Direct access to side chain N,N'-diaminoalkylated derivatives of basic amino acids suitable for solid-phase peptide synthesis

Article abstract of DOI:10.1007/s00726-012-1336-5

A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N'-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of N ^α-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative N ^α -(9H-fluorenyl-9-methoxycarbonyl)-N ^β,N ^β ′-bis[2-(tert-butoxycarbonylamino)ethyl]-l-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00726-012-1336-5

Amino Acids (2013) 44:321–333
DOI 10.1007/s00726-012-1336-5
ORIGINAL ARTICLE
Direct access to side chain N,N0-diaminoalkylated derivatives
of basic amino acids suitable for solid-phase peptide synthesis
•
Jean-Philippe Pitteloud Nina Bionda
•
Predrag Cudic
Received: 9 March 2012 / Accepted: 29 May 2012 / Published online: 20 June 2012
Ó Springer-Verlag 2012
Abstract A simple and efficient one-pot procedure that
enables rapid access to orthogonally protected N,N0-diam-
inoalkylated basic amino acid building blocks fully com-
patible with standard Boc and Fmoc solid-phase peptide
synthesis is reported. Described synthetic approach
includes double reductive alkylation of Na-protected dia-
mino acids with N-protected amino aldehydes in the pres-
ence of sodium cyanoborohydride. This approach allows
preparation of symmetrical, as well as unsymmetrical,
basic amino acid derivatives with branched side-chains that
can be further modified, enhancing their synthetic utility.
The suitability of the synthesized branched basic amino
acid building blocks for use in standard solid-phase peptide
synthesis has been demonstrated by synthesis of an indo-
licidin analogue in which the lysine residue was substituted
with the synthetic derivative Na-(9H-fluorenyl-9-methox-
Keywords N-alkylation ꢀ Reductive amination ꢀ One-pot
procedure ꢀ Branched basic amino acids ꢀ Solid-phase
peptide synthesis ꢀ Indolicidin
Abbreviations
AcOH
ACN
Aloc
Boc
Acetic acid
Acetonitrile
Allyloxycarbonyl
Tert-butoxycarbonyl
Cationic antimicrobial peptide
Benzyloxycarbonyl
Circular dichroism
Cell-penetrating peptide
Dichloromethane
CAMP
Cbz
CD
CPP
DCM
EDT
EtOAc
Fmoc
FA
1,2-Ethanedithiol
Ethyl acetate
b
b
0
ycarbonyl)-N ,N -bis[2-(tert-butoxycarbonylamino)ethyl]-
L-2,3-diaminopropionic acid. This substitution resulted in
an analogue with more ordered secondary structure in
2,2,2-trifluoroethanol and enhanced antibacterial activity
without altering hemolytic activity.
9-Fluorenylmethoxycarbonyl
Formic acid
HPLC
High-performance liquid
chromatography
LPS
Lypopolysaccharide
MALDI-TOF MS Matrix-assisted laser desorption
ionization time of flight mass
spectrometry
MAP
MBHA
MeOH
MIC
Mtt
Multiantigen peptide system
4-Methylbenzhydrylamine
Methanol
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1336-5) contains supplementary
material, which is available to authorized users.
Minimal inhibitory concentration
4-Methoxytrityl
J.-P. Pitteloud ꢀ N. Bionda ꢀ P. Cudic (&)
Torrey Pines Institute for Molecular Studies,
11350 SW Village Parkway, Port St. Lucie, FL 34987, USA
e-mail: pcudic@tpims.org
NMR
PBS
Nuclear magnetic resonance
Phosphate buffered saline
Solid-phase peptide synthesis
Trifluoroacetic acid
SPPS
TFA
TFE
N. Bionda
Department of Chemistry and Biochemistry,
Florida Atlantic University, Boca Raton, FL 33431, USA
2,2,2-Trifluoroethanol
Triisopropylsilane
TIS
123

322
J.-P. Pitteloud et al.
addition, the reactivity of the lysine side-chain amino
group was also exploited during the preparation of neo-
glycoproteins through mono- and di-reductive amination
using thioglycosides bearing an x-aldehydoaglycon (Lee
and Lee 1980). Interestingly, the acid hydrolysis of the
obtained neoglycoprotein led to the identification of Ne,Ne0-
di(2-aminoethyl)-L-lysine.
TLC
tR
Thin-layer chromatography
Retention time
Trityl
Trt
Introduction
The presence of basic amino acids has been also proved
crucial for many cationic antimicrobial peptides (CAMPs)
(Jenssen et al. 2006; Findlay et al. 2010) and cell-penetrating
peptides (CPPs) (Green and Loewenstein 1988; Derossi et al.
1994; Oehlke et al. 1998). For the former, cationicity pro-
motes the initial electrostatic interactions of the CAMP to the
negatively charged phospholipid membranes of bacteria and
other pathogens, conferring them with selectivity against
microbes compared with host tissues. The presence of
properly spaced positive charges has proved to be important
for binding and neutralizing bacterial lipopolysaccharides
(LPS), the primary cause of the Gram-negative septic shock
(David 2001). A net positive charge at physiological pH has
also been a key factor for the membrane translocation
properties exhibited by CPPs (Futaki et al. 2001; Wender
et al. 2000; Patel et al. 2007). Typically, the cationic CPPs
contain clusters of primarily arginine and lysine residues.
Motivated by the importance and versatility of lysine
and the growing needs for polyfunctional amino acid
building blocks suitable for the synthesis of complex and
diverse peptides on solid-phase, herein we report direct
access to novel orthogonally protected N,N0-diaminoalky-
lated amino acid derivatives (Fig. 1). Our approach con-
sists of the one-pot double reductive alkylation (Levadala
et al. 2004; Bartholoma et al. 2009) of commercially
available Na-protected diamino acids (lysine, ornithine,
2,4-diaminobutyric acid, and 2,3-diaminopropionic acid)
with convenient N-protected aliphatic amino aldehydes in
the presence of sodium cyanoborohydride (NaBH3CN)
(Borch et al. 1971). The suitability of our synthetic basic
amino acid building blocks possessing branched side-
chains for utilization in SPPS has been demonstrated
through the substitution of the sole lysine residue in the
naturally occurring antimicrobial peptide indolicidin
(ILPWKWPWWPWRR-NH2) as a model peptide (Selsted
et al. 1992). The effect of the substitution on the confor-
mation, antibacterial activity, and human erythrocyte tox-
icity of the indolicidin analogue has also been described.
Peptides and proteins play ubiquitous key roles in many
biological systems and have been recognized for their clin-
ical potentials. At present, therapeutic peptides exist for a
wide variety of human diseases (Stevenson 2009). However,
despite the great potential, there are still some limitations for
developing peptides as drugs per se. In particular, these
limitations are high susceptibility to enzymatic proteolysis
and poor penetration of biological barriers, leading to low
oral bioavailability and short in vivo half-lives. For these
reasons, efforts have been directed towards the synthesis of
naturally occurring peptide mimics that display enhanced
pharmacokinetic and biological properties (Liskamp et al.
