Solid-phase route to Fmoc-protected cationic amino acid building blocks

Article abstract of DOI:10.1007/s00726-012-1239-5

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work Aconvenient, inexpensive route to multiple-charged amino acid building blocks with v

Full text of DOI:10.1007/s00726-012-1239-5

Amino Acids (2012) 43:1633–1641
DOI 10.1007/s00726-012-1239-5
ORIGINAL ARTICLE
Solid-phase route to Fmoc-protected cationic amino acid
building blocks
•
Jacob Dahlqvist Clausen Lars Linderoth
•
•
Hanne Mørck Nielsen Henrik Franzyk
Received: 19 October 2011 / Accepted: 31 January 2012 / Published online: 23 February 2012
Ó Springer-Verlag 2012
Abstract Diamino acids are commonly found in bioac-
tive compounds, yet only few are commercially available
as building blocks for solid-phase peptide synthesis. In the
present work a convenient, inexpensive route to multiple-
charged amino acid building blocks with varying degree of
hydrophobicity was developed. A versatile solid-phase
protocol leading to selectively protected amino alcohol
intermediates was followed by oxidation to yield the
desired di- or polycationic amino acid building blocks in
gram-scale amounts. The synthetic sequence comprises
loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a
freshly prepared trityl bromide resin, followed by ring
opening with an appropriate primary amine, on-resin
Nb-Boc protection of the resulting secondary amine,
exchange of the Na-protecting group, cleavage from the
resin, and finally oxidation in solution to yield the target
c-aza substituted building blocks having an Fmoc/Boc
protection scheme. This strategy facilitates incorporation of
multiple positive charges into the building blocks provided
that the corresponding partially protected di- or polyamines
are available. An array of compounds covering a wide
variety of c-aza substituted analogs of simple neutral amino
acids as well as analogs displaying high bulkiness or poly-
cationic side chains was prepared. Two building blocks were
incorporated into peptide sequences using microwave-
assisted solid-phase peptide synthesis confirming their gen-
eral utility.
Keywords Aziridine ꢀ Cationic amino acids ꢀ
Hydrophobic amino acids ꢀ Peptides ꢀ Solid-phase synthesis
Introduction
Traditionally, native peptides have been deemed less
appealing as pharmaceuticals due to their inherent draw-
backs: (1) short duration of action caused by low in vivo
stability and fast renal clearance, (2) lack of oral bio-
availability, (3) difficult and costly synthesis (Nestor 2009;
Otvos 2008), or (4) inefficient recombinant expression.
Nevertheless, in recent years, peptide-based drugs have
attracted considerable attention as improved synthetic
methods and medicinal chemistry advances have alleviated
many of their shortcomings. Stability towards proteolysis
may be achieved by e.g., incorporation of unnatural amino
acids, backbone modification or N-terminal acylation
(Bellmann-Sickert et al. 2011). Further, PEGylation and
conjugation to fatty acids have proved beneficial for a
prolonged action due to slow release from subcutaneous
depots into circulation as well as improved clearance pro-
files (Bellmann-Sickert et al. 2011; Pollaro and Heinis
2010). Together with reduced production prices, this has
led to a revived interest in synthetic peptides, which now
account for a substantial fraction of compounds undergoing
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1239-5) contains supplementary
material, which is available to authorized users.
J. D. Clausen ꢀ H. Franzyk (&)
Department of Drug Design and Pharmacology,
Faculty of Health and Medical Sciences,
University of Copenhagen, Universitetsparken 2,
2100 Copenhagen, Denmark
e-mail: hf@farma.ku.dk
J. D. Clausen ꢀ H. M. Nielsen
Department of Pharmacy, Faculty of Health and Medical
Sciences, University of Copenhagen, Universitetsparken 2,
2100 Copenhagen, Denmark
L. Linderoth
Novo Nordisk A/S, Novo Nordisk Park, 2760 Maløv, Denmark
˚
123

1634
J. D. Clausen et al.
clinical trials in many pharmaceutical companies (Haberl
et al. 2009; Stevenson 2009). In the last decade, several
peptide therapeutics have in fact been marketed towards
blockbuster indications such as diabetes, cancer, autoim-
mune inflammatory and cardiovascular diseases, CNS-
related conditions as well as infections (Bellmann-Sickert
and Beck-Sickinger 2010; Bellmann-Sickert et al. 2011;
Briand and Muller 2010; Jenssen 2009; Nestor 2009; Otvos
2008). Other continuously evolving areas concern drug
delivery with peptide carriers, i.e., cell-penetrating peptides
capable of targeting, interacting with and crossing extra-
cellular as well as subcellular membranes (D’Souza et al.
2011; Fischer et al. 2005; Foerg and Merkle 2008; Foged
and Nielsen 2008; Mae and Langel 2006; Vives et al. 2008;
Wender et al. 2008; Won et al. 2011).
Dap-OH, Na-Fmoc-Nb-benzyl-Dap-OH, Na-Fmoc-Nb-(2-
hydroxyethyl)-Dap-OH, and Na-Fmoc-Nb-(isopropyl,
Boc)-Lys-OH), but usually at high cost.
Protocols for reductive amination of Dap (Chhabra et al.
2002; Flouret et al. 1995) and lysine (Flouret et al. 1995;
Huang et al. 2007; Schierholt and Lindhorst 2009; Stef-
anowicz et al. 2010) have been reported. Although this
direct route is compatible with Fmoc protection, the start-
ing Fmoc-Dap-OH is expensive or requires synthesis from
asparagine via several steps (Akaji and Aimoto 2001).
