Synthesis of triamino acid building blocks with different lipophilicities

Article abstract of DOI:10.1371/journal.pone.0124046

To obtain different amino acids with varying lipophilicity and that can carry up to three positive charges we have developed a number of new triamino acid building blocks. One set of building blocks was achieved by aminoethyl extension, via reductive amination, of the side chain of ortnithine, diaminopropanoic and diaminobutanoic acid. A second set of triamino acids with the aminoethyl extension having hydrocarbon side chains was synthesized from diaminobutanoic acid. The aldehydes needed for the extension by reductive amination were synthesized from the corresponding Fmoc-L-2-amino fatty acids in two steps. Reductive amination of these compounds with Boc-L-Dab-OH gave the C4-C8 alkyl-branched triamino acids. All triamino acids were subsequently Boc-protected at the formed secondary amine to make the monomers appropriate for the N-terminus position when performing Fmoc-based solid-phase peptide synthesis.

Full text of DOI:10.1371/journal.pone.0124046

RESEARCH ARTICLE
Synthesis of Triamino Acid Building Blocks
with Different Lipophilicities
Jyotirmoy Maity, Dmytro Honcharenko, Roger Strömberg*
Department of Biosciences and Nutrition, Novum, Karolinska Institute (KI), Stockholm, Sweden
* Roger.Stromberg@ki.se
Abstract
To obtain different amino acids with varying lipophilicity and that can carry up to three posi-
tive charges we have developed a number of new triamino acid building blocks. One set of
building blocks was achieved by aminoethyl extension, via reductive amination, of the side
chain of ortnithine, diaminopropanoic and diaminobutanoic acid. A second set of triamino
acids with the aminoethyl extension having hydrocarbon side chains was synthesized from
diaminobutanoic acid. The aldehydes needed for the extension by reductive amination
were synthesized from the corresponding Fmoc-L-2-amino fatty acids in two steps. Reduc-
tive amination of these compounds with Boc-L-Dab-OH gave the C4-C8 alkyl-branched tria-
mino acids. All triamino acids were subsequently Boc-protected at the formed secondary
amine to make the monomers appropriate for the N-terminus position when performing
Fmoc-based solid-phase peptide synthesis.
OPEN ACCESS
Citation: Maity J, Honcharenko D, Strömberg R
(2015) Synthesis of Triamino Acid Building Blocks
with Different Lipophilicities. PLoS ONE 10(4):
e0124046. doi:10.1371/journal.pone.0124046
Academic Editor: Stephan Neil Witt, Louisiana State
University Health Sciences Center, UNITED STATES
Received: November 21, 2014
Accepted: March 10, 2015
Published: April 14, 2015
Copyright: © 2015 Maity et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Lipophilicity has immense importance for pharmacological properties. Drug molecules are re-
quired to have lipophilic properties to accomplish a desired pharmacokinetic profile [1]. Oligo-
nucleotides and peptides having inadequate affinity with the lipid bilayer of plasma
membranes are conjugated with lipophilic parts to enhance their cellular uptake [2]. Antisense
oligonucleotides were conjugated to cholesterol and bile acids to enhance lipophilicity and to
improve liver specific drug targeting and hepatocellular uptake efficiency [3]. Arginine-based
double-tailed lipid-peptide conjugates with a positive charge were synthesized as a potent nu-
cleic acid transporter [4]. Cationic lipid-mediated nucleic acids delivery has emerged as a posi-
tive move towards delivering genes into mammalian cells. Various cationic liposomes have
been used for gene delivery to mammalian cells in vitro and in vivo [5]. Arginine-rich peptide
sequence with peptide amphiphiles at its N-terminus had shown spontaneous assembly forma-
tion of various nanostructures in aqueous solution. Micelles of these peptides were loaded with
the anti-tumor drug doxorubicin and delivery of the drug into HeLa cells was observed [6]. Ad-
dition of a lipid tail at the N-terminus of the antimicrobial peptide tridecaptin A1 was found to
enhance the biological activity. Some simpler analogues were also found to show antimicrobial
activity against Gram-negative bacteria [7]. Di- or tri-peptide analogues, when lipidated with a
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Grant from Alphabeta AB to RS. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: A patent application by
Alphabeta AB for various peptoid ligands includes the
building blocks presented. RS is a minor share holder
of AlphaBeta Ab (<1%). The patent application
(Strömberg R., Honcharenko D., Maity J., Nilsson L,
Juneja, A, Johansson J, Presto J, Dolfe L,
Kurudenkandy F R, Fisahn A. Novel ligands for
PLOS ONE | DOI:10.1371/journal.pone.0124046 April 14, 2015
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New Triamino Acids
prevention of neurotoxicity of the Alzheimer's disease
related amyloid-beta peptide. Patent applications
EP14151655.9 and PCT: SMK/FP7071327.) includes
the amino acids presented in this manuscript. This
does not alter the authors' adherence to all PLOS
ONE policies on sharing data and materials, as
detailed online in the guide for authors http://www.
PLOSone.org/static/editorial.action#competing. There
are no restrictions on sharing of data and/
C12-18 lipid at the C-terminus of the peptides, exhibited enhanced antimicrobial activity com-
pared to their basic di- or tri-peptides [8].
Lysine is one of the naturally occurring amino acids that have an aliphatic side chain with a
primary amine functionality at the terminus. Besides the high level of safe supplemental intake
of L-lysine [9], it has been used therapeutically to restrain herpes simplex [10] and found to be
effective in the treatment of stress-related intestinal disorders [11]. A triamino acid, 4-L-azaly-
sine and its analogues have been found to retain engaging pharmacological properties. It
showed inhibitory activity towards the growth of E. Coli. 9723 and a broad range of lactic acid
bacteria [12]. It has been found to be efficient as a metabolic inhibitor of arylesterase [13]. Tria-
mino acids at the N-terminus part of peptoid ligands targeting the α-helical conformation of
the amyloid-β peptide (Aβ) related to Alzheimers disease have also been shown to improve
their antineurotoxicity [14]. The biological significance and potential medicinal importance of
triamino acids and amino acids/peptides with hydrocarbon tails encouraged us to extend the
arsenal of amino acids with such functionalities by synthesis of some new triamino acid build-
ing blocks with as well as without a hydrocarbon branching.
or materials.
We here describe the strategy for synthesis of two sets of triamino acids. One set is varied
with respect to distance between the alpha-carbon and the secondary amine of the side chain
and the other set of compounds have aliphatic hydrocarbon tails of different length adjacent to
the terminus amino functionality of the side chain (Fig 1). When these amino acid monomers
are incorporated at the N-terminus of any potential peptides/peptoids, the amino groups will
be partially/fully protonated depending on pH of the solution and the pKa value of the respec-
tive amines, and together with the hydrocarbon chain of the branched derivatives this creates a
cationic/hydrophobic microenvironment at the N-terminus of the peptide/peptoid. In addition
to this, the hydrocarbon chain of the branched derivatives introduces additional lipophilic
character, creating a cationic environment along with lipophilicity at the N-terminus of the
peptides/peptoids. Protecting groups of these triamino acids have been manipulated in such a
way that the final monomers would be suitable for their incorporation at the N-terminus end
of a peptide/peptoid sequence by the Fmoc-strategy through solid-phase peptide synthesis,
while still enabling further functionalization of the side chain.
Results and Discussion
We chalked out synthetic schemes for the target molecules largely from commercially available,
stereochemically pure and suitably protected starting materials. We found that reductive ami-
nation reaction between Nα-Fmoc-protected amino aldehydes and side chain primary amine of
the Nα-Boc-protected diamino carboxylic acids would provide us the basic structural moiety of
the target molecules. Originating the functionality of the aldehydes to carboxylic acids gave us
a plan to start the synthetic pathway from the corresponding stereochemically pure N-pro-
tected Nα-amino-carboxylic acids.