2011). For instance, replacing a proteolytically cleavable
peptide bond with bioisosteric [peptoids (Zuckermann et al.
1992; Fowler and Blackwell 2009; Yoo et al. 2010), aza-
peptides (Gante et al. 1995; Zega 2005), urea-peptidomi-
metics (Boeijen and Liskamp 1999), b-peptidosulfonamides
(de Jong et al. 2002), depsipeptides (Cudic and Stawikowski
2007)] but less labile chemical entities afforded peptide
analogs (pseudopeptides) with increased stability and bio-
logical activity. Other approaches have focused on the
introduction of constrained amino acid mimetics that can
imitate specific secondary structural features, conferring
them with enhanced pharmacological properties (Toniolo
2004). The cyclization of biologically active peptides
(Katsara et al. 2006; Karskela et al. 2006), the incorporation
of non-proteinogenic amino acids (e.g. D-amino acids,
N-alkylated amino acids, b-amino acids) (Gentilucci et al.
´
2010), as well as peptide glycosylation (Sola and Griebenow
2010) have also met success in improving the physico-
chemical and pharmacological properties of peptides.
Among the amino acid building blocks routinely
employed in solid-phase peptide synthesis (SPPS), basic
amino acids such as lysine, ornithine, diaminobutyric acid,
and diaminopropionic acid have been extensively used for
the synthesis of complex structures due to the reactivity of
their side-chain primary amines (Fig. 1). For example, the
availability of orthogonally protected lysine has been cru-
cial in the synthesis of densely branched multiantigen
peptide systems (MAPs) (Sebestik et al. 2011; Sadler and
Tam 2002), constrained cyclic peptides (Tolle et al. 1982;
Katsara et al. 2006; Zhang and Taylor 1996; Yu and Taylor
1996), a-helical bundle proteins (Hahn et al. 1990; Mutter
et al. 1992), and collagen-like helical structures (Fields and
Prockop 1996; Fields et al. 1993; Grab et al. 1996). In
Materials and methods
General methods
Unless otherwise specified, all chemicals and solvents were
purchased from Sigma-Aldrich (St. Louis, MO, USA) and
123

Direct access to side chain N,N0-diaminoalkylated derivatives of basic amino acids
323
Fig. 1 Common applications of
basic amino acids and design of
novel N,N0-diaminoalkylated
amino acid building blocks
O
O
H
N
H2N
PG
SPPS/branching
Alkylation
OH
OH
Amidation
Cyclization
Cross-linking
m
m
H2N
N
X
m = 1 (Diaminopropionic acid, Dap)
= 2 (Diaminobutyric acid, Dab)
= 3 (Ornithine, Orn)
= 4 (Lysine, Lys)
X
X= NH-Boc, NH-Fmoc, NH-Cbz, N3
were of analytical reagent grade or better. All amino acid
precursors were purchased from Chem-Impex (Wood Dale,
IL, USA) and used without further purification. Marfey’s
reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanineamide, FDAA)
was purchased from TCI America (Portland, OR, USA).
3-(N-benzyloxycarbonyl) aminopropionaldehyde was pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). The controlled addition of solutions was performed
by a ChemyxÒ (Stafford, TX, USA) Fusion 200 syringe
pump. Thin layer chromatography (TLC), used for moni-
toring reactions, was performed on Whatman precoated
silica gel F-254 plates (Whatman Inc., Piscataway, NJ,
USA) and visualized by ultraviolet light and/or staining
with ninhydrin solution. Silica gel 60 (EMD Chemicals,
Germany; particle size 0.040–0.063 mm, 230–400 Mesh
ASTM) was used for flash chromatography. Reverse-phase
flash chromatography was performed on a CombiFlashÒ
CompanionÒ using RediSep Rf GoldÒ C18 columns. The
peptides were synthesized using a PS3 automated peptide
synthesizer (Protein Technologies Inc., Tucson, AZ, USA).
Mass spectrometric analyses (MS) of the amino acids and
peptides were performed on MALDI–TOF Voyager-DETM
STR (Applied Biosystems, Foster City, CA, USA). The
purity of our analogues and peptides ([95 %) was assessed
on an analytical Agilent Infinity 1260 high-performance
liquid chromatography (HPLC) system (Agilent Inc., Santa
Clara, CA, USA) equipped with a UV/Vis detector. For
reverse-phase HPLC analysis, a C18 monomeric column
dichroism (CD) spectra were recorded on a JASCO 810
spectropolarimeter (Easton, MD) using a quartz cell of
0.1 cm optical path length. Spectra were measured over a
wavelength range of 180–250 nm with an instrument
scanning speed 200 nm/min and a response time of 1 s.
The concentrations of peptides were 0.125 mg/mL and
0.25 mg/mL. Microbial strains were purchased from
American Type Culture Collection (ATCC, Manassas,
VA). Dehydrated culture media and agar, and polystyrene
plates used for antimicrobial assays were purchased from
BD (Franklin Lakes, NJ). Control antibiotics were pur-
chased from Sigma Aldrich (St. Louis, MO). Antimicrobial
activity assays were carried out in standard, sterile 96-well
plates, and minimal inhibitory concentration (MIC) values
were determined by measuring turbidity at 600 nm using a
Stat Fax 2100 Microplate reader (Awareness Technology
Inc., Palm City, FL). Human red blood cells (hRBCs) were
purchased from Innovative Research (Novi, MI). Human
serum was purchased from Sigma Aldrich (St. Louis, MO).
NMR analysis
1
The H and 13C NMR spectra were recorded at 400 and
100 MHz, respectively, on a Bruker Avance 400 MHz
NMR spectrometer (Billerica, MA). Samples were pre-
pared in tubes with a diameter of 5 mm employing 0.5 mL
of the indicated deuterated solvent. The chemical shifts (d)
are reported in units of parts per million (ppm) downfield
1
˚
(Grace Vydac, 250 9 4.6 mm, 5 lm, 120 A), 1 mL/min
from tetramethylsilane. The assignment of the H NMR
signals was based on 1H-1H and 13C-1H correlation
flow rate, and elution method of 98 % A for 0.5 min fol-
lowed by linear gradient of 2–85 % B over 32 min were
used, where A was 0.1 % TFA in H2O and B was 0.08 %
TFA in ACN. Eluting products were detected by UV at 214
and 267 nm. The enantiomeric integrity of the synthetic
analogues was determined by derivatization with Marfey’s
reagent and HPLC analysis of the corresponding products.
Rink amide 4-methylbenzhydrylamine (MBHA) resin
(substitution level 0.66 mmol/g) was purchased from
NovabiochemÒ (EMD Chemicals, NJ, USA). Circular
spectrometry.
