Structure-Activity Relationships in Nucleotide Oligomerization Domain-1 (Nod1)-Agonistic γ-Glutamyl-diaminopimelic Acid Derivatives

Abstract

N-acyl-γ-glutamyl-diaminopimelic acid is a prototype ligand for Nod1. We report a detailed SAR of C₁₂-γ-D-Glu-DAP. Analogues with glutaric or γ-aminobutyric acid replacing the glutamic acid show greatly attenuated Nod1-agonistic activity. Substitution of the meso-diaminopimelic (DAP) acid component with monoaminopimelic acid, L- or D-lysine, or cadaverine also results in reduced activity. The free amine on DAP is crucial. However, the N-acyl group on the D-glutamyl residue can be substituted with N-alkyl groups with full preservation of activity. The free carboxylates on the DAP and Glu components can also be esterified, resulting in more lipophilic, but active analogues. Transcriptomal profiling showed a dominant upregulation of IL-19, IL-20, IL-22, and IL-24, which may explain the pronounced Th2-polarizing activity of these compounds, and also implicate cell signaling mediated by TREM-1. These results may explain the hitherto unknown mechanism of synergy between Nod1- and TLR-agonists, and are likely to be useful in designing vaccine adjuvants.

Introduction

Cellular pattern recognition receptors (PRRs) are ubiquitous innate immune sensors that recognize specific molecular patterns present in molecules that are broadly shared by pathogens, but are structurally distinct from host molecules^1–4 PRRs include not only membrane-bound Toll-like receptors (TLRs), members of which are expressed on the cell surface, as well as the endocytoplasmic reticulum, but also cytosolic receptors encompassing the Nod and RIG-I families of proteins.^4;5 Nod1 and Nod2 are prototype members of the nucleotide oligomerization domain (NOD) and ligand-recognizing leucine-rich repeat (NLR)- containing proteins which serve to signal the presence of intracytoplasmic peptidoglycan fragments by sensing diaminopimelic acid peptides and muramyl dipeptides, respectively.^6–9 Our interest in understanding the structural determinants of the biological properties of ligands of PRRs stems from the potential value in harnessing innate immune stimulatory properties in marshalling, and specifically directing subsequent adaptive immune responses with desired immunophenotypes and profiles.^10;11 We had previously reported SAR studies on TLR2 and TLR7,^12;13 and whilst studies on several other TLR ligands continue, our attention is focused also on the intracytoplasmic NOD family of receptors, especially Nod1. Whereas the muramyldipeptide chemotype has been extensively studied since the early 1970s^14–19 (preceding by several decades the discovery of Nod2 as its primary receptor^9;20), structure-activity relationships and signaling pathways involving Nod1-agonistic γ-glutamyl-meso-diaminopimelic acid peptides^21;22 have remained sparse. Investigators at the Fujisawa Pharmaceutical Company reported in the early 1980s that D-lactoyl-L-alanyl- γ-D-glutamyl-(L)-meso-diaminopimelyl-(L)-glycine and heptanoyl-γ-D-glutamyl-(L)-meso-diaminopimelyl- D-alanine enhanced host defense in murine models of fatal E. coli sepsis.^23–27 Subsequent to the discovery that the γ-glutamyl-meso-diaminopimelic acid (iE-DAP) fragment is the crucial pharmacophore mediating recognition of peptidoglycan fragments by Nod1,^21;22 synthetic, lipophilic, N-acyl glutamyl derivatives have been shown to be potent Nod1 agonists.^28;29 N-dodecanoyl-γ-D-glutamyl-diaminopimelic acid (C₁₂-iE-DAP) is now available commercially as a reference Nod1 agonist. Also noteworthy is work by Boons and colleagues who have synthesized and evaluated DAP-containing muramyl peptides.^30;31

We were keen to explore the structural space around iE-DAP scaffold for the reason that Nod1 agonists are associated with the mobilization of adaptive immune responses with a dominant Th2 bias, which is undesirable when a strong CD8+ cytotoxic T lymphocytic response is required.³² We hypothesized that covalent coupling to iE-DAP of the TLR7/8-agonistic imidazoquinolines^12;33 that we have found to be extraordinarily Th1-polarizing^10;11 could lead to vaccine adjuvants with balanced Th1/Th2 immunostimulatory phenotypes. In order to accomplish this goal, it was necessary to first determine optimal positions on the iE-DAP backbone that would permit the generation of dual-active ‘hybrid’ molecules, combining the TLR7-agonistic imidazoquinoline and NOD1-active iE-DAP chemotypes in a single covalently coupled construct. We report in this paper an SAR study of Nod1-agonistic iE-DAP derivatives. Secondary screens including transcriptomal profiling in ex vivo models using whole human blood have revealed a possible basis for the pronounced Th2 bias of this chemotype.

Results and Discussion

The construction of orthogonally protected γ-D-glutamyl-diaminopimelic acid synthons (Scheme 1) allowed convenient access to a number of analogues wherein the carboxyl and amino substituents on both amino acids could be varied (Schemes 2–4). For the sake of consistency, we designate the α-amino group on the D-Glu as N, and the γ-amino group on the diaminopimelic acid (DAP) residue as N′. We used the commercially available diaminopimelic acid (6; mixture of 4 isomers), since derivatives of all four isomers have been shown to be active.^28;29 Esters of DAP proved surprisingly difficult to obtain by a number of conventional methods, necessitating sequential N-Boc protection of the amines, esterification (as benzyl or ethyl) of the carboxylic acids, deprotection of the N-Boc groups, followed by mono-N-Boc protection using stoichiometric equivalents of Boc₂O. N-Fmoc-D-Glu-α-benzyl/allyl esters (4, 5 respectively) were coupled to the mono-N-Boc-diaminopimelic acid diesters (9 or 10) using standard protocols to afford the orthogonally protected synthons (11–13; Scheme 1). N-Fmoc (D-Glu) deprotection, followed by acylation with lauroyl chloride and subsequent deprotection of the N-Boc and O-Bn groups yielded C₁₂-iE-DAP (16; Scheme 2), the activity of which was identical to commercially available reference compound with EC₅₀ values of ~30 pM (Fig. 1A).

Scheme 1.

Reagents: i. ROH, HBTU, TEA, DMAP, DMF; ii. (a) CF₃COOH (b) FmocCl, Na₂CO₃, Dioxane, H₂O; iii. (a) (Boc)₂O, TEA, H₂O (b) R′OH, HBTU, DMAP, TEA, DMF; iv. (a) HCl-dioxane (b) (Boc)₂O (1eq.), TEA, MeOH; v. PS-Carbodiimide, PS-DMAP, CH₂Cl₂.

Scheme 2.

Reagents: i. 30% Piperidine, CH₂Cl₂; ii. C₁₁H₂₃COCl, pyridine, DMAP; iii. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH; iv. (a) CF₃COOH (b) C₁₁H₂₃COCl, TEA, CH₂Cl₂; v. H₂, Pd(OH)₂/C, AcOH, MeOH, 60 psi; vi. CF₃COOH; vii. (a) C₁₁H₂₃COCl, pyridine, CH₂Cl₂ (b) H₂, Pd(OH)₂/C, MeOH, 60 psi; viii. C₁₁H₂₃CHO (1eq.), MP-CNBH₃, CH₂Cl₂, MeOH, AcOH; ix. RCHO(excess), MP-CNBH₃, CH₂Cl₂, MeOH, AcOH; x. 1H-Pyrazole-1-carboxamidine.HCl, pyridine, MW irradiation, 60°C; xi. H₂, Pd(OH)₂/C, MeOH, 60 psi; xii. N,N′-Di-Boc-1HPyrazole- 1-carboxamidine•HCl, pyridine, THF, 50°C.

Scheme 4.

Reagents: i. 30% Piperidine, CH₂Cl₂; ii. (a) (Boc)₂O, TEA, MeOH (b) H₂, Pd(OH)₂/C, MeOH, 60 psi (c) C₁₁H₂₃SH, HBTU, DMAP, TEA, DMF; iii. CF₃COOH; iv. C₁₁H₂₃COCl, pyridine; v. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH.

Figure 1.

A. NF-κB induction activity in stable transfectants of HEK-293 cells expressing human Nod1. B. Rank-order plot of potencies (EC₅₀ values) of iE-DAP analogues.

The N′-acyl (DAP) compound (18), the N,N′-diacyl (D-Glu, and DAP, respectively) analogue (20), as well as N-alkyl (22) and N,N-dialkyl analogues (26–28) were also synthesized from synthon 11 (Scheme 2). Our initial attempts at converting the N′ amine to a guanidine group resulted in an undesired cyclization-elimination reaction to form the aminoimidazolone 30; this was obviated by the use of N,N′-di- Boc-1H-pyrazole-1-carboxamidine as the guanidination reagent, affording the desired product 32 (Scheme 2).

Next, analogues were synthesized from 12 with the acyl group on the α-amine of D-Glu transposed to the α-carboxylic acid group as either the undecanoyl ester (35), the 1-aminoundecane-derived amide (37), as well as the N-acyl long-chain ester 41 (Scheme 3). The N-guanidino ester analogue 43, as well as the α-D-Glu ethyl ester derivative of C₁₂-iE-DAP (45) were also obtained from 12 (Scheme 3). The α-D-Glu thioester DAP ethyl ester analogue (48) and DAP ethyl ester derivative of C₁₂-iE-DAP (50) were synthesized from synthon 13 (Scheme 4).

Scheme 3.

Reagents: i. (a) 30% Piperidine, CH₂Cl₂ (b) (Boc)₂O, TEA, MeOH; ii. (a) Chlorotris (triphenylphosphine) rhodium(I), EtOH, H₂O, reflux (b) C₁₁H₂₃OH, HBTU, DMAP, TEA, DMF; iii. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH; iv. (a) Chlorotris(triphenylphosphine) rhodium(I), EtOH, H₂O, reflux (b) C₁₁H₂₃NH₂, HBTU, TEA, DMAP, CH₂Cl₂; v. (a) Chlorotris (triphenylphosphine) rhodium(I), EtOH, H₂O, reflux (b) ROH, HBTU, DMAP, DMF; vi. (a) 30% Piperidine, CH₂Cl₂ (b) C₁₁H₂₃COCl, pyridine, DMAP; vii. (a) 30% Piperidine, CH₂Cl₂ (b) 1H-Pyrazole-1- carboxamidine•HCl, pyridine, MW irradiation, 60°C.

In order to examine the importance of the α-amino and carboxyl groups of D-Glu, γ-aminobutyric acid (des-carboxyl, 53) or glutaric acid (des-amino, 56) were coupled to the DAP derivative 9 as depicted in Scheme 5. Similarly, suitably protected mono-aminopimelic acid 58, D- and L-lysine derivatives (62–65; Scheme 6), and commercially available N-Boc-cadaverine and D, L-α-amino-ε-caprolactam were used to examine the roles of the amine and carboxyl groups of DAP (Scheme 7).

Scheme 5.

Reagents: i. Fmoc-GABA-OH, PS-Carbodiimide, PS-DMAP, CH₂Cl₂; ii. (a) 30% Piperidine, CH₂Cl₂ (b) C₁₁H₂₃COCl, pyridine; iii. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH; iv. Glutaric anhydride, CH₂Cl₂; v. C₁₁H₂₃OH, HBTU, DMAP, TEA, DMF.

Scheme 6.

Reagents: i. BnOH, p-TSA, reflux; ii. (a) BnOH, HBTU, DMAP, DMF (b) 30% Piperidine, CH₂Cl₂; iii. (a) PhCH₂OCOCl, Na₂CO₃, Dioxane, H₂O (b) CF₃COOH; iv. (a) (Boc)₂O, TEA, MeOH (b) H₂, Pd(OH)₂/C, MeOH, 60 psi.

Scheme 7.

Reagents: i. PS-Carbodiimide, PS-DMAP, TEA, CH₂Cl₂; ii. 30% Piperidine, CH₂Cl₂; iii. C₁₁H₂₃COCl, pyridine; iv. H₂, Pd(OH)₂/C, MeOH, 60 psi; v. CF₃COOH.

The N-acyl on the D-Glu residue appears to be optimal for Nod1-agonistic activity since the transposition of the acyl group to the N′ position on DAP (18), or diacylation at N and N′ (20) resulted in substantial decrease in activity (Table 1). The substitution of the N-acyl (C₁₂) group with N-alkyl (22) led to virtually identical activity (27 pM versus 23 pM, Table 1). Compounds with C₈ or C₁₆ N,N-dialkyl groups (26, 28) displayed attenuated potency while the C₁₂ N,N-dialkyl compound 27 displayed a rather dramatic increase in potency, with residual partial activity evident even at very high dilutions (Table 1, 27Fig. 1). Compound was the most potent analogue identified in this study. The primary amines on both the DAP and D-Glu segments were found to be crucial; the guanidino analogues 32 and 43, as well as the aminoimidazolone analogue 30 exhibited diminished activity.

Table 1.

EC₅₀ values of hNod1 agonistic activities of analogues

Cmpd	Structure	EC50 (nM)
16		0.027
18		0.55
20		0.58
22		0.023
26		26: 0.20
28		28: 0.12
27		0.0015
30		1.31
32		0.622
35		0.066
37		0.826
41		0.134
43		0.238
45		0.069
48		0.181
50		0.145
53		0.504
56		0.39
67		>100
69		>100
71		1.73
73		>100
75		0.37
77		0.57
79		0.96

We next examined the importance of the free α-carboxyl group on D-Glu. The potencies of the undecanoyl ester of iE-DAP (35), as well as the ethyl ester of C₁₂-iE-DAP (45) were comparable to that of the reference compound, C₁₂-iE-DAP (16). The undecanoyl ester of C₁₂-iE-DAP (41), and the thioester analogue 48 also retained modest activity. The 1-aminoundecane-derived amide analogue (37), however, was substantially less active. Augmented activity in 35 and 45 appears to be related to neither net charge nor bulk hydrophobicity, both of which could be reasonably expected to modulate transmembrane permeation, and consequent presentation of the ligand to the intracellular Nod1 receptor. The net charge at physiological pH of 35 and 37 is zero, whilst that of 41 and 45 is −1. We surmised that the higher potency of 45 relative to 41 could be due to the more facile hydrolytic cleavage by intracellular esterases of the shorter, ethyl ester of 45, but our observation that the rate and kinetics of NF-κB induction for both compounds were very similar (data not shown) is not in agreement with our conjecture, and we do not yet understand the basis for the difference in potencies of these analogues. Substitution of the N-acyl D-Glu fragment with either N-acyl γ-aminobutyric acid (53, des-α-carboxyl analogue), or the undecanoyl ester of glutaric acid (56, des-α-amino analogue) resulted only in considerable loss in Nod1-agonistic activity. Taken together, the above SAR data suggest that considerable ‘plasticity’ exists in the recognition of the D-Glu fragment. Indeed, a plot of the potencies of these analogues displays an unexpectedly diffuse, near-uniform distribution of EC₅₀ values (Fig. 1B), unlike the well-demarcated and discrete SAR that we had previously observed for TLR2-¹³ and TLR7-active¹² compounds.

In contradistinction to the broad and diffuse SAR for D-Glu fragment, the DAP portion of the molecule is far more stringent in determining Nod1 agonism. Whereas esterification of carboxyl groups of DAP (50) is tolerated, substitution with mono-aminopimelic acid (67, des-amino) or cadaverine (69, des-dicarboxyl) resulted in abrogation of activity. The meso-diaminopimelic acid-containing peptidoglycan is specific for Gram-negative bacteria, and the corresponding homologue in Gram-positive organisms is L-lysine coupled via its α-amine to γ-D-glutamyl residue (77), the activity of which is ~10-fold less than that of 16.^34;35 The D-lysine analogue 73 is bereft of any activity. The unnatural ε-amine-coupled lysine analogues were also evaluated. The D-lysine analogue 75 was found to be four-fold more active than 79, its L-lysine congener.

