Development of an Intein-Inspired Amide Cleavage Chemical Device

Article abstract of DOI:10.1021/acs.joc.5b02399

A photoresponsive amide cleavage device was developed based on the asparagine imidation-mediated cleavage of peptide bonds during intein-mediated protein splicing. The chemical environment of the protein splicing process was mimicked by the incorporation of geminal dimethyl groups and a secondary amine unit in asparagine scaffold. Furthermore, the resulting photoresponsive device could induce the phototriggered cleavage of an amide bond by the protection of the secondary amine unit with an o-nitrobenzyloxycarbonyl group.

Full text of DOI:10.1021/acs.joc.5b02399

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Note
Development of an intein-inspired amide cleavage chemical device
Chiaki Komiya, Keisuke Aihara, Ko Morishita, Hao Ding, Tsubasa Inokuma, Akira Shigenaga, and Akira Otaka
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b02399 • Publication Date (Web): 08 Dec 2015
Downloaded from http://pubs.acs.org on December 9, 2015
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Development of an intein-inspired amide cleavage chemical device
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Chiaki Komiya‡, Keisuke Aihara‡, Ko Morishita, Hao Ding, Tsubasa Inokuma, Akira Shigenaga, and
Akira Otaka*
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‡These authors contributed equally.
Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima
University, Tokushima 770ꢀ8505, Japan
*Eꢀmail: aotaka@tokushimaꢀu.ac.jp
Abstract
A photoꢀresponsive amide cleavage device was developed based on the asparagine imidationꢀmediated
cleavage of peptide bonds during inteinꢀmediated protein splicing. The chemical environment of the
protein splicing process was mimicked by the incorporation of geminal dimethyl groups and a
secondary amine unit in asparagine scaffold. Furthermore, the resulting photoꢀresponsive device could
induce the photoꢀtriggered cleavage of an amide bond by the protection of the secondary amine unit
with an oꢀnitrobenzyloxycarbonyl group.
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Intein proteins,1 which are found in a wide range of unicellular organisms,2 mediate the selfꢀsplicing
of inteinꢀcontaining proteins to produce inteinꢀremoved splicing proteins through sequential N–S(or O),
S(or O)–S(or O) and S(or O)–Nꢀacyl transfers. The third S(or O)–Nꢀacyl transfer step in this process
starts from the imide cyclization of an asparagine (Asn) residue at the intein Cꢀterminus, which is
followed by the transfer of an O(or S)–peptidyl unit to the liberated amino group.3 The progress of this
sequence of reactions depends on several requirements, including (i) enhancement of the nucleophilicity
of the amide side chain of the Asn residue; (ii) activation of the scission peptide bond; and (iii)
appropriate arrangement of the functional groups involved in the reactions. An analysis of the structural
basis for this reaction4 indicated that the appropriate arrangement of several functional units, including a
water molecule, assist in the cleavage of the amide via an acidꢀbaseꢀcatalyzed mechanism (Figure 1).4a
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Figure 1. Mechanism of acidꢀbaseꢀcatalyzed amide cleavage.
Methodologies for photoꢀinduced amide bond cleavage5 and conformational change6 have provided
powerful tools for the spatiotemporal control of the functions of peptide/proteins. We previously
developed a stimulusꢀresponsive processing residue (Spr)7 based on a trimethylꢀlock system.8,9 This Spr
system has shown utility in the field of chemical biology and has potential for realꢀlife application.7a,c,e
In conjunction with our studies on Spr, we also explored the development of an alternative new scaffold.
In this context, the result of a mechanistic study4 of the third step of the reaction mentioned above
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inspired us to design a new amide bond cleavage device with a modified Asn structure. In this way, it
was envisioned that the modifications shown in Figure 2 would provide the structural features necessary
to affect the cleavage of an amide bond. The incorporation of a pendant secondary amine would provide
an intramolecular base,10 which could enhance the nucleophilicity of the amide nitrogen. Furthermore,
the incorporation of geminal dimethyl groups would lead to a ThorpeꢀIngold effect,11–13 which would
fix the conformation of the intein system and assist in the formation of the succinimide ring. Lastly, the
masking of the basic character of the secondary amine with a photoꢀsensitive Nꢀprotecting group,14 such
as oꢀnitrobenzyloxycarbonyl (oNBnoc), could provide a simple platform for the development of
stimulusꢀresponsive amide bond cleavage device.
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Figure 2. Design of an inteinꢀmediated UVꢀresponsive amide cleavage device.
Our work toward preparation of a pendant secondary amine capable of responding to UV irradiation
started from Nꢀethylethylenediamine (1) (Scheme 1). The reaction of 1 with tertꢀbutyloxycarbonyl
anhydride (Boc2O) in THF allowed for the selective protection of the primary amino group. The
subsequent reaction of the secondary amine group with pꢀnitroformate 215 and triethylamine (Et3N) in
THF afforded the requisite compound 3 in quantitative yield (over two steps). The trifluoroacetic acid
(TFA)ꢀmediated removal of the Boc protecting group from 3 gave the pendant amine unit 4a. The
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synthesis of the Bocꢀprotected pendant unit 4b proceeded via the trifluoroacetylation of the primary
amino group of 1, followed by the introduction of a Boc group and the subsequent hydrolysis of the
trifluoroacetyl protecting group.
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Scheme 1. Synthesis of the pendant secondary amine 4a and 4b
Reagents and conditions: (i) Boc2O, THF; (ii) 2, Et3N, THF, quant. (two steps); (iii) TFA, CH2Cl2; (iv)
ethyltrifluoroacetate, CH2Cl2; (v) Boc2O, CH2Cl2; (vi) K2CO3, MeOH, H2O
Scheme 2. (a) Synthesis of the Inteinꢀinspired amide cleavage device 10a and 10b and (b) the
preparation of a model peptide.
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Reagents and conditions: (i) TFA, TES, CH2Cl2; (ii) AllocCl, NaHCO3, THF, H2O; (iii) LiOH, THF,
H2O; (iv) Ac2O, THF; (v) allyl alcohol, 60% (five steps); (vi) 4a or 4b, PyBrop, DIEA, CH2Cl2; (vii)
Pd(PPh3)4, Nꢀmethylaniline, THF then FmocOSu, DIPEA
PhFlꢀdiMeꢀAsp(OMe)ꢀOMe 516 was synthesized over three steps from LꢀAspartic acid (6) following
Goodman’s procedure (Scheme 2). The deprotection of the PhFl group in 5 with a mixture of TFA and
triethyl silane (TES) in CH2Cl2, followed by the protection of the resulting amine with
allylchloroformate (AllocꢀCl) gave AllocꢀdiMeꢀAsp(OMe)ꢀOMe 7. The subsequent hydrolysis of the
two methyl esters of 7 with LiOH in a mixture of THF and H2O gave the corresponding carboxylic acid,
which was reacted with Ac2O in THF at reflux temperature, followed by an alcoholysis reaction with
allyl alcohol to give the αꢀallylester AllocꢀdiMeꢀAsp(OH)ꢀOAllyl 8 in 60% isolated yield (over five
steps). The reaction of the UVꢀresponsive amine 4a with the stericallyꢀcrowded βꢀcarboxylic acid of 8
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was accomplished using bromotripyrrolidinophosphonium hexafluorophosphate (PyBrop) and
diisopropylethyl amine (DIPEA) in CH2Cl2 to yield the fully protected Asn derivative
AllocꢀdiMeꢀAsn(EtꢀNꢀoNBnoc)ꢀOAllyl 9a in 75% isolated yield. The conversion of 9a to the
corresponding 9ꢀfluorenylmethyloxycarbonyl (Fmoc)ꢀprotected derivative for Fmoc solidꢀphase peptide
synthesis (SPPS) was achieved by the deprotection of the allyl and allyloxycarbonyl (Alloc) groups by
the treatment of 9a with Pd(PPh)4 and Nꢀmethylaniline, followed by the Fmoc protection of the resulting
amine to give FmocꢀdiMeꢀAsn(EtꢀNꢀoNBnoc)ꢀOH 10a in 92% isolated yield. The Bocꢀprotected
material FmocꢀdiMeꢀAsn(EtꢀNꢀBoc)ꢀOH 10b was also prepared in a similar manner in 70% isolated
yield from 9b.
