Novel amino acids: Synthesis of furoxan and sydnonimine containing amino acids and peptides as potential nitric oxide releasing motifs

Article abstract of DOI:10.1039/c3ob41047a

The incorporation of furoxan and sydnonimine ring systems into amino acid side chains is demonstrated with the preparation of four novel amino acids which carry these nitric oxide-releasing motifs. N-((4-Nitrophenoxy)carbonyl)-3- phenylsydnonimine 9 and bis(phenylsulfonyl)furoxan 10 are the key intermediates for introducing the heterocycle side chains onto appropriate amine and alcohol functionalities respectively. Furoxan 5 and 7 both displayed NO release based on determination of nitrite production. Orthogonal amino acid protecting group strategies were deployed to demonstrate that the amino acids could be incorporated into peptide frameworks. By way of demonstration the amino acids were placed centrally into several tripeptide motifs. Griess test assays showed that these amino acids released NO in the presence of γ-glutathione (GST). is

Full text of DOI:10.1039/c3ob41047a

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Novel amino acids: synthesis of furoxan and
sydnonimine containing amino acids and peptides as
potential nitric oxide releasing motifs
Cite this: Org. Biomol. Chem., 2013, 11,
4657
Andrew Nortcliffe, Nigel P. Botting† and David O’Hagan*
The incorporation of furoxan and sydnonimine ring systems into amino acid side chains is demonstrated
with the preparation of four novel amino acids which carry these nitric oxide-releasing motifs. N-((4-
Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9 and bis(phenylsulfonyl)furoxan 10 are the key inter-
mediates for introducing the heterocycle side chains onto appropriate amine and alcohol functionalities
respectively. Furoxan 5 and 7 both displayed NO release based on determination of nitrite production.
Orthogonal amino acid protecting group strategies were deployed to demonstrate that the amino acids
could be incorporated into peptide frameworks. By way of demonstration the amino acids were placed
centrally into several tripeptide motifs. Griess test assays showed that these amino acids released NO in
the presence of γ-glutathione (GST).
Received 20th May 2013,
Accepted 28th May 2013
DOI: 10.1039/c3ob41047a
www.rsc.org/obc
Introduction
Nitric oxide (NO) has a well documented role in many bio-
logical functions, including cardiovascular disease,1 cancer
biology2 and neurotransmission.3 NO plays a critical role in
cell signaling, where small changes in NO levels can have a
large affect on cellular biochemistry.4 As such the use of NO as
a therapeutic, particularly in cancer treatments continues to
attract attention. For example, the NO release analogue of
the non-steroidal anti-inflammatory drug, Sulindac, showed
increased cytotoxicity in PC-3 prostate cancer cells compared
to an analogue unable to release NO.5
In recent years the ability to use NO as a therapy has taken
two approaches. The first addresses molecules that affect the
endogenous production of NO by modulation of nitric oxide
synthase (NOS). Thus compounds such as 7-nitroindazole 1
act as selective neuronal-NOS (nNOS) inhibitors reducing
intracellular NO levels,6 and dihydropteridine 2 acts as an NOS
activator upregulating NO levels.7
A second approach has focused on the exogenous increase
of circulating NO through the use of functional groups which
are metabolized with concomitant NO release. Heterocycles
such as furoxans 3 and sydnonimines 4 are known to be
metabolized in such a manner.8 The NO release properties of
furoxans was first reported over thirty years ago.9 A release
mechanism was proposed by Feelisch et al.,10 in 1992 which
proposed the nucleophilic attack of thiolate ions as the key
step involved in the liberation of NO. Drug-furoxan hybrids
display wide ranging biological effects.11 Although the sydno-
nimines 4 are less well studied in terms of their mechanism of
NO release, they are also recognized as an important class of
NO donors.12
There is an increasing focus on the development of
peptide-based therapies in medicinal chemistry research and a
demand for access to non-natural amino acid residues, and
particularly those which may have a therapeutic role.13 Given
the growing interest in developing NO hybrid drugs, we set out
to design and synthesise a series of amino acids carrying
the furoxan and sydnonimine motifs as pro-NO release com-
pounds capable of exogenously producing NO. These amino
acids could then be used for bespoke peptide synthesis. In this
paper we present the synthesis of the free amino acids 5–8.
University of St Andrews, School of Chemistry and Centre for Biomolecular Sciences,
North Haugh, St Andrews, Fife KY16 9ST, UK. E-mail: do1@st-andrews.ac.uk;
Fax: +44 (0)1334 463800; Tel: +44 (0)1334 467171
†Nigel Botting died on June 4th 2011.
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The routes to each allow for their incorporation into peptide
motifs as exemplified by several tripeptide syntheses.
Scheme 1 Synthesis of 10. Reagents and conditions: i. bromoacetonitrile, NaI,
K2CO3, CH3CN, r.t., 16 h, 87%; ii. isopentyl nitrite, Et2O, r.t., dark, 16 h, then HCl
(g), r.t., 15 min, 94%; iii. 4-nitrophenyl chloroformate, Et3N, CH2Cl2, −20 °C to
r.t., 16 h, 64%.
Results and discussion
Synthesis of NO donor building block motifs
N-((4-Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9 and bis-
(phenylsulfonyl)furoxan 10 were prepared as the key synthetic
precursors for incorporation of the heterocyclic motifs into the
amino acid side chains.
Scheme 2 Synthesis of sydnonimine modified aromatic amino acids 16a and
16b. Reagents and conditions: i. 9, CH3CN, reflux, 18 h, 16a = 89%, 16b = 85%.
Synthesis of the amino acids
It was envisaged that the heterocyclic cores could be tagged via
the phenol substituent of L-tyrosine, and through the amino
substituent of 4-amino-L-phenylalanine. Thus these amino
acids had to be prepared in a suitable protected form for coup-
ling to the heterocyclic motifs 9 and 10. The protected amino
acids 14 and 15 were prepared from tyrosine16 and 4-nitrophe-
nylalanine17 respectively as previously described. The coupling
reactions to the sydnonimines was achieved by reaction of 14
or 15 with 9 to give the protected amino acids 16 and 16a in
excellent yields as illustrated in Scheme 2.
Analogously treatment of 14 with DBU and 10 furnished
the desired tyrosine-furoxan ether 17 in 86% yield following
conditions described by Fruttero et al (Scheme 3).18 Amine 15
proved unreactive to 10 despite a variety of attempts under
different conditions. And thus we could not prepare the corres-
ponding amine analogue in the furoxan series.
Carbamate 9 was prepared from aniline 11 in three steps as
illustrated in Scheme 1. N-Alkylation of 11 with bromoaceto-
nitrile furnished aminoacetonitrile 12 in good yield. Nitrosation
with isopentyl nitrite followed by acid-catalysed ring closure
provided 3-phenylsydnonimine hydrochloride 13.14 In our
hands, the reported procedure by Turnbull and Beal furnished
mixtures of the desired product 13 and the hydrochloride salt
of 12. The procedure was modified such that isopentyl nitrite
was increased in equivalence, and the nitrosation reaction
time extended. The optimized reaction generated 13 in excel-
lent yield. The resultant 3-phenylsydnonimine 13 was acylated
using 4-nitrophenyl chloroformate to generate carbamate 9.
Bis(phenylsulfonyl)furoxan 10 was prepared in a three steps
according to the procedure of Wang et al.15
This coupling strategy was explored with L-serine and 2,3-
diaminopropionic acid. Thus the protected amino acids 1819
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and 1920 were successfully acylated to generate carbamate 20a accomplished with NaOH (1 : 1 THF–H2O) providing carboxylic
and urea 20b in 60% and 55% yields respectively as illustrated acids 23 and 24 also in good yields. A one-pot procedure,
in Scheme 4.
as shown in Scheme 5, which combined these two deprotec-
With this series of heterocycle-appended amino acids avail- tion steps, gave the free amino acid HCl salts 5 and 6. This
able in their protected form, selective deprotections were now straightforward strategy was also applied to the doubly pro-
explored as a prerequisite for peptide synthesis. tert-Butoxycar- tected amino acid 17 derived from tyrosine, and orthogonal
bonyl deprotection of 16 and 16a with HCl proved straightfor- deprotection furnished the amine HCl salt 25 or the free car-
ward and furnished the desired amine HCl salts 21 and 22 in boxylic acid 26 in good yields (Scheme 6). Again a two step,
quantitative yields. Hydrolysis of the methyl ester was one pot reaction allowed preparation of the free amino acid 7
as its hydrochloride salt. In all cases the side chain hetero-
cyclic motifs were sufficiently unreactive and survived these
standard deprotection protocols.
Boc deprotection of 20a–b, progressed very cleanly to give
the amine hydrochlorides 27 and 28, however, base treatment
of 20a promoted an elimination reaction and gave significant
decomposition to dehydroalanine 29. With 20b there was no
recoverable product and we assume it was similarly susceptible
to elimination (Scheme 7).
Due to the susceptibility of the carbamate 20a to base
mediated elimination, an alternative approach was taken
to differentially deprotect the carboxylic acid functionality.
Accordingly L-serine was protected as its tert-butoxycarbonyl
tert-butyl ester 30 in a two step protocol.21,22 Acylation with 9
provided the sydnonimine 31 in modest yield and then a
global deprotection with trifluoroacetic acid furnished the free
amino acid 8 as its trifluoroacetate salt. Reprotection of the
amine with Boc2O gave the N-protected amino acid 32 now
with a free carboxylic acid group (Scheme 8).
Scheme 3 Synthesis of furoxan modified tyrosine 17. Reagents and conditions:
i. 10, DBU, CH2Cl2, r.t. 2 h, 86%.
Amino acid derivatives 16 and 17 were modified to install
orthogonal protecting groups. Thus deprotection of 16 and 17
generate amines 21 and 25 respectively. These intermediates
were progressed without purification to their Fmoc or Alloc
carbamates, to give sydnonimines 16b and 16c and furoxans
17a and 17b. Base mediated deprotections furnished the Fmoc
protected free carboxylic acids 33 and 45 and Alloc protected
free carboxylic acids 34 and 36 as summarized in Schemes
9 and 10.
Scheme 4 Synthesis of sydnonimine modified protected amino acids 20a and
20b: Reagents and conditions: i. 9, CH3CN, reflux, 18 h, 20a = 60%, 20b = 55%.
Scheme 5 Selective deprotection of sydnonimine modified amino acids 16 and 16b: Reagents and conditions: i. 4H HCl in dioxane, CH2Cl2, r.t., 1 h, 21 and 22 =
quant.; ii. NaOH, THF–H2O (1 : 1), r.t. 15 min, 23 = 95%, 24 = 88%; iii. NaOH, THF–H2O (1 : 1), r.t. 15 min, then 4H HCl in dioxane, CH2Cl2, r.t., 1 h, 6 = 77%, 7 = 75%.
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Treatment of 16 with HCl, followed by trituration with 37–40 in excellent yield with no observable racemization as
diethyl ether gave the amine hydrochloride, which was directly judged by 1H NMR. These intermediate dipeptides could be
protected with Fmoc-Cl or Alloc-Cl. Subsequent purification deprotected and then coupled under standard conditional
allowed the five step transformation from tyrosine to 16b and through the amine terminus to generate the various tri-
16c to be carried out with only one chromatography step. peptides 41–44 as illustrated in Scheme 11.
Amino acids 5 and 7 were then introduced into two tripeptide
Assay of NO/NO2− production from the amino acids 5 and 7
sequences to exemplify their utility in peptide synthesis. Coup-
ling of 23 and 26 to L-valine and L-phenylalanine methyl esters
The release of nitric oxide from amino acids 5 and 7 was deter-
in separate experiments using HATU furnished dipeptides
mined by examining the formation of nitrite. It has been
previously reported that the formation of nitrite (NO2−), is a
reliable measure of the NO release, in the presence of excess
thiol.10 Equimolar production of superoxide (O2˙) along with
NO is reported for sydnonimines.24 To this affect, the Griess
−
assay for NO2 was used to determine the production of NO
from amino acids 5 and 7, and as a control, L-tyrosine. Amino
acids 5, 7 (1 mmol, [final], 400 μM) in a solution of DMSO–
dH2O (2 : 8) were analysed after incubation with a glutathione
(GST) buffer containing superoxide dismutase (SOD) ([GST]
6 mmol, final [GST] 600 μM, pH 7.6, [SOD] 4557 u mL−1).
−
Amino acids 5 and 7 showed an NO2 release of 30.4
3.9 μmol and 44.8 3.2 μmol respectively. The amount of NO
required to induce a cellular response change is on the pico-
molar to nanomolar range.25,26 Control incubations performed
Scheme 6 Selective deprotection of sydnonimine modified amino acids
under identical conditions in the absence of GST and SOD
gave no significant NO production. This is in agreement with
28a–b. Reagents and conditions: i. 4H HCl in dioxane, CH2Cl2, r.t., 1 h, 27 and
28 quant.; ii. NaOH, THF–H2O (1 : 1), r.t. 15 min, 29 = 70%.