General procedure for the double reductive alkylation
of Na-protected diamino acids
A solution of Na-protected diamino acid (1–4) in MeOH
(90 mL/mmol) containing 1–6 % AcOH was treated with a
methanolic solution of the corresponding amino aldehyde
(3.0 eq.). After stirring for 1 h at room temperature, a
123

324
J.-P. Pitteloud et al.
solution of NaBH3CN in MeOH (1 mL) was added drop-
wise for 2 h. The resulting clear solution was stirred until
TLC and MALDI-TOF MS analyses of representative ali-
quots confirmed the complete conversion to the desired di-
alkylated building block. The reaction mixture was con-
centrated by rotary evaporation and mixed with EtOAc
before washing with water. The organic phase was sepa-
rated and the aqueous layer extracted three times with fresh
EtOAc. The combined organics were dried over anhydrous
sodium sulfate, filtered, and evaporated under vacuum. The
crude residues were purified by flash chromatography
(silica gel, DCM/MeOH/AcOH 85:10:5) or reverse-phase
flash chromatography (C-18 silica gel, 5–85 % gradient of
ACN in H2O containing 0.1 FA) to give the following pure
di-alkylated products (5–15):
6.7 Hz, 1H, Fmoc-CH2a), 4.30 (00t00, J = 8.9 Hz, 1H, Fmoc-
CH2b), 4.21 (t, J = 6.8 Hz, 1H, Fmoc-CH), 4.08 (d,
J = 6.5 Hz, 1H, Orn-Ha), 3.39–3.32 (m, 4H, arms-CH2),
3.11 (br.s, 6H, Orn-H2c ? arms-CH2), 1.86 (t, J = 6.7 Hz,
1H, Orn-H1b), 1.74 (br.s, 3H, Orn-H2b ? Orn-H2c), 1.40 (s,
18H, Boc-CH3); 13C NMR (101 MHz) d 176.04, 159.10,
158.60, 145.37, 145.15, 142.61, 128.83, 128.81, 128.19,
128.16, 126.31, 126.15, 120.95, 81.23, 68.06, 62.05, 55.37,
55.13, 54.39, 36.98, 30.22, 28.71, 21.77; MALDI-TOF MS,
calculated m/z 640.77, found m/z 641.61 [M ? H]?,
663.59 [M ? Na]?.
a
e
e
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis[2-
(tert-butoxycarbonylamino)ethyl]-L-lysine 8. HPLC:
1
tR = 25.720 min; H NMR (400 MHz, Methanol-d4) d
7.79 (d, J = 7.3 Hz, 2H, Fmoc-H), 7.67 (t, J = 7.8 Hz,
3H, Fmoc-H), 7.39 (t, J = 7.3 Hz, 3H, Fmoc-H), 7.30 (t,
J = 7.2 Hz, 3H, Fmoc-H), 4.40 (dd, J = 10.2, 5.1 Hz,
1H, Fmoc-CH2a), 4.31 (dd, J = 8.8, 4.2 Hz, 1H, Fmoc-
CH2b), 4.22 (t, J = 7.5 Hz, 1H, Fmoc-CH), 4.20–4.14
(m, 1H, Lys-Ha), 3.41 (d, J = 5.5 Hz, 4H, arms-CH2),
3.35–3.26 (m, 4H, arms-CH2), 3.26–3.15 (m, 2H, Lys-
a
b
b
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis[2-
(tert-butoxycarbonylamino)ethyl]-L-2,3-diaminopropionic
acid 5. HPLC: tR = 26.272 min; 1H NMR (400 MHz,
Methanol-d4) d 7.81 (d, J = 7.5 Hz, 2H, Fmoc-H), 7.67 (d,
J = 7.4 Hz, 2H, Fmoc-H), 7.40 (t, J = 7.4 Hz, 2H, Fmoc-
H), 7.31 (t, J = 7.4 Hz, 2H, Fmoc-H), 4.69 (dd, J = 8.6,
5.3 Hz, 1H, Dap-Ha), 4.47 (dd, J = 10.4, 7.1 Hz, 1H,
Fmoc-CH2a), 4.37 (dd, J = 10.2, 7.2 Hz, 1H, Fmoc-CH2b),
4.25 (t, J = 6.9 Hz, 1H, Fmoc-CH), 3.81 (dd, J = 13.6,
H2e), 1.98–1.84 (m, 1H, Lys-H1b), 1.84–1.63 (m, 3H, Lys-
H2b ? Lys-H2d), 1.44 (s, 20H, Boc-CH3 ? Lys-H2c); 13C
NMR (101 MHz) d 175.45, 159.22, 158.71, 145.33,
145.15, 142.60, 128.81, 128.18, 128.15, 126.26, 126.17,
120.94, 81.39, 67.99, 55.38, 54.78, 54.69, 36.72, 32.20,
28.69, 24.23, 23.85; MALDI-TOF MS, calculated m/z
654.79, found m/z 656.00 [M ? H]?.
5.3 Hz, 1H, Dap-H1b), 3.57 (dd, J = 13.5, 9.1 Hz, 1H, Dap-
H2b), 3.44 (s, 8H, arms-CH2), 1.43 (s, 18H, Boc-CH3); 13C
NMR (101 MHz) d 171.81, 159.34, 158.83, 145.05,
142.62, 128.89, 128.19, 126.21, 121.00, 81.48, 68.59,
56.25, 50.88, 36.73, 28.68; MALDI-TOF MS, calculated
m/z 612.71, found m/z 613.94 [M ? H]?, 635.92
[M ? Na]?.
a
b
b
0
N -(tert-butoxycarbonyl)-N ,N -bis[2-(9H-fluorenyl-9-
ylmethoxycarbonylamino)ethyl]-L-2,3-diaminopropionic
acid 9. HPLC: tR = 29.495 min; 1H NMR (400 MHz,
DMSO-d6) d 7.87 (d, J = 7.3 Hz, 4H, Fmoc-H), 7.66 (d,
J = 7.4 Hz, 4H, Fmoc-H), 7.39 (t, J = 7.4 Hz, 4H, Fmoc-
H), 7.31 (t, J = 7.4 Hz, 4H, Fmoc-H), 7.20 (t, J = 5.7 Hz,
2H, Fmoc-CO–NH), 6.94 (d, J = 7.7 Hz, 1H, Boc- CO–
NH), 4.27 (d, J = 7.0 Hz, 4H, Fmoc-CH2), 4.19 (t,
J = 6.9 Hz, 2H, Fmoc-CH), 4.03 (‘‘q’’, J = 7.5 Hz, 1H,
Dap-Ha), 3.05 (‘‘q’’, J = 6.6 Hz, 4H, arms-CH2), 2.80 (dd,
a
c
c
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis[2-
(tert-butoxycarbonylamino)ethyl]-L-2,4-diaminobutyric
acid 6. HPLC: tR = 25.679 min; 1H NMR (400 MHz,
Methanol-d4) d 7.79 (d, J = 7.4 Hz, 2H, Fmoc-H), 7.66 (t,
J = 6.3 Hz, 2H, Fmoc-H), 7.39 (t, J = 7.4 Hz, 2H, Fmoc-
H), 7.31 (t, J = 7.3 Hz, 2H, Fmoc-H), 4.37 (d, J = 6.7 Hz,
2H, Fmoc-CH2), 4.23 (t, J = 6.7 Hz, 1H, Fmoc-CH), 4.13
(t, J = 6.3 Hz, 1H, Dab-Ha), 3.40–3.30 (m, 4H, arms-
CH2), 3.22–2.88 (m, 6H, Dab-H2c ? arms-CH2), 2.25–2.07
J = 13.7, 5.7 Hz, 1H, Dap-H1b), 2.72 (dd, J = 13.3,
8.1 Hz, 1H, Dap-H2b), 2.64–2.51 (m, 4H, arms-CH2), 1.34
(s, 9H, Boc-CH3); 13C NMR (101 MHz) d 173.22, 163.20,
156.23, 155.43, 143.91, 143.87, 140.71, 127.60, 127.06,
125.16, 120.09, 78.12, 65.45, 54.82, 53.29, 52.19, 46.76,
38.31, 28.17; MALDI-TOF MS, calculated m/z 734.84,
found m/z 735.99 [M ? H]?, 757.99 [M ? Na]?.
