The stereoselective synthesis of aziridine analogues of diaminopimelic acid (DAP) and their interaction with dap epimerase

Article abstract of DOI:10.1039/b513409a

Aziridine analogues of diaminopimelic acid (DAP) have been prepared stereoselectively for the first time and evaluated as inhibitors of DAP epimerase. (2R,3S,3′S)-3-(3′-Aminopropane)aziridine-2,3′- dicarboxylate 4 was synthesised and shown to be a reversible inhibitor of DAP epimerase with an IC₅₀ value of 2.88 mM. (2S,4S)- and (2S,4R)-2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid (LL-azi-DAP 14 and DL-azi-DAP 29) were made as pure diastereomers, and both were shown to be irreversible inhibitors of DAP epimerase. LL-Azi-DAP 14 selectively binds to Cys-73 of the enzyme active site whereas DL-azi-DAP 29 binds to Cys-217 via attack of sulfhydryl on the methylene of the inhibitor aziridine ring. These observations are consistent with the two base mechanism proposed for the epimerisation of LL-DAP 1 and meso-DAP 2 by DAP epimerase. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b513409a

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A R T I C L E
The stereoselective synthesis of aziridine analogues of
diaminopimelic acid (DAP) and their interaction with dap epimerase†
Christopher M. Diaper,a Andrew Sutherland,b Bindu Pillai,c Michael N. G. James,c
Paul Semchuk,d John S. Blancharde and John C. Vederas*a
a Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
E-mail: john.vederas@ualberta.ca; Fax: (780) 4922134; Tel: (780) 4924575
b WestChem, Department of Chemistry, University of Glasgow, Glasgow, United Kingdom
G12 8QQ
c Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H6
d Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta, Canada
T6G 2H7
e Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx, New York, 10461
Received 22nd September 2005, Accepted 24th October 2005
First published as an Advance Article on the web 15th November 2005
Aziridine analogues of diaminopimelic acid (DAP) have been prepared stereoselectively for the first time and
evaluated as inhibitors of DAP epimerase. (2R,3S,3ꢀS)-3-(3ꢀ-Aminopropane)aziridine-2,3ꢀ-dicarboxylate 4 was
synthesised and shown to be a reversible inhibitor of DAP epimerase with an IC50 value of 2.88 mM. (2S,4S)- and
(2S,4R)-2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid (LL-azi-DAP 14 and DL-azi-DAP 29) were made as
pure diastereomers, and both were shown to be irreversible inhibitors of DAP epimerase. LL-Azi-DAP 14 selectively
binds to Cys-73 of the enzyme active site whereas DL-azi-DAP 29 binds to Cys-217 via attack of sulfhydryl on the
methylene of the inhibitor aziridine ring. These observations are consistent with the two base mechanism proposed
for the epimerisation of LL-DAP 1 and meso-DAP 2 by DAP epimerase.
Introduction
The worldwide increase in bacterial resistance has led to renewed
interest in the development of new antibiotics,1,2 including
methods of inhibiting microbial cell wall biosynthesis.3 As meso-
diaminopimelic acid (meso-DAP 2) is a precursor to L-lysine
and an important component of the peptidoglycan layer of
Gram-negative bacteria,4 it has attracted considerable interest
for the possible development of new antibiotics.5 Crucially, the
biosynthesis of meso-DAP is restricted to bacteria5 and plants
and is absent in mammals, which require L-lysine in their diet.6
Therefore specific inhibitors of DAP enzymes are likely to be
antimicrobial agents with low mammalian toxicity.
One of the key enzymes in the DAP pathway is DAP
epimerase, an unusual enzyme that interconverts LL-DAP 1 and
meso-DAP 2 without the use of co-factors, metals, or reducible
keto or imino functionality (Scheme 1).7
This process is mechanistically analogous to the other PLP-
Scheme 1 The biosynthetic pathways to meso-DAP and proposed
independent amino acid racemases.8,9 It is proposed to involve
transition state for the inter-conversion of LL-DAP 1 and meso-DAP
two active site cysteine residues, one of which as a thiolate,
acts as a general base while the other, as a thiol, acts as a
general acid.10 Initial tritium labeling studies showed that the
a-proton of DAP is exchanged during epimerisation.7 Further
investigations using 3-fluoro-DAP isomers demonstrated that
the mechanism involves development of anionic character at
the a-carbon and that the substrate is rigidly held in the
enzyme active site.11 In 1990, the irreversible inactivation of
DAP epimerase was reported12 using a crude mixture of all
possible diastereomers of 2-(4-amino-4-carboxybutyl)aziridine-
2-carboxylic acid (azi-DAP) 3. Enzymatic digestion studies
2 by DAP epimerase.
determined that Cys-73 was alkylated by the aziridino moiety,
thereby demonstrating its presence in the active site (Scheme 2).13
Recently a diastereomeric mixture of oxa analogues of azi-DAP
† Electronic supplementary information (ESI) available: experimental
details for the preparation of 7, 8, 18, 21, 22 and 23; 600 MHz NOE
assignment of stereochemistry for 26; 600 MHz 1H NMR determination
of dr’s for 26 and 28; HPLC analysis of 14 precursor; MS/MS analysis
enzymatic digests of DAP epimerase inhibited with either 14 or 29. See
DOI: 10.1039/b513409a
Scheme 2 Proposed mechanism for the irreversible inhibition of DAP
epimerase by azi-DAP 3.
4 4 0 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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was found to irreversibly inhibit DAP epimerase, presumably
due to thiol opening of the epoxide moiety.14
The crystal structure of inactive DAP epimerase from
Haemophilus influenzae has been solved15 and subsequently
refined16 to reveal a disulfide linkage between Cys-73 and Cys-
217 at the interface of two structurally superimposable domains.
Based on this observation it was proposed that in the active
reduced form of the enzyme, these conserved residues are the key
catalytic units required for activity. This led to detailed kinetic
studies of DAP epimerase.10 Assignment of the specific roles of
Cys-217 and Cys-73 in the active site could be confirmed by the
interaction of pure 3-fluoro-DAP isomers with the single DAP
epimerase mutants C73A, C73S, C217A and C217S.17
The substrates LL-DAP 1 and meso-DAP 2 for DAP epimerase
differ from the other PLP-independent amino acid racemases8,18
(e.g. glutamate racemase,19–21 aspartate racemase,22 proline
racemase23,24) in that they contain two stereocentres. DAP
epimerase has very strict requirements for its substrates,5 and
only accepts DAP isomers with the L configuration at the distal
site.7,25 Neither D nor L isomers of lysine or a-aminopimelic acid
are substrates or good inhibitors, thereby demonstrating that
both carboxyl and amino groups must be present.26
Unfortunately, the crystal structures of the inactive disulfide
form of DAP epimerase15,16 do not allow reliable modeling of
the substrate in the collapsed active site. Interestingly, no crystal
structures of other PLP-independent amino acid racemases have
been obtained thus far with a substrate analog correctly bound
in the active site to illustrate catalytic function. It therefore
appeared that formation of a complex of DAP epimerase with
pure diastereomers of the aziridine analogues 3 and 4 could
provide further insight into the organisation of the enzyme
active site. In particular, crystal structures of the DAP epimerase
with stereochemically pure isomers of azi-DAP 14 and 29 could
afford a detailed picture of the active site complex for each
substrate isomer and the arrangement of residues for catalysis
of this unusual reaction. In this report we describe the first
stereoselective synthesis of highly reactive aziridine analogues 4,
14 and 29 and analysis of their interaction with DAP epimerase.
Results
Stereoselective synthesis of DAP aziridine analogue 4
It seemed that aziridine 4 could be prepared by aziridi-
nation of a suitable a,b-unsaturated ester or amide bear-
ing a chiral auxiliary.27 In 1993 we reported the aziridina-
tion of N-enoylbornane[10,2]sultams by the oxidative addi-
tion of N-aminophthalimide mediated by lead tetraacetate
with excellent diastereoselectivity.28 This approach was subse-
quently found to be compatible with other auxiliaries such
as oxoalkenyloxazolidinone29 and camphor.30 It appeared that
a similar approach using the 3-amino-2-ethyl-3,4-dihydro-
quinazolin-4-one 1231 would lead to the stereoselective synthesis
of the internal N-quinazolinyl aziridine 7. The desired aziridine
4 could then be accessed by N–N bond reduction using dissolved
metal in ammonia (Scheme 3).32
Scheme 3 The synthesis of internal aziridine DAP analogues. Reagents
and conditions: (i) XcCOCH2PO(OEt)2, DBU, LiCl, MeCN (85%);
(ii) TFA, CH2Cl2 (94%); (iii) 12, Pb(OAc)4, HMDS, CH2Cl2 (72%);
(iv) TFA, CH2Cl2 (99%); (v) LiOH, MeOH–H2O (85%); (vi) Li, NH3
=
(48%); (vii) MeO2CCH PPh3, THF (88%); (viii) TFA, CH2Cl2 (88%);
(ix) 12, Pb(OAc)4, HMDS, CH2Cl2 (83%); (x) TFA, CH2Cl2 (quant.);
(xi) LiOH, MeOH–H2O, (84%); (xii) Li, NH3 (48%).
1
the observed H NMR coupling constant of 4.8 Hz for the
2ꢀ-H proton.36 Removal of the Boc group followed by base
hydrolysis and reductive cleavage of the N–N bond with Li–
NH3 affords the target molecule 4 in 40% yield over three steps
(Scheme 3).
