A backbone amide protecting group for overcoming difficult sequences and suppressing aspartimide formation

Article abstract of DOI:10.1002/psc.2877

A backbone amide bond protecting group, 2-hydroxy-4-methoxy-5-nitrobenzyl (Hmnb), improved the synthesis of aggregation and aspartimide-prone peptides. Introduction of Hmnb is automated and carried out during peptide assembly by addition of 4-methoxy-5-nitrosalicylaldehyde to the peptidyl-resin and on-resin reduction to the secondary amine. Acylation of the hindered secondary amine is aided by the formation of an internal nitrophenol ester that undergoes a favourable O,N intramolecular acyl transfer. This activated ester participates in the coupling and generally gives complete reaction with standard coupling conditions. Hmnb is easily available in a single preparative step from commercially available material. Different methods for removing the amide protecting group were explored. The protecting group is labile to acidolysis, following reduction of the nitro group to the aniline. The two main uses of backbone protection of preventing aspartimide formation and of overcoming difficult sequences are demonstrated, first with the synthesis of a challenging aspartimide-prone test sequence and then with the classic difficult sequence ACP (65-74) and a 23-mer homopolymer of polyalanine.

Full text of DOI:10.1002/psc.2877

Special Issue Article
Received: 14 January 2016
Revised: 18 February 2016
Accepted: 29 February 2016
Published online in Wiley Online Library: 18 April 2016
(wileyonlinelibrary.com) DOI 10.1002/psc.2877
A backbone amide protecting group for
overcoming difficult sequences and
suppressing aspartimide formation
Abu-Baker M. Abdel-Aal,a George Papageorgiou,a Richard Raz,a
Martin Quibell,a Fabienne Burlinaa,b,c,d and John Offera*
A backbone amide bond protecting group, 2-hydroxy-4-methoxy-5-nitrobenzyl (Hmnb), improved the synthesis of aggregation
and aspartimide-prone peptides. Introduction of Hmnb is automated and carried out during peptide assembly by addition
of 4-methoxy-5-nitrosalicylaldehyde to the peptidyl-resin and on-resin reduction to the secondary amine. Acylation of the
hindered secondary amine is aided by the formation of an internal nitrophenol ester that undergoes a favourable O,N
intramolecular acyl transfer. This activated ester participates in the coupling and generally gives complete reaction with
standard coupling conditions. Hmnb is easily available in a single preparative step from commercially available material.
Different methods for removing the amide protecting group were explored. The protecting group is labile to acidolysis, following
reduction of the nitro group to the aniline. The two main uses of backbone protection of preventing aspartimide formation and of
overcoming difficult sequences are demonstrated, first with the synthesis of a challenging aspartimide-prone test sequence and
then with the classic difficult sequence ACP (65-74) and a 23-mer homopolymer of polyalanine. Copyright © 2016 European
Peptide Society and John Wiley & Sons, Ltd.
Keywords: difficult sequence; aspartimide formation; O,N acyl transfer; backbone amide protection; Hmb; nitro reduction; reductive
amination; Fmoc SPPS
[8]. The solid phase is not, however, a universal solution; poor
solubility can manifest on solid phase as the difficult sequence
Introduction
Backbone amide bond protection is the reversible alkylation of
a peptide bond and the powerful solubilizing effect of this mod-
ification to peptides is well established. The improved synthesis
of long, challenging peptides through the application of back-
bone protection has been the most convincing demonstration
of its utility [1–3]. The concept and beneficial properties of this
substitution were originally introduced through the
dimethoxybenzyl (Dmb) group (Figure 1) that masked the
amide–amide hydrogen bonding potential for a peptide chain
and thereby caused dramatically increased solubility of pep-
tides [4–6]. The same concept is now generally exploited to
overcome the difficult sequences phenomena in solid phase
peptide synthesis (SPPS) and to prevent aspartimide formation
during Fmoc SPPS. This is achieved using a range of commer-
cially available amide protecting groups, installed during the
synthesis and removed during the final cleavage. Although
adding another hydrophobic group to a peptide might be ex-
pected to decrease solubility, the major factor driving peptide
insolubility is structurally based through formation of β À sheet
architecture [7]. Therefore, the simple masking of intermittent
backbone amides prevents the formation of interchain hydro-
gen bonds, inhibits interchain aggregation and favours an
open-chain, disordered structure, with a concomitant improve-
ment in solubility.
