Synthesis and purification of aggregation-prone hydrophobic peptides by the incorporation of an Fmoc dipeptide with the peptide bond protected with a modified 2-hydroxy-4-methoxybenzyl (Hmb) group

Article abstract of DOI:10.1016/j.tetlet.2008.04.055

The dipeptide Fmoc-Val-(2-hydroxy-4-methoxybenzyl)Gly-OBzl was synthesized and the 2-hydroxyl group carbamoylated to give a Boc-N(CH₃)CH₂CH₂N(CH₃)CO-, (Boc-Nmec-) modification of the 2-hydroxy-4-methoxybenzyl (Hmb) group. After catalytic hydrogenation and purification, the resulting dipeptide Fmoc-Val-(Boc-Nmec-Hmb)Gly-OH was used in solid phase peptide synthesis. During treatment with TFA, the peptide was released from the resin and the Boc group cleaved. The peptide could then be purified with an alkylated peptide bond carrying a cationic charge that both increased the solubility of the peptide during the purification steps and facilitated analysis by MALDI-TOF mass spectrometry. The Nmec group was cleaved by intramolecular cyclization under slightly alkaline conditions, followed by cleavage of the Hmb group by TFA to give the fully deprotected peptide.

Full text of DOI:10.1016/j.tetlet.2008.04.055

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3921–3924
Synthesis and purification of aggregation-prone hydrophobic
peptides by the incorporation of an Fmoc dipeptide with
the peptide bond protected with a modified 2-hydroxy-
4-methoxybenzyl (Hmb) group
*
´
Karolina Wahlstro¨m, Ove Planstedt, Anders Unden
Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
Received 13 March 2008; revised 31 March 2008; accepted 9 April 2008
Available online 12 April 2008
Abstract
The dipeptide Fmoc-Val-(2-hydroxy-4-methoxybenzyl)Gly-OBzl was synthesized and the 2-hydroxyl group carbamoylated to give a
Boc-N(CH3)CH2CH2N(CH3)CO–, (Boc-Nmec-) modification of the 2-hydroxy-4-methoxybenzyl (Hmb) group. After catalytic hydroge-
nation and purification, the resulting dipeptide Fmoc-Val-(Boc-Nmec-Hmb)Gly-OH was used in solid phase peptide synthesis. During
treatment with TFA, the peptide was released from the resin and the Boc group cleaved. The peptide could then be purified with an
alkylated peptide bond carrying a cationic charge that both increased the solubility of the peptide during the purification steps and facil-
itated analysis by MALDI-TOF mass spectrometry. The Nmec group was cleaved by intramolecular cyclization under slightly alkaline
conditions, followed by cleavage of the Hmb group by TFA to give the fully deprotected peptide.
Ó 2008 Elsevier Ltd. All rights reserved.
Aggregation of the peptide chain during peptide synthe-
sis is a major problem in both Boc and Fmoc chemistries. It
results in incomplete acylation of the growing peptide
chain and is the major source of deletion peptides. Incom-
plete acylation is usually caused by b-sheet formation,
which has been studied extensively. Several methods to
reduce the extent of the problem have been put for-
ward.1–6 Although much research has been devoted to solv-
ing problems associated with peptide aggregation during
the synthesis of the peptide, relatively few studies have con-
centrated on the difficulties encountered after synthesis.
RP-HPLC of poorly soluble peptides is a major practical
problem resulting in low loading of the peptide onto the
column, poor resolution and low yields. In addition,
hydrophobic and aggregation-prone peptides are often
very difficult to characterize by mass spectrometry because
of poor ionization. These difficulties are especially severe
during the purification of transmembrane sequences of
integral membrane proteins, which contain long stretches
of hydrophobic amino acid residues. In this case, the intro-
duction of a C-terminal thioester building block containing
several arginine residues has been reported to result in a
large improvement in solubility.7,8 This strategy is, how-
ever, dependent on the use of Boc chemistry to synthesize
the C-terminal thioesters and the in situ neutralization
method to counteract aggregation during the coupling
steps; thus, this method is not directly relevant to Fmoc
chemistry. The same concept has already been exploited
by Choma et al. using a ‘solubilising tail’ to be used in both
Boc and Fmoc chemistries.9–11 Addition of cationic charges
to the terminals of the peptide chain is, however, unlikely
to have a major effect on b-sheet formation.
An efficient general strategy to counteract b-sheet
formation during the synthesis of a peptide is reversible
modification of selected peptide bonds.12,13 Reversible
protection of the peptide backbone using the
*
Corresponding author. Tel.: +46 8 164268; fax: +46 8 16 1371.
´
E-mail address: andersu@neurochem.su.se (A. Unden).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.055

