A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.5b01532

Sirtuins are important regulators of lysine acylation, which is implicated in cellular metabolism and transcriptional control. This makes the sirtuin class of enzymes interesting targets for development of small molecule probes with pharmaceutical potential. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation, and two known inhibitors, suramin and SirReal2, were profiled against substrates containing ε-N-acyllysine modifications of varying length.

Full text of DOI:10.1021/acs.jmedchem.5b01532

Article
pubs.acs.org/jmc
A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate
Screening and Inhibitor Evaluation
Iacopo Galleano,† Matthias Schiedel,‡ Manfred Jung,‡ Andreas S. Madsen,† and Christian A. Olsen*,†
†Center for Biopharmaceuticals and Department of Drug Design & Pharmacology, University of Copenhagen, Universitetsparken 2,
DK-2100, Copenhagen, Denmark
‡Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universitat Freiburg, Albertstraße 25, 79104 Freiburg im Breisgau, Germany
̈
S
* Supporting Information
ABSTRACT: Sirtuins are important regulators of lysine
acylation, which is implicated in cellular metabolism and
transcriptional control. This makes the sirtuin class of enzymes
interesting targets for development of small molecule probes
with pharmaceutical potential. To achieve detailed profiling
and kinetic insight regarding sirtuin inhibitors, it is important
to have access to efficient assays. In this work, we report
readily synthesized fluorogenic substrates enabling enzyme-
economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long
chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation,
and two known inhibitors, suramin and SirReal2, were profiled against substrates containing ε-N-acyllysine modifications of
varying length.
for deacylase activity against a series of acyl groups using this
peptide sequence, and finally report evaluation of both
inhibition of the deacetylase and longer chain deacylase activity
of suramin as well as the recently reported SIRT2 inhibitor
SirReal2.
INTRODUCTION
■
Humans have seven isoforms of the NAD+-dependent sirtuin
enzymes (SIRT1−7) that are homologous to yeast Sir2 (silent
information regular 2).1−3 Although these enzymes were
originally classified as class-III histone deacetylases (HDACs),
it has become evident that several members of this class should
rather be denoted lysine deacylases (KDACs) as targeting of
several ε-N-acyllysine posttranslational modifications (PTMs)
have been discovered.4−6 Thus, SIRT1−3 appear to be robust
deacetylases but are not restricted to histone deacetylation as
SIRT2 is primarily cytoplasmic and SIRT3 is mitochondrial.2,7
Sirtuin 5 hydrolyzes ε-N-acyllysine modifications that are based
on the dicarboxyl-derived groups malonyl (Kmal), succinyl
(Ksuc), and glutaryl (Kglut) modifications.8−11 Sirtuin 6 is a
histone deacetylase12,13 but was also recently shown to
demyristoylate TNF-α.14 This led to a number of studies
involving sirtuin-mediated hydrolysis of ε-N-acyllysine mod-
ifications of varying hydrocarbon length.15−17 Interestingly,
SIRT1−3 consistently exhibited potent activity against long
chain acyl groups (e.g., C8−C16) in these studies, which may
indicate that the physiologically relevant substrate space of
these enzymes is broader than previously anticipated.
Furthermore, a recent kinetic study revealed that NAD+
dependence and inhibition of sirtuin deacylase activity by
nicotinamide were dependent upon the identity of the acyl
substrate.18 Efficient assay platforms to assess inhibitors against
both sirtuin deacetylase and long chain deacylase activity are
therefore desirable.
RESULTS AND DISCUSSION
■
Preliminary Substrate Evaluation. We have previously
reported efficient and enzyme-economical assay protocols for
continuous assays employing SIRT5.10 These were based on
fluorogenic Ac-Leu-Gly-Lys-AMC peptide substrates [Ac-LGK-
AMC (1); Figure 1a], which were applied in a trypsin-coupled
assay format where release of the C-terminally linked 7-amino-
4-methylcoumarin (AMC) fluorophore is monitored continu-
ously. This design, however, was not directly transferable to
substrates containing long chain acyl groups due to impaired
aqueous solubility. Furthermore, known water-soluble sequen-
ces (QPKK (2) and TARK (3); Figure 1a), of which the former
is commercially available in sirtuin deacetylase assay kits,
proved incompatible with continuous assaying due to
premature peptide cleavage by trypsin. Inspired by recent
work demonstrating posttranslational acylation of K259 in
dihydrolipoamide acetyltransferase [DLAT (4)],19 we envi-
sioned that the amino acids 256−259 of this protein could
provide the desired solubility without encountering problems
with trypsin cleavage. The deacylase activities of SIRT2 and
SIRT6 were thus initially tested against substrates 1, 2a−d, 3a−
d, and 4a−d (Figure 1b) since previous studies have shown
In the present article, we describe the discovery of a highly
efficient fluorogenic substrate for assessment of SIRT2
deacylase activity. We furthermore provide results of screening
Received: October 1, 2015
© XXXX American Chemical Society
A
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Figure 1. (a) Structures of substrates. (b) End-point deacylation data represented as conversion of substrate in micromolar based on a standard
curve correlating relative fluorescence units (RFU) to concentration of AMC. The bar graphs represent conversion of substrate given in μM
standard deviation based on at least two assays performed in duplicate.
high efficiency of SIRT2 against long chain ε-N-acyllysine
substrates and that SIRT6 preferentially cleaves these
modifications as well.14,16
acyl groups of varying length combined with differences in
branching (4e,f) and oxidation state. Thus, α,β-unsaturated
(4h−j), β-hydroxylated (4k−m), and β-keto (4n−p) function-
alized substrates were included in the series (please consult the
Supporting Information, Scheme S1 for syntheses of the
carboxylic acids). Because of a combination of synthetic access
as well as potential biological relevance, and because both
stereoisomers exist as acyl-CoA species, it was decided to use
the racemic β-hydroxy acids for the substrates in this initial
screen (4k−m).
Compounds 4a−p were then tested as substrates for sirtuins
1−3 and 6 (Figure 2). Sirtuins 1 and 3 exhibited insignificant
cleavage of all substrates 4e−p; however, this was also reflected
in lower conversions of ETDK substrates 4a−d compared to
QPKK (2a−d) and TARK (3a−d) for both SIRT1 and SIRT3.
Thus, the ETDK sequence appears to be particularly efficient
for SIRT2-mediated long chain deacylation, whereas the QPKK
and the TARK sequence were preferred for all deacylation
reactions employing SIRT1, SIRT3, and SIRT6 (Figure 2).
Interestingly, SIRT2 was able to hydrolyze unsaturated (4i,j),
β-hydroxylated (4l,m), and β-keto (4o,p) substrates with chain
lengths of C10 and C12. For the unsaturated acyl groups (4i,j),
the substrate conversion even rivaled that of SIRT2-mediated
deacetylation of any tested peptide sequence (1a−4a).
Whether this is just an in vitro finding associated with the
efficient long chain deacylase activity of SIRT2 remains to be
determined. Nevertheless, these observations are interesting
and warrant further investigation of a possible physiological
existence of proteins that are modified by unsaturated or
oxidized acyl groups of varying lengths.
Expectedly, SIRT6 consistently cleaved the long acyl chains
(Kdec, Klau, and Kmyr) but not ε-N-acetyllysine (Kac). The basic
substrates (QPKK and TARK sequences) appeared to be more
efficiently cleaved by SIRT6 than the acidic ETDK sequence.
On the other hand, ETDK substrates containing long chains
(4b−d) gave rise to the highest conversions exhibited by
SIRT2 (Figure 1b). Interestingly, the opposite trend was
observed for deacetylase activity of SIRT2, which was lower for
the ETDK substrate (4a) compared to both QPKK (2a) and
TARK (3a). This is in agreement with recent data indicating
different kinetic behavior of the enzymes depending on
substrate acyl group.18 On the basis of these findings, the
ETDK sequence was selected for further evaluation along two
different avenues: (1) cleavage of potential ε-N-acyllysine
modifications and (2) development of an efficient continuous
SIRT2 deacylase assay.
Substrate Synthesis and Evaluation. It has been
speculated that reactive acyl-CoA species may nonspecifically
modify proteins without enzymatic catalysis.20 With the high
conversions recorded for deacylation applying the ETDK
sequence, we therefore decided to apply this as a universal
scaffold for screening of both long and short chain ε-N-
acyllysine modifications inspired by acyl-CoA species from
various biochemical pathways (Scheme 1). The substrates were
prepared by acylation of a common precursor, compound 8,
prepared by coupling Ac-Glu(tBu)-Thr(tBu)-Asp(tBu)-OH to
compound 6, which was obtained employing a POCl3-mediated
AMC fluorophore coupling to the C-terminal of lysine
(5).10,21,22 In the substrate collection, it was decided to include
Optimization of Continuous SIRT2 Assay. To gain
insight regarding the kinetics associated with SIRT2 cleavage of
B
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a
Scheme 1. Substrate Synthesis and Structures of Acyl Modifications (4a−p)
a
Reagents and conditions: (a) POCl3 (3.5 equiv), pyridine (10.3 equiv), 7-amino-4-methylcoumarin (AMC, 1.1 equiv), THF 0 °C → rt, 2 h (88%);
(b) TFA−CH2Cl2 (15:85), 0 °C → rt, 3 h (94%); (c) Ac-Glu(tBu)-Thr(tBu)-Asp(tBu)-OH (0.95 equiv), HATU (1.05 equiv), lutidine (2.2 equiv),
CH2Cl2, rt, 2 h (80%); (d) Fmoc deprotection with either (i) tris(2-aminoethyl)amine (20 equiv), CH2Cl2, rt, 210 min (4a) or (ii) piperidine (2
equiv), CH2Cl2−acetonitrile (20:15) 0 °C → rt, 14 h (4b−p); (e) (i) Acylation as outlined in the Experimental Section, (ii) CF3COOH-CH2Cl2-
H2O (50:48:2).
Figure 2. End-point deacylation data for incubation of substrates 4a−p with sirtuins 1−3 and 6. The bar graphs represent conversion of substrate
given in μM standard deviation based on at least two assays performed in duplicate.
substrates with varying acyl length, we detemined the rates of
deacylation at varying substrate concentrations and fitted the
data to the Michaelis−Menten equation. However, we found
these experiments to be incompatible with the concentration of
C
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bovine serum albumin (BSA; 1 mg/mL) in the standard HDAC
assay buffer used for the initial substrate screening, which might
relate to sequestering of the lipidated substrates. Because
SIRT2 is predominantly localized to the cytoplasm,3 a lower
pH would also be more physiologically relevant23 and we
therefore changed our buffer to one inspired by a previously
applied HDAC assay buffer24 (50 mM HEPES, pH 7.4, 0.05
mg/mL BSA, 100 mM KCl, 0.001% Tween-20, 200 μM
TCEP). Gratifyingly, this buffer proved suitable for all
substrates (4a−d), and we thus determined the rates of
deacylation at varying substrate concentrations and fitted the
data to the Michaelis−Menten equation. This was achieved
employing an assay format where the secondary, fluorophore-
releasing enzyme (trypsin) is included during the deacylation
reaction. To avoid premature cleavage of the sirtuin during
these assays, we therefore performed an optimization of SIRT2
to trypsin concentration enabling linear curves for the
fluorophore release, which indicates steady-state kinetics.
