Synthesis and evaluation of N⁸-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase

Abstract

Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible acetylation of polyamines remains poorly understood. Although eukaryotic N⁸-acetylspermidine deacetylase activity has already been detected and studied, the specific enzyme responsible for this activity has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine amidohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently solved crystal structure of this polyamine deacetylase revealed the formation of an “L”-shaped active site tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating narrow, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N⁸-acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of the synthesized compounds exhibit modest potency, with IC₅₀ values in the mid-micromolar range, but compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhibitory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpolyamine function in both prokaryotes and eukaryotes.

1. Introduction

Polyamines such as putrescine, spermidine, and spermine are ubiquitous in living organisms and implicated in numerous essential biological processes.¹ For instance, polyamine concentrations affect the cell cycle progression through tightly regulated biosynthetic pathways.^{1, 2} At the molecular level, since polyamines are polycations, they can bind to nucleic acids and modulate DNA-protein interactions.² Given the importance of polyamines in different cellular processes, various enzymes of polyamine metabolism have been studied as potential drug targets.^{3, 4} For example, since upregulation of polyamine biosynthesis is a hallmark of certain cancers,^{5, 6} inhibitors of ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase have been evaluated in approaches to cancer chemotherapy. Depletion of putrescine and spermidine was achieved in vitro by cell treatment with the irreversible ODC inhibitor α-difluoromethylornithine (DFMO),7 leading to inhibition of cell growth.⁸ Although DFMO failed in clinical trials as a cancer chemotherapeutic agent, it was approved by the FDA for the treatment of parasitic infections such as African sleeping sickness.⁹

While most of the enzymes of polyamine metabolism have been extensively studied, enzymes involved in the reversible acetylation of polyamines are less well understood. The acetylation of polyamines decreases their overall charge, which is believed to regulate their function in vivo. Indeed, acetylated polyamines destabilize nucleosome structure, whereas the corresponding free polyamines bind to DNA and facilitate condensation.^{10, 11} In eukaryotes, spermidine can be either N¹- or N⁸-acetylated by two distinct enzymes: a cytoplasmic spermidine/spermine N¹-acetyltransferase¹² or a nuclear spermidine N⁸-acetyltransferase.¹³ Despite similar structures, N¹- and N⁸-acetylspermidine are metabolized differently once formed: N¹-acetylspermidine is catabolized to putrescine by a cytosolic polyamine oxidase,¹⁴ and N⁸-acetylspermidine is hydrolyzed to generate spermidine by a specific cytosolic N⁸-acetylspermidine deacetylase that is unable to use N¹-acetylspermidine as a substrate.¹⁵

Even though the N⁸-acetylspermidine deacetylase activity has been characterized and studied in vivo and in subcellular fractions, no eukaryotic polyamine deacetylase has been identified to date. However, a prokaryotic polyamine deacetylase has been reported: acetylpolyamine amidohydrolase (APAH) from Mycoplana ramosa.¹⁶ Notably, APAH has broader substrate specificity in comparison with the mammalian enzyme. As shown in Figure 1, APAH substrates include both small and large acetylpolyamines such as acetylputrescine, acetylcadaverine, N¹- and N⁸-acetylspermidine, and N¹-acetylspermine.^{16, 17} Bacterial APAH is a dimeric zinc-dependent hydrolase^{16, 17} as recently confirmed in crystal structure determinations of APAH from M. ramosa¹⁸ and from B. pseudomallei.¹⁹ As previously proposed,²⁰ APAH adopts the α/β fold first observed for the binuclear manganese metalloenzyme arginase²¹ and also shared by the histone deacetylases (HDACs).^22–24 Key active site residues required for the chemical mechanism of deacetylation are conserved between APAH and HDACs.²⁵ In contrast with the HDACs, the dimerization of APAH results in the formation of a narrow “L”-shaped active site at the dimer interface,¹⁸ conferring specificity for slender, flexible substrates rather than the large peptide substrates typically processed by HDACs. This structural feature now guides the design of specific inhibitors of APAH.

Figure 1.

Substrates of APAH from Mycoplana ramosa: (A) acetylputrescine, (B) acetylcadaverine, (C) N¹-acetylspermidine, (D) N⁸-acetylspermidine, and (E) N¹-acetylspermine.

Inhibitors of APAH will be helpful tools for the exploration of both prokaryotic and eukaryotic acetylpolyamine function in vivo. For instance, inhibitors of the mammalian N⁸-acetylspermidine deacetylase have been described earlier, some of which exhibit low nanomolar inhibitory activity.^{26, 27} So far, the HDAC inhibitor M344²⁸ and the trifluoromethylketone analogue of l-arginine²⁹ are the only compounds reported to inhibit the bacterial polyamine deacetylase in the low and mid-micromolar range, respectively.^{18, 29} Here, we report the synthesis of new polyamine derivatives as well as new synthetic routes for some of the previously described mammalian N⁸-acetylspermidine deacetylase inhibitors.^{26, 27} All compounds synthesized in the current study are analogues of N⁸-acetylspermidine bearing different functional groups targeting Zn²⁺ coordination. We also report the inhibitory potency of these compounds against M. ramosa APAH.

2. Results and discussion

2.1. Inhibitor design

The X-ray crystal structure of inactive H159A APAH complexed with N⁸-acetylspermidine (PDB accession code 3Q9C) illustrates the molecular details of substrate recognition in the enzyme active site.¹⁸ Key interactions are made by the N4 secondary amino group of N⁸-acetylspermidine, which donates a hydrogen bond to E117 and makes a cation-π interaction with F225; the N1 primary amino group, which donates a hydrogen bond to E106 in the other monomer of the homodimer; the amide NH group, which donates a hydrogen bond to the backbone carbonyl of G167; and the amide carbonyl group, which coordinates to the Zn²⁺ ion and accepts a hydrogen bond from Y323. Based on the mechanism of catalysis by the related HDACs,^30–32 a nucleophilic Zn²⁺-bound water molecule is activated by metal coordination and general base H159 (Figure 2). Nucleophilic attack at the scissile carbonyl group of the substrate results in the formation of a tetrahedral intermediate stabilized by metal coordination and hydrogen bond interactions with surrounding residues. The collapse of this intermediate is enabled by H159, which serves as a general acid catalyst in this step of the mechanism,³¹ leading to the formation of products spermidine and acetate.²⁵

Figure 2.

Mechanism of APAH.

Based on the structural features important for substrate recognition and catalysis, including the tetrahedral structure of the transition state flanking the tetrahedral intermediate, we designed and synthesized potential APAH inhibitors based on the N⁸-acetylspermidine substrate-like scaffold. As shown in Figure 3, compounds I–X all share a common 1,3-diaminopropane moiety to preserve key enzyme-substrate hydrogen bond interactions with the polyamine N1 and N4 groups (Figure 2). However, each compound differs in the nature of its head group designed to mimic substrate or tetrahedral transition state binding to the active site Zn²⁺ ion. Most of these Zn²⁺-binding groups have been successfully incorporated into effective inhibitors of HDACs^{29, 33–39} and other metallohydrolases.^40–43

Figure 3.

Structures of target compounds as potential APAH inhibitors. These compounds are analogues of the substrate N⁸-acetylspermidine (Figure 1D).

2.2. Chemistry

Syntheses of compounds I–X are summarized in Schemes 1–4. Compounds I–X were each synthesized from key intermediates 3 or 4. As shown in Scheme 1, 3 and 4 were obtained in two steps. In the first step, 1,3-diaminopropane was N-alkylated with an alkylbromide, either 5-bromopent-1-ene or 7-bromohept-1-ene, to yield alkylamines 1 and 2, respectively. This reaction was performed with an excess of 1,3-diaminopropane (10 equivalents, neat) to favor the monoalkylation of the unprotected diamine over polyalkylation. Monoalkylamines 1 and 2 were then quantitatively N-protected with tert-butoxycarbonyl (Boc) groups with di-tert-butyl pyrocarbonate (Boc₂O).

Scheme 1.

Synthesis of intermediates 3 and 4. Reagents and conditions: (a) 1,3-diaminopropane (neat, 10 eq), 0°C (1 h) then room temperature (RT) (2 h); (b) Boc₂O (3 eq) in CH₂Cl₂, 0°C then RT (overnight).

Scheme 4.

Synthesis of compounds VII–X. Reagents and conditions: (a) 9-BBN (2.5 eq) in THF, 0°C then RT (20 h), then NaOH, H₂O₂, 0°C then RT (30 min); (b) CBr₄ (2 eq), PPh₃ (2 eq) in THF, 0°C then RT (overnight); (c) KSAc (6 eq) in MeCN, RT (overnight); (d) anhydrous HCl (4 N) in dioxane, RT (2 h); (e) NaOMe (2 eq) in MeOH, RT (2 h); (f) anhydrous HCl (1 N) in AcOEt, RT (5 h); (g) NaSMe (6 eq) in EtOH, 60°C (overnight); (h) m-CPBA (3 eq) in CH₂Cl₂, 0°C then RT (2 h); (i) [Ir(cod)Cl]₂ (5 mol %), dppm (10 mol %), pinacolborane (1.9 eq), RT (24 h); (j) aqueous HCl (6 N), reflux (24 h).

