Synthesis of N-succinyl-L, L-diaminopimelic Acid mimetics via selective protection

Article abstract of DOI:10.2174/092986610790780387

The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient preparation of mono-N-acylated 2,6-diaminop

Full text of DOI:10.2174/092986610790780387

Protein & Peptide Letters, 2010, 17, 405-409
405
Synthesis of N-Succinyl-L,L-Diaminopimelic Acid Mimetics Via Selective
Protection
V. Vanꢀk1, J. Pícha1, M. Budꢀ ínskꢁ1, M. ꢂanda1, J. Jiráꢃek1, R.C. Holz2 and J. Hlaváꢃek1,*
1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám.
2, 166 10 Prague 6, Czech Republic; 2Department of Chemistry, Loyola University Chicago, 1068 W Sheridan Rd, Chi-
cago, IL 60626, USA
Abstract: The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-
diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient prepara-
tion of mono-N-acylated 2,6-diaminopimelic acid (DAP) derivatives bearing the different carboxyl groups or lipophilic
moieties on their amino group.
Keywords: Bacterial enzyme, N-succinyl-L,L-diaminopimelic acid (SDAP), synthesis of SDAP analogues, selective protec-
tion.
INTRODUCTION
toxicity against only bacteria, providing a previously unde-
scribed class of antimicrobial agents [7,12].
Bacterial infections are a significant and growing medical
problem around the World [1] in part, because an increasing
number of disease-causing microbes have become resistant
to existing antibiotics [2-5]. The World Health Organization
(WHO) has reported that level of antibiotics resistance is
increasing at an alarming rate (resistance towards tetracy-
clines has increased from 0% in 1948 to 98% in 1998). To-
day, 1500 people die each hour from an infectious disease,
half of these children under five years of age [4]. These find-
ings have stimulated a sustained search for new potent an-
timicrobial agents against drug resistant bacterial strains
[5,6].
DapE's catalyze the hydrolysis of N-succinyl-L,L-
diaminopimelic acid (SDAP) forming L,L-diaminopimelic
acid and succinate “Scheme (1)”. With an intention to dis-
cover and develop new antimicrobial agents that target DapE
enzyme, we focused our effort to the synthesis of potential
DapE inhibitors based on the substrate SDAP.
O-
O
O
H
N
H
+
H
H3N
O-
OH
O
O-
DapE's
O-
O
H
+
O
+
H
O
H3N
-O
H3N
Based on bacterial genetic information, the meso-
diaminopimelate (mDAP)/lysine biosynthetic pathway offers
several potential anti-bacterial targets that have yet to be
explored [7-9]. One of the products of this pathway, lysine,
is required in protein synthesis and is also used in the pepti-
doglycan layer of Gram-positive bacterial cell walls. A sec-
ond product, meso-diaminopimelic acid (mDAP) is an essen-
tial component of the peptidoglycan cell wall in Gram-
negative bacteria, providing a link between polysaccharide
strands. It has been shown that deletion of the gene encoding
for one of the enzymes in the (mDAP)/lysine biosynthetic
pathway, the dapE-encoded N-succinyl-L,L-diaminopimelic
acid desuccinylase (DapE; EC 3.5.1.18), is lethal to Helico-
bacter pylori and Mycobacterium smegmatis [10,11]. Even
in the presence of lysine supplemented media H. pylori was
unable to grow. Therefore, DapE's are essential for cell
growth and proliferation and are part of a biosynthetic path-
way that is the only source for lysine in bacteria. Since there
are no similar biosynthetic pathways in mammals, inhibitors
that target one or more of the enzymes in the (mDAP)/lysine
biosynthetic pathway are hypothesized to exhibit selective
-O
O
O
Scheme 1.
The most eligible structural alternation of SDAP from
enzyme mechanistic and synthetic standpoints (i.e. those
potentially inducing inhibitory properties) is the succinate
moiety. Altering the structure of the N-linked succinate moi-
ety will likely inhibit the enzymes ability to cleave the adja-
cent amide bond. Therefore, we have prepared compounds
bearing different N-linked acyl side chains terminated with
(i) a carboxyl group or (ii) a lipophilic moiety.
RESULTS
In order to provide an efficient and reliable synthetic ap-
proach for the preparation of large series of unsymmetrical
diaminopimelic acid (DAP) derivatives with N-linked acyl
side chains, a synthetic method was sought that (i) did not
require large amounts of expensive starting materials, (ii)
gave the desired product after a minimum number of steps,
and (iii) did not demand complicated purification procedures
of the product as previously reported [13-16]. The first at-
tempts were performed with RS,RS-2,6-diaminopimelic acid
with an intention to re-synthesize the most successful DAP
derivatives from dapE inhibitory assay in optical pure form
*Address correspondence to this author at the Institute of Organic Chemis-
try and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; Tel: +420
220183378; Fax: +420 220183578; E-mail: honzah@uochb.cas.cz
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