33
Reaction of phosphonate ester XcCOCH2PO(OEt)2 with
N,N-di-Boc glutamate semialdehyde 534 using a Horner–
Wadsworth–Emmons type coupling35 gives exclusively the trans-
alkene in 85% yield. One of the Boc protecting groups could
then be removed by TFA to generate the aziridine precursor 6 in
94% yield. Aziridination of 6 with quinazolin-4-one 12 proceeds
with optimum selectivity at a temperature of −40 ◦C to give
a diastereomeric ratio of 9 : 1. The major isomer 7 can be
isolated by recrystallisation in 72% yield. Efforts to prove the
absolute stereochemistry of aziridine 7 by X-ray crystallography
were unsuccessful. However, it has been demonstrated that
addition to N-enoylbornane[10,2]sultams occurs by syn attack
from the re face of the a-carbon.28 Moreover, syn attack would
give an aziridine with trans geometry which is consistent with
In addition to testing the single diastereomer 4 with DAP
epimerase, it was desirable to examine interaction of the enzyme
with another diastereomer to provide insight into stereochemical
preferences for the active site. Hence, a mixture of diastere-
omers 4 and 11 was prepared from N,N-di-Boc glutamate
semialdehyde 5 via the trans-alkene 837 in an analogous manner
(Scheme 3).
Inhibition study of aziridines 4 and 11 with DAP epimerase
Although several assays for DAP epimerase have been
developed,38 the activity of DAP epimerase is easily monitored
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1
4 4 0 3

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using a coupled enzyme assay. At pH 7.8, meso-DAP 2 is
generated from LL-DAP 1 by DAP epimerase and is then
transformed by DAP dehydrogenase to produce L-THDP and
NADPH (Scheme 1), which can be measured spectrophotomet-
rically at 340 nm.7,39 The enantiopure LL-DAP 1 is prepared
by the photolysis of a suitably functionalised diacyl peroxide,40
which is then subjected to hydrolysis to remove the protecting
groups.
Aziridine 4 was initially tested as a substrate and as an in-
hibitor of DAP dehydrogenase, the enzyme used for analysis. As
expected, this compound is neither a substrate nor an inhibitor
of the dehydrogenase at a concentration of 1 mM. Disappoint-
ingly, aziridine 4 is also a very weak inhibitor of DAP epimerase
with an IC50 value of only 2.88 mM. A time dependence study
showed 4 to be a fully reversible inhibitor. These results show
that unlike azi-DAP 3, which rapidly inactivates DAP epimerase,
presumably by cysteine attack at the methylene position of the
aziridine ring, both secondary carbons at the aziridine ring
positions of 4 are more stable towards the enzyme, resulting
in poor reversible binding. The mixture of trans-stereoisomers
4 and 11 was also tested against DAP epimerase. Inhibition
by this mixture is similar to the results obtained for the pure
diastereomer, suggesting analogous modes of ineffective binding
for both compounds.
Stereoselective synthesis of azi-DAP
Although a number of synthetic methods have been developed
for the asymmetric synthesis of disubstituted aziridines,41,42
examples of their application in the synthesis of the 1-carboxy-
1-alkyl aziridine functionality required for the synthesis of azi-
DAP are limited.43–47 A number of synthetic strategies were
explored in the stereospecific synthesis of azi-DAP. Wenker ring
closure of (2S,6S)-2,6-diamino-6-(hydroxymethyl)pimelic acid
13,48,49 was unsuccessful (Scheme 4). Attempts to utilise the
chiral auxiliary methodology used in the construction of the
internal aziridine 4 were frustrated by problems encountered
in the synthesis of the required N-enoylbornane[10,2]sultam
precursor. This led us to investigate the use of chiral 3-
acetoxyaminoquinazolinones,31 which exert reagent controlled
diastereoselectivity in the aziridination of prochiral alkenes such
as 16 (Scheme 4). Boc protected unsaturated a-aminopimelic
acids have been reported,37,50 and it was proposed that aziridi-
nation of the Cbz protected analogue with chiral aminoquina-
zolinones 19–21 would allow the rapid preparation of a single
diastereomer of the fully protected azi-DAP derivative 17.
The required unsaturated a-aminopimelic ester 16 was con-
structed in one-pot by photolysis of the hydroxamate ester of N-
Cbz-L-glutamic acid a-methyl ester 15 with methyl a-thiophenol
methylacrylate using a tungsten lamp at room temperature to
give a 68% yield over three steps (Scheme 4).50
The 3-acetoxy derivatives of the known aminoquinazolin-
4(3H)-ones 19,47 20,51 and the previously unreported 21 were
investigated as aziridinating agents. No reaction is observed
during titanium mediated aziridination31 using the hydroxy
aminoquinazolin-4(3H)-one 20. Aziridination using 21 and
TFA31 gives low conversion (22%) and poor selectivity (3 :
1 dr), whereas the reaction of 19 in the presence of HMDS31
proceeds with moderate yield and selectivity (49%, dr 6 : 1). The
mixture of diastereomers 17 can be saponified with base and
then reduced using lithium in ammonia at −78 ◦C, to produce
azi-DAP 14 as a 6 : 1 mixture of diastereomers at the quaternary
carbon along with the unexpected fully saturated e-methyl-a-
aminopimelic acid 18 (1 : 1 dr at the carbon bearing methyl).52
The structure of 18 was confirmed by independent synthesis
(see ESI†). Separation of 14 and 18 is possible by careful
preparative TLC under basic conditions (azi-DAP undergoes
very rapid decomposition on exposure to acid). To our chagrin,
extensive attempts to separate the minor azi-DAP diastereomer
from 14, or to purify their corresponding diacid salts or the fully
Scheme 4 The synthesis of azi-DAP. Reagents and conditions: (i)
i-BuOCOCl, NMM, THF; (ii) NEt3, 2-mercaptopyridine N-oxide, THF;
(iii) methyl a-thiophenol methylacrylate, ht (tungsten lamp), THF (68%
over 3 steps); (iv) 19, Pb(OAc)4, HMDS, CH2Cl2 (49%); (v) LiOH,
MeOH–H2O (quant.); (vi) Li, NH3.
protected intermediates 17 using flash chromatography, HPLC
or recrystallisation all failed.
To improve the selectivity of the aziridination and thus obviate
the problematic isomer separation, we adopted an intramolec-
ular variant of the aminoquinazolin-4(3H)-one methodology
that had been reported to give complete diastereoselectivity
during the aziridination of O-cinnamoyl esters.53 This neces-
sitated the modification of our synthetic strategy to couple
(R)-aminoquinazolin-4(3H)-one 22 to the 1,2-disubstituted a,b-
unsaturated acid 24 (Scheme 5).
The acid 24 could be synthesised in two steps from the
diester 16 by basic hydrolysis and regioselective esterification
of the diacid using catalytic p-toluene sulfonic acid (PTSA)
in anhydrous methanol.54 Although the selective esterification
proceeds in >95% yield (1H NMR), the isolated yields after
purification by flash chromatography are significantly lower
(30–60%). Use of polymer bound PTSA55 circumvents this
problem and allows the quantitative recovery of the product 24.
The coupling of acid 24 to (R)-aminoquinazolin-4(3H)-one 22
proceeds in satisfactory 62% yield using DCC–DMAP. However,
several cycles of flash chromatography are required to achieve
the complete removal of the unwanted urea by-product. Switch-
ing to resin bound N-benzyl-Nꢀ-cyclohexylcarbodiimide reagent
(PS-DCC)56 permits facile purification without significant loss
of yield, as demonstrated by the coupling of acid 24 to the (S)-
aminoquinazolin-4(3H)-one 23 to give 27 in 51% after a single
pass through a plug of silica.
4 4 0 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1

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is formed concomitantly with azi-DAP 29. The temperature of
the reaction has little effect on the product ratio. The reaction
to form 18 may proceed via reductive aziridine ring cleavage to
generate an anion ato the carboxylate followed by elimination of
nitrogen and further reduction of the resulting conjugated olefin.
Separation of the crude reaction mixture could be achieved using
preparative TLC under basic conditions, albeit in low isolated
yield (23% for azi-DAP 29). It was later found that HPLC pu-
rification of the diacid salt is not necessary prior to the reductive
deprotection. Therefore pure azi-DAP 14 could be prepared
directly from 26 with an improved yield of 30% over the final
two steps. As expected, azi-DAP isomers 14 and 29 are extremely
sensitive compounds that are stable in strong base, but degrade
rapidly under neutral or acidic conditions. The presence of
multiple internal nucleophiles (four carboxylate oxygens and the
distal amino group) assists rapid intramolecular aziridine ring
opening. In solid form as carboxylate salts, 14 and 29 decompose
gradually at room temperature, with half lives of about 1 to
2 weeks. Although storage at −20 ◦C retards decomposition,
they are only stable for prolonged periods under cryogenic
(−80 ◦C) conditions. Unfortunately, lack of stability in aqueous
media precludes evaluation of their antimicrobial activity.
Inhibition of DAP epimerase by azi-DAP isomers 14 and 29
Inhibition studies of DAP epimerase by Higgins et al. using
a mixture of all azi-DAP diastereomers generated in situ
from the corresponding a-(fluoromethyl)diaminopimelic acids
demonstrated that azi-DAP is an irreversible inhibitor. They
were unable to determine Ki or ki values due to the extremely fast
conversion of the enzyme-inhibitor complex to the inactivated
enzyme.12,58 Using the coupled enzyme assay we were able to
confirm that both diastereomers of azi-DAP are irreversible
inhibitors, and show both time and concentration dependence.