We developed an alternative route for the synthesis of compounds I, II, and III compared with that previously published.^{26, 27} The new synthetic route allows for more flexibility in generating additional compounds from intermediate 4. As depicted in Scheme 2, compounds I, II, and III were synthesized from carboxylic acid 5. Oxidative cleavage of alkene 4 into 5 was performed by the Sharpless method with ruthenium chloride as catalyst and sodium periodate as oxidant^{44, 45} in solvent system H₂O/AcOEt/MeCN (3/2/2).⁴⁶ N,N’-carbonyldiimidazole (CDI) mediated coupling of carboxylic acid 5 with N-O-dimethylamine quantitatively led to Weinreb amide 6. N-methoxy-N-methylamides are well-known reagents for the synthesis of ketones from carboxylic acids in the presence of Grignard or organolithium reagents.⁴⁷ Using this strategy, methylketone 7 was synthesized from Weinreb amide 6 with an excess of methylmagnesium bromide (5 equivalents). CDI-activated carboxylic acid 5 was reacted with unprotected hydroxylamine to form the corresponding hydroxamic acid 8.⁴⁸ Deprotection of compounds 5, 7, and 8 with anhydrous HCl (4 N) in dioxane at room temperature led to target compounds I, II, and III as dihydrochloride salts.

Scheme 2.

Synthesis of compounds I–III. Reagents and conditions: (a) RuCl₃ (4 mol %), NaIO₄ (4.5 eq) in H₂O/AcOEt/MeCN (3/2/2), RT (3 h); (b) anhydrous HCl (4 N) in dioxane, RT (2 h); (c) CDI (2 eq) in CH₂Cl₂, RT (1 h), then MeNHOMe•HCl (2 eq), RT (overnight); (d) MeMgBr (5 eq) in THF, 0°C (2 h) then RT (1 h); (e) CDI (1.5 eq) in THF, RT (1 h), then NH₂OH•HCl (2 eq), RT (overnight).

Alkene 4 also served as a key common intermediate for the synthesis of compounds IV, V, and VI as shown in Scheme 3. As for the synthesis of carboxylic acid 5, introduction of the aldehyde functionality was achieved by an oxidative cleavage of alkene 4. This was done sequentially by first oxidizing 4 to the corresponding cis-diol with osmium tetroxide as catalyst in the presence of N-methylmorpholine N-oxide (NMO) in dioxane/H₂O (4/1) as solvent⁴⁹ until completion of the reaction (3 hours). The cis-diol derived from 4 was not isolated, but directly cleaved to aldehyde 9 by reaction with sodium periodate. Nucleophilic trifluoromethylation of aldehyde 9 was achieved using Ruppert’s reagent (trifluoromethyltrimethylsilane, TMSCF₃)⁵⁰ and a source of fluoride, tetra-n-butylammonium fluoride (TBAF), as initiator under usual conditions.^{51, 52} Trifluoromethyl carbinol 10 was then easily oxidized to the corresponding trifluoromethyl ketone 11 by Dess-Martin periodinane (DMP).^{53, 54} Alkene 4 was quantitatively oxidized to epoxide 12 using m-chloroperbenzoic acid (m-CPBA). Regioselective ring opening of unsymetrically substituted epoxide 12 was achieved using lithium bromide and acetic acid yielding α-bromohydrin 13.⁵⁵ Subsequent alcohol oxidation of 13 with DMP led to the formation of α-bromoketone 14.⁵³ Bromide 14 was then treated with potassium thioacetate to afford thioester 15. Oxirane 12 also served as a precursor for the synthesis of α-methoxyketone derivative VI. Ring opening of its epoxide moiety with sodium methoxide in MeOH regioselectively formed the corresponding α-methoxyalcohol 16, after which oxidation with DMP afforded α-methoxyketone 17.⁵³ Deprotection of compounds 11, 15, and 17 with anhydrous HCl (4 N in dioxane, or 1 N in AcOEt) at room temperature led to target compounds IV, V, and VI as dihydrochloride salts. Synthesis of the α-mercaptoketone from thioester 15 was attempted. Successful thioacetate group alcoholysis in MeOH under basic conditions (sodium methoxide) gave the corresponding N-Boc protected α-mercaptoketone, but no pure deprotected α-mercaptoketone could be isolated after deprotection under acidic conditions.

Scheme 3.

Synthesis of compounds IV–VI. Reagents and conditions: (a) NMO (2.5 eq), OsO₄ (2.5 mol %) in dioxane/H₂O (4/1), RT (3 h), then NaIO₄ (2.5 eq), RT (20 min); (b) TMSCF₃ (3 eq), TBAF (10 mol %) in THF, RT (2 h), then TBAF (in THF containing ca. 5% H₂O, 1.5 eq), RT (45 min); (c) DMP (4 eq) in CH₂Cl₂, RT (overnight); (d) anhydrous HCl (4 N) in dioxane, RT (2 h); (e) m-CPBA (2 eq) in CH₂Cl₂, 0°C then RT (21 h); (f) LiBr (3.2 eq), AcOH (3 eq) in THF, RT (overnight); (g) DMP (3 eq), RT (3 h); (h) KSAc (6 eq) in MeCN, RT (overnight); (i) NaOMe (6 eq) in MeOH, RT (24 h), (j) DMP (6 eq) in CH₂Cl₂, RT (24 h); (k) anhydrous HCl (1 N) in AcOEt, RT (6 h).

As shown in Scheme 4, alkene 3 was used as precursor for compounds VII–X. Alkene 3 hydroboration with 9-borabicyclo[3.3.1]nonane (9-BBN)⁵⁶ followed by oxidation with H₂O₂ under basic conditions (NaOH) quantitatively afforded alcohol 18. Subsequent bromination of 18 was achieved using carbon tetrabromide (CBr₄) in the presence of triphenylphosphine (PPh₃). Corresponding alkyl bromide 19 was then treated with potassium thioacetate to afford 20, as for the synthesis of thioesther 15. Thioacetate group alcoholysis in MeOH under basic conditions (sodium methoxide) gave thiol 21. Alkyl bromide 19 also served a precursor for the synthesis of sulfone derivative IX. Nucleophilic substitution of bromine by sodium thiomethoxide in EtOH afforded thioether 22, after which oxidation with m-CPBA led to sulfone 23. Hydroboration of alkene 3 with pinacolborane using [Ir(cod)Cl]₂ as catalyst and 1,1-bis(diphenylphosphino)methane (dppm) as ligand under usual conditions selectively gave terminal boronic ester 24.⁵⁷ Complete deprotection of 24 was achieved in aqueous HCl (6 N) under reflux affording compound X as a dihydrochloride salt. Deprotection of compounds 20, 21, and 23 with anhydrous HCl (4 N in dioxane, or 1 N in AcOEt) at room temperature led to target compounds VII, VIII, and IX as dihydrochloride salts.

2.3. Enzyme inhibition

All N⁸-acetylspermidine analogues were tested in vitro for APAH inhibition. Results are summarized in Table 1. Three compounds exhibit very poor inhibitory potency against APAH: carboxylic acid I, thioester VII, and sulfone IX, with IC₅₀ values in the millimolar range. In contrast, sulfone and thioester analogues of SAHA are effective inhibitors of HDAC in the mid- or low micromolar range, respectively,^{33, 58} and carboxylic acid I was previously shown to be a potent inhibitor of the mammalian N⁸-acetylspermidine deacetylase in the low micromolar range.²⁷

Table 1.

Inhibitory potency of compounds I–X against M. ramosa APAH

Compound	Head-group	IC50 (µM)
I	-COOH	1800 ± 200
II	-COCH3	160 ± 10
III	-CONHOH	0.39 ± 0.03
IV	-COCF3	0.27 ± 0.03 38 ± 6
V	-COCH2SAc	39 ± 10 4000 ± 1000
VI	-COCH2OCH3	380 ± 50
VII	-SAc	1900 ± 200
VIII	-SH	26 ± 3
IX	-SO2CH3	10000 ± 3000
X	-B(OH)2	230 ± 40

A second set of compounds are modest inhibitors of APAH, with inhibitory potencies in the mid-micromolar range. In increasing order of potency, these compounds are: α-methoxyketone VI, boronic acid X, ketone II, thioester V, and thiol VIII. Ketone II is a modest inhibitor or the bacterial polyamine deacetylase but a mid-nanomolar selective inhibitor of the mammalian N⁸-acetylspermidine deacetylase.²⁶ The efficacy and specificity of ketone I have also been demonstrated in vivo, and this compound was used to probe the function of the mammalian polyamine deacetylase.⁵⁹ In general, the incorporation of each of these functional groups in the design of HDAC inhibitors resulted in the generation of highly potent compounds with inhibitory potency in the nanomolar range.^{33, 35–39} However, in contrast to our N⁸-acetylspermidine analogues, HDAC inhibitors are designed based on the combination of a metal-binding group, a linker, and an active site-capping group. For a given Zn²⁺-binding group, optimization of the linker and the capping group to optimize interactions with the mouth of the active site cleft is usually required to achieve exceptional inhibitory potency.