Employing these conditions, we then determined the
Michaelis−Menten parameters (Figure 3 and Table 1).
As previously reported using an LC-MS-based assay with
longer peptide fragments,17 the substrate affinity for SIRT2 was
significantly higher for Kmyr compared to Kac, as indicated by 2
orders of magnitude difference in Km values (Table 1).
Not surprisingly, the enzymatic efficiencies (kcat × Km
−1
)
obtained in the current assay are lower than those reported
with longer peptide fragments,17 as also recently reported for
SIRT5-based assays.25 However, the protocol developed here is
highly economical with respect to enzyme consumption and
substrates are readily available in few synthetic steps. The
differences in enzymatic efficiencies between substrates
depended primarily on differences in Km, but a 3−7-fold
lower kcat value for Kac was also observed compared to the
values for Kdec, Klau, and Kmyr (Table 1).
Investigation of the Inhibitory Effect of Suramin and
SirReal2. With this new efficient and enzyme economical
SIRT2 assay, we could then measure the potency of SIRT2
inhibitors against SIRT2-mediated hydrolysis of substrates with
various lengths at the substrate Km values, which would enable
comparison of inhibitory activities between substrates. For
these experiments, we chose suramin (9; Figure 4),10,26,27
which is a well-known inhibitor of sirtuins 1, 2, and 5, together
with the recently reported SIRT2 inhibitor, SirReal2 (10;
Figure 4).28
For SirReal2 (10), limited solubility in the buffer prohibited
full inhibition curves due to the need for large amounts of
DMSO. The values recorded for inhibition at the highest
concentration applied in this assay indicated somewhat lower
potency than originally reported (0.4 μM),28 which is not
surprising because we used different substrates and applied
them at their respective Km values in the present study.
Interestingly, however, these conditions enable comparison of
inhibitor potencies against substrates with different chain
lengths, which is relevant because nicotinamide inhibition of
sirtuins was recently shown to vary in response to acyl chain
length of the substrates.18 For SirReal2, we observed similar
inhibition of Kac, Kdec, and Klau deacylation but significantly
lower effect against the demyristoylation activity (Figure 4 and
Table 2). Suramin (9) was therefore included in the inhibitor
evaluation, and the full dose−response experiments performed
with this inhibitor recapitulated the substrate-specific inhibition
indicated by the SirReal2 results (Figure 4 and Table 2). These
initial observations regarding an acyl group preference of SIRT
inhibitors are intriguing and the substrate design developed
herein should enable further kinetic evaluation as well as effects
on substrates containing additional types of acyl groups in
future studies.
CONCLUSION
Figure 3. Progress curves for SIRT2-mediated deacylation of
substrates 4a and 4b and Michaelis−Menten plots for SIRT2-
dependent deacylation of substrates 4a−d based on at least two
assays performed in duplicate.
■
In the present study, we have identified a fluorophore-
conjugated peptide sequence that enables continuous, trypsin-
coupled assaying of SIRT2-mediated deacylase activity. Such
a
Table 1. Kinetic Parameters for SIRT2 Deacylation of Fluorogenic Substrates 4a−4d
kcat (s−1
)
kcatKm (s−1 M−1
)
−1
substrate
Km (μM)
4a (ETDKac)
4b (ETDKdec
750 90
6.0 0.8
4.5 0.6
1.8 0.1
(6.1 0.1) × 10−3
(2.2 0.1) × 10−2
(3.4 0.3) × 10−2
(1.50 0.05) × 10−2
(8.1 0.4) × 101
(3.8 0.4) × 103
(7.5 0.05) × 103
(8.5 0.8) × 103
)
4c (ETDKlau
)
4d (ETDKmyr
)
a
The Km value for NAD+ measured with substrate 4d at 20 μM concentration was determined to be 39 2 μM. Michaelis−Menten plot is shown in
Supporting Information, Figure S1.
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EXPERIMENTAL SECTION
■
General. All final substrates for biochemical investigation were
purified by preparative reversed-phase HPLC on a C18 Phenomenex
Luna column [250 mm × 20 mm, 5 μm, 100 Å] using an Agilent 1260
LC system equipped with a diode array UV detector and a gradient of
eluent I (water−MeCN−TFA, 95:5:0.1) and eluent II (0.1% TFA in
acetonitrile) rising linearly from 0% to 95% of eluent II during t = 5−
45 min with a flow rate of 20 mL/min. All compounds were purified to
>95% homogeneity as determined by analytical HPLC analysis, and
lyophilization provided white fluffy solids, which were reconstituted in
DMSO (>20 mM) before use. The concentrations were determined
by UV spectroscopy using the extinction coefficient of Ac-Lys-AMC
(ε326 = 17780 M−1 cm−1).29 Analytical reversed-phase HPLC was
performed on a C18 Phenomenex Luna column [150 mm × 4.60 mm,
3 μm, 100 Å] using an Agilent 1100 LC system equipped with a diode
array UV detector. UPLC-MS analyses were performed on a Waters
Acquity ultra high-performance liquid chromatography system
equipped with a C18 Phenomenex Kinetex column [50 mm × 2.1
mm, 1.7 μm, 100 Å]. A gradient with eluent III (0.1% HCOOH in
water) and eluent IV (0.1% HCOOH in acetonitrile) rose linearly
from 0% to 95% of eluent IV during t = 0.00−5.20 min.
General Acylation Procedure A. The desired acid (10 mg, 0.06
mmol) was dissolved in anhydrous CH2Cl2 (2 mL). Then HATU (23
mg, 0.06 mmol) and lutidine (12 mg, 0.12 mmol) were added and the
resulting suspension was stirred for 10 min, before the mixture was
cooled to 0 °C and Ac-Glu(tBu)-Thr(tBu)-Asp(tBu)-Lys-AMC (8, 40
mg, 0.05 mmol) was added. After the reaction reached completion as
judged by LC-MS analysis, the solution was partitioned between
CH2Cl2 (6 mL) and brine (5 mL). The aqueous phase was back
extracted with CH2Cl2 (3 × 5 mL), and the combined organic layer
was washed with aqueous HCl 0.5 M (2 × 10 mL). The acidic layer
was back extracted with CH2Cl2 (2 × 8 mL), and the combined
organic phase was washed with satd aqueous NaHCO3 (2 × 10 mL).
The basic aqueous phase was also extracted with CH2Cl2 (2 × 10 mL),
and the resulting combined organic phase was washed with brine (30
mL). Again, the aqueous phase was back extracted with CH2Cl2 (2 ×
10 mL), and the final combined organic layer was dried over MgSO4,
filtered, and concentrated in vacuo to give a residue, which was stirred
in CF3COOH−CH2Cl2−H2O (2 mL, 50:48:2) for 90 min. The
solution was then concentrated under reduced pressure and the crude
residue was precipitated from ice-cold diethyl ether (15 mL). The
crude residue was purified by reversed-phase preparative HPLC to
afford the desired product.
Figure 4. Structures of suramin (9) and SirReal2 (10) as well as dose−
response curves for their inhibition of SIRT2-mediated deacylation of
substrates 4a−c (based on at least two individual assays performed in
duplicate).
Table 2. Inhibition of SIRT2-Mediated Deacylation by
a
Suramin and SirReal2
Suramin (9)
IC50 SD [Ki (μM)]
SirReal2 (10) inhibition at
20 μM (%)
substrate
4a (ETDKac)
4b (ETDKdec
11 1 [5.7 0.4]
13 1 [7 2]
22 1 [11 1]
95 1 [49 3]
55
3
)
67 10
4c (ETDKlau
)
51 13
4d (ETDKmyr
)
no inhibition
General Acylation Procedure B. Ac-Glu(tBu)-Thr(tBu)-Asp-
(tBu)-Lys-AMC (8, 40 mg, 0.05 mmol) and i-Pr2EtN (18 mg, 0.14
mmol) were suspended in anhydrous CH2Cl2 (2 mL) at 0 °C, and the
desired anhydride, acid chloride, or NHS-ester (0.07 mmol) was
added. The reaction reached completion as judged by LC-MS analysis,
and the clear solution was diluted with CH2Cl2 (6 mL) and washed
with aqueous HCl 0.5 M (2 × 5 mL). The aqueous phase was back
extracted with CH2Cl2 (3 × 5 mL), and the combined organic layer
was washed with brine (10 mL). Again, the aqueous phase was
extracted with CH2Cl2 (3 × 8 mL) and the resulting combined organic
layer was dried over MgSO4, filtered, and concentrated to dryness in
vacuo. The resulting residue was dissolved in CF3COOH−CH2Cl2−
H2O (2 mL, 50:48:2) and stirred for 90 min. The solution was then
concentrated under reduced pressure and the crude residue was
precipitated from ice-cold diethyl ether (15 mL). The crude residue
was purified by reversed-phase preparative HPLC to afford the desired
product.
Ac-Glu-Thr-Asp-Lys(Ac)-AMC (4a). Yield, 12 mg (36% from 7).
1H NMR (DMSO-d6) δ 10.21 (s, 1H, NHAMC), 8.23 (d, J = 7.7, 1H,
NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu), 7.99 (d, J = 7.5, 1H, NHLys), 7.78
(m, 2H, NHεLys, H8AMC), 7.71 (d, J = 8.7, 1H, H5AMC), 7.68 (d, J =
8.0, 1H, NHThr), 7.52 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.1,
1H, H3AMC), 4.61 (q, J = 7.4, 1H, HαAsp), 4.31 (m, 2H, HαLys, HαGlu),
4.25 (dd, J = 7.9, 4.4, 1H, HαThr), 4.01 (m, 1H, HβThr), 3.00 (q, J =
6.6, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7,
7.4, 1H, HβAsp‑B), 2.39 (d, J = 1.0, 3H, 4AMC-CH3), 2.33−2.21 (m, 2H,
a
Ki values were approximated from the IC50 values by using the
Cheng−Prusoff equation [Ki = IC50/(1 + [substrate]/Km)].
substrates are useful for investigation of substrate acyl group
specificity, which is an important research area in rapid
development. A small collection of substrates containing
unprecedented acyl groups that are potentially available
through reaction of lysine side chains with the corresponding
acyl-CoA species was synthesized, and new enzymatic activities
of sirtuins were demonstrated in vitro using this collection.
These findings now warrant further scrutiny to determine
whether they are physiologically relevant. Furthermore, we
established assay protocols for evaluation of sirtuin inhibitors in
a continuous manner, which may reveal kinetic and mechanistic
insight.
