Sterically hindered Cα,α-disubstituted α-amino acids: Synthesis from α-nitroacetate and incorporation into peptides

Article abstract of DOI:10.1021/jo015809o

The preparation of sterically hindered and polyfunctional C^α,α-disubstituted α-amino acids (ααAAs) via alkylation of ethyl nitroacetate and transformation into derivatives ready for incorporation into peptides are described. Treatment of ethyl nitroacetate with N,N-diisopropylethylamine (DIEA) in the presence of a catalytic amount of tetraalkylammonium salt, followed by the addition of an activated alkyl halide or Michael acceptor, gives the doubly C-alkylated product in good to excellent yields. Selective nitro reduction with Zn in acetic acid or hydrogen over Raney Ni gives the corresponding amino ester that, upon saponification, can be protected with the fluorenylmethyloxycarbonyl (Fmoc) group. The first synthesis of an orthogonally protected, tetrafunctional C^α,α-disubstituted analogue of aspartic acid, 2,2-bis(tert-butylcarboxymethyl)glycine (Bcmg), is described. Also, the sterically demanding C^α,α-dibenzylglycine (Dbg) has been incorporated into a peptide using solid-phase synthesis. It was found that once sterically congested Dbg is at the peptide N-terminus, further chain extension becomes very difficult using uronium or phosphonium salts (PyAOP, PYAOP/HOAt, HATU). However, preformed amino acid symmetrical anhydride couples to N-terminal Dbg in almost quantitative yield in nonpolar solvent (dichloroethane-DMF, 9:1).

Full text of DOI:10.1021/jo015809o

7
118
J . Org. Chem. 2001, 66, 7118-7124
Ster ica lly Hin d er ed Cr,r-Disu bstitu ted r-Am in o Acid s: Syn th esis
fr om r-Nitr oa ceta te a n d In cor p or a tion in to P ep tid es
Yanwen Fu, Lars G. J . Hammarstr o¨ m, Tod J . Miller, Frank R. Fronczek,
Mark L. McLaughlin,* and Robert P. Hammer*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
rphammer@lsu.edu
Received May 31, 2001
The preparation of sterically hindered and polyfunctional CR,R-disubstituted R-amino acids (RRAAs)
via alkylation of ethyl nitroacetate and transformation into derivatives ready for incorporation
into peptides are described. Treatment of ethyl nitroacetate with N,N-diisopropylethylamine (DIEA)
in the presence of a catalytic amount of tetraalkylammonium salt, followed by the addition of an
activated alkyl halide or Michael acceptor, gives the doubly C-alkylated product in good to excellent
yields. Selective nitro reduction with Zn in acetic acid or hydrogen over Raney Ni gives the
corresponding amino ester that, upon saponification, can be protected with the fluorenylmethyl-
R,R
oxycarbonyl (Fmoc) group. The first synthesis of an orthogonally protected, tetrafunctional C
-
disubstituted analogue of aspartic acid, 2,2-bis(tert-butylcarboxymethyl)glycine (Bcmg), is described.
R,R
Also, the sterically demanding C -dibenzylglycine (Dbg) has been incorporated into a peptide using
solid-phase synthesis. It was found that once sterically congested Dbg is at the peptide N-terminus,
further chain extension becomes very difficult using uronium or phosphonium salts (PyAOP, PyAOP/
HOAt, HATU). However, preformed amino acid symmetrical anhydride couples to N-terminal Dbg
in almost quantitative yield in nonpolar solvent (dichloroethane-DMF, 9:1).
In tr od u ction
The discovery of CR,R-disubstituted R-amino acids
RRAAs, 1) in biological systems and their propensity to
Sch em e 1
(
induce secondary structure even into short peptides has
resulted in an increased interest in novel methods for
their synthesis.1 The most common methods of RRAA
preparation include the Bucherer-Bergs and the Streck-
,2
These and other limitations of the hydantoin approach
have led to the development of alternative syntheses of
RRAAs, including glycine anion and cation equivalents.
3
er syntheses. We have found the Bucherer-Bergs method
7
(Scheme 1) convenient and effective for preparation of a
number of hydantoins 2 that can be hydrolyzed to RRAAs.
Also, we and others have developed milder methods for
cleaving the hydantoin ring so that side-chain protected
RRAAs can be obtained directly from hydantoin hydroly-
Nitroacetate esters have long been recognized as poten-
tially useful synthetic intermediates for the preparation
of amino acids as well as other biologically significant
compounds.8 Alkylations of nitroacetate esters have
usually resulted in racemic monoalkylations, and the
predominant products were often results of C- and/or
O-alkylation.8 The previously reported C-dialkylation of
ethyl nitroacetate with benzyl bromide employed an
4
sis. However, several more sterically hindered hydan-
toins, including dibenzyl hydantoin 2 (R ) Bn), have been
,9
impossible to hydrolyze to the free amino acids even
5
under the most extreme conditions. Also, we have found
1
0
that the Bucherer-Bergs route cannot be extended to a
number of noncyclic difunctionalized ketones due to their
resistance to hydantoin formation.6
inorganic base, and the reaction took a prolonged time.
Electrochemically generated anions have also been used
on a small scale to perform mono- and dialkylations of
*
To whom correspondence should be addressed. Telephone: (225)
78-4025. Facsimile: (225) 578-3458.
1) For a review of the role of RRAAs in natural bioactive peptides,
see: Nagaraj, R.; Balaram, P. Acc. Chem. Res. 1981, 14, 356-362.