Furthermore, the covalent attachment of both isomers to the
enzyme can be demonstrated by electrospray mass spectroscopy
(MS).
The original studies by Higgins et al. using tryptic digests of
inhibited DAP epimerase combined with Edman degradation
of the purified fragments reported that the enzyme is attacked
at Cys-73 by the crude mixture of all diastereomers of azi-
DAP.13 Subsequent studies suggested17 that inhibition of DAP
epimerase with pure LL-azi-DAP 14 should result in selective
attachment to Cys-73, whereas DL-azi-DAP 29 would react with
Cys-217. In order to test this proposal, a trypsin digestion
was done on DAP epimerase inhibited with excess pure azi-
DAP isomers. HPLC purification of the fragments followed by
MS/MS analysis displays a surprisingly low level of sequence
coverage (40% for inhibition with 14 and 32% with 29). In each
case, a fragment incorporating the Cys-73 unit (residues 62–78)
could be sequenced and shown to contain free Cys-73 or Cys-
73 with azi-DAP covalently attached. No fragment containing
Cys-217 could be identified after HPLC of the crude digest.
In order to study the interaction of Cys-217 with azi-DAP,
an alternative digestion protocol was required. As mentioned
above, covalent attachment of an azi-DAP molecule to DAP
epimerase could be observed for both 14 and 29 by MS. Al-
though treatment with cyanogen bromide followed by trypsin
digestion allowed the detection of a suitable fragment (residues
210–230) of the parent (not inhibited) DAP epimerase, it
was found that the reaction of cyanogen bromide resulted
in the cleavage of azi-DAP from the enzyme inactivated by
29. Presumably a mechanism similar to the cleavage of the
peptide chain adjacent to methionine residues occurs under
these conditions. The inactivation of DAP epimerase by azi-DAP
occurs by nucleophilic ring opening of the aziridine to generate
30, which has a sulfide linkage between the enzyme and the
inhibitor.13 Nucleophilic attack of the sulfur on cyanogen bro-
mide followed by intramolecular displacement of the resulting
positively charged sulfonium species 31 from the nearby peptide
Scheme 5 The stereoselective synthesis of LL- and DL-azi-DAP.
Reagents and conditions: (i) LiOH, MeOH–H2O (82%); (ii) PS-PTSA,
MeOH (95%); (iii) 22, DCC, DMAP, CH2Cl2 (62%); (iv) 23, PS-DCC,
DMAP, CH2Cl2 (51%); (v) Pb(OAc)4, HMDS, CH2Cl2 (26 55%, 28 87%);
(vi) Na2CO3, MeOH–H2O (from 28 69%); (vii) Li, NH3 (14 30% over 2
steps, 29 23%).
Intramolecular aziridination of 27 progresses rapidly at 0 ◦C
to give the aziridine 28 with complete diastereoselectivity (>99 :
1 dr, see ESI†). The stereochemical outcome of this reaction can
be determined by comparison of the observed NOE interactions
with energy minimised models of the two possible reaction prod-
ucts (see ESI†). Preparation of the aziridine 26 under analogous
conditions is slightly less selective (98 : 2 dr). Fortunately, the
minor diastereomer could be removed by careful purification
using flash column chromatography to give a >99 : 1 dr.
Hydrolysis of the fully protected azi-DAP 28 using lithium hy-
droxide or sodium hydroxide results in decomposition. However,
sodium carbonate57 in methanol–water is sufficiently mild to
permit quantitative conversion to the corresponding salt of the
diacid (1H NMR). Final deprotection using lithium or sodium
in ammonia at −78 ◦C results in the formation of both the
desired azi-DAP isomer 29 as the major product and the fully
saturated e-methyl-a-aminopimelic acid 18 in a 5.6 : 1 ratio (1H
NMR). The undesired 18 is again formed as a 1 : 1 mixture of
diastereomers at the carbon bearing the methyl. Reductions with
sub-stoichiometric quantities of lithium on carefully purified
(RP-HPLC) carboxylate salts of the diacid intermediate demon-
strate that the fully saturated e-methyl-a-aminopimelic acid 18
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1
4 4 0 5

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bond affords the dealkylated peptide 33, which can subsequently
undergo hydrolysis (Scheme 6).
react with the DAP epimerase. The use of a such a large excess
of inhibitor was initially prompted by the desire to completely
and rapidly inactivate the enzyme for crystallographic studies
prior to its facile aerobic oxidation to form the internal disulfide
between Cys-73 and Cys-217.
Fortunately, preliminary X-ray crystallography studies of
DAP epimerase separately inhibited with the individual azi-
DAP isomers show LL-azi-DAP 14 bound exclusively to Cys-73,
and DL-azi-DAP 29 attached only to Cys-217. In both cases,
inactivation proceeds as predicted by attack of thiolate on the
aziridine methylene. After complete refinement and structural
analysis, these three dimensional structures will be the subject
of a separate report.
In conclusion, we have described the first stereoselective
synthesis of highly sensitive aziridine analogues of DAP 4,
14 and 29. The results show that internal aziridine analogue
4 is a reversible inhibitor of DAP epimerase. The LL-azi-
DAP 14 and DL-azi-DAP 29 rapidly and irreversibly inactivate
DAP epimerase, as can be seen from a coupled enzyme assay
with DAP dehydrogenase as well as by MALDI-TOF MS
analysis. Proteolytic digestion of the enzyme inactivated by
each inhibitor with thermolysin/trypsin followed by MS/MS
provides excellent sequence coverage (89%) and allows analysis
of fragments incorporating both cysteines of interest. MS/MS
analysis of the digest of each inactivated DAP epimerase shows
that the individual azi-DAP isomers bind irreversibly to Cys-73
and Cys-217. The use of excess inhibitor to assist preparation
of samples for X-ray crystallographic analysis results in some
unexpected alkylation of the cysteine residue that is expected to
act as the protonating thiol. This may be due to the presence
of very small quantities (<1.0%; undetectable by NMR) of
the “wrong” isomer of azi-DAP. Preliminary X-ray analysis
of inhibited DAP epimerase crystals showed that LL-azi-DAP
14 binds selectively to Cys-73 and DL-azi-DAP 29 to Cys-
217. The covalent attachment of azi-DAP is consistent with
the proposed mechanism of inhibition i.e. attack of thiolate at
the methylene position of the aziridine ring. Furthermore, the
observed selectivity of azi-DAP inhibition is consistent with the
two base mechanism proposed for the epimerisation of LL-DAP
1 and DL-DAP 2 by DAP epimerase, more specifically that Cys-
73 acts as a thiolate base to deprotonate LL-DAP 1, and Cys-217
in the case of meso-DAP 2.17
Scheme 6 Proposed mechanism for the cleavage of covalently linked
azi-DAP from cysteine residues of DAP epimerase on treatment with
cyanogen bromide.
An analogous process was previously observed during
cyanogen bromide cleavage of a protease inhibited by N-
iodoacetyl peptides.59 Fortunately, inactivation of DAP
epimerase with azi-DAP 29 followed by treatment with ther-
molysin and trypsin provides excellent sequence coverage (89%)
of the resulting fragments by MS/MS. More importantly, frag-
ments incorporating both cysteines of interest could be analysed
using this protocol (Table 1). Analysis of DAP epimerase
inhibited with a large excess (10–100 fold) of azi-DAP 29 reveals
that in addition to the expected alkylation of Cys-217, attack
also occurs at Cys-73 to some extent. In principle, it is possible
that a single diasteroisomer of azi-DAP (i.e. 29) reacts with both
cysteines. However, the excess of inhibitor could possibly be
contaminated by very small amounts (<1.0%) of the other azi-
DAP diastereomer 14, which is not easily detectable and could
Experimental
General methods
All reactions were performed under dry argon. All solvents
were purified and distilled according to Perrin et al.60 Flash
chromatography employed silica gel 60 (Silicycle, 230–420 mesh)
and was performed according to the Still procedure.61 One or
more of the following methods were used for visualisation:
UV fluorescence, iodine staining, phosphomolybdic acid/ceric
sulfate, potassium permangante or ninhydrin. Melting points
were determined on a Bu¨chi apparatus using open-end capillary
tubes and areuncorrected. NMR spectra wererecorded on Inova
Varian 300, 400, 500 and 600 MHz instruments. IR spectra
were determined with a Nicolet Magna 750 FT-IR spectrometer.
Mass spectra (MS) were recorded with a Micromass ZabSpec
Hybrid Sector-TOF instrument (electrospray ionization (ES)).
Optical rotations were measured on a Perkin-Elmer 241 po-
larimeter at 26 ◦C with a micro cell (100 mm; 0.9 mL) or a
standard cell (100 mm, 8 mL) respectively. UV spectroscopy
was performed on a GBC Cintra 40 UV spectrometer. [a]D are
given in units of 10−1 deg cm2 g−1. Microanalyses were completed
at the University of Alberta Microanalytical Laboratory. All
Table 1 MS/MS analysis of trypsin/thermolysin digest of DAP
epimerase inhibited with azi-DAP 29. Italic: undetected residues. Bold:
sequence coverage. Boxed: fragments containing Cys-73 and Cys-217
1
literature compounds had IR, H NMR, 13C NMR and mass
spectra consistent with assigned structures.
