Selective O-acylation of unprotected N-benzylserine methyl ester and O,N-acyl transfer in the formation of cyclo[Asp-Ser] diketopiperazines

Article abstract of DOI:10.1016/j.tet.2010.10.007

The synthesis of two diastereomeric cyclo[Asp-N-Bn-Ser] diketopiperazines (2a and 2b) was investigated. Initial formation of the Boc-aspartyl-N-benzyl serine isopeptide methyl esters (4a and 4b) was observed, which derive from the selective O-acylation of

Full text of DOI:10.1016/j.tet.2010.10.007

Tetrahedron 66 (2010) 9528e9531
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Selective O-acylation of unprotected N-benzylserine methyl ester and O,N-acyl
transfer in the formation of cyclo[Asp-Ser] diketopiperazines
,
b
Mattia Marchini a, Michele Mingozzi a, Raffaele Colombo a, Cesare Gennari a , Marco Durini ,
*
,
Umberto Piarulli b
*
a
ꢀ
Universita degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, Via G. Venezian 21 I-20133 Milano, Italy
b
ꢀ
Universita degli Studi dell’Insubria, Dipartimento di Scienze Chimiche e Ambientali, Via Valleggio 11 I-22100 Como, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2010
Received in revised form 28 September 2010
Accepted 4 October 2010
Available online 16 October 2010
The synthesis of two diastereomeric cyclo[Asp-N-Bn-Ser] diketopiperazines (2a and 2b) was in-
vestigated. Initial formation of the Boc-aspartyl-N-benzyl serine isopeptide methyl esters (4a and 4b)
was observed, which derive from the selective O-acylation of unprotected (S)- or (R)-N-benzylserine. This
unexpected O-acylation is preferred over the formation of the tertiary amide and the resulting ester bond
is stable in solution to O,N-acyl transfer. The O,N-acyl migration is then triggered by cleavage of the Boc
protecting group and treatment with base, which also promotes immediate cyclization to the
diketopiperazines.
Keywords:
O-Acyl isopeptides
O,N-Acyl transfer
N-Benzylserine
Diketopiperazines
Peptide synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
COOH
O
COOH
O
H
O
H
O
N
N
O,N-Acyl transfer reactions have recently been reported as
a valuable method for the efficient synthesis of difficult peptide
sequences and racemization-free segment condensation.1 This
methodology, named the ‘depsipeptide technique’2 or ‘O-acyl iso-
peptide methodology’,3 consists of the extension of a peptide se-
N
Ph
N
Ph
R or S
R or S
NHBoc
OH
DKP-2a
DKP-1a = S
DKP-1b
= S
DKP-2b = R
= R
quence, from a suitable point, via functionalization of the b-hydroxy
group of a N-protected serine or threonine derivative. After the final
coupling (or cleavage from the resin) and deprotection of the Ser or
Thr nitrogen, the O,N-acyl transfer restores the native peptide.
We have recently reported the synthesis of a new bifunctional
diketopiperazine scaffold (DKP-1, Scheme 1), formally derived from
(S)-aspartic acid and either (S)- or (R)-2,3-diaminopropionic acid
(DKP-1a and DKP-1b), and bearing a carboxylic acid and an amino
functionality.4 Depending on the relative configuration of the two
stereocenters, these were conveniently used as peptide secondary
structure inducers in linear or cyclic peptides.4
Scheme 1. Structure of bifunctional diketopiperazine scaffolds DKP-1a and DKP-1b
and their precursors DKP-2a and DKP-2b.
hydroxymethyl diketopiperazines DKP-2a and DKP-2b as suitable
precursors (Scheme 1). Transformation of the hydroxy group to the
protected amine of the final scaffold was accomplished via a Mit-
sunobu-type reaction (HN3, DIAD, Ph3P) followed by azide re-
duction and in situ Boc protection (Me3P, Boc-ON).4 DKP-2a and
DKP-2b were in turn synthesized from (S)-aspartic acid and either
(S)- or (R)-serine.4
Herein we report on our recent findings regarding the synthesis
of diketopiperazines DKP-2a and DKP-2b, and in particular: (i) the
preferential O-acylation of N-benzylserine methyl ester by pro-
tected aspartic acid, and (ii) the puzzling mechanism of the sub-
sequent O,N-acyl transfer with immediate diketopiperazine ring
closure.
