Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self-assembly

Article abstract of DOI:10.1002/psc.2402

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water-soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile con

Full text of DOI:10.1002/psc.2402

Research Article
Received: 3 November 2011
Revised: 13 December 2011
Accepted: 12 January 2012
Published online in Wiley Online Library: 23 March 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2402
Bidirectional solid phase synthesis of a model
oligoglycine bolaamphiphile and purification
by rapid self-assembly
a,b
b
Venthan B. Naidoo and Marina Rautenbach *
We utilised a simple bidirectional (N!C and C!N) solid phase synthesis strategy entailing conventional solid phase peptide
synthesis and fragment condensation with a water-soluble carbodiimide to synthesise a model anionic glycylglycine bolaam-
phiphile containing a suberic acid linker moiety, namely N,N′-suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine
was purified using its inherent ability to rapidly self-assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Mon-
itoring of the rapid assembly process corroborated our visual observation and confirmed packing-directed self-assembly
rather than non-specific aggregation or precipitation. The progress of suberoyldiglycylglycine self-assembly was observed
to be via the formation of oligomers in the solution, which then self-assembled to form layered b-sheet type macrostructures.
Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated
after 5–7 days. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: bolaamphiphile; solid phase synthesis; purification; rapid self-assembly
N′-suberoyldiglycylglycine (Figure 1), one of the bolaamphiphiles
created by Grigoryan et al. [10] using solution phase synthesis.
Introduction
Fuoss and Edelson [1] first introduced the term ‘bolaform electro-
lyte’ in 1951 for a hydrophobic chain connecting two ionic moie-
ties. Essentially, a bolaamphiphile is a molecule consisting of two
hydrophilic species connected by a hydrophobic tether. Since its
discovery, many bio-inspired peptidolipidic bolaamphiphiles
have been synthesised and characterised [2–9]. These bolaam-
phiphiles display two very unique properties, namely, if designed
in a particular way, they tend to have biological activity as well as
the propensity to self-assemble into a variety of nanostructure,
microstructure and macrostructure. Bolaamphiphiles have also
been the subject of much attention because of their application
in functionalised nanomaterials, as nanotubes, as nanovesicles,
as templates to cast nanowires, as nanocarriers of drugs and
many other bionanotechnological applications. For selected
reviews on bolaamphiphiles and their applications, refer to
References [2–9].
Solution phase methods depend heavily on the unpredictable
solubility properties of the peptide intermediates, whereas solid
phase synthesis offers the ease of intermediate purification
from starting materials and by-products by simple filtration
and washing steps of resin-bound intermediates. Furthermore,
the ease of synthesising very complex peptides, selectively pro-
tected on their functional side chains, and even large peptide
libraries on solid phase makes it a methodology of choice in
peptide library synthesis.
Our synthetic strategy was based on the SPPS protocol de-
veloped by Atherton and Sheppard [21,22] where the peptide
is anchored with an acid-labile ester bond and chain elonga-
tion proceeds from the C-terminus towards the N-terminus.
The SPPS protocol employed a protection scheme in which a
base-labile Fmoc group, requiring only mild removal conditions
[23,24], is used to protect the a-amino group of the incoming
amino acid. The solid phase synthesis of a bolaamphiphile with
dicarboxylic acid linker requires an adaptation of conventional
SPPS synthetic protocol to a bidirectional synthetic route
Oligoglycine bolaamphiphiles, introduced by Grigoryan et al.
[10] as possible antistaphylococcic agents, have gained much
prominence in the field of self-assembly [11–20]. In 1996, Shimizu
et al. [11] observed vesicle assembly in microtubular structures
made up of synthetic oligoglycine-based bolaamphiphiles. These
dicarboxylic glycylglycine bolaamphiphiles self-assemble in a va-
riety of ways such as the formation of vesicles, fibres, tubes and
spheroids [12]. The formation of these assembly patterns is pH
dependent in the aqueous dispersions [13]. Matsui et al. [15,16],
using glycylglycine bolaamphiphiles, indicated the possibility of
organising peptide nanotubes into bundles through the use of
metal coordination bridges, as well as creating nanowires.
Taking into account that many peptides have a natural ten-
dency to self-assemble, we investigated the application of
SPPS-derived bolaamphiphiles. To achieve this, we have adapted
a solid phase synthetic route to synthesise a model bolaamphi-
*
Correspondence to: Marina Rautenbach, BIOPEP Peptide Group, Department of
Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, South
Africa. E-mail: mra@sun.ac.za
a UNESCO Associated Centre for Macromolecules and Materials, Division of Poly-
mer Science, Department of Chemistry, University of Stellenbosch, Private Bag
X1, Matieland 7600, South Africa
b BIOPEP Peptide Group, Department of Biochemistry, University of Stellenbosch,
Private Bag X1, Matieland 7602, South Africa
Abbreviations used: E-SEM, environmental scanning electron microscopy;
ESMS, electrospray ionisation mass spectrometry; FTIR, Fourier transform infra-
red spectroscopy.