2) For examples of RRAAs in short, highly structured peptides,
(6) It was observed that a number of symmetrical difunctionalized
ketones, such as 1,3-dihydroxy 2-propanone, 4-oxoheptanedinitrile, 1,5-
diamino-3-pentanone, dimethyl 3-oxoglutarate, dimethyl 4-oxopime-
late, and 4-ketopimelic acid do not form hydantoins to an appreciable
extent under standard Bucherer-Bergs conditions. Hammarstr o¨ m, L.
G. J . Synthetic Peptides: Design, Structure & Biological Function.
Dissertation, Louisiana State University, 2001; Ch. 2, pp 20-43.
(7) (a) Zanardi, F.; Battistini, L.; Rassu, G.; Cornia, M.; Casiraghi,
G. J . Chem. Soc., Perkin Trans. 1 1995, 2471-2475. (b) Charette, A.
B.; Gagnon, A.; J anes, M.; Mellon, C. Tetrahedron Lett. 1998, 39, 5147-
5150. (c) Ezquerra, J .; Pedregal, C.; Moreno-Ma n˜ as, M.; Roglans, A.
Tetrahedron Lett. 1993, 34, 8535-8538.
5
(
(
see: (a) Yokum, T. S.; Gauthier, T. J .; Hammer, R. P.; McLaughlin,
M. L. J . Am. Chem. Soc. 1997, 119, 1167-1168. (b) Rossi, P.; Felluga,
F.; Tecilla, P.; Formaggio, F.; Crisma, M.; Toniolo, C.; Scrimin, P. J .
Am. Chem. Soc. 1999, 121, 6948-6949.
(
3) (a) Wheeler, H. L.; Hoffman, C. Am. Chem. J . 1911, 45, 368-
83. (b) Dyker, G. Angew. Chem., Int. Ed. Engl. 1997, 36, 1700-1702
and references therein.
4) (a) Wysong, C. L.; Yokum; T. S.; Morales, G. A.; Gundry, R. L.;
3
(
(8) Shipchandler, M. T. Synthesis 1979, 666-686.
McLaughlin, M. L.; Hammer, R. P. J . Org. Chem. 1996, 61, 7650-
(9) (a) Dauzonne, D.; Royer, R. Synthesis 1987, 4, 399-401. (b)
Gogte, V. N.; Natu, A. A.; Pore, V. S. Synth. Commun. 1987, 17, 1421-
1429. (c) Boyd, R. N.; Kelly, R. J . J . Am. Chem. Soc. 1952, 74, 4600-
4602. (d) Kaji, E.; Zen, S. Bull. Chem. Soc. J pn. 1973, 46, 337-338.
(10) Kang, F. A.; Yin, H. Y.; Yin, C. L. Chin. Sci. Bull. 1997, 42,
1628-1631.
7
3
652. (b) Kubik, S.; Meissner, R. S.; Rebek, J . Tetrahedron Lett. 1994,
5, 6635-6638.
(
5) Hydrolysis of hydantoin 2 (R ) Bn) with either strong base (6
M KOH, 20 psi, 180 °C, 2 days) or strong acid (10 N HBr, reflux, 2
days) gave starting material as the only isolable product.
1
0.1021/jo015809o CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/21/2001

CR,R-Disubstituted R-Amino Acids
J . Org. Chem., Vol. 66, No. 21, 2001 7119
Sch em e 2
Ta ble 1. Alk yla tion of Eth yl Nitr oa ceta te 3 to
Cr,r-Dia lk yla ted Nitr oa ceta tes 4
time temp prod. yield,
methoda (h)
(°C)
no.
%
b
entry
electrophile
PhCH2Br
p-O2NPhCH2Br
p-NCPhCH2Br
1
2
3
4
5
6
7
8
9
A
B
B
B
B
A
A
C
C
C
1
2
2
25-60c 4a 63
0-25
0-25
0-25
50
60
60
25
25
25
4b 75
4c 82
4d 72
4e 79
4f
4f
4g 70
4h 89
4i
nitroacetate esters.11 The strategy and conditions pro-
p-CH3O2CPhCH2Br
(CH3)3CO2CCH2Br
CH2dCHCH2Br
CH dCHCH I
2
24
24
24
30
48
48
posed herein employ the commercially available ethyl
nitroacetate (3) along with 2 equiv each of a tertiary
amine base and an electrophile to provide a variety of
traced
e
f
2
2
45
CH2dCHSO2Ph
CH2dCHCO2t-Bu
CH2dCHCN
R,R
novel C -dialkyl nitroacetate esters (Scheme 2). Fur-
1
0
87
thermore, methods for preparing Fmoc-protected RRAAs,
including tetrafunctional RRAAs, suitable for solid-phase
peptide synthesis are elaborated.
The main application of these protected RRAAs will be
for the synthesis of peptide analogues which are confor-
mationally constrained (limited φ and ψ space) into turn,
a
Method A: Solvent: DMF; 1.5 M 3, 0.15 M Bu4NBr, 2.1 equiv
of DIEA, 2.1, equiv of electrophile added last. Method B: Solvent:
DMF; 1.5 M 3, 0.15 M Bu4NBr, 2.1, equiv of electrophile, 2.1 equiv
of DIEA added last. Method C: Solvent: CH3CN; 1.5 M 3, 0.15 M
Et4NBr, 2.1 equiv of DIEA, 2.1, equiv of electrophile added last.
b
All percentage yields refer to isolated, analytically pure products.