406 Protein & Peptide Letters, 2010, Vol. 17, No. 3
Vanꢀk et al.
later or to resolve them. In our hands, the direct N-acylation
of RS,RS-2,6-diaminopimelic acid did not give good yields,
mostly due to poor solubility of the starting material in a
reaction mixture [17]. Therefore, the synthetic methods were
developed based on the condensation of different anhydrides
or acids with suitably protected monoamine 2 “Scheme (2)”
and monoamine 6 “Scheme (3)”.
Treatment of 2 with various anhydrides furnished N-
acylated compounds 3a-3k, while in the preparation of 3l
and 3m, the free carboxylic acid was coupled to 2 using Py-
BOP as a condensation agent (Table 1). Above intermediates
were easily purified by flash chromatography on silica gel
(40-63 μm, Fluka) using elution with linear gradient of etha-
nol in chloroform. Finally, all of the precursors were fully
deprotected by hydrogenolysis under mild conditions, fol-
lowed by purification on reverse-phase HPLC providing pure
target compounds 4a-4m (Table 1).
The first key intermediate 2 was synthesized by first pro-
tecting the free amino groups with Boc (tert-
butyloxycarbonyl) followed by esterification of the carbox-
yls using N,N´-diisopropyl-O-benzylisourea in DMF (N,N-
dimethylformamide) under mild conditions, affording the
benzyl ester 1. The Boc groups were removed by acid cata-
lyzed hydrolysis with TFA. Treatment of the raw product
with one equivalent of Z-Cl and TEA in THF provided the
mono-protected intermediate 2. 1H NMR (600 MHz, DMSO-
d6): 1.37 (2H, m, CH2); 1.43 and 1.56 (2H, m, CH2); 1.62
and 1.68 (2H, m, CH2); 3.31 (1H, m, N-CH-CO); 4.04 (1H,
m, N-CH-CO); 5.03-5.11 (6H, m, 3x O-CH2-); 7.30-7.36
(15H, m, 3x C6H5); 13C NMR (150.9 MHz, DMSO-d6): two
diastereoisomers – some carbon signals are doubled, 21.96
and 21.99 (CH2); 30.60 and 30.64 (CH2); 34.18 (CH2); 54.00
and 54.07 (NCH<); 54.18 and 54.19 (N-CH<); 65.68, 65.70
and 66.06 (3x O-CH2); 127.90, 127.92, 128.02, 128.15,
128.20, 128.52 and 128.59 (aromatic –CH=, 3x C6H5);
136.13, 136.36 and 137.08 (aromatic >C=, 3x C6H5); 156.38
(N-CO-O); 172.43, 172.45, 175.72 and 175.73 (2x C-CO-O).
HRMS (ESI) calcd for C29H33N2O6 [M+H]+ 505.2333;
found: 505.2336.
For target compounds containing double bond in the N-
linked side chain, different protection groups were employed
“Scheme (3)”. First, both amino groups of RS,RS-2,6-
diaminopimelic acid were protected by reaction with Z-Cl.
Heating of the resulting intermediate with two equivalents of
N, N´-diisopropyl-O-tert-butylisourea afforded the fully pro-
tected compound 5. The Z protecting groups were removed
by hydrogenolysis and reaction of the amine with one
equivalent of Boc2O furnished the desired intermediate 6. 1H
NMR (600 MHz, CDCl3): 1.44 (9H, bs, t-Bu); 1.46 (18H, s,
2x t-Bu); 1.45 (2H, m, CH2); 1.53 and 1.70 (2H, m, CH2);
1.63 and 1.80 (2H, m, CH2); 3.29 (1H, dd, J = 7.4 and 5.4,
N-CH-CO); 4.17 (1H, m, N-CH-CO); 5.05 (1H, bd, J = 8.0,
NH); 13C NMR (150.9 MHz, CDCl3): two diastereoisomers
– some carbon signals are doubled, 21.26 and 21.35 (CH2);
27.97 (3x CH3, t-Bu); 28.02 (3x CH3, t-Bu); 28.30 (3x CH3,
t-Bu); 32.61 and 32.67 (CH2); 34.58 and 34.60 (CH2); 53.72
and 53.77 (N-CH<); 54.76 and 54.78 (N-CH<); 79.55 (>C<,
t-Bu); 80.94 (>C<, t-Bu); 81.76 (>C<, t-Bu); 155.34 (NH-
NH2
NH2
O
NHBoc
NHBoc
OBzl
NH
NH2
O
Z
c, d
a, b
HO
OH
O
BzlO
HN
OBzl
Bzl
O
O
R
O
O
R
1
2
NH2
NH
HN
Z
f
e
HO
OH
BzlO
OBzl
O
O
O
O
4a-m
3a-m
Scheme 2. Reagents, conditions, and yields: (a) Boc2O, Na2CO3, water, dioxane, 0 °C, 1 h then at rt overnight (94%); (b) N,N´-diisopropyl-
O-benzylisourea, benzene, DMF, 80°C, 8 h (83%); (c) TFA, dichloromethane, water, 1 h at rt; (d) benzyl chloroformate, TEA, THF, 0°C 1 h
then at rt overnight (37% over two steps); (e) Method A: anhydride, chloroform, 50°C 4 h, (yields see Table 1), Method B: carboxylic acid,
PyBOP, TEA, dichloromethane, rt overnight, (yields see Table 1); (f) 10% Pd/H2, methanol, rt overnight, RP-HPLC.
NH2
NH2
O
NH
NH
Z
Z
NH2
NHBoc
OtBu
a, b
tBuO
OtBu
c, d
tBuO
HO
OH
O
O
O
O
O
5
6
R
R
HN
NHBoc
OtBu
HN
NH2
O
f
tBuO
HO
OH
e
O
O
O
8a,b
7a,b
Scheme 3. Reagents, conditions, and yields: (a) benzyl chloroformate, Na2CO3, water, and dioxane, 0°C, 1h then at rt overnight (64%); (b)
N,N´-diisopropyl-O-tert-butylisourea, dioxane, 80°C, 8 h (48%); (c) 10% Pd/H2, methanol, rt overnight (90%); (d) Boc2O, TEA, dioxane,
0°C, 1 h then at rt overnight (30%); (e) Method B: carboxylic acid, PyBOP, TEA, dichloromethane, rt overnight, (yields see Table 2); (f)
TFA, dichloromethane, water, 1 h rt, RP-HPLC.

Synthesis of N-Succinyl-L,L-Diaminopimelic Acid Mimetics
Protein & Peptide Letters, 2010, Vol. 17, No. 3 407
Table 1. N-Acylation of Intermediates 2 and 6 with Yields and Analytical Data of the Protected Intermediates 3a-3m, 7a, 7b and
Final Products 4a- 4m and 8a, 8ba
Method
ESI MSb
HPLCc in min
R
Intermediate
Calc/found (m/z)
Yield
3a
4a
4a
5.21
d
O
O
O
O
O
O
A
2
C34H38N2O9
618.35/641.25
[M+Na]+
C12H20N2O6
288.30 (289.13)
[M+H]+
OH
OH
OH
(73%)
3b
4b
4b
4.55
e
A
2
C35H40N2O9
632.34/655.24
[M+Na]+
C13H22N2O7
318.33 (319,14) [M+H]+
(71%)
3c
4c
4c
6.69
e
A
2
C36H43N2O9
646.29/647.18
[M+H]+
C14H24N2O7
332.16 (333.11) [M+H]+
(86%)
4d
4d
O
3d
F F
A
2
C11H14F4N2O7
362.24 (363.07) [M+H]+
C33F4H32N2O9
676.29/699.19
[M+Na]+
OH
5.12
e
(87%)
F F
O
4e
4e
7.51
d
A
2
3e
C11H20N2O5
260.29 (261.14) [M+H]+
C33H39N2O7 574.27/575.36
[M+H]+
(82%)
O
4f
4f
6.89
f
A
2
3f
C12H22N2O5
274.32 (275.15) [M+H]+
C34H40N2O7
588.28/589.11 [M+H]+
(87%)
O
A
2
3g
4g
4g
3.11
d
C31H34N2O7
546.24/547.19 [M+H]+
C9H16N2O5
232.24 (233.11) [M+H]+
(85%)
O
3h
4h
4h
8.14
d
A
2
C33H38N2O7
574.26/575.22
[M+H]+
C11H20N2O5
260.29 (261.14) [M+H]+
(90%)
O
3i
4i
4i
7.81
f
A
2
C34H40N2O7
588.28/589.08
[M+H]+
C12H22N2O5
274.32 (275.15) [M+H]+
(91%)
O
4j
4j
6.55
f
3j
A
2
C14H18N2O5
294.31 (295.15) [M+H]+
C36H36N2O7
608.25/609.15
[M+H]+
(88%)
O
3k
4k
4k
4.33
d
O
O
A
2
C34H36N2O9
616.24/617.14
[M+H]+
C12H18N2O7
302.29 (303.11) [M+H]+
OH
(18 %)
3l
4l
4l
3.73
f
B
2
OC2H5
C34H38N2O9
618.26/619.21
[M+H]+
C12H20N2O7
304.30 (305.13) [M+H]+
(80%)
O
O

408 Protein & Peptide Letters, 2010, Vol. 17, No. 3
Vanꢀk et al.
(Table 1) contd…..
Method
ESI MSb
HPLCc in min
R
Intermediate
Calc/found (m/z)
Yield
4m
4m
4.41
f
O
3m
B
C13H22N2O7
318.33 (319.21) [M+H]+
C35H40N2O9
632.27/633.11
[M+H]+
OCH3
2
(91%)
O
O
8a
8a
8.98
f
7a
B
C12H18N2O7
302.29 (303.11) [M+H]+
C25H42N2O9
514.29/515.10
[M+H]+
O
6
(71 %)
OCH3
7b
8b
8b
5.06
f
B
6
O O
C24H41N3NaO8
499.37/522.27
[M+Na]+
C11H17N3O6
287.27 (288.11) [M+H]+
NH2
(65 %)
a All of DAP derivatives had a correct C,H,N elemental analysis in the range of 0.3%. b Determined with an ESI technique using Agilent 5975B MSD equipment (Agilent Technolo-
gies, Santa Clara, Ca, USA). c In the analytical RP HPLC a TSP instrument with an SP 8800 pump, an SP 4290 integrator, TSP Spectra 100 UV detector at 220 nm and 5μm Supelco
15 x 0.4 cm column (Supelco, Bellefonte, PA, USA) with flow 1ml/min were used. An isocratic analysis with 0.05% TFA/aq (d), 2.5% CH3CN in 0.05% TFA/aq (e) and 5% CH3CN
in 0.05% TFA/aq (f) was applied. For preparative RP HPLC using the same instruments, the 10 μm Vydac 25 x 1 cm column (Grace Davison Discovery Sciences, Hesperia, CA,
USA) with flow 3 ml/min and a gradient of 5%-50% ACN in 0.05% TFA, 120 min at 230 nm was applied.
CO-O); 171.89 (O-CO-); 175.23 and 175.30 (O-CO-).
HRMS (ESI) calcd for C20H38N2O6 [M+H]+ 403.2803;
found: 403.2803.
PyBOP
=
(Benzotriazol-1-yloxy)-tris(pyrrolidino)
phosphonium hexafluorophosphate
RP HPLC = Reverse phase high performance liquid
chromatography
The PyBOP-mediated reaction of 6 with two fumaric or
maleic acid derivatives afforded 7a and 7b, which were puri-
fied by column chromatography similarly to derivatives 3a-
3m. Full deprotection was accomplished by hydrolysis with
TFA providing target compounds 8a and 8b (Table 1).
TEA
TFA
THF
Z
=
=
=
=
Triethylamine
Trifluoroacetic acid
Tetrahydrofurane
Benzyloxycarbonyl
CONCLUSIONS
We developed a simple methodology for quick and effi-
cient preparation of mono-N-acylated 2,6-diaminopimelic
acid derivatives providing a suitable route for the large scale
preparation of series of DAP compounds. The unambiguous
acylation of unsymmetrically protected key intermediates 2
or 6, afforded 3a-3m and 7a, 7b as the protected derivatives
of the target compounds 4a- 4m and 8a, 8b in moderate to
high yields. Moreover, these target compounds were ob-
tained in high purity after only a one-step deprotection proc-
ess under mild conditions (hydrogenolysis or acidic condi-
tions).
ACKNOWLEDGEMENTS
This work was supported by the National Science Foun-
dation (CHE-0652981, RCH), the Academy of Sciences of
the Czech Republic (GAV No. IAA400550614 and Research
Project No. Z4 055 0506) and the Ministry of Education,
Youth and Sports of the Czech Republic (LC060777, Re-
search Centre for Chemical Genetics).
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ABBREVIATIONS
Boc
=
=
=
=
tert-Butyloxycarbonyl
Benzyl
Bzl
[3]
DAP
DapE
2,6-Diaminopimelic acid
[4]
[5]
[6]
Mono-N-succinyl-L,L-diaminopimelic acid
desuccinylase
DMF
=
=
=
N,N-dimethylformamide
ESIMS
HRMS
Electro spray ionization mass spectrometry
High resolution mass spectrometry

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Received: July 10, 2009
Revised: September 07, 2009
Accepted: October 02, 2009