a
phile, bis(N -amido-glycylglycine)-1,8-octane dicarboxylate or N,
J. Pept. Sci. 2012; 18: 317–325
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

NAIDOO AND RAUTENBACH
Acid (TFA) labile
ester bond
A
PyBOP, DIPEA, HOBt
Solvent: DMF
O
O
H
R
R
O
O
N
(CH ) COOH
2 6
NH2
N
H
N
H
O
O
O
HOOC(CH ) COOH
Diglycyl-peptide synthesised on resin
with normal Fmoc-chemistry using
PyBOP, DIPEA and HOBt in coupling
reactions
2 6
Activation with EDC at pH5
Solvent: 12.5mM phosphate buffer
suberic acid
Remove excess EDC and solvent
by filtration
B
O
O
H
N
H
N
O
H
HO
(
CH2
)
6
OH
N
R
O
N
H
N
(CH
2)
6CO-EDC
O
H
O
N
O
O
O
H
O
O
Cleavage of product from
resin with 95% aqueous
TFA
H2N
Coupling at pH8
O
N
H
O
Remove excess reagents
and urea by filtration. Wash
and vacuum-dry resin
Solvent: 0.2M phosphate buffer
O
H
O
H
N
R
O
O
N
CH2
)
6
(
N
N
H
H
O
O
O
O
Figure 1. A scheme depicting the general solid phase synthesis strategy employed used for the synthesis from the resin-bound diglycyl moiety to
resin-bound diglycyl suberic acid moiety (A) and finally to N,N′-suberoyldiglycylglycine (B).
(
Figure 1). The first part of the synthesis is in the C!N direc-
the possibility of pH control of the activation and coupling reac-
tions, thus also eliminating the necessity of protecting the incom-
ing carboxyl group [32].
With the diglycine bolaamphiphile, N,N′-suberoyldiglycylglycine
(Figure 1), serving as model, we report here a new solid phase syn-
thesis strategy, ideal solvent conditions for rapid self-assembly and
purification using its self-assembly character. To confirm that puri-
fication indeed occurred via self-assembly, we also assessed the
rapid self-assembly with electrospray mass spectrometry (ESMS)
and environmental scanning electron microscopy (E-SEM).
tion leaving the second carboxyl group of dicarboxylic acid
linker open for the following part of the synthesis in the
N!C direction. The reversal of the synthesis direction to the
N!C direction poses some problems, and early studies in pep-
tide synthesis chemistry showed that most of the N!C synthe-
sis strategies resulted in optically impure peptides [25]. Numer-
ous synthetic strategies [26–32] have since been introduced to
address this anomaly. We report here on the use of one of the
strategies [32] in combination with SPPS to synthesise peptide
bolaamphiphiles. The choice of method depended on the solu-
bility of the peptide fragment and the identity of the N-terminal
residue being attached in the N!C direction.
Materials and Methods
To synthesise in the N!C direction, the resin-bound carboxyl
group must be activated and the carboxyl group of the incoming
amino acid must be reversibly protected to prevent polymerisa-
tion. In situ activation of the carboxyl group of the resin-bound
linker by, for example, methods using phosphonium/uronium/
immonium or carbodiimide activation reagents yields high cou-
pling efficiencies if the a-carboxyl group of the incoming amino
acid is reversibly protected (reviewed in [33]). Phosphonium/uro-
nium/immonium-based reagents react with the a-carboxyl
groups in the presence of a base to form the highly active benzo-
triazolyl ester or analogous ester [33,34]. Combining the catalyst
HOBt with carbodiimides would also lead to an extremely active
benzotriazolyl ester of the carboxyl group [33,34]. HOBt and a
number of its analogues are also the so-called configuration-
trapping agents limiting racemisation to less than 0.4% for most
amino acids [33,35].
Materials
W
W
NovaSyn KA (0.15 mEq/g), NovaSyn TGT (0.2–0.26 mEq/g),
W
HOBt and PyBOP were from Novabiochem Co. (Luzern, Switzer-
land). Fmoc-Gly-OH was from Advanced ChemTech (Louisville,
USA). DMF (99.5%), glacial acetic acid, diethyl ether (99.5%),
DCM (99%), methanol (99%), glycylglycine (>98%), sodium dihy-
drogen phosphate dihydrate (99%) and disodium hydrogen
phosphate dihydrate (99%) were from Merck (Darmstadt,
Germany). 2-Methylbutan-2-ol (t-amyl alcohol; 98%) was from
BDH Chemicals (Poole, UK). DIPEA, TFA (>98%), piperidine
(98%), suberic acid (>98%), pyridine (98%), EDC and aluminium
oxide (>98%) were from Sigma Chemical Co. (St. Louis, MI,
USA). Acetonitrile (HPLC-grade, UV cut-off 190 nm) was from
Romil LTD (Cambridge, UK). Analytical grade water was prepared
W
by filtering glass distilled water through a Millipore Milli-Q water
The reversible protection of the a-carboxyl group of the incom-
ing amino acid could be quite complex if introduced into the
base-labile Fmoc-synthesis strategy [23,24]. Pre-activation of the
carboxyl group by forming a more stable active ester, such as
an OPfp [36] should eliminate the necessity of protecting the
a-carboxyl group of the incoming amino acid. Alternatively, the
use of the water-soluble and stable carbodiimide, EDC, offers
purification system (Millipore Headquarters 290 Concord Road,
Billerica, MA 01821 USA).