2
,12
c
Spontaneous exotherm to 60 °C. d Major product is monoallylated
helix or extended conformations.
Incorporation of
e
RRAAs such as Aib,13 Deg, and Dpg into peptides is
product, ethyl 2-nitro-4-pentenoate. Bu4NI is substituted for
Bu4NBr. f Bu4NBr and Et4NBr give the same yield.
generally accomplished by standard carbodiimide proto-
cols14 or in more difficult cases coupling is accomplished
1
5
(Table 1). DIEA was employed based on evidence which
suggests that ethyl nitroacetate anion exhibits high
nucleophilic reactivity in the presence of a quaternary
ammonium cation that does not tend to form a strong
ion pair.11 The reactivity of the anion was further
increased when a catalytic amount of tetraalkylammo-
nium salt was added to the reaction. The benzyl bromides
with preformed acid fluorides, or in situ activation with
1
6
uronium (e.g., HATU) or phosphonium salts (e.g.,
PyBOP).17 More sterically hindered RRAAs such as Dbg
are much more difficult to incorporate. For example,
reported acylation of the N-terminus of Dbg only gave a
low to moderate coupling yield after prolonged reaction
time.18 In this paper, we report an efficient method for
1
9
gave rapid conversion to dialkylated products 4 in a
matter of minutes to hours. Reactions were initially
exothermicsbenzyl bromides having electron-withdraw-
ing substituents (Table 1, entries 2-4) needed to be
cooled initially to control the reaction. Unlike benzyl
bromides, dialkylation with tert-butyl 2-bromoacetate
(Table 1, entry 5) required longer reaction time at slightly
readily incorporating amino acid residues to sterically
hindered N-terminus of Dbg.
Resu lts a n d Discu ssion
r
Syn th esis of N -F m oc-P r otected rrAAs. We have
found that treatment of ethyl nitroacetate with 2 equiv
of DIEA in the presence of catalytic amounts of tetra-
alkylammonium salts, followed by the addition of an
activated alkyl halide or Michael acceptor, gave the
disubstituted nitroacetate ester in good to excellent yields
2
0
elevated temperature (50 °C) to provide 4e in 79% yield.
Allyl bromide gave only trace amounts of the diallylated
product 4f even after prolonged reaction times and
heating. The major product of this reaction was the mono-
C-allylated derivative, ethyl 2-nitro-4-pentenoate (65%).
Higher yields of 4f could be obtained using allyl iodide
as the electrophile; however, this reaction still required
(
11) Niyazymbetov, M. E.; Evans, D. H. J . Org. Chem. 1993, 58,
7
79-783.
(
12) (a) Balaram, P. Curr. Opin. Struct. Biol. 1992, 2, 845-851. (b)
2
4 h at 60 °C to provide product, which was still admixed
Toniolo, C.; Benedetti, E. Macromolecules 1991, 24, 4004-4009. (c)
Toniolo, C. J assen. Chim. Acta 1993, 11, 10-16. (d) Wysong, C. L.;
Yokum, T. S.; McLaughlin, M. L.; Hammer, R. P. CHEMTECH 1997,
with monoallylated ester. Double Michael-type addition
to a variety of activated alkenes provided tetrafunctional
nitroesters 4g-i in good yields.
2
7, 26-33.
13) Abbreviations: Aib, 2-aminoisobutyric acid; CCA, R-cyano-4-
hydroxycinnamic acid; DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene; Deg,
(
The high yields for these dialkylation reactions make
this a particularly viable route to symmetrically substi-
tuted RRAAs (vide infra) with or without side chain
functionality. As examples of the utility of these R-amino
acid precursors, the dibenzyl derivative 4a and di-tert-
R,R
R,R
C
-diethylglycine; DIEA, N,N-diisopropylethylamine; Dpg, C -dipro-
pylglycine; FAB-MS, fast atom bombardment mass spectrometry;
HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HOAt,
1
-hydroxy-7-azabenzotriazole; MALDI-MS, matrix assisted laser de-
sorption ionization mass spectrometry; PAL, 5-(4-aminomethyl-3,5-
dimethoxyphenoxy)valeric acid; PEG-PS, poly(ethylene glycol)-poly-
styrene graft; PyAOP, 7-azabenzotriazol-1-yloxytris(pyrrolidino)phos-
phonium hexafluorophosphate; PyBOP, 1-benzotriazolyoxytris(pyrro-
lidino)phosphonium hexafluorophosphate; TFA, trifluoroacetic acid;
TPS, triisopropylsilane.
R
butyl acetate derivate 4e were converted to N -fluoren-
R,R
ylmethyloxycarbonyl (Fmoc)-protected
C
-dibenzyl-
glycine (Dbg) 7a and 2,2-bis(tert-butylcarboxymethyl)-
glycine (Bcmg(OtBu) -OH) 7e, respectively (Scheme 3).
(14) (a) Moretto, V.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Balaram,
2
H.; Balaram, P. Macromolecules 1989, 22, 2939-2944. (b) Prasad, S.;
First, several methods were investigated for reduction
of the nitro group to an amino group. Nitro ester 4a was
readily reduced with Zn/acetic acid to afford the amino
ester 5a in high yield (Table 2, entry 1). Results for
catalytic hydrogenation approaches to reduce the nitro
Rao, R. B.; Balaram, P.; Biopolymer 1995, 35, 11-20.