Na-(tert-butoxycarbonyl)-Ne,Ne0-bis[3-(9H-fluorenyl-9-
ylmethoxycarbonylamino)propyl]-L-lysine 10. HPLC:
tR = 28.586 min; 1H NMR (400 MHz, Methanol-d4) d
7.76 (d, J = 7.5 Hz, 4H, Fmoc-H), 7.60 (d, J = 7.6 Hz,
4H, Fmoc-H), 7.36 (t, J = 7.5 Hz, 4H, Fmoc-H), 7.28 (t,
J = 7.2 Hz, 4H, Fmoc-H), 4.37 (d, J = 6.7 Hz, 4H, Fmoc-
(m, 1H, Dab-H1b), 2.04–1.95 (m, 1H, Dab-H2b), 1.43 (s,
18H, Boc-CH3); 13C NMR (101 MHz) d 158.61, 158.36,
145.37, 142.63, 128.81, 128.18, 126.21, 120.96, 80.80,
67.99, 55.17, 54.73, 53.12, 48.47, 28.75, 20.95; MALDI-
TOF MS, calculated m/z 626.74, found m/z 627.72
[M ? H]?.
a
d
d
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis[2-
(tert-butoxycarbonylamino)ethyl]-L-ornithine 7. HPLC:
tR = 25.742 min; 1H NMR (400 MHz, Methanol-d4) d
7.79 (d, J = 7.5 Hz, 2H, Fmoc-H), 7.66 (dd, J = 11.0,
7.6 Hz, 2H, Fmoc-H), 7.39 (t, J = 7.4 Hz, 2H, Fmoc-H),
7.31 (t, J = 7.4 Hz, 2H, Fmoc-H), 4.40 (dd, J = 10.4,
123

Direct access to side chain N,N0-diaminoalkylated derivatives of basic amino acids
325
CH2), 4.16 (t, J = 6.8 Hz, 2H, Fmoc-CH), 4.10 (dd,
J = 8.4, 4.5 Hz, 1H, Lys-Ha), 3.18 (t, J = 6.2 Hz, 4H,
arms-CH2), 3.14–2.96 (m, 6H, arms-CH2 ? Lys-H2e),
13C NMR (101 MHz) d 174.36, 159.04, 157.97, 138.27,
129.49, 129.04, 128.83, 81.27, 67.61, 55.97, 52.82, 50.61,
38.73, 28.66, 25.69; MALDI-TOF MS, calculated m/z
586.68, found m/z 587.71 [M ? H]?.
1.92–1.76 (m, 5H, Lys-H1b ? arms-CH2-CH2-CH2), 1.74–
1.59 (m, 3H, Lys-H2b ? Lys-H2d), 1.42 (s, 11H, Boc-
a
b
b
0
N -(benzyloxycarbonyl)-N ,N -bis[2-(tert-butoxycar-
bonylamino)ethyl]-L-2,3-diaminopropionic acid 14.
CH3 ? Lys-Hc); 13C NMR (101 MHz) d 175.74, 159.19,
1
2
HPLC: tR = 22.377 min; H NMR (400 MHz, Methanol-
d4) d 7.42–7.25 (m, 5H, Cbz-H), 5.11 (s, 2H, Cbz-CH2),
4.27 (t, J = 7.2 Hz, 1H, Dap-Ha), 3.29 (br. s, 4H, arms-
158.13, 145.22, 142.63, 128.81, 128.74, 128.12, 126.02,
120.98, 80.63, 67.73, 54.32, 53.87, 51.78, 38.60, 32.30,
28.73, 25.51, 24.06, 23.85; MALDI-TOF MS, calculated
m/z 804.97, found m/z 806.05 [M ? H]?.
CH2), 3.25–3.20 (m, 2H, Dap-H2b), 3.18–2.99 (m, 4H,
arms-CH2), 1.43 (s, 18H, Boc-CH3). 13C NMR (101 MHz)
d 174.80, 158.53, 158.48, 138.06, 129.52, 129.09, 128.97,
80.67, 67.92, 57.30, 54.96, 52.10, 37.80, 28.81; MALDI-
TOF MS, calculated m/z 524.61, found m/z 525.90
[M ? H]?.
a
b
b
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis[3-
(benzyloxycarbonylamino)propyl]-L-2,3-diaminopropionic
1
acid 11. HPLC: tR = 26.577; H NMR (400 MHz, Meth-
anol-d4) d 7.77 (d, J = 7.4 Hz, 2H, Fmoc-H), 7.63 (t,
J = 8.1 Hz, 2H, Fmoc-H), 7.37 (t, J = 7.3 Hz, 2H, Fmoc-
H), 7.28 (dd, J = 9.0, 5.6 Hz, 12H, Fmoc-H ? Cbz-H),
5.02 (s, 4H, Cbz-CH2), 4.36 (dd, J = 6.9, 3.6 Hz, 2H,
Fmoc-CH2), 4.30 (t, J = 7.6 Hz, 1H, Dap-Ha), 4.19 (t,
a
b
b
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis(3-
azidopropyl)-L-2,3-diaminopropionic acid 15. HPLC:
1
tR = 23.570 min; H NMR (400 MHz, DMSO-d6) d 7.89
(d, J = 7.4 Hz, 2H, Fmoc-H), 7.71 (dd, J = 7.4, 2.9 Hz,
2H, Fmoc-H), 7.57 (d, J = 8.4 Hz, 1H, Fmoc-CO–NH),
7.42 (t, J = 7.3 Hz, 2H, Fmoc-H), 7.32 (td, J = 7.4,
1.2 Hz, 2H, Fmoc-H), 4.29 (dd, J = 7.0, 3.6 Hz, 2H,
Fmoc-CH2), 4.23 (q, J = 6.9, 6.1 Hz, 1H, Fmoc-CH), 4.12
(td, J = 8.2, 5.9 Hz, 1H, Dap-Ha), 3.32 (t, J = 6.7 Hz, 4H,
J = 6.6 Hz, 1H, Fmoc-CH), 3.28–3.22 (m, 1H, Dap-H1b),
3.24–3.03 (m, 9H, Dap-H2b ? arms-CH2), 1.87 (s, 4H,
arms-CH2-CH2-CH2); 13C NMR (101 MHz, MeOD) d
174.38, 159.06, 158.49, 145.28, 145.12, 142.61, 142.58,
138.25, 129.48, 129.02, 128.82, 128.16, 126.19, 120.95,
68.28, 67.61, 60.39, 55.77, 52.68, 50.95, 38.70, 25.70;
MALDI-TOF MS, calculated m/z 708.80, found m/z 709.99
[M ? H]?, 731.98 [M ? Na]?.