Scheme 7 Selective deprotection of furoxan modified amino acids 17. Reagents and conditions: i. 4H HCl in dioxane, CH2Cl2, r.t., 1 h, 25 = quant.; ii. NaOH, THF–
H2O (1 : 1), r.t. 15 min, 26 = 90%; iii. (a) NaOH, THF–H2O (1 : 1), r.t. 15 min, then 4H HCl in dioxane, CH2Cl2, r.t., 1 h, 27 = 80%.
Scheme 8 Deprotection of sydnonimine modified amino acid 31. Reagents and conditions: i. 9, CH3CN, reflux, 18 h, 31 = 60%; ii. TFA, CH2Cl2, r.t. 16 h, 8 = quant.;
iii. Boc2O, NaHCO3, H2O–dioxane (1 : 1), r.t. 16 h, 32 = 72%.
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the reported release of NO from furoxans and sydnonimines in
aqueous conditions, No measurable NO production was
observed from tyrosine.
Conclusion
The design and synthesis of a series of amino acids is demon-
strated, carrying the furoxan and sydnonimine ring systems.
The amino acids, with these heterocycles appended to their
side chains proved amenable to orthogonal deprotection and
synthesis strategies and they were successfully coupled cen-
trally into tripeptide sequences. Amino acids 5 and 7 are
shown to be exogenous pro-NO-release compounds based on
their nitrite production in the presence of GST. Thus incorpor-
ation of these amino acids into appropriate bioactive peptides
presents a strategy for the intracellular localization and target-
ing of NO delivery.
−
−
−
−
NO2
production
NO2
production
NO2
NO2
production
(μmol) (pH
7.6 buffer)
production
as % (pH
7.6 buffer)
(μmol) (GST as % (GST
Compound buffer)
buffer)
7
5
44.8 ( 3.2)
30.4 ( 3.9)
0.0 ( 0.1)
11.2
7.5
0
3.2 ( 1.1)
5.2 ( 5.5)
0.0 ( 0.1)
<1%
1.3%
0
Tyrosine
Scheme 9 Protecting group manipulation of amino acid 16. Reagents and
conditions: i. 4H HCl in dioxane, CH2Cl2, r.t., 1 h; ii. chloroformate, THF, Et3N, 0 °C
to r.t. 1 h, 85–90%; iii. NaOH, THF–H2O (1 : 1), r.t. 15 min, 80%.
Scheme 10 Protecting group manipulation of amino acid 17. Reagents and
conditions: i. 4H HCl in dioxane, CH2Cl2, r.t., 1 h; ii. chloroformate, THF, Et3N, 0 °C
to r.t. 1 h, 81–90%; iii. NaOH, THF–H2O (1 : 1), r.t. 15 min, 77–80%.
Scheme 11 Peptide synthesis with amino acid 31: Reagents and conditions: i. L-Val-OMe or L-Phe-OMe, HATU, DiPEA, DMF, 0 °C to r.t., 91–95%; ii. (a) 4H HCl in
dioxane, CH2Cl2, r.t., 1 h; iii. Fmoc-L-Arg(Pbf)-OH or Fmoc-L-Asp(OtBu)-OH, HATU, DiPEA, DMF, 0 °C to r.t., 80–88%.
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solution was extracted into EtOAc (3 × 20 mL), and the com-
bined organic extracts were washed with brine (20 mL) and
dried over Na2SO4. The mixture was filtered and the solvent
Experimental
General
All reagents and solvents were of highest grade from commer- removed under reduced pressure to yield the carboxylic acid.
cial sources, unless otherwise specified. Analytical thin layer
General procedure D for the synthesis of amino acids 5,
6 and 7
chromatography (TLC) experiments were performed on
0.20 mm glass-backed silica gel 60 F254 plates (MERCK). The
compounds were visualised using a 254 nm UV fluorescence Sodium hydroxide (2 equiv.) was added to a solution of pro-
lamp and visualised with potassium permanganate or cerium tected amino acid (1 equiv.) in THF (2 mL) and water (2 mL).
molybdate and heating with a heat gun. Flash column chrom- The resulting solution was stirred for 30 min and cooled to
atography was conducted using silica gel 60 as the stationary 0 °C. The solution was acidified (pH = 2) with aq HCl (2 N)
phase and solvents indicated. Melting points were determined with continued stirring. The cooled solution was extracted
in open capillary tubes using an Electrothermal Melting Point with EtOAc (3 × 20 mL), and the combined organic extracts
apparatus and are uncorrected. Infrared spectra were recorded were evaporated at reduced pressure. The residue was dis-
on a Perkin-Elmer Paragon series 1000 FTIR spectrometer. The solved in CH2Cl2 (4 mL) and cooled to 0 °C. HCl in dioxane
samples were prepared as thin films between NaCl plates. (4 M, ca. 30 equiv.) was added dropwise and the solution
Absorption maxima are given in wavenumbers (cm−1).
stirred at 0 °C for 1 h. The solvent was then evaporated under
NMR spectra were recorded on Bruker Advance 400 or 500 reduced pressure and the residue triturated with diethyl ether
instruments. 1H and 13C NMR spectra were recorded using to give the amino acid as its hydrochloride salt.
deuterated solvent as the lock and residual solvent as the
General procedure E for the synthesis of Fmoc and Alloc
amines, 16b, 16c, 17a and 17b
internal standard. Chemical shifts are reported in parts per
million (ppm) and coupling constants (J) are given in Hertz
(Hz). The abbreviations for the multiplicity of the proton, HCl in dioxane (4 M, ca. 40 equiv.) was added to a solution of
signals are as follows: s singlet, d doublet, dd doublet of doub- Boc-amino acid in CH2Cl2 cooled to 0 °C and stirred for 3 h.
lets, ddd doublet of doublet of doublets, triplet, dq double of Evaporation of the solvent under reduced pressure and tritura-
quartets, tt triplet of triplets, m multiplet, br s broad singlet. tion with Et2O furnished the desired amine HCl salt. The salt
When necessary, resonances were assigned using two-dimen- was suspended in THF (10–15 mL) and cooled to 0 °C. Chloro-
sional experiments (COSY, HMBC and HSQC). Mass analyses formate (1.5 equiv.) and triethylamine (3 equiv.) were added
were recorded on a Micromass LCT TOF or LTQ Orbitrap XL and the solution was stirred for 1 h and then the resultant sus-
mass spectrometer using ESI in positive mode or negative pension diluted with EtOAc (20 mL). The solution was washed
mode. Triethylamine and diisopropylethylamine were distilled with aqueous HCl (2 N, 30 mL) and brine (30 mL) and dried
from KOH and stored over KOH under argon.
over Na2SO4. The mixture was filtered and the solvent removed
under reduced pressure to yield a white residue which was dis-
solved in CH2Cl2 and evaporated onto silica gel. Purification
by silica gel chromatography gave the desired carbamate.
General procedure A for the synthesis of sydnonimine
carbamate or urea, 16, 16a, 20, 20a, and 21
Alcohol or amine (1 equiv.) and N-((4-nitrophenoxy)carbonyl)-
3-phenylsydnonimine (1 equiv.) were suspended in acetonitrile
General procedure F for the synthesis of peptides 37–44
and heated at reflux for 18 h. The solvent was removed under HATU (3 equiv.) was added to a solution of amine (1.1 equiv.)
reduced pressure and the residue dissolved in CH2Cl2 (10 mL) and acid (1 equiv.) in dry DMF (5 mL) cooled to 0 °C. The solu-
and evaporated onto silica gel. Silica gel chromatography pro- tion was stirred for 15 min and diisopropylethylamine
vided the desired sydnonimine carbamate or urea.
(4 equiv.) was added. The solution was allowed to warm to
room temperature and stirred for 18 h. The resultant solution
was diluted with EtOAc (50 mL) and water (50 mL). The
organic layer was separated and washed with aqueous HCl
General procedure B for the synthesis of amine
hydrochlorides, 21, 22, 25, 27 and 28
A solution of HCl in dioxane (4 M, ca. 40 equiv.) was added to (2 N, 20 mL), saturated NaHCO3 (20 mL) and brine (20 mL).
a solution of Boc-amino acid in cooled to 0 °C and CH2Cl2 and The organic layer was separated and dried over Na2SO4, filtered
stirred for 3 h. Evaporation of the solvent under reduced and the solvent removed under reduced pressure. The resul-
pressure and trituration with Et2O furnished the desired tant residue was dissolved in CH2Cl2 and evaporated onto
amine as its hydrochloride salt.
silica gel. Purification by silica gel chromatography provided
the desired peptide.
2-(Phenylamino)acetonitrile 12. Bromoacetonitrile (6.27 g,
3.64 mL, 52 mmol), potassium carbonate (8.3 g, 60.1 mmol)
General procedure C for the synthesis of carboxylic acids,
23, 24, 26, 33, 34, 35 and 36
Sodium hydroxide (2 equiv.) was added to a solution of methyl and sodium iodide (3.7 g, 24.7 mmol) were added to a solution
ester (1 equiv.) in THF–H2O (1 : 1) and stirred for 30 min. The of freshly distilled aniline 11 (4.55 g, 4.46 mL, 48.9 mmol) in
solution was cooled to 0 °C and acidified (pH = 2) with dry acetonitrile (100 mL) and the suspension stirred at room
aqueous HCl (2 N) with continued stirring. The cooled temperature for 12 h in the absence of light. The suspension
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was filtered and partitioned between Et2O (100 mL) and dis- provided 16 as a pale yellow crystalline solid (772 mg,
tilled water (100 mL). The organic layer was separated and 1.60 mmol, 89%). Rf 0.30 (50 : 50, EtOAc–PE, UV, cerium moly-
washed with distilled water (50 mL) and brine (50 mL), dried bdate stain). [α]2D0 +31.0 (c = 1.0, CHCl3). M.p. 58–60 °C. 1H
over Na2SO4, filtered and the solvent removed under reduced NMR (500 MHz; CDCl3) δ 8.17 (s, CH aromatic, 1H), 7.79 (d,
pressure to yield 3-(phenylamino)acetonitrile 12 as a colour- CH aromatic, J = 8.0 Hz, 2H), 7.71 (t, CH aromatic, J = 8.4 Hz,
less oil which crystallised on standing (5.62 g, 42.6, 87%): m.p. 1H), 7.65 (d, CH aromatic, J = 8.0 Hz, 2H), 7.13–7.09 (m, CH
40–42 °C, [Lit. 40 °C (Sakai)22]. 1H NMR (300 MHz; CDCl3) δ aromatic, 4H), 5.02 (d, NH Boc, J = 8.4 Hz, 1H), 4.55 (dd, α-H,
7.24–7.17 (m, CH aromatic, 2H), 6.81 (tt, CH aromatic, J = J = 13.7 Hz and 6.0 Hz, 1H), 3.69 (s, CO2CH3, 3H), 3.08 (dd,
8.4 Hz and 1.1 Hz, 1H), 6.65–6.61 (m, CH aromatic, 2H) CHAHB, J = 14.0 Hz and 5.7 Hz, 1H), 3.03 (dd, CHAHB, J =
and 3.99–3.95 (m, CH2, NH, 3H). 13C NMR (100 MHz; CDCl3) 13.8 Hz and 6.1 Hz, 1H), 1.40 (s, Boc CH3 × 3, 9H). 13C NMR
δ 145.3, 129.8, 120.2, 117.3, 113.8 and 32.8. m/z (ES+) 155 (125 MHz; CDCl3) δ 175.7, 172.4, 160.4, 155.2, 151.1, 133.7,
([M + Na]+, 100%).
133.3, 132.7, 130.7, 130.0, 121.9, 121.5, 103.4, 80.0, 54.4, 52.3,
3-Phenylsydnonimine hydrochloride 13. Isopentyl nitrite 37.6, 28.3. νmax (NaCl/thin film)/cm−1 1744, 1678, 1586, 1471,
(1.41 g, 1.6 mL, 0.012, 12.0 mmol) was added to a stirring solu- 1367, 1282, 1213, 971, 1018. m/z (ES+) 505 ([M + Na]+, 100%).
tion of 12 (660 mg, 5.0 mmol) in Et2O (5 mL) the resulting sus- HRMS m/z (ES+) calcd for C24H26N4NaO7 [M + Na]+ requires
pension was stirred for 16 h in the absence of light. Dry HCl 505.1699, found 505.1672.
