Development of Peptide-Based Sirtuin Defatty-Acylase Inhibitors Identified by the Fluorescence Probe, SFP3, That Can Efficiently Measure Defatty-Acylase Activity of Sirtuin

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Development of Peptide-Based Sirtuin Defatty-Acylase Inhibitors

Identiﬁed by the Fluorescence Probe, SFP3, That Can Eﬃciently

Measure Defatty-Acylase Activity of Sirtuin

Mitsuyasu Kawaguchi, Naoya Ieda, and Hidehiko Nakagawa*

Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan

S

* Supporting Information

ABSTRACT: Sirtuins (SIRTs) are a family of nicotinamide adenine

dinucleotide-dependent histone deacetylases that serve as epigenetic

regulators of many physiological processes. Recent studies have shown

that in addition to their well-known deacetylase activity, sirtuins also

exhibit deacylase activity, such as demyristoylase activity. Here, we

show that our previously reported sirtuin ﬂuorescence probe, SFP3, can

measure the defatty-acylase activity of SIRT1−3, enabling selective

assay of the deacylase activity. We further utilized this ﬁnding to

develop the ﬁrst inhibitors of SIRT2 defatty-acylase activity. Notably,

most previously reported sirtuin inhibitors, including compound TM,

AGK2, and SirReal2, showed almost no SIRT2 defatty-acylase-

inhibitory activity, but are essentially speciﬁc deacetylase inhibitors.

These results suggest that the active sites catalyzing the deacetylase and

defatty-acylase activities of sirtuins may be independent.

N-terminal glycine myristoylation and cysteine palmitoylation,

but little is known about the biological signiﬁcance of lysine

fatty acylation.¹⁵Recent reports suggest that SIRT6 demyr-

istoylase activity regulates the secretion of TNF-α⁹and that

SIRT2 defatty-acylase activity regulates the localization of K-

Ras4a oncoprotein and promotes cellular transformation.¹⁶

Interestingly, a study of the SIRT6 G60A mutant, which

possesses defatty-acylase activity but lacks deacetylase activity,

demonstrated that the deacetylase and defatty-acylase activities

of SIRT6 regulate independent cellular functions, for example,

having roles in transcriptional regulation and protein secretion,

respectively.¹⁷

Many SIRT2 “deacetylase” inhibitors have been re-

ported,¹⁸⁻²²but their “defatty-acylase”-inhibitory activities

have hardly been examined.²³⁻²⁷This is an important lacuna

because speciﬁc defatty-acylase inhibitors would be useful not

only for basic biological studies but also as candidate

therapeutic agents, for example, for inﬂammation and cancer.

Therefore, in this study, we set out ﬁrst to establish an assay

system for measuring SIRT defatty-acylase activity and, second,

to use the developed assay to discover SIRT2 defatty-acylase

inhibitors based on the structure of the SIRT2 inhibitor

RIK^TfaRY reported by Suga and co-workers.²⁸To evaluate

SIRT2 defatty-acylase activity, we used two indicators, that is,

our recently reported sirtuin ﬂuorescence probe, SFP3,²⁹and a

ﬂuorescence probe, p53(Myr)-AMC, developed to monitor

demyristoylase activity. A study of a series of newly synthesized

INTRODUCTION

■

Human sirtuins (SIRT1−7) are a family of nicotinamide

adenine dinucleotide (NAD⁺)-dependent histone deacetylases

that function as epigenetic regulators, and their activities are

correlated with cellular energy levels, that is, the NAD⁺−

NADH ratio regulates their activities.^1,2Their catalytic

activities toward histone proteins and nonhistone proteins,

including transcription factors and metabolic enzymes, play

important roles in many physiological and pathological

functions, including metabolic regulation, stabilization of

genomic DNA, stress responses, and cancers.³⁻⁶

For a long time, it was thought that sirtuins only catalyze

deacetylation reactions of histones and the adenine diphos-

phate-ribosylation reaction. However, more recently, it was

found that some sirtuins (SIRT4−7) exhibit quite weak

deacetylase activity but have relatively strong deacylase

activities instead; for example, SIRT4 shows lipoamidase

activity, SIRT5 shows desuccinylase and demalonylase

activities, SIRT6 shows demyristoylase activity, and SIRT7

shows desuccinylase activity.⁷⁻¹⁰In addition, SIRT1−3, which

has strong deacetylase activity, can also eﬃciently remove long-

chain fatty acid-derived acyl groups at the ε-amino group of

lysine residues, that is, they have defatty-acylase activity.¹¹

These intriguing ﬁndings were supported by X-ray crystallo-

graphic data showing the existence of a large hydrophobic

pocket in SIRT2 and SIRT6 that can accommodate the

myristoyl group.^12,13

Protein fatty acylation is important for anchoring proteins to

the cell membrane and plays signiﬁcant roles in cell signaling

and protein−protein interactions.¹⁴Early studies focused on

Received: February 18, 2019

Published: May 22, 2019

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DOI: 10.1021/acs.jmedchem.9b00315

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Chart 1. Structures of Dabcyl Derivatives Used in This Study

a

Scheme 1. Syntheses of Fmoc-Lys(Dabcyls)-OH 4, 5, and 6

a

Reagents and conditions: (a) NaNO₂, 1 N HCl, H₂O, 0 °C and then N,N-diethylaniline, H₂O/AcOH, 0 °C to room temperature (R.T.), 52%; (b)

N-hydroxysuccinimide, EDCI·HCl, dimethylformamide (DMF), R.T., 9.2%; (c) Fmoc-Lys-OH·HCl, N,N-diisopropylethylamine (DIPEA), DMF,

R.T., 35%; (d) NaNO₂, 1 N HCl, H₂O, 0 °C and then phenol, 2 N NaOH, 0 °C to R.T., 30%; (e) MOM-Cl, DIPEA, DMF, 0 °C to R.T., 86%; (f)

2 N NaOH, EtOH, 70 °C and then 2 N HCl, 69%; (g) N-hydroxysuccinimide, EDCI·HCl, DMF, R.T., 59%; (h) Fmoc-Lys-OH·HCl, DIPEA,

DMF, R.T., 83%; (i) nitrosobenzene, AcOH, R.T., 90%; (j) N-hydroxysuccinimide, EDCI·HCl, DMF, R.T., 51%; and (k) Fmoc-Lys-OH·HCl,

DIPEA, DMF, R.T., 86%.

inhibitors (S2DMi-1−14), as well as previously reported

inhibitors, showed that the SIRT2-inhibitory activities

evaluated with the two probes were strongly correlated with

each other, whereas they were not correlated with the

deacetylase-inhibitory activities. Thus, our ﬂuorescence probe

SFP3 can eﬃciently measure SIRT2 defatty-acylase activity.

Interestingly, most of the previously reported inhibitors,

including compound TM and SirReal2, showed only very

weak SIRT2 defatty-acylase-inhibitory activity (IC₅₀> 2.0 and

>10 μM, respectively),¹⁹whereas our newly synthesized

inhibitors, S2DMi-6, S2DMi-7, and S2DMi-9, showed potent

defatty-acylase-inhibitory activity (IC₅₀= 0.019−0.022 μM).

Thus, the new inhibitors are expected to be useful as both

research tools and candidate therapeutic agents.

CHEMISTRY

■

We ﬁrst prepared dabcyl derivatives (1−6), whose carboxyl

groups are linked to the ε-amino group of Fmoc-Lys-OH

(Chart 1). Fmoc-Lys(Dabcyl)-OH (1), Fmoc-Lys(Dabcyl-

EH)-OH (2), and Fmoc-Lys(Dabcyl-PH)-OH (3) were

prepared as previously described.²⁹The synthetic routes to

S2DMi-1−14 and their intermediates used in this study are

shown in scheme 1−4. Scheme 1 shows the preparation of

Fmoc-Lys(diEtDabcyl-EH)-OH (4), Fmoc-Lys(OH-Dabcyl-

EH)-OH (5), and Fmoc-Lys(Dabcyl-EH-H)-OH (6). Azo

coupling reaction between 4-aminobenzeneacetic acid (7) and

N,N-diethylaniline or phenol aﬀorded azobenzene 8 or 10,

respectively. Aminobenzeneacetic acid (7) was also coupled

with nitrosobenzene to aﬀord azobenzene 14. The phenol

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a

Scheme 2. Syntheses of S2DMi-1−8, S2DMi-13, and S2DMi-14

a

Reagents and conditions: (a) Fmoc solid-phase synthesis using Sieber amide resin; (b) 1% TFA, 1% triethylsilane, CH₂Cl₂, R.T., 49% for S2DMi-

1-protected, 82% for S2DMi-2-protected, 84% for S2DMi-3-protected, 64% for S2DMi-4-protected, 42% for S2DMi-6-protected, quant. for

S2DMi-7-protected, 98% for S2DMi-8-protected, quant. for S2DMi-13-protected, 83% for S2DMi-14-protected; (c) 50% TFA, CH₂Cl₂, R.T.,

65% for S2DMi-1, 79% for S2DMi-2, 874% for S2DMi-3, 42% for S2DMi-4, 22% for S2DMi-6, 60% for S2DMi-7, 39% for S2DMi-8, 58% for

S2DMi-13, 30% for S2DMi-14; (d) Fmoc solid-phase synthesis using Fmoc-NH-SAL resin; and (e) 90% TFA, 5% H₂O, 5% triethylsilane, R.T.,

11%.

a

Scheme 3. Synthesis of S2DMi-9

a

Reagents and conditions: (a) myristoyl chloride, Na₂CO₃, dioxane, H₂O, 0 °C to R.T., 78%; (b) Lawesson’s reagent, THF, R.T., 69%; (c) Fmoc

solid-phase synthesis using Sieber amide resin; (d) 1% TFA, 1% triethylsilane, CH₂Cl₂, R.T., 99%; and (e) 50% TFA, CH₂Cl₂, R.T., 35%.

group of compound 10 was protected with methoxymethyl

(MOM) to give compound 12 in two steps. The carboxyl

groups of compounds 8, 12, and 14 were activated in the

presence of N-hydroxysuccinimide and 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDCI) to aﬀord succini-

midyl ester compounds 9, 13, and 15, respectively.

Condensation reaction of each succinimidyl ester compound

with the ε-amino group of Fmoc-Lys-OH gave the desired

Fmoc-Lys(diEtDabcyl-EH)-OH (4), Fmoc-Lys(OH-Dabcyl-

EH)-OH (5), and Fmoc-Lys(Dabcyl-EH-H)-OH (6).

Scheme 2 shows the syntheses of S2DMi-1−8, S2DMi-13,

and S2DMi-14. Fmoc solid-phase synthesis using Sieber amide

resin with modiﬁed lysine residues (1−6) gave protected

S2DMi-1−4, S2DMi-6−8, S2DMi-13, and S2DMi-14.

Protecting groups were removed by treatment with TFA.

The crude peptides were puriﬁed by high-performance liquid

chromatography (HPLC) to aﬀord >95% pure S2DMi-1−4,

S2DMi-6−8, S2DMi-13, and S2DMi-14. Fmoc solid-phase

synthesis using Fmoc-NH-SAL resin also gave S2DMi-5.

The preparation of S2DMi-9 is shown in Scheme 3. The

amino group of Fmoc-Lys-OH was acylated with myristoyl

chloride to aﬀord compound 17. The electron-rich amide

group was converted to a thioamide group by using Lawesson’s

reagent to aﬀord compound 18. Fmoc solid-phase synthesis

gave protected S2DMi-9, which was deprotected under acidic

conditions to aﬀord crude S2DMi-9. The crude peptide was

puriﬁed by HPLC to aﬀord S2DMi-9.

Scheme 4 illustrates the syntheses of S2DMi-10−12.

Condensation of the amino group of Cbz-Lys-OH with

azobenzene 13 gave compound 20. Compound 20 was

condensed with H-Ala-NH₂in the presence of COMU to

aﬀord compound 21. Removal of the MOM group of

compound 21 under acidic conditions gave S2DMi-10.

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Scheme 4. Syntheses of S2DMi-10−12

a

Reagents and conditions: (a) 13, DIPEA, DMF, R.T., 74%; (b) H-Ala-NH₂·HCl, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-

morpholino-carbenium hexaﬂuorophosphate (COMU), DIPEA, DMF, R.T., 84%; (c) 50% triﬂuoroacetyl (TFA), CH₂Cl₂, R.T., 76%; (d) benzyl

bromide, Cs₂CO₃, tetrabutylammonium iodide (TBAI), DMF, R.T., 84%; and (e) phenethyl bromide, Cs₂CO₃, TBAI, DMF, R.T., 42%.

Alkylation of S2DMi-10 with benzyl bromide or phenethyl

bromide gave S2DMi-11 or S2DMi-12, respectively.

a one-step sirtuin activity probe.²⁹As shown in Figure 1a,

SFP3 consists of ﬂuorescein, H3K9 peptide, and dabcyl

propanoic acid (Dabcyl-PH) quencher attached to the ε-

amino group of the lysine residue. In the presence of sirtuin

and NAD⁺, Dabcyl-PH of SFP3 is cleaved by SIRT1−3 and 6,

aﬀording a strongly ﬂuorescent product, SFP0. Recently, Jung

and co-workers reported that SIRT2 has a unique “selectivity

pocket”.²⁰Here, we hypothesized that the hydrophobic Dabcyl

quencher and long-chain fatty acyl groups are both recognized

by the same selectivity pocket of SIRT2 (Figure 1b). If this is

the case, the Dabcyl moiety might compete with long-chain

fatty acyl groups for binding to the selectivity pocket, that is, it

might be a speciﬁc competitive inhibitor of SIRT2 defatty-

acylase activity. Thus, we anticipated that SFP3 might

eﬃciently measure the defatty-acylase activity of SIRT2. To

test these ideas, we synthesized a series of new peptide-based

SIRT2 inhibitors bearing a Dabcyl moiety and evaluated their

SIRT2-inhibitory activity by means of the SFP3 assay.

In designing the new compounds, we were concerned that

the Dabcyl-PH group might be cleaved by SIRT2, as in the

case of SFP3 (Figure 1a). As our previously reported

ﬂuorescence probes SFP1 and SFP2, which contain Dabcyl

and Dabcyl-EH, respectively, instead of the Dabcyl-PH moiety

in SFP3, were unreactive to SIRT2,²⁹we focused on Dabcyl or

Dabcyl-EH instead of Dabcyl-PH to decrease the electron

density of the amide group with the aim of making the

compounds inert to SIRT2-mediated cleavage (Figure 1c).

The preparation of p53(Myr)-AMC is shown in Scheme 5.

Condensation of the carboxyl group of Cbz-Lys(Boc)-OH

with 7-amino-4-methylcoumarin (22) gave compound 23. The

Boc group was removed by the treatment with TFA, and the

formed amino group was condensed with myristoyl chloride to

aﬀord compound 25. The Cbz group was removed by catalytic

reduction using Pd/C under H₂, and the formed amino group

was condensed with Ac-Arg(Pbf)-His(Trt)-Lys(Boc)-OH

tripeptide (29) to aﬀord compound 27. Protecting groups

were removed by treatment with TFA, and the crude peptide

was puriﬁed by HPLC to aﬀord >95% pure p53(Myr)-AMC.

Scheme 6 illustrates the syntheses of H4K16-NH₂, H4K16-

Ac, and H4K16-Myr. Fmoc solid-phase synthesis using the

Sieber amide resin with Boc-protected, acetylated, or

myristoylated lysine residues gave protected H4K16-NH₂,

H4K16-Ac, and H4K16-Myr. Protecting groups were removed

by treatment with TFA. The crude peptides were puriﬁed by

HPLC to aﬀord >95% pure H4K16-NH₂, H4K16-Ac, and

H4K16-Myr.