(15) (a) Wenschuh, H.; Beyermann, M.; Krause, E.; Carpino, L. A.;
Bienert, M. Tetrahedron Lett. 1993, 34, 3733-3736. (b) Wenschuh, H.;
Beyermann, M.; Krause, E.; Brudel, M.; Winter, R.; Sch u¨ mann, M.;
Carpino, L. A.; Bienert, M. J . Org. Chem. 1994, 59, 3275-3280.
(16) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201-203.
17) Frerot, E.; Coste, J .; Pantaloni, A.; Dufour, M. N.; J ouin, P.
Tetrahedron 1991, 47, 259-270.
18) (a) Cotton, R.; Hardy, P. M.; Langran-Goldsmith, A. E. Int. J .
(
(19) For a crystal structure of 4a , see: Miller, T. J .; Fu, Y.; Fronczek,
F. R.; Hammer, R. P. Acta Cryst. C. 2000, C56, e 574-575.
(20) For an ORTEP of 4e and its crystallographic data, see Sup-
porting Information. The author has also deposited atomic coordinates
with the Cambridge Crystallographic Data Centre.
(
Pept. Protein Res. 1986, 28, 245-253. (b) Torrini, I.; Mastropietro, G.;
Pagani Zecchini, G.; Paglialunga Paradisi, M.; Lucente, G.; Spisani,
S. Arch. Pharm. 1998, 331, 170-176.

7
120 J . Org. Chem., Vol. 66, No. 21, 2001
Fu et al.
Sch em e 3
F igu r e 1. Intramolecular hydrogen bonding in hydroxyl-
amine.
To make the desired Fmoc-protected amino acids, the
ester of the R-carboxylate must first be removed. For
amino ester 5a this was readily accomplished by sapon-
fication under strongly basic conditions (2 M KOH) in
refluxing H O-EtOH (v/v, 2:1) to give the free RRAA 1a
2
in 88% yield (Scheme 3). Unlike saponification of 5a ,
hydrolysis of the ethyl ester of triester 5e required more
precise conditions to avoid concurrent tert-butyl ester
hydrolysis. Selective hydrolysis was realized by treatment
of 5e with 3 equiv of lithium hydroxide in H O-EtOH
2
Ta ble 2. Red u ction of Cr,r-Disu bstitu ted Nitr o Ester s 4a ,
e, a n d 4h
conditionsa
Zn/AcOH
(v/v, 1:2) at 50 °C to give the side-chain protected RRAA
1e in 82% yield. Acid-catalyzed hydrolysis of 5e with HCl,
nucleophilic dealkylation, and saponfication using KOH
failed to selectively hydrolyze the ethyl ester. The low
selectivity of ethyl vs tert-butyl ester hydrolysis in this
compound is most likely due to the steric congestion of
the ethyl ester resulting from its proximity to the
4
entry nitro ester
product yield %b
1
2
3
4
5
6
4a
4a
4e
4h
4h
4h
5a
6a
5e
5h
6h
6h
81
73
92
92
97
97
HCO2NH4,10% Pd-C
H2/Raney Ni
H2/Raney Ni
H2/10% Pd-C
HCO2NH4,10% Pd-C
R
quaternary R-carbon. The free RRAAs are N -protected
to allow for SPPS. Protection of the amine with Fmoc-Cl
using an in situ silylation procedure24 provided Fmoc-
a
Hydrogenation over Pd or Ni were carried out in hydrogena-
tion flask at 50 psi; reduction with Zn/AcOH or HCO2NH4 in the
Dbg-OH (7a ) in 80% yield and Fmoc-Bcmg(OtBu)
2
-OH
presence of Pd-C was performed in a regular round-bottom flask.
(7e) in 71% yield. The synthetic route allows facile
b
All yields refer to isolated pure products.
synthesis of sterically hindered RRAAs, even those with
sensitive side-chain functionality, in good yields (57% for
group were mixed. Reduction of the nitro group of CR,R
-
7
a from 4a , 54% for 7e from 4e).
disubstituted nitro triester 4h , an analogue of 4e, with
hydrogen (50 psi) over T-1 Raney Ni afforded the desired
amino triester 5h in excellent yield (Table 2, entry 4). In
contrast, Pd/C-catalyzed hydrogenation or catalytic hy-
drogen transfer with ammonium formate in the presence
of Pd resulted in almost quantitative conversion of 4h to
the corresponding hydroxylamine 6h (Table 2, entries 5
and 6). Similarly catalytic transfer hydrogenation of 4a
also produced the hydroxylamine product 6a (data not
shown). It has been suggested21 that Pd catalysts are
preferable for partial reduction of aliphatic nitro groups
to hydroxylamines. Boger et al.22 reported hydrogenation
of tertiary aliphatic nitro groups over a variety of
catalysts. They found that hydrogenation over Raney Ni
provided the amino derivatives with variable amounts
of corresponding hydrocarbon from loss of the nitro group,
In cor p or a tion of Dbg in to P ep tid es Usin g SP P S.
Dbg has been previously incorporated into peptides using
solution-phase peptide synthesis, and the prevalent
method was using 2-trifluoromethyl-4,4-dibenzyloxazolin-
18a,25
5
-one as the Dbg precusor.