(gas) was bubbled through the solution for 15 min. The sus-
(S)-N-((4-(2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxo-
pension was cooled in an ice bath for 15 min, filtered and propyl)phenyl)carbamoyl)-3-phenylsydnonimine 16a. General
dried under high vacuum to give 3-phenylsydnonimine. HCl procedure A was followed using 15 (186 mg, 0.632 mmol)
13 as a pale pink solid (927 mg, 4.70 mmol, 94%): m.p. Silica gel chromatography, eluting with CH2Cl2 and acetone
179–181 °C, [Lit. 180–181 °C (Daeniker)].23 1H NMR (400 MHz; (100 : 0 to 90 : 10) provided 16a as a yellow solid (243 mg,
d6-DMSO) δ 10.10 (s, NH·HCl, 2H), 8.71 (s, CH aromatic, 1H), 0.506 mmol, 88%). Rf 0.19 (50 : 50, EtOAc–PE, UV, cerium
8.06 (d, CH aromatic, J = 7.1, 2H), 7.86–7.77 (m, CH aromatic, molybdate stain). [α]2D0 +30.3, c = 1.0, CHCl3, m.p. 62–64 °C. 1H
3H). 13C NMR (100 MHz; d6-DMSO) δ 170.4, 134.5, 133.8, NMR (500 MHz; CDCl3) δ 8.25 (s, CH aromatic, 1H), 7.80–7.77
131.3, 123.6, 103.2. νmax (thin film)/cm−1 2365, 1684, 1653, (m, CH aromatic, 2H), 7.71–7.60 (m, CH aromatic, 3H), 7.45 (d,
1616, 1288, 1111. m/z (ES+) 162 ([M + H]+, 100%). HRMS m/z CH aromatic, J = 8.5 Hz, 2H), 7.28 (s, NH urea, 1H), 7.05–7.00
(ES+) calcd for C8H8N3O [M + H] requires 162.0667, found (m, CH aromatic, 2H), 5.10–5.01 (m, NH Boc, 1H), 4.53 (dd,
162.0663.
α-H, J = 13.3 Hz and 5.8 Hz, 1H), 3.69 (s, CO2CH3, 3H),
N-((4-Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9. Triethyl- 3.08–2.94 (m, CH2, 2H), 1.40 (s, Boc CH3 × 3, 9H). 13C NMR
amine (1.18 g, 1.65 mL, 11.7 mmol) was added dropwise to a (100 MHz; CDCl3) δ 173.4, 172.5, 159.4, 155.2, 138.5, 134.0,
stirring solution of 13 (1.00 g, 5.07 mmol) and p-nitrophenyl 133.0, 130.5, 130.1, 129.7, 121.5, 119.0, 103.4, 80.1, 54.5, 52.2,
chloroformate (1.53 g, 7.61 mmol) in CH2Cl2 (20 mL) at 37.7, 28.3. νmax (NaCl/thin film)/cm−1 1740, 1708, 1650, 1591,
−20 °C. The solution was allowed to warm to room tempera- 1517, 1411, 1365, 1244, 1166. m/z (ES+) 504 ([M + Na]+, 100%).
ture overnight with continued stirring and was partitioned HRMS m/z (ES+) calcd for C24H27N5NaO6 [M + Na]+ requires
between CH2Cl2 (100 mL) and distilled water (100 mL). The 504.1859, found 504.1861.
organic layer was separated and washed with distilled water
(S)-4-(4-(2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-
(50 mL) and brine (50 mL), dried over Na2SO4, filtered and the phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide 17. DBU
solvent removed in vacuo to give a pale yellow solid which was (544 μL, 3.64 mmol) was added to a solution of 14 (537 mg,
purified by silica gel chromatography (CH2Cl2–acetone; 100 : 0 1.82 mmol) in CH2Cl2 (10 mL) and the solution stirred vigor-
to 95 : 5) provide N-((4-nitrophenoxy)carbonyl)-3-phenylsydnoni- ously. Bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide 10 (666 mg,
mine 9 as a white amorphous solid (1.04 g, 3.19 mmol, 64%). 1.82 mmol) was added and stirring continued for 2 h. The
Rf 0.69 (90 : 10, CH2Cl2). M.p. (CH3CN) 198–200 °C. 1H NMR reaction mixture was washed sequentially with water (20 mL),
(400 MHz; CDCl3) δ 8.27 (d, CH aromatic, J = 9.0 Hz, 2H), 8.20 HCl (2 N, 2 × 15 mL) and brine (20 mL), and then the organic
(s, CH aromatic, 1H) 7.84 (d, CH aromatic, J = 8.4 Hz, 2H), layer was dried over Na2SO4. The mixture was filtered and the
7.79–7.76 (m, CH aromatic, 1H), 7.73–7.20 (m, CH aromatic, solvent removed under reduced pressure to give the product
2H) and 7.40 (d, CH aromatic, J = 9.0 Hz, 2H). 13C NMR which was purified by silica gel chromatography, eluting with
(100 MHz; CDCl3) δ 176.1, 159.3, 157.4, 146.9, 144.9, 133.9, CH2Cl2 and hexane (50 : 50 to 100 : 0) to give 17 as a white
131.1 125.3, 122.6, 121.8, 103.9. νmax (thin film)/cm−1 1700, solid (805 mg, 1.57 mmol, 86%). Rf 0.54 (30 : 70, EtOAc–PE,
1671, 1587, 1576, 1513, 1490, 1345, 1276, 1216, 1187, 976, 765. UV, KMnO4 stain). [α]2D0 +29.2 (c
m/z (ES+) 349 ([M + Na]+, 100%). HRMS m/z (ES+) calcd for 129–131 °C. 1H NMR (500 MHz; CDCl3) δ 8.06 (d, CH aromatic,
C15H10N4NaO5 [M + Na]+ requires 349.0549, found 349.0555.
J = 8.2 Hz, 2H), 7.76 (tt, CH aromatic, J = 7.5 Hz and 1.2, 1H),
= 1.0, CHCl3). M.p.
(S)-N-((4-(2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxo- 7.62 (t, CH aromatic, J = 8.2 Hz, 2H), 7.22–7.18 (m, CH aro-
3
propyl)phenyloxy)carbonyl)-3-phenylsydnonimine 16. General matic, 4H), 5.09 (d, NH Boc, J = 8.1 Hz, 1H), 4.57 (dd, α-H, J =
procedure A was followed using N-tert-butoxycarbonyl-(S)-tyro- 13.9 Hz and 6.5 Hz, 1H), 3.69 (s, CO2CH3, 3H), 3.14 (dd,
sine methyl ester 14 (532 mg, 1.80 mmol). Silica gel chromato- CHAHB, J = 13.9 Hz and 5.8 Hz, 1H), 3.03 (dd, CHAHB, J =
graphy, eluting with CH2Cl2 and acetone (100 : 0 to 90 : 10) 13.9 Hz and 6.3 Hz, 1H), 1.39 (s, Boc CH3 × 3, 9H). 13C NMR
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(125 MHz; CDCl3) δ 172.1, 158.3, 155.0, 151.6, 137.8, 135.9, ([M + H]+, 100%). HRMS m/z (ES+) calcd for C19H19N4O5
135.0, 130.9, 129.8, 128.6, 119.9, 110.7, 80.1, 54.4, 52.4, 37.7, [M + H]+ requires 383.1355, found 383.1360.
28.3. νmax (NaCl/thin film)/cm−1 1746, 1713, 1619, 1535, 1449,
(S)-N-((4-(2-Amino-3-methoxy-3-oxopropyl)phenyl)-carbamoyl)-
1366, 1275, 1200, 1167, 1019. m/z (ES+) 542 ([M + Na]+, 100%). 3-phenylsydnonimine hydrochloride 22. General procedure B
HRMS m/z (ES+) calcd for C23H25N3NaO9S [M + Na]+ requires was followed using (S)-N-((4-(2-((tert-butoxycarbonyl)amino)-3-
542.1209, found 542.1210.
methoxy-3-oxopropyl)phenyl)carbamoyl)-3-phenylsydnonimine
(S)-N-((2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropoxy)- 16a (100 mg, 0.208 mmol) and generating 22 (87 mg,
carbonyl)-3-phenylsydnonimine 20a. General procedure D Was 0.208 mmol, quant.) as an orange solid. [α]2D0 +5.2 (c = 0.8,
followed using N-(tert-butoxycarbonyl)-L-serine methyl ester 18 CH3OH). M.p. 160 °C (decomp.). 1H NMR (500 MHz; d6-
(250 mg, 1.141 mmol). Purification over silica, eluting with DMSO) δ 9.61 (s, NH urea, 1H), 9.11 (s, CH aromatic, 1H), 8.65
CH2Cl2 and acetone (100 : 0 to 90 : 10), gave 20a as a colourless (br, s, NH2·HCl, 3H), 8.13 (m, CH aromatic, 2H), 7.86–7.74 (m,
crystalline solid (272 mg, 0.670 mmol, 60%). Rf 0.24 (90 : 10, CH aromatic, 3H), 7.54 (d, CH aromatic, J = 8.3 Hz, 2H), 7.18
CH2Cl2–CH3OH, UV, cerium molybdate stain). [α]2D0 +17.9 (c = (d, CH aromatic, J = 8.3 Hz, 2H), 4.25–4.19 (m, α-H, 1H), 3.68
1
1.0, CHCl3). M.p. 40–41 °C. H NMR (300 MHz; CDCl3) δ 8.12 (s, CO2CH3, 3H) and 3.18–3.00 (m, CH2, 2H). 13C NMR
(s, CH aromatic, 1H), 7.81 (d, CH aromatic, J = 8.2 Hz, 2H), (75 MHz; d6-DMSO) δ 176.0, 169.4, 159.5, 139.2, 133.5, 133.4,
7.75–7.69 (m, CH aromatic, 3H), 5.52 (d, NH Boc, J = 8.4 Hz, 133.1, 130.4, 127.2, 122.6, 118.5, 106.2, 53.2, 52.6, 35.2. νmax
1H), 4.61–4.55 (m, CH2, 2H), 4.41–4.35 (m, α-H, 1H), 3.77 (s, (NaCl/thin film)/cm−1 3334, 1738, 1706, 1650, 1591, 1517,
CO2CH3, 3H), 1.44 (s, Boc CH3 × 3, 9H). 13C NMR (100 MHz; 1365, 1274, 1246, 1165. m/z (ES+) 382 ([M + H]+, 100%). HRMS
CDCl3) δ 175.1, 170.5, 160.1, 155.3, 133.6, 133.1, 130.5, 121.4, m/z (ES+) calcd for C19H20N5O4 [M + H]+ requires 382.1515,
103.0, 89.8, 65.2, 53.1, 52.5, 28.1. νmax (NaCl/thin film)/cm−1 found 382.1520.
2978, 1793, 1712, 1585, 1390, 1377, 1282, 1069, 972. m/z (ES+)
429 ([M + Na]+, 100%). HRMS m/z (ES+) calcd for C18H22N4- phenoxy)carbonyl)-3-phenylsydnonimine 23. General pro-
NaO7 [M + Na]+ requires 429.1386, found 429.1390.
cedure C was followed using 16 (100 mg, 0.207 mmol) and gen-
(S)-N-((4-(2-((tert-Butoxycarbonyl)amino)-2-carboxyethyl)-
(S)-N-((2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropoxy)- erating 23 as a white solid (92 mg, 0.197 mmol, 95%): Rf 0.63
carbamoyl)-3-phenylsydnonimine 20b. General procedure A (100 : 1, EtOAc–AcOH, UV, cerium molybdate stain). [α]2D0 −24.8
was followed using methyl 3-amino-2-(tert-butoxycarbonyl- (c = 0.1, CH3OH). M.p. 115–118 °C. 1H NMR (500 MHz; d6-
amino)propanoate 19 (250 mg, 1.141 mmol). Purification over DMSO) δ 8.62 (s, CH aromatic, 1H), 8.06 (d, CH aromatic, J =
silica gel, eluting with CH2Cl2 and acetone (100 : 0 to 90 : 10), 7.6 Hz, 2H), 7.80–7.70 (m, CH aromatic, 3H), 7.20 (d, CH aro-
gave 20b as a yellow crystalline solid (254 mg, 0.627 mmol, matic, J = 8.3 Hz, 2H), 7.10 (d, CH aromatic, J = 8.3 Hz, 2H),
55%). Rf 0.13 (95 : 5, CH2Cl2–acetone, UV, KMnO4). [α]2D0 +18.1, 6.51 (br, s, NH Boc, 1H), 4.01–3.96 (m, α-H, 1H), 3.06 (dd,
c = 1.0, CHCl3. M.p. 150 °C (decomp.). 1H NMR (500 MHz, CHAHB, J = 14.5 Hz and 4.9 Hz, 1H), 2.88 (dd, CHAHB, J = 13.4
CDCl3) δ 8.16 (s, CH aromatic, 1H), 7.79 (d, CH aromatic, J = Hz and 8.1 Hz, 1H) and 1.34 (s, Boc CH3 × 3, 9H). 13C NMR
8.8 Hz, 2H), 7.69 (t, CH aromatic, J = 7.3 Hz, 1H), 7.63 (t, CH (125 MHz; d6-DMSO) δ 174.9, 174.4, 158.0, 155.1, 150.3, 135.0.
aromatic, J = 7.8 Hz, 2H), 7.37 (br, s, NH-urea, 1H), 5.87 (t, NH 133.6, 133.0, 130.2, 129.9, 122.4, 121.2, 104.7, 77.7, 55.8, 36.2,
Boc, J = 7.2 Hz, 1H), 4.41–4.38 (m, α-H, 1H), 3.76–3.63 (m, CH2, 28.2. νmax (NaCl/thin film)/cm−1 1727, 1623, 1580, 1502, 1469,
CO2CH3, 5H) and 1.41 (s, Boc CH3 × 3, 9H). 13C NMR 1365, 1295, 1250, 1187, 1166. m/z (ES+) 491 ([M + Na]+, 100%).
(125 MHz, CDCl3)
δ
172.9, 171.6, 162.1, 155.6, 134.0, HRMS m/z (ES+) calcd for C23H24N4NaO5 [M + Na]+ requires
132.9, 130.5, 121.6, 102.4, 79.9, 54.8, 52.5, 42.2, 28.3. νmax 491.1543, found 491.1540.
(NaCl/thin film)/cm−1 1760, 1600, 1477, 1450, 1325, 1145,
(S)-N-((4-(2-((tert-Butoxycarbonyl)amino)-2-carboxyethyl)-
1032, 980. m/z (ES+) 428 ([M + Na]+, 100%). HRMS m/z (ES+) phenoxy)carbamoyl)-3-phenylsydnonimine 24. General pro-
calcd for C18H23N5NaO6 [M + Na]+ requires 428.1546, found cedure C was followed using 16a (100 mg, 0.208 mmol), and
428.1550.
generating 24 as a yellow solid (85 mg, 0.183 mmol, 88%): Rf
(S)-N-((4-(2-Amino-3-methoxy-3-oxopropyl)phenoxy)-carbonyl)- 0.19 (80 : 20, CH2Cl2–acetone, UV, cerium molybdate stain).
3-phenylsydnonimine hydrochloride 21. General procedure B [α]2D0 +8.0 (c = 1.0, CH3OH). M.p. 151–154 °C. 1H NMR
was followed using 16 (92 mg, 0.191 mmol) to generate 21 as a (500 MHz; CDCl3) δ 9.46 (s, NH urea, 1H), 8.55 (s, CH aromatic,
white solid (78 mg, 0.191 mmol, quant.). [α]2D0 +2.6 (c = 1.0, 1H), 7.86 (d, CH aromatic, J = 7.9 Hz, 2H), 7.72 (t, CH aromatic,
CH3OH). M.p. 117–119 °C. 1H NMR (500 MHz; d6-DMSO) J = 7.3 Hz, 1H), 7.66 (t, CH aromatic, J = 7.6 Hz, 2H), 7.36 (d,
δ 8.82–8.75 (m, CH aromatic, NH2·HCl, 4H), 8.09 (d, CH aro- CH aromatic, J = 8.0 Hz, 2H), 7.07 (d, CH aromatic, J = 8.0 Hz,
matic, J = 7.6 Hz, 2H), 7.83–7.71 (m, CH aromatic, 3H), 7.28 (d, 2H), 5.16 (d, NH Boc, J = 7.7, 1H), 4.78 (dd, α-H, J = 13.4 Hz
CH aromatic, J = 8.4 Hz, 2H), 7.13 (d, CH aromatic, J = 8.4 Hz, and 6.2 Hz, 1H), 3.15–3.05 (m, CH2, 1H), 1.45 (s, Boc CH3 × 3,
2H), 4.29–4.23 (m, α-H, 1H), 3.68 (s, CO2CH3, 3H), 3.22 (dd, 9H). 13C NMR (125 MHz; CDCl3) δ 176.8, 175.9, 155.8, 155.3,
CHAHB, J = 14.2 Hz and 5.7 Hz, 1H), and 3.12 (dd, CHAHB, J = 138.0, 133.5, 133.3, 130.6, 130.4, 129.9, 121.7, 119.0, 80.0, 54.8,
14.2 Hz and 7.1 Hz, 1H). 13C NMR (125 MHz; d6-DMSO) 37.7, 28.4. νmax (NaCl/thin film)/cm−1 1710, 1650, 1590, 1517,
δ 173.7, 169.3, 157.4, 150.9, 133.4, 133.2, 131.3, 130.3, 122.6, 1411, 1365, 1313, 1245, 1167, 963. m/z (ES+) 490 ([M + H]+,
121.8, 105.4, 53.1, 52.6, 35.1. νmax (NaCl/thin film)/cm−1 1780, 100%). HRMS m/z (ES+) calcd for C23H25N5NaO6 [M + H]+
1734, 1515, 1313, 1247, 1227, 1068, 868. m/z (ES+) 383 requires 490.1703, found 490.1711.
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(S)-N-((4-(2-Amino-2-carboxyethyl)phenyl)carbonyl)-3-phenyl- CH aromatic, 4H), 5.02 (d, NH Boc, J = 8.7, 1H), 4.62 (dd, α-H,
sydnonimine hydrochloride 5. General procedure D was fol- 3J = 11.9 Hz and 6.3 Hz, 1H), 3.24 (dd, CHAHB, J = 14.3 Hz and
lowed using 16 (100 mg, 0.207 mmol) and generating 5 as a 5.6 Hz, 1H), 3.09 (dd, CHAHB, J = 14.3 Hz and 6.6 Hz, 1H) and
white powder (65 mg, 0.160 mmol, 77%). [α]D20 −0.6 (c = 1.0, 1.42 (s, Boc CH3 × 3, 9H). 13C NMR (75 MHz; d6-DMSO) δ
CH3OH). M.p. 108–111 °C 1H NMR (500 MHz; CDCl3) δ 8.73 (s, 175.5, 158.4, 155.4, 151.6, 137.9, 135.9, 134.7, 131.0, 129.8,
CH aromatic, 1H), 8.51 (br, s, NH2·HCl, 3H), 8.03 (d, CH aro- 128.7, 120.0, 110.7, 80.6, 54.2, 37.2, 28.3. νmax (NaCl/thin film)/
matic, J = 8.0 Hz, 2H), 7.79 (tt, CH aromatic, J = 7.5 Hz and cm−1 1717, 1619, 1535, 1505, 1445, 1368, 1267, 1167, 1019. m/z
2.3 Hz, 1H), 7.74 (t, CH aromatic, J = 8.1 Hz, 2H), 7.31 (d, CH (ES+) 528 ([M + Na]+, 100%). HRMS m/z (ES+) calcd for
aromatic, J = 8.5 Hz, 2H), 7.12 (d, CH aromatic, J = 8.5 Hz, 2H), C22H23N3NaO9S [M + Na]+ requires 528.1053, found 528.2050.
4.16 (dd, α-H, 3J = 11.6 Hz and 5.9 Hz, 1H) and 3.15 (d, CH2, J =
(S)-4-(4-(2-Amino-2-carboxyethyl)phenoxy)-3-(phenylsulfonyl)-
6.3 Hz, 2H). 13C NMR (125 MHz; d6-DMSO) δ 174.6, 170.8, 1,2,5-oxadiazole 2-oxide 7. General procedure D was followed
158.3, 151.4, 133.9, 133.7, 132.0, 130.8, 130.8, 123.1, 122.3, using 17 (100 mg, 0.193 mmol) and providing 7 as a white
105.8, 55.4, 35.5. νmax (NaCl/thin film)/cm−1 3036, 1755, powder (68 mg, 0.154 mmol, 80%). [α]2D0 −0.52 (c = 0.7,
1629, 1597, 1544, 1507, 1368, 1256, 1216, 1193. m/z (ES+) 391 CH3OH). M.p. 250 °C (decomp). 1H NMR (300 MHz; d6-DMSO)
([M + Na]+, 100%). HRMS m/z (ES+) calcd for C18H16N4NaO5 δ 8.57 (br, s, NH2·HCl, 3H), 8.05 (d, CH aromatic, J = 8.4 Hz,
[M + Na]+ requires 391.1018, found 391.1022.
2H), 7.92 (tt, CH aromatic, J = 7.5 Hz and 2.2 Hz, 1H), 7.78 (t,
(S)-N-((4-(2-Amino-2-carboxyethyl)phenyl)carbamoyl)-3-phenyl- CH aromatic, J = 8.0 Hz, 2H), 7.42–7.36 (m, CH aromatic, 4H),
sydnonimine hydrochloride 6. General procedure D was fol- 4.08 (t, α-H, J = 7.2 Hz, 1H), 3.21 (d, CH2, J = 7.2, 2H). 13C NMR
lowed using 16a (100 mg, 0.208 mmol) and generating 6 as a (75 MHz; d6-DMSO) δ 174.6, 158.4, 152.1, 137.2, 135.4, 134.1,
yellow solid (63 mg, 0.156 mmol, 75%). [α]2D0 −1.6, c = 1.0, 131.7, 130.6, 129.0, 120.0, 108.8, 53.8, 35.3. νmax (NaCl/thin
MeOH. M.p. 200 °C (decomp). 1H NMR (300 MHz; d6-DMSO) δ film)/cm−1 1727, 1623, 1534, 1449, 1352, 1256, 1201, 1163,
9.59 (s, NH urea, 1H), 9.11 (s, CH aromatic, 1H), 8.42 (br, s, 1083. m/z (ES+) 406 ([M + H]+, 100%). HRMS m/z (ES+) calcd for
NH2·HCl, 3H), 8.13 (m, CH aromatic, 2H), 7.83 (tt, CH aro- C17H16N3O7S [M + H]+ requires 406.0709, found 406.0713.
matic, J = 7.5 Hz and 1.5 Hz, 1H), 7.76 (d, CH aromatic, J =
(S)-N-((2-Amino-3-methoxy-3-oxopropoxy)carbonyl)-3-phenyl-
7.8 Hz, 2H), 7.54 (d, CH aromatic, J = 8.5 Hz, 2H), 7.16 (d, CH sydnonimine hydrochloride 27. General procedure B was fol-
aromatic, J = 8.5 Hz, 2H), 4.10–4.07 (m, α-H, 1H) and 3.11–3.06 lowed using 20a (100 mg, 0.246 mmol), and providing 27
(m, CH2, 2H). 13C NMR (75 MHz; d6-DMSO) δ 174.8, 170.3, (84 mg, 0.246 mmol, quant.) as an off-white solid. [α]D20 +9.4
152.7, 139.0, 134.1, 133.5, 130.4, 130.0, 129.8, 123.3, 117.8, (c = 0.9, CH3OH). M.p. 102–104 °C. 1H NMR (300 MHz; d6-
106.2, 53.2, 35.8. νmax (NaCl/thin film)/cm−1 1700, 1648, 1520, DMSO) δ 8.95 (br, s, NH2·HCl, 3H), 8.78 (s, CH aromatic, 1H),
1409, 1360, 1240, 970. m/z (ES+) 390 ([M + H]+, 100%). HRMS 8.12–8.04 (m, CH aromatic, 2H), 7.83–7.71 (m, CH aromatic,
m/z (ES+) calcd for C23H25N5NaO6 [M + H]+ requires 390.1178, 3H), 4.54–4.40 (m, CH2, α-H, 3H), 3.78 (s, CO2CH3, 3H).
found 490.1711.
13C NMR (100 MHz; d6-DMSO) δ 173.1, 167.6, 157.5, 133.3,
(S)-4-(4-(2-Amino-3-methoxy-3-oxopropyl)phenoxy)-3-(phenyl- 130.3, 122.5, 105.4, 62.8, 53.1, 51.5. νmax (NaCl/thin film)/cm−1
sulfonyl)-1,2,5-oxadiazole 2-oxide hydrochloride 25. General 1682, 1583, 1519, 1471, 1366, 1313, 1213 1187, 969. m/z (ES+)
procedure B was followed using 17 (100 mg, 0.193), and gener- 307 ([M + Na]+, 100%). HRMS m/z (ES+) calcd for C13H15N4O5
ating 25 as a white solid (87 mg, 0.193 mmol, quant.): [M + Na]+ requires 307.1042, found 307.1049.
[α]2D0 +9.3 (c = 0.9, CH3OH). M.p. 174–177 °C. 1H NMR (300 MHz;
(S)-N-((4-(2-Amino-3-methoxy-3-oxopropyl)phenoxy)-carbamoyl)-
d6-DMSO) δ 8.72 (br, s, NH2·HCl, 3H), 8.05 (d, CH aromatic, J = 3 phenylsydnonimine hydrochloride 28. General procedure B
8.1 Hz, 2H), 7.92 (tt, CH aromatic, J = 7.5 Hz and 1.5 Hz, 1H), was followed with 20b (100 mg, 0.246 mmol) generating 28 as
7.77 (d, CH aromatic, J = 7.9 Hz, 2H), 7.41–7.35 (m, CH aro- an off-white solid (84 mg, 0.246 mmol, quant.). [α]2D0 +11.0 (c =
1
matic, 4H), 4.31 (t, α-H, J = 6.1 Hz, 1H), 3.68 (s, CO2CH3, 3H), 1.0, CH3OH). M.p. 150–152 °C. H NMR (500 MHz, d6-DMSO)
3.23 (dd, CHAHB, J = 14.2 Hz and 6.1 Hz, 1H) and 3.14 (dd, δ 9.25 (1H, s, CH-7), 8.89–8.79 (br, s, NH2·HCl, 3H), 8.14 (d, CH
CHAHB, J = 14.2 Hz and 7.3 Hz, 1H). 13C NMR (75 MHz; d6- aromatic, J = 7.7 Hz, 2H), 7.84–7.77 (m, CH aromatic, 3H),
DMSO) δ 169.3, 158.4, 151.8, 136.9, 136.3, 133.1, 131.1, 130.1, 4.14–4.09 (br, s, α-H, 1H) and 3.75–3.71 (m, CH2, CO2CH3, 5H).
128.5, 119.8, 111.2, 53.0, 52.6, 35.0. νmax (NaCl/thin film)/cm−1 13C NMR (125 MHz, d6-DMSO) δ 176.5, 168.8, 167.7, 134.3,
1742, 1617, 1503, 1357, 1275, 1275, 1161, 1017, 990. m/z (ES+) 133.3, 130.9, 123.4, 107.5, 53.0, 52.2, 39.9. νmax (NaCl/thin
420 ([M + H]+, 100%). HRMS m/z (ES+) calcd for C18H18N3O7S film)/cm−1 1744, 1609, 1546, 1514, 1495, 1416, 1315, 1237,
[M + H]+ requires 420.0865, found 420.0870.
1050, 823. m/z (ES+) 306 ([M + Na]+, 100%). HRMS m/z (ES+)
(S)-4-(4-(2-((tert-Butoxycarbonyl)amino)-2-carboxyethyl)-phenoxy)- calcd for C13H16N5O6 [M + H]+ requires 306.1202, found
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide 26. General pro- 306.1220.
cedure C was followed using 17 (100 mg, 0.193 mmol) and gen-
(S)-N-((3-(tert-Butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxo-
erating 26 as a white solid (92 mg, 0.197 mmol, 90%): Rf 0.48 propoxy)carbonyl)-3-phenylsydnonimine 31. General pro-
(100 : 1, EtOAc–AcOH, UV, KMnO4 stain): [α]2D0 +6.4 (c = 1.0, cedure A was followed using 30 (200 mg, 0.766 mmol).
1
CH3OH). M.p. 66–68 °C. H NMR (500 MHz; CDCl3) δ 8.10 (d, Purification over silica gel, eluting with CH2Cl2 and acetone
CH aromatic, J = 8.4 Hz, 2H), 7.89 (tt, CH aromatic, J = 6.7 and (100 : 0 to 90 : 10) gave 31 as an orange gum (206 mg,
1.2 Hz, 1H), 7.64 (t, CH aromatic, J = 7.7 Hz, 2H), 7.29–7.25 (m, 0.459 mmol, 60%). Rf 0.21 (95 : 5, CH2Cl2–acetone, UV, cerium
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molybdate stain). [α]2D0 +34.6 (c = 1.0, CHCl3). 1H NMR silica gel, eluting with CH2Cl2 and acetone (100 : 0 to 90 : 10)
(300 MHz; CDCl3) δ 8.17 (s, CH aromatic, 1H), 7.83 (d, CH gave 16b as a white crystalline solid (361 mg, 0.598 mmol.
aromatic, J = 8.6 Hz, 2H), 7.72 (tt, CH aromatic, J = 8.7 Hz and 90%). Rf 0.58 (90 : 10, CH2Cl2–acetone, UV, cerium molybdate
1.2 Hz, 1H) 7.66 (t, CH aromatic, J = 7.9 Hz, 2H), 5.51 (d, NH stain). [α]2D0 +33.7 (c = 1.0, CHCl3). M.p. 74–76 °C. 1H NMR
Boc, J = 8.7, 1H), 4.55 (dd, CHAHB, J = 10.9 Hz and 3.8 Hz, 1H), (400 MHz, CDCl3) δ 8.15 (CH aromatic, 1H), 7.80–7.65 (m, CH
4.46 (m, α-H, 1H), 4.31 (dd, CHAHB J = 10.9 Hz and 3.2 Hz, Fmoc, CH aromatic, 7H) 7.59 (d, CH Fmoc, J = 7.6 Hz, 2H),
1H), 1.45 (s, Boc CH3 × 3, 9H), 1.43 (s, tBu ester CH3 × 3, 9H). 7.40 (t, CH Fmoc, J = 7.5 Hz, 2H), 7.31 (tt, CH Fmoc, J = 7.5 Hz
13C NMR (100 MHz; CDCl3) δ 174.9, 169.1, 160.5, 155.5, 133.7, and 1.0 Hz, 2H), 7.16 (d, CH aromatic, J = 8.6 Hz, 2H), 7.09 (d,
133.3, 130.6, 121.4, 103.1, 82.4, 79.7, 65.9, 53.7, 28.3, 28.1. νmax CH aromatic, J = 8.6 Hz, 2H) 5.29 (d, NH Fmoc, J = 8.0 Hz, 1H),
(NaCl/thin film)/cm−1 1722, 1589, 1500, 1458, 1368, 1152, 4.67 (ddd, α-H, J = 13.6 Hz, 8.0 Hz and 5.6 Hz, 1H), 4.45 (dd,
1030, 972, 846. m/z (ES+) 471 ([M + Na]+, 100%). HRMS m/z Fmoc CHAHB, J = 10.6 Hz and 7.2 Hz, 1H), 4.36 (dd, Fmoc
(ES+) calcd for C21H28N4NaO7 [M + Na]+ requires 471.1856, CHAHB, J = 10.0 Hz and 6.8 Hz, 1H), 4.22 (t, CH Fmoc, J =
found 471.1862.
7.1 Hz, 1H), 3.74 (s, CO2CH3, 3H) and 3.12 (d, CH2, J = 5.7 Hz,
(S)-N-(((2-Amino-2-carboxyethoxy)carbonyl)-3-phenylsydnoni- 2H). 13C NMR (100 MHz, CDCl3) δ 175.7, 171.9, 160.7,
mine trifluoroacetate 8. Trifluoroacetic acid (2 mL) was added 155.7, 151.2, 143.9, 143.8, 141.3, 133.7, 133.3, 132.5, 130.7,
to a solution of 31 (200 mg, 0.447 mmol) in CH2Cl2 (2 mL) at 130.1, 127.7, 127.1, 125.2, 125.1, 122.0, 121.6, 120.0, 103.4,
0 °C. The resultant solution was stirred for 18 h and the 67.0, 54.8, 52.4, 47.2, 37.5. νmax (NaCl/thin film)/cm−1 1742,
solvent evaporated to yield 8 (183 mg, 0.447 mmol, quant.) as 1720, 1675, 1587, 1508, 1471, 1450, 1366, 1281, 1213, 1190,
an orange gum. Rf 0.14 (90 : 10, CH2Cl2–acetone, UV, KMnO4). 1084. m/z (ES+) 627 ([M + Na]+, 100%). HRMS m/z (ES+) calcd
[α]2D0 +3.9 (c = 1.0, CH3OH). 1H NMR (400 MHz, d6-DMSO) for C34H28N4NaO7 [M
δ 8.62 (s, CH aromatic, 1H), 8.51 (br, s, NH2·CF3CO2H, 627.1841.
+
Na]+ requires 627.1856, found
3H), 8.08 (d, CH aromatic, J = 8.0 Hz, 2H), 7.81–7.72 (m,
(S)-N-((4-(2-((Allyloxycarbonyl)amino)-3-methoxy-3-oxopropyl)-
CH aromatic, 3H) and 4.42–4.31 (m, CH2, α-H, 3H). 13C NMR phenyloxy)carbonyl)-3-phenylsydnonimine 16c. General pro-
(100 MHz, d6-DMSO) δ 174.5, 174.2, 168.8, 159.1, 133.5, cedure E was followed using 16 (100 mg, 0.208 mmol), allyl
130.7, 130.2, 122.4, 118.4, 104.5, 62.8, 51.7. νmax (NaCl/ chloroformate (35 mg, 0.311 mmol) and triethylamine (63 mg,
thin film)/cm−1 1736, 1566, 1487, 1477, 1360, 1010, 956, 90 μL, 0.622 mmol). Purification over silica gel eluting with
822. m/z (ES+) 315 ([M + Na]+, 100%). HRMS m/z (ES+) calcd CH2Cl2 and acetone (100 : 0 to 90 : 10) gave 16c as a white solid
for C12H12N4NaO5 [M
315.0700.
+
Na]+ requires 315.0705, found (82 mg, 0.176, 85%). Rf 0.62 (90 : 10, CH2Cl2–acetone). [α]2D0
+23.7 (c = 1.0, CHCl3). M.p. 101–104 °C. 1H NMR (500 MHz,
(S)-N-((2-((tert-Butoxycarbonyl)amino)-3-oxopropoxy)-carbonyl)- CDCl3) δ 8.16 (s, CH aromatic, 1H), 7.80 (d, CH aromatic, J =
3-phenylsydnonimine 32. Boc2O (134 mg, 0.616 mmol) and 8.4 Hz, 2H), 7.76–7.64 (m, CH aromatic, 3H), 7.17–7.10 (m, CH
NaHCO3 (52 mg, 0.616 mmol) were added to a solution of 8 aromatic, 4H), 5.96–5.82 (m, CH Alloc, 1H), 5.29 (d, CH
(100 mg, 0.246 mmol) in H2O–dioxane (1 : 1, 2 mL) and stirred Alloc, J = 17.1, 1H), 5.21 (d, CH Alloc, J = 10.1, 1H), 4.65
for 12 h at room temperature. The solution was extracted into (dd, α-H, J = 13.7 Hz and 6.0 Hz, 1H), 4.56 (d, CH2 Alloc, J =
Et2O (2 mL) and the aqueous layer separated. The solution was 5.6 Hz, 2H), 3.72 (s, CO2CH3, 3H) and 3.11 (d, CH2, J =
cooled to 0 °C and acidified (pH = 2) with aqueous HCl (2 N) 5.5, 2H). 13C NMR (125 MHz, CDCl3) δ 175.7, 172.0, 160.4,
with continued stirring. The cooled solution was extracted into 155.6, 151.1, 133.7, 133.4, 132.6, 132.4, 130.7, 130.0, 122.0,
EtOAc (3 × 2 mL), and the combined organic extracts were 121.5, 117.7, 103.2, 65.8, 54.7, 52.4, 37.5. νmax (NaCl/thin film)/
washed with brine (2 mL) and dried over Na2SO4. The mixture cm−1 1718, 1585, 1508, 1495, 1448, 1403, 1368, 1260, 1213,
was filtered and the solvent removed under reduced pressure 1051, 941. m/z (ES+) 489 ([M + Na]+, 100%). HRMS m/z (ES+)
to yield 32 as an orange gum (70 mg, 0.177 mmol, 72%). Rf calcd for C23H22N4NaO7 [M + Na]+ requires 489.1386, found
0.22 (100 : 1, EtOAc–AcOH, UV, KMnO4). [α]2D0 +27.2 (c = 0.5, 489.1392.
1
CH3OH). H NMR (500 MHz, CD3CN) δ 8.68 (s, CH aromatic,
(S)-N-(((4-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-
1H), 8.01 (d, CH aromatic, J = 8.7 Hz, 2H), 7.81–7.72 (m, CH carboxyethyl)phenoxy)carbonyl)-3-phenylsydnonimine 33.
aromatic, 3H) and 4.59–4.352 (m, CH2, α-H, 3H), 1.53 (s, Boc General procedure C was followed using 16b (218 mg,
CH3 × 3, 9H). 13C NMR (125 MHz, CD3CN) δ 171.7, 170.5, 0.360 mmol). The residue was dissolved in CH2Cl2 and evapor-
168.6, 153.3, 134.4, 133.0, 130.7, 122.8, 107.5, 85.5, 61.9, 55.5, ated onto silica gel. Purification by silica gel chromatography,
27.5. νmax (NaCl/thin film)/cm−1 1751, 1710, 1630, 1557, 1494, eluting with CH2Cl2 and MeOH (97 : 3) furnished 33 as a pale
1451, 1403, 1216, 1050. m/z (ES+) 415 ([M + Na]+, 100%). HRMS yellow solid (170 mg, 0.289 mmol, 80%): Rf 0.29 (90 : 10,
m/z (ES+) calcd for C17H20N4NaO7 [M + Na]+ requires 415.1230, CH2Cl2–acetone, UV, cerium molybdate stain). [α]2D0 +5.8 (c =
1
found 415.1236.