The best two APAH inhibitors showing potency in the nanomolar range are hydroxamate III and trifluoromethylketone IV (Table 1). Hydroxamates have been extensively studied as metal-coordinating groups for the design of metalloenzyme inhibitors, such as the FDA-approved HDAC inhibitor SAHA for anti-cancer chemotherapy.⁶⁰ Hydroxamates form a stable bidentate five-membered ring complex with the catalytic Zn²⁺ ion that contributes to high affinity. Hydroxamate III exhibits an IC₅₀ value of 390 nM against APAH. This compound is also a potent inhibitor of mammalian N⁸-acetylspermidine deacetylase activity, with an apparent K_i of 1 nM measured with subcellular extracts.²⁷

Trifluoromethylketones are well-known to exist as gem-diol hydrate in aqueous solution. This was further demonstrated for compound IV with ¹³C and ¹⁹F NMR in D₂O. Indeed, a single peak at −85.1 ppm for ¹⁹F NMR and a quadruplet at 93.6 ppm clearly indicates that the trifluoromethylketone group is hydrated in water. This gem-diol form mimics the tetrahedral intermediate in zinc metalloenzyme-catalyzed hydrolytic reactions, as first demonstrated for carboxypeptidase A.^{61, 62} Surprisingly, as shown in Figure 4, the data obtained for compound IV fit better with a two-site binding model, whereas the concentration-response curve of most of the other compounds were typical of a one-site binding model as for hydroxamate III. The biphasic curve obtained for inhibition by trifluoromethylketone IV yields IC₅₀ values of 270 nM and 38 µM. Similar behavior is also observed for compound V, with IC₅₀ values of 39 µM and 4 mM. While such biphasic inhibition is also observed in other systems, e.g., the inhibition of smooth muscle endothelin-converting enzyme by the metalloprotease inhibitor phosphoramidon,⁶³ the reason for this uncommon inhibition mode against APAH remains unclear. This type of dose-response curve is usually observed when a ligand binds to a receptor existing in two different affinity states,⁶⁴ or to a receptor or a transporter in two different sites.⁶⁵ However, the X-ray crystal structure of the APAH-IV complex reveals the binding of the gem-diol form of the inhibitor solely in the enzyme active site (study in progress), and we have not observed any other notable features, e.g., time-dependent inhibition, in our measurements. Thus, it is possible that monomers A and B of APAH exist in two different affinity states with regard to the binding of IV.

Figure 4.

Inhibition of APAH by: (A) hydroxamate III and (B) trifluoromethylketone IV.

3. Conclusions

In summary, we have designed and synthesized a series of N⁸-acetylspermidine analogues bearing different functional groups targeting Zn²⁺ coordination interactions in the active site of APAH. Most analogues studied are modest inhibitors, but two – compounds III and IV, bearing hydroxamate and trifluoromethylketone groups, respectively – exhibit nanomolar inhibitory potency. Future work on the optimization of these leads may facilitate the development of even better APAH inhibitors. Moreover, compounds III and IV may also be useful tools for probing the function of acetylpolyamines in both eukaryotes and prokaryotes, and in searching for the as-yet unidentified mammalian N⁸-acetylspermidine deacetylase.

4. Experimental Section

4.1. Chemistry

4.1.1. General Procedures

All reagents were of at least 95% purity and purchased from Fisher Scientific, Alfa Aesar or Sigma Aldrich. All solvents were of HPLC grade and purchased from Fisher Scientific or Sigma Aldrich. For reactions requiring anhydrous conditions, solvents (THF, MeCN, and MeOH) were purchased as anhydrous grade from Fisher Scientific (except CH₂Cl₂, which was freshly distilled under N₂ from P₂O₅). Reactions were monitored by TLC with Sigma Aldrich aluminum plates (silica gel with fluorescent indicator, 60 Å, 200 µm) and visualized by staining with a ninhydrin solution or under UV light when necessary. Flash column chromatography was performed using Fisher Scientific silica gel 60 (230–400 mesh). Melting points were determined using a Mel-Temp Electrothermal apparatus and were uncorrected. High-resolution mass spectrometry (HRMS) was performed on a Waters LC-TOF mass spectrometer (model LCT-XE Premier) using electrospray ionization (ESI) in positive mode. For compound X, the boronic acid moiety was derivatized by adding (+)-pinanediol to enable analysis by mass spectrometry.

¹H and ¹³C NMR spectra were recorded on Bruker DMX 360 and DRX 500 spectrometers operating at 360 and 500 MHz, respectively, for ¹H NMR and at 90.6 and 125.6 MHz, respectively, for ¹³C NMR. ¹⁹F NMR spectra were recorded at 282.4 MHz on a Bruker DMX 360 spectrometer, and ¹¹B NMR spectra at 128 MHz on a Bruker DMX 400 spectrometer. ¹H and ¹³C NMR chemical shifts (δ) are reported in ppm relative to the residual solvent peak. NMR coupling constants (J) are reported in Hz, and multiplicities are denoted as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quadruplet; and m, multiplet.

4.1.2. N¹-(pent-4-enyl)propane-1,3-diamine (1)

To 35.2 mL of 1,3-diaminopropane (422 mmol) at 0°C and under argon was added dropwise 5-bromopent-1-ene (5.0 mL, 42.2 mmol). The solution was stirred at 0°C one hour, and two additional hours at room temperature. The reaction mixture was then partitioned between AcOEt (250 mL), brine (40 mL), saturated aqueous NaHCO₃ (40 mL), and H₂O (40 mL). The aqueous layer was extracted with AcOEt and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by flash column chromatography on SiO₂ with CHCl₃/MeOH/NH₄OH gradients afforded pure alkyl diamine 1 (5.64 g, 94%) as a slightly yellow oil. ¹H NMR (360 MHz, CDCl₃) δ: 5.81-5.70 (m, 1H), 4.96 (dd, J = 1.8 Hz, J = 17.3 Hz, 1H), 4.90 (dd, J = 1.8 Hz, J = 10.1 Hz, 1H), 2.72 (t, J = 6.6 Hz, 2H), 2.63 (t, J = 6.8 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 2.06-2.00 (m, 2H), 1.63-1.41 (m, 7H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 138.2, 114.4, 49.3, 47.7, 40.3, 33.5, 31.3, 29.0. HRMS (ESI) calcd for C₈H₁₉N₂ [M + H]⁺ 143.1548, found 143.1549.

4.1.3. N¹-(hept-6-enyl)propane-1,3-diamine (2)

Alkylation of 1,3-diaminopropane (27.4 mL, 328 mmol) with 7-bromohept-1-ene (5 mL, 32.8 mmol) was performed under the same conditions as for 1, and afforded after purification alkyl diamine 2 as a slightly yellow oil (5.31 g, 95%). ¹H NMR (360 MHz, CDCl₃) δ: 5.79-5.68 (m, 1H), 4.93 (dd, J = 1.1 Hz, J = 17.3 Hz, 1H), 4.87 (dd, J = 1.1 Hz, J = 10.1 Hz, 1H), 2.70 (t, J = 6.8 Hz, 2H), 2.60 (t, J = 6.8 Hz, 2H), 2.53 (t, J = 6.8 Hz, 2H), 1.99 (m, 2H), 1.57 (m, 2H), 1.45 (m, 2H), 1.36-1.24 (m, 4H), 1.15 (s, 3H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 139.0, 114.3, 50.2, 48.0, 40.7, 34.0, 33.7, 30.1, 28.9, 26.9. HRMS (ESI) calcd for C₁₀H₂₃N₂ [M + H]⁺ 171.1861, found 171.1853.