Bolaamphiphile Synthesis
In our synthesis, in situ activation of the carboxyl groups of Fmoc-
W
Gly-OH and suberic acid linker was accomplished using PyBOP ,
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 317–325

NOVEL SPPS AND PURIFICATION BY SELF-ASSEMBLY OF PEPTIDE BOLAAMPHIPHILE
a relatively low-cost reagent, HOBt as catalyst and DIPEA as base
25].
The self-assembled solution was centrifuged using a bench top
PicoFuge Microcentrifuge (Stratagene, La Jolla, CA, USA) for 5 min
W
[
and the supernatant carefully removed. The resulting pellet was
washed with cold analytical quality water using the centrifugation
procedure for further analysis or dissolved in 50% acetonitrile and
lyophilised.
Part A: synthesis of R-O-Gly -CO(CH ) COOH (Figure 1A)
2
2 6
The synthesis was accomplished on a polydimethylacrylamide
resin encapsulated in Kieselguhr (R). Normal SPPS, based on the
Fmoc chemistry [23,24], was used with freshly distilled amine-free
DMF as solvent and 20% high quality distilled piperidine in DMF
as base for removal of the Fmoc group after each coupling step.
The first Gly residue was coupled to the resin with an acid-labile
ester bond. Activation of the incoming carboxyl group was ac-
Analysis of Synthesis and Self-assembled Products
Electrospray ionisation MS was performed using a Micromass tri-
ple quadrupole mass spectrometer fitted with an electrospray
ionisation source (Micromass UK Ltd., Manchester, UK). The car-
rier solvent was 50% (v/v) acetonitrile in analytical grade water
delivered at a flow rate of 20 ml/min during each analysis using
a Pharmacia LKB 2249 pump (Pharmacia, Freiburg, Germany).
Ten microlitre of the sample solution (0.2 mg in 1.0 ml of a 50%
CH CN : 0.05% aqueous TEA solution or 50% CH CN) was intro-
W
complished by using PyBOP , as activation agent, with freshly
W
distilled DIPEA as base and HOBt as catalyst. The PyBOP reagent
was used as follows: a fivefold molar equivalent (in terms of resin
capacity) of both Fmoc-Gly-OH (or suberic acid) and HOBt were
dissolved in a minimum amount of DMF, fivefold molar equiva-
W
lent PyBOP was dissolved separately in a minimum of DMF
3
3
and mixed with a tenfold molar equivalent DIPEA and both were
mixed thoroughly with the resin. In our shake flask method, the
total volume of DMF was limited to <1.5 ml/g of resin. The reac-
tion time of the coupling steps is approximately 60 min for cou-
pling of the Gly residues and overnight for the dicarboxylic acid
linker. The coupling time depended on the completeness of acyl-
ation as determined with the Kaiser [37] and Fmoc tests [24] after
the coupling/deblocking of the Gly residues and a picric acid test
duced into the ionisation source using a Rheodyne injector
Rheodyne/IDEX, Rohnert Park, CA, USA). A capillary voltage of
(
3
.5 kV was applied throughout, and the source temperature was
ꢀ
set at 80 C. The skimmer lens offset was 5 V, and the cone volt-
age was varied between 50 and 60 V. Data acquisition was in
the negative or positive mode, scanning the first analyser (MS1)
through m/z = 100 to 1500 at a scan rate of 675 amu/s. Averaging
spectra across the elution peak and subtracting the background
produced representative spectra. The instrument was calibrated
using the ion spectrum of polyethylene glycol acquired under
similar conditions.
[38] after the coupling of the dicarboxylic acid linker. After the
coupling of the linker, the resin was washed with DMF and
diethyl ether then thoroughly dried under vacuum.
ꢂ
To obtain the fragment patterns, the [M + H] molecular spe-
Part B: synthesis of N,N′-suberoyldiglycylglycine (Figure 1B)
1
cies was selected in MS and subjected to collisionally induced
The unprotected glycylglycine fragment was attached to R-O-Gly
CO(CH COOH (Figure 1A) using a two-step carbodiimide method
33]. A ten times molar equivalent of EDC was dissolved in 250ml
analytical grade water and added to the resin swollen in 250 ml of
5 mM phosphate buffer (pH 5.0). After 15 min of activation, the
2
-
decomposition. These experiments were conducted using the
same ionisation parameters as before. Argon was introduced into
2 6
)
[
ꢂ3
the collision cell at (1.8 ꢁ 0.2) ꢃ 10 mb, and the collision energy
setting was 30 eV. Data acquisition was in the negative mode,
2
scanning the second analyser (MS
a scan rate of 220 amu/s.
2
) through m/z = 10 to 450 at
excess EDC was removed from the resin and the suspension of ac-
tivated resin was added dropwise to 1.5 ml glycylglycine in 0.2 M
phosphate buffer (pH 8.0). The resin mixture was gently shaken
for 24–36 h, where after it was washed on a sintered glass filter with
water, t-amyl alcohol, acetic acid, again t-amyl alcohol and finally
with peroxide-free diethyl ether and then thoroughly dried under
vacuum. To monitor the coupling reaction, a sample of the resin
NMR of crude preparation and purified bolaamphiphile in
DMSO-d were recorded on a Varian VXR 300-MHz instrument
Agilent Technologies, Inc. Santa Clara, CA, USA). Assignments
6
(
were made empirically.