0.5, CH3OH). M.p. 148–150 °C (decomp.) H NMR (500 MHz,
(S)-N-(((4-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3- d6-DMSO) δ 8.59 (s, CH aromatic, 1H), 8.05 (d, CH aromatic,
methoxy-3-oxopropyl)phenoxy)carbonyl)-3-phenylsydnonimine J = 7.8 Hz, 2H), 7.87 (d, CH Fmoc, J = 6.7 Hz, 2H), 7.77 (tt,
16b. General procedure E was followed using 16 (320 mg, CH aromatic, J = 7.5 Hz and 2.5 Hz, 1H), 7.71 (t, CH aromatic,
0.664 mmol), Fmoc chloroformate (258 mg, 0.994 mmol) and J = 8.0 Hz, 2H), 7.66 (t, CH Fmoc, J = 7.9 Hz, 2H),
triethylamine (201 mg, 280 μL, 2.00 mmol). Purification over 7.41–7.37 (m, CH Fmoc, 2H), 7.33–7.28 (m, CH Fmoc, 2H),
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7.23 (d, CH aromatic, J = 8.2 Hz, 2H), 6.99 (d, CH aromatic, J = 1357, 1260, 1165. m/z (ES+) 664 ([M + Na]+, 100%). HRMS m/z
8.2 Hz, 2H), 4.27–4.23 (m, Fmoc CHAHB, 1H), 4.20–4.15 (m, (ES+) calcd for C33H27N3NaO9S [M + Na]+ requires 664.1366,
Fmoc CHAHB, CH Fmoc, 2H), 4.07 (dd, α-H, J = 13.6 Hz and found 664.1359.
8.6 Hz, 1H), 3.11 (dd, CHAHB, J = 13.6 Hz and 4.0 Hz, 1H) and
2.90 (dd, CHAHB, J = 13.3 Hz and 9.0 Hz, 1H). 13C NMR phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide 17b.
(125 MHz, d6-DMSO) δ 174.9, 173.4, 158.7, 155.6, 150.3, 143.8, General procedure was followed using 17 (227 mg,
(S)-4-(4-(2-((Allyloxycarbonyl)amino)-3-methoxy-3-oxopropyl)-
E
140.6, 135.0, 133.6, 133.0, 130.2, 129.9 127.6, 127.0, 125.3, 0.437 mmol), allyl chloroformate (80 mg, 70 μL, 0.655 mmol)
125.2, 122.4, 121.2, 120.0, 104.7, 65.4, 56.3, 46.6, 36.3. νmax and triethylamine (132 mg, 180 μL, 1.31 mmol). Purification
(NaCl/thin film)/cm−1 1712, 1584, 1508, 1471, 1450, 1368, over silica gel eluting with EtOAc and hexanes (0 : 100 to
1296, 1212, 982. m/z (ES+) 613 ([M + Na]+, 100%). HRMS m/z 15 : 85) provided 17b as a white solid (178 mg, 0.354 mmol,
(ES+) calcd for C33H26N4NaO7 [M + Na]+ requires 613.1699, 85%). Rf 0.62 (50 : 50, EtOAc–petrol). [α]2D0 +29.3 (c = 1.0,
1
found 613.1677.
CHCl3). M.p. 78–80 °C. H NMR (500 MHz, CDCl3) δ 8.09 (d,
(S)-N-((4-(2-((Allyloxy)amino)-2-carboxyethyl)-phenoxy)carbonyl)- CH aromatic, J = 8.3 Hz, 2H), 7.79 (tt, CH aromatic, J = 6.7, 1.2
3-phenylsydnonimine 34. General procedure C was followed Hz, 1H), 7.64 (t, CH aromatic, J = 7.9 Hz, 2H), 7.31–7.22 (m, CH
using 16c (50 mg, 0.107 mmol) to generate 34 as a white solid aromatic, 4H), 5.94–5.82 (m, CH Alloc, 1H), 5.28 (dd, CH Alloc,
(39 mg, 0.09 mmol, 80%). Rf 0.28 (90 : 10, CH2Cl2–acetone, UV, J = 17.2 Hz and 1.2 Hz, 1H), 5.21 (dd, CH Alloc, J = 10.2 Hz and
KMnO4). [α]2D0 +26.0 (c = 0.5, CH3OH). M.p. 113–116 °C. 1H 1.2 Hz, 1H), 4.68–4.35 (m, CH2 Alloc, α-H, 3H), 3.72 (s,
NMR (500 MHz, CD3CN) δ 8.57 (s, CH aromatic, 1H), 8.06 (d, CO2CH3, 3H), 3.21–3.05 (m, CH2, 2H). 13C NMR (125 MHz,
CH aromatic, J = 7.6 Hz, 2H), 7.93 (t, CH aromatic, J = 7.4 Hz, CDCl3) δ 171.7, 158.3, 155.5, 151.6, 137.9, 135.9, 134.7, 132.5,
1H), 7.85 (t, CH aromatic, J = 8.2 Hz, 2H), 7.41 (d, CH aromatic, 130.8, 129.8, 128.6, 119.9, 118.0, 110.7, 65.9, 54.7, 52.5, 37.7.
J = 8.4 Hz, 2H), 7.24 (d, CH aromatic, J = 8.4 Hz, 2H), 6.06–5.92 νmax (NaCl/thin film)/cm−1 1745, 1614, 1536, 1505, 1449, 1439,
(m, CH Alloc, NH Alloc, 2H), 5.38 (d, CH Alloc, J = 17.7 Hz, 1314, 1257, 1211, 1167, 1019. m/z (ES+) 526 ([M + Na]+, 100%).
1H), 5.29 (d, CH Alloc, J = 10.3, 1H), 4.63–4.57 (m, CH2 Alloc, HRMS m/z (ES+) calcd for C22H21N3NaO9S [M + Na]+ requires
2H), 4.54 (ddd, α-H, J = 14.0 Hz, 9.0 Hz, and 5.0 Hz, 1H), 3.33 526.0896, found 526.0902.
(dd, CHAHB, J = 14.0 Hz and 5.4 Hz, 1H), 3.08 (dd, CHAHB, J =
(S)-4-(4-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-
14.0 Hz and 9.0 Hz, 1H). 13C NMR (125 MHz, CD3CN) δ 173.0, oxopropyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide
172.0, 163.2, 155.9, 151.6, 136.2, 133.3, 130.2, 133.6, 133.5, 35. General procedure C was followed using 17a (226 mg,
130.5, 122.5, 121.7, 116.6, 102.7, 65.2, 55.2, 36.3. νmax (NaCl/ 0.352 mmol). The product was uptaken in CH2Cl2 and evapor-
thin film)/cm−1 1647, 1605, 1580, 1494, 1451, 1400, 1256, ated onto silica gel. Purification by silica gel chromatography,
1200, 1050. m/z (ES+) 475 ([M + Na]+, 100%). HRMS m/z (ES+) eluting with CH2Cl2 and MeOH (97 : 3) furnished 35 as a
calcd for C22H20N4NaO7 [M + Na]+ requires 475.1230, found white solid (177 mg, 0.281 mmol, 80%). Rf 0.33 (100 : 1,
475.1238.
EtOAc–AcOH, UV, cerium molybdate stain). [α]2D0 +3.9 (c =
1
(S)-4-(4-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3- 0.5, CH3OH). M.p. 189–191 °C. H NMR (500 MHz, d6-DMSO)
methoxy-3-oxopropyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxa- δ 8.04 (d, CH aromatic, J = 7.7 Hz, 2H), 7.90 (t, CH aromatic,
diazole 2-oxide 17a. General procedure E was followed using J = 7.4 Hz, 1H), 7.86 (d, CH Fmoc, J = 7.5 Hz, 2H), 7.79–7.74
17 (500 mg, 0.963 mmol), Fmoc chloroformate (374 mg, (m, CH Fmoc, NH Fmoc, 3H), 7.63 (t, CH Fmoc, J = 7.7 Hz,
1.445 mmol) and triethylamine (292 mg, 410 μL, 2.00 mmol). 2H), 7.41–7.34 (m, CH aromatic, CH Fmoc, 4H), 7.32–7.27
Purification over silica gel eluting with EtOAc and hexanes (m, CH aromatic, CH Fmoc, 4H), 4.22–4.15 (m, CH2 Fmoc, CH
(0 : 100 to 15 : 85) provided 17a as a white solid (556 mg, Fmoc, α-H, 4H), 3.13 (dd, CHAHB, J = 13.9 Hz and 4.4 Hz, 1H)
0.887 mmol, 90%): Rf 0.50 (50 : 50, EtOAc–PE, UV, cerium moly- and 3.13 (dd, CHAHB, J = 13.6 Hz and 5.9 Hz, 1H). 13C
1
bdate stain). [α]2D0 +25.4, (c = 1.0, CHCl3). M.p. 141–143 °C. H NMR (125 MHz, d6-DMSO) δ 173.7, 159.0, 156.5, 151.7,
NMR (500 MHz, CDCl3) δ 8.10 (d, CH aromatic, J = 8.3 Hz, 2H), 144.2, 141.2, 137.9, 136.8, 131.1, 130.5, 129.0, 128.1, 127.6,
7.79–7.76 (m, CH Fmoc, CH aromatic, 3H), 7.63 (t, CH aro- 125.7, 120.6, 119.9, 111.7, 66.1, 55.8, 47.0, 36.2. νmax (NaCl/
matic, J = 8.2 Hz, 2H), 7.57 (dd, CH Fmoc, J = 7.4 and 4.4 Hz, thin film)/cm−1 1689, 1623, 1537, 1501, 1446, 1353, 1161,
2H), 7.40 (t, CH Fmoc, J = 7.2 Hz, 2H), 7.32 (t, CH Fmoc, J = 1022. m/z (ES+) 650 ([M + Na]+, 100%). HRMS m/z (ES+) calcd
7.6 Hz, 2H). 7.22 (d, CH aromatic, J = 8.5 Hz, 2H), 7.15 (d, CH for C32H25N3NaO9S [M + Na]+ requires 650.1209, found
aromatic, J = 8.5 Hz, 2H), 5.34 (d, NH Fmoc, J = 8.1 Hz, 1H), 650.1215.
4.67 (dd, α-H, J = 13.6 Hz and 6.0 Hz, 1H), 4.47 (dd, Fmoc
(S)-4-(4-(2-((Allyloxycarbonyl)amino)-2-carboxyethyl)phenoxy)-
CHAHB, J = 10.6 Hz and 7.2 Hz, 1H), 4.37 (dd, Fmoc CHAHB, J = 3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide 36. General pro-
10.6 Hz and 6.7 Hz, 1H), 4.20 (t, CH Fmoc, J = 7.2 Hz, 1H), 3.73 cedure C was followed using 17b (100 mg, 0.199 mmol) and
(3H, s, CH2−18), 3.18 (dd, CHAHB, J = 14.0 Hz and 5.8 Hz, 1H), generating 36 as a white solid (78 mg, 0.159 mmol, 90%). Rf
3.09 (dd, CHAHB, J = 14.0 Hz and 6.2 Hz, 1H). 13C NMR 0.41 (100 : 1, EtOAc–AcOH, UV, KMnO4). [α]2D0 +23.5 (c = 0.7,
1
(125 MHz, CDCl3) δ 171.7, 158.3, 155.6, 151.6, 144.0, 143.7, CH3OH). M.p. 138–141. H NMR (500 MHz, CD3CN) δ 8.18 (d,
141.3, 137.9, 135.9, 130.9, 129.8, 128.7, 127.8, 127.1, 125.1, CH aromatic, J = 7.9 Hz, 2H), 7.98 (t, CH aromatic, J = 7.6 Hz,
125.0, 120.1, 119.9, 110.7, 66.9, 54.7, 52.5, 47.2, 37.7. νmax 1H), 7.83 (t, CH aromatic, J = 7.9 Hz, 2H), 7.47–7.43 (m, CH
(NaCl/thin film)/cm−1 1746, 1692, 1625, 1539, 1506, 1450, aromatic, 2H) 7.36 (d, CH aromatic, J = 7.5 Hz, 2H), 6.05–5.95
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(m, CH Alloc, NH Alloc, 2H), 5.36 (d, CH Alloc, J = 17.1 Hz, 1438, 1368, 1215, 1167, 1050. m/z (ES+) 652 ([M + Na]+, 100%).
1H), 5.28 (d, CH Alloc, J = 10.7 Hz, 1H), 4.59 (d, CH2 Alloc, J = HRMS m/z (ES+) calcd for C33H35N5NaO8 [M + Na]+ requires
5.3 Hz, 2H), 4.54 (ddd, α-H, J = 15.0 Hz, 9.1 Hz and 5.0 Hz, 652.2383, found 652.2390.
1H), 3.34 (dd, CHAHB, J = 15.0 Hz and 5.0 Hz, 1H) and 3.09
Dipeptide 39. General procedure F was followed using
(dd, CHAHB, J = 15.0 Hz and 9.0 Hz, 1H). 13C NMR (125 MHz, HATU (113 mg, 0.297 mmol), valine methyl ester hydrochlo-
CD3CN) δ 172.2, 158.9, 155.5, 151.7, 137.6, 136.1, 136.0, 133.2, ride (18 mg, 0.109 mmol), 26 (50 mg, 0.099 mmol) and diiso-
131.0, 129.9, 128.7, 119.9, 116.6, 111.3, 65.1, 54.9, 36.2. νmax propylethylamine (distilled, 51 mg, 70 μL, 0.296 mmol).
