The synthesis of diaminopimelic acid containing peptidoglycan fragments using metathesis cross coupling

Article abstract of DOI:10.1016/j.tetlet.2005.01.062

Properly protected diaminopimelic acid (DAP), a component of peptidoglycan of Gram-negative bacteria, was prepared by a metathesis cross coupling between properly protected allyl and vinyl glycine derivatives using Grubb's second-generation catalyst follo

Full text of DOI:10.1016/j.tetlet.2005.01.062

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1675–1678
The synthesis of diaminopimelic acid containing
peptidoglycan fragments using metathesis cross coupling
Abhijit Roy Chowdhury and Geert-Jan Boons*
Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
Received 9 December 2004;revised 11 January 2005;accepted 13 January 2005
Available online 1 February 2005
Abstract—Properly protected diaminopimelic acid (DAP), a component of peptidoglycan of Gram-negative bacteria, was prepared
by a metathesis cross coupling between properly protected allyl and vinyl glycine derivatives using GrubbÕs second-generation
catalyst followed by reduction of the double bond of the resulting compound. The DAP derivatives were used in the solution-
and polymer-supported synthesis of biological active peptides.
ꢀ 2005 Published by Elsevier Ltd.
Peptidoglycan (PGN) is an essential component of the
cell wall of virtually all bacteria that preserves cell integ-
rity by withstanding the internal osmotic pressure. It is
also responsible for the maintenance of cell shape and
is intimately involved in cell division. PGN is especially
abundant in Gram-positive bacteria, in which it ac-
counts for approximately half of the cell wall mass.
On the other hand, Gram-negative bacteria contain only
a relatively thin PGN layer found in the periplasmic
space.1,2
OH
OH
O
O
O
HO
O
O
O
NHAc
NHAc
H
N
O
O
NH2
O
N
H
2
O
O
NH
N
H
3
HO
PGN are large polymers composed of alternating b-(1–
4)-linked N-acetylglucosamine (GlcNAc) and N-acetyl-
muramic acid (MurNAc) residues, which are cross
linked by short peptide bridges (Fig. 1). These peptides
consist of four or five alternating L- and D-amino acids,
which are attached to the carboxylic acid of MurNAc.
The third amino acid of the peptides moiety of PGN
of Gram-positive bacteria is commonly lysine while
Gram-negative bacteria have a diaminopimelic acid
(DAP) residue at this position.
O
H2N
R
Figure 1. R = H (Lys) Gram-positive PGN, R = COOH (DAP) Gram-
negative PGN.
ligand for receptors of the innate immune system of
eukaryotes.7,8
For example, CD14 and Toll-like receptor 2 (TLR2),
which are expressed on the surface of host monocytes
and macrophages, can bind to PGN resulting in the
secretion of several pro-inflammatory cytokines through
the activation of transcription factors such as nuclear
factor jB (NF-jB). These cytokines provide a first line
of defense against invading pathogens. Over production
of these mediators is known, however, to result in the
clinical symptoms of septic shock.9,10 Recently, it has
been shown that nucleotide-binding oligomerization
domain proteins 1 and 2 (NOD1 and 2), which are intra-
cellular pattern recognition receptors, can detect small
The biosynthesis of PGN is a well-recognized target for
antibiotic development.3–6 For example, penicillins,
cephalosporins, and vancomycin all act by inhibiting
key steps in the assembly of the PGN layers. Recently,
PGN has also attracted considerable attention as a
Keywords: Peptidoglycan;Amino acid;Cross metathesis;
Diaminopimelic acid.
*
meso-2,6-
Corresponding author. Tel.: +1 706 542 9161;fax: +1 706 542
4412;e-mail: gjboons@ccrc.uga.edu
0040-4039/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2005.01.062

1676
A. Roy Chowdhury, G.-J. Boons / Tetrahedron Letters 46(2005) 1675–1678
O
O
fragments of PGN initiating a pro-inflammatory re-
sponse.11–14
R1HN
R1HN
OR2
OR2
1) NO2PhSeCN, Bu3P, THF
2) 30% H2O2
In order to determine the molecular details of PGN-
mediated cytokine production, well-defined synthetic
fragments of PGN are required.15 Furthermore,
synthetic analogs of PGN part structures may provide
a lead compound for the development of novel
antibiotics.