(
~5–10 mg) was taken after 24 and 36 h, washed and treated with
Environmental Scanning Electron Microscopy
TFA (as described later). The TFA-product mixture was then re-
moved and evaporated under nitrogen. The residual product was
made up in 50% acetonitrile and analysed using ESMS.
For imaging with E-SEM, a 5 ml sample of the self-assembled stock
solution of the bolaamphiphile was placed on a support film
coated with silver and mounted on a specimen stub and then
air dried and viewed in E-SEM mode using a Peltier cooling stage.
Removal of peptides from the solid phase resin
ꢀ
The stage was cooled to 5 C at a pressure of 5–6.5 Torr, at a rel-
The peptides were cleaved from the resin with 95% TFA and 5%
ative humidity of between 75 and 100%. By adjusting the water
vapour pressure in the chamber and the temperature of the cool-
ing stage, it was possible to increase or decrease the water level
in and on the surface of the sample. Images were obtained using
the gaseous secondary electron detector. All samples were
viewed using a Philips XL 30 FEG-SEM (FEI, Hillsboro, Oregon,
USA) at 15 kV accelerating voltage. The peptide fibres isolated
from 7-day or older assembled samples were washed thoroughly
with deionised water, and excess water was blotted off with filter
paper. A 4-nm layer of Au/Pd alloy was deposited onto the sam-
ples using an E5100 Polaron sputter coater (Polaron Equipment
Ltd. Hertfordshire, UK). The samples were viewed in the high vac-
uum mode at 10–15 kV accelerating voltage.
H
2
O as scavenger. The peptide resin was treated for 3–6 h with
four-bed volumes 5% H O in TFA. After cleavage, the resin was
removed by filtration and washed with 5% H O in TFA, acetic acid
2
2
and analytical grade water. The combined filtrate containing the
bolaamphiphile was dried under high vacuum on a BÜCHI Rota-
vapor (BÜCHI Labortechnik AG, Switzerland) at 40–50 C, resus-
pended in 50% acetonitrile and lyophilised.
ꢀ
Purification of the Synthesis Product
The synthesis product was purified using the unique self-assembly
properties of the bolaamphiphile product. A number of ꢁ10 mM solu-
tions were prepared in 0.1% TFA and left unperturbed for 1–7days.
J. Pept. Sci. 2012; 18: 317–325 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

NAIDOO AND RAUTENBACH
Fourier Transform Infrared Spectroscopy
phosphate buffer concentration, favours the activation of the free
carboxyl group of the linker and minimises phosphate competition
for EDC. Adjustment to pH 8 by addition of the excess phosphate at
the time of the addition of glycylglycine peptide fragment prevents
the activation of the incoming carboxyl groups on the incoming
peptide by the excess carbodiimide. This allows the preferential for-
mation of the peptide bond between the activated carboxyl groups
of the acid and the amino group of the incoming peptide fragment.
To limit the activation of the carboxyl group of the incoming pep-
tide, the excess EDC was removed by repeated washing of the resin
after the initial activation step. The high concentration of 0.2M
phosphate in the second step favours the reaction of EDC with
phosphate, resulting in the formation of a urea derivative. Phos-
phate thus quenches any residual EDC further minimising activa-
tion of the carboxyl group of the incoming peptide.
Self-assembled samples were carefully isolated and washed with
analytical quality water by centrifugation to remove traces of TFA
as confirmed with IR (absence of TFA bands that interferes with
amide I signals). A lyophilised self-assembly sample was analysed
by means of photoacoustic Fourier transform IR (FTIR) spectros-
copy. The photoacoustic detector used was an MTEC model 300
unit (MTec Photoacoustics, Inc, Ames, Iowa, USA) that was cou-
pled to a Perkin Elmer Paragon 1000 (Perkin Elmer, Waltham,
MA, USA). The following parameters were used for the determi-
nation of each spectrum: mirror velocity, 0.1 cm/s; resolution,
ꢂ1
8
cm ; source aperture, maximum; number of scans, 128; sample
reference, carbon black; and detector gas atmosphere, helium. A
typical scan required 15 min. The IR spectrum was scanned from
ꢂ1
wavenumber 4000 to 450 cm and was mathematically adjusted
The crude and purified peptide bolaamphiphile was charac-
terised by ESMS (Figures 2 and 3). ESMS of the crude product
to compensate for the photoacoustic effect.
2 2 6
showed limited contamination by HO-Gly -CO(CH ) COOH at
m/z = 286, indicating incomplete coupling of the second Gly-Gly
unit (Figure 2A). H-NMR of the crude synthetic compound also
Results and Discussion
1
A number of groups [10–16] have synthesised either the model
bolaamphiphile, N,N′-suberoyldiglycylglycine, or oligoglycine ana-
logues using solution phase synthesis. However, this synthetic
strategy has many limitations especially in terms of versatility of
peptide design specifically related to asymmetric bolaamphiphiles.