4.1.4. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-(pent-4-enyl)propane-1,3-diamine (3)

To a solution of alkyl diamine 1 (5.40 g, 38.0 mmol) in dry CH₂Cl₂ (150 mL) at 0°C and under argon was added dropwise Boc₂O (26.2 mL, 114 mmol). The solution was allowed to warm gradually to room temperature and was stirred overnight. The reaction mixture was concentrated in vacuo and the resulting residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford pure 3 as a colorless oil (12.7 g, quantitative). ¹H NMR (500 MHz, CDCl₃) δ: 5.78-5.70 (m, 1H), 5.10 (bs, 1H), 4.96 (dd, J = 2.0 Hz, J = 17.3 Hz, 1H), 4.91 (dd, J = 2.0 Hz, J = 10.0 Hz, 1H), 3.19 (t, J = 6.3 Hz, 2H), 3.08 (t, J = 7.5 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H), 1.97 (m, 2H), 1.62-1.52 (m, 4H), 1.40 (s, 9H), 1.38 (s, 9H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 156.1 (2C), 137.9, 115.0, 79.5, 79.0, 46.5, 44.0, 37.7, 31.0, 28.5 (7C), 27.6. HRMS (ESI) calcd for C₁₈H₃₄N₂NaO₄ [M + Na]⁺ 365.2416, found 365.2410.

4.1.5. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-(hept-6-enyl)propane-1,3-diamine (4)

Boc-protection of alkyl diamine 2 (5.1 g, 29.9 mmol) with Boc₂O (20.6 mL, 89.7 mmol) was performed under the same conditions as for 3, and afforded 4 as a colorless oil (11.1 g, quantitative). ¹H NMR (500 MHz, CDCl₃) δ: 5.63 (m, 1H), 5.26 (bs, 1H), 4.84 (dd, J = 1.6 Hz, J = 17.2 Hz, 1H), 4.79 (dd, J = 1.6 Hz, J = 10.1 Hz, 1H), 3.11 (t, J = 6.0 Hz, 2H), 3.00 (t, J = 6.0 Hz, 2H), 2.95 (t, J = 6.3 Hz, 2H), 1.91 (apparent q (dt), J = 7.2 Hz, 2H), 1.55-1.50 (m, 2H), 1.42-1.33 (m, 2H), 1.32 (s, 9H), 1.30 (s, 9H), 1.29-1.24 (m, 2H), 1.19-1.11 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 155.9 (2C), 138.5, 114.3, 79.2, 78.6, 46.8, 43.7, 37.6, 33.5, 28.4, 28.8 (7C), 28.2, 26.2. HRMS (ESI) calcd for C₂₀H₃₈N₂NaO₄ [M + Na]⁺ 393.2729, found 393.2727.

4.1.6. N,N’-Bis(tert-butoxycarbonyl)-6-[(3-aminopropyl)amino]hexanoic acid (5)

To a solution of alkene 4 (4.00 g, 10.8 mmol) in 200 mL of a H₂O/AcOEt/MeCN (3/2/2) mixture were added successively ruthenium(III) chloride hydrate (35%, 256 mg, 432 µmol) and sodium periodate (10.4 g, 48.6 mmol). After stirring at room temperature for 3 hours, the reaction mixture was quenched by the addition of isopropanol (80 mL), the suspension filtered over a celite pad, and the filtrate concentrated in vacuo. The aqueous phase was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was further purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford carboxylic acid 5 as a colorless oil (4.11 g, 98%). ¹H NMR (500 MHz, CDCl₃) δ: 8.79 (bs, 1H), 5.37 (bs, 1H), 3.21 (t, J = 6.3 Hz, 2H), 3.12 (t, J = 6.6 Hz, 2H), 3.07 (t, J = 6.0 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.66-1.60 (m, 4H), 1.54-1.48 (m, 2H), 1.43 (s, 9H), 1.42 (s, 9H), 1.33-1.27 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 178.6, 156.5 (2C), 79.7, 79.4, 46.9, 44.2, 38.0, 34.0, 28.6 (7C), 28.2, 26.5, 24.5. HRMS (ESI) calcd for C₁₉H₃₆N₂NaO₆ [M + Na]⁺ 411.2471, found 411.2473.

4.1.7. N’,N’’-Bis(tert-butoxycarbonyl)-6-[(3-aminopropyl)amino]-N-methoxy-N-methylhexanamide (6)

To a solution of carboxylic acid 5 (2.08 g, 5.35 mmol) in dry CH₂Cl₂ (80 mL) under argon was added CDI (1.74 g, 10.7 mmol). After one hour at room temperature, N,O-dimethylhydroxylamine hydrochloride (1.04 g, 10.7 mmol) was added to the solution. After stirring overnight, the reaction mixture was diluted CH₂Cl₂ (100 mL), washed with 0.1 M HCl (3 × 50 mL), water, and brine. The organic layer was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was further purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford Weinreb amide 6 (2.31 g, quantitative) as a colorless oil. ¹H NMR (360 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.65 (s, 3H), 3.22 (t, J = 6.1 Hz, 2H), 3.15 (s, 3H), 3.14-3.03 (m, 4H), 2.39 (t, J = 7.2 Hz, 2H), 1.67-1.58 (m, 4H), 1.56-1.46 (m, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.34-1.26 (m, 2H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 174.6, 156.1 (2C), 79.6, 79.1, 61.3, 46.9, 44.1, 37.8, 32.3, 31.9, 28.6 (7C), 28.3, 26.8, 24.5. HRMS (ESI) calcd for C₂₁H₄₂N₃O₆ [M + H]⁺ 432.3074, found 432.3091.

4.1.8. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]heptan-2-one (7)

To a solution of Weinreb amide 6 (1.36 g, 3.15 mmol) in dry THF (25 mL) at 0°C and under argon was added dropwise methylmagnesium bromide (1.0 M in THF, 15.8 mL, 15.8 mmol). The solution was stirred 2 hours at 0°C and then one hour at room temperature. The reaction mixture was cooled down to 0°C, quenched by the addition of saturated aqueous NH₄Cl (20 mL), diluted with AcOEt (100 mL), and the aqueous phase was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was further purified by flash column chromatography with hexanes/AcOEt gradients to afford ketone 7 as a colorless oil (1.12 g, 92%). ¹H NMR (500 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.17 (t, J = 6.5 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 2.06 (s, 3H), 1.61-1.56 (m, 2H), 1.55-1.49 (m, 2H), 1.48-1.41 (m, 2H), 1.39 (s, 9H), 1.37 (s, 9H), 1.22-1.16 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 208.9, 156.1 (2C), 79.5, 79.0, 46.8, 43.9, 43.6, 37.7, 29.9, 28.5 (7C), 28.3, 26.4, 23.5. HRMS (ESI) calcd for C₂₀H₃₈N₂O₅Na [M + Na]⁺ 409.2678, found 409.2670.

4.1.9. N’,N’’-Bis(tert-butoxycarbonyl)-6-[(3-aminopropyl)amino]-N-hydroxyhexanamide (8)

To a solution of carboxylic acid 5 (600 mg, 1.54 mmol) in dry THF (20 mL) under argon was added CDI (376 mg, 2.32 mmol). After one hour at room temperature, hydroxylamine hydrochloride (215 mg, 3.09 mmol) was added to the solution. After stirring overnight, the reaction mixture was diluted with a 5% KHSO₄ aqueous solution (60 mL) and extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was further purified by flash column chromatography on SiO₂ with AcOEt/MeOH gradients to afford hydroxamic acid 8 (550 mg, 88%) as a slightly yellow oil. ¹H NMR (500 MHz, DMSO-d₆) δ: 10.29 (bs, 1H), 8.61 (bs, 1H), 6.72 (bs, 1H), 3.08 (apparent q (2t), J = 7.5 Hz, 4H), 2.88 (apparent q (dt), J = 6.5 Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H), 1.58-1.54 (m, 2H), 1.52-1.46 (m, 2H), 1.44-1.39 (m, 2H), 1.38 (s, 9H), 1.37 (s, 9H), 1.21-1.15 (m, 2H). ¹³C NMR (125.6 MHz, DMSO-d₆) δ: 169.0, 155.6, 154.6, 78.2, 77.4, 46.3, 44.3, 37.6, 32.2, 28.2, 28.1 (6C), 27.6, 25.9, 24.9. HRMS (ESI) calcd for C₁₉H₃₇N₃O₆Na [M + Na]⁺ 426.2580, found 426.2576.