Alternatively, strategies based on readily accessible pre-
cursors of N-alkylated Dap derivatives may be employed.
However, as nucleophilic conditions are involved, Na-
benzyloxycarbonyl (Z) protection is usually employed
during the key step, e.g., ring opening of Na-Z-serine-b-
lactone constitutes a relatively efficient way of producing
Na-Z-protected intermediates (Cassidy and Poulter 1996;
Kretsinger and Schneider 2003; Moura and Pinto 2007;
Ratemi and Vederas 1994), albeit it involves subsequent
protecting group manipulations to give the desired Na-
Fmoc-protected building blocks. Likewise, 4-nitrophenyl-
sulfonyl (p-nosyl)-activated aziridines readily undergo ring
opening with amines (Hu 2004; Olsen et al. 2007), and
recently this has been exploited in solid-phase synthesis
(SPS) (Ottesen et al. 2009). Attachment to the resin via a
trityl linker (Crestey et al. 2008) proved favorable since
ring opening of the resulting sterically congested activated
chiral aziridine (i.e., resin 2 in Fig. 1) proceeded regiose-
lectively (Ottesen et al. 2009), whereas solution-phase ring
opening of tert-butyl N-(o-nosyl)-aziridine-2-carboxylate
with benzylamine afforded the desired product and its
regioisomer in a 3.4:1 ratio (Turner et al. 2001). The ver-
satility of the chosen solid-phase synthesis strategy (Fig. 1)
appears only limited by the diversity of primary amine
nucleophiles, and by the ease of the on-resin protection of
the resulting secondary amine (Scheme 1).
Unnatural amino acids may be applied as sequence
modifications that confer enhanced protease resistance as
well as improved pharmacological properties to a bioactive
peptide. For this purpose, a plethora of amino acids dis-
playing variations of side chain structure have been
introduced as commercial building blocks for solid-phase
peptide synthesis (SPPS). Development of novel readily
accessible amino acids is essential for continuous expan-
sion of the possible chemical space for peptide design.
While a vast number of hydrophobic and bulky amino
acids have been widely employed, unnatural diamino acids
are scarcely represented in modified peptides, the homo-
logs of lysine being the only exceptions. However, since
rare, complex non-essential diamino acids are commonly
found in biologically active natural products (Nestor
2009), they appear to constitute a relevant, but quite
unexplored motif for optimization of peptide leads.
Structural diversity in a,b-diamino acids has been intro-
duced via enantioselective synthesis based on catalytic
asymmetric reactions, chiral auxiliaries as well as on uti-
lization of the chiral pool, e.g., extensive functional group
manipulation of cheap natural amino acids (Viso et al.
2005; Wang et al. 2010). Thus, efficient methodologies
leading to chiral 3-alkyl or 3-aryl substituted 2,3-diamin-
obutyric acids have been reported, but most routes suffer
from incompatibility with protecting groups suitable for
standard fluorenylmethyloxycarbonyl (Fmoc)-SPPS, which
ideally requires an Na-Fmoc and side-chain N-butyloxy-
carbonyl (Boc) protection scheme.
To investigate the scope of this protocol, several
building blocks (Fig. 2) displaying di- and polyamino-
functionalized side chains were selected as targets, e.g.,
R
NBoc
β
α
N
O Trt
OH
p
FmocHN
COOH
N
Ns
Instead of carefully establishing additional chiral cen-
ters, our current focus is on establishing a general route to
cationic mimics of neutral amino acids via formal insertion
of an azamethylene moiety into their side chains. It was
envisioned that a convenient way to construct such build-
ing blocks involved the formal alkylation of a suitable
L-2,3-diaminopropanoic acid (Dap) derivative or precursor.
A few such partially or fully N-protected building blocks
are commercially available (e.g., Na-Fmoc-Nb-methyl-
1
2
OH
H2N
COOMe
R
3a R = Trt
3b R = pNs
Fig. 1 Retro-synthesis of Nb-alkylated L-Dap building blocks from L-
serine methyl ester
123

Solid-phase route to Fmoc-protected cationic amino acid building blocks
1635
conditions in the SPS approach. To confirm their synthetic
utility, selected building blocks were to be incorporated
into modified peptide sequences using SPPS.
a,b
c,d
N
O Trt
HO Trt
p
Ns
e,f
2
R
R
Results and discussion
BocN
BocN
p
O Trt
Ns
Solid-phase synthesis of cationic amino acid building
blocks
O Trt
FmocHN
N
H
4
5
An array of primary amines was chosen to meet our aim of
designing amino acids that display a wide variety of cat-
ionic side chains comprising a few resembling those of
essential amino acids except for the c-aza substitution
(Fig. 2). This strategy also facilitates incorporation of
multiple positive charges provided that appropriate par-
tially protected di- and polyamines are available. The
selected array of building blocks covers a high diversity, as
it comprises simple diamino acids (1a, 1b and 1e), high
bulkiness (1c, 1d and 1f) as well as polycationic side chains
(1g and 1h).
Thus, to obtain Na-Fmoc-protected amino acid building
blocks with Boc protection of the remaining amino
group(s), an 8-step protocol was developed (Scheme 1).