The synthesis of the model bolaamphiphile required an adaptation
of conventional SPPS synthetic protocol to a bidirectional synthetic
route (Figure 1). In our synthesis of the symmetric N,N′-suberoyldi-
glycylglycine (Figure 1), the coupling of the first amino acid residue,
Gly, with an acid-labile ester bond to the resin was >99% according
to an analytical Fmoc test [24]. The elongation of each of the pep-
tide chains in the N!C direction was successful as monitored by
the Kaiser test [37]. The overnight coupling of the dicarboxylic acid
linkers led to >99% coupling efficiency in each case as determined
by an analytical picric acid test [38].
indicated that there was incomplete coupling in the last step of
the original synthetic sequences. Signals observed at 2.0–2.2 p.p.m.
in the a-CH of the diacid linker were diagnostic for indicating the in-
2
complete coupling of the peptide fragment.
Purification of N,N′-suberoyldiglycylglycine
For the purification of N,N′-suberoyldiglycylglycine from the
crude synthetic product, we exploited its inherent ability to self-
assemble. A number of solvents were assessed for self-assembly,
but diluted aqueous TFA proved to be exceptional in the promo-
tion of rapid self-assembly. A solution of the crude preparation of
approximately 10 mM, based on the critical micellular concentra-
tion N,N′-suberoyldiglycylglycine [12], was prepared in 0.1% TFA
and was left undisturbed at room temperature for up to 7 days.
Rapid visual self-assembly took place, and we were able to isolate
self-assembled purified product via centrifugation within a day,
but the best overall yields were obtained after 5 days.
NMR of the self-assembled fraction of the N,N′-suberoyldiglycyl-
glycine synthesis product confirmed that the self-assembled frac-
tion contained both glycylglycine moieties in the major compound.
ESMS analysis of the self-assembled product and the supernatant
from the purification verified the efficiency of this extremely simple
purification protocol. Refer to Figure 2A and B and the following
discussion for more detail on the ESMS analyses. We confirmed that
With the activation of the free carboxyl group of the linker on
the resin, it was possible to synthesise the second peptide unit in
the normal C!N direction and couple it in our solid phase
method as protected or unprotected peptide fragment in the
N!C direction. Although we were able to pre-activate the free
carboxyl group of the resin-bound linker by forming its Pfp ester,
the unpredictable solubility of some of the incoming peptides,
such as glycylglycine in suitable organic solvents, made this a less
W
attractive option. Using PyBOP as activation agent in this step
would necessitate the incoming peptide fragment to be pro-
tected in order to limit polymerisation. Unprotected glycylglycine
dipeptide was readily available, but it was highly soluble in water
and poorly soluble in most organic solvents. By using Gly as N-
terminal residue of the peptide unit coupled in the N!C direc-
tion, we avoided the problem of racemisation and increased
our choice of activation agents. Having investigated the phos-
phonium, carbodiimide and active ester activation procedures
for the N!C direction synthesis step, we decided on a modified
two-step carbodiimide method, using the water-soluble carbodii-
mide, EDC (Figure 1). The choice of method hinged solely on the
solubility of the peptide fragment being attached in the N!C
direction, as well as the presence/absence of a chiral N-terminal res-
idue. The EDC method, commonly used in peptide fragment con-
densation reactions [32], utilises the water-soluble properties of
the carbodiimide with activation and coupling proceeding at differ-
ent pH values (pH 5 and 8) in order to limit unwanted side reactions
and polymerisation. The first step at pH 5, together with low
the major contaminant, diglycylsuberic acid or HO-Gly
2 2
-CO(CH )
6
COOH at m/z = 286, remained in the solution and could thus easily
be removed (Figure 2A). The purified mass yield using the rapid
self-assembly was 88%, indicating a high yield synthesis and suc-
cessful purification protocol. In our group, we were also able to suc-
cessfully synthesise Leu-containing and Lys-containing asymmetric
analogues of the model bolaamphiphile, all with >70% yields, us-
ing our novel bidirectional solid phase methodology to couple
the diglycyl unit (results not shown). However, only the anionic ana-
logues and bolaamphiphiles with aliphatic residues responded well
to self-assembly dependent purification in 0.1% TFA.
Characterisation of Purified N,N′-suberoyldiglycylglycine
The high purity and correct molecular mass of N,N′-suberoyldigly-
ꢂ
cylglycine was confirmed with ESMS as we observed [M ꢂ H]
with m/z = 401 (C H N O requires 401.3967) and a TFA adduct
16 26 4 8
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 317–325

NOVEL SPPS AND PURIFICATION BY SELF-ASSEMBLY OF PEPTIDE BOLAAMPHIPHILE
[
M-H]
-
8
74.0
7.0
4
00.6
100
100
A
C
Contaminant
termination product)
(
286.7
1
13.0
2
28.9
4
01.0
4
2.2
130.9
167.2 172.0
211.7
3
43.7
5
14.6
281.9
300
367.0
350
0
0
50
100
150
200
250
400
450
m/z
113
4
00.6
100
74
229
B
D
86
O
O
H
N
H
N
HO
CH CH CH CH CH CH
O
2
2
2
2
2
2
N
N
[M-H+TFA]
-
(adduct)
H
H
O
O
O
O
5
14.5
326
172
[
M-H+2TFA] (adduct)
3
28.7
25.6
131
2
6
28.5
282
0
200
400
600
800
1000
367
m/z
Figure 2. Negative mode ESMS spectra of the products from the self-assembly purification step: supernatant (A) and self-assembled product in pellet
(
B). The spectrum in C depicts the CID fragmentation pattern of [M ꢂ H]ꢂ after selection in MS
1
. Figure 2D shows the proposed fragment identification.
