sabine.Flitsch@manchester.ac.uk; Fax: 01316504719; Tel: 01316504719
Received 3rd May 2005, Accepted 31st May 2005
First published as an Advance Article on the web 16th June 2005
Hydroxymethylphenoxy linkers that are commonly used in
solid phase peptide synthesis are surprisingly susceptible to
efficient cleavage by the protease chymotrypsin with a broad
range of amino acid residues being tolerated at the scissile
bond; this enzyme-cleavable linker system has been applied
to peptide and glycopeptide synthesis.
Chymotrypsin is well known to be specific for phenylalanine
and tyrosine residues in the P1 site5 and thus the PheAsp bond
should present an excellent substrate site for this protease. The
ester bond of the Wang linker, on the other hand, was flanked by
a negatively charged amino acid (Asp) in the pseudo P1 position
(i.e. the position that would be occupied by the P1 residue in
the corresponding amide) and such a substrate would not be
expected to be cleaved by chymotrypsin. Also surprising was the
stability of dipeptide 2 as the major product in the reaction
mixture: even if 2 was initially formed by ester hydrolysis,
one would expect it to be hydrolysed rapidly in solution by
chymotrypsin. This was further investigated by incubation of
a pure sample of 2 with chymotrypsin in solution; however,
no hydrolysis product 3 was observed, possibly due to the
insolubility of 2 in aqueous buffer.
The current trend for miniaturisation and automation of syn-
thesis and analysis at the interface of chemistry and biology has
resulted in an increasing demand for synthetic tools that can
be used on solid supports, and which are also biocompatible.
Biocatalysts are particularly attractive tools because they can
operate under mild, physiological aqueous conditions with
very high chemo-, regio- and stereo-selectivity. Thus, several
groups have described enzyme-cleavable linkers which allow
for the release of a variety of different compounds from solid
supports through enzyme-mediated hydrolysis.1–3 In general,
such linkers have been specifically designed and synthesized to
incorporate enzyme recognition motifs, some of which remain
as tags in the compound library. The need for such motifs
has limited the application of enzyme-cleavable linkers. In this
paper, we describe the unexpected discovery that the protease
chymotrypsin can cleave ester linkers that are commonly
employed in solid phase peptide synthesis, such as the Wang
linker (hydroxymethylphenoxyacetic acid, HMPA). Cleavage is
not limited to peptides containing C-terminal aromatic amino
acids normally associated with chymotrypsin recognition motifs
but can be used for a wide range of peptide sequences. The
application of such enzyme-cleavable linkers to solid phase
peptide and glycopeptide synthesis is also described.
The interesting lability of the commonly used Wang linker to
chymotrypsin-catalyzed hydrolysis had not been reported before
and was further investigated. First of all, the C-terminal amino
acid residue attached to the Wang linker was replaced by amino
acids with diverse functionalities to investigate the tolerance of
chymotrypsin at the ‘pseudo P1’ site of the ester. A series of
resin-bound Fmoc-L-amino acids incorporating the acid-labile
Wang linker was synthesized and subjected to treatment with
chymotrypsin (2 mg ml−1 in 0.1 M potassium phosphate buffer
pH 8) for 16 h (Table 1). Release of the Fmoc-protected amino
acid was monitored by high performance liquid chromatography
after the specified reaction time. The results show that complete
hydrolysis was achieved for Fmoc-L-amino acids containing
hydrophobic (entries 1, 2 and 3), polar (entry 4) and charged
(entries 5 and 6) side-chains. FmocGly 5g appeared to be the
only substrate obtained in poor conversion (entry 7).6 Hydrolysis
was also possible, but much more sluggish, with substrates
containing larger hydrophobic protecting groups (e.g. entry 8).
The Wang linker is popular in Fmoc peptide synthesis
because of its acid lability. However, acid lability did not
seem to be important for chymotrypsin hydrolysis, since the
base-labile HMBA (hydroxymethylbenzoic acid) linker was also
readily cleaved by the enzyme (Table 1, entries 9–11). Thus,
chymotrypsin can be employed to cleave both acid- and base-
labile ester linkers and might be useful for orthogonal linker
strategies.
In the present studies, the chymotrypsin-catalyzed cleavage
of peptides such as 1 (Scheme 1), which were linked through a
standard Wang linker to PEGA1900 [a copolymer of poly(ethylene
glycol) and polyacrylamide], was investigated. PEGA was
chosen as the support because it has been shown to provide
fully accessible functionalized sites for enzyme catalysis.4 Rather
unexpectedly, the predominant hydrolysis product of 1 was the
dipeptide FmocPheAsp 2 as opposed to the expected FmocPhe
3, with 2 and 3 formed in a ratio of 2 : 1 respectively (Scheme 1).
Work reported by Bordusa and colleagues7 has shown that
chymotrypsin can accept activated esters of aromatic alcohols,
in which the leaving group (which would be the Wang linker in
structure 4) binds to the aromatic pocket (S1 site). To investigate
if the present linker presents such a substrate mimetic, the non-
aromatic hydroxyoctanoic acid (HOA) was investigated as a
third ester linker system. Again, enzymatic hydrolysis appeared
to be unaffected by the change in linker structure (entries 12–14),
and suggests that the selective ester cleavage by chymotrypsin in
4 might be a result of the much more labile ester bond rather
than due to any additional recognition of the aromatic group of
the HMPA or HMBA linker.8
A great advantage of biocatalysis over chemical synthesis
is that reactions can often be highly stereoselective. Thus,
chymotrypsin is known to be highly specific for L-amino acids.
Such stereospecificity was also observed for the present linker
cleavage: whereas the L-amino acid in 4a was efficiently cleaved
Scheme 1 Enzymatic hydrolysis of solid-supported FmocPheAsp.
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 0 5 – 2 5 0 7
2 5 0 5
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