DEPBT as coupling reagent to avoid racemization in a solution-phase synthesis of a kyotorphin derivative

Article abstract of DOI:10.1055/s-0033-1341068

The synthesis of IbKTP-NH₂ with reduction of racemization up to no detectable level has been achieved using 3-(diethoxyphosphoryloxy)-1,2,3- benzotriazin-4(3H)-one (DEPBT). Among all coupling systems tested, only DEPBT-mediated amide bond formation between N^α-acylated tyrosine and arginine without loss of optical purity. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0033-1341068

PAPER
▌A
paper
DEPBT as Coupling Reagent To Avoid Racemization in a Solution-Phase
Synthesis of a Kyotorphin Derivative
DEPBT Prevents Racemization in Kyotorphin Synthesis
Vasanthakumar G. Ramu, Eduard Bardaji, Montserrat Heras*
Innovation Laboratory in Organic Chemistry Processes and Products (LIPPSO), Department of Chemistry, University of Girona, Campus
Montilivi, 17071 Girona, Spain
Fax +34(972)418150; E-mail: montserrat.heras@udg.edu
Received: 14.02.2014; Accepted after revision: 06.03.2014
and a large excess of 1-hydroxybenzotriazole (HOBt)
(Figure 1) as racemization suppressor at low temperature.5
Nevertheless, we aimed to improve the optical purity of
IbKTP-NH2 (1) for further studies. In the present work,
Abstract: The synthesis of IbKTP-NH2 with reduction of racemi-
zation up to no detectable level has been achieved using 3-
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT).
Among all coupling systems tested, only DEPBT-mediated amide
bond formation between Nα-acylated tyrosine and arginine without we describe our synthetic efforts to solve the racemization
loss of optical purity.
problem of compound 1.
Key words: amide bond formation, coupling reagent, DEPBT,
kyotorphin, racemization
CO2Et
CN
N
N
N
N
PF6
N
N
N
O
PF6
Kyotorphin (KTP) is an endogenous dipeptide (Tyr-Arg)1
found in the brain of several mammals, including hu-
mans.2 Kyotorphin is a potent analgesic only when it is de-
livered directly to the central nervous system,3 due to its
reduced ability to cross the blood-brain barrier (BBB). As
a part of our research oriented to enhance the BBB cross-
ing properties of KTP, new kyotorphin derivatives were
designed and synthesized. Unlike original KTP, these new
peptides revealed to be highly analgesic following sys-
temic administration,4,5 and displayed safer side effects
profile,6 which make them a promising alternative to opi-
oid drugs.
PF6
P
N
O
N
O
N
Me2N
BOP
P
NMe2
Me2N
HBTU
NMe2
NMe2
PyOxim
O
O
N
OEt
N
N
P
Me
OEt
O
N
C
N
N
Me
DEPBT
EDC
O
O
N
N
N
OH
CN
N
N
EtO
N
N
OH
OH
Oxyma
HOBt
HODhbt
The compound IbKTP-NH2 (1) was one of the kyotorphin
derivatives that gave the best results. This new peptide 1
combines C-terminal amidation with chemical conjuga-
tion to ibuprofen at N-terminal position. It was synthe-
sized at gram-scale following standard solution-phase
peptide synthesis as depicted in Scheme 1. Briefly, the
synthetic strategy started with the coupling reaction be-
tween (S)-ibuprofen and protected methyl L-tyrosinate
followed by saponification of compound 2 to give the free
carboxylic acid 3. The latter was then coupled with L-ar-
ginine amide and the tert-butyl protecting group in the
peptide 4 side chain was removed by treatment with TFA
followed by repetitive lyophilization in aqueous HCl (3
times) in order to get the final IbKTP-NH2 (1) as the hy-
drochloride salt. However, compound 1 was obtained as a
diastereomeric mixture caused by the partial racemization
at the tyrosine stereogenic carbon during the arginine cou-
pling reaction step. As previously reported, the racemiza-
tion was reduced until a diastereomeric ratio of 87:13
using the coupling reagent benzotriazol-1-yloxytris(di-
methylamino)phosphonium hexafluorophosphate (BOP)
Figure 1 Coupling reagents and racemization suppressant agent
structures
It is well established that during amide coupling reactions,
racemization is prone to occur using Nα-acyl-protected
amino acids than their Nα-carbamate-protected counter-
parts.7 The significant amount of racemization produced
during the amide bond formation between amino acid 3
and amidated arginine was probably due to the Nα-acyl
group (ibuprofen) of the compound 3 (Scheme 1). In fact,
in our previous synthesis of endogenous kyotorphin
(KTP) and amidated kyotorphin (KTP-NH2) derivative,
no racemization was detected in the amide bond formation
using BOP as coupling reagent. In this case, tyrosine was
Nα-protected with Boc.4
The racemization pathway of Nα-acyl protected amino
acids is well studied and it has been attributed to the facile
formation of optically labile azlactone intermediates
through the mechanism illustrated in Scheme 2. This
mechanism involves a base-catalyzed ring closure to
azlactone. The α-proton in the azlactone is rather acidic
and in the presence of a base can be deprotonated and re-
protonated causing its racemization. Both enantiomers of
azlactone can acylate amines, but the resulting product
SYNTHESIS 2014, 46, 000A–000F
Advanced online publication: 02.04.2014
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0033-1341068; Art ID: SS-2014-Z0109-OP
© Georg Thieme Verlag Stuttgart · New York