Fig 1. Schematic structures of the triamino acid building blocks. (P1-P3 = protecting groups).
doi:10.1371/journal.pone.0124046.g001
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New Triamino Acids
The synthetic route chosen is based on that we wished to protect the terminus N7 amine po-
sition with a 9-fluorenylmethyloxycarbonyl (Fmoc) group, for possible further extension at
this position. Accordingly we wanted the other primary and secondary amino functionalities
N2 and N5 respectively, to be protected with a tert-butyloxycarbonyl (Boc) group so that these
can be simultaneously removed upon final deprotection when performing Fmoc-based solid-
phase synthesis. For incorporation into a peptide chain the Boc-protected aldehyde and an
N2-Fmoc protection could instead be used. Conversion of Fmoc-protected amino acids into
their corresponding Fmoc-protected amino aldehydes has been accomplished by two major ap-
proaches. One is through reduction of the acids into alcohols, followed by oxidation and a sec-
ond is by the synthesis of Weinreb amides, followed by reduction [15–20]. Another noticeable
approach is via synthesis of amino esters, where acids were converted into their corresponding
ethyl esters by treatment with ethanol and sulphuric acid, followed by reduction with diisobu-
tylaluminium hydride (DIBAL) under inert condition [21]. Synthetic procedures for the syn-
thesis of these types of chiral aldehyde building blocks are also available in literature [22].
Our strategy for the synthesis of amino aldehydes was through the synthesis of thioesters of
the available amino acids [23–25], followed by reduction at neutral condition. We commenced
our synthetic pathway with commercially available chiral (S)-N-Fmoc-2-amino-2-alkylacetic
acids (1, 2 and 3; Fig 2). The synthetic route was chosen so that the chirality of these molecules
would be intact. Ethyl thioesters were readily derived from the corresponding carboxylic acids
using 1.1 equiv. of N,N'-dicyclohexylcarbodiimide (DCC) in dichloromethane at room temper-
ature [26]. Use of 3–10 mol% of 4-(N,N-dimethylamino)pyridine (DMAP) for this type of es-
terification reaction has been suggested to accelerate the rate of the reaction between carboxylic
acids and thiols, and also to suppress side product formation [26]. The desired products were
obtained in good yields when we performed these reactions at room temperature using 0.25
equiv. of DMAP. Reactions were smooth and complete conversion occurred in two hours. A
facile work up procedure followed by purification gave us the corresponding N-Fmoc-
2-amino-ethyl thioesters 4, 5 and 6 respectively.
α-Amino aldehydes are widely used chiral synthons in organic chemistry but they have a
tendency to racemize under acidic or basic conditions and also during chromatographic purifi-
cation over silica gel. This directed us towards milder conditions for the reduction reaction
with a simple work up procedure. We dissolved the ethanethiol esters in acetone at room tem-
perature and treated them with triethylsilane in presence of catalytic 10% Pd/C to convert the
thioester functionality into an aldehyde [27]. The reactions were allowed to proceed for two
hours. A simple work up protocol and purification of the crude reaction mixture by column
chromatography gave the N-Fmoc-2-amino-aldehydes 7, 8 and 9 respectively (Fig 2).
After synthesis of the chiral aldehydes our next task was to attempt the key step of our syn-
thetic pathway, i.e. the reductive amination reaction of these chiral aldehydes with a chiral
Fig 2. Synthesis of alkyl branched amino aldehydes. (i) EtSH, DCC, DMAP, dichloromethane at rt, 2 h (ii) Triethylsilane, 10% Pd/C, acetone at rt, 2 h.
doi:10.1371/journal.pone.0124046.g002
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New Triamino Acids
Fig 3. Synthesis of triamino acid building blocks with varied lipophilic tails. (i) NaBH3CN, 1% AcOH in methanol, rt, 18 h (ii) (Boc)2O, water:dioxane (v/
v, 1:1), 10% aq. soln. of Na2CO3, rt, 18 h.
doi:10.1371/journal.pone.0124046.g003
diamino carboxylic acid. Reductive amination reaction is a multipurpose and convenient meth-
od for the preparation of amines in organic synthesis [28]. A variety of organocatalysts, com-
plexes of transition metals or boron, tin and silicon reagents are available for this reaction. We
selected sodium cyanoborohydride (NaBH3CN) as a suitable reagent due to its earlier applica-
tions for reductive alkylation reaction in amino acid chemistry and/ or peptide chemistry
[29–33]. N2-Boc-2,4-diamino-butanoic acid (10) was subjected to a reductive amination reac-
tion with aldehyde 7 at room temperature in a solvent mixture of acetic acid/ methanol (1:99,
v/v) where NaBH3CN was used as the catalyst for the reaction (Fig 3). The reaction mixture
was stirred for 18 h at room temperature for the production of the desired compound (with re-
tained chirality at the C-2 and C-7 centre). The progress of the reaction was monitored with
TLC and, after a work up process, the crude reaction mixture was purified by silica gel column
chromatography to give compound 11 in good yield. The triamino acid was then further pro-
tected with Boc at the secondary amine by treatment with Boc-anhydride in a solvent mixture
of water and dioxane (1:1, v/v), containing 10% Na2CO3 aqueous solution. This reaction af-
forded the final product (2S,2'S)-N2,N4-bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethy-
loxycarbonyl)-2'-aminohexyl]-2,4-diaminobutanoic acid (14).
Similar procedures were used for synthesis of triamino acids branched with longer (C6 and
C8) alkyl chains. The diamino acid 10 was treated with the chiral aldehydes 8 and 9 in presence
of NaBH3CN to produce the triamino carboxylic acids 12 and 13, respectively. Subsequent Boc
protection gave the monomers 15 and 16, respectively (Fig 3).
Three different strategies for the synthesis of 4-L-azalysine have been described. The first
strategy involved L-serine as the starting material which was converted into methyl (S)-oxazoli-
dine-4-carboxylate in three steps. In next five steps, it was converted into the end product via
Garner’s aldehyde. A second strategy involved the use of L-asparagine that was converted into
N-protected 2,3-diaminopropionic ester, followed by reductive amination with N-Boc-2-ami-
noacetaldehyde. Strategic manipulation of protecting groups produced the final desired prod-
uct in another four steps [34,35]. The third strategy was a solid-phase dependent procedure to
synthesize di- or polycationic amino acid building blocks. In this protocol, protected aziridine-
2-methanol was loaded onto a trityl bromide resin, followed by ring opening with a variety of
primary amines. After detachment of the product from solid support, the primary alcohol was
converted into a carboxylic acid [36]. For synthesis of the triamino acids with different dis-
tances between the alpha-carbon and the secondary amine of the side chain we opted for a
short route starting with the respective amino acids, ortnithine, diaminopropanoic and diami-
nobutanoic acid. We have reported on synthesis of a couple of these triamino acids but with a
different choice of protection scheme [14, 37]. As with the branched derivative we wished to
have building blocks for termination of a peptide/peptoid with possibility for extension on the
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New Triamino Acids
side chain and therefore an Fmoc-protection on the side chain amino group and the N2-Boc
protection was used on the diamino acid.
The synthetic pathway is similar to that for the compounds with aliphatic branching except
that N-Fmoc-glycinal (19) was used instead of the branched aminoaldehydes. The N-Fmoc-
glycinal was synthesized from of the inexpensive starting material 3-aminopropane-1,2-diol
(17) which was Fmoc protected to form 18 and then oxidatively cleaved with periodate to give
19 (Fig 4) [38].