arms-CH2), 2.76 (dd, J = 13.3, 6.1 Hz, 1H, Dap-H1b), 2.66
(dd, J = 13.3, 8.1 Hz, 1H, Dap-H2b), 2.53–2.38 (m, 4H,
arms-CH2), 1.70–1.54 (m, 4H, arms-CH2-CH2-CH2); 13C–
NMR (101 MHz) d 172.96, 155.90, 143.83, 143.78,
140.73, 127.63, 127.03, 125.21, 120.10, 65.70, 54.59,
52.37, 50.45, 48.71, 46.66, 25.79; MALDI-TOF MS, cal-
culated m/z 492.53, found m/z 493.77 [M ? H]?, 515.75
[M ? Na]?.
One-pot synthesis of unsymmetrically N,N0-diam-
inoalkylated amino acid derivative 17 via tandem double
reductive alkylation.
a
e
e
0
N -(9H-fluorenyl-9-ylmethoxycarbonyl)-N ,N -bis[3-
(benzyloxycarbonylamino)propyl]-L-lysine 12. HPLC:
tR = 26.333 min; 1H NMR (400 MHz, Methanol-d4) d
7.79 (d, J = 7.5 Hz, 2H, Fmoc-H), 7.66 (dd, J = 9.8,
7.6 Hz, 2H, Fmoc-H), 7.38 (t, J = 7.5 Hz, 2H, Fmoc-H),
7.35–7.23 (m, 12H, Fmoc-H ? Cbz-H), 5.06 (s, 4H, Cbz-
CH2), 4.40 (dd, J = 10.4, 6.8 Hz, 1H, Fmoc-CH2a), 4.32
(dd, J = 10.5, 7.0 Hz, 1H, Fmoc-CH2b), 4.21 (t,
J = 6.6 Hz, 1H, Fmoc-CH), 4.21–4.15 (m, 1H, Lys-Ha),
3.19 (t, J = 6.4 Hz, 4H, arms-CH2), 3.10 (t, J = 8.1 Hz,
4H, arms-CH2), 3.11–2.96 (m, 2H, Lys-H2e), 1.91–1.78 (m,
A solution of 1a (100 mg, 0.31 mmol) in MeOH con-
taining 6 % AcOH (32 mL) was treated simultaneously
with methanolic solutions of 2-(N-tertbutoxycarbonyl)am-
inoacetaldehyde (59 mg, 0.37 mmol) and NaBH3CN
(16 mg, 0.25 mmol) for 4 h. Upon stirring at room tem-
perature for additional 4 h, solutions of 3-azidopropional-
dehyde (46 mg, 0.46 mmol) and NaBH3CN (16 mg,
0.25 mmol) in MeOH were added simultaneously for 4 h
and the reaction mixture stirred overnight at room tem-
perature. The reaction mixture was concentrated by rotary
evaporation and mixed with EtOAc before washing with
water. The organic phase was separated and the aqueous
layer extracted 3 times with EtOAc. The combined organic
layers were dried over anhydrous sodium sulfate, filtered,
and evaporated under vacuum. HPLC of the crude reaction
mixture revealed the presence of three major products at tR
23.57, 25.01, and 26.27 min. The mixture of products was
5H, Lys-H1b ? arms-CH2-CH2-CH2), 1.78–1.55 (m, 3H,
Lys-H2b ? Lys-H2d), 1.51–1.35 (m, 2H, Lys-H2c); 13C NMR
(101 MHz) d 175.69, 159.20, 158.66, 145.33, 145.13,
142.58, 138.21, 129.51, 129.09, 128.84, 128.81, 128.18,
128.15, 126.25, 126.15, 120.96, 67.96, 67.69, 54.83, 53.81,
51.73, 38.63, 32.23, 25.52, 23.88, 23.79; MALDI-TOF MS,
calculated m/z 750.88, found m/z 752.08 [M ? H]?.
a
b
b
0
N -(tert-butoxycarbonyl)-N ,N -bis[3-(benzyloxycar-
bonylamino)propyl]-L-2,3-diaminopropionic acid 13.
1
HPLC: tR = 22.678 min; H NMR (400 MHz, Methanol-
d4) d 7.39–7.25 (m, 10H, Cbz-H), 5.07 (s, 4H, Cbz-CH2),
4.27 (t, J = 6.8 Hz, 1H, Dap-Ha), 3.31–3.28 (m, 2H, Dap-
H2b), 3.22 (t, J = 6.3 Hz, 8H, arms-CH2), 1.91 (p, J = 8.4,
7.6 Hz, 4H, arms-CH2-CH2-CH2), 1.45 (s, 9H, Boc-CH3).
123

326
J.-P. Pitteloud et al.
separated by reverse-phase flash chromatography (5–85 %
gradient of ACN in H2O, both containing 0.1 % FA) to
give pure unsymmetrically substituted product 17 (65 mg,
38 %) as a white powder.
were loaded with 90 lL of mid-logarithmic phase cells
with initial OD600 0.001 of the tested microorganism and
10 lL aliquots of twofold serial dilutions of the control
antibiotics or tested peptide analogues. Controls on each
plate were media without bacteria, bacterial inoculum
without antimicrobials added and bacterial inoculum to
which control antibiotics were added. In the case of Gram-
positive bacteria controls were methicillin and vancomycin
and in the case of Gram-negative bacteria ampicillin and
ciprofloxacin. Concentrations of these antibiotics were the
same as of the tested peptides, in the range from 1 to
128 lg/mL. All samples were loaded in duplicates. Plates
were incubated at 37 °C overnight with gentle shaking. The
inhibition of the bacterial growth was determined by
Na-(9H-fluorenyl-9-ylmethoxycarbonyl)-Nb-(3-azido-
b
0
propyl)-N -(2-(tert-butoxycarbonylamino)-ethyl)-L-2,3-
diaminopropionic acid 17. HPLC: tR = 25.010 min; 1H
NMR (400 MHz, DMSO-d6) d 7.89 (d, J = 7.5 Hz, 2H,
Fmoc-H), 7.72 (d, J = 7.4 Hz, 2H, Fmoc-H), 7.58 (d,
J = 8.0 Hz, 1H, Fmoc-CO–NH), 7.41 (t, J = 7.4 Hz, 2H,
Fmoc-H), 7.32 (t, J = 7.4 Hz, 2H, Fmoc-H), 6.64 (t,
J = 5.4 Hz, 1H, Boc-CO–NH), 4.32–4.17 (m, 3H, Fmoc-
CH2 ? Fmoc-CH), 4.11 (q, J = 7.8 Hz, 1H, Dap-Ha), 3.32
(t, J = 6.8 Hz, 2H, arm-CH2–N3), 2.99 (q, J = 6.7 Hz,
2H, arm-CH2–NHBoc), 2.79 (dd, J = 13.4, 6.1 Hz, 1H,
measuring OD600
.