B
V. G. Ramu et al.
PAPER
O
O
O
O
HO
O
a
b
NH
NH
CO2H
Ot-Bu
Ot-Bu
95%
98%
2
3
c
NH
NH
59%
H2N
H2N
HN
HN
O
O
H2N
H2N
d,e
70%
N
N
H
O
H
O
O
NH
O
NH
OH
Ot-Bu
IbKTP-NH2 (1)
4
Scheme 1 Synthetic strategy for the preparation of IbKTP-NH2 (1). Reagents and conditions: (a) H-Tyr(t-Bu)OMe (1 equiv), BOP (1.1 equiv),
N-methylmorpholine (NMM, 3 equiv), DMF, r.t., 6 h; (b) LiOH (2.5 equiv) THF–MeOH–H2O (1:2:2), r.t., overnight; (c) H-Arg-NH2·2HCl (1
equiv), BOP (1 equiv), HOBt (6 equiv), NMM (3 equiv), DMF, –15 °C to r.t., 21 h; (d) TFA–DMF (1:1), r.t., 2 h; (e) HCl (1 M), lyophilization
(3 times).
will be a mixture of epimeric peptides. Carbamate type Taking this result as a starting point, the use of HBTU and
Nα-protecting groups decrease the likelihood of azlactone EDC (Figure 1) instead of BOP as coupling reagents was
formation.7 However, in the intermediate 3, the α-acyl first examined in combination with the racemization sup-
group derivate from ibuprofen does not play the role of a pressant HOBt. Both HBTU, an uronium-type coupling
protecting group because it is part of the compound struc- reagent, and EDC, a carbodiimide type coupling reagent,
ture and, consequently is not possible to change the acyl are commonly used in solution- and solid-phase peptide
group by a carbamate group such as Boc. To circumvent synthesis, and are remarkably efficient in terms of reactiv-
the loss of optical integrity in the amide bond formation of ity and racemization minimization.8,9 Moreover, EDC has
intermediate 4, we had to find the adequate coupling reac- been largely used in solution synthesis of complex natural
tion, which could suppress completely the racemization.
products; this reagent and its urea by-product are water
soluble and facilitate product purifications.10 In our hands,
the use of these reagents gave compound 4 in good yield,
but the racemization was more pronounced than observed
previously (entries 2 and 3). Next, we investigated the use
of 2-cyano-2-(hydroxyimino)acetate (Oxyma, Figure 1)
as racemization suppressant instead of HOBt. Oxyma has
been recently reported as a high efficient additive mainly
in combination with carbodiimide-type coupling reagents
for peptide bond formation both in solution and solid-
phase synthesis.11 Compared with HOBt, Oxyma showed
greater capacity to suppress racemization and enhanced
coupling rates. In addition, Oxyma is less hazardous than
HOBt.12 Thus, EDC in combination with Oxyma was ex-
amined as coupling reagent for the amide bond formation
of dipeptide 4. Nevertheless, the optical purity obtained
for compound 4 was lower compared to our best result
with BOP/HOBt and just a little bit better than those ob-
tained with EDC/HOBt even when the reaction was per-
formed at low temperature (entries 4 and 5).
