(Acyloxy)alkoxy moiety as amino acids protecting group for the synthesis of (R,R)-2,7 diaminosuberic acid via RCM

Article abstract of DOI:10.2174/092986612803521666

A new synthetic pathway is described to prepare asymmetrically protected 2,7-diaminosuberic acid. This strategy exploits (acyloxy)alkoxy promoiety as protecting group and RCM reaction using second generation Grubbs catalyst and provides the trans isomer of (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino) -2-(tert-butoxycarbonylamino)-8-methoxy-8-oxooct-4-enoic acid, which was in turn reduced to obtain (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(tert- butoxycarbonylamino)-8-methoxy-8-oxooctanoic acid.

Full text of DOI:10.2174/092986612803521666

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Protein & Peptide Letters, 2012, 19, 1245-1249
1245
(Acyloxy)Alkoxy Moiety as Amino Acids Protecting Group for the Synthe-
sis of (R,R)-2,7 Diaminosuberic Acid via RCM
Adriano Mollica*,1, Federica Feliciani1, Azzurra Stefanucci2, Evgeny A. Fadeev3 and
Francesco Pinnen1
1Dipartimento di Scienze del Farmaco, Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti
Italy; 2Dipartimento di Chimica, Università degli Studi di Roma "La Sapienza", P.le Aldo Moro 5, I-00185 Roma, Italy;
3Department of Chemistry, University of California Irvine, 1212 Natural Sciences Building 1, Irvine, CA 92697, U.S.A.
Abstract: A new synthetic pathway is described to prepare asymmetrically protected 2,7-diaminosuberic acid. This strat-
egy exploits (acyloxy)alkoxy promoiety as protecting group and RCM reaction using second generation Grubbs catalyst
and provides the trans isomer of (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(tert-butoxycarbonylamino)-8-
methoxy-8-oxooct-4-enoic acid, which was in turn reduced to obtain (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbony-
lamino)-2-(tert-butoxycarbonylamino)-8-methoxy-8-oxooctanoic acid.
Keywords: Grubbs catalyst, ring closing metathesis, (acyloxy)alkoxy, diaminosuberic acid, orthogonal protecting groups.
INTRODUCTION
structure, for convenient manipulation during solution and/or
solid phase peptide synthesis, the two amino and carboxylic
groups of the suberic acid residue must be differentially pro-
tected. Previously described syntheses of 2,7-diaminosuberic
acid involve Kolbe dimerization of two glutamic acid units.
This approach is useful only for the synthesis of symmetrical
compounds and was also used to prepare asymmetrically
protected 2,7-diaminosuberic acid [11] through the dimeriza-
tion of two differentially protected glutamic acid residues.
As expected, this procedure yields a statistical mixture con-
taining two undesired symmetric dimers besides a desired
unsymmetrical dimer, which must be isolated. Other ap-
proaches were accomplished i) by a copper-mediated or-
ganozinc/haloalkyne coupling [12], ii) by Ring-Closing Al-
kyne Metathesis (RCAM) reaction [13] and iii) by using 1,2-
benzenedimethanol as protecting groups of C-terminus of
allylglycine residues [14]. Recently, Grubbs and co-workers
The (acyloxy)alkoxy promoiety [1-3] was originally
taken into account for its lability to the esterase metabolism
and for this feature designed for the synthesis of prodrugs,
cleavable in vivo but stable in anhydrous acidic conditions.
These properties have stimulated us to explore the use of this
moiety as simultaneous N- and C-terminal protecting group
in peptide synthesis, alternative to the conventional orthogo-
nal protection. In this context, to verify this potentiality of
the (acyloxy)alkoxy promoiety we developed the synthesis
of diaminosuberic acid, a well known and structurally inter-
esting natural bivalent amino acid. The synthesis provides
the ring closing metathesis (RCM) strategy combined with
the (acyloxy)alkoxy as protecting group. The key synthon 6
of the process is illustrated in Figure (1).