The sequence involves simple activation of an inexpensive
trityl alcohol resin, loading of an activated aziridine alco-
hol followed by four efficient on-resin transformations,
R
BocN
g
h
1a-h
OH
FmocHN
6
Scheme 1 Reagents and conditions: (a) AcBr (10 Eq), DCM,
30 min, rt; (b) (S)-1-(p-nosyl)aziridine-2-methanol (1.5 Eq, 3b),
DIPEA (3 Eq), DCM, 15 min, rt; (c) amine (R-NH2) (4 Eq), DCE,
15 min, MW, 80°C; (d) Boc2O (4 Eq), DCE, 30 min, MW, 120°C;
(e) 2-mercaptoethanol (10 Eq), DBU (5 Eq), DMF, 2 h, rt; (f) Fmoc-
OSu (5 Eq), DIPEA (5 Eq), THF, 16 h, rt; (g) TFA/DCM (2:98),
45 min, rt; (h) NaOCl (5 Eq), TEMPO (1 Eq), KBr (0.1 Eq), acetone/
5% NaHCO3 (aq), 1 h, 0°C
c-aza-lysine derivative 1g (Chhabra et al. 2002). Hence,
synthesis of this series of Na-Fmoc-protected amino acid
building blocks for SPPS was undertaken using general
Fig. 2 Array of Nb-substituted
Dap building blocks for SPPS
displaying high diversity:
varying degree of
1d
1e
BocN
BocN
hydrophobicity (i.e., 1a, 1b, and
1e), high bulkiness (i.e., 1c, 1d,
and 1f), or multiple charges
(i.e., 1g and 1h)
OH
OH
FmocHN
FmocHN
O
O
1f
1c
BocN
BocN
NH2
H2N
OH
OH
FmocHN
FmocHN
NH2
H2N
O
O
N
O
Trt
H2N
NH2
p
Ns
NHBoc
2
H2N
NHBoc
NBoc
BocN
NH2
BocN
BocN
OH
OH
O
1b
FmocHN
FmocHN
1g
NHBoc
O
Boc
N
N
NHBoc
Boc
BocN
BocN
FmocHN
OH
OH
FmocHN
O
O
1h
1a
123

1636
J. D. Clausen et al.
thus minimizing loss of product as well as time spent on
purification since only simple washing procedures were
required between each step. Finally, the desired crude
building blocks were obtained after cleavage under mild
conditions and a subsequent oxidation of the corresponding
alcohol.
Table 1 The outcome of target Dap building block synthesis
Compound
Nb-alkyl
substituent (R)
Scale
(mmol)
Overall
yield (%)
1a
1b
1c
1d
1e
1f
Propyl
5.7
5.7
4.5
5.6
4.6
4.5
11.0
5.6
54
77
76
64
63
67
71
33
Isobutyl
Cyclohexylmethyl
Adamant-1-ylmethyl
Benzyl
(S)-1-Tritylaziridine-2-methanol (3a; Fig. 1) (Baldwin
et al. 1993; McKeever and Pattenden 2003; Utsunomiya
et al. 1993) was prepared from L-serine methyl ester on a
100-g scale (*tenfold up-scaling cf. literature procedure)
resulting in an overall yield of 63% via esterification to the
methyl ester followed by N-tritylation, O-mesylation, and
ring closure to give the corresponding aziridine methyl
ester, which then was reduced. All conversions required
only purification by simple non-chromatographic work-up
or crystallization (Baldwin et al. 1993; Ottesen et al. 2009).
The key intermediate (S)-1-(p-nosyl)aziridine-2-methanol
(3b) was in turn obtained from 3a on a large scale
([60 mmol) that together with a slightly modified proce-
dure resulted in an improved yield (75%). Then, 3b was
loaded onto freshly prepared polystyrene trityl bromide
resin (Crestey et al. 2008) to give resin-bound aziridine 2,
which was subjected to microwave-assisted nucleophilic
ring opening with primary amines. The resulting secondary
amine intermediate was readily Boc-protected with Boc2O
under microwave-assisted conditions to give resin 4
(Scheme 1). Removal of the nosyl group with 2-mercap-
toethanol/DBU and subsequent Fmoc protection with
Fmoc-OSu/DIPEA gave resin 5 from which protected
amino alcohol 6 was cleaved under mild conditions (2%
TFA in DCM) compatible with its side chain Boc protec-
tion. Finally, oxidation of 6 with NaOCl/TEMPO/KBr in a
heterogeneous mixture of acetone and 5% aqueous
NaHCO3 yielded the final building blocks 1a–h.
Naphth-1-ylmethyl
N-Boc-(2-aminoethyl)
Boc3(spermine)
1g
1h
appear to be correlated with high uptake efficiency in
human cells (Patel et al. 2007; Stewart et al. 2008).
Recently, designed 8-meric peptides were reported to be
capable of efficiently transporting a fluorescent cargo even
across the highly negative membranes of mitochondria.
The uptake of such mitochondria-penetrating peptides
(MPPs) was found to depend distinctly on the ratio and
distribution of hydrophobic and basic residues (Kelley
et al. 2011; Yousif et al. 2009). Hence, we chose the overall
most promising peptide sequence [FrFxK]2 (F = L-Phen-
ylalanine = Phe, r = D-Arginine, Fx = L-Cyclohexylala-
nine = Cha, K = L-Lysine) reported from those studies as
the template for preliminary model sequences incorporat-
ing our amino acids displaying similar hydrophobic side
chains as well as conferring additional positive charge to
the peptide. Several residues (e.g., Phe and Arg) constitute
sites for possible replacement with the present synthetic
cationic amino acids.