O
O
R1
H
R1
O
H
R1
base
base
O
O
X
R
N
N
N
H
O
R
R
R2
O
Y
O
R1
O
H2N
R1
H
N
O
O
R
N
Y
N
H
O
R2
R
Scheme 2 Racemization of Nα-acylamino acid derivatives
As mentioned above, in a previous work, racemization of
compound 4 was reduced until 87:13 ratio using the com-
bination BOP/HOBt (Figure 1) at low temperature (Table
1, entry1).
Synthesis 2014, 46, A–F
© Georg Thieme Verlag Stuttgart · New York

PAPER
DEPBT Prevents Racemization in Kyotorphin Synthesis
C
Table 1 Study of the Coupling Conditions between Compound 3 and H-Arg-NH2
NH
H2N
HN
O
O
H2N
HO
O
N
H
NH
O
NH
Ot-Bu
O
Ot-Bu
H-Arg-NH2•2HCl
coupling conditions
4
3
Entry
1c
Coupling reagent
Temp (°C)
–15
Time (h)
21
Yield (%)a
59
drb
87:13
BOP (1 equiv)
HOBt (6 equiv)
2d
3d
4d
5d
6d
7d
HBTU (1 equiv)
HOBt (1 equiv)
0 to r.t.
0 to r.t.
0 to r.t.
–15
1
12
4
73
71
70
71
78
76
68:32
70:30
78:22
80:20
78:22
EDC (1 equiv)
HOBt (1 equiv)
EDC (2 equiv)
Oxyma (2 equiv)
EDC (2 equiv)
Oxyma (2 equiv)
12
2
PyOxim (1equiv)
Oxyma (1 equiv)
0 to r.t.
0 to r.t.
DEPBT (1.1 equiv)
2
>98
a Yields of isolated products.
b Determined by 1H NMR spectroscopy.
c Reaction was performed in DMF using NMM (3 equiv) as base.
d Reaction was performed in DMF using i-Pr2NEt (DIPEA, 3 equiv) as base.
We also tested the viability of O-[(cyano(ethoxycarbon- demonstrated remarkable reactivity and reduces dramati-
yl)methyliden)amino]yloxytri(pyrrolidino)phosphonium cally the degree of racemization in amide bond formation,
hexafluorophosphate (PyOxim, Figure 1) a phosphonium- even when a strong base is required,21 especially in the so-
type coupling reagent derived from Oxyma that was re- lution synthesis of complex and racemization-sensitive
ported to exhibit higher control of optical purity over their natural products.22,23 When our target coupling reaction
HOBt counterparts such as BOP, in solution- as well as in was run with DEPBT in the absence of additives, dipep-
solid-phase peptide synthesis.13 In our case, the combina- tide 4 was obtained in good yield. Comparison of 1H NMR
tion PyOxm/Oxyma as coupling reagent led to compound spectrum of compound 4 synthesized using BOP/HOBt
4 in good yield, but with similar optical purity as the other with the one obtained using DEPBT clearly shows the
tests (entry 6,). Later, our attention was focused on 3- elimination of signal splitting (Figure 2), and confirmed
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
that coupling had occurred with no detectable epimeriza-
(DEPBT) developed by Ye et al. in the nineties.14,15 tion (entry 7). This clearly shows the advantageous role of
DEPBT is a phosphonate-type coupling reagent derived DEPBT on chiral integrity control of compound 4 over the
from the racemization suppressant 3-hydroxy-1,2,3-ben- other coupling systems tested. This significant perfor-
zotriazin-4(3H)-one (HODhbt, Figure 1).16 This latter was mance of DEPBT in amide peptide bond formation and
developed contemporaneously with HOBt and other ben- control on chiral integrity of compound 4 has to be related
zotriazole-type additives, but although proved to be supe- with the higher reactivity of the intermediate HODhbt and
rior to all in terms of coupling efficiency and inhibit the nature of the diethyl phosphite ester involved in its for-
racemization capacity,17,18 its widespread use was limited mation, in agreement with the proposed DEPBT-mediated
due to ring-opening side reaction.19,20 Even so, DEPBT coupling mechanism.15
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, A–F

D
V. G. Ramu et al.
PAPER
Figure 2 (A) 1H NMR spectrum (region 4–9 ppm) of 4 synthesized with DEPBT. (B) 1H NMR spectrum (region 4–9 ppm) of 4 synthesized
with BOP/HOBt.