Retrosynthetic analysis of N-Boc-aminomethyl diketopiper-
azines DKP-1a and DKP-1b identified the two diastereomeric
* Corresponding authors. Tel.: þ39 031 238 6444; fax: þ39 031 238 6449 (U.P.);
tel.: þ39 02 5031 4091; fax: þ39 02 5031 4072 (C.G.); e-mail addresses: cesar-
e.gennari@unimi.it (C. Gennari), umberto.piarulli@uninsubria.it (U. Piarulli).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.10.007

M. Marchini et al. / Tetrahedron 66 (2010) 9528e9531
9529
OH
also (iv) regular peptide coupling agents (EDC, HOAT).10 In general,
these methodologies were applied to either unfunctionalized or
protected functionalized N-alkyl amino acids, although, in one
case, a coupling to hydroxy-unprotected N-neopentylserine was
claimed11 and in another case several amino acids were coupled to
O
BocHN
COO
N
COOCH3
3a = S
OOC
OOC
Ph
BocHN
Ph
COOH
+
HATU, i Pr2NEt
3b
= R
N-benzylhomoserine without preliminary protection of the g-hy-
CH2Cl2
72%
droxy group.8b In order to avoid the introduction of an additional
protecting group orthogonal to those already present in the two
amino acid fragments, we investigated the direct coupling of the
aspartic acid derivative to N-benzylserine methyl ester. This was
realized using Carpino’s reagent HATU, to form the dipeptides 3 in
good yield (72%).4a However, a closer inspection of the spectro-
scopic properties of those derivatives revealed that, instead, the
formation of the isopeptides 4 had occurred via the selective acyl-
OH
O
BocHN
O
N
H
COOCH3
HN
COOCH3
4a = S
4b
Ph
(S) or (R)
= R
a) CF3COOH, CH2Cl2
b) sat. aq. NaHCO3 , EtOAc
81%
(over 2 steps)
ation of the unprotected
benzylserine.
b-hydroxy group of either (S)- or (R)-N-
Diagnostic of this outcome were the NMR spectra: (i) in the 1H
COO
O
NMR spectrum, the OeCH2 protons of serine were rather deshiel-
H
O
N
ded [4a:
d 4.32e4.48, m, 2H (CDCl3); 4b: d 4.30, dd, 1H and d 4.39,
N
Ph
dd, 1H (CD2Cl2)]; (ii) in the HMBC spectrum of both compounds,
a long range coupling (through three bonds) was clearly evident
between the OeCH2 protons of serine and the a-carbonyl carbon of
OH
2a = S
aspartic (Fig. 1).
2b
= R
To further prove this, the isopeptide 4 was capped with acetic
anhydride and, in this case, a long range coupling (through three
bonds) between the benzylic CH2 protons of benzylserine and the
acetyl carbonyl carbon was detected. More surprisingly, both iso-
peptides 4 underwent O,N-acyl transfer and immediate ring closure
to form diketopiperazines 2, after deprotection with TFA and sub-
sequent treatment with NaHCO3 in a basic biphasic medium
(EtOAc/H2O). The structure of diketopiperazine 2a (cis) was secured
by X-ray structural determination.4a
Puzzled by this behavior, we decided to investigate, using the
isopeptide 4b as a substrate, the conditions promoting the O,N-acyl
transfer/cyclization reactions and the relevant mechanism. In fact,
despite the presence of a nucleophilic nitrogen, the isopeptide 4b
was stable both in the solid state and in solution (dichloro-
methane). In methanol, complete degradation of the product was
Scheme 2. Coupling of N-(tert-butoxycarbonyl)-(2S)-aspartic acid
either (S)- or (R)-N-benzylserine methyl ester.