Thus, in order to prove the synthetic utility of the
present amino acid building blocks, two of these (1e and
1g) were incorporated into 12-meric analogs (Table 2) of
the above selected peptide sequence using Fmoc-based
SPPS. By performing the syntheses on an automated
microwave-assisted peptide synthesizer, their compatibility
with such conditions was also shown. The sequences were
prepared as C-terminal amides using a Rink amide resin,
while DIC/HOAt was employed as coupling system. As the
applicability of cationic peptides is often investigated by
cellular uptake studies using detection by fluorescence, the
Using the appropriate primary amines as nucleophiles in
the sequence depicted in Scheme 1, the eight building
blocks were obtained in moderate to high overall yields
(33–77%; Table 1). The differences in the overall yields
appear not to be correlated to steric hindrance of the pri-
mary amines. However, when multiple Boc protecting
groups are present (e.g., in 1g and 1h) an alternative work-
up procedure should be used to avoid a diminished yield
due to partial loss of Boc groups (otherwise only 34% yield
was obtained for 1g). Yet, the combination of a high-
loading resin and a building block of considerable size (i.e.,
as in the synthesis of 1h) may also contribute to a signif-
icant lowering of the yield.
Table 2 Modified 12-mer peptides based on a sequence exhibiting
mitochondrial penetration
Peptide Sequence
Charge Residues MW
7
TAMRA-
1eRFxKFR1eRFxKFR-
NH2
?9
12
2,264.9
Incorporation of cationic hydrophobic amino acids
into peptides
8
TAMRA-
F1gFxKFRF1gFxKFR-
NH2
?9
12
2,153.7
Membrane-interacting peptides generally contain numer-
ous basic residues as a high number of positive charges
123

Solid-phase route to Fmoc-protected cationic amino acid building blocks
1637
˚
was from NovaBiochem. DMF dried over 4 A molecular
N-terminal required labelling with a fluorescent cargo.
Polarity and charge of fluorescence probes may confer
significantly changed overall properties to the labelled
peptides, however, only a negatively charged probe (e.g.,
carboxyfluorescein) is expected to impede the uptake effi-
ciency of the peptides (Yousif et al. 2009). Thus, the
positively charged 5(6)-carboxytetramethylrhodamine
(TAMRA) was conjugated manually to the N-terminal
upon SPPS of the sequences (Table 2). After purification
by HPLC, the two peptides were obtained in purities above
95%.
sieves (H2O \ 0.01%) was from Fluka, and anhydrous
˚
CH2Cl2 was distilled from P2O5 and kept over 4 A
molecular sieves. Water for reversed-phase HPLC was
filtered through a 0.22 lm membrane filter (Millipak40,
Millipore). Other solvents were analytical or HPLC grade
and were used as received. Microwave-assisted ring
opening of 2 in the syntheses of 1a–h was carried out in
an initiator single-mode microwave cavity producing
controlled irradiation at 2.45 GHz (Biotage AB, Uppsala).
The reactions were run in sealed vessels (10–20 mL), and
magnetic stirring was used. Variable power was employed
to reach the temperature desired (80 and 120°C, respec-
tively) (within 1–2 min) and then to maintain it during the
period of time programmed. Temperature was monitored
by an IR sensor focused on a point on the reactor vial; the
sensor was calibrated to an internal solution reaction
temperature by the manufacturer. Preparation of resin-
bound peptides was performed on a CEM Liberty
microwave peptide synthesizer, and the final introduction
of TAMRA was performed manually at rt. Analytical
DAD–HPLC was performed using a Phenomenex Luna
C18(2), 3 lm, 4.6 9 150 mm column eluted at a rate of
0.8 mL/min. Injection volumes were 20 lL of a 1 mg/mL
solution, and separations were performed at 40°C. Solvent
mixtures A (H2O–MeCN–TFA 95:5:0.1) and B (MeCN–
H2O–TFA 95:5:0.1) were used. A linear gradient 0–80%
B during 5 min followed by a linear rise to 100% B
during 25 min was used for elution. Building blocks were
detected with UV at k = 269 nm (detection of the Fmoc
group). Preparative HPLC separations of peptides were
carried out on an XBridgeTM Prep C18, 5 lm, OBDTM
50 9 250 mm column. Solvent A: H2O; Solvent B:
MeCN; Solvent C: 1% TFA in H2O were used in a gra-
dient rising linearly from 30 to 50% of B during 40 min
with 10% C constantly at a flow rate of 60 mL/min. The
fractions of interest were analyzed by LCMS. Fractions of
[95% purity were pooled and lyophilized. Vacuum liquid
chromatography (VLC) was performed using Merck silica
Conclusion
The main advantage of the present protocol is that it allows
for a fast convenient gram-scale parallel solid-phase syn-
thesis of arrays of cationic amino acid building blocks
suitably protected for applications in peptide modification
using standard SPPS methods. Thus, we have obtained
eight different Na-Fmoc-protected cationic amino acid
building blocks using the serine-derived (S)-1-(p-no-
syl)aziridine-2-methanol as the common key intermediate
that was readily obtained in multi-gram scale from an
inexpensive starting material and cheap chemicals.
It is expected that the currently improved access to this
relatively unexplored type of amino acid building blocks
may promote further developments in drug-related peptide
research, as incorporation in different classes of mem-
brane-interacting peptides, e.g., cell-penetrating peptides or
antimicrobial peptides may indeed prove beneficial due to
their cationic-hydrophobic character. Such unnatural amino
acids may also be introduced as a means of enhancing
stability of peptides towards proteolytic degradation.