Primary assay readouts using cell-culture systems may not always accurately reflect in vivo behavior owing to a variety of reasons, including differential plasma protein binding (that we ourselves have observed and characterized),³⁶ and we therefore wished to confirm that the 27 was indeed more potent than 16, the reference Nod1 agonist, in ex vivo assays employing whole human blood. Although Nod1 agonists have previously been shown to enhance neutrophil recruitment in mice,³⁴ a recent report employing synthetic, non-acylated, γ-glutamyl-meso-diaminopimelic acid dipeptide indicated that Nod1-specific responses could not be elicited in isolated human neutrophils.³⁷ Using an ex vivo system using fresh, whole human blood that we have established for immunoprofiling innate immune stimuli,^10;13;38 both p38 MAPK³⁹ and CD11b⁴⁰ were upregulated in a dose-dependent manner, with 27 being more potent than 16 (Fig. 2). Indeed, the non-acylated, γ-glutamyl-meso-diaminopimelic acid dipeptide was inactive in our primary and secondary screens (data not shown), emphasizing the requirement of an N-acyl functionality on glutamic acid.

Figure 2.

Dose-responses of p38 MAP kinase (A) and CD11b (B) induction in the granulocytic population in whole human blood by 16 and 27 determined by flow cytometry.

Recent findings using murine models of immunization indicate that Nod1 engagement drives B and T cell immunity with a predominant Th2 polarization, and additional Th17 priming.³² However, mechanisms underlying the induction of strong Th2-biased immune responses have remained obscure. Given the significant differences between species-dependent recognition of innate immune ligands,¹¹ we desired to evaluate whether surrogate markers of Th2-skewed responses could be observed in human ex vivo model systems, in the hope that these studies may also help uncover signaling events determining Th2 polarization. Transcriptomal profiling experiments using human blood revealed a very prominent upregulation of Class II helical cytokines by 27, especially members of the IL-10 superfamily⁴¹ including IL-19, IL-20, IL-22, and IL-24 (Table 2); these cytokines, especially IL-19,^42–45 have been shown to drive Th2-polarized responses. We have never before observed this particular constellation of cytokines in our earlier¹⁰ (and ongoing) immunoprofiling studies, and we are excited in no small measure that quantifying the induction of IL-19 and related cytokines may have predictive value as biomarkers and surrogate signatures for Th2 induction in immunoprofiling vaccine adjuvants, and may perhaps supplant the necessity of long and laborious immunization experiments which typically involve large numbers of animals. Also observed, as could be expected in the context of Th17-induction,³² was the upregulation of IL-17. Similar responses were observed with 16, but of lower magnitude, consistent with the lower potency of 16 (data not shown).

Table 2.

Interleukin Transcriptomal Responses in Human PBMCs exposed to 20 μg/mL of 27

Probe Set ID	Fold change	Regulation	Gene Symbol	Gene Title
220745_at	30.39	up	IL19	interleukin 19
205207_at	29.54	up	IL6	interleukin 6 (interferon, beta 2)
210118_s_at	14.34	up	IL1A	interleukin 1, alpha
220322_at	11.10	up	IL1F9	interleukin 1 family, member 9
216876_s_at	10.56	up	IL17A	interleukin 17A
216244_at	8.27	up	IL1RN	interleukin 1 receptor antagonist
212659_s_at	8.19	up	IL1RN	interleukin 1 receptor antagonist
216243_s_at	7.54	up	IL1RN	interleukin 1 receptor antagonist
207901_at	6.13	up	IL12B	interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40)
207906_at	5.99	up	IL3	interleukin 3 (colony-stimulating factor, multiple)
212657_s_at	4.94	up	IL1RN	interleukin 1 receptor antagonist
39402_at	4.45	up	IL1B	interleukin 1, beta
205067_at	4.14	up	IL1B	interleukin 1, beta
224071_at	4.00	up	IL20	interleukin 20
221165_s_at	3.87	up	IL22	interleukin 22
207433_at	3.62	up	IL10	interleukin 10
211269_s_at	3.62	up	IL2RA	interleukin 2 receptor, alpha
206341_at	3.57	up	IL2RA	interleukin 2 receptor, alpha
206569_at	3.41	up	IL24	interleukin 24
227997_at	3.17	up	IL17RD	interleukin 17 receptor D
222223_s_at	3.15	up	IL1F5	interleukin 1 family, member 5 (delta)
211506_s_at	2.29	up	IL8	interleukin 8
224262_at	2.05	up	IL1F10	interleukin 1 family, member 10 (theta)
206295_at	2.03	up	IL18	interleukin 18 (interferon-gammainducing factor)
1555431_a_at	4.05	down	IL31RA	interleukin 31 receptor A
220056_at	3.69	down	IL22RA1	interleukin 22 receptor, alpha 1
207008_at	2.75	down	IL8RB	interleukin 8 receptor, beta
220663_at	2.06	down	IL1RAPL1	interleukin 1 receptor accessory proteinlike 1
205227_at	2.02	down	IL1RAP	interleukin 1 receptor accessory protein

Further analyses of gene transcripts induced by 27 yielded the unexpected finding that exposure of human blood to 27 results in the activation of pathways involving signaling mediated by Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1; Fig. 3). TREM-1 is a recently-discovered receptor expressed on the cell surface of monocytes and neutrophils, and plays an important role in amplifying myeloid cell-activated inflammatory responses.^46–48 Our observation of the upregulation of TREM-1 signaling components may explain the hitherto unknown mechanism of synergy between Nod1- and TLRagonists. ^32;34;49;50

Figure 3.

Pathway analysis of transcriptomal activation patterns in human PBMCs exposed to 20 μg/mL of 16 and 27. The dashed line corresponds to the threshold of −log (base 10) value for P = 0.05 (after Benjamini-Hochberg multiple testing correction).

The overarching objective that led to our work on Nod1 ligands was not so much to synthesize higher potency compounds, but rather to understand SAR that would permit us to covalently couple other innate immune ligands^12;13;33 to the iE-DAP scaffold in order to test the hypothesis that such hybrids may imbue a Th1-Th2 balanced, yet effective vaccine adjuvants. The studies reported here indicate that various substitutions and modifications on the γ-D-glutamyl moiety are tolerated whereas the diaminopimelic acid fragment is far more stringent. The α-carboxylic acid of D-Glu appears to be optimal for coupling with other innate immune ligands, and work in this area is in progress. Secondary and tertiary screens, including transcriptomal profiling have shed light on the possible mechanisms underlying the strong Th2 bias of Nod1 ligands, and of synergy with other innate immune stimuli.

Experimental Section

Chemistry

All of the solvents and reagents used were obtained commercially and used as such unless noted otherwise. Moisture- or air-sensitive reactions were conducted under nitrogen atmosphere in oven-dried (120 °C) glass apparatus. The solvents were removed under reduced pressure using standard rotary evaporators. Flash column chromatography was carried out using RediSep Rf ‘Gold’ high performance silica columns on CombiFlash Rf (Teledyne-Isco, Lincoln, NE) instrument unless otherwise mentioned, while thin-layer chromatography was carried out on silica gel CCM pre-coated aluminum sheets. Purity for all final compounds was confirmed to be at least 97% by LC-MS using a Zorbax Eclipse Plus 4.6 mm x 150 mm, 5 μm analytical reverse phase C₁₈ column with H₂O-isopropanol or H₂O-CH₃CN gradients (with 0.1% CF₃COOH in both mobile phases) and an Agilent ESI-TOF mass spectrometer (mass accuracy of 5 ppm) operating in the positive ion (or negative ion, as appropriate) acquisition mode.

Synthesis of Compound 2: (R)-1-Benzyl 5-tert-butyl 2-((tert-butoxycarbonyl)amino) pentanedioate

To a solution of 1 (1 g, 3.29 mmol) in anhydrous DMF, were added HBTU (1.38 g, 3.62 mmol), triethylamine (0.5 mL, 3.62 mmol), benzyl alcohol (682 μL, 6.59 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in ethyl acetate and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (10% EtOAc/hexanes) to obtain the compound 2 (1.14 g, 88 %). ¹H NMR (500 MHz, CDCl₃) δ 7.39 – 7.31 (m, 5H), 5.16 (dt, J = 23.2, 11.5 Hz, 3H), 4.35 (dd, J = 13.0, 8.2 Hz, 1H), 2.35 – 2.22 (m, 2H), 2.13 (td, J = 13.3, 7.0 Hz, 1H), 1.92 (tt, J = 14.7, 7.5 Hz, 1H), 1.43 (s, 18H). ¹³C NMR (126 MHz, CDCl₃) δ 172.09, 172.06, 155.39, 131.57, 118.82, 80.74, 79.93, 65.95, 53.11, 31.60, 28.30, 28.06, 27.89, 27.68. MS (ESI) calculated for C₂₁H₃₁NO₆, m/z 393.22, found 416.22 (M + Na)⁺.

Synthesis of Compound 3: (R)-1-Allyl 5-tert-butyl 2-((tert-butoxycarbonyl)amino) pentanedioate

To a solution of 1 (1 g, 3.29 mmol) in anhydrous DMF, were added HBTU (1.38 g, 3.62 mmol), triethylamine (0.5 mL, 3.62 mmol), allyl alcohol (450 μL, 6.59 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in ethyl acetate and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (10% EtOAc/hexanes) to obtain the compound 3 (1.0 g, 90 %). ¹H NMR (500 MHz, CDCl₃) δ 5.91 (ddt, J = 16.3, 10.6, 5.8 Hz, 1H), 5.34 (dd, J = 17.2, 1.3 Hz, 1H), 5.26 (dd, J = 10.4, 0.7 Hz, 1H), 5.11 (d, J = 8.0 Hz, 1H), 4.64 - 4.63 (m, 2H), 4.33 (dd, J = 13.2, 8.3 Hz, 1H), 2.38 - 2.26 (m, 2H), 2.17-2.11 (m, 1H), 1.96-1.88 (m, 1H), 1.45 (s, 9H), 1.44 (s, 9 H). ¹³C NMR (126 MHz, CDCl₃) δ 172.05, 172.02, 155.35, 131.53, 118.78, 80.70, 79.89, 65.91, 53.07, 31.56, 28.26, 28.02, 27.85, 27.64. MS (ESI) calculated for C₁₇H₂₉NO₆, m/z 343.20, found 366.19 (M + Na)⁺.

Synthesis of Compound 4: (R)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzyloxy)- 5-oxopentanoic acid

The solution of 2 (1 g, 2.54 mmol) in trifluoroacetic acid was stirred for 1 hour, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt. To the solution of trifluoroacetate salt in dioxane, the aqueous solution of sodium carbonate (810 mg, 7.64 mmol) was added at 10 °C. The reaction mixture was stirred at room temperature for 10 min, followed by the addition of FmocCl (722 mg, 2.79 mmol) solution in dioxane. The reaction mixture was stirred at room temperature for 16 hrs, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethyl acetate and water, and the solution was acidified using 10% HCl until pH was ~1. The aqueous layer was washed with ethyl acetate. The ethyl acetate was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (40% EtOAc/hexanes) to obtain the compound 4 (1.0 g, 91 %). ¹H NMR (500 MHz, CDCl₃) δ 7.73 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.4 Hz, 2H), 7.33 (qd, J = 15.0, 7.2 Hz, 9H), 5.47 (d, J = 8.2 Hz, 1H), 5.19 – 5.06 (m, 2H), 4.53 – 4.30 (m, 3H), 4.16 (dd, J = 15.6, 8.8 Hz, 1H), 2.48 – 2.12 (m, 3H), 1.94 (dt, J = 14.5, 7.7 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 177.59, 171.91, 156.27, 143.99, 143.84, 141.50, 141.49, 135.19, 128.88, 128.81, 128.56, 127.95, 127.30, 125.29, 125.26, 120.21, 120.20, 67.73, 67.34, 53.42, 47.32, 29.91, 27.64. MS (ESI) calculated for C₂₇H₂₅NO₆, m/z 459.16, found 482.17 (M + Na)⁺.

Synthesis of Compound 5: (R)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5- oxopentanoic acid

The solution of compound 3 (1 g, 2.91 mmol) in trifluoroacetic acid was stirred for 1 hour, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt. To the solution of trifluoroacetate salt in dioxane, the aqueous solution of sodium carbonate (926 mg, 8.74 mmol) was added at 10 °C. The reaction mixture was stirred at room temperature for 10 min, followed by the addition of FmocCl (830 mg, 3.2 mmol) solution in dioxane. The reaction mixture was stirred at room temperature for 16 hours, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethyl acetate and water, and the solution was acidified using 10% HCl until pH was ~1. The aqueous layer was washed with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (40% EtOAc/hexanes) to obtain the compound 5 (1.1 g, 92 %). ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.1 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 5.90 (ddd, J = 22.5, 11.0, 5.7 Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.30 (dd, J = 27.7, 13.7 Hz, 2H), 4.65 (d, J = 5.5 Hz, 2H), 4.58 – 4.35 (m, 3H), 4.21 (t, J = 6.8 Hz, 1H), 2.58 – 2.17 (m, 3H), 2.07 – 1.92 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 172.70, 172.54, 155.72, 155.57, 135.49, 128.74, 128.57, 128.43, 80.07, 67.19, 67.14, 53.27, 32.24, 28.47, 28.45, 21.48, 21.19, 21.08. MS (ESI) calculated for C₂₃H₂₃NO₆, m/z 409.15, found 432.14 (M + Na)⁺.

Synthesis of Compound 7: Dibenzyl 2,6-bis((tert-butoxycarbonyl)amino)heptanedioate

To a solution of compound 6 (1 g, 5.25 mmol) in water, were added di-tert-butyl dicarbonate (4.58 g, 21.0 mmol) and triethylamine (2.92 mL, 21.0 mmol). The reaction mixture was stirred for 6 hours, followed by removal of the solvent under reduced pressure to obtain the residue. To the solution of residue in anhydrous DMF, were added HBTU (4.38 g, 11.55 mmol), triethylamine (1.6 mL, 11.55 mmol), benzyl alcohol (1.63 mL, 15.75 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 8 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in ethyl acetate and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (8% EtOAc/hexanes) to obtain the compound 7 (2.48 g, 83%). ¹H NMR (500 MHz, CDCl₃) δ 7.44 – 7.29 (m, 10H), 5.13 (dt, J = 12.4, 8.3 Hz, 6H), 4.29 (s, 2H), 1.85 – 1.71 (m, 2H), 1.68 (s, 2H), 1.49 – 1.31 (m, 20H). ¹³C NMR (126 MHz, CDCl₃) δ 172.70, 172.54, 155.72, 155.57, 135.49, 128.74, 128.57, 128.43, 80.07, 67.19, 67.14, 53.27, 32.24, 28.47, 28.45, 21.48, 21.19, 21.08. MS (ESI) calculated for C₃₁H₄₂N₂O₈, m/z 570.29, found 593.27 (M + Na)⁺.