Whereas focus on activation of sirtuins has traditionally been
dominating the drug discovery efforts in the field, the ability to
inhibit these enzymes will be important to clarify the roles of
sirtuins as either antitumor targets or tumor suppressors.
Efficient assay protocols for screening regimes as well as kinetic
investigations are therefore of pertinent interest and the results
reported herein provide progress in this direction.
E
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HγGlu), 1.92 (m, 1H, HβGlu‑A), 1.86 (s, 3H, CH3CONHGlu), 1.77 (s,
3H, CH3CONHεLys), 1.73 (m, 2H, HβLys‑A, HβGlu‑A), 1.68−1.58 (m,
1H, HβLys‑B), 1.44−1.31 (m, 3H, HδLys, HγLys‑A), 1.27 (m, 1H,
HγLys‑B), 1.05 (d, J = 6.3, 3H, HγThr). 13C NMR (151 MHz, DMSO) δ
174.0 (CδGlu), 171.9 (COγAsp), 171.6 (COαGlu), 171.2 (COαLys),
170.7 (COαAsp), 169.9 (COαThr), 169.7 (CONHGlu), 169.1 (CON-
HεLys), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.1
(C7AMC), 125.9 (C5AMC), 115.4 (C6AMC), 115.2 (C4aAMC), 112.4
(C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9 (CαThr), 53.9 (CαLys),
52.2 (CαGlu), 49.6 (CαAsp), 38.4 (CεLys), 35.8 (CβAsp), 31.3 (CβLys),
30.3 (CγGlu), 28.8 (CδLys), 27.1 (CβGlu), 22.9 (CγLys), 22.6
HPLC gradient 10−40% eluent II in eluent I (25 min total run time),
tR = 18.6 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys(Myr)-AMC (4d). Yield, 31 mg (73%, only
part of the crude residue was purified). 1H NMR (600 MHz, DMSO-
d6) δ 12.43 (s, 2H, COOHAsp, COOHGlu), 10.21 (s, 1H, NHAMC), 8.22
(d, J = 7.7, 1H, NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu), 7.96 (d, J = 7.5,
1H, NHLys), 7.79 (d, J = 2.0, 1H, H8AMC), 7.76−7.68 (m, 3H, NHThr
,
NHεLys, H5AMC), 7.54 (dd, J = 8.7, 1.9, 1H, H6AMC), 6.26 (d, J = 1.1,
1H, H3AMC), 5.04 (s, 1H, OHThr), 4.58 (q, J = 7.0, 1H, HαAsp), 4.32
(m, 2H, HαGlu, HαLys), 4.23 (dd, J = 7.9, 4.3, 1H, HαThr), 4.06−3.99
(m, 1H, HβThr), 3.01 (q, J = 6.5, 2H, HεLys), 2.71 (dd, J = 16.6, 5.7,
1H, HβAsp‑A), 2.58 (dd, J = 16.5, 7.1, 1H, HβAsp‑B), 2.39 (d, J = 1.0, 3H,
4AMC-CH3), 2.31−2.23 (m, 2H, HγGlu), 2.00 (t, J = 7.5, 2H,
CH2CONHεLys), 1.95−1.84 (m, 4H, CH3CONHGlu, HβGlu‑A), 1.80−
1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.68−1.59 (m, 1H, HβLys‑B), 1.47−1.12
(m, 26H, CH3(CH2)11, HδLys, HγLys), 1.05 (d, J = 6.3, 3H, HγThr), 0.84
(t, J = 7.0, 3H, CH3(CH2)12). 13C NMR (151 MHz, DMSO) δ 174.0
(COδGlu), 172.1 (CONHεLys/COγAsp), 172.0 (CONHεLys/COγAsp),
171.7 (COαGlu), 171.1 (COLys), 170.7 (COαAsp), 169.9 (COThr), 169.5
(CONHGlu), 159.98, 153.6 (C8aAMC), 153.0 (C4AMC), 142.1 (C7AMC),
125.8 (C5AMC), 115.4 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC),
105.8 (C8AMC), 66.6 (CβThr), 58.0 (CαThr), 53.8 (CαLys), 52.1 (CαGlu),
49.7 (CαAsp), 38.2 (CεLys), 36.1 (CβAsp), 35.4 (CH2CONHεLys), 31.3
(CH3(CH2)11/CδLys/CγLys), 31.2 (CβLys), 30.3 (CγGlu), 29.1−28.7
(9C, CH3(CH2)11/CδLys/CγLys), 27.1 (CβGlu), 25.3−22.8 (2C,
CH3(CH2 )1 1/CδL ys/CγLy s), 22.4 (CH3 CONHG lu ), 22.1
(CH3(CH2)11/CδLys/CγLys), 19.3 (CγThr), 18.0 (4AMC-CH3), 13.9
(CH3CONHεLys), 22.5 (CH3CONHGlu), 19.2 (CγThr), 18.0 (4AMC
-
CH3). HRMS calcd for C33H44N6NaO13+ [M + Na]+, 755.2864; found,
755.2851. UPLC-MS, tR = 1.25 min.
Ac-Glu-Thr-Asp-Lys(Dec)-AMC (4b). Yield, 27 mg (67% from 8).
1H NMR (DMSO-d6) δ 12.27 (br s, 2H, COOHAsp, COOHGlu), 10.21
(s, 1H, NHAMC), 8.22 (d, J = 7.7, 1H, NHAsp), 8.12 (d, J = 7.7, 1H,
NHGlu), 7.98 (d, J = 7.5, 1H, NHLys), 7.78 (d, J = 2.0, 1H, H8AMC),
7.70 (m, 3H, NHThr, NHεLys, H5AMC), 7.52 (dd, J = 8.7, 2.0, 1H,
H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 5.04 (br s, 1H, OHThr), 4.61 (q,
J = 7.3, 1H, HαAsp), 4.31 (m, 2H, HαGlu, HαLys), 4.25 (dd, J = 7.9, 4.3,
1H, HαThr), 4.02 (m, 1H, HβThr), 3.01 (q, J = 6.6, 3H, HεLys), 2.74
(dd, J = 16.6, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.3, 1H, HβAsp‑B),
2.39 (d, J = 1.1, 3H, 4AMC-CH3), 2.31−2.22 (m, 2H, HγGlu), 2.00 (t, J
= 7.5, 2H, CH2CONHεLys), 1.96−1.89 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.78−1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.63 (m, 1H,
HβLys‑B), 1.46−1.14 (m, 18H, HγLys, HδLys, CH3(CH2)7), 1.05 (d, J =
6.3, 3H, HγThr), 0.83 (t, J = 7.0, 3H, CH3(CH2)8). 13C NMR (151
MHz, DMSO) δ 174.0 (COδGlu), 172.0 (2C, COγAsp, CONHεLys),
171.6 (COαGlu), 171.1 (COαLys), 170.7 (COαAsp), 169.9 (COαThr),
169.6 (CONHGlu), 160.0 (4AMC-CH3), 153.6 (C8aAMC), 153.0
(C4AMC), 142.1 (C7AMC), 125.8 (C5AMC), 115.4 (C6AMC), 115.1
(C4aAMC), 112.3 (C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9 (CαThr),
53.8 (CαLys), 52.2 (CαGlu), 49.6 (CαAsp), 38.2 (CεLys), 35.9 (CβAsp),
35.4 (CH2CONHεLys), 31.3 (CH3(CH2)7/CδLys), 31.2 (CβLys), 30.3
(CγGlu), 28.9−28.7 (5C, CH3(CH2)7/CδLys), 27.1 (CβGlu), 25.3−22.8
(2C, CH3(CH2)7/CδLys), 22.4 (CH3CONHGlu), 22.1 (CγLys), 19.2
(CγThr), 18.0 (4AMC-CH3), 13.9 (CH3(CH2)8). MS calcd for
+
(CH3(CH2)12). MS calcd for C45H69N6O13 [M + H]+, 901.5; found,
901.5. HRMS calcd for C45H68N6NaO13+ [M + Na]+, 923.4742; found,
923.4748. Analytical HPLC gradient 15−50% eluent II in eluent I (25
min total run time), tR = 19.2 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys(isovaleryl)-AMC (4e). The title compound
was synthesized according to general acylation procedure A. Reagents:
isovaleric acid (6 mg, 0.06 mmol, 1.2 equiv), HATU (23 mg, 0.06
mmol, 1.3 equiv), lutidine (12 mg, 0.12 mmol, 2.6 equiv). Reaction
time: 4 h. Purification of the crude residue by preparative HPLC
afforded Ac-Glu-Thr-Asp-Lys(isovaleryl)-AMC (4e, 22 mg, 61% from
1
7) as a colorless fluffy solid after lyophilization. H NMR (600 MHz,
DMSO-d6) δ 12.24 (br s, 2H, COOHAsp, COOHGlu), 10.21 (s, 1H,
NHAMC), 8.22 (d, J = 7.7, 1H, NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu),
7.98 (d, J = 7.5, 1H, NHLys), 7.78 (d, J = 2.0, 1H, H8AMC), 7.74−7.66
(m, 3H, NHThr, NHεLys, H5AMC), 7.52 (dd, J = 8.7, 2.0, 1H, H6AMC),
6.26 (d, J = 1.2, 1H, H3AMC), 5.03 (br s, 1H, OHThr), 4.61 (q, J = 7.4,
1H, HαAsp), 4.35−4.28 (m, 2H, HαGlu, HαLys), 4.25 (dd, J = 8.0, 4.4,
1H, HαThr), 4.02 (m, 1H, HβThr), 3.02 (q, J = 6.7, 2H, HεLys), 2.75
(dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.4, 1H, HβAsp‑B),
2.40 (d, J = 1.1, 3H, 4AMC-CH3), 2.31−2.22 (m, 2H, HγGlu), 1.95−1.88
(m, 4H, CHCH2CONHεLys,HβGlu‑A), 1.86 (s, 3H, CH3CONHGlu),
1.78−1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.67−1.60 (m, 1H, HβLys‑B),
1.43−1.31 (m, 4H, HδLys, HγLys), 1.05 (d, J = 6.3, 3H, HγThr), 0.82
(dd, J = 6.3, 1.7, 6H, (CH3)2CHCH2CONHεLys). 13C NMR (151
MHz, DMSO) δ 174.0 (COδGlu), 171.9 (COγAsp), 171.6 (COαGlu),
171.3 (CONHεLys), 171.1 (COαLys), 170.6 (COαAsp), 169.9
(COαThr), 169.6 (CONHGlu), 160.0 (C2AMC), 153.6 (C8aAMC),
153.1 (C4AMC), 142.0 (C7AMC), 125.9 (C5AMC), 115.4 (C6AMC),
115.1 (C4aAMC), 112.3 (C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9
(CαThr), 53.8 (CαLys), 52.1 (CαGlu), 49.6 (CαAsp), 44.8
(CH2CONHεLys), 38.2 (CεLys), 35.8 (CβAsp), 31.3 (CβLys), 30.2
(CγGlu), 28.9 (CδLys), 27.0 (CβGlu), 25.5 (CHCH2CONHεLys), 22.8
(CγLys), 22.4 (CH3CONHGlu), 22.3 ((CH3)2CHCH2CONHεLy+s), 19.2
(CγThr), 18.0 (4AMC-CH3). HRMS calcd for C36H50N6NaO13 [M +
Na]+, 797.3334; found, 797.3340. Analytical HPLC gradient 0−38%
eluent II in eluent I (20 min total run time), tR = 13.1 min (>95%
purity, UV230).