Incorporation of Nb-alkylated Dap residues in peptides
may also generate surface patches that allow for strong
polar interactions that are able to interfere with hydro-
phobic protein–protein interactions. Finally, Nb-alkylated
Dap derivatives and analogs displaying di- and polycat-
ionic side chains constitute unique building blocks for
combinatorial chemistry, and for construction of dendri-
mers with a higher degree of amine functionalization than
e.g., traditional lysine dendrimers.
1
gel 60 (particle size 0.015–0.040 mm). H NMR and 13C
NMR spectra were recorded at 300 and 75 MHz,
respectively, using CD3OD as solvent. Multiplicities of
1H NMR signals are reported as follows: s, singlet; bs,
broad singlet; d, doublet; dd, doublet of doublets; ddd,
doublet of doublet of doublets; t, triplet; m, multiplet. All
target amino acid building blocks were characterized by
HRMS performed on an Agilent Technologies 1200 Ser-
Experimental
General
ies LC/MSD-TOF instrument using a C18 column
(5?95% MeCN in H2O, 15 min), while HRMS of pep-
tides was performed using a Bruker MicrOTOF-Q II
Quadropol MS Detector in positive mode. Optical rota-
tions were measured with a Perkin–Elmer model 241
polarimeter.
Starting materials were purchased from commercial sup-
pliers and used without further purification. Trityl alcohol
polystyrene resin (loading: 1.75 mmol/g) was obtained
from IRIS Biotech. Rink amide AM resin (0.71 mmol/g)
123

1638
J. D. Clausen et al.
(S)-[1-(4-Nitrophenylsulfonyl)aziridin-2-yl]methanol
(3b)
times). Then Boc2O (4 Eq) in DCE (4 mL/g resin) was
added to the resin in 20 mL MW reaction vials, and then
the mixture was heated by MW to 120°C for 30 min. The
resin was transferred to a 300 mL manual reaction vessel
with baffles/coarse glass frit/screw cap/Teflon stopcock
mounted on a 180° variable-speed flask shaker and washed
and drained (DMF, MeOH, DCM; each 3 9 100 mL).
1,8-Diazabicycloundec-7-ene (DBU; 5 Eq) in dry DMF
(3 mL/g resin) and 2-mercaptoethanol (10 Eq) in dry DMF
(3 mL/g resin) were added to the resin which then was
shaken at rt for 90 min. The resin was drained and treated
once more with DBU (5 Eq) and 2-mercaptoethanol (10
Eq) in dry DMF for 30 min. The resin was washed and
drained (DMF, MeOH, DCM; each 3 9 100 mL).
9-Fluorenylmethyl-succinimidyl carbonate (Fmoc-OSu) (5
Eq) in dry THF (12 mL/g resin) was added to the resin pre-
swollen in dry THF (4 mL/g resin) with diisopropylethyl-
amine (DIPEA) (5 Eq) added. The mixture was shaken at rt
for 16 h. The resin was washed and drained (DMF, MeOH,
DCM; each 39). The product was cleaved off the resin by
treatment with 2% TFA in DCM (10 mL/g resin) for
30 min and then repeated (10 mL/g resin) for 15 min. The
resin was eluted with additional DCM (10 mL/g resin), and
the pooled eluates were concentrated in vacuo with toluene
to give the crude product as orange oil. The crude was
suspended in a mixture of acetone (10 mL/mmol) and 5%
aqueous NaHCO3 (5 mL/mmol). Upon cooling to 0°C,
KBr (0.1 Eq) and 2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO; 1 Eq) was added to the mixture. Then, an
aqueous NaOCl solution (10–15%; 5 Eq) was added
dropwise. The mixture was stirred vigorously at 0°C until
TLC showed full conversion (approx. 1 h). Acetone was
removed in vacuo, and the resulting aqueous phase was
acidified with 10% citric acid to pH *5–6, and then the
product was extracted with EtOAc (3 9 25 mL/mmol).
The combined organic phases were washed with brine
(25 mL/mmol), dried (Na2SO4), filtered and concentrated
in vacuo to yield the crude as a yellow oil, which was
purified by VLC ([8 cm; stepwise gradient ending with
3:2:0.01 Heptane–EtOAc–AcOH) to yield the desired
building block as pale amorphous foam (34–76%). Purity
was analyzed by reversed-phase analytical HPLC.
(S)-1-tritylaziridin-2-methanol (3a; 20 g, 63.4 mmol) dis-
solved in CHCl3–MeOH (3:1; 40 mL) was added dropwise
to TFA (47 mL, 634 mmol) at 0°C. The resulting mixture
was stirred at 0°C for 1 h. Toluene (100 mL) and MeOH
(100 mL) were added to the reaction mixture, which then
was concentrated in vacuo (39) to remove TFA. The res-
idue was dissolved in EtOAc (100 mL), which was
extracted with water (2 9 100 mL). The combined aque-
ous phases were treated with NaHCO3 (*20 g) to pH *8
and then added EtOAc (100 mL). Then 4-nitrobenzene-1-
sulfonyl chloride (13.35 g, 60.2 mmol) in EtOAc (100 mL)
was added dropwise to the two-phase mixture, which was
stirred for 1 h at rt. The phases were separated, and the
aqueous phase was diluted with water (100 mL) and
extracted with additional EtOAc (3 9 100 mL). The
combined organic phases were washed with brine
(200 mL), dried (Na2SO4), filtered and concentrated in
vacuo to yield the crude as an orange oil, which was
purified by VLC ([8 cm; stepwise gradient ending with
3:2:0.01 Heptane–EtOAc–AcOH) to yield the desired
product as a slightly yellowish solid. Yield 12.3 g (75%).