Methyl (1S,2R)-N-{(2ꢀE,6ꢀS)-[N,N-di-tert-butoxycarbonyl-6ꢀ-
amino]pentan-2ꢀ-endioate}bornane-10,2-sultam. LiCl (0.22 g,
4 4 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1

View Online
5.2 mmol) was suspended in MeCN (10 mL). A solu-
tion of (1S,2R)-N-(2ꢀ-diethoxyphosphonoacetyl)bornane-10,2-
sultam33 (2.04 g, 5.2 mmol) in MeCN (15 mL) was added along
with DBU (0.79 g, 5.2 mmol). Aldehyde 5 (1.49 g, 4.3 mmol) in
MeCN (20 mL) was then added and the reaction mixture was
allowed to stir for 2 h. The reaction mixture was concentrated in
vacuo. The resulting residue was dissolved in EtOAc (100 mL),
washed with H2O (2 × 70 mL), dried (MgSO4) and concentrated
in vacuo. Purification by flash column chromatography, eluting
with 65 : 35 hexane–EtOAc gave the title compound (2.15 g,
85%) as a white foam. [a]D −65.8 (c 0.5, CHCl3); mmax(CHCl3
cast)/cm−1 2978, 1793, 1746, 1685, 1368, 1331, 1269, 1237, 1220,
1166, 1135; dH (300 MHz, CDCl3) 0.96 and 1.18 (each 3H, 2 × s,
8-H3 and 9-H3), 1.31–1.51 (20H, m, 2 × Ot-Bu and 5-H2), 1.89,
2.01–2.14 and 2.26–2.39 (9H, m, 3-H2, 4-H, 6-H2, 4ꢀ-H2 and 5-
H2), 3.41 (1H, d, J = 13.5 Hz, 10-HH), 3.50 (1H, d, J = 13.5 Hz,
10-HH), 3.71 (3H, s, OMe), 3.91 (1H, t, J = 6.3 Hz, 2-H), 4.86
(1H, dd, J = 9.0, 4.2 Hz, 6ꢀ-H), 6.56 (1H, d, J = 15.1 Hz, 2ꢀ-
H), 7.06 (1H, dt, J = 15.1 Hz, 6.7, 3ꢀ-H); dC (75.5 MHz, CDCl3)
19.9, 20.9, 26.5, 28.0, 28.7, 29.4, 32.9, 38.5, 44.7, 47.8, 48.5, 52.2,
53.2, 57.7, 65.2, 83.4, 121.5, 149.2, 152.0, 164.0, 170.9; m/z (ES)
607.2668 (MNa+), C28H44N2O9NaS requires 607.2665.
Methyl (1S,2R)-N-{(2ꢀE,6ꢀS)-[N-tert-butoxycarbonyl-6ꢀ-amino]-
pentan-2ꢀ-endioate}bornane-10,2-sultam 6. To a solution of
methyl (1S,2R)-N-{(2ꢀE,6ꢀS)-[N,N-di-tert-butoxycarbonyl-6ꢀ-
amino]pentan-2ꢀ-endioate}bornane-10,2-sultam (1.85 g, 3.17
mmol) in CH2Cl2 (20 mL) was added a solution of TFA
(0.47 g, 3.80 mmol) in CH2Cl2 (10 mL). After 4 h, the reaction
mixture was concentrated in vacuo. Purification by flash column
chromatography, eluting with 6 : 4 hexane–EtOAc gave the title
compound 6 (1.44 g, 94%) as a viscous oil. [a]D −41.5 (c 0.4,
CHCl3); mmax(CHCl3 cast)/cm−1 3373, 2960, 1744, 1713, 1367,
1331, 1167, 544; dH (300 MHz, CDCl3) 0.95 and 1.14 (each 3H,
2 × s, 8-H3 and 9-H3), 1.32–1.44 (11H, m, Ot-Bu and 5-H2),
1.71–2.19 and 2.22–2.41 (9H, m, 3-H2, 4-H, 6-H2, 4ꢀ-H2 and
5-H2), 3.41 (1H, d, J = 12.2 Hz, 10-HH), 3.49 (1H, d, J =
12.2 Hz, 10-HH), 3.73 (3H, s, OMe), 3.90 (1H, dd, J = 7.2,
4.5 Hz, 2-H), 4.30 (1H, br q, J = 4.0 Hz, 6ꢀ-H), 5.04 (1H, br d, J
= 7.2 Hz, NH), 6.53 (1H, dt, J = 15.2, 1.6 Hz, 2ꢀ-H), 7.00 (1H,
dt, J = 15.2, 6.8 Hz, 3ꢀ-H); dC (75.5 MHz, CDCl3) 19.9, 20.9,
26.5, 28.3, 28.4, 31.1, 32.9, 38.5, 44.8, 47.8, 48.5, 52.5, 53.1, 53.2,
65.2, 83.3, 121.7, 148.6, 155.3, 163.9, 172.8; m/z (ES) 507.2136
(MNa+), C23H36N2O7NaS requires 507.2141.
and 8-H(Q)), 8.12 (1H, d, J = 8.0 Hz, 5-H(Q)); dC (125 MHz,
CDCl3) 10.7, 19.9, 20.8, 26.3, 27.2, 28.3, 28.8, 32.8, 38.0, 44.6,
46.2, 46.7, 48.8, 48.9, 52.4, 53.1, 53.3, 53.5, 65.6, 79.8, 121.3,
126.2, 126.3, 126.6, 126.9, 133.6, 146.1, 155.5, 159.9, 164.0,
172.9; m/z (ES) 694.3 (MH+); Found: C, 58.75; H, 6.65; N,
10.04. C33H45N5O8S requires C, 59.02; H, 6.71; N, 10.43%.
(2R,3S,3ꢀS)-3-[3ꢀ -Aminopropane)aziridine-2,3ꢀ-dicarboxylate
4. Aziridine 7 (0.283 g, 0.47 mmol) was dissolved in CH2Cl2
(10 mL) and a solution of TFA (0.192 g, 1.88 mmol) in
CH2Cl2 (5 mL) was added. After 15 h, the reaction mixture
was concentrated in vacuo. The resulting residue was washed
with 1 : 1 Et2O–hexane (2 × 20 mL). The resulting white
solid (0.287 g, 0.47 mmol) was filtered, dried (MgSO4) and
dissolved in MeOH (5 mL). A solution of LiOH·H2O (0.053 g,
1.41 mmol) in H2O (2 mL) was added. After 14 h, the reaction
mixture was concentrated in vacuo. The resulting oil was washed
with Et2O (2 × 20 mL) producing a white solid. Purification
using cellulose chromatography, eluting with 7 : 3 MeOH–H2O
gave a white solid (0.132 g, 0.35 mmol). Pre-distilled ammonia
(20 mL) was added to the flask and the reaction mixture was
cooled to −78 ◦C. Lithium (∼0.05 g) was added until a deep
blue colour persisted. After 1 h, the reaction mixture was
quenched by the addition of NH4Cl (∼50 mg). The reaction
mixture was warmed to room temperature and diluted with
H2O (20 mL). The reaction mixture was washed with EtOAc
(2 × 20 mL). The aqueous layer was then concentrated in
vacuo. Purification by flash column chromatography, eluting
with 95 : 5 n-PrOH–NH4OH gave the title compound 4 as a
white solid (32 mg, 40% for three steps). [a]D +4.0 (c 4.5, H2O);
mmax(microscope)/cm−1 2944, 1737, 1617, 1508, 1419, 1215,
1124; dC (300 MHz, D2O) 1.60–1.80 (m, 2H, 1ꢀ-H2), 1.86–2.14
(m, 2H, 2ꢀ-H2), 4.05 (br m, 1H, 3-H), 4.11 (m, 1H, 2-H), 4.27
(m, 1H, 3ꢀ-H); dC (75.5 MHz, D2O) 27.3, 28.7, 71.6, 72.2, 73.8,
172.8; m/z (ES) 211.1 (MNa+).
Dimethyl (2R,3S,3ꢀS) and (2S,3R,3ꢀS)-1-(2-ethyl-3,4-dihydro-
quinazolin-4-one)-3-[3ꢀ-(tert-butoxycarbonylamino) butanoate]-
aziridine-2-carboxylate 9 and 10. A solution of Pb(OAc)4
(0.264 g, 0.62 mmol) in CH2Cl2 (5 mL) was added to a solution
of quina◦zolin-4-one32 12 (0.098 g, 0.52 mmol) in CH2Cl2 (5 mL)
at −25 C. After 30 min, a solution of alkene37 8 (0.187 g,
0.62 mmol) in CH2Cl2 (5 mL) along with HMDS (0.084 g,
0.52 mmol) was added. After 2 h, the reaction mixture was
warmed to room temperature and filtered under gravity. The
organic layer was washed with a saturated solution of NaHCO3
(30 mL), H2O (30 mL), dried (MgSO4) and concentrated in
vacuo. Purification by flash column chromatography, eluting
with 7 : 3 hexane–EtOAc gave an inseparable mixture of the title
compounds 9 and 10 (0.21 g, 83%) as a white foam. mmax(CHCl3
cast)/cm−1 3349, 2977, 1737, 1713, 1677, 1596, 1367, 1202, 1163;
dH (300 MHz, CDCl3) 1.37 (3H, t, J = 10.7 Hz, CH3CH2), 1.40
and 1.43 (9H, 2 × s, 2 × Ot-Bu), 1.75–2.10 (4H, m, 1ꢀ-H2 and
2ꢀ-H2), 2.72 (1H, m, CH3CHH), 3.00 (1H, m, CH3CHH), 3.15
(1H, dd, J = 4.9, 2.5 Hz, 3-H), 3.59 (3H, m, OMe), 3.58–3.76
(4H, m, 2-H and OMe), 4.26 (1H, br q, J = 4.6 Hz, 3ꢀ-H), 5.20
(1H, 2 × br d, J = 6.9 Hz, NH), 7.38 (1H, m, 6-H(Q)), 7.56–7.68
(2H, m, 7-H(Q) and 8-H(Q)), 8.14 (1H, m, J = 8.7 Hz, 5-H(Q));
dC (75.5 MHz, CDCl3) 10.6, 27.0, 27.4, 28.35, 29.0, 47.3, 51.7,
52.5, 52.9, 53.3, 80.9, 121.2, 126.3, 127.0, 133.7, 146.1, 156.2,
160.1, 166.8, 172.9; m/z (ES) 511.2165 (MNa+). C24H32N4O7Na
requires 511.2169.
