Expeditious synthesis of enantiopure, orthogonally protected bis-α-amino acids (OPBAAs) and their use in a study of Nod1 stimulation

Article abstract of DOI:10.1002/asia.201403173

A convenient approach towards the synthesis of orthogonally protected chiral bis-a-amino acids (OPBAAs) is described. The key transformations include: (1) a highly stereoselective conjugation (alkylation) of the Sch?llkopf bislactim ethers and oxazolidinyl alkyl halides to build a backbone skeleton; and (2) our orthogonal protection strategy. A series of enantiopure OPBAAs bearing a variety of alkyl chain as a spacer; two stereogenic centers; and three protecting groups were prepared as examples. These versatile molecules were applied to the synthesis of biologically interesting di- or tri-peptide analogues, including chiral iE-meso-DAP and A-iE-meso-DAP, for the study of Nod1 activation in the innate immune response.

Full text of DOI:10.1002/asia.201403173

DOI: 10.1002/asia.201403173
Full Paper
Peptides
Expeditious Synthesis of Enantiopure, Orthogonally Protected Bis-
a-Amino Acids (OPBAAs) and their Use in a Study of Nod1
Stimulation
Po-Ting Chen,[a] Cheng-Kun Lin,[a] Chih-Ju Tsai,[a] Duen-Yi Huang,[b] Fu-Yao Nien,[c] Wan-
Wan Lin,[b, d] and Wei-Chieh Cheng*[a, c]
Abstract: A convenient approach towards the synthesis of
orthogonally protected chiral bis-a-amino acids (OPBAAs) is
described. The key transformations include: (1) a highly ste-
reoselective conjugation (alkylation) of the Schçllkopf bis-
lactim ethers and oxazolidinyl alkyl halides to build a back-
bone skeleton; and (2) our orthogonal protection strategy. A
series of enantiopure OPBAAs bearing a variety of alkyl
chain as a spacer; two stereogenic centers; and three pro-
tecting groups were prepared as examples. These versatile
molecules were applied to the synthesis of biologically inter-
esting di- or tri-peptide analogues, including chiral iE-meso-
DAP and A-iE-meso-DAP, for the study of Nod1 activation in
the innate immune response.
Introduction
hibits 1000 times more potency than SK&F 107647 for induc-
tion of colony-stimulating activity.[3]
Many natural or synthetic molecules containing a bis-a-amino
acid (BAA) moiety exhibit an increased structural stability com-
pared to a disulfide-linked bis-a-amino acid[1] and show inter-
esting biological activities. For example, the dimer of HP5b
(Figure 1),[2] isolated from natural human leukocytes, exhibits
hematopoietic inhibition and stimulates hematopoiesis,[3] al-
though the instability of the disulfide linkage limits its biologi-
Bacterial cell wall peptidoglycan (PGN) consists of linear
polysaccharide chains with alternating N-acetylglucosamine
(GlcNAc) and N-acetylmuramic acid (MurNAc) moieties, which
are cross-linked by short peptide chains to support bacterial
cell shapes and growth.[5] In the PGN of mycobacteria, gram-
negative bacteria, and some gram-positive bacteria, a type of
BAA called (2S, 6R)-2,6-diaminopimelic acid (meso-DAP) is re-
sponsible for peptide cross-linking, the final stage of cell wall
biosynthesis catalyzed by transpeptidases.[6] Interestingly, PGN
fragments containing chiral meso-DAP such as g-d-Glu-meso-
DAP (iE-meso-DAP) or l-Ala-g-d-Glu-meso-DAP (A-iE-meso-DAP)
(Figure 1) have been found to stimulate innate immune re-
sponses through recognition with the human intracellular nu-
cleotide-binding oligomerization domain-containing protein
1 (Nod1), to induce the activation of the NF-kB pathway and
the production of inflammatory cytokines.[7] In addition, a BAA-
containing peptide, heptanoyl-g-d-Glu-meso-DAP-d-Ala (FK-
565), is a synthetic immunostimulant which enhances host de-
fense against microbial infection, and shows antiviral activi-
ties.[8] Chiral BAAs are therefore important building blocks, but
their direct purification from mixtures of stereomers or clean
isolation from natural sources is extremely difficult.[9] Thus, syn-
thetic chemistry approaches are the only means by which pure
samples can be obtained for biological study.
cal application. Fortunately,
a molecule surrogate, SK&F
107647, containing a 2,7-diaminosuberic acid (DAS) moiety as
a spacer, has a greatly improved molecular stability and biolog-
ical activity (Figure 1).[4] Furthermore, its analog bearing the
shorter spacer 2,5-diaminoadipic acid (DAA) instead of DAS ex-
[a] P.-T. Chen,+ C.-K. Lin,+ C.-J. Tsai, Prof. Dr. W.-C. Cheng
The Genomics Research Center
Academia Sinica
No. 128, Academia Road Sec. 2, Nankang District, Taipei, 11529 (Taiwan)
Fax: (+886)2-27899931
E-mail: wcheng@gate.sinica.edu.tw
[b] Dr. D.-Y. Huang, Dr. W.-W. Lin
Department of Pharmacology, College of Medicine
National Taiwan University
No. 1, Jen-Ai Road, Section 1, Taipei, 10051 (Taiwan)
[c] F.-Y. Nien, Prof. Dr. W.-C. Cheng
Department of Chemistry
National Cheng Kung University
No. 1, University Road, Tainan City, 701 (Taiwan)
Structurally, chiral OPBAAs consist of 1) two stereocenters;
2) three protecting groups (one for a carboxyl group and two
for amino groups); and 3) a varied and flexible alkyl spacer be-
tween two a-amino acid moieties (Figure 1). To date, several
methods toward the preparation of BAAs have been report-
ed,[10] for example, using Grubbs’ cross metathesis to combine
two a-amino acids, but several problems exist among some of
[d] Dr. W.-W. Lin
Graduate Institute of Medical Sciences
Taipei Medical University
No. 250, Wuxing Street, Taipei, 11031 (Taiwan)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.201403173.
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orthogonal protection strategy is
as follows: one carboxyl group
will be protected as an ethyl
ester, which can be easily depro-
tected under basic hydrolysis;
the neighboring amino group
will be protected by the benzy-
loxycarbonyl (Cbz) group, which
can be easily removed via cata-
lytic hydrogenation; and the
other amino group will be pro-
tected by the acid-labile tert-bu-
toxycarbonyl (Boc) group. Nota-
bly, there is one free carboxylic
acid, which can directly undergo
further chemical transformation.
Results and Discussion
The synthesis of orthogonally
protected meso-DAP (D, n=3,
Scheme 1) was first pursued, as
our model study to examine the
feasible conditions of transfor-
mations. Following Chen’s proto-
col with slight modifications,[14]
Figure 1. Bioactive and natural molecules containing bis-a-amino acids and the general structure of OPBAAs.
Schçllkopf bis-lactim ether
A
these synthetic routes such as length of the alkyl spacer, lack
of an orthogonal protecting group strategy, lack of stereoselec-
tivity, and contamination by homo-coupling side-products.
Herein, we report an asymmetric synthetic method and an or-
thogonal protection strategy for the preparation of various
enantiopure OPBAAs bearing a variety of alkyl chains between
two stereocenters. These versatile OPBAAs will be utilized as
building blocks in the synthesis of biologically interesting di-
or tri-peptide analogues for the study of human Nod1 activa-
tion in the innate immune response through the NF-kB path-
way.