Reductive amination reaction with compound 19 and the respective Nα-Boc-diamino acids
10, 20, 21 in acetic acid/methanol (1:99, v/v) using NaBH3CN at room temperature resulted in
the triamino acid derivatives 22–24. After work up and purification by column chromatogra-
phy Boc protection on the secondary amine was achieved by treatment with (Boc)2O to afford
the products (S)-N2,N3-bis-tert-butoxycarbonyl-N3-[N-(9-fluorenylmethyloxycarbonyl)-
2-aminoethyl]-2,3-diaminopropanic acid (25), (S)-N2,N4-bis-tert-butoxycarbonyl-N4-[N-
(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,4-diaminobutanoic acid (26) and (S)-N2,N5-
bis-tert-butoxycarbonyl-N5-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,3-diamino-
pentanoic acid (27).
Conclusion
We have revealed a facile synthetic procedure for the preparation of suitably protected tria-
mino acids in decent to good yields. Thus an extension of the available arsenal of triamino
acids building blocks with varying lipophilicity and that can carry up to three positive charges
is provided. Starting from N-Fmoc-2-alkyl amino acids (1–3) with varied chain length of the
alkyl group, we converted them into the corresponding aminoaldehydes (7–9) in two steps.
These aldehydes were protected and suitable for reductive amination reaction with the pro-
tected diamino acid Nα-Boc-L-Dab (10). The resulting alkyl branched triamino acids were
Boc-protected to obtain the final monomers (14–16). In addition another series of triamino
acids with different distances between the alpha-carbon and the secondary amine of the side
chain (25–27) were made by reductive amination with N-Fmoc-glycinal (19) and a series of
Fig 4. Synthesis of triamino acid building blocks with different distances between the α-amino group and the secondary amine. (i) Fmoc-OSu,
MeOH/ pyridine, rt, 18 h (ii) NaIO4, THF, rt, 8 h (iii) NaBH3CN, 1% AcOH in methanol, rt, 18 h (iv) (Boc)2O, water:dioxane (v/v, 1:1), 10% aq. Na2CO3, rt, 18 h.
doi:10.1371/journal.pone.0124046.g004
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New Triamino Acids
Nα-Boc diamino acids (20, 10, 21). This facile and variable procedure provided novel amino
acids with hydrocarbon branching of the aminoethyl extension and convenient synthesis of
triamino acids with different distances between the alpha-carbon and the secondary amine.
The final monomers were suitably protected for their incorporation at the N-terminus of a
peptide/peptoid sequence by Fmoc-based solid-phase synthesis while enabling further functio-
nalization of the side chain when still attached to the support.
Experimental Section
Melting points of the compounds were recorded using Büchi Melting Point apparatus (B-545)
and were uncorrected. Reactions under anhydrous conditions were carried out under a nitro-
gen atmosphere. Column chromatography was performed with silica gel 60 (particle size
0.040–0.063 mm, 230–400 mesh, Aldrich) and analytical grade solvents. Thin layer chromatog-
raphy (TLC) was conducted on glass plates coated with silica gel 60 F254, obtained from Merck.
TLC plates were visualized by UV light (254 or 360 nm) and/or by staining with I2 by keeping
the plates in an iodine chamber. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Bruker DRX-400 in CDCl3/CD3OD at 25°C. Chemical shifts are reported
in ppm relative to TMS (Me4Si as internal standard, δ = 0 ppm) or the deuterated solvent as
the internal standard for 1H and 13C NMR. Coupling constants (J values) are given in Hz. The
following abbreviations are used in connection with NMR spectra: s = singlet, br s = broad sin-
glet, d = doublet, dd = double doublet, t = triplet, q = quartet and m = multiplet. Structural as-
signments are based on DEPT-135, COSY and HMQC where applicable. HRMS (ESI-TOF)
was recorded on a Micromass LCT either in positive ion mode or in negative ion mode. Specific
rotation measurements were performed using an Autopol IV Automatic Polarimeter (Rudolph
Research Analytical). RP-HPLC analysis of final building blocks is reported in S1 Supporting
Information. THF, CH2Cl2, toluene, hexane, and Et2O were dried by standard procedures and
stored over molecular sieves (4 Å). N-(9-Fluorenylmethyloxycarbonyl)-2-aminoacetaldehyde
(19) [38], (S)-N-(9-fluorenylmethoxycarbonyl)-2-aminohexanal (7) [25] and (S)-N2-tert-
butoxycarbonyl-N5-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,5-diaminopentanoic
acid (24) [38] were synthesized as reported (see S1 Supporting Information). All other solvents
and chemicals were of reagent grade and used without further purification. 2-Alkyl amino
acids 1, 2 and 3 were purchased from Watanabe Chemical Industries, Ltd., Japan and Boc-pro-
tected amino acids 10, 20 and 21 were purchased from IRIS Biotech, Germany. Other reagents
were obtained from common commercial sources and used as received.
General procedure for synthesis of ethylthio esters (4, 5 and 6)
To a solution of Fmoc-amino acids (1–3, 1.3 mmol) in anhydrous dichloromethane (DCM,
20 mL) at rt, ethanethiol (5 mmol) was added dropwise, followed by addition of solid DCC
(1.6 mmol) and (DMAP, 0.25 mmol) under inert atmosphere. The reaction mixture was stirred
for 2 h at rt. Progress of the reaction was monitored by TLC. Upon complete conversion of the
starting material into product, water (20 mL) was added into the reaction mixture and the lay-
ers were separated. The organic layer was collected and washed with brine (2 x 10 mL), dried
over Na2SO4 and concentrated to dryness under reduced pressure to get the crude materials.
Pure products (4–6) were obtained by eluting the crude through a short column of silica gel.
(S)-S-Ethyl N-(9-fluorenylmethoxycarbonyl)-2-aminohexanthioate (4). Compound 4
was purified by flash column chromatography using 0 to 25% ethyl acetate (EtOAc) in hexane as
eluent to afford a white amorphous solid (0.36 g, 71%); m.p. 116–117°C. Rf = 0.54 (EtOAc/hex-
ane, 1:5, v/v). 1H NMR (400 MHz, CDCl3): δ = 7.69 (d, J = 7.6 Hz, 2 H, Ar-H), 7.54 (t, J = 7.2 Hz,
2 H, Ar-H), 7.33 (t, J = 7.6 Hz, 2 H, Ar-H), 7.24 (t, J = 7.6 Hz, 2 H, Ar-H), 5.13 (d, J = 8.0 Hz,
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New Triamino Acids
1 H, NH), 4.44–4.40 (m, 1 H, NCOOCH2aCH), 4.36–4.30 (m, 2 H, NCOOCH2bCH, 2-CH), 4.17
(t, J = 6.8 Hz, 1 H, NCOOCH2CH), 2.81 (q, J = 7.2 Hz, 2 H, COSCH2CH3), 1.85–1.80 (m, 1 H,
3-CH2a), 1.57–1.51 (m, 1 H, 3-CH2b), 1.30–1.23 (m, 4 H, 4-CH2, 5-CH2), 1.18 (t, J = 7.2 Hz, 3 H,
COSCH2CH3), 0.83 (t, J = 6.8 Hz, 3 H, 6-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 201.1
(COS), 156.0 (NCOO), 144.1, 143.9, 141.5 (C-Ar), 127.9, 127.2, 125.2, 120.1 (CH-Ar), 67.2
(NCOOCH2CH), 61.1 (C-2), 47.4 (NCOOCH2CH), 32.8 (C-3), 27.5 (C-4), 23.4 (SCH2CH3),
22.4 (C-5), 14.6 (SCH2CH3), 14.0 (C-6) ppm. MS-ESI (m/z): calcd. for C23H28NO3S [M+H]+
398.1784; found 398.1779.