RIK^TfaRY and compounds M and TM were synthesized in

accordance with the previous reports.^28,19

RESULTS

■

We previously reported a ﬂuorescence resonance energy

transfer-based ﬂuorescence probe, SFP3, designed to establish

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a

Scheme 5. Synthesis of p53(Myr)-AMC

a

Reagents and conditions: (a) Cbz-Lys(Boc)-OH, COMU, DIPEA, DMF, R.T., 38%; (b) 50% TFA, CH₂Cl₂, R.T., 77%; (c) myristoyl chloride,

DIPEA, CH₂Cl₂, R.T., 75%; (d) Pd/C, H₂, MeOH, CH₂Cl₂, R.T., quant.; (e) 29, COMU, DIPEA, DMF, R.T, 70%; (f) TFA, CH₂Cl₂, R.T., 58%;

(g) Fmoc solid-phase synthesis using 2-chlorotrityl chloride resin; and (h) 1% TFA, CH₂Cl₂, 87%.

a

Scheme 6. Syntheses of H4K16-NH₂, H4K16-Ac, and H4K16-Myr

a

Reagents and conditions: (a) Fmoc solid-phase synthesis using Sieber amide resin; (b) 1% TFA, 1% triethylsilane, CH₂Cl₂, R.T., quant. for

H4K16-NH₂-protected, 98% for H4K16-Ac-protected, and 95% for H4K16-Myr-protected; (c) 50% TFA, CH₂Cl₂, R.T., 61% for H4K16-NH₂,

70% for H4K16-Ac, and 65% for H4K16-Myr.

Thus, we designed and synthesized S2DMi-1−14, which

contain Ac-Arg-Ile-Lys-Arg-Tyr-NH₂(Ac-RIKRY-NH₂), Ac-

Ser-Asp-Lys-Thr-Ile-NH₂(Ac-SDKTI-NH₂), or Cbz-KA-NH₂,

in which the ε-amino group of lysine is acylated by Dabcyl,

Dbcyl-EH, or Dabcyl-PH, or a thiomyristoyl group (Chart 2).

We also prepared RIK^TfaRY as a reference compound (Chart

2).²⁸We examined the SIRT2-inhibitory activity by using

SFP3 as a substrate and found that RIK^TfaRY, S2DMi-1, and

S2DMi-3 showed moderate SIRT2-inhibitory activity (Table

1, IC₅₀= 0.32, 0.43, and 0.30 μM, respectively), whereas

S2DMi-2 showed about 9-fold stronger inhibitory activity

(IC₅₀= 0.036 μM) than RIK^TfaRY. Thus, the Dabcyl-EH

scaﬀold appears to be suitable as a SIRT2-inhibitory core

structure. We next checked the stability of S2DMi-1−3 in the

presence of SIRT2 and NAD⁺. HPLC analysis showed that

S2DMi-1 and S2DMi-3 aﬀorded complex degradation

together with deacylation products, Dabcyl and Dabcyl-PH,

respectively (Figure 2a,e), whereas S2DMi-2 was stable,

remaining >99% intact after 4 h incubation (Figure 2c). We

next examined the time dependence of the SIRT2-inhibitory

activity of S2DMi-1−3. The inhibitory activities of S2DMi-1

and S2DMi-3 toward SIRT2 decreased time-dependently

(Figure 3b,f), whereas that of S2DMi-2 remained nearly

constant for 60 min (Figure 2d), in accordance with the HPLC

analyses. These results conﬁrm that the phenylacetyl group of

Dabcyl-EH is resistant to deacylation by SIRT2 and thus

should be suitable for use as an inhibitory core structure.

We next evaluated S2DMi-4−14, together with known

SIRT2 inhibitors, including compound M, compound TM,

AGK2, nicotinamide (NAM), and SirReal2 (Chart 2) in the

SFP3 assay. S2DMi-5−7 all showed potent inhibitory activities

(Table 1, IC₅₀= 0.068, 0.019, and 0.019 μM, respectively). In

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Figure 1. (a) Schematic illustration of our hypothesis that the Dabcyl-PH group of our sirtuin ﬂuorescence probe SFP3 is accommodated in the

SIRT2 selectivity pocket and is cleaved by SIRT2 defatty-acylase activity. (b) Design of SIRT2 defatty-acylase inhibitors with a SIRT2 recognition

peptide and cleavage-resistant Dabcyl and Dabcyl-EH moieties. (c) The relative rate of reaction of the acylated amino group of lysine with NAD⁺in

the SIRT2 catalytic pocket is expected to depend on the electron density of the amide group.

contrast, S2DMi-4 and S2DMi-8, which contain the SDKTI

peptide instead of RIKRY in S2DMi-1 and S2DMi-7, showed

about 80-fold weaker inhibitory activity (IC₅₀= >2.0 and 1.5

μM, respectively), suggesting that the peptide sequence is

critical for SIRT2 inhibition. S2DMi-9, which has a

thiomyristolated lysine peptide, showed similar inhibitory

activity (IC₅₀= 0.022 μM) to S2DMi-6 and S2DMi-7,

suggesting that it acts as a mechanism-based inhibitor.

However, the thiomyristoyl group of S2DMi-9 is expected to

form a covalent bond with NAD⁺resulting in strong and

lasting inhibition, whereas S2DMi-6, S2DMi-7, and S2DMI-9

are expected to have a diﬀerent inhibitory mechanism. This

speculation was supported by the results of HPLC analyses and

examination of the time dependence of inhibitory activity.

Indeed, while HPLC analysis showed that S2DMi-6 and

S2DMi-9 were both stable in the presence of SIRT2 and

NAD⁺(Figure 2g,i), their SIRT2-inhibitory characteristics

were diﬀerent. That is, the inhibitory activity of S2DMi-6 was

essentially constant (Figure 2h), whereas that of S2DMi-9

increased time-dependently (Figure 2j), suggesting the

accumulation of the S2DMi-9−NAD⁺complex. On the other

hand, S2DMi-10−12 showed very weak inhibitory activities in

spite of having the Dabcyl-EH scaﬀold, like S2DMi-6.

Surprisingly, compound TM, a selective and potent SIRT2

inhibitor, showed almost no inhibitory activity toward SIRT2

in the SFP3 assay. These results suggest that the peptide

sequences Cbz-Lys-Ala-NH₂(S2DMi-10−12) and Cbz-Lys

(compound M and TM) are not suitable for inhibiting SIRT2-

mediated SFP3 cleavage. This idea is supported by the results

for S2DMi-13 and 14, which have truncated (Ac-Ile-Lys-Arg-

NH₂) and K-Ras4a peptide (Ac-Val-Lys-Ile-Lys-Lys-NH₂)

sequences with the phenylacetyl scaﬀold, like S2DMi-7;¹⁶

these compounds exhibited weaker inhibitory activities than

S2DMi-7 (IC₅₀= 0.063 and 0.12 μM, respectively). In

addition, compound 6 (Chart 1) itself exhibited no inhibitory

activity toward SIRT2, suggesting that the Dabcyl-EH scaﬀold

is not suﬃcient for SIRT2 inhibition (Figure S5). Thus, the

RIKRY sequence was considered to be the most suitable

peptide among those examined. Finally, the known SIRT2

inhibitors AGK2 and SirReal2 showed almost no inhibitory

activity in the SFP3 assay (IC₅₀> 50 and >10 μM,

respectively), though NAM showed moderate inhibitory

activity (IC₅₀= 11 μM); this is consistent with the fact that

NAM is a common byproduct of both deacetylation and

deacylation mediated by SIRT2.

Since compound TM, AGK2, and SirReal2 are reported to

be potent SIRT2 deacetylase inhibitors (IC₅₀= 0.028, 3.5, and

0.14 μM, respectively),^19,21but exhibited very weak inhibitory

activity in the SFP3 assay, the activity evaluated by SFP3 is

likely to be distinct from the SIRT2 deacetylase activity.

Therefore, we hypothesized that the enzymatic activity

detected by SFP3 is SIRT2’s defatty-acylase activity. To

conﬁrm this, we synthesized p53(Myr)-AMC as another

demyristoylase ﬂuorescence probe (Figure 3a). p53(Myr)-

AMC was hydrolyzed by SIRT2, releasing p53(NH₂)-AMC

(Figure 3b), and the SIRT2 demyristoylase activity could be

detected in terms of a ﬂuorescence increment upon subsequent

enzymatic reaction with trypsin (Figure 3a,c). Thus, we used

p53(Myr)-AMC to evaluate the SIRT2 demyristoylase-

inhibitory activity of the above compounds and reported

inhibitors. S2DMi-2, S2DMi-5−7, S2DMi-9, S2DMi-13, and

S2DMi-14 were potent/moderate SIRT2 demyristoylase

inhibitors (Table 1, IC₅₀= 0.13, 0.19, 0.044, 0.051, 0.044,

0.25, and 0.52 μM, respectively), whereas other S2DMis were

weak inhibitors (IC₅₀> 1.1 μM). Compounds M, TM, and

AGK2 showed almost no inhibitory activity, though NAM was

inhibitory (IC₅₀= 20 μM). The IC₅₀values obtained with

p53(Myr)-AMC were strongly correlated to those obtained

with SFP3 (correlation coeﬃcient: 0.993 (Figure 4a)). The

ﬁnding that the activity evaluated by the SFP3 assay well

reﬂects the demyristoylase activity of SIRT2 strongly supports

our above hypothesis, indicating that SFP3 is available as a

speciﬁc activity probe for SIRT2 defatty-acylase activity.

In addition, we measured the inhibitory activities of the

above compounds toward the enzymatic reaction of SIRT2

with H4K16-Myr and H4K16-Ac peptides, which is similar to

a native SIRT2 substrate, lacking a ﬂuorophore. S2DMi-6,

S2DMi-7, and S2DMi-9 inhibited the demyristoylation

reaction in a dose-dependent manner, whereas compound

TM, AGK2, and SirReal2 showed no demyristoylation-

inhibitory activity (Figures 5 and S5). These results are

consistent with those in the SFP3 and p53(Myr)-AMC assays.

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Chart 2. Structures of Candidate SIRT2 Defatty-acylase Inhibitors Synthesized in This Study (S2DMi-1−14) and Reported

a

SIRT2 Inhibitors, Compounds M, TM, AGK2, Nicotinamide (NAM), and SirReal2

a

Absorption spectra of these compounds are shown in Figure S1.

Although the inhibitory activities of S2DMi-6, S2DMi-7, and

S2DMi-9 were weaker than in SFP3 and p53(Myr)-AMC

assays, this may be at least partly because the concentrations of

H4K16-Myr (SIRT2 substrate) and SIRT2 were much higher.

Overall, these results demonstrate that S2DMi-6, S2DMi-7,

and S2DMi-9 inhibited SIRT2 demyristoylase activity, whereas

reported SIRT2 inhibitors, including compound TM, AGK2,

and SirReal2, did not aﬀect this activity even in the assay with

the native peptide substrate.

Next, we tried to directly compare the SIRT2 deacetylase

and demyristoylase-inhibitory activities of S2DMi-6, S2DMi-7,

and S2DMi-9 by utilizing the H4K16-Ac peptide. In the result,

it was found that S2DMi-6, S2DMi-7, and S2DMi-9 inhibited

the deacetylation reaction more potently than the demyr-

istoylation reaction in a dose-dependent manner (Figure S7).

We speculate that this result is due to the diﬀerence of K_m

values of two peptide substrates, H4K16-Ac and H4K16-Myr

(K_m= 21.1 and 0.202 μM for H4K16-Ac and H4K16-Myr,

respectively; Figure S8), meaning that much higher concen-

trations of S2DMi-6, S2DMi-7, and S2DMi-9 are necessary to

inhibit the SIRT2 demyristoylase activity because H4K16-Myr

can bind with SIRT2 more tightly than H4K16-Ac.

We further tested the SIRT2 deacetylase-inhibitory activity

of the above compounds by using the commercially available

FLUOR DE LYS SIRT2 assay kit. RIK^TfaRY, compound M,

AGK2, and SirReal2 showed potent to moderate SIRT2-

inhibitory activities, corresponding well with the reported

values (Table 1, IC₅₀= 0.067, 0.14, 8.9, and 0.70 μM,

respectively).¹⁹⁻²¹S2DMi-1−14 exhibited moderate SIRT2-

inhibitory activities (IC₅₀= 0.11−1.2 μM), which were weaker

than those in the SFP3 assay. Notably, the IC₅₀values in the

FLUOR DE LYS assay and the SFP3 assay showed no

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Table 1. SIRT2-Inhibitory Activities (IC₅₀) and/or % Inhibition Values of RIK^TfaRY, S2DMi-1−14, Compounds M, TM,

AGK2, Nicotinamide (NAM), and SirReal2 Evaluated by Using SFP3, p53(Myr)-AMC (deMyr), and FLUOR DE LYS SIRT2

g

Assay Kits (deAc)

SIRT2 (μM) SFP3

SIRT2 (μM) deMyr

SIRT2 (μM) deAc

c

RIK^TfaRY

S2DMi-1

S2DMi-2

S2DMi-3

S2DMi-4

S2DMi-5

S2DMi-6

S2DMi-7

S2DMi-8

S2DMi-9

S2DMi-10

S2DMi-11

S2DMi-12

S2DMi-13

S2DMi-14

compound M

compound TM

AGK2

0.32 0.030

0.43 0.023

0.036 0.006

0.30 0.022

1.1 0.14

1.6 0.46

0.13 0.021

0.067 0.006, 0.031

1.6 0.44

0.17 0.022

1.1 0.11

b

>2.0, 23%

>2.0, 2.0%

a

b

>2.0, 7.8%

1.2 0.10

0.068 0.007

0.019 0.007

0.019 0.006

1.5 0.10

0.19 0.020

0.044 0.005

0.051 0.006

0.26 0.020

0.11 0.011

0.18 0.013

0.58 0.038

0.11 0.013

0.19 0.013

0.19 0.020

0.40 0.024

0.11 0.010

0.12 0.015

b

>2.0, 17%

0.022 0.003

0.044 0.004

a

b

>2.0, 18%

>2.0, 13%

5.3%

3.2%

a

b

a

b

>2.0, 1.4%

>2.0, 2.9%

0.063 0.008

0.12 0.011

0.25 0.020

0.52 0.068

a

b

>2.0, 2.8%

>2.0, 4.2%

5.2%

4.2%

>1.0, 42%

a

b

d

0.14 0.014, 0.028

8.9 2.3, 3.5

e

>50

NAM

SirReal2

11 1.6

>10

20 2.2

>10

38 1.8

0.70 0.061, 0.14

f

a

b

c

d

e

f

g

Inhibition (%) at 2 μM. Inhibition (%) at 1 μM. Data from ref 28. Data from ref 19. Data from ref 21. Data from ref 20. The data indicate

IC₅₀(mean standard deviation (S.D.), n = 3) of at least two experiments. For details, see Figures S2 −S4.

correlation (correlation coeﬃcient: 0.135 (Figure 4b)),

suggesting that these two catalytic activities of SIRT2 are

independent and that SFP3 can eﬃciently measure defatty-

acylase activity of SIRT2. None of the compounds tested

showed inhibitory activity toward Developer II solution

(Figure S14), indicating that the inhibitory activities in the

FLUOR DE LYS assay represent SIRT2 deacetylase-inhibitory

activities.

