Stereoselective syntheses of 4-oxa diaminopimelic acid and its protected derivatives via aziridine ring opening

Article abstract of DOI:10.1021/ol701742x

Regio- and stereoselective aziridine ring opening with oxygen nucleophiles derived from serine and threonine provides a route to stereochemically pure 4-oxa-2,6-diaminopimelic acid (oxa-DAP) and its methyl-substituted derivatives. Oxa-DAP is a substrate of DAP epimerase, a key enzyme for biosynthesis of L-lysine and formation of peptidoglycan precursors. Orthogonally protected analogues of lanthionine and/β-methyllanthionine wherein oxygen replaces sulfur were prepared that could be used for solid-supported peptide synthesis to make oxa derivatives of lantibiotics.

Full text of DOI:10.1021/ol701742x

ORGANIC
LETTERS
2007
Vol. 9, No. 21
4211-4214
Stereoselective Syntheses of 4-Oxa
Diaminopimelic Acid and Its Protected
Derivatives via Aziridine Ring Opening
Hongqiang Liu, Vijaya R. Pattabiraman, and John C. Vederas*
Department of Chemistry, UniVersity of Alberta, Edmonton, Alberta, Canada T6G 2G2
john.Vederas@ualberta.ca
Received July 21, 2007
ABSTRACT
Regio- and stereoselective aziridine ring opening with oxygen nucleophiles derived from serine and threonine provides a route to stereochemically
pure 4-oxa-2,6-diaminopimelic acid (oxa-DAP) and its methyl-substituted derivatives. Oxa-DAP is a substrate of DAP epimerase, a key enzyme
for biosynthesis of L-lysine and formation of peptidoglycan precursors. Orthogonally protected analogues of lanthionine and â-methyllanthionine
wherein oxygen replaces sulfur were prepared that could be used for solid-supported peptide synthesis to make oxa derivatives of lantibiotics.
Bacterial enzymes on the biosynthetic route to L-lysine also
generate key precursors for the peptidoglycan cell wall layer.1
meso-Diaminopimelic acid (meso-DAP) (1b) occurs in the
peptidoglycan of virtually all Gram-negative bacteria, whereas
its metabolic product, L-lysine, is used analogously in many
Gram-positive organisms.2 The enzymes on the pathway
provide an opportunity for development of new antibiotics
with low mammalian toxicity because mammals do not make
DAP and require L-lysine in their diet.1 One of them, DAP
epimerase, catalyzes the reversible conversion of LL-DAP
(1a) to meso-DAP (1b). Its unusual mechanism does not rely
on metals or cofactors but rather utilizes two cysteine residues
as a thiol-thiolate pair to deprotonate the R-carbon of the
Figure 1. Interconversion of LL-DAP and meso-DAP by DAP
epimerase, and structures of oxa-DAP, lanthionine, and â-methyl-
lanthionine.
(1) For recent reviews, see: (a) Vederas, J. C. Can. J. Chem. 2006, 84,
1197-1207 and references cited therein. (b) Cox, R. J.; Sutherland, A.;
Vederas, J. C. Bioorg. Med. Chem. 2000, 8, 843-872.
(2) (a) van Heijenoort, J. Nat. Prod. Rep. 2001, 18, 503-519. (b)
Wiseman, J. S.; Nichols, J. S. J. Biol. Chem. 1984, 259, 8907-8914. (c)
Diaper, C. M.; Sutherland, A.; Pillai, B.; James, M. N. G.; Semchuk, P.;
Blanchard, J. S.; Vederas, J. C. Org. Biomol. Chem. 2005, 3, 4402-4411.
(d) Pillai, B.; Cherney, M. M.; Diaper, C. M.; Sutherland, A.; Blanchard,
J. S.; Vederas, J. C.; James, M. N. G. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 8668-8673.
substrate and reprotonate from the opposite side (Figure 1).2c,d
DAP epimerase exhibits very strict substrate specificity.
meso-Lanthionine (3a), which has a sulfur atom instead of
10.1021/ol701742x CCC: $37.00
© 2007 American Chemical Society
Published on Web 09/12/2007