(S)-S-Ethyl N-(9-fluorenylmethoxycarbonyl)-2-aminooctanthioate (5). Compound 5
was purified by flash column chromatography using 0 to 25% EtOAc in hexane as eluent to afford
a white amorphous solid (0.33 g; 86%); m.p. 96–99°C. Rf = 0.56 (EtOAc/hexane, 1:5, v/v). 1H
NMR (400 MHz, CDCl3): δ = 7.70 (d, J = 7.6 Hz, 2 H, Ar-H), 7.55 (t, J = 7.2 Hz, 2 H, Ar-H), 7.33
(t, J = 7.6 Hz, 2 H, Ar-H), 7.25 (t, J = 7.6 Hz, 2 H, Ar-H), 5.11 (d, J = 8.4 Hz, 1 H, NH), 4.44–4.40
(m, 1 H, NCOOCH2aCH), 4.36–4.30 (m, 2 H, NCOOCH2bCH, 2-CH), 4.17 (t, J = 6.8 Hz, 1 H,
NCOOCH2CH), 2.82 (q, J = 7.2 Hz, 2 H, COSCH2CH3), 1.84–1.78 (m, 1 H, 3-CH2a), 1.57–1.49
(m, 1 H, 3-CH2b), 1.26–1.16 (m, 11 H, 4-CH2, 5-CH2, 6-CH2, 7-CH2, COSCH2CH3), 0.81 (t,
J = 6.8 Hz, 3 H, 8-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 201.1 (COS), 156.0 (NCOO),
144.1, 143.9, 141.5 (C-Ar), 127.9, 127.2, 125.2, 120.1 (CH-Ar), 67.2 (NCOOCH2CH), 61.1 (C-2),
47.4 (NCOOCH2CH), 33.1 (C-3), 31.7 (C-4), 29.0 (C-5), 25.3 (C-6), 23.4 (SCH2CH3), 22.7 (C-7),
14.7 (SCH2CH3), 14.2 (C-8) ppm. MS-ESI (m/z): calcd. for C25H32NO3S [M+H]+ 426.2103;
found 426.2101.
(S)-S-Ethyl N-(9-fluorenylmethoxycarbonyl)-2-aminodecan-thioate (6). Compound 6
was purified by flash column chromatography using 0 to 20% EtOAc in hexane as eluent to afford
a white amorphous solid (0.39 g; 87%); m.p. 92–93°C. Rf = 0.59 (EtOAc/hexane, 1:5, v/v). 1H
NMR (400 MHz, CDCl3): δ = 7.69 (d, J = 7.6 Hz, 2 H, Ar-H), 7.54 (t, J = 7.2 Hz, 2 H, Ar-H), 7.33
(t, J = 7.6 Hz, 2 H, Ar-H), 7.24 (t, J = 7.6 Hz, 2 H, Ar-H), 5.13 (d, J = 8.4 Hz, 1 H, NH), 4.44–4.39
(m, 1 H, NCOOCH2aCH), 4.36–4.30 (m, 2 H, NCOOCH2bCH, 2-CH), 4.17 (t, J = 6.8 Hz, 1 H,
NCOOCH2CH), 2.81 (q, J = 7.2 Hz, 2 H, COSCH2CH3), 1.84–1.77 (m, 1 H, 3-CH2a), 1.56–1.50
(m, 1 H, 3-CH2b), 1.26–1.16 (m, 15 H, 4-CH2, 5-CH2, 6-CH2, 7-CH2, 8-CH2, 9-CH2,
COSCH2CH3), 0.80 (t, J = 6.8 Hz, 3 H, 10-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 201.1
(COS), 155.9 (NCOO), 144.0, 143.9, 141.5 (C-Ar), 127.9, 127.2, 125.2, 120.1 (CH-Ar), 67.2
(NCOOCH2CH), 61.1 (C-2), 47.4 (NCOOCH2CH), 33.1 (C-3), 32.0 (C-4), 29.5, 29.3, 25.4 (C-5,
C-6, C-7, C-8), 23.4 (SCH2CH3), 22.8 (C-9), 14.6 (C-10), 14.2 (SCH2CH3) ppm. MS-ESI (m/z):
calcd. for C27H36NO3S [M+H]+ 454.2416; found 454.2413.
General procedure for synthesis of aliphatic amino aldehydes (7, 8
and 9)
Ethylthio esters (4–6, 0.35 mmol) were dissolved in dry acetone (6 mL) under inert atmosphere.
10% Pd/C was added to the solution followed by addition of triethylsilane (0.56 mmol) whereup-
on the mixture was stirred at rt. Progress of the reaction was monitored by TLC. After 2 h, the re-
action was stopped by passing it through a short pad of celite and washed with acetone (3 x 6
mL). The combined organic layers was evaporated to dryness under reduced pressure and dis-
solved in ethylacetate (15 mL). After washing the organic layer with brine (2 x 8 mL) it was dried
over Na2SO4 and concentrated under reduced pressure to get the crude products. Subsequent
purification by silica gel column chromatography yielded compounds 7–9.
(S)-N-(9-Fluorenylmethoxycarbonyl)-2-aminooctanal (8). Compound 8 was purified by
flash column chromatography using 0 to 60% EtOAc in hexane as eluent to afford a white
amorphous solid (0.26 g; 92%); m.p. 75–76°C. Rf = 0.28 (EtOAc/hexane, 1:5, v/v). 1H NMR
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New Triamino Acids
(400 MHz, CDCl3): δ = 9.59 (s, 1 H, CHO), 7.77 (d, J = 7.6 Hz, 2 H, Ar-H), 7.60 (d, J = 7.2 Hz,
2 H, Ar-H), 7.40 (t, J = 7.2 Hz, 2 H, Ar-H), 7.32 (t, J = 7.6 Hz, 2 H, Ar-H), 5.30 (d, J = 6.4 Hz, 1
H, NH), 4.43 (d, J = 6.8 Hz, 2 H, NCOOCH2CH), 4.34–4.29 (m, 1 H, 2-CH), 1.94–1.89 (m, 1
H, NCOOCH2CH), 1.66–1.58 (m, 1 H, 3-CH2a), 1.33–1.26 (m, 9 H, 3-CH2b, 4-CH2, 5-CH2,
6-CH2, 7-CH2), 0.87 (t, J = 7.2 Hz, 3 H, 8-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 199.3
(CHO), 156.3 (NCOO), 143.8, 141.4 (C-Ar), 127.7, 127.1, 125.0, 120.0 (CH-Ar), 67.0
(NCOOCH2CH), 60.3 (C-2), 47.2 (NCOOCH2CH), 31.5 (C-3), 29.2 (C-4), 29.0 (C-5), 25.0 (C-
6), 22.5 (C-7), 14.0 (C-8) ppm. MS-ESI (m/z): calcd. for C23H28NO3 [M+H]+ 366.2064; found
366.2069.