(3S)-3,6-dihydro-2,5-diethoxy-3-isopropyl-pyrazine (1) was pre-
pared from l-valine in 60% overall yield over four steps. Like-
wise, the enantiomer, (3R)-3,6-dihydro-2,5-diethoxy-3-isopropyl-
pyrazine (2), was also prepared from d-valine.
As illustrated in Scheme 2, l-glutamic acid was converted
into the protected glutamic acid 3 in 99% yield over two steps
via esterification in the presence of chlorotrimethylsilane
(TMSCl) in methanol and N-Boc protection under basic condi-
tions.[15] After the reduction with lithium aluminum hydride
and N,O-ketalization with 2,2-dimethoxypropane of compound
3, a halogenation of the corresponding primary alcohol was
performed under Appel’s conditions[16] to give oxazolidinyl
alkyl bromide 4 in a good yield (66%). Similarly, 5 and 6 were
also prepared from the corresponding l-aspartic acid and d-
glutamic acid, respectively.
Scheme 1 depicts our synthetic strategy. We propose gener-
ation of one desired stereocenter via the chiral Schçllkopf bis-
lactim ether method,[11] wherein two enantiopure templates A
are easily prepared from l- or d-valine.[12] The other stereocen-
ter (in the chiral oxazolidinyl alkyl halides B) can be directly
prepared from the available a-amino acid chiral pools.[13] Our
Next, the diastereoselective alkylation of lithiated 1 with 4
(Scheme 3) was carried out to generate the adduct 7 (65%).
The stereochemistry of the
newly created stereocenter (C2)
of
7 was confirmed by 2D-
NOSEY NMR experiments.[17] Se-
lective hydrolysis of 7 under vari-
ous acidic conditions was inves-
tigated, and the optimized con-
ditions were found to be treat-
ment with 0.5n HCl in THF at
08C for 2 h, which gave a single
product, followed by N-Cbz pro-
tection to give 9 in 70% yield
over two steps. Harsher condi-
Scheme 1. Strategy for the general synthesis of OPBAAs.
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Scheme 2. Synthesis of oxazolidinyl alkyl halides 4–6.
Scheme 4. Preparation of oxazolidinyl alkyl bromides 14–16.
Table 1. Correlations between substrates A/B, intermediates C and prod-
ucts D.
Entry
A/B
C (adduct)
Yield (%)[a]
D (OPBAA)
Yield (%)[b]
1
2
3
4
5
6
7
8
1/5
1/4
1/6
2/4
17
7
55
65
62
71
61
60
58
60
24
11
29
34
40
36
–
42
37
45
45
21
22
23
18
19
20
[c]
–
2/6
28
25
26
27
1/16
1/15
1/14
Scheme 3. Synthesis of orthogonally protected meso-DAP 11.
[a] Two steps (coupling and hydrogenation) for entries 6–8. [b] Calculated
yields from C (adduct). [c] Not prepared.
tions resulted in complete deprotection to give 8 and/or poor
selectivity.
Selective deprotection of the oxazolidine ring in 8 was per-
formed over 36 h in the presence of PTSA at room tempera-
ture to generate alcohol 10 (80%) without affecting the N-Boc
moiety.[18] TEMPO oxidation gave acid 11 (72%), our first
OPBAA example. This model study and feasible reaction condi-
tions encouraged us to apply this strategy to prepare various
interesting OPBAAs.
In order to vary the spacer in the oxazolidinyl alkyl halide
(Scheme 4), alcohols 12 (n=2) and 13 (n=1) prepared from l-
glutamic acid and l-aspartic acid, respectively, were oxidized
under Swern conditions and then condensed with various tri-
phenyl phosphonium ylides in a Wittig reaction, followed by
bromination to generate the corresponding oxazolidinyl alkyl
bromides 14–16 with high yields (ca. 90%).
Figure 2. Structures of all synthetic adducts C in this study.
With two chiral templates 1 and 2 and six oxazolidinyl alkyl
bromides 4–6 and 14–16 in hand, a series of chiral adducts C
(eight examples) and chiral OPBAAs (seven examples), as
shown in Table 1, were successfully prepared based on the
conditions previously discovered in Scheme 3. Notably, when
14–16 were used, hydrogenation of an unsaturated spacer in
adducts C was performed after conjugation of A and B (see en-
tries 6–8, Table 1). All chemical structures of adducts and
OPBAAs are shown in Figure 2 and Figure 3; the variety in
Figure 3. Structures of all OPBAAs D in this study.
structure and high yields obtained emphasizes the feasibility
and flexibility of our synthetic approach.
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To exemplify the utility of these chiral OPBAAs, biologically
interesting small peptides containing a chiral OPBAA were de-
signed, synthesized, and evaluated towards human Nod1 stim-
ulation.
from 11 by using 31 instead of 30, and the overall yield was
32% over five steps (Scheme 5). Notably, the purification of di-
peptide 36 and tripeptide 40 through silica gel column chro-
matography was easier than that of corresponding 33 and 34,
as the former pair possessed a primary alcohol group instead
of a carboxylic acid. These molecules were selected for synthe-
sis and testing in order to understand the effect of the moiety
(R=CH2OH or COOH) on agonist behavior.
Currently, peptidoglycan fragments possessing the iE-meso-
DAP moiety, such as dipeptide (iE-meso-DAP) or tripeptide (A-
iE-meso-DAP), have been reported as human Nod1 agonists.
Our chemistry and various OPBAAs allowed us not only to pre-
pare bioactive peptides including 33 (iE-meso-DAP) and 34 (A-
iE-meso-DAP) but also to examine whether small peptides with
a different spacer length in OPBAA affect human Nod1 stimula-
tion activity or not.
Biological Evaluation
Eight enantiopure di- or tri-peptides 33–40, prepared from the
OPBAAs 11, 24–27, as well as commercially available mix iE-
DAP 41 (a mixture of g-d-Glu-d-mDAP and g-d-Glu-l-mDAP,
used for comparison purposes) were evaluated for their capaci-
ty to stimulate human Nod1. As shown in Figure 5 for dipep-
As illustrated in Scheme 5, the activated glutamic acid deriv-
atives 30 and 31 were prepared from d-Glu(OBn)-OH.[19] N-Boc
deprotection of OPBAA 11 with trifluoroacetic acid (TFA), fol-
lowed by conjugation to 30, basic hydrolysis of the methyl
ester, N-Boc deprotection of the adduct 32, and hydrogenoly-
sis, gave the desired molecule 33 in an overall 37% yield over
five steps. Likewise, a series of dipeptides were prepared using
our various OPBAAs or intermediates, and all structures are
shown in Figure 4. In addition, tripeptide 34 was also prepared
Figure 5. Stimulation of human Nod1 by dipeptide-based molecules includ-
ing mix iE-DAP (reference), 33, 35, 37, 38, and 39. HEK293T cells were trans-
fected with human Nod1 plasmid and kB reporter. The indicated amount of
each compound was added to the cells for 24 h, and the ability of each
compound to activate NF-kB was determined by luciferase reporter assay.
