Automated synthesis of backbone protected peptides

Article abstract of DOI:10.1039/c4cc03065f

The synthesis of peptides rich in aggregation prone sequences can be improved with backbone protection. We report the automated introduction of backbone protection to a peptide. This new method was applied in a fully-automated synthesis, giving improved handling, quality and yield of several challenging target sequences. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03065f

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Automated synthesis of backbone protected
peptides†
Cite this: Chem. Commun., 2014,
50, 8316
Abu-Baker M. Abdel-Aal, George Papageorgiou, Martin Quibell and John Offer*
Received 24th April 2014,
Accepted 10th June 2014
DOI: 10.1039/c4cc03065f
www.rsc.org/chemcomm
The synthesis of peptides rich in aggregation prone sequences can and have been successfully applied to enable synthesis of difficult
be improved with backbone protection. We report the automated sequences11,12 and long peptides.13 However, their use is unfortu-
introduction of backbone protection to a peptide. This new method nately limited to those sequences containing conveniently positioned
was applied in a fully-automated synthesis, giving improved handling, X-Ser or X-Thr. Backbone amide protection was first investigated
quality and yield of several challenging target sequences.
(Dmb 2 Scheme 1) for its dramatic effect on peptide solubility.6 The
effect of Dmb 2 on improving peptide solubility was explored in
Solid phase peptide synthesis (SPPS) is a powerful technology for detail by Narita and co-workers.14 Current opinion considers that the
the chemical synthesis of peptides and small proteins. However, poor solubility observed for many peptide sequences in solution
access to many targets is often complicated and sometimes reappears on the solid phase as difficult peptides and backbone
precluded by the occurrence of so-called difficult sequences.1,2 protection acts in both cases by disrupting structure formation.1
When encountered during SPPS difficult sequences are associated A fully solvated peptide-resin should give the best coupling kinetics
with a collapse of the swollen resin volume, incomplete acylation and evidence from NMR and I.R. studies on aggregating peptides
and in the case of Fmoc/tBu synthesis, incomplete deprotection on solid phase supports this model.15,16 Many novel backbone
steps, extending over several residues.3,4 As the cause of difficult protection strategies have been developed but the increased steric
sequences is intermolecular chain association, double coupling hindrance that accompanies the introduction of a secondary amine
provides no improvement. Pioneering work by Sheppard and into a sequence prohibits quantitative coupling using standard
co-workers demonstrated that introducing proline into an aggregating conditions (except with glycine) and has limited the wider adoption
peptide sequence, before the onset of aggregation prevented inter- of these new backbone protection strategies.17 A solution to over-
chain association by removal of hydrogen bonding.1 By extension, come this obstacle harnessed an intramolecular acyl transfer. This
they also demonstrated that reversible alkylation of the peptide was achieved by the use of 2-hydroxy-4-methoxybenzyl (Hmb) 3
backbone suppressed interchain association and was a general which can be considered as a simple modification of Dmb 2
solution to the difficult sequence problem.5 Reversible substitution (Scheme 1). Acylation of the exposed 2-hydroxy position gave
of the amide bond was called ‘backbone protection’ when a phenyl ester, positioned for acyl migration through a con-
introduced by Weygand and co-workers because of similarities strained, six-membered ring. This procedure gave quantitative
to the protecting group strategies then in development.6 How- coupling under favourable conditions. However, the kinetics of acyl
ever, it is only rarely necessary to protect the amide bond itself.7 transfer were slow and variable between residues. Additionally, the
Amongst the many backbone protection groups available the optimised non-standard conditions used, consisting of a symmetric
most widely used are the commercially available pseudopro- anhydride in dichloromethane, were difficult to automate.5 Ideally,
lines (c-Pro 1 Scheme 1).8 Pseudoprolines have revolutionized for practical convenience the coupling onto the secondary amine
Fmoc/tBu synthesis by enabling the synthesis of previously
intractable peptides, unobtainable even by in situ neutralisation
Boc protocols.9 Their key advantage is that they can be intro-
duced with great convenience as dipeptide building blocks10
Scheme 1 Peptide backbone protection groups. (1) Pseudoproline(c-Pro),
R = H, Ser; R = CH3, Thr. (2) Dimethoxylbenzyl (Dmb); (3) 2-hydroxy-4-
methoxybenzyl (Hmb), (4) 2-hydroxy-4-methoxy-5-methylsulfinyl benzyl
(Hmsb).
MRC National Institiute for Medical Research, The Ridgeway, London, UK.