4.1.10. N,N’-Bis(tert-butoxycarbonyl)-6-[(3-aminopropyl)amino]hexanal (9)

To a solution of alkene 4 (2.71 g, 7.31 mmol) in 150 mL of a dioxane/H₂O (4/1) mixture were successively added NMO monohydrate (2.47 g, 18.3 mmol) dissolved in 6 mL of water, and osmium tetroxide (47 mg, 185 µmol). TLC monitoring showed completion after 3 hours at room temperature. Sodium periodate (3.91 g, 18.3 mmol) was added to the solution. After stirring for 20 min, the reaction mixture was quenched with isopropanol (50 mL), the suspension filtered over a celite pad, and the filtrate concentrated in vacuo. The aqueous phase was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was further purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford aldehyde 9 as a colorless oil (2.64 g, 97%). ¹H NMR (500 MHz, CDCl₃) δ: 9.63 (s, 1H), 5.25 (bs, 1H), 3.11 (t, J = 7.0 Hz, 2H), 3.02 (t, J = 6.7 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H), 2.31 (t, J = 6.9 Hz, 2H), 1.55-1.49 (m, 4H), 1.42-1.37 (m, 2H), 1.32 (s, 9H), 1.30 (s, 9H), 1.21-1.15 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 202.1, 155.9 (2C), 79.3, 78.7, 46.6, 43.8, 43.6, 37.5, 28.3 (7C), 27.8, 26.2, 21.6. HRMS (ESI) calcd for C₁₉H₃₆N₂O₅Na [M + Na]⁺ 395.2522, found 395.2509.

4.1.11. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptan-2-ol (10)

To a solution of aldehyde 9 (1.53 g, 4.11 mmol) in dry THF (20 mL) at room temperature and under argon were added successively TMSCF₃ (1.82 mL, 12.3 mmol) and anhydrous TBAF (1.0 M in THF, 410 µL, 410 µmol). After 2 hours at room temperature, TBAF (1.0 M in THF containing ca. 5% H₂O, 6.2 mL, 6.20 mmol) was added dropwise and the solution was stirred for 45 min and concentrated in vacuo. The corresponding residue was partitioned between AcOEt (100 mL) and saturated aqueous NaHCO₃ (50 mL) and the aqueous phase was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. Purification by flash column chromatography on SiO₂ with hexanes/AcOEt gradients afforded trifluoromethylalcohol 10 as a colorless oil (1.49 g, 82%). ¹H NMR (360 MHz, CDCl₃) δ: 5.32 (bs, 1H), 4.46 (bs, 1H), 3.79-3.73 (m, 1H), 3.11 (t, J = 6.5 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.97 (t, J = 6.5 Hz, 2H), 1.58-1.48 (m, 6H), 1.46-1.38 (m, 2H), 1.34 (s, 9H), 1.32 (s, 9H), 1.25-1.14 (m, 2H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 156.3 (2C), 125.5 (q, J = 281.8 Hz), 79.6, 79.1, 69.7 (q, J = 29.0 Hz), 46.9, 43.9, 37.6, 29.5, 28.3 (7C), 28.1, 26.4, 24.6. ¹⁹F NMR (338.8 MHz) δ: −79.8. HRMS (ESI) calcd for C₂₀H₃₇N₂NaO₅F₃ [M + Na]⁺ 465.2552, found 465.2553.

4.1.12. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptan-2-one (11)

To a solution of trifluoromethylalcohol 10 (1.40 g, 3.16 mmol) in dry CH₂Cl₂ (50 mL) at room temperature and under argon was added DMP (5.37 g, 12.7 mmol). The reaction mixture was stirred overnight at room temperature, then cooled down to 0°C, quenched with the addition of an aqueous sodium thiosulfate solution (0.5 M) saturated with NaHCO₃ (150 mL), and the aqueous layer was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford trifluoromethylketone 11 as a slightly yellow oil (1.32 g, 95%). ¹H NMR (500 MHz, CDCl₃) (ketone/hydrate : 1/0) δ: 5.26 (bs, 1H), 3.11 (t, J = 6.7 Hz, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 7.1 Hz, 2H), 1.60-1.53 (m, 4H), 1.45-1.39 (m, 2H), 1.33 (s, 9H), 1.30 (s, 9H), 1.23-1.17 (m, 2H). ¹³C NMR (500 MHz, CDCl₃) δ: 191.2 (q, J = 35.2 Hz), 156.1 (2C), 115.5 (q, J = 291.4 Hz), 79.5, 78.8, 46.6, 43.4, 37.6, 36.1, 28.3 (7C), 28.1, 25.9, 22.0. ¹⁹F NMR (338.8 MHz) δ: −79.4. HRMS (ESI) calcd for C₂₀H₃₅N₂O₅F₃Na [M + Na]⁺ 463.2396, found 463.2397.

4.1.13. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-[5-(oxiran-2-yl)pentyl]propane-1,3-diamine (12)

To a solution of alkene 4 (2.89 g, 7.80 mmol) in dry CH₂Cl₂ (150 mL) under argon at 0°C was added dropwise m-CPBA (77%, 3.50 g, 15.6 mmol) in 40 mL dry CH₂Cl₂. The solution was allowed to reach room temperature and stirred until completion of the reaction (21 hours) as shown by TLC. Saturated aqueous NaHCO₃ (50 mL) was added to the reaction mixture and the aqueous layer was extracted with AcOEt. Combined organic extracts were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford epoxide 12 (3.01 g, quantitative) as a colorless oil. ¹H NMR (360 MHz, CDCl₃) δ: 5.28 (bs, 1H), 3.05 (t, J = 7.2 Hz, 2H), 2.97-2.88 (m, 4H), 2.71-2.67 (m, 1H), 2.54-2.52 (m, 1H), 2.26-2.24 (m, 1H), 1.51-1.46 (m, 2H), 1.38-1.26 (m, 6H), 1.27 (s, 9H), 1.24 (s, 9H), 1.18-1.14 (m, 2H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 155.8 (2C), 79.0, 78.4, 51.8, 46.6, 46.5, 43.6, 37.3, 32.1, 28.3, 28.2 (7C), 26.4, 25.5. HRMS (ESI) calcd for C₂₀H₃₉N₂O₅ [M + H]⁺ 387.2859, found 387.2854.

4.1.14. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-1-bromoheptan-2-ol (13)

To a solution of epoxide 12 (2.10 g, 5.43 mmol) in dry THF (25 mL) under argon were added successively lithium bromide (1.51 g, 17.4 mmol) and glacial acetic acid (930 µL, 16.2 mmol) dropwise. After stirring at room temperature overnight, saturated aqueous NaHCO₃ (30 mL) was added to the reaction mixture. The aqueous layer was extracted with AcOEt and the combined organic extracts were dried over Na₂SO₄, filtered and concentrated in vacuo. Purification by flash column chromatography on SiO₂ with hexanes/AcOEt gradients afforded α-bromohydrin 13 (2.31 g, 91%) as a colorless oil. ¹H NMR (360 MHz, CDCl₃) δ: 5.26 (bs, 1H), 3.71-3.64 (m, 1H), 3.38 (dd, J = 4.0 Hz, J = 10.1 Hz, 1H), 3.30 (dd, J = 6.5 Hz, J = 10.1 Hz, 1H), 3.13 (t, J = 6.5 Hz, 2H), 3.05-2.96 (m, 5H), 1.58-1.53 (m, 2H), 1.48-1.39 (m, 4H), 1.37-1.31 (m, 2H), 1.36 (s, 9H), 1.34 (s, 9H), 1.22-1.15 (m, 2H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 156.0 (2C), 79.4, 78.9, 70.7, 46.9, 43.9, 39.8, 37.6, 34.9, 28.4 (7C), 27.9, 26.6, 25.2. HRMS (ESI) calcd for C₂₀H₃₉N₂O₅BrNa [M + Na]⁺ 489.1940, found 489.1931.

4.1.15. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-1-bromoheptan-2-one (14)

To a solution of α-bromohydrin 13 (2.10 g, 4.49 mmol) in dry CH₂Cl₂ (50 mL) at room temperature and under argon was added DMP (5.72 g, 13.5 mmol). After 3 hours, the reaction mixture was cooled down to 0°C, quenched with the addition of an aqueous sodium thiosulfate solution (0.5 M) saturated with NaHCO₃ (100 mL), and the aqueous layer was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford α-bromoketone 14 as a colorless oil (2.03 g, 97%). ¹H NMR (360 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.78 (s, 2H), 3.12 (t, J = 6.5 Hz, 2H), 3.04-2.95 (m, 4H), 2.54 (t, J = 7.2 Hz, 2H), 1.56-1.47 (m, 4H), 1.45-1.36 (m, 2H), 1.33 (s, 9H), 1.31 (s, 9H), 1.21-1.12 (m, 2H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 201.7, 155.9 (2C), 79.3, 78.7, 46.6, 43.8, 39.5, 37.6, 34.2, 28.3, 27.8 (7C), 26.1, 23.4. HRMS (ESI) calcd for C₂₀H₃₇N₂O₅BrNa [M + Na]⁺ 487.1784, found 487.1794.