We examined whether S2DMi-6, S2DMi-7, and S2DMi-9

permeate the cell membrane and inhibit sirtuin activities in

cells. For such purposes, we set out to evaluate HeLa cells

viability because it is reported that SIRT2-selective inhibitors,

such as compound TM, inhibit HeLa cells growth via a

degradation of c-Myc protein, which is associated with SIRT2

deacetylation activity.¹⁹In addition, it was found that S2DMi-

6, S2DMi-7, and S2DMi-9 strongly inhibit SIRT2 deacetylase

activity, and their activities are almost the same as that of

compound TM (Table 1). Cell viability assay showed that

S2DMi-6, S2DMi-7, and S2DMi-9 did not inhibit cell growth

even at 50 μM; in contrast, compound TM strongly inhibited

cell growth, which is consistent with the previous report

(Figure S16).¹⁹This result suggests that S2DMi-6, S2DMi-7,

and S2DMi-9 cannot permeate the cell membrane or they are

unstable in cellular systems.

We ﬁnally set out to elucidate the inhibition mechanism of

S2DMi-6 and S2DMi-9. Michaelis−Menten and Lineweaver−

Burk plots indicated that S2DMi-6 and S2DMi-9 show mixed

inhibition of SIRT2-mediated hydrolysis of SFP3 (Figure 6).

In contrast, S2DMi-6 and S2DMi-9 showed noncompetitive

inhibition with respect to NAD⁺(Figures 6 and S17).

To examine the selectivity of the above compounds, we

evaluated their inhibitory activities toward SIRT1, 3, and 6 by

using the SFP3 assay. The potent inhibitors of the SIRT2

defatty-acylase activity, such as S2DMi-2, S2DMi-5−7,

S2DMi-9, S2DMi-13, and S2DMi-14, also showed moder-

ate/potent inhibitory activities toward SIRT1 (Table 2) and

SIRT3 (Table 2). In contrast, these inhibitors, except S2DMi-

9, exhibited much weaker inhibitory activity toward SIRT6

(Table 2). We also evaluated the inhibitory activities toward

SIRT1 and 3 demyristoylase activities by using p53(Myr)-

AMC because it was found that p53(Myr)-AMC can detect

SIRT1 and SIRT3 demyristoylase activities in the same

manner as SIRT2 (Figure S15). S2DMi-2, S2DMi-5−7,

S2DMi-9, S2DMi-13, and S2DMi-14 also potently inhibited

SIRT1 and 3 demyristoylase activities, and their inhibitory

activities were similar to those obtained in the SFP3 assay

(Table 2). It was surprising that the synthesized inhibitors

showed little selectivity for SIRT2 over other SIRTs, even

though they contained the SIRT2-selective peptide sequence

RIKRY.²⁸It is possible that the binding of fatty-acylated

peptide induces a conformational change that alters the

substrate recognition. This is plausible because binding of a

thiomyristoylated peptide, BHJH-TM1, with SIRT2 induced a

substantial conformational change.¹²It was also suggested that

the structures of the hydrophobic pockets of SIRT1−3 are

quite similar, but diﬀerent from that of SIRT6, which seems

consistent with the results in Table 2.

DISCUSSION AND CONCLUSIONS

■

Our results here show that our previously reported

ﬂuorescence probe SFP3 can eﬃciently measure the defatty-

acylase activity of SIRT2. The inhibitory activities of the

synthesized inhibitors in the SFP3 assay were strongly

correlated with those toward p53(Myr)-AMC, which can

monitor SIRT1−3 demyristoylase activities. Importantly, the

SIRT2 defatty-acylase-inhibitory activities of the synthesized

compounds were not correlated with their SIRT2 deacetylase-

inhibitory activities, suggesting that these two enzymatic

activities are independent of each other. Notably, most

previously reported inhibitors, except RIK^TfaRY, showed

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Figure 2. (a, c, e, g, i) HPLC stability analyses of 20 μM S2DMi-1−3, S2DMi-6, and S2DMi-9 in the presence or absence of 350 nM SIRT2 and

500 μM NAD⁺for 4 h at 37 °C. Reactions were performed in SIRT assay buﬀer I (50 mM Tris−HCl (pH 8.0), 150 mM NaCl, 1 mM dithiothreitol

(DTT), and 0.05% Triton X-100), containing 0.2% dimethyl sulfoxide (DMSO). HPLC conditions: A/B = 80:20 (initial) → 0:100 (20 min) →

0:100 (23 min) with a linear gradient, A = 0.1% TFA Milli-Q, B = 0.1% TFA CH₃CN. In (i), a red arrow, a black arrow, and an arrowhead indicate

S2DMi-9, NAD⁺, and DTT, respectively. Absorbance was monitored at 510 nm (S2DMi-1−3), 350 nm (S2DMi-6), or 254 nm (S2DMi-9). (b, d,

f, h, j) Time dependence of SIRT2-inhibitory activities of S2DMi-1−3, S2DMi-6, and S2DMi-9 (indicated concentrations). The results are shown

as mean S.D. (n = 3).

Figure 3. (a) Application of p53(Myr)-AMC ﬂuorescence probe for measuring SIRT2 demyristoylase activity. (b) HPLC analyses of enzymatic

reactions of 50 μM p53(Myr)-AMC and 500 μM NAD⁺with 175 nM SIRT2 after 1 h. Absorbance at 325 nm was monitored. (c) Enzymatic

reactions were performed in 50 μM p53(Myr)-AMC and 500 μM NAD⁺with or without 70 nM SIRT2 for 3 h, and then 1 mM NAM and 100 nM

trypsin were added. Fluorescence intensity (F.I.) was measured with an ARVO X5 plate reader (ﬁlters: Ex = 355/40 nm, Em = 460/25 nm) for 20

min at 37 °C. The results are shown as mean S.D. (n = 3).

almost no SIRT2 defatty-acylase-inhibitory activity. Therefore,

our compounds, such as S2DMi-6, S2DMi-7, and S2DMi-9,

are the ﬁrst potent SIRT2 defatty-acylase inhibitors, having

IC₅₀values of nM order; however, they inhibit SIRT2

deacetylase activity more potently than the demyristoylase

one from the native-peptide-based assay (Figures 5 and S7),

whose characteristics are needed to be improved in the future

to develop SIRT defatty-acylase-speciﬁc inhibitors.

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hydrocarbon, such as a thiomyristoyl group, or aromatic rings,

as in Dabycl-EH, at the ε-amino group of the lysine residue is

critical for SIRT2 defatty-acylase inhibition. Third, the

phenylacetyl scaﬀold is resistant to hydrolysis by SIRT and

thus should be suitable for the development of SIRT inhibitors.

Modiﬁcation with a thioamide moiety might be useful because

most potent SIRT2 inhibitors have a thioamide moiety and

bind to NAD⁺(mechanism-based inhibitors).^19,22A recent

study has shown that SIRT2 can deacylate benzoylated lysine,

supporting our result that the Dabcyl moiety of S2DMi-1 was

cleaved by SIRT2.³⁰Therefore, the phenylacetyl scaﬀold

appears to be resistant to various SIRT2 deacylase activities,

including deacetylase, demyristoylase, and debenzoylase

activities.

It is noteworthy that compound TM, SirReal2, and AGK2

had almost no eﬀect on SIRT2 defatty-acylase activity.

Considering that S2DMi-6 and S2DMi-9 exhibited mixed-

type inhibition, it seems likely that binding of substrates

bearing a long-chain fatty acyl group, such as SFP3 and

p53(Myr)-AMC, induces a quite dramatic conformational

change of the substrate recognition pocket. As a consequence,

compound TM, SirReal2, and AGK2 may no longer be able to

bind, and therefore these inhibitors act as speciﬁc inhibitors of

SIRT2’s deacetylase activity.

Figure 4. (a) Correlation between SIRT2 defatty-acylase-inhibitory

activity evaluated by the SFP3 assay and the demyristoylase-inhibitory

activity evaluated by the p53(Myr)-AMC assay for the indicated

compounds (RIK^TfaRY, S2DMi-1, S2DMi-2, S2DMi-5−7, S2DMi-9,

S2DMi-13, and S2DMi-14). (b) Correlation between SIRT2 defatty-

acylase-inhibitory activities evaluated by SFP3 and deacetylase-

inhibitory activities evaluated by the FLUOR DE LYS SIRT2 assay

kit for the indicated compounds (RIK^TfaRY, S2DMi-1−3, S2DMi-5−

9, S2DMi-13, and S2DMi-14). The data from Table 1 were plotted.

Three key ﬁndings of this study are as follows. First, our

ﬂuorescence probe SFP3 could measure the defatty-acylase

activity of SIRT2. Therefore, it should be useful to screen small

chemicals that selectively inhibit defatty-acylase activity over

deacetylase activity of various sirtuins. Second, the SIRT2

recognition peptide sequence RIKRY is extremely important

for potent inhibitory activity, as in S2DMi-2, S2DMi-5−7, and

S2DMi-9. It is noteworthy that compound TM did not inhibit

defatty-acylase activity, even though it is reported to be a

potent SIRT2 inhibitor and contains a thiomyristoyl group that

should be accommodated in the hydrophobic selectivity

pocket. These results suggest that the combination of a

suitable peptide sequence and attachment of a hydrophobic

S2DMi-6, S2DMi-7, and S2DMi-9 lacked selectivity for

SIRT2 over SIRT1 and 3 despite the above hypothesis that

Dabcyl moieties can occupy the “SIRT2 selectivity pocket”.

This is because a substrate recognition pocket of SIRT2 is very

ﬂexible and the pocket structures bound with acetylated

peptide and bound with myristoylated peptide are quite

diﬀerent to ﬁt the shape to each substrate; in addition, the

structures of the hydrophobic pockets of SIRT1−3 are

reported to be all similar.¹²Therefore, this means that the

Figure 5. (a) SIRT2 inhibition assay using a peptide substrate, H4K16-Myr. Generation of H4K16-NH₂in the presence of SIRT2 inhibitors was

monitored with HPLC. (b) HPLC analyses of the enzymatic reactions of 100 μM H4K16-Myr, 500 μM NAD⁺, and 175 nM SIRT2 in the presence

or absence of SIRT2 inhibitors (S2DMi-6, S2DMi-7, S2DMi-9, compound TM, SirReal2 (10 μM), AGK2 (50 μM), and NAM (500 μM)) after 2

h. Absorbance at 280 nm was monitored. A ﬁlled arrowhead, an open arrowhead, and an arrow indicate S2DMi-6, S2DMi-7, and AGK2,

respectively. (c) SIRT2 inhibition (%) at the indicated concentrations of SIRT2 inhibitors. The results are mean S.D. (n = 3).

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Table 2. SIRT1, 3, and 6 Inhibitory Activities (IC₅₀) and/or % Inhibition Values of RIK^TfaRY, S2DMi-1−14, Compounds M,

e

TM, AGK2, NAM, and SirReal2 Evaluated by Using SFP3 and p53(Myr)-AMC (deMyr)

SIRT1

SIRT3

SIRT6

SFP3 (μM)

deMyr (μM)

SFP3 (μM)

deMyr (μM)

SFP3 (μM)

a

c

a

RIK^TfaRY

S2DMi-1

S2DMi-2

S2DMi-3

S2DMi-4

S2DMi-5

S2DMi-6

S2DMi-7

S2DMi-8

S2DMi-9

S2DMi-10

S2DMi-11

S2DMi-12

S2DMi-13

S2DMi-14

compound M

compound TM

AGK2

>2.0, 16%

32%

0.54 0.062

0.77 0.072

0.11 0.036

1.5 0.12

58%

3.1%

13%

40%

13%

11%

28%

43%

32%

a

c

a

>2.0, 22%

42%

34%

a

0.40 0.11

1.7 0.16

0.098 0.009

0.33 0.035

c

a

42%

11%

a

c

a

c

a

>2.0, 8.9%

3.2%

>2.0, 10%

−1.1%

a

1.1 0.30

0.14 0.011

0.050 0.004

0.071 0.009

0.18 0.020

0.055 0.007

0.077 0.004

0.48 0.052

0.19 0.027

0.34 0.045

a

0.12 0.092

0.048 0.006

a

c

a

c

a

>2.0, 18%

31%

>2.0, 14%

5.6%

2.6%

0.33 0.018

0.026 0.001

0.046 0.004

0.13 0.015

0.25 0.069

a

c

a

c

a

8.8%

20%

5.9%

2.6%

3.8%

a

c

a

c

a

1.3%

2.8%

−0.08%

5.6%

−7.4%

a

c

a

c

a

>2.0, 2.0%

6.3%

>2.0, 7.0%

−0.13%

N.D.

−0.32%

a

0.27 0.031

1.4 0.21

0.084 0.008

0.43 0.032

N.D.

−2.3%

28%

a

N.D.

1.7%

0.64%

−3.5%

−3.3%

N.D.

120 13

−2.2%

a

c

a

c

a

−0.59%

1.7%

a

c

a

c

a

0.81%

2.7%

−0.90%

27%

−1.2%

b

6.8%

10%

9.1%

d

NAM

SirReal2

33 1.7

>10

N.D.

>10

10 0.72

N.D.

a

b

c

d

e

Inhibition (%) at 2 μM. Inhibition (%) at 50 μM. Inhibition (%) at 1 μM. Data from ref 29. The data indicate IC₅₀(mean S.D., n = 3) of at

least two experiments. N.D. = not determined. For details, see Figures S9 −S13.

Figure 6. Competition analyses of S2DMi-6 and S2DMi-9 with SFP3. (a, d) Lineweaver−Burk plots of enzymatic cleavage of 0.625, 1.25, and 2.5

μM SFP3 in the presence of 35 nM SIRT2, 500 μM NAD⁺, and 0, 15, 20, or 30 nM S2DMi-6 or S2DMi-9. The initial velocity, v, was calculated

from the changes of ﬂuorescence intensity (F.I./min). The results are shown as mean S.D. (n = 3). (b, e) Michaelis−Menten plots of the data

shown in (a) and (d). (c, f) K_mand V_maxvalues were calculated from the data in (b) and (e).

SIRT2 selectivity pocket might be beneﬁcial just for the

development of SIRT2-selective deacetylase inhibitors, but not

for SIRT2 defatty-acylase inhibitors. Given this speculation, we

expect that it will be diﬃcult to develop subtype-selective

defatty-acylase inhibitors. However, selective SIRT2 defatty-

acylase inhibitors are of interest because it was recently

reported that SIRT2 defatty-acylase activity promotes carcino-

genesis through the regulation of Ras localization.¹⁶Therefore,

SIRT2 defatty-acylase inhibitors are candidate therapeutic

agents for cancers, with a novel mechanism of action, which

would be independent of SIRT2 deacetylase function, whose

inhibition also has an anticancer action via c-Myc degrada-

tion.¹⁹The defatty-acylase activity of SIRT6 regulates TNF-α

secretion and R-Ras2 localization,^9,31so it seems likely that the

defatty-acylase activities of other sirtuins also have signiﬁcant

biological functions distinct from those of their deacetylase

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mg, 0.47 mmol, 1.0 equiv) in DMF (1 mL). The reaction mixture was

stirred at R.T. for 15 min and then AcOEt was added. The organic

layer was washed with sodium phosphate buﬀer (pH ∼ 4) and brine,

dried over Na₂SO₄, and evaporated. The residue was puriﬁed by

column chromatography on silica gel (eluent: CH₂Cl₂/MeOH = 97/3

→ 95/5) to aﬀord Fmoc-Lys(OH-Dabcyl-EH)-OH (5) (252 mg,

activities. Thus, there is a clear need for development of potent

and selective inhibitors of the diﬀerent activities of individual

members of the SIRT family.