problem. Difficult sequences are defined as a reproducible,
sequence-dependent collapse of the swollen resin volume ac-
companied by incomplete acylation and for Fmoc peptide syn-
thesis, incomplete deprotection extending over several
residues [9,10]. Although difficult sequences are also
*
Correspondence to: John Offer, The Francis Crick Institute, Mill Hill Laboratory, The
Ridgeway, London NW7 1AA, UK. E-mail: john.offer@crick.ac.uk
a The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, London
NW7 1AA, UK
b Sorbonne Universités, UPMC Université Paris 06, LBM, 4 place Jussieu, 75005,
Paris, France
c
CNRS, UMR 7203, LBM, Paris, France
d Département de Chimie, LBM, ENS, PSL Research University, 24 Rue Lhomond,
75005, Paris, France
Abbreviations: Aa, amino acid; Boc, tert-butyloxycarbonyl; CHCA, α-cyano-
4-hydroxycinnamic acid; DCM, dichloromethane; DIC, 1,3-diisopropylcarb-
odiimide; DIEA, N,N-diisopropylethylamine; Dmb, 2,4-dimethoxybenzyl;
DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethyoxycarbonyl; HCTU,
O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
Hmb, 2-hydroxy-4-methoxybenzyl; HOBt, 1-hydroxybenzotriazole; MALDI,
matrix-assisted light desorption ionization; MW, microwave; Pbf, 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl; RP-HPLC, reversed phase HPLC;
r.t., room temperature; SPPS, solid-phase peptide synthesis; tBu, tert-butyl;
TFA, trifluoroacetic acid; TES, triethylsilane; Trt, trityl.
Attaching peptides to a solid phase is a general solution to
improve their solubility. It solvates the protected peptides at
concentrations they would not achieve unassisted in solution
J. Pept. Sci. 2016; 22: 360–367
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

A BACKBONE AMIDE PROTECTING GROUP
Figure 1. Backbone amide protecting groups discussed.
encountered with Boc synthesis they are addressed to a great
extent by the in situ neutralization protocols developed by Kent
and co-workers [11]. Difficult sequences can vary widely from
mildly challenging to complete synthetic failure. In the extreme
cases, such as alanine homooligopeptides, which exacerbate
the phenomenon, the peptide becomes an insoluble aggregate
attached to the resin incapable of further reaction [12]. The
cause of difficult sequences is interchain aggregation from
amide–amide hydrogen bonding. Introduction of the tertiary
amides of sarcosine and proline into polyalanine test sequences
before the onset of aggregation significantly delayed aggrega-
tion [13]. Inserting Dmb into the sequence also prevented ag-
gregation. In addition, the protecting group could be
removed in the cleavage step. However, this improvement
was frustrated by another obstacle: the difficulty to acylate
the sterically hindered secondary amine formed by the N-
substitution. A solution to this problem was intimated in the
salicylimine models of the Kemp group [14,15] and the
salicylamide reactions of Brenner [16] that explored the effi-
cient nature of intramolecular acyl transfer as a way to over-
come hindered acylation. These concepts were subsequently
incorporated into the design of the backbone protecting group,
2-hydroxy-4-methoxybenzyl (Hmb) (Figure 1). The hydroxyl
group of Hmb was first acylated through internally base cata-
lyzed coupling followed by intramolecular acyl transfer to the
secondary amine [17]. However, although complete acylation
was now observed for a wide range of amino acid pairings that
formed the Hmb-substituted amide bond, non-standard cou-
pling conditions of symmetric anhydride and dichloromethane
(DCM) solvent were required with coupling times ranging from
1 to 48 h for complete acyl transfer [18,19].