Methyl (1S,2R,2ꢀR,3ꢀS,3ꢀꢀS)-{1ꢀ-(2-ethyl-3,4-dihydroquinazolin-
4-one)-3ꢀ-[3ꢀꢀ-(N-tert-butoxycarbonylamino)butanoate] aziridine-
2ꢀ-carboxylate} bornane-10,2-sultam 7.
A
solution of
Pb(OAc)4 (0.78 g, 1.75 mmol) in CH2Cl2 (10 mL) was added
to a solution of quinazolin-4-one32 12 (0.29 g, 1.52 mmol)
in CH2Cl2 (10 mL) at −40 ◦C. After 30 min, a solution
of sultam 6 (0.81 g, 1.67 mmol) in CH2Cl2 (15 mL) along
with HMDS (0.246 g, 1.52 mmol) was added. After 4 h, the
reaction mixture was warmed to room temperature and filtered
under gravity. The organic layer was washed with a saturated
solution of NaHCO3 (50 mL), H2O (50 mL), dried (MgSO4)
and concentrated in vacuo. Purification by flash column
chromatography, eluting with 7 : 3 hexane–EtOAc gave a 9 : 1
mixture of the two diastereomeric aziridines. Recrystallisation
from EtOAc–hexane gave the title compound 7 (0.736 g, 72%)
as a white solid. Mp 132–134 ◦C (EtOAc–hexane); [a]D −214.6
(c 0.8, CHCl3); mmax(CHCl3 cast)/cm−1 3320, 2975, 1741, 1713,
1675, 1595, 1520, 1472, 1456, 1367, 1334, 1272, 1240, 1165,
1136, 534; dH (300 MHz, CDCl3) 0.98 and 1.26 (each 3H, 2
× s, 8-H3 and 9-H3), 1.32–1.44 (14H, m, Ot-Bu, CH2CH3 and
5-H2), 1.63–1.99 and 2.05–2.21 (9H, m, 3-H2, 4-H, 6-H2, 1ꢀꢀ-H2
and 2ꢀꢀ-H2), 2.74 (1H, m, CHHCH3), 3.01 (1H, m, CHHCH3),
3.45 (1H, d, J = 13.8 Hz, 10-HH), 3.57 (1H, d, J = 13.8 Hz,
10-HH), 3.70–3.77 (5H, m, OMe, 2-H and 3ꢀ-H), 3.84 (1H, d,
J = 4.8 Hz, 2ꢀ-H), 4.35 (1H, br m, 3ꢀꢀ-H), 5.17 (1H, br d, J =
8.0 Hz, NH), 7.30 (1H, m, 6-H(Q)), 7.58–7.68 (2H, m, 7-H(Q)
Dimethyl (2R,3S,3ꢀS) and (2S,3R,3ꢀS)-1-(2-ethyl-3,4-dihydro-
quinazolin-4-one)-3-[3ꢀ-aminobutanoate]aziridine-2-carboxylate.
A mixture of aziridines 9 and 10 (0.1 g, 0.2 mmol) were dissolved
in CH2Cl2 (5 mL) and a solution of TFA (0.26 g, 2.2 mmol)
in CH2Cl2 (2 mL) was added. After 10 h, the reaction mixture
was concentrated in vacuo. The resulting residue was washed
with 1 : 1 Et2O–hexane (2 × 20 mL). The resulting white solid
was filtered and dried to give an inseparable mixture of the
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1
4 4 0 7

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title compounds (0.105 g, quant.). mmax(CHCl3 cast)/cm−1 2957,
1735, 1672, 1597 1202, 1134; dH(300 MHz, CDCl3) 1.39 (3H,
t, J = 7.3 Hz, CH3CH2), 1.90–2.21 (4H, m, 1ꢀ-H2 and 2ꢀ-H2),
2.91 (1H, m, CH3CHH), 3.03 (1H, m, CH3CHH), 3.27 (1H, t,
J = 4.7 Hz, 3-H), 3.61 (3H, m, OMe), 3.71 (1H, m, 2-H), 3.76
(3H, s, OMe), 4.26 (1H, br q, J = 5.5 Hz, 3ꢀ-H), 7.44 (1H, m,
6-H(Q)), 7.68–7.82 (2H, m, 7-H(Q) and 8-H(Q)), 8.10 (1H, d, J
= 8.3 Hz, 5-H(Q)); dC (75.5 MHz, CDCl3) 10.5, 25.6, 26.6, 31.0,
51.3, 52.7, 53.5, 53.6, 59.5, 122.4, 127.2, 128.7, 136.0, 153.3,
161.8, 166.4, 169.6; m/z (ES) 389.1827. C19H25N4O5 requires
389.1825.
PhCH2), 5.22 (1H, d, J = 7.2 Hz, NH), 5.49 (1H, s, C CHH),
=
=
6.12 (1H, s, C CHH), 7.36 (5H, m, Ph); dC (125 MHz, CDCl3)
24.1, 31.4, 32.3, 51.9, 52.4, 53.7, 67.0, 125.3, 128.0, 128.1, 128.4,
136.2, 139.7, 155.7, 167.3, 172.7; m/z (ES) 372.1421 (MNa+).
C18H23NO6Na requires 372.1423.
Dimethyl (2S,4ꢀS) and (2R,4ꢀS)-1-(2-[(1S)-1-tert-butyldimeth-
ylsilyloxyethyl]-3,4-dihydroquinazolin-4-one)-2-[4ꢀ-(benzyloxy-
carbonylamino)pentanoate]aziridine-2-carboxylate 17. A sol-
ution of Pb(OAc)4 (0.421 g, 0.94 mmol) in CH2Cl2 (20 mL)
was added to a solution of quinazolin-4-one51 19 (0.263 g,
0.82 mmol) in CH2Cl2 (10 mL) at −40 ◦C. After 30 min, a
solution of alkene 16 (0.33 g, 0.94 mmol) in CH2Cl2 (5 mL)
along with HMDS (0.133 g, 0.82 mmol) was added. After 6 h,
the reaction mixture was warmed to room temperature and
filtered under gravity. The organic layer was washed with a
saturated solution of NaHCO3 (60 mL), H2O (60 mL), dried
(MgSO4) and concentrated in vacuo. Purification by flash
column chromatography, eluting with 65 : 35 hexane–EtOAc
gave an inseparable mixture of the title compounds 17 (0.27 g,
49%) as a viscous oil. mmax (CHCl3 cast)/cm−1 3351, 2977, 2952,
1714, 1677, 1597, 1472, 1164; dH (300 MHz, CDCl3) (for major
diastereomer only) 0.05 (3H, s, t-BuSi(CH3)CH3), 0.23 (3H, s,
t-BuSi(CH3)CH3), 0.92 (9H, s, t-Bu), 1.58 (3H, d, J = 6.3 Hz,
CH3CHOTBDMS), 1.62–2.04 (5H, m, 1ꢀ-HH, 2ꢀ-H2, 3ꢀ-H2),
2.31 (1H, m, 1ꢀ-HH), 2.74 (1H, br d, J = 4.8 Hz, 3-HH), 3.20
(1H, br s, 3-HH), 3.49 (3H, s, OMe), 3.71 (3H, s, OMe), 4.36
(1H, q, J = 3.5 Hz, 4ꢀ-H), 5.10 (2H, s, PhCH2), 5.40 (1H, q, J =
6.3 Hz, CH3CHOTBDMS), 5.44 (1H, br d, J = 6.1 Hz, NH),
7.27–7.44 (6H, m, Ph and 6-H(Q)), 7.64–7.72 (2H, m, 7-H(Q)
and 8-H(Q)), 8.09 (1H, br t, J = 8.5 Hz, 5-H(Q)); m/z (ES)
689.2979 (MNa+). C34H46N4O8NaSi requires 689.2983.
(2R,3S,3ꢀS) and (2S,3R,3ꢀS)-1-(2-Ethyl-3,4-dihydroquinzolin-
4-one)-3-[3ꢀ-aminopropane]aziridine-2,3ꢀ-dicarboxylate. A mix-
ture of dimethyl (2R,3S,3ꢀS) and (2S,3R,3ꢀS)-3-[3ꢀ-aminobutan-
4ꢀ-oate]-1-(2-ethyl-3,4-dihydroquinazolin-4-one)
aziridine-2-
carboxylate (0.1 g, 0.2 mmol) were dissolved in MeOH (5 mL)
and a solution of LiOH·H2O (0.026 g, 0.62 mmol) in H2O
(2 mL) was added. After 14 h, the reaction mixture was
concentrated in vacuo. The resulting oil was washed with Et2O
(2 × 20 mL) producing a white solid. Purification using cellulose
chromatography, eluting with 7 : 3 MeOH–H2O gave the title
compound (0.061 g, 84%) as a white solid. mmax(microscope)/
cm−1 2939, 1655, 1610, 1595, 1414, 1206, 558; dH(300 MHz,
D2O) 1.30 (3H, m, CH3CH2), 1.60–2.20 (4H, m, 1ꢀ-H2 and
2ꢀ-H2), 2.91–3.30 (2H, m, CH3CH2), 3.64 (1H, m, 3-H), 3.79
(1H, m, 2-H), 4.10 (1H, br m, 3ꢀ-H), 7.44 (2H, m, 6-H(Q) and
7-H(Q)), 7.80 (1H, m, 8-H(Q)), 8.12 (1H, m, 5-H(Q)); m/z (ES)
373.1674 (MH+). C17H19Li2N4O5 requires 373.1676.