4
00.56
group or an OH group (Figure 2C and D). The product ion with
m/z= 228.7 was attributed to the fission of the C–C bond neighbouring
the amide carbonyl of the linker.
1
00
%
0
A
The NMR analysis of the purified N,N′-suberoyldiglycylglycine
also indicated a very high purity and was consistent with the
structure of this compound and gave the following results:
d ( 300MHz; DMSO-d ; TMS) 1.21–1.29 (4H, m, 2 ꢃ g-CH ), 1.43–1.55
H
6
2
5
14.45
(
4H, m, 2 ꢃ b-CH ), 2.13 (4H, t, J=7.4Hz, 2ꢃ a-CH ), 3.45 (4H, d,
2
2
J = 5.5 Hz, 5 ꢃ 2), 3.51 (4H, d, J = 5.5 Hz, 2 ꢃ 2); 8.13 (4H, t ꢃ 2,
G
G
2
28.65 325.62
J = 5.4 Hz, 2 ꢃ 3
G
and 2 ꢃ 6
G
); d
C
6
(75 MHz; DMSO-d ; TMS). 24.97
(
suberic b ꢃ 2) b 28.40 (suberic b ꢃ 2); 35.17 suberic g ꢃ 2); 41.84
2
00
400
600
800
1000
1200
(CH
2
COO ꢃ 2); 40.93 (CONHCH
2
CO ꢃ 2); 169.81 (CH
2
CH
2
CONH ꢃ 2);
171.68 (CH
2
COO ꢃ 2); 173.09 (CONHCH
2
CO ꢃ 2).
-
1204.41
[3M-H]
-
4
00.56 [M-H]
monomer
8
02.36 [2M-H]
dimer
-
1
00
B
100
C
100
D
trimer
Probing the Rapid Self-assembly of Purified N,N′-
suberoyldiglycylglycine
1
212.89
During the self-assembly dependent purification of the anionic
bolaamphiphile, we found that self-assembly was rapidly initi-
ated in the 0.1% TFA solution. To eliminate the probability of
non-specific precipitation or aggregation in the presence of the
TFA, we decided to probe and revisit the self-assembly process
and macro-assemblies of this model bolaamphiphile.
ESMS was conducted over a period of 7 h on the purified
bolaamphiphile dissolved in 0.1% TFA. The results revealed the
formation of ESMS stable dimers and trimers in the solution, indi-
cating higher-order oligomeric structures and the onset of self-
assembly (Figure 3). We confirmed the rapid self-assembly after
%
%
+
%
+
K
1225.43
8
+
40.57
1184.35
+
Na
8
24.45
3
78.23
3
43.60
0
0
0
350
400
m/z
450
750 775 800 825 850
m/z
1180 1200 1220
m/z
Figure 3. Negative mode ESMS spectra of N,N′-suberoyldiglycylglycineat
at initiation of self-assembly (A) and after 7 h, showing the monomers
remaining in solution (B) and detection of ESMS stable dimers (C) and tri-
mers (D) in solution.
7
h by using E-SEM (Figure 4A). According to earlier reports
[11,12,14], the time required for self-assembly was 7 to 14 days,
(
m/z = 514.5) (compare Figure 2B with 2A). Collisionally induced
however, we observed macro-assemblies and nanostructures
within a day. A distinct feature of bolaamphiphiles is that the ini-
tiation of self-assembly is usually marked by the formation of
monolayers [39]. Layering was evident from the electron
fragmentation of the m/z= 401 molecular ion in the self-assembled
fraction gave the expected fragmentation pattern, namely fission at
the amide bonds and other product ions from the loss of a carboxyl
J. Pept. Sci. 2012; 18: 317–325 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

NAIDOO AND RAUTENBACH
A
A
B
B
Figure 5. Scanning electron microscopy images of the self-assembly
structures of N,N′-suberoyldiglycylglycine after 5–7 days with (A) self-as-
sembly structure with multiple elongated microtubes and (B) larger mi-
crotubular structure. Samples were sputter-coated Au/Pd alloy.
C
formed protrusions into the solvent away from the layered
structure (Figure 4B and C). The formation of the curved tubu-
lar structures from monolayers could be due to strain and tilts
induced by the hydrophobic interactions and the hydrogen-
bonded network. The tight hydrophobic interaction of the
hexamethylene chain promoted by the ‘dehydration’ effect
of the TFA [40] of the flanking peptide moieties and low pH
of the solution probably resulted in the shortening of the
intralayer hydrogen bonds, and this, in turn, induced strain
and a tilt in the molecular arrangement making the b-sheet
network more convex [16]. This tilt could lead the b-sheet
structures to fold and form nanotubes [16].