E-mail: joffer@nimr.mrc.ac.uk; Tel: +44 (0)20 8816 2082
† Electronic supplementary information (ESI) available: Synthesis and character-
ization of peptides, experimental conditions. See DOI: 10.1039/c4cc03065f
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should be performed under standard conditions. Clearly, with a sulfoxide substituted salicylaldehyde 8 in hand we first needed to
more reactive internal ester the acylation/migration step could prove that the salicylaldehyde could be installed quantitatively with
be accelerated. This had previously been achieved by modifying the sulfoxide intact. In contrast to many aldehydes where imine
Hmb with an electron withdrawing group para to the 2-hydroxy formation is slow, salicylaldehydes form stable imines rapidly.21
position.18–20 Based on these considerations, we chose a variant
Formation of the imine with a single equivalent of 8 gave an
of the sulfoxide modified Hmb previously reported by us Hmsb intense yellow colouration, the resin was washed to remove excess
4 (Scheme 2B).18,20 The use of sulfoxide, resolves the problem of how salicylaldehyde and treated with an additional single equivalent of
to introduce an electron withdrawing group to the backbone protec- salicylaldehyde before thoroughly washing with dimethylformamide
tion without making the modification irreversibly stable to acid, as it (DMF). NaBH4 in DMF was added and the strong yellow colouration
can be mildly reduced to the acid labile thioether. For a chemical of the Schiff base quickly faded. Analytical HPLC of a test cleavage
reaction to be generally applicable to the solid phase it has to be showed the presence of a single product with the correct mass of the
quantitative. Ede and co-workers had demonstrated introduction of target peptide with backbone protection attached, bearing intact
Hmb to a resin-bound peptide by exploiting the unusually stable sulfoxide 4 (ESI,† Fig. S1).
Schiff base,21 formed between a salicylaldehyde and resin-bound
This technology was demonstrated by the improved preparation of
primary amine, stable to washing and subsequently quantitatively a ‘difficult sequence’ derived from influenza virus Hemagglutinin,
reduced on resin.22 Therefore a successful demonstration of quanti- reported by Sampson and co-workers (Fig. 1).11 For comparison, the
tative on-resin reductive amination of salicylaldehydes, with a group test sequence was synthesised without backbone protection on rink
capable of assisting its own acylation under standard conditions amide resin (Fig. 1A). The peptide aggregated around the tenth residue
would allow automation of backbone protection installation.
(Ser10) and provided a poor quality crude product with many deletion
Synthesis of the salicylaldehyde 8 (Scheme 2) was simplified impurities (Fig. 1A). The use of microwave gave a marginally improved
from an earlier route and obtained in a good overall yield from product, however it still contained major deletion sequences (Fig. 1C).
commercial starting materials (ESI†). Chemoselective removal In contrast, fully automated addition of Hmsb 4 backbone protection to
of the methylether by boron tribromide gave salicylaldehyde 7. For Ala9, followed by a standard coupling cycle for the addition of
automation to work the sulfoxide group had to be present for imine Lys8 provided a greatly improved crude product (Fig. 1(B & D)). The
formation on the solid phase and survive the conditions of results using conventional automated synthesis and peptide coupling
reductive amination (Scheme 2B). Previously, we had introduced agents O-(6-chlorobenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
backbone protection by synthesizing an amino acid building block hexafluorophosphate. (HCTU)/DIEA, 30 min or microwave (DIC/
and oxidizing the thioether to the sulfoxide after preparation of the 1-hydroxybenzotriazole (HOBt), 10 min) were comparable.
building block.18 However, oxidation is difficult to perform quanti-
tatively on-resin and is also not compatible with all amino acids.
Therefore thioether 7 was cautiously oxidized to the sulfoxide 8 in
the presence of the unprotected aldehyde function. With the
Fig. 1 Synthesis of a difficult peptide sequence from influenza virus
Hemagglutinin.11 Analytical HPLC traces of crude product prepared using:
(A) conventional automated SPPS, (a = target peptide, b = deletion of Met1-
Glu2-Asp3, c = deletion of Met1-Glu2, d = deletion of Glu1, e = t-butylated
product). (B) Conventional automated SPPS with backbone protection at Ala9.
Scheme 2 (A) Synthesis of Hmsb backbone protection. (i) DMF, POCl3, 0 1C;
(ii) ClCH2CH2Cl, 80 1C; (iii) BBr3ꢀSMe2, CH2Cl2, 0 1C; (iv) m-CPBA, CHCl3, ꢁ10 1C.
(B) Automated introduction of backbone protection to peptide on solid phase.
(i) Imine formation; salicylaldehyde 8 1.1 equivalent to resin loading; (ii) DMF
wash; (iii) NaBH4/DMF; (iv) FmocAA, HCTU/DIEA, 30 min; (v) SPPS.