(2R,3S,3ꢀS) and (2S,3R,3ꢀS)-3-[3ꢀ-Aminopropane)aziridine-
2,3ꢀ-dicarboxylate 4 and 11. Pre-distilled ammonia (20 mL)
was added to a flask containing (2R,3S,3ꢀS) and (2S,3R,3ꢀS)-
3-[3ꢀ -aminopropane]-1-(2-ethyl-3,4-dihydroquinazolin-4-one)-
aziridine-2,3ꢀ-dicarboxylate (0.12 g, 0.32 mmol) and the reaction
mixture was cooled to −78 ◦C. Lithium (∼0.05 g) was added
until a deep blue colour persisted. After 1 h, the reaction mixture
was quenched by the addition of solid NH4Cl (∼50 mg). The
reaction mixture was warmed to room temperature and diluted
with H2O (20 mL). The reaction mixture was washed with
EtOAc (2 × 20 mL). The aqueous layer was then concentrated
in vacuo. Purification by flash column chromatography, eluting
with 95 : 5 n-PrOH–NH4OH gave an inseparable mixture of
the title compounds 4 and 11 (29 mg, 48%) as a white solid.
mmax(microscope)/cm−1 2943, 1737, 1617, 1508, 1419, 1215,
1124; dH (300 MHz, D2O) 1.60–1.80 (2H, m, 1ꢀ-H2), 1.86–2.14
(2H, m, 2ꢀ-H2), 4.05 (1H, br m, 3-H), 4.11 (1H, m, 2-H), 4.27
(1H, m, 3ꢀ-H); m/z (ES) 211.1 (MNa+).
(2S )-2-Benzyloxycarbonylamino-6-methylene-heptanedioic
acid. LiOH·H2O (300 mg, 7.2 mmol) was added to a solution
of 16 (0.98 g, 2.8 mmol) in 3 : 1 MeOH–H2O (40 mL) at room
temperature and stirred for 16 h. The mixture was concentrated
in vacuo. The residue was taken up into H2O and was then
extracted with EtOAc. The aqueous layer was then acidified
to pH 2 (1 M HCl) and was then extracted with EtOAc (×4).
The organic fractions were collected, dried (MgSO4) and
concentrated in vacuo to give the title compound (0.77 g, 82%).
[a]D −2.5 (c 1.3, CH3OH); mmax (CH2Cl2 cast)/cm−1 3033, 2957,
1709, 1630, 1530, 1420, 1235, 1062, 738, 698; dH (500 MHz,
CDCl3) 1.47–1.52 (2H, m, 4-H2), 1.77–1.85 (2H, m, 3-HH),
2.31 (2H, br t, J = 7.0 Hz, 5-H2), 4.44 (1H, q, J = 6.0 Hz,
2-H), 5.09 (2H, s, PhCH2), 5.36 (1H, d, J = 8.0 Hz, NH), 5.63
=
=
(1H, s, C CHH), 6.25 (1H, s, C CHH), 7.28–7.34 (5H, m,
Ph); dC (125 MHz, CDCl3)23.7, 31.5, 53.6, 67.1, 128.1, 128.2,
128.5, 136.1, 139.1, 155.8, 172.2, 177.7; m/z (ES) calcd. for
C16H19NO6Na 344.1105 (MNa+), found 344.1105.
Dimethyl (2S)-N-benzyloxycarbonyl-2-amino-6-methylhept-6-
ene-dioate 16. Ester 15 (0.817 g, 2.77 mmol) was dissolved in
degassed THF (20 mL) and cooled to 0 ◦C. NMM (0.28 g,
2.77 mmol) and isobutyl chloroformate (0.378 g, 2.77 mmol)
were both added. After 15 min, a solution of N-hydroxy-
2-thiopyridine (0.423 g, 3.33 mmol) and NEt3 (0.464 mL,
3.33 mmol) in degassed THF (15 mL) was added and argon
was bubbled through the reaction mixture. (N.B. At this stage
the reaction mixture was covered with aluminium foil). After
1 h, a degassed solution of methyl a-thiophenol methylacrylate50
(1.44 g, 6.92 mmol) in THF (4 mL) was added and the reaction
mixture was irradiated with a tungsten lamp (250 W) for 30 min
with cooling at 20 ◦C. The reaction mixture was diluted with
CH2Cl2 (100 mL) and washed with a saturated solution of
NaHCO3 (2 × 50 mL), brine (100 mL), dried (MgSO4) and
concentrated in vacuo. Purification by flash column chromatog-
raphy, eluting with 7 : 3 hexane–EtOAc gave the title compound
16 (0.66 g, 68%) as a viscous oil. [a]D +8.6 (c 0.7, CHCl3);
mmax(CHCl3 cast)/cm−1 3348, 2952, 1719, 1630, 1439, 1209, 1057;
dH (300 MHz, CDCl3) 1.42–1.52 (2H, m, 4-H2), 1.64 (1H, m, 3-
HH), 1.83 (1H, m, 3-HH), 2.29 (2H, br t, J = 7.8 Hz, 5-H2), 3.71
(6H, s, 2 × OMe), 4.38 (1H, q, J = 6.0 Hz, 2-H), 5.08 (2H, s,
(2S )-2-Benzyloxycarbonylamino-6-methylene-heptanedioic
acid 1-methyl ester 24. Polymer bound p-toluenesulfonic acid
(30–60 mesh, 2.0–3.5 mmol g−1, 700 mg) was added to a solution
of (2S)-2-benzyloxycarbonylamino-6-methylene-heptanedioic
acid (0.77 g, 2.4 mmol) in anhydrous MeOH (20 mL) at room
temperature and shaken for 16 h. The solids were removed by
filtration and washed extensively with MeOH. The resulting
solution was concentrated in vacuo to give a 95 : 5 mixture
of the title compound 24 and diester 16 (0.80 g, quant.) as a
colourless oil. [a]D +13.3 (c 0.5, CHCl3); mmax(CH2Cl2 cast)/cm−1
3323, 2953, 1705, 1527, 1455, 1437, 1346, 1216, 1060, 754;
dH(500 MHz, CDCl3) 1.48–1.57 (2H, m, 4-CH2), 1.63–1.68
(1H, m, 3-CHH), 1.82–1.85 (1H, m, 3-CHH), 2.28–2.32 (2H,
m, 5-H2), 3.72 (3H, s, OCH3), 4.39 (1H, q, J = 5.5 Hz, CHN),
5.09 (2H, s, PhCH2), 5.27 (1H, d, J = 7.5 Hz, NH), 5.62 (1H, s,
=
=
C CHH), 6.27 (1H, s, C CHH), 7.28–7.34 (5H, m, Ph); dC
(125 MHz, CDCl3) 24.0, 30.9, 32.1, 52.4, 53.6, 67.0, 127.4,
128.1, 128.2, 128.5, 136.2, 139.2, 155.9, 171.7, 172.9; m/z (ES)
calcd. for C17H21NO6Na 358.1263 (MNa+), found 358.1261.
4 4 0 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1

View Online
H2O); mmax(H2O cast)/cm−1 3205, 3057, 1628, 1415, 1114; dH
(400 MHz, D2O) 1.09–1.15 (1H, m, CHCH2CHH), 1.47–1.57
(2H, m, CHCH2CHH, CHCHH) 1.61 (1H, bs, CHHN),
1.80–1.90 (2H, m, CHCH2), 1.92 (1H, bs, CHHN), 2.15–2.21
(1H, m, CCHH), 3.65–3.71 (1H, m, CHN); dC (100 MHz, D2O)
21.8, 31.6, 32.3, 55.0, 62.8, 175.8, 179.8; m/z (ES) calcd. for
C8H13N2O4 201.0870 (M+), found 201.0869.