are now commercially available as pseudoprolines of Ser, Thr
and protected pairings with DmbGly. The success of these
reagents is due to their simple introduction using standard cou-
pling conditions. However, they are restricted to substitution
sites containing Ser, Thr and Gly (and in some cases Cys), and
not all difficult sequences contain one of these residues at a
convenient position before the onset of aggregation. In con-
trast, Hmb can theoretically be added at any position on the
peptide backbone. Although complete acylation of Hmb
substituted amino acids can be achieved, the use of Hmb-
substituted dipeptide derivatives was also explored to develop
a more readily usable reagent akin to pseudoprolines and
Dmb dipeptides. However, on activation, the phenolic hydroxyl
of Hmb-mono and dipeptide derivatives formed a poorly reac-
tive 4,5-dihydro-8-methoxy-1,4-benzoxapin-2(3H)-one preclud-
ing their use as dipeptide reagents [21]. Ideally, a broadly
applicable amide-bond protecting group should be easily intro-
duced with simple reagents, be rapidly acylated under typical
reaction conditions and be easily removed as required. Observa-
tions from Hmb-substituted amino acid and dipeptide studies
suggested the possibility to improve the acyl-transfer properties
through introduction of an electron-withdrawing group para to
the 2-hydroxyl of Hmb, and this was explored with Hmsb and
related compounds (Figure 2). The resulting internal ester
formed during coupling was now more activated and exhibited
accelerated acyl transfer permitting the use of standard cou-
plings. However, the Hmsb protecting group was now largely
stable to trifluoroacetic acid (TFA) acidolysis. Reductive standard
conversion of sulfoxide to sulfide restored acid lability [21].
Many peptides have been prepared using Hmsb in an auto-
mated fashion [22,23].
The use of backbone protection became widespread with the
availability of commercial dimethyloxazolidine derivatives of
serine and threonine (pseudoprolines) [20], Dmb-substituted
dipeptide derivatives [2,3] and Hmb-substituted amino acid an-
alogues (Figure 1). The former derivatives side-stepped the
need to acylate a sterically hindered secondary amine on the
solid phase through their introduction as dipeptide building
blocks [20]. Virtually all the protected dipeptide combinations
Accelerated acyl transfer has also been demonstrated for back-
bone protection using a nitro group as the electron-withdrawing
group. The nitro group is much more electron-withdrawing than
sulfoxide and therefore should show improved self-acylation prop-
erties over Hmsb. It has been studied as 2-hydroxy-6-nitrobenzyl
[24] (Figure 1), which was photolabile and 2-hydroxy-4-methoxy-
5-nitrobenzyl (Hmnb) [21] (Figure 2), which was stable to photolysis
and acid. We envisioned that the Hmnb group would become acid
Figure 2. Backbone protecting group Hmnb, intramolecular O,N acyl transfer assisted by para nitro group.
J. Pept. Sci. 2016; 22: 360–367
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ABDEL-AAL ET AL.
Table 1. On-resin reduction of nitro group of Hmnb
Materials and Methods
Entry
1
Nitro reduction method
% Nitro reductiona
All reagents and solvents were purchased from Novabiochem®
Merck-Millipore or Sigma-Aldrich. Fmoc-Gly/Ala/Ile/Leu/Val-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Gln-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys
(Boc)-OH, Fmoc-Tyr(OtBu)-OH and Boc-Val-OH were used. Peptides
were prepared using DIC/HOBt (CEM Liberty Microwave Synthe-
sizer) or HCTU/DIEA (CS Bio 336 automated synthesizer) activation
for Fmoc/t-Bu chemistry. Following peptide cleavage, the TFA-
cleavage cocktail was filtered; the filtrate sparged (N2) and pep-
tides were precipitated with Et2O (Na-dried, 4 °C). Peptides were
Na2S2O4, K2CO3, TBAHS,
5%
DCM-water (10 eq, r.t. 12 h)
CrCl2, DMF (2x2.5 eq, r.t. 6 h)
CrCl2, DMF (2.5 eq, r.t. 12 h)
CrCl2, DMF (2.5 eq, r.t. 6 h)
CrCl2, DMF (2.5 eq, r.t. 2 h)
CrCl2, DMF (2.5 eq, 40 °C, 2 h)
CrCl2, DMF (1.0 eq, 40 °C, 2 h)
CrCl2, DMF (2.5 eq, 70 °C, 2 h)
CrCl2, DMF (2.5 eq, 70 °C, 1 h)
2
3
4
5
6
7
8
9
100%
75%
60%
30%
65%
8%
purified by semi-preparative HPLC on
a RP-C18 column
100%
100%
(22 × 250mm, Vydac) using linear gradients of CH3CN in 0.1%
TFA/H2O with a flow rate of 5 mlminÀ1. HPLC gradients were pre-
pared using solvent A as 0.1% TFA aqueous solution and solvent B
as 90% CH3CN in 0.1% TFA aqueous solution. Detection was
performed at 214 nm. Peptides were characterized by matrix-
assisted laser desorption ionization time-of-flight mass spectrome-
try (MALDI-TOF MS) on a BRUKER microflex using CHCA matrix
(10 mg mlÀ1 in CH3CN/H2O/TFA, 50 : 50: 0.1v/v). The ion positive
reflector mode was used.
aEstimated from analytical HPLC.
labile if the nitro group was converted (after acyl transfer) to an an-
iline group by reduction on the solid phase. The Hmnb precursor, 2-
hydroxy-4-methoxy-5-nitrobenzaldehyde, is available by a single,
simple preparative step from 2-hydroxy-4-methoxybenzaldehyde.