a CH2 group at the 4-position, is not accepted by the enzyme.3
Significant differences in bond lengths and bond angles
apparently cannot be accommodated in the very tight active
site of this epimerase. However, it seemed that replacement
of the CH2 at the 4-position in DAP with an oxygen atom
could generate a substrate or inhibitor as the predicted bond
lengths (1.43 vs 1.54 Å) and bond angles (109°28′ vs
111°43′) of C-C-C and C-O-C bonds are more compa-
rable.4 In this context, we decided to develop a stereoselective
method for the synthesis of ether-bridged bisamino acids as
there were no general methods for the synthesis of such
compounds.5 Ideally, development of such methodology
would give access not only to oxa-DAP but also to
orthogonally protected analogues. The latter could be used
in peptide synthesis to mimic lanthionine and â-methyl-
lanthionine (Figure 1, 3a and 3b) in lantibiotics, such as nisin
and lacticin 3147.6 These potent antimicrobial agents are
effective at nanomolar concentrations against a range of
Gram-positive bacteria, including organisms resistant to
conventional antibiotics.
to solvolysis reactions in the corresponding alcohol as
solvent. In order to use serine and threonine as nucleophiles
in lower concentration, the reactivity of the hydroxyl needs
to be enhanced. This could possibly be accomplished via
disruption of the hydrogen bond between the oxygen lone
pair and hydrogen of monoprotected neighboring amine.9
Bisprotection of the amino group could lead to favorable
reversal of hydrogen bonding to increase the electron density
on the oxygen, thereby improving its nucleophilicity (Figure
3). The phthalimido (Pht) group was chosen for diprotection
Our synthetic strategy involves ring opening of serine- and
threonine-derived aziridines bearing an electron-withdrawing
group on the nitrogen by the hydroxyl side chain of suitably
protected amino acids in the presence of a Lewis acid catalyst
(Figure 2).
Figure 3. H-Bonding patterns and effects on nucleophilicity of
hydroxyls of protected serine or threonine derivatives.
of the amino group because of the ease of preparation and
removal.10 To activate the aziridine for regioselective ring
opening at the â-position, the electron-withdrawing p-nitro-
benzyloxycarbonyl group (pNZ) was selected for nitrogen
protection.8a
The ring opening of pNZ-aziridinocarboxylate ester 4a
with Pht-Ser-OMe 5a (5.0 equiv) and BF3‚OEt2 (0.1 equiv)
affords the desired product 6a. Although the reaction is
sluggish and requires 2 days for completion, the yield (56%)
is comparable to those using oxygen nucleophiles as solvent
(Table 1, entry 1).8 Encouraged by this positive result, the
Figure 2. Aziridine ring opening with oxygen nucleophiles.
The chemistry of aziridine ring opening with stoichiomet-
ric quantities of a variety of nucleophiles in the presence of
Lewis acids is well-documented in the literature.7 However,
due to poor nucleophilicity of the hydroxyl group, examples8
of ring openings with oxygen nucleophiles are usually limited
Table 1. Optimization of Aziridine Ring Opening Conditions
(3) Lam, L. K. P.; Arnold, L. D.; Kalantar, T. H.; Kelland, J. G.; Lane-
Bell, P. M.; Palcic, M. M.; Pickard, M. P.; Vederas, J. C. J. Biol. Chem.
1988, 263, 11814-11819.
entry nu/E+/BF3‚OEt2 solvent
temp
rt
rt
time
% yield
1
2
3
4
5/1/0.1
2/1/0.2
2/1/0.2
2/1/0.5
CHCl3
CHCl3
toluene 110 °C 5 h
toluene 110 °C 1.5 h
2 days
4 days
57
60
70
72
(4) (a) Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen,
A. G.; Taylor, R. J. Chem. Soc., Perkin Trans. 1987, 2, S1-S19. (b) Blukis,
V.; Kasai, P. H.; Myers, R. J. J. Chem. Phys. 1963, 38, 2753-2760.
(5) For synthesis of a mixture of oxa-DAP isomers, see: Zahn, H.;
Dietrich, R.; Gerstner, W. Chem. Ber. 1955, 88, 1734-1746.
(6) For selected reviews, see: (a) Chatterjee, C.; Paul, M.; Xie, L.; van
der Donk, W. A. Chem. ReV. 2005, 105, 633-684. (b) McAuliffe, O.; Ross,
R. P.; Hill, C. FEMS Microbiol. ReV. 2001, 25, 285-308. (c) Sahl, H. G.;
Bierbaum, G. Annu. ReV. Microbiol. 1998, 52, 41-79.
reaction was examined with respect to the solvent, temper-
ature, and reagent stoichiometries (Table 1). Although
(7) For reviews, see: (a) Hu, X. E. Tetrahedron 2004, 60, 2701-2743
and references cited therein. (b) Tanner, D. Angew. Chem., Int. Ed. Engl.
1994, 33, 599-619. (c) Zhou, P.; Chen, B.; Davis, F. A. Aziridines and
Epoxides in Organic Synthesis; Yudin, A. K., Ed.; Wiley-VCH: Weinheim,
Germany, 2006; p 89.
(8) (a) McKeever, B.; Pattenden, G. Tetrahedron 2003, 59, 2071-2712.
(b) Nakajima, K.; Neya, M.; Yamada, S.; Okawa, K. Bull. Chem. Soc. Jpn.
1982, 55, 3049-3050. (c) BhanuPrasad, B. A.; Sekar, G.; Singh, V. K.
Tetrahedron Lett. 2000, 41, 4677-4679. (d) Ho, M.; Wang, M.; Pham, D.
T. Tetrahedron Lett. 1991, 32, 1283-1286.
(9) Enhanced nucleophilicity of the hydroxyl groups via intramolecular
H-bonding has been offered as an explanation for glycosidation of serine
and threonine. (a) Szabo, L.; Li, Y.; Polt, R. Tetrahedron Lett. 1991, 5,
585-588. (b) Polt, R.; Jennifer, T. L.; Li, Y.; Hurby, V. J. Am. Chem. Soc.
1992, 114, 10249-10258.
(10) Bose, A. K. Organic Syntheses; Wiley & Sons: New York, 1973;
Collect. Vol. V, pp 973-974.
4212
Org. Lett., Vol. 9, No. 21, 2007