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K. Wahlstro¨m et al. / Tetrahedron Letters 49 (2008) 3921–3924
N-(2-hydroxy-4-methoxybenzyl) (Hmb) group was intro-
duced by Johnson et al.14 With this method, selected resi-
dues are introduced as N,O-bis-Fmoc-N-(2-hydroxy-4-
methoxybenzyl) amino acids, the Fmoc groups are cleaved
with piperidine, and the coupling of the next residue to the
relatively unreactive secondary amino group is facilitated
by an intramolecular N)O acyl transfer. Hmb-protected
peptides attached to a solid phase are less prone to b-sheet
formation and coupling of the remaining residues usually
proceeds with significantly enhanced efficiency.15,16 This
method has an important additional advantage and can,
through the use of a modified protocol, be used to facilitate
the purification of synthetic peptides. If the phenolic
hydroxyl groups are acetylated prior to cleavage of the
peptide with TFA, the peptide can be isolated and purified
with acetylated Hmb groups still attached to the peptide
backbone.17 This increases the solubility of peptides that
are prone to b-sheet formation and thereby improves the
purification yield. This has been demonstrated by the syn-
thesis and purification of the notoriously difficult b-sheet-
prone amyloid b (1–43) peptide.18 However, there are some
potential difficulties associated with this strategy. During
solid phase peptide synthesis (SPPS) the phenolic hydroxyl
group of Hmb is acylated, thereby consuming up to 1 equiv
of activated amino acid in each coupling step. Further-
more, the extent of O-acetylation of the Hmb group at
the end of the synthesis cannot be monitored and in the
synthesis of amyloid b (1–43) this acetylation step was
therefore performed by an extended overnight treatment
with anhydride/diisopropylethylamine. This prolonged
treatment can be expected to lead to acetylation of the p-
nitrogen of protected histidine side chains, and possibly
even other, less reactive groups such as amide nitrogens.
Acetylation of Hmb groups will also increase the hydro-
phobicity of the Hmb-protected peptide, which generally
tends to reduce the solubility of the peptide and thereby
render the purification more difficult.
tide chain as dipeptides. This strategy is limited to dipep-
tides with C-terminal glycine residues, as activation of
chiral residues in peptides is prone to racemization. In this
context, however, glycine is an important amino acid as it
is one of the six most frequently occurring residues in
hydrophobic transmembrane sequences, often occurring
in pairs with valine and isoleucine residues.19
One advantage of using dipeptides with modified Hmb
groups would be avoidance of the modification of the
Hmb groups at the end of the synthesis, however, this
requires that the modified Hmb groups are stable to both
piperidine and TFA yet can be rapidly removed under mild
reaction conditions.
Phenolic carbamates are known to be very stable to both
nucleophiles, bases, and acids.20–22 We therefore investi-
gated the possibility of using Hmb-protected dipeptides
where the phenolic oxygen in Hmb is protected with a
Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl group
(Boc-Nmec, compound 1, Scheme 1). This protecting
group had previously been investigated as an intermediate
in the synthesis of cyclization-activated prodrugs of pheno-
23
lic compounds, and we have recently used Boc-Nmec as
a protective group for tyrosine residues in SPPS (unpub-
lished results). During cleavage of the peptide from the
resin with TFA, the Boc group was removed but the pro-
tonated methyl-(2-methylamino-ethyl) carbamoyl group
remained attached to the Hmb group 3. In this form, pep-
tide will generally have a greatly increased solubility in
aqueous solutions as a result of both the b-sheet-disrupting
effect of a benzylated peptide bond and the solubilizing
effect of the cationic charge. After purification, the peptide
was exposed to slightly alkaline reaction conditions during
which an intramolecular cyclization reaction produced the
Hmb-protected peptide 4 and N,N0-dimethylimidazoldione
(5). The Hmb group can be removed with TFA. In the fully
deprotected form, the peptide 6 could be isolated either by
an additional chromatographic step or, for aggregation-
prone peptides, simply by precipitation.
Despite these potential difficulties, synthesis of peptides
with modified Hmb groups that are stable during the TFA
cleavage step is a very attractive concept, as it can greatly
facilitate both the synthesis and the purification of many
hydrophobic and b-sheet-prone peptides.
We therefore synthesized Fmoc-Val-(Boc-Nmec-
Hmb)Gly-OH (12) as outlined in Scheme 2. The Hmb
derivative of the benzyl ester of glycine 9 was obtained
by the reductive alkylation of H-Gly-OBzl (7) with
hydroxymethoxybenzyl aldehyde (8) in ethanol, and the
free base was isolated as a crystalline compound. Acylation
One method that can be used to avoid some of these
problems is to introduce modified Hmb groups into a pep-
Scheme 1. General scheme for the use of Fmoc dipeptides with peptide bonds protected with modified 2-hydroxy-4-methoxybenzyl groups, R@CH(CH3)2.
(a) Peptide synthesis; (b) TFA, RP-HPLC purification; (c) DMF/water, N-methylmorpholine; (d) TFA, diisopropylsilane.