(S)-N-(9-Fluorenylmethoxycarbonyl)-2-aminodecanal (9). Compound 9 was purified by
flash column chromatography using 0 to 80% EtOAc in hexane as eluent to afford a white amor-
phous solid (0.36 g; 84%); m.p. 69–70°C. Rf = 0.35 (EtOAc/hexane, 1:5, v/v). 1H NMR (400
MHz, CDCl3): δ = 9.58 (s, 1 H, CHO), 7.77 (d, J = 7.6 Hz, 2 H, Ar-H), 7.60 (d, J = 7.6 Hz, 2 H,
Ar-H), 7.40 (t, J = 7.6 Hz, 2 H, Ar-H), 7.32 (t, J = 7.6 Hz, 2 H, Ar-H), 5.31 (d, J = 6.8 Hz, 1 H,
NH), 4.43 (d, J = 6.8 Hz, 2 H, NCOOCH2CH), 4.34–4.29 (m, 1 H, 2-CH), 4.23 (t, J = 6.8 Hz, 1 H,
NCOOCH2CH), 1.93–1.89 (m, 1 H, 3-CH2a), 1.65–1.58 (m, 1 H, 3-CH2b), 1.33–1.26 (m, 12 H,
4-CH2, 5-CH2, 6-CH2, 7-CH2, 8-CH2, 9-CH2), 0.88 (t, J = 7.2 Hz, 3 H, 10-CH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 199.3 (CHO), 156.0 (NCOO), 143.8, 143.7, 141.3 (C-Ar), 127.7, 127.1,
125.0, 120.0 (CH-Ar), 67.0 (NCOOCH2CH), 60.3 (C-2), 47.2 (NCOOCH2CH), 31.8, 29.3, 29.2,
25.0, 22.6 (C-3, C-4, C-5, C-6, C-7, C-8, C-9), 14.1 (C-10) ppm. MS-ESI (m/z): calcd. for
C25H32NO3 [M+H]+ 394.2377; found 394.2390.
General procedure for synthesis of triamino acids 11, 12 and 13
Boc-L-Dab-OH (10, 0.5 mmol) was dissolved in 1% acetic acid (AcOH) in methanol (MeOH,
10 mL) and kept stirring at rt. The respective amino aldehydes (7–9, 0.46 mmol) were added
into the reaction mixture slowly followed by addition of NaBH3CN (1.14 mmol). The reaction
mixture was stirred at rt for 18 h. The progress of the reaction was monitored by TLC. On attain-
ing maximum conversion, the reaction mixture was evaporated to dryness and was dissolved in
ethylacetate (20 mL). Organic layer was washed with water (10 mL) and brine (10 mL x 2), dried
over Na2SO4 and evaporated to dryness under reduced pressure to get crude compounds. Pure
compounds (11–13) were obtained by purification of the crude by silica gel column chromatog-
raphy (Fig 3).
(2S,2'S)-N2-(tert-Butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-
hexyl]-2,4-diaminobutanoic acid (11). Compound 11 was purified by flash column chroma-
tography using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to afford a
white amorphous solid (0.265 g; 50%); m.p. 93–97°C. Rf = 0.34 (MeOH/AcOH/DCM, 7.5:1:91.5,
v/v/v) 1H NMR (400 MHz, CDCl3): δ = 7.72 (d, J = 7.2 Hz, 2 H, Ar-H), 7.59 (d, J = 7.2 Hz, 2 H,
Ar-H), 7.36 (t, J = 7.2 Hz, 2 H, Ar-H), 7.27 (d, J = 7.6 Hz, 2 H, Ar-H), 5.89 (br s, 1 H, NHCOO),
5.79 (br s, 1 H, NHCOO), 4.40–4.34 (m, 1 H, 2-CH), 4.25–4.17 (m, 2 H, NCOOCH2CH), 4.02–
3.96 (m, 1 H, NCOOCH2CH), 3.88–3.85 (m, 1 H, 2'-CH), 3.26–3.13 (m, 2 H, 4-CH2) 3.03–2.93
(m, 2 H, 1'-CH2), 2.12–2.01 (m, 3 H, 3-CH2a, 3'-CH2), 1.75–1.68 (m, 1 H, NH), 1.43–1.32 (m, 14
H, 3-CH2b, 4'-CH2, 5'-CH2, C(CH3)3), 0.90–0.86 (m, 3 H, 6'-CH3) ppm. 13C NMR (100 MHz,
CDCl3): δ = 177.6 (COOH), 156.7, 155.7 (2 x NCOO), 144.1, 141.3 (C-Ar), 127.7, 127.1, 125.5,
125.4, 120.0 (CH-Ar), 79.7 (C(CH3)3), 67.1 (NCOOCH2CH), 51.4 (C-2), 50.8 (C-1'), 49.6 (C-2'),
48.7 (C-4), 47.3 (NCOOCH2CH), 32.2, 31.7, 31.0 (C-3, C-3', C-4'), 28.5 (C(CH3)3), 22.4 (C-5'),
14.1 (C-6') ppm. MS-ESI (m/z): calcd. for C30H40N3O6 [M-H]- 538.2923; found 538.2918.
(2S,2'S)-N2-(tert-Butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-aminooc-
tyl]-2,4-diaminobutanoic acid (12). Compound 12 It was purified by flash column
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New Triamino Acids
chromatography using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to
afford a white amorphous solid (0.253 g; 57%); m.p. 72–76°C. Rf = 0.46 (MeOH/AcOH/
DCM, 7.5:1:91.5, v/v/v) 1H NMR (400 MHz, CDCl3): δ = 7.73 (d, J = 6.8 Hz, 2 H, Ar-H), 7.60
(t, J = 6.0 Hz, 2 H, Ar-H), 7.36 (t, J = 6.4 Hz, 2 H, Ar-H), 7.29–7.23 (m, 2 H, Ar-H), 5.89 (br s,
1 H, NHCOO), 5.79 (br s, 1 H, NHCOO), 4.39–4.34 (m, 1 H, 2-CH), 4.22–4.15 (m, 2 H,
NCOOCH2CH), 4.03–3.86 (m, 2 H, NCOOCH2CH, 2'-CH), 3.29–3.06 (m, 2 H, 4-CH2),
3.00–2.91 (m, 2 H, 1'-CH2), 2.10–1.96 (m, 3 H, 3-CH2, 3'-CH2a), 1.72–1.69 (m, 1 H, NH),
1.38–1.09 (m, 18 H, C(CH3)3, 3'-CH2b, 4'-CH2, 5'-CH2, 6'-CH2, 7'-CH2), 0.91–0.78 (m, 3 H,
8'-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 177.7 (COOH), 156.7, 155.7 (2 x NCOO),
144.2, 141.4 (C-Ar), 127.7, 127.2, 125.5, 125.4, 120.0 (CH-Ar), 79.7 (C(CH3)3), 67.1
(NCOOCH2CH), 53.6 (C-2), 51.4 (C-1'), 50.7 (C-2'), 48.7 (C-4), 47.2 (NCOOCH2CH), 32.0,
31.0, 29.1 (C-3, C-3', C-4'), 28.5 (C(CH3)3), 26.3 (C-5'), 26.2 (C-6'), 22.7 (C-7'), 14.2 (C-
8') ppm. HRMS (ESI-TOF): calcd. for C32H44N3O6 [M–H]– 566.3236, found 566.3226.