Dap-H1b), 2.71 (dd, J = 13.3, 8.3 Hz, 1H, Dap-H2b),
2.60–2.40 (m, 4H, arm-CH2–N3 ? arm-CH2–NHBoc),
1.61 (dq, J = 13.4, 6.8 Hz, 2H, arm-CH2–CH2–CH2–N3),
1.35 (s, 9H, Boc-CH3); 13C NMR (101 MHz) d 172.93,
155.97, 155.64, 143.80, 143.76, 140.70, 127.62, 127.04,
125.27, 125.24, 120.09, 77.57, 65.77, 54.79, 53.09, 52.37,
50.33, 48.69, 46.63, 37.72, 28.20, 25.93; MALDI-TOF MS,
calculated m/z 552.62, found m/z 553.85 [M ? H]?.
Hemolytic activity
Human red blood cells (hRBCs) were diluted with PBS to
2 %. Into each well of the clear, flat-bottom 96-well plate,
50 lL of the hRBCs was placed followed by addition of
50 lL of peptide solution to final peptide concentrations of
8–64 lg/mL. Assays were performed in triplicate, and each
experiment was repeated twice. As a positive control,
50 lL of Triton X-100 in water was used at a final con-
centration of 0.5 % (v/v). As a negative control, 50 lL of
water and PBS was used. Plates were incubated for 1 h at
37 °C after which 100 lL of PBS was added to each well,
and the plates were centrifuged for 10 min at
1,000g. Supernatants (150 lL) were transferred into a new
plate, and absorbance at 405 nm was measured. The data
were corrected for background to obtain solely hemolytic
activity of the peptides. The degree of hemolysis was
expressed in percent relative to total hemolysis caused by
Triton-X-100.
Synthesis of indolicidin and its modified analogue
Indo-5
Both indolicidin and its analogue Indo-5 were synthesized
using Fmoc-protected amino acids on a Rink amide
4-methylbenzhydrylamine (MBHA) resin (substitution
level 0.66 mmol/g) by solid-phase Fmoc- methodology on a
PS3 automated peptide synthesizer. Cleavage of the pep-
tides from the solid support was carried out by Reagent K
containing TIS (TFA/H2O/thioanisole/phenol/EDT/TIS
81.5:5:5:5: 2.5:1 v/v) for 2 h at room temperature. The crude
peptides were precipitated, washed with cold methyl tert-
butyl ether, and purified on an Agilent Infinity 1260 HPLC
system using a preparative Vydac C18 column (Grace Vy-
Results and discussion
˚
dac, 250 9 22 mm, 10 lm, 120 A) with an elution method
The main goal of the work described here was to synthesize
novel orthogonally protected branched basic amino acids
suitable for the construction of complex peptides on a solid
support. The versatility and general applicability of the
described synthetic route were demonstrated by utilization
of a variety of amino acid and aldehyde starting materials,
Table 1. In order to meet the requirements of standard
solid-phase peptide synthesis, Fmoc, Boc, Cbz carbamates,
and azide (Lundquist and Pelletier 2001) as amine pro-
tecting groups were used.
The treatment of Na-Fmoc-2,3-diaminopropionic acid
(1a) with excess 2-(N-tert-butoxycarbonyl) aminoacetal-
dehyde (3 eq.) in the presence of NaBH3CN afforded the
protected dialkylated amino acid 5 in 85 % yield (entry 1,
consisting of a linear gradient of 2–85 % B over 32.5 min,
where A was 0.1 % TFA in H2O and B was 0.08 % TFA in
ACN. The final purity of the peptides ([98 %) was assessed
by analytical reverse-phase HPLC using conditions descri-
bed in the general methods. The molecular masses were
determined using MALDI-TOF MS.
Antimicrobial activity
The assessment of antibacterial activity and determination
of MIC values was done using standard micro dilution
broth method according to the Clinical and Laboratory
Standards Institute (CLSI) guidelines (CLSI 2011) using
96-well flat-bottomed polystyrene microtiter plates. Plates
123

Direct access to side chain N,N0-diaminoalkylated derivatives of basic amino acids
327
Table 1). The acidic conditions employed (6 % AcOH in
MeOH, pH *4) were optimal for the complete solubiliza-
tion of the diamino acid precursor and the stability of the
reducing agent. Moreover, the acid-labile Boc-protecting
group displayed complete stability after prolonged stirring
and post-reaction manipulations. Similar treatment of
longer, offering less sterically hindered reaction sites, Na-
Fmoc amino acid precursors 2a–4a resulted in the corre-
sponding branched basic amino acids 6–8 in excellent
yields and purities (entries 2–4). The direct use of ornithine
derivative 3a as a hydrochloric salt did not affect the
reaction outcome under the optimized conditions.
Employing the same approach, the synthesis of analogues
bearing a Na-Boc protecting group was explored as well. As
in the aforementioned cases, no steric effect on the reaction
yield was observed. The reaction of 4b with 3-(N-9-fluor-
enylmethoxycarbonyl) aminopropionaldehyde (Reggelin
et al. 2006) resulted in 89 % yield of the desired product 10
(entry 6). Shortening the length of both building blocks, 1b
and 2-(N-9-fluorenylmethoxycarbonyl) aminoacetaldehyde
(Diness et al. 2004) did not affect the reaction outcome, and
product 9 was obtained in 80 % yield (entry 5).
The orthogonality of both Fmoc and Boc protecting
groups present in analogues 5–10 allows the modification of
the branched side-chains while on solid support, a desirable
feature during the construction of complex peptides. How-
ever, the acidic conditions required for the removal of Boc
limit the use of more acid labile protecting groups (e.g. Trt
or Mtt) and acid labile resins (e.g. trityl, Sieber amide, Rink
acid). Also, it is worth noting that the incorporation of a
second side-chain containing primary amine offers the
possibility of exploiting such branched amino acid building
blocks as lysine mimics bearing two positive charges under
physiological conditions (vide infra).