+
C41H61N6O13 [M + H]+, 845.4; found, 845.3. HRMS calcd for
+
C41H60N6NaO13 [M + Na]+, 867.4116; found, 867.4124. Analytical
HPLC gradient 10−35% eluent II in eluent I (25 min total run time),
tR = 15.0 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys(Lau)-AMC (4c). Yield, 30 mg (73%, only
1
part of the crude residue was purified). H NMR (600 MHz, DMSO-
d6) δ 12.29 (br s, 2H, COOHAsp, COOHGlu), 10.21 (s, 1H, NHAMC),
8.22 (d, J = 7.7, 1H, NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu), 7.97 (d, J =
7.5, 1H, NHLys), 7.78 (d, J = 2.0, 1H, H8AMC), 7.75−7.66 (m, 3H,
NHThr, NHεLys, H5AMC), 7.52 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J
= 1.1, 1H, H3AMC), 5.03 (br s, 1H, OHThr), 4.60 (q, J = 7.3, 1H,
HαAsp), 4.37−4.28 (m, 2H, HαGlu, HαLys), 4.24 (dd, J = 7.9, 4.3, 1H,
HαThr), 4.02 (m, 1H, HβThr), 3.01 (q, J = 6.6, 2H, HεLys), 2.74 (dd, J =
16.6, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.3, 1H, HβAsp‑B), 2.40 (d, J
= 1.0, 3H, 4AMC-CH3), 2.31−2.23 (m, 2H, HγGlu), 1.99 (t, J = 7.5, 2H,
CH2CONHεLys), 1.96−1.83 (m, 4H, CH3CONHGlu, HβGlu‑A), 1.79−
1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.67−1.59 (m, 1H, HβLys‑B), 1.46−1.13
(m, 22H, CH3(CH2)9, HδLys, HγLys), 1.05 (d, J = 6.3, 3H, HγThr), 0.84
(t, J = 7.0, 3H, CH3(CH2)10). 13C NMR (151 MHz, DMSO) δ 174.0
(COδGlu), 172.0 (2C, COγAsp, CONHεLys), 171.6 (COαGlu), 171.1
(COαLys), 170.7 (COαAsp), 169.9 (COThr), 169.6 (CONHGlu), 160.0
(C2AMC), 153.6 (C8aAMC), 153.0 (C4AMC), 142.1 (C7AMC), 125.8
(C5AMC), 115.4 (C6AMC), 115.1 (C4aAMC), 112.3 (C3AMC), 105.8
(C8AMC), 66.7 (CβThr), 58.0 (CαThr), 53.8 (CαLys), 52.1 (CαGlu), 49.6
(CαAsp), 38.2 (CεLys), 35.9 (CβAsp), 35.4 (CH2CONHεLys), 31.3
(CH3(CH2)9/CδLys/CγLys), 31.2 (CβLys), 30.3 (CγGlu), 29.0−28.7 (7C,
CH3(CH2)9/CδLys/CγLys), 27.1 (CβGlu), 25.3−22.8 (2C, CH3(CH2)9/
CδLys/CγLys), 22.4 (CH3CONHGlu), 22.1 (CH3(CH2)9/CδLys/CγLys),
19.2 (CγThr), 18.0 (4AMC-CH3), 13.9 (CH3(CH2)10). MS calcd for
Ac-Glu-Thr-Asp-Lys((S)-2-methylbutyryl)-AMC (4f). The title
compound was synthesized according to general acylation procedure
A. Reagents: (S)-2-methylbutyric acid (6 mg, 0.06 mmol, 1.2 equiv),
HATU (23 mg, 0.06 mmol, 1.3 equiv), lutidine (12 mg, 0.12 mmol,
2.6 equiv). Reaction time: 7 h. Purification of the crude residue by
preparative HPLC afforded Ac-Glu-Thr-Asp-Lys((S)-2-methylbutyr-
+
C43H65N6O13 [M + H]+, 873.5; found, 873.5. HRMS calcd for
+
C43H64N6NaO13 [M + Na]+, 895.4429; found, 895.4436. Analytical
F
DOI: 10.1021/acs.jmedchem.5b01532
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
yl)-AMC (4f, 22 mg, 61% from 7) as a colorless fluffy solid after
lyophilization. H NMR (600 MHz, DMSO-d6) δ 12.26 (br s, 2H,
(4h, 17 mg, 49% from 7) as a colorless fluffy solid after lyophilization.
1
1H NMR (600 MHz, DMSO-d6) δ 12.24 (br s, 2H, COOHAsp
,
COOHAsp, COOHGlu), 10.22 (s, 1H, NHAMC), 8.22 (d, J = 7.7, 1H,
NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu), 7.98 (d, J = 7.5, 1H, NHLys), 7.78
(d, J = 1.9, 1H, H8AMC), 7.74−7.66 (m, 3H, NHεLys, NHThr, H5AMC),
7.52 (dd, J = 8.7, 1.9, 1H, H6AMC), 6.26 (d, J = 0.9, 1H, H3AMC), 5.05
(br s, 1H, OHThr), 4.61 (q, J = 7.3, 1H, HαAsp), 4.35−4.28 (m, 2H,
HαGlu, HαLys), 4.25 (dd, J = 7.9, 4.4, 1H, HαThr), 4.05−3.98 (m, 1H,
HβThr), 3.02 (m, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59
(dd, J = 16.7, 7.4, 1H, HβAsp‑B), 2.40 (s, 3H, 4AMC-CH3), 2.31−2.24
(m, 2H, HγGlu), 2.08 (dq, J = 13.5, 6.7, 1H, CH(CH3)CONHεLys),
1.96−1.88 (m, 1H, HβGlu‑A), 1.86 (s, 3H, CH3CONHGlu), 1.79−1.70
(m, 2H, HβGlu‑B, HβLys‑A), 1.68−1.59 (m, 1H, HβLys‑B), 1.49−1.19 (m,
6H, CH2CH(CH3)CONHεLys, HγLys, HδLys), 1.05 (d, J = 6.3, 3H,
HγThr), 0.93 (d, J = 6.8, 3H, CH3CH2CH(CH3)CONHεLys), 0.75 (t, J
= 7.4, 3H, CH3CH2CH(CH3)CONHεLys). 13C NMR (151 MHz,
DMSO) δ 175.3 (CONHεLys), 174.0 (COδGlu), 171.9 (COγAsp), 171.6
(COαGlu), 171.1 (COαLys), 170.6 (COαAsp), 169.9 (COαThr), 169.6
(CONHGlu), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC), 142.0
(C7AMC), 125.9 (C5AMC), 115.4 (C6AMC), 115.1 (C4aAMC), 112.3
(C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9 (CαThr), 53.9 (CαLys),
52.1 (CαGlu), 49.6 (CαAsp), 41.3 (CH(CH3)CONHεLys), 38.2 (CεLys),
35.8 (CβAsp), 31.3 (CβLys), 30.3 (CγGlu), 28.9 (CδLys), 27.0 (CβGlu),
26.8 (CH2CH(CH3)CONHεLys), 22.8 (CγLys), 22.4 (CH3CONHGlu),
19.2 (CγThr), 18.0 (4AMC-CH3), 17.6 (CH3CH2CH(CH3)CONHεLys),
11.8 (CH3CH2 CH(CH3)CONHεL ys). HRMS calcd for
COOHGlu), 10.22 (s, 1H, NHAMC), 8.22 (d, J = 7.7, 1H, NHAsp), 8.12
(d, J = 7.7, 1H, NHGlu), 7.99 (d, J = 7.5, 1H, NHLys), 7.82 (t, J = 5.6,
1H, NHεLys), 7.77 (d, J = 1.9, 1H, H8AMC), 7.72 (d, J = 8.7, 1H,
H5AMC), 7.68 (d, J = 8.0, 1H, NHThr), 7.52 (dd, J = 8.7, 2.0, 1H,
H6AMC), 6.60−6.52 (m, 1H, CHCHCONHεLys), 6.27 (d, J = 1.1, 1H,
H3AMC), 5.86 (dd, J = 15.3, 1.7, 1H, CHCONHεLys), 5.04 (br s, 1H,
OHThr), 4.61 (q, J = 7.4, 1H, HαAsp), 4.35−4.28 (m, 2H, HαGlu
,
HαLys), 4.25 (dd, J = 8.0, 4.4, 1H, HαThr), 4.01 (m, 1H, HβThr), 3.08
(q, J = 6.7, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J
= 16.7, 7.4, 1H, HβAsp‑B), 2.40 (d, J = 1.0, 3H, 4AMC-CH3), 2.31−2.22
(m, 2H, HγGlu), 1.96−1.88 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.79−1.70 (m, 5H, CH3CHCHCONHεLys, HβGlu‑B
,
,
HβLys‑A), 1.68−1.59 (m, 1H, HβLys‑B), 1.46−1.33 (m, 3H, HδLys
HγLys‑A), 1.32−1.21 (m, 1H, HγLys‑B), 1.05 (d, J = 6.3, 3H, HγThr). 13
C
NMR (151 MHz, DMSO) δ 173.9 (COδGlu), 171.9 (COγAsp), 171.5
(COαGlu), 171.1 (COαLys), 170.6 (COαAsp), 169.9 (COαThr), 169.6
(CONHGlu), 164.8 (CONHεLys), 160.0 (C2AMC), 153.6 (C8aAMC),
153.1 (C4AMC), 142.0 (C7AMC), 137.3 (CHCHCONHεLys), 125.9
(CHCONHεLys), 125.9 (C5AMC), 115.3 (C6AMC), 115.1 (C4aAMC),
112.3 (C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9 (CαThr), 53.8
(CαLys), 52.1 (CαGlu), 49.6 (CαAsp), 38.2 (CεLys), 35.8 (CβAsp), 31.2
(CβLys), 30.2 (CγGlu), 28.8 (CδLys), 27.0 (CβGlu), 22.8 (CγLys), 22.4
(CH3CONHGlu), 19.2 (CγThr), 18.0 (4AMC-CH3), 17.3
(CH3CHCHCONHεLys). The cis- isomer could be detected (approx
+
C36H50N6NaO13 [M + Na]+, 797.3334; found, 797.3339. Analytical
1
+
10%, based on H NMR). HRMS calcd for C35H46N6NaO13 [M +
Na]+, 781.3021; found, 781.3007. Analytical HPLC gradient 0−20%
eluent II in eluent I (40 min total run time), tR = 25.1 min (>95%
purity, UV230).