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With the requisite Asn derivatives in hand, we proceeded to synthesize two model peptides
(HꢀYGGFLꢀXꢀSGFLYGFꢀNH2 11a and 11b: X = Asn derivatives) to examine the selfꢀprocessing
properties of the peptides. The Fmoc protected amino acids were condensed on NovaSyn® TGR resin
using diisopropylcarbodiimide (DIPCDI) and 1ꢀhydroxybenztriazole (HOBt) in dimethylformamide
(DMF) except for 10a and 10b. The condensation of compound 10a was achieved using
1ꢀ[bisꢀ(dimethylamino)methylene]1Hꢀ1,2,3ꢀtriazolo[4,5ꢀβ]pyridineꢀ3ꢀoxide
hexafluorophosphate
(HATU) and DIEA in Nꢀmethylpyrrolidone (NMP). The completed peptide resins were subsequently
exposed to a mixture of TFAꢀethanedithiol (EDT)ꢀmꢀcresolꢀthioanisoleꢀH2O at room temperature for 2 h
to give a mixture of two peptides with mass values identical to that of the desired material (Figure 3).
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Figure 3. Possible mechanism for the generation of the byproduct 18.
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The origin of the two peptides was attributed to the formation of the succinimide species 12 during the
activation of 10a, followed by the aminolysis of the succinimide ring by the amino group of the growing
peptide chain. Given that one of the two possible electrophilic sites in the succinimide ring of 12 was
sterically crowded by the neighboring geminal dimethyl groups, it was anticipated that the major
product of this reaction would be desired αꢀpeptide 11a. We envisaged that the benzyl protection of the
amide nitrogen in 10a would prevent the formation of the succinimide ring.17 However, our attempts to
synthesize the dimethoxybenzyl (DMB)ꢀprotected Asn derivative 13 resulted in failure (Scheme 3).
Although the condensation of the Nꢀdimethoxybenzyl diamine derivative 14a with 8 gave the desired
amide 15 in 50% yield, the subsequent exchange of the protecting groups resulted in the release of the
pendant amine molecule 14a. The failure of this reaction was attributed to the nucleophilic attack of the
αꢀcarboxylate on the substituted amide. To confirm the structure of peptide 18 as the isoꢀpeptide form,
the αꢀamide protected derivative 16, suitable for the straightforward preparation of 18, was prepared
from the reaction of the βꢀallyl ester 17 with 14a (Scheme 3).
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Scheme 3. Synthetic approach to 13 and the synthesis of the isomeric peptide 18
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Reagents and conditions: (i) 2,4ꢀdimethoxybenzaldehyde, Na2SO4, MeOH; NaBH4; (ii) MsCl, Et3N,
CH2Cl2 then 14a, 50%; (iii) MsCl, Et3N, THF then 14a, 60%; (iv) Pd(PPh3)4, Nꢀmethylaniline, THF
then FmocOSu, DIPEA, quant.
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The resulting imidationꢀtolerant βꢀcarboxylic derivative 16 was also incorporated into a peptide resin
in a manner similar to that employed for 11a. The subsequent deprotection of the resin afforded the
βꢀpeptide 18. This result clearly indicated that the major and minor products of the succinimide
ringꢀopening reaction described above were 11a and 18, respectively. This result is shown in the HPLC
chart in Figure 3 for the αꢀ and βꢀpeptides, respectively. A peptide sample without a UVꢀresponsive
group was also synthesized using the Bocꢀprotected secondary amine 10b in a manner similar to that
used for 11a. This peptide behaved in the same way as the corresponding system containing 10a, in that
the deprotection of the protected resin afforded a mixture of αꢀ and βꢀpeptides.
Peptides 19 and 20 were also prepared to determine the effects of the geminal dimethyl groups and
secondary amine on the outcome of the transformation.18 The Asnꢀprotected derivative without geminal
dimethyl groups was prepared by the coupling of the βꢀcarboxylic acid of FmocꢀAsp(OH)ꢀOAllyl19 with
NꢀDMBꢀN’ꢀBocꢀN’ꢀethyl ethylenediamine 14b, followed by the removal of the allyl ester. The
Fmocꢀbased incorporation of the resulting amino acid into the resin was followed by an acidic
deprotection step to afford the desired peptide 19, where the Asn residue had been successfully modified
with a pendant secondary amine without the need for the protection of the secondary amine. Notably, no
significant side reactions, including the hydrolysis of the peptide bonds, were observed during the acidic
deprotection and HPLC purification stages. The Asnꢀincorporated peptide 20 was also synthesized as a
separate reference compound using standard Fmoc protocols.
We initially investigated the selfꢀprocessing of these synthetic peptides, as shown in Figure 4. Peptide
samples were dissolved in a buffered solution (6 M guanidine hydrochloride (Gn·HCl)ꢀ0.2 M
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phosphate), where they were monitored for peptide bond cleavage by HPLC analysis. As expected, the
presence of both the secondary amine as an intramolecular base and the geminal methyl groups as an
inducer of cyclization greatly facilitated the cleavage of the peptide bond (Figure 4, Entries 1–3). When
a mixture of the materials was held at pH 7.4 for 24 h at 37 °C, almost all of the samples went to
completion to afford a mixture of split peptides consisting of Nꢀhalf imide peptide 21, Cꢀhalf peptide 22
and the succinimide ringꢀopened peptides 23 and 24. The results of these comparison experiments
clearly show that modifications capable of mimicking the environments involved in the inteinꢀinduced
cleavage of an amide bond were responsible for the envisioned artificial amide bond cleavage reaction
(Figure 4, Entry 4).
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Figure 4. Selfꢀprocessing reactions of the model peptides. *Internal standard.
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Encouraged by the potential utility of 10a as a stimulusꢀresponsive processing device, we next
examined the photoꢀresponsive cleavage of the peptide bond in the synthetic peptide 11a (Figure 5).
When the oNBnocꢀprotected peptide 11a was incubated for 24 h at 37 °C in a mixture of 6 M Gn·HCl
and 0.2 M phosphate at pH 7.4 without UV irradiation, the material remained almost completely intact.
The irradiation of the reaction mixture with UV light led to the removal of the oNBnoc group from the
secondary amine unit to produce peptide 11b, which was split to the processing peptides with about
80% cleavage after 24 h. These results clearly indicated that 11a could serve as a stimulusꢀresponsive
processing device and an alternative to the Spr system based on a trimethyl lock.
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Figure 5. Photoꢀresponsive peptide bond cleavage. *Internal standard. **Not peptidyl compounds,
provably derived from deprotected oNBnoc group with UV irradiation.
In conclusion, we achieved the development of the new amide bond cleavage device modeled on the
inteinꢀmediated protein splicing. The design concept of the device is derived from the mimicking
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chemical environments involved in the protein splicing. Although preparation of the device and its
incorporation into peptides are laborious, an important issue in this work is that the incorporation of
geminal dimethyl groups and a secondary amine unit in asparagine scaffold well imitate the splicing
system. Furthermore, protection of the secondary amine with the photoꢀremoval group allowed the
device to cleave the amide bond in response to photoꢀirradiation.
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Experimental Section
General Information
All reactions were carried out under an atmosphere of argon. All commercial reagents were used
without further purification. For column chromatography, silica gel (spherical, natural, 63ꢀ210 ꢁm) was
used. The progress of reactions was monitored by thin layer chromatography using precoated silica gel
glass plates (0.25 mm) with F254 indicator. Mass spectra (ESIꢀMS) were obtained using a ToF mass
1
13
spectrometer. HꢀNMR and CꢀNMR spectra were measured using a 300 or 400 MHz spectrometer at
room temperature unless otherwise noted. Chemical shifts were calibrated to the solvent signal.
Multiplicities are given as s (singlet), d (doublet), br d (broad doublet), t (triplet), br t (broad triplet), q
(quartet), m (multiplet) or br m (broad multiplet). For HPLC separation, a Cosmosil 5C18ꢀARꢀII
analytical column (4.6 × 250 mm, flow rate 1.0 mL/min) or a Cosmosil 5C18ꢀARꢀII semiꢀpreparative
column (10 × 250 mm, flow rate 3.0 mL/min) was employed and eluting products were detected by UV
at 220 nm. A solvent system consisting of 0.1% TFA aqueous solution (v/v, solvent A) and 0.1% TFA
in MeCN (v/v, solvent B) was used for HPLC elution. IR spectra and optical rotations were measured
using a polarimeter (concentration in g/100 mL), respectively. Photolysis was performed with the
filtered output (>365 nm) of a 3000 mW/cm2 HGꢀXe lamp.