b-allyl ester with
2. Results and discussion
As we outlined in the introduction, the synthesis of DKP-2
was conveniently planned starting from suitably protected N-(tert-
butoxycarbonyl)-(2S)-aspartic acid
b
-allyl ester5 and either (S)-
or (R)-N-benzylserine methyl ester.6 Coupling of the protected
aspartic acid with the secondary amine of benzylserine was then
envisaged (Scheme 2). Several protocols have been employed for
the formation of the peptide linkage on N-benzyl amino acids, in-
cluding (i) pentafluorophenyl esters,7 (ii) mixed anhydrides (by
reaction with iso-butyl chloroformate),8 (iii) acyl chlorides,9 and
Fig. 1. HMBC spectra of isopeptides 4a (CDCl3) and 4b (CD2Cl2), highlighting the long range coupling (through three bonds) between the OeCH2 protons of Ser and the
carbon of Asp.
a-carbonyl

9530
M. Marchini et al. / Tetrahedron 66 (2010) 9528e9531
observed after 72 h, which could be attributed to the trans-
esterification of the aspartate -allyl ester and to cleavage of the
b
isopeptide, giving rise to N-(tert-butoxycarbonyl)-(2S)-aspartic acid
dimethylester and N-benzylserine methyl ester. However, no
isomerization to the dipeptide 3b was observed.
Since the O,N-acyl transfer indeed occurred during the forma-
tion of diketopiperazine 2b upon nitrogen deprotection, the Boc
group of isopeptide 4b was cleaved by reaction with TFA to give the
bis-TFA salt 5b, which was fully characterized (Scheme 3). Also in
this case, the HMBC spectrum confirmed that no O,N-acyl shift had
occurred. Its reactivity was then investigated. A solution of the bis-
TFA salt 5b in CD2Cl2 was monitored by 1H NMR spectroscopy, and
no O,N-acyl migration was observed after 48 h; conversely, when
dissolved in methanol, complete cleavage of the isopeptide was
detected in 6 h, giving rise to (2S)-aspartic acid
b-allyl ester a-
methyl ester and N-benzylserine methyl ester. However, again no
isomerization to the dipeptide was observed.
Fig. 2. 1H NMR monitoring of the transformation of isopeptide 5b into the DKP-2b
OOC
OOC
H3N
(CD3OD/4 equiv Et3N).
CF3COOH
CH2Cl2
O
O
Based on these experimental observations, a reasonable mech-
anistic explanation involves a rate limiting O,N-acyl transfer with
immediate ring closure to DKP, so that no dipeptide intermediate
can be detected (Scheme 3). On a preparative scale, the synthesis of
DKP-2b from bis-TFA salt 5b was better performed (85% isolated
BocHN
O
O
HN
COOCH3
H2N
COOCH3
Ph
2 CF3COO
Ph
4b
5b
yield) with iPr2EtN (4 equiv) in PrOH.
i
COO
O
3. Conclusions
CH3OH
Base
H2N
O
NHBn
O
In conclusion, the synthesis of two diastereomeric cyclo[Asp-N-
Bn-Ser] diketopiperazines (2a and 2b) has been studied in detail. A
peculiar pathway was observed with initial formation of the Boc-
aspartyl-N-benzyl serine isopeptide methyl ester (4), which derives
from the selective O-acylation of unprotected N-benzylserine. This
unexpected O-acylation is preferred over the formation of the ter-
tiary amide and the resulting ester bond is stable in solution to O,N-
acyl transfer. The O,N-acyl migration is then triggered by cleavage of
the Boc protecting group and treatment with base, which also pro-
motes immediate cyclization to the diketopiperazine (2). We tend to
believe that also in other cases, where the formation of dipeptides
involving N-substituted, unprotected serine derivatives was repor-
ted,8b,11 probably the actual products were the isopeptides.
MeO
H
COO
COO
O
H
O
O
N
HN
H
NBn
N
Ph
OH
O
OH
2b
Scheme 3. Deprotection of isopeptide 4b and suggested mechanism for DKP-2b
formation.
Upon addition of 4 equiv of base (Et3N or iPr2EtN) to a solution
of bis-TFA salt 5b in methanol, ring closure occurred rapidly and
was virtually complete after 2 h. However, when monitoring the
reaction by 1H NMR spectroscopy (CD3OD/4 equiv Et3N), the di-
peptide resulting from the O,N-acyl shift was never detected, and
only the signals of the starting bis-TFA salt 5b and the resulting
DKP-2b (trans) were identified. As can be clearly seen in Fig. 2, the
4. Experimental section
4.1. General
All manipulations requiring anhydrous conditions were carried
out in flame-dried glassware, with magnetic stirring and under
a nitrogen atmosphere. All commercially available reagents were
used as received. Anhydrous solvents were purchased from com-
mercial sources and withdrawn from the container by syringe,
under a slight positive pressure of nitrogen. (2S)-Aspartic acid
two dd at
benzylserine in the isopeptide 5b, decreased in intensity with
time, while the dd at 3.93 and 4.02, corresponding to the same
d 4.32 and d 4.45, belonging to the OeCH2 protons of
d
d
OeCH2 protons in DKP-2b, proportionally increased. The same
holds for the two d of the benzylic CH2 protons, which in the
isopeptide 5b resonate at
to 4.12 and
5.38, and for the serine CaꢀH, which moves from
3.6 to 3.77.
The same kinetic experiment was performed (CD3OD/4 equiv
Et3N) with the bis-TFA salt derived from isopeptide 4a and in this
case the O,N-acyl shift/ring closure occurred even faster, all of the
bis-TFA salt being converted into the diketopiperazine DKP-2a (cis)
after 25 min.
The same transformation [bis-TFA salt 5b to DKP-2b (trans)] was
followed by 1H NMR spectroscopy in CD2Cl2 (containing 4 equiv of
Et3N). Also in this case, despite the much reduced reaction rate
(only 42% conversion was observed after 15 h), no product of the
O,N-acyl shift was ever detected.
d
3.73 and
d
3.87, while in DKP-2b shift
b-allyl ester hydrochloride, N-(tert-butoxycarbonyl)-(2S)-aspartic
acid b
-allyl ester,5 (S)-serine methyl ester hydrochloride,12 (S)-N-
d
d
d
d
benzylserine methyl ester6 were prepared according to literature
procedures and their analytical data were in agreement with those
already published. The synthesis of 4-allyl 1-[(2R)-2-(benzyl-
amino)-3-methoxy-3-oxopropyl] N-(tert-butoxycarbonyl)-L-aspar-
tate (4a), and its spectroscopic characterization was already
reported4a although its structure was attributed to the corre-
sponding dipeptide 3a. Reactions were monitored by analytical thin
layer chromatography using 0.25 mm pre-coated silica gel glass
plates (DURASIL-25 UV254) and compounds visualized using UV
fluorescence, aqueous potassium permanganate or ninhydrin. Flash
column chromatography was performed according to the method

M. Marchini et al. / Tetrahedron 66 (2010) 9528e9531
9531
of Still and co-workers13 using Chromagel 60 ACC (40e63
gel. Proton NMR spectra were recorded on a spectrometer operat-
ing at 400.16 MHz. Proton chemical shifts are reported in parts per
mm) silica
1H), 3.10 (dd, J1¼18.53 Hz, J2¼6.10 Hz, 1H); 13C NMR (100 MHz,
CD2Cl2):
d
¼170.1, 166.3, 165.9, 131.2, 130.5, 130.0, 129.3, 118.9, 66.5,
32
62.7, 56.1, 53.9, 53.7, 53.4, 53.2, 52.9, 50.6, 49.4, 33.4; [
a
]
ꢀ8.7
D
million (
d
) with the solvent reference relative to tetramethylsilane
(CHCl3, c 1.50); HRMS (ESI) m/z calcd for [C18H25N2O6]þ: 365.17071
[MþH]þ; found: 365.17033.