EXPERIMENTAL SECTION

■

1

0.39 mmol) in 83% yield as a yellow solid. H NMR (500 MHz,

General Information. Proton nuclear magnetic resonance spectra

(¹H NMR) were recorded on a JEOL JNM-ECZ500R spectrometer

in the indicated solvent. Chemical shifts (δ) are reported in parts per

million relative to the internal standard, tetramethylsilane. Electro-

spray ionization (ESI) mass spectra (MS) were recorded on a JEOL

JMS-T100LC mass spectrometer equipped with a nanospray ion

source. Ultraviolet−visible-light absorption spectra were recorded on

a UV-1800 spectrophotometer. Analytical HPLC was performed with

a Shimadzu pump system equipped with a reversed-phase ODS

column (Inertsil ODS-3 4.6 × 150 mm², GL Science) at a ﬂow rate of

1.0 mL/min. Semipreparative HPLC puriﬁcation was performed with

a JASCO PU-2086 pump system equipped with a reversed-phase

ODS column (Inertsil ODS-3 20 × 250 mm², GL Science) at a ﬂow

rate of 10 mL/min. Microplate ﬂuorescence assay was performed on

an ARVO X5 plate reader (PerkinElmer). Recombinant human

SIRT1 was purchased from R&D Systems, SIRT2 and SIRT3 were

purchased from ATGen, and SIRT6 was purchased from BioVision.

The FLUOR DE LYS SIRT2 assay kit (BML-LI179-0005) was

purchased from Enzo Life Sciences, Inc. Amino acids, Fmoc-NH-

Sieber amide resin, Fmoc-NH-SAL resin, and 2-chlorotrityl chloride

resin were purchased from Watanabe Chemical Industries, Ltd.

SirReal2 was purchased from Cayman Chemical. Cell Counting Kit-8

was purchased from Dojindo. All other reagents and solvents were

purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd.

(TCI), Wako Pure Chemical Industries, or Nacalai Tesque and used

without further puriﬁcation. Flash column chromatography was

performed using silica gel 60 (particle size 0.032−0.075) supplied

by Taiko Shoji Ltd. The purities of the synthesized compounds were

assessed by HPLC, and purity was ≥95% (Figure S18). The

enantiomerical purities of compounds 1−6 were assessed by HPLC

using CHIRALPAK IA-3 (Daicel), and enantiomerical purity was

≥99% (Figure S19).

DMSO-d₆): δ 1.28−1.46 (m, 4H), 1.54−1.76 (m, 2H), 3.00−3.09

(m, 2H), 3.42 (s, 3H), 3.49 (s, 2H), 3.85−3.96 (m 1H), 4.17−4.23

(m, 1H), 4.23−4.28 (m, 2H), 7.19 (d, 2H, J = 9.0 Hz), 7.32 (dd, 2H,

J = 7.5, 7.5 Hz), 7.38−7.46 (m, 4H), 7.62 (d, 1H, J = 7.3 Hz), 7.72 (d,

2H, J = 7.5 Hz), 7.79 (d, 2H, J = 8.2 Hz), 7.86 (d, 2H, J = 8.4 Hz),

7.89 (d, 2H, J = 7.5 Hz), 8.11 (t, 1H, J = 5.5 Hz).

Synthesis of Fmoc-Lys(Dabcyl-EH-H)-OH (6). To a solution of

Fmoc-Lys-OH·HCl (496 mg, 1.2 mmol, 1.2 equiv) and DIPEA (533

μL, 3.1 mmol, 3.0 equiv) in DMF (6 mL) was added a solution of 15

(344 mg, 1.0 mmol, 1.0 equiv) in DMF (2 mL). The reaction mixture

was stirred at R.T. for 10 min and then AcOEt was added. The

organic layer was washed with sodium phosphate buﬀer (pH ∼ 4) and

brine, dried over Na₂SO₄, and evaporated. The residue was puriﬁed by

column chromatography on silica gel (eluent: CH₂Cl₂/MeOH = 97/3

→ 95/5) to aﬀord Fmoc-Lys(Dabcyl-EH-H)-OH (6) (521 mg, 0.88

1

mmol) in 86% yield as a yellow solid. H NMR (500 MHz, DMSO-

d₆): δ 1.26−1.48 (m, 4H), 1.56−1.76 (m, 2H), 3.01−3.10 (m, 2H),

3.51 (s, 2H), 3.88−3.95 (m 1H), 4.18−4.25 (m, 1H), 4.25−4.31 (d,

2H, J = 7.0 Hz), 7.32 (dd, 2H, J = 7.4, 7.5 Hz), 7.41 (dd, 2H, J = 7.5,

7.5 Hz), 7.46 (d, 1H, J = 7.8 Hz), 7.54−7.65 (m, 4H), 7.72 (d, 2H, J

= 7.5 Hz), 7.82−7.92 (m, 6H), 8.12 (t, 1H, J = 5.4 Hz).

Synthesis of 4-[(4-(Diethylamino)phenyl)diazenyl]-

benzeneacetic Acid (diEtDabcyl-EH (8)). To a solution of 4-

aminobenzeneacetic acid (7) (453 mg, 3.0 mmol, 1.0 equiv) in 1 N

HCl (20 mL) was added dropwise a solution of NaNO₂(310 mg, 4.5

mmol, 1.5 equiv) in H₂O (2 mL) at 0 °C. The reaction mixture was

stirred at 0 °C for 30 min and then added dropwise to a solution of

N,N-diethylaniline (575 μL, 3.6 mmol, 1.2 equiv) in AcOH/H₂O

(10/5 mL) at 0 °C. Stirring was continued at R.T. for 2.5 h, and then

2 N NaOH was added to adjust the pH to 4. The resulting precipitate

was collected by ﬁltration and washed with cold water and Et₂O to

aﬀord diEtDabcyl-EH (8) (485 mg, 1.6 mmol) in 52% yield as a

brown solid. MS (ESI⁺): 312 [M + H]⁺.

Synthesis of 2,5-Dioxopyrrolidin-1-yl 4-[(4-(diethylamino)-

phenyl)diazenyl]benzeneacetate (diEtDabcyl-EH-SE (9)). To a

solution of 8 (480 mg, 1.5 mmol, 1.0 equiv) and EDCI•HCl (1480

mg, 7.7 mmol, 5.0 equiv) in dry DMF (8 mL) was added N-

hydroxysuccinimide (887 mg, 7.7 mmol, 5.0 equiv). The reaction

mixture was stirred at R.T. for 3 h, and then AcOEt and sodium

phosphate buﬀer (pH ∼ 4) were added. The organic layer was

separated, washed with brine, dried over Na₂SO₄, and evaporated.

The residue was puriﬁed by column chromatography on silica gel

(eluent: n-hexane/AcOEt = 2/1 → 1/1) to aﬀord diEtDabcyl-EH-SE

(9) (58 mg, 0.14 mmol) in 9.2% yield as an orange solid. MS (ESI⁺):

409 [M + H]⁺.

Synthesis of RIK^TfaRY. RIK^TfaRY was synthesized in accordance

with the previous report.²⁸Purity by HPLC: 98.1% (230 nm); t_R=

11.6 min (A/B = 95:5 → 90:10 (5 min) → 0:100 (20 min); A: 0.1%

TFA Milli-Q, B: 0.1% TFA CH₃CN).

Syntheses of Compounds M and TM. Compounds M and TM

were synthesized in accordance with the previous report.¹⁹Purity by

HPLC: 97.6% (254 nm); t_R= 22.3 min for compound M, 97.5% (254

nm); t_R= 24.1 min for compound TM (A/B = 50:50 → 0:100 (20

min) → 0:100 (25 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN).

Syntheses of Fmoc-Lys(Dabcyl)-OH (1), Fmoc-Lys(Dabcyl-

EH)-OH (2), and Fmoc-Lys(Dabcyl-PH)-OH (3). These com-

pounds were synthesized in accordance with our previous report.²⁹

Synthesis of Fmoc-Lys(diEtDabcyl-EH)-OH (4). To a solution

of Fmoc-Lys-OH·HCl (60 mg, 0.15 mmol, 1.05 equiv) and DIPEA

(74 μL, 0.43 mmol, 3.0 equiv) in DMF (1 mL) was added 5 (58 mg,

0.14 mmol, 1.0 equiv). The reaction mixture was stirred at R.T. for 30

min and then AcOEt was added. The organic layer was washed with

sodium phosphate buﬀer (pH ∼ 4) and brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: CH₂Cl₂/MeOH = 99/1 → 97/3) to aﬀord Fmoc-

Lys(diEtDabcyl-EH)-OH (4) (33 mg, 0.050 mmol) in 35% yield as a

Synthesis of 4-[(4-Hydroxyphenyl)diazenyl]benzeneacetic

Acid (OH-Dabcyl-EH (10)). To a solution of 4-aminobenzeneacetic

acid (7) (756 mg, 5.0 mmol, 1.0 equiv) in 2 N HCl (15 mL) and

H₂O (5 mL) was added dropwise a solution of NaNO₂(414 mg, 6.0

mmol, 1.2 equiv) in H₂O (1 mL) at 0 °C. The reaction mixture was

stirred at 0 °C for 30 min, and then phenol (470 mg, 5.0 mmol, 1.0

equiv) in 2 N NaOH (5 mL) was added at 0 °C. The reaction mixture

was stirred at 0 °C for 1.5 h. The precipitate was collected by

ﬁltration, and the residue was puriﬁed by column chromatography on

silica gel (eluent: CH₂Cl₂/MeOH = 95/5) to aﬀord OH-Dabcyl-EH

1

yellow solid. H NMR (500 MHz, DMSO-d₆): δ 1.15 (t, 6H, J = 7.1

Hz), 1.27−1.46 (m, 4H), 1.55−1.76 (m, 2H), 3.00−3.09 (m, 2H),

3.44 (q, 4H, J = 7.1 Hz), 3.86−3.94 (m 1H), 4.18−4.24 (m, 1H),

4.25−4.31 (m, 2H), 6.77 (d, 2H, J = 9.4 Hz), 7.32 (dd, 2H, J = 7.5,

7.5 Hz), 7.37−7.44 (m, 4H), 7.62 (d, 1H, J = 8.0 Hz), 7.69 (d, 2H, J

= 8.3 Hz), 7.71−7.75 (m, 4H), 7.89 (d, 2H, J = 7.5 Hz), 8.09 (t, 1H, J

= 5.6 Hz); MS (ESI⁺): 662 [M + H]⁺.

Synthesis of Fmoc-Lys(OH-Dabcyl-EH)-OH (5). To a solution

of Fmoc-Lys-OH·HCl (226 mg, 0.56 mmol, 1.2 equiv) and DIPEA

(244 μL, 1.4 mmol, 3.0 equiv) in DMF (5 mL) was added 13 (185

1

(10) (385 mg, 1.5 mmol) in 30% yield as a yellow solid. H NMR

(500 MHz, DMSO-d₆): δ 3.67 (s, 2H), 6.94 (d, 2H, J = 8.7 Hz), 7.44

(d, 2H, J = 8.4 Hz), 7.76 (d, 2H, J = 8.4 Hz), 7.79 (d, 2H, J = 8.7 Hz),

10.31 (brs, 1H); MS (ESI⁻): 255 [M − H]⁻.

Synthesis of Methoxymethyl 4-[(4-(Methoxymethoxy)-

phenyl)diazenyl]benzeneacetate (11). To a solution of 10 (385

mg, 1.5 mmol, 1.0 equiv) and DIPEA (1310 μL, 7.5 mmol, 5.0 equiv)

in DMF (6 mL) was added dropwise MOM-Cl (342 μL, 3.0 mmol,

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DOI: 10.1021/acs.jmedchem.9b00315

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Journal of Medicinal Chemistry

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2.0 equiv) at 0 °C. The reaction mixture was stirred at 0 °C for 15

min and at R.T. for 13 h and then diluted with AcOEt, washed with

sodium phosphate buﬀer (pH ∼ 4) and brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: n-hexane/AcOEt = 5/1 → 4/1) to aﬀord 11 (445

(93 mg, 0.15 mmol), Fmoc-Lys(Dabcyl-EH)-OH (94 mg, 0.15

mmol), Fmoc-Lys(Dabcyl-PH)-OH (65 mg, 0.10 mmol), Fmoc-

Lys(OH-Dabcyl-EH)-OH (98 mg, 0.15 mmol), Fmoc-Lys(Dabcyl-

EH-H)-OH (89 mg, 0.15 mmol), Fmoc-Val-OH (51 mg, 0.15 mmol),

Fmoc-Lys(Boc)-OH (70 mg, 0.15 mmol), Fmoc-Ile-OH (53 mg, 0.15

mmol), Fmoc-Thr(tBu)-OH (60 mg, 0.15 mmol), Fmoc-Asp(tBu)-

OH (62 mg, 0.15 mmol), and Fmoc-Ser(tBu)-OH (58 mg, 0.15

mmol). The Fmoc group was removed twice by using 20% piperidine

in DMF (2 mL) at R.T. for 2 and 10 min. The N-terminal amino acid

was acetylated with 25% Ac₂O in dichloromethane (DCM) (2 mL)

for 5 min. For cleavage of the synthetic peptide without loss of side-

chain-protecting groups, the resin was mixed with 1% TFA and 1%

triethylsilane (TES) in DCM (2 mL) for 2 min (15 times); then, the

ﬁltrate was collected in sat. NaHCO₃aq. (50 mL). The organic phase

was separated, washed with brine, dried over Na₂SO₄, and evaporated

to aﬀord the following compounds.

1

mg, 1.3 mmol) in 86% yield as a yellow solid. H NMR (500 MHz,

DMSO-d₆): δ 3.39 (s, 3H), 3.42 (s, 3H), 3.86 (s, 2H), 5.22 (s, 2H),

5.31 (s, 2H), 7.21 (d, 2H, J = 8.8 Hz), 7.49 (d, 2H, J = 8.3 Hz), 7.83

(d, 2H, J = 8.3 Hz), 7.89 (d, 2H, J = 8.8 Hz); MS (ESI⁺): 345 [M −

H]⁺.

Synthesis of 4-[(4-(Methoxymethoxy)phenyl)diazenyl]-

benzeneacetic Acid (12). To a solution of 11 (445 mg, 1.3

mmol, 1.0 equiv) in EtOH (2 mL) was added 2 N NaOH (2 mL, 3.0

equiv). The mixture was stirred at 70 °C for 1.5 h, cooled to 0 °C,

acidiﬁed with 2 N HCl, and extracted with AcOEt. The combined

organic layer was washed with brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: CH₂Cl₂/MeOH = 98/2) to aﬀord 12 (268 mg, 0.89

S2DMi-1-protected (39 mg, 0.025 mmol), 49% yield as an orange

solid. MS (ESI⁺): 1609 [M + Na]⁺.

1

mmol) in 69% yield as a yellow solid. H NMR (500 MHz, DMSO-

S2DMi-2-protected (66 mg, 0.041 mmol), 82% yield as an orange

solid. MS (ESI⁺): 1601 [M + H]⁺.

d₆): δ 3.42 (s, 3H), 3.69 (s, 2H), 5.31 (s, 2H), 7.21 (d, 2H, J = 9.0

Hz), 7.46 (d, 2H, J = 8.3 Hz), 7.81 (d, 2H, J = 8.3 Hz), 7.89 (d, 2H, J

= 9.0 Hz).

S2DMi-3-protected (68 mg, 0.042 mmol), 84% yield as an orange

solid. MS (ESI⁺): 1637 [M + Na]⁺.

S2DMi-4-protected (33 mg, 0.032 mmol), 64% yield as an orange

solid. MS (ESI⁺): 1045 [M + Na]⁺.