The sterically hindered
Dbg inhibits efficient peptide bond formation. In our
work, the coupling of the carboxyl group of Dbg using
PyAOP proved relatively efficient, but acylation of the
N-terminal Dbg turned out to be very difficult. The same
problem was also encountered in solution-phase synthesis
18
of peptides containing a Dbg residue. In our studies,
coupling of Fmoc-Lys(Boc)-OH onto the N-terminus of
Dbg on PAL resin using PyAOP or PyAOP/HOAt were
ineffective (Table 3, entries 1, 2), even though PyAOP
has been shown to be efficient for acylation of RRAAs with
2
6
less-hindered side chains, such as dipropylglycine and
Aib.27 The acylation of Dbg using HATU also gave a low
yield as determined by UV analysis of the Fmoc-depro-
tection (Table 3, entry 3).
while reduction using H
2
2 4
-Pd/C, HCO NH -Pd/C, and
H
2
-PtO
2
afforded mixtures including the hydroxylamine
R,R
and hydrocarbon. The observed partial reduction of C
-
disubstituted nitro ester 4h into the hydroxylamine ester
may be attributed to the relatively low activity of Pd
catalysts and the intramolecular hydrogen bonding which
enhanced the stability of the hydroxylamine derivative
Several other groups had previously found that sec-
ondary amino acids acylate poorly with in situ reagents
(23) For an ORTEP of 5e and its crystallographic data, see Sup-
porting Information. The author has also deposited atomic coordinates
with the Cambridge Crystallographic Data Centre.
(Figure 1). On the basis of these experimental results,
reduction of nitro triester 4e was carried out using H
2
/
(24) Bolin, D. R.; Sytwu, I.-I.; Humiec, F.; Meienhofer, J . Int. J . Pept.
Ni at 50 psi, and the desired amino ester 5e was obtained
Protein Res. 1989, 33, 353-359.
(25) (a) Hardy, P. M.; Goldsmith, A.; Cotton, R. In Peptides,
as a clear crystalline solid23 in excellent yield (Table 2,
Proceedings of the 18th European Peptide Symposium; Ragnarsson,
U., Ed.; Almqvist & Wiksell: Stockholm, 1984; pp 121-124. (b) Valle,
G.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Lelj, F.; Barone, V.;
Fraternali, F.; Hardy, P. M.; Langran-Goldsmith, A.; Maia, H. L. S. J .
Chem. Soc., Perkin Trans. 2 1990, 8, 1481-1487.
(26) Coupling of R-amino acids to resin-bound dipropylglycine with
PyAOP/DIEA at 50 °C gives > 90% yield. Fu, Y.; Hammer, R. P.
Unpublished results.
entry 3).
(21) (a) Freifelder, M. In Catalytic Hydrogenation in Organic
Synthesis, Procedures and Commentary; J ohn Wiley & Sons: New
York, 1978; pp 26-39. (b) Rylander, P. In Catalytic Hydrogenation in
Organic Synthesis; Academic Press: New York, 1979; pp113-125.
(
22) Boger, D. L.; Lerner, R. A.; Cravatt, B. F. J . Org. Chem. 1994,
(27) Albericio, F.; Cases, M.; Alsina, J .; Triolo, S. A.; Carpino, L. A.;
Kates, S. A. Tetrahedron Lett. 1997, 38, 4853-4856.
5
9, 5078-5079.

CR,R-Disubstituted R-Amino Acids
J . Org. Chem., Vol. 66, No. 21, 2001 7121
Ta ble 3. Cou p lin g of F m oc-Lys(Boc)-OH on to H-Dbg-Ala -Dp g-Glu (OtBu )-P AL-P EG-P S
entry
coupling method
PyAOP
PyAOP-HOAt
HATU
symmetrical anhydride
symmetrical anhydride
symmetrical anhydride
base
solvent
time (h)
temp °C
yield (%)a
1
2
3
4
5
6
DDIEA
DIEA
DIEA
no base
no base
no base
DCE-DMF (1:1)
DCE-DMF (1:1)
DMF
DCE-DMF (9:1)
DCE-DMF (1:1)
DMF
8
8
8
3
3
3
50
25
25
50
50
50
20
25
18
95
81
52
b
a
The coupling yield was determined by UV analysis of the Fmoc-deprotection.15b For entries 4-6, the preformed Fmoc-Lys(Boc)-
b
symmetrical anhydride was used in the coupling. Symmetrical anhydride was prepared by treatment of 2 equiv of Fmoc-Lys(Boc)-OH
with 1 equiv of DCC in CH2Cl2 at room temperature for 2 h. DCU was removed by filtration. After removal of CH2Cl2 by evaporation, the
symmetrical anhydride was taken up in the described solvent for coupling.
F igu r e 2. Potential anchimeric assistance provided by the
H-bond donation of the symmetrical anhydride.
(DCC/HOBt; HOBt/HOAt-derived uronium or phospho-
nium salts), but more readily react with symmetrical
anhydrides in nonpolar solvents such as dichloro-
methane.2 To our surprise, coupling of the symmetrical
anhydride of Fmoc-Lys(Boc)-OH with the N-terminus of
Dbg in DCE-DMF (v/v, 9:1) in the absence of base gave
an almost quantitative yield (Table 3, entry 4). It is not
as yet understood why the symmetrical anhydride method
is so much more efficient at acylating the sterically
hindered Dbg residue than methods based on uronium
or phosphonium salts. One possiblility is that the sym-
metrical anyhdride may possess an enhanced reactivity
in the aprotic nonpolar solvent for which anchimeric
assistance from H-bonding is more pronounced (Figure
8,29
2
). The results bear this out in that coupling of the
symmetrical anhydride in DCE-DMF (9:1) gives a 95%
coupling yield (Table 3, entry 4). In contrast reaction of
the symmetrical anhydride in the more H-bond accepting
solvent mixture of DCE-DMF (1:1) or in pure DMF
(Table 3, entries 5, 6), gave systematic decreases in
coupling efficiency. The fact that the nonpolar solvent
swells the PAL-PEG-PS resin more efficiently than pure
DMF may also attribute to the successful coupling. The
pentapeptide containing Dbg was prepared using the
optimized coupling conditions and cleaved from the resin
by 88% TFA and analyzed by reverse-phase HPLC
F igu r e 3. (A) HPLC profile of crude peptide (H-Lys-Dbg-Ala-
Dpg-Glu-NH
). Column: Delta-Pak C18, 15 µm 300 Å, 8 × 100
mm; eluent A: 0.05% TFA in H O; eluent B: 0.05% TFA in
2
2
(Figure 3).