(TMS) employed as the internal standard. The following abbrevia-
tions are used to describe spin multiplicity: s¼singlet, d¼doublet,
t¼triplet, q¼quartet, m¼multiplet, br¼broad signal, dd¼doublet of
doublet. Carbon NMR spectra were recorded on a spectrometer
operating at 100.63 MHz, with complete proton decoupling. Carbon
4.1.3. Allyl [(2S,5R)-4-benzyl-5-(hydroxymethyl)-3,6-dioxopiperazin-
2-yl]acetate (2b). The bis-trifluoro acetate salt 5b (1.08 g,
i
i
1.82 mmol, 1 equiv) was dissolved in PrOH (20 mL) and Pr2EtN
(0.9 mL, 5.6 mmol, 4 equiv) was added at rt. The reaction was stir-
red for 40 h at rt, monitoring the formation of DKP by TLC (EtOAc/
hexane: 8/2). The solution was then concentrated under reduced
pressure and the residue was purified by flash chromatography on
silica gel (Petroleum ether/EtOAc, 75:25) to afford the desired
product (2b) as a white foam (514.1 mg, 85% yield). Rf¼0.1 (AcOEt/
hexane: 8/2); IR (film): nmax 3364, 3032, 2942, 1738, 1651, 1452,
1383, 1329, 1273, 1183, 1129; 1H NMR (400 MHz, CD2Cl2):
chemical shifts are reported in ppm (d) relative to TMS with the
respective solvent resonance as the internal standard. Infrared
spectra were recorded on a standard FT-IR and peaks are reported
in cmꢀ1. Optical rotation values were measured on an automatic
polarimeter with a 1 dm cell at the sodium D line. High resolution
mass spectra (HRMS) were performed on a hybrid quadrupole time
of flight mass spectrometer equipped with an ESI ion source. A
Reserpine solution 100 pg/ml (about 100 count/s), 0.1% HCOOH/
CH3CN 1:1 was used as reference compound (Lock Mass). FAB mass
spectra were recorded using a glycerol matrix.
d
¼7.43e7.25 (m, 5H), 7.20 (bs, 1H), 6.02e5.83 (m, 1H), 5.39e5.20
(m, 3H), 4.70e4.55 (m, 3H), 4.12 (d, J¼15.21 Hz, 1H), 4.01 (dd,
J1¼11.81 Hz, J2¼1.90 Hz, 1H), 3.90 (dd, J1¼11.80 Hz, J2¼3.05 Hz, 1H),
3.81 (br, 1H), 3.21 (dd, J1¼17.40 Hz, J2¼3.95 Hz, 1H), 2.86 (dd,
4.1.1.
4-Allyl 1-[(2R)-2-(benzylamino)-3-methoxy-3-oxopropyl]
-aspartate (4b). To a solution of -allyl
N-(tert-butoxycarbonyl)-
L
b
J1¼17.40 Hz, J2¼8.00 Hz, 1H); 13C NMR (100 MHz, CD2Cl2):
¼170.8,
d
(2S)-N-(tert-butoxycarbonyl) aspartate ester (2.24 g, 8.2 mmol,
1.1 equiv) in DMF (70 mL), under a nitrogen atmosphere and at 0 ꢁC,
HATU (3.39 g, 8.94 mmol, 1.2 equiv), HOAt (1.21 g, 8.94 mmol,
168.2, 166.6, 135.9, 131.8, 128.8, 127.9, 127.9, 127.8, 118.3, 117.9, 65.7,
61.9, 61.6, 51.1, 47.3, 37.1; [
a
]
20 ꢀ35.3 (CHCl3, c 1.00); HRMS (ESI) m/z
D
calcd for [C17H20N2NaO5]þ: 355.12644 [MþNa]þ; found: 355.12590.
i
1.2 equiv) and Pr2EtN (5.1 mL, 29.8 mmol, 4 equiv) were added
successively. After 30 min, a solution of (R)-N-benzylserine methyl
ester (1.56 g, 7.45 mmol, 1 equiv) in DMF (10 mL) was added. The
reaction mixture was then stirred at 0 ꢁC for 1 h and at rt for 24 h.