(NaCl/thin film)/cm−1 2525, 1658, 1537, 1451, 1404, 1260, Purification over silica gel, eluting with EtOAc and hexanes
1201, 1112, 1020. m/z (ES+) 512 ([M + Na]+, 100%). HRMS m/z (10 : 90 to 25 : 75) generated dipeptide 39 as a white solid
(ES+) calcd for C21H19N3NaO9S [M + Na]+ requires 512.0740 (58 mg, 0.094 mmol, 95%). Rf 0.60 (90 : 10, CH2Cl2–MeOH, UV,
found 512.0745.
cerium molybdate stain). [α]2D0 −9.2 (c = 0.1, CHCl3). M.p.
Dipeptide 37. General procedure F was followed using 126–128 °C. 1H NMR (500 MHz, CDCl3) δ 8.15–8.09 (d, CH aro-
HATU (442 mg, 1.16 mmol), valine methyl ester hydrochloride matic, J = 8.3 Hz, 2H), 7.78 (t, CH aromatic, J = 7.6 Hz, 1H),
(49 mg, 0.352 mmol), 23 (150 mg, 0.320 mmol) and diisopro- 7.67–7.63 (m, CH aromatic, 2H), 7.30–7.26 (m, CH aromatic,
pylethylamine (distilled, 200 mg, 270 μL, 1.55 mmol). Purifi- 2H), 7.23–7.19 (m, CH aromatic, 2H), 6.45–6.36 (m, NH amide,
cation over silica gel eluting with CH2Cl2 and MeOH (100 : 0 to 1H), 5.05 (br, s, NH Boc, 1H), 4.46 (dd, Val α-H, J = 8.8 Hz and
97 : 3) generated dipeptide 37 as a white solid (169 mg, 5.0 Hz, 1H), 4.38–4.33 (m, TyrHet α-H, 1H), 3.71–3.68 (s,
0.291 mmol, 91%). Rf 0.62 (90 : 10, CH2Cl2–CH3OH, UV, CO2CH3, 3H), 3.15–3.10 (m, TyrHet-CHAHB, 1H), 3.08–3.02 (m,
cerium molybdate stain). [α]2D0 +29.4, (c = 1.0, CHCl3). M.p. TyrHet-CHAHB, 1H), 2.16–2.07 (m, Val CH, 1H), 1.42 (s, Boc
139–140 °C. 1H NMR (500 MHz, CHCl3) δ 8.17 (s, CH aromatic, CH3 × 3, 9H), 0.89 (d, Val CH3, J = 6.9 Hz, 3H), 0.86 (d, Val CH3,
1H), 7.79 (d, CH aromatic, J = 7.9 Hz, 2H), 7.69 (tt, CH J = 6.9 Hz, 3H). 13C NMR (125 MHz, CD3CN) δ 171.8, 170.8,
aromatic, J = 7.5 Hz and 1.2 Hz, 1H), 7.69 (t, CH aromatic, J = 158.3, 160.6, 155.5, 151.7, 152.8, 137.9, 137.8, 135.8, 135.6,
7.7 Hz, 2H), 7.68 (d, CH aromatic, J = 7.5 Hz, 2H), 7.16 (d, CH 135.5, 135.3, 130.9, 129.0, 128.6, 119.9, 119.3, 110.8, 80.5, 57.3,
aromatic, J = 7.8 Hz, 2H), 7.09 (d, CH aromatic, J = 8.4 Hz, 2H), 55.7, 52.2, 37.2, 31.2, 28.3, 18.6, 17.7. νmax (NaCl/thin film)/
6.55 (br, s, NH amide, 1H), 5.01 (br, s, NH Boc, 1H), 4.43 (dd, cm−1 3019, 2927, 1741, 1699, 1678, 1618, 1533, 1505, 1449,
Val α-H, J = 8.6 Hz and 5.1 Hz, 1H), 4.36–4.28 (m, TyrHet α-H, 1438, 1368, 1215, 1168, 1018. m/z (ES+) 641 ([M + Na]+, 100%).
1H), 3.66 (s, CO2CH3, 3H), 3.07–2.93 (m, TyrHet CH2, 2H), HRMS m/z (ES+) calcd for C28H34N4NaO10S [M + Na]+ requires
2.10–2.04 (m, Val CH, 1H), 1.38 (s, Boc CH3 × 3, 9H), 0.85 (d, 641.1893, found 641.1900.
Val CH3, J = 7.0 Hz, 3H) and 0.82 (d, Val CH3, J = 7.0 Hz, 3H).
Dipeptide 40. General procedure F was followed using
13C NMR (125 MHz, CDCl3) δ 175.7, 171.8, 171.2, 160.3, 155.5, HATU (113 mg, 0.297 mmol), phenylalanine methyl ester
151.0, 133.7, 133.4, 133.3, 130.6, 130.1, 121.9, 121.3, 103.4, hydrochloride (23 mg, 0.109 mmol), 26 (50 mg, 0.099 mmol)
80.2, 57.3 55.8, 52.1, 37.1, 31.2, 28.3, 18.8, 17.8. νmax (NaCl/ and diisopropylethylamine (distilled, 51 mg, 70 μL,
thin film)/cm−1 1743, 1647, 1593, 1545, 1495, 1451, 1367, 0.296 mmol). Purification over silica gel, eluting with EtOAc
1251, 1215, 1050. m/z (ES+) 604 ([M + Na]+, 100%). HRMS m/z and hexanes (10 : 90 to 25 : 75) gave dipeptide 40 as a white
(ES+) calcd for C29H35N5NaO8 [M + Na]+ requires 604.2383, solid (60 mg, 0.094 mmol, 95%). Rf 0.62 (90 : 10, CH2Cl2–
found 604.2395.
MeOH, UV, cerium molybdate stain). [α]2D0 −22.0 (c = 0.1,
Dipeptide 38. General procedure F with HATU (442 mg, CHCl3). M.p. 118–121 °C. 1H NMR (500 MHz, CDCl3) δ
1.163 mmol), was followed using phenylalanine methyl ester 8.18–8.12 (m, CH aromatic, 2H), 7.81 (tt, CH aromatic, J =
hydrochloride (76 mg, 0.352 mmol), 23 (150 mg, 0.320 mmol) 7.5 Hz and 1.2 Hz, 1H), 7.67 (t, CH aromatic, J = 8.0 Hz, 2H),
and diisopropylethylamine (distilled, 200 mg, 270 μL, 7.28–7.20 (m, CH aromatic, 7H), 7.05 (d, CH aromatic, J =
1.55 mmol). Purification over silica gel, eluting with EtOAc 7.4 Hz, 2H), 6.34 (d, NH amide, J = 7.2 Hz, 1H), 4.99 (br, s, NH
and hexanes (10 : 90 to 25 : 75) generated dipeptide 38 as an off Boc, 1H), 4.81 (dd, Phe α-H, J = 13.3 Hz and 6.4, 1H), 4.37–4.33
white solid (191 mg, 0.304 mmol, 95%). Rf 0.58 (90 : 10, (m, TyrHet α-H, 1H), 3.72 (s, CO2CH3, 3H), 3.14–3.02 (m, Phe
CH2Cl2–CH3OH, UV, cerium molybdate stain). [α]2D0 +17.6, (c = CH2, TyrHet CH2, 4H), 1.43 (s, Boc CH3 × 3, 9H). 13C NMR
0.7, CHCl3). M.p. 159–160 °C 1H NMR (500 MHz, CDCl3) δ 8.27 (125 MHz, CDCl3) δ 171.4, 170.5, 158.4, 155.3, 151.6, 138.0,
(s, CH aromatic, 1H), 7.85 (d, CH aromatic, J = 7.7 Hz, 2H), 135.9, 135.5, 135.3, 130.9, 129.8, 129.2, 128.6, 128.6, 127.2,
7.76 (t, CH aromatic, J = 7.4 Hz, 1H), 7.76 (t, CH aromatic, J = 120.0, 110.7, 80.3, 53.3, 52.4, 51.6, 37.9, 37.6, 28.1. νmax (NaCl/
7.7 Hz, 2H), 7.30–7.14 (m, CH aromatic, 7H), 7.07 (d, CH aro- thin film)/cm−1 2960, 2924, 2853, 1741, 1658, 1617, 1535,
matic, J = 7.6 Hz, 2H), 6.55 (d, NH amide, J = 7.9 Hz, 1H), 5.01 1505, 1450, 1366, 1259, 1202, 1167, 1019. m/z (ES+) 667
(d, NH Boc, J = 8.1 Hz, 1H), 4.82 (dd, Phe α-H, J = 13.4 Hz and ([M + H]+, 100%). HRMS m/z (ES+) calcd for C32H35N4O10S
6.3, 1H), 4.40–4.33 (m, TyrHet α-H, 1H), 3.70 (s, CO2CH3, 3H), [M + H]+ requires 667.2074, found 667.2078.
3.14–3.03 (m, Phe CH2, TyrHet CH2, 4H), and 1.43 (Boc CH3 ×
Tripeptide 41. General procedure B using dipeptide 37
3, 9). 13C NMR (125 MHz, CDCl3) δ 175.7, 171.4, 170.9, 160.2, (66 mg, 0.098 mmol) was followed to give the product as a
155.3, 151.0, 135.8, 133.7, 133.3, 133.3, 130.6, 130.1, 129.2, white solid. The residue was used in general procedure F with
128.5, 127.1, 121.9, 121.6, 103.5, 80.2, 55.6, 53.4, 52.3, 37.9, Fmoc-Arg(Pbf)-OH (208 mg, 0.320 mmol), HATU (442 mg,
37.4, 28.2. νmax (NaCl/thin film)/cm−1 1733, 1677, 1587, 1508, 1.297 mmol) and diisopropylethylamine (distilled, 166 mg,
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220 μL, 1.28 mmol). Purification over silica gel, eluting with 127.8, 127.5, 127.1, 127.0, 125.0, 124.9, 122.2, 121.6, 120.1,
CH2Cl2 and methanol (99 : 1) provided tripeptide 41 as a white 120.0, 103.6, 81.6, 67.0, 54.0, 53.4, 52.4, 51.6, 47.1, 37.8, 37.2,
solid (312 mg, 0.281 mmol, 85%). Rf 0.59 (90 : 10, CH2Cl2– 36.8, 28.0. νmax (NaCl/thin film)/cm−1 1734, 1670, 1603, 1588,
CH3OH, UV, cerium molybdate stain). [α]2D0 +110 (c = 0.6, 1513, 1495, 1451, 1368, 1216, 1050. m/z (ES+) 923 ([M + H]+,
1
CHCl3). M.p. 115–119 °C. H NMR (500 MHz, CDCl3) δ 8.27 (s, 100%). HRMS m/z (ES+) calcd for C51H50N6O11 [M + H]+
CH aromatic, 1H), 7.79 (d, CH aromatic, J = 7.7 Hz, 2H), 7.74 requires 923.3610, found 923.3614.
(d, CH Fmoc, J = 7.5 Hz, 2H), 7.63 (t, CH aromatic, J = 7.5 Hz,
Tripeptide 43. General procedure B was followed using
1H), 7.55 (t, CH aromatic, J = 7.9 Hz, 2H), 7.52 (d, CH Fmoc, J = dipeptide 39 (62 mg, 0.098 mmol) to give a white solid. The
7.5 Hz, 2H), 7.30 (t, CH Fmoc, J = 7.5 Hz, 2H), 7.21–7.16 (m, residue was used in general procedure F with Fmoc-Asp(OtBu)-
CH Fmoc, CH aromatic, 4H), 7.10 (br, s, NH sulfonamide, 1H), OH (41 mg, 0.098 mmol), HATU (113 mg, 0.297 mmol) and di-
7.02 (d, CH aromatic, J = 8.0 Hz, 2H), 6.54–6.15 (m, NH amide, isopropylethylamine (distilled, 39 mg, 50 μL, 0.297 mmol).
NH guanidine, NH Fmoc, 4H), 4.74 (dd, TyrHet α-H, J = 14.4 Hz Purification over silica gel, eluting with EtOAc and hexanes
and 8.0 Hz, 1H), 4.36 (dd, Val α-H, J = 7.5 Hz and 5.8 Hz, (10 : 90 to 25 : 75) provided tripeptide 43 as a white solid
1H), 4.31–4.23 (m, CH2 Fmoc, Arg α-H, 3H), 4.07 (t, CH Fmoc, (72 mg, 0.078 mmol, 80%. Rf 0.65 (90 : 10, CH2Cl2–CH3OH,
J = 6.8 Hz, 1H), 3.62 (s, CO2CH3, 3H), 3.26–3.10 (m, Arg UV, cerium molybdate stain). [α]2D0 −22.0 (c = 0.1, CHCl3). M.p.