Our initial efforts focussed upon identification of a mild, peptide
synthesis compatible, reductive method that would convert the aro-
matic nitro group to aniline and explore the acid lability of the group
under different cleavage conditions. The use of a TFA/TMSBr/
thioanisole mixture is attractive as it is a much stronger acid treat-
ment than TFA alone but the mixture remains volatile and can be
simply removed by sparging as for TFA [25].
2-Hydroxy-4-methoxy-5-nitrobenzaldehyde
2-Hydroxy-4-methoxybenzaldehyde (24.2 g, 159 mmol) was dis-
solved in glacial acetic acid (48 ml) and heated on a water bath
for 10 min. The solution was treated dropwise with concentrated
HNO3 (8.6ml, 191 mmol). When reaction ceased, the solution was
cooled and a red precipitate formed. The solid was filtered, washed
with cold acetic acid, and dried. Recrystallization from EtOH
(charcoal) gave the desired product, 2-hydroxy-4-methoxy-5-
nitrobenzaldehyde (11.24 g, 36%), as pale fluffy needles, mp
1
172–173 °C. H NMR (400 MHz, CDCl3) δ, 11.75 (1H, s, OH), 9.78
Table 2. Cleavage of backbone-modified peptide
(1H, s, CHO), 8.30 (1H, s, 6-H), 6.56 (1H, s, 3-H), 4.01 (3H, s, OCH3).
The filtrate from the original reaction mixture was carefully diluted
with water and a brown solid precipitated; this was filtered, washed
with cold water and dried. Flash chromatography [EtOAc-hexanes
(2 : 3) to (1 : 1)] gave two products. First eluted product was a pale yel-
Peptide
Peptide cleavage
Ratio of 6a–b to 7
5a
TFA/TES/H2O
TFA/TMSBr/thioanisole/EDT
TFA/TES/H2O
1 : 0
0 : 1
3 : 1
0 : 1
5b
1
low solid (4.73 g). H NMR indicated that it was mostly unreacted
TFA/TMSBr/thioanisole/EDT
starting material contaminated with some 3-methoxy-6-nitrophenol.
Scheme 1. Incorporating Hmnb into synthesis of aspartimide-prone sequence VKDGYL. (i) Imine formation; 2-hydroxy-4-methoxy-2-nitrobenzaldehyde 1.1
equivalent to resin loading, followed by DMF wash; (ii) NaBH4/DMF; (iii) Fmoc-Asp(OtBu)-OH, DIC/HOBt 30 min, continue SPPS; (iv) Nitro reduction: CrCl2/DMF
(2.5 eq, 70 °C, 2 h); (v) TFA/ TES/ H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h); (vi) TFA/ TMSBr/thioanisole/EDT (100 : 10 : 5 : 2.5 v/v, 1 h).
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A BACKBONE AMIDE PROTECTING GROUP
The second eluted product was 2-hydroxy-4-methoxy-3-nitrobenzaldehyde
[1.35 g, 4% as light brown needles, mp 144–146 (EtOH), 1H NMR
(400 MHz, CDCl3): δ 11.73 (1H, s, OH), 9.79 (1H, s, CHO), 7.65 (1H, d,
J= 9.2Hz, 6-H), 6.67 (1H, d, J = 8.7Hz, 5-H), 3.98 (3H, s, OCH3)].
20% piperidine in DMF in two stages with an initial 3 min followed
by 7-min treatment. All coupling reactions were performed with
fivefold excess Fmoc-Aa-OH/HCTU/DIEA (1.1: 1.0: 1.0) for 30 min.
Hmnb was installed onto Ala68 by imine formation and reduction:
2-Hydroxy-4-methoxy-5-nitrobenzaldehyde was added to the
peptidyl-resin to form the imine (30 min, 1.1 eq, 0.01 M) followed
by DMF flow wash. The imine 9 was reduced with NaBH4 in DMF
(15 min, ×5 eq, 0.1M) to amine 10 followed by DMF flow wash.