CH CN; gradient: 10-60% B over 50 min; flow rate 1 mL/
3
min. (B) MALDI-MS spectrum of the main peak on HPLC.
Con clu sion
In summary, we have outlined the synthesis of steri-
of previously unavailable RRAAs with and without side
chain functionality. Also, we have presented the first
example of Dbg being incorporated into peptides via solid-
phase approach and an efficient method for readily
incorporating amino acid residues onto the sterically
hindered N-terminus of RRAAs. This facile and conve-
nient coupling method may be extended to a variety of
sterically hindered RRAAs for solid-phase or solution-
phase peptide synthesis.
cally hindered RRAAs via the mild dialkylation of ni-
troacetate. The method has been shown to be successful
for the synthesis of highly sterically encumbered RRAAs
(
Fmoc-Dbg-OH), as well as the synthesis of chemically
R
sensitive RRAAs, such as the N - and side-chain-protected
tetrafunctional derivatives (Fmoc-Bcmg(OtBu) -OH). This
methodology should prove invaluable for the preparation
2
(28) (a) Coupling to N-MePhe derivative: Rich, D. H.; Bhatnagar,
P.; Mathiaparanam, P.; Grant, J . A.; Tam, J . P. J . Org. Chem. 1978,
Exp er im en ta l Section
4
3, 296-302. (b) Resin-bound N-benzyl(“BAL”)-amino acid: J ensen,
K. J .; Alsina, J .; Songster, M. F.; Vagner, J .; Albericio, F., Barany, G.
Gen er a l Meth od s. All melting points were measured with
a Fisher-J ohns melting point apparatus and are uncorrected.
H and C spectra were recorded on a 250 and 500 MHz
spectrometers. Flash column chromatography was performed
J . Am. Chem. Soc. 1998, 120, 5441-5452, and refs cited therein.
(29) For an excellent review of coupling methods for sterically
1
13
hindered amino acids, see: Humphrey, J . M.; Chamberlain, A. R.
Chem. Rev. 1997, 2243-2266

7
122 J . Org. Chem., Vol. 66, No. 21, 2001
Fu et al.
using silica gel 60 (230-400 mesh). Elemental analyses were
performed by M-H-W Laboratories (Phoenix, AZ). TLC was
performed on Merck silica gel 60 F254 plates and visualized by
UV lamp. The peptide was synthesized in a two-necked round-
bottom flask, one neck fitted with frit suitable for filtration. A
Brinkmann rotary evaporator and a Labquake shaker were
used for rotating and shaking the flask containing resin. UV
analysis of the Fmoc-deprotection was performed on a Gilford
UV spectrometer. The peptide was purified on a Waters HPLC
system (Delta-Pak C18 column, 15 µm 300 Å, 8 × 100 mm for
analytical scale, flow rate 1 mL/min; 25 × 100 mm for
preparative scale, flow rate 10 mL/min). Peptide mass was
verified by mass spectrometry on a Bruker ProFLEX III
MALDI-MS.
C
22
H
23NO
8
: C, 61.53; H, 5.40; N, 3.26. Found: C, 61.66; H,
5.62; N 3.26.
Bis(ter t-bu tyl) 3-Eth ylca r boxy-3-n itr oglu ta r a te (4e).
This compound was prepared from tert-butyl bromoacetate in
79% yield by using the same procedure as described for 4b.
1
H NMR (250 MHz, CDCl ) δ 4.25 (q, J ) 7.2 Hz, 2H), 3.42 (d,
3
J ) 16.8 Hz, 2H), 3.30 (d, J ) 16.8 Hz, 2H), 1.42 (s, 18 H),
1.24 (t, J ) 7.2 Hz, 3H); 13C NMR (60 MHz, CDCl
) δ 167.4,
165.0, 90.0, 82.6, 63.5, 39.9, 28.1, 13.9; Anal. Calcd for C16
NO : C, 53.18; H, 7.53; N, 3.88. Found: C, 53.04; H, 7.33; N
3.81.
