Optimized M24B Aminopeptidase Inhibitors for CARD8 Inflammasome Activation

Abstract

Inflammasomes are innate immune signaling platforms that trigger pyroptotic cell death. NLRP1 and CARD8 are related human inflammasomes that detect similar danger signals, but NLRP1 has a higher activation threshold and triggers a more inflammatory form of pyroptosis. Both sense the accumulation of intracellular peptides with Xaa-Pro N-termini, but Xaa-Pro peptides on their own without a second danger signal only activate the CARD8 inflammasome. We recently reported that a dual inhibitor of the Xaa-Pro-cleaving M24B aminopeptidases PEPD and XPNPEP1 called CQ31 selectively activates the CARD8 inflammasome by inducing the build-up of Xaa-Pro peptides. Here, we performed structure–activity relationship studies on CQ31 to develop the optimized dual PEPD/XPNPEP1 inhibitor CQ80 that more effectively induces CARD8 inflammasome activation. We anticipate that CQ80 will become a valuable tool to study the basic biology and therapeutic potential of selective CARD8 inflammasome activation.

Graphical Abstract

INTRODUCTION

NLRP1 and CARD8 are related intracellular pattern-recognition receptors (PRRs) that detect similar danger-associated signals and assemble into innate immune signaling platforms called inflammasomes (Figure 1).¹ Both inflammasomes recruit and activate the cysteine protease caspase-1 (CASP1), which in turn cleaves and activates the pore-forming protein gasdermin D (GSDMD) to meditate a lytic form of cell death called pyroptosis.^2,3 The NLRP1 inflammasome, but not the CARD8 inflammasome, also activates CASP1 to cleave and activate the cytokines IL-1β and −18 and thereby induces a more inflammatory form of pyroptosis. Consistent with its more inflammatory nature, NLRP1 is more tightly controlled than CARD8.^2,4-7

Figure 1.

Overview of NLRP1 and CARD8 inflammasome activation. The domain structures of human NLRP1 and CARD8 are shown, and the autoproteolysis sites are indicated. The proteasome-mediated degradation of the NT fragments of NLRP1 and CARD8 releases the CT fragments from autoinhibition. CT fragments are then restrained as part of ternary complexes consisting of the CT fragment, a FL PRR, and DPP9. Acceleration of NT degradation and/or disruption of the ternary complex, which can be caused by DPP8/9 inhibition, releases the CT fragments to form an inflammasome. The NLRP1 inflammasome activates a form of CASP1 that cleaves and activates the inflammatory cytokines IL-1β and IL-18 as well as GSDMD. The N-terminus of GSDMD forms pores in the cell membrane, releasing the activated inflammatory cytokines and triggering pyroptosis. The CARD8 inflammasome activates a form of CASP1 that cleaves GSDMD and triggers lytic cell death but does not process pro-IL-1β/18. Potent DPP8/9 inhibitors (e.g., VbP) activate both inflammasomes, whereas weak DPP8/9 inhibitors, such as the Xaa-Pro peptides that accumulate upon inhibition of PEPD and XPNPEP1, only activate the CARD8 inflammasome.

Although they have distinct activation thresholds and inflammatory outputs, NLRP1 and CARD8 have similar domain structures and activation mechanisms. Both proteins undergo autoproteolysis in their function-to-find domains (FIINDs) to create N-terminal (NT) and C-terminal (CT) polypeptide chains that remain non-covalently associated (Figure 1).⁸ In this structure, the autoinhibitory NT fragments prevent the inflammatory CT fragments from self-oligomerizing to nucleate inflammasome formation. The proteasome-mediated degradation of the NT fragment releases the CT fragment from autoinhibition,^9,10 but freed CT fragments are next sequestered in a ternary complex with one copy of the full-length (FL) PRR and one copy of either DPP8 or 9 (DPP8/9).^5,6,11 Potent DPP8/9 inhibitors, including ValboroPro (VbP), destabilize these ternary complexes and thereby activate both inflammasomes (Figure 1).^6,12-15 In addition, DPP8/9 are serine proteases that cleave Xaa-Pro dipeptides (where Xaa is any amino acid) from the N-termini of proteasome-generated polypeptides,¹⁶ and inhibition of this activity also appears to modulate NT degradation through poorly characterized mechanisms (Figure 1).

Humans also have four M24B metallo-aminopeptidases, PEPD, XPNPEP1, 2, and 3, which can cleave the NT amino acid from peptides with proline in the second position. PEPD is thought to primarily cleave Xaa-Pro dipeptides,¹⁷ whereas XPNPEP1-3 catabolizes longer Xaa-Pro-containing substrates.¹⁸ Collectively, these aminopeptidases play key roles in the catabolism of peptides into amino acids and in the maturation and/or degradation of various peptide hormones.¹⁹ We recently reported a small molecule called CQ31 that inhibits the PEPD, and to a lesser extent, XPNPEP1 (we did not assess XPNPEP2 or 3 inhibition because these enzymes were not expressed in the cell types studied) (Figures 1, and 2).⁷ We found that CQ31 causes the accumulation of various Xaa-Pro-containing peptides, which in turn weakly inhibit DPP8/9 in such a way that sufficiently derepresses CARD8 but not NLRP1. Thus, CQ31 selectively activates the CARD8 inflammasome without simultaneously activating the NLRP1 inflammasome.

Figure 2.

Structures of representative inhibitors with (2S,3R)-3-amino-2-hydroxy acid pharmacophores.

Selective CARD8 inflammasome activators are not only useful research tools to study CARD8 biology but also have potential therapeutic values. For example, such agents can in theory kill CARD8-expressing cancer cells without triggering the highly inflammatory NLRP1 response in normal tissues.^7,15 In addition, these compounds could potentially enhance the elimination of HIV-1 reservoirs by stimulating pyroptosis in HIV-1 infected cells.²⁰ In our original report describing the discovery of CQ31, we did not attempt to improve the activity of this lead molecule. Here, we report optimized M24B aminopeptidase inhibitors for CARD8 inflammasome activation.

RESULTS AND DISCUSSION

Chemistry.

To generate a diverse compound library of CQ31 analogues, we designed a highly diastereoselective synthetic strategy using the chemistry of tert-butanesulfinamide (Scheme 1). The enolate addition of methyl 2-((tert-butoxycarbonyl)oxy)acetate to (S)-tert-butanesulfinylimines provided the enantiopure 9 with a (2S,3R) absolute stereochemistry in high yields (66–87%). Notably, this methodology has been used to prepare the side chain of Taxol and has a wide substrate scope.²¹ Sequential hydrolysis of the methyl ester, coupling with proline methyl ester, and global deprotection afforded the NT analogues (Table 1) of CQ31 in good overall yields (52–76%). Moreover, we expanded the CT analogues (Tables 2-4) by using intermediates of CQ31 (11 and 12) with the indicated protecting groups to facilitate different deprotections (Scheme 2).

Scheme 1.

Syntheses of NT Analogues of CQ31

Table 1.

Inhibition of PEPD and XPNPEP1 by NT Analogues^a

Cmpd	R1	PEPD IC50 (μM ± SEM)b	XPNPEP1 IC50 (μM ± SEM)b
CQ31		0.67 ± 0.05	122 ± 2.5
CQ42		7.6 ± 0.74	241 ± 16
CQ43		1.3 ± 0.47	115 ± 8.8
CQ45		1.5 ± 0.28	57 ± 7.8
CQ59		1.8 ± 0.20	190 ± 19
CQ63		0.53 ± 0.06	92 ± 19
CQ75		0.30 ± 0.05	115 ± 9.4
CQ47		12 ± 2.0	33 ± 3.0
CQ62		4.2 ± 0.39	66 ± 8.5
CQ64		2.8 ± 0.69	203 ± 40
CQ68		1.7 ± 0.20	69 ± 9.0
CQ69		1.8 ± 0.23	47 ± 7.1
CQ70		2.0 ± 0.06	65 ± 4.9

^aThe inhibition of recombinant PEPD and XPNPEP1 activities were determined using alanine release and H-Lys(abz)-Pro-Pro-pNA cleavage assays, respectively.

^bIC₅₀ values are means ± SEM of three biological replicates.

Table 2.

Inhibition of PEPD and XPNPEP1 by CT Analogues^a

Cmpd	R2	PEPD IC50 (μM ± SEM)b	XPNPEP1 IC50 (μM ± SEM)b
CQ31		0.67 ± 0.05	122 ± 2.5
CQ35		0.84 ± 0.17	74 ± 15
CQ36		1.0 ± 0.09	324 ± 14
CQ38		2.2 ± 0.74	154 ± 22
CQ39		>100	190 ± 16
CQ76		>100	38 ± 6.0
CQ52		51 ± 0.37	57 ± 2.0
CQ72		>100	96 ± 31
MeBs	-	>100	298 ± 45
CQ49		0.28 ± 0.004	53 ± 5.2
CQ53		>100	12 ± 2.5
CQ55		1.6 ± 0.37	303 ± 79
CQ99		6.2 ± 0.48	67 ± 8.4
CQ95		0.29 ± 0.13	140 ± 13

^aThe inhibition of recombinant PEPD and XPNPEP1 activities were determined using alanine release and H-Lys(abz)-Pro-Pro-pNA cleavage assays, respectively.

^bIC₅₀ values are means ± SEM of three biological replicates.

Table 4.

Inhibition of PEPD and XPNPEP1 by Amide Analogues of CQ80^a

Cmpd	R3	PEPD IC50 (μM ± SEM)b	XPNPEP1 IC50 (μM ± SEM)b
CQ96		7.5 ± 1.4	10 ± 3.2
CQ109		4.0 ± 0.72	11 ± 3.9
CQ110		1.5 ± 0.11	14 ± 4.0
CQ111		14 ± 6.8	84 ± 8.2
CQ112		66 ± 8.7	95 ± 5.8
CQ113		0.56 ± 0.08	2.9 ± 0.48
CQ114		0.72 ± 0.41	7.0 ± 2.9
CQ115		3.7 ± 0.90	14 ± 6.0
CQ116		5.7 ± 1.0	3.4 ± 0.94
CQ117		7.6 ± 2.0	2.2 ±0.10
CQ118		10 ± 2.2	2.0 ± 0.18
CQ119		14 ± 4.3	1.7 ± 0.27

^aInhibition of recombinant PEPD and XPNPEP1 activities was determined using alanine release and H-Lys(abz)-Pro-Pro-pNA cleavage assays, respectively.

^bIC₅₀ values are means ± SEM of three biological replicates.

Scheme 2.

Syntheses of CT Analogues of CQ31

Structure–Activity Relationship Studies.

CQ31 is an esterified (to improve the cell penetrance, discussed below) analogue of the pseudo-dipeptide (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-proline (AHMH-Pro or CQ04, Figure 2). The NT 3-amino-2-hydroxy acid pharmacophore is found in bestatin and amastatin, natural product aminopeptidase inhibitors that were also previously used as the foundation for rational design of the XPNPEP1 inhibitor apstatin (Figure 2).²² CQ31 inhibits PEPD and XPNPEP1 with half-maximal inhibitory concentration (IC₅₀) values of 0.67 and 122 μM, respectively. CQ31’s selectivity for PEPD over XPNPEP1 is not surprising given the substrate preferences of these enzymes (Figure 1). Although CQ31 only weakly inhibits XPNPEP1, we found that XPNPEP1 blockade contributed to CQ31-induced CARD8 activation as knockout of both PEPD and XPNPEP1 was required to generate CQ31-resistant cells.⁷ Based on these data, we reasoned that CQ31 analogues that more potently inhibited both PEPD and XPNPEP1 would more effectively activate the CARD8 inflammasome. As mentioned above, the human genome contains two additional M24B aminopeptidases (XPNPEP2 and 3) that may also contribute to inflammasome repression, but these were not expressed in the cell types we studied previously.⁷

We first synthesized analogues of CQ31 with different NT side chains (Schemes 1 and S1, Table 1). These compounds were then tested for their ability to inhibit the enzymatic activities of purified PEPD and XPNPEP1 as well as to induce cell death in wild-type (WT) MV4;11 cells, which express a functional CARD8 inflammasome. Notably, we also tested the compounds in CASP1 knockout MV4;11 cells to confirm that cell death was due to inflammasome activation. Most of these CQ31 analogues had similar or slightly reduced PEPD and XPNEP1 inhibitory activity (Table 1, Figure S1a,b), and all had reduced cytotoxicity (Figure 3). Notably, three compounds (CQ42, CQ47, and CQ62) were markedly less potent inhibitors of PEPD (IC₅₀s > 4 μM) and almost completely inactive against MV4;11 cells. One of these inactive compounds, CQ47, was the most active of this series against XPNPEP1 (IC₅₀ = 33 μM), further highlighting the importance of potent PEPD blockade. On that note, only one compound, CQ75, retained PEPD inhibition but lost cytotoxicity, likely due to the hydrophilic hydroxyl group compromising cell permeability (Table 1 and Figure 3). Overall, these data indicated that PEPD and XPNPEP1 broadly accommodate many NT side chains, but that modification on this part of the molecule was unlikely to substantially improve bioactivity.

Figure 3.

Cytotoxicity of NT analogues of CQ31. WT and CASP1^−/− MV4;11 cells were treated with the indicated compounds (100 μM to 15.2 nM, three-fold dilutions) for 24 h before CTG analysis.

Instead, we next wanted to explore various CT analogues, including proline ring modifications, other natural amino acids, and carboxylic acid isosteres (Scheme 2). We found that several substituents on the 4-position on the proline ring (CQ35-38) slightly reduced activity, and a cis-4-OH group (CQ39) rendered the compound completely inactive (Table 2 and Figures 4, and S2a,b). As expected, we found that analogues with CT amino acids other than proline (CQ52 and CQ72) were very weak PEPD and XPNPEP1 inhibitors and triggered CASP1-independent cell death similar to bestatin methyl ester (MeBs, Figure 2, Table 2 and Figure 4), likely due to inhibition of aminopeptidases outside the M24B family. In addition, we discovered that removal of the proline carboxylate (CQ76) abrogated all activity. Analogues in which the carboxylate was replaced with amides (CQ49 and CQ95) retained similar PEPD/XPNPEP1 inhibition and cytotoxicity as CQ31, but other carboxylate isosteres (CQ53, CQ55, and CQ99) were completely inactive in cells. These data collectively indicated that the proline in the second position was critical for activity, and furthermore that we were unlikely to find a dipeptide-based inhibitor that was significantly more potent than CQ31.

Figure 4.

Cytotoxicity of CT analogues of CQ31. WT and CASP1^−/− MV4;11 cells were treated with the indicated compounds (100 μM to 15.2 nM, three-fold dilutions) for 24 h before CTG analysis.

As mentioned above, PEPD and XPNPEP1 are thought to cleave the NT amino acids preceding proline dipeptides and longer oligopeptides, respectively.^17,18 We next wanted to verify the substrate preferences of PEPD and XPNPEP1 by measuring the release of NT alanine residues before proline from polypeptides of increasing lengths (i.e., AP, APA, and APPA) (Figure 5a). As expected, PEPD cleaved the AP dipeptide but not the APPA tetrapeptide, whereas XPNPEP1 cleaved the tetrapeptide but not the dipeptide. However, both enzymes cleaved the APA tripeptide to some extent, suggesting that tripeptide-based compounds might be more effective dual PEPD/XPNPEP1 inhibitors and CARD8 inflammasome activators.

Figure 5.

Pseudo-tripeptides analogues of CQ31 are dual PEPD and XPNPEP1 inhibitors. (a) PEPD and XPNPEP1 activities were assessed using the alanine assay as depicted. Briefly, peptides of varying lengths with NT alanines (AP, APA, and APPA) were incubated with PEPD or XPNPEP1, and the released alanine was determined by measuring the H₂O₂ generated after a sequential transformation by alanine transaminase and pyruvate oxidase. (b) Structures of analogues CQ50 and CQ80. The percent inhibition of PEPD and XPNPEP1 enzymatic activity or cell line viability (as determined by CTG) after treatment with the indicated compounds (1 μM for PEPD, MV4;11, and OCI-AML2 assays; and 20 μM for XPNPEP1 assay). (c,d) Inhibition of recombinant PEPD (c) or XPNPEP1 (d) activity by the indicated compounds. (e–g) Viability of MV4;11 WT (e,f) or CASP1^−/− (g) cells after treatment with the indicated compounds (24 h) as assessed by CTG (e, g) or Cytotox-Flour assays (f). (h) OCI-AML2 cells were treated with indicated compounds (6.25 μM) incubated for 6 h before staining with PI. The PI uptake was recorded for 6 h. Data in (b) (n = 3) are means of biological replicates; and data in (c–h) (n = 3) are means ± SEM of biological replicates.

We next synthesized longer peptide-like compounds with the same NT AHMH-Pro residues as CQ31 (Table 3). As expected, the pseudo-tetrapeptides CQ50 (AHMH-Pro-Pro-Ala-OMe) and apstatin (Figure 2) were less potent PEPD inhibitors than CQ31 (IC₅₀s > 10 μM vs 0.67 μM) but were much more potent XPNPEP1 inhibitors (IC₅₀s of 8.1 and 18 μM, respectively, vs 122 μM) (Table 3 and Figure 5b-d). Consistent with their weak PEPD inhibition, CQ50 only activated the CARD8 inflammasome at high doses (IC₅₀ = 35 μM), and apstatin was not cytotoxic at all (Figure 5e,f). In contrast to these pseudo-tetrapeptides, we found that several pseudo-tripeptides (CQ78-CQ81) inhibited PEPD with potencies similar to CQ31 (IC₅₀ values ~ 0.7–1.2 μM) but inhibited XPNPEP1 more effectively than CQ31 (IC₅₀ values = 20–50 μM) (Table 3 and Figure 5b-d). Notably, these pseudo-tripeptides were more cytotoxic than CQ31 to MV4;11 cells as well as OCI-AML2 cells, which also express a functional CARD8 inflammasome.¹⁵ In particular, CQ80 (AHMH-Pro-Leu-OMe) and CQ78 (AHMH-Pro-Val-OMe) induced CASP1-dependent cell death in MV4;11 cells with IC₅₀s of 0.27 and 0.45 μM, respectively, and are therefore approximately an order of magnitude more cytotoxic than CQ31 (IC₅₀ = 3.8 μM) (Table 3 and Figure 5e-g). Consistent with this increased potency, we also observed that CQ78 and CQ80 caused more rapid lytic cell death in OCI-AML2 cells compared to CQ31, as measured by the uptake of propidium iodide (PI, Figure 5h). Thus, these pseudo-tripeptides, and especially CQ80, are more effective inflammasome activators than CQ31.

Table 3.

Inhibition of Enzyme Activity and Cell Viability by CT Analogues^a

cmpd	P1	PEPD IC50 (μM ± SEM)b	XPNPEP1 IC50 (μM ± SEM)b	MV4;11 WT IC50 (μM ± SEM)b	MV4;11 CASP1−/− IC50 (μM)b
CQ31		0.67 ± 0.05	122 ± 2.5	2.2 ± 0.61	>100
apstatin		26 ± 6.4	18 ± 1.7	>20	>20
CQ50	l-Pro-Ala-OMe	11 ± 1.2	8.1 ± 1.1	35 ± 6.5	>100
CQ78	l-Val-OMe	1.2 ± 0.15	41 ± 10	0.45 ± 0.06	>20
CQ79	l-Phe-OMe	0.72 ± 0.05	48 ± 1.6	0.66 ± 0.04	>20
CQ80	l-Leu-OMe	0.91 ± 0.08	43 ± 4.2	0.27 ± 0.03	>20
CQ81	l-Ile-OMe	0.84 ± 0.08	23 ± 6.6	0.60 ± 0.06	>20

^aInhibition of recombinant PEPD and XPNPEP1 activities was determined using alanine release and H-Lys(abz)-Pro-Pro-pNA cleavage assays, respectively. Cell viability was assessed using CTG assay after 24 h treatments.

^bIC₅₀ values are means ± SEM of three biological replicates.

CQ80 Selectively Activates CARD8.

We next sought to confirm that CQ80 indeed induces CARD8 inflammasome activation in several cell types. As expected, we found that both CQ31 and CQ80 induced PI uptake in WT but not CASP1^−/− or CARD8^−/−, MV4;11 cells (Figure 6a). Similarly, both CQ31 and CQ80 induced cell death in WT but not CARD8^−/−, OCI-AML2 cells (Figure 6b). Moreover, CQ31 and CQ80 induced lactate dehydrogenase (LDH) release and GSDMD cleavage, two hallmarks of pyroptosis, in WT, but not CARD8^−/−, MV4;11, OCI-AML2, and THP-1 cells (THP-1 cells also express the CARD8 inflammasome components¹⁵) (Figure 6c-e). Lastly, human resting T cells are a primary cell type with a functional CARD8 inflammasome,²³ and as expected, both CQ31 and CQ80 induced GSDMD cleavage in these cells as well (Figure 6f). Thus, CQ80 activates the CARD8 inflammasome in both cancer cell lines and normal primary cells.

Figure 6.

CQ80 selectively activates the CARD8 inflammasome. (a) Indicated MV4;11 cells were incubated compounds (6.25 μM) for 6 h before monitoring for the PI uptake. (b) Indicated OCI-AML2 cells were treated with CQ31 or CQ80 for 24 h before assessing cell viability by CTG. (c–f) Indicated cells were treated with CQ31 or CQ80 (20 μM) for 24 h before LDH release and immunoblot analyses. (g) N/TERT-1 immortalized keratinocytes were treated with VbP (10 μM) or the indicated CQ compounds (20 μM) for 24 h before CTG analyses. (h,i) BMDMs from C57BL/6J mice (h) or RAW264.7 cells (i) were treated with VbP (10 μM), CQ31 (20 μM), or CQ80 (20 μM) for 24 h before LDH release and/or immunoblot analyses. Data in (a–e,g,i) (n = 3) are means ± SEM of biological replicates. ***p < 0.001, **p < 0.01, and *p < 0.05 by two-sided Students t test. n.s., not significant.

We next wanted to test if CQ80 triggers any NLRP1 inflammasome activation. Human-immortalized N/TERT-1 keratinocytes express a functional human NLRP1, but not a CARD8, inflammasome.^14,24 We therefore treated these cells with VbP (which activates both NLRP1 and CARD8), CQ31, CQ80, and the other pseudo-tetrapeptides before evaluating cell viability. We found that only VbP induced cell death in N/TERT-1 keratinocytes (Figure 6g). We next applied VbP, CQ31, and CQ80 to mouse primary bone marrow-derived macrophages (BMDMs), which express mouse NLRP1A and NLRP1B allele 2,²⁵ and mouse RAW264.7 cells, which express mouse NLRP1B allele 1. Only VbP induced pyroptosis in these mouse cells, as determined by LDH release and GSDMD cleavage assays (Figure 6h,i). It should be noted that rodents do not have a CARD8 homolog. Overall, these data show that CQ80 does not activate the human or rodent NLRP1 inflammasomes.

Mechanistic Studies.

We reasoned that CQ80, like CQ31, inhibits PEPD and XPNPEP1, causing the accumulation of XP-containing peptides that weakly inhibit DPP8/9 and thereby activate CARD8 (Figure 1).⁷ Consistent with this mechanism, we found that neither CQ80 nor CQ31 induced any additional pyroptosis in DPP8/9^−/− THP-1 cells, as determined by LDH release and GSDMD cleavage assays (Figure 7a). In addition, we found that the PEPD and XPNPEP1 single THP-1 cell knockouts were sensitive to CQ80 and CQ31, but that PEPD/XPNPEP1 double knockout THP-1 cells were completely resistant (Figure 7b). Thus, CQ80, like CQ31, blocks both PEPD and XPNPEP1 in cells, leading to the inhibition of DPP8/9 and downstream CARD8 inflammasome activation.

Figure 7.

CQ80 disrupts the CARD8-DPP9 ternary complex. (a,b) Indicated THP-1 cells were incubated compounds CQ31 or CQ80 (20 μM) for 24 h before LDH release and immunoblot analyses. (c) Schematic of the dTAG-based ternary complex disruption assay. Briefly, HEK 293T cells stably expressing CASP1 and GSDMD (HEK 293T^CASP1+GSDMD) are transiently co-transfected with two constructs encoding a (1) chimeric protein in which dTAG (FKBP12 with an F36V mutation) is fused N terminus of CARD8 ZU5-UPA-CARD domains (dTAG-CARD8^ZUC) and the (2) isolated uncleavable CARD8 FIIND^S297A domain. Treatment of these cells with the dTAG13 ligand induces the degradation of the NT fragment of the dTAG-CARD8^ZUC protein, releasing the CARD8 CT to form a ternary complex with a co-expressed FIIND^S297A and endogenous DPP9. Small molecules that disrupt this complex induce the formation of the CARD8 inflammasome and pyroptosis. (d) HEK 293T^CASP1+GSDMD were transiently transfected with plasmids encoding dTAG-CARD8^ZUC and CARD8 FIIND^S297A. Cells were treated with dTAG-13 (500 nM), VbP (10 μM), CQ31 (20 μM), CQ80 (20 μM), or VP-OMe (1 mM) for 6 h before LDH release and immunoblot analyses. (e) CARD8^−/− THP-1 cells stably containing the DOX-inducible CARD8 E274R mutant were treated with or without DOX (100 ng/mL, 16 h) and then treated with VbP (10 μM), CQ31 (20 μM), CQ80 (20 μM), VP-OMe (1 mM), or MeBs (10 μM) for 6 h. Cell death was assessed using LDH release and immunoblot analyses. Data in (a–d) (n = 3) are means ± SEM of biological replicates. ***p < 0.001, **p < 0.01, and *p < 0.05 by two-sided Students t test. n.s., not significant.

We next wanted to determine if these inhibitors primarily activated the CARD8 inflammasome by destabilizing the ternary complex or by accelerating the NT degradation. We previously developed a method to evaluate ternary complex destabilization in cells using the degradation tag (dTAG) system^26,27 (Figure 7c). In this assay, we first generate HEK 293T cells that stably express CASP1 and GSDMD (HEK 293T^CASP1+GSDMD cells). We then ectopically co-express the ZU5-UPA-CARD region of CARD8 with an NT dTAG (dTAG-CARD8^ZUC) together with autoproteolysis-defective S297A mutant CARD8 FIIND domain (FIIND^SA) in these cells. Treatment of these cells with the small molecule dTAG-13 induces the degradation of the NT region of dTAG-CARD8^ZUC, releasing the CARD8^CT to form ternary complexes with endogenous DPP9 and the co-expressed FIIND^SA. As previously reported, we found that VbP destabilizes these ternary complexes and induces pyroptosis (Figure 7d). Similarly, we found that CQ31 and CQ80, as well as high concentrations of the esterified VP-OMe dipeptide itself, similarly induce more pyroptosis after dTAG-13 treatment (Figure 7d). Thus, Xaa-Pro peptides destabilize the DPP9-CARD8 ternary complexes in cells. We should note that NLRP1, but not CARD8, directly interacts with the DPP9 active site in the ternary complex,^5,11,28 and that only VbP, and not Xaa-Pro peptides or CQ31, appreciably destabilizes the DPP9-NLRP1 ternary complex in vitro.⁷ Interestingly, CQ31 synergizes with distinct stresses that accelerate NLRP1 NT degradation to induce more pyroptosis, indicating that Xaa-Pro peptide accumulation contributes to, but is not sufficient for, NLRP1 activation.²⁷

We next wanted to determine if CQ80 also induces CARD8 NT degradation. Before describing these studies, we should note that we recently demonstrated the aminopeptidase inhibitor MeBs profoundly accelerates NT degradation by inducing the accumulation of many (non-XP) peptides that interfere with protein folding and stability.²⁶ As the CQ compounds also induce the accumulation of peptides, albeit with different sequences,⁷ we reasoned that they might accelerate NT degradation via a similar mechanism. We previously developed an assay to assess NT degradation that leverages the CARD8 E274R mutant that does not bind to DPP8/9.^6,26,27 Briefly, we generated CARD8^−/− THP-1 cells containing a doxycycline (DOX)-inducible CARD8 E274R mutant; treatment of these cells with DOX induces pyroptosis as DPP8/9 cannot repress the CARD8 CT that released during normal protein homeostasis. Even though these cells are undergoing pyroptosis, agents that dramatically accelerate NT degradation, like MeBs, still induce considerably more cell death (Figure 7e).²⁶ In contrast, we found that VbP, the CQ compounds, and VP-OMe did not cause substantially more death in this assay, suggesting that these molecules do not dramatically accelerate degradation, at least not to the same extent as MeBs. It should be noted that prior studies have suggested that VbP does accelerate NT degradation (at least over long intervals),^14,26,29 and thus, a more sensitive assay will likely need to be developed to conclusively demonstrate whether these compounds have any impact on NT degradation. Indeed, these data show that CQ31 and CQ80 do not trigger as much degradation as MeBs, perhaps because they do not induce the accumulation of as many peptides.

In Vitro Stability Investigations.

The data above demonstrate that CQ80 is an improved selective CARD8 inflammasome activator. However, we recognized its CT methyl ester, which was added to enable cell penetrance, might be rapidly hydrolyzed in biological systems and limit its bioactivity. Although such esters are not necessarily incompatible with activity in living systems (e.g., MeBs is more active than bestatin in mice⁹), we next wanted to create an active CQ80 analogue that lacked this potential metabolic liability.

We previously showed that CQ31 is completely de-esterified to form CQ04 in cell culture after 24 h.⁷ Here, we first tested the stability of CQ31 in cell culture supernatants, which we reasoned should mimic cell culture. Indeed, we found that CQ31 was rapidly converted to CQ04 within 30 min (Figure S4a,b). We next synthesized CQ31 analogues with tert-butyl and cyclopentyl esters (CQ34 and CQ94, respectively), which are potentially more stable to esterase-mediated hydrolysis. We found that CQ34 was considerably more stable than CQ31 or CQ94 in cell culture supernatants but was nevertheless completely hydrolyzed after 8 h (Figure S4c,d). Interestingly, CQ31, CQ34, and CQ94 all inhibited PEPD and XPNPEP1 similarly, but that CQ34 and CQ94 were slightly more toxic to MV4;11 cells (Figure S4e-g). Thus, these data suggested that increasing stability improved bioactivity.

We next tested CQ80 for stability in cell culture supernatants. We found that it was de-esterified (to the free acid CQ92) far more slowly than CQ31 (~50% remained intact after 24 h), perhaps accounting for some of its increased bioactivity (Figure 8a). Notably, CQ92 is a more potent inhibitor of both PEPD and XPNPEP1 than CQ80 (Figure S4h,i), much like CQ04 is more potent than CQ31. Thus, even though CQ80 is active against PEPD/XPNPEP1 on its own, it is also likely converted to the more active CQ92 inside cells. However, we wanted to test if replacement of the ester was possible. As the tert-butyl and cyclopentyl esters above did not prevent hydrolysis, we instead decided to synthesize amide analogues of CQ80 rather than esters (Table 4). Notably, we found that several amide analogues with aliphatic or aromatic groups at the C-terminus (CQ116-119) were particularly potent XPNPEP1 inhibitors (IC₅₀s < 5 μM), albeit with reduced PEPD inhibitory activity. As expected, we found that a representative of these compounds (CQ116) was completely stable in cell culture supernatants (Figure 8a). Moreover, we found that several of these compounds induced CASP1-dependent pyroptosis at only slightly higher concentrations than CQ80 (Figure 8b). Overall, these data show that CQ80 is relatively more stable than CQ31, and that its C-terminus can be modified to remove the ester without completely abrogating its bioactivity.

Figure 8.

Exploration of CT amide analogues of CQ80. (a) LC/MS analyses of CQ92 (the acid of CQ80), CQ80, and CQ116 (retention times were 2.6 min for CQ92, 2.82 min for CQ80, and 3.24 min for CQ116). CQ80 and CQ116 were incubated in the conditioned medium from MV4;11 cells for 24 h at 37 °C before LC/MS analyses (denoted as “24 h” on diagram). Data in (a) (n = 3) are means ± SEM of biological replicates. (b) Indicated MV4;11 cells were treated with indicated compounds (10 μM to 1.5 nM, three-fold dilutions) for 24 h before CTG analysis.

CONCLUSIONS

Here, we introduce CQ80, an improved dual inhibitor of PEPD/XPNPEP1 that selectively activates the CARD8 inflammasome at high nanomolar concentrations. Although PEPD and XPNPEP1 have evolved to cleave Xaa-Pro peptides of different lengths, we discovered that both enzymes can hydrolase Xaa-Pro-Xaa tripeptides to some extent. Using this knowledge, we designed pseudo-tripeptide inhibitors consisting of the amastatin NT pharmacophore preceding a proline residue and a hydrophobic amino acid. These pseudo-tripeptides sufficiently inhibited both PEPD and XPNPEP1 in cells to rapidly trigger CARD8, but not NLRP1, inflammasome activation. We should note that we were unable to generate highly potent (i.e., low nanomolar) inhibitors against either enzyme, perhaps because they both have broad substrate scopes (except for the proline residue) and therefore do not tightly bind any specific sequences. However, CQ80 effectively activates the CARD8 inflammasome in many cell types, and we anticipate that it will become a valuable research tool to study this important innate immune sensor. Moreover, we expect that CQ80 will form the foundation for future medicinal chemistry studies to develop an inflammasome-activating drug to treat human disease.

EXPERIMENTAL SECTION

Materials and Methods.

All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Chemical reagents were purchased from Aldrich, Acros, or Fisher at the highest commercial quality and used without further purification, unless otherwise stated. Other reagents used for assays include VbP (Tocris 3719), bestatin methyl ester (MeBs; Sigma, 200485), apstatin (SCBT, sc-201309), dTAG-13 (R&D Systems, 6605/5), and FuGENE HD (Promega, E2311). Reactions were monitored by thin-layer chromatography (TLC) carried out on MilliporeSigma glass TLC plates (silica gel 60 coated with F₂₅₄, 250 μm) using UV light for visualization and aqueous ammonium cerium nitrate/ammonium molybdate or basic aqueous potassium permanganate as the developing agent. NMR spectra were recorded on a Bruker Ultrashield Plus Avance III 500 MHz or Bruker Avance III 600 MHz NMR. The spectra were calibrated by using residual undeuterated solvents (for ¹H NMR) and deuterated solvents (for ¹³C NMR) as internal references: undeuterated chloroform (δ_H = 7.26 ppm) and CDCl₃ (δ_C = 77.16 ppm); undeuterated methanol (δ_H = 3.31 ppm) and methanol-d₄ (δ_C = 49.00 ppm). The following abbreviations are used to designate multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad. High-resolution mass spectra (HRMS) were recorded on a Waters Micromass LCT Premier XE TOF liquid chromatography–mass spectrometry (LC–MS). Purity of the compounds were assayed using a Waters Acquity ultraperformance liquid chromatography system equipped with a SQ Detector and an ELS Detector. The gradient solvent consisted of 0.1% TFA and 5–95% acetonitrile in water over 8 min maintaining a constant flow rate of 0.30 mL/min, and all final compounds were ≥95%.

General Synthetic Methods.

Diastereoselective Enolate Addition of Methyl 2-((tert-Butoxycarbonyl)oxy)acetate to (S)-tert-Butanesulfinylimines (Method A).

A solution of methyl 2-((tert-butoxycarbonyl)oxy)acetate 8 (951 mg, 5.0 mmol) in dry tetrahydrofuran (THF) (15 mL) maintained under an atmosphere of argon was cooled to −78 °C and then treated with LiHMDS (5.0 mL, 1.0 M solution in THF, 5.0 mmol). The reaction mixture was stirred for 1 h at the same temperature before imine 7 (1.0 mmol) in THF (1.0 mL) was added slowly. The mixture was allowed to stir for 5 h before it was quenched with saturated aq NH₄Cl (30 mL). The aqueous phase was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous MgSO₄, filtered, and concentrated under vacuum. The residue was passed through a short plug of silica gel with EtOAc/hexane = 1:6, v/v → 1:1, v/v to give the desired methyl ester 9 as a colorless oil, white powder, or solid.

Hydrolysis of the Methyl Ester, Coupling with Proline Methyl Ester and Global Deprotection (Method B).

To the solution of methyl ester 9 (1.0 mmol) in 1,4-dioxane/H₂O (1:1), 100 mL was added NaOH (60 mg, 1.5 mmol), and the reaction mixture was stirred at 22 °C for 1 h. The mixture was acidified to pH 3–4 with Dowex 50W X8 resin. The resin was filtered and washed with CH₂Cl₂. The aqueous phase was extracted with CH₂Cl₂ (3 × 100 mL). The combined organic phases were washed with brine (100 mL), dried over anhydrous MgSO₄, filtered, and concentrated under vacuum to give a colorless oil, which was used for the next step without further purifications. To a solution of crude oil from the last step in CH₂Cl₂ (20 mL) were sequentially added l-proline methyl ester hydrochloride (199 mg, 1.2 mmol), HATU (457 mg, 1.2 mmol), and 4-methylmorpholine (253 mg, 275 μL, 2.5 mmol) at 0 °C. The reaction mixture was allowed to stir for another 3 h before it was quenched by addition of saturated aq NaHCO₃ solution (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL). The organic layers were combined, washed with brine (50 mL), dried over Na₂SO₄, and concentrated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, acetone/hexane = 1:4, v/v → 1:1, v/v) to give the desired amide as a colorless oil. To a stirred solution of the obtained oil in MeOH (30 mL) was added HCl (5 mL, 3.0 M solution in MeOH, 15 mmol) at 0 °C. The reaction mixture was warmed to 22 °C and stirred for 24 h at the same temperature. The mixture was concentrated under vacuum, and the residue was purified by recrystallization from MeOH/diethyl ether to give 10 as a white solid.

Coupling with Proline Methyl Ester and Cbz Deprotection (Method C).

To a solution of carboxylic acid 12 (295 mg, 1.0 mmol) in CH₂Cl₂ (30 mL) were sequentially added l-proline analogues 13 (1.2 mmol), HATU (457 mg, 1.2 mmol), and 4-methylmorpholine (253 mg, 275 μL, 2.5 mmol) at 0 °C. The reaction mixture was allowed to stir for another 4 h before it was quenched by addition of saturated aqueous NaHCO₃ solution (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (3 × 30 mL). The organic layers were combined, washed with brine (30 mL), dried over Na₂SO₄, and concentrated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, acetone/hexanes = 1:4, v/v → 1:1, v/v) to give the desired amide as a colorless oil. To a stirred solution of the obtained oil in MeOH (20 mL) were added sequentially AcOH (50 μL) and Pd/C (53.2 mg, 0.05 mmol, 10 wt %) at 22 °C. The resultant mixture was stirred under H₂ (1 atm) at that temperature for 2 h before it was diluted with EtOAc (30 mL) and passed through a plug of Celite. To the filtrate was added HCl (500 μL, 2.0 M in Et₂O, 1.0 mmol), and the solvent was removed under vacuum. The residue was purified by recrystallization from MeOH/diethyl ether to give 14 as a white solid.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-4-phenylbutanoate (9b; Method A).

The product is obtained as white powder; yield 72%; ¹H NMR (600 MHz, chloroform-d): δ 7.31–7.20 (m, 5H), 4.23 (dd, J = 10.5, 6.9 Hz, 1H), 4.02 (dt, J = 8.6, 6.8 Hz, 1H), 3.63 (s, 3H), 3.50 (dd, J = 14.0, 8.4 Hz, 1H), 3.24 (dd, J = 13.9, 6.6 Hz, 1H), 1.57 (s, 9H), 1.06 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 173.52, 155.36, 137.52, 129.84, 128.72, 127.09, 84.17, 71.57, 60.21, 53.77, 52.18, 35.81, 28.44, 22.70. HRMS (m/z): [M + Na]⁺ calcd for C₂₀H₃₁NO₆NaS⁺, 436.1770; found, 436.1769.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-6,6,6-trifluorohexanoate (9c; Method A).

The product is obtained as white powder; yield 75%; ¹H NMR (600 MHz, chloroform-d): δ 5.01 (d, J = 2.6 Hz, 1H), 3.85 (ddd, J = 10.9, 8.5, 5.9 Hz, 1H), 3.75 (s, 3H), 3.42 (d, J = 10.4 Hz, 1H), 2.55–2.45 (m, 1H), 2.30–2.20 (m, 1H), 1.99 (dddd, J = 14.1, 10.7, 8.5, 5.4 Hz, 1H), 1.96–1.86 (m, 1H), 1.51 (s, 9H), 1.17 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 168.19, 152.63, 129.65, 127.82, 125.99, 124.16, 84.14, 76.36, 58.02, 56.72, 52.65, 31.03, 30.84, 30.64, 30.45, 27.76, 26.50, 26.49, 26.47, 26.45, 22.68. HRMS (m/z): [M + Na]⁺ calcd for C₁₆H₂₈NO₆F₃NaS⁺, 442.1487; found, 442.1486.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-5,5-dimethylhexanoate (9d; Method A).

The product is obtained as white powder; yield 82%; ¹H NMR (600 MHz, chloroform-d): δ 5.62 (s, 1H), 4.31 (dd, J = 11.2, 4.4 Hz, 1H), 4.01 (d, J = 11.8 Hz, 1H), 3.73 (s, 3H), 2.79 (t, J = 13.1 Hz, 1H), 1.53 (s, 9H), 1.40 (d, J = 14.2 Hz, 1H), 1.20 (s, 9H), 1.02 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 173.54, 156.75, 84.48, 73.87, 60.82, 52.09, 48.68, 42.12, 30.52, 30.41, 28.42, 23.38. HRMS (m/z): [M + Na]⁺ calcd for C₁₈H₃₅NO₆NaS⁺, 416.2083; found, 416.2090.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-4-methylpentanoate (9e; Method A).

The product is obtained as white powder; yield 70%; ¹H NMR (600 MHz, chloroform-d): δ 4.62 (s, 1H), 4.33 (t, J = 7.9 Hz, 1H), 3.75 (s, 3H), 3.52 (dd, J = 9.1, 6.4 Hz, 1H), 2.75–2.52 (m, 1H), 1.52 (s, 9H), 1.25 (s, 9H), 1.06 (d, J = 7.0 Hz, 3H), 1.02 (d, J = 6.7 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 174.07, 155.79, 83.84, 76.95, 72.10, 60.32, 58.12, 52.29, 29.05, 28.36, 22.90, 21.78, 21.60. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₃₁NO₆S⁺, 388.1762; found, 388.1770.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)hept-6-enoate (9f; Method A).

The product is obtained as white powder; yield 77%; ¹H NMR (600 MHz, chloroform-d): δ 5.77 (ddd, J = 16.6, 10.4, 7.3 Hz, 1H), 5.08–4.95 (m, 2H), 4.31 (d, J = 6.4 Hz, 1H), 3.77 (dd, J = 10.0, 4.8, 4.3 Hz, 1H), 3.73 (s, 3H), 2.53 (s, 1H), 2.23–2.13 (m, 1H), 2.11–1.99 (m, 1H), 1.71 (dd, J = 9.4, 5.7 Hz, 1H), 1.50 (s, 9H), 1.21 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 173.31, 155.22, 137.01, 115.77, 84.08, 71.86, 60.15, 52.06, 51.05, 31.29, 29.67, 28.24, 22.82. HRMS (m/z): [M + Na]⁺ calcd for C₁₇H₃₁NO₆NaS⁺, 400.1770; found, 400.1780.

Methyl (2S,3R)-3-(Adamantan-1-yl)-2-((tert-butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl) -amino)propanoate (9h; Method A).

The product is obtained as white powder; yield 66%; ¹H NMR (600 MHz, chloroform-d): δ 5.30 (br s, 1H), 3.70 (s, 3H), 3.67–3.63 (m, 1H), 3.42–3.38 (m, 1H), 1.85–1.80 (m, 3H), 2.04–1.99 (m, 3H), 1.71–1.60 (m, 6H), 1.54–1.50 (m, 3H), 1.49 (s, 9H), 1.16 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 169.75, 152.66, 83.37, 74.36, 67.32, 56.93, 52.46, 39.53, 36.77, 36.58, 28.56, 27.85, 22.84. HRMS (m/z): [M + Na]⁺ calcd for C₂₃H₃₉NO₆NaS⁺, 480.2404; found, 480.2396.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-3-cyclopropyl-propanoate (9i; Method A).

The product is obtained as white powder; yield 75%; ¹H NMR (600 MHz, chloroform-d): δ 5.09 (d, J = 2.9 Hz, 1H), 3.72 (s, 3H), 3.57 (d, J = 9.8 Hz, 1H), 2.92 (dd, J = 9.8, 3.0 Hz, 1H), 1.50 (s, 9H), 1.16 (s, 9H), 1.15–1.10 (m, 1H), 0.81–0.74 (m, 1H), 0.68–0.60 (m, 2H), 0.43–0.34 (m, 1H). ¹³C NMR (151 MHz, CDCl₃): δ 168.56, 152.86, 83.59, 77.37, 64.31, 56.47, 52.43, 27.81, 22.65, 14.80, 6.00, 4.93. HRMS (m/z): [M + Na]⁺ calcd for C₁₆H₂₉NO₆NaS⁺, 386.1613; found, 386.1628.

Methyl (2S,3R)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-3-phenylprop-anoate (9j; Method A).

The product is obtained as white powder; yield 87%; ¹H NMR (600 MHz, chloroform-d): δ 7.41 (d, J = 7.4 Hz, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H), 5.24 (d, J = 2.9 Hz, 1H), 4.99 (dd, J = 9.9, 2.9 Hz, 1H), 4.15 (d, J = 9.8 Hz, 1H), 3.77 (s, 3H), 1.41 (s, 9H), 1.18 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 168.40, 152.43, 138.15, 128.80, 128.30, 127.34, 83.69, 77.83, 60.55, 56.92, 52.71, 27.72, 22.61. HRMS (m/z): [M + Na]⁺ calcd for C₁₉H₂₉NO₆NaS⁺, 422.1612; found, 422.1613.

Methyl (2S,3S)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-3-(furan-2-yl)-propanoate (9k; Method A).

The product is obtained as white powder; yield 84%; ¹H NMR (600 MHz, chloroform-d): δ 7.38 (dd, J = 2.0, 0.9 Hz, 1H), 6.51 (dt, J = 3.3, 1.0 Hz, 1H), 6.33 (dd, J = 3.3, 1.8 Hz, 1H), 5.48 (d, J = 2.9 Hz, 1H), 5.02 (ddd, J = 10.7, 2.9, 1.0 Hz, 1H), 3.90 (d, J = 10.7 Hz, 1H), 3.78 (s, 3H), 1.46 (s, 9H), 1.20 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 168.04, 152.40, 151.15, 142.82, 110.82, 109.34, 83.76, 75.65, 56.97, 56.41, 52.76, 27.76, 22.58. HRMS (m/z): [M + Na]⁺ calcd for C₁₇H₂₇NO₇NaS⁺, 412.1421; found, 412.1406.

Methyl (2S,3R)-3-(Benzofuran-6-yl)-2-((tert-butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)-amino)propanoate (9l; Method A).

The product is obtained as white powder; yield 72%; ¹H NMR (600 MHz, chloroform-d): δ 7.60 (d, J = 1.8 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.26 (dd, J = 8.6, 2.0 Hz, 1H), 6.70 (dd, J = 2.2, 0.9 Hz, 1H), 5.19 (d, J = 3.1 Hz, 1H), 5.02 (dd, J = 9.8, 3.0 Hz, 1H), 4.11 (d, J = 9.8 Hz, 1H), 3.70 (s, 3H), 1.32 (s, 9H), 1.12 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 168.44, 154.78, 152.43, 145.74, 132.86, 127.82, 123.61, 120.38, 111.67, 106.96, 83.70, 78.14, 60.68, 56.92, 52.71, 27.71, 22.61. HRMS (m/z): [M + Na]⁺ calcd for C₂₁H₂₉NO₇NaS⁺, 462.1562; found, 462.1560.

Methyl (2S,3S)-2-((tert-Butoxycarbonyl)oxy)-3-(((S)-tert-butylsulfinyl)amino)-3-(thiophen-2-yl)propanoate (9m; Method A).

The product is obtained as white powder; yield 75%; ¹H NMR (600 MHz, chloroform-d): δ 7.41 (dt, J = 2.5, 1.1 Hz, 1H), 7.31 (dd, J = 5.1, 3.0 Hz, 1H), 7.13 (dd, J = 5.1, 1.4 Hz, 1H), 5.37 (d, J = 2.8 Hz, 1H), 5.08 (ddd, J = 10.1, 2.8, 1.0 Hz, 1H), 3.93 (d, J = 10.1 Hz, 1H), 3.77 (s, 3H), 1.45 (s, 9H), 1.19 (s, 9H). ¹³C NMR (151 MHz, CDCl₃): δ 168.29, 152.59, 139.47, 126.67, 126.49, 123.70, 83.80, 76.95, 57.92, 56.87, 52.71, 27.75, 22.65. HRMS (m/z): [M + Na]⁺ calcd for C₁₇H₂₇NO₆NaS₂⁺, 428.1178; found, 428.1193.

Methyl ((2S,3R)-3-Amino-2-hydroxy-4-phenylbutanoyl)-l-prolinate (10b; Method B).

The product is obtained as white powder; yield 57%; HPLC purity: 95.81%; ¹H NMR (600 MHz, methanol-d₄): δ 7.45–7.18 (m, 5H), 4.93 (d, J = 8.1 Hz, 0.17H), 4.49 (dd, J = 20.4, 9.3 Hz, 0.17H), 4.40 (br s, 0.83H), 4.28 (d, J = 9.0 Hz, 0.83H), 3.85–3.76 (m, 1H), 3.70 (s, 3H), 3.29–2.74 (m, 4H), 2.35–1.74 (m, 4H). ¹³C NMR (151 MHz, MeOD, more than 16 ¹³C signals for compound 10b were observed due to the presence of different rotameric species): δ 174.04, 173.92, 170.93, 170.51, 136.75, 130.35, 130.16, 130.10, 130.07, 128.59, 128.49, 69.66, 68.67, 60.56, 60.39, 57.83, 56.35, 53.32, 52.86, 49.57, 48.27, 48.10, 38.91, 35.65, 35.24, 32.14, 29.73, 25.71, 22.73. HRMS (m/z): [M + H]⁺ calcd for C₁₆H₂₃N₂O₄⁺, 307.1658; found, 307.1673.

Methyl ((2S,3R)-3-Amino-6,6,6-trifluoro-2-hydroxyhexanoyl)-l-prolinate (10c; Method B).

The product is obtained as a white solid; yield 55%; HPLC purity: 98.71%; ¹H NMR (600 MHz, methanol-d₄): δ 4.78 (d, J = 8.6 Hz, 0.19H), 4.51 (d, J = 3.7 Hz, 0.81H), 4.49 (dd, J = 8.7, 4.3 Hz, 1H), 3.83 (dd, J = 10.0, 7.2 Hz, 0.81H), 3.77–3.74 (m, 0.81H), 3.74 (s, 0.57H), 3.72 (s, 2.43H), 3.71–3.62 (m, 0.38H), 3.62–3.57 (m, 0.19H), 3.55 (dd, J = 6.9, 3.5 Hz, 0.81H), 2.47–2.33 (m, 2H), 2.34–2.24 (m, 1H), 2.19–1.86 (m, 5H). ¹³C NMR (151 MHz, MeOD, more than 12 ¹³C signals for compound 10c were observed due to the presence of different rotameric species): δ 175.04, 174.11, 171.23, 130.95, 129.11, 127.29, 125.46, 69.79, 68.41, 61.30, 60.69, 53.67, 53.58, 52.93, 49.57, 48.49, 48.39, 32.20, 31.02, 30.82, 30.63, 30.43, 29.95, 25.84, 23.46, 22.44. HRMS (m/z): [M + H]⁺ calcd for C₁₂H₂₀N₂O₄F₃⁺, 313.1375; found, 313.1373.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5,5-dimethylhexanoyl)-l-prolinate (10d; Method B).

The product is obtained as a white solid; yield 67%; HPLC purity: 96.23%; ¹H NMR (600 MHz, methanol-d₄): δ 4.75 (dd, J = 8.7, 1.8 Hz, 0.14H), 4.49 (dd, J = 8.7, 4.5 Hz, 0.86H), 4.42 (d, J = 3.9 Hz, 0.86H), 4.39 (d, J = 2.2 Hz, 0.14H), 3.87–3.81 (m, 1H), 3.74 (s, 0.42H), 3.73 (s, 2.58H), 3.69–3.62 (m, 1H), 3.62–3.57 (m, 0.14H), 3.55 (dd, J = 5.4, 3.9 Hz, 0.86H), 2.35–2.23 (m, 1H), 2.17–1.86 (m, 3H), 1.81 (dd, J = 14.9, 5.4 Hz, 0.86H), 1.78 (dd, J = 14.9, 6.4 Hz, 0.14H), 1.46 (dd, J = 15.0, 5.5 Hz, 0.86H), 1.42 (dd, J = 15.4, 5.1 Hz, 0.14H), 1.02 (s, 7.74H), 1.01 (s, 1.26H). ¹³C NMR (151 MHz, MeOD, more than 14 ¹³C signals for compound 10d were observed due to the presence of different rotameric species): δ 175.25, 174.24, 171.63, 171.17, 72.29, 70.20, 61.41, 60.64, 52.98, 52.97, 52.00, 51.75, 49.57, 48.51, 43.97, 43.76, 31.19, 31.15, 29.94, 29.82, 29.80, 25.85, 22.44. HRMS (m/z): [M + H]⁺ calcd for C₁₄H₂₇N₂O₄⁺, 287.1971; found, 287.1972.

Methyl ((2S,3R)-3-Amino-2-hydroxy-4-methylpentanoyl)-l-prolinate (10e; Method B).

The product is obtained as a white solid; yield 65%; HPLC purity: 96.80%; ¹H NMR (600 MHz, methanol-d₄): δ 4.79 (d, J = 7.8 Hz, 0.17H), 4.63 (d, J = 3.2 Hz, 0.83H), 4.59 (br s, 0.17H), 4.50 (dd, J = 8.7, 4.6 Hz, 0.83H), 3.90–3.83 (m, 0.83H), 3.75 (s, 0.51H), 3.73 (s, 2.49H), 3.74–3.69 (m, 0.83H), 3.67–3.53 (m, 0.34H), 3.33 (br s, 0.17H), 3.19 (dd, J = 7.8, 3.1 Hz, 0.83H), 2.47–2.37 (m, 0.17H), 2.35–2.25 (m, 0.83H), 2.25–1.86 (m, 4H), 1.14 (d, J = 6.6 Hz, 0.51H), 1.10 (d, J = 6.7 Hz, 2.49H), 1.09 (d, J = 6.7 Hz, 2.49H), 1.06 (d, J = 6.7 Hz, 0.51H). ¹³C NMR (151 MHz, MeOD, more than 12 ¹³C signals for compound 10e were observed due to the presence of different rotameric species): δ 175.23, 174.10, 171.97, 171.55, 68.83, 67.08, 61.31, 60.67, 60.18, 60.11, 53.09, 52.97, 48.44, 46.72, 32.28, 29.93, 28.88, 28.78, 25.91, 22.49, 19.57, 19.15, 18.65. HRMS (m/z): [M + H]⁺ calcd for C₁₂H₂₃N₂O₄⁺, 259.1658; found, 259.1654.

Methyl ((2S,3R)-3-(Adamantan-1-yl)-3-amino-2-hydroxypropanoyl)-l-prolinate (10h; Method B).

The product is obtained as a white solid; yield 72%; HPLC purity: 95.74%; ¹H NMR (600 MHz, methanol-d₄): δ 4.80 (d, J = 1.8 Hz, 0.90H), 4.76 (d, J = 9.3 Hz, 0.10H), 4.69 (br s, 0.10H), 4.50 (dd, J = 8.7, 4.8 Hz, 0.90H), 3.86–3.79 (m, 0.90H), 3.78 (s, 0.30H), 3.73 (s, 2.70H), 3.71–3.65 (m, 0.90H), 3.65–3.54 (m, 0.20H), 3.35 (br s, 0.10H), 3.06 (br s, 0.90H), 2.45–2.37 (m, 0.10H), 2.34–2.25 (m, 1H), 2.26–2.17 (m, 0.20H), 2.12–2.02 (m, 5.90H), 2.03–1.96 (m, 1.80H), 1.85–1.63 (m, 10H). ¹³C NMR (151 MHz, MeOD): δ 174.20, 172.55, 64.91, 62.62, 60.73, 52.97, 49.85, 49.57, 39.43, 39.09, 37.49, 37.33, 29.86, 29.55, 25.93. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₃₁N₂O₄⁺, 351.2284; found, 351.2291.

Methyl ((2S,3R)-3-Amino-3-cyclopropyl-2-hydroxypropanoyl)-l-prolinate (10i; Method B).

The product is obtained as a white solid; yield 76%; HPLC purity: 98.96%; ¹H NMR (500 MHz, methanol-d₄): δ 4.79 (d, J = 8.2 Hz, 0.19H), 4.58 (d, J = 4.5 Hz, 0.81H), 4.51 (d, J = 3.6 Hz, 0.19H), 4.48 (dd, J = 8.7, 4.3 Hz, 0.81H), 3.89–3.81 (m, 0.81H), 3.76 (s, 0.57H), 3.80–3.73 (m, 0.81H), 3.72 (s, 2.43H), 3.70–3.52 (m, 0.38H), 2.72 (dd, J = 10.8, 3.7 Hz, 0.19H), 2.65 (dd, J = 10.5, 4.4 Hz, 0.81H), 2.34–2.23 (m, 0.81H), 2.23–2.15 (m, 0.19H), 2.13–1.87 (m, 3H), 1.20–1.07 (m, 1H), 0.80–0.64 (m, 2H), 0.55–0.35 (m, 2H). ¹³C NMR (126 MHz, MeOD, more than 12 ¹³C signals for compound 10i were observed due to the presence of different rotameric species): δ 174.76, 174.09, 171.59, 171.33, 71.51, 70.21, 61.17, 60.71, 60.62, 60.23, 53.08, 52.86, 48.42, 32.20, 29.98, 25.86, 22.64, 11.71, 11.62, 4.94, 4.67, 4.64, 4.30. HRMS (m/z): [M + H]⁺ calcd for C₁₂H₂₁N₂O₄⁺, 257.1501; found, 257.1497.

Methyl ((2S,3R)-3-Amino-2-hydroxy-3-phenylpropanoyl)-l-prolinate (10j; Method B).

The product is obtained as a white solid; yield 67%; HPLC purity: 98.99%; ¹H NMR (600 MHz, methanol-d₄): δ 7.54–7.43 (m, 5H), 4.69 (d, J = 6.4 Hz, 0.81H), 4.56 (d, J = 6.4 Hz, 0.19H), 4.54 (d, J = 6.6 Hz, 0.81H), 4.52 (d, J = 6.7 Hz, 0.19H), 3.73 (s, 0.57H), 3.74–3.67 (m, 0.81H), 3.61 (s, 2.43H), 3.56–3.50 (m, 0.19H), 3.39–3.33 (m, 0.19H), 3.30–3.23 (m, 0.81H), 2.27–2.14 (m, 0.81H), 2.04–1.98 (m, 0.19H), 1.97–1.84 (m, 3.43H), 1.82–1.70 (m, 0.57H). ¹³C NMR (151 MHz, MeOD, more than 15 ¹³C signals for compound 10j were observed due to the presence of different rotameric species): δ 173.86, 173.69, 170.96, 170.89, 135.08, 134.95, 130.69, 130.57, 130.35, 130.24, 129.10, 128.62, 72.81, 71.51, 60.76, 60.61, 59.26, 58.47, 53.23, 52.79, 48.26, 47.93, 31.81, 29.94, 25.77, 22.68. HRMS (m/z): [M + H]⁺ calcd for C₁₅H₂₁N₂O₄⁺, 293.1501; found, 293.1496.

Methyl ((2S,3S)-3-Amino-3-(furan-2-yl)-2-hydroxypropanoyl)-l-prolinate (10k; Method B).

The product is obtained as a white solid; yield 58%; HPLC purity: 95.70%; ¹H NMR (600 MHz, methanol-d₄): δ 7.65 (dd, J = 1.9, 0.8 Hz, 0.18H), 7.64 (dd, J = 1.9, 0.8 Hz, 0.82H), 6.60 (d, J = 3.4 Hz, 0.82H), 6.56 (d, J = 3.5 Hz, 0.18H), 6.51 (dd, J = 3.4, 1.9 Hz, 1H), 4.79 (d, J = 7.0 Hz, 0.82H), 4.72 (d, J = 6.5 Hz, 0.18H), 4.67 (d, J = 7.1 Hz, 0.82H), 4.65 (d, J = 7.1 Hz, 0.18H), 4.43 (dd, J = 8.8, 4.5 Hz, 0.82H), 4.39 (dd, J = 8.3, 2.3 Hz, 0.18H), 3.80–3.76 (m, 0.82H), 3.75 (s, 0.54H), 3.65 (s, 2.46H), 3.62–3.55 (m, 0.18H), 3.49–3.40 (m, 1H), 2.29–2.20 (m, 0.82H), 2.16–2.06 (m, 0.36H), 2.03–1.90 (m, 2.46H), 1.90–1.82 (m, 0.36H). ¹³C NMR (151 MHz, MeOD, more than 13 ¹³C signals for compound 10k were observed due to the presence of different rotameric species): δ 174.16, 173.65, 170.52, 170.37, 147.89, 145.32, 145.21, 112.25, 112.16, 111.90, 111.51, 71.15, 69.71, 60.73, 53.15, 52.76, 52.11, 49.57, 48.19, 48.12, 32.07, 29.98, 25.80, 22.72. HRMS (m/z): [M + H]⁺ calcd for C₁₃H₁₉N₂O₅⁺, 283.1294; found, 283.1296.

Methyl ((2S,3R)-3-Amino-3-(benzofuran-6-yl)-2-hydroxypropanoyl)-l-prolinate (10l; Method B).

The product is obtained as a white solid; yield 57%; HPLC purity: 98.27%; ¹H NMR (600 MHz, methanol-d₄): δ 7.86 (d, J = 2.2 Hz, 0.18H), 7.84 (d, J = 2.2 Hz, 0.82H), 7.81 (d, J = 1.9 Hz, 0.82H), 7.77 (d, J = 1.8 Hz, 0.18H), 7.62 (d, J = 8.6 Hz, 0.18H), 7.61 (d, J = 8.5 Hz, 0.82H), 7.47 (dd, J = 8.6, 1.9 Hz, 0.82H), 7.42 (dd, J = 8.5, 1.8 Hz, 0.18H), 6.93 (d, J = 1.3 Hz, 0.82H), 6.91 (d, J = 1.4 Hz, 0.18H), 4.76 (d, J = 6.9 Hz, 0.82H), 4.66 (d, J = 6.9 Hz, 0.18H), 4.64 (d, J = 6.9 Hz, 0.82H), 4.57 (d, J = 6.9 Hz, 0.18H), 4.38 (dd, J = 8.7, 4.2 Hz, 0.82H), 4.12 (br d, J = 8.6 Hz, 0.18H), 3.73 (s, 0.54H), 3.73–3.63 (m, 1H), 3.62–3.57 (m, 0.18H), 3.50 (s, 2.46H), 3.27–3.20 (m, 0.82H), 2.23–2.14 (m, 0.82H), 2.01–1.80 (m, 2.64H), 1.78–1.61 (m, 0.54H). ¹³C NMR (151 MHz, MeOD, more than 17 ¹³C signals for compound 10l were observed due to the presence of different rotameric species): δ 173.86, 173.60, 171.07, 170.93, 156.66, 156.58, 148.19, 147.89, 129.63, 129.56, 129.54, 125.16, 124.72, 122.53, 121.80, 112.98, 112.89, 107.81, 107.64, 73.00, 71.73, 60.72, 60.57, 59.34, 58.69, 53.22, 52.66, 48.26, 47.89, 31.69, 29.91, 25.76, 25.72, 22.64. HRMS (m/z): [M + H]⁺ calcd for C₁₇H₂₁N₂O₅⁺, 333.1450; found, 333.1442.

Methyl ((2S,3S)-3-Amino-2-hydroxy-3-(thiophen-2-yl)-propanoyl)-l-prolinate (10m; Method B).

The product is obtained as a white solid; yield 52%; HPLC purity: 96.00%; ¹H NMR (600 MHz, methanol-d₄): δ 7.63 (dd, J = 3.1, 1.3 Hz, 0.82H), 7.61 (dd, J = 3.0, 1.3 Hz, 0.18H), 7.57 (dd, J = 5.0, 2.8 Hz, 0.18H), 7.56 (dd, J = 5.0, 2.9 Hz, 0.82H), 7.30 (dd, J = 5.1, 1.3 Hz, 0.82H), 7.23 (dd, J = 5.1, 1.3 Hz, 0.18H), 4.71 (d, J = 6.2 Hz, 0.18H), 4.69 (d, J = 6.3 Hz, 0.82H), 4.67 (d, J = 6.3 Hz, 0.82H), 4.52 (d, J = 6.5 Hz, 0.18H), 4.42 (dd, J = 8.7, 4.2 Hz, 0.82H), 4.24 (dd, J = 8.5, 2.0 Hz, 0.18H), 3.74 (s, 0.54H), 3.76–3.71 (m, 1.82H), 3.66 (s, 2.46H), 3.61–3.53 (m, 0.18H), 3.44–3.39 (m, 0.18H), 3.38–3.35 (m, 0.82H), 2.28–2.19 (m, 0.82H), 2.10–2.05 (m, 0.18H), 2.01–1.88 (m, 2.64H), 1.87–1.77 (m, 0.36H). ¹³C NMR (151 MHz, MeOD, more than 13 ¹³C signals for compound 10m were observed due to the presence of different rotameric species): δ 173.97, 173.74, 171.02, 170.93, 135.72, 135.67, 128.69, 128.30, 127.69, 127.56, 126.50, 125.94, 72.40, 71.09, 60.75, 60.69, 55.13, 54.21, 53.20, 52.82, 48.25, 48.00, 31.95, 29.97, 25.80, 25.70. HRMS (m/z): [M + H]⁺ calcd for C₁₃H₁₉N₂O₄S⁺, 299.1066; found, 299.1052.

Methyl (2S,4S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexano-yl)-4-fluoropyrrolidine-2-carboxylate (14a; Method B).

The product is obtained as a white solid; yield 57%; HPLC purity: 95.09%; ¹H NMR (600 MHz, methanol-d₄): δ 5.46–5.30 (m, 1H), 4.77 (dd, J = 7.0, 4.1 Hz,, 0.85H), 4.74 (dd, J = 7.0, 4.1 Hz, 0.15H), 4.44 (d, J = 3.8 Hz, 0.85H), 4.42 (d, J = 2.2 Hz, 0.15H), 4.12–4.03 (m, 0.85H), 4.03–3.95 (m, 0.85H), 3.95–3.89 (m, 0.15H), 3.87–3.77 (m, 0.15H), 3.75 (s, 0.45H), 3.74 (s, 2.55H), 3.72–3.67 (m, 0.15H), 3.59–3.53 (m, 0.85H), 2.59–2.41 (m, 2H), 1.84–1.46 (m, 3H), 1.01 (d, J = 6.4 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H). ¹³C NMR (151 MHz, MeOD, more than 13 ¹³C signals for compound 14a were observed due to the presence of different rotameric species): δ 174.54, 172.76, 171.78, 171.76, 94.40, 93.22, 92.05, 90.89, 70.62, 69.31, 59.99, 59.13, 55.68, 55.52, 54.96, 54.81, 53.22, 53.06, 52.89, 39.50, 39.32, 38.96, 36.50, 36.36, 30.74, 25.22, 25.16, 23.07, 22.78, 22.58, 22.29. HRMS (m/z): [M + H]⁺ calcd for C₁₃H₂₄N₂O₄F⁺, 291.1720; found, 291.1733.

Methyl (S)-5-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-5-azaspiro[2.4]heptane-6-carboxylate (14b; Method B).

The product is obtained as a white solid; yield 64%; HPLC purity: 97.33%; ¹H NMR (600 MHz, methanol-d₄): δ 4.94 (d, J = 7.5 Hz, 0.15H), 4.65 (dd, J = 8.6, 4.5 Hz, 0.85H), 4.44 (d, J = 5.1 Hz, 0.85H), 4.38 (d, J = 4.7 Hz, 0.15H), 3.81 (dd, J = 10.4, 5.1 Hz, 0.85H), 3.76 (s, 0.45H), 3.74 (s, 2.55H), 3.72–3.63 (m, 0.45H), 3.56 (dd, J = 10.0, 3.0 Hz, 0.85H), 3.50 (dd, J = 8.3, 4.8 Hz, 0.85H), 2.32 (dd, J = 12.8, 8.7 Hz, 1H), 1.88 (dd, J = 12.9, 4.5 Hz, 0.85H), 1.83–1.69 (m, 1.15H), 1.67–1.48 (m, 3H), 1.00 (d, J = 6.5 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.76–0.52 (m, 4H). ¹³C NMR (151 MHz, MeOD, more than 15 ¹³C signals for compound 14b were observed due to the presence of different rotameric species): δ 175.00, 173.72, 171.52, 171.20, 70.28, 69.10, 61.99, 60.85, 55.87, 55.63, 53.15, 53.11, 52.94, 52.91, 40.32, 39.42, 39.15, 37.96, 30.73, 25.19, 25.14, 23.20, 22.83, 22.60, 22.28, 22.24, 22.16, 15.72, 12.28, 9.34, 6.74. HRMS (m/z): [M + H]⁺ calcd for C₁₅H₂₇N₂O₄⁺, 299.1971; found, 299.1968.

Methyl (2S,4S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexano-yl)-4-methylpyrrolidine-2-carboxylate (14c; Method B).

The product is obtained as a white solid; yield 67%; HPLC purity: 96.90%; ¹H NMR (600 MHz, methanol-d₄): δ 4.44 (dd, J = 9.4, 7.9 Hz, 1H), 4.38 (d, J = 4.5 Hz, 1H), 4.08 (t, J = 8.4 Hz, 1H), 3.73 (s, 3H), 3.47 (dt, J = 9.9, 5.3 Hz, 1H), 3.15 (t, J = 10.0 Hz, 1H), 2.50 (dt, J = 13.2, 7.1 Hz, 1H), 2.44–2.35 (m, 1H), 1.77 (dt, J = 13.2, 7.2 Hz, 1H), 1.60 (ddd, J = 14.2, 8.4, 5.9 Hz, 1H), 1.56–1.48 (m, 2H), 1.12 (d, J = 6.5 Hz, 3H), 1.00 (d, J = 6.5 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.21, 171.40, 69.02, 61.12, 55.20, 53.25, 52.91, 39.26, 37.99, 35.16, 25.18, 23.17, 22.18, 16.80. HRMS (m/z): [M + H]⁺ calcd for C₁₄H₂₇N₂O₄⁺, 287.1971; found, 287.1983.

Methyl (2S,4S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexano-yl)-4-hydroxypyrrolidine-2-carboxylate (14d; Method B).

The product is obtained as a white solid; yield 47%; HPLC purity: 97.93%; ¹H NMR (600 MHz, methanol-d₄): δ 4.96 (dd, J = 8.6, 1.8 Hz, 0.16H), 4.64 (dd, J = 9.3, 3.5 Hz, 0.84H), 4.51–4.45 (m, 0.84H), 4.43 (d, J = 4.1 Hz, 0.84H), 4.41–4.38 (m, 0.16H), 4.38 (d, J = 2.9 Hz, 0.16H), 3.99 (dd, J = 11.0, 5.0 Hz, 1H), 3.76 (s, 0.48H), 3.73 (s, 2.52H), 3.67 (dd, J = 7.2, 2.8 Hz, 0.16H), 3.57 (dd, J = 10.8, 2.0 Hz, 1H), 3.55–3.52 (m, 0.84H), 2.40 (ddd, J = 13.8, 9.4, 4.7 Hz, 1H), 2.17–2.09 (m, 1H), 1.83–1.74 (m, 1H), 1.74–1.62 (m, 1H), 1.60–1.51 (m, 1H), 1.02 (d, J = 6.6 Hz, 2.52H), 1.00 (d, J = 6.5 Hz, 2.52H), 0.99 (d, J = 6.6 Hz, 0.48H), 0.98 (d, J = 6.6 Hz, 0.48H). ¹³C NMR (151 MHz, MeOD, more than 13 ¹³C signals for compound 14d were observed due to the presence of different rotameric species): δ 175.18, 173.52, 172.11, 171.91, 70.86, 70.44, 69.07, 68.55, 59.99, 59.19, 56.93, 55.88, 53.10, 52.94, 40.59, 39.60, 39.30, 38.07, 25.24, 25.21, 23.11, 22.85, 22.50, 22.31. HRMS (m/z): [M + H]⁺ calcd for C₁₃H₂₅N₂O₅⁺, 289.1763; found, 289.1767.

(2S,3R)-3-Amino-2-hydroxy-5-methyl-1-(pyrrolidin-1-yl)hexan-1-one (14e; Method B).

The product is obtained as a white solid; yield 77%; HPLC purity: 99.30%; ¹H NMR (600 MHz, chloroform-d): δ 4.86 (d, J = 3.3 Hz, 1H), 4.25–4.17 (m, 1H), 4.11–4.03 (m, 1H), 4.04–3.97 (m, 1H), 3.99–3.91 (m, 2H), 3.80 (dd, J = 3.4, 1.8 Hz, 1H), 2.54–2.45 (m, 2H), 2.44–2.35 (m, 1H), 2.29–2.19 (m, 1H), 2.7– 2.00 (m, 2H), 1.47 (d, J = 6.6 Hz, 6H). ¹³C NMR (151 MHz, MeOD): δ 171.26, 68.76, 52.87, 47.77, 47.38, 39.62, 26.99, 25.19, 24.97, 22.98, 22.51. HRMS (m/z): [M + H]⁺ calcd for C₁₁H₂₃N₂O₂⁺, 215.1760; found, 215.1762.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-leucinate (14f; Method B).

The product is obtained as a white solid; yield 75%; HPLC purity: 99.30%; ¹H NMR (600 MHz, methanol-d₄): δ 4.49 (dd, J = 9.6, 4.8 Hz, 1H), 4.20 (d, J = 4.1 Hz, 1H), 3.73 (s, 3H), 3.49 (ddd, J = 8.1, 6.2, 4.1 Hz, 1H), 1.81–1.61 (m, 5H), 1.53–1.44 (m, 1H), 0.99 (d, J = 6.5 Hz, 3H), 0.98 (d, J = 4.9 Hz, 3H), 0.97 (d, J = 4.9 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.35, 173.60, 71.12, 53.20, 52.84, 52.18, 41.14, 39.22, 26.02, 25.25, 23.19, 22.94, 22.30, 21.95. HRMS (m/z): [M + H]⁺ calcd for C₁₄H₂₉N₂O₄⁺, 289.2127; found, 289.2125.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-phenylalaninate (14g; Method B).

The product is obtained as a white solid; yield 72%; HPLC purity: 98.46%; ¹H NMR (600 MHz, methanol-d₄): δ 7.32–7.21 (m, 5H), 4.73 (dd, J = 8.3, 5.7 Hz, 1H), 4.18 (d, J = 3.8 Hz, 1H), 3.71 (s, 3H), 3.44 (ddd, J = 7.9, 6.2, 3.7 Hz, 1H), 3.20 (dd, J = 13.9, 5.8 Hz, 1H), 3.13 (dd, J = 13.9, 8.3 Hz, 1H), 1.74–1.65 (m, 1H), 1.54 (ddd, J = 14.3, 8.1, 6.3 Hz, 1H), 1.35 (ddd, J = 14.3, 7.9, 6.4 Hz, 1H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 173.34, 173.14, 137.91, 130.24, 129.67, 128.12, 70.80, 54.86, 53.16, 52.87, 38.86, 38.01, 25.23, 22.96, 22.36. HRMS (m/z): [M + H]⁺ calcd for C₁₇H₂₇N₂O₄⁺, 323.1971; found, 323.1982.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-pyrrolidine-2-carboxamide (14h; Method C).

The product is obtained as a white solid; yield 45%; HPLC purity: 99.23%; ¹H NMR (500 MHz, methanol-d₄): δ 4.45 (d, J = 2.9 Hz, 1H), 4.43 (dd, J = 8.2, 4.6 Hz, 1H), 3.82 (dt, J = 10.1, 6.5 Hz, 1H), 3.66 (dt, J = 10.1, 6.5 Hz, 1H), 3.54 (dd, J = 7.2, 3.2 Hz, 1H), 2.34–2.22 (m, 1H), 2.12–1.95 (m, 3H), 1.83–1.73 (m, 1H), 1.69–1.60 (m, 1H), 1.56–1.47 (m, 1H), 1.01 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 176.96, 171.91, 68.79, 61.51, 52.71, 39.67, 38.88, 30.98, 25.94, 25.21, 22.89, 22.64. HRMS (m/z): [M + H]⁺ calcd for C₁₂H₂₄N₃O₃⁺, 258.1818; found, 258.1812.

N-(((S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-pyrrolidin-2-yl)methyl)methanesulfon-amide (14i; Method C).

The product is obtained as a white solid; yield 62%; HPLC purity: 98.32%; ¹H NMR (600 MHz, methanol-d₄): δ 4.42 (d, J = 2.2 Hz, 1H), 4.20–4.14 (m, 1H), 3.77 (dt, J = 10.2, 6.9 Hz, 1H), 3.59–3.51 (m, 3H), 3.14 (dt, J = 13.9, 2.8 Hz, 1H), 2.93 (s, 3H), 2.19–2.09 (m, 1H), 2.8–2.00 (m, 1H), 1.99–1.89 (m, 2H), 1.81–1.72 (m, 1H), 1.70–1.62 (m, 1H), 1.59–1.48 (m, 1H), 1.00 (d, J = 6.5 Hz, 6H). ¹³C NMR (151 MHz, MeOD): δ 171.97, 68.12, 58.96, 52.72, 48.51, 44.58, 40.06, 39.72, 28.23, 25.22, 25.18, 22.75, 22.71. HRMS (m/z): [M + H]⁺ calcd for C₁₃H₂₈N₃O₄S⁺, 322.1801; found, 322.1798.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-methoxypyrrolidine-2-carboxamide (14j; Method C).

The product is obtained as a white solid; yield 51%; HPLC purity: 95.63%; ¹H NMR (500 MHz, methanol-d₄): δ 4.42 (d, J = 3.5 Hz, 1H), 4.26 (dd, J = 8.0, 5.5 Hz, 1H), 3.87–3.77 (m, 1H), 3.70 (s, 3H), 3.70–3.64 (m, 1H), 3.54–3.48 (m, 1H), 2.28–2.19 (m, 1H), 2.16–2.07 (m, 1H), 2.06–1.94 (m, 2H), 1.87–1.70 (m, 2H), 1.66–1.56 (m, 1H), 1.56–1.46 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 171.90, 171.32, 69.00, 64.34, 59.64, 52.79, 48.57, 39.65, 30.74, 26.01, 25.23, 22.99, 22.50. HRMS (m/z): [M + H]⁺ calcd for C₁₃H₂₆N₃O₄⁺, 288.1923; found, 288.1916.

(2S,3R)-1-((S)-2-(1H-Tetrazol-5-yl)pyrrolidin-1-yl)-3-amino-2-hydroxy-5-methylhexan-1-one (14k; Method C).

The product is obtained as a white solid; yield 38%; HPLC purity: 96.40%; ¹H NMR (600 MHz, methanol-d₄): δ 5.43 (dd, J = 8.2, 3.2 Hz, 1H), 4.45 (d, J = 3.2 Hz, 1H), 3.95–3.84 (m, 2H), 3.51 (dd, J = 7.2, 3.2 Hz, 1H), 2.47–2.38 (m, 1H), 2.22–2.10 (m, 3H), 1.77–1.68 (m, 1H), 1.60–1.48 (m, 2H), 0.99 (d, J = 6.5 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 171.96, 68.97, 53.33, 52.77, 48.33, 39.54, 32.07, 25.67, 25.17, 22.94, 22.45. HRMS (m/z): [M + H]⁺ calcd for C₁₂H₂₃N₆O₂⁺, 283.1882; found, 283.1884.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-isopentylpyrrolidine-2-carboxamide (14l; Method C).

The product is obtained as a white solid; yield 53%; HPLC purity: 95.08%; ¹H NMR (600 MHz, methanol-d₄): δ 4.39 (d, J = 3.2 Hz, 1H), 4.39–4.37 (m, 1H), 3.80 (dt, J = 10.0, 6.8 Hz, 1H), 3.64 (dt, J = 10.0, 6.9 Hz, 1H), 3.45–3.41 (m, 1H), 3.26–3.18 (m, 2H), 2.28–2.21 (m, 1H), 2.11–2.03 (m, 1H), 2.01–1.93 (m, 1H), 1.92–1.86 (m, 1H), 1.81–1.75 (m, 1H), 1.66–1.57 (m, 2H), 1.50–1.42 (m, 1H), 1.43–1.37 (m, 2H), 1.00 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.6 Hz, 6H). ¹³C NMR (151 MHz, MeOD): δ 179.79, 174.19, 61.91, 52.44, 49.57, 48.65, 39.34, 38.76, 30.87, 26.83, 26.08, 25.38, 23.88, 23.05, 22.83, 22.81, 22.71. HRMS (m/z): [M + H]⁺ calcd for C₁₇H₃₄N₃O₃⁺, 328.2600; found, 328.2596.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-prolyl-l-prolyl-l-alaninate (14m; Method C).

The product is obtained as a white solid; yield 37%; HPLC purity: 97.72%; ¹H NMR (600 MHz, methanol-d₄): δ 4.73 (dd, J = 8.5, 4.7 Hz, 1H), 4.47–4.43 (m, 2H), 4.37 (dd, J = 14.7, 7.3 Hz, 1H), 3.85–3.78 (m, 2H), 3.71 (s, 3H), 3.69–3.61 (m, 2H), 3.54 (td, J = 7.2, 2.9 Hz, 1H), 2.40–2.30 (m, 1H), 2.28–2.17 (m, 1H), 2.16–1.96 (m, 6H), 1.82–1.72 (m, 1H), 1.71–1.62 (m, 1H), 1.56–1.47 (m, 1H), 1.38 (d, J = 7.3 Hz, 3H), 1.00 (d, J = 5.4 Hz, 3H), 0.99 (d, J = 5.3 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.54, 174.06, 172.45, 171.54, 68.57, 61.28, 60.19, 52.73, 48.67, 48.48, 39.40, 30.42, 29.23, 25.92, 25.88, 25.20, 22.79, 22.66, 17.25. HRMS (m/z): [M + H]⁺ calcd for C₂₁H₃₇N₄O₆⁺, 441.2713; found, 441.2694.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-prolyl-l-valinate (14n; Method C).

The product is obtained as white powder; yield 57%; HPLC purity: 95.54%; ¹H NMR (600 MHz, methanol-d₄): δ 4.54 (dd, J = 8.3, 5.3 Hz, 1H), 4.41 (d, J = 3.4 Hz, 1H), 4.38 (d, J = 5.9 Hz, 1H), 3.83–3.76 (m, 1H), 3.71 (s, 3H), 3.68–3.61 (m, 1H), 3.46 (ddd, J = 7.6, 6.6, 3.4 Hz, 1H), 2.32–2.23 (m, 1H), 2.11–2.03 (m, 1H), 2.02–1.86 (m, 3H), 1.82–1.72 (m, 1H), 1.60 (ddd, J = 14.1, 7.6, 6.7 Hz, 1H), 1.56–1.41 (m, 2H), 1.32–1.23 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.56, 173.46, 172.03, 69.50, 61.46, 58.39, 52.64, 52.42, 48.62, 40.14, 38.43, 30.60, 26.32, 26.01, 25.31, 23.01, 22.61, 16.05. HRMS (m/z): [M + H]⁺ calcd for C₁₈H₃₄N₃O₅⁺, 372.2498; found, 372.2513.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-prolyl-l-phenylalaninate (14o; Method C).

The product is obtained as a white solid; yield 52%; HPLC purity: 99.54%; ¹H NMR (600 MHz, methanol-d₄): δ 7.32–7.27 (m, 2H), 7.26–7.17 (m, 3H), 4.66 (dd, J = 7.7, 6.1 Hz, 1H), 4.47 (dd, J = 8.5, 5.0 Hz, 1H), 4.42 (d, J = 3.0 Hz, 1H), 3.81–3.74 (m, 1H), 3.67 (s, 3H), 3.67–3.60 (m, 1H), 3.48 (dd, J = 7.2, 3.0 Hz, 1H), 3.13 (dd, J = 13.9, 6.1 Hz, 1H), 3.05 (dd, J = 13.9, 7.7 Hz, 1H), 2.27–2.18 (m, 1H), 2.06–1.87 (m, 3H), 1.82–1.71 (m, 1H), 1.67–1.58 (m, 1H), 1.53–1.45 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.28, 172.06, 137.98, 130.36, 129.49, 127.90, 69.13, 61.56, 55.44, 52.65, 52.64, 48.53, 40.05, 38.27, 30.54, 25.90, 25.28, 22.96, 22.66. HRMS (m/z): [M + H]⁺ calcd for C₂₂H₃₄N₃O₅⁺, 420.2498; found, 420.2510.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-prolyl-l-leucinate (14p; Method C).

The product is obtained as a white solid; yield 55%; HPLC purity: 98.50%; ¹H NMR (600 MHz, methanol-d₄): δ 4.49 (dd, J = 8.7, 5.0 Hz, 1H), 4.45 (dd, J = 9.2, 6.0 Hz, 1H), 4.42 (d, J = 3.4 Hz, 1H), 3.83–3.77 (m, 1H), 3.71 (s, 3H), 3.69–3.63 (m, 1H), 3.48 (dd, J = 7.2, 3.3 Hz, 1H), 2.32–2.24 (m, 1H), 2.11–2.03 (m, 1H), 2.03–1.94 (m, 2H), 1.82–1.72 (m, 2H), 1.67–1.58 (m, 3H), 1.52–1.45 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.53, 174.51, 172.02, 69.37, 61.50, 52.67, 52.66, 52.23, 48.61, 41.42, 40.07, 30.59, 25.98, 25.85, 25.30, 23.32, 22.99, 22.62, 21.84. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₃₆N₃O₅⁺, 386.2655; found, 386.2640.

Methyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-prolyl-l-isoleucinate (14q; Method C).

The product is obtained as a white solid; yield 58%; HPLC purity: 95.15%; ¹H NMR (600 MHz, methanol-d₄): δ 4.55 (dd, J = 8.4, 5.0 Hz, 1H), 4.45 (d, J = 3.4 Hz, 1H), 4.33 (d, J = 5.8 Hz, 1H), 3.85–3.79 (m, 1H), 3.72 (s, 3H), 3.70–3.64 (m, 1H), 3.53 (dd, J = 7.2, 3.4 Hz, 1H), 2.32–2.25 (m, 1H), 2.21–2.12 (m, 1H), 2.11–2.03 (m, 1H), 2.02–1.92 (m, 3H), 1.84–1.70 (m, 2H), 1.68–1.58 (m, 1H), 1.57–1.46 (m, 1H), 1.00 (d, J = 6.5 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.66, 173.43, 171.72, 68.77, 61.49, 59.39, 52.83, 52.49, 48.65, 39.54, 31.85, 30.67, 25.96, 25.22, 22.91, 22.56, 19.55, 18.52. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₃₆N₃O₅⁺, 386.2655; found, 386.2648.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-amino-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14r; Method C).

The product is obtained as a white solid; yield 45%; HPLC purity: 99.24%; ¹H NMR (600 MHz, methanol-d₄): δ 4.47 (d, J = 2.9 Hz, 1H), 4.45 (dd, J = 8.7, 4.8 Hz, 1H), 4.39 (dd, J = 10.4, 4.7 Hz, 1H), 3.86–3.79 (m, 1H), 3.73–3.67 (m, 1H), 3.57 (dd, J = 7.2, 2.8 Hz, 1H), 2.33–2.24 (m, 1H), 2.11–2.03 (m, 1H), 2.03–1.94 (m, 2H), 1.81–1.70 (m, 2H), 1.71–1.61 (m, 2H), 1.62–1.54 (m, 1H), 1.57–1.49 (m, 1H), 1.01 (d, J = 7.0 Hz, 3H), 0.98 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 177.44, 174.34, 171.95, 68.68, 61.95, 52.97, 52.80, 42.00, 39.47, 30.72, 25.99, 25.90, 25.19, 23.56, 22.92, 22.62, 21.86. HRMS (m/z): [M + H]⁺ calcd for C₁₈H₃₅N₄O₄⁺, 371.2658; found, 371.2660.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-(dimethylamino)-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14s; Method C).

The product is obtained as a white solid; yield 44%; HPLC purity: 98.75%; ¹H NMR (500 MHz, methanol-d₄): δ 4.88 (dd, J = 10.3, 4.1 Hz, 1H), 4.54–4.41 (m, 1H), 4.26 (d, J = 3.6 Hz, 1H), 3.79–3.67 (m, 1H), 3.64–3.54 (m, 1H), 3.15–3.12 (m, 1H), 3.12 (s, 3H), 2.93 (s, 3H), 2.29–2.17 (m, 1H), 2.09–1.98 (m, 1H), 1.98–1.85 (m, 2H), 1.84–1.70 (m, 2H), 1.65–1.54 (m, 1H), 1.51–1.37 (m, 2H), 1.36–1.26 (m, 1H), 0.97 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 174.26, 174.02, 173.60, 73.13, 61.54, 51.73, 48.53, 43.17, 41.93, 37.51, 36.16, 30.37, 26.27, 25.74, 25.68, 23.73, 23.52, 22.85, 21.96. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₃₉N₄O₄⁺, 399.2971; found, 399.2979.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-(isopentylamino)-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14t; Method C).

The product is obtained as a white solid; yield 42%; HPLC purity: 96.48%; ¹H NMR (600 MHz, methanol-d₄): δ 4.47 (d, J = 2.9 Hz, 1H), 4.47–4.43 (m, 1H), 4.35 (dd, J = 9.9, 5.4 Hz, 1H), 3.84–3.78 (m, 1H), 3.72–3.66 (m, 1H), 3.57 (dd, J = 7.1, 2.8 Hz, 1H), 3.24 (dd, J = 12.8, 7.4 Hz, 1H), 3.16 (dd, J = 13.3, 7.2 Hz, 1H), 2.33–2.22 (m, 1H), 2.10–1.90 (m, 3H), 1.82–1.57 (m, 5H), 1.57–1.48 (m, 2H), 1.47–1.36 (m, 2H), 1.01 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.43, 174.23, 171.86, 68.69, 61.85, 53.39, 52.79, 48.68, 42.17, 39.49, 39.30, 38.68, 30.75, 26.83, 25.96, 25.90, 25.20, 23.43, 22.89, 22.82, 22.79, 22.64, 22.10. HRMS (m/z): [M + H]⁺ calcd for C₂₃H₄₅N₄O₄⁺, 441.3441; found, 441.3448.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-(methoxyamino)-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14u; Method C).

The product is obtained as a white solid; yield 32%; HPLC purity: 97.35%; ¹H NMR (600 MHz, methanol-d₄): δ 4.46 (dd, J = 8.2, 6.2 Hz, 1H), 4.28 (d, J = 3.4 Hz, 1H), 4.27–4.24 (m, 1H), 3.78–3.73 (m, 1H), 3.59 (dd, J = 9.9, 7.2 Hz, 1H), 3.15 (ddd, J = 7.6, 6.2, 3.5 Hz, 1H), 2.30–2.22 (m, 1H), 2.10–2.02 (m, 1H), 1.98–1.85 (m, 2H), 1.83–1.69 (m, 2H), 1.68–1.59 (m, 1H), 1.57–1.48 (m, 1H), 1.49–1.41 (m, 1H), 1.36–1.24 (m, 4H), 0.97 (d, J = 6.6 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 174.22, 173.71, 171.18, 72.93, 64.20, 61.68, 51.77, 50.85, 49.57, 43.10, 41.99, 30.41, 26.29, 25.72, 23.47, 23.28, 22.86, 22.20. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₃₇N₄O₅⁺, 401.2764; found, 401.2764.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-(azetidin-1-yl)-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14v; Method C).

The product is obtained as a white solid; yield 27%; HPLC purity: 96.41%; ¹H NMR (600 MHz, methanol-d₄): δ 4.50–4.48 (m, 1H), 4.48–4.46 (m, 1H), 4.46–4.40 (m, 1H), 4.32–4.25 (m, 1H), 4.29 (d, J = 3.6 Hz, 1H), 4.05–3.94 (m, 2H), 3.79–3.72 (m, 1H), 3.64–3.57 (m, 1H), 3.18–3.14 (m, 1H), 2.35–2.22 (m, 2H), 2.09–2.00 (m, 1H), 1.99–1.85 (m, 1H), 1.84–1.73 (m, 1H), 1.67–1.52 (m, 2H), 1.51–1.43 (m, 1H), 1.38–1.31 (m, 1H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, MeOD): δ 173.79, 173.71, 173.27, 61.60, 52.16, 51.80, 49.57, 48.43, 41.51, 30.52, 26.25, 25.83, 25.73, 23.50, 23.48, 22.85, 22.11, 16.27. HRMS (m/z): [M + H]⁺ calcd for C₂₁H₃₉N₄O₄⁺, 411.2971; found, 411.2981.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-((N,N-dimethylsulfamoyl)-amino)-4-methyl-1-oxopentan-2-yl)-pyrrolidine-2-carboxamide (14w; Method C).

The product is obtained as a white solid; yield 29%; HPLC purity: 98.14%; ¹H NMR (600 MHz, methanol-d₄): δ 4.61–4.58 (m, 0.21H), 4.48 (d, J = 2.6 Hz, 0.79H), 4.44 (dd, J = 8.5, 4.8 Hz, 0.79H), 4.38 (d, J = 9.3 Hz, 0.21H), 4.35 (dd, J = 9.5, 5.4 Hz, 0.79H), 4.26 (d, J = 4.0 Hz, 0.21H), 3.79 (dd, J = 10.3, 6.3 Hz, 0.79H), 3.69 (dd, J = 10.0, 5.9 Hz, 1H), 3.62 (dd, J = 7.2, 2.5 Hz, 0.79H), 3.57 (ddd, J = 12.0, 10.1, 7.1 Hz, 0.21H), 3.53–3.46 (m, 0.21H), 2.79 (s, 4.74H), 2.76 (s, 1.26H), 2.33–2.24 (m, 1H), 2.23–2.16 (m, 0.21H), 2.10–1.94 (m, 2.79H), 1.84–1.70 (m, 2H), 1.70–1.63 (m, 1H), 1.63–1.48 (m, 3H), 1.02 (d, J = 4.7 Hz, 2.37H), 1.01 (d, J = 4.4 Hz, 2.37H), 0.98 (d, J = 4.7 Hz, 0.63H), 0.96 (d, J = 4.4 Hz, 0.63H), 0.96 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H). ¹³C NMR (151 MHz, MeOD, more than 20 ¹³C signals for compound 14w were observed due to the presence of different rotameric species): δ 178.25, 177.89, 174.50, 173.78, 172.62, 172.21, 70.32, 68.44, 63.26, 62.22, 55.44, 54.80, 52.86, 52.67, 49.57, 48.79, 43.07, 42.72, 39.76, 39.53, 39.10, 39.00, 33.04, 30.63, 26.45, 26.08, 26.00, 25.34, 25.23, 23.68, 23.66, 22.96, 22.88, 22.84, 22.78, 22.45, 22.12, 21.80. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₄₀N₅O₆S⁺, 478.2699; found, 478.2688.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-((2-fluoroethyl)amino)-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14x; Method C).

The product is obtained as a white solid; yield 47%; HPLC purity: 96.66%; ¹H NMR (600 MHz, methanol-d₄): δ 4.51–4.47 (m, 1H), 4.45 (d, J = 2.9 Hz, 1H), 4.44–4.38 (m, 3H), 3.81 (dt, J = 10.2, 6.7 Hz, 1H), 3.68 (dt, J = 10.1, 7.0 Hz, 1H), 3.60–3.44 (m, 3H), 2.28 (dt, J = 13.4, 7.2 Hz, 1H), 2.10–1.90 (m, 2H), 1.82–1.46 (m, 7H), 1.01 (d, J = 6.6 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 175.07, 174.29, 174.13, 83.73, 82.40, 73.08, 61.96, 53.21, 51.71, 43.57, 41.91, 41.16, 40.99, 30.42, 26.34, 25.85, 25.75, 23.48, 22.92, 21.96. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₃₈N₄O₄F⁺, 417.2877; found, 417.2889.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-1-(tert-butylamino)-4-methyl-1-oxopentan-2-yl)pyrrolidine-2-carboxamide (14y; Method C).

The product is obtained as a white solid; yield 51%; HPLC purity: 96.71%; ¹H NMR (600 MHz, methanol-d₄): δ 4.45 (d, J = 2.4 Hz, 1H), 4.44–4.42 (m, 0.75H), 4.41 (dd, J = 8.3, 5.7 Hz, 0.25H), 4.37–4.27 (m, 1H), 3.86–3.76 (m, 1H), 3.68 (dt, J = 10.0, 6.7 Hz, 1H), 3.61–3.52 (m, 1H), 2.31–2.20 (m, 1H), 2.13–1.90 (m, 3H), 1.79–1.61 (m, 3H), 1.61–1.47 (m, 3H), 1.34 (s, 2.25H), 1.32 (s, 6.75H), 1.01 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.6 Hz, 2.25H), 0.91 (d, J = 6.6 Hz, 0.75H). ¹³C NMR (126 MHz, MeOD, more than 22 ¹³C signals for compound 14y were observed due to the presence of different rotameric species): δ 174.08, 174.05, 173.91, 173.81, 173.75, 173.52, 73.25, 73.11, 62.06, 61.85, 53.64, 52.24, 52.07, 51.69, 43.48, 43.22, 42.01, 41.57, 41.57, 38.89, 30.49, 30.28, 28.93, 28.86, 26.49, 26.32, 26.12, 25.86, 25.75, 25.72, 23.50, 22.91, 22.86, 22.13, 21.65. HRMS (m/z): [M + H]⁺ calcd for C₂₂H₄₃N₄O₄⁺, 427.3284; found, 427.3298.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-4-methyl-1-oxo-1-(phenethylamino)pentan-2-yl)pyrrolidine-2-carboxamide (14z; Method C).

The product is obtained as a white solid; yield 53%; HPLC purity: 97.80%; ¹H NMR (600 MHz, methanol-d₄): δ 7.30–7.25 (m, 2H), 7.24–7.16 (m, 3H), 4.44 (d, J = 3.2 Hz, 1H), 4.43 (dd, J = 5.6, 2.6 Hz, 1H), 3.83–3.77 (m, 1H), 3.67 (dt, J = 9.8, 6.6 Hz, 1H), 3.55 (dd, J = 7.2, 3.0 Hz, 1H), 3.53–3.46 (m, 1H), 3.39–3.34 (m, 1H), 2.83–2.76 (m, 2H), 2.28–2.20 (m, 1H), 2.08–2.1 (m, 1H), 2.02–1.94 (m, 1H), 1.95–1.88 (m, 1H), 1.81–1.72 (m, 1H), 1.69–1.60 (m, 2H), 1.58–1.42 (m, 4H), 1.01 (d, J = 6.6 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 174.66, 174.14, 174.02, 140.39, 129.86, 129.47, 127.35, 73.07, 61.86, 53.28, 51.73, 43.48, 42.03, 41.82, 38.88, 36.29, 30.40, 26.33, 25.78, 25.75, 23.50, 23.45, 22.90, 22.01. HRMS (m/z): [M + H]⁺ calcd for C₂₆H₄₃N₄O₄⁺, 475.3284; found, 475.3297.

(S)-1-((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-N-((S)-4-methyl-1-((2-(naphthalen-2-yl)ethyl)amino)-1-oxopentan-2-yl)-pyrrolidine-2-carboxamide (14aa; Method C).

The product is obtained as a white solid; yield 56%; HPLC purity: 95.99%; ¹H NMR (600 MHz, methanol-d₄): δ 7.82–7.78 (m, 3H), 7.66 (br s, 1H), 7.47–7.37 (m, 3H), 4.42 (d, J = 3.1 Hz, 1H), 4.38 (dd, J = 8.4, 5.4 Hz, 1H), 4.30 (dd, J = 9.9, 5.4 Hz, 1H), 3.79–3.72 (m, 1H), 3.70–3.62 (m, 1H), 3.61–3.56 (m, 1H), 3.56–3.51 (m, 1H), 3.50–3.43 (m, 1H), 3.04–2.93 (m, 3H), 2.19–2.07 (m, 1H), 2.00–1.85 (m, 2H), 1.82–1.71 (m, 2H), 1.72–1.60 (m, 1H), 1.60–1.42 (m, 2H), 1.42–1.33 (m, 1H), 1.01 (d, J = 6.5 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.98–0.93 (m, 1H), 0.93–0.85 (m, 1H), 0.84 (d, J = 6.6 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 174.76, 174.07, 174.02, 137.87, 135.02, 133.75, 129.10, 128.62, 128.59, 128.34, 128.31, 126.98, 126.41, 72.98, 61.81, 53.30, 51.65, 43.46, 41.99, 41.40, 36.24, 30.30, 26.29, 25.71, 23.47, 23.38, 22.90, 21.92. HRMS (m/z): [M + H]⁺ calcd for C₃₀H₄₅N₄O₄⁺, 525.3441; found, 525.3459.

(S)-N-((S)-1-((2-(1H-indol-6-yl)ethyl)amino)-4-methyl-1-oxopentan-2-yl)-1-((2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl)-pyrrolidine-2-carboxamide (14ab; Method C).

The product is obtained as a white solid; yield 53%; HPLC purity: 99.57%; ¹H NMR (600 MHz, methanol-d₄): δ 7.55 (dt, J = 8.0, 1.0 Hz, 1H), 7.32 (dt, J = 8.1, 0.9 Hz, 1H), 7.10–7.06 (m, 2H), 7.00 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.44 (d, J = 3.0 Hz, 1H), 4.41 (dd, J = 8.5, 5.3 Hz, 1H), 4.33 (dd, J = 9.9, 5.4 Hz, 1H), 3.78 (dt, J = 10.0, 6.8 Hz, 1H), 3.63 (dt, J = 9.9, 6.7 Hz, 1H), 3.61–3.53 (m, 2H), 3.47–3.42 (m, 1H), 2.98–2.92 (m, 2H), 2.22–2.15 (m, 1H), 2.04–1.90 (m, 1H), 1.88–1.79 (m, 1H), 1.80–1.72 (m, 1H), 1.69–1.60 (m, 2 H), 1.59–1.43 (m, 3H), 1.00 (d, J = 6.5 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.98–0.92 (m, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 174.65, 174.16, 174.04, 138.11, 131.33, 128.80, 123.53, 122.31, 119.61, 119.31, 113.02, 112.25, 73.02, 61.85, 53.33, 51.66, 43.47, 41.90, 41.16, 30.34, 26.32, 25.97, 25.78, 25.73, 23.47, 23.45, 22.89, 21.95. HRMS (m/z): [M + H]⁺ calcd for C₂₈H₄₄N₅O₄⁺, 514.3393; found, 514.3383.

(S)-N-((S)-1-((2-([1,1’-Biphenyl]-4-yl)ethyl)amino)-4-methyl-1-ox-opentan-2-yl)-1-((2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl)-pyrrolidine-2-carboxamide (14ac; Method C).

The product is obtained as a white solid; yield 55%; HPLC purity: 99.48%; ¹H NMR (600 MHz, methanol-d₄): δ 7.60–7.56 (m, 2H), 7.56–7.52 (m, 2H), 7.43–7.38 (m, 2H), 7.33–7.27 (m, 3H), 4.43 (d, J = 3.0 Hz, 1H), 4.43–4.39 (m, 1H), 4.33 (dd, J = 9.9, 5.4 Hz, 1H), 3.81–3.74 (m, 1H), 3.67–3.60 (m, 1H), 3.58–3.50 (m, 2H), 3.45–3.38 (m, 1H), 2.89–2.82 (m, 2H), 2.24–2.17 (m, 1H), 2.04–1.96 (m, 1H), 1.96–1.84 (m, 2H), 1.80–1.71 (m, 1H), 1.70–1.59 (m, 2H), 1.58–1.41 (m, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, MeOD): δ 174.74, 174.13, 174.09, 142.16, 140.49, 139.55, 130.42, 129.82, 128.18, 128.01, 127.79, 73.02, 61.88, 53.33, 51.72, 48.62, 43.53, 42.01, 41.64, 38.88, 35.82, 30.40, 26.33, 25.80, 25.74, 23.46, 22.91, 22.01. HRMS (m/z): [M + H]⁺ calcd for C₃₂H₄₇N₄O₄⁺, 551.3597; found, 551.3613.

tert-Butyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-pro-linate (15a; Method C).

The product is obtained as white powder; yield 77%; HPLC purity: 99.47%; ¹H NMR (600 MHz, methanol-d₄): δ 4.61 (dd, J = 8.6, 1.7 Hz, 0.14H), 4.40 (d, J = 4.4 Hz, 0.86H), 4.37 (dd, J = 8.7, 4.7 Hz, 1H), 3.82 (dt, J = 10.4, 7.0 Hz, 0.86H), 3.71–3.65 (m, 1H), 3.65–3.55 (m, 0.28H), 3.53–3.46 (m, 0.86H), 2.32–2.23 (m, 1H), 2.16–2.11 (m, 0.14H), 2.09–1.86 (m, 2.86H), 1.81–1.69 (m, 1H), 1.67–1.56 (m, 1H), 1.56–1.49 (m, 1H), 1.49 (s, 1.26H), 1.47 (s, 7.74H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H). ¹³C NMR (151 MHz, MeOD, more than 16 ¹³C signals for compound 15a were observed due to the presence of different rotameric species): δ 173.87, 173.02, 171.60, 171.24, 83.18, 83.06, 70.55, 69.29, 62.02, 61.43, 53.02, 52.55, 48.44, 48.41, 39.60, 39.43, 32.23, 29.98, 28.20, 28.15, 25.78, 25.25, 23.15, 22.82, 22.73, 22.62, 22.36, 22.26. HRMS (m/z): [M + H]⁺ calcd for C₁₆H₃₁N₂O₄⁺, 315.2284; found, 315.2288.

Cyclopentyl ((2S,3R)-3-Amino-2-hydroxy-5-methylhexanoyl)-l-prolinate (15b; Method C).

The product is obtained as a white solid; yield 67%; HPLC purity: 98.68%; ¹H NMR (600 MHz, methanol-d₄): δ 5.21–5.16 (m, 1H), 4.72–4.67 (m, 0.13H), 4.43 (dd, J = 9.0, 4.4 Hz, 0.87H), 4.41 (d, J = 3.0 Hz, 0.87H), 4.38 (d, J = 3.0 Hz, 0.13H), 3.86–3.79 (m, 0.87H), 3.70 (dt, J = 10.2, 6.4 Hz, 1H), 3.66–3.55 (m, 0.26H), 3.54–3.47 (m, 0.87H), 2.34–2.23 (m, 1H), 2.15–2.09 (m, 0.13H), 2.09–2.00 (m, 1.87H), 1.99–1.92 (m, 1H), 1.92–1.83 (m, 2H), 1.82–1.59 (m, 8H), 1.58–1.47 (m, 1H), 1.00 (d, J = 6.4 Hz, 3H), 0.98 (d, J = 6.4 Hz, 3H). ¹³C NMR (151 MHz, MeOD, more than 17 ¹³C signals for compound 15b were observed due to the presence of different rotameric species): δ 174.43, 173.55, 171.57, 171.21, 79.89, 79.78, 70.43, 69.02, 61.59, 60.94, 53.04, 48.46, 48.43, 39.47, 39.30, 33.57, 33.54, 33.47, 33.42, 32.25, 29.95, 25.86, 25.22, 24.68, 24.65, 23.06, 22.78, 22.64, 22.41, 22.30. HRMS (m/z): [M + H]⁺ calcd for C₁₇H₃₁N₂O₄⁺, 327.2284; found, 327.2282.

Antibodies.

Antibodies used include GSDMD rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), CARD8 C-terminus rabbit polyclonal Ab (Abcam, Ab24186), PARP rabbit polyclonal Ab (Cell Signaling Tech, 9542), GAPDH rabbit monoclonal Ab (Cell Signaling Tech, 14C10), mouse GSDMD rabbit monoclonal Ab [EPR19828] (Abcam, ab209845), DPP9 rabbit polyclonal (Abcam, ab42080), PEPD rabbit monoclonal Ab (EPR16959; Abcam, ab197890), XPNPEP1 (Abcam, ab123929), GSDMD rabbit monoclonal Ab [EPR20829-408] (Abcam, ab215203), MYC tag rabbit monoclonal Ab (Cell Signaling Tech, 2278), HA tag rabbit monoclonal Ab (Cell Signaling Tech, 3724), IRDye 800CW donkey anti-rabbit (925-32211), IRDye 680RD donkey anti-rabbit (925-68073), IRDye 800CW donkey anti-mouse (925-32212), IRDye 680RD donkey anti-mouse (925-68072), and IRDye 800CW donkey anti-goat (925-32214).

Cell Culture.

HEK293T, THP-1, and RAW264.7 cells were purchased from ATCC. MV4;11 and OCI-AML2 cells were purchased from DSMZ. N/TERT1 cells were a gift from the Rheinwald Lab (Dickson et al., 2000). HEK293T and RAW264.7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with l-glutamine and 10% fetal bovine serum (FBS). N/TERT1 cells were grown in keratinocyte serum-free medium supplemented with 1× penicillin/streptomycin, bovine pituitary extract (25 μg/mL), and epidermal growth factor (EGF) (0.2 ng/mL). All other cell lines were grown in Roswell Park Memorial Institute (RPMI) medium 1640 with l-glutamine and 10% FBS. All cells were grown at 37 °C in a 5% CO₂ atmosphere incubator. Cell lines were regularly tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza).

Mouse BMDM Isolation and Culture.

Bone marrow was harvested from the femurs and tibias of 7–12-week-old C57BL/6J mice. Briefly, femurs and tibias were harvested from mice and crushed with a mortar and pestle in cold 1× PBS supplemented with 2.5% FBS. The mixture was passed through a 70 μm nylon cell strainer. RBCs were lysed for 4–5 min on ice in 1× RBC lysis buffer (Biolegend), and cells were centrifuged at 300g for 5 min at 4 °C. The cell pellet was washed in cold 1× PBS supplemented with 2.5% FBS before being passed again through in a 70 μm nylon cell strainer and counted. Counted cells were plated on non-tissue culture 10 cm plates at 5–10 × 10⁶ cells per plate in DMEM supplemented with 10% FBS and 15–20% L-cell media. Cells were incubated at 37 °C for 6 days before replating and assaying, as indicated. These procedures were approved by the Memorial Sloan Kettering Cancer Center IACUC.

Human Primary Cell Isolation and Culture.

Isolated human primary cells were obtained from Astarte Biologics. All cells were >90% purity, as validated by flow cytometry by Astarte Biologics. T cells were thawed in RPMI-1640 medium supplemented with 10% FBS and cultured in RPMI-1640 medium, 10% FBS, and 30 U/mL IL-2 (PeproTech).

Cloning.

CASP1^−/− MV4;11 cells;¹³ CARD8^−/− MV4;11, THP-1, OCI-AML2 cells;¹³ DPP8/9^−/− THP-1;¹³ and PEPD^−/−, XPNPEP1^−/−, and PEPD/XPNPEP1^−/− THP-1 cells were generated previously.¹³ Plasmids for CARD8 variants, CASP1, GSDMD, and dTAG-CARD8^ZUC were cloned, as described previously.^5,6,15

Protein Purification.

Human prolidase (PEPD) was generated and purified previously.⁷ XPNPEP1 in pET15-b containing a 6× His tag was expressed in Escherichia coli Rosetta DE3 cells. Cells were induced with IPTG for 16 h at 37 °C, pelleted, and lysed in buffer A [20 mM Tris–HCl (pH 7.9), 500 mM NaCl, and 10% (v/v) glycerol] by sonication. Affinity purification chromatography was performed using TALON resin, according to previously published protocols (Li et al., 2008).

Substrate Assays.

For the PEPD assay, a solution of substrate (Ala-Pro) was prepared in DMSO. 24 μL of 50 nM recombinant human PEPD was added to a 384-well, black, clear-bottom plate (Corning) with 1 μL of dipeptide Ala-Pro (final concentration 40 μM). Alanine liberated was measured as the increasing fluorescence signal (Resorufin, Ex/Em: 535/587 nm) recorded at 25 °C using an l-alanine assay kit (Abcam, ab83394) at 25 °C according to manufacturer’s instructions. For the AMC reporter assays, experiments were performed with cell lysates. 5 μL of solution of the substrate (2.5 mM Ala-AMC) was added to the mixture of 10 μL of HEK293T cell lysates (2.5 mg/mL) and 10 μL of the indicated compounds in a 384-well, black, clear-bottom plate (Corning) to initiate the reaction. Substrate cleavage was measured as the increasing fluorescence signal (Ex/Em: 380/460 nm) recorded at 25 °C for 20 min. XPNPEP1 enzymes (XPNPEP1 3.5 nM) were plated on a black 384-well clear bottom plate and treated with the indicated doses of compounds. The H-Lys(abz)-Pro-Pro-pNA substrate was added to a final concentration of 100 μM, and fluorescence was monitored (Ex/Em: 320/410) for 60 min.

CellTiter-Glo Cell Viability and CytoTox-Fluor Cell Death Assays.

Cells were plated (2000 cells per well) in white, 384-well clear-bottom plates (Corning) using an EL406 Microplate Washer/Dispenser (BioTek) in 25 μL of the final volume of medium. To the cell plates were added compounds at different concentrations using a pintool (CyBio), and the plates were allowed to incubate in the incubator. After incubation for indicated times, the CytoTox-Fluor reagent (Promega, G9262) was added according to the manufacturer’s protocol. The assay plates were then incubated for another 30 min before fluorescence was recorded using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Next, the CellTiter-Glo (CTG) reagent (Promega, G7573) was subsequently added to the assay plates following the manufacturer’s protocol. Assay plates were shaken on an orbital shaker for 2 min and incubated at 25 °C for 10 min. Luminescence was then read using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).

PI Flux Analysis.

2 × 10⁴ MV4;11 or OCI-AML2 cells were plated in a 384-well, black, clear-bottom plate (Corning) in RPMI medium. Cells were then treated, as indicated, and PI was added at a final concentration of 10 μM. PI fluorescence was measured (Ex/Em: 535/617 nm) and recorded at 37 °C every 5 min in a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). The obtained measurements were baseline corrected to vehicle/DMSO and normalized to maximum response.

LDH Cytotoxicity and Immunoblotting Assays.

HEK293T cells were transiently transfected and treated with inhibitors, as indicated. MV4;11, OCI AML2, THP-1, and RAW264.7 cells were plated in 12-well culture plates at 5 × 10⁵ cells/well and treated, as indicated. Supernatants were analyzed for LDH activity using the Pierce LDH Cytotoxicity Assay Kit (Life Technologies). LDH activity was quantified relative to a lysis control where cells were lysed in 80 μL of a 9% Triton X-100 solution. For immunoblotting, cells were washed 2× in PBS (pH = 7.4), resuspended in PBS, and lysed by sonication. Protein concentrations were determined and normalized using the DCA Protein Assay kit (Bio-Rad). The samples were separated by SDS-PAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-Cor).

Transient Transfections.

HEK293T cells were plated in six-well culture plates at 5 × 10⁵ cells/well in DMEM. The next day, the indicated plasmids were mixed with an empty vector to a total of 2.0 μg of DNA in 125 μL of Opti-MEM and transfected using FuGENE HD (Promega), according to the manufacturer’s protocol.

dTAG-CARD8 Assay.

HEK293T cells stably expressing CASP1 and GSDMD were seeded at 1.5 × 10⁵ cells per well in 12-well tissue culture dishes. After 48 h, the cells were transfected with plasmids encoding dTAG–CARD8-ZUC (0.5 μg), CARD8 FIIND-S297A (0.3 μg), and RFP (0.2 μg) with FuGENE HD, according to the manufacturer’s instructions (Promega). After 24 h, cells were treated with DMSO, dTAG13 (500 nM), and indicated compounds for 6 h prior to LDH release and immunoblot analyses.

Statistical Analysis.

Two-sided Student’s t tests were used for significance testing unless stated otherwise. P values less than 0.05 were considered to be significant. Graphs and error bars represent means ± SEM of three independent experiments, unless stated otherwise. The investigators were not blinded in all experiments. All statistical analyses were performed using Microsoft Excel and GraphPad Prism 9..

ABBREVIATIONS

AHMH-Pro

(2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl-proline

ALAT

alanine transaminase

BMDMs

bone marrow-derived macrophages

CARD8

caspase activation and recruitment domain-containing 8

CASP1

caspase-1

CT

C terminal

DPP8/9

dipeptidyl peptidase 8/9

dTAG

degradation tag

FIINDs

function-to-find domains

GSDMD

gasdermin D

HATU

hexafluorophosphate azabenzotriazole tetramethyl uronium

HRP

horseradish peroxidase

HRMS

high-resolution mass spectra

IC₅₀

half-maximal inhibitory concentration

IL-1β

interleukin-1β

IL-18

interleukin-18

LDH

lactate dehydrogenase

NLRP1

nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1

NT

N terminal

PEPD

peptidase D

POX

pyruvate oxidase

PRRs

pattern-recognition receptors

SAR

structure–activity relationship

TLC

thin-layer chromatography

UPLC

ultraperformance liquid chromatography

WT

wild-type

XPNPEP1

X-prolyl aminopeptidase 1