Ala67 was coupled directly to the Hmnb alkylated peptidyl-resin un-
der standard coupling conditions followed by DCM flow wash, 1 h
shaking in DCM, DMF flow wash, 30 min re-swelling in DMF. Gln66
was added as Fmoc-Gln-OH without trityl protection. On comple-
tion of the sequence the resin was washed with DCM and dried un-
der vacuum yielding 565 mg of peptide-resin 11. Test cleavage of
the Hmnb-ACP peptide was performed using TFA/TES/H2O
(95 : 2.5: 2.5 v/v, rt. 1.5h). Analytical HPLC gave a single peak as
H-Val-Lys-Asp-Gly-Tyr-Leu-NH2 (7)
The VKDGYL peptide was assembled on H-Rink amide ChemMatrix®
resin (Sigma-Aldrich) (150 mg, resin loading 0.4–0.6 mmol/g) using
DIC/HOBt activation (CEM Liberty1™ Single Channel Microwave Pep-
tide Synthesizer). Fmoc deprotection was performed using 20% pi-
peridine in DMF in two stages with an initial 0.5 min followed by a
longer 3-min treatment (7 ml, 40 W, 75°C). Coupling reagents were
as follows: Fmoc-Aa-OH/terminal Boc-Val-OH (0.2 M in DMF), DIC
(0.8 M in DMSO) and HOBt (0.5 M in DMF). All coupling reactions were
performed with fivefold excess Fmoc-Aa-OH/DIC/HOBt (1.0 : 0.8 : 2.0)
and terminal Boc-Val-OH for 10 min, 25 W, 75 °C. Hmnb was installed
onto Gly4: 2-hydroxy-4-methoxy-5-nitrobenzaldehyde was added to
the peptidyl resin (10min, 1.1eq, 0.02M, 25W, 50°C) to form the im-
ine followed by DMF flow wash. The imine was reduced with NaBH4
in DMF [2×15min, 5eq, 0.1M, r.t.] followed by DMF flow wash. Asp3
was added using standard coupling. Synthesis yielded 230 mg of
dried peptide-resin. Test cleavage of the peptide-resin 4 was per-
formed using TFA/TES/H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h). Analytical HPLC
gave a major peak as the target peptide with the Hmnb backbone
protecting group characterized by MALDI-TOF MS, m/z= 874.3 (first
isotope), calc. m/z ([M + H]+) = 874.4. The MS profile showed a series
of ions distinctive for nitro containing compound corresponding to
the loss of one and two oxygens [26].
The peptide-resin was swollen in DMF, filtered and mixed with
freshly prepared solution of chromium(II) chloride in DMF (CrCl2,
0.04 M). Reaction conditions are reported in Table 1. The peptidyl-
resin was washed with DMF (3 × 5 ml), DCM (3 × 5 ml) and dried.
The peptide-resin was cleaved using a mixture of TFA/H2O/TES
(100 : 5 : 2.5 v/v, 1 ml, 1.5 h). Analytical HPLC traces of the crude prod-
uct gave a single peak as the target peptide with retention of the
Ahmb group (6a) characterized by MALDI-TOF MS, m/z= 866.0 (first
isotope), calc. m/z ([M + Na]+) = 866.4. A portion of the peptide-resin
(25 mg)
was
cleaved
with
TFA/TMSBr/thioanisole/EDT
(100 : 10 : 5 : 2.5 v/v, 1.5 h). Analytical HPLC traces of the crude product
exhibit a single peak as the target peptide 7 without auxiliary charac-
terized by MALDI-TOF MS, m/z= 693.0 (first isotope), calc. m/z ([M
+ Na]+) = 693.0. HPLC conditions: RP-C18, 5–35% CH3CN in
0.1% TFA over 30 min, 1 ml minÀ1. HPLC purification yielded
2.6 mg of peptide, 34.4% yield (purity > 98%).
Alternatively, the peptide-resin (50 mg) was acetylated using
acetic anhydride (0.16 mmol, 16 μl, 10 eq) and DIEA (0.08 mmol,
15 μl, 5 eq) in 300 μl DMF for 30 min. The solution was drained and
the peptide-resin treated with 20% piperidine/DMF, washed with
DMF (3 × 5 ml), DCM (3 × 5 ml) and dried. Portions of the peptidyl-
resin were cleaved using a mixture of TFA/H2O/TES (100 : 5 : 2.5 v/v,
1ml, 1.5h) or TFA/TMSBr/thioanisole/EDT (100 : 10 : 5 : 2.5 v/v, 1 ml,
1.5 h). A summary of different cleavage conditions attempted
on the peptide with Ac-Ahmb or Ahmb is given in Table 2.