Eth yl 2,2-Bis(a llyl)-2-n itr oa ceta te (4f). This compound
3
27
H -
8
3
0
was previously prepared by Pd-mediated approaches. In this
work 4f was prepared from allyl iodide by using the same
procedure as described for 4a followed by flash chromatogra-
Eth yl 2-Ben zyl-2-n itr o-3-p h en ylp r op a n oa te (4a ). To a
solution of ethyl nitroacetate 3 (1.0 g, 7.51 mmol) in dry DMF
1
phy (hexanes-ether, 1:3) to provide 4f in 45% yield. H NMR
+
-
(5 mL) were added DIEA (2.0 g, 15.4 mmol) and Bu
4
N Br
3
(250 MHz, CDCl ) δ 5.68-5.57 (m, 2H), 5.24-5.17 (m, 4H),
(0.24 g, 0.75 mmol). To this clear yellow solution was added
4.27 (q, J ) 7.2 Hz, 2H), 2.97-2.90 (m, 4H), 1.29 (t, J ) 7.2
Hz, 3H); 13C NMR (60 MHz, CDCl
) δ 166.8, 129.5, 121.6, 95.0,
benzyl bromide (2.6 g, 15.4 mmol), and the reaction spontane-
ously warmed to 60 °C over 5 min. After 20 min, DIEA‚HBr
precipitated out of solution. After 1 h, the solution was filtered
3
+
62.9, 38.2, 14.1; MS (FAB) calcd for C10
236.1, found 236.1. Anal. Calcd for C10
H
H
15NO
15NO
4
Na (M + Na)
4
: C, 56.32; H,
to remove DIEA‚HBr, the salt was washed with Et
and the combined filtrates were washed with H
O (5 × 50 mL).
The organic layer was dried (Na SO ) and evaporated at 0 °C
to provide a yellow oil, which was of sufficient purity to be
used in further reactions or could be purified to homogeneity
2
O (100 mL),
7.09; N, 6.57. Found: C, 56.41; H, 6.98; N 6.53.
2
Eth yl 2,2-Bis(2-p h en ylsu lfon yl eth yl)-2-n itr oa ceta te
(4g). To a solution of ethyl nitroacetate (0.4 g, 3.0 mmol) in
acetonirile (5 mL) were added DIEA (0.79 g, 6.15 mmol) and
tetraethylammonium bromide (0.09 g, 0.3 mmol). To this
solution was added phenyl vinyl sulfone (1.03 g, 6.15 mmol)
dropwise, and the solution was stirred at room temperature
for 30 h. The acetonitrile was then removed in vacuo, and the
residue was dissolved in ethyl acetate (25 mL). The resulting
2
4
by silica gel chromatography using Et
2
O-pentane (1:4) to
10
provide a white solid. Yield, 1.48 g (63%); mp 81-82 °C (lit.
1
mp 79-80 °C); H NMR (250 MHz, DMSO-d
6
) δ 7.38-7.22 (m,
1
0H), 4.10 (q, J ) 7.1 Hz, 2H), 3.52 (d, J ) 14.2 Hz, 2H), 3.46
1
3
(d, J ) 14.2 Hz, 2H), 1.07 (t, J ) 7.1 Hz, 3H); C NMR (60
solution was washed with 10% aqueous K
and dried over anhydrous Na SO , and the solvent was
removed in vacuo to give 4g as a light yellow solid. Yield, 0.98
2
CO
3
(2 × 10 mL)
MHz, CDCl ) δ 166.5, 133.4, 130.3, 128.8, 128.0, 97.4, 62.9,
3
2
4
+
40.2, 13.7; HRMS (FAB) calcd for C18
314.1392, found 314.1395. Anal. Calcd for C18
68.99; H, 6.11; N, 4.47. Found: C, 69.09; H, 5.94; N, 4.32.
Eth yl 2-Nitr o-2-(4-n itr oben zyl)-3-(4-n itr op h en yl)p r o-
p a n oa te (4b). To a solution of ethyl nitroacetate 3 (1.0 g, 7.51
H
19NO
4
(M + H)
1
H
19NO
4
:
C,
g (70%). mp 88-89 °C; H NMR (250 MHz, CDCl
3
) δ 7.92-
7.59 (m, 10H), 4.25 (q, J ) 7.1 Hz, 2H), 3.17-3.10 (m, 4H),
1
3
2.60-2.53 (m, 4H), 1.26 (t, J ) 7.1 Hz, 3H); C NMR (60 MHz,
CDCl ) δ 164.3, 138.1, 134.2, 129.5, 127.8, 92.8, 63.6, 49.3, 26.6,
13.3; HRMS (FAB) calcd for C20
found 470.0936. Anal. Calcd for C20
3
+
-
+
mmol) in dry DMF (5 mL) was added Bu
4
N Br (0.24 g, 0.75
H
23NO
8
S
2
(M + H) 470.0977,
8 2
mmol) and 4-nitrobenzyl bromide (3.3 g, 15.4 mmol). To this
cooled solution, DIEA (2.0 g, 15.4 mmol) was added dropwise,
and the solution was allowed to stir for 2 h. The resulting
solution was diluted with diethyl ether (100 mL) and washed
successively with 1 N HCl (2 × 15 mL), saturated aqueous
sodium carbonate solution (2 × 15 mL; to remove mono-
alkylated product), and water (5 × 15 mL). The solvent was
removed in vacuo, and the resulting light yellow solid was
recrystallized from ethanol to provide 2.27 g (75%) of 4b as
H23NO S : C, 51.16; H,
4.94; N, 2.98; S, 13.66. Found: C, 51.11; H, 5.07; N, 3.00; S,
13.51.