4.1.16. S-{N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-2-oxoheptyl}thioacetate (15)

To a solution of α-bromoketone 14 (1.72 g, 3.70 mmol) in dry MeCN (25 mL) at room temperature under argon was added potassium thioacetate (2.54 g, 22.2 mmol). After stirring overnight, the reaction mixture was diluted with water and the aqueous layer was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by flash column chromatography on SiO₂ with hexanes/AcOEt gradients afforded thioester 15 (1.65 g, 97%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.68 (s, 2H), 3.18 (t, J = 6.5 Hz, 2H), 3.08-3.02 (m, 4H), 2.50 (t, J = 7.5 Hz, 2H), 2.32 (s, 3H), 1.62-1.54 (m, 4H), 1.49-1.43 (m, 2H), 1.40 (s, 9H), 1.38 (s, 9H), 1.25-1.18 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 203.8, 194.3, 156.1 (2C), 79.5, 79.0, 46.7, 43.9, 41.5, 39.0, 37.7, 30.2, 28.5 (7C), 28.4, 26.3, 23.4. HRMS (ESI) calcd for C₂₂H₄₀N₂O₆SNa [M + Na]⁺ 483.2505, found 483.2517.

4.1.17. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-1-methoxyheptan-2-ol (16)

Epoxide 12 (1.33 g, 3.44 mmol) was dissolved in a 0.5 M solution of sodium methoxide in MeOH (41.2 mL, 20.6 mmol). The reaction mixture was stirred under argon at room temperature until completion of the reaction (24 hours) as shown by TLC. After removal of the solvent in vacuo, the residue was partitioned between AcOEt (100 mL) and water (25 mL), and the aqueous layer was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Purification by flash column chromatography on SiO₂ with hexanes/AcOEt gradients afforded alcohol 16 (1.34 g, 93%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ: 5.33 (bs, 1H), 3.66-3.60 (m, 1H), 3.27-3.24 (m, 4H), 3.15-3.10 (m, 3H), 3.02-2.96 (m, 4H), 2.72 (bs, 1H), 1.55-1.50 (m, 2H), 1.43-1.35 (m, 4H), 1.33 (s, 9H), 1.31 (s, 9H), 1.29-1.23 (m, 2H), 1.20-1.14 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 156.0 (2C), 79.2, 78.7, 77.1, 69.9, 58.8, 46.9, 43.7, 37.4, 33.1, 28.3 (7C), 28.2, 26.8, 25.2. HRMS (ESI) calcd for C₂₁H₄₃N₂O₆ [M + H]⁺ 419.3121, found 419.3124.

4.1.18. N,N’-Bis(tert-butoxycarbonyl)-7-[(3-aminopropyl)amino]-1-methoxyheptan-2-one (17)

To a solution of alcohol 16 (1.19 g, 2.84 mmol) in dry CH₂Cl₂ (50 mL) at room temperature and under argon was added DMP (7.24 g, 17.1 mmol). After 24 hours, the reaction mixture was cooled down to 0°C, quenched with the addition of an aqueous sodium thiosulfate solution (0.5 M) saturated with NaHCO₃ (150 mL), and the aqueous layer was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford α-methoxyketone 17 as a colorless oil (1.08 g, 91%). ¹H NMR (500 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.94 (s, 2H), 3.35 (s, 3H), 3.18 (t, J = 6.5 Hz, 2H), 3.08-3.02 (m, 4H), 2.38 (t. J = 7.5 Hz, 2H), 1.61-1.52 (m, 4H), 1.49-1.43 (m, 2H), 1.39 (s, 9H), 1.37 (s, 9H), 1.29-1.23 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 208.5, 156.24 (2C), 79.5, 79.0, 77.6, 59.3, 46.8, 43.9, 38.6, 37.7, 28.4 (7C), 28.3, 26.4, 23.0. HRMS (ESI) calcd for C₂₁H₄₀N₂O₆Na [M + Na]⁺ 439.2784, found 439.2772.

4.1.19. N,N’-Bis(tert-butoxycarbonyl)-5-[(3-aminopropyl)amino]pentan-1-ol (18)

To a solution of alkene 3 (3.00 g, 8.76 mmol) in dry THF (150 mL) at 0°C and under argon was added dropwise 9-BBN (0.5 M in THF, 43.8 mL, 21.9 mmol). The solution was allowed to warm up to room temperature and stirred for 20 hours. The reaction mixture was then cooled down to 0°C, and aqueous NaOH (6 M, 30 mL) was added, followed by the dropwise addition of H₂O₂ (30%, 15 mL). After stirring 30 min at room temperature, the solution was concentrated in vacuo. The aqueous phase was diluted with 50 mL of water, and extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford alcohol 18 as a colorless oil (3.15 g, quantitative). ¹H NMR (500 MHz, CDCl₃) δ: 5.35 (bs, 1H), 3.54 (t, J = 6.5 Hz, 2H), 3.16 (t, J = 6.5 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 3.01 (t, J = 6.5 Hz, 2H), 2.74 (bs, 1H), 1.61-1.55 (m, 2H), 1.53-1.43 (m, 4H), 1.38 (s, 9H), 1.36 (s, 9H), 1.29-1.23 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ:156.2 (2C), 79.5, 79.0, 62.4, 47.0, 43.9, 37.6, 32.3, 28.4 (7C), 27.8, 23.0. HRMS (ESI) calcd for C₁₈H₃₆N₂O₅Na [M + Na]⁺ 383.2522, found 383.2508.

4.1.20. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-(5-bromopentyl)propane-1,3-diamine (19)

To a solution of alcohol 18 (3.00 g, 8.32 mmol) in dry THF (100 mL) at 0°C were added successively CBr₄ (5.52 g, 16.6 mmol) and PPh₃ (4.37 g, 16.7 mmol). The reaction mixture was allowed to reach room temperature and stirred overnight. After removal of the solvent in vacuo, purification of the residue by flash column chromatography on SiO₂ with hexanes/AcOEt gradients afforded alkyl bromide 19 (3.38 g, 96%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.23 (t, J = 6.5 Hz, 2H), 3.17 (t, J = 6.5 Hz, 2H), 3.08 (t, J = 6.5 Hz, 2H), 3.02 (t, J = 6.5 Hz, 2H), 1.83-1.77 (m, 2H), 1.61-1.56 (m, 2H), 1.51-1.44 (m, 2H), 1.39 (s, 9H), 1.37 (s, 9H), 1.38-1.31 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 156.0 (2C), 79.5, 78.9, 46.7, 43.8, 37.7, 33.6, 32.4, 28.4 (7C), 27.7, 25.4. HRMS (ESI) calcd for C₁₈H₃₆N₂O₄Br [M + H]⁺ 423.1858, found 423.1866.

4.1.21. S-{N,N’-Bis(tert-butoxycarbonyl)-5-[(3-aminopropyl)amino]pentyl} thioacetate (20)

Reaction of alkyl bromide 19 (1.70 g, 4.02 mmol) and potassium thioacetate (2.75 g, 24.1 mmol) under the same conditions as for compound 15 afforded after purification thioester 20 (1.58 g, 94%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.07 (t, J = 6.5 Hz, 2H), 2.96 (t, J = 6.5 Hz, 2H), 2.91 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.14 (s, 3H), 1.51-1.47 (m, 2H), 1.44-1.38 (m, 2H), 1.38-1.32 (m, 2H), 1.28 (s, 9H), 1.26 (s, 9H), 1.19-1.13 (m, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 195.3, 155.8 (2C), 79.1, 78.5, 46.5, 43.6, 37.5, 30.3, 29.0, 28.7, 28.2 (7C), 27.8, 25.7. HRMS (ESI) calcd for C₂₀H₃₈N₂O₅SNa [M + Na]⁺ 441.2399, found 441.2406.

4.1.22. N,N’-Bis(tert-butoxycarbonyl)-5-[(3-aminopropyl)amino]pentane-1-thiol (21)

To a solution of thioester 20 (550 mg, 1.31 mmol) in dry MeOH (10 mL) under argon was added sodium methoxide (0.5 M solution in MeOH, 5.3 mL, 2.65 mml). The solution was stirred at room temperature for 2 hours, quenched by the addition of 10% aqueous citric acid (100 mL), and the aqueous phase was extracted with AcOEt. Combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford thiol 21 as a colorless oil (485 mg, 98%). ¹H NMR (500 MHz, CDCl₃) δ: 5.19 (bs, 1H), 3.07 (t, J = 6.0 Hz, 2H), 2.98 (t, J = 6.3 Hz, 2H), 2.92 (t, J = 6.3 Hz, 2H), 2.35 (apparent q (dt), J = 7.3 Hz, 2H), 1.51-1.41 (m, 4H), 1.37-1.32 (m, 2H), 1.29 (s, 9H), 1.26 (s, 9H), 1.23-1.17 (m, 2H), 1.16 (t, J = 7.3 Hz, 1H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 155.8 (2C), 79.1, 78.5, 46.6, 43.7, 37.5, 33.4, 28.2 (7C), 27.7, 25.3, 24.2. HRMS (ESI) calcd for C₁₈H₃₆N₂O₄SNa [M + Na]⁺ 399.2293, found 399.2293.