K. Wahlstro¨m et al. / Tetrahedron Letters 49 (2008) 3921–3924
3923
Scheme 2. Synthesis of Fmoc-Val-(Boc-Nmec-Hmb)Gly-OH. (a) Sodium borohydride in ethanol; (b) Fmoc-L-Val-OH/N,N0-diisopropyl carbodiimide in
DCM; (c) p-nitrophenylchloroformate/diisopropylethylamine in DCM; (d) Mono-Boc-N,N0-dimethylethylenamine/diisopropylethylamine in DCM; (e)
H2/10% Pd on charcoal in ethanol.
of 9 with Fmoc-Val-OH/N,N0-diisopropylcarbodiimide
gave the dipeptide Fmoc-Val-(Hmb)Gly-OBzl (10) as a
solid foam after purification by dry flash chromatography.
The phenolic hydroxyl group was activated with p-nitro-
phenylchloroformate followed by treatment with mono-
Boc-N,N0-dimethylethylenamine to give carbamate 11.
Introduction of the Boc-Nmec group resulted in a large
increase in retention time of product 11 in TLC systems,
and facilitated purification by dry flash chromatography.
The benzyl ester was removed by catalytic hydrogenation,
and the extent of the reaction was followed by TLC. The
Fig. 1. (A) RP-HPLC elution profile of the Nmec-Hmb backbone-
reaction was terminated when traces of 9-methylfluorene
were detected by TLC. After purification by dry flash chro-
matography, the protected dipeptide 12 was obtained as a
protected amyloid b (35–42) peptide; MV(NmecHmb)GGVVIA-OH B.
Amyloid b (35–42) (MV(Hmb)GGVVIA-OH) after the treatment of
the purified peptide for 8 h with 30% DMF/water and 10 equiv of
N-methylmorpholine.
1
solid foam in an overall yield of 45%. The H NMR and
13C NMR spectra were in accordance with the expected
structure (see Supplementary data).
and the product was precipitated five times with diethyl
ether. The resulting Hmb-protected peptide was redis-
solved in acetonitrile/water, lyophilized, and purified by
RP-HPLC.
Incorporation of dipeptide 12 into peptides during solid
phase peptide synthesis generally proceeded more slowly
than for Fmoc amino acids, but acylation of a b-branched
residue such as valine was complete after 4 h when coupled
as HOAt esters in NMP.
In order to estimate the effect of dipeptide 12 on the sol-
ubility of a very aggregation-prone peptide, amyloid b (35–
42) (MVGGVVIA) was synthesized (Fig. 1). After cleavage
from the resin, the peptide MV(NmecHmb)GGVVIA-OH
had a solubility of more than 50 mg/ml in 50% acetoni-
trile/water while the unprotected peptide was almost insol-
uble (solubility <1 mg/ml).
The rate of the intramolecular cyclization reaction had
been investigated previously for a hydroxyanisole deriva-
tive in aqueous solutions by Saari and co-workers, and
the half-life at pH 7.4 was found to be 36.3 min.23 Water
is, however, generally a very poor solvent for hydrophobic,
b-sheet-prone peptides and we therefore required a more
general solvent system. The cyclization reaction was slow
in the aprotic solvent DMF, but we found that DMF/water
was a better solvent system. The reaction was studied using
the model peptide KG(HmbNmec)VAKKKA-OH, which
was designed to have high solubility even without a proton-
ated Nmec group. The cyclization proceeded smoothly in
4 h in 30% DMF/H2O using N-methylmorphiline as base.
DMF was removed by evaporation under reduced pressure
The Nmec-Hmb group also facilitated analysis of the
peptides by MALDI-TOF mass spectrometry. For the
unmodified amyloid b (35–42) only weak signals with poor
signal-to-noise ratios were obtained. This is probably the
result of rapid aggregation of the peptide on the matrix,
as we observed precipitation of the peptide within 15–
30 min during the preparation of the sample. To a lesser
extent, the same problems were encountered for amyloid
b (35–42) where Gly37 was modified with the Hmb group.
In contrast, we always recorded robust signals and excel-
lent signal-to-noise ratios for the same peptide protected
with Nmec-Hmb. However, peptides modified with the
Nmec-Hmb group were not completely stable in
MALDI-TOF mass spectrometry. Even for purified pep-
tides that appeared as homogenous peaks in HPLC, there
was always partial cleavage of the carbamate group, as
peptides with molecular masses of 114 less than expected
were evident.
In conclusion, the problems associated with post-syn-
thetic handling of peptides are frequently underestimated.
These problems are inherent properties of the particular
peptide sequence and are usually very difficult to counter-
act without reversible covalent modifications of the

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frequently occur in large stretches of hydrophobic peptides,
it is therefore likely that the use of the protected dipeptides
discussed in this study will result in a major improvement
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peptides.
Supplementary data
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Experimental details for the synthesis of Fmoc-Val-
(Boc-Nmec-Hmb)Gly-OH. 13C NMR for Fmoc-Val-(Boc-
Nmec-Hmb)Gly-OH. Supplementary data associated with
this article can be found, in the online version, at
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