Large-scale loading of resin
AcBr (25.9 mL, 350 mmol) was carefully added to trityl
alcohol resin (20.0 g, 1.75 mmol/g) pre-swollen in dry
DCM (100 mL) at rt into a 300 mL manual reaction vessel
with baffles/coarse glass frit/screw cap/Teflon stopcock
mounted on a 180° variable-speed flask shaker. The reac-
tion mixture was shaken at rt for 30 min and then washed
with dry DCM (3 9 100 mL, each for 5 min) to remove
excess AcBr. A solution of DIPEA (18.3 mL, 105 mmol)
and 3b (13.56 g, 52.5 mmol) in DCM (100 mL) was added
to the Trt-Br resin. The resulting mixture was shaken for
15 min at rt. The resin was drained and washed (DMF,
MeOH, DCM; each for 3 times). Upon draining, the resin
was transferred to a round-bottomed flask and dried in
vacuo to give resin-bound (S)-1-(p-nosyl)aziridine-2-
methanol (2; 30.65 g, 1.14 mmol/g) (Crestey et al. 2008).
Na-Fmoc-Nb-(Propyl, Boc)-L-Dap-OH (1a)
General procedure for SPS of amino acid building
blocks (GP)
Preparation according to GP using propan-1-amine
(1.85 mL, 22.6 mmol). Yield: 1.44 g (54%) as pale foam.
Purity: 99%. [a]2D0 = -16.8° (c = 0.53, MeOH); 1H NMR
(CD3OD, 300 MHz): d 7.76 (d, J = 7.5 Hz, 2H), 7.63 (bd,
J = 7.0 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.28 (t,
J = 7.5 Hz, 2H), 4.50 (dd, J = 5.0, 8.0 Hz, 1H), 4.33 (m,
2H), 4.17 (t, J = 6.5 Hz, 1H), 3.63–3.79 (m, 1H),
3.30–3.54 (m, 1H; hidden in part under the solvent peak),
The appropriate amine (4 Eq) was added to resin-bound
(S)-1-(p-nosyl)aziridine-2-methanol pre-swollen in dichlo-
roethane (DCE) (6 mL/g resin) in one or more microwave
reactors (each of 20 mL). The mixture was heated by
microwave (MW) to 80°C for 15 min. The resin was
transferred to a syringe (50 mL) fitted with a PP filter, and
then washed and drained (DMF, MeOH, DCM; each 3
123

Solid-phase route to Fmoc-protected cationic amino acid building blocks
1639
3.04–3.30 (m, 2H), 1.53 (m, 2H), 1.44 (s, 9H), 0.83 (t,
J = 7.5 Hz, 3H). 13C NMR (CD3OD, 75 MHz): d 173.7,
158.2, 156.9/157.7, 144.9/145.0 (2C), 142.4 (2C), 128.6
(2C), 128.0 (2C), 126.1 (2C), 120.8 (2C), 81.5/81.2, 68.0,
54.6, 50.8/51.1, 49.7, 48.3, 28.7 (3C), 22.1/22.8, 11.5.
HRMS: (m/z): [M ? Na]? calcd for C26H32N2O6,
491.2152; found, 491.2158; DM = 1.2 ppm.
NMR (CD3OD, 300 MHz): d 7.76 (d, J = 7.5 Hz, 2H),
7.64 (d, J = 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t,
J = 7.5 Hz, 2H), 4.54–4.69 (m, 2H), 4.23–4.40 (m, 2H),
4.18 (t, J = 7.0 Hz, 1H), 3.74–3.86 (m, 1H), 3.34–3.50 (m,
1H), 3.10 (m, 1H), 2.83 (m, 1H), 1.92 (bs, 3H), 1.66 (m,
6H), 1.50 (bm, 6H), 1.46* (s, 9H), 1.42 (s, 9H), * = Minor
rotamer. 13C NMR (CD3OD, 75 MHz): d 173.8/173.7,
158.2, 157.3/158.0, 145.0 (2C), 142.4 (2C), 128.6 (2C),
128.0 (2C), 126.1 (2C), 120.8 (2C), 81.4, 68.0, 60.9/61.2,
54.3/53.6, 51.9/52.3, 48.3, 42.0/42.3 (3C), 38.0 (3C), 37.2/
37.0, 29.8 (3C), 28.7/28.7 (3C). HRMS: (m/z): [M ? Na]?
calcd for C34H42N2O6, 597.2935; found, 597.2912;
DM = 3.8 ppm.