The mixture was then diluted with EtOAc (200 mL) and the organic
phase was washed with 1 M KHSO4 (2ꢂ70 mL), aqueous NaHCO3
(2ꢂ70 mL), and brine (2ꢂ50 mL), dried over Na2SO4, and volatiles
were removed under reduced pressure. The residue was purified by
flash chromatography on silica gel (Petroleum ether/EtOAc, 75:25)
to afford the desired product (4b) as a transparent oil (2.73 g, 72%
yield). Rf¼0.3 (hexane/EtOAc 6:4); IR (film): nmax 3362, 2978, 1740,
1500, 1455, 1368, 1167, 1053; 1H NMR (400 MHz, CD2Cl2):
Acknowledgements
We thank the European Commission (Marie Curie Early Stage
Research Training Fellowship ‘Foldamers’ MEST-CT-2004-515968)
and Ministero dell’Universitae della Ricerca (PRIN prot. 2008J4YNJY)
for financial support.
ꢀ
References and notes
1. Coin, I. J. Pept. Sci. 2010, 16, 223e230.
2. Carpino, L. A.; Krause, E.; Sferdean, C. D.; Schumann, M.; Fabian, H.; Bienert, M.;
Beyermann, M. Tetrahedron Lett. 2004, 45, 7519e7523.
d
¼7.37e7.20 (m, 5H), 5.95e5.82 (m, 1H), 5.41 (d, J¼7.86 Hz, 1H),
5.34e5.18 (m, 2H), 4.61e4.48 (m, 3H), 4.39 (dd, J1¼10.92 Hz,
J2¼4.62 Hz, 1H), 4.30 (dd, J1¼10.92 Hz, J2¼4.86 Hz, 1H), 3.86 (d,
J¼13.13 Hz, 1H), 3.71 (s, 3H), 3.70 (d, J¼13.14 Hz, 1H), 3.51 (t,
J¼4.74 Hz, 1H), 2.94 (dd, J1¼17.17 Hz, J2¼4.68 Hz, 1H), 2.82 (dd,
J1¼17.03 Hz, J2¼4.79 Hz, 1H), 1.42 (s, 9H); 13C NMR (100 MHz,
3. (a) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Dos Santos, S.; Mandal, B.;
Mimna, R.; Murat, K.; Patiny, L.; Saucede, L.; Tuchscherer, G. Angew. Chem., Int.
Ed. 2004, 43, 4172e4178; (b) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso,
Y. Chem. Commun. 2004, 124e125; (c) Kiewitz, S. D.; Kakizawa, T.; Kiso, Y.;
Cabrele, C. J. Pept. Sci. 2008, 14, 1209e1215.
~
4. (a) Ressurreic¸ ao, A. S. M.; Bordessa, A.; Civera, M.; Belvisi, L.; Gennari, C.;
~
Piarulli, U. J. Org. Chem. 2008, 73, 652e660; (b) Ressurreic¸ ao, A. S. M.; Vidu, A.;
CD2Cl2):
d
¼172.4, 170.6, 139.7, 131.9, 128.3, 128.2, 127.1, 118.1, 65.9,
Civera, M.; Belvisi, L.; Potenza, D.; Manzoni, L.; Ongeri, S.; Gennari, C.; Piarulli,
U. Chem.dEur. J. 2009, 15, 12184e12188; (c) Delatouche, R.; Durini, M.; Civera,
M.; Belvisi, L.; Piarulli, U. Tetrahedron Lett. 2010, 51, 4278e4280.
5. Webster, K. L.; Maude, A. B.; O’Donnell, M. E.; Mehrotra, A. P.; Gani, D. J. Chem.
Soc., Perkin Trans. 1 2001, 1673e1695.