Bis(ter t-bu tyl)-4-eth ylcar boxy-4-n itr opim elate (4h ). This
compound was prepared from tert-butyl acrylate in 89% yield
1
by using the same procedure as described for 4g. H NMR (250
MHz, DMSO-d ) δ 4.24 (q, J ) 7.1 Hz, 2H), 2.41-2.20 (m, 8H),
6
1.40 (s, 18H), 1.21 (t, J ) 7.1 Hz, 3H); 13C NMR (60 MHz,
DMSO-d ) δ 170.3, 165.8, 94.5, 80.3, 62.9, 29.0, 28.3, 27.6, 13.5;
6
1
+
white crystals. mp 194-196 °C; H NMR (250 MHz, DMSO-
HRMS (FAB) calcd for C H NO (M + H) 390.2128, found
390.2118. Anal. Calcd for C H NO : C, 55.51; H, 8.02; N,
18 31 8
1
8
31
8
d
6
) δ 8.21 (d, J ) 8.7 Hz, 4H), 7.51 (d, J ) 8.7 Hz, 4H), 4.11(q,
J ) 7.1 Hz, 2H), 3.74 (d, J ) 14.2 Hz, 2H), 3.70 (d, J ) 14.2
3.60. Found: C, 55.31; H, 7.83; N, 3.87.
4-E t h ylca r b oxy-4-n it r op im elon it r ile (4i). This com-
pound was prepared from acrylonitrile in 87% yield by using
1
3
Hz, 2H), 1.05 (t, J ) 7.1 Hz, 3H); C NMR (60 MHz, DMSO-
) δ 165.0, 147.1, 141.1, 131.6, 123.4, 96.9, 63.0, 39.6, 13.3;
d
6
+
11
HRMS (FAB) calcd for C18
4
1
H
17
N
H
3
O
8
(M + H) 404.1094, found
the same procedure as described for 4g. mp 52-53 °C (lit.
1
04.1094. Anal. Calcd for C18
17
N
3
O
8
: C, 53.60; H, 4.25; N,
mp 52-53 °C); H NMR (250 MHz, DMSO-d ) δ 4.27 (q, J )
6
1
3
0.42. Found: C, 53.40; H, 4.51; N 10.31.
7.1 Hz, 2H), 2.70-2.54 (m, 8H), 1.24 (t, J ) 7.1 Hz, 3H);
C
E t h yl 2-(4-Cya n ob en zyl)-2-n it r o-3-(4-cya n op h en yl)-
p r op a n oa te (4c). This compound was prepared from 4-cy-
NMR (60 MHz, DMSO-d ) δ 164.7, 118.9, 93.2, 63.5, 28.5, 13.4,
6
+
11.7; HRMS (FAB) calcd for C H N O Na (M + Na) 262.0804,
1
0
13
3
4
anobenzyl bromide in 82% yield by using the same procedure
10 13 3 4
found 262.0809. Anal. Calcd for C H N O : C, 50.20; H, 5.48;
1
as described for 4b. mp 138-139 °C; H NMR (250 MHz,
N, 17.56. Found: C, 50.07; H, 5.36; N, 17.61.
DMSO-d
6
) δ 7.83 (d, J ) 8.3 Hz, 4H), 7.41 (d, J ) 8.3 Hz, 4H),
Eth yl 2,2-Diben zylglycin e Ester (5a ). To a solution of
nitro ester 4a (1.0 g, 3.19 mmol) in glacial AcOH (15 mL) at
15 °C was added Zn dust (0.63 g, 9.57 mmol) in several portions
over 1 h. The resulting mixture was allowed to warm to room
temperature and vigorously stirred for 24 h. The solids were
filtered off and washed with 1.0 M HCl (2 × 5 mL), and the
combined filtrates were concentrated to an oil. This oil was
dissolved in a mixture of ethyl acetate (10 mL) and aqueous 1
M NaOH (15 mL) and stirred for 24 h. The organic layer was
4
)
.08 (q, J ) 7.1 Hz, 2H), 3.76 (d, J ) 14.2 Hz, 2H), 3.69 (d, J
1
3
14.2 Hz, 2H), 1.03 (t, J ) 7.1 Hz, 3H); C NMR (60 MHz,
) δ 165.0, 139.0, 132.3, 131.2, 118.5, 110.7, 96.9, 62.9,
DMSO-d
6
+
3
3
6
9.9, 13.3; HRMS (FAB) calcd for C20
H
17
N
3
O
H
4
(M + H)
17 3 4
64.1297, found 364.1298. Anal. Calcd for C20
N O : C,
6.10.; H, 4.72; N, 11.56. Found: C, 65.93; H, 4.97; N 11.55.
Eth yl 2,2-Bis(m eth ylcar boxyben zyl)-2-n itr oacetate (4d).
This compound was prepared from methyl 4-(bromomethyl)-
benzoate in 72% yield by using the same procedure as
separated, dried (MgSO ), and evaporated in vacuo to provide
4
1
described for 4b. mp 116-117 °C; H NMR (250 MHz, DMSO-
amino ester 5a as a white solid (0.73 g, 81% yield). mp 93-95
1
d
6
) δ 8.01-7.97 (m, 4H), 7.26-7.21 (m, 4H), 4.14 (q, J ) 7.2
°C; H NMR (250 MHz, DMSO-d ) δ 7.30-7.16 (m, 10H), 4.0
6
1
3
Hz, 2H), 3.92 (s, 6H), 3.53 (s, 4H), 1.13 (t, J ) 7.2 Hz, 3H);
NMR (60 MHz, DMSO-d
1
C
6
) δ 165.8, 165.2, 138.7, 130.6, 129.2,
(30) (a) Lopez, A.; Moreno-Manas, M.; Pleixats, R.; Roglans, A. An.
29.0, 96.9, 62.8, 52.1, 39.9, 13.3; HRMS (FAB) calcd for C22
H
23
-
Quim. Int. Ed. 1997, 93, 355-362. (b) Ferroud, D.; Genet, J . P.; Muzart,
J . Tetrahedron Lett. 1984, 25, 4379-