OH
1 R1 = Z, R2 = Bn
5 R1 = Z, R2 = Bn (60%)
6 R1 = Z, R2 = t-Bu (55%)
7 R1 = Fmoc, R2 = Bn (52%)
8 R1 = Boc, R2 = t-Bu (55%)
2 R1 = Z, R2 = t-Bu
3 R1 = Fmoc, R2 = Bn
4 R1 = Boc, R2 = t-Bu
Scheme 1. Synthesis of vinyl glycine derivatives.
Although lysine-containing compounds can be conve-
niently synthesized by standard solution- or polymer-
supported approaches,16,17 the chemical synthesis of
DAP containing fragments is hampered by difficulties
in obtaining this unusual amino acid. For example,
Vederas and co-workers attempted the synthesis of
DAP by a ring closing metathesis (RCM) of N-(benzyl-
cine were formed which could be easily separated by
silica gel column chromatography. The amount of
homo-dimerized allyl glycine varied from 17% to 26%.
Surprisingly, a prolonged reaction time failed to increase
the yield of the cross coupling. The latter finding indi-
cates that the homo-dimerized allyl glycine is not acti-
vated by the GrubbÕs catalyst to give compound 13.
Indeed, when a mixture of the homo-dimer of allyl
and vinyl glycine 5 were subjected to metathesis condi-
tions for two days, no cross coupling product 13 was
formed. Fortunately, the use of an excess of vinyl
glycine resulted in a significant improvement of the
yield.
oxycarbonyl)-L-vinyl
glycine
and
N-(tert-butyl-
carbonyl)-D-allylglycine that were esterified to ethylene
glycol. This approach failed, however, due to the
formation of an, a,b-unsaturated ester during vinyl
glycine preparation. DAP was successfully synthesized
by multi-step approach in which the key C–C bond-
forming step was an ene reaction of methyl glyoxylate
with
methyl
N-(benzyloxycarbonyl)-L-allylglycine
catalyzed by a chiral Cu-catalyst to give a 96/4 mixture of
diastereoisomers.18 Several other lengthy synthetic
approaches have been reported, which required separa-
tion of enantiomers or epimers.19–24
Previously, it has been shown that protecting groups
may influence the outcome of cross metathesis reac-
tions.25 Therefore, several different allyl and vinyl gly-
cines were used containing benzyl carbamate (Z), 9-
fluorenylmethylcarbamate (Fmoc) and tert-butyl carba-
mate (Boc) as amino protecting groups and benzyl (Bn),
tert-butyl (t-Bu) and methyl esters for carboxylic acid
protection. The reaction combinations of vinyl (5–8)
and allyl glycine (9–12) were chosen in such a manner
that the resulting DAP derivatives (13–18) had an
appropriate protecting group pattern for the synthesis
of biologically relevant peptides. As can be seen in
Table 1, the protecting groups had a small effect on
the yields of the reactions ranging from 52% to 60%.
In each case, the use of an excess of vinyl glycine led
to an improvement in the yield of the cross metathesis
product.
We envisaged that DAP could be conveniently prepared
by a cross metathesis reaction between readily available
allyl and vinyl glycine followed by a reduction of the
double bond of the resulting compound. High selectivity
in olefin cross metathesis reactions has been achieved
when two olefins have significantly different reactivi-
ties.25 In this respect, it has been shown that a cross
metathesis of vinyl glycine with terminal alkenes using
GrubbÕs first-generation catalyst gives mainly the recov-
ery of the vinyl glycine, homo-dimerization of the alkene
and a very low yield of cross coupling product.26 On the
other hand, the use of allyl glycines gave reasonable
yields of cross metathesis products when applied under
similar reaction conditions. The lower reactivity of vinyl
glycines is probably due to a combination of steric and
electronic factors. The introduction of the GrubbÕs sec-
ond-generation catalyst has made it possible to perform
cross metathesis reactions with sterically more demand-
ing substrates. Therefore, it was expected that using this
catalyst, a reaction of properly protected vinyl with allyl
glycine would give a cross coupling product in good
yield.