Figure 4. Environmental scanning electron microscopy images of the
self-assembly process of N,N′-suberoyldiglycylglycine over 24 h with (A)
self-assembly into monolayer and multilayers after 7 h; (B) self-assembly
into nanotubes after 24 h; (C) magnified images of areas in white squares
After 7 days in the acidic bolaamphiphile solution, we ob-
served, with the use of E-SEM, assemblies of elongated micro-
tubes with approximately 3.7 mm outer diameter (Figure 5A).
(2.5 ꢃ 2.5 mm) in B showing the nanotubes.
micrographs obtained after 7 h (Figure 4A). Apart from layering,
the self-assembling bolaamphiphile folded leading to protrusions
into the solvent environment (Figure 4A). This rapid self-assembly
is attributed to the promotion of strong intralayer hydrogen
bonding through the use of 0.1% TFA. The solvent also promotes
more effective hydrophobic interaction of the hexamethylene
chain by ‘drying’ out the flanking peptide backbone, i.e. it out-
competes the water molecules attached to the peptide backbone
B
A
[40]. Kogiso et al. [14] showed with X-ray diffraction that the
amides and two carboxylic acid groups of an analogous diglycyl
bolaamphiphile with a dodecadiacid linker formed a b-sheet lay-
ered assembly via eight amide–amide hydrogen bonds and two
acid–acid hydrogen bonds.
After 24 h, E-SEM imaging revealed the growth of multiple
nanotubes, from the surface of the layered macrostructure. These
nanotubes, with outer and outer and inner diameters of 235 ꢁ 9
and 135 ꢁ 6 nm, respectively, probably assembled from the pro-
trusions formed by the folded monolayers. They typically also
2924
3
086
4
000
3000
2000
1000
-1
Wavenumber (cm )
1
700
1650
1600
-1
Wavenumber (cm )
Figure 6. FTIR of the N,N′-suberoyldiglycylglycine microtubular struc-
tures and fibres isolated after 5 days (A) and second derivative IR spectra
of the bolaamphiphile in the amide I region (B).
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 317–325

NOVEL SPPS AND PURIFICATION BY SELF-ASSEMBLY OF PEPTIDE BOLAAMPHIPHILE
Self assembly of
bolaamphiphiles into
layered -sheets
with a tilt
Bolaamphiphile
oligomer formation in
acidic solution
Folding of tilted
-
sheets to form
nanotubes
Association of
microtubes to form
larger tubular
structures or
macroscopic
bundles
Growth and
association of
nanotubes to form
microtubes
Figure 7. A schematic representation of the major steps in self-assembly of N,N′-suberoyldiglycylglycine.
These microtubes could assemble to form the larger microtubes
In general, the observed tubular structures of N,N′-suberoyldi-
glycylglycine (bis(N -amido-glycylglycine)-1,8-octane dicarboxy-
a
(
10.5 ꢁ 1mm outer diameter) we also observed in the self-assembly
fraction (Figure 5B). More interlayer and intralayer hydrogen
bonding would result in the extension of the b-sheet network, and
this translates into longer and wider tubes [41]. Alternatively, the
assembly of multiple nanotubes via intertubular hydrogen bonds
into macroscopic bundles would also lead to microtubular structures
with wider diameters [16].
late) that formed in the acidic environment correlated with
crystalline tubules that the group of Matsui [13,16] found for
a
the self-assembly of a closely related bolaamphiphile, bis(N -
amido-glycylglycine)-1,7-heptane dicarboxylate, at an acidic pH.
However, the organisation into macroscopic bundles [16] took
place in the absence of metal ions and may be dependent on
the TFA, although we were unable to find any IR bands indicating
that TFA may be acting as cross-linking agent.
FTIR spectroscopy of the microtubes showed a strong CH an-
2
tisymmetric and weaker symmetric stretching band at 2925 and
ꢂ1
2
852 cm , respectively (Figure 6A). This may suggest a gauche-
including conformation rather than an all trans zigzag conforma-
tion [42–44] of the hexamethylene spacer and ‘bended’ spacer in
the tubular structures. However, highly similar CH stretching
2
Conclusions
bands in this model bolaamphiphile were interpreted by Kogiso
The SPPS method combined with fragment condensation using
water-soluble EDC not only represents a very simple, cheap effi-
cient way to attach peptide fragments in the N!C direction
but also addresses the issue of solubility of the peptide fragments
for use in the synthesis of large bolaamphiphilic peptide libraries.
The advantages of our bidirectional solid phase synthesis method
are its flexibility, versatility in terms of bolaamphiphile design (i.e.
to synthesise both symmetrical and asymmetrical peptide
bolaamphiphiles) and the possibility to apply it to a convergent
synthesis protocol. Such an approach provides countless possibil-
ities for the synthesis of ‘peptido’-organic compounds and even
compounds with multiple peptide arms in which the direction
of the peptide bond is reversed in a part of the compound.