The basal NF-kB activity under Nod1 transfection without ligand stimulation
was 2.61ꢁ0.37 (meanꢁSE) fold on average.
tide-based molecules toward human Nod1 agonist study,
enantiopure iE-meso-DAP (33, m=1) was found to be the
most potent Nod1 dipeptid ligand tested, more potent than
mix iE-DAP (41). This suggests that the chirality of DAP pro-
foundly affects human Nod1 stimulating activity, and is consis-
tent with previously reported results.[7b,20] Surprisingly, only the
molecule containing the meso-DAP typed BAA such as 33 (m=
1) is human Nod1 agonist (Figure 5). By contrast, compounds
containing shorter (m=0) or longer (m=2, 3, or 4) spacers
were inactive toward the human Nod1 agonist study, even at
the concentrations up to 10 mgmLꢀ1 (Figure 5). These results
suggest that subtle structural modifications on the backbone
of BAA apparently influence the human Nod1 stimulation ac-
tivity.
Scheme 5. Preparation of bioactive peptides 33 (iE-meso-DAP) and 34 (A-iE-
meso-DAP).
Next, dipeptides 33 and 36 and tripeptides 34 and 40, shar-
ing the common meso-DAP typed BAA, were chosen to evalu-
ate their ability of human Nod1-dependent NF-kB activation,
and the results are shown in Figure 6. The enantiopure tripep-
tide 34 (A-iE-meso-DAP) showed a clear trend in potency and
was a more active Nod1 agonist than others, even at lower
concentrations (0.1 mgmLꢀ1). At a concentration of 1 mgmLꢀ1,
Figure 4. Structures of six dipeptides and two tripeptides.
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toward the human Nod1 receptor will be sought in order to
understand the pathogen–host interaction, and the results will
be published in due course.
Experimental Section
General Information
All solvents and reagents were obtained commercially and used
without further purification. 1H NMR spectra were recorded on
a Bruker AVANCE 600 spectrometer in deuterated solvents such as
chloroform-d (d=7.24), [D4]MeOH (d=3.31), and deuterium oxide
(d=4.80) at ambient temperature. 13C NMR spectra were obtained
with a Bruker AVANCE 600 spectrometer and were assigned ac-
cording to chloroform-d (d=77.23 ppm of central line). Chemical
shifts are given in ppm (d) and coupling constants (J) are given in
Hz. The splitting patterns are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), and dd (double of doublets).
High resolution mass spectra were obtained on a Bruker Daltonics
BioTOF III spectrometer (ESI-MS). Optical rotations were measured
with a PerkinElmer Model 341 polarimeter. Flash column chroma-
tography was carried out using Merck Kieselgel Si60 (40–63 mm).
Reactions were monitored by analytical thin-layer chromatography
(TLC) in silica gel 60 F254 plates and visualized by exposure to ul-
traviolet light at 254 nm and/or immersion in a staining solution
(p-anisaldehyde or acidic ninhydrin, phosphomolybdic acid or po-
tassium permanganate) followed by heating on a hot plate. Con-
centration refers to rotary evaporation.
Figure 6. Stimulation of human Nod1 by 33, 34, 36, and 40. HEK293T cells
were transfected with human Nod1 plasmid and kB reporter. The indicated
amount of each compound was added to the cells, and the ability of each
compound to activate NF-kB was determined by luciferase reporter assay.
The basal NF-kB activity under Nod1 transfection without ligand stimulation
was 3.24ꢁ0.54 (meanꢁSE) fold on average.
34 exhibited three times the potency of 33. Interestingly, when
the internal carboxylic acid (R=COOH) in DAP was changed to
the hydroxymethyl group (R=CH2OH), the activity decreased
(33 vs. 36 and 34 vs. 40), implying that the internal carboxylic
acid contributes more to human Nod1 recognition than the
hydroxymethyl group. Notably, the activity trend of tripeptide
40 was similar to that of dipeptide 33 in the concentration
range from 0.1 to 100 mgmLꢀ1 (Figure 6), indicating that the
additional l-Ala residue in tripeptide 40 could compensate for
the loss of stimulation activity caused from the functional
group conversion from R=COOH to CH2OH. From a synthetic
chemistry point of view, tripeptide 40 is easier to prepare than
dipeptide 33, a finding that establishes it as the molecular
template of choice for the exploration of new tripeptide-based
human Nod1 agonists.
(S)-tert-Butyl 4-(3-((2R,5S)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)propyl)-2,2-dimethyloxazolidine-3-carboxyl-
ate (7)
To a solution of compound 1 (100 mg, 0.47 mmol) in THF (4 mL) n-
butyllithium (201 mL, 0.52 mmol, 2.5m in hexanes) was added drop-
wise at ꢀ788C. After stirring for 30 min at ꢀ788C, a solution of 4
(166 mg, 0.52 mmol) in THF (2 mL) was added dropwise and the
resulting mixture was stirred for 3 h. The mixture was quenched
with saturated aqueous solution of ammonium chloride, and
evaporated under vacuum. The residue was extracted with EtOAc/
H2O, and the layers were separated. The organic phase was
washed with saturated aqueous solution of sodium bicarbonate,
brine, and dried over anhydrous MgSO4. The solvent was evaporat-
ed under reduced pressure, and the residue was purified by
column chromatography (10% EtOAc in hexanes, silica gel) to give
7 as a light yellow oil (138 mg, 0.31 mmol, 65%). ½aꢂ2D5 = +6.5 (c=
8.9, CHCl3); 1H NMR (600 MHz, CDCl3) (two rotamers were ob-
served): d=4.03–4.11 (4H, m), 3.94 (1H, s, br), 3.67–3.85 (4H, m),
2.21–2.23 (1H, m), 1.74–1.90 (4H, m), 1.50–1.60 (4H, m), 1.41–1.43
(13H, m), 1.22 (6H, t, J=7.2 Hz), 0.99 (3H, d, J=7.2 Hz), 0.64 ppm
(3H, d, J=7.2 Hz); 13C NMR (150 MHz, CDCl3): d=163.4, 163.3,
163.3, 163.2, 152.3, 152.0, 93.7, 93.2, 80.1, 79.5, 67.2, 66.7, 61.0,
60.8, 60.7, 57.9, 57.5, 55.5, 55.3, 34.2, 34.0, 33.8, 32.7, 32.1, 31.9,
28.6, 27.7, 27.0, 24.8, 23.5, 19.2, 16.8, 14.5 ppm; HRMS (ESI): m/z
calcd for C24H43N3O5 +H+: 454.3275 [M+H+]; found: 454.3271.
Conclusions
A concise, stereoselective approach for the preparation of
structurally diverse OPBAAs bearing two stereocenters, three
orthogonally protecting groups, and a spacer of different
length between two a-amino acid moieties has been devel-
oped. Using these versatile and enantiopure building blocks,
a series of small peptides inspired from peptidoglycan frag-
ments were synthesized and then studied for their ability to
activate human Nod1 receptor in the innate immune response
through the NF-kB pathway. Preliminary structure–activity rela-
tionship (SAR) results showed that only the dipeptides contain-
ing a specific length of BAAs are recognized as a potent
human Nod1 agonist. By contrast, shorter or longer spacers
than DAP abolished the molecular recognition with the human
Nod1 receptor. When the internal carboxylic acid was convert-
ed into the hydroxymethyl moiety in iE-meso-DAP or A-iE-
meso-DAP, human Nod1 agonistic activity decreased dramati-
cally, indicating that the carboxylic acid group plays an impor-
tant role in human Nod1 recognition. This chemistry and the
chiral building blocks and intermediates described herein will
be used to synthesize various peptidoglycan fragments to
study their biological functions in the future. Additionally,
a more detailed and clearer SAR study of tripeptide analogues
(S)-tert-Butyl 4-((R)-4-(((benzyloxy)carbonyl)amino)-5-ethoxy-
5-oxopentyl)-2,2-dimethyloxazolidine-3-carboxylate (9)
To a solution of compound 7 (100 mg, 0.22 mmol) in THF (1.5 mL)
was added 0.5m HCl aqueous solution (0.5 mL) and reacted at 08C
for 2 h. After the reaction was completed (checked by TLC), aque-
ous sat. NaHCO3 was added until pH>8. Benzyl chloroformate was
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25
added to the mixture and reacted at room temperature for 2 h.