4.1.23. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-[5-(methylthio)pentyl]propane-1,3-diamine (22)

To a solution of alkyl bromide 19 (1.22 g, 2.88 mmol) in EtOH (15 mL) was added sodium thiomethoxide (1.21 g, 17.3 mmol). The reaction mixture was stirred overnight at 60°C under argon. After removal of the solvent under vacuo, the residue was partitioned between CH₂Cl₂ (50 mL) and saturated aqueous NaHCO₃ (25 mL), and the aqueous layer was extracted with CH₂Cl₂. Combined organic extracts were washed with water, brine, dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by flash column chromatography on SiO₂ with hexanes/AcOEt gradients afforded thioether 22 (1.10 g, 98%) as a colorless oil. ¹H NMR (360 MHz, CDCl₃) δ: 5.21 (bs, 1H), 3.20 (t, J = 6.5 Hz, 2H), 3.13-3.05 (m, 4H), 2.46 (t, J = 7.4 Hz, 2H), 2.06 (s, 3H), 1.66-1.54 (m, 4H), 1.53-1.46 (m, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.38-1.29 (m, 2H). ¹³C NMR (90.6 MHz, CDCl₃) δ: 156.1 (2C), 79.6, 79.1, 46.9, 44.1, 37.8, 34.3, 29.0, 28.5 (7C), 28.2, 26.1, 15.6. HRMS (ESI) calcd for C₁₉H₃₉N₂O₄S [M + H]⁺ 391.2631, found 391.2633.

4.1.24. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-[5-(methylsulfonyl)pentyl]propane-1,3-diamine (23)

To a solution of thioether 22 (500 mg, 1.28 mmol) in dry CH₂Cl₂ (10 mL) under argon at 0°C was added dropwise m-CPBA (77%, 861 mg, 3.84 mmol) in 10 mL dry CH₂Cl₂. The solution was allowed to reach room temperature and stirred until completion of the reaction (2 hours) as shown by TLC. Saturated aqueous NaHCO₃ (10 mL) was added to the reaction mixture and the aqueous layer was extracted with AcOEt. Combined organic extracts were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford sulfone 23 (524 mg, 97%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ: 5.14 (bs, 1H), 3.07 (t, J = 7.0 Hz, 2H), 3.01 (t, J = 6.5 Hz, 2H), 2.92 (t, J = 6.5 Hz, 2H), 2.87 (t, J = 7.0 Hz, 2H), 2.75 (s, 3H), 1.73-1.67 (m, 2H), 1.53-1.48 (m, 2H), 1.44-1.40 (m, 2H), 1.31-1.26 (m, 2H), 1.30 (s, 9H), 1.27 (s, 9H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 155.9 (2C), 79.3, 78.6, 54.3, 46.3, 43.6, 40.2, 37.5, 28.2 (7C), 28.0, 25.4, 22.0. HRMS (ESI) calcd for C₁₉H₃₈N₂O₆SNa [M + Na]⁺ 445.2348, found 445.2344.

4.1.25. N¹,N³-Bis(tert-butoxycarbonyl)-N¹-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pentyl]propane-1,3-diamine (24)

To a solution of [Ir(cod)Cl]₂ (196 mg, 292 µmol) and dppm (225 mg, 585 µmol) in dry CH₂Cl₂ (150 mL) under argon was added pinacolborane (1.65 mL, 11.3 mmol) and alkene 3 (2.00 g, 5.84 mmol) in 30 mL dry CH₂Cl₂. After stirring 24 hours at room temperature, the reaction mixture was cooled to 0°C, quenched with the addition of 60 mL of water, and the aqueous phase extracted with Et₂O. Combined organic extracts were dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on SiO₂ with hexanes/AcOEt gradients to afford 24 as a colorless oil (2.25 g, 82%). ¹H NMR (500 MHz, CDCl₃) δ: 5.24 ppm (bs, 1H), 3.16 (t, J = 6.6 Hz, 2H), 3.04-2.99 (m, 4H), 1.59-1.54 (m, 2H), 1.45-1.39 (m, 2H), 1.37 (s, 9H), 1.35 (s, 9H), 1.35-1.30 (m, 2H), 1.21-1.15 (m, 2H), 1.15 (s, 12H), 0.68 (t, J = 7.8 Hz, 2H). ¹³C NMR (125.6 MHz, CDCl₃) δ: 156.0 (2C), 82.6 (2C), 79.3, 78.8, 47.0, 43.8, 37.7, 29.5, 28.5 (7C), 28.3, 24.8 (4C), 23.8, 11.2 (bs). ¹¹B NMR (128 MHz, CDCl₃) δ: 33.0 ppm. HRMS (ESI) calcd for C₂₄H₄₈N₂O₅B [M + H]⁺ 471.3605, found 471.3615.

4.1.26. 6-[(3-aminopropyl)amino]hexanoic acid dihydrochloride (I)

A solution of compound 5 (300 mg, 772 µmol) in anhydrous 4 N HCl in dioxane (20 mL) was stirred at room temperature under argon for 2 hours. The reaction mixture was cooled down to 0°C and, diluted with Et₂O (20 mL). The precipitate was filtered, washed with cold Et₂O, and dried in vacuo to afford carboxylic acid I (dihydrochloride salt) as a white solid (188 mg, 93%). mp: 172-174 °C. ¹H NMR (500 MHz, D₂O) δ: 3.20 (t, J = 8.0 Hz, 2H), 3.17-3.11 (m, 4H), 2.45 (t, J = 7.4 Hz, 2H), 2.17-2.11 (m, 2H), 1.79-1.72 (m, 2H), 1.71-1.65 (m, 2H), 1.49-1.43 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 178.8, 47.6, 44.4, 36.6, 33.5, 25.2, 25.1, 23.8, 23.7. HRMS (ESI) calcd for C₉H₂₁N₂O₂ [M + H]⁺ 189.1603, found 189.1604.

4.1.27. 7-[(3-aminopropyl)amino]heptan-2-one dihydrochloride (II)

Deprotection of compound 7 (320 mg, 828 µmol) was performed under the same conditions as for I to afford ketone II (dihydrochloride salt) as a white powder (202 mg, 94%). mp: 206-209 °C (dec.). ¹H NMR (500 MHz, D₂O) δ: 3.14 (t, J = 8.0 Hz, 2H), 3.10 (t, J = 8.0 Hz, 2H), 3.06 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 2.19 (s, 3H), 2.11-2.05 (m, 2H), 1.71-1.65 (m, 2H), 1.60-1.54 (m, 2H), 1.38-1.31 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 216.9, 47.5, 44.3, 42.7, 36.5, 29.2, 25.2, 25.0, 23.6, 22.5. HRMS (ESI) calcd for C₁₀H₂₃N₂O [M + H]⁺ 187.1810, found 187.1813.

4.1.28. 6-[(3-aminopropyl)amino]-N-hydroxyhexanamide dihydrochloride (III)

Deprotection of compound 8 (350 mg, 867 µmol) was performed under the same conditions as for I to afford hydroxamic acid III (dihydrochloride salt) as a white powder (230 mg, 96%). mp: 135-139 °C. ¹H NMR (500 MHz, D₂O) δ: 3.19 (t, J = 8.0 Hz, 2H), 3.16-3.10 (m, 4H), 2.23 (t, J = 7.3 Hz, 2H), 2.16-2.10 (m, 2H), 1.77-1.71 (m, 2H), 1.70-1.64 (m, 2H), 1.45-1.39 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 173.1, 47.6, 44.4, 36.6, 32.0, 25.2, 25.0, 24.3, 23.7. HRMS (ESI) calcd for C₉H₂₂N₃O₂ [M + H]⁺ 204.1712, found 204.1722.

4.1.29. 7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptan-2-one dihydrochloride (IV)

Deprotection of compound 11 (350 mg, 795 µmol) was performed under the same conditions as for I to afford trifluoromethylketone IV (dihydrochloride salt) as a white powder (234 mg, 94%). mp: 228–231 °C (dec.). ¹H NMR (500 MHz, D₂O) (ketone/hydrate : 0/1) δ: 3.20 (t, J = 8.0 Hz, 2H), 3.17-3.12 (m, 4H), 2.18-2.11 (m, 2H), 1.88 (t, J = 8.2 Hz, 2H), 1.80-1.74 (m, 2H), 1.62-1.55 (m, 2H), 1.51-1.45 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 123.6 (q, J = 286.4 Hz), 93.6 (q, J = 31.4 Hz), 47.7, 44.4, 36.6, 33.7, 25.7, 25.3, 23.7, 20.7. ¹⁹F NMR (338.8 MHz) δ: −85.1. HRMS (ESI) calcd for C₁₀H₂₀N₂OF₃ [M + H]⁺ 241.1528, found 241.1534.