Synthesis of 2, 5-Dioxopyrrolidin-1-yl 4-[(4-

(methoxymethoxy)phenyl)diazenyl]benzeneacetate (13). To

a solution of 12 (268 mg, 0.89 mmol, 1.0 equiv) and EDCI•HCl (855

mg, 4.5 mmol, 5.0 equiv) in dry DMF (5 mL) was added N-

hydroxysuccinimide (513 mg, 4.5 mmol, 5.0 equiv). The reaction

mixture was stirred at R.T. for 5 h, and then AcOEt and sodium

phosphate buﬀer (pH ∼ 4) were added. The organic layer was

separated, washed with brine, dried over Na₂SO₄, and evaporated.

The residue was puriﬁed by column chromatography on silica gel

(eluent: n-hexane/AcOEt = 2/1 → 1/1) to aﬀord 13 (205 mg, 0.52

mmol) in 59% yield as an orange solid. ¹H NMR (500 MHz, DMSO-

d₆): δ 2.82 (s, 4H), 3.42 (s, 3H), 3.85 (s, 2H), 5.31 (s, 2H), 7.22 (d,

2H, J = 8.8 Hz), 7.56 (d, 2H, J = 8.3 Hz), 7.85 (d, 2H, J = 8.3 Hz),

7.89 (d, 2H, J = 8.8 Hz).

S2DMi-6-protected (34 mg, 0.041 mmol), 42% yield as a yellow

solid. MS (ESI⁺): 1640 [M + Na]⁺.

S2DMi-7-protected (78 mg, 0.050 mmol), quantitative yield as a

yellow solid. MS (ESI⁺): 1580 [M + Na]⁺.

S2DMi-8-protected (49 mg, 0.049 mmol), 98% yield as a yellow

solid. MS (ESI⁺): 994 [M + H]⁺.

S2DMi-13-protected (47 mg, 0.050 mmol), quantitative yield as a

yellow solid. MS (ESI⁺): 953 [M + Na]⁺.

S2DMi-14-protected (45 mg, 0.042 mmol), 83% yield as a yellow

solid. MS (ESI⁺): 1101 [M + Na]⁺.

Syntheses of S2DMi-1−4, S2DMi-6−8, S2DMi-13, and

S2DMi-14. To a solution of S2DMi-1−4, S2DMi-6−8, S2DMi-13,

or S2DMi-14-protected in CH₂Cl₂(3 mL) was added TFA (3 mL).

The reaction mixture was stirred for 2 h at R.T. and then evaporated.

The residue was puriﬁed by reversed-phase HPLC to aﬀord the

following compounds.

S2DMi-1·3TFA (22 mg, 0.0161 mmol), 65% yield as a red solid.

Purity by HPLC: 96.1% (254 nm); t_R= 14.4 min (A/B = 95:5 →

90:10 (5 min) → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); high-resolution mass spectrometry (HRMS) (ESI⁺): calcd:

1027.5954; found: 1027.5994 [M + H]⁺(+3.92 ppm).

S2DMi-2·3TFA (45 mg, 0.0325 mmol), 79% yield as a red solid.

Purity by HPLC: 99.7% (254 nm); t_R= 8.6 min (A/B = 80:20 →

0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN);

HRMS (ESI⁺): calcd: 1041.6110; found: 1041.6087 [M + H]⁺(−2.27

ppm).

S2DMi-3·3TFA (51 mg, 0.037 mmol), 87% yield as a red solid.

Purity by HPLC: 99.7% (254 nm); t_R= 8.7 min (A/B = 80:20 →

0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN);

HRMS (ESI⁺): calcd: 1055.6267; found: 1055.6257 [M + H]⁺(−0.93

ppm).

S2DMi-4·TFA (13 mg, 0.0134 mmol), 42% yield as a red solid.

Purity by HPLC: 95.9% (254 nm); t_R= 14.8 min (A/B = 95:5 →

90:10 (5 min) → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); HRMS (ESI⁺): calcd: 877.4184; found: 877.4192 [M +

Na]⁺(+0.92 ppm).

Synthesis of 4-(2-Phenyldiazenyl)benzeneacetic Acid

(Dabcyl-EH-H (14)). To a solution of nitrosobenzene (535 mg, 5.0

mmol, 1.0 equiv) in AcOH (6 mL) was added dropwise a solution of

7 (907 mg, 6.0 mmol, 1.2 equiv) in AcOH (6 mL) at R.T. The

reaction mixture was stirred at R.T. for 21 h and then evaporated. The

residue was puriﬁed by column chromatography on silica gel (eluent:

CH₂Cl₂/MeOH = 95/5) to aﬀord Dabcyl-EH-H (14) (1087 mg, 4.5

1

mmol) in 90% yield as a yellow solid. H NMR (500 MHz, DMSO-

d₆): δ 3.71 (s, 2H), 7.49 (d, 2H, J = 8.3 Hz), 7.55−7.64 (m, 3H), 7.85

(d, 2H, J = 8.2 Hz), 7.89 (d, 2H, J = 8.0 Hz).

Synthesis of 2,5-Dioxopyrrolidin-1-yl 4-4-(2-

Phenyldiazenyl)benzeneacetate (Dabcyl-EH-H-SE (15)). To a

solution of 14 (480 mg, 2.0 mmol, 1.0 equiv) and EDCI•HCl (1920

mg, 10 mmol, 5.0 equiv) in dry DMF (10 mL) was added N-

hydroxysuccinimide (1150 mg, 10 mmol, 5.0 equiv). The reaction

mixture was stirred at R.T. for 11 h, and then AcOEt and sodium

phosphate buﬀer (pH ∼ 4) were added. The organic layer was

separated, washed with brine, dried over Na₂SO₄, and evaporated.

The residue was puriﬁed by column chromatography on silica gel

(eluent: n-hexane/AcOEt = 2/1 → 1/1) to aﬀord Dabcyl-EH-H-SE

1

(15) (344 mg, 1.0 mmol) in 51% yield as an orange solid. H NMR

(500 MHz, DMSO-d₆): δ 2.82 (s, 4H), 4.27 (s, 2H), 7.56−7.64 (m,

5H), 7.88−7.92 (m, 4H).

Fmoc Solid-Phase Syntheses of S2DMi-1−4, S2DMi-6−8,

S2DMi-13, S2DMi-14-Protected. The peptide chain was synthe-

sized using Fmoc solid-phase chemistry on Fmoc-NH-Sieber amide

resin (96 mg, 0.050 mmol) in a PD-10 Empty Column at R.T. Each

assembly step was done by activating Fmoc-amino acid (0.15 mmol)

with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuor-

ophosphate (HBTU) (57 mg, 0.15 mmol), 1-hydroxybenzotriazole

hydrate (HOBt·H₂O) (23 mg, 0.15 mmol), and DIPEA (53 μL, 0.30

mmol) in DMF (1.5 mL) for 1 h. The Fmoc-amino acids used in this

synthesis were as follows: Fmoc-Tyr(tBu)-OH (69 mg, 0.15 mmol),

Fmoc-Arg(Pbf)-OH (98 mg, 0.15 mmol), Fmoc-Lys(Dabcyl)-OH

S2DMi-6·2TFA (11 mg, 0.0089 mmol), 22% yield as a yellow

solid. Purity by HPLC: 98.7% (254 nm); t_R= 10.2 min (A/B = 80:20

→ 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN);

HRMS (ESI⁺): calcd: 1014.5637; found: 1014.5682 [M + H]⁺(+4.37

ppm).

S2DMi-7·2TFA (37 mg, 0.030 mmol), 60% yield as a yellow solid.

Purity by HPLC: 97.7% (254 nm); t_R= 10.1, 11.4 min (A/B = 80:20

→ 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN);

HRMS (ESI⁺): calcd: 998.5688; found: 998.5670 [M + H]⁺(−1.87

ppm).

M

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Journal of Medicinal Chemistry

Article

S2DMi-8 (16 mg, 0.019 mmol), 39% yield as a yellow solid. Purity

by HPLC: 98.2% (254 nm); t_R= 8.7, 10.5 min (A/B = 80:20 → 0:100

(20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN); HRMS

(ESI⁺): calcd: 848.3919; found: 848.3908 [M + Na]⁺(−1.28 ppm).

S2DMi-13·TFA (23 mg, 0.029 mmol), 58% yield as a yellow solid.

Purity by HPLC: 98.2% (254 nm); t_R= 8.8, 10.6 min (A/B = 80:20

→ 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN);

HRMS (ESI⁺): calcd: 679.4044; found: 679.4049 [M + Na]⁺(+0.71

ppm).

S2DMi-14·2TFA (14 mg, 0.013 mmol), 30% yield as a yellow

solid. Purity by HPLC: 98.8% (254 nm); t_R= 7.3, 8.8 min (A/B =

80:20 → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); HRMS (ESI⁺): calcd: 878.5616; found: 878.5630 [M +

H]⁺(+1.53 ppm).

DMF (1.5 mL) for 1 h. The Fmoc-amino acids used in this synthesis

were as follows: Fmoc-Tyr(tBu)-OH (69 mg, 0.15 mmol), Fmoc-

Arg(Pbf)-OH (98 mg, 0.15 mmol), Fmoc-Lys(thioMyr)-OH (98 mg,

0.17 mmol), and Fmoc-Ile-OH (53 mg, 0.15 mmol). The Fmoc group

was removed twice by using 20% piperidine in DMF (2 mL) at R.T.

for 2 and 10 min. The N-terminal amino acid was acetylated with 25%

Ac₂O in DCM (2 mL) for 5 min. For cleavage of the synthetic peptide

without loss of side-chain-protecting groups, the resin was mixed with

1% TFA and 1% TES in DCM (2 mL) for 2 min (15 times), and the

ﬁltrate was collected in sat. NaHCO₃aq. (50 mL). The organic phase

was separated, washed with brine, dried over Na₂SO₄, and evaporated

to aﬀord S2DMi-9-protected (77 mg, 0.049 mmol) in 99% yield as a

white solid. MS (ESI⁺): 1563 [M + H]⁺.

Synthesis of S2DMi-9. To a solution of S2DMi-9-protected (37

mg, 0.0237 mmol) in CH₂Cl₂(3 mL) was added TFA (3 mL). The

reaction mixture was stirred for 2 h at R.T. and then evaporated. The

residue was puriﬁed by reversed-phase HPLC to aﬀord S2DMi-9·

2TFA (10 mg, 0.0083 mmol) in 35% yield as a white solid. Purity by

HPLC: 98.8% (254 nm); t_R= 11.9 min (A/B = 70:30 → 0:100 (20

min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN); HRMS (ESI⁺):

calcd: 1002.6650; found: 1002.6639 [M + H]⁺(−1.14 ppm).

Synthesis of Cbz-Lys(OH-Dabcyl-EH)-OH (20). To a solution

of Cbz-Lys-OH (19) (320 mg, 1.1 mmol, 1.1 equiv) and DIPEA (543

μL, 3.1 mmol, 3.0 equiv) in DMF (8 mL) was added 13 (413 mg, 1.0

mmol, 1.0 equiv). The reaction mixture was stirred at R.T. for 2.5 h,

and then AcOEt was added. The organic layer was washed with

sodium phosphate buﬀer (pH ∼ 4) and brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: CH₂Cl₂/MeOH = 96/4) to aﬀord Cbz-Lys(OH-

Dabcyl-EH)-OH (20) (435 mg, 0.77 mmol) in 74% yield as a yellow

solid. ¹H NMR (500 MHz, DMSO-d₆): δ 1.28−1.45 (m, 4H), 1.53−

1.74 (m, 2H), 3.01−3.08 (m, 2H), 3.42 (s, 3H), 3.49 (s, 2H), 3.85−

3.95 (m 1H), 5.00−5.06 (m, 2H), 5.30 (s, 2H), 7.20 (d, 2H, J = 9.0

Hz), 7.25−7.39 (m, 5H), 7.43 (d, 2H, J = 8.2 Hz), 7.53 (d, 1H, J =

8.0 Hz), 7.78 (d, 2H, J = 8.2 Hz), 7.87 (d, 2H, J = 8.0 Hz), 8.09 (t,

1H, J = 5.4 Hz).

Fmoc Solid-Phase Synthesis of S2DMi-5. The peptide chain

was synthesized using Fmoc solid-phase chemistry on Fmoc-NH-SAL

resin (72 mg, 0.050 mmol) in a PD-10 Empty Column at R.T. Each

assembly step was done by activating Fmoc-amino acid (0.15 mmol)

with HBTU (57 mg, 0.15 mmol), HOBt·H₂O (23 mg, 0.15 mmol),

and DIPEA (53 μL, 0.30 mmol) in DMF (1.5 mL) for 1 h. The

Fmoc-amino acids used in this synthesis were as follows: Fmoc-

Tyr(tBu)-OH (69 mg, 0.15 mmol), Fmoc-Arg(Pbf)-OH (98 mg, 0.15

mmol), Fmoc-Lys(diEtDabcyl-EH)-OH (33 mg, 0.05 mmol), and

Fmoc-Ile-OH (53 mg, 0.15 mmol). The Fmoc group was removed

twice by using 20% piperidine in DMF (2 mL) at R.T. for 2 and 10

min. The reaction mixture after the condensation with Fmoc-

Lys(diEtDabcyl-EH)-OH and N-terminal amino acid was treated with

25% Ac₂O in DCM (2 mL) for 5 min. For cleavage of the synthetic

peptide, the resin was gently stirred in TFA/H₂O/TES (1800/100/

100 μL) for 90 min, and then the ﬁltrate was collected and evaporated

to aﬀord crude S2DMi-5 (60 mg). The crude product was puriﬁed by

reversed-phase HPLC to aﬀord S2DMi-5·3TFA (8.0 mg, 0.0057

mmol) in 11% yield as a red solid. Purity by HPLC: 95.8% (254 nm);

t_R= 8.2 min (A/B = 80:20 → 0:100 (20 min); A: 0.1% TFA Milli-Q,

B: 0.1% TFA CH₃CN); HRMS (ESI⁺): calcd: 1069.6423; found:

1069.6418 [M + H]⁺(−0.46 ppm).

Synthesis of Fmoc-Lys(Myr)-OH (17). To a solution of Fmoc-

Lys-OH·HCl (16) (609 mg, 1.4 mmol, 1.0 equiv) and Na₂CO₃(458

mg, 4.3 mmol, 3.0 equiv) in H₂O/dioxane (10 mL/8 mL) was added

dropwise a solution of myristoyl chloride (466 μL, 1.7 mmol, 1.2

equiv) in dioxane (8 mL) at 0 °C. The reaction mixture was stirred

under N₂for 3 h at R.T. and then diluted with CH₂Cl₂. The organic

phase was washed with 1 N HCl and brine, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: CH₂Cl₂/MeOH = 3/97 → 10/90) to aﬀord Fmoc-

Lys(Myr)-OH (17) (653 mg, 1.1 mmol) in 78% yield as a white

Synthesis of Cbz-Lys(OH-Dabcyl-EH)-Ala-NH₂(21). To a

solution of Cbz-Lys(OH-Dabcyl-EH)-OH (20) (432 mg, 0.77

mmol, 1.0 equiv), COMU (395 mg, 0.92 mmol, 1.2 equiv), and

DIPEA (402 μL, 2.3 mmol, 3.0 equiv) in DMF (6 mL) was added H-

Ala-NH₂·HCl (115 mg, 0.92 mmol, 1.2 equiv). The reaction mixture

was stirred at R.T. for 12 h, and then AcOEt was added. The

precipitate was collected to aﬀord Cbz-Lys(OH-Dabcyl-EH)-Ala-

1

NH₂(21) (409 mg, 0.64 mmol) in 84% yield as a yellow solid. H

NMR (500 MHz, DMSO-d₆): δ 1.20 (d, 3H, J = 7.0 Hz), 1.23−1.70

(m, 6H), 2.98−3.09 (m, 2H), 3.42 (s, 3H), 3.50 (s, 2H), 3.92−3.99

(m 1H), 4.16−4.23 (m, 1H), 5.02 (s, 2H), 5.30 (s, 2H), 7.00 (brs,

1H), 7.21 (d, 2H, J = 9.0 Hz), 7.26−7.38 (m, 6H), 7.40−7.46 (m,

3H), 7.78 (d, 2H, J = 8.3 Hz), 7.88 (d, 2H, J = 7.9 Hz), 8.09 (t, 1H, J

= 5.5 Hz).