(2S )-2-Benzyloxycarbonylamino-6-methylene-heptanedioic
acid 7-{(2R)-2-[1-(3-amino-4-oxo-3,4-dihydroquinazolin-2-yl)-
ethoxy]-ethyl} ester 1-methyl ester 25. A solution of DCC
(25 mg, 0.12 mmol) and DMAP (2 mg, 160 lmol) in CH2Cl2
(2 mL) was added to a solution of 22 (27 mg, 100 lmol) and
monoester 24 (36 mg, 100 lmol) in CH2Cl2 (2 mL) at room
temperature and was stirred for 1 h. The resulting precipitate
was removed by filtration and the mixture was concentrated in
vacuo. Purification by flash chromatography, eluting with 2 : 1
CHCl3–EtOAc gave the title compound 25 (40 mg, 62%) as a
colourless oil. [a]D +7.8 (c 1.6, CH2Cl2); mmax(CH2Cl2 cast)/cm−1
3326, 2933, 1717, 1676, 1599, 1526, 1454, 1299, 1248, 1213,
1179, 1109, 1058, 775, 697; dH(500 MHz, CDCl3) 1.49–1.56
(2H, m, 4-CH2), 1.62–1.67 (1H, m, 3-CHH), 1.69 (3H, d, J
= 6.5 Hz, CH3CH), 1.81–1.87 (1H, m, 3-CHH), 2.26–2.31
(2H, m, 5-H2), 3.72 (3H, s, OCH3), 3.83 (2H, t, J = 4.5 Hz,
CO2CH2CH2), 4.31–4.34 (2H, m, CO2CH2CH2), 4.34–4.40
(1H, m, CHN), 5.05 (1H, q, J = 6.5 Hz, CH3CH), 5.10 (2H, s,
PhCH2), 5.42 (1H, d, J = 8.5 Hz, NH), 5.49 (2H, s, NH2),
(2S )-2-Benzyloxycarbonylamino-6-methylene-heptanedioic
acid 7-{(2S)-2-[1-(3-amino-4-oxo-3,4-dihydroquinazolin-2-yl)-
ethoxy]-ethyl} ester 1-methyl ester 27. Polymer bound N-
benzyl-Nꢀ-cyclohexylcarbodiimide (1.3 mmol g−1, 7.5 g) and
DMAP (70 mg, 0.57 mmol) were added to a solution of 23
(760 mg, 3.0 mmol) and monoester 24 (678 mg, 2.0 mmol) in
CH2Cl2 (60 mL) at room temperature and was shaken for 48 h.
The resin was removed by filtration was washed extensively
with CH2Cl2. The solvent was removed in vacuo and the residue
was purified by passage through a plug of silica, eluting with
1 : 1 EtOAc–hexane to give the title compound 27 (588 mg,
51%) as a colourless oil. [a]D −2.3 (c 4.7, CH2Cl2); mmax (CH2Cl2
cast)/cm−1 3324, 2927, 1716, 1674, 1623, 1599, 1530, 1246, 1213,
1180, 1109, 1058, 775, 736, 697; dH(500 MHz, CDCl3)1.46–1.54
(2H, m, 4-CH2), 1.62–1.67 (1H, m, 3-CHH), 1.68 (3H, d,
J = 7.0 Hz, CH3CH), 1.80–1.82 (1H, m, 3-CHH), 2.26–2.31
(2H, m, 5-H2), 3.72 (3H, s, OCH3), 3.82 (2H, t, J = 4.5 Hz,
CO2CH2CH2), 4.30–4.34 (2H, m, CO2CH2CH2), 4.35–4.39
(1H, m, CHN), 5.06 (1H, q, J = 6.5 Hz, CH3CH), 5.09 (2H, s,
=
=
5.54 (1H, s, C CHH), 6.11 (1H, s, C CHH), 7.28–7.34 (5H,
m, Ph), 7.47–7.50 (1H, m, ArCH), 7.74–7.75 (2H, m, ArCH),
8.26–8.28 (1H, m, ArCH); dC (125 MHz, CDCl3) 16.8, 24.1,
29.8, 31.4, 32.2, 52.4, 53.8, 63.9, 67.1, 75.9, 120.3, 125.7, 126.5,
127.1, 127.9, 128.2, 128.6, 134.1, 136.3, 139.7, 146.4, 153.9,
156.0, 160.6, 166.8, 172.9; m/z (ES) calcd. for C29H35N4O8
567.2449 (M+), found 567.2446.
(2S)-2-Benzyloxycarbonylamino-5-[(1R,7R)-7-methyl-2,11-
dioxo-4,5,7,15-tetrahydro-3,6-dioxa-8,15a,15b-triaza-naphthyl
[a]cyclopropa[c]cyclodecen-1a-yl]pentanoic acid methyl ester 26.
Pb(OAc)4 (147 mg, 0.33 mmol) was added to a solution of the
alkene 25 (183 mg, 0.32 mmol) and HMDS (110 lL, 0.52 mmol)
in CH2Cl2 (10 mL) at 0 ◦C. The mixture was then stirred for
15 min. The resulting mixture was purified by passage through
a plug of silica, eluting with 1 : 1 EtOAc–CH2Cl2 to give the
=
PhCH2), 5.42 (3H, bs, NH, NH2), 5.53 (1H, s, C CHH), 6.10
(1H, s, C CHH), 7.28–7.34 (5H, m, Ph), 7.45–7.48 (1H, m,
=
ArCH), 7.73–7.74 (2H, m, ArCH), 8.24–8.26 (1H, m, ArCH);
dC (125 MHz, CDCl3) 16.8, 24.0, 31.3, 32.1, 33.9, 53.87, 63.8,
67.0, 75.8, 120.2, 125.6, 126.4, 127.0, 127.8, 128.1, 128.5, 134.1,
136.3, 139.6, 146.2, 153.9, 160.5, 166.7, 172.8; m/z (ES) calcd.
for C29H35N4O8 567.2449 (M+), found 567.2449.
title compound 26 (157 mg, 86%) as a white foam. [a]D
−
(2S)-2-Benzyloxycarbonylamino-5-[(1R,7S)-7-methyl-2,11-
dioxo-4,5,7,15-tetrahydro-3,6-dioxa-8,15a,15b-triaza-naphthyl-
[a]cyclopropa[c]cyclodecen-1a-yl]pentanoic acid methyl ester 28.
Pb(OAc)4 (442 mg, 1.0 mmol) was added to a solution of alkene
27 (545 mg, 0.96 mmol) and HMDS (0.35 mL, 1.7 mmol) in
CH2Cl2 (25 mL) at 0 ◦C. The mixture was then stirred for
15 min. The resulting mixture was then purified by passage
through a plug of silica, eluting with 1:1 EtOAc/CH2Cl2 to give
28.6 (c 1.6, CH2Cl2); mmax(CH2Cl2 cast)/cm−1 3336, 2951, 1738,
1682, 1592, 1526, 1273, 1217, 1172, 736, 697; dH (500 MHz,
CD2Cl2) 1.49 (3H, d, J = 6.0 Hz, CH3CH), 1.55–1.56 (2H,
m, 4-CH2), 1.74–1.77 (1H, m, 3-CHH), 1.91–1.92 (1H, m,
3-CHH), 2.64–2.67 (2H, m, 5-H2), 3.13 (1H, bs, CHHN), 3.29
(1H, bs, CHHN), 3.65 (1H, m, CO2CH2CHH), 3.74 (3H, s,
OCH3), 3.92–3.97 (1H, m, CO2CH2CHH), 4.04–4.10 (1H, m,
CO2CHHCH2), 4.38–4.41 (1H, m, CHN), 4.82–4.86 (1H, m,
CO2CHHCH2), 5.10 (2H, s, PhCH2), 5.14 (1H, q, J = 6.0 Hz,
CH3CH), 5.38 (1H, d, J = 8.0 Hz, NH), 7.30–7.37 (5H, m, Ph),
7.45–7.48 (1H, m, ArCH), 7.65–7.67 (1H, m, ArCH), 7.71–7.74
(1H, m, ArCH), 8.17–8.19 (1H, m, ArCH); dC (125 MHz,
CD2Cl2) 21.3, 32.2, 33.0, 45.9, 52.7, 56.1, 64.4, 67.3, 68.2, 72.1,
122.0, 126.7, 127.3, 127.6, 128.3, 128.5, 128.9, 134.3, 146.0,
155.4, 160.0, 166.8, 173.0; m/z (ES) calcd. for C29H35N4O8
565.2293 (M+), found 565.2299.
the title compound 28 (300 mg, 55%) as a white foam. [a]D
+
191.2 (c 3.8, CH2Cl2); mmax (CH2Cl2 cast)/cm−1 3330, 2953, 1738,
1682, 1592, 1526, 1273, 1216, 1171, 736, 697; dH (500 MHz,
CD2Cl2) 1.49 (3H, d, J = 6.0 Hz, CH3CH), 1.55–1.60 (2H,
m, 4-CH2), 1.74–1.76 (1H, m, 3-CHH), 1.93–1.95 (1H, m,
3-CHH), 2.67–2.72 (2H, m, 5-H2), 3.13 (1H, d, J = 3.5 Hz,
CHHN), 3.29 (1H, dd, J = 3.5, 1.0 Hz, CHHN), 3.63–3.67
(1H, m, CO2CH2CHH), 3.74 (3H, s, OCH3), 3.91–3.95 (1H, m,
CO2CH2CHH), 4.10–4.14 (1H, m, CO2CHHCH2), 4.39–4.40
(1H, m, CHN), 4.84–4.87 (1H, m, CO2CHHCH2), 5.10 (2H, s,
PhCH2), 5.16 (1H, q, J = 6.5 Hz, CH3CH), 5.42 (1H, d, J =
8.0 Hz, NH), 7.30–7.39 (5H, m, Ph), 7.45–7.48 (1H, m, ArCH),
7.65–7.67 (1H, m, ArCH), 7.71–7.74 (1H, m, ArCH), 8.17–8.19
(1H, m, ArCH); dC (125 MHz, CD2Cl2) 21.3, 32.3, 32.8, 46.0,
52.6, 56.1, 64.4, 67.3, 68.2, 72.1, 122.0, 126.7, 127.3, 127.6,
128.3, 128.5, 128.9, 134.3, 146.0, 155.4, 160.0, 166.8, 173.0; m/z
(ES) calcd. for C29H35N4O8 565.2293 (M+), found 565.2292.
(2S)-2-[(4S)-4-Amino-4-carboxybutyl]aziridine-2-carboxylic
acid (LL-azi-DAP) 14.