reaction yields are not improved dramatically, optimization
shows that using 0.5 equiv of BF3‚OEt2 catalyst in refluxing
toluene (Table 1, entry 4) shortens the reaction time
significantly from 4 days to 1.5 h and affords the best yield.11
The scope of the reaction could be explored using several
primary and secondary hydroxyl side chain containing amino
acids as the nucleophiles with pNZ-protected, â-substituted,
and â-unsubstituted aziridines. In all cases, the reaction
proceeds smoothly with the optimized conditions to give a
single isomer of the oxygen-bridged bisamino acids in
moderate to good yields (Scheme 1).12
with a sterically demanding cyclic secondary alcohol as the
nucleophile to afford 6i and 6j in good yields. As derivatives
6b and 6f are protected oxygen mimics of lanthionine and
â-methyllanthionine isomers found in lantibiotics,6 it seemed
desirable to investigate orthogonal protecting groups that are
suitable for Fmoc solid phase peptide synthesis. These could
then be used for the preparation of analogues of lantibiotics.14
Hence, the reaction was examined using pNZ15 and allyl
protection for the aziridine component and with Fmoc and
t-butyl protecting groups on the serine nucleophile. All four
protecting groups can be manipulated selectively on solid
support. However, all attempts to use Fmoc-Ser-OtBu as
nucleophile under the optimized conditions (toluene, 110 °C)
led to a complex mixture of products, presumably due to
the instability of the t-Bu group. Fortunately, milder condi-
tions using 0.2 equiv of BF3‚OEt2 in ethanol-free CHCl3 at
40 °C could be employed. Using these conditions, the
orthogonally protected oxygen analogues 7b and 7c of
lanthionine and â-methyllanthionine, respectively, can be
obtained in modest yields. These compounds are suitable for
direct incorporation onto solid support after removal of t-Bu
group (Table 2). The lower yields can be attributed to the
Scheme 1. Preparation of Bisamino Acid Ether Derivatives
Table 2. Synthesis of Orthogonally Protected Oxygen Analogs
of Lanthionine and â-Methyllanthionine
entry
R1
product
% yield
1
2
3
H
H
CH3
(2S,6S)-7a
(2R,6S)-7b
(2R,3S,6S)-7c
36
37
40
decreased nucleophilicity of the oxygen lone pairs resulting
from unfavorable H-bonding as described in Figure 3, as
well as decreased temperature. Nonetheless, the yields in
these cases are still preparatively useful.
In order to verify the stereochemical integrity of the
aziridine ring opening, 13C NMR spectra of a mixture of
stereochemically pure 7a and 7b were examined. Appearance
of individual peaks for each diastereomer at different parts
per million values (Figure 4) clearly illustrate the stereo-
chemical integrity of the ring opening.
Having successfully obtained protected LL- and meso-oxa-
DAP derivatives 6a and 6b, we attempted global removal
of the protecting groups using 6 N HCl. Unfortunately, this
provides only partially deprotected product with the pNZ
group still attached, probably due to the acidic stability of
pNZ.16 Simple hydrogenolytic deprotection of the remaining
Interestingly, the sec-sec dimethyl ethers 6g and 6h could
be formed stereochemically pure and in good yields. Reac-
tions leading to sec-sec ethers are often reported to undergo
stereocenter scrambling.13 Ring opening is also successful
(11) A side product was isolated in 6% yield resulting from the attack
of OH on the pNZ to form a benzyl ether. For such a side reaction, see:
Osterkamp, F.; Wehlan, H.; Koert, U.; Wiesner, M.; Raddatz, P.; Goodman,
S. Tetrahedron 1999, 55, 10713-10734.
(12) Intramolecular aziridine rearrangement with formation of oxazoline
(5%) was observed during the synthesis of 6e. For rearrangement of
acylaziridines to oxazolines using Lewis acids, see: (a) Ferraris, D.; Druey,
W. J., III; Cox, C.; Lectka, T. J. Org. Chem. 1998, 63, 4568-4569. (b)
Tomasini, C.; Vecchione, A. Org. Lett. 1999, 1, 2153-2156.
(13) Kuethe, J. T.; Marcoux, J. F.; Wong, A.; Wu, J.; Hillier, M. C.;
Dormer, P. G.; Davies, I. W.; Hughes, D. L. J. Org. Chem. 2006, 71, 7378-
7390.
(14) Pattabiraman, V. R.; Stymiest, J. L.; Derksen, D. J.; Martin, N. I.;
Vederas, J. C. Org. Lett. 2007, 9, 699-702.
(15) Isidro-Llobet, A.; Guasch-Camell, J.; Alvarez, M.; Albericio, F. Eur.
J. Org. Chem. 2005, 14, 3031-3039.
(16) Shields, J. E.; Carpenter, F. H. J. Am. Chem. Soc. 1961, 83, 3066-
3070.
Org. Lett., Vol. 9, No. 21, 2007
4213

Figure 4. Portions of the 125 MHz 13C NMR spectra of
diastereomers 7a, 7b, and a mixture of 7a and 7b at 27 °C in CDCl3.
Figure 5. Time course of epimerization of LL-DAP (1a) and LL-
oxa-DAP (8a) catalyzed by DAP epimerase. As both meso- and
LL-isomers are substrates/products and there is an isotope effect
for removal of R-hydrogen, the conversion to deuterated product
slows as the reaction proceeds.
pNZ gives the completely deprotected LL- (8a) and meso-
oxa-DAP (2a) isomers required for evaluation with DAP
epimerase (Scheme 2).
DAP epimerase accommodates the substitution of oxygen
atom for a methylene group at the 4-position in its natural
substrate, the presence of a polar oxygen atom appears to
disrupt the tight hydrophobic interactions in the closed
enzyme active site.2d
Scheme 2. Deprotection to Obtain LL-Oxa-DAP
In conclusion, a facile, regio- and stereoselective procedure
to prepare ether-linked bisamino acids via aziridine ring
opening with oxygen nucleophiles has been developed. LL-
Oxa-DAP (8a), obtained via the new methodology, was
identified as an effective substrate for DAP epimerase, in
contrast to lanthionine (the 4-thia analogue of DAP).
Furthermore, analogues of lanthionine and â-methyllanthion-
ine with orthogonal protecting groups amenable for solid
phase peptide synthesis have been prepared which could be
readily incorporated as building blocks into cyclic peptides
and lantibiotics.
a Isolated yields over two steps after cationic ion exchange
column.
The enzymatic studies with LL-oxa-DAP (8a) employed
DAP epimerase from Haemophilus influenzae.1,2c,d The
enzymatic reactions were done in deuterated solvents in order
to monitor the exchange of R-hydrogen with a deuterium
by the epimerase using NMR and mass spectrometry.17
Incubation of LL-oxa-DAP (8a) with DAP epimerase in
deuterated Tris buffer results in change of the integral for
the R-hydrogen in the 1H NMR spectrum due to incorpora-
tion of deuterium at the R-position. This demonstrates that
DAP epimerase accepts LL-oxa-DAP (8a) as a substrate.
Initial velocity for the enzymatic reaction could be obtained
by monitoring the change in integral values for the R-proton
of 8a and 1a in separate reactions as a function of time. LL-
Oxa-DAP (8a) exhibits an initial velocity that is 26% of that
of the natural substrate LL-DAP (1a) (Figure 5). Although
Acknowledgment. We thank Matthew Clay, Sandra
Marcus, and Mark Miskolzie (Department of Chemistry, Uni-
versity of Alberta) for providing LL-DAP, DAP epimerase,
and assistance with NMR. This work was supported by
Merck Frosst Canada Ltd., the Natural Sciences and Engi-
neering Research Council of Canada (NSERC), the Canada
Research Chair in Bioorganic and Medicinal Chemistry, and
the Alberta Heritage Foundation for Medical Research
(AHFMR).
Supporting Information Available: Experimental pro-
cedures and spectral data for all compounds synthesized and
DAP epimerase assay. This material is available free of
charge via the Internet http://pubs.acs.org.
(17) (a) Campbell, I. D. Fresenius Z. Anal. Chem. 1986, 324, 437-441.
(b) Petersen, B. O.; Krah, M.; Duss, J.; Thomsen, K. K. Eur. J. Biochem.
2000, 267, 361-369.
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Org. Lett., Vol. 9, No. 21, 2007