Synthesis of S2DMi-10. To a solution of Cbz-Lys(OH-Dabcyl-

EH)-Ala-NH₂(21) (409 mg, 0.64 mmol) in CH₂Cl₂(9 mL) was

added TFA (9 mL). The reaction mixture was stirred at R.T. for 1 h

and then evaporated. The residue was puriﬁed by column

chromatography on silica gel (eluent: CH₂Cl₂/MeOH = 97/3 →

92/8) to aﬀord S2DMi-10 (289 mg, 0.49 mmol) in 76% yield as an

orange solid. ¹H NMR (500 MHz, DMSO-d₆): δ 1.20 (d, 3H, J = 7.1

Hz), 1.24−1.70 (m, 6H), 2.98−3.08 (m, 2H), 3.48 (s, 2H), 3.92−

3.99 (m 1H), 4.16−4.24 (m, 1H), 5.02 (s, 2H), 6.93 (d, 2H, J = 8.7

Hz), 7.00 (brs, 1H), 7.26−7.38 (m, 6H), 7.39−7.43 (m, 3H), 7.74 (d,

2H, J = 8.3 Hz), 7.78 (d, 2H, J = 9.7 Hz), 8.07 (t, 1H, J = 5.5 Hz),

10.26 (s, 1H); purity by HPLC: 95.4% (254 nm); t_R= 13.6 min (A/B

= 80:20 → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); HRMS (ESI⁺): calcd: 589.2775; found: 589.2779 [M +

H]⁺(+0.72 ppm).

1

solid. H NMR (500 MHz, DMSO-d₆): δ 0.84 (t, 3H, J = 7.0 Hz),

1.16−1.74 (m, 28H), 2.02 (t, 2H, J = 7.4 Hz), 2.95−3.06 (m, 2H),

3.86−3.94 (m, 1H), 4.18−4.31 (m, 3H), 7.33 (dd, 2H, J = 7.3, 7.4

Hz), 7.42 (dd, 2H, J = 7.4, 7.4 Hz), 7.60 (d, 1H, J = 8.0 Hz), 7.70−

7.79 (m, 3H), 7.89 (d, 2H, J = 7.5 Hz).

Synthesis of Fmoc-Lys(thioMyr)-OH (18). To a solution of

Fmoc-Lys(Myr)-OH (17) (139 mg, 0.24 mmol, 1.0 equiv) in THF

(5 mL) was added Lawesson’s reagent (97 mg, 0.24 mmol, 1.0 equiv).

The reaction mixture was stirred at R.T. for 2 h under N₂and then

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: CH₂Cl₂/MeOH = 3/97 → 5/95) to aﬀord Fmoc-

Lys(thioMyr)-OH (18) (98 mg, 0.17 mmol) in 69% yield as a white

1

solid. H NMR (500 MHz, DMSO-d₆): δ 0.88 (t, 3H, J = 8.0 Hz),

1.19−2.02 (m, 28H), 2.59 (t, 2H, J = 7.5 Hz), 3.60−3.71 (m, 2H),

3.86−3.94 (m, 1H), 4.23 (t, 1H, J = 6.8 Hz), 4.35−4.61 (m, 3H),

5.38 (d, 1H, J = 7.0 Hz), 7.32 (dd, 2H, J = 7.4, 7.5 Hz), 7.37−7.44

(m, 3H), 7.55−7.63 (m, 2H), 7.77 (d, 2H, J = 7.5 Hz); (ESI⁺): 594

[M + H]⁺.

Fmoc Solid-Phase Synthesis of S2DMi-9-Protected. The

peptide chain was synthesized using Fmoc solid-phase chemistry on

Fmoc-NH-Sieber amide resin (96 mg, 0.050 mmol) in a PD-10

Empty Column at R.T. Each assembly step was done by activating

Fmoc-amino acid (0.15 mmol) with HBTU (57 mg, 0.15 mmol),

HOBt·H₂O (23 mg, 0.15 mmol), and DIPEA (53 μL, 0.30 mmol) in

Synthesis of S2DMi-11. To a solution of S2DMi-10 (59 mg, 0.10

mmol, 1.0 equiv), Cs₂CO₃(39 mg, 0.12 mmol, 1.2 equiv), and TBAI

(3.7 mg, 0.010 mmol, 0.10 equiv) in DMF (2 mL) was added benzyl

bromide (18 μL, 0.15 mmol, 1.5 equiv). The reaction mixture was

stirred at R.T. for 2 h and then AcOEt was added. The organic layer

N

DOI: 10.1021/acs.jmedchem.9b00315

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Journal of Medicinal Chemistry

Article

was washed with sodium phosphate buﬀer (pH ∼ 4) and brine, dried

over Na₂SO₄, and evaporated. The residue was puriﬁed by column

chromatography on silica gel (eluent: CH₂Cl₂/MeOH = 97/3 → 93/

7) to aﬀord S2DMi-11 (57 mg, 0.084 mmol) in 84% yield as a light-

yellow solid. ¹H NMR (500 MHz, DMSO-d₆): δ 1.20 (d, 3H, J = 7.0

Hz), 1.22−1.69 (m, 6H), 2.96−3.08 (m, 2H), 3.49 (s, 2H), 3.92−

3.99 (m 1H), 4.16−4.24 (m, 1H), 5.02 (s, 2H), 5.23 (s, 2H), 7.00

(brs, 1H), 7.21 (d, 2H, J = 9.0 Hz), 7.25−7.46 (m, 12H), 7.49 (d, 2H,

J = 7.3 Hz), 7.78 (d, 2H, J = 8.4 Hz), 7.84−7.91 (m, 3H), 8.08 (t, 1H,

J = 5.5 Hz); purity by HPLC: 95.9% (254 nm); t_R= 16.0, 18.3 min

(A/B = 80:20 → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); HRMS (ESI⁺): calcd: 701.3064; found: 701.3049 [M +

Na]⁺(−2.01 ppm).

Synthesis of S2DMi-12. To a solution of S2DMi-10 (59 mg, 0.10

mmol, 1.0 equiv), Cs₂CO₃(49 mg, 0.15 mmol, 1.5 equiv), and TBAI

(3.7 mg, 0.010 mmol, 0.10 equiv) in DMF (2 mL) was added

phenethyl bromide (41 μL, 0.30 mmol, 3.0 equiv). The reaction

mixture was stirred at R.T. for 48 h, and then AcOEt was added. The

organic layer was washed with sodium phosphate buﬀer (pH ∼ 4) and

brine, dried over Na₂SO₄, and evaporated. The residue was puriﬁed by

column chromatography on silica gel (eluent: CH₂Cl₂/MeOH = 97/3

→ 94/6) to aﬀord S2DMi-12 (29 mg, 0.042 mmol) in 42% yield as a

light-yellow solid. ¹H NMR (500 MHz, DMSO-d₆): δ 1.20 (d, 3H, J =

7.0 Hz), 1.22−1.44 (m, 4H), 1.45−1.56 (m, 1H), 1.58−1.68 (m, 1H),

2.97−3.06 (m, 2H), 3.08 (t, 2H, J = 6.8 Hz), 3.49 (s, 2H), 3.92−3.99

(m 1H), 4.16−4.24 (m, 1H), 4.31 (t, 2H, J = 6.8 Hz), 5.02 (s, 2H),

7.00 (brs, 1H), 7.13 (d, 2H, J = 9.0 Hz), 7.22−7.26 (m, 1H), 7.27−

7.38 (m, 10H), 7.40−7.45 (m, 3H), 7.77 (d, 2H, J = 8.3 Hz), 7.83−

7.90 (m, 3H), 8.08 (t, 1H, J = 5.8 Hz); purity by HPLC: 97.6% (254

nm); t_R= 13.7, 16.6 min (A/B = 70:30 → 0:100 (20 min); A: 0.1%

TFA Milli-Q, B: 0.1% TFA CH₃CN); HRMS (ESI⁺): calcd:

693.3401; found: 693.3389 [M + H]⁺(−1.65 ppm).

Synthesis of 26. To a solution of 25 (85 mg, 0.13 mmol) in

MeOH/CH₂Cl₂(8 mL/7 mL) was added Pd/C. The reaction

mixture was stirred at R.T. under H₂for 1.5 h and then ﬁltered

through Celite. The ﬁltrate was evaporated to aﬀord 26 (69 mg, 0.13

1

mmol) in quantitative yield as a white solid. H NMR (500 MHz,

DMSO-d₆): δ 0.85 (t, 3H, J = 6.8 Hz), 1.16−1.51 (m, 27H), 1.60−

1.70 (m, 1H), 1.99 (t, 2H, J = 7.5 Hz), 2.40 (s, 3H), 2.95−3.05 (m,

2H), 6.26 (s, 1H),7.56 (dd, 1H, J = 2.1, 8.6 Hz), 7.68−7.74 (m, 2H),

7.84 (d, 1H, J = 2.1 Hz); MS (ESI⁺): 514 [M + H]⁺.

Synthesis of 27. To a solution of Ac-R(Pbf)H(Trt)K(Boc)-OH

(29) (50 mg, 0.047 mmol, 1.0 equiv), COMU (30 mg, 0.070 mmol,

1.5 equiv), and DIPEA (16 μL, 0.093 mmol, 2.0 equiv) in DMF (2

mL) was added 26 (29 mg, 0.056 mmol, 1.2 equiv). The reaction

mixture was stirred at R.T. for 1 h and then AcOEt was added. The

organic layer was washed with sodium phosphate buﬀer (pH ∼ 4),

sat. NaHCO₃, and brine, dried over Na₂SO₄, and evaporated. The

residue was puriﬁed by column chromatography on silica gel (eluent:

CH₂Cl₂/MeOH = 3/97 → 8/92) to aﬀord 27 (51 mg, 0.032 mmol)

in 70% yield as a white solid. MS (ESI⁺): 1571 [M + H]⁺.

Synthesis of Ac-R(Pbf)H(Trt)K(Boc)-OH (29). Attachment of

the ﬁrst amino acid to 2-chlorotrityl chloride resin (63 mg, 0.10

mmol) was performed by reacting Fmoc-Lys(Boc)-OH (70 mg, 0.15

mmol) and DIPEA (87 μL, 0.50 mmol) with the resin in CH₂Cl₂(2

mL) for 2 h using a PD-10 Empty Column. The peptide chain was

synthesized using Fmoc solid-phase chemistry at R.T. Each assembly

step was done by activating Fmoc-His(Trt)-OH (186 mg, 0.30 mmol)

or Fmoc-Arg(Pbf)-OH (195 mg, 0.30 mmol) with HBTU (113 mg,

0.30 mmol), HOBt·H₂O (46 mg, 0.30 mmol), and DIPEA (105 μL,

0.60 mmol) in DMF (2 mL) for 1 h. The Fmoc group was removed

with 20% piperidine in DMF (2 mL) at R.T. twice, for 2 min and 10

min. The N-terminal amino acid was acetylated with 25% Ac₂O in

CH₂Cl₂(2 mL) for 5 min. For cleavage of the synthetic peptide

without loss of side-chain-protecting groups, the resin was mixed with

1% TFA in CH₂Cl₂(2 mL) for 1 min (6 times, monitored with TLC),

and then the ﬁltrate was collected in 10% pyridine in MeOH (2 mL),

after which the solution was evaporated. The residue was dissolved in

AcOEt. This solution was washed with 0.1 N HCl and brine, dried

over Na₂SO₄, and evaporated to aﬀord Ac-R(Pbf)H(Trt)K(Boc)-

OH (29) (94 mg, 0.087 mmol) in 87% yield as a white solid. MS

(ESI⁺): 1076 [M + H]⁺.

Synthesis of p53(Myr)-AMC. To a solution of 27 (51 mg, 0.032

mmol) in CH₂Cl₂(5 mL) was added TFA (7 mL). The reaction

mixture was stirred at R.T. for 2.5 h and then evaporated. The residue

was puriﬁed by reversed-phase HPLC to aﬀord p53(Myr)-AMC (25

mg, 0.019 mmol) in 58% yield as a white solid. ¹H NMR (500 MHz,

DMSO-d₆): δ 0.84 (t, 3H, J = 6.8 Hz), 1.12−1.78 (m, 38H), 1.87 (s,

3H), 2.00 (t, 2H, J = 7.5 Hz), 2.41 (s, 3H), 2.70−2.81 (m, 2H),

2.93−3.20 (m, 6H), 4.10−4.18 (m, 1H), 4.22−4.31 (m, 1H), 4.31−

4.39 (m, 1H), 4.54−4.64 (m, 1H), 6.30 (s, 1H), 7.36 (s, 1H), 7.44 (d

1H, J = 8.8 Hz), 7.58−7.63 (m, 1H), 7.68−7.78 (m, 4H), 7.80−7.87

(m, 2H), 8.08 (d, 1H, J = 6.8 Hz), 8.18 (d, 1H, J = 7.0 Hz), 8.26 (d,

1H, J = 7.8 Hz), 8.37 (d, 1H, J = 6.7 Hz), 8.90 (s, 1H); purity by

HPLC: 97.2% (254 nm); t_R= 9.97 min (A/B = 70: 30 → 0: 100 (20

min), A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN,); HRMS (ESI⁺):

calcd: 977.6300; found: 977.6318 [M + H]⁺(+1.82 ppm).

Synthesis of H4K16-NH₂-Protected. The peptide chain was

synthesized using Fmoc solid-phase chemistry on Fmoc-NH-Sieber

amide resin (49 mg, 0.025 mmol) in a PD-10 Empty Column at R.T.

Each assembly step was done by activating Fmoc-amino acid (0.075

mmol) with HBTU (29 mg, 0.075 mmol), HOBt·H₂O (12 mg, 0.075

mmol), and DIPEA (27 μL, 0.15 mmol) in DMF (1.0 mL) for 1 h.

The Fmoc-amino acids used in this synthesis were as follows: Fmoc-

Trp(Boc)-OH (40 mg, 0.075 mmol), Fmoc-Arg(Pbf)-OH (49 mg,

0.075 mmol), Fmoc-His(Trt)-OH (47 mg, 0.075 mmol), Fmoc-

Lys(boc)-OH (35 mg, 0.075 mmol), Fmoc-Ala-OH (25 mg, 0.075

mmol), and Fmoc-Gly-OH (22 mg, 0.075 mmol). The Fmoc group

was removed twice by using 20% piperidine in DMF (2 mL) at R.T.

for 2 and 10 min. The N-terminal amino acid was acetylated with 25%

Ac₂O in DCM (2 mL) for 5 min. For cleavage of the synthetic peptide

without loss of side-chain-protecting groups, the resin was mixed with

Synthesis of 23. To a solution of Cbz-Lys(Boc)-OH (495 mg, 1.1

mmol, 1.0 equiv), COMU (758 mg, 1.8 mmol, 1.5 equiv), and DIPEA

(617 μL, 3.5 mmol, 3.0 equiv) in DMF (6 mL) was added 7-amino-4-

methylcoumarin (22) (207 mg, 1.2 mmol, 1.1 equiv) at R.T. The

reaction mixture was stirred at R.T. for 8 h and then extracted with

AcOEt. The organic layer was washed with sodium phosphate buﬀer

(pH ∼ 4), 0.1 N HCl, and brine, dried over Na₂SO₄, and evaporated.