HPLC gradient 0−25% eluent II in eluent I (25 min total run time), tR
= 17.0 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys(glutaryl)-AMC (4g). The title compound
was synthesized according to general acylation procedure B. The
reaction was performed using 35 mg (0.04 mmol) of Ac-Glu(tBu)-
Thr(tBu)-Asp(tBu)-Lys-AMC. Reagents: glutaric anhydride (7 mg,
0.06 mmol, 1.5 equiv), i-Pr2EtN (16 mg, 0.12 mmol, 3.0 equiv).
Reaction time: 1 h. Purification of the crude residue by preparative
HPLC afforded Ac-Glu-Thr-Asp-Lys(glutaryl)-AMC (4g, 20 mg, 61%
Ac-Glu-Thr-Asp-Lys((E)-dec-2-enoyl)-AMC (4i). The title com-
pound was synthesized according to general acylation procedure A.
Reagents: (E)-dec-2-enoic acid (9 mg, 0.06 mmol, 1.2 equiv), HATU
(23 mg, 0.06 mmol, 1.3 equiv), lutidine (12 mg, 0.12 mmol, 2.6 equiv).
Reaction time: overnight. Purification of the crude residue by
preparative HPLC afforded Ac-Glu-Thr-Asp-Lys((E)-dec-2-enoyl)-
AMC (4i, 19 mg, 48% from 7) as a colorless fluffy solid after
1
from 7) as a colorless fluffy solid after lyophilization. H NMR (600
1
MHz, DMSO-d6) δ 12.16 (br s, 3H, COOHAsp, COOHGlu
,
lyophilization. H NMR (600 MHz, DMSO-d6) δ 12.25 (br s, 2H,
HOOC(CH2)3CONHεLys), 10.21 (s, 1H, NHAMC), 8.22 (d, J = 7.7,
1H, NHAsp), 8.11 (d, J = 7.7, 1H, NHGlu), 7.99 (d, J = 7.5, 1H,
NHαLys), 7.79−7.74 (m, 2H, H8AMC, NHεLys), 7.70 (d, J = 8.7, 1H,
H5AMC), 7.69 (d, J = 8.0, 1H, NHThr) 7.52 (dd, J = 8.7, 2.0, 1H,
H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 5.04 (br s, 1H, OHThr), 4.61
(m, 1H, HαAsp), 4.35−4.28 (m, 2H, HαLys, HαGlu), 4.25 (dd, J = 8.0,
4.4, 1H, HαThr), 4.01 (m, 1H, HβThr), 3.01 (m, 2H, HεLys), 2.75 (dd, J
= 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.4, 1H, HβAsp‑B), 2.40 (d,
J = 1.1, 3H, 4AMC-CH3), 2.30−2.23 (m, 2H, HγGlu), 2.17 (t, J = 7.4,
2H, CH2(CH2)2CONHεLys), 2.06 (t, J = 7.5, 2H, CH2CONHεLys),
1.95−1.88 (m, 1H, HβGlu‑A), 1.86 (s, 3H, CH3CONHGlu), 1.78−1.59
(m, 5H, HβGlu‑B, HβLys‑A, CH2CH2CONHεLys, HβLys‑B), 1.43−1.22 (m,
4H, HγLys, HδLys), 1.05 (d, J = 6.3, 3H, HγThr). 13C NMR (151 MHz,
DMSO) δ 174.2 (HOOC(CH2)3CONHεLys), 174.0 (COδGlu), 171.9
(COγAsp), 171.6 (CONHεLys/COαGlu), 171.4 (CONHεLys/COαGlu),
171.1 (COαLys), 170.7 (COαAsp), 169.9 (COαThr), 169.6
(CH3CONHGlu), 160.0 (C2AMC), 153.6 (C8aAMC), 153.1 (C4AMC),
142.0 (C7AMC), 125.9 (C5AMC), 115.4 (C4aAMC), 115.2 (C6AMC),
112.4 (C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9 (CαThr), 53.8
(CαLys), 52.1 (CαGlu), 49.6 (CαAsp), 38.3 (CδLys), 35.8 (CβAsp), 34.5
(CH2CONHεLys), 33.0 (CH2(CH2)2CONHεLys), 31.3 (CβLys), 30.2
(CγGlu), 28.8 (CδLys), 27.0 (CβGlu), 22.8 (CγLys), 22.4
(CH3CONHGlu), 20.7 (CH2CH2CONHεLys), 19.2 (CγThr), 18.0
COOHAsp, COOHGlu), 10.21 (s, 1H, NHAMC), 8.22 (d, J = 7.7, 1H,
NHThr), 8.12 (d, J = 7.7, 1H, NHGlu), 7.99 (d, J = 7.5, 1H, NHLys), 7.83
(t, J = 5.6, 1H), 7.77 (d, J = 2.0, 1H, H8AMC), 7.73−7.67 (m, 2H,
NHThr, H5AMC), 7.52 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.56 (dt, J = 15.3,
6.9, 1H, CHCHCONHεLys), 6.26 (d, J = 1.1, 1H, H3AMC), 5.84 (d, J =
15.4, 1H, CHCONHεLys), 5.04 (br s, 1H, OHThr), 4.61 (q, J = 7.4, 1H,
HαAsp), 4.36−4.29 (m, 2H, HαGlu, HαLys), 4.25 (dd, J = 8.0, 4.3, 1H,
HαThr), 4.05−3.98 (m, 1H, HβThr), 3.08 (q, J = 6.7, 2H, HεLys), 2.75
(dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.4, 1H, HβAsp‑B),
2.40 (d, J = 1.1, 3H, 4AMC-CH3), 2.30−2.24 (m, 2H, HγGlu), 2.08 (q, J
= 6.9, 2H, CH3(CH2)5CH2), 1.96−1.89 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.79−1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.68−1.60 (m,
1H, HβLys‑B), 1.46−1.19 (m, 14H, HγLys, HδLys, CH3(CH2)5CH2), 1.05
(d, J = 6.3, 3H, HγThr), 0.85 (t, J = 7.0, 3H, CH3(CH2)6). 13C NMR
(151 MHz, DMSO) δ 174.0 (COδGlu), 171.9 (COγAsp), 171.6
(COαGlu), 171.1 (COαLys), 170.6 (COαAsp), 169.9 (COαThr), 169.6
(CONHGlu), 164.9 (CONHεLys), 160.0 (C2AMC), 153.6 (C8aAMC),
153.0 (C4AMC), 142.2 (CHCHCONHεLys/C7AMC), 142.0
(CHCHCONHεLys/C7AMC), 125.9 (C5AMC), 124.4 (CHCONHεLys),
115.4 (C6AMC), 115.1 (C4aAMC), 112.4 (C3AMC), 105.8 (C8AMC), 66.7
(CβThr), 57.9 (CαThr), 53.8 (CαLys), 52.1 (CαGlu), 49.6 (CαAsp), 38.3
(CεLys), 35.8 (CβAsp), 31.2 (3C, CH3(CH2)5CH2, CβLys), 30.3 (CγGlu),
28.8−27.9 (4C, CH3(CH2)5, CδLys), 27.0 (CβGlu), 22.8 (CH3(CH2)5/
CγLys), 22.4 (CH3CONHGlu), 22.1 (CH3(CH2)5/CγLys), 19.2 (CγThr),
18.0 (4AMC-CH3), 13.9 (CH3(CH2)6). HRMS calcd for
+
(4AMC-CH3). HRMS calcd for C36H48N6NaO15 [M + Na]+,
827.3075; found, 827.3062. Analytical HPLC gradient 0−30% eluent
II in eluent I (20 min total run time), tR = 11.9 min (>95% purity,
UV230).
Ac-Glu-Thr-Asp-Lys(crotonyl)-AMC (4h). The title compound
was synthesized according to general acylation procedure B. Reagents:
crotonyl chloride (7 mg, 0.07 mmol, 1.5 equiv), i-Pr2EtN (18 mg, 0.14
mmol, 3.0 equiv). Reaction time: 1 h. Purification of the crude residue
by preparative HPLC afforded Ac-Glu-Thr-Asp-Lys(crotonyl)-AMC
+
C41H58N6NaO13 [M + Na]+, 865.3960; found, 865.3964. Analytical
HPLC gradient 15−40% eluent II in eluent I (25 min total run time),
tR = 14.7 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys((E)-dodec-2-enoyl)-AMC (4j). The title
compound was synthesized according to general acylation procedure
A. Reagents: (E)-dodec-2-enoic acid (11 mg, 0.06 mmol, 1.2 equiv),
HATU (23 mg, 0.06 mmol, 1.3 equiv), lutidine (12 mg, 0.12 mmol,
G
DOI: 10.1021/acs.jmedchem.5b01532
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2.6 equiv). Reaction time: overnight. Purification of the crude residue
in aliquots (∼3 mg/run) by preparative HPLC afforded Ac-Glu-Thr-
Asp-Lys((E)-dodec-2-enoyl)-AMC (4j, 6 mg, 39% from 7) as a
colorless fluffy solid after lyophilization. 1H NMR (600 MHz, DMSO-
d6) δ 12.23 (br s, 2H, COOHAsp, COOHGlu), 10.23 (s, 1H, NHAMC),
8.23 (d, J = 7.7, 1H, NHAsp), 8.13 (d, J = 7.7, 1H, NHGlu), 7.99 (d, J =
7.5, 1H, NHLys), 7.84 (t, J = 5.6, 1H, NHεLys), 7.78 (d, J = 1.9, 1H,
H8AMC), 7.75−7.66 (m, 2H, NHThr, H5AMC), 7.52 (dd, J = 8.7, 1.9, 1H,
H6AMC), 6.56 (dt, J = 15.2, 6.9, 1H, CHCHCONHGlu), 6.26 (d, J =
1.1, 1H, H3AMC), 5.84 (d, J = 15.4, 1H, CHCONHGlu), 5.05 (br s, 1H,
Ac-Glu-Thr-Asp-Lys((R/S)-3-hydroxydecanoyl)-AMC (4l). The
title compound was synthesized according to general acylation
procedure A. Reagents: 3-hydroxydecanoic acid (10 mg, 0.06 mmol,
1.2 equiv), HATU (23 mg, 0.06 mmol, 1.3 equiv), lutidine (12 mg,
0.12 mmol, 2.6 equiv). Reaction time: overnight. Purification of the
crude residue by preparative HPLC afforded Ac-Glu-Thr-Asp-Lys((R/
S)-3-hydroxydecanoyl)-AMC (4l, 24 mg, 60% from 7) as a colorless
1
fluffy solid after lyophilization. H NMR (600 MHz, DMSO-d6) δ
12.24 (br s, 2H, COOHAsp, COOHGlu), 10.20 (d, J = 1.6, 1H,
NHAMC), 8.22 (d, J = 7.7, 1H, NHAsp), 8.11 (d, J = 7.7, 1H, NHGlu),
7.98 (d, J = 6.4, 1H, NHLys), 7.77 (d, J = 1.4, 1H, H8AMC), 7.74 (t, J =
5.5, 1H, NHεLys), 7.72−7.66 (m, 2H, NHThr, H5AMC), 7.52 (dd, J =
8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 5.04 (br s, 1H,
OHThr), 4.61 (q, J = 7.3, 1H, HαAsp), 4.37−4.28 (m, 2H, HαGlu
,
HαLys), 4.25 (dd, J = 7.9, 4.3, 1H, HαThr), 4.06−3.98 (m, 1H, HβThr),
3.08 (q, J = 6.6, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59
(dd, J = 16.7, 7.4, 1H, HβAsp‑B), 2.40 (s, 3H, 4AMC-CH3), 2.31−2.23
(m, 2H, HγGlu), 2.07 (q, J = 6.9, 2H, CH3(CH2)7CH2), 1.96−1.89 (m,
OHThr), 4.61 (q, J = 7.4, 1H, HαAsp), 4.35−4.28 (m, 2H, HαGlu
,
HαLys), 4.26 (dd, J = 8.0, 4.4, 1H, HαThr), 4.02 (m, 1H, HβThr), 3.79−
3.72 (m, 1H, (CH2)6CH(OH)CH2), 3.08−2.96 (m, 2H, HεLys), 2.75
(dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.4, 1H, HβAsp‑B),
2.39 (d, J = 1.1, 3H, 4AMC-CH3), 2.30−2.23 (m, 2H, HγGlu), 2.11 (d, J
= 6.6, 2H, CH2CONHεLys), 1.96−1.89 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.79−1.69 (m, 2H, HβGlu‑B, HβLys‑A), 1.68−1.58 (m,
1H, HβLys‑B), 1.44−1.14 (m, 16H, HγLys, HδLys, CH3(CH2)6CH(OH)-
CH2), 1.05 (d, J = 6.3, 3H, HγThr), 0.84 (t, J = 7.0, 3H, CH3(CH2)6).
13C NMR (151 MHz, DMSO) δ 174.0 (COδGlu), 171.9 (COγAsp),
171.6 (COαGlu), 171.1 (COαLys), 170.8 (CONHεLys), 170.6
(COαAsp), 169.9 (COαThr), 169.6 (CONHGlu), 160.0 (C2AMC),
153.6 (C8aAMC), 153.0 (C4AMC), 142.0 (C7AMC), 125.9 (C5AMC),
115.4 (C6AMC), 115.1 (C4aAMC), 112.4 (C3AMC), 105.8 (C8AMC), 67.4
(CH(OH)CH2CONHεLys), 66.7 (CβThr), 57.9 (CαThr), 53.8 (CαLys),
52.1 (CαGlu), 49.6 (CαAsp), 43.6 (CH2CONHεLys), 38.2 (CεLys), 36.9
(CH3(CH2)6), 35.8 (CβAsp), 31.2 (2C, CH3(CH2)6, CβLys), 30.3
(CγGlu), 29.1−28.7 (3C, CH3(CH2)6, CδLys), 27.0 (CβGlu), 25.0
(CH3(CH2)6), 22.8 (CγLys), 22.4 (CH3CONHGlu), 22.1 (CH3(CH2)6),
19.2 (CγThr), 18.0 (4AMC-CH3), 14.0 (CH3(CH2)6). HRMS calcd for
1H, HβGlu‑A), 1.86 (s, 3H, CH3CONHGlu), 1.79−1.69 (m, 2H, HβGlu‑B
HβLys‑A), 1.69−1.59 (m, 1H, HβLys‑B), 1.46−1.17 (m, 18H, HγLys
,
,
HδLys, CH3(CH2)7), 1.05 (d, J = 6.3, 3H, HγThr), 0.85 (t, J = 7.0, 3H,
CH3(CH2)8). 13C NMR (151 MHz, DMSO) δ 174.0 (COδGlu), 171.9
(COγAsp), 171.6 (COαGlu), 171.1 (COαLys), 170.6 (COαAsp), 169.9
(COαThr), 169.6 (CONHGlu), 164.9 (CONHεLys), 160.0 (C2AMC),
153.6 (C8aAMC), 153.0 (C4AMC), 142.2 (CHCHCONHεLys), 142.1
(C7AMC), 125.9 (C5AMC), 124.5 (CHCONHεLys), 115.4 (C6AMC),
115.1 (C4aAMC), 112.3 (C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 57.9
(CαThr), 53.8 (CαLys), 52.1 (CαGlu), 49.6 (CαAsp), 38.3 (CεLys), 35.8
(CβAsp), 31.3−31.2 (3C, CH3(CH2)7CH2, CβLys,), 30.3 (CγGlu), 28.9−
27.9 (6C, CH3(CH2)7, CδLys), 27.0 (CβGlu), 22.8 (CγLys), 22.4
(CH3CONHGlu), 22.1 (CH3(CH2)7), 19.2 (CγThr), 18.0 (4AMC-CH3),
+
13.9 (CH3(CH2)8). HRMS calcd for C43H62N6NaO13 [M + Na]+,
893.4273; found, 893.4275. Analytical HPLC gradient 15−40% eluent
II in eluent I (25 min total run time), tR = 14.7 min (>95% purity,
UV230).
Ac-Glu-Thr-Asp-Lys((R/S)-3-hydroxybutyryl)-AMC (4k). The
title compound was synthesized according to general acylation
procedure A. Reagents: (R/S)-3-hydroxy-butyric acid (5 mg, 0.05
mmol, 1.0 equiv), HATU (19 mg, 0.05 mmol, 1.1 equiv), lutidine (10
mg, 0.09 mmol, 2.0 equiv). Reaction time: after 2 h, 3-hydroxy-butiryc
acid (1 mg, 0.01 mmol, 0.2 equiv), HATU (4 mg, 0.01 mmol, 0.2
equiv), and lutidine (1 mg, 0.01 mmol, 0.2 equiv) were preincubated
in CH2Cl2 (200 μL) then added to the reaction mixture. The reaction
mixture was then stirred for 1 h. Purification of the crude residue by
preparative HPLC afforded Ac-Glu-Thr-Asp-Lys((R/S)-3-hydroxy-
butyryl)-AMC (4k, 21 mg, 59% from 7) as a colorless fluffy solid
+
C41H60N6NaO14 [M + Na]+, 883.4065; found, 883.4070. Analytical
HPLC gradient 10−45% eluent II in eluent I (25 min total run time),
tR = 11.5 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys((R/S)-3-hydroxydodecanoyl)-AMC (4m).
The title compound was synthesized according to general acylation
procedure A. Reagents: (R/S)-3-hydroxydodecanoic acid (12 mg, 0.06
mmol, 1.2 equiv), HATU (23 mg, 0.06 mmol, 1.3 equiv), lutidine (12
mg, 0.12 mmol, 2.6 equiv). Reaction time: overnight. Purification of
the crude residue in aliquots (∼5 mg/run) by preparative HPLC
afforded Ac-Glu-Thr-Asp-Lys((R/S)-3-hydroxydodecanoyl)-AMC
(4m, 22 mg, 53% from 7) as a colorless fluffy solid after lyophilization.
1
after lyophilization. H NMR (600 MHz, DMSO-d6) δ 12.22 (br s,
2H, COOHAsp, COOHGlu), 10.21 (d, J = 1.4, 1H, NHAMC), 8.22 (d, J =
7.7, 1H, NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu), 7.98 (d, J = 7.4, 1H,
NHLys), 7.77 (d, J = 1.9, 1H, H8AMC), 7.75 (t, J = 5.6, 1H, NHεLys),
7.71 (d, J = 8.7, 1H, H5AMC), 7.68 (d, J = 8.0, 1H, NHThr), 7.52 (dd, J
= 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 4.61 (q, J = 7.4,
1H, HαAsp), 4.32 (m, 2H, HαGlu, HαLys), 4.25 (dd, J = 8.0, 4.4, 1H,
HαThr), 4.01 (m, 1H, HβThr), 3.93 (m, 1H, CH3CH(OH)CH2), 3.02
(m, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J =
16.7, 7.4, 1H, HβAsp‑B), 2.40 (d, J = 1.1, 3H, 4AMC-CH3), 2.31−2.22
(m, 2H, HγGlu), 2.17 (dd, J = 13.8, 7.2, 1H, CH2,ACONHεLys), 2.06
(ddd, J = 13.8, 5.9, 1.7, 1H, CH2,BCONHεLys), 1.95−1.88 (m, 1H,
1H NMR (600 MHz, DMSO-d6) δ 12.26 (br s, 2H, COOHAsp
,
COOHGlu), 10.20 (d, J = 1.8, 1H, NHAMC), 8.22 (d, J = 7.7, 1H,
NHAsp), 8.12 (d, J = 7.7, 1H, NHGlu), 7.99 (d, J = 7.2, 1H, NHLys), 7.78
(d, J = 1.4, 1H, H8AMC), 7.74 (t, J = 5.3, 1H, NHεLys), 7.72−7.67 (m,
2H, NHThr, H5AMC), 7.52 (dd, J = 8.7, 1.8, 1H, H6AMC), 6.26 (d, J =
1.1, 1H, H3AMC), 5.04 (br s, 1H, OHThr), 4.61 (q, J = 7.3, 1H, HαAsp),
4.35−4.28 (m, 2H, HαGlu, HαLys), 4.25 (dd, J = 8.0, 4.3, 1H, HαThr),
4.05−3.99 (m, 1H, HβThr), 3.78−3.72 (m, 1H, CH(OH)-
CH2CONHεLys), 3.02 (m, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H,
HβAsp‑A), 2.59 (dd, J = 16.7, 7.3, 1H, HβAsp‑B), 2.40 (d, J = 1.0, 3H,
4AMC-CH3), 2.30−2.23 (m, 2H, HγGlu), 2.11 (d, J = 6.6, 2H,
CH2CONHεLys), 1.96−1.88 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.79−1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.67−1.59 (m,
1H, HβLys‑B), 1.43−1.14 (m, 20H, CH3(CH2)8, HγLys, HδLys), 1.05 (d,
J = 6.3, 3H, HγThr), 0.84 (t, J = 7.0, 3H, CH3(CH2)8). 13C NMR (151
MHz, DMSO) δ 174.0 (COδGlu), 171.9 (COγAsp), 171.6 (COαAsp),
171.1 (COαLys), 170.8 (CONHεLys), 170.7 (COαAsp), 169.9
(COαThr), 169.6 (CONHGlu), 160.0 (C2AMC), 153.6 (C8aAMC),
153.0 (C4AMC), 142.0 (C7AMC), 125.9 (C5AMC), 115.4 (C6AMC),
115.1 (C4aAMC), 112.4 (C3AMC), 105.8 (C8AMC), 67.4 (CH(OH)-
CH2CONHεLys), 66.7 (CβThr), 57.9 (CαThr), 53.8 (CαLys), 52.1
(CαGlu), 49.6 (CαAsp), 43.6 (CH2CONHεLys), 38.2 (CεLys), 36.9
HβGlu‑A), 1.86 (s, 3H, CH3CONHGlu), 1.79−1.69 (m, 2H, HβLys‑A
,
HβGlu‑B), 1.63 (m, 1H, HβLys‑B), 1.43−1.22 (m, 5H, HγLys), 1.05 (d, J =
6.3, 3H, HγThr), 1.02 (dd, J = 6.2, 3.8, 3H, CH3CH(OH)CH2). 13C
NMR (151 MHz, DMSO) δ 174.0 (COδGlu), 171.9 (CγAsp), 171.6
(COαGlu), 171.1 (COαLys), 170.7 (COαAsp), 170.6 (CONHεLys),
169.9 (COαThr), 169.6 (CONHGlu), 160.0 (C2AMC), 153.6 (C8aAMC),
153.1 (C4AMC), 142.0 (C7AMC), 125.9 (C5AMC), 115.4 (C6AMC), 115.2
(C4aAMC), 112.4 (C3AMC), 105.8 (C8AMC), 66.7 (CβThr), 63.8
(CH3CH(OH)CH2), 57.9 (CαThr), 53.9 (CαLys), 52.1 (CαGlu), 49.6
(CαAsp), 45.3 (CH2CONHεLys), 38.2 (CεLys), 35.8 (CβAsp), 31.2
(CβLys), 30.2 (CγGlu), 28.8 (CδLys), 27.0 (CβGlu), 23.3 (CH3CH-
(OH)CH2), 22.8 (CγLys), 22.4 (CH3CONHGlu), 19.2 (CγThr), 18.0
(4AMC-CH3). HRMS calcd for C35H48N6NaO14+ [M + Na]+, 799.3126;
found, 799.3139. Analytical HPLC gradient 0−35% eluent II in eluent
I (20 min total run time), tR = 12.4 min (>95% purity, UV230).
(CH3(CH2)8), 35.8 (CβAsp), 31.3 (CβLys/CH3(CH2)8), 31.2 (CβLys
/
CH3(CH2)8), 30.3 (CγGlu), 29.1−28.7 (5C, CH3(CH2)8, CδLys), 27.0
(CβGlu), 25.0 (CH3(CH2)8), 22.8 (CγLys), 22.4 (CH3CONHGlu), 22.1
(CH3(CH2)8), 19.2 (CγThr), 18.0 (4AMC-CH3), 14.0 (CH3(CH2)8).
H
DOI: 10.1021/acs.jmedchem.5b01532
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+
HRMS calcd for C43H64N6NaO14 [M + Na]+, 911.4378; found,
(C2AMC), 153.6 (C8aAMC), 153.0 (C4AMC), 142.0 (C7AMC), 125.9
(C5AMC), 115.4 (C6AMC), 115.1 (C4aAMC), 112.4 (C3AMC), 105.8
(C8AMC), 66.7 (CβThr), 57.9 (CαThr), 53.8 (CαLys), 52.1 (CαGlu), 50.5
(CH2CONHεLys), 49.6 (CαAsp), 42.0 (CH2COCH2CONHεLys), 38.5
(CεLys), 35.8 (CβAsp), 31.2−31.1 (CβLys, CH3(CH2)5), 30.3 (CγGlu),
28.6−28.4 (3C, CH3(CH2)5s, CδLys), 27.0 (CβGlu), 22.9−22.8
(CH3(CH2)5, CγLys), 22.4 (CH3CONHGlu), 22.0 (CH3(CH2)5), 19.2
(CγThr), 18.0 (4AMC-CH3), 13.9 (CH3(CH2)6). HRMS calcd for
911.4384. Analytical HPLC gradient 15−40% eluent II in eluent I (25
min total run time), tR = 11.0 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys(acetoacetyl)-AMC (4n). The title com-
pound was synthesized according to general acylation procedure B.
The reaction was performed on 35 mg of Ac-Glu(tBu)-Thr(tBu)-
Asp(tBu)-Lys-AMC (0.04 mmol). Reagents: N-hydroxysuccinimidyl
acetoacetate (10 mg, 0.05 mmol, 1.2 equiv), i-Pr2EtN (10 mg, 0.08
mmol, 2.0 equiv). Reaction time: 1 h. Purification of the crude residue
by preparative HPLC afforded Ac-Glu-Thr-Asp-Lys(acetoacetyl)-
+
C41H58N6NaO14 [M + Na]+, 881.3909; found, 881.3915. Analytical
HPLC gradient 15−40% eluent II in eluent I (25 min total run time),
tR = 9.9 min (>95% purity, UV230).
1
AMC (4n, 21 mg, 61% from 7, keto−enol ratio 85:15 based on H
NMR) as a colorless fluffy solid after lyophilization. H NMR (600
1
Ac-Glu-Thr-Asp-Lys(3-oxododecanoyl)-AMC (4p). The title
compound was synthesized according to general acylation procedure
A. Reagents: 3-oxododecanoic acid (12 mg, 0.06 mmol, 1.2 equiv),
HATU (23 mg, 0.06 mmol, 1.3 equiv), lutidine (12 mg, 0.12 mmol,
2.6 equiv). Reaction time: the reaction mixture was stirred overnight,
then 3-oxododecanoic acid (2 mg, 0.01 mmol, 0.2 equiv), HATU (5
mg, 0.01 mmol, 0.2 equiv), and lutidine (1 mg, 0.01 mmol, 0.2 equiv)
were preincubated for 5 min in anhyd CH2Cl2 (250 μL), then added
to the reaction and stirred for 3 h more. Purification of the crude
residue in aliquots (∼3 mg/run) by preparative HPLC afforded Ac-
Glu-Thr-Asp-Lys(3-oxododecanoyl)-AMC (the crude residue was
partitioned in small batches and then purified: 4p, 14 mg, 51% from
7, keto−enol ratio 85:15, based on 1H NMR) as a colorless fluffy solid
MHz, DMSO-d6) δ 12.23 (br s, 2H, COOHAsp, COOHGlu), 10.22 (s,
1H, NHAMC), 8.22 (d, J = 7.7, 1H, NHAsp), 8.11 (d, J = 7.7, 1H,
NHGlu), 8.03−7.96 (m, 2H, NHεLys, NHLys), 7.78 (d, J = 2.0, 1H,
H8AMC), 7.71 (d, J = 8.7, 1H, H5AMC), 7.68 (d, J = 8.0, 1H, NHThr),
7.52 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 4.61
(q, J = 7.4, 1H, HαAsp), 4.36−4.28 (m, 2H, HαLys, HαGlu), 4.25 (dd, J =
8.0, 4.4, 1H, HαThr), 4.02 (td, J = 6.4, 1.8, 1H, HβThr), 3.26 (s, 2H,
CH2CONHεLys), 3.05 (q, J = 6.7, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7,
1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.4, 1H, HβAsp‑B), 2.40 (d, J = 1.1, 3H,
4AMC-CH3), 2.31−2.23 (m, 2H, HγGlu), 2.11 (s, 3H,
CH3COCH2CONHεLys), 1.96−1.88 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.80−1.70 (m, 2H, HβLys‑A, HβGlu‑B), 1.68−1.60 (m,
1H, HβLys‑B), 1.45−1.33 (m, 3H, HδLys, HγLys‑A), 1.33−1.21 (m, 1H,
HγLys‑B), 1.05 (d, J = 6.3, 3H, HγThr). 13C NMR (151 MHz, DMSO) δ
203.1 (COCH2CONHεLys), 174.0 (COδGlu), 171.9 (COγAsp), 171.6
(COαGlu), 171.1 (COαLys), 170.7 (COαAsp), 169.9 (COαThr), 169.6
(CONHGlu), 165.9 (CONHεLys), 160.0 (C2AMC), 153.6 (C8aAMC),
153.1 (C4AMC), 142.0 (C7AMC), 125.9 (C5AMC), 115.4 (C6AMC), 115.2
(C4aAMC), 112.4 (C3AMC), 105.8 (C8AMC), 66.8 (CβThr), 57.9 (CαThr),
53.8 (CαLys), 52.1 (CαGlu), 51.3 (CH2CONHεLys), 49.6 (CαAsp), 38.5
(CεLys), 35.8 (CβAsp), 31.2 (CβLys), 30.2 (CγGlu), 29.9
(CH3COCH2CONHεLys), 28.6 (CδLys), 27.0 (CβGlu), 22.8 (CγLys),
22.4 (CH3CONHGlu), 19.2 (CγThr), 18.0 (4AMC-CH3). HRMS calcd
1
after lyophilization. H NMR (600 MHz, DMSO-d6) δ 12.23 (br s,
2H, COOHAsp, COOHGlu), 10.22 (s, 1H, NHAMC), 8.22 (d, J = 7.7,
1H, NHAsp), 8.11 (d, J = 7.7, 1H, NHGlu), 7.98 (t, J = 6.1, 2H, NHLys
,
NHεLys), 7.78 (d, J = 2.0, 1H, H8AMC), 7.71 (d, J = 8.7, 1H, H5AMC),
7.68 (d, J = 8.0, 1H, NHThr), 7.52 (dd, J = 8.7, 2.0, 1H, H6AMC), 6.26
(d, J = 1.1, 1H, H3AMC), 5.04 (d, J = 4.6, 1H, OHThr), 4.61 (q, J = 7.4,
1H, HαAsp), 4.36−4.28 (m, 2H, HαLys, HαGlu), 4.26 (dd, J = 8.0, 4.4,
1H, HαThr), 4.05−3.98 (m, 1H, HβThr), 3.24 (s, 2H, CH2CONHεLys),
3.04 (q, J = 6.6, 2H, HεLys), 2.75 (dd, J = 16.7, 5.7, 1H, HβAsp‑A), 2.59
(dd, J = 16.7, 7.4, 1H, HβAsp‑B), 2.45 (t, J = 7.3, 2H,
CH2COCH2CONHεLys), 2.40 (d, J = 1.0, 3H, 4AMC-CH3), 2.30−
2.24 (m, 2H, HγGlu), 1.96−1.89 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.79−1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.68−1.60 (m,
1H, HβLys‑B), 1.45−1.14 (m, 18H, HγLys, HδLys, CH3(CH2)7CH2), 1.05
(d, J = 6.3, 3H, HγThr), 0.84 (t, J = 7.0, 3H, CH3(CH2)8). 13C NMR
(151 MHz, DMSO) δ 205.0 (COCH2CONHεLys), 174.0 (COδGlu),
171.9 (COγAsp), 171.6 (COαGlu), 171.1 (COαLys), 170.6 (COαAsp),
169.9 (COαThr), 169.6 (CONHGlu), 165.9 (CONHεLys), 160.0
(C2AMC), 153.6 (C8aAMC), 153.0 (C4AMC), 142.0 (C7AMC), 125.9
(C5AMC), 115.4 (C6AMC), 115.1 (C4aAMC), 112.4 (C3AMC), 105.8
(C8AMC), 66.7 (CβThr), 57.9 (CαThr), 53.8 (CαLys), 52.1 (CαGlu), 50.5
(CH2CONHεLys), 49.6 (CαAsp), 42.0 (CH2COCH2CONHεLys), 38.5
(CεLys), 35.8 (CβAsp), 31.3 (CH3(CH2)7), 31.2 (CβLys), 30.2 (CγGlu),
+
for C35H46N6NaO14 [M + Na]+, 797.2970; found, 797.2949.
Analytical HPLC gradient 0−30% eluent II in eluent I (20 min total
run time), tR = 12.8 min (>95% purity, UV230).
Ac-Glu-Thr-Asp-Lys(3-oxodecanoyl)-AMC (4o). The title com-
pound was synthesized according to general acylation procedure A.
Reagents: 3-oxodecanoic acid (10 mg, 0.06 mmol, 1.2 equiv), HATU
(23 mg, 0.06 mmol, 1.3 equiv), lutidine (12 mg, 0.12 mmol, 2.6 equiv).
Reaction time: the reaction mixture was stirred overnight, then 3-
oxodecanoic acid (5 mg, 0.03 mmol, 0.6 equiv), HATU (11 mg, 0.03
mmol, 0.6 equiv), lutidine (6 mg, 0.06 mmol, 1.2 equiv) were
preincubated for 5 min in anhyd CH2Cl2 (250 μL), then added to the
reaction. After 2 h, 3-oxodecanoic acid (2 mg, 0.01 mmol, 0.2 equiv),
HATU (6 mg, 0.01 mmol, 0.2 equiv), and lutidine (3 mg, 0.03 mmol,
0.6 equiv) were preincubated in anhyd CH2Cl2 (100 μL) for 5 min
then added to the reaction and stirred for 1 h. Purification of the crude
residue by preparative HPLC afforded Ac-Glu-Thr-Asp-Lys(3-
oxodecanoyl)-AMC (4o, 17 mg, 42% from 7, keto−enol ratio 85:15,
28.9−28.4 (5C, CδLys, CH3(CH2)7), 27.0 (CβGlu), 22.9−22.8 (CγLys
,
CH3(CH2)7), 22.4 (CH3CONHGlu), 22.1 (CH3(CH2)7), 19.2 (CγThr),
18.0 (4AMC-CH3), 13.9 (CH3(CH2)8). HRMS calcd for
+
C43H62N6NaO14 [M + Na]+, 909.4222; found, 909.4225. Analytical
1
1
HPLC gradient 15−50% eluent II in eluent I (25 min total run time),
tR = 12.2 min (>95% purity, UV230).
based on H NMR) as a colorless fluffy solid after lyophilization. H
NMR (600 MHz, DMSO-d6) δ 12.26 (br s, 2H, COOHAsp
,
Fluorescence Measurements. All microtiter plates were analyzed
using a PerkinElmer Enspire plate reader with excitation at 360 nm
and detecting emission at 460 nm. Fluorescence measurements (RFU)
were converted to [AMC] concentrations based on an [AMC]−
fluorescence standard curve and all data analysis was performed using
GraphPad Prism.
Fluorogenic Sirtuin Substrate Screening. The initial screening
for substrate deacylation activity was performed in Tris buffer (see
Supporting Information) with end-point fluorophore release by
trypsin. For a final volume of 25 μL per well, acyl substrates (1a,
2a−d, 3a−d, and 4a−p, 50 μM) and NAD+ (500 μM) were added to
each well followed by a solution of sirtuin enzyme (250 nM). The
reaction was incubated at 37 °C for 60 min, then 25 μL of a solution of
trypsin and nicotinamide (25 μL, 5.0 mg/mL and 4 mM, respectively;
final concentrations 2.5 mg/mL and 2 mM, respectively) was added,
and the assay development was allowed to proceed for 90 min at room
COOHGlu), 10.22 (s, 1H, NHAMC), 8.22 (d, J = 7.7, 1H, NHAsp),
8.11 (d, J = 7.7, 1H, NHGlu), 8.02−7.95 (m, 2H, NHεLys, NHLys), 7.78
(d, J = 2.0, 1H, H8AMC), 7.74−7.67 (m, 2H, NHThr, H5AMC), 7.52 (dd,
J = 8.7, 2.0, 1H, H6AMC), 6.26 (d, J = 1.2, 1H, H3AMC), 5.04 (br s, 1H,
OHThr), 4.61 (q, J = 7.3, 1H, HαAsp), 4.36−4.28 (m, 2H, HαLys
,
HαGlu), 4.25 (dd, J = 8.0, 4.3, 1H, HαThr), 4.06−3.98 (m, 1H, HβThr),
3.25 (s, 2H, CH2CONHεLys), 3.04 (q, J = 6.7, 2H, HεLys), 2.75 (dd, J =
16.7, 5.7, 1H, HβAsp‑A), 2.59 (dd, J = 16.7, 7.3, 1H, HβAsp‑B), 2.45 (t, J
= 7.3, 2H, CH2COCH2CONHεLys), 2.40 (d, J = 1.0, 3H, 4AMC-CH3),
2.30−2.23 (m, 2H, HγGlu), 1.95−1.90 (m, 1H, HβGlu‑A), 1.86 (s, 3H,
CH3CONHGlu), 1.79−1.70 (m, 2H, HβGlu‑B, HβLys‑A), 1.68−1.60 (m,
1H, HβLys‑B), 1.46−1.14 (m, 14H, CH3(CH2)5CH2, HγLys, HδLys), 1.05
(d, J = 6.3, 3H, HγThr), 0.83 (t, J = 7.1, 3H, CH3(CH2)6). 13C NMR
(151 MHz, DMSO) δ 205.0 (COCH2CONHεLys), 174.0 (COδGlu),
171.9 (COγAsp), 171.6 (COαGlu), 171.1 (COαLys), 170.7 (COαAsp),
169.9 (COαThr), 169.6 (CONHGlu), 165.9 (CONHεLys), 160.0
I
DOI: 10.1021/acs.jmedchem.5b01532
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
temperature before fluorescence analysis. The data were analyzed to
afford [AMC] relative to control wells.
ABBREVIATIONS USED
■
ADPR, adenosine diphosphate ribose; AMC, 7-amino-4-
methylcoumarin; BSA, bovine serum albumin; Dec, decanoyl;
DIC, N,N′-diisopropylcarbodiimide; DLAT, dihydrolipoamide
acetyltransferase; Glut, glutaryl; H3, histone 3 protein; H4,
histone 4 protein; HATU, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; HDAC,
histone deacetylase; HOBt, hydroxybenzotriazole; HPLC, high
performance liquid chromatography; h, hour; KDAC, lysine
deacylase; Kac, ε-N-acetyllysine; Kcr, ε-N-crotonyllysine; Kdec, ε-
N-decanoyllysine; Kglut, ε-N-glutaryllysine; Klau, ε-N-laurylly-
sine; Kmal, ε-N-malonyllysine; Kmyr, ε-N-myristoyllysine; Ksuc, ε-
N-succinyllysine; Lau, lauryl; MS, mass spectrometry; Myr,
myristoyl; NAD, nicotinamide adenine dinucleotide; NMR,
nuclear magnetic resonance; RFU, relative fluorescence units;
rt, room temperature; SIRT, sirtuin; SPPS, solid-phase peptide
synthesis; Suc, succinyl; TFA, trifluoroacetic acid; tR, retention
time; UPLC, ultrahigh performance liquid chromatography;
VLC, vacuum liquid chromatography
Determination of Kinetic Parameters for SIRT2-mediated
Deacylation. Rate experiments for determination of kinetic
parameters (substrates 4a−d and NAD+) were performed in HEPES
buffer (see Supporting Information) in a final volume of 50 μL per
well, where either acyl substrate in 2-fold dilution series [2.5−0.039
mM (for 2a), 100−0.78 μM (for 2b), and 50−0.78 μM (for 2c−d)
with NAD+ (500 μM)] or NAD+ in 2-fold dilution series [250−7.8
μM with substrate 4d (20 μM)] were incubated with trypsin (40.0 ng/
μL) and SIRT2 [500 nM (for 4a) and 60 nM (for 4b−d)]. In situ
fluorophore release was monitored immediately by fluorescence
readings recorded continuously every 10−15 s for 60 min at 25 °C
to obtain initial rates ν0 (nM s−1) for each concentration. The data
were fitted to the Michaelis−Menten equation to afford Km (μM) and
kcat (s−1) values.
Inhibition of SIRT2-Mediated Deacylase Activities. Hydrolase
activities of sirtuin 2 against substrates 4a−d were evaluated in HEPES
buffer under varying concentrations of suramin (9) or SirReal2 (10)
with end-point fluorophore release by trypsin. The sirtuin 2 enzyme
(1000 nM for 4a, 10 nM for 4b−d) was incubated with the relevant
substrate at the substrate Km value (4a at 750 μM, 4b at 6 μM, 4c at
4.5 μM, 4d at 1.8 μM), NAD+ (200 μM), and inhibitor [suramin (9):
1000−1.6 μM for 4d, 1000−0.32 μM for 4a−c in 5-fold dilution series
(final DMSO concentration ≤1%); SirReal2 (10): 20−0.16 μM in a 5-
fold dilution series (final DMSO concentration ≤5%)] in a total
volume of 25 μL. The reaction was incubated at 37 °C for 60 min, and
then a solution of trypsin and nicotinamide (25 μL to give final
concentrations of 0.2 mg/mL and 2 mM, respectively) was added. The
assay development was allowed to proceed for 15 min at room
temperature before fluorescence analysis. The data were fitted to the
concentration−response equation with variable Hill slope to obtain
IC50 values.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +45 35326708. E-mail: cao@sund.ku.dk.
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by The University of Copenhagen,
The Lundbeck Foundation (Young Group Leader Fellowship,
C.A.O.), The Danish Independent Research Council
Production and Technical Sciences (Sapere Aude grant no.
12-132328, A.S.M.), and The Carlsberg Foundation (equip-
ment). We thank the COST action CM1406 (EPICHEMBIO)
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Weissleder, R.; Shirihai, O. S.; Ellisen, L. W.; Espinosa, J. M.;
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