1
CH2NH, TyrHet CHAHB, 3H) 3.04–2.98 (m, TyrHet CHAHB, 1H), 135–139 °C (decomp.). H NMR (500 MHz, CDCl3) δ 8.17–8.08
2.86 (s, Pbf CH2, 2H), 2.58 (s, Pbf CH3, 3H), 2.50 (s, Pbf CH3, (m, CH aromatic, 2H), 7.81–7.44 (m, CH Fmoc, CH aromatic,
3H), 2.05–2.01 (m, Pbf CH3, Val CH, 4H), 1.73–1.65 (m, ArgCH- 3H), 7.63 (t, CH aromatic, J = 7.6 Hz, 2H), 7.60–7.55 (m, CH
CHAHB, 1H), 1.60–1.53 (m, ArgCHCHAHB, 1H), 1.41–1.35 (m, Fmoc, 2H), 7.43–7.37 (m, CH Fmoc, 2H), 7.35–7.25 (m, CH
ArgCH2CH2CH2, Pbf CH3 × 2, 8H) and 0.83–0.80 (6H, m, 2 × Fmoc, CH aromatic, 4H), 7.13–7.10 (m, CH aromatic, 2H),
CH3−54). 13C NMR (125 MHz, CDCl3) δ 175.5, 172.9, 171.8, 7.02–6.99 (m, NH amide, 1H), 6.43 (br, s, NH amide), 5.78 (dd,
171.4, 159.9, 158.7, 156.6, 156.4, 150.6, 144.0, 143.6, 141.2, NH Fmoc, J = 19.0, 8.3, 1H), 4.64 (t, TyrHet α-H, J = 6.7 Hz, 1H),
141.1, 138.4, 133.9, 133.6, 133.2, 133.0, 132.3, 130.5, 130.2, 4.49–4.34 (m, CH2 Fmoc, Asp α-H, Val α-H, 4H), 4.20 (t, Fmoc
127.7, 127.1, 125.3, 125.2, 124.6, 121.8, 121.6, 119.9, 117.6, CH, J = 6.7 Hz, 1H), 3.78–3.71 (s, CO2CH3, 3H), 3.17–3.04 (m,
103.4, 86.3, 67.1, 55.0, 54.7, 53.5, 52.2, 47.0, 43.5, 39.9, 36.8, TyrHet CH2, 2H), 2.85–2.80 (m, Asp CHAHB, 1H), 2.61 (dd, Asp
31.0, 29.1, 28.5, 25.5, 19.4, 19.0, 18.0 and 12.5. νmax (NaCl/thin CHAHB, J = 17.0 Hz and 5.8 Hz, 1H), 2.14–2.08 (m, Val CH, 1H),
film)/cm−1 1725, 1651, 1553, 1451, 1404, 1369, 1212, 1107, 1.41 (s, tBu ester CH3 × 3, 9H) and 0.86–0.83 (m, Val CH3, 6H).
1019, 974, 851. m/z (ES+) 1112 ([M + H]+, 100%). HRMS m/z 13C NMR (125 MHz, CDCl3) δ 171.8, 171.0, 170.7, 169.9, 160.7,
(ES+) calcd for C58H65N9O12S [M + H]+ requires 1112.4546, 158.4, 156.1, 152.8, 151.4, 141.3, 141.6, 137.8, 137.0, 135.8,
found 1112.4546.
135.6, 135.2, 135.1, 131.0, 129.8, 129.1, 128.6, 127.7, 127.1,
Tripeptide 42. General procedure B was followed using 125.0, 120.1, 119.4, 110.7, 82.1, 67.2, 57.4, 54.4, 52.2, 51.2,
dipeptide 37 (201 mg, 0.098 mmol) to give a white solid. The 47.1, 37.1, 36.9, 31.0, 28.0, 18.9, 17.9. νmax (NaCl/thin film)/
residue was used in general procedure F with Fmoc-Asp(OtBu)- cm−1 1733, 1700, 1656, 1648, 1581, 1553, 1539, 1494, 1450,
OH (132 mg, 0.320 mmol), HATU (442 mg, 1.297 mmol) and 1404, 1367, 1260, 1181. m/z (ES+) 913 ([M + H]+, 100%). HRMS
diisopropylethylamine (distilled, 166 mg, 220 μL, 1.28 mmol). m/z (ES+) calcd for C46H50N5O13S [M + H]+ requires 912.3125,
Purification by silica gel chromatography, eluting with CH2Cl2 found 912.3126.
and MeOH (99 : 1) provided tripeptide 42 as a white solid
Tripeptide 44. General procedure B was followed using
(250 mg, 0.272 mmol, 85%). Rf 0.59 (90 : 10, CH2Cl2–CH3OH, dipeptide 40 (66 mg, 0.098 mmol) to give a white solid. The
UV, cerium molybdate stain). [α]2D0 +157 (c = 1.0, CHCl3). M.p. residue was used in general procedure F with Fmoc-Arg(Pbf)-
116–117 °C. 1H NMR (500 MHz, CDCl3) δ 8.20 (s, CH aromatic, OH (41 mg, 0.098 mmol), HATU (113 mg, 0.297 mmol) and
1H), 7.82 (d, CH aromatic, J = 7.9 Hz, 2H), 7.75–7.67 (m, CH diisopropylethylamine (distilled, 39 mg, 50 μL, 0.297 mmol).
Fmoc, CH aromatic, 3H), 7.63–7.52 (m, CH Fmoc, CH aromatic, Purification by silica gel chromatography, eluting with ethyl
4H), 7.38–7.20 (m, CH Fmoc, CH aromatic, 7H), 7.09–7.05 (m, acetate and hexanes (10 : 90 to 25 : 75) provided tripeptide 44
CH aromatic, 4H), 6.91 (d, CH aromatic, J = 8.1 Hz, 2H), 6.74 as a white solid (103 mg, 0.086 mmol, 85%). Rf 0.62 (90 : 10,
(d, NH amide, J = 8.1 Hz, 1H), 6.60 (d, NH amide, J = 7.6 Hz, CH2Cl2–CH3OH, UV, cerium molybdate stain), [α]2D0 −9.2 (c =
1H), 5.96 (d, NH Fmoc, J = 8.5 Hz, 1H), 4.77 (dd, Phe α-H, J = 0.1, CHCl3) m.p. 109–110 °C. 1H NMR (500 MHz, CDCl3) δ
13.4 Hz and 6.7 Hz, 1H), 4.64 (dd, TyrHet α-H, J = 15.0 Hz and 8.11–8.05 (m, CH aromatic, 2H), 7.71–7.70 (m, CH Fmoc, CH
7.9 Hz, 1H), 4.47–4.40 (m, CH2 Fmoc, Arg α-H, 3H), 4.19 (t, CH aromatic, 3H), 7.62–7.53 (m, CH Fmoc, CH aromatic, 4H),
Fmoc, J = 6.5 Hz, 1H), 3.67 (s, CO2CH3, 3H), 3.15–3.08 (m, 7.39–7.15 (m, CH Fmoc, CH aromatic, 8H), 7.14–7.02 (m, CH
TyrHet CHAHB, Phe CHAHB, 2H), 3.00 (dd, Phe CHAHB, J = 13.8 aromatic, 5H), 6.94 (br, s, NH amide, 1H), 6.36 (br, s, NH gua-
Hz and 6.9 Hz, 1H), 2.91 (dd, TyrHet CHAHB, J = 14.2 Hz and nidine, 2H), 5.95 (br, s, NH Fmoc, 1H), 4.70 (dd, Phe α-H, J =
8.2 Hz, 1H), 2.62 (dd, Asp CHAHB, J = 16.5 Hz and 5.6 Hz, 1H), 13.9 Hz and 6.7 Hz, 1H), 4.57 (t, TyrHet α-H, J = 7.1 Hz, 1H),
2.56 (dd, Asp CHAHB, J = 16.5 Hz and 7.2 Hz, 1H) and 1.39 (s, 4.39–4.22 (m, CH2 Fmoc, Arg α-H, 3H), 4.11 (t, Fmoc CH, J =
tBu ester CH3 × 3, 9H). 13C NMR (125 MHz, CDCl3) 175.8, 7.4 Hz, 1H), 3.60 (s, CO2CH3, 3H), 3.17–2.85 (m, ArgCH2NH,
171.5, 170.8, 170.8, 170.1, 160.6, 156.4, 151.0, 143.8, 143.7, Pbf CH2, TyrHet CH2, Phe CH2, 8H), 2.61 (s, Pbf CH3, 3H), 2.51
141.3, 135.8, 133.7, 133.4, 133.4, 130.5, 130.1, 129.2, 128.6, (s, Pbf CH3, 3H), 2.07 (s, Pbf CH3, 3H), 1.82–1.74 (m,
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ArgCHCHAHB, 1H), 1.65–1.54 (m, ArgCHCHAHB, 1H), 1.47–1.40
(m, ArgCH2CH2CH2 Pbf CH3 × 2, 8H). 13C NMR (125 MHz,
CDCl3) δ 172.6, 171.7, 170.9, 159.0, 158.3, 156.6, 156.4, 151.3,
143.8, 143.7, 141.2, 138.5, 138.0, 136.0, 135.8, 135.4, 132.4,
132.3, 130.8, 129.8, 129.2, 128.6, 128.8, 128.7, 127.7, 127.1,
125.2, 124.8, 120.0, 119.9, 117.7, 110.7, 86.5, 67.1, 56.8, 53.7,
55.5, 52.4, 47.0, 43.2, 43.2, 37.7, 36.8, 30.3, 28.6, 25.2, 19.4,
18.1 12.5. νmax (NaCl/thin film)/cm−1 1752, 1730, 1635, 1600,
7 C. J. Suckling, C. L. Gibson, J. K. Huggan, R. R. Morthala,
B. Clarke, S. Kununthur, R. M. Wadsworth, S. Daff and
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1566.
8 L. J. Ignarro, C. Napoli and J. Loscalzo, Circ. Res., 2002, 90,
21–28.
9 P. Ghosh, B. Ternai and M. Whitehouse, Med. Res. Rev.,
1981, 1, 159–187.
1540, 1491, 1450, 1270, 1175, 1040. m/z (ES+) 1197 ([M + H]+, 10 M. Feelisch, K. Schönafingeri and H. Noack, Biochem. Phar-
100%). HRMS m/z (ES+) calcd for C61H65N8O14S [M + H]+
macol., 1992, 44, 1149–1157.
requires 1197.4057, found 1197.4062.
11 (a) A. Gasco, R. Fruttero, G. Sorba, A. Di Stilo and
R. Calvino, Pure Appl. Chem., 2004, 76, 973–981;
(b) M. R. Miller and I. L. Megson, Br. J. Pharmacol., 2007,
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Chem., 2003, 40, 943–947; (b) X. Tang, T. Cai and
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2006, 15, 363–369; (d) M. Vahermo, T. Suominen,
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NO determination assay
Procedure adapted from Del Grosso et al.27 The amino acid
(1 mmol) was dissolved in DMSO–dH2O (2 : 8); Buffer solution
was added (pH 7.6, containing 6 mmol GST, 4557 u mL−1
,
superoxide dismutase). The mixture was incubated at room
temperature for 1 h. Griess reagent A (1% sulfanilamide, 5%
H3PO4 in dH2O) was added and incubated for 5 min. Griess
reagent B (N-1-naphthyl)ethylenediamine dihydrochloride in
dH2O) was added and the mixture incubated for 10 min. The
final concentration of amino acid was 400 μM. UV absorbance
at 568 nm was measured using a Multiskan FC Microplate
Photometer and calibrated using a standard curve prepared
from a standard solution of NaNO2 to give nitrite concen-
tration. Measurements were made in triplicate.
13 M. Steinberg, Clin. Ther., 2009, 31, 2312–2331.
14 E. N. Beal and K. Turnbull, Synth. Commun., 1992, 22,
673–676.
15 T. Wang, Y.-H. Zhang, X.-W. Kong, Y.-S. Lai, H. Ji,
Y.-P. Chen and S.-X. Peng, Chem. Biodivers., 2009, 6, 466–
474.
16 J. M. Richter, B. W. Whitefield, T. J. Maimone, D. W. Lin,
M. P. Castroviejo and P. S. Baran, J. Am. Chem. Soc., 2007,
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Acknowledgements
We thank CRUK for a Studentship (AN), Dr Matt Smith (St
Andrews University) for assistance with the NO release assay,
Dr Terry K. Smith (St Andrews University) for use of a Micro-
plate reader and the EPSRC National Mass Spectrometry
Service Centre (Swansea).
17 M.-A. Lassoie, F. Broeders, P. Collart, L. Defrère, F. de Lave-
leye-Defais, T. Demaude, A. Gassama, G. Guillaumet,
J.-C. Hayez, L. Kiss, L. Knerr, J.-M. Nicolas, S. Norsikian,
L. Quéré, S. Routier, V. Verbois and L. Provins, Bioorg. Med.
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18 R. Fruttero, M. Crosetti, K. Chegaev, S. Guglielmo,
A. Gasco, F. Berardi, M. Niso, R. Perrone, M. A. Panaro
and N. A. Colabufo, J. Med. Chem., 2010, 53, 5467–
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