(C) Automated microwave assisted SPPS. (D) Automated microwave-assisted
SPPS backbone protection at Ala9. (E) MALDI-MS of target peptide, a, (calculated
mass [M + H]+ = 1481.6 m/z) peptide cleavage conditions: TFA/TMSBr/
thioanisole/ethanedithiol (1.0 : 0.05 : 0.05 : 0.025 v/v), 1.0 h. HPLC condi-
tions: RP-C18, 0–50% (0.1% TFA, 90% CH3CN) in 30 min, 1 mL minꢁ1
.
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However, with conventional automated synthesis an additional
Communication
dichloromethane mix/wash step for 1 h gave a noticeable improve-
ment to the initial product purity, presumably by favouring intra-
molecular acyl migration.5,23 The recovered yield after preparative
HPLC was much higher, approximately 30%, for both cases, in
contrast to 3% for conventional SPPS and 8% for microwave assisted
SPPS, reflecting the less challenging purification task and the
practical advantages of using backbone protection. The Hmsb
backbone protecting group requires the sulfoxide to be reduced to
a thioether for clean deprotection. Previously we used ammonium
iodide and dimethylsulfide,18 but we found trimethylsilyl bromide
(TmsBr) and thioanisole in the cleavage cocktail more convenient.24
All the peptides were cleaved using the same cleavage cocktail for
comparison of the crude product (Scheme 3). The backbone protec-
tion can also be retained on the side-chain deprotected peptide for
its beneficial solubility effects.
In the absence of an a priori method to predict an aggregation-
prone sequence, a pragmatic approach would be to include a
synthetic cycle to protect every sixth residue and prevent any
potential aggregation. Polyalanine (with a C-terminal Val) is the
prototypical difficult sequence, identified by Merrifield and
co-workers and frequently used since as a benchmark.25–27
Furthermore, homooligomers of alanine have become the sub-
ject of interest because of their biological relevance; as they are
one of the most common homopeptide repeats and expanded
polyalanine repeats are central to several neurodegenerative
diseases.28 Aggregation begins at the 5th alanine added from
C-terminus and the addition of further alanine residues becomes
increasingly troublesome.26 The crude product at any of the later
stages would be highly insoluble and difficult to analyse or purify.
In contrast, automated introduction of two appropriately cited
Hmsb backbone protecting groups (at positions Ala8 and Ala14)
using microwave protocols on a Tentagel resin not only prevented
aggregation during SPPS, but also by retention on the cleaved crude
peptide provided a fully soluble, chemically defined, analogue that
is readily analysed and shown to be of high quality with confirmed
molecular weight. Circular dichroism of the product gave a spectra
Fig. 2 Automated synthesis of the polyalanine octadecapeptide. Fmoc-
Ala7HmsbAla6HmsbAla4Val-OH (A). Analytical HPLC trace of crude pro-
duct HPLC conditions: RP-C18, 30–50% B (0.1% TFA, 90% CH3CN) in
30 min. 1 mL minꢁ1. (B) MALDI-MS of product (calculated mass [M + Na]+
=
1965.8 m/z [M + K]+ = 1981.8 m/z). (C) Circular dichroism of polyalanine
containing backbone protection forming random coil (dotted). Aggregated
beta-sheets of polyalanine (solid) without backbone protection.
characteristic for random coil (Fig. 2C). Cleavage of the Hmsb protection at Leu679 using the automated procedure with trypto-
groups from the polyalanine product removed the solubilising phan as the following residue. The results demonstrate successful
properties afforded by backbone protection, yielding an insoluble, incorporation of backbone protection and improved synthesis
but chemically homogenous product.
(ESI,† Fig. S2).
Both previous examples had possessed alanine at the position
We have demonstrated that the introduction of backbone
for backbone protection. We therefore synthesised an additional protection into a difficult sequence can be fully automated and
example, the highly conserved epitope of gp41 that binds tightly delivers a significant improvement in yield and quality of previously
to the broadly neutralising 4E10 antibody and previously synthe- aggregation prone peptides. The ability to freely add reversible
sised with in situ neutralisation Boc cycles29 We added backbone amide protection along the peptide backbone has been a long-term
goal for peptide chemists, both to overcome difficult sequences and
also to solubilise the peptide in solution. The site of backbone
protection is no longer restricted to only two residues. This study
has demonstrated the suitability of salicylaldehydes for automated
introduction to solid phase by the inclusion of an imine formation/
reduction cycle. The Hmbs group actively participates in acylation
using an activated acyl transfer so that its reactivity resembles a
primary amine more than a secondary amine. With the demonstra-
tion of efficient automated introduction, backbone protection
can be used preventively at every sixth residue for routine
Scheme 3 Mechanism of removal of Hmsb backbone protection. TFA/
TMSBr/thioanisole/ethanedithiol (1.0 : 0.05 : 0.05 : 0.025 v/v).
8318 | Chem. Commun., 2014, 50, 8316--8319
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