The residue was puriﬁed by column chromatography on silica gel

(eluent: n-hexane/AcOEt = 3/2 → 1/1) to aﬀord 23 (240 mg, 0.45

1

mmol) in 38% yield as a white solid. H NMR (500 MHz, DMSO-

d₆): δ 1.20−1.42 (m, 13H), 1.58−1.72 (m, 2H), 2.40 (s, 3H), 2.85−

2.92 (m, 2H), 4.08−4.16 (m 1H), 5.03 (s, 2H), 6.27 (s, 1H), 6.76 (t,

1H, J = 5.0 Hz), 7.29−7.40 (m, 5H), 7.50 (dd, 1H, J = 1.6, 8.6 Hz),

7.63 (d, 1H, J = 6.0 Hz), 7.73 (d, 1H, J = 8.6 Hz), 7.77 (d, 1H, J = 1.6

Hz), 10.47 (s, 1H).

Synthesis of 24. To a solution of 23 (238 mg, 0.44 mmol) in

CH₂Cl₂(6 mL) was added TFA (6 mL) at R.T. The reaction mixture

was stirred at R.T. for 1 h and then evaporated. The residue was

dissolved in AcOEt and the solution was basiﬁed with 1 N NaOH.

The organic phase was washed with brine, dried over Na₂SO₄, and

evaporated to aﬀord 24 (150 mg, 0.34 mmol) in 77% yield as a white

solid. ¹H NMR (500 MHz, DMSO-d₆): δ 1.26−1.46 (m, 4H), 1.58−

1.73 (m, 2H), 2.41 (s, 3H), 4.10−4.18 (m 1H), 5.04 (s, 2H), 6.27 (s,

1H), 7.15−7.42 (m, 6H), 7.51 (dd, 1H, J = 1.6, 8.7 Hz), 7.67 (d, 1H,

J = 7.7 Hz), 7.73 (d, 1H, J = 8.7 Hz), 7.78 (d, 1H, J = 1.6 Hz); MS

(ESI⁺): 438 [M + H]⁺.

Synthesis of 25. To a solution of 24 (76 mg, 0.17 mmol, 1.0

equiv) and DIPEA (76 μL, 0.44 mmol, 2.5 equiv) in CH₂Cl₂(10 mL)

was added myristoyl chloride (56 μL.0.21 mmol, 1.2 equiv) at R.T.

The reaction mixture was stirred at R.T. for 30 min and then

evaporated. The residue was puriﬁed by column chromatography on

silica gel (eluent: n-hexane/AcOEt = 1/1 → AcOEt) to aﬀord 25 (85

1

mg, 0.13 mmol) in 75% yield as a white solid. H NMR (500 MHz,

CDCl₃): δ 0.86 (t, 3H, J = 6.7 Hz), 1.18−1.31 (m, 22H), 1.38−1.50

(m, 2H), 1.56−1.64 (m, 3H), 1.68−1.80 (m, 1H), 2.16 (t, 2H, J = 7.5

Hz), 2.41 (s, 3H), 3.18−3.40 (m, 2H), 4.24−4.32 (m, 1H), 5.12 (s,

2H), 5.58−5.70 (m, 2H), 6.19 (s, 1H), 7.28−7.38 (m, 5H), 7.48−

7.55 (m, 2H), 7.66 (brs, 1H), 9.06 (brs, 1H).

O

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Journal of Medicinal Chemistry

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1% TFA and 1% TES in DCM (2 mL) for 2 min (15 times), and the

ﬁltrate was collected in sat. NaHCO₃aq. (50 mL). The organic phase

was separated, washed with brine, dried over Na₂SO₄, and then

evaporated to aﬀord H4K16-NH₂-protected (58 mg, 0.026 mmol) in

quantitative yield as a white solid. MS (ESI⁺): 2263.9 [M + Na]⁺.

Synthesis of H4K16-Ac-Protected. The peptide chain was

synthesized using Fmoc solid-phase chemistry on Fmoc-NH-Sieber

amide resin (98 mg, 0.050 mmol) in a PD-10 Empty Column at R.T.

Each assembly step was done by activating Fmoc-amino acid (0.15

mmol) with HBTU (57 mg, 0.15 mmol), HOBt·H₂O (23 mg, 0.15

mmol), and DIPEA (53 μL, 0.30 mmol) in DMF (1.5 mL) for 1 h.

The Fmoc-amino acids used in this synthesis were as follows: Fmoc-

Trp(Boc)-OH (79 mg, 0.15 mmol), Fmoc-Arg(Pbf)-OH (98 mg,

0.15 mmol), Fmoc-His(Trt)-OH (93 mg, 0.15 mmol), Fmoc-

Lys(Ac)-OH (63 mg, 0.15 mmol), Fmoc-Ala-OH (50 mg, 0.15

mmol), and Fmoc-Gly-OH (44 mg, 0.15 mmol). The Fmoc group

was removed twice by using 20% piperidine in DMF (2 mL) at R.T.

for 2 and 10 min. The N-terminal amino acid was acetylated with 25%

Ac₂O in DCM (2 mL) for 5 min. For cleavage of the synthetic peptide

without loss of side-chain-protecting groups, the resin was mixed with

1% TFA and 1% TES in DCM (2 mL) for 2 min (15 times), and the

ﬁltrate was collected in sat. NaHCO₃aq. (50 mL). The organic phase

was separated, washed with brine, dried over Na₂SO₄, and then

evaporated to aﬀord H4K16-Ac-protected (107 mg, 0.049 mmol) in

98% yield as a white solid.

Synthesis of H4K16-Myr-Protected. The peptide chain was

synthesized using Fmoc solid-phase chemistry on Fmoc-NH-Sieber

amide resin (49 mg, 0.025 mmol) in a PD-10 Empty Column at R.T.

Each assembly step was done by activating Fmoc-amino acid (0.075

mmol) with HBTU (29 mg, 0.075 mmol), HOBt·H₂O (12 mg, 0.075

mmol), and DIPEA (27 μL, 0.15 mmol) in DMF (1.0 mL) for 1 h.

The Fmoc-amino acids used in this synthesis were as follows: Fmoc-

Trp(Boc)-OH (40 mg, 0.075 mmol), Fmoc-Arg(Pbf)-OH (49 mg,

0.075 mmol), Fmoc-His(Trt)-OH (47 mg, 0.075 mmol) Fmoc-

Lys(Myr)-OH (44 mg, 0.075 mmol), Fmoc-Ala-OH (25 mg, 0.075

mmol), and Fmoc-Gly-OH (22 mg, 0.075 mmol). The Fmoc group

was removed twice by using 20% piperidine in DMF (2 mL) at R.T.

for 2 and 10 min. The N-terminal amino acid was acetylated with 25%

Ac₂O in DCM (2 mL) for 5 min. For cleavage of the synthetic peptide

without loss of side-chain-protecting groups, the resin was mixed with

1% TFA and 1% TES in DCM (2 mL) for 2 min (15 times), and the

ﬁltrate was collected in sat. NaHCO₃aq. (50 mL). The organic phase

was separated, washed with brine, dried over Na₂SO₄, and then

evaporated to aﬀord H4K16-Myr-protected (56 mg, 0.024 mmol) in

95% yield as a white solid. MS (ESI⁺): 2352 [M + H]⁺.

Synthesis of H4K16-NH₂. To a solution of H4K16-NH₂-

protected (58 mg, 0.026 mmol) in CH₂Cl₂(3 mL) was added

TFA (3 mL). The reaction mixture was stirred for 3 h at R.T. and

then evaporated. The residue was puriﬁed by reversed-phase HPLC to

aﬀord H4K16-NH₂·4TFA (25 mg, 0.015 mmol) in 61% yield as a

white solid. Purity by HPLC: 98.4% (254 nm); t_R= 6.17 min (A/B =

80:20 → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); HRMS (ESI⁺): calcd: 1195.6475; found: 1195.6447 [M +

2H]²⁺(−2.33 ppm).

Synthesis of H4K16-Ac. To a solution of H4K16-Ac-protected

(107 mg, 0.049 mmol) in CH₂Cl₂(3 mL) was added TFA (3 mL).

The reaction mixture was stirred for 3 h at R.T. and then evaporated.

The residue was puriﬁed by reversed-phase HPLC to aﬀord H4K16-

Ac·3TFA (54 mg, 0.034 mmol) in 70% yield as a white solid. Purity

by HPLC: 99.6% (254 nm); t_R= 6.66 min (A/B = 80:20 → 0:100 (20

min); A: 0.1% TFA Milli-Q, B: 0.1% TFA CH₃CN); HRMS (ESI⁺):

calcd: 1237.65809; found: 1237.65929 [M + 2H]²⁺(+0.97 ppm).

Synthesis of H4K16-Myr. To a solution of H4K16-Myr-

protected (56 mg, 0.024 mmol) in CH₂Cl₂(3 mL) was added

TFA (3 mL). The reaction mixture was stirred for 3 h at R.T. and

then evaporated. The residue was puriﬁed by reversed-phase HPLC to

aﬀord H4K16-Myr·3TFA (27 mg, 0.016 mmol) in 65% yield as a

white solid. Purity by HPLC: 99.3% (254 nm); t_R= 11.9 min (A/B =

80:20 → 0:100 (20 min); A: 0.1% TFA Milli-Q, B: 0.1% TFA

CH₃CN); HRMS (ESI⁺): calcd: 1405.8459; found: 1405.8494 [M +

2H]²⁺(+2.51 ppm).

In Vitro Assay. Absorption Measurement. Absorption spectra

were measured in a quartz cuvette (4 × 4 × 40 mm³) on a UV-1800

instrument (Shimadzu, Japan). Spectra of RIK^TfaRY and S2DMi-1−

14 were measured in Dulbecco’s phosphate-buﬀered saline (Sigma),

and the ﬁnal concentration of each was 5 μm (0.2% DMSO).

Evaluation of SIRT2-Inhibitory Activity by the SFP3 Assay.

RIK^TfaRY, S2DMi-1−14, compound 6, compound M, compound

TM, AGK2, NAM, and SirReal2 were each dissolved in DMSO or

Milli-Q (for NAM) and diluted as required with SIRT assay buﬀer I

(Tris−HCl (pH 8.0), containing 150 mM NaCl, 1 mM DTT, and

0.05% Triton X-100) (×4 conc. 0.8% DMSO or 2% DMSO (for

AGK2 and NAM)). SIRT2 (ﬁnal 35 nM, 10 μL) was ﬁnally added to

a solution of 2.5 μM SFP3 (10 μL), 500 μM NAD⁺(10 μL), and the

indicated concentrations of SIRT2 inhibitor (10 μL, ﬁnal 0.2%

DMSO or 0.5% DMSO (for AGK2 and NAM)) in SIRT assay buﬀer

I. Fluorescence intensity was measured at 5 min intervals with an

ARVO X5 plate reader (ﬁlters: Ex = 485/14 nm, Em = 535/25 nm)

for 60 min at 37 °C. IC₅₀values were calculated by using GraphPad

Prism6. The results are shown as mean S.D. (n = 3).

Evaluation of SIRT2-Inhibitory Activity by the p53(Myr)-

AMC (deMyr Activity) Assay. RIK^TfaRY, S2DMi-1−14, compound

6, compound M, compound TM, AGK2, NAM, and SirReal2 were

each dissolved in DMSO or Milli-Q (for NAM) and diluted as

required with SIRT assay buﬀer I (×4 conc. 2% DMSO). SIRT2 (ﬁnal

70 nM, 5 μL) was ﬁnally added to a solution of p53(Myr)-AMC

(ﬁnal 50 μM, 5 μL), NAD⁺(ﬁnal 500 μM, 5 μL), and the indicated

concentrations of SIRT2 inhibitor (ﬁnal 0.5% DMSO, 5 μL) in SIRT

assay buﬀer I. Plates were incubated at R.T. for 3 h, and then a

solution (20 μL) of trypsin (ﬁnal 100 nM) and nicotinamide (ﬁnal 1

mM) in SIRT assay buﬀer I was added. Fluorescence intensity was

measured at 5 min intervals with an ARVO X5 plate reader (ﬁlters: Ex

= 355/40 nm, Em = 460/25 nm) for 20 min at 37 °C. IC₅₀values

were calculated by using GraphPad Prism6. The results are shown as

mean S.D. (n = 3).

Evaluation of SIRT2-Inhibitory Activity by the FLUOR DE

LYS SIRT2 Kit Assay. RIK^TfaRY, S2DMi-1−14, compound 6,

compound M, compound TM, AGK2, NAM, and SirReal2 were

dissolved in DMSO or Milli-Q (for NAM) and diluted as required

with sirtuin assay buﬀer II (50 mM Tris−HCl (pH 8.0), containing

137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mg/mL bovine serum

albumin (BSA)) (×4 conc. 2% DMSO). SIRT2 (ﬁnal 2 U, 5 μL) was

ﬁnally added to a solution of deacetylase substrate (ﬁnal 50 μM; 5

μL), NAD⁺(ﬁnal 500 μM; 5 μL), and the indicated concentrations of

SIRT2 inhibitor (ﬁnal 0.5% DMSO, 5 μL) in sirtuin assay buﬀer II.

Plates were incubated at R.T. for 2 h; then, a solution (20 μL) of

Developer II and nicotinamide (ﬁnal 2 mM) was added to each well.

Fluorescence intensity was measured at 5 min intervals with an ARVO

X5 plate reader (ﬁlters: Ex = 355/40 nm, Em = 460/25 nm) for 30

min at 37 °C. IC₅₀values were calculated by using GraphPad Prism6.

The results are shown as mean S.D. (n = 3).

Evaluation of Inhibitory Activity against Developer II by

the FLUOR DE LYS SIRT2 Kit Assay. RIK^TfaRY, S2DMi-1−14,

compound M, compound TM, AGK2, NAM, and SirReal2 were

dissolved in DMSO or Milli-Q (for NAM) and diluted as required

with sirtuin assay buﬀer II (50 mM Tris−HCl (pH 8.0), containing

137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mg/mL BSA) (×4 conc.

2% DMSO). 1/200 diluted Developer II (ﬁnal 1/400 dilution; 20 μL)

was ﬁnally added to a solution of deacetylated standard substrate

(ﬁnal 50 μM; 10 μL) and SIRT2 inhibitor (ﬁnal 1 μM, 0.5% DMSO,

10 μL) in sirtuin assay buﬀer II. Fluorescence intensity was measured

at 5 min intervals with an ARVO X5 plate reader (ﬁlters: Ex = 355/40

nm, Em = 460/25 nm) for 20 min at 37 °C. The results are shown as

mean S.D. (n = 3).

Evaluation of SIRT1-Inhibitory Activity by the SFP3 Assay.

RIK^TfaRY, S2DMi-1−14, compound 6, compound M, compound

TM, AGK2, NAM, and SirReal2 were dissolved in DMSO or Milli-Q

(for NAM) and diluted as required with SIRT assay buﬀer I (Tris−

HCl (pH 8.0), containing 150 mM NaCl, 1 mM DTT, 0.05% Triton

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DOI: 10.1021/acs.jmedchem.9b00315

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

X-100) (×4 conc. 0.8% DMSO or 2% DMSO (for AGK2 and

nicotinamide)). SIRT1 (ﬁnal 7 nM, 10 μL) was ﬁnally added to a

solution of 2.5 μM SFP3 (10 μL), 500 μM NAD⁺(10 μL), and the

indicated concentrations of SIRT2 inhibitor (10 μL, ﬁnal 0.2%

DMSO or 0.5% DMSO (for AGK2 and nicotinamide)) in SIRT assay

buﬀer I. Fluorescence intensity was measured at 5 min intervals with

an ARVO X5 plate reader (ﬁlters: Ex = 485/14 nm, Em = 535/25

nm) for 60 min at 37 °C. IC₅₀values were calculated by using

GraphPad Prism6. The results are shown as mean S.D. (n = 3).