A solution of Na2CO3 (55 mg,
0.56 mmol) in H2O (3 mL) was added to a solution of aziridine
26 (72 mg, 0.13 mmol) in MeOH (9 mL) at room temperature.
The mixture was then stirred for 48 h and then concentrated in
vacuo. Pre-distilled ammonia (10 mL) was added to a flask and
the reaction mixture was cooled to −78 ◦C. Lithium was added
portionwise until a deep blue colour persisted. After 5 min the
reaction mixture was quenched by the addition of solid NH4Cl.
The reaction mixture was warmed to room temperature and
the ammonia was allowed to evaporate under an atmosphere
of argon. Purification by preparative TLC (UniplateTM silica
gel HLF plates with organic binder, 5% aq NH4OH in MeOH,
products removed from silica using 20% aq NH4OH in MeOH)
was followed by concentration in vacuo. The residue was
dissolved in water (1 mL) and filtered through a 0.45 lm filter
to remove any remaining silica. Concentration in vacuo gave the
title compound 14 (8 mg, 30%) as a white solid. [a]D +3.3 (c 0.40,
(2R)-2-[(4S)-4-Benzyloxycarbonylamino-4-carboxybutyl]-
1-{2-[(1S)-1-(2-hydroxy-ethoxy)-ethyl]-4-oxo-4H-quinazolin-
3-yl}-aziridine-2-carboxylic acid.
A solution of Na2CO3
(108 mg, 1.1 mmol) in H2O (4 mL) was added to a solution
of aziridine 28 (140 mg, 0.25 mmol) in MeOH (12 mL) at
room temperature. The mixture was then stirred for 48 h
and then concentrated in vacuo. The residue was taken up
into H2O (50 mL) and purified by RP-HPLC [Gracevydac
reverse phase polymer column (259VHP810), eluting with 9:1
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 4 0 2 – 4 4 1 1
4 4 0 9

View Online
i-PrOH/50 mM NH4OH, detection at 220 nm, 450 lL injection
volume of 2 mg mL−1 solution, flow rate 1 mL min−1, tR 7.2 min,
(see supporting information)] to give the title compound
(97 mg, 69%) as a white solid. [a]D + 124.0 (c 2.1, MeOH); dH
(500 MHz, CD3OD) 1.44–1.48 (2H, m, 4-CH2), 1.55 (3H, d,
J = 6.0 Hz, CH3CH), 1.60–1.64 (1H, m, 3-CHH), 1.72–1.74
(1H, m, 3-CHH), 1.89–1.93 (1H, m, 5-CHH), 2.56–2.61 (1H,
m, 5-H2), 2.81 (1H, d, J = 2.0 Hz, CHHN), 3.63 (1H, bs,
CHHN), 3.69–3.81 (4H, m, OCH2CH2OH), 4.06–4.08 (1H, m,
CHN), 5.06 (2H, s, PhCH2), 5.16 (1H, q, J = 6.5 Hz, CH3CH),
7.30–7.36 (5H, m, Ph), 7.45–7.51 (1H, m, ArCH), 7.63–7.64
(1H, m, ArCH), 7.70–7.73 (1H, m, ArCH), 8.11–8.13 (1H, m,
ArCH); dC (125 MHz, CD3OD) 19.7, 22.9, 33.9, 34.4, 48.5,
62.8, 67.4, 68.2, 71.6, 74.2, 122.8, 127.2, 127.4, 128.8, 128.9,
129.4, 134.8, 138.5, 147.0, 158.1, 159.9, 161.6, 172.2 179.3;
m/z (ES) calcd. for C28H32N4O9Na 591.2062 (MNa+), found
591.2068.
20.9, 30.9, 55.2, 175.4; m/z (ES) calcd. for C7H15N2O4 191.1026
(M+), found 191.1026.
Inhibition Studies with DAP Epimerase Using Coupled Assay
DAP epimerase activity was monitored using a coupled assay
with DAP dehydrogenase which detects the production of
NADPH at 340 nm. The assay was performed in a 1 mL
quartz cuvette filled with buffer solution (0.1 M tris-HCl, 1 mM
EDTA, 1 mM DTT, pH 7.8), 0.4 mM L,L-DAP, 0.3 mM
NADP+ and 0.06 units of DAP dehydrogenase. One unit of DAP
epimerase activity corresponds to the production of one lmol
of NADPH per minute. For inhibition studies with aziridine
analogue 4, the assay buffer contained varying concentrations of
the aziridine, 0.4 mM L,L-DAP, 0.3 mM NADP+, 50 mU of DAP
dehydrogenase and 15 mU of DAP epimerase. These results were
compared against a control performed simultaneously, where the
Tris-HCl buffer replaced the inhibitor. The DAP epimerase used
in the study of aziridines 4 and 11 was isolated from Escherichia
coli mutant BL21(DE3) pLysS using a modified procedure7,39
and DAP dehydrogenase was purified from Bacillus sphaericus
IFO 3525 as previously reported.62 The DAP epimerase used in
the study of azi-DAP was isolated from Haemophilus influenza
as described previously.15
(2R)-2-[(4S)-4-amino-4-carboxybutyl]aziridine-2-carboxylic
acid (DL-azi-DAP) 29 and (2S)-2-amino-6-methyl-heptanedioic
acid 18. Pre-distilled ammonia (10 mL) was added to a
flask containing (2R)-2-[(4S)-4-benzyloxycarbonylamino-4-
carboxybutyl]-1-{2-[(1S)-1-(2-hydroxy-ethoxy)-ethyl]-4-oxo-4H-
quinazolin-3-yl}-aziridine-2-carboxylic acid (25 mg, 44 lmol)
and the reaction mixture was cooled to −78 ◦C. Lithium was
added portionwise until a deep blue colour persisted. After
5 min the reaction mixture was quenched by the addition of
solid NH4Cl (10 mg). The reaction mixture was warmed to
room temperature and the ammonia was allowed to evaporate
under a stream of argon. Purification by prep TLC (UniplateTM
silica gel HLF plates with organic binder, 5% aq NH4OH in
MeOH, products removed from silica using 20% aq NH4OH in
MeOH) was followed by concentration in vacuo. The residue
was dissolved in water (1 mL) and filtered through a 0.45 lm
filter to remove any remaining silica. Concentration in vacuo
gave the title compounds 29 (2.5 mg, 28%) and 18 (∼0.5 mg)
as white solids. aziridine 29: Rf 0.52; [a]D − 11.3 (c 0.35, H2O);
mmax(H2O cast)/cm−1 3401, 2940, 1628, 1411, 1348, 1197, 1147,
1122; dH (500 MHz, D2O) 1.24–1.29 (1H, m, CHCH2CHH),
1.53–1.58 (1H, m, CHCH2CHH). 1.81–1.90 (2H, m, CHCH2),
1.92 (1H, bs, CHHN), 2.19 (1H, bs, CHHN), 2.20–2.22 (1H, m,
CCHH), 3.71–3.73 (1H, m, CHN); dC (125 MHz, D2O) 21.8,
30.5, 31.7, 31.9, 54.9, 62.7, 175.9, 177.7; m/z (ES) calcd. for
C8H13N2O4 201.0870 (M+), found 201.0870.
Labeling of DAP Epimerase with azi-DAP
A solution of azi-DAP in water (1–5 mg mL−1) was added in
excess (10–100 equiv) to a solution of DAP epimerase (0.54 mg
mL−1) in buffered solution (0.1 M tris-HCl, 1 mM EDTA, 1 mM
DTT, pH 7.8) at room temperature and allowed to stand for
16 h. An aliquot (50 lL) was removed and added to a 10 mM
solution of DTT in ammonium bicarbonate (50 lL, pH 8) and
the mixture was heated at 37 ◦C for 30 min. An aliquot was
removed and purified by RP-HPLC using a JupiterTM reverse
˚
phase 1 mm Microbore column (5 l, C18, 300 A), eluting with a
linear gradient over 50 min from 0.05% aqueous TFA to 0.05%
TFA in MeCN, column temperature 70 ◦C, detection at 210 nm,
injection volume 50 lL, flow rate 100 lL min−1, tR 29 min.
Acknowledgements
Financial support was provided by the Natural Sciences and
Engineering Research Council of Canada, the Canada Research
Chair in Bioorganic and Medicinal Chemistry, Merck Frosst
Canada and the Alberta Heritage Foundation for Medical
Research (postdoctoral fellowship for A. S.).
Acid 18 as
a mixture of diastereomers: Rf 0.61;
mmax(microscope)/cm−1 3050, 2940, 1693, 1583, 1513, 1441, 1408;
dH (300 MHz, D2O) 1.10 (3H, d, J = 11.5 Hz, CH3), 1.32–1.51
(3H, m, CHH, CH2), 1.58–1.65 (1H, m, CHH), 1.81–1.92 (2H,
m, CH2), 2.38–2.46 (1H, m, CHCH3), 3.73 (1H, t, J = 6.5 Hz,
CHN), dC (100 MHz, D2O) 17.8 & 17.9, 23.2 & 23.3, 31.2 & 31.2,
33.9, 41.6 & 41.7, 55.5 & 55.6, 175.7, 185.1; m/z (ES) calcd. for
C8H14NO4 188.0917 (M+), found 188.0918.
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A
solution of (2S)-4-[(4S)-4-benzyloxycarbonylamino-4-
methoxycarbonyl-butyrylperoxy)-2-tert-butoxycarbonylamino-
4-oxo-butyric acid tert-butyl ester40 (92 mg, 0.19 mmol) in
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View Online
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