The residue was extracted with EtOAc. The combined organic
phases were washed with saturated sodium bicarbonate aqueous
solution, brine, dried over anhydrous MgSO4 and concentrated.
The residue was purified by flash column chromatography (10%
EtOAc in hexanes, silica gel) to give 9 as a colorless oil (73 mg,
0.15 mmol, 70%). ½aꢂ2D5 = +3.6 (c=4.3, CHCl3); 1H NMR (600 MHz,
CDCl3) (two rotamers were observed): d=7.28–7.33 (5H, m), 5.47–
5.31 (1H, m), 5.07 (2H, s), 4.16–4.32 (3H, m), 3.64–3.87 (3H, m),
1.68–1.83 (4H, m), 1.50–1.60 (4H, m), 1.42–1.43 (11H, m), 1.35–
1.26 ppm (5H, m); 13C NMR (150 MHz, CDCl3): d=172.5, 156.1,
156.0, 152.5, 151.9, 136.5, 136.4, 129.2, 128.7, 128.4, 128.3, 127.1,
125.5, 93.9, 93.4, 80.3, 79.7, 67.2, 67.1, 67.0, 61.6, 61.6, 57.3, 57.2,
54.0, 33.5, 32.8, 32.4, 28.6, 28.5, 27.8, 27.0, 24. 7, 23.4, 22.1, 22.0,
14.3 ppm; HRMS (ESI): m/z calcd for C25H38N2O7 +H+: 479.2752
[M+H+]; found: 479.2758.
used as an alkylating agent, in 55% yield as a colorless oil. ½aꢂD
=
+12.8 (c=1.1, CHCl3); 1H NMR (600 MHz, CDCl3) (two rotamers
were observed): d=4.04–4.12 (4H, m), 3.94 (1H, s, br), 3.64–3.88
(4H, m), 2.15–2.21 (1H, m), 1.65–1.84 (4H, m), 1.51–1.58 (4H, m),
1.38–1.47 (11H, m), 1.20 (6H, t, J=7.2 Hz), 0.96 (3H, d, J=7.2 Hz),
0.64 ppm (3H, d, J=7.2 Hz); 13C NMR (150 MHz, CDCl3): d=163.4,
163.2, 162.9, 152.3, 152.0, 93.8, 93.3, 79.9, 79.4, 67.4, 67.1, 61.0,
60.9, 60.7, 60.6, 57.7, 57.4, 55.4, 55.1, 32.1, 30.9, 28.6, 27.7, 26.9,
24.7, 23.4, 19.2, 16.8, 14.5 ppm; HRMS (ESI): m/z calcd for
C23H41N3O5 +H+: 440.3119 [M+H+]; found: 440.3121.
(S)-tert-Butyl 4-(4-((2R,5S)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)butyl)-2,2-dimethyloxazolidine-3-carboxylate
(18)
The title compound 18 was synthesized by the procedure as de-
scribed for the preparation of compound 7 except for using 16 as
an alkylating agent and the adduct was subsequently hydrogenat-
ed [20% Pd(OH2)/C, MeOH, rt, 1 h] in 60% yield as a colorless oil.
½aꢂ2D5 = +27.1 (c=0.9, CHCl3); H NMR (600 MHz, CDCl3) (two rotam-
(2R,6S)-Ethyl 2-(((benzyloxy)carbonyl)amino)-6-((tert-butoxy-
carbonyl)amino)-7-hydroxyheptanoate (10)
1
To a solution of 9 (1.0 g, 2.09 mmol) in MeOH (10 mL) was added
p-toluenesulfonic acid monohydrate (39 mg, 0.21 mmol) and sever-
al drops of H2O. After stirring for 36 h at room temperature, MeOH
was removed under vacuum. The residue was purified by flash
column chromatography (50% EtOAc in hexanes, silica gel) to give
10 as a colorless oil (733 mg, 1.67 mmol, 80%). ½aꢂ2D5 =ꢀ9.7 (c=1.0,
CHCl3); 1H NMR (600 MHz, CDCl3) (two rotamers were observed):
d=7.29–7.34 (5H, m), 5.41 (1H, d, J=7.2 Hz), 5.08 (2H, s), 4.83
(1H, d, J=7.2 Hz), 4.34–4.36 (1H, m), 4.16–4.19 (2H, m), 3.50–3.58
(3H, m), 1.81–1.83 (1H, m), 1.61–1.65 (2H, m), 1.34–1.39 (12H, m),
1.24–1.27 ppm (3H, m); 13C NMR (150 MHz, CDCl3): d=172.5, 156.1,
156.0, 152.5, 151.9, 136.5, 136.4, 129.2, 128.7, 128.4, 128.3, 127.1,
125.5, 93.9, 93.4, 80.3, 79.7, 67.2, 67.1, 67.0, 61.63, 61.55, 57.3, 57.2,
54.0, 33.5, 32.8, 32.4, 28.6, 28.5, 27.8, 27.0, 24.7, 23.4, 22.1, 22.0,
14.3 ppm; HRMS (ESI): m/z calcd for C22H34N2O7 +H+: 439.2439
[M+H+]; found: 439.2443.
ers were observed): d=4.01–4.11 (4H, m), 3.90 (1H, s, br), 3.65–
3.90 (4H, m), 2.19–2.21 (1H, m), 1.65–1.70 (4H, m), 1.46–1.56 (4H,
m), 1.35–1.46 (11H, m), 1.10–1.32 (10H, m), 0.99 (3H, d, J=7.2 Hz),
0.64 ppm (3H, d, J=7.2 Hz); 13C NMR (150 MHz, CDCl3): d=163.4,
163.1, 152.3, 151.9, 93.7, 93.1, 80.0, 79.4, 67.1, 66.8, 60.8, 60.6, 60.5,
57.9, 57.4, 55.4, 34.3, 34.2, 33.6, 33.0, 31.9, 30.4, 28.6, 27.7, 26.9,
26.5, 26.3, 24.7, 24.6, 23.4, 19.2, 16.7, 14.5, 14.5 ppm; HRMS (ESI):
m/z calcd for C25H47N3O5 +H+: 468.3432 [M+H+]; found: 468.3431.
(S)-tert-Butyl 4-(5-((2R,5S)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)pentyl)-2,2-dimethyloxazolidine-3-carboxyl-
ate (19)
The title compound 19 was synthesized by the procedure as de-
scribed for the preparation of compound 7 except for using 15 as
an alkylating agent and the adduct was subsequently hydrogenat-
ed [20% Pd(OH2)/C, MeOH, rt, 1 h] in 58% yield as a colorless oil.
(2S,6R)-6-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
nyl)amino)-7-ethoxy-7-oxoheptanoic acid (11)
1
½aꢂ2D5 = +9.6 (c=3.2, CHCl3); H NMR (600 MHz, CDCl3) (two rotam-
ers were observed): d=4.03–4.14 (4H, m), 3.94 (1H, s, br), 3.66–
3.88 (4H, m), 2.23–2.25 (1H, m), 1.66–1.72 (4H, m), 1.50–1.55 (4H,
m), 1.43–1.45 (11H, m), 1.17–1.30 (12H, m), 1.00 (3H, d, J=7.2 Hz),
0.67 ppm (3H, d, J=7.2 Hz); 13C NMR (150 MHz, CDCl3): d=163.6,
163.2, 152.4, 152.1, 93.8, 93.2, 80.1, 79.5, 67.3, 66.9, 60.9, 60.7, 60.6,
58.0, 57.6, 55.6, 34.3, 33.8, 33.9, 31.9, 29.7, 28.7, 27.8, 27.0, 26.6,
26.5, 24.8, 24.7, 23.5, 19.3, 16.8, 14.6, 14.6 ppm; HRMS (ESI): m/z
calcd for C26H47N3O5 +H+: 482.3588 [M+H+]; found: 482.3592.
To the solution of the 10 (50 mg, 0.11 mmol) in dichloromethane
(1 mL) and water (5 mL) was added (diacetoxyiodo)benzene
(177 mg, 0.55 mmol) and 2,2,6,6-tetramethyl-1-piperidinyloxy
(31 mg, 0.02 mmol). After stirring at room temperature for 1 h, the
reaction was quenched with 10% aqueous Na2S2O3. The organic
layer was washed with 10% aqueous Na2S2O3, brine, dried over an-
hydrous MgSO4, and concentrated. The residue was purified by
flash column chromatography (5% MeOH and 0.3% formic acid in
dichloromethane, silica gel) to give 11 as a pale yellow oil (35 mg,
0.08 mmol, 72%). ½aꢂ2D5 = +2.1 (c=1.1, CHCl3); 1H NMR (600 MHz,
CDCl3): d=7.28–7.33 (5H, m), 5.46–5.50 (1H, m), 5.12–5.17 (1H, m),
5.06–5.11 (2H, m), 4.09–4.32 (4H, m), 1.81–1.86 (2H, m), 1.67–1.76
(2H, m), 1.41 (11H, s, br), 1.12–1.25 ppm (3H, m); 13C NMR
(150 MHz, CDCl3): d=176.0, 172.5, 156.3, 156.0, 136.4, 128.7, 128.4,
128.4, 80.5, 67.3, 61.8, 53.8, 53.1, 32.3, 32.0, 28.5, 21.2, 14.3 ppm;
HRMS (ESI): m/z calcd for C22H32N2O8 +H+: 453.2231 [M+H+];
found: 453.2233.
(S)-tert-Butyl 4-(6-((2R,5S)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)hexyl)-2,2-dimethyloxazolidine-3-carboxylate
(20)
The title compound 20 was synthesized by the procedure as de-
scribed for the preparation of compound 7 except for using 14 as
an alkylating agent and the adduct was subsequently hydrogenat-
ed [20% Pd(OH2)/C, MeOH, rt, 1 h] in 60% yield as a colorless oil.
1
½aꢂ2D5 = +10.5 (c=2.2, CHCl3); H NMR (600 MHz, CDCl3) (two rotam-
ers were observed): d=4.02–4.15 (4H, m), 3.94–3.97 (1H, m), 3.66–
3.88 (4H, m), 2.24–2.25 (1H, m), 1.67–1.73 (4H, m), 1.51–1.55 (4H,
m), 1.43–1.45 (11H, m), 1.18–1.28 (14H, m), 1.01 (3H, d, J=7.2 Hz),
0.67 ppm (3H, d, J=7.2 Hz); 13C NMR (150 MHz, CDCl3): d=163.6,
163.2, 152.4, 152.1, 93.8, 93.2, 80.1, 79.5, 67.3, 66.9, 60.8, 60.7, 60.6,
58.0, 57.6, 55.7, 55.6, 52.5, 34.4, 34.3, 33.8, 33.1, 31.9, 31.9, 29.7,
29.6, 28.7, 27.8, 27.0, 26.6, 26.5, 24.8, 24.6, 23.5, 19.3, 16.8, 14.6,
(S)-tert-Butyl 4-(2-((2R,5S)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)ethyl)-2,2-dimethyloxazolidine-3-carboxylate
(17)
The title compound 17 was synthesized by the procedure as de-
scribed for the preparation of compound 7, except that 5 was
&
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Chem. Asian J. 2014, 00, 0 – 0
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6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
14.6 ppm; HRMS (ESI): m/z calcd for C27H49N3O5 +H+: 496.3745
[M+H+]; found: 496.3737.
m), 4.11–4.19 (2H, m), 1.91 (2H, s, br), 1.69–1.76 (2H, m), 1.42 (9H,
s), 1.17–1.26 ppm (3H, m); 13C NMR (150 MHz, CDCl3): d=175.7,
175.4, 172.9, 172.4, 172.1, 156.8, 156.4, 155.9, 136.3, 136.0, 128.7,
128.4, 128.3, 82.0, 80.5, 67.7, 67.3, 61.9, 61.8, 54.5, 54.2, 53.9, 53.7,
53.2, 53.0, 52.8, 29.9, 28.7, 28.6, 28.5, 14.3 ppm; HRMS (ESI): m/z
calcd for C21H30N2O8 +H+: 439.2075 [M+H+]; found: 439.2079.
(R)-tert-Butyl 4-(3-((2R,5S)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)propyl)-2,2-dimethyloxazolidine-3-carboxyl-
ate (21)
The title compound 21 was synthesized by the procedure as de-
scribed for the preparation of compound 7 except for using 6 as
an alkylating agent in 62% yield as a colorless oil. ½aꢂ2D5 = +1.1 (c=
3.2, CHCl3); 1H NMR (600 MHz, CDCl3) (two rotamers were ob-
served): d=4.02–4.13 (4H, m), 3.94 (1H, s, br), 3.67–3.87 (4H, m),
2.22–2.24 (1H, m), 1.76–1.81 (1H, m), 1.66–1.68 (1H, m), 1.50–1.55
(4H, m), 1.42–1.44 (13H, m), 1.21–1.25 (8H, m), 0.99 (3H, d, J=
7.2 Hz), 0.67 ppm (3H, br); 13C NMR (150 MHz, CDCl3): d=163.5,
163.4, 163.3, 152.3, 152.0, 93.8, 93.3, 80.1, 79.5, 67.4, 66.8, 61.0,
60.8, 60.7, 60.6 57.9, 57.6, 55.7, 55.4, 34.3, 33.9, 32.8, 32.1, 31.9,
28.7, 27.7, 27.0, 24.8, 23.5, 21.6, 16.8, 14.6, 14.5 ppm; HRMS (ESI):
m/z calcd for C24H43N3O5 +H+: 454.3275 [M+H+]; found: 454.3278.
(2S,7R)-7-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
nyl)amino)-8-ethoxy-8-oxooctanoic acid (25)
The title compound 25 was synthesized by the procedure as de-
scribed for the preparation of compound 11 except for using 18 as
a starting material over four steps in 37% yield as a pale yellow oil.