Evaluation of SIRT1-Inhibitory Activity by the p53(Myr)-

AMC Assay (deMyr Activity). RIK^TfaRY, S2DMi-1−14, compound

6, compound M, compound TM, AGK2, and SirReal2 were dissolved

in DMSO and diluted as required with SIRT assay buﬀer I (×4 conc.

2% DMSO). SIRT1 (ﬁnal 14 nM, 5 μL) was ﬁnally added to a

solution of p53(Myr)-AMC (ﬁnal 50 μM, 5 μL), NAD⁺(ﬁnal 500

μM, 5 μL), and the indicated concentrations of SIRT2 inhibitor (ﬁnal

0.5% DMSO, 5 μL) in SIRT assay buﬀer I. Plates were incubated at

R.T. for 3 h, and then a solution (20 μL) of trypsin (ﬁnal 100 nM)

and nicotinamide (ﬁnal 1 mM) in SIRT assay buﬀer I was added.

Fluorescence intensity was measured at 5 min intervals with an ARVO

X5 plate reader (ﬁlters: Ex = 355/40 nm, Em = 460/25 nm) for 20

min at 37 °C. IC₅₀values were calculated by using GraphPad Prism6.

The results are shown as mean S.D. (n = 3).

Evaluation of SIRT3-Inhibitory Activity by the SFP3 Assay.

RIK^TfaRY, S2DMi-1−12, compound M, compound TM, AGK2, and

NAM were dissolved in DMSO or Milli-Q (for NAM) and diluted as

required with SIRT assay buﬀer I (Tris−HCl (pH 8.0), containing

150 mM NaCl, 1 mM DTT, 0.05% Triton X-100) (×4 conc. 0.8%

DMSO or 2% DMSO (for AGK2 and NAM)). SIRT3 (ﬁnal 60 nM,

10 μL) was ﬁnally added to a solution of 2.5 μM SFP3 (10 μL), 500

μM NAD⁺(10 μL), and the indicated concentrations of SIRT2

inhibitor (10 μL, ﬁnal 0.2% DMSO or 0.5% DMSO (for AGK2 and

NAM)) in SIRT assay buﬀer I. Fluorescence intensity was measured

at 5 min intervals with an ARVO X5 plate reader (ﬁlters: Ex = 485/14

nm, Em = 535/25 nm) for 60 min at 37 °C. IC₅₀values were

calculated by using GraphPad Prism6. The results are shown as mean

S.D. (n = 3).

Evaluation of SIRT3-Inhibitory Activity by the p53(Myr)-

AMC Assay (deMyr Activity). RIK^TfaRY, S2DMi-1−12, compound

M, compound TM, and AGK2 were dissolved in DMSO and diluted

as required with SIRT assay buﬀer I (×4 conc. 2% DMSO). SIRT3

(ﬁnal 120 nM, 5 μL) was ﬁnally added to a solution of p53(Myr)-

AMC (ﬁnal 50 μM, 5 μL), NAD⁺(ﬁnal 500 μM, 5 μL), and the

indicated concentrations of SIRT2 inhibitor (ﬁnal 0.5% DMSO, 5 μL)

in SIRT assay buﬀer I. Plates were incubated at R.T. for 3 h, and then

a solution (20 μL) of trypsin (ﬁnal 100 nM) and nicotinamide (ﬁnal 1

mM) in SIRT assay buﬀer I was added. Fluorescence intensity was

measured at 5 min intervals with an ARVO X5 plate reader (ﬁlters: Ex

= 355/40 nm, Em = 460/25 nm) for 20 min at 37 °C. IC₅₀values

were calculated by using GraphPad Prism6. The results are shown as

mean S.D. (n = 3).

Evaluation of SIRT6-Inhibitory Activity by the SFP3 Assay.

RIK^TfaRY, S2DMi-1−14, compound M, compound TM, and SirReal2

were dissolved in DMSO and diluted as required with SIRT assay

buﬀer I (Tris−HCl (pH 8.0), containing 150 mM NaCl, 1 mM DTT,

0.05% Triton X-100) (×4 conc. 0.8% DMSO). SIRT6 (ﬁnal 48 nM,

10 μL) was ﬁnally added to the solution of 2.5 μM SFP3 (10 μL), 500

μM NAD⁺(10 μL), and the indicated concentrations of SIRT2

inhibitor (10 μL, ﬁnal 0.2% DMSO) in SIRT assay buﬀer I.

Fluorescence intensity was measured at 5 min intervals with an ARVO

X5 plate reader (ﬁlters: Ex = 485/14 nm, Em = 535/25 nm) for 60

min at 37 °C. IC₅₀values were calculated by using GraphPad Prism6.

The results are shown as mean S.D. (n = 3).

HPLC Analysis of the Stability of SIRT2 Inhibitors in the

Presence of SIRT2. S2DMi-1−3, S2DMi-6, and S2DMi-9 were each

dissolved in DMSO (10 mM) and diluted as required with SIRT assay

buﬀer I (200 μM, 2% DMSO). SIRT2 (ﬁnal 350 nM) was added to

the solutions of SIRT2 inhibitors (ﬁnal 20 μM, 0.2% DMSO) and

NAD⁺(ﬁnal 500 μM) in SIRT assay buﬀer I. Each mixture was

incubated at 37 °C. Aliquots (20 μL) were taken at 0 and 4 h and

analyzed by HPLC. HPLC conditions: A/B = 80:20 (initial) → 0:100

(20 min) → 0:100 (23 min) with a linear gradient, A = 0.1% TFA

Milli-Q, B = 0.1% TFA CH₃CN. Absorbance was monitored at 510

nm (S2DMi-1−3), 350 nm (S2DMi-6), or 254 nm (S2DMi-9).

Competition Assay with SFP3 (Lineweaver−Burk Plots and

Michaelis−Menten Plots). SIRT2 (ﬁnal 35 nM, 10 μL) was added

to a solution of SFP3 (ﬁnal 0.625, 1.25, and 2.5 μM, 10 μL) and 500

μM NAD⁺and S2DMi-6 or S2DMi-9 (ﬁnal 15, 20, and 30 nM, 0.2%

DMSO, 10 μL) in SIRT assay buﬀer I. Fluorescence intensity was

measured at 1 min intervals with an ARVO X5 plate reader (ﬁlters: Ex

= 485/14 nm, Em = 535/25 nm) for 30 min at 37 °C. The K_mand

V_maxvalues were calculated by using GraphPad Prism6. The rate of

ﬂuorescence increase (v; ﬂuorescence intensity/min) over 20 min

(from 10 to 30 min) was calculated, and a double-reciprocal plot of

initial velocity as a function of substrate concentration, 1/v against 1/

[SFP3], was drawn. The results are shown as mean S.D. (n = 3)

Competition Assay with NAD⁺(Lineweaver−Burk Plots and

Michaelis−Menten Plots). SIRT2 (ﬁnal 35 nM, 10 μL) was added

to a solution of SFP3 (ﬁnal 2.5 μM, 10 μL) and NAD⁺(6.7, 20, and

60 μM) and S2DMi-6 or S2DMi-9 (ﬁnal 15, 20, and 30 nM, 0.2%

DMSO, 10 μL) in SIRT assay buﬀer I. Fluorescence intensity was

measured at 1 min intervals with an ARVO X5 plate reader (ﬁlters: Ex

= 485/14 nm, Em = 535/25 nm) for 30 min at 37 °C. The K_mand

V_maxvalues were calculated by using GraphPad Prism6. The rate of

ﬂuorescence increase (v; ﬂuorescence intensity/min) over 10 min

(from 10 to 30 min) was calculated, and a double-reciprocal plot of

initial velocity as a function of substrate concentration, 1/v against 1/

[SFP3], was drawn. The results are shown as mean S.D. (n = 3)

HPLC Analysis of SIRT2-Mediated Demyristoylation of

H4K16-Myr and Inhibition Assay. H4K16-Myr (ﬁnal 100 μM,

0.5% DMSO) was incubated in SIRT assay buﬀer I without 1 mM

DTT, containing 500 μM NAD⁺and 175 nM SIRT2 at 37 °C for 2 h

in the presence or absence of SIRT2 inhibitors (S2DMi-6, S2DMi-7,

S2DMi-9 (0.1, 1, 10 μM, 0.5% DMSO), compound TM (10 μM,

0.5% DMSO), AGK2 (50 μM, 0.5% DMSO), SirReal2 (10 μM, 0.5%

DMSO), NAM (500 μM, 0.5% DMSO)). The reactions were stopped

using 20 μL of 1 N HCl in MeOH. Aliquots (20 μL) were taken and

analyzed by HPLC. HPLC conditions: A/B = 80:20 (initial) → 0:100

(20 min) → 0:100 (25 min) with a linear gradient, A = 0.1% TFA

Milli-Q, B = 0.1% TFA CH₃CN. Absorbance was monitored at 280

nm.

HPLC Analysis of SIRT2-Mediated Deacetylation of H4K16-

Ac and Inhibition Assay. H4K16-Ac (ﬁnal 100 μM, 0.5% DMSO)

was incubated in SIRT assay buﬀer I without 1 mM DTT, containing

500 μM NAD⁺and 175 nM SIRT2 at 37 °C for 30 min in the

presence or absence of SIRT2 inhibitors (S2DMi-6, S2DMi-7,

S2DMi-9 (0.01, 0.1, 1, 10 μM, 0.5% DMSO), and NAM (500 μM,

0.5% DMSO)). The reactions were stopped using 20 μL of 1 N HCl

in MeOH. Aliquots (20 μL) were taken and analyzed by HPLC.

HPLC conditions: A/B = 80:20 (initial) → 0:100 (20 min) → 0:100

(25 min) with a linear gradient, A = 0.1% TFA Milli-Q, B = 0.1% TFA

CH₃CN. Absorbance was monitored at 280 nm.

Determination of Kinetic Parameters of H4K16-Ac and

H4K16-Myr toward SIRT2. For kinetics analysis, 70 nM (for

H4K16-Ac) or 10 nM (for H4K16-Myr) SIRT2 was incubated in 20

μL of SIRT assay buﬀer I without 1 mM DTT, containing 500 μM

NAD⁺with H4K16-Ac or H4K16-Myr at varied concentrations at 37

°C for 600 s (for H4K16-Ac) or 320 s (for H4K16-Myr). Peptide

concentrations used for H4K16-Ac were 1, 2, 4, 8, 16, 32, and 64 μM

and those for H4K16-Myr were 0.25, 0.5, 1, 2, 4, 8, and 16 μM. The

reactions were stopped using 10 μL of 2 N HCl in MeOH. Aliquots

(20 μL) were taken and analyzed by HPLC. HPLC conditions: A/B =

80:20 (initial) → 0:100 (20 min) → 0:100 (25 min) with a linear

HPLC Analysis of SIRT2-Mediated Cleavage of p53(Myr)-

AMC. p53(Myr)-AMC (ﬁnal 50 μM, 0.5% DMSO) was incubated in

SIRT assay buﬀer I, containing 500 μM NAD⁺and 175 nM SIRT2 at

37 °C. Aliquots (20 μL) were taken at 0 and 1 h and analyzed by

HPLC. HPLC conditions: A/B = 99:1 (initial) → 0:100 (20 min) →

0:100 (23 min) with a linear gradient, A = 0.1% FA Milli-Q, B = 0.1%

FA CH₃CN. Absorbance was monitored at 325 nm.

Q

DOI: 10.1021/acs.jmedchem.9b00315

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

gradient, A = 0.1% TFA Milli-Q, B = 0.1% TFA CH₃CN. The product

and substrate peaks were quantiﬁed by integrating the area at 280 nm,

which were converted to initial velocity (μM/s). The velocities were

plotted against the peptide concentrations, and then K_mand V_max

values were calculated by using GraphPad Prism6.

Cell Viability Assay Using CCK-8 Reagent. HeLa cells (1.0 ×

10⁴cells) were grown on a PLL-coated 96-well microplate (IWAKI)

overnight in 100 μL of Dulbecco’s modiﬁed Eagle’s medium

(DMEM) (10% fetal bovine serum and penicillin (100 units/mL)

and streptomycin (100 μg/mL)) in a humidiﬁed incubator containing

5% CO₂in air at 37 °C for 24 h. Various concentrations (ﬁnal 1.56,

3.13, 6.25, 6.25, 12.5, 25, 50 μM) of compounds (compound TM,

S2DMi-6, S2DMi-7 and S2DMi-9) or DMSO (control) in DMEM

(2% DMSO, 10 μL) were added to each well and further incubated

for 72 h. CCK-8 reagent (10 μL) was added to each well and

incubated at 37 °C for 2 h, and then absorption at 450 nm was

measured with an ARVO X5 plate reader.

Dabcyl, 4-(4-(dimethylamino)phenylazo)benzoic acid; NAM,

nicotinamide; DTT, dithiothreitol

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ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

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Absorbance spectra of synthesized compounds; SIRT

inhibition curves of synthesized compounds and known

SIRT2 inhibitors toward SIRT1−3, and 6 in SFP3,

p53(Myr)-AMC, and FLUOR DE LYS SIRT2 kit

assays; Michaelis−Menten plots of H4K16-Ac and

H4K16-Myr against SIRT2; SIRT2 deacetylase inhib-

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using HeLa cells; competition analysis of S2DMi-6 and

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compounds 1−6 (PDF)

Molecular formula strings and enzyme inhibition data

(CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: deco@phar.nagoya-cu.ac.jp. Tel: +81-52-836-3407.

Fax: +81-52-836-3407.

■

ORCID

(13) Wang, Y.; Fung, Y. M. E.; Zhang, W.; He, B.; Chung, M. W. H.;

Jin, J.; Hu, J.; Lin, H.; Hao, Q. Deacylation Mechanism by SIRT2

Revealed in the 1′-SH-2′-O-Myristoyl Intermediate Structure. Cell

Chem. Biol. 2017, 24, 339−345.

Hidehiko Nakagawa: 0000-0001-8435-4401

Notes

The authors declare no competing ﬁnancial interest.

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Dynamic Protein Acylation: New Substrates, Mechanisms, and Drug

Targets. Trends Biochem. Sci. 2017, 42, 566−581.

ACKNOWLEDGMENTS

■

The authors thank Drs. Hiroyuki Yamakoshi and Seiichi

Nakamura at Nagoya City University for help and advice for

the chiral column HPLC experiment and thank all of the

members of H.N.’s laboratory for fruitful discussions. This

work was supported in part by JSPS KAKENHI Grant

Numbers 26893223, 15K18899 (M.K.) and 16H05103

(H.N.), as well as by the Hori Sciences and Arts Foundation

(M.K.), Mochida Memorial Foundation for Medical and

Pharmaceutical Research (M.K.), a Grant-in-Aid for Scientiﬁc

Research on Innovative Areas from MEXT (26111012, H.N.),

and a grant from Daiichi Sankyo Foundation of Life Science

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ABBREVIATIONS

SIRT, sirtuin; NAD, nicotinamide adenine dinucleotide; TNF-

α, tumor necrosis factor α; AMC, 7-amino-4-methylcoumarin;

■

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DOI: 10.1021/acs.jmedchem.9b00315

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.9b00315

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Development of Peptide-Based Sirtuin Defatty-Acylase Inhibitors Identified by the Fluorescence Probe, SFP3, That Can Efficiently Measure Defatty-Acylase Activity of Sirtuin

DOI: 10.1021/acs.jmedchem.9b00315

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Full text of DOI:10.1021/acs.jmedchem.9b00315

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