Finally, removal of tert-butyl protecting group of 4 by tive confirms the racemization-free coupling. Additional-
treatment with 4 M HCl in 1,4-dioxane instead of TFA ly, the final deprotection step of compound 4 has also
(Scheme 1) directly provided the target kyotorphin deriv- been improved using 4 M HCl in 1,4-dioxane instead of
ative 1 as its hydrochloride salt in 80% yield (Scheme 3). TFA.
This deprotection procedure improved the reaction yield
and eliminated repetitive lyophilization steps with HCl
necessary to exchange trifluoroacetate counterion from
the final dipeptide 1.
All commercially available chemicals were used as purchased with-
out further purification. NMR spectra were recorded on a Bruker
Ultrashield Avance III 400 spectrometer. 1H NMR spectra were re-
corded at 400 MHz and 13C NMR spectra and DEPT experiments
were determined at 100 MHz. Spectra recorded in CDCl3 were ref-
erenced to residual CHCl3 at 7.26 ppm for proton and 77.0 ppm for
carbon. Spectra recorded in DMSO-d6 were referenced to residual
DMSO at 2.50 ppm for proton and 39.5 ppm for carbon. High reso-
lution mass spectra (HRMS) were determined under conditions of
In conclusion, we have improved the synthesis of kyotor-
phin derivative 1 by reducing the racemization up to no
detectable level at its key coupling reaction step; among
all coupling reagent systems tested only DEPBT afforded
the desired result. 1H NMR spectra of kyotorphin deriva-
NH
NH
H2N
H2N
HN
HN
O
O
4 M HCl, 1,4-dioxane
r.t., 1 h
H2N
H2N
N
H
N
H
O
O
NH
O
NH
80%
O
Ot-Bu
OH
1
4
Scheme 3 Removal of tert-butyl group of compound 4
Synthesis 2014, 46, A–F
© Georg Thieme Verlag Stuttgart · New York

PAPER
DEPBT Prevents Racemization in Kyotorphin Synthesis
E
electrospray ionization (ESI) on a Bruker Micro Q-Tof instrument
using a hybrid quadrupole time-of-flight mass spectrometer. Ana-
lytical TLC was performed on precoated TLC plates, silica gel 60
F254 (Merck). The spots on the TLC plates were visualized with
UV/visible light (254 nm) and/or with a solution of KMnO4 (1.5
g/100 mL H2O). HPLC analyses were carried out using a Dionex
P680 instrument. Separations were achieved on an analytical C18
Kromasil reversed phase column (4.6 mm × 40 mm; 3.5 μm particle
size). The compounds were eluted using a linear gradient of 0–
100% MeCN in 0.1% TFA at flow rate of 1.0 mL/min. The absor-
bance was measured at 220 nm. The HPLC retention time of each
compound was determined when the peak was at its maximum
height.
J = 8.5 Hz, 2 H, CHaryl), 7.15 (d, J = 8.1 Hz, 2 H, CHaryl), 8.21 (d,
J = 8.4 Hz, 1 H, NH), 12.63 (br, 1 H, OH).
13C NMR (100 MHz, DMSO-d6): δ = 18.7 (q), 22.2 (q, 2 C), 28.5
(q, 3 C), 29.6 (d), 36.1 (t), 44.2 (t), 44.3 (d), 53.2 (d), 77.6 (s), 123.4
(d, 2 C), 127.1 (d, 2 C), 128.6 (d, 2 C), 129.7 (d, 2 C), 132.3 (s),
139.0 (s), 139.1 (s), 153.4 (s), 173.0 (s), 173.2 (s).
HRMS (ESI): m/z calcd for C26H35NO4 + Na [M + Na]+: 448.2458;
found: 448.2476; m/z calcd for C26H36NO4 [M + H]+: 426.2639;
found: 426.2653.