½aꢂ2D5 = +2.0 (c=2.2, CHCl3); H NMR (600 MHz, CDCl3) (two rotam-
1
ers were observed): d=7.28–7.33 (5H, m), 5.41–5.43 (1H, m), 5.08–
5.12 (1H, m), 5.06 (2H, s), 4.09–4.33 (4H, m), 1.74–1.85 (2H, m),
1.60–1.66 (2H, m), 1.41 (9H, s), 1.31–1.38 (4H, m), 1.17–1.25 ppm
(3H, m); 13C NMR (150 MHz, CDCl3): d=176.5, 172.7, 156.2, 155. 9,
136.4, 128.7, 128.4, 128.3, 80.4, 67.2, 61.7, 53.9, 53.4, 32.7, 32.4,
28.5, 24.9, 24.9, 14.3 ppm; HRMS calcd for C23H34N2O8 +H+:
467.2388 [M+H+]; found: 467.2386.
(S)-tert-Butyl 4-(3-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)propyl)-2,2-dimethyloxazolidine-3-carboxyl-
ate (22)
(2S,8R)-8-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
nyl)amino)-9-ethoxy-9-oxononanoic acid (26)
The title compound 22 was synthesized by the procedure as de-
scribed for the preparation of compound 7 except for using 4 as
an alkylating agent in 71% yield as a colorless oil. ½aꢂ2D5 = +4.3 (c=
5.3, CHCl3); 1H NMR (600 MHz, CDCl3) (two rotamers were ob-
served): d=4.01–4.12 (4H, m), 3.93 (1H, s, br), 3.66–3.86 (4H, m),
2.21–2.23 (1H, m), 1.64–1.90 (4H, m), 1.48–1.53 (4H, m), 1.40–1.43
(13H, m), 1.19–1.24 (8H, m), 0.99 (3H, d, J=7.2 Hz), 0.65 ppm (3H,
s, br); 13C NMR (150 MHz, CDCl3): d=163.4, 163.4, 163.2, 162.7,
152.3, 152.0, 93.7, 93.2, 80.1, 79.5, 67.3, 66.8, 61.01, 60.96, 60.8,
60.7, 60.5, 57.9, 57.6, 55.6, 55.3, 34.3, 33.9, 32.8, 32.1, 31.9, 28.6,
27.7, 26.9, 24.8, 23.5, 21.6, 19.8, 19.3, 17.8, 17.7, 16.8, 14.6 ppm;
HRMS (ESI): m/z calcd for C24H43N3O5 +H+: 454.3275 [M+H+];
found: 454.3269.
The title compound 26 was synthesized by the procedure as de-
scribed for the preparation of compound 11 except for using 19 as
a starting material over four steps in 45% yield as a pale yellow oil.
½aꢂ2D5 = +2.4 (c=3.3, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.27–
7.33 (5H, m), 5.38–5.39 (1H, m), 5.10–5.12 (1H, m), 5.08 (2H, s),
4.09–4.35 (3H, m), 3.62–3.71 (1H, m), 1.78–1.80 (2H, m), 1.62–1.63
(2H, m), 1.42 (9H, s), 1.30–1.34 (6H, m), 1.18–1.26 ppm (3H, m);
13C NMR (150 MHz, CDCl3): d=176.9, 176.6, 176.33, 173.3, 172.8,
172.5, 157.0, 156.7, 156.2, 155.8, 136.4, 136.4, 136.1, 128.7, 128.4,
128.3, 128.2, 81.7, 80.3, 67.7, 67.2, 61.7, 61.6, 54.8, 54.6, 54.04,
53.99, 53.5, 52.6, 52.5, 32.7, 32.6, 32.4, 29.9, 28.8, 28.5, 25.2, 25.1,
25.1, 14.4 ppm; HRMS (ESI): m/z calcd for C24H36N2O8 +H+:
481.2544 [M+H+]; found: 481.2549.
(R)-tert-Butyl 4-(3-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihy-
dropyrazin-2-yl)propyl)-2,2-dimethyloxazolidine-3-carboxyl-
ate (23)
(2S,9R)-9-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
nyl)amino)-10-ethoxy-10-oxodecanoic acid (27)
The title compound 27 was synthesized by the procedure as de-
scribed for the preparation of compound 11 except for using 20 as
a starting material over four steps in 45% yield as a pale yellow oil.
½aꢂ2D5 = +1.7 (c=2.9, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.27–
7.33 (5H, m), 5.34–5.35 (1H, m), 5.08 (2H, s), 5.02–5.03 (1H, m),
4.09–4.33 (4H, m), 1.77–1.79 (2H, m), 1.61–1.63 (2H, m), 1.42 (9H,
s), 1.31–1.35 (8H, m), 1.18–1.27 ppm (3H, m); 13C NMR (150 MHz,
CDCl3): d=176.8, 176.6, 173.4, 172.9, 172.5, 156.7, 156.5, 156.2,
155.8, 136.4, 128.7, 128.4, 128.3, 128.2, 81.5, 80.3, 67.6, 67.2, 61.7,
54.8, 54.1, 53.5, 52.6, 32.8, 32.5, 29.9, 29.0, 28.5, 25.3, 25.7, 25.0,
14.4 ppm; HRMS (ESI): m/z calcd for C25H38N2O8 +H+: 495.2701
[M+H+]; found: 495.2703.
The title compound 23 was synthesized by the procedure as de-
scribed for the preparation of compound 7 except for using 6 as
an alkylating agent in 61% yield as a colorless oil. ½aꢂ2D5 =ꢀ5.7 (c=
2.8, CHCl3); 1H NMR (600 MHz, CDCl3) (two rotamers were ob-
served): d=4.02–4.13 (4H, m), 3.93 (1H, s, br), 3.65–3.86 (4H, m),
2.21–2.23 (1H, m), 1.71–1.77 (3H, m), 1.49–1.53 (4H, m), 1.41–1.44
(13H, m), 1.22 (8H, t, J=7.2 Hz), 0.98 (3H, d, J=7.2 Hz), 0.66 ppm
(3H, br); 13C NMR (150 MHz, CDCl3): d=163.5, 163.4, 163.3, 152.3,
152.0, 93.7, 93.2, 80.1, 79.5, 67.3, 66.8, 61.0, 60.8, 60.7, 57.9, 57.5,
55.5, 55.4, 34.2, 34.0, 33.8, 32.7, 32.1, 31.9, 28.6, 27.7, 27.0, 24.8,
23.5, 21.6, 19.3, 16.8, 14.6, 14.5 ppm; HRMS (ESI): m/z calcd for
C24H43N3O5 +H+: 454.3275 [M+H+]; found: 454.3271.
(2R,6S)-6-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
(2S,5R)-5-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
nyl)amino)-7-ethoxy-7-oxoheptanoic acid (28)
nyl)amino)-6-ethoxy-6-oxohexanoic acid (24)
The title compound 28 was synthesized by the procedure as de-
scribed for the preparation of compound 11 except for using 23 as
a starting material over four steps in 42% yield as a pale yellow oil.
½aꢂ2D5 =ꢀ1.6 (c=10.8, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.28–
7.33 (5H, m), 5.51–5.52 (1H, m), 5.14–5.19 (1H, m), 5.05–5.12 (2H,
m), 4.09–4.32 (4H, m), 1.83–1.86 (2H, m), 1.67–1.76 (2H, m), 1.41
The title compound 24 was synthesized by the procedure as de-
scribed for the preparation of compound 11 except for using 17 as
a starting material over four steps in 34% yield as a pale yellow oil.