Na-Fmoc-Nb-(Isobutyl, Boc)-L-Dap-OH (1b)
Preparation according to GP using 2-methylpropan-1-
amine (2.25 mL, 22.6 mmol). Yield: 2.10 g (77%) as pale
1
foam. Purity: 98%. [a]2D0 = -18.9° (c 0.53, MeOH); H
NMR (CD3OD, 300 MHz): d 7.77 (d, J = 7.5 Hz, 2H),
7.64 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.28 (t,
J = 7.5 Hz, 2H), 4.55 (dd, J = 5.0, 9.0 Hz, 1H), 4.33 (m,
2H), 4.18 (t, J = 7.0 Hz, 1H), 3.65–3.82 (m, 1H),
3.28–3.53 (m, 1H; hidden in part under the solvent peak),
3.06–3.22 (m, 1H), 2.91–3.04 (m, 1H), 1.90 (n,
J = 7.0 Hz, 1H), 1.45* (s, 9H), 1.43 (s, 9H), 0.85 (d,
J = 6.5 Hz, 6H), 0.84* (d, J = 6.5 Hz, 6H), * = Minor
rotamer. 13C NMR (CD3OD, 75 MHz): d 173.7, 158.2,
157.0/157.8, 145.0/145.2 (2C), 142.4 (2C), 128.6 (2C),
128.0 (2C), 126.1 (2C), 120.8 (2C), 81.5/81.3, 68.0, 56.3/
56.7, 54.4/54.2, 50.1/49.7, 48.3, 29.0/28.4, 28.7 (3C), 20.5
(2C). HRMS: (m/z): [M ? Na]? calcd for C27H34N2O6,
505.2309; found, 505.2329; DM = 3.9 ppm.
Na-Fmoc-Nb-(Benzyl, Boc)-L-Dap-OH (1e)
Preparation according to GP using benzylamine (2.00 mL,
18.2 mmol). Yield: 1.47 g (63%) as pale foam. Purity:
1
97%. [a]2D0 = -13.4° (c 0.52, MeOH); H NMR (CD3OD,
300 MHz):
d 7.76 (d, J = 7.5 Hz, 2H), 7.64 (d,
J = 7.0 Hz, 2H), 7.16–7.40 (m, 9H), 4.53 (dd, J = 5.0,
9.0 Hz, 1H), 4.42–4.63 (m, 1H), 4.24–4.40 (m, 3H), 4.17 (t,
J = 7.0 Hz, 1H), 3.69 (m, 1H), 3.41 (m, 1H; hidden in part
under the solvent peak), 1.48* (s, 9H), 1.38 (s, 9H),
* = Minor rotamer. 13C NMR (CD3OD, 75 MHz): d
173.5, 158.2, 157.2/157.6, 144.9/145.0 (2C), 142.4 (2C),
138.8/139.3, 129.5 (2C), 128.6 (2C), 128.6, 128.4, 128.2,
128.0 (2C), 126.1 (2C), 120.8 (2C), 82.0/81.8, 68.0, 54.3,
51.6/52.6, 48.8, 48.3, 28.6 (3C). HRMS: (m/z): [M ? Na]?
calcd for C30H32N2O6, 539.2152; found, 539.2168;
DM = 2.9 ppm.
Na-Fmoc-Nb-(Cyclohexylmethyl, Boc)-L-Dap-OH (1c)
Preparation according to GP using cyclohexylmethanamine
(2.33 mL, 17.9 mmol). Yield: 1.77 g (76%) as pale foam.
1
Purity: 99%. [a]2D0 = -15.9° (c 0.52, MeOH); H NMR
Na-Fmoc-Nb-(Naphth-1-ylmethyl, Boc)-L-Dap-OH (1f)
(CD3OD, 300 MHz): d 7.77 (d, J = 7.5 Hz, 2H), 7.64 (d,
J = 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t,
J = 7.5 Hz, 2H), 4.54 (dd, J = 5.0, 9.0 Hz, 1H), 4.33 (m,
2H), 4.18 (t, J = 7.0 Hz, 1H), 3.64–3.81 (m, 1H),
3.26–3.53 (m, 1H; hidden in part under the solvent peak),
3.07–3.24 (m, 1H), 2.92–3.06 (m, 1H), 1.54–1.75 (m, 6H),
1.44 (s, 9H), 1.43* (s, 9H), 1.11–1.31 (m, 3H), 0.81–0.98
(m, 3H), * = Minor rotamer. 13C NMR (CD3OD,
75 MHz): d 173.7, 158.2, 157.0/157.8, 145.0/145.0 (2C),
142.4 (2C), 128.6 (2C), 128.0 (2C), 126.1 (2C), 120.8 (2C),
81.5/81.3, 68.0, 55.2/55.5, 54.4/54.3, 50.2/49.7, 48.3, 37.9/
38.6, 32.0, 31.9/31.8, 28.7 (3C), 27.6, 27.0 (2C). HRMS:
(m/z): [M ? Na]? calcd for C30H38N2O6, 545.2622; found,
545.2647; DM = 4.5 ppm.
Preparation according to GP using naphthalen-1-ylmetha-
namine (2.63 mL, 17.9 mmol). Yield: 1.70 g (67%) as pale
foam. Purity: 98%. [a]2D0 = -5.2° (c 1.01, MeOH); 1H
NMR (CD3OD, 300 MHz): d 7.16–7.80 (m, 15H),
4.80–5.10 (m, 2H), 4.54 (m, 1H), 4.31 (m, 2H), 4.17 (t,
J = 6.5 Hz, 1H), 3.22–3.70 (m, 2H; hidden in part under
the solvent peak), 1.49 (s, 9H), 1.40* (s, 9H), * = Minor
rotamer. 13C NMR (CD3OD, 75 MHz): d 173.6, 158.1,
156.9/157.5, 145.0/145.0 (2C), 142.4 (2C), 135.2, 133.6/
133.9, 132.6/132.4, 129.7/129.8, 129.0/129.2, 128.6 (2C),
128.0 (2C), 127.1, 126.7, 126.2, 126.1 (2C), 125.8, 124.2/
123.8, 120.8 (2C), 82.0, 67.9/68.3, 54.1, 50.4, 48.3,
48.1–48.6, 28.7 (3C). HRMS: (m/z): [M ? Na]? calcd for
C34H34N2O6, 589.2309; found, 589.2326; DM = 2.8 ppm.