Figure 3. Analytical HPLC and MALDI-TOF mass spectra of crude
aspartimide-prone peptide VKDGYL : (A) With Hmnb after cleavage of 4
with TFA/TES/H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h), calc. m/z = 874.4 for [M + H]+
ion (distinctive NO2 ion pattern [26]) † non-aspartimide side-product, HPLC
H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH, ACP (65-74),
peptide 14
conditions: RP-C18, 5–35 % CH3CN in 0.1% TFA over 30 min, 1 ml minÀ1
.
(B) Peptide 6a with Ahmb after on-resin nitro-reduction with CrCl2 and
cleavage of 5a with TFA/TES/H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h), calc.
m/z = 866.4 for [M + Na]+ ion. * peptide 7. (C) Peptide VKDGYL 7 after
cleavage of 5a with TFA/TMSBr/thioanisole/EDT (100 : 10 : 5 : 2.5 v/v, 1.5 h),
calc. m/z = 693.0 for [M + H]+ ion.
The ACP (65-74) peptide was assembled on Fmoc-Gly-NovaSyn®
TGT resin (Novabiochem®) (526mg, resin loading 0.19mmol/g)
using HCTU/DIEA activation for Fmoc/t-Bu chemistry (CS Bio 336
automated synthesizer). Fmoc-deprotection was performed using
J. Pept. Sci. 2016; 22: 360–367
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wileyonlinelibrary.com/journal/jpepsci

ABDEL-AAL ET AL.
Scheme 2. Ac-Ahmb versus Ahmb (i) N-acetylation: Ac2O (10 eq); DIEA (5 eq) in DMF for 30 min followed by 20% piperidine/DMF to remove acetylation of
the Ac-Ahmb hydroxyl. (ii) TFA/TES/H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h); (iii) TFA/TMSBr/thioanisole/EDT (100 : 10 : 5 : 2.5 v/v, 1 h).
the target peptide with retention of Hmnb characterized by MALDI-
TOF MS, m/z = 1266.6 (first isotope), calc. m/z ([M + Na]+) = 1266.5.
The peptide-resin (250 mg) was swollen in DMF, filtered and
mixed with a freshly prepared solution of chromium(II) chloride
(CrCl2, 0.04 M, 3.1ml, 2.5 eq) at 70 °C for 2 h. The reaction mixture
was drained, and the peptide resin was thoroughly washed with
DMF (3× 10 ml), DCM (3×10ml) and dried.
The peptide-resin (50mg) was cleaved using a mixture of
TFA/H2O/TES (100 : 5 : 2.5 v/v, 1 ml, 1.5 h). Analytical HPLC traces of
the crude product showed a single peak as the target Ahmb-ACP
peptide 13 characterized by MALDI-TOF MS, m/z = 1236.7 (first
isotope), calc. m/z ([M + Na]+) = 1236.5. Alternatively, the peptide-
resin (50mg) was cleaved with TFA/TMSBr/thioanisole/EDT
(100 : 10 : 5 : 2.5 v/v, 1.5h). Analytical HPLC showed a single peak
as the target ACP (65-74) peptide 14 characterized by MALDI-TOF
MS, m/z = 1085.6 (first isotope), calc. m/z ([M + Na]+) = 1086.5. HPLC
purification yielded 3.2 mg of peptide, 21.3% yield (purity> 98%).
in the preceding texts (CS Bio 336 automated synthesizer). Back-
bone protection was inserted after Fmoc deprotection of Ala7,
Ala13 and Ala19. Each backbone-protecting group was introduced
in two steps. 2-Hydroxy-4-methoxy-5-nitrobenzaldehyde dis-
solved in DMF was added to peptide-resin (1.1 eq, 0.01 M,
30 min) followed by DMF wash. Reduction with NaBH4 dissolved
in DMF (filtered, PVDF 0.2 μm) (5 eq, 0.1 M, 15 min) was followed
by thorough DMF wash. Synthesis yielded 190 mg of dried
peptide-resin. A portion of the peptide-resin (50 mg) was
cleaved using TFE/DCM (1 : 1 v/v, 1 ml, 1.5 h) and HPLC purifica-
tion (30–50% B in 30 min) yielded 13 mg of peptide, (86% yield).