½aꢂ2D5 = +3.9 (c=1.7, CHCl3); 1H NMR (600 MHz, CDCl3): d=7.28–
7.33 (5H, m), 5.56 (1H, s), 5.20 (1H, s), 5.08 (2H, s), 4.28–4.35 (2H,
Chem. Asian J. 2014, 00, 0 – 0
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7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Paper
(11H, s, br), 1.16–1.25 ppm (3H, m); 13C NMR (150 MHz, CDCl3): d=
176.1, 175.6, 172.6, 172.2, 157.0, 156.3, 155.9, 155.7, 136.3, 135.9,
128.6, 128.3, 128.3, 128.1, 81.9, 80.3, 67.7, 67.2, 61.7, 61.6, 54.7,
54.4, 53.8, 53.2, 53.1, 32.10, 32.05, 31.9, 31.8, 28.4, 28.3, 21.4, 21.26,
14.3 ppm; HRMS (ESI): m/z calcd for C22H32N2O8 +H+: 453.2231
[M+H+]; found: 453.2234.
1.96–2.07 (1H, m), 1.83–1.93 (3H, m), 1.68–1.77 (1H, m), 1.54 (3H,
d, J=7.14 Hz), 1.42–1.47 ppm (2H, m); 13C NMR (150 MHz, D2O):
d=178.9, 177.9, 174.9, 174.7, 170.4, 54.9, 54.9, 54.6, 49.2, 32.3, 31.2,
30.1, 27.4, 21.2, 16.5 ppm; HRMS (ESI): m/z calcd for C15H26N4O8 +
H+: 391.1823 [M+H+]; found: 391.1825.
(2R,5S)-2-Amino-5-((R)-4-amino-4-carboxybutanamido)hexa-
(2R,6R)-6-(((Benzyloxy)carbonyl)amino)-2-((tert-butoxycarbo-
nedioic acid (35)
nyl)amino)-7-ethoxy-7-oxoheptanoic acid (29)
The title compound 35 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 24 as
a starting material over five steps as a white solid. ½aꢂ2D5 =ꢀ8.2 (c=
3.3, D2O); 1H NMR (600 MHz, D2O): d=4.18–4.19 (1H, m), 3.74–3.81
(2H, m), 2.48–2.53 (2H, m), 2.12–2.21 (2H, m), 1.69–1.95 ppm (4H,
m); 13C NMR (150 MHz, D2O): d=178.3, 178.2, 174.3, 174.3, 174.2,
174.1, 174.0, 54.8, 54.7, 54.7, 54.6, 54.4, 54.3, 54.2, 54.1, 31.6, 31.2,
27.4, 27.3, 27.1, 26.3, 26.2 ppm; HRMS (ESI): m/z calcd for
C11H19N3O7 +H+: 306.1296 [M+H+]; found: 306.1294.
The title compound 29 was synthesized by the procedure as de-
scribed for the preparation of compound 11 except for using 21 as
a starting material over four steps in 36% yield as a pale yellow oil.
½aꢂ2D5 =ꢀ4.6 (c=2.2, CHCl3); H NMR (600 MHz, CDCl3) (two rotam-
1
ers were observed): d=7.28–7.32 (5H, m), 5.52–5.53 (1H, m), 5.21–
5.31 (1H, m), 5.05–5.11 (2H, m), 3.62–4.31 (4H, m), 1.67–1.83 (4H,
m), 1.39–1.41 (11H, m), 1.13–1.25 (3H, m); 13C NMR (150 MHz,
CDCl3): d=176.3, 175.4, 173.1, 172.7, 172.6, 172.2, 157.3, 156.5,
156.3, 156.2, 156.0, 136.4, 136.0, 128.6, 128.4, 128.4, 128.2, 81.8,
80.5, 80.3, 67.8, 67.3, 61.8, 61.6, 54.5, 53.8, 53.7, 53.2, 52.7, 32.3,
32.1, 31.9, 31.8, 28.5, 21.4, 21.2, 14.3 ppm; HRMS (ESI): m/z calcd
for C22H32N2O8 +H+: 453.2231 [M+H+]; found: 453.2233.
(2R,6S)-2-Amino-6-((R)-4-amino-4-carboxybutanamido)-7-hy-
droxyheptanoic acid (36)
The title compound 36 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 10 as
a starting material over five steps as a white solid. ½aꢂ2D5 =ꢀ12.5
(2R,6S)-2-Amino-6-((R)-4-amino-4-carboxybutanamido)hepta-
nedioic acid (33, iE-meso-DAP)
1
(c=2.5, D2O); H NMR (600 MHz, D2O): d=3.87–3.95 (1H, m), 3.76
To a solution of compound 11 (50 mg, 0.11 mmol) in dichlorome-
thane (3 mL) was added trifluoroacetic acid (1 mL) at 08C. After
stirring for 1 h, trifluoroacetic acid was removed under vacuum.
Triethylamine (33 mg, 0.33 mmol) and compound 39 (47 mg,
0.13 mmol) were added to the residue in dichloromethane (10 mL)
at 08C. After stirring for 16 h at room temperature, the mixture
was washed with aqueous 1m HCl, aqueous sat. NaHCO3, brine,
dried over anhydrous MgSO4, and concentrated. The residue was
purified by flash column chromatography (50% EtOAc in hexanes,
silica gel) to give 32 as a white solid (48 mg, 0.08 mmol, 70%). To
a solution of 32 (50 mg, 0.09 mmol) in MeOH (4 mL) was added
lithium hydroxide (21 mg, 0.9 mmol) and 5 drops of H2O. After stir-
ring at room temperature for 14 h, trifluoroacetic acid (1 mL) was
added at 08C and stirred at room temperature for 1 h. The mixture
was concentrated under vacuum and then purified by flash
column chromatography (33% ammonium hydroxide in 1-propa-
nol, silica gel). The purified product was dissolved in MeOH (5 mL),
and 20% palladium hydroxide on activated charcoal (9 mg) was
added to the mixture. The resulting mixture was stirred under 5
atm pressure of H2 gas at room temperature for 1 h. The mixture
was filtered through Celite, and the filtrate was evaporated. The
residue was purified by flash column chromatography (33% am-
monium hydroxide in 1-propanol, silica gel) to provide 33 as
a white solid. ½aꢂ2D5 =ꢀ6.5 (c=0.8, D2O); 1H NMR (600 MHz, D2O):
d=4.24–4.27 (1H, m), 3.76–3.83 (2H, m), 2.48–2.51 (2H, m), 2.14–
2.20 (2H, m), 1.83–1.97 (3H, m), 1.73–1.79 (1H, m), 1.41–1.58 ppm
(2H, m); 13C NMR (150 MHz, D2O): d=177.6, 174.4, 174.4, 173.8,
54.5, 54.2, 31.4, 31.1, 30.6, 29.9, 26.2, 21.1 ppm; HRMS (ESI): m/z
calcd for C12H21N3O7 +H+: 320.1452 [M+H+]; found: 320.1455.
(1H, t, J=6.1 Hz), 3.72 (1H, t, J=6.3 Hz), 3.59 (1H, dd, J=4.7,
11.6 Hz), 3.49 (1H, dd, J=6.7, 11.6 Hz), 2.42–2.44 (2H, m), 2.11–2.15
(2H, m), 1.79–1.91 (2H, m), 1.57–1.61 (1H, m), 1.37–1.47 ppm (3H,
m); 13C NMR (150 MHz, D2O): d=174.7, 174.6, 173.9, 63.5, 54.6,
54.1, 50.9, 31.6, 30.1, 29.6, 26.4, 20.8 ppm; HRMS (ESI): m/z calcd
for C12H23N3O6 +H+: 306.1660 [M+H+]; found: 306.1657.
(2R,7S)-2-Amino-7-((R)-4-amino-4-carboxybutanamido)octa-
nedioic acid (37)
The title compound 37 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 25 as
a starting material over five steps as a white solid. ½aꢂ2D5 =ꢀ2.5 (c=
1
0.2, D2O); H NMR (600 MHz, D2O): d=4.15–4.17 (1H, m), 3.73–3.81
(2H, m), 2.46–2.49 (2H, m), 2.13–2.21 (2H, m), 1.80–1.91 (3H, m),
1.69–1.72 (1H, m), 1.35–1.45 ppm (4H, m); 13C NMR (150 MHz,
D2O): d=179.1, 174.9, 174.1, 174.0, 55.0, 54.9, 54.6, 54.1, 31.2, 31.0,
30.9, 30.2, 30.2, 26.3, 24.9, 24.8, 24.0, 23.9 ppm; HRMS (ESI): m/z
calcd for C13H23N3O7 +H+: 334.1609 [M+H+]; found: 334.1608.
(2R,8S)-2-Amino-8-((R)-4-amino-4-carboxybutanamido)nona-
nedioic acid (38)
The title compound 38 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 26 as
a starting material over five steps as a white solid. ½aꢂ2D5 =ꢀ2.3 (c=
1
1.0, D2O); H NMR (600 MHz, D2O): d=4.13–4.15 (1H, m), 3.72–3.81
(2H, m), 2.45–2.49 (2H, m), 2.11–2.21 (2H, m), 1.78–1.90 (3H, m),
1.65–1.69 (1H, m), 1.32–1.48 ppm (6H, m); 13C NMR (150 MHz,
D2O): d=179.4, 175.0, 174.1, 174.0, 55.2, 54.8, 54.1, 31.2, 31.1, 30.3,
27.9, 26.3, 24.8, 24.0 ppm; HRMS (ESI): m/z calcd for C14H25N3O7 +
H+: 348.1765 [M+H+]; found: 348.1767.
(2R,6S)-2-Amino-6-((R)-4-((S)-2-aminopropanamido)-4-carbox-
ybutanamido)heptanedioic acid (34, A-iE-DAP)
The title compound 34 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 31 as
a coupling partner over five steps as a white solid. ½aꢂ2D5 =ꢀ0.4 (c=
(2R,9S)-2-Amino-9-((R)-4-amino-4-carboxybutanamido)deca-
nedioic acid (39)
1
1.3, D2O); H NMR (600 MHz, D2O): d=4.21–4.23 (1H, m), 4.12–4.18
The title compound 39 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 27 as
(2H, m), 3.74–3.76 (1H, m), 2.37–2.40 (2H, m), 2.12–2.21 (1H, m),
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a starting material over five steps as a white solid. ½aꢂ2D5 =ꢀ2.0 (c=
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1
1.5, D2O); H NMR (600 MHz, D2O): d=4.13–4.15 (1H, m), 3.74–3.81
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(2H, m), 2.47–2.50 (2H, m), 2.11–2.24 (2H, m), 1.78–1.92 (3H, m),
1.65–1.71 (1H, m), 1.32–1.48 ppm (8H, m); 13C NMR (150 MHz,
D2O): d=179.7, 175.1, 174.2, 174.1, 55.4, 54.8, 54.2, 31.32, 31.29,
30.3, 28.1, 27.9, 26.5, 25.0, 24.1 ppm; HRMS (ESI): m/z calcd for
C15H27N3O7 +H+: 362.1922 [M+H+]; found: 362.1921.
(2R,6S)-2-Amino-6-((R)-4-((S)-2-aminopropanamido)-4-carbox-
ybutanamido)-7-hydroxyheptanoic acid (40)
The title compound 40 was synthesized by the procedure as de-
scribed for the preparation of compound 33 except for using 10 as
a starting material and 31 as a coupling partner over five steps as
a white solid. ½aꢂ2D5 =ꢀ3.4 (c=1.0, D2O); 1H NMR (600 MHz, D2O):
d=4.18–4.21 (1H, m), 4.11–4.14 (1H, m), 3.87–3.91 (1H, m), 3.74–
3.76 (1H, m), 3.59–3.62 (1H, m), 3.50–3.53 (1H, m), 2.31–2.41 (2H,
m), 2.11–2.19 (1H, m), 1.80–2.02 (3H, m), 1.60–1.68 (1H, m), 1.56
(3H, d, J=7.08 Hz), 1.36–1.51 ppm (3H, m); 13C NMR (150 MHz,
D2O): d=177.8, 175.4, 174.7, 170.8, 63.54, 63.50, 54.7, 54.6, 51.0,
49.2, 32.4, 30.1, 29.7, 27.6, 20.9, 16.6 ppm; HRMS (ESI): m/z calcd
for C15H28N4O7 +H+: 377.2031 [M+H+]; found: 377.2030.
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HEK293T Bioassay for Nod1 Stimulation: The NF-kB reporter plas-
mid containing three kB-binding sites (pGL2-ELMA-luciferase) was
provided by Dr. S. L. Hsieh (Yang-Ming University, Taipei, Taiwan).
The expression vector pCMV-Flag-Nod1 was a gift from Dr. John
Reed (Sanford-Burnham Institute for Medical Research, La Jolla, Cal-
ifornia, USA). Ligand dependent NF-kB activation was determined
by using 5ꢁ104 HEK293T cells transfected with pCMV-Flag-Nod1,
NF-kB reporter plasmid and b-galactosidase expression vector
(pSVlacZ) using the Lipofectamine 2000 reagent (Invitrogen) for
24 h. After treatment of the various ligands for another 24 h, cells
were lysed in reporter lysis buffer (Promega). Subsequently, the ly-
sates were reacted with commercial luciferase substrate provided
in the luciferase assay system kit (Promega) and assayed by using
a microplate luminometer (Packard, Meriden, CT, USA). Luciferase
activity values were normalized to transfection efficiency moni-
tored by b-galactosidase expression and presented as the folds of
luciferase activity of the control group without pCMV-Flag-Nod1
transfection.
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Acknowledgements
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We thank Academia Sinica, and Ministry of Science and Tech-
nology for financial support.
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Keywords: amino acids · immunology · innate immunity ·
Nod1 · Schçllkopf bis-lactim ethers · synthetic methods
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Received: October 13, 2014
Published online on && &&, 0000
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FULL PAPER
Peptides
Give it a nod: A concise preparation of
structurally diverse OPBAAs bearing
three orthogonally protecting groups
and a spacer of varied length has been
developed. Using these versatile build-
ing blocks, a series of small peptides in-
spired from peptidoglycan fragments
were synthesized and then studied for
their ability to activate human Nod1 re-
ceptor in the innate immune response
through the NF-kB pathway.
Po-Ting Chen, Cheng-Kun Lin,
Chih-Ju Tsai, Duen-Yi Huang,
Fu-Yao Nien, Wan-Wan Lin,
Wei-Chieh Cheng*
&& – &&
Expeditious Synthesis of Enantiopure,
Orthogonally Protected Bis-a-Amino
Acids (OPBAAs) and their Use in
a Study of Nod1 Stimulation
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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