Na-Fmoc-Nb-(Adamant-1-ylmethyl, Boc)-L-Dap-OH (1d)
Na-Fmoc-c-aza-L-Lys(Boc2)-OH (1g)
Preparation according to GP using 1-adamantylmethan-
amine (2.00 g, 12.1 mmol). Yield: 2.04 g (64%) as pale
Preparation using tert-butyl 2-aminoethylcarbamate
(5.00 g, 31.2 mmol) according to GP until cleavage, upon
1
foam. Purity: 99%. [a]2D0 = -21.0° (c 0.53, MeOH); H
123

1640
J. D. Clausen et al.
Fluorophore conjugation and purification
which the resin was drained into a DCM solution (50 mL)
with an equivalent amount (relative to TFA) of DIPEA
added. The organic phase was washed (1 9 sat. NaHCO3,
2 9 H2O, each 25 mL). The crude was concentrated in
vacuo before the final oxidation step. Yield: 4.42 g (71%)
as pale foam. Purity: 97%. [a]2D0 = -13.5° (c 0.53,
MeOH); 1H NMR (CD3OD, 300 MHz): d 7.77 (d,
J = 7.5 Hz, 2H), 7.64 (d, J = 7.0 Hz, 2H), 7.37 (t,
J = 7.0 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 4.54 (dd,
J = 4.5, 9.0 Hz, 1H), 4.33 (m, 2H), 4.19 (t, J = 6.5 Hz,
1H), 3.68–3.85 (m, 1H), 3.12–3.56 (m, 5H; hidden in part
under the solvent peak), 1.45 (s, 9H), 1.41 (s, 9H). 13C
NMR (CD3OD, 75 MHz): d 173.8, 158.2, 157.5/157.0,
144.9/145.1 (2C), 142.4 (2C), 128.6 (2C), 128.0 (2C),
126.1 (2C), 120.8 (2C), 81.8/81.6, 80.0, 68.0/68.3, 54.5/
54.6, 50.5/49.8, 48.9, 48.3, 28.8 (3C), 28.7 (3C). HRMS:
(m/z): [M ? Na]? calcd for C30H39N3O8, 592.2629; found,
592.2629; DM = 0.0 ppm.
Upon deprotection the N-terminal of each of the complete
oligomer sequences, they were conjugated manually in a
10 mL syringe fitted with a PP-filter to 5(6)-carboxyte-
tramethylrhodamine (TAMRA; 3 Eq) with HOAt (3 Eq)
and DIC (3 Eq) in NMP overnight at rt. The resin was
washed (NMP, MeOH, DCM; each 39). The conjugated
sequences were finally deprotected and cleaved in a single
step with TFA–H2O–triisopropylsilane (95:2.5:2.5) for 3 h
at rt. The peptides were precipitated in ice-cold Et2O and
after centrifugation (3,200 rpm for 30 s), the ether layer
was decanted off. The pellet was re-solubilized in 250 mL
5% MeCN, 0.1% TFA in water and directly purified by
preparative reversed-phase HPLC, monitoring conjugate
elution at 214 nm. The fractions of interest were analyzed
by LCMS. Fractions of [95% purity were pooled and
lyophilized. HRMS: 7 (m/z): [M ? 5H]5? calcd for
C
117H168N31O16?, 453.6712; found, 453.6712; DM =
0.0 ppm; 8 (m/z): [M ? 5H]5? calcd for C113H160N27O16
,
?
Na-Fmoc-Nb-(Spermine, Boc4)-L-Dap-OH (1h)
431.2568; found, 431.2576; DM = 1.8 ppm.
Preparation according to GP using N1,N5,N10-tri-Boc-
spermine (7.30 g, 14.5 mmol). Yield: 1.66 g (33%) as pale
foam. Purity: 98%. [a]2D0 = -5.6° (c 1.01, MeOH); 1H
NMR (CD3OD, 300 MHz): d 7.79 (d, J = 7.5 Hz, 2H),
7.65 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t,
J = 7.5 Hz, 2H), 4.50 (dd, J = 5.0, 8.5 Hz, 1H), 4.34 (m,
2H), 4.20 (t, J = 7.0 Hz, 1H), 3.65–3.82 (m, 1H),
3.28–3.56 (m, 1H; hidden in part under the solvent peak),
3.08–3.28 (m, 10H), 3.02 (t, J = 6.5 Hz, 2H), 1.59–1.83
(m, 4H), 1.38–1.56 (m, 4H), 1.46 (s, 9H), 1.44 (s, 18H),
1.42 (s, 9H). 13C NMR (CD3OD, 75 MHz): d 173.6, 158.2,
156.7-157.4 (4C), 145.0/145.1 (2C), 142.4 (2C), 128.7
(2C), 128.0 (2C), 126.1 (2C), 120.8 (2C), 81.6/81.5, 80.8
(2C), 79.8, 68.0/68.8, 54.5, 48.3, 47.7, 46.1/46.9 (5C), 38.9,
30.2, 29.5, 28.8 (12C), 27.0, 26.7. HRMS: (m/z):
[M ? Na]? calcd for C48H73N5O12, 934.5148; found,
934.5189; DM = 4.3 ppm.
Acknowledgments We thank Novo Nordisk for supporting a Drug
Research Academy scholarship (JDC), as well as Brødrene Hart-
manns Fond and the Drug Research Academy for financial support
and a materials grant, respectively.
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