The peptide was characterized by MALDI-TOF MS, m/z = 2247.9,
calc. m/z ([M + Na]+) = 2247.2.
Results and Discussion
Aspartimide formation is the most serious side reaction of Fmoc
SPPS, caused by repetitive piperidine treatments of Asp-containing
peptides. Prolonged and repeated base treatment of Asp-Xaa se-
quences causes nucleophilic attack of the amide-backbone onto
the β-carboxy of protected aspartic acid. The aspartimide can un-
dergo nucleophilic ring opening caused by piperidine or water
Synthesis of H-Ala22Val-OH
The peptide was assembled on Fmoc-Val-NovaSyn® TGT resin
(Novabiochem®) (170mg, resin loading 0.19 mmol/g) using stan-
dard HCTU/DIEA activation for Fmoc/t-Bu chemistry as described
Scheme 3. Automated synthesis of ACP (65-74) using Hmnb backbone amide protecting group. (i) Imine formation; Hmnb 1.1 equivalent to resin loading
followed by DMF wash; (ii) NaBH4/DMF; (iii) standard SPPS HCTU/DIEA coupling cycle, DCM 1 h, standard SPPS; (iv) nitro reduction: CrCl2/DMF (2.5 eq, 70 °C,
2 h); (v) TFA/TES/H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h); (vi) TFA/TMSBr/thioanisole/EDT (100 : 10 : 5 : 2.5 v/v, 1 h).
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J. Pept. Sci. 2016; 22: 360–367

A BACKBONE AMIDE PROTECTING GROUP
during subsequent steps producing further by-products (L/D-β-
aspartyl and D-aspartyl peptides) that are difficult or impossible to
separate from the product [27]. Aspartimide formation is exacer-
bated by alternative bases such as DBU sometimes used for
deprotection and by microwave synthesis. Backbone protection
has been demonstrated to abolish aspartimide formation
completely [28]. The main contemporary approach is protection
of the Asp-Gly amide, by using commercially available derivatives
such as the Fmoc-Asp(OtBu)-(Dmb)Gly-OH building block [2]. We
have chosen a well-established model for aspartimide formation
derived from a scorpion toxin II peptide variant. Backbone protec-
tion is theoretically a universal solution to the aspartimide forma-
tion problem. It is also the only case where backbone
protection is performing its eponymous role by suppressing
reaction of the amide bond. Additionally, although the amide
bond is generally considered stable, alkylation of the cysteine
amide can favour N,S acyl transfer under ligation conditions
and should be avoided [29]. We have developed a generally
applicable protocol based on Hmnb backbone protection to
abrogate aspartimide formation (Scheme 1, Figure 3). The pep-
tide was assembled on H-Rink amide ChemMatrix® resin using
DIC/HOBt activation for microwave-assisted Fmoc/t-Bu chem-
istry. The protocol is fully automated, and only three cycles were
added to the standard SPPS to achieve backbone modification at
Asp-Gly; imine formation, imine reduction and post-synthesis nitro
reduction. All reactions are simple, quantitative, performed in
DMF and compatible with standard SPPS. Imine formation was
achieved with the addition of a slight excess of 2-hydroxy-4-
methoxy-5-nitrobenzaldehyde onto peptidyl-resin 1. Like most
salicyaldehydes, 2-hydroxy-4-methoxy-5-nitrobenzaldehyde forms
exceptionally stable imines [22,24,30]. Furthermore, the imine 2
was smoothly reduced to secondary amine using 5 equivalents of so-
dium borohydride. Imine formation is associated with a noticeable
change of the resin colour to bright yellow, and imine reduction is ac-
companied by a loss of colour and evolution of hydrogen gas. All
couplings were performed using standard SPPS reagents with single
coupling of all amino acids to give the Hmnb substituted product 4.
We observed that Hmnb backbone protection is stable to
acid, even anhydrous HF. Test cleavage of the peptide-resin 4 gave
a crude product containing the Hmnb modified target peptide
(Figure 3). A minor, later running side-product was also observed
with identical mass. We considered that reduction of the nitro af-
ter completion of the peptide synthesis to aniline would change
the electronic properties of the benzyl group and could increase
the acid lability of the backbone protection.
peptide synthesis performed on polyethylenglycol or polystyrene
resins.