Synthesis of asparagine derivatives
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2ꢀNitrobenzyl {2ꢀ[(tertꢀbutoxycarbonyl)amino]ethyl}ethyl
carbamate 3. To a solution of
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Nꢀethylethylenediamine (1) (5.37 mL, 50.0 mmol) in THF (100 mL) was added a solution of Boc2O
(3.27 g, 15.0 mmol) in THF (30 mL) dropwise at 0 °C. The reaction mixture was stirred at room
temperature for 5 h, and then concentrated in vacuo. The obtained residue was subsequently diluted with
EtOAc and sat. NaHCO3 aq. The obtained mixture was extracted three times with EtOAc. The combined
organic layer was washed with brine, dried over Na2SO4, filtrated and concentrated in vacuo. The
obtained crude carbamate (2.82 g, 15.0 mmol, quant., pale yellow powder) was used for a next step
without further purification.
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The obtained carbamate (2.82 g) in THF (30 mL) was treated with Et3N (1.62 mL, 11.6 mmol)
followed by 2ꢀnitrobenzyl 4ꢀnitrophenyl carbonate 215 (3.69 g, 11.6 mmol). The reaction mixture was
stirred at room temperature for 4 h, and then concentrated in vacuo and diluted with EtOAc and 5%
KHSO4 aq. The obtained mixture was extracted three times with EtOAc. The combined organic layer
was washed with sat. NaHCO3 aq. and brine, dried over Na2SO4, filtrated and concentrated in vacuo.
The crude material was purified by column chromatography (nꢀhexane/EtOAc = 8/1 then 1/1) to afford
oNBnocꢀdiamine 3 (4.26 g, 11.6 mmol, quant.) as yellow oil. IR (CHCl3): vmax, cm−1: 1364, 1477, 1529,
1701, 2875, 2977, 3358; 1HꢀNMR (DMSOꢀd6, 100 °C, 300 MHz) δ = 1.09 (3H, t, J = 7.0 Hz), 1.38 (9H,
s), 3.11 (2H, dt, J = 6.6 and 6.6 Hz), 3.22–3.35 (4H, m), 5.39 (2H, s), 6.35–6.47 (1H, br m), 7.60 (1H,
dd, J = 8.1 and 7.5 Hz), 7.67 (1H, d, J = 7.3 Hz), 7.76 (1H, dd, J = 7.3 and 7.5 Hz), 8.05 (1H, d, J = 8.1
Hz); 13CꢀNMR (DMSOꢀd6, 60 °C, 75 MHz) δ = 13.1, 28.0, 41.9, 62.7, 77.5, 124.3, 128.7, 128.8, 132.1,
133.6, 147.2, 154.5, 155.3; HRMS (ESIꢀTOF) m/z calcd for C17H25N3NaO6 ([M + Na]+): 390.1641,
found: 390.1643.
2ꢀNitrobenzyl (2ꢀaminoethyl)(ethyl)carbamate 4a. Carbamate 3 (2.00 g, 2.72 mmol) in CH2Cl2 (1.36
mL) was treated with trifluoroacetic acid (1.36 mL). The reaction mixture was stirred at room
temperature for 45 min and concentrated in vacuo. After dilution of the resulting residue with EtOAc
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and sat. NaHCO3 aq, the solution was extracted three times with EtOAc. The combined organic layer
was washed with brine, dried over Na2SO4, filtrated and concentrated in vacuo. Crude oNBoc amine 4a
(1.45 g) was obtained as yellow powder. The obtained crude 4a was used for preparation of 9a and 14a
without further purification.
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tertꢀButyl (2ꢀaminoethyl)(ethyl)carbamate 4b. To a stirred mixture of Nꢀethylethylenediamine 1 (2.39
mL, 22.7 mmol) in CH2Cl2 (50 mL) was added ethyl trifluoroacetate (3.46 mL, 22.7 mmol) in CH2Cl2
(50 mL) dropwise over 40 min at 0 °C. The reaction mixture was stirred at room temperature for 1 h and
then concentrated in vacuo. After dilution of the resulting residue with CH2Cl2 (100 mL), to the solution
was added Boc2O (4.95 g, 22.7 mmol) in CH2Cl2 (5.0 mL) at 0 °C. The reaction mixture was stirred at
room temperature for 1.5 h and then diluted with EtOAc and sat. NaHCO3 aq. The solution was
extracted three times with EtOAc. The combined organic layer was washed with brine, dried over
MgSO4, filtrated and concentrated in vacuo. The obtained crude material in MeOH (90 mL) and H2O
(10 mL) was treated with K2CO3 (2.00 g). The reaction mixture was refluxed for 2 h and then
concentrated in vacuo. The mixture was extracted three times with EtOAc. The combined organic layer
was washed with H2O and brine, dried over MgSO4, filtrated and concentrated in vacuo. Crude Boc
amine 4b (4.27 g) was obtained as pale yellow oil. The obtained crude 4b was used for preparation of
9b and 14b without further purification.
(S)ꢀ3ꢀ{[(Allyloxy)carbonyl]amino}ꢀ2,2ꢀdimethylsuccinic
acid
4ꢀ(allyl)ester
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and
(S)ꢀ4ꢀ(allyloxy)ꢀ2ꢀ{[(allyloxy)carbonyl]amino}ꢀ3,3ꢀdimethylꢀ4ꢀoxobutanoic acid 17 To a solution of
PhFlꢀdiMeꢀAsp(OMe)ꢀOMe 516 (1.83 g, 4.05 mmol) in CH2Cl2 (10.2 mL) was added triethyl silane
(1.43 mL, 14.2 mmol) followed by trifluoroacetic acid (10.2 mL) at 0 °C. The reaction mixture was
stirred at room temperature for 1 h and concentrated in vacuo. The obtained mixture was diluted with 1
M HCl aq. The precipitate was filtrated and washed with MeOH. The filtrate was concentrated in vacuo
and the resulting crude amine was used for a next step without further purification.
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The crude amine in THF (12.5 mL) and H2O (8.96 mL) was treated with NaHCO3 (2.51 g, 29.9
mmol) followed by allylchloroformate (63.6 ꢁL, 5.98 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 9 h and diluted with H2O and EtOAc. The mixture was extracted three times with
EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtrated and
concentrated in vacuo. The obtained crude AllocꢀdiMeꢀAsp(OMe)ꢀOMe 7 was used for next step
without further purification.
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The crude 7 in THF (10.9 mL) and H2O (30 mL) was treated with 1 M LiOH aq. (17.4 mL, 17.4
mmol) at 0 °C. The reaction mixture was stirred at room temperature for 10 h and diluted with CH2Cl2.
The aqueous layer was washed three times with CH2Cl2 and then acidified (pH ≈ 3) with 3 M HCl aq.
To the aqueous layer was added EtOAc and NaCl. The mixture was extracted three times with EtOAc.
The combined organic layer was washed with brine, dried over Na2SO4, filtrated and concentrated in
vacuo. Crude carboxylic acid (1.22 g) was obtained as colorless oil. 600 mg of it was used for next step
without further purification.
The stirred mixture of obtained crude carboxylic acid (600 mg) in THF (2.43 mL) was treated with
Ac2O (616 ꢁL, 6.56 mmol). The reaction mixture was refluxed for 18 h and then concentrated in vacuo.
To the obtained crude anhydride was added allylalcohol (7.5 mL). The reaction mixture was stirred at
room temperature for 23 h and concentrated in vacuo. The obtained crude material was purified by
column chromatography (chloroform/MeOH = 400/1 then 50/1) to afford 8 (449 mg, 1.47 mmol, 61%
over five steps from 5) as colorless oil and 17 (131 mg, 0.459 mmol, 19% over five steps from 5) as
colorless oil. 8: [α]28 ꢀ12.3 (c 1.56, CHCl3); IR (CHCl3): vmax, cm−1: 1330, 1519, 1713, 2886, 2942,
D
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3084, 3349; HꢀNMR (CDCl3, 400 MHz) δ = 1.26 (3H, s), 1.37 (3H, s), 4.58–4.67 (5H, m), 5.21–5.27
(2H, m), 5.29–5.36 (2H, m), 5.66 (br d, J = 9.6 Hz), 5.85–5.98 (2H, m); 13CꢀNMR (CDCl3, 75 MHz) δ =
22.2, 23.3, 45.7, 59.8, 66.0, 66.4, 118.2, 118.7, 131.9, 132.5, 156.5, 175.1, 175.3; HRMS (ESIꢀTOF)
m/z calcd for C13H19N1NaO6 ([M + Na]+): 308.1110, found 308.1115. 17: [α]28 ꢀ11.8 (c 2.40, CHCl3),
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IR (CHCl3): vmax, cm−1:932, 1251, 1525, 1724, 2886, 2944, 2984, 3088, 3350; HꢀNMR (CDCl3, 400
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(1H, br d, J = 10.4), 5.81–5.98 (2H, m); CꢀNMR (CDCl3, 75 MHz) δ = 22.1, 23.3, 45.6, 59.9, 66.3,
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66.4, 118.2, 119.3, 131.3, 132.6, 156.4, 170.2, 181.5; HRMS (ESIꢀTOF) m/z calcd for C25H19N1NaO6
([M + Na]+): 308.1110, found: 308.1121.