(2S,2'S)-N2-(tert-Butyloxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-
decyl]-2,4-diaminobutanoic acid (13). Compound 13 was purified by flash column chroma-
tography using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to afford a
white amorphous solid (0.325 g; 60%); m.p. 49–53°C. Rf = 0.29 (MeOH/AcOH/DCM, 7.5:1:91.5,
v/v/v) 1H NMR (400 MHz, CDCl3): δ = 7.73 (d, J = 7.6 Hz, 2 H, Ar-H), 7.60 (t, J = 6.8 Hz, 2 H,
Ar-H), 7.36 (t, J = 7.6 Hz, 2 H, Ar-H), 7.29–7.25 (m, 2 H, Ar-H), 5.90–5.86 (m, 1 H, NHCOO),
4.70–4.80 (m, 1 H, NHCOO), 4.35–4.31 (m, 1 H, 2-CH), 4.27–4.16 (m, 2 H, NCOOCH2CH),
4.04–3.86 (m, 2 H, NCOOCH2CH, 2'-CH), 3.27–3.14 (m, 2 H, 4-CH2), 3.02–2.92 (m, 2 H, 1'-
CH2), 2.10–2.04 (m, 3 H, 3-CH2, 3'-CH2a), 1.77–1.57 (m, 1 H, 3'-CH2b), 1.38–1.19 (m, 22 H,
C(CH3)3, 3'-CH2b, 4'-CH2, 5'-CH2, 6'-CH2, 7'-CH2, 8'-CH2, 9'-CH2) 0.90–0.86 (m, 3 H, 10'-
CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 177.6 (COOH), 156.7, 156.0 (2 x NCOO), 144.2,
144.1, 141.4 (C-Ar), 127.9, 127.8, 127.2, 125.4, 120.0 (CH-Ar), 79.9 (C(CH3)3), 67.2
(NCOOCH2CH), 54.6 (C-2), 51.5 (C-1'), 49.6 (C-2'), 48.8 (C-4), 47.3 (NCOOCH2CH), 32.0,
31.0, 30.8, 29.6, 29.4 (C-3, C-3', C-4', C-5', C-6'), 28.5 (C(CH3)3), 26.3 (C-7'), 22.8 (C-8'), 21.0
(C-9'), 14.2 (C-10') ppm. HRMS (ESI-TOF): calcd. for C34H48N3O6 [M–H]– 594.3549, found
594.3455.
General procedure for Boc protection (14, 15 and 16)
The respective triamino acids (11–13, 0.22 mmol) were dissolved in a solvent mixture of
water and dioxane (1:1, v/v, 10 mL) and then stirred at 0–5°C using an ice bath. Solid
Na2CO3 (0.45 mmol) was added into the reaction mixture, followed by addition of Boc anhy-
dride [(Boc)2O, 0.42 mmol]. The ice bath was removed after 1 hour and the reaction mixture
was stirred at rt for 18 h. The progress of the reaction was monitored by TLC. After complete
reaction, the temperature of the reaction mixture was set to 0–5°C and water (10 mL) was
added. 1 M HCl was added into the reaction mixture dropwise to adjust the pH of the solu-
tion (to pH 3). The product was extracted with ethylacetate (15 mL x 3). The combined ethy-
lacetate layers was washed with water (10 mL x 2) and brine (10 mL), dried over Na2SO4 and
evaporated to dryness under reduced pressure. The crude compounds were purified by silica
gel column chromatography to afford compounds 14–16.
(2S,2'S)-N2,N4-Bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-
amino-hexyl]-2,4-diaminobutanoic acid (14). Compound 14 was purified by flash column
chromatography using 0 to 100% EtOAc in hexane containing 1% AcOH as eluent to afford a white
amorphous solid (0.245 g; 88%); m.p. 49–52°C. Rf = 0.47 (EtOAc/AcOH/hexane, 75:1.24, v/v/v) 1H
NMR (400 MHz, CDCl3): δ = 7.75 (d, J = 7.6 Hz, 2 H, Ar-H), 7.57 (d, J = 6.8 Hz, 2 H, Ar-H), 7.38
(t, J = 7.6 Hz, 2 H, Ar-H), 7.29 (t, J = 6.8 Hz, 2 H, Ar-H), 4.50–4.32 (m, 2 H, NCOOCH2CH),
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New Triamino Acids
4.25–4.15 (m, 2 H, 2-CH, NCOOCH2CH), 3.73–3.54 (m, 3 H, 2'-CH, 1'-CH2), 3.42–3.38 (m, 1 H,
4-CH2a), 3.32–3.30 (m, 1 H, 4-CH2b), 3.14–2.89 (m, 2 H, 3-CH2), 2.12–2.00 (m, 2 H, 3'-CH2),
1.44–1.26 (m, 22 H, 2 x C(CH3)3, 4'-CH2, 5'-CH2), 0.92–0.86 (m, 3 H, 6'-CH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 173.8 (COOH), 156.8, 156.7, 156.2 (3 x NCOO), 143.9, 141.3 (C-Ar),
127.7, 127.1, 125.2, 125.0, 120.0 (CH-Ar), 82.1, 81.1, 80.2 (2 x C(CH3)3), 66.9, 66.3,
(NCOOCH2CH), 51.2 (C-2), 50.6 (C-1'), 49.6 (C-2'), 47.3 (NCOOCH2CH), 43.6, 43.1 (C-4),
33.2, 32.9, 32.0, 31.1 (C-3, C-3'), 28.4 (2 x C(CH3)3), 27.9 (C-4'), 22.6 (C-5'), 14.0 (C-6') ppm.
[α]24D = –2.0 (c 0.1, MeOH). HRMS (ESI-TOF): calcd. for C35H48N3O8 [M–H]– 638.3447,
found 638.3454.
(2S,2'S)-N4-Bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-
octyl]-N2,2,4-diaminobutanoic acid (15). Compound 15 was purified by flash column
chromatography using 0 to 100% EtOAc in hexane containing 1% AcOH as eluent to afford
a colourless sticky solid (0.232 g; 88%). Rf = 0.53 (EtOAc/AcOH/hexane, 75:1.24, v/v/v) 1H
NMR (400 MHz, CDCl3): δ = 7.75 (d, J = 7.6 Hz, 2 H, Ar-H), 7.58 (d, J = 6.8 Hz, 2 H, Ar-H), 7.38
(t, J = 7.2 Hz, 2 H, Ar-H), 7.29 (t, J = 7.2 Hz, 2 H, Ar-H), 4.50–4.35 (m, 2 H, NCOOCH2CH),
4.22–4.11 (m, 2 H, 2-CH, NCOOCH2CH), 3.80–3.62 (m, 3 H, 2'-CH, 1'-CH2), 3.66–3.60 (m, 1
H, 4-CH2a), 3.49–3.44 (m, 1 H, 4-CH2b), 3.22–2.88 (m, 2 H, 3-CH2), 2.09–2.02 (m, 2 H, 3'-CH2),
1.44 (s, 18 H, 2 x C(CH3)3), 1.33–1.24 (m, 8 H, 4'-CH2, 5'-CH2, 6'-CH2, 7'-CH2), 0.92–0.84 (m,
3 H, 8'-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 173.7 (COOH), 157.0, 156.24, 156.18, 155.6
(3 x NCOO), 144.1, 141.3 (C-Ar), 127.8, 127.2, 125.3, 125.1, 120.1 (CH-Ar), 82.0, 80.9, 80.8, 80.2
(2 x C(CH3)3), 66.3, 66.1 (NCOOCH2CH), 51.4 (C-2), 50.7 (C-1'), 47.4 (NCOOCH2CH), 44.0,
43.0 (C-2', C-4), 33.5, 33.2, 31.8, 31.6, 30.5, 29.8, 29.4, 29.3 (C-3, C-3', C-4', C-5', C-6'), 28.5 (2 x C
(CH3)3), 22.7 (C-7'), 14.2 (C-8') ppm. [α]22D = –2.6 (c 0.3, MeOH). HRMS (ESI-TOF): calcd. for
C37H52N3O8 [M–H]– 666.3760, found 666.3755.
(2S,2'S)-N2,N4-Bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-
amino-decyl]-2,4-diaminobutanoic acid (16). Compound 16 was purified by flash column
chromatography using 0 to 90% MeOH in dichloromethane containing 1% AcOH as eluent to
afford a white amorphous solid (0.365 g; 79%); m.p. 44–46°C. Rf = 0.58 (EtOAc/AcOH/hexane,
75:1.24, v/v/v) 1H NMR (400 MHz, CDCl3): δ = 7.75 (d, J = 7.6 Hz, 2 H, Ar-H), 7.57 (d, J = 6.8
Hz, 2 H, Ar-H), 7.38 (t, J = 7.6 Hz, 2 H, Ar-H), 7.29 (t, J = 6.8 Hz, 2 H, Ar-H), 4.46–4.38 (m, 2
H, NCOOCH2CH), 4.19–4.16 (m, 2 H, 2-CH, NCOOCH2CH), 3.82–3.71 (m, 3 H, 2'-CH, 1'-
CH2), 3.60–3.32 (m, 2 H, 4-CH2), 3.22–2.88 (m, 2 H, 3-CH2), 2.10–2.00 (m, 2 H, 3'-CH2), 1.44
(s, 18 H, 2 x C(CH3)3), 1.26 (m, 12 H, 4'-CH2, 5'-CH2, 6'-CH2, 7'-CH2, 8'-CH2, 9'-CH2), 0.87
(t, J = 6.8 Hz, 3 H, 10'-CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 174.0 (COOH), 156.8,
156.7, 156.1, 155.7 (3 x NCOO), 144.0, 141.3 (C-Ar), 127.7, 127.1, 125.2, 125.0, 120.0 (CH-Ar),
81.9, 81.0, 80.2 (2 x C(CH3)3), 66.8, 66.1 (NCOOCH2CH), 51.3 (C-2), 50.6, 49.6 (C-1'), 47.4
(NCOOCH2CH), 43.9, 42.9 (C-4, C-2'), 33.4, 31.9, 30.4, 29.5, 29.3 (C-3, C-3', C-4', C-5', C-6',
C-7',), 28.4 (2 x C(CH3)3), 25.8 (C-8'), 22.7 (C-9'), 14.1 (C-10') ppm. [α]24D = –4.0 (c 0.1,
MeOH). HRMS (ESI-TOF): calcd. for C39H56N3O8 [M–H]– 694.4073, found 694.4063.
General procedure for synthesis of triamino acids 22, 23 and 24
The respective N-Boc-L-diamino acids (20, 10, 21, 0.5 mmol) were dissolved under stirring in
anhydrous methanol (10 mL, containing 1% of AcOH). N-(9-Fluorenylmethoxycarbonyl)glyc-
inal (19, 0.46 mmol) was added to the reaction mixture under a nitrogen atmosphere, followed
by the addition of sodium cyanoborohydride (1.14 mmol). The reaction mixture was stirred at
room temperature for 18 h and the progress of the reaction was monitored by TLC. The sol-
vents were evaporated in vacuo, and the residue was dissolved in ethyl acetate (25 mL). The or-
ganic layer was washed with water (15 mL) and brine (2 x 15 mL), dried over Na2SO4, filtered
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New Triamino Acids
and concentrated under reduced pressure. The crude products were purified by eluting them
with a solution of chloroform-methanol through a short silica gel column (Fig 4) to yield
22–24 respectively.
(S)-N2-tert-Butoxycarbonyl-N3-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-
2,3-diaminopropionic acid (22). Compound 22 was purified by flash column chromatogra-
phy using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to afford a
colourless sticky solid (0.2 g; 43%). Rf = 0.14 (MeOH/AcOH/DCM, 10:1:89, v/v/v) 1H NMR
(400 MHz, CD3OD): δ = 7.68 (d, J = 7.2 Hz, 2 H, Ar-H), 7.53 (d, J = 7.2 Hz, 2 H, Ar-H), 7.28 (t,
J = 7.2 Hz, 2 H, Ar-H), 7.19 (t, J = 7.2 Hz, 2 H, Ar-H), 4.28 (d, J = 6.8 Hz, 2 H, NCOOCH2CH),
4.09 (t, J = 6.8 Hz, 1 H, NCOOCH2CH), 4.03 (t, J = 6.0 Hz, 1 H, 2-CH), 3.35–3.32 (m, 2 H,
NCH2CH2NHFmoc), 3.15–3.14 (m, 2 H, 3-CH2), 3.08–3.05 (m, 2 H, NCH2CH2NHFmoc),
1.32 (s, 9 H, C(CH3)3) ppm. 13C NMR (100 MHz, CD3OD): δ = 177.4 (COOH), 159.3, 158.0
(2 x NCOO), 145.2, 142.6 (C-Ar), 128.8, 128.1, 126.1, 120.9 (CH-Ar), 81.0 (C(CH3)3), 68.1
(NCOOCH2CH), 52.8 (C-2), 51.0 (C-3), 49.1 (NCH2CH2NHFmoc), 48.5 (NCOOCH2CH),
38.6 (NCH2CH2NHFmoc), 28.7 (C(CH3)3) ppm. HRMS (ESI-TOF): calcd. for C25H30N3O6
[M–H]– 468.2140, found 468.2147.
(S)-N2-tert-Butoxycarbonyl-N4-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-
2,4-diaminobutanoic acid (23). Compound 23 was purified by flash column chromatography
using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to afford a colourless
sticky material (0.2 g; 42%). Rf = 0.20 (MeOH/AcOH/DCM, 10:1:89, v/v/v) 1H NMR (400 MHz,
CD3OD): δ = 7.67 (d, J = 7.6 Hz, 2 H, Ar-H), 7.52 (d, J = 7.6 Hz, 2 H, Ar-H), 7.27 (t, J = 7.6 Hz, 2
H, Ar-H), 7.19 (t, J = 7.6 Hz, 2 H, Ar-H), 4.27 (d, J = 6.8 Hz, 2 H, NCOOCH2CH), 4.08 (t, J = 6.8
Hz, 1 H, NCOOCH2CH), 3.90 (t, J = 6.0 Hz, 1 H, 2-CH), 3.34–3.32 (m, 2 H, NCH2CH2NHFmoc),
2.98–2.97 (m, 4 H, 3-CH2, NCH2CH2NHFmoc), 2.06–1.99 (m, 1 H, 4-CH2a), 1.90–1.80 (m, 1 H,
4-CH2b, merged with other peak), 1.32 (s, 9 H, C(CH3)3) ppm. 13C NMR (100 MHz, CD3OD):
δ = 179.9 (COOH), 159.2, 159.0 (2 x NCOO), 145.2, 142.6 (C-Ar), 128.8, 128.1, 126.1, 120.9
(CH-Ar), 80.7 (C(CH3)3), 68.1 (NCOOCH2CH), 54.9 (C-2), 48.9 (C-3), 48.3 (NCOOCH2CH),
46.5 (C-4), 38.5 (NCH2CH2NHFmoc), 31.2 (NCH2CH2NHFmoc), 28.7 (C(CH3)3) ppm. HRMS
(ESI-TOF): calcd. for C26H32N3O6 [M–H]– 482.2297, found 482.2286.
General procedure for synthesis of final monomers 25, 26 and 27
The respective triamino acids (22–24, 0.22 mmol) were dissolved in a solvent mixture of 1,
4-dioxane and water (1:1, v/v, 10 mL) under stirring at rt. After cooling in an ice-water bath
solid Na2CO3 (0.45 mmol) was added, followed by the addition of di-tert-butyl dicarbonate
(0.42 mmol). The ice-water bath was removed after 1 h and the reaction mixture was stirred at
room temperature for 18 h. Progress of the reaction was monitored by TLC and after complete
consumption of starting material, the reaction mixture was chilled in an ice-water bath, water
was added and the pH of the solution was adjusted to pH 3 by dropwise addition of 1 M HCl.
The product was extracted with ethyl acetate (15 mL x 3). The organic phase was washed with
water (15 mL) and brine (2 x 15 mL), dried over Na2SO4, filtered and concentrated to dryness
under reduced pressure to get a crude product. Pure compounds (25–27) were obtained by
passing them through column of silica gel and eluting with solvent gradient of EtOAc in hex-
ane containing 1% acetic acid.
(S)-N2,N3-Bis-tert-butoxycarbonyl-N3-[N-(9-fluorenylmethyloxycarbonyl)-2-ami-
noethyl]-2,3-diaminopropionic acid (25). Compound 25 was purified by flash column chro-
matography using 0 to 90% EtOAc in hexane containing 1% AcOH as eluent to afford a white
amorphous solid (0.084 g; 67%); m.p. 76–80°C. Rf = 0.20 (EtOAc/AcOH/hexane, 80:1:19, v/v/v) 1H
NMR (400 MHz, CDCl3): δ = 7.67 (d, J = 7.2 Hz, 2 H, Ar-H), 7.51 (d, J = 7.2 Hz, 2 H, Ar-H), 7.31
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New Triamino Acids
(t, J = 7.2 Hz, 2 H, Ar-H), 7.21 (t, J = 7.2 Hz, 2 H, Ar-H), 4.45–4.30 (m, 3 H, 2-CH, NCOOCH2CH),
4.17–4.09 (m, 1 H, NCOOCH2CH), 3.50 (br s, 2 H, NCH2CH2NHFmoc), 3.37–3.14 (m, 4 H,
3-CH2, NCH2CH2NHFmoc), 1.36 (s, 18 H, 2 x C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3): δ =
173.8 (COOH), 157.1, 156.7, 156.3, 155.7 (3 x NCOO), 144.0, 141.4 (C-Ar), 127.8, 127.2, 125.2,
120.1 (CH-Ar), 81.7, 81.4, 80.5 (2 x C(CH3)3), 67.6, 67.1 (NCOOCH2CH), 54.1, 53.0 (C-2), 50.1,
49.5, 48.6, 47.6 (C-3, NCH2CH2NHFmoc), 47.3 (NCOOCH2CH), 40.7, 40.0 (NCH2CH2NHFmoc),
28.4 (2 x C(CH3)3) ppm. [α]24D = –11.0 (c 0.1, MeOH). HRMS (ESI-TOF): calcd. for C30H38N3O8
[M–H]– 568.2664, found 568.2670.
(S)-N2,N4-Bis-tert-butoxycarbonyl-N4-[N-(9-fluorenylmethyloxycarbonyl)-2-ami-
noethyl]-2,4-diaminobutanoic acid (26). Compound 26 was purified by flash column chro-
matography using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to afford
a white amorphous solid (0.085 g; 66%); m.p. 71–76°C. Rf = 0.28 (EtOAc/AcOH/hexane, 80:1:19,
v/v/v) 1H NMR (400 MHz, CDCl3): δ = 7.69–7.67 (d, J = 7.6 Hz, 2 H, Ar-H), 7.53 (d, J = 7.2
Hz, 2 H, Ar-H), 7.32 (td, J = 7.2, 3.6 Hz, 2 H, Ar-H), 7.22 (td, J = 7.6, 2.0 Hz, 2 H, Ar-H), 4.45–
4.44 (m, 1 H, NCOOCH2CH), 4.24–4.19 (m, 1 H, 2-CH), 4.13–4.10 (m, 2 H, NCOOCH2CH),
3.77–3.70 (m, 1 H, 3-CH2a), 3.30–3.25 (m, 3 H, NCH2aCH2NHFmoc), 2.98–2.94 (m, 1 H,
NCH2bCH2NHFmoc), 2.87–2.83 (m, 1 H, 3-CH2b), 2.04–1.94 (m, 1H, 4-CH2a), 1.76–1.71
(m, 1 H, 4-CH2b), 1.39–1.35 (2s, 18 H, 2 x C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3): δ =
173.0 (COOH), 158.4, 156.9, 155.6 (3 x NCOO), 144.1, 141.4 (C-Ar), 127.8, 127.2, 125.3, 120.1
(CH-Ar), 82.8, 81.7, 80.5 (2 x C(CH3)3), 67.1, 66.9 (NCOOCH2CH), 51.2 (C-2), 50.3 (C-3),
47.4 (NCOOCH2CH), 46.3, 40.7, 34.4, 29.8 (C-4, NCH2CH2NHFmoc), 28.5, 28.4 (2 x C
(CH3)3) ppm. [α]22D = –2.3 (c 0.3, MeOH). HRMS (ESI-TOF): calcd. for C31H40N3O8 [M–H]–
582.2821, found 582.2816.
(S)-N2,N5-Bis-tert-butoxycarbonyl-N5-[N-(9-fluorenylmethyloxycarbonyl)-2-ami-
noethyl]-2,5-diaminopentanoic acid (27). Compound 27 was purified by flash column
chromatography using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent
to afford a white sticky solid material (0.097 g; 74%). Rf = 0.17 (EtOAc/AcOH/hexane, 80:1:19,
v/v/v) 1H NMR (400 MHz, CDCl3): δ = 7.67 (d, J = 7.6 Hz, 2 H, Ar-H), 7.45 (d, J = 7.6 Hz, 2 H,
Ar-H), 7.30 (t, J = 7.6 Hz, 2 H, Ar-H), 7.21 (t, J = 7.6 Hz, 2 H, Ar-H), 4.43–4.27 (m, 3 H,
NCOOCH2CH, C-2), 4.11 (t, J = 6.8 Hz, 1 H, NCOOCH2CH), 3.26–3.02 (m, 6 H, 3-CH2,
NCH2CH2NHFmoc), 1.74–1.71 (m, 1 H, 4-CH2a), 1.56–1.51 (m, 3 H, 4-CH2b, 5-CH2), 1.36
(s, 18 H, 2 x C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3): δ = 175.3 (COOH), 157.0, 156.8,
155.8 (3 x NCOO), 144.0, 141.4 (C-Ar), 127.8, 127.1, 125.2, 120.1 (CH-Ar), 80.7, 80.5, 80.2 (2 x
C(CH3)3), 67.0, 66.8 (NHCOOCH2CH), 54.4, 53.0, 47.3, 46.8 (C-2, NCH2CH2NHFmoc), 46.4
(NHCOOCH2CH), 40.5, 40.1, 29.9 (C-5, NCH2CH2NHFmoc), 28.5 (2 x C(CH3)3), 27.0, 24.6,
24.2 (C-3, C-4) ppm. [α]24D = +3.0 (c 0.1, MeOH). HRMS (ESI-TOF): calcd. for C32H42N3O8
[M–H]– 596.2977, found 596.2984.
Supporting Information
S1 Supporting Information. Experimental procedures for compounds 7, 19 and 25 as well
as 1H NMR and 13C NMR spectra of compounds 4–9, 11–16 and 22–27; RP-HPLC chro-
matograms of purified compounds 14–16 and 25–27.
(PDF)
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New Triamino Acids
Acknowledgments
We gratefully acknowledge financial support from the Swedish Research Council. We also
thank Prof. Göran Widmalm and Wangchuk Rabten at Stockholm University for assistance
with the polarimeter.
Author Contributions
Conceived and designed the experiments: JM RS. Performed the experiments: JM DH. Ana-
lyzed the data: JM DH RS. Contributed reagents/materials/analysis tools: JM DH RS. Wrote
the paper: JM DH RS.
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