O
Xaa1-C=C-Xaa2
NH
[15] reported
a
convenient synthesis of (S,S)-2,7-
diaminosuberic acid. Notwithstanding the usefulness of the
previous approaches, the problems related to the develop-
ment of alternative synthetic methods which would lead to
the preparation of unambiguous diastereomeric and selec-
tively protected 2,7-diaminosuberic acid derivatives, have
not yet been solved. In this paper we report a new method for
the synthesis of orthogonally protected 2,7-diaminosuberic
acid with a well-defined stereochemistry. The methodology,
reported in Scheme 1, combines the Grubbs approach [16]
and the (acyloxy)alkoxy promoiety used as novel protecting
group.
O
O
O
Figure 1. The key synthon for the synthesis of 6
The diaminosuberic acid is considered an isosteric di-
carba analogue of cystine, where the sulphur atoms have
been replaced by an olefinic bridge, and it has been used to
improve the chemical stability of biologically active peptides
such as analogues of calcitonin [4], oxytocin [5-7], and
somatostatin [8-10]. Despite the apparent semplicity of its
MATERIAL AND METHODS
Reagents
*Address correspondence to this author at the Faculty of Pharmacy, De-
partment of Pharmaceutical Sciences, University of Chieti-Pescara “G. d’
Annunzio”, Via dei Vestini 31, 66100, Chieti, Italy;
All the starting materials and reagents were purchased
from Sigma-Aldrich. Column chromatography was per-
Tel/Fax +3908713554476; E-mail: a.mollica@unich.it
1875-5305/12 $58.00+.00
© 2012 Bentham Science Publishers

1246 Protein & Peptide Letters, 2012, Vol. 19, No. 12
Mollica et al.
O
H
O
Boc
N
H
O
NO2
Cs+
+
Boc
N
-
O
O
a
O
O
I
O
O
O
NO2
1
2
O
H
O
O
Boc
N
c
H
b
O
O
O
O
Boc
O
N
O
O
N
H
NH
O
O
3
4
O
H
Boc
N
O
OH
H
Fmoc
NH
Boc
N
f
OH
Fmoc
NH
d,e
O
O
O
6
O
5
Scheme 1. Reagents and conditions: a) DMF, 2 h 0°C then r.t. overnight; b) D-Allyl-Gly-OMe.HCl, NMM, HOBt, DMF, 2 h 0°C then r.t.
overnight under N2; c) Grubbs’ catalyst second generation, CH2Cl2, reflux, 1 h; d) NaHCO3, MeOH, H2O, r.t., 7 h; e) Fmoc-OSu, NaHCO3
ss, DMF, r.t., 30 min; f) H2, Pd/C, MeOH, r.t., overnight.
1
formed on glass columns loaded with silica gel. Routine H
NMR spectra were determined in CDCl3 or DMSO solution
with a Varian 300 MHz and chemical shifts were referred to
TMS as internal standard. For establishing the olefin con-
figuration 1D Noesy and 2D gCosy experiments on product
5 were carried on a Varian Unity Inova 800 MHz NMR
spectrometer. The mass spectra were performed on a TOF-
ESI spectrometer. TLC were performed on silica gel Merck
60 F254 plates.
glycinyloxy)methyl]carbonyl]-D-allyl-glycine methyl ester
(3) and the side product Boc-D-allyl-Gly-D-allyl-Gly-OMe
in a 7:3 ratio. After purification by silica gel column chroma-
tography, 3 was subjected to RCM cyclization in the pres-
ence of the second generation of Grubbs’ catalyst, in the
conditions previously reported [18], monitoring the reaction
by TLC. This reaction provided the cyclic compound 4 as
unique E isomer, which was in turn hydrolyzed by treatment
with NaHCO3 (1 eq.) in MeOH/H2O for 7 h and promptly
reacted with Fmoc-OSu giving the (2R,7R,E)-7-(((9H-
fluoren-9-yl)methoxy)carbonylamino)-2-(tert-butoxycarbo-
nylamino)-8-methoxy-8-oxooct-4-enoic acid 5. Catalytic
hydrogenation on Pd/C (10% grade) in MeOH yielded the
(2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-
(tert-butoxycarbonylamino)-8-methoxy-8-oxooctanoic
acid 6.