Therefore, it was important to demonstrate that the
described reaction conditions are compatible with other
combinations of orthogonal protecting groups. The reaction
of Na-Fmoc-protected substrates 1a and 4a with 3-(N-
benzyloxycarbonyl) aminopropionaldehyde yielded the
corresponding dialkylated products 11 and 12 in 86 and
Table 1 Double reductive alkylation of Na-protected diamino acids with selected N-protected amino aldehydes
H
O
O
R2
N
CHO
n-1
H
N
H
N
NaBH3CN
R1
OH
R1
OH
MeOH/AcOH
m
m
NH2
N
n
n
1 m=1 (Dap)
Series: a R1 = Fmoc
b R1 = Boc
2 m=2 (Dab)
NH
HN
3
4
m=3 (Orn)
m=4 (Lys)
c
R1 = Cbz
R2
R2
Entry
Substrate
R1
n
R2
Product
Yield
(%)a
1
1a
2a
3a
4a
1b
4b
1a
4a
1b
1c
1a
Fmoc
Fmoc
Fmoc
Fmoc
Boc
2
2
2
2
2
3
3
3
3
2
3
Boc
Boc
Boc
Boc
5
6
85
89
87
88
80
89
86
89
82
81
65b
2
3
7
4
8
5
Fmoc
Fmoc
Cbz
Cbz
Cbz
Boc
N3
9
6
Boc
10
11
12
13
14
15
7
Fmoc
Fmoc
Boc
8
9
10
11
Cbz
Fmoc
The protected diamino acid dissolved in MeOH/AcOH was treated with the corresponding aldehyde (3.0 eq.) followed by a solution of
NaBH3CN in MeOH and stirring at room temperature
a
starting diamino acid as limiting reagent
b
purified by RP-flash chromatography with a gradient 5–100 % B, where A was 0.1 % FA in H2O and B was 0.1 % FA in ACN
123

328
J.-P. Pitteloud et al.
89 %, respectively (entries 7 and 8). Identical treatment of
Na-Boc-2,3-diaminopropionic acid 1b with excess 3-(N-
benzyloxycarbonyl) aminopropionaldehyde gave com-
pound 13 in 82 % yield (entry 9). No effect on the reaction
yield was observed upon switching the protecting groups of
starting materials. The reaction of Na-Cbz-Dap-OH 1c with
2-(N-tert-butoxycarbonyl) aminoacetaldehyde afforded
analogue 14 in 81 % yield and high purity. The stability
displayed by Cbz protecting group towards the conditions
commonly employed in Fmoc SPPS, makes analogues 11
and 12 suitable for the construction of peptides that can be
further modified upon release of the protected peptide from
hyperacid labile resins (e.g. Rink acid, HMPB, 2-chloro-
trityl) or resins susceptible to nucleophilic displacement
(e.g. HMBA) (White and Chan 2003; Novabiochem Cat-
alog 2010/2011; Tsubery et al. 2000; White and Yudin
2011). However, taking into consideration that the solid-
phase methodology is a standard approach for routine
peptide synthesis, modifications of peptide’s basic amino
acid sequence(s) on the solid support is highly desirable.
The unique and versatile properties offered by the azide
group made it a very attractive option for functionalization
of the poly-amino acid building blocks that will allow
further solid-phase modifications.
building blocks in a single step, a solution of 1a in MeOH/
AcOH was treated simultaneously with methanolic solu-
tions of 2-(N-tert-butoxycarbonyl)aminoacetaldehyde
(1.2 eq.) and NaBH3CN (0.8 eq.) for a period of 4 h
(Scheme 1). Upon stirring at room temperature for addi-
tional 4 h, the presence of monoalkylated derivative 16
along with unreacted starting 1a and homo-dialkylated 5
was confirmed by TLC. Consequently, the simultaneous
addition of 3-azidopropionaldehyde (1.5 eq.) and a second
portion of NaBH3CN (0.8 eq.) for 4 h yielded a mixture of
the desired mixed-dialkylated product 17 (52 %) along
with homo-dialkylated byproducts 5 (25 %) and 15 (22 %)
(based on RP-HPLC analysis of the crude reaction mix-
ture). The mixture of products 5, 15, and 17 (tR 26.27,
23.57, and 25.01 min) was efficiently separated by reverse-
phase flash chromatography to give unsymmetrically
substituted product 17 in overall 38 % yield. Although the
synthesis of mixed dialkylated amino acid building blocks
has been previously reported (Levadala et al. 2004), the
nature of the appended groups (heterocyclic, carboxylic
acid) in described derivatives prevents their further modi-
fication upon incorporation into the peptide sequences. In
contrast, branched amino acid-building blocks 15 and 17
are fully compatible with the standard Boc and Fmoc-SPPS
allowing their further synthetic exploitation in preparation
of diverse and unusual peptide sequences.
For example, the azide group can be considered as a
temporarily protected amine that can be selectively trans-
formed into a free amine by treatment with an adequate
aryl/alkyl phosphine in the presence of water (Lundquist
and Pelletier 2002). Also, efficient conditions have been
described for the direct conversion of azides to various
carbamates, including allyloxycarbamate (Aloc) (Ariza
et al. 1999). More importantly, organic azides have been
increasingly popular for their chemoselective and mild
reaction with terminal alkynes via 1,3-dipolar cycloaddi-
tion (Hu¨isgen reaction) in the presence of copper (I) salts
(Rostovtsev et al. 2002; Tornøe et al. 2002). The extraor-
dinary level of selectivity, reliability, simplicity, and scope
displayed by this reaction place it as a gold standard in the
field of ‘click chemistry’ (Kolb et al. 2001). Furthermore,
modifications of this reaction in which the toxic copper is
not necessary have been reported to be highly compatible
in the study of biological processes in living systems
(Chang et al. 2010; Ning et al. 2008; Lutz 2008). Other
applications of azides in organic synthesis have been
It has been reported previously that activation of car-
boxylic acids by dialkoxyboranes may result in the for-
mation of amides and lactams (Collum et al. 1978). Also,
the tandem amination-lactamization of ortho-carbox-
ybenzaldehyde with amines and amino esters in the pres-
ence of NaBH(OAc)3 has been suggested to take place via
carboxyl group activation by a borane or borate interme-
diate (Abdel-Magid et al. 1994). However, in our case no
cyclic byproducts were detected under the applied reaction
conditions. In addition, the stereochemical integrity of
diamino acids 1–4 was not compromised by the alkylation
reactions, as indicated by RP-HPLC analysis of product 6
as a model system, upon derivatization with Marfey0s
reagent (Bhushan and Bru¨ckner 2004) (Online Resource).
Synthesis of modified indolicidin analogues
In order to test the suitability of synthesized branched basic
amino acid building blocks for use in standard automated
Fmoc-solid-phase peptide synthesis, we have prepared an
analogue of the cationic antimicrobial peptide indolicidin,
as a model peptide, in which its sole lysine residue was
replaced with N,N0-diaminoalkylated lysine analogue 5
(Indo-5). Indolicin (H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp–
Trp-Pro-Trp-Arg–Arg-NH2) is a 13 amino acid C-terminus
amidated peptide isolated from the granules of bovine
neutrophils and it is the smallest known, naturally
¨
reviewed as well (Brase et al. 2005). Taking all these into
consideration, we have synthesized symmetrical diazido 15
as well as unsymmetrical monoazido 17 amino acid
derivatives.