Ibu-Tyr(t-Bu)-Arg-NH2 (4)
To a solution of compound 3 (340 mg, 0.8 mmol) in DMF (4 mL)
was added DIPEA (0.43 mL, 2.5 mmol) and the resulting mixture
was allowed to stir for 0.5 h at 0 °C. Next, DEPBT (264 mg, 0.88
mmol) and H-Arg-NH2·2HCl (197 mg, 0.8 mmol) were added suc-
cessively. The resulting reaction mixture was stirred 1 h at 0 °C and
the stirring was continued at r.t. for another hour. Upon completion
of the reaction (HPLC monitoring), the reaction mixture was diluted
with EtOAc (15 mL). The resulting solution was washed with aq 1
M HCl (3 × 7 mL), sat. aq NaHCO3 (3 × 7 mL), and brine (10 mL).
The organic layer was dried (MgSO4), filtered, and concentrated in
vacuo to obtain compound 4; yield: 353 mg (76%); colorless solid;
mp 122–124 °C; HPLC: tR = 7.96 min, 98.0% pure.
Methyl (S)-2-[(S)-2-(4-Isobutylphenyl)propanamido]-3-(4-tert-
butoxyphenyl)propanoate (2)
To a solution of (S)-ibuprofen (206 mg, 1 mmol) in DMF (4 mL)
was added 0.33 mL (2 mmol) of NMM, and the resulting mixture
was allowed to stir for 0.5 h at r.t. Next, BOP (442 mg, 1.1 mmol)
and H-Tyr(t-Bu)-OMe·HCl (288 mg, 1 mmol) were added succes-
sively. The resulting reaction mixture was stirred overnight at r.t.
Upon completion of the reaction (TLC, eluent: n-hexane–EtOAc,
1:1), the reaction mixture was diluted with EtOAc (15 mL). The re-
sulting solution was washed with sat. aq NaHCO3 (3 × 7 mL), H2O
(10 mL), 1 M aq KHSO4 (3 × 7 mL), and brine (10 mL). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo. The
resulting residue was purified by SiO2 flash chromatography (n-
hexane–EtOAc, 10:1 to 5:1) to afford compound 2; yield: 417 mg
(95%); colorless oil; Rf = 0.41 (n-hexane–EtOAc, 1:1); HPLC: tR =
10.16 min; 99.0% pure.
1H NMR (400 MHz, DMSO-d6): δ = 0.84 [d, J = 6.6 Hz, 6 H,
CH(CH3)2], 1.10 (d, J = 7.0 Hz, 3 H, CH3-βibuprofen), 1.25 [s, 9 H,
C(CH3)3], 1.35–1.55 (m, 3 H of CH2CH2arginine), 1.61–1.66 (m, 1 H
of CH2CH2arginine), 1.78 [nonet, J = 6.7 Hz, 1 H, CH(CH3)2], 2.37 (d,
J = 7.0 Hz, 2 H, ArCH2ibuprofen), 2.72 (dd, J = 13.9 Hz, J′ = 9.9 Hz, 1
H of the CH2-βtyrosine), 2.99–3.08 (m, 3 H, CH2NH and 1 H of the
CH2-βtyrosine), 3.59 (q, J = 7.0 Hz, 1 H, CH-αibuprofen), 4.14 (q, J = 7.8
Hz, 1 H, CH-αarginine), 4.48–4.54 (m, 1 H, CH-αtyrosine), 6.83 (d,
J = 8.4 Hz, 2 H, CHaryl), 7.00–7.06 (m, 2 H, D2O exch. and 2 H,
CHaryl), 7.08 (br, 2 H, D2O exch.), 7.13–7.16 (m, 4 H, CHaryl), 7.35
(br, 1 H, D2O exch.), 7.68 (t, J = 5.2 Hz, 1 H, NH, D2O exch.), 8.03
(d, J = 8.0 Hz, 1 H, NH, D2O exch.), 8.21 (d, J = 8.2 Hz, 1 H, NH,
D2O exch.).
13C NMR (100 MHz, DMSO-d6): δ = 18.5 (q), 22.2 (q, 2 C), 24.9
(t), 28.5 (q, 3 C), 29.2 (t), 29.6 (d), 36.8 (t), 39.9 (t), 44.2 (t), 44.4
(d), 51.9 (d), 53.9 (d), 77.5 (s), 123.3 (d, 2 C), 127.1 (d, 2 C), 128.6
(d, 2 C), 129.8 (d, 2 C), 132.7 (s), 139.0 (s), 139.1 (s), 153.3 (s),
156.9 (s), 171.1 (s), 173.1 (s), 173.3 (s).