Having established a convenient approach for the prep-
aration of properly protected DAP derivatives, attention
was focused on the synthesis of biologically active PGN
fragments. Recently, it was shown that the dipeptide i-
Glu-DAP can induce activation of the transcription fac-
tor NF-jB in HEK-293 cells transfected with the gene
encoding for NOD1.11 We envisaged that hydrogena-
tion of 14 over Pd–C would lead to a reduction of the
double bond with concomitant removal of the benzyl
carbamate group. Coupling of the resulting compound
with an appropriately protected isoglutamic acid 20
would, after removal of the protecting groups, give the
targeted dipeptide (Scheme 2). Thus, the double bond
of 14 was reduced with concomitant removal of benzyl
carbamate by catalytic hydrogenation over Pd–C to af-
ford compounds 19 in an almost quantitative yield. The
resulting compound was coupled with isoglutamic acid
20 using N,N,N0,N0-tetramethyl-O-(7-azabenzotriazol-
1-yl)uronium hexafluorophosphate (HATU) as the
Several protected vinyl glycine (5–8) derivatives were
prepared by treatment of corresponding homoserines
(1–4) with 2-nitrophenyl selenocyanate and tributyl-
phosphine in THF followed by the addition of hydrogen
peroxide (Scheme 1).27,28 A reaction of 5 with a stoichio-
metric quantity of 9 in the presence of GrubbÕs second-
generation catalyst gave, after a reaction time of 24 h,
the expected cross coupling product 13 in an acceptable
yield of 46% (Table 1). Apart from this compound,
unreacted vinyl glycine and homo-dimerized allyl gly-

A. Roy Chowdhury, G.-J. Boons / Tetrahedron Letters 46(2005) 1675–1678
Table 1. Cross metathesis reactions of vinyl and allyl glycine derivatives
1677
O
O
R3HN
Grubbs 2 nd. gen cat.
O
R
OR4
R3HN
R
R1HN
OR4
S
DCM, rt.
OR2
+
O
S
OR2
R1HN
5-8
9-12
13-18
Substrates
R1
R2
R3
R4
Comp./yield, %
5:9 (1:1)
Z
Bn
Bn
Boc
Boc
Boc
Boc
Boc
Boc
Fmoc
Fmoc
Boc
Boc
Me
Me
t-Bu
t-Bu
Bn
13/46
13/60
14/42
14/55
15/34
15/49
16/31
16/59
17/61
18/64
5:9 (1.8:1)
6:10 (1:1)
6:10 (1.8:1)
7:11 (1:1)
7:11 (1.8:1)
8:12 (1:1)
8:12 (1.8:1)
5:11 (1.8:1)
7:10 (1.8:1)
Z
Z
t-Bu
Z
t-Bu
Bn
Fmoc
Fmoc
Boc
Boc
Z
Bn
Bn
Bn
t-Bu
t-Bu
Bn
Bn
Bn
Fmoc
Bn
t-Bu
activation reagent to give the dipeptide 21 in a yield of
86%. The Fmoc protecting group of 21 can be selectively
removed for further extension. However, target com-
pound 2229 was obtained by a two-step deprotection
procedure involving cleavage of the tert-butyl carbamate
and tert-butyl ester by treatment with 20% TFA in
DCM followed by the simultaneous removal of the
Fmoc and benzyl ester by catalytic hydrogenation over
Pd–C to give 22 in an overall yield of 84%.
Thus, catalytic hydrogenation of compound 16 over
Pt–C resulted in a clean reduction of the double bond
and removal of the benzyl ester to give DAP derivative
23 in a yield of 98%. Next, the dipeptide D-Ala-D-Ala
attached to a hyper acid sensitive Sieber amide
linker was prepared using classical Fmoc chemistry
and
benzotriazole-1-yl-oxy-tris-pyrrolidino-phospho-
nium hexafluorophosphate (PyBOP)/N-hydroxybenzo-
triazole(HOBT)/N-ethyldiisopropyl amine (DIPEA) as
the coupling reagent. In the next step of peptide
assembly, DAP derivative 23 was incorporated using
the same activation reagent to give a polymer bound
tripeptide. Treatment of this derivative with 20%
piperidine in DMF led to the removal of the Fmoc
group as judged by a positive Kaiser test. Subsequently,
an isoglutamine moiety was incorporated using Fmoc-
D-isoglutamine and PyBOP/HOBT/DIPEA as the
coupling reagent. Finally, removal of the Fmoc protect-
ing group of the tetrapeptide and coupling of Fmoc-L-
Ala gave the fully assembled peptide. The Fmoc
protecting group was removed using standard condi-
tions and the terminal amine acetylated with acetic
anhydride to give peptide 24. The peptide was cleaved
from the solid support using 2% TFA and 1% triisopro-
pyl silane (TIS). Under these conditions the side chain
protecting groups were not removed which
facilitated the purification of the compound. Finally,
global deprotection with 20% TFA gave the target
pentapeptide 25.
Derivative 16 is suitably protected for use in solid sup-
ported synthesis of DAP containing peptides. To illus-
trate the use of this compound, pentapeptide 25,30
which is a fragment of PGN of Gram-negative bacteria,
was prepared by
(Scheme 3).
a
polymer-supported approach
O
O
O
OH
BocHN
Pd/C, H2,
BocHN
R
R
Ot-Bu
Ot-Bu
+
EtOH, DCM, H2O
O
O
O
S
S
R
FmocHN
Ot-Bu
Ot-Bu
OBn
H2N
ZHN
14
20
19
O
O
H2N
BocHN
R
R
OH
Ot-Bu
1) 20% TFA, DCM
2) 10% Pd/C, H2
EtOH, DCM, H2O
O
O
HATU, DIEA
S
S
In conclusion, the unusual amino acid, diaminopimelic
acid, could conveniently be prepared by a metathesis
reaction between appropriately protected vinyl and allyl
glycine derivatives followed by reduction of the double
bond of the resulting compounds. By careful selection
of protecting groups, the latter reaction selectively re-
moved a benzyl carbamate or benzyl ester to give
DAP derivatives that were used in the solution- or poly-
mer-supported synthesis of biologically active PGN part
structures.
OH
Ot-Bu
(84% over two steps)
O
NH
O
NH
O
O
R
H2N
22
FmocHN
R
OH
OBn
21
Scheme 2. Synthesis of biological active Glu-(7S,11R)-7-11 diamino-
pimelic acid.

1678
A. Roy Chowdhury, G.-J. Boons / Tetrahedron Letters 46(2005) 1675–1678
O
O
7. Dziarski, R. Cell. Mol. Life Sci. 2003, 60, 1793–1804.
8. Akira, S.;Takeda, K. Nature Rev. Immunol. 2004, 4, 499–
511.
BocHN
BocHN
R
R
OtBu
OtBu
3% Pt/C, H2,
9. Athman, R.;Philpott, D. Curr. Opin. Microbiol. 2004, 7,
25–32.
EtOH, DCM, H2O
O
O
10. Van Amersfoort, E. S.;Van Berkel, T. J. C.;Kuiper, J.
Clin. Microbiol. Rev. 2003, 16, 379.
S
S
OBn
OH
11. Girardin, S. E.;Travassos, L. H.;Herve, M.;Blanot, D.;
Boneca, I. G.;Philpott, D. J.;Sansonetti, P. J.;Mengin-
Lecreulx, D. J. Biol. Chem. 2003, 278, 41702–41708.
12. Chamaillard, M.;Girardin, S. E.;Viala, J.;Philpott, D. J.
Cell. Microbiol. 2003, 5, 581–592.
FmocHN
FmocHN
16
23
O
O-PS
=
H2N
P
13. Girardin, S. E.;Boneca, I. G.;Carneiro, L. A.;Antignac,
A.;Jehanno, M.;Viala, J.;Tedin, K.;Taha, M. K.;
Labigne, A.;Zahringer, U.;Coyle, A. J.;DiStefano, P. S.;
Bertin, J.;Sansonetti, P. J.;Philpott, D. J. Science 2003,
300, 1584–1587.
NH2
a) Fmoc-D-Ala, PyBOP, HOBt, DIPEA, then 20% piperidine in DMF
b) Fmoc-D-Ala, PyBOP, HOBt, DIPEA, then 20% piperidine in DMF
c) 23, PyBOP, HOBt, DIPEA, then 20% piperidine in DMF
d) Fmoc-D-isoGln, PyBOP, HOBt, DIPEA, then 20% piperidine in DMF
e) Fmoc-L-Ala, PyBOP, HOBt, DIPEA, then 20% piperidine in DMF
f) 10% Ac2O, 5% DIPEA, DMF
14. Girardin, S. E.;Hugot, J. P.;Sansonetti, P. J.
Immunol. 2003, 24, 652–658.
Trends
15. Siriwardena, A.;Jorgensen, M. R.;Wolfert, M. A.;
Vandenplas, M. L.;Moore, J. N.;Boons, G. J. J. Am.
Chem. Soc. 2001, 123, 8145–8146.
tBuO
O
16. VanNieuwenhze, M. S.;Mauldin, S. C.;Zia-Ebrahimi,
M.;Winger, B. E.;Hornback, W. J.;Saha, S. L.;Aikins, J.
A.;Blaszczak, L. C. J. Am. Chem. Soc. 2002, 124, 3656–
3660.
17. Kubasch, N.;Schmidt, R. R. Eur. J. Org. Chem. 2002,
2710–2726.
18. Gao, Y.;Lane-Bell, P.;Vederas, J. C. J. Org. Chem. 1998,
63, 2133–2143.
19. Collier, P. N.;Patel, I.;Taylor, R. J. K. Tetrahedron Lett.
2001, 42, 5953–5954.
NHBoc
O
O
H
N
H
N
NH
P
AcHN
N
H
N
H
O
O
O
O
NH2
24
HO
O
a) 2% TFA, 1% TIS in DCM
b) 20% TFA in DCM
20. Sutherland, A.;Vederas, J. C. Chem. Commun. 2002, 224–
225.
NH2
O
21. Davis, F. A.;Srirajan, V. J. Org. Chem. 2000, 65, 3248–
3251.
22. Jurgens, A. R. Tetrahedron Lett. 1992, 33, 4727–4730.
23. Williams, R. M.;Liu, J. W. J. Org. Chem. 1998, 63, 2130–
2132.
O
H
N
H
N
NH2
AcHN
N
H
N
H
24. Roberts, J. L.;Chan, C. Tetrahedron Lett. 2002, 43, 7679–
7682.
O
O
O
O
NH2
25
25. Chatterjee, A. K.;Choi, T. L.;Sanders, D. P.;Grubbs, R.
H. J. Am. Chem. Soc. 2003, 125, 11360–11370.
26. Biagini, S. C. G.;Gibson, S. E.;Keen, S. P. J. Chem. Soc.,
Perkin Trans. 1 1998, 2485–2499.
Scheme 3. Polymer-supported synthesis of DAP containing peptide
derived from Gram-negative PGN.
27. Pellicciari, R.;Natalini, B.;Marinozzi, M. Synth. Com-
mun. 1988, 18, 1715–1721.
Acknowledgements
28. Salituro, G. M.;Townsend, C. A. J. Am. Chem. Soc. 1990,
112, 760–770.
This research was supported by the Institute of General
Medicine of the National Institutes of Health
(GM065248).
26
29. Analytical data of compound 22: ½aꢀD +5.7, 1H NMR
(500 MHz, CD3OD): d 4.32 (1H, q, a-CH, DAP), 3.94
(1H, t, a-CH, Glu), 3.86 (1H, q, a-CH, DAP), 2.47 (2H, c-
CH2, Glu), 2.10–2.19 (1H, m, b-CH2, Glu), 2.04–2.10 (1H,
m, b-CH2, Glu), 1.78–1.96, 1.65–1.69, 1.45–1.57 (6H, m,
b,c,d-CH2CH2CH2, DAP). 13C NMR (125 MHz,
CD3OD): 175.12, 174.45, 53.92, 53.71, 53.34, 32.14,
32.01, 31.22, 31.11, 27.24, 22.65. HRMS-MALDI-TOF
calcd for C12H21N3O7 (M+Na): 342.1380, found 342.0795.
References and notes
1. Rietschel, E. T.;Schletter, J.;Weidemann, B.;El-Samalo-
uti, V.;Mattern, T.;Zahringer, U.;Seydel, U.;Brade, H.;
Flad, H. D.;Kusumoto, S.;Gupta, D.;Dziarski, R.;
Ulmer, A. J. Microb. Drug Resist. 1998, 4, 37–44.
2. van Heijenoort, J. Glycobiology 2001, 11, 25R–36R.
3. Koch, A. L. Clin. Microbiol. Rev. 2003, 16, 673–687.
4. Weidenmaier, C.;Kristian, S. A.;Peschel, A. Curr. Drug
Targets 2003, 4, 643–649.
5. Lazar, K.;Walker, S. Curr. Opin. Chem. Biol. 2002, 6,
786–793.
6. Cox, R. J.;Sutherland, A.;Vederas, J. C. Bioorg. Med.
Chem. 2000, 8, 843–871.
26
30. Analytical data of compound 25: ½aꢀD +26.25, 1H NMR
(500 MHz, CD3OD): d 4.21–4.29 (4H, m, a-CH, Glu, a-
CH, DAP, 2 · a-CH, Ala), 3.71–3.74 (1H, m, a-CH, Ala),
3.20 (1H, q, a-CH, DAP), 2.38–2.40 (2H, m, c-CH2, Glu),
2.12–2.15 (1H, m, b-CH2, Glu), 2.03 (3H, s, NHCOCH3),
1.79–1.98 (5H, m, b-CH2, Glu, b-CH2, DAP, d-CH2,
DAP), 1.45–1.50 (2H, m, c-CH2, DAP), 1.34–1.42 (9H, m,
3 · CH3, Ala). HRMS-MALDI-TOF calcd for
C23H40N8O9 (M+Na): 595.6122, found 595.4370.