Synthesis of Compound 8: Diethyl 2,6-bis((tert-butoxycarbonyl)amino)heptanedioate

To a solution of compound 6 (1 g, 5.25 mmol) in water, were added di-tert-butyl dicarbonate (4.58 g, 21.0 mmol) and triethylamine (2.92 mL, 21.0 mmol). The reaction mixture was stirred for 6 hours, followed by removal of the solvent under reduced pressure to obtain the residue. To the solution of residue in anhydrous DMF, were added HBTU (4.38 g, 11.55 mmol), triethylamine (1.6 mL, 11.55 mmol), ethyl alcohol (0.92 mL, 15.75 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 6 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in ethyl acetate and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (8% EtOAc/hexanes) to obtain the compound 8 (2.0 g, 85%). ¹H NMR (500 MHz, CDCl₃) δ 5.17 – 4.99 (m, 2H), 4.30 – 4.08 (m, 6H), 1.84 – 1.74 (m, 2H), 1.72 – 1.55 (m, 4H), 1.48 – 1.31 (m, 18H), 1.25 (t, J = 7.1 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 173.24, 173.06, 156.07, 155.92, 80.33, 68.61, 61.80, 53.58, 39.17, 32.74, 32.67, 30.80, 29.38, 28.81, 28.79, 28.71, 24.19, 23.45, 21.82, 21.47, 14.64, 14.52. MS (ESI) calculated for C₂₁H₃₈N₂O₈, m/z 446.26, found 469.26 (M + Na)⁺.

Synthesis of Compound 9: Dibenzyl 2-amino-6-((tert-butoxycarbonyl)amino)heptanedioate

The compound 7 (2 g, 3.5 mmol) was dissolved in 10 mL of HCl-dioxane and stirred for 2 hours. The solvent was then removed under vacuum to obtain the hydrochloride salt, which was then dissolved in methanol. The triethylamine was added to solution until pH ~ 7, followed by gradual addition of di-tert-butyl dicarbonate (764 mg, 3.5 mmol). The reaction mixture was stirred for 30 minutes, followed by evaporation of the solvent under reduced pressure to obtain the crude product, which was then purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain the compound 9 (640 mg, 39%). ¹H NMR (500 MHz, CDCl₃) δ 7.45 – 7.28 (m, 10H), 5.23 – 5.09 (m, 4H), 5.03 (t, J = 7.3 Hz, 1H), 4.41 – 4.23 (m, 1H), 3.41 (td, J = 8.1, 5.2 Hz, 1H), 1.85 – 1.50 (m, 6H), 1.49 – 1.34 (m, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 176.17, 176.14, 173.01, 155.83, 136.10, 135.84, 129.25, 129.09, 129.06, 128.88, 128.78, 80.36, 67.46, 67.16, 67.14, 54.70, 54.68, 53.80, 53.74, 34.73, 34.66, 32.77, 32.74, 28.77, 21.93. MS (ESI) calculated for C₂₆H₃₄N₂O₆, m/z 470.24, found 471.24 (M + H)⁺.

Synthesis of Compound 10: Diethyl 2-amino-6-((tert-butoxycarbonyl)amino)heptanedioate

The compound 8 (1 g, 2.24 mmol) was dissolved in 5 mL of HCl-dioxane and stirred for 2.5 hours. The solvent was then removed under vacuum to obtain the hydrochloride salt, which was then dissolved in methanol. The triethylamine was added to solution until pH ~ 7, followed by gradual addition of di-tert-butyl dicarbonate (490 mg, 2.24 mmol). The reaction mixture was stirred for 30 minutes, followed by evaporation of the solvent under reduced pressure to obtain the crude product, which was then purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain the compound 10 (271 mg, 35%). ¹H NMR (500 MHz, CDCl₃) δ 5.31 (dd, J = 31.4, 7.9 Hz, 1H), 4.28 – 4.16 (m, 5H), 3.96 (bs, 1H), 2.04 – 1.88 (m, 2H), 1.88 – 1.76 (m, 1H), 1.73 – 1.62 (m, 1H), 1.62 – 1.43 (m, 2H), 1.42 (s, 9H), 1.32 – 1.24 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 176.13, 176.08, 172.94, 155.59, 80.00, 61.50, 61.08, 54.40, 53.50, 53.44, 34.56, 34.53, 32.65, 32.62, 28.50, 21.73, 21.67, 14.42, 14.37. MS (ESI) calculated for C₁₆H₃₀N₂O₆, m/z 346.21, found 347.21 (M + H)⁺.

Synthesis of Compound 11: (5R)-Tribenzyl 1-(9H-fluoren-9-yl)-18,18-dimethyl-3,8,16-trioxo- 2,17-dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate

To a solution of 4 (500 mg, 1.08 mmol) in anhydrous dichloromethane, were added 9 (522 mg, 1.11 mmol), polystyrene bound carbodiimide (967 mg, 1.19 mmol) and a catalytic amount of polystyrene bound DMAP. The reaction mixture was stirred at room temperature for 4 hours, followed by filtration to remove the solid resin. The filtrate was evaporated under vacuum to obtain the residue which was then purified using column chromatography (35% EtOAc/hexanes) to obtain the compound 11 (748 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.3 Hz, 2H), 7.39 – 7.30 (m, 19H), 6.59 – 6.49 (m, 0.50H), 6.28 (s, 0.50H), 5.71 (dd, J = 25.1, 7.7 Hz, 1H), 5.24 – 5.04 (m, 7H), 4.56 (d, J = 6.6 Hz, 1H), 4.50 – 4.33 (m, 3H), 4.33 – 4.15 (m, 2H), 2.31 – 2.16 (m, 2H), 2.03 – 1.90 (m, 2H), 1.85 – 1.75 (m, 2H), 1.70 – 1.51 (m, 2H), 1.40 – 1.37 (m, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 172.65, 172.57, 172.16, 172.09, 171.97, 171.82, 156.53, 155.78, 144.10, 143.85, 141.51, 141.48, 135.55, 135.36, 128.85, 128.83, 128.81, 128.73, 128.64, 128.60, 128.53, 128.45, 127.92, 127.30, 125.36, 120.18, 80.12, 67.59, 67.41, 67.33, 67.24, 53.63, 53.53, 53.33, 52.21, 47.37, 47.34, 32.32, 32.22, 31.73, 28.93, 28.51, 21.54, 21.43, 21.28, 21.13. MS (ESI) calculated for C₅₃H₅₇N₃O₁₁, m/z 911.40, found 934.39 (M + Na)⁺.

Synthesis of Compound 12: (5R)-5-Allyl 10,14-dibenzyl 1-(9H-fluoren-9-yl)-18,18-dimethyl- 3,8,16-trioxo-2,17-dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate

To a solution of 5 (500 mg, 1.22 mmol) in anhydrous dichloromethane, were added 9 (585 mg, 1.24 mmol), polystyrene bound carbodiimide (1.09 g, 1.34 mmol) and a catalytic amount of polystyrene bound DMAP. The reaction mixture was stirred at room temperature for 4 hours, followed by filtration to remove the solid resin. The filtrate was evaporated under reduced pressure to obtain the residue which was then purified using column chromatography (35% EtOAc/hexanes) to obtain the compound 12 (830 mg, 79%). ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 6.6 Hz, 2H), 7.46 – 7.30 (m, 14H), 6.69 – 6.30 (m, 1H), 5.91 (dd, J = 19.3, 12.8 Hz, 1H), 5.72 (dd, J = 20.3, 7.6 Hz, 1H), 5.35 (d, J = 17.2 Hz, 1H), 5.27 (d, J = 10.4 Hz, 1H), 5.22 – 5.04 (m, 5H), 4.70 – 4.56 (m, 3H), 4.52 – 4.36 (m, 3H), 4.35 – 4.19 (m, 2H), 2.38 – 2.20 (m, 3H), 1.98 (dd, J = 19.2, 8.3 Hz, 1H), 1.91 – 1.56 (m, 4H), 1.44 (s, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 172.66, 172.56, 172.20, 172.12, 171.99, 171.85, 156.56, 155.83, 144.09, 143.86, 141.51, 135.54, 135.45, 131.59, 128.83, 128.71, 128.64, 128.51, 127.94, 127.31, 125.35, 120.19, 119.40, 119.36, 119.32, 80.13, 67.43, 67.33, 67.23, 66.42, 53.61, 53.53, 53.33, 52.24, 47.37, 32.41, 32.34, 31.84, 31.75, 31.61, 29.00, 28.51, 21.46, 21.27, 21.14. MS (ESI) calculated for C₄₉H₅₅N₃O₁₁, m/z 861.38, found 884.33 (M + Na)⁺.

Synthesis of Compound 13: (5R)-5-Benzyl 10,14-diethyl 1-(9H-fluoren-9-yl)-18,18-dimethyl- 3,8,16-trioxo-2,17-dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate

To a solution of 4 (195 mg, 0.425 mmol) in anhydrous dichloromethane, were added 10 (150 mg, 0.43 mmol), polystyrene bound carbodiimide (367 mg, 0.467 mmol) and a catalytic amount of polystyrene bound DMAP. The reaction mixture was stirred at room temperature for 4 hours, followed by filtration to remove the solid resin. The filtrate was evaporated under reduced pressure to obtain the residue which was then purified using column chromatography (30% EtOAc/hexanes) to obtain the compound 13 (232 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ 7.79 – 7.69 (m, 2H), 7.59 (dd, J = 21.4, 11.5 Hz, 2H), 7.55 – 7.26 (m, 9H), 6.55 (dd, J = 27.8, 7.3 Hz, 1H), 6.31 (dd, J = 18.6, 7.1 Hz, 1H), 5.75 (dd, J = 27.6, 7.7 Hz, 1H), 5.27 – 5.05 (m, 3H), 4.61 – 4.33 (m, 4H), 4.28 – 4.04 (m, 6H), 2.31 – 2.20 (m, 2H), 2.04 – 1.93 (m, 1H), 1.89 – 1.52 (m, 5H), 1.46 – 1.36 (m, 9H), 1.34 – 1.21 (m, 7H). ¹³C NMR (126 MHz, CDCl₃) δ 156.50, 144.11, 143.88, 141.52, 135.38, 131.11, 129.02, 128.87, 128.76, 128.66, 128.59, 127.93, 127.31, 125.37, 120.19, 80.07, 68.37, 67.61, 67.29, 61.75, 61.57, 53.29, 52.13, 47.36, 38.92, 32.48, 32.37, 31.74, 30.56, 29.13, 28.53, 23.94, 23.20, 21.44, 14.37, 14.28, 11.12. MS (ESI) calculated for C₄₃H₅₃N₃O₁₁, m/z 787.37, found 810.37 (M + Na)^+.

Synthesis of Compound 14: Dibenzyl 2-((R)-4-amino-5-(benzyloxy)-5-oxopentanamido)-6- ((tert-butoxycarbonyl)amino)heptanedioate

The compound 11 (500 mg, 0.55 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure. The crude was purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain the compound 14 (347 mg, 92%). ¹H NMR (400 MHz, CDCl₃) δ 7.46 – 7.31 (m, 15H), 6.57 (dt, J = 36.1, 8.8 Hz, 1H), 5.25 – 5.05 (m, 7H), 4.59 (d, J = 5.6 Hz, 1H), 4.28 (s, 1H), 3.52 (dd, J = 8.2, 5.1 Hz, 1H), 2.36 (qt, J = 21.1, 6.8 Hz, 2H), 2.13 (dd, J = 12.5, 6.0 Hz, 1H), 1.93 – 1.50 (m, 7H), 1.49 – 1.32 (m, 11H). ¹³C NMR (101 MHz, CDCl₃) δ 176.03, 172.79, 172.67, 136.10, 135.85, 129.15, 129.12, 129.01, 128.94, 128.85, 128.76, 67.53, 67.32, 54.38, 54.01, 53.70, 52.40, 33.12, 33.00, 32.17, 30.48, 30.31, 28.82, 21.78, 21.59. MS (ESI) calculated for C₃₈H₄₇N₃O₉, m/z 689.33, found 690.33 (M + H)^+.

Synthesis of Compound 15: (15R)-Tribenzyl 2,2-dimethyl-4,12,17-trioxo-3-oxa-5,11,16- triazaoctacosane-6,10,15-tricarboxylate

To a solution of 14 (200 mg, 0.29 mmol) in pyridine, were added lauroyl chloride (82.6 μL, 0.34 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 6 hours, followed by evaporation of the solvent under reduced pressure to obtain the residue, which was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (50% EtOAc/hexanes) to obtain the compound 15 (209 mg, 83%). ¹H NMR (500 MHz, CDCl₃) δ 7.42 – 7.27 (m, 15H), 6.98 (dd, J = 16.0, 7.8 Hz, 0.50H), 6.57 – 6.46 (m, 1.50H), 5.22 – 5.07 (m, 7H), 4.76 – 4.70 (m, 0.50H), 4.61 – 4.51 (m, 1.50H), 4.31 – 4.20 (m, 1H), 2.28 – 2.16 (m, 5H), 1.98 – 1.57 (m, 11H), 1.50 – 1.36 (m, 6H), 1.32 – 1.20 (m, 17H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.68, 172.61, 172.46, 172.38, 172.33, 172.24, 172.20, 172.12, 172.04, 135.57, 135.45, 135.40, 128.85, 128.84, 128.82, 128.71, 128.65, 128.62, 128.58, 128.53, 128.48, 128.47, 80.10, 67.53, 67.23, 53.39, 52.29, 51.86, 36.83, 36.77, 32.53, 32.40, 32.12, 31.58, 29.84, 29.82, 29.70, 29.56, 29.51, 29.18, 28.54, 25.86, 25.79, 22.90, 14.34. MS (ESI) calculated for C₅₀H₆₉N₃O₁₀, m/z 871.50, found 894.50 (M + Na)⁺.

Synthesis of Compound 16: 2-Amino-6-((R)-4-carboxy-4-dodecanamidobutanamido) heptanedioic acid

G3The compound 15 (50 mg, 57.3 μmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 16 (35 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.46 – 4.30 (m, 2H), 3.78 – 3.68 (m, 1H), 2.35 (t, J = 5.6 Hz, 2H), 2.27 – 2.10 (m, 3H), 1.88 (ddd, J = 24.9, 20.5, 16.8 Hz, 4H), 1.75 – 1.66 (m, 1H), 1.65 – 1.47 (m, 4H), 1.36 – 1.21 (m, 16H), 0.87 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.64, 175.50, 175.02, 173.29, 55.13, 54.97, 53.42, 53.16, 37.09, 36.99, 33.25, 33.04, 32.52, 32.45, 32.38, 31.64, 31.53, 31.47, 30.92, 30.82, 30.65, 30.50, 28.82, 28.58, 28.30, 27.14, 27.11, 23.89, 23.06, 22.71, 22.63, 14.59. MS (ESI) calculated for C₂₄H₄₃N₃O₈, m/z 501.31, found 502.33 (M + H)⁺.

Synthesis of Compound 17: (5R)-Tribenzyl 1-(9H-fluoren-9-yl)-3,8,16-trioxo-2-oxa-4,9,15- triazaheptacosane-5,10,14-tricarboxylate

The compound 11 (100 mg, 0.10 mmol) was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt. To the solution of trifluoroacetate salt in anhydrous dichloromethane, were added lauroyl chloride (39 μL, 0.16 mmol), triethylamine (30 μL, 0.22 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 2 hours, followed by complete removal of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (40% EtOAc/hexanes) to obtain the compound 17 (103 mg, 95%). ¹H NMR (400 MHz, CDCl₃) δ 7.83 – 7.70 (m, 2H), 7.68 – 7.52 (m, 2H), 7.46 – 7.27 (m, 19H), 6.27 – 6.06 (m, 1H), 5.93 – 5.74 (m, 1H), 5.30 – 5.04 (m, 6H), 4.67 – 4.28 (m, 5H), 4.28 – 4.18 (m, 1H), 2.33 – 2.26 (m, 2H), 2.22 – 2.13 (m, 2H), 2.01 (dd, J = 15.1, 7.4 Hz, 1H), 1.92 – 1.79 (m, 2H), 1.79 – 1.66 (m, 3H), 1.64 – 1.56 (m, 2H), 1.48 – 1.17 (m, 19H), 0.90 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.48, 172.36, 172.06, 172.02, 156.61, 143.74, 141.50, 135.39, 128.84, 128.70, 128.47, 127.92, 127.30, 125.35, 120.17, 67.55, 67.41, 67.31, 53.83, 51.87, 51.64, 47.35, 36.69, 32.19, 32.11, 31.62, 29.83, 29.69, 29.54, 29.46, 25.81, 22.88, 21.67, 14.32. MS (ESI) calculated for C₆₀H₇₁N₃O₁₀, m/z 993.51, found 1016.53 (M + Na)⁺.

Synthesis of Compound 18: 2-((R)-4-Amino-4-carboxybutanamido)-6-dodecanamido) heptanedioic acid

The compound 17 (70 mg, 0.07 mmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (50% MeOH/CH₂Cl₂) to obtain the compound 18 (32 mg, 92%). ¹H NMR (500 MHz, MeOD) δ 4.34 (s, 2H), 3.78 – 3.69 (m, 2H), 2.50 (t, J = 17.3 Hz, 2H), 2.23 (t, J = 7.4 Hz, 2H), 2.17 – 2.06 (m, 2H), 1.86 – 1.66 (m, 5H), 1.64 – 1.55 (m, 2H), 1.50 – 1.41 (d, J = 15.2 Hz, 2H), 1.39 – 1.19 (m, 14H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.56, 53.81, 36.98, 33.24, 32.21, 30.92, 30.82, 30.64, 30.47, 27.13, 25.50, 23.90, 23.51, 14.59. MS (ESI) calculated for C₂₄H₄₃N₃O₈, m/z 501.31, found 502.32 (M + H)⁺.

Synthesis of Compound 19: Dibenzyl 2-amino-6-((R)-5-(benzyloxy)-4-dodecanamido-5- oxopentanamido)heptanedioate

The compound 15 (200 mg, 0.23 mmol) was dissolved in 10 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of compound 19 (203 mg, quantitative yield). MS (ESI) calculated for C₄₅H₆₁N₃O₈, m/z 771.44, found 772.48 (M + H)⁺.

Synthesis of Compound 20: 2-((R)-4-Carboxy-4-dodecanamidobutanamido)-6-dodecanamidoheptanedioic acid

To a solution of 19 (50 mg, 56.4 μmol) in pyridine, were added lauroyl chloride (16.1 μL, 67.7 μmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 6 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product. The crude was purified using column chromatography (35% EtOAc/hexanes) to obtain the pure product, which was dissolved in methanol and subjected to catalytic hydrogenolysis using Pd(OH)₂/C at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the compound 20 (34.3 mg, 89%). ¹H NMR (500 MHz, MeOD) δ 4.45 – 4.25 (m, 3H), 2.34 (t, J = 6.7 Hz, 2H), 2.28 – 2.07 (m, 5H), 1.98 – 1.79 (m, 3H), 1.70 (dd, J = 19.9, 14.8 Hz, 2H), 1.64 – 1.55 (m, 4H), 1.49 – 1.39 (m, 3H), 1.37 – 1.15 (m, 31H), 0.87 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, MeOD) δ 176.64, 175.98, 175.72, 175.21, 53.63, 36.99, 33.35, 33.25, 32.27, 30.92, 30.83, 30.65, 30.49, 28.88, 28.73, 27.10, 23.89, 23.54, 14.59. MS (ESI) calculated for C₃₆H₆₅N₃O₉, m/z 683.47, found 682.49 (M – H)⁻.

Synthesis of Compound 21: (15R)-Tribenzyl 2,2-dimethyl-4,12-dioxo-3-oxa-5,11,16- triazaoctacosane-6,10,15-tricarboxylate

To a solution of 14 (190 mg, 0.28 mmol) in anhydrous dichloromethane (10 mL) and methanol (2 mL), were added dodecyl aldehyde (51.5 mg, 0.28 mmol), 3 – 4 drops of acetic acid, and macroporous resin bound sodium cyanoborohydride (150 mg, 0.33 mmol). The solution was stirred for 8 hours, followed by filtration to remove the resin. The solvent was removed under vacuum to obtain the residue, which was purified using column chromatography (15% EtOAc/hexanes) to obtain the compound 21 (124 mg, 51%). ¹H NMR (500 MHz, CDCl₃) δ 7.43 – 7.27 (m, 15H), 5.25 – 5.09 (m, 6H), 4.50 (dd, J = 12.7, 7.7 Hz, 1H), 4.35 – 4.16 (m, 1H), 2.73 – 2.64 (m, 1H), 2.62 – 2.13 (m, 4H), 2.02 (t, J = 20.0 Hz, 1H), 1.91 – 1.72 (m, 3H), 1.71 – 1.50 (m, 4H), 1.41 (d, J = 12.6 Hz, 7H), 1.38 – 1.17 (m, 25H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.53, 172.11, 155.94, 155.65, 155.52, 135.36, 134.56, 130.91, 128.62, 128.43, 128.29, 79.94, 68.16, 67.97, 67.06, 60.40, 59.91, 53.22, 52.10, 47.94, 32.38, 32.20, 32.04, 31.95, 31.43, 30.99, 30.37, 29.64, 29.58, 29.36, 29.09, 28.92, 28.66, 28.34, 28.12, 27.43, 27.00, 23.76, 23.00, 22.72, 21.45, 21.24, 14.16. MS (ESI) calculated for C₅₀H₇₁N₃O₉, m/z 857.52, found 858.53 (M + H)⁺.

Synthesis of Compound 22: 2-Amino-6-((R)-4-carboxy-4-(dodecylamino)butanamido) heptanedioic acid

The compound 21 (50 mg, 58.3 μmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 22 (35 mg, quantitative yield in 2 steps). ¹H NMR (500 MHz, MeOD) δ 4.40 (d, J = 3.5 Hz, 1H), 3.88 – 3.56 (m, 3H), 3.21 – 3.07 (m, 1H), 3.06 – 2.94 (m, 2H), 2.53 (t, J = 6.7 Hz, 2H), 2.14 (d, J = 6.0 Hz, 2H), 1.91 (s, 3H), 1.79 – 1.43 (m, 6H), 1.43 – 1.20 (m, 15H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 175.33, 62.44, 62.15, 54.58, 53.37, 46.97, 46.32, 45.72, 45.40, 44.90, 33.10, 32.83, 32.34, 31.97, 31.13, 30.78, 30.68, 30.52, 30.24, 27.61, 27.46, 26.59, 26.28, 23.75, 23.09, 22.55, 22.34, 14.44. MS (ESI) calculated for C₂₄H₄₅N₃O₇, m/z 487.33, found 488.34 (M + H)⁺.

Synthesis of Compound 23: (15R)-Tribenzyl 2,2-dimethyl-16-octyl-4,12-dioxo-3-oxa-5,11,16- triazatetracosane-6,10,15-tricarboxylate

To a solution of 14 (100 mg, 0.15 mmol) in anhydrous dichloromethane (10 mL) and methanol (5 mL), were added octyl aldehyde (56.5 μL, 0.36 mmol), 3 – 4 drops of acetic acid, and macroporous resin bound sodium cyanoborohydride (163.5 mg, 0.36 mmol). The reaction mixture was then stirred for 10 hours, followed by filtration to remove the resin. The solvent was removed under vacuum to obtain the compound 23 as crude. MS (ESI) calculated for C₅₄H₇₉N₃O₉, m/z 913.58, found 914.60 (M + H)⁺.

Synthesis of Compound 24: (15R)-Tribenzyl 16-dodecyl-2,2-dimethyl-4,12-dioxo-3-oxa-5,11,16- triazaoctacosane-6,10,15-tricarboxylate

To the solution of 14 (100 mg, 0.15 mmol) in anhydrous dichloromethane (10 mL) and methanol (5 mL), were added dodecyl aldehyde (66.8 mg, 0.36 mmol), 3 – 4 drops of acetic acid, and macroporous resin bound sodium cyanoborohydride (163.5 mg, 0.36 mmol). The solution was then stirred for 14 hours, followed by filtration to remove the resin. The solvent was removed under vacuum to obtain the residue, which was purified using column chromatography (15% EtOAc/hexanes) to obtain the compound 24 (105 mg, 71%). ¹H NMR (500 MHz, CDCl₃) δ 7.45 – 7.28 (m, 15H), 5.28 (dd, J = 12.0, 3.7 Hz, 1H), 5.23 – 5.01 (m, 6H), 4.44 (bs, 1H), 4.34 – 4.11 (m, 2H), 3.22 – 2.85 (m, 4H), 2.58 – 2.24 (m, 4H), 1.85 – 1.74 (m, 2H), 1.72 – 1.53 (m, 6H), 1.48 – 1.34 (m, 11H), 1.34 – 1.12 (m, 37H), 0.97 – 0.82 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.69, 172.23, 171.94, 171.40, 162.45, 135.64, 135.61, 134.64, 131.10, 129.21, 129.16, 129.03, 129.01, 128.92, 128.89, 128.82, 128.78, 128.62, 128.45, 128.38, 128.35, 80.10, 68.36, 68.11, 67.20, 63.20, 62.48, 53.51, 53.40, 52.72, 52.55, 38.92, 32.71, 32.12, 30.55, 29.82, 29.69, 29.63, 29.61, 29.54, 29.22, 29.20, 29.13, 28.51, 27.11, 24.94, 23.93, 23.20, 22.90, 21.88, 21.71, 14.34. MS (ESI) calculated for C₆₂H₉₅N₃O₉, m/z 1025.71, found 1026.73 (M + H)⁺.

Synthesis of Compound 25: (15R)-Tribenzyl 16-hexadecyl-2,2-dimethyl-4,12-dioxo-3-oxa- 5,11,16-triazadotriacontane-6,10,15-tricarboxylate

To a solution of 14 (100 mg, 0.15 mmol) in anhydrous dichloromethane (10 mL) and methanol (5 mL), were added hexadecanal (86.4 mg, 0.36 mmol), 3 – 4 drops of acetic acid, and macroporous resin bound sodium cyanoborohydride (163.5 mg, 0.36 mmol). The solution was then stirred for 14 hours, followed by filtration to remove the resin. The solvent was removed under vacuum to obtain the residue, which was purified using column chromatography (12% EtOAc/hexanes) to obtain the compound 25 (112 mg, 68%). ¹H NMR (500 MHz, CDCl₃) δ 7.45 – 7.28 (m, 15H), 5.31 – 4.96 (m, 7H), 4.48 (d, J = 5.2 Hz, 1H), 4.26 – 4.18 (m, 1H), 3.24 – 2.06 (m, 9H), 1.77 – 1.55 (m, 8H), 1.46 – 1.33 (m, 11H), 1.33 – 1.07 (m, 53H), 0.88 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.62, 172.24, 172.11, 171.63, 155.80, 155.69, 135.57, 131.10, 128.93, 128.83, 128.64, 128.57, 128.47, 128.41, 80.10, 68.36, 67.24, 67.18, 63.06, 62.51, 53.47, 53.37, 52.40, 52.14, 38.92, 32.82, 32.32, 32.14, 30.55, 29.92, 29.87, 29.86, 29.76, 29.74, 29.70, 29.58, 29.42, 29.36, 29.12, 28.51, 27.25, 25.07, 23.93, 23.19, 22.90, 21.78, 21.65, 14.34, 14.27. MS (ESI) calculated for C₇₀H₁₁₁N₃O₉, m/z 1137.83, found 1138.85 (M + H)⁺.

Synthesis of Compound 26: 2-Amino-6-((R)-4-carboxy-4-(dioctylamino)butanamido) heptanedioic acid

The crude of compound 23 was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the residue which was further purified using column chromatography to obtain the trifluoroacetate salt of compound 26 (60 mg, 64%). ¹H NMR (500 MHz, 10% CDCl₃ in MeOD) δ 4.34 (dd, J = 9.2, 4.6 Hz, 1H), 3.91 – 3.79 (m, 2H), 3.21 – 3.00 (m, 4H), 2.51 (ddd, J = 29.4, 15.8, 6.3 Hz, 2H), 2.20 – 2.09 (m, 1H), 2.08 – 1.95 (m, 1H), 1.95 – 1.78 (m, 3H), 1.78 – 1.39 (m, 8H), 1.34 – 1.16 (m, 19H), 0.82 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, 10% CDCl₃ in MeOD) δ 175.14, 174.73, 174.41, 171.93, 53.92, 53.35, 33.09, 32.93, 32.48, 31.97, 31.09, 30.27, 30.21, 30.19, 27.71, 25.66, 23.75, 23.42, 22.53, 14.55. MS (ESI) calculated for C₂₈H₅₃N₃O₇, m/z 543.39, found 544.41 (M + H)⁺.

Synthesis of Compound 27: 2-Amino-6-((R)-4-carboxy-4-(didodecylamino)butanamido) heptanedioic acid

The compound 24 (50 mg, 0.048 mmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 27 (37 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.43 – 4.37 (m, 1H), 3.87 (dd, J = 29.3, 7.5 Hz, 2H), 3.20 (dd, J = 19.5, 10.2 Hz, 2H), 3.15 – 3.05 (m, 2H), 2.73 – 2.46 (m, 3H), 2.27 – 1.99 (m, 3H), 1.98 – 1.83 (m, 3H), 1.72 (s, 5H), 1.63 – 1.43 (m, 3H), 1.31 (d, J = 38.3 Hz, 33H), 0.88 (t, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, MeOD) δ 33.24, 30.92, 30.80, 30.69, 30.64, 30.34, 27.78, 23.90, 14.60. MS (ESI) calculated for C₃₆H₆₉N₃O₇, m/z 655.51, found 656.51 (M + H)⁺.

Synthesis of Compound 28: 2-Amino-6-((R)-4-carboxy-4-(dihexadecylamino)butanamido) heptanedioic acid

The compound 25 (50 mg, 0.043 mmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 28 (38 mg, quantitative yield). ¹H NMR (500 MHz, 20% CDCl₃ in MeOD) δ 4.48 – 4.20 (m, 2H), 3.86 – 3.73 (m, 3H), 3.29 – 3.04 (m, 4H), 2.72 – 2.44 (m, 3H), 2.31 – 1.80 (m, 7H), 1.80 – 1.48 (m, 8H), 1.46 – 1.12 (m, 46H), 0.89 (t, J = 6.9 Hz, 6H). ¹³C NMR (126 MHz, 20% CDCl₃ in MeOD) δ 31.40, 29.14, 29.11, 29.09, 29.04, 28.97, 28.94, 28.88, 28.82, 28.53, 26.03, 22.10, 13.07. MS (ESI) calculated for C₄₄H₈₅N₃O₇, m/z 767.64, found 768.65 (M + H)⁺.

Synthesis of Compound 30: (2R)-5-((4-(2-Amino-4-oxo-4,5-dihydro-1H-imidazol-5-yl)-1- carboxybutyl)amino)-2-dodecanamido-5-oxopentanoic acid

To a solution of 19 (100 mg, 0.13 mmol) in pyridine, was added 1H-pyrazole-1-carboxamidine.HCl (55.2 mg, 0.37 mmol). The reaction mixture was heated in microwave at 65 °C for 1 hour. The solvent was removed under vacuum and the residue was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (30% MeOH/CH₂Cl₂) to obtain the compound 30 (38 mg, 58%). ¹H NMR (500 MHz, MeOD) δ 4.40 – 4.27 (m, 2H), 4.21 – 4.18 (m, 1H), 2.34 (t, J = 5.9 Hz, 2H), 2.24 (dd, J = 13.3, 6.9 Hz, 2H), 2.20 – 1.95 (m, 2H), 1.95 – 1.65 (m, 5H), 1.64 – 1.54 (m, 2H), 1.54 – 1.43 (m, 2H), 1.37 – 1.21 (m, 15H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.67, 176.56, 176.20, 175.20, 61.16, 54.19, 54.01, 53.67, 37.11, 37.03, 33.35, 33.25, 32.54, 32.46, 31.92, 31.80, 30.92, 30.81, 30.65, 30.51, 28.87, 27.09, 23.90, 22.56, 22.33, 22.19, 14.60. MS (ESI) calculated for C₂₅H₄₃N₅O₇, m/z 525.32, found 524.32 (M – H)⁻.

Synthesis of Compound 32: 2-((R)-4-Carboxy-4-dodecanamidobutanamido)-6-guanidinoheptanedioic acid

To a solution of 19 (50 mg, 0.06 mmol) in THF, was added N,N′-di-Boc-1H-pyrazole-1- carboxamidine.HCl (57.4 mg, 0.19 mmol) and pyridine (1 mL). The reaction mixture was then stirred at 50 °C for 3 hours. The solvent was removed under reduced pressure and the residue was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 1 hour, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the crude, which was purified using column chromatography (8% MeOH/CH₂Cl₂). The product was dissolved in 5 mL of trifluoroacetic acid and the reaction mixture was stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 32 (30 mg, 63%). ¹H NMR (500 MHz, MeOD) δ 4.34 (s, 2H), 4.00 (s, 1H), 2.38 – 2.28 (m, 2H), 2.24 (dd, J = 14.4, 7.0 Hz, 2H), 2.18 – 2.08 (m, 1H), 2.07 – 1.80 (m, 4H), 1.79 – 1.65 (m, 2H), 1.61 – 1.55 (m, 2H), 1.55 – 1.36 (m, 3H), 1.36 – 1.19 (m, 14H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, 5% CDCl₃ in MeOD) δ 176.46, 175.71, 175.05, 158.34, 57.03, 54.25, 37.11, 33.35, 33.20, 33.11, 32.83, 32.64, 30.88, 30.78, 30.62, 30.51, 29.28, 27.06, 23.86, 22.90, 22.58, 14.59. MS (ESI) calculated for C₂₅H₄₅N₅O₈, m/z 543.33, found 544.35 (M + H)⁺.

Synthesis of Compound 33: (6R)-6-Allyl 11,15-dibenzyl 2,2,19,19-tetramethyl-4,9,17-trioxo- 3,18-dioxa-5,10,16-triazaicosane-6,11,15-tricarboxylate

The compound 12 (250 mg, 0.29 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure. The residue was dissolved in dichloromethane, followed by the addition of di-tert-butyl dicarbonate (95 mg, 0.43 mmol) and triethylamine (80 μL, 0.58 mmol). The reaction mixture was stirred at room temperature for 1 hour, followed by removal of the solvent under reduced pressure. The crude was purified using column chromatography (30% EtOAc/Hexanes) to obtain the compound 33 (147 mg, 69%). ¹H NMR (500 MHz, CDCl₃) δ 7.41 – 7.29 (m, 10H), 5.97 – 5.82 (m, 1H), 5.30 (ddd, J = 37.2, 23.6, 5.8 Hz, 3H), 5.21 – 5.06 (m, 5H), 4.68 – 4.51 (m, 3H), 4.38 – 4.20 (m, 2H), 2.31 (dd, J = 14.5, 7.3 Hz, 2H), 2.25 – 2.13 (m, 1H), 1.99 – 1.54 (m, 6H), 1.43 (s, J = 2.9 Hz, 20H). ¹³C NMR (126 MHz, CDCl₃) δ 172.67, 172.58, 172.32, 172.18, 171.85, 156.06, 155.83, 135.55, 135.53, 135.47, 132.32, 132.24, 131.68, 128.83, 128.81, 128.69, 128.64, 128.61, 128.51, 128.46, 119.23, 119.13, 80.34, 80.17, 80.08, 77.48, 77.23, 76.98, 67.37, 67.27, 67.22, 67.18, 66.26, 53.34, 53.16, 53.04, 52.25, 52.18, 32.47, 32.24, 31.85, 31.76, 31.64, 29.88, 29.26, 29.01, 28.86, 28.50. MS (ESI) calculated for C₃₉H₅₃N₃O₁₁, m/z 739.37, found 762.37 (M + Na)⁺.

Synthesis of Compound 34: (6R)-11,15-Dibenzyl 6-undecyl 2,2,19,19-tetramethyl-4,9,17-trioxo- 3,18-dioxa-5,10,16-triazaicosane-6,11,15-tricarboxylate

To a solution of compound 33 (80 mg, 0.10 mmol) in ethanol:water (9:1), was added Wilkinson’s catalyst (10 mg, 0.01 mmol). The reaction mixture was refluxed for 3 hours at 90 °C, followed by the removal of the solvent under reduced pressure. The crude was purified using column chromatography (5% MeOH/CH₂Cl₂) and dried under vacuum. To the solution of afforded product (52 mg, 0.07 mmol) in anhydrous DMF, were added 1-undecanol (30 μL, 0.15 mmol), HBTU (33.7 mg, 0.09 mmol), triethylamine (20 μl, 0.15 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred at room temperature for 14 hours, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethyl acetate and washed with water. The organic fraction was dried over anhydrous sodium sulfate, filtered, concentrated and purified using column chromatography (35% EtOAc/hexanes) to obtain the compound 34 (44.4 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ 7.40 – 7.30 (m, 10H), 5.27 (dd, J = 22.5, 8.4 Hz, 1H), 5.20 – 4.96 (m, 5H), 4.65 – 4.49 (m, 1H), 4.41 – 4.20 (m, 2H), 4.16 – 4.07 (m, 2H), 2.30 (t, J = 6.8 Hz, 2H), 2.24 – 2.13 (m, 1H), 1.93 – 1.72 (m, 3H), 1.67- 1.59 (m, 3H), 1.43 (d, J = 2.7 Hz, 20H), 1.35 – 1.18 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.93, 170.59, 170.19, 154.13, 133.94, 127.20, 127.03, 126.85, 115.67, 78.69, 78.48, 65.63, 64.37, 51.77, 51.70, 51.47, 51.42, 50.64, 50.59, 30.87, 30.48, 30.17, 28.18, 28.07, 27.91, 27.82, 27.09, 26.90, 24.36, 21.27, 12.95. MS (ESI) calculated for C₄₇H₇₁N₃O₁₁, m/z 853.51, found 876.47 (M + Na)⁺.

Synthesis of Compound 35: 2-Amino-6-((R)-4-amino-5-oxo-5-(undecyloxy)pentanamido) heptanedioic acid

The compound 34 (40 mg, 46.9 μmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 35 (32.8 mg, quantitative yield). ¹H NMR (500 MHz, CDCl₃) δ 4.40 (dd, J = 9.2, 4.8 Hz, 1H), 4.32 – 4.22 (m, 2H), 4.11 (dd, J = 11.8, 6.0 Hz, 1H), 3.82 – 3.74 (m, 1H), 2.53 (t, J = 6.9 Hz, 2H), 2.23 (dt, J = 13.3, 6.7 Hz, 1H), 2.18 – 2.09 (m, 1H), 1.90 (ddd, J = 21.0, 13.9, 5.3 Hz, 3H), 1.80 – 1.67 (m, 3H), 1.64 – 1.46 (m, 2H), 1.43 – 1.22 (m, 16H), 0.91 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 176.90, 172.93, 70.30, 57.13, 56.15, 56.04, 55.92, 55.86, 35.59, 34.66, 34.59, 33.84, 33.76, 33.26, 33.17, 32.99, 32.88, 32.08, 29.78, 29.71, 29.39, 26.26, 25.15, 24.95, 16.95. MS (ESI) calculated for C₂₃H₄₃N₃O₇, m/z 473.31, found 474.31 (M + H)⁺.

Synthesis of Compound 36: Dibenzyl 2-((tert-butoxycarbonyl)amino)-6-((R)-4-((tert-butoxycarbonyl) amino)-5-oxo-5-(undecylamino)pentanamido)heptanedioate

To a solution of 33 (240 mg, 0.32 mmol) in ethanol:water (9:1), was added Wilkinson’s catalyst (30 mg, 0.03 mmol). The reaction mixture was refluxed for 3 hours at 90 °C, followed by removal of the solvent under reduced pressure. The crude was purified using column chromatography (5% MeOH/CH₂Cl₂). To the solution of afforded product (193 mg, 0.27 mmol) in anhydrous dichloromethane, were added undecylamine (118 μL, 0.55 mmol), HBTU (157 mg, 0.41 mmol), triethylamine (77 μl, 0.55 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred at room temperature for 14 hours, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethyl acetate and washed with water. The organic fraction was dried over anhydrous sodium sulfate, filtered, concentrated and purified using column chromatography (25% EtOAc/hexanes) to obtain the compound 36 (188 mg, 81%). ¹H NMR (500 MHz, CDCl₃) δ 7.44 – 7.27 (m, 10H), 6.65 – 6.49 (m, 1H), 5.63 (d, J = 8.1 Hz, 1H), 5.62 – 5.52 (m, 1H), 5.23 – 4.92 (m, 5H), 4.60 – 4.45 (m, 2H), 4.27 (d, J = 8.2 Hz, 1H), 4.09 (d, J = 6.7 Hz, 1H), 3.31 – 3.14 (m, 2H), 2.38 – 2.22 (m, 1H), 2.00 – 1.53 (m, 5H), 1.49 – 1.32 (m, 23H), 1.31 – 1.21 (m, 16H), 0.90 – 0.84 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 174.07, 173.52, 172.99, 172.68, 171.76, 171.40, 157.18, 156.56, 135.51, 135.42, 128.88, 128.81, 128.74, 128.66, 128.59, 128.50, 128.38, 80.44, 80.17, 67.43, 67.28, 67.19, 53.47, 53.21, 52.65, 52.28, 39.89, 39.73, 32.98, 32.75, 32.47, 32.11, 31.96, 31.51, 30.67, 29.82, 29.76, 29.54, 28.53, 27.12, 22.89, 22.03, 21.87, 21.48, 21.35, 14.34. MS (ESI) calculated for C₄₇H₇₂N₄O₁₀, m/z 852.52, found 875.53 (M + Na)⁺.

Synthesis of Compound 37: 2-Amino-6-((R)-4-amino-5-oxo-5-(undecylamino)pentanamido) heptanedioic acid

The compound 36 (40 mg, 46.9 μmol) was dissolved in methanol, followed by the addition of Pd(OH)2/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 37 (32.8 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.47 – 4.32 (m, 1H), 3.92 – 3.79 (m, 1H), 3.64 – 3.54 (m, 1H), 3.28 – 3.13 (m, 2H), 2.56 – 2.34 (m, 2H), 2.19 – 2.01 (m, 2H), 2.00 – 1.60 (m, 4H), 1.58 – 1.41 (m, 4H), 1.30 (d, J = 21.4 Hz, 16H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 175.94, 175.78, 174.26, 174.03, 169.72, 55.49, 53.98, 53.77, 53.47, 40.75, 33.08, 32.29, 31.95, 31.82, 31.51, 31.31, 30.75, 30.46, 30.32, 28.74, 28.49, 28.04, 23.74, 22.62, 22.07, 14.44. MS (ESI) calculated for C₂₃H₄₄N₄O₆, m/z 472.33, found 473.33 (M + H)⁺.

Synthesis of Compound 38: (5R)-10,14-Dibenzyl 5-undecyl 1-(9H-fluoren-9-yl)-18,18-dimethyl- 3,8,16-trioxo-2,17-dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate

To a solution of 12 (100 mg, 0.11 mmol) in ethanol:water (9:1), was added Wilkinson’s catalyst (10 mg, 0.01 mmol). The reaction mixture was refluxed for 3 hours at 90 °C, followed by removal of the solvent under reduced pressure. The crude was purified using column chromatography (5% MeOH/CH₂Cl₂) and dried under vacuum. To the solution of afforded product (70 mg, 85.2 μmol) in anhydrous DMF, were added 1-undecanol (35 μL, 170 μmol), HBTU (38.8 mg, 102.2 μmol) and a catalytic amount of DMAP. The reaction mixture was stirred at room temperature for 14 hours, followed by removal of the solvent under reduced pressure. The crude was dissolved in ethyl acetate and washed with water. The organic fraction was dried over anhydrous sodium sulfate, filtered, concentrated and purified using column chromatography (15% EtOAc/hexanes) to obtain the compound 38 (63 mg, 75%). ¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 6.4 Hz, 2H), 7.50 – 7.26 (m, 14H), 6.64 (s, 0.50H), 6.37 (s, 0.50H), 5.67 (dd, J = 32.4, 7.9 Hz, 1H), 5.21 – 4.99 (m, 5H), 4.62 – 4.53 (m, 1H), 4.47 – 4.31 (m, 3H), 4.30 – 4.19 (m, 2H), 4.13 (td, J = 6.5, 2.6 Hz, 2H), 2.34 – 2.16 (m, 3H), 1.99 – 1.89 (m, 1H), 1.87 – 1.74 (m, 2H), 1.72 – 1.56 (m, 6H), 1.39 (d, J = 20.0 Hz, 10H), 1.33 – 1.20 (m, 15H), 0.90 (dt, J = 13.9, 7.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.77, 172.70, 172.64, 144.54, 144.29, 141.96, 135.98, 129.25, 129.14, 129.06, 128.95, 128.88, 128.36, 127.73, 125.79, 120.62, 80.57, 67.85, 67.75, 67.67, 66.58, 53.76, 52.66, 47.80, 32.91, 32.54, 32.19, 30.23, 30.13, 29.96, 29.85, 29.59, 29.13, 28.94, 26.41, 23.32, 21.58, 14.77. MS (ESI) calculated for C₅₇H₇₃N₃O₁₁, m/z 975.52, found 998.46 (M + Na)⁺.

Synthesis of Compound 39: (5R)-10,14-Dibenzyl 5-ethyl 1-(9H-fluoren-9-yl)-18,18-dimethyl- 3,8,16-trioxo-2,17-dioxa-4,9,15-triazanonadecane-5,10,14-tricarboxylate

To a solution of 12 (100 mg, 0.11 mmol) in ethanol:water (9:1), was added Wilkinson’s catalyst (10 mg, 0.01 mmol). The reaction mixture was refluxed for 3 hours at 90 °C, followed by removal of the solvent under reduced pressure. The crude was purified using column chromatography (5% MeOH/CH₂Cl₂) and dried under vacuum. To the solution of afforded product (75 mg, 91.3 μmol) in anhydrous DMF, were added ethyl alcohol (11 μL, 182 μmol), HBTU (41.6 mg, 109.5 μmol) and a catalytic amount of DMAP. The reaction mixture was stirred at room temperature for 14 hours, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethyl acetate and washed with water. The organic fraction was dried over anhydrous sodium sulfate, filtered, concentrated and purified using column chromatography (15% EtOAc/hexanes) to obtain the compound 39 (58.9 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 7.79 – 7.69 (m, 2H), 7.57 – 7.52 (m, 2H), 7.57 – 7.26 (m, 14H), 6.61 (d, J = 6.9 Hz, 0.50H), 6.37 (t, J = 6.9 Hz, 0.50H), 5.65 (dd, J = 29.3, 7.3 Hz, 1H), 5.23 – 5.00 (m, 5H), 4.64 – 4.54 (m, 1H), 4.45 – 4.37 (m, 2H), 4.29 – 4.15 (m, 4H), 2.36 – 2.17 (m, 3H), 1.98 – 1.88 (m, 1H), 1.87 – 1.74 (m, 2H), 1.73 – 1.62 (m, 2H), 1.48 – 1.33 (m, 10H), 1.33 – 1.23 (m, 5H). ¹³C NMR (126 MHz, CDCl₃) δ 172.67, 172.18, 171.99, 156.58, 155.85, 144.11, 143.88, 141.52, 135.55, 135.46, 131.10, 129.00, 128.83, 128.71, 128.64, 128.53, 127.95, 127.31, 125.36, 120.20, 80.12, 68.37, 67.43, 67.24, 61.98, 53.55, 53.35, 52.22, 47.39, 38.92, 32.45, 31.79, 30.56, 29.12, 28.82, 28.52, 23.94, 23.20, 21.47, 21.29, 21.14, 14.36, 14.29, 11.17. MS (ESI) calculated for C₄₈H₅₅N₃O₁₁, m/z 849.38, found 872.37 (M + Na)⁺.

Synthesis of Compound 40: (15R)-10,6-Dibenzyl 15-undecyl 2,2-dimethyl-4,12,17-trioxo-3-oxa- 5,11,16-triazaoctacosane-6,10,15-tricarboxylate

The compound 38 (50 mg, 0.05 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure. The residue (0.05 mmol) was dissolved in pyridine, followed by the addition of lauroyl chloride (14.6 μL, 0.06 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (18% EtOAc/hexanes) to obtain the compound 40 (36.4 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 7.74 – 7.29 (m, 10H), 7.18 – 7.06 (m, 1H), 6.59 – 6.41 (m, 2H), 5.31 – 5.08 (m, 5H), 4.73 – 4.63 (m, 1H), 4.54 (ddd, J = 12.1, 10.0, 5.6 Hz, 2H), 4.30 – 4.20 (m, 1H), 4.15 – 4.07 (m, 2H), 2.38 – 2.14 (m, 5H), 1.97 – 1.54 (m, 17H), 1.41 (d, J = 19.6 Hz, 7H), 1.34 – 1.19 (m, 26H), 0.98 – 0.81 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.71, 172.50, 172.32, 172.18, 135.62, 128.86, 128.82, 128.65, 128.52, 128.46, 80.11, 67.31, 67.24, 66.15, 53.36, 52.27, 51.86, 36.87, 32.12, 29.82, 29.72, 29.56, 29.52, 29.44, 28.70, 28.55, 25.99, 25.90, 22.90, 14.34. MS (ESI) calculated for C₅₄H₈₅N₃O₁₀, m/z 935.62, found 958.59 (M + Na)⁺.

Synthesis of Compound 41: 2-Amino-6-((R)-4-dodecanamido-5-oxo-5-(undecyloxy) pentanamido)heptanedioic acid

The compound 40 (30 mg, 32.0 μmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 41 (24.6 mg, quantitative yield). ¹H NMR (500 MHz, CDCl₃) δ 4.51 – 4.26 (m, 2H), 4.21 (ddd, J = 19.6, 10.9, 5.9 Hz, 1H), 4.15 – 3.95 (m, 2H), 2.51 – 2.13 (m, 4H), 2.12 – 1.48 (m, 21H), 1.48 – 1.38 (m, 2H), 1.37 – 1.05 (m, 21H), 0.96 – 0.80 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 168.30, 68.63, 50.65, 50.48, 50.31, 50.14, 49.97, 39.16, 32.38, 30.79, 30.15, 29.86, 29.83, 29.37, 26.21, 24.17, 23.44, 23.14, 14.53, 14.51, 11.41. MS (ESI) calculated for C₃₅H₆₅N₃O₈, m/z 655.48, found 654.51 (M – H⁺)⁻.

Synthesis of Compound 43: 2-Amino-6-((R)-4-guanidino-5-oxo-5-(undecyloxy)pentanamido) heptanedioic acid

The compound 38 (50 mg, 0.05 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure. The crude mixture was purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain the free primary amine derivative, which was dissolved in pyridine, followed by the addition of 1H-pyrazole-1-carboxamidine.HCl (22.2 mg, 0.15 mmol). The sealed reaction mixture was then heated in microwave at 65 °C for 1 hour. The solvent was removed under vacuum and the residue was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 4 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the crude product which was purified using column chromatography (30% MeOH/CH₂Cl₂) to obtain the free carboxylic acid derivative, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 43 (20.3 mg, 47% in 4 steps). ¹H NMR (500 MHz, MeOD) δ 4.43 – 4.08 (m, 5H), 3.58 (bs, 1H), 2.46 – 2.27 (m, 3H), 2.12 – 1.90 (m, 2H), 1.84 (bs, 3H), 1.66 (dd, J = 13.7, 6.9 Hz, 2H), 1.53 – 1.41 (m, 2H), 1.40 – 1.19 (m, 15H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 67.16, 33.09, 30.76, 30.68, 30.49, 30.39, 29.66, 26.97, 23.75, 14.46. MS (ESI) calculated for C₂₄H₄₅N₅O₇, m/z 515.33, found 516.31 (M + H)⁺.

Synthesis of Compound 44: (15R)-10,6-Dibenzyl 15-ethyl 2,2-dimethyl-4,12,17-trioxo-3-oxa- 5,11,16-triazaoctacosane-6,10,15-tricarboxylate

The compound 39 (100 mg, 0.11 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure to obtain the residue which was dissolved in pyridine, followed by the addition of lauroyl chloride (41.9 μL, 0.18 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (35% EtOAc/hexanes) to obtain the compound 44 (65.4 mg, 69%). ¹H NMR (500 MHz, CDCl₃) δ 7.73 – 7.27 (m, 10H), 7.06 (dd, J = 17.7, 8.8 Hz, 1H), 6.65 – 6.53 (m, 1H), 6.47 – 6.36 (m, 1H), 5.20 – 5.05 (m, 4H), 4.53 – 4.50 (m, 3H), 4.33 – 4.09 (m, 4H), 2.35 – 2.11 (m, 5H), 1.93 – 1.69 (m, 4H), 1.68 – 1.65 (m, 3H), 1.47 – 1.33 (m, 9H), 1.32 – 1.17 (m, 18H), 0.92 – 0.83 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.42, 172.26, 135.58, 131.11, 129.02, 128.82, 128.65, 128.52, 128.47, 80.10, 68.37, 67.24, 61.95, 53.43, 52.32, 51.83, 38.93, 36.85, 32.12, 31.64, 30.56, 29.83, 29.71, 29.56, 29.51, 29.13, 28.55, 25.89, 23.94, 23.20, 22.91, 14.34, 14.28, 11.18. MS (ESI) calculated for C₄₅H₆₇N₃O₁₀, m/z 809.48, found 832.51 (M + Na)⁺.

Synthesis of Compound 45: 2-Amino-6-((R)-4-dodecanamido-5-ethoxy-5-oxopentanamido) heptanedioic acid

The compound 44 (30 mg, 37.0 μmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 45 (23.4 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.35 – 4.26 (m, 2H), 4.08 (qd, J = 7.1, 3.1 Hz, 2H), 3.73 (d, J = 11.0 Hz, 1H), 2.32 – 2.24 (m, 2H), 2.20 – 2.01 (m, 3H), 1.92 – 1.74 (m, 4H), 1.74 – 1.31 (m, 6H), 1.30 – 1.07 (m, 18H), 0.81 (dt, J = 13.9, 4.4 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 173.46, 62.57, 53.58, 53.23, 37.02, 36.92, 33.23, 33.13, 32.83, 31.46, 30.91, 30.82, 30.63, 30.46, 30.44, 28.53, 28.23, 27.18, 27.11, 23.89, 14.65, 14.59. MS (ESI) calculated for C₂₆H₄₇N₃O₈, m/z 529.33, found 528.36 (M – H)⁻.

Synthesis of Compound 46: Diethyl 2-((R)-4-amino-5-(benzyloxy)-5-oxopentanamido)-6-((tert-butoxycarbonyl) amino)heptanedioate

The compound 13 (100 mg, 0.12 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure to obtain the crude product which was purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain the compound 46 (66 mg, 92%). MS (ESI) calculated for C₂₈H₄₃N₃O₉, m/z 565.30, found 588.30 (M + Na)⁺.

Synthesis of Compound 48: Diethyl 2-amino-6-((R)-4-amino-5-oxo-5-(undecylthio) pentanamido)heptanedioate

To a solution of compound 46 (50 mg, 0.08 mmol) in dichloromethane, were added di-tert-butyl dicarbonate (28.9 mg, 0.13 mmol) and triethylamine (24.6 μl, 0.17 mmol). The reaction mixture was stirred for 30 minutes, followed by complete removal of the solvent under reduced pressure. The residue was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was then subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in anhydrous DMF, followed by the addition of 1-undecanethiol (40 μL, 0.17 mmol), HBTU (40.2 mg, 0.10 mmol), triethylamine (24.7 μL, 0.17 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred at room temperature for 14 hours, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethyl acetate and washed with water. The organic fraction was dried over anhydrous sodium sulfate, filtered, concentrated and purified using column chromatography (30% EtOAc/hexanes) to obtain the compound 47 (41 mg, 63%). The compound 47 (40 mg, 0.05 mmol) was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 48 (41 mg, quantitative yield). ¹H NMR (500 MHz, 20% CDCl₃ in MeOD) δ 4.32 – 4.26 (m, 1H), 4.26 – 4.13 (m, 3H), 4.09 (q, J = 7.1 Hz, 2H), 3.91 (dt, , J = 2.0, 6.5 Hz, 1H), 2.95 (t, J = 7.3 Hz, 2H), 2.50 – 2.35 (m, 2H), 2.28 – 1.69 (m, 6H), 1.69 – 1.59 (m, 1H), 1.57 – 1.35 (m, 4H), 1.34 – 1.11 (m, 21H), 0.80 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, 20% CDCl₃ in MeOD) δ 197.50, 174.15, 63.80, 62.71, 62.66, 59.98, 53.86, 53.83, 53.78, 53.74, 33.18, 31.98, 31.79, 31.65, 31.19, 31.16, 30.84, 30.71, 30.58, 30.30, 29.94, 28.52, 28.44, 23.85, 22.77, 22.63, 14.64, 14.59, 14.56. MS (ESI) calculated for C₂₇H₅₁N₃O₆S, m/z 545.35, found 568.36 (M + Na)⁺.

Synthesis of Compound 50: (2R)-5-((6-Amino-1,7-diethoxy-1,7-dioxoheptan-2-yl)amino)-2- dodecanamido-5-oxopentanoic acid

To a solution of 46 (36 mg, 0.06 mmol) in pyridine, were added lauroyl chloride (22.6 μL, 0.09 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (18% EtOAc/hexanes) to obtain the compound 49 (39.0 mg, 82%). The compound 49 (30 mg, 0.04 mmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was then dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 50 (26.4 mg, quantitative yield in two steps). ¹H NMR (500 MHz, MeOD) δ 4.45 – 4.37 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 4.16 (q, J = 6.6 Hz, 2H), 4.05 – 3.97 (m, 1H), 2.34 (d, J = 2.6 Hz, 2H), 2.28 – 2.11 (m, 3H), 2.01 – 1.80 (m, 4H), 1.78 – 1.40 (m, 6H), 1.40 – 1.19 (m, 21H), 0.90 (dt, J = 13.6, 4.7 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.49, 175.06, 174.90, 173.29, 170.44, 63.70, 62.49, 53.88, 53.80, 53.33, 53.13, 52.83, 49.52, 49.35, 49.18, 49.01, 48.84, 48.67, 48.50, 36.96, 36.87, 33.10, 32.79, 32.09, 32.04, 31.95, 31.02, 30.89, 30.78, 30.68, 30.51, 30.38, 28.84, 28.47, 27.01, 26.97, 23.76, 22.65, 22.42, 22.32, 14.44. MS (ESI) calculated for C₂₈H₅₁N₃O₈, m/z 557.37, found 558.40 (M + H)⁺.

Synthesis of Compound 51: Dibenzyl 2-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) butanamido)-6-((tert-butoxycarbonyl)amino)heptanedioate

To a solution of 9 (221 mg, 0.47 mmol) in anhydrous dichloromethane, were added 4-Fmoc-amino-butyric acid (150 mg, 0.46 mmol), polystyrene bound carbodiimide (382.11 mg, 0.47 mmol) and a catalytic amount of polystyrene bound DMAP. The reaction mixture was stirred at room temperature for 8 hours, followed by filtration to remove the solid resin. The filtrate was evaporated under vacuum to obtain the residue which was then purified using column chromatography (50% EtOAc/hexanes) to obtain the compound 51 (230 mg, 63%). ¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 7.4 Hz, 2H), 7.44 – 7.25 (m, 14H), 6.47 (d, J = 7.2 Hz, 1H), 5.21 – 5.00 (m, 6H), 4.61 – 4.50 (m, 1H), 4.46 – 4.33 (m, 2H), 4.31 – 4.15 (m, 2H), 3.29 – 3.11 (m, 2H), 2.26 – 2.12 (m, 2H), 1.76 (dd, J = 19.9, 6.1 Hz, 4H), 1.70 – 1.52 (m, 2H), 1.38 (d, J = 13.3 Hz, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 172.70, 172.59, 172.45, 172.29, 157.10, 155.76, 144.15, 141.53, 135.54, 128.85, 128.82, 128.72, 128.64, 128.57, 128.45, 127.88, 127.25, 125.28, 120.16, 80.19, 67.42, 67.34, 67.24, 66.76, 53.34, 52.18, 47.49, 40.21, 33.33, 32.45, 32.30, 31.65, 28.53, 26.21, 21.59, 21.28. MS (ESI) calculated for C₄₅H₅₁N₃O₉, m/z 777.36, found 800.33 (M + Na)⁺.

Synthesis of Compound 52: Dibenzyl 2-((tert-butoxycarbonyl)amino)-6-(4-dodecanamidobutanamido) heptanedioate

The compound 51 (140 mg, 0.18 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure to obtain the residue which was dissolved in pyridine, followed by the addition of lauroyl chloride (64.0 μL, 0.27 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (30% EtOAc/hexanes) to obtain the compound 52 (100 mg, 75%). ¹H NMR (500 MHz, CDCl₃) δ 7.41 – 7.29 (m, 10H), 6.84 – 6.76 (m, 1H), 5.97 – 5.88 (m, 1H), 5.26 – 5.09 (m, 5H), 4.59 – 4.50 (m, 1H), 4.26 (d, J = 8.5 Hz, 1H), 3.40 – 3.30 (m, 1H), 3.25 – 3.17 (m, 1H), 2.29 – 2.22 (m, 2H), 2.17 – 2.14 (m, 2H), 1.87 – 1.76 (m, 4H), 1.66 (s, 4H), 1.63 – 1.58 (m, 2H), 1.42 (d, J = 14.4 Hz, 9H), 1.33 – 1.20 (m, 16H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 174.14, 173.13, 172.73, 172.62, 172.43, 172.27, 155.92, 155.80, 135.57, 128.84, 128.69, 128.65, 128.56, 128.46, 80.12, 67.27, 53.39, 52.34, 38.80, 37.10, 33.62, 32.30, 32.12, 31.57, 29.84, 29.82, 29.72, 29.57, 29.55, 28.55, 26.06, 25.93, 22.90, 21.66, 21.34, 14.34. MS (ESI) calculated for C₄₂H₆₃N₃O₈, m/z 737.46, found 760.41 (M + Na)⁺.

Synthesis of Compound 53: 2-Amino-6-(4-dodecanamidobutanamido)heptanedioic acid

The compound 52 (50 mg, 0.06 mmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 53 (38 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.39 (dd, J = 9.3, 4.7 Hz, 1H), 3.80 – 3.73 (m, 1H), 3.20 (t, J = 7.0 Hz, 2H), 2.32 – 2.24 (m, 2H), 2.21 – 2.14 (m, 2H), 2.00 – 1.68 (m, 7H), 1.66 – 1.42 (m, 5H), 1.36 – 1.24 (m, 14H), 0.90 (dd, J = 13.1, 6.2 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.66, 175.79, 175.46, 173.04, 54.83, 53.43, 39.81, 37.36, 34.18, 33.23, 32.45, 32.39, 31.61, 31.48, 30.90, 30.80, 30.63, 30.52, 27.24, 26.85, 23.89, 23.07, 22.75, 14.58. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 458.39 (M + H)⁺.

Synthesis of Compound 54: 5-((1,7-Bis(benzyloxy)-6-((tert-butoxycarbonyl)amino)-1,7- dioxoheptan-2-yl)amino)-5-oxopentanoic acid

To a solution of 9 (100 mg, 0.21 mmol) in anhydrous dichloromethane, was added glutaric anhydride (25.5 mg, 0.22 mmol). The reaction mixture was stirred at room temperature for 1 hour, followed by removal of the solvent under reduced pressure. The residue was then purified using column chromatography (5% MeOH/CH₂Cl₂) to obtain the compound 54 (118 mg, 95%). ¹H NMR (500 MHz, CDCl₃) δ 7.73 – 7.29 (m, 10H), 6.87 – 6.34 (m, 1H), 5.22 – 5.08 (m, 4H), 4.60 (bs, 1H), 4.32 – 3.92 (m, 1H), 2.54 – 2.17 (m, 4H), 2.13 – 1.54 (m, 6H), 1.52 – 1.20 (m, 13H). ¹³C NMR (126 MHz, CDCl₃) δ 176.46, 172.80, 172.69, 172.41, 135.41, 131.11, 129.01, 128.86, 128.84, 128.74, 128.69, 128.54, 80.46, 68.37, 67.51, 67.43, 67.15, 53.35, 52.10, 51.82, 38.92, 35.67, 35.08, 33.61, 33.49, 32.99, 32.44, 31.80, 31.66, 31.00, 30.55, 29.91, 29.13, 28.53, 28.31, 23.93, 23.20, 21.55, 21.21, 20.61, 19.92. MS (ESI) calculated for C₃₁H₄₀N₂O₉, m/z 584.27, found 607.26 (M + Na)⁺.

Synthesis of Compound 55: Dibenzyl 2-((tert-butoxycarbonyl)amino)-6-(5-oxo-5-(undecyloxy) pentanamido)heptanedioate

To a solution of 54 (90 mg, 0.15 mmol) in anhydrous DMF, were added 1- undecanol (63 μL, 0.30 mmol), HBTU (70 mg, 0.18 mmol), triethylamine (43 μL, 0.30 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred at room temperature for 12 hours, followed by removal of the solvent under reduced pressure. The residue was dissolved in ethylacetate and washed with water. The organic fraction was dried over anhydrous sodium sulfate, filtered, concentrated and purified using column chromatography (10% EtOAc/hexanes) to obtain the compound 55 (86.4 mg, 78%).¹H NMR (500 MHz, CDCl₃) δ 7.40 – 7.29 (m, 10H), 6.15 (d, J = 6.3 Hz, 1H), 5.20 – 5.02 (m, 5H), 4.62 – 4.54 (m, 1H), 4.32 – 4.19 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 2.40 – 2.32 (m, 2H), 2.30 – 2.22 (m, 2H), 2.00 – 1.90 (m, 2H), 1.87 – 1.57 (m, 7H), 1.43 (s, 9H), 1.33 – 1.19 (m, 17H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.49, 173.48, 172.70, 172.57, 172.42, 172.27, 172.26, 155.85, 155.70, 135.55, 135.42, 128.86, 128.82, 128.73, 128.66, 128.55, 128.47, 80.20, 80.09, 67.43, 67.35, 67.25, 64.90, 53.31, 52.03, 51.99, 35.39, 33.47, 32.49, 32.26, 32.11, 31.96, 31.84, 29.80, 29.79, 29.73, 29.54, 29.47, 28.82, 28.52, 26.11, 22.89, 21.48, 21.20, 20.97, 14.33. MS (ESI) calculated for C₄₂H₆₂N₂O₉, m/z 738.45, found 761.43 (M + Na)⁺.

Synthesis of Compound 56: 2-Amino-6-(5-oxo-5-(undecyloxy)pentanamido)heptanedioic acid

The compound 55 (35 mg, 0.04 mmol) was dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the residue, which was dissolved in 5 mL of trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 56 (26 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.34 (dd, J = 8.7, 5.0 Hz, 1H), 4.02 (t, J = 6.7 Hz, 2H), 3.82 – 3.73 (m, 1H), 2.34 (t, J = 7.5 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.97 – 1.80 (m, 5H), 1.74 – 1.63 (m, 1H), 1.62 – 1.42 (m, 4H), 1.29 – 1.25 (m, 16H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 175.63, 175.60, 175.35, 175.32, 175.12, 172.76, 65.83, 54.68, 54.54, 53.40, 53.36, 35.86, 34.37, 33.19, 32.40, 32.32, 31.51, 31.39, 30.85, 30.84, 30.78, 30.59, 30.51, 29.86, 27.16, 23.86, 22.99, 22.73, 22.27, 14.60. MS (ESI) calculated for C₂₃H₄₂N₂O₇, m/z 458.30, found 459.32 (M + H)⁺.

General procedure for the syntheses of compounds (66–79)

Synthesis of compound 66: Dibenzyl 2-((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- (benzyloxy)-5-oxopentanamido)heptanedioate

To a solution of 4 (100 mg, 0.21 mmol) in dichloromethane, were added 58 (78.8 mg, 0.22 mmol), polystyrene bound carbodiimide (170 mg, 0.22 mmol), triethylamine (60 μL, 0.43 mmol) and a catalytic amount of polystyrene bound DMAP. The reaction mixture was stirred at room temperature for 8 hours, followed by filtration to remove the solid resin. The filtrate was evaporated under vacuum to obtain the residue which was then purified using column chromatography (55% EtOAc/hexanes) to obtain the compound 66 (154 mg, 89%). ¹H NMR (500 MHz, CDCl_3,) δ 7.74 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 7.0 Hz, 2H), 7.41 – 7.24 (m, 19H), 6.47 (d, J = 7.8 Hz, 0.50H), 6.19 (d, J = 7.8 Hz, 0.50H), 5.67 (dd, J = 20.4, 8.0 Hz, 1H), 5.20 – 5.03 (m, 6H), 4.59 (ddd, J = 13.0, 7.5, 5.3 Hz, 1H), 4.49 – 4.33 (m, 3H), 4.18 (t, J = 6.8 Hz, 1H), 2.27 – 2.19 (m, 5H), 2.02 – 1.77 (m, 3H), 1.63 – 1.61 (m, 1H), 1.38 – 1.16 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.17, 171.06, 170.13, 170.04, 169.40, 153.91, 141.79, 141.42, 139.22, 133.04, 126.56, 126.55, 126.53, 126.51, 126.46, 126.31, 126.24, 126.21, 126.13, 126.09, 125.63, 125.00, 123.03, 117.90, 65.30, 65.07, 64.95, 64.08, 51.35, 50.01, 45.06, 31.74, 29.88, 26.66, 26.14, 22.55, 22.27. MS (ESI) calculated for C₄₈H₄₈N₂O₉, m/z 796.34, found 819.31 (M + Na)⁺.

Synthesis of compound 67: 2-((R)-4-carboxy-4-dodecanamidobutanamido)heptanedioic acid

The compound 66 (147 mg, 0.18 mmol) was dissolved in 10 mL of dichloromethane, followed by the addition of 3 mL of piperidine. The reaction mixture was stirred for 15 minutes, followed by removal of the solvent under reduced pressure to obtain the residue, which was dissolved in pyridine, followed by the addition of lauroyl chloride (64.0 μL, 0.27 mmol) and a catalytic amount of DMAP. The reaction mixture was stirred for 4 hours, followed by evaporation of the solvent under reduced pressure. The residue was then dissolved in dichloromethane and washed with water. The organic solvent was dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to obtain the crude product which was purified using column chromatography (25% EtOAc/hexanes) to obtain the protected intermediate of compound 67 (100 mg, 73%). The protected intermediate (50 mg, 0. 06 mmol) was then dissolved in methanol, followed by the addition of Pd(OH)₂/C. The solution was subjected to catalytic hydrogenolysis at 60 psi hydrogen pressure for 2 hours, followed by filtration through celite bed. The solvent was removed under reduced pressure to obtain the compound 67 (29 mg, quantitative yield). ¹H NMR (500 MHz, MeOD) δ 4.42 – 4.28 (m, 2H), 2.33 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.24 – 2.18 (m, 2H), 2.18 – 2.10 (m, 1H), 1.93 (dt, J = 13.8, 6.3 Hz, 1H), 1.85 – 1.82 (m, 1H), 1.72 – 1.54 (m, 5H), 1.41 (dd, J = 14.5, 7.4 Hz, 3H), 1.30 – 1.26 (m, 15H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 177.59, 176.63, 176.63, 175.03, 54.04, 53.66, 37.03, 34.85, 33.36, 33.24, 32.50, 32.45, 30.91, 30.81, 30.64, 30.48, 28.97, 28.79, 27.07, 26.66, 25.78, 23.90, 14.60. MS (ESI) calculated for C₂₄H₄₂N₂O₈, m/z 486.29, found 485.29 (M – H)⁻.

In the case of N-Boc protected intermediates (compound 68, 72, 76, 78), the compound after hydrogenolysis was dissolved in trifluoroacetic acid and stirred for 30 minutes, followed by removal of the solvent by purging nitrogen and drying under vacuum to obtain the trifluoroacetate salt of the compound 69, 73, 77, 79.

Synthesis of compound 68: (R)-Benzyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((5- ((tert-butoxycarbonyl)amino)pentyl)amino)-5-oxopentanoate

¹H NMR (500 MHz, CDCl₃) δ 7.77 (d, J = 7.5 Hz, 2H), 7.71 (dt, J = 7.3, 3.6 Hz, 1H), 7.61 (t, J = 6.8 Hz, 2H), 7.54 (dd, J = 5.7, 3.3 Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.37 – 7.30 (m, 5H), 5.18 (q, J = 12.1 Hz, 2H), 4.46 – 4.32 (m, 3H), 4.25 – 4.17 (m, 2H), 3.42 (dd, J = 5.5, 3.9 Hz, 1H), 3.18 (ddd, J = 20.5, 13.5, 7.0 Hz, 2H), 3.07 (s, 2H), 2.20 – 2.18 (m, 3H), 1.52 – 1.38 (m, 10H), 1.37 – 1.27 (m, 7H). ¹³C NMR (126 MHz, CDCl₃) δ 168.20, 141.65, 135.35, 131.45, 129.30, 129.14, 129.03, 128.87, 128.24, 127.59, 125.59, 120.48, 79.88, 68.70, 67.86, 50.32, 50.15, 49.98, 47.61, 39.76, 39.20, 30.83, 30.05, 29.41, 28.88, 24.34, 24.21, 23.47. MS (ESI) calculated for C₃₇H₄₅N₃O₇, m/z 643.33, found 666.34 (M + Na)⁺.

Synthesis of compound 69: (R)-5-((5-Aminopentyl)amino)-2-dodecanamido-5-oxopentanoic acid

¹H NMR (400 MHz, MeOD) δ 4.21 (dd, J = 8.4, 4.9 Hz, 1H), 3.30 – 3.18 (m, 2H), 2.92 (t, J = 6.7 Hz, 2H), 2.27 – 2.20 (m, 4H), 2.08 (td, J = 13.5, 6.5 Hz, 1H), 1.94 (td, J = 13.6, 7.3 Hz, 1H), 1.72 – 1.39 (m, 8H), 1.37 – 1.20 (m, 16H), 0.88 (dd, J = 12.0, 5.4 Hz, 3H). ¹³C NMR (101 MHz, 10% CDCl₃ in MeOD) δ 174.03, 53.83, 38.93, 37.98, 36.02, 32.27, 31.46, 29.16, 29.04, 28.87, 27.91, 26.29, 25.40, 22.64, 22.17, 13.26. MS (ESI) calculated for C₂₂H₄₃N₃O₄, m/z 413.33, found 414.37(M+H)⁺.

Synthesis of compound 70: (2R)-Benzyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo- 5-((2-oxoazepan-3-yl)amino)pentanoate

¹H NMR (500 MHz, CDCl₃, Ethyl Acetate) δ 7.76 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.35 (qd, J = 14.7, 7.3 Hz, 9H), 6.98 – 6.90 (m, 1H), 6.06 (bs, 1H), 5.88 – 5.81 (m, 1H), 5.23 – 5.13 (m, 2H), 4.54 – 4.48 (m, 1H), 4.46 – 4.33 (m, 3H), 4.21 (t, J = 7.1 Hz, 1H), 3.30 – 3.18 (m, 2H), 2.37 – 2.18 (m, 3H), 1.99 – 1.97 (m, 1H), 1.84 – 1.76 (dd, J = 14.6, 9.4 Hz, 1H), 1.62 (bs, 3H), 1.49 – 1.35 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 175.57, 172.14, 171.16, 156.34, 144.15, 143.99, 141.49, 135.45, 128.85, 128.71, 128.54, 127.90, 127.30, 125.43, 120.17, 67.51, 67.29, 53.98, 53.91, 52.46, 47.38, 42.38, 32.37, 32.32, 31.76, 31.70, 29.05, 28.10, 27.98, 27.93. MS (ESI) calculated for C₃₃H₃₅N₃O₆, m/z 569.25, found 592.23 (M + Na)⁺.

Synthesis of compound 71: (2R)-2-Dodecanamido-5-oxo-5-((2-oxoazepan-3-yl)amino)pentanoic acid

¹H NMR (500 MHz, MeOD) δ 4.54 (dd, J = 11.4, 1.5 Hz, 1H), 4.41 – 4.31 (m, 1H), 3.30 – 3.13 (m, 3H), 2.34 (tt, J = 6.4, 5.0 Hz, 2H), 2.27 – 2.11 (m, 3H), 2.02 – 1.72 (m, 5H), 1.65 – 1.49 (m, 3H), 1.43 – 1.22 (m, 16H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 177.24, 176.35, 176.30, 174.12, 174.02, 53.35, 49.46, 49.29, 42.47, 36.94, 33.37, 33.27, 33.10, 32.21, 32.16, 30.78, 30.77, 30.67, 30.51, 30.50, 30.36, 29.94, 29.16, 28.87, 28.80, 26.92, 23.75, 14.45. MS (ESI) calculated for C₂₃H₄₁N₃O₅, m/z 439.30, found 462.31 (M + Na)⁺.

Synthesis of compound 72: (R)-Benzyl 2-((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 5-(benzyloxy)-5-oxopentanamido)-6-((tert-butoxycarbonyl)amino)hexanoate

¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.2 Hz, 2H), 7.45 – 7.28 (m, 14H), 6.32 (d, J = 6.9 Hz, 1H), 5.71 (d, J = 8.0 Hz, 1H), 5.22 – 5.08 (m, 4H), 4.59 (t, J = 10.9 Hz, 2H), 4.40 (d, J = 6.9 Hz, 3H), 4.20 (t, J = 6.9 Hz, 1H), 3.02 (bs, 2H), 2.24 (s, 3H), 1.95 (dd, J = 15.4, 7.9 Hz, 1H), 1.83 (ddd, J = 15.7, 10.6, 5.3 Hz, 1H), 1.67 (d, J = 8.6 Hz, 1H), 1.41 (s, 11H), 1.36 – 1.23 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 172.37, 172.01, 171.81, 156.51, 156.28, 144.10, 143.86, 141.53, 135.48, 135.36, 128.87, 128.84, 128.78, 128.73, 128.63, 128.56, 127.94, 127.31, 125.34, 120.20, 120.18, 79.31, 67.61, 67.36, 67.27, 53.63, 52.36, 47.37, 40.17, 32.26, 32.02, 29.71, 28.62, 22.50. MS (ESI) calculated for C₄₅H₅₁N₃O₉, m/z 777.36, found 800.35 (M + Na)⁺.

Synthesis of compound 73: (R)-6-Amino-2-((R)-4-carboxy-4-dodecanamidobutanamido) hexanoic acid

¹H NMR (500 MHz, MeOD) δ 4.31 (dd, J = 9.1, 4.4 Hz, 1H), 4.26 (dd, J = 9.6, 4.3 Hz, 1H), 2.89 – 2.79 (m, 2H), 2.26 (t, J = 7.2 Hz, 2H), 2.21 – 2.08 (m, 3H), 1.88 – 1.75 (m, 2H), 1.68 – 1.48 (m, 5H), 1.47 – 1.08 (m, 18H), 0.80 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.55, 174.81, 53.42, 53.24, 40.56, 36.97, 33.10, 33.04, 32.34, 30.79, 30.77, 30.68, 30.52, 30.50, 30.38, 28.64, 27.95, 27.00, 23.78, 23.75, 14.45. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 458.33 (M + H)⁺.

Synthesis of compound 74: (R)-Benzyl 6-((R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 5-(benzyloxy)-5-oxopentanamido)-2-(((benzyloxy)carbonyl)amino)hexanoate

¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H), 7.61 – 7.52 (m, 2H), 7.38 – 7.25 (m, 19H), 5.80 (bs, 1H), 5.71 (d, J = 7.8 Hz, 1H), 5.45 – 5.41 (m, 1H), 5.19 – 5.09 (m, 4H), 5.06 (s, 2H), 4.44 – 4.30 (m, 4H), 4.16 (t, J = 6.8 Hz, 1H), 3.14 (dt, J = 13.8, 6.8 Hz, 2H), 2.17 – 2.11 (m, 3H), 1.93 (dd, J = 17.2, 11.2 Hz, 1H), 1.80 (dt, J = 10.2, 7.3 Hz, 1H), 1.70 – 1.62 (m, 1H), 1.49 – 1.38 (m, 2H), 1.33 – 1.24 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 172.41, 172.02, 156.59, 156.24, 144.10, 143.81, 141.53, 141.48, 136.40, 135.46, 135.37, 128.86, 128.84, 128.74, 128.72, 128.62, 128.53, 128.41, 128.30, 127.94, 127.29, 125.35, 125.31, 120.20, 67.58, 67.37, 67.28, 67.20, 53.88, 53.75, 47.34, 39.29, 32.59, 32.34, 29.00, 28.82, 22.53. MS (ESI) calculated for C₄₈H₄₉N₃O₉, m/z 811.35, found 834.34 (M + Na)⁺.

Synthesis of compound 75: (R)-2-Amino-6-((R)-4-carboxy-4-dodecanamidobutanamido) hexanoic acid

¹H NMR (500 MHz, MeOD) δ 4.34 (dd, J = 9.1, 4.9 Hz, 1H), 3.80 (t, J = 6.1 Hz, 1H), 3.22 (dt, J = 13.3, 6.7 Hz, 1H), 3.13 (dt, J = 13.5, 6.7 Hz, 1H), 2.30 – 2.12 (m, 5H), 1.97 – 1.79 (m, 3H), 1.64 – 1.56 (m, 2H), 1.52 (dd, J = 13.8, 6.7 Hz, 2H), 1.49 – 1.39 (m, 2H), 1.30 – 1.26 (d, J = 17.6 Hz, 16H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.53, 174.85, 54.57, 53.08, 39.85, 36.89, 33.27, 33.10, 31.40, 30.78, 30.68, 30.52, 30.50, 30.34, 30.00, 28.69, 26.96, 23.76, 23.40, 14.46. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 456.33 (M – H)⁻.

Synthesis of compound 76: (S)-Tert-butyl 2-((R)-4-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-oxopentanamido)-6-(((benzyloxy)carbonyl)amino) hexanoate

¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H), 7.61 – 7.54 (m, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.35 – 7.25 (m, 12H), 6.30 (dd, J = 84.8, 7.6 Hz, 1H), 5.74 (dd, J = 17.5, 7.9 Hz, 1H), 5.14 (t, J = 12.2 Hz, 2H), 5.08 – 5.01 (m, 2H), 4.88 (bs, 1H), 4.42 – 4.30 (ddt, J = 31.5, 17.9, 7.6 Hz, 4H), 4.17 (t, J = 6.8 Hz, 1H), 3.14 (dd, J = 12.7, 6.4 Hz, 2H), 2.31 – 2.13 (m, 3H), 1.98 (dd, J = 14.5, 7.7 Hz, 1H), 1.76 (ddd, J = 15.5, 10.2, 5.3 Hz, 1H), 1.64 – 1.61 (m, 1H), 1.51 – 1.44 (m, 2H), 1.44 – 1.42 (m, 9H), 1.37 – 1.27 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 172.11, 171.84, 171.77, 156.72, 156.51, 144.12, 143.85, 141.50, 136.79, 135.36, 128.85, 128.73, 128.70, 128.57, 128.28, 127.92, 127.30, 125.35, 120.19, 120.17, 82.33, 67.57, 67.31, 66.76, 53.67, 52.76, 47.34, 40.77, 32.41, 32.16, 29.58, 28.70, 28.19, 22.29. MS (ESI) calculated for C₄₅H₅₁N₃O₉, m/z 777.36, found 800.36 (M + Na)⁺.

Synthesis of compound 77: (S)-6-Amino-2-((R)-4-carboxy-4-dodecanamidobutanamido) hexanoic acid

¹H NMR (500 MHz, MeOD) δ 4.35 (bs, 2H), 2.91 (t, J = 7.4 Hz, 2H), 2.32 (t, J = 6.7 Hz, 2H), 2.22 (t, J = 7.5 Hz, 2H), 2.17 – 2.08 (m, 1H), 1.96 – 1.87 (m, 2H), 1.77 – 1.55 (m, 5H), 1.48 (dd, J = 15.2, 7.6 Hz, 2H), 1.30 – 1.27 (m, 16H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.53, 175.19, 53.81, 40.68, 37.07, 33.41, 33.23, 32.47, 32.36, 30.92, 30.81, 30.65, 30.50, 29.17, 28.86, 28.12, 27.10, 23.95, 23.89, 14.60. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 458.32 (M + H)⁺.

Synthesis of compound 78: (S)-Tert-butyl 6-((R)-4-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-oxopentanamido)-2-((tert-butoxycarbonyl)amino) hexanoate

¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 7.5 Hz, 2H), 7.61 – 7.55 (m, 2H), 7.41 – 7.25 (m, 9H), 5.89 (bs, 1H), 5.78 (d, J = 7.9 Hz, 1H), 5.16 (q, J = 12.2 Hz, 2H), 5.07 (d, J = 8.1 Hz, 1H), 4.37 (ddd, J = 28.8, 10.6, 7.1 Hz, 3H), 4.19 (t, J = 7.0 Hz, 1H), 3.19 (ddt, J = 25.6, 13.1, 6.5 Hz, 2H), 2.18 (t, J = 10.6 Hz, 2H), 2.01 – 1.92 (m, 1H), 1.78 – 1.62 (m, 3H), 1.58 (ddd, J = 11.4, 9.3, 5.7 Hz, 1H), 1.53 – 1.45 (m, 2H), 1.41 (d, J = 8.0 Hz, 18H), 1.38 – 1.27 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.29, 170.24, 170.19, 154.78, 153.95, 142.30, 142.03, 139.72, 139.67, 133.58, 127.06, 126.95, 126.81, 126.13, 126.12, 125.48, 123.57, 123.54, 118.40, 118.38, 80.27, 78.07, 65.77, 65.53, 52.05, 52.00, 45.54, 37.78, 31.12, 30.81, 27.32, 26.97, 26.73, 26.40, 20.95. MS (ESI) calculated for C₄₂H₅₃N₃O₉, m/z 743.38, found 766.36 (M + Na)⁺.

Synthesis of compound 79: (S)-2-Amino-6-((R)-4-carboxy-4-dodecanamidobutanamido) hexanoic acid

¹H NMR (500 MHz, MeOD) δ 4.35 (dd, J = 8.9, 4.9 Hz, 1H), 3.74 (t, J = 5.9 Hz, 1H), 3.24 – 3.12 (m, 2H), 2.30 – 2.13 (m, 5H), 1.95 – 1.80 (m, 3H), 1.66 – 1.37 (m, 7H), 1.31 – 1.27 (m, 15H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 176.28, 175.26, 174.74, 173.09, 54.56, 53.07, 39.70, 36.88, 33.15, 32.90, 31.17, 30.61, 30.59, 30.50, 30.35, 30.31, 30.23, 30.16, 29.73, 28.66, 26.78, 23.59, 23.11, 14.54. MS (ESI) calculated for C₂₃H₄₃N₃O₆, m/z 457.32, found 458.35 (M + H)⁺.

NF-κB induction

The induction of NF-κB was quantified using HEK-Blue^™ cells as previously described by us.^10;51 HEK293 cells stably transfected with human Nod1 and alkaline phosphatase (sAP) were obtained from InvivoGen (San Diego, CA), and were maintained in HEK-Blue^™ Selection medium containing zeocin and normocin. Stable expression of secreted alkaline phosphatase (sAP) under control of NF-κB/AP-1 promoters is inducible by Nod1 agonists, and extracellular sAP in the supernatant is proportional to Nod1-mediated NF-κB induction. HEK-Blue cells were incubated at a density of ~10⁵ cells/mL in a volume of 80 μL/well, in 384-well, flat-bottomed, cell culture-treated microtiter plates until confluency was achieved, and subsequently graded concentrations of compounds. sAP was assayed spectrophotometrically using an alkaline phosphatase-specific chromogen (present in the HEK-detection medium as supplied by the vendor) at 620 nm.

Experiments involving human blood

Human blood was obtained from healthy adults by antecubital venipuncture in accordance with University of Kansas Human Subjects Experimentation protocols (Protocol # HSCL 12397).

Phosflow^™ flow cytometric assay for p38MAPK

Assays were performed as described by us previously.^10;38;52 Briefly, 1 mL aliquots of fresh whole blood, anticoagulated with heparin were incubated with 25 μL an equal volume of graded concentrations of compounds diluted in saline for 15 minutes at 37°C. Erythrocytes were lysed and leukocytes were fixed in one step by mixing 200 μL of the samples in 4 mL pre-warmed Whole Blood Lyse/Fix Buffer (Becton-Dickinson Biosciences, San Jose, CA). After washing the cells at 500 g for 8 minutes in buffer, the cells were permeabilized in ice-cold methanol for 30 min, washed twice in phosphate-buffered saline and transferred to a Millipore MultiScreen BV 1.2μ filter plate and stained with either phycoerythrin (PE)-conjugated mouse anti-p38MAPK (pT180/pY182; BD Biosciences) mAb, or a matched PE-labeled mouse IgG₁ κ isotype control mAb for 60 minutes. The cells were washed twice in the plate by aspiration as per protocols supplied by the vendor. Cytometry was performed using a BD FACSArray instrument in the single-color mode for PE acquisition on 20,000 gated events. Post-acquisition analyses were performed using FlowJo v 7.0 software (Treestar, Ashland, OR).

CD11b flow cytometric assay

Assays were performed as described by us previously.^10;38;52 Briefly, 1 mL aliquots of fresh anticoagulated whole blood were incubated with 25 μL of graded dilutions of the compounds for 1 hour at 37°C. Negative (saline) controls were included in each experiment. Samples were placed on ice for 15 minutes before 20 μL of anti-CD11b/Mac-1 antibody (Becton-Dickinson) were added to each sample tube and allowed to incubate on ice for 30 minutes. This 0°C incubation step prevented internalization of antibody, and ensured staining of only extracellularly expressed CD11b. Erythrocytes were lysed and leukocytes were fixed in one step by mixing 200 μL of the samples in 4 mL pre-warmed Whole Blood Lyse/Fix Buffer (Becton-Dickinson Biosciences, San Jose, CA). After washing the cells twice at 200 g for 5 minutes in CBA buffer, the cells were transferred to a 96-well plate. Flow cytometry was performed using a BD FACSArray instrument in the single-color mode for PE acquisition on 20,000 gated events. Post-acquisition analyses were performed using FlowJo v 7.0 software.

Transcriptomal profiling in whole human blood

Assays were performed as described by us previously.¹⁰ Briefly, peripheral blood mononuclear cells (PBMCs) isolated from fresh, heparin-anticoagulated human blood was stimulated with 20 μg/mL of the compounds for two hours, and total RNA was extracted from treated and negative control PBMC samples with QIAamp RNA Blood Mini Kit (Qiagen). 4 μg of each of the RNA samples was used for transcriptomal profiling, employing the Human Genome GeneChip U133 plus 2.0 oligonucleotide array (Affymetrix, Santa Clara, CA). Established standard protocols at the KU Genomics Facility were performed on cRNA target preparation, array hybridization, washing, staining and image scanning. The microarray data were collected using the Affymetrix GeneChip Command Console Software (AGCC) and then subjected to quality assessment before further analyses. QC criteria included low background, low noise, detection of positive controls, and a 5′/3′ ratio of < 3.0. To facilitate direct comparison of gene expression data between different samples, the GeneChip data were first subjected to preprocessing, which included scaling of data from all chips to a target intensity value of 500 (in Affymetrix Expression Console Software), and further normalization in GeneSpring GX (Agilent Technologies, Santa Clara, CA). Prior to identifying target genes, genes that were detected as non-expressed in all samples, i.e., those with absence (A) cells were filtered out. Genes whose expression was changed by our compounds by at least 2-fold (compared to the negative control) were identified to be differentially expressed. Pathway analyses of gene expression were performed using IPA® (Ingenuity Systems, Redwood, CA).

Abbreviations

AP-1

Activator protein-1

CD

Cluster of differentiation

DAP

Diaminopimelic acid

DMAP

4- Dimethylaminopyridine

EC₅₀

Half-maximal effective concentration

ESI-TOF

Electrospray ionizationtime of flight

FmocCl

Fluorenylmethyloxycarbonyl chloride

Glu

Glutamic Acid

HBTU

O-Benzotriazole- N,N,N,N′-tetramethyl uronium hexafluorophosphate

HEK

Human embryonic kidney

iE-DAP

γ-D-Glutamyl-diaminopimelic acid

IL

Interleukin

NF-κB

Nuclear factor-κB

Nod1

Nucleotide oligomerization domain-1

Nod2

Nucleotide oligomerization domain-2

NLR

Nod like receptor

p38MAPK

p38 Mitogen activated protein kinase

PBMCs

Peripheral blood mononuclear cells

PE

Phycoerythrin

PRRs

Pattern recognition receptors

RIG-I

Retinoic acid inducible gene-I

RNA

Ribonucleic acid

sAP

Secreted alkaline phosphatase

SAR

Structure activity relationship

Th1

Helper T lymphocyte, type 1

Th2

Helper T lymphocyte, type 2

TLR

Toll like receptor

TREM-1

Triggering Receptor Expressed on Myeloid Cells-1