Allyl
(S)ꢀ2ꢀ{[(allyloxy)carbonyl]amino}ꢀ4ꢀ{[2ꢀethyl(2ꢀnitrobenzyloxycarbonyl)aminoethyl]amino}ꢀ3,3ꢀdimeth
ylꢀ4ꢀoxobutanoate 9a. To a solution of 8 (34.5 mg, 0.121 mmol) in CH2Cl2 (605 ꢁL) were added crude
4a (93.8 mg), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrop) (152 mg, 0.454 mmol)
and N,Nꢀdiisopropylethylamine (DIPEA) (77.2 ꢁL, 0.454 mmol) at 0 °C. The reaction mixture was
stirred at room temperature for 34 h and then diluted with EtOAc and 5% KHSO4 aq. The solution was
extracted three times with EtOAc. The combined organic layer was washed with sat. NaHCO3 aq., dried
over Na2SO4, filtrated and concentrated in vacuo. The obtained crude material was purified with column
chromatography (nꢀhexane/EtOAc = 1/1 then 1/2) to afford amide 9a (48.0 mg, 90.4 ꢁmol, 75%) as
colorless oil. [α]28D ꢀ2.9 (c 1.25, CHCl3); IR (CHCl3): vmax, cm−1: 1268, 1342, 1427, 1526, 1650, 1703,
2875, 2939, 2973, 3079, 3361; 1HꢀNMR (DMSOꢀd6, 70 °C, 300 MHz) δ = 1.08 (3H, t, J = 7.1 Hz), 1.13
(3H, s), 1.14 (3H, s), 3.19–3.35 (6H, m), 4.46–4.59 (5H, m), 5.13–5.24 (2H, m), 5.24–5.36 (2H, m), 5.4
(2H, s), 5.81–5.98 (2H, m), 7.16 (1H, br d, J = 2.9 Hz), 7.46–7.55 (1H, br m), 7.60 (1H, dd, J = 7.1, 8.0
13
Hz), 7.69 (1H, br d, J = 7.2 Hz), 7.78 (1H, dd, J = 7.1, 7.2 Hz), 8.07 (1H, d, J = 8.0 Hz); CꢀNMR
(DMSOꢀd6, 70 °C, 75 MHz) δ =13.0, 21.1, 22.6, 37.7, 41.8, 44.1, 45.3, 59.7, 62.7, 64.4, 64.5, 116.6,
117.4, 124.2, 128.7, 128.9, 131.9, 132.0, 133.1, 147.3, 154.5, 155.7, 169.7, 174.6; HRMS (ESIꢀTOF)
m/z calcd for C25H34N4NaO9 ([M + Na]+): 557.2223, found: 557.2244.
(S)ꢀ2ꢀ{[(9HꢀFluorenꢀ9ꢀyl)methoxycarbonyl]amino}ꢀ4ꢀ{[2ꢀethyl(2ꢀnitrobenzyloxycarbonyl)amino
ethyl]amino}ꢀ3,3ꢀdimethylꢀ4ꢀoxobutanoic acid 10a. To a stirred mixture of 9a (20.2 mg, 37.4 ꢁmol) in
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THF were added Pd(PPh3)4 (6.49 mg, 5.61 ꢁmol) and Nꢀmethylaniline (40.8 ꢁL, 0.374 mmol). The
reaction mixture was stirred at room temperature for 6 h. To the reaction mixture were added DIPEA
(15.3 ꢁL, 89.8 ꢁmol) and FmocOSu (15.1 mg, 44.9 ꢁmol) at 0 °C. The reaction mixture was stirred at
room temperature for 10 h and diluted with EtOAc and 5% KHSO4 aq. The solution was extracted three
times with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtrated and
concentrated in vacuo. The obtained crude material was purified with column chromatography
(CHCl3/MeOH = 150/1 then 30/1) to afford carboxylic acid 10a (21.7 mg, 34.3 ꢁmol, 92%) as pale
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yellow oil. [α]28 ꢀ1.1 (c 1.30, CHCl3); IR (CHCl3): vmax, cm−1: 930, 1249, 1367, 1530, 1672, 1726,
D
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2875, 2934, 2975, 3079, 3350; HꢀNMR (CDCl3, 50 °C, 300 MHz) δ = 1.03–1.20 (6H, m), 1.25 (3H, s),
3.30 (2H, q, J = 7.1 Hz), 3.34–3.57 (4H, m), 4.18 (1H, t, J = 6.8 Hz), 4.37 (2H, d, J = 6.8 Hz), 4.42–4.58
(1H, m), 5.47 (2H, s), 6.03–6.13 (1H, m), 6.79–6.94 (1H, m), 7.24–7.61 (9H, m), 7.71 (2H, d, J = 7.5
13
Hz), 7.98 (1H, d, J = 7.9 Hz); CꢀNMR (CDCl3, 75 MHz) δ =13.9, 23.2, 23.6, 40.7, 43.0, 45.5, 45.9,
47.3, 60.0, 64.7, 67.5, 120.1, 125.2, 125.3, 127.2, 127.9, 128.9, 129.1, 129.3, 132.3, 133.8, 141.4, 143.8,
143.9, 147.9, 149.0, 156.9, 157.6, 172.3; HRMS (ESIꢀTOF) m/z calcd for C33H37N4O9 ([M + H]+):
633.2561, found: 633.2549.
Allyl
(S)ꢀ2ꢀ{[(allyloxy)carbonyl]amino}ꢀ4ꢀ{[2ꢀ(tertꢀbutoxy
carbonyl)(ethyl)aminoethyl]amino}ꢀ3,3ꢀdimethylꢀ4ꢀoxobutanoate 9b. Amide 9b was prepared from
carboxylic acid 8 (72.0 mg, 0.252 mmol) and crude 4b (96 mg) in a manner similar to that described for
preparation of 9a. 9b (109 mg, 0.239 mmol, 95%) was obtained as colorless oil. [α]28 ꢀ6.7 (c 2.18,
D
1
CHCl3); IR (CHCl3): vmax, cm−1: 1341, 1431, 1525, 1709, 2880, 2934, 2975, 3317; HꢀNMR (CDCl3,
400 MHz) δ = 1.10 (3H, t, J = 7.0 Hz), 1.25 (3H, s), 1.35 (3H, s), 1.46 (3H, s), 3.21 (2H, q, J = 7.0 Hz),
3.28–3.50 (4H, br m), 4.32 (2H, d, J = 9.2 Hz), 4.57 (2H, ddd, J = 1.6, 1.6, 5.6 Hz), 4.60 (2H, ddd, J =
1.6, 1.6, 5.6 Hz), 5.16–5.25 (2H, m), 5.26–5.35 (2H, m), 5.82–5.97 (2H, m), 6.38 (2H, br d, J = 9.2 Hz),
7.05–7.20 (1H, br m); 13CꢀNMR (CDCl3, 75 MHz) δ =13.9, 23.5, 24.6, 28.6, 41.1, 43.0, 44.5, 45.5,
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61.3, 65.9, 80.3, 117.6, 118.6, 131.8, 132.9, 156.5, 157.6, 170.6, 176.3; HRMS (ESIꢀTOF) m/z calcd for
C22H37N3NaO7 ([M + Na]+): 478.2529, found: 478.2539.
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(S)ꢀ2ꢀ{[(9HꢀFluorenꢀ9ꢀyl)methoxycarbonyl]amino}ꢀ4ꢀ{[2ꢀ(tertꢀbutoxycarbonyl)(ethyl)aminoethyl]
amino}ꢀ3,3ꢀdimethylꢀ4ꢀoxobutanoic acid 10b. Carboxylic acid 10b was prepared from amide 9b (88.8
mg, 0.195 mmol) in a manner similar to that described for 10a. 10b (72.2 mg, 0.136 mmol, 70%) was
obtained as pale yellow oil. [α]28D ꢀ1.0 (c 2.16, CHCl3); IR (CHCl3): vmax, cm−1: 1366, 1450, 1479, 1531,
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1709, 2875, 2934, 2975, 3329; HꢀNMR (CDCl3, 400 MHz) δ = 1.11 (3H, t, J =7.2 Hz), 1.24 (3H, s),
1.36 (3H, s), 1.46 (9H, s), 3.22 (2H, q, J = 7.2 Hz), 3.3–3.47 (4H, m), 4.22 (1H, t, J = 7.2 Hz), 4.30 (2H,
d, J = 7.2 Hz), 4.56 (1H, br d, J = 8.0 Hz), 6.11–6.26 (1H, br m), 7.31 (2H, dd, J = 7.6, 7.6 Hz), 7.39
(2H, dd, J = 9.2, 7.6 Hz), 7.57–7.65 (2H, m), 7.75 (2H, d, J = 9.2 Hz), 7.77–7.85 (1H, br m); 13CꢀNMR
(CDCl3, 75 MHz) δ =13.8, 23.4, 23.7, 28.5, 41.5, 43.2, 45.3, 45.5, 47.3, 60.1, 67.4, 80.8, 120.1, 125.3,
127.2, 127.8, 141.4, 143.8, 144.0, 156.8, 157.8, 172.1, 178.9; HRMS (ESIꢀTOF) m/z calcd for
C30H39N3NaO7 ([M + Na]+): 576.2686, found: 576.2672.
2ꢀNitrobenzyl {2ꢀ[(2,4ꢀdimethoxybenzyl)amino]ethyl}(ethyl)carbamate 14a. Crude amine 4a (1.24 g)
in MeOH (8.9 mL) was treated with 2,4ꢀdimethoxybenzaldehyde (1.23 g, 7.41 mmol), AcOH (278 ꢁL,
4.86 mmol) and Na2SO4 (3.29 g, 46.3 mmol). The reaction mixture was stirred at room temperature for
2 h. To the reaction mixture was added NaBH4 (700 mg, 18.5 mmol) at 0 °C. The reaction mixture was
additionally stirred at room temperature for 1 h and then diluted with sat. NaHCO3 aq. The mixture was
extracted three times with EtOAc. The combined organic layer was washed with brine, dried over
Na2SO4, filtrated and concentrated in vacuo. The obtained crude material was purified with column
chromatography (nꢀhexane/EtOAc = 2/1 then EtOAc/MeOH 3/1) to afford DMBꢀoNBnoc amine 14a
(1.84 g, 4.41 mmol, 95% over two steps) as light brown oil. IR (CHCl3): vmax, cm−1: 1343, 1423, 1465,
1
1529, 1613, 1701, 2836, 2935, 3340; HꢀNMR (DMSOꢀd6, 80 °C, 300 MHz) δ =1.09 (3H, t, J = 7.0
Hz), 1.38 (9H, s), 3.11(2H, dt, J = 6.6, 6.6 Hz), 3.21ꢀ3.37 (4H, m), 5.39 (2H, s), 6.33ꢀ6.48 (1H, br m),
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7.60 (1H, dd, J = 8.1, 7.6 Hz), 7.68 (1H, d, J = 7.3 Hz), 7.76 (1H, dd, J = 7.3, 7.5 Hz), 8.05 (1H, d, J =
8.1 Hz); 13CꢀNMR (DMSOꢀd6, 60 °C, 75 MHz) δ =13.1, 28.0, 38.5, 41.9, 46.1, 62.7, 77.5, 124.3, 128.7,
128.8, 132.1, 133.6, 147.2, 154.5, 155.3; HRMS (ESIꢀTOF) m/z calcd for C21H28N3O6 ([M + Na]+):
418.1978, found: 418.1988.
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tertꢀButyl {2ꢀ[(2,4ꢀdimethoxybenzyl)amino]ethyl}(ethyl)carbamate 14b. DMBꢀBoc amine 14b was
prepared from amine crude 4b (500 mg) and 2,4ꢀdimethoxybenzaldehyde (221 mg, 1.33 mmol) in a
manner similar to that described for 14a. 14b (396 mg, 1.17 mmol, 88%) was obtained as yellow oil. IR
1
(CHCl3): vmax, cm−1: 1156, 1366, 1463, 1507, 1613, 1690, 2837, 2933, 2973, 3342; HꢀNMR
(DMSOꢀd6, 60 °C, 300 MHz) δ = 1.02 (3H, t, J = 7.0 Hz), 1.38 (9H, s), 2.69 (2H, t, J = 6.8 Hz), 3.17
(2H, q, J = 7.0 Hz), 3.25 (2H, t, J = 6.8 Hz), 3.70 (2H, s), 3.76 (3H, s), 3.78 (3H, s), 6.59 (1H, br s), 6.48
(1H, dd, J = 8.3, 2.2 Hz), 6.55 (1H, d, J = 2.2 Hz), 7.18 (1H, d, J = 8.3 Hz); 13CꢀNMR (CDCl3, 75 MHz)
δ = 13.6, 28.5, 42.5, 46.6, 47.3, 48.7, 55.4, 55.5, 70.6, 77.2, 79.4, 98.6, 103.8, 120.2, 130.6, 158.7,
160.3; HRMS (ESIꢀTOF) m/z calcd for C18H31N2O4 ([M + H]+): 339.2284, found: 339.2281.
Allyl
(S)ꢀ2ꢀ[(allyloxycarbonyl)amino]ꢀ4ꢀ[(2,4ꢀdimethoxybenzyl)(2ꢀethylꢀ2ꢀnitrobenzyloxycarbonyl
aminoethyl)amino]ꢀ3,3ꢀdimethylꢀ4ꢀoxobutanoate 15. To a solution of Carboxylic acid 8 (118 mg, 0.413
mmol) in CH2Cl2 (1.5 mL) was added Et3N (173 ꢁL, 1.24 mmol) followed by the addition of MsCl
(38.4 ꢁL, 0.496 mmol) in CH2Cl2 (100 ꢁL) at 0 °C. The reaction mixture was stirred at same
temperature for 2 h. After addition of 14a (199 mg, 0.476 mmol) in CH2Cl2 (1.5 mL) to the reaction
mixture at 0 °C, The resulting mixture was stirred at room temperature for additional 17 h and then
diluted with EtOAc and 5% KHSO4 aq. The solution was extracted three times with EtOAc. The
combined organic layer was washed with sat. NaHCO3 aq. and brine, dried over Na2SO4, filtrated and
concentrated in vacuo. The obtained crude material was purified with column chromatography
(nꢀhexane/EtOAc = 13/7 then 3/2) to afford amide 15 (140 mg, 0.204 mmol, 50%) as colorless oil.
[α]29 ꢀ0.4 (c 0.80, CHCl3); IR (CHCl3): vmax, cm−1:1208, 1423, 1477, 1528, 1614, 1707, 2838, 2939,
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2972, 3084, 3314, 3443; 1HꢀNMR (DMSOꢀd6, 100 °C, 300 MHz) δ = 1.04 (3H, t, J = 7.0 Hz), 1.27 (3H,
s), 1.33 (3H, s), 3.21(3H, q, J = 7.0 Hz), 3.26–3.44 (4H, m), 3.76 (3H, s), 3.78 (3H, s), 4.50–4.64 (7H,
m), 5.12–5.24 (2H, m), 5.26–5.38 (4H, m), 5.77–6.11 (2H, m), 6.49 (1H, dd, J = 8.4, 2.0 Hz), 6.56 (1H,
d, J = 2.0 Hz), 6.85 (1H, br d, J = 9.3 Hz), 6.99 (1H, d, J = 8.4 Hz), 7.54–7.63 (2H, m), 7.71 (1H, t, J =
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7.5 Hz), 8.03 (1H, d, J = 8.1 Hz); CꢀNMR (CDCl3, 75 MHz, rotamer)20 δ = 13.2, 13.9, 24.2, 25.3,
25.5, 29.6, 42.4, 43.1, 43.6, 43.7, 44.1, 44.3, 45.5, 46.4, 47.6, 55.1, 55.3, 63.2, 63.5, 63.7, 65.7, 98.4,
104.0, 116.6, 117.0, 117.4, 117.5, 117.9, 118.1, 124.8, 128.2, 128.3, 128.4, 128.5, 128.7, 131.8, 131.9,
132.7, 133.1, 133.2, 133.5, 133.6, 155.0, 155.4, 156.8, 157.9, 160.2, 160.3, 170.7, 176.2, 176.3, 176.4;
HRMS (ESIꢀTOF) m/z calcd for C34H44N4NaO11 ([M + Na]+): 707.2904, found: 707.2924.
Synthesis of α-amide protected derivatives 16 and 27
Allyl
(S)ꢀ3ꢀ(allyloxycarbonyl)aminoꢀ4ꢀ{[2,4ꢀdimethoxybenzyl][2ꢀ(ethylꢀ2ꢀnitrobenzyloxycarbonyl
amino)ethyl]amino}ꢀ2,2ꢀdimethylꢀ4ꢀoxobutanoate 25. Carboxylic acid 17 (90.6 mg, 0.318 mmol) in
THF (3.1 mL) was treated with Et3N (133 ꢁL, 0.953 mmol). Following to addition of MsCl (29.5 ꢁL,
0.381 mmol) at 0 °C, the reaction mixture was stirredat same temperature for 30 min. After addition of
14a (199 mg, 0.476 mmol) to the reaction mixture at 0 °C. The resulting solution was stirred at room
temperature for additional 20 h and then diluted with EtOAc and 5% KHSO4 aq. The solution was
extracted three times with EtOAc. The combined organic layer was washed with sat. NaHCO3 aq. and
brine, dried over Na2SO4, filtrated and concentrated in vacuo. The obtained crude material was purified
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with column chromatography (nꢀhexane/EtOAc = 2/1 then 1/1) to afford amide 25 (130 mg, 0.190
mmol, 60%) as colorless oil. [α]28 ꢀ16.8 (c 1.03, CHCl3); IR (CHCl3): vmax, cm−1: 1209, 1343, 1426,
7
8
D
9
1
1509, 1529, 1645, 1709, 2939, 2976, 3196; HꢀNMR (DMSOꢀd6, 100 °C, 300 MHz) δ = 1.04 (3H, br t,
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J = 6.1 Hz), 1.15 (3H, s), 1.24 (3H, s), 3.13–3.62 (6H, br m), 3.76 (6H, s), 4.33–4.64 (6H, m), 4.74–4.97
(1H, br m), 5.08–5.23 (2H, m), 5.23–5.34 (2H, m), 5.37 (2H, s), 5.78–6.02 (2H, m), 6.45 (1H, d, J = 8.2
Hz), 6.55 (1H, s), 6.85 (1H, br d, J = 9.0 Hz), 7.03 (1H, d, J = 8.2 Hz), 7.52–7.67 (2H, m), 7.73 (1H, dd,
J = 7.4, 7.4 Hz), 8.04 (1H, dd, J = 8.1 Hz); 13CꢀNMR (DMSOꢀd6, 100 °C, 75 MHz) δ = 12.7, 21.0, 22.5,
41.6, 43.3, 43.4, 44.9, 45.5, 54.8, 55.1, 55.8, 62.4, 64.1, 64.4, 98.4, 104.7, 116.5, 116.8, 123.9, 128.4,
128.7, 131.6, 132.2, 132.8, 133.1, 147.3, 154.2, 155.1, 157.9, 159.8, 169.3, 174.6; HRMS (ESIꢀTOF)
m/z calcd for C34H44N4NaO11 ([M + Na]+): 707.2904 found: 707.2933.
(S)ꢀ3ꢀ{[(9HꢀFluorenꢀ9ꢀyl)methoxycarbonyl]amino}ꢀ4ꢀ{[2,4ꢀdimethoxybenzyl][2ꢀethyl(2ꢀnitrobenzyl
oxycarbonyl)aminoethyl]amino}ꢀ2,2ꢀdimethylꢀ4ꢀoxobutanoic acid 16. Carboxylic acid 16 was prepared
from amide 25 (55.5 mg, 81.1 ꢁmol) in a manner similar to that described for 10a. 16 (63.0 mg, 80.5
ꢁmol, quant.) was obtained as pale yellow amorphous. [α]28 ꢀ8.2 (c 1.49, CHCl3); IR (CHCl3): vmax
,
D
1
cm−1:1342, 1452, 1508, 1526, 1613, 1645, 1708, 2853, 2931, 2961, 3068, 3421; HꢀNMR (DMSOꢀd6,
100 °C, 300 MHz) δ = 0.97 (3H, t, J = 7.0 Hz), 1.04 (3H, s), 1.14 (3H, s), 3.12–3.43 (6H, br m), 3.66
(3H, s), 3.68 (3H, s), 4.01–4.57 (5H, br m), 4.75 (1H, br d, J = 1.8 Hz), 5.31 (2H, s), 6.34 (1H, dd, J =
8.3, 2.0 Hz), 6.47 (1H, d, J = 2.0 Hz), 6.84–7.07 (2H, m), 7.16–7.29 (2H, m), 7.34 (2H, dd, J = 7.5, 7.1
13
Hz), 7.46–7.72 (5H, m), 7.79 (2H, d, J = 7.5 Hz), 7.97 (1H, 7.9 Hz); CꢀNMR (DMSOꢀd6, 75 MHz,
rotamer)20 δ = 13.1, 13.7, 20.8, 21.0, 24.3, 42.2, 42.3, 43.1, 44.9, 45.8, 45.9, 46.7, 55.0, 55.4, 55.6, 55.9,
63.2, 65.9, 98.1, 98.3, 104.3, 104.5, 116.4, 117.0, 120.2, 124.7, 125.4, 127.0, 127.7, 128.7, 129.0, 132.3,
132.6, 134.1, 134.2, 140.7, 143.5, 143.7, 143.8, 154.3, 154.7, 156.0, 156.3, 158.0, 158.2, 159.7, 160.2,
169.9, 177.5, 177.7; HRMS (ESIꢀTOF) m/z calcd for C42H46N4NaO11 ([M + Na]+): 805.3061, found:
805.3063.
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Allyl
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(S)ꢀ3ꢀ[(allyloxycarbonyl)amino]ꢀ4ꢀ{[2ꢀethyl(tertꢀbutoxycarbonyl)aminoethyl][2,4ꢀdimethoxybenzyl]ami
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no}ꢀ2,2ꢀdimethylꢀ4ꢀoxobutanoate 26. Amide 26 was prepared from 17 (63.5 mg, 0.223 mmol) and 14b
(113 mg) in a manner similar to 25. 26 (74.6 mg, 0.122 mmol, 55%) was obtained as colorless oil. [α]28
D
ꢀ1.8 (c 1.07, CHCl3); IR (CHCl3): vmax, cm−1: 1366, 1507, 1646, 1693, 1723, 2832, 2875, 2934, 2975;
1HꢀNMR (DMSOꢀd6, 100 °C, 300 MHz) δ = 0.88–1.11 (3H, m,), 1.16 (3H, s), 1.13 (3H, s), 1.38 (9H),
3.03–3.60 (6H, br m), 3.77(3H, s), 3.79 (3H, s), 4.38–4.65 (6H, m), 4.84 (1H, br d, J = 9.0 Hz), 5.11–
5.24 (2H, m), 5.24–5.41 (2H, m), 5.75–6.06 (2H, m), 6.48 (1H, br d, J = 7.7 Hz), 6.57 (1H, s), 6.83 (1H,
13
br d, J = 9.0 Hz), 7.03 (1H, br d, J = 7.7 Hz); CꢀNMR (DMSOꢀd6, 100 °C, 75 MHz) δ = 12.8, 21.1,
22.6, 27.6, 41.3, 43.5, 44.8, 45.5, 54.8, 55.0, 55.8, 64.1, 64.4, 78.1, 98.3, 104.7, 116.4, 116.7, 128.7,
132.2, 132.8, 153.9, 155.0, 157.8, 159.8, 169.2, 174.6; HRMS (ESIꢀTOF) m/z calcd for C31H47N3NaO9
([M + Na]+): 628.3210, found: 628.3234.
(S)ꢀ3ꢀ{[(9HꢀFluorenꢀ9ꢀyl)methoxycarbonyl]amino}ꢀ4ꢀ{[2ꢀethyl(tertꢀbutoxycarbonyl)aminoethyl]
[2,4ꢀdimethoxybenzyl]amino}ꢀ2,2ꢀdimethylꢀ4ꢀoxobutanoic acid 27. Carboxylic acid 27 was prepared
from amide 26 (55.9 mg, 92.3 ꢁmol) in a manner similar to that described for 10a. 27 (64.7 mg, 92.0
ꢁmol, quant.) was obtained as pale yellow amorphous. [α]25 ꢀ1.5 (c 0.80, CHCl3); IR (CHCl3): vmax
,
D
cm−1: 1160, 1210, 1455, 1508, 1616, 1643, 1692, 1718, 2928, 2973, 3277; 1HꢀNMR (DMSOꢀd6, 100 °C,
300 MHz) δ = 0.10 (3H, t, J = 7.1 Hz), 1.12 (3H, s), 1.22 (1H, s), 1.38 (9H, s), 3.10–3.44 (6H, br m),
3.72 (3H, s), 3.77 (3H, s), 4.07–4.25 (1H, br m), 4.25–4.41 (2H, br m), 4.41–4.61 (2H, br m) 4.81 (1H,
br d, J = 6.2 Hz), 6.42 (1H, dd, J = 8.3, 2.0 Hz), 6.55 (1H, d, J = 2.0 Hz), 7.25–7.36 (2H, m), 7.40 (2H,
dd, J = 7.3, 7.5 Hz), 7.67 (2H, d, J = 7.0 Hz), 7.84 (2H, d, J = 7.3 Hz); 13CꢀNMR (DMSOꢀd6, 75 MHz,
rotamer)23 δ =13.5, 20.8, 20.9, 24.1, 24.3, 28.0, 42.8, 43.6, 44.8, 45.8, 46.6, 46.7, 55.0, 55.2, 55.3, 55.7,
65.9, 66.0, 78.5, 78.7, 98.2, 98.3, 104.3, 104.4, 116.4, 116.9, 120.1, 125.3, 125.4, 127.0, 127.3, 127.7,
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128.9, 140.7, 143.5, 143.6, 143.8, 156.0, 156.3, 157.8, 158.0, 159.6, 159.9, 169.9, 177.6, 177.7; HRMS
(ESIꢀTOF) m/z calcd for C39H49N3NaO9 ([M + Na]+): 726.3367, found: 726.3365.
Synthesis of 29 for preparation of peptide 19
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Allyl N2ꢀ{[(9HꢀFluorenꢀ9ꢀyl)methoxy]carbonyl}ꢀN4ꢀ{2ꢀ[(tertꢀbutoxycarbonyl)(ethyl)amino]ethyl}ꢀN4
ꢀ(2,4ꢀdimethoxybenzyl)ꢀLꢀasparaginate 28. To a stirred mixture of FmocꢀLꢀAsp(OH)ꢀOAllyl19 (350 mg,
0.886 mmol) and 14b (250 mg, 0.739 mmol) in CH2Cl2 (10 mL) was added EDC·HCl (170 mg, 0.886
mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h and diluted with EtOAc
and 5% KHSO4 aq. The solution was extracted three times with EtOAc. The combined organic layer
was washed with brine, sat. NaHCO3 aq. and brine, dried over MgSO4, filtrated and concentrated in
vacuo. The obtained crude material was purified with column chromatography (nꢀhexane/EtOAc = 1/1)
to afford amide 28 (500 mg, 0.698 mmol, 95%) as pale yellow oil. [α]28 22.8 (c 2.13, CHCl3); IR
D
1
(CHCl3): vmax, cm−1: 1289, 1454, 1506, 1642, 1690, 1725, 2838, 2934, 2972, 3438; HꢀNMR
(DMSOꢀd6, 100 °C, rotamer, 300 MHz) δ =1.01 (3H, t, J = 7.0 Hz), 1.39 (9H, s), 2.89–2.95 (2H, m),
3.09–3.23 (4H, m), 3.33 (2H, br t, J = 6.3 Hz), 3.75 (3H, s), 3.79 (3H, s), 4.20–4.28 (1H, m), 4.30–4.36
(2H, m), 4.38–4.51 (2H, m), 4.56–4.63 (3H, m), 5.18 (1H, dd, J = 10.4, 1.5 Hz), 5.31 (1H, dd, J = 17.2,
1.5 Hz), 5.89 (1H, ddt, J = 17.2, 10.4, 5.3 Hz), 6.47 (1H, br d, J = 8.2 Hz), 6.58 (1H, s), 7.02 (1H, d, J =,
8.2 Hz), 7.05–7.21 (1H, m), 7.31 (2H, dd, J = 7.5, 7.1 Hz), 7.41 (2H, dd, J = 7.5, 7.1 Hz), 7.67 (2H, d, J
= 7.3 Hz), 7.85 (2H, d, J = 7.5 Hz); 13CꢀNMR (DMSOꢀd6, 75 MHz, rotamer)20 δ =13.3, 13.8, 28.0, 34.0,
34.7, 41.5, 42.1, 42.7, 43.6, 44.7, 44.9, 46.3, 46.6, 50.5, 50.9, 55.1, 55.2, 55.3, 55.4, 64.9, 65.0, 65.8,
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78.4, 78.9, 98.2, 98.5, 104.3, 104.5, 116.3, 117.4, 117.6, 120.1, 125.2, 127.1, 127.6, 128.2, 128.4, 128.9,
132.3, 132.4, 140.7, 143.7, 155.7, 155.8, 157.9, 159.7, 160.2, 169.1, 169.6, 171.1, 171.3; HRMS
(ESIꢀTOF) m/z calcd for C40H49N3NaO9 ([M + Na]+):738.3367, found: 738.3389.
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N2ꢀ{[(9HꢀFluorenꢀ9ꢀyl)methoxy]carbonyl}ꢀN4ꢀ{2ꢀ[(tertꢀbutoxycarbonyl)(ethyl)amino]ethyl}ꢀN4ꢀ(2,4ꢀd
imethoxybenzyl)ꢀLꢀasparagine 29. To a solution of amide 28 (450 mg, 0.629 mmol) in THF (6.0 mL)
was added Pd(PPh3)4 (72.7 mg, 62.9 ꢁmol) and Nꢀmethylaniline (685 ꢁL, 6.29 mmol). The reaction
mixture was stirred at room temperature for 1 h and concentrated in vacuo. The obtained crude material
was purified with column chromatography (nꢀhexane/EtOAc = 1/1 then EtOAc/MeOH = 10/1) to afford
carboxylic acid 29 (394 mg, 0.583 mmol, 93%) as pale yellow amorphousness. [α]28 39.5 (c 1.26,
D
CHCl3); IR (CHCl3): vmax, cm−1: 1289, 1506, 1610, 1643, 1690, 1718, 2843, 2972, 3314, 3427;
1HꢀNMR (DMSOꢀd6, 120 °C, rotamer, 300 MHz) δ =1.02 (3H, t, J = 7.0 Hz), 1.40 (9H, s), 2.86–2.96
(2H, m), 3.15 (2H, q, J = 7.0 Hz), 3.19–3.27 (2H, br m), 3.30–3.42 (2H, br m), 3.75 (3H, s), 3.80 (3H,
s), 4.20–4.29 (1H, m), 4.29–4.35 (2H, m), 4.42–4.58 (3H, m), 6.47 (1H, dd, J = 8.4, 2.2 Hz), 6.58 (1H,
d, J = 2.2 Hz), 6.86 (1H, br d, J = 7.5 Hz), 7.04 (1H, d, J = 8.4 Hz), 7.31 (2H, dd, J = 7.5, 7.0 Hz), 7.41
13
(2H, dd, J = 7.5, 7.0 Hz), 7.68 (2H, d, J = 7.5 Hz), 7.84 (2H, d, J = 7.5 Hz); CꢀNMR (DMSOꢀd6, 75
MHz, rotamer)20 δ =13.2, 13.8, 28.0, 33.9, 34.6, 41.5, 42.2, 42.8, 43.6, 44.0, 44.4, 44.9, 45.3, 46.4, 46.6,
50.5, 50.8, 55.1, 55.2, 55.3, 65.8, 66.3, 78.4, 78.7, 78.9, 98.1, 98.5, 104.3, 104.5, 116.4, 117.1, 120.1,
125.2, 127.1, 127.6, 128.2, 128.4, 128.7, 140.7, 143.8, 154.1, 154.6, 155.7, 155.8, 157.9, 159.7, 160.1,
169.4, 169.8, 172.9, 173.2; HRMS (ESIꢀTOF) m/z calcd for C37H45N3NaO9 ([M + Na]+): 698.3054,
found: 698.3051.
General procedures for peptide synthesis
Fmocꢀbased solidꢀphase peptide synthesis (Fmoc SPPS). On NovaSyn® TGR resin (0.22 mmol
amine/g) were coupled Fmoc protected naturally occurring amino acid derivatives (5.0 eq., a protective
group of a side chain: tꢀBu for serine and tyrosine) in the presence of N,N’ꢀdiisopropylcarbodiimide
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(DIC, 5.0 eq.) and 1ꢀhydroxybenzotriazole hydrate (HOBt·H2O, 5.5 eq.) in DMF for 2 h. Coupling of
asparagine derivatives 10a, 16, 10b or 27 (2 eq.) was performed using
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Oꢀ(7ꢀazabenzotriazolꢀ1ꢀyl)ꢀN,N,N’,N’ꢀtetramethyluronium hexafluorophosphate (1.95 eq.) and
N,Nꢀdiisopropylethyamine (4.0 eq.) for 2 h. For Fmoc removal of the peptide resin, 20% (v/v) piperidine
in DMF (10 min) was employed.
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TFA
cleavage.
The
resulting
completed
resin
was
treated
with
TFA/mꢀcresol/1,2ꢀethanedithiol/thioanisole/H2O (80/5/5/5/5 (v/v)) for 2 h at room temperature
otherwise noted. After filtration of the resin, cooled Et2O was added to the filtrate, and the resulting
precipitate was collected by centrifugation. The obtained precipitate was washed with Et2O and was
purified by semiꢀpreparative HPLC to give peptides.
Preparation of model peptides 11a, 18, 11b, 19 and 20
Peptides were synthesized according to the section “Fmocꢀbased solidꢀphase peptide synthesis”
Fmoc SPPS using 10a. Peptide 11a (major peak): a white lyophilized powder (2.08 mg, 1.19 ꢁmol,
6.2%); retention time = 16.2 min (Analytical HPLC conditions: linear gradient of solvent B in solvent
A, 38 to 50% over 30 min); retention time = 24.2 (Semiꢀpreparative HPLC conditions: linear gradient of
solvent B in solvent A, 38 to 50% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C86H113N17O21 ([M +
2H]2+): 859.9, found: 859.7. Peptide 18 (minor peak): a white lyophilized powder (1.30 mg, 0.699 ꢁmol,
3.9%); retention time = 17.6 min (Analytical HPLC conditions: linear gradient of solvent B in solvent
A, 38 to 50% over 30 min); retention time = 22.9 (Semiꢀpreparative HPLC conditions: linear gradient of
solvent B in solvent A, 38 to 50% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C86H113N17O21 ([M +
2H]2+): 859.9, found: 859.8.
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Fmoc SPPS using 16 isomer. Peptide 18: retention time = 16.3 min (Analytical HPLC conditions:
linear gradient of solvent B in solvent A, 38 to 50% over 30 min); LRMS (ESIꢀTOF) m/z calcd for
C86H113N17O21 ([M + 2H]2+): 859.9, found: 859.8.
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Fmoc SPPS using 10b. Peptide 11b (major peak): a white lyophilized powder (0.75 mg, 0.411 ꢁmol,
8.2%); retention time = 18.9 min (Analytical HPLC conditions: linear gradient of solvent B in A, 25 to
40% over 30 min); retention time = 21.4 (Semiꢀpreparative HPLC conditions: linear gradient of solvent
B in solvent A, 27 to 42% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C78H108N16O17 ([M + 2H]2+):
770.4, found: 770.2.
Peptide 30 (minor peak): retention time = a white lyophilized powder (0.39 mg, 0.214 ꢁmol, 4.3%);
20.3 min (Analytical HPLC conditions: linear gradient of solvent B in solvent A, 25 to 40% over 30
min); retention time = 23.0 (Semiꢀpreparative HPLC conditions: linear gradient of solvent B in solvent
A, 27 to 42% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C78H108N16O17 ([M + 2H]2+): 770.4, found:
770.3.
Fmoc SPPS using 27. Peptide 30: retention time = 20.3 min (Analytical HPLC conditions: linear
gradient of solvent B in solvent A, 25 to 40% over 30 min); LRMS (ESIꢀTOF) m/z calcd for
C78H108N16O17 ([M + 2H]2+): 770.4, found: 770.2.
Fmoc SPPS using 29. Peptide 19: a white lyophilized powder (12.0 mg, 6.70 ꢁmol, 50%); retention
time = 20.4 min (Analytical HPLC conditions: linear gradient of solvent B in solvent A, 30 to 40% over
30 min); retention time = 24.3 (Semiꢀpreparative HPLC conditions: linear gradient of solvent B in
solvent A, 27 to 41% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C76H104N16O17 ([M + 2H]2+): 756.4,
found: 756.3.
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Fmoc SPPS using FmocꢀAsn(OtBu)ꢀOH Peptide 20: a white lyophilized powder (8.32 mg, 5.26 ꢁmol,
53%); retention time = 23.5 min (Analytical HPLC conditions: linear gradient of solvent B in solvent A,
5 to 60% over 30 min); retention time = 28.0 (Semiꢀpreparative HPLC conditions: linear gradient of
solvent B in solvent A, 28 to 42% over 30 min); LRMS (ESIꢀTOF) m/z calcd for C72H95N15O17 ([M +
H]+): 1440.7, found: 1441.0.
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Self-processing of peptide 11b, 19 and 20
Selfꢀprocessing of peptide 11b. A solution of model peptide 11b (45.0 ꢁg, 25.9 nmol) and benzene
sulfonic acid sodium salt (internal standard, 20.7 ng, 0.115 nmol) in phosphate buffer (0.2 M, pH 7.4
and 7.9, 550 ꢁL) containing 6 M guanidine hydrochloride was incubated at 37 °C and the reaction was
monitored by analytical HPLC. Analytical HPLC conditions: a linear gradient of solvent B in solvent A,
1 to 60% over 30 min.
The remaining substrate was calculated based on peak areas (= A) of HPLC as follow. At = 0 indicates
peak areas at beginning of the reaction (t = 0).
ꢂꢃꢄꢅꢃꢆꢇꢈꢆꢉ / ꢂꢊꢋꢆꢉꢇꢋꢈꢌ ꢃꢆꢈꢋꢍꢈꢇꢍ
ꢀ ꢁ
Remaining substrate % =
× 100
ꢎꢏꢐ
ꢎꢏꢐ
ꢂ
/ ꢂ
ꢊꢋꢆꢉꢇꢋꢈꢌ ꢃꢆꢈꢋꢍꢈꢇꢍ
ꢃꢄꢅꢃꢆꢇꢈꢆꢉ
11b: retention time = 21.9 min.
23 or 24: retention time = 16.4 min; LRMS (ESIꢀTOF) m/z calcd for C38H57N8O9 ([M + H]+): 769.4,
found: 769.3.
23 or 24: retention time = 17.4 min; LRMS (ESIꢀTOF) m/z calcd for C38H57N8O9 ([M + H]+): 769.4,
found: 769.3.
22: retention time = 20.1 min; LRMS (ESIꢀTOF) m/z calcd for C40H53N8O9 ([M + H]+): 789.4, found:
789.2.
Benzene sulfonic acid sodium salt (internal standard): retention time = 7.5 min.
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Selfꢀprocessing of peptide 19
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Procedure of selfꢀprocessing of 19 was conducted as similar to that described for 11b (pH = 7.9).
19: retention time = 21.9 min.
31 or 32: retention time = 15.6 min; LRMS (ESIꢀTOF) m/z calcd for C36H53N8O9 ([M + H]+): 741.4,
found: 741.3.
31 or 32: retention time = 16.3 min; LRMS (ESIꢀTOF) m/z calcd for C36H53N8O9 ([M + H]+): 741.4,
found: 741.3.
Selfꢀprocessing of peptide 20. Procedure of selfꢀprocessing of 20 was conducted as similar to that
described for 11b (pH = 7.9). Almost no splitted peptide was observed within 24 h.
20: retention time = 23.5 min.
Photo-responsible amide bond cleavage of peptide 11a
Photoꢀresponsible peptide 11a (45.0 ꢁg, 24.2 nmol) and benzene sulfonic acid sodium salt (internal
standard, 3.00 ng, 16.7 pmol) in phosphate buffer (0.2 M, pH 7.4, 515 ꢁL) containing 6 M guanidine
hydrochloride was incubated at 37 °C for 24 h, and the reaction mixture was then irradiated by UV
(>365 nm) for 1 min. The resulting solution was incubated at 37 °C. The reaction was monitored by
analytical HPLC. Analytical HPLC conditions: a linear gradient of solvent B in solvent A, 1 to 60%
over 30 min.
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11a: retention time = 28.2 min.
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Supporting Information Available:
This material is available free of charge via the Internet at http://pubs.acs.org. 1H and 13C NMR spectra
for new compounds.
Acknowledgements
This research was supported in part by a GrantꢀinꢀAid for Scientific Research (KAKENHI).
Reference
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