An important finding in this study is that the inherent ability of
some small molecules to self-assemble can be used as a purifica-
tion method for such compounds. Crystallisation has long been
an established purification method in organic chemistry. Some
organic components, however, are difficult to crystallise, espe-
cially larger molecules such as peptides and polypeptides. How-
ever, many bio-inspired organic compounds and peptides have
been observed to self-assemble [2–20,41,44,50–61], but the pos-
sibility of rapid self-assembly as purification method for peptides
and analogous compounds has gone, until now, unrecognised.
et al. [12] as an indication of high trans conformational popula-
ꢂ1
tions. Strong amide I bands at 1656 and 1620 cm combined
ꢂ1
with the weaker band at 1688 cm indicate a hydrogen-bonded
network, possibly a mixture of parallel and antiparallel b-sheet
type network [41,45–48] (Figure 6B). Furthermore, N–H stretching
ꢂ1
ꢂ1
bands at 3301 cm (amide stretch A) and 3083 cm (amide
stretch B) together with the amide II bands at 1554 and
ꢂ1
1
576 cm indicate strong intermolecular amide–amide bonds
(Figure 6A) [11–13,42,46–49]. The detected amide stretch A band
was comparable with that for polyglycine I and the amide stretch
B band to that of the acid crystal of this model bolaamphiphile as
observed by Kogiso et al. [12]. The significant red-shifted carboxyl
ꢂ1
acid bands at 1706 and 1722 cm (Figure 6B) also corroborated
observations that the terminal glycine a-COOH groups partici-
pate in bifurcated and lateral acid–acid hydrogen bonds in the
intermolecular network that leads to the formation of tubes
[11–13,41,49]. These bands were also highly comparable with
those found by Kogiso et al. [12] for the acid crystal of this model
bolaamphiphile. Our FTIR results substantiated previous findings
that self-assembly takes place via the formation of b-sheet type
network [11,13,41] and that layer formation is a result of intra-
layer hydrogen bonding via the terminal a-carboxyl groups [41].
J. Pept. Sci. 2012; 18: 317–325 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

NAIDOO AND RAUTENBACH
1
1 Shimizu T, Kogiso M, Masuda M. Vesicle assembly into microtubes.
Nature 1996; 383: 487–488.
2 Kogiso M, Ohnishi S, Yase K, Masuda M, Shimizu T. Dicarboxylic oligo-
peptide bolaamphiphiles: proton-triggered self-assembly of micro-
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DOI:10.1021/la9802419
The choice of dilute aqueous TFA as the acidic solvent medium
for the self-assembly of the model anionic bolaamphiphile signif-
icantly reduced self-assembly time and allowed the purification
of the compound of interest from a crude mixture. Monitoring
of the rapid assembly process corroborated our visual observa-
tion and confirmed programmed self-assembly rather than non-
specific aggregation or precipitation. Also, we obtained a better
understanding of self-assembly in 0.1% TFA and found that there
is a progression from oligomers in the solution to layered b-sheet
structures from which nanotubes and finally microtubes, consist-
ing of a multitude of elongated smaller microtubes, grew over
the self-assembly process (Figure 7).
To our knowledge, this is the first study using rapid self-assembly
induced by TFA for purification of an anionic oligoglycine
bolaamphiphile from a crude synthetic mixture. These results in-
dicated that the acidic environment combined with the dehydra-
tion effect induced by TFA lead to highly specific self-assembly
that can be compared with crystallisation and can therefore func-
tion as a purification method for analogous anionic peptides and
bolaamphiphiles.
1
1
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1
83–189.
1
1
5 Matsui H, Pan S, Gologan B, Jonas SH. Bolaamphiphile nanotube-
templated metallized wires. J. Phys. Chem. B, 2000; 104: 9576–9579.
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macroscopic bundles. Langmuir 2001; 17: 7918–7922. DOI:10.1021/
la010910+
7 Gao BX, Djalali R, Haboosheh A. Peptide nanotubes: simple separation
using size-exclusion columns and use as templates for fabricating
one-dimensional single chains of Au nanoparticles. Adv. Mater. 2005;
1
1
7: 1753–1757. DOI:10.1002/adma.200500357
18 Kameta N, Mizuno G, Masuda M, Minamikawa H, Kogiso M, Shimizu T.
Molecular monolayer nanotubes having 7–9 nm inner diameters
covered with different inner and outer surfaces. Chem. Lett. 2007;
3
6: 896–897. DOI:10.1246/cl.2007.896
1
2
9 Kameta N, Masuda M, Mizuno G, Morii N, Shimizu T. Supramolecular
nanotube endo sensing for a guest protein. Small 2008; 5: 561–565.
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0 Kameta N, Yoshida K, Masuda M, Shimizu T. Supramolecular nano-
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Acknowledgements
This work was supported by the National Research Foundation
SIDA grant to M. R. The authors would like to acknowledge the
following people: Vijay Bandu (Centre for Electron Microscopy,
University of Kwazulu-Natal) for his assistance with the E-SEM,
Johan Eygelaar and Paul Cloete from Roediger Agencies for their
assistance with the photoacoustic FTIR analyses, the late Hendrik
S. C. Spies (NMR Central Analytical Facility, Stellenbosch Univer-
sity) and Martin Bredenkamp (formerly from the Chemistry De-
partment, Stellenbosch University) for their assistance with the
NMR analyses/interpretation and Marthinus J van der Merwe (for-
merly from the LC-MS Central Analytical Facility, Stellenbosch
University) for his assistance with the ESMS analyses.
2
2
2
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J. Pept. Sci. 2012; 18: 317–325 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci