Synthesis of Triamino Acid Building Blocks with Different Lipophilicities

Jyotirmoy Maity; Dmytro Honcharenko; Roger Strömberg

doi:10.1371/journal.pone.0124046

Abstract

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.

Citation: Maity J, Honcharenko D, Strömberg R (2015) Synthesis of Triamino Acid Building Blocks with Different Lipophilicities. PLoS ONE 10(4): e0124046. https://doi.org/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

Data Availability: 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 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/or materials.

Introduction

Lipophilicity has immense importance for pharmacological properties. Drug molecules are required to have lipophilic properties to accomplish a desired pharmacokinetic profile [1]. Oligonucleotides 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 nucleic acid transporter [4]. Cationic lipid-mediated nucleic acids delivery has emerged as a positive 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 formation 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]. Addition 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 C_12-18 lipid at the C-terminus of the peptides, exhibited enhanced antimicrobial activity compared 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-azalysine 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]. Triamino 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 building blocks with as well as without a hydrocarbon branching.

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 respective 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.

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Fig 1. Schematic structures of the triamino acid building blocks.

(P₁-P₃ = protecting groups).

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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 amination reaction between N^α-Fmoc-protected amino aldehydes and side chain primary amine of the N^α-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-protected N^α-amino-carboxylic acids.

The synthetic route chosen is based on that we wished to protect the terminus N7 amine position 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 approaches. One is through reduction of the acids into alcohols, followed by oxidation and a second 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 diisobutylaluminium hydride (DIBAL) under inert condition [21]. Synthetic procedures for the synthesis 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 temperature [26]. Use of 3–10 mol% of 4-(N,N-dimethylamino)pyridine (DMAP) for this type of esterification 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.

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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.

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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 purification 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 temperature 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 synthetic pathway, i.e. the reductive amination reaction of these chiral aldehydes with a chiral diamino carboxylic acid. Reductive amination reaction is a multipurpose and convenient method for the preparation of amines in organic synthesis [28]. A variety of organocatalysts, complexes 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 applications 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 reaction 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 retained 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 protected 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 afforded the final product (2S,2'S)-N²,N⁴-bis(tert-butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminohexyl]-2,4-diaminobutanoic acid (14).

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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.

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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)-oxazolidine-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-aminoacetaldehyde. Strategic manipulation of protecting groups produced the final desired product 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 distances 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 diaminobutanoic 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 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].

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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.

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Reductive amination reaction with compound 19 and the respective N^α-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 chromatography 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-diaminopentanoic acid (27).

Conclusion

We have revealed a facile synthetic procedure for the preparation of suitably protected triamino 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 protected diamino acid N^α-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 N^α-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 functionalization 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 nitrogen 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 chromatography (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 singlet, d = doublet, dd = double doublet, t = triplet, q = quartet and m = multiplet. Structural assignments 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-protected 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 layers 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. R_f = 0.54 (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 = 8.0 Hz, 1 H, NH), 4.44–4.40 (m, 1 H, NCOOCH_2aCH), 4.36–4.30 (m, 2 H, NCOOCH_2bCH, 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-CH_2a), 1.57–1.51 (m, 1 H, 3-CH_2b), 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. R_f = 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, NCOOCH_2aCH), 4.36–4.30 (m, 2 H, NCOOCH_2bCH, 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-CH_2a), 1.57–1.49 (m, 1 H, 3-CH_2b), 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. R_f = 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, NCOOCH_2aCH), 4.36–4.30 (m, 2 H, NCOOCH_2bCH, 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-CH_2a), 1.56–1.50 (m, 1 H, 3-CH_2b), 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) whereupon the mixture was stirred at rt. Progress of the reaction was monitored by TLC. After 2 h, the reaction 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 dissolved 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. R_f = 0.28 (EtOAc/hexane, 1:5, v/v). ¹H NMR (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-CH_2a), 1.33–1.26 (m, 9 H, 3-CH_2b, 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 amorphous solid (0.36 g; 84%); m.p. 69–70°C. R_f = 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-CH_2a), 1.65–1.58 (m, 1 H, 3-CH_2b), 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 attaining 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 chromatography (Fig 3).

(2S,2'S)-N²-(tert-Butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminohexyl]-2,4-diaminobutanoic acid (11).

Compound 11 was purified by flash column chromatography 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. R_f = 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-CH_2a, 3'-CH₂), 1.75–1.68 (m, 1 H, NH), 1.43–1.32 (m, 14 H, 3-CH_2b, 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-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminooctyl]-2,4-diaminobutanoic acid (12).

Compound 12 It was purified by flash column 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. R_f = 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'-CH_2a), 1.72–1.69 (m, 1 H, NH), 1.38–1.09 (m, 18 H, C(CH₃)₃, 3'-CH_2b, 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-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminodecyl]-2,4-diaminobutanoic acid (13).

Compound 13 was purified by flash column chromatography 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. R_f = 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'-CH_2a), 1.77–1.57 (m, 1 H, 3'-CH_2b), 1.38–1.19 (m, 22 H, C(CH₃)₃, 3'-CH_2b, 4'-CH₂, 5'-CH₂, 6'-CH₂, 7'-CH₂, 8'-CH_2, 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 anhydride [(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 solution (to pH 3). The product was extracted with ethylacetate (15 mL x 3). The combined ethylacetate 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-[N^2'-(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. R_f = 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), 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-CH_2a), 3.32–3.30 (m, 1 H, 4-CH_2b), 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]₂₄^D = –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-[N^2'-(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%). R_f = 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-CH_2a), 3.49–3.44 (m, 1 H, 4-CH_2b), 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]₂₂^D = –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-[N^2'-(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. R_f = 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]₂₄^D = –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)glycinal (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 solvents were evaporated in vacuo, and the residue was dissolved in ethyl acetate (25 mL). The organic layer was washed with water (15 mL) and brine (2 x 15 mL), dried over Na₂SO₄, filtered 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 chromatography using 0 to 15% MeOH in dichloromethane containing 1% AcOH as eluent to afford a colourless sticky solid (0.2 g; 43%). R_f = 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%). R_f = 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-CH_2a), 1.90–1.80 (m, 1 H, 4-CH_2b, 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 hexane containing 1% acetic acid.

(S)-N²,N³-Bis-tert-butoxycarbonyl-N³-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,3-diaminopropionic acid (25).

Compound 25 was purified by flash column chromatography 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. R_f = 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 (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]₂₄^D = –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-aminoethyl]-2,4-diaminobutanoic acid (26).

Compound 26 was purified by flash column chromatography 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. R_f = 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-CH_2a), 3.30–3.25 (m, 3 H, NCH_2aCH₂NHFmoc), 2.98–2.94 (m, 1 H, NCH_2bCH₂NHFmoc), 2.87–2.83 (m, 1 H, 3-CH_2b), 2.04–1.94 (m, 1H, 4-CH_2a), 1.76–1.71 (m, 1 H, 4-CH_2b), 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]₂₂^D = –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-aminoethyl]-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%). R_f = 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-CH_2a), 1.56–1.51 (m, 3 H, 4-CH_2b, 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]₂₄^D = +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 chromatograms of purified compounds 14–16 and 25–27.

https://doi.org/10.1371/journal.pone.0124046.s001

(PDF)

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. Analyzed the data: JM DH RS. Contributed reagents/materials/analysis tools: JM DH RS. Wrote the paper: JM DH RS.

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Figures

Abstract

Introduction

Results and Discussion

Conclusion

Experimental Section

General procedure for synthesis of ethylthio esters (4, 5 and 6)

(S)-S-Ethyl N-(9-fluorenylmethoxycarbonyl)-2-aminohexanthioate (4).

(S)-S-Ethyl N-(9-fluorenylmethoxycarbonyl)-2-aminooctanthioate (5).

(S)-S-Ethyl N-(9-fluorenylmethoxycarbonyl)-2-aminodecan-thioate (6).

General procedure for synthesis of aliphatic amino aldehydes (7, 8 and 9)

(S)-N-(9-Fluorenylmethoxycarbonyl)-2-aminooctanal (8).

(S)-N-(9-Fluorenylmethoxycarbonyl)-2-aminodecanal (9).

General procedure for synthesis of triamino acids 11, 12 and 13

(2S,2'S)-N2-(tert-Butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-aminohexyl]-2,4-diaminobutanoic acid (11).

(2S,2'S)-N2-(tert-Butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-aminooctyl]-2,4-diaminobutanoic acid (12).

(2S,2'S)-N2-(tert-Butyloxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-aminodecyl]-2,4-diaminobutanoic acid (13).

General procedure for Boc protection (14, 15 and 16)

(2S,2'S)-N2,N4-Bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-hexyl]-2,4-diaminobutanoic acid (14).

(2S,2'S)-N4-Bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-octyl]-N2,2,4-diaminobutanoic acid (15).

(2S,2'S)-N2,N4-Bis(tert-butoxycarbonyl)-N4-[N2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-decyl]-2,4-diaminobutanoic acid (16).

General procedure for synthesis of triamino acids 22, 23 and 24

(S)-N2-tert-Butoxycarbonyl-N3-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,3-diaminopropionic acid (22).

(S)-N2-tert-Butoxycarbonyl-N4-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,4-diaminobutanoic acid (23).

General procedure for synthesis of final monomers 25, 26 and 27

(S)-N2,N3-Bis-tert-butoxycarbonyl-N3-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,3-diaminopropionic acid (25).

(S)-N2,N4-Bis-tert-butoxycarbonyl-N4-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,4-diaminobutanoic acid (26).

(S)-N2,N5-Bis-tert-butoxycarbonyl-N5-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,5-diaminopentanoic acid (27).

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 chromatograms of purified compounds 14–16 and 25–27.

Acknowledgments

Author Contributions

References

(2S,2'S)-N²-(tert-Butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminohexyl]-2,4-diaminobutanoic acid (11).

(2S,2'S)-N²-(tert-Butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminooctyl]-2,4-diaminobutanoic acid (12).

(2S,2'S)-N²-(tert-Butyloxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-aminodecyl]-2,4-diaminobutanoic acid (13).

(2S,2'S)-N²,N⁴-Bis(tert-butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-hexyl]-2,4-diaminobutanoic acid (14).

(2S,2'S)-N⁴-Bis(tert-butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-octyl]-N²,2,4-diaminobutanoic acid (15).

(2S,2'S)-N²,N⁴-Bis(tert-butoxycarbonyl)-N⁴-[N^2'-(9-fluorenylmethyloxycarbonyl)-2'-amino-decyl]-2,4-diaminobutanoic acid (16).

(S)-N²-tert-Butoxycarbonyl-N³-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,3-diaminopropionic acid (22).

(S)-N²-tert-Butoxycarbonyl-N⁴-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,4-diaminobutanoic acid (23).

(S)-N²,N³-Bis-tert-butoxycarbonyl-N³-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,3-diaminopropionic acid (25).

(S)-N²,N⁴-Bis-tert-butoxycarbonyl-N⁴-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,4-diaminobutanoic acid (26).

(S)-N²,N⁵-Bis-tert-butoxycarbonyl-N⁵-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-2,5-diaminopentanoic acid (27).

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 chromatograms of purified compounds 14–16 and 25–27.