Symmetrical derivative 15 was synthesized by con-
trolled addition of 3-azidopropionaldehyde (3 eq.) (Boyer
1951; Davies et al. 1967) and NaBH3CN to a solution of 1a
in MeOH/AcOH (65 % yield, entry 11). In an attempt to
synthesize unsymmetrically substituted amino acid
123

Direct access to side chain N,N0-diaminoalkylated derivatives of basic amino acids
329
lysine residue in indolicidin will increase its net positive
charge and disrupt the balance between the charge and
hydrophobicity without altering the number of peptide
bonds.
O
NH
Fmoc
Boc NH
Fmoc
OH
Both indolicidin and its analogue Indo-5 were suc-
cessfully synthesized using Fmoc-protected amino acids
and Rink amide MBHA resin following standard Fmoc-
SPPS protocols on an automated peptide synthesizer. Upon
cleavage of the peptides from the solid support and puri-
fication by HPLC, 98 % pure peptides were obtained. The
antimicrobial activities of both indolicidin and its analogue
Indo-5 were tested against multidrug-resistant strains of
Staphylococcus aureus [ATCC 33591] and Escherichia
coli [ATCC 29181], and the results are summarized in
Table 2. Interestingly, the synthetic analogue Indo-5,
bearing an additional positive charge, showed somewhat
improved activity against both bacterial strains (MIC
32 lg/mL) in comparison with the natural product indo-
licidin (MIC 64 lg/mL). These results are in agreement
with previous reports in which an increase in net positive
charge translates into enhanced indolicidin’s antimicrobial
activity (Nan et al. 2009a, b). However, the obtained MIC
data showed that a decrease in overall hydrophobicity, as
indicated by the HPLC retention times (Table 2), did not
correlate with their antibacterial activities (Lee et al. 2004;
Chen et al. 2007), with less hydrophobic Indo-5 being
more active. Not surprisingly, indolicidin and Indo-5
exhibited comparable hemolytic activities, confirming the
dependence of its hemolytic activity with specific trypto-
phans and the arginines in the sequence (Ando et al. 2010).
The structural features of indolicidin and Indo-5 were
monitored by CD spectroscopy, Fig. 2. The CD spectra
were recorded in water as well as in less polar solvent
systems such as 50 and 100 % TFE. Typically, TFE is used
as a membrane mimicking solvent, and it is known to
induce formation of the stable conformations in peptides
which are otherwise unstructured in aqueous solutions
(Sonnichsen et al. 1992; Roccatano et al. 2002; Otvos
1997).
NH2
1a
O
NaBH3CN
MeOH/AcOH
H
O
H
N
OH
NH
5
NH
Boc
16
H
NaBH3CN
N3
O
O
H
N
Fmoc
OH
5
15
N
The CD spectrum of indolicidin measured in water was
similar to those previously reported, displaying a single
negative band at 201 nm, characteristic of an unordered
conformation (Falla et al. 1996; Ando et al. 2010). How-
ever, the proposed secondary structure of indolicidin has
been somewhat controversial, and poly-L-Pro II helix and
b-turn structures were reported as well (Hsu et al. 2005;
Ladokhin et al. 1999; Andrushchenko et al. 2006).
NH
Boc
N3
17
Scheme 1 One-pot synthesis of unsymmetrically substituted N,N0-
diaminoalkylated amino acid building block via tandem double
reductive alkylation
An increase in the TFE concentration resulted in a slight
shift of the minimum from 201 to 198 nm suggesting no
major conformational change. In the case of the indolicidin
analogue Indo-5, the CD spectrum recorded in water
showed an intense negative band at 201 nm along with a
weak positive band at 227 nm, reminiscent of a poly-L-Pro
occurring, linear cationic antimicrobial peptide. In general,
the antibacterial activity of cationic peptides can be mod-
ulated through alteration of the peptide’s hydrophobicity
and/or net charge. Therefore, incorporation of 5 instead of
123

330
J.-P. Pitteloud et al.
% Hemolysise
Table 2 Retention time, charge, antimicrobial and hemolytic activity of indolicidin (Indo) and its analogue (Indo-5)
Peptides
tR (min)a
Net charge
MIC (lg/mL)b
S. aureusc
E. colid
Indo
Indo-5
a
19.05
18.47
?4
?5
64
32
64
32
20
18
Analytical RP-HPLC conditions described in general methods
See ‘‘Materials and methods’’ section
ATCC 33591
b
c
d
e
ATCC 29181
Determined at MIC concentration of peptides and expressed in percent relative to total hemolysis caused by Triton-X-100
(a)
(b)
O
O
H
H
N
ILPW
N
WPWWPWRR
NH2
ILPW
WPWWPWRR
NH2
N
N
H
H
N
NH3
NH3
NH3
Fig. 2 Structures and CD spectra of a indolicidin, and b synthetic analogue Indo-5 in water (solid line), 50 % TFE (dashed line), and 100 %
TFE (dotted line)
II helix structure. However, the observed decrease in the
intensity of the CD spectrum of Indo-5 in 50 and 100 %
TFE suggests appearance of a more ordered secondary
structure (Otvos 1997). Obtained results suggest that the
substitution of the lysine residue with our branched basic
amino acids, such as 5, can induce changes in the peptide’s
secondary structure.
diaminoalkylated amino acid building blocks suitable for
standard Boc and Fmoc-solid-phase peptide synthesis. The
synthetic strategy includes double reductive alkylation of
commercially available Na-protected diamino acids with N-
protected amino aldehydes in the presence of sodium
cyanoborohydride. The mild reaction conditions required
for this reaction proceeds without giving rise to side-
products. The advantages of this approach include versatile
starting materials and access to symmetrically and
unsymmetrically branched basic amino acid building
blocks. In addition, the possibility of further modification
of these derivatives in solution or on solid support made
them well suited for potential combinatorial chemistry
Conclusion
We have devised a simple and efficient one-pot procedure
that enables rapid access to orthogonally protected N,N0-
123

Direct access to side chain N,N0-diaminoalkylated derivatives of basic amino acids
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diversity. Successful synthesis of an indolicidin analogue
in which Lys residue was substituted with 5 demonstrated
applicability of the prepared branched amino acid building
blocks in standard SPPS. This substitution resulted in an
indolicidin analogue with increased net positive charge,
more ordered secondary structure, improved antibacterial
activity, and with hemolytic activity comparable to the
parent natural product.
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Taking into consideration the increasing interest for
peptides with unusual structural features due to their
improved biological properties, the described synthesis of
N,N0-diaminoalkylated basic amino acids is of particular
practical value.
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Acknowledgments We acknowledge support of the N,N0-diam-
inoalkylated basic amino acid building blocks work described herein
by the Torrey Pines Institute for Molecular Studies (TPIMS) start-up
fund to P.C. We are especially thankful to Dr. Stanislaw F. Wnuk and
Yong Liang from Florida International University for their assistance
with NMR experiments. Also, we thank our colleague Dr. Mare Cudic
for helpful comments and Ms. Karen Gottwald for editing the text.
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