EXPERIMENTAL
The synthesis of selectively protected (2R,7R)-2,7-
diaminosuberic acid (6) is reported in the Scheme 1. The
reaction between Boc-D-allyl-Gly-OH as cesium salt and 1-
iodomethyl p-nitrophenyl carbonate [17], provided a mixture
of Boc-(D-allyl-glycinyloxy)methyl-p-nitrophenyl carbonate
(2) (70%) and the expected side product Boc-D-allyl-Gly-
OpNP (30%). The crude mixture was reacted as such with D-
allyl-Gly-OMe.HCl, prepared according to published proce-
dure [17]. This step provided a mixture of Boc-[[(D-allyl-
Synthesis of Compound 1
Boc-D-allyl-Gly-OH.DCHA (2.11 g, 5.32 mmol) was
dissolved in deionized water (100 mL) and acidified with 2N

(Acyloxy)Alkoxy Moiety as Amino Acids Protecting Group
Protein & Peptide Letters, 2012, Vol. 19, No. 12 1247
HCl. The solution was then extracted with EtOAc (3 x 50
mL) and washed with sat. NaCl (50 mL). The pooled organic
layers were dried over Na2SO4 and evaporated under reduced
pressure to give Boc-D-allyl-Gly-OH. Yield 1.15 g (quantita-
tive). Boc-D-allyl-Gly-OH (0.60 g, 2.80 mmol) was then
dissolved in MeOH (20 mL) and Cs2CO3 (0.46 g, 1.39
mmol) was added. The mixture was stirred at room tempera-
ture for 1 h and then evaporated under reduced pressure to
give 1, which was used without further purification. Yield:
0.91 g (quantitative).
2H, CH=CH), 5.69 (m, 2H, O-CH2-O). ESI-MS Calcd. for
C16H26N2O7 [M+H]+ 359.2, Found 359.7. Anal. Calcd for
C16H26N2O7: C, 53.62; H, 7.31; N, 7.82; O, 31.25. Found: C,
53.05; H, 7.52; N, 7.61; O, 31.43.
Synthesis of Compound 5
NaHCO3 (0.01 g, 0.14 mmol) in H2O (1 mL) was added
to a solution of 4 (0.05 g, 0.14 mmol) in MeOH (5 mL) and
the reaction was stirred at room temperature monitoring on
TLC (CH2Cl2/EtOAc 8:2). After 7 h, the solvent was re-
moved under reduced pressure, the crude residue was redis-
solved with sat. NaHCO3 (1.5 mL) and the solution was
cooled to 0 ºC. Fmoc-OSu (0.05 g, 0.14 mmol) in DMF (1
mL) was then added and the reaction was stirred at room
temperature. After 30 min, the mixture was diluted with H2O
and extracted with EtOAc. The aqueous layer was acidified
to pH 5 at 0 ºC with 2N HCl and then extracted twice with
EtOAc. The pooled organic layers were dried over anhy-
drous Na2SO4 and evaporated under reduced pressure. The
crude product was purified by silica gel column chromatog-
raphy (EtOAc/CH2Cl2 8:2 to EtOAc/MeOH 95:5). Overall
Synthesis of Compound 2
A solution of Boc-D-allyl-Gly-OCs (0.98 g, 2.97 mmol)
in 15 mL of anhydrous DMF was added dropwise under N2
to an ice-cold stirred solution of 1-iodomethyl-p-nitrophenyl
carbonate (0.96 g, 2.97 mmol) in anhydrous DMF (15 mL).
The reaction was stirred under N2 at 0 ºC for 2 h then at
room temperature overnight, monitoring the completeness on
silica gel TLC (hexane/EtOAc 8:2). The DMF was removed
under reduced pressure and the residue was redissolved in
EtOAc (250 mL) and washed with sat. NaHCO3 (2 x 100
mL), and sat. NaCl (100 mL). The organic layer was dried
over anhydrous Na2SO4 and evaporated under reduced pres-
sure to give the crude title compound which was used in the
next step without further purification.
1
yield of two steps: 0.014 g (11%). H NMR (DMSO-d6) ꢀ
1.34 (s, 9H, Boc), 2.28-2.34 (m, 4H, D-allyl-Gly ꢁCH2), 3.56
(s, 3H, OCH3), 3.92 (m, 2H, D-allyl-Gly, ꢂCH), 4.20-4.26
(m, 3H, CH=CH and CH Fmoc), 5.44 (br, 2H, CH2 Fmoc),
7.18 (d, 1H, NH-Boc), 7.30-7.88 (m, 9H, aromatics Fmoc
and d at 7.55 NH-Fmoc). ESI-MS Calcd. for C29H34N2O8
[M+Na]+ 561.2, Found 561.2. Anal. Calcd for C29H34N2O8:
C, 64.67; H, 6.36; N, 5.20; O, 23.76. Found: C, 64.83; H,
6.12; N, 5.44; O, 23.81.
Synthesis of Compound 3
NMM (0.26 mL, 2.40 mmol) was added to an ice-cold
stirred solution containing 2 (0.98 g, 2.40 mmol), D-allyl-
Gly-OMe.HCl (0.40 g, 2.40 mmol) and HOBt (0.32 g, 2.40
mmol) in anhydrous DMF (30 mL). After stirring under N2
at 0 ºC for 2 h and at room temperature overnight, the DMF
was removed under reduced pressure. The residue was dis-
solved in CH2Cl2 (200 mL) and washed with 10% aqueous
citric acid (2 x 50 mL), H2O (100 mL), sat. NaHCO3 (2 x 50
mL) and sat. NaCl (100 mL). The organic layer was dried
over anhydrous Na2SO4 and evaporated under reduced pres-
sure. The crude title compound was purified on silica gel
column (CH2Cl2 100% to CH2Cl2/EtOAc 85:15). Rf = 0.4
(CH2Cl2/EtOAc 9:1). Yield 0.27 g (28%). 1H NMR (CDCl3)
ꢀ 1.43 (s, 9H, Boc), 2.40-2.53 (m, 4H, D-allyl-gly ꢁCH2),
3.75 (s, 3H, OCH3), 4.37 (m, 2H, D-allyl-gly ꢂCH), 4.95 (d,
1H, NH-Boc), 5.03-5.23 (m, 4H, CH2=CH), 5.35 (d, 1H,
NH), 5.57 (m, 2H, CH2=CH), 5.75 (q, 2H, O-CH2-O). ESI-
MS Calcd. for C18H28N2O8 [M+H]+ 401.2, Found 401.8. A-
nal. Calcd for C18H28N2O8: C, 53.99; H, 7.05; N, 7.00; O,
31.96. Found: C, 54.22; H, 7.31; N, 7.15; O, 31.75.
Synthesis of Compound 6
A solution of 5 (0.004 g, 0.007 mmol) in MeOH (5 mL)
was stirred at room temperature under H2 atmosphere in the
presence of Pd/C (1 mg). After 2h, a second portion of cata-
lyst (1 mg) was added and the mixture was kept overnight
under the same conditions. The catalyst was filtered off and
the solvent removed under reduced pressure to give the pure
1
compound 6. Yield 0.002 g (50%). H NMR (DMSO-d6)
ꢀ1.24-1.25 (m, 4H,2 x ꢃCH2)1.34 (s, 9H, Boc), 1.54 (m, 4H,
2 x ꢁCH2) 3.58 (s, 3H, OCH3), 3.85 (m, 2H, ꢂCH), 4.10-
4.23 (m, 3H, CH2 and CH-Fmoc), 7.22 (d, 1H, NH-Boc),
7.30-7.88 (m, 9H, aromatics and NH-Fmoc). ESI-MS Calcd.
for C29H36N2O8 [M+Na]+ 563.2, Found 563.6. Anal. Calcd
for C29H36N2O8: C, 64.43; H, 6.71; N, 5.18; O, 23.68. Found:
C, 64.75; H, 6.32; N, 5.44; O, 23.35.