Reduction of the nitro group of Hmnb opens the possibilities
for further chemical modifications of the aniline. We wanted to
explore the effect of aniline acetylation on the acid lability of
the backbone protection during cleavage (Scheme 2). Peptide-
resins with Ahmb and Ac-Ahmb (5a–b) were cleaved using
two peptide cleavage methods: the standard TFA mixture and
a TFA/TMSBr/thioanisole mixture (Table 2). The two protecting
groups demonstrated different stability towards standard TFA
mixture. For instance, TFA cleavage of 5a gave predominantly the
target peptide with Ahmb (a small amount of completely
Several methods have been reported for peptide compati-
ble solid-phase reduction of nitroaromatic compounds includ-
ing the use of sodium dithionite [31] and CrCl2 [32]. The
dithionite method requires the use of an aqueous solution,
unsuitable for most resins. We tried different solvent mixtures
with DMF, EtOH and dioxane at different ratios but no reduc-
tion was observed. We also tried using dithionite in a
DCM/H2O solvent system with a phase transfer catalyst (
Table 1, entry 1). At best, this gave 5% reduction. The CrCl2
reduction was simply performed by mixing the peptide resin
with 2.5 equivalents of freshly prepared solution of CrCl2 in DMF
overnight, followed by a thorough DMF wash. Reduction (60–
75%) was observed, and reaction was complete after repeat treat-
ment of the resin with CrCl2 (Table 1, entries 2–4). Hari and Miller
used higher equivalents in their work [32]. After optimization of re-
action conditions (entries 5–9), 1 h reaction at 70 °C gave complete
conversion (entry 9). This method should also be compatible with
Figure 4. Analytical HPLC traces and MALDI-TOF mass spectra of crude
ACP (65-74) peptide: (A) With retention of Hmnb after cleavage of 11 with
TFA/TES/H2O (95 : 2.5 : 2.5 v/v, rt. 1.5 h), calc. m/z = 1266.5 for [M + Na]+ ion.
(B) Peptide 13 with Ahmb auxiliary after on-resin nitro-reduction with
CrCl2 and cleavage with TFA/TES/H2O (95 : 2.5 : 2.5 v/v, 0 °C, 1.5 h), calc.
m/z = 1236.5 for [M + Na]+ ion. (C) ACP (65-74) peptide 14 after cleavage of
12 with TFA/TMSBr/thioanisole/EDT (100 : 10 : 5 : 2.5 v/v, 1.5 h), calc.
m/z = 1086.5 for [M + Na]+ ion.
J. Pept. Sci. 2016; 22: 360–367
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

ABDEL-AAL ET AL.
Polyalanine sequences are some of the most aggregation-prone
sequences known [12,13]. With polyalanine (C-terminal Val) aggre-
gation onset is early, starting after addition of the fifth residue.
Hmnb was inserted at the fifth residue and at every sixth residue
subsequently (Figure 5). The successful installation of three Hmnb
groups by salicyldimine reductive amination cycles suggests that
this reaction belongs to the exclusive set of solid phase applicable
reactions. The handling and characterization of polyalanine are usu-
ally very difficult; however, with backbone protection a 23 mer of
polyalanine has good solubility in standard HPLC solvents and
can be easily characterized by MALDI-TOF MS.
Conclusion
We have developed a versatile backbone protection, Hmnb, which
is easily available and can be introduced into a peptide synthesis in
an automated fashion. Hmnb was installed on the peptidyl-resin by
an imine formation/reduction cycle. Both imine formation and re-
duction go to completion and give satisfactory incorporation on
the solid phase. The nitro group assisted the acylation of the newly
formed secondary amine by activating the intramolecular acyl
transfer from the hydroxyl group to the secondary amine. Removal
of Hmnb backbone protection was accomplished by conversion of
Hmnb to the aniline analogue Ahmb on resin. Although both Hmnb
and to a lesser extent Ahmb were stable to standard TFA treatment,
Ahmb could be completely removed by cleavage with a mixture of
TFA and TMSBr/thioanisole.
Figure 5. (A) MALDI-TOF mass spectrum and (B) crude analytical HPLC trace
of polyalanine 23-mer peptide (H-Ala6HmnbAla6HmnbAla6HmnbAla4Val-OH)
after cleavage with TFE/DCM (1 : 1 v/v), calc. m/z = 2247.2 for [M + Na]+ ion.
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J. Pept. Sci. 2016; 22: 360–367
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