20
65.6, 59.1, 52.0, 51.8, 50.0, 36.7, 28.0; [
a
]
þ11 (CHCl3, c 1.00);
D
HRMS (ESI) m/z calcd for [C23H33N2O8]þ: 465.22314 [MþH]þ;
found: 465.22267.
6. Thompson, C. M.; Frick, J. A.; Green, D. L. C. J. Org. Chem. 1990, 55, 111e116.
7. Boger, D. L.; Zhou, J. J. Am. Chem. Soc. 1993, 115, 11426e11433.
8. (a) Torrini, I.; Mastropietro, G.; Pagani Zecchini, G.; Paglialunga Paradisi, M.;
Lucente, G.; Spisani, S. Arch. Pharmacol. 1998, 331, 170e176; (b) Marastoni, M.;
Bazzaro, M.; Bortolotti, F.; Tomatis, R. Bioorg. Med. Chem. 2003, 11, 2477e2483.
9. (a) Aicher, T. D.; Anderson, R. C.; Gao, J. P.; Shetty, S. S.; Coppola, G. M.; Stanton,
J. L.; Knorr, D. C.; Sperbeck, D. M.; Brand, L. J.; Vinluan, C. C.; Kaplan, E. L.;
Dragland, C. J.; Tomaselli, H. C.; Islam, A.; Lozito, R. J.; Liu, X. L.; Maniara, W. M.;
Fillers, W. S.; DelGrande, D.; Walter, R. E.; Mann, W. R. J. Med. Chem. 2000, 43,
236e249; (b) Yoon, U. C.; Jin, Y. X.; Oh, S. W.; Park, C. H.; Park, J. H.; Campana, C.
F.; Cai, X. L.; Duesler, E. N.; Mariano, P. S. J. Am. Chem. Soc. 2003, 125,
10664e10671.
4.1.2. (2S)-4-(Allyloxy)-1-{[(2R)-2-(benzylammonio)-3-methoxy-3-
oxopropyl]oxy}-1,4-dioxobutan-2-aminium
bis(trifluoroacetate)
(5b). To a solution of 4b (845 mg, 1.82 mmol) in CH2Cl2 (14.4 mL)
was added trifluoroacetic acid (7.2 mL, 4 mL/mmol). The reaction
mixture was stirred for 3 h at rt and then concentrated at reduced
pressure. The excess TFA was azeotropically removed from the
residue with toluene. Diethyl ether was added to the residue and
the resulting suspension was evaporated under reduced pressure to
give the isopeptide bis-TFA salt (5b) as a white foamy solid in
quantitative yield. Rf¼0.37 (DCM/MeOH 95:5); IR (film): nmax 2921,
2850, 1759, 1681, 1539, 1456, 1392, 1202, 1137; 1H NMR (400 MHz,
10. (a) Veerman, J. J. N.; Bon, R. S.; Hue, B. T. B.; Girones, D.; Rutjes, F. P. J. T.; van
Maarseveen, J. H.; Hiemstra, H. J. Org. Chem. 2003, 68, 4486e4494; (b) Tullberg,
M.; Grotli, M.; Luthman, K. J. Org. Chem. 2007, 72, 195e199.
11. Seebach, D.; Sommerfeld, T. L.; Jiang, Q. Z.; Venanzi, L. M. Helv. Chim. Acta 1994,
CD2Cl2):
d
¼7.59e7.37 (m, 5H), 6.01e5.86 (m, 1H), 5.41e5.25 (m,
77, 1313e1330.
2H), 4.78e4.59 (m, 4H), 4.48e4.37 (m, 2H), 4.29 (d, J¼12.98 Hz,1H),
4.08e4.01 (br, 1H), 3.89e3.81 (s, 3H), 3.17 (dd, J¼18.33 , 3.91 Hz,
12. Huang, Y.; Dalton, D. R.; Carroll, P. J. J. Org. Chem. 1997, 62, 372e376.
13. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923e2925.