4.1.30. 7-{[(3-aminopropyl)amino]-2-oxoheptyl} thioacetate dihydrochloride (V)

Deprotection of compound 15 (300 mg, 651 µmol) was performed under the same conditions as for I to afford thioacetate derivative V (dihydrochloride salt) as a white powder (210 mg, 97%). mp: 211–213 °C (dec.). ¹H NMR (500 MHz, D₂O) δ: 4.00 (s, 2H), 3.23 (t, J = 8.0 Hz, 2H), 3.19 (t, J = 7.8 Hz, 2H), 3.16 (t, J = 7.8 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.49 (s, 3H), 2.21-2.15 (m, 2H), 1.82-1.76 (m, 2H), 1.74-1.68 (m, 2H), 1.50-1.43 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 209.8, 200.1, 47.7, 44.5, 41.1, 39.3, 36.7, 29.5, 25.3, 25.1, 23.8, 22.7. HRMS (ESI) calcd for C₁₂H₂₅N₂O₂S [M + H]⁺ 261.1637, found 261.1626.

4.1.31. 7-[(3-aminopropyl)amino]-1-methoxyheptan-2-one dihydrochloride (VI)

A solution of compound 17 (240 mg, 576 µmol) in anhydrous 1 N HCl in AcOEt (30 mL) was stirred at room temperature under argon for 6 hours. The suspension was diluted with hexanes (20 mL). The precipitate was filtered, washed with AcOEt and hexanes, and dried in vacuo to afford α-methoxyketone VI (dihydrochloride salt) as an off-white solid (151 mg, 91 %). mp: 216–218 °C (dec.). ¹H NMR (500 MHz, D₂O) δ: 4.35 (s, 2H), 3.44 (s, 3H), 3.19 (t, J = 8.0 Hz, 2H), 3.15 (t, J = 7.9 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.55 (t, J = 7.3 Hz, 2H), 2.16-2.10 (m, 2H), 1.77-1.71 (m, 2H), 1.68-1.62 (m, 2H), 1.45-1.39 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 212.8, 76.5, 58.7, 47.6, 44.4, 37.8, 36.6, 25.3, 25.2, 23.7, 22.3. HRMS (ESI) calcd for C₁₁H₂₅N₂O₂ [M + H]⁺ 217.1916, found 217.1925.

4.1.32. S-{5-[(3-aminopropyl)amino]pentyl} thioacetate dihydrochloride (VII)

Deprotection of compound 20 (300 mg, 717 µmol) was performed under the same conditions as for I to afford thioacetate derivative VII (dihydrochloride salt) as a white powder (196 mg, 94%). mp: 260–262 °C (dec.). ¹H NMR (500 MHz, D₂O) δ: 3.19 (t, J = 8.0 Hz, 2H), 3.15 (t, J = 7.9 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 2.41 (s, 3H), 2.16-2.10 (m, 2H), 1.78-1.72 (m, 2H), 1.69-1.64 (m, 2H), 1.52-1.46 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 202.2, 47.6, 44.4, 36.6, 30.1, 28.4, 28.1, 25.0, 24.7, 23.7. HRMS (ESI) calcd for C₁₀H₂₃N₂OS [M + H]⁺ 219.1531, found 219.1532.

4.1.33. 5-[(3-aminopropyl)amino]pentane-1-thiol dihydrochloride (VIII)

A solution of compound 21 (208 mg, 552 µmol) in anhydrous 1 N HCl in AcOEt (40 mL) was stirred at room temperature under argon for 5 hours. The reaction mixture was concentrated in vacuo and resuspended in hexanes (20 mL). The precipitate was filtered, washed with hexanes, and dried in vacuo to afford thiol derivative VIII (dihydrochloride salt) as a white solid (129 mg, 93%). mp: 284–286 °C (dec.). ¹H NMR (500 MHz, D₂O) δ: 3.24 (t, J = 8.0 Hz, 2H), 3.21-3.15 (m, 4H), 2.65 (t, J = 7.1 Hz, 2H), 2.21-2.15 (m, 2H), 1.82-1.76 (m, 2H), 1.76-1.70 (m, 2H), 1.59-1.52 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 47.8, 44.5, 36.7, 32.4; 25.0, 24.5, 23.8, 23.5. HRMS (ESI) calcd for C₈H₂₁N₂S [M + H]⁺ 177.1425, found 177.1424.

4.1.34. N¹-[5-(methylsulfonyl)pentyl]propane-1,3-diamine dihydrochloride (IX)

Deprotection of compound 23 (300 mg, 710 µmol) was performed under the same conditions as for I to afford sulfone IX (dihydrochloride salt) as a white powder (202 mg, 97%). mp: 202–204 °C. ¹H NMR (500 MHz, D₂O) δ: 3.28 (t, J = 7.8 Hz, 2H), 3.15 (t, J = 7.8 Hz, 2H), 3.11-3.07 (m, 7H), 2.12-2.05 (m, 2H), 1.89-1.82 (m, 2H), 1.78-1.71 (m, 2H), 1.58-1.51 (m, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 53.1, 47.3, 44.3, 39.5, 36.4, 24.9, 24.3, 23.6, 20.9. HRMS (ESI) calcd for C₉H₂₃N₂O₂S [M + H]⁺ 223.1480, found 223.1483.

4.1.35. 5-[(3-aminopropyl)amino]pentylboronic acid dihydrochloride (X)

The protected compound 24 (300 mg, 638 µmol) was stirred to reflux with 6N aqueous HCl (10 mL) for 24 hours. After cooling to room temperature, the reaction mixture was washed with Et₂O (20 mL) and the aqueous phase was concentrated in vacuo to give boronic acid X as an off-white solid (148 mg, 89%). mp: 288–290 °C (dec.). ¹H NMR (500 MHz, D₂O) δ: 3.18 (t, J = 8.0 Hz, 2H), 3.15 (t, J = 7.9 Hz, 2H), 3.10 (t, J = 7.8 Hz, 2H), 2.16-2.10 (m, 2H), 1.76-1.70 (m, 2H), 1.50-144 (m, 2H), 1.43-1.37 (m, 2H), 0.83 (t, J = 7.6 Hz, 2H). ¹³C NMR (125.6 MHz, D₂O) δ: 47.8, 44.4, 36.6, 28.3, 25.3, 23.7, 23.1, 13.8 (bs). ¹¹B NMR (128 MHz, D₂O) δ: 32.4. HRMS (ESI) calcd for C₁₈H₃₆N₂O₂B [M + (+)-pinanediol - 2H₂O + H]⁺ 323.2870, found 323.2869.

4.2. APAH expression, purification, and activity assay

APAH was expressed in Escherichia coli BL21(DE3) cells (Novagen Inc.) and purified as previously described.¹⁸ The inhibition of APAH by the synthesized diamine derivatives was analyzed using a fluorimetric assay, as previously described.¹⁸ Activity was measured using the commercially available Fluor-de-Lys deacetylase fluorogenic substrate (BML-KI104, Enzo Life Sciences). Briefly, deacetylation of the substrate molecule allows a protease developer to cleave the peptide bond linking the C-terminal fluorophore, resulting in a fluorescence shift. Assays were run at 25 °C and contained 250 nM enzyme, 150 µM substrate, and 0–100 mM inhibitor in assay buffer (25 mM Tris (pH 8.2), 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl₂ (100 µM tris-(2-carboxyethyl)phosphine was added only for the assay of thiol compound VIII) in a final volume of 50 µL. After 30 min, reactions were quenched by adding 100 µM M344 (Sigma Aldrich) and the appropriate Fluor-de-Lys developer (BML-KI105, Enzo Life Sciences, 50 µL). Fluorescence was measured after 45 min using a Fluoroskan II plate reader (excitation = 355 nm, emission = 460 nm). Assays for each concentration of inhibitor were performed in triplicate in separate experiments. IC₅₀ values for each compound were determined using the software Graphpad Prism (2008).

Abbreviations

APAH

acetylpolyamine amidohydrolase

dppm

bis(diphenylphosphino)methane

9-BBN

9-borabicyclo[3.3.1]nonane

DMP

Dess-Martin periodinane

DFMO

α-difluoromethylornithine

Boc₂O

di-tert-butyl pyrocarbonate

ESI

electrospray ionization

HRMS

high-resolution mass spectrometry

HDAC

histone deacetylase

m-CPBA

m-chloroperbenzoic acid

NMO

N-methylmorpholine N-oxide

CDI

N,N’-carbonyldiimidazole

ODC

ornithine decarboxylase

RT

room temperature

Boc

tert-butoxycarbonyl

TBAF

tetra-n-butylammonium fluoride

TMSCF₃

trifluoromethyltrimethylsilane

PPh₃

triphenylphosphine