HRMS (ESI): m/z calcd for C32H49N6O4 [M + H]+: 581.3810; found:
581.3811.
1H NMR (400 MHz, CDCl3): δ = 0.90 [d, J = 6.4 Hz, 6 H,
CH(CH3)2], 1.32 [s, 9 H, C(CH3)3], 1.48 (d, J = 7.2 Hz, 3 H, CH3-
βibuprofen), 1.85 [nonet, J = 6.7 Hz, 1 H, CH(CH3)2], 2.45 (d,
J = 7.2 Hz, 2 H, ArCH2ibuprofen), 2.94 (dd, J = 13.9 Hz, J′ = 6.2 Hz, 1
H of the CH2-βtyrosine), 3.02 (dd, J = 13.9 Hz, J′ = 5.7 Hz, 1 H of the
CH2-βtyrosine), 3.52 (q, J = 7.2 Hz, 1 H, CH-αibuprofen), 3.64 (s, 3 H,
OCH3), 7.75 (app q, J = 5.9 Hz, 1 H, CH-αtyrosine), 5.76 (d, J = 7.7
Hz, 1 H, NH), 6.79–6.84 (m, 4 H, CHaryl), 7.09 (d, J = 8.1 Hz, 2 H,
CHaryl), 7.14 (d, J = 8.1 Hz, 2 H, CHaryl).
13C NMR (100 MHz, CDCl3): δ = 18.1 (q), 22.3 (q, 2 C), 28.8 (q, 3
C), 30.1 (d), 37.0 (t), 45.0 (t), 46.6 (d), 52.1 (q), 53.1 (d), 78.3 (s),
124.1 (d, 2 C), 127.3 (d, 2 C), 129.52 (d, 2 C), 129.53 (d, 2 C), 130.5
(s), 137.6 (s), 140.7 (s), 154.3 (s), 171.8 (s), 174.0 (s).
HRMS (ESI): m/z calcd for C22H37N3O6 + Na [M + Na]+: 462.2575;
found: 462.2577; m/z calcd for C22H38N3O6 [M + H]+: 440.2755;
found: 440.2739.
Ibu-Tyr-Arg-NH2 (1)
Compound 4 (320 mg, 0.55 mmol) was dissolved in 4 M HCl in 1,4-
dioxane (15 mL) and stirred for 1 h at r.t. After completion of the
reaction (HPLC monitoring), the solvent was evaporated in vacuo
and the resulting residue was triturated with Et2O (15 mL), filtered,
washed with Et2O (2 × 10 mL) and n-pentane (2 × 10 mL), and dried
to obtain compound 1; yield: 230 mg (80%); colorless solid; mp
140–142 °C; HPLC: tR = 7.32 min, 99.9% pure.
(S)-2-[(S)-2-(4-Isobutylphenyl)propanamido]-3-(4-tert-butoxy-
phenyl)propanoic Acid (3)
To a solution of compound 2 (395 mg, 0.9 mmol) in THF–MeOH–
H2O (1:2:2, 7 mL) was added LiOH·H2O (94.5 mg (2.25 mmol) and
the resulting mixture was stirred for 1 h at r.t. Upon completion of
the reaction (TLC, eluent: n-hexane–EtOAc, 1:1), the pH of the so-
lution was adjusted to 2 with the addition of aq 1 M HCl. The result-
ing solution was extracted with CH2Cl2 (3 × 10 mL). The combined
organic layers were dried (MgSO4), filtered, and concentrated in
vacuo. The crude material was triturated with n-pentane (4 mL), fil-
tered, washed with n-pentane (1 mL), and dried to obtain compound
3; yield: 375 mg (98%); colorless solid; mp 57–58 °C; Rf = 0.18
(CHCl3–MeOH–NH3, 12:2:0.2); HPLC: tR = 9.41 min, 99.9% pure.