Double Bond Geometry Assignment
The key intermediate 4 was analyzed on a Varian Unity
Inova 800 MHz NMR spectrometer. Resonance assignments
were made using standard 1D proton (s2pul), selective NO-
ESY1D [19] and gCOSY pulse sequences. Spectral window
for all experiments was set at 9000 Hz. J coupling constant
between the olefin CH protons is 14 Hz, suggesting E con-
figuration. NOESY1D experiment was performed with selec-
tive excitation of resonances at 3.78 ppm, 2.86 ppm, 2.70
ppm, 2.10 ppm and 1.45 ppm, For each setting of the selec-
tive excitation a series of measurements were performed
with eight settings of mixing time at 0.05, 0.1, 0.2, 0.3, 0.5,
0.7, 1, 2 seconds. Both methylene protons 1 and 4 show
strong NOE correlations with protons 2 and 3 respectively
Synthesis of Compound 4
To a stirred refluxing solution of 3 (0.25 g, 0.62 mmol) in
CH2Cl2 (230 mL), benzylidene[1,3-bis-(2,4,6-trimethyl-
phenyl)-2-imidazolidinylidene]-dichloro(phenylmethylene)-
(tricyclohexylphosphine)ruthenium (Grubbs’ second genera-
tion catalyst) (0.05 g, 0.06 mmol) was added and the reaction
was monitored by TLC (CH2Cl2:EtOAc 8:2). After 1 h the
solvent was evaporated and the crude product was purified
on silica gel column (CH2Cl2/EtOAc 95:5 to CH2Cl2/EtOAC
85:15). Rf = 0.5 (CH2Cl2/AcOEt 8:2). Yield 0.17 g (77%). 1H
NMR (CDCl3) ꢀ 1.44 (s, 9H, Boc), 2.07-2.88 (m, 4H, D-
allyl-Gly ꢁCH2), 3.77 (s, 3H, OCH3), 4.48 (m, 2H, D-allyl-
Gly ꢂCH), 5.26 (d, 1H, NH-Boc), 5.35 (d, 1H, NH), 5.45 (m,

1248 Protein & Peptide Letters, 2012, Vol. 19, No. 12
Mollica et al.
(Figure 2). This observation confirmed the olefin E configu-
ABBREVIATIONS
ration.
Boc
=
=
=
=
=
tert-Butyloxycarbonyl
Benzyloxycarbonyl
O
Cbz
H
O
O
H
DCHA
Fmoc
Dicyclohexylamin
N
O
9-Fluorenylmethoxycarbonyl
2
O
H
H
H
Fmoc-OSu
N-(9-Fluorenylmethoxycarbonyloxy)succi-
nimide
H
H
1
H
O
HOBt
NMM
RCM
TLC
=
=
=
=
1-Hydroxybenzotriazole
N-Methylmorpholine
H
4
H
H
3
NH
Ring-Closing Metathesis
Thin layer chromatography
Boc
Figure 2. Arrows indicate strong NOESY signals for compound 5.
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RESULT AND DISCUSSION
In this paper we report a novel synthetic pathway to ob-
tain (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
2-(tert-butoxycarbonylamino)-8-methoxy-8-oxooctanoic acid
(6) using (acyloxy)alkoxy promoiety as novel protecting
group. However (Acyloxy)alkoxy promoiety is cleavable in
mild alkaline conditions, this group was useful for the simul-
taneous protection of amino acid N and C terminal functions
during synthetic steps. Thus (Acyloxy)alkoxy was proved to
be completely stable in acidic anhydrous environment used
in solution phase peptide synthesis. This strategy was in our
knowledge still unexplored and this work represents the first
successful attempt to extend this methodology to the synthe-
sis of diaminosuberic acid derivatives to solve the problems
related to the development of alternative synthetic methods
which lead to the preparation of unambiguous diastereomeric
and selectively protected (2R,7R)-diaminosuberic acid de-
rivative.
[3]
[4]
[5]
[6]
[7]
[8]
The protecting groups of 6 can be readly manipulated and
should prove useful for both solution-phase and solid-phase
cyclic peptide synthesis. Efforts to apply this methodology in
the synthesis of cyclic peptides based on endogenous pep-
tides such as endomoriphins, enkephalins and chemotactic
derivatives [e.g. 20-29] are being pursued in this laboratory
and will be reported in due course. In conclusion, (R,R)-2,7-
diaminosuberic acid orthogonally protected 6 can be readly
synthesized from Boc-(R)-allylglycine in few steps. Despite
the low overall yield, the synthesis proceeded straightfor-
ward to give only the trans isomer 5 as intermediate. Current
investigations are directed to improve the yields of the criti-
cal steps to render the procedure suitable for preparing
multigram quantities of 5 and 6.
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[11]
[12]
[13]
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
ACKNOWLEDGEMENT
[14]
Declared none.

(Acyloxy)Alkoxy Moiety as Amino Acids Protecting Group
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Received: February 16, 2012
Revised: March 30, 2012
Accepted: April 11, 2012