1H NMR (400 MHz, DMSO-d6): δ = 0.84 [d, J = 6.8 Hz, 6 H,
CH(CH3)2], 1.15 (d, J = 7.2 Hz, 3 H, CH3-βibuprofen), 1.34–1.52 (m,
3 H of the CH2CH2arginine), 1.60–1.68 (m, 1 H of the CH2CH2arginine),
1.78 [nonet, J = 6.7 Hz, 1 H, CH(CH3)2], 2.37 (d, J = 7.1 Hz, 2 H,
ArCH2ibuprofen), 2.65 (dd, J = 13.8 Hz, J′ = 9.5 Hz, 1 H of the CH2-
βtyrosine), 2.91 (dd, J = 13.8 Hz, J′ = 4.3 Hz, 1 H of the CH2-βtyrosine),
3.03 (app q, J = 6.5 Hz, 2 H, CH2NH), 3.60 (q, J = 6.9 Hz, 1 H, CH-
αibuprofen), 4.15 (app q, J = 7.6 Hz, 1 H, CH-αarginine), 4.41–4.46 (m, 1
H, CH-αtyrosine), 6.63 (d, J = 8.4 Hz, 2 H, CHaryl), 7.0 (d, J = 8.4 Hz,
2 H, CHaryl), 7.02 (d, J = 8.1 Hz, 2 H, CHaryl), 7.08 (br, 1 H, D2O
exch.), 7.13 (d, J = 8.1 Hz, 2 H, CHaryl), 7.27 (br, 2 H, D2O exch.),
7.61 (t, J = 5.5 Hz, 1 H, NH, D2O exch.), 7.95 (d, J = 8.0 Hz, 1 H,
NH, D2O exch.), 8.07 (d, J = 8.3 Hz, 1 H, NH, D2O exch.), 9.20 (br,
1 H, D2O exch.).
1H NMR (400 MHz, DMSO-d6): δ = 0.84 [d, J = 6.6 Hz, 6 H,
CH(CH3)2], 1.14 (d, J = 7.0 Hz, 3 H, CH3-βibuprofen), 1.26 [s, 9 H,
C(CH3)3], 1.79 [nonet, J = 6.7 Hz, 1 H, CH(CH3)2], 2.39 (d, J = 7.1
Hz, 2 H, ArCH2ibuprofen), 2.80 (dd, J = 13.7 Hz, J′ = 9.9 Hz, 1 H of
the CH2-βtyrosine), 3.02 (dd, J = 13.8 Hz, J′ = 4.7 Hz, 1 H of the CH2-
β
tyrosine), 3.58 (q, J = 7.0 Hz, 1 H, CH-αibuprofen), 4.39 (ddd,
J = 9.7 Hz, J′ = 8.5 Hz, J′′ = 4.7 Hz, 1 H, CH-αtyrosine), 6.84 (d, J =
8.5 Hz, 2 H, CHaryl), 7.04 (d, J = 8.1 Hz, 2 H, CHaryl), 7.08 (d,
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, A–F

F
V. G. Ramu et al.
PAPER
13C NMR (100 MHz, DMSO-d6): δ = 18.7 (q), 22.2 (q, 2 C), 25.0
(t), 29.3 (d), 29.7 (t), 36.6 (t), 40.4 (t), 44.3 (d), 44.4 (t), 51.9 (d),
54.1 (d), 114.8 (d, 2 C), 127.1 (d, 2 C), 127.9 (s), 128.7 (d, 2 C),
130.2 (d, 2 C), 139.1 (s), 139.2 (s), 155.8 (s), 156.8 (s), 171.2 (s),
173.2 (s), 173.5 (s).
HRMS (ESI): m/z calcd for C28H41N6O4 [M + H]+: 525.3184; found:
525.3188.
(6) Ribeiro, M. M. B.; Santos, S. S.; Sousa, D. S. C.; Oliveira,
M.; Santos, S. M.; Heras, M.; Bardaji, E.; Tavares, I.;
Castanho, M. A. R. B. Amino Acids 2013, 45, 171.
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Zahradnik, R., Eds.; Springer-Verlag: Berlin, 1994.
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Acknowledgment
Marie Curie Industry-Academia Partnerships and Pathways (Euro-
pean Commission) is acknowledged for funding and fellowship to
Vasanthakumar G. Ramu (FP7-PEOPLE-2007-3-1-IAPP, project
PEP2BRAIN-230654). We thank Prof. Miguel A. R. B. Castanho,
Scientific Coordinator of the PEP2BRAIN project, for helpful sug-
gestions.
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M. Org. Lett. 1999, 1, 91.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
n
nfomartit
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Synthesis 2014, 46, A–F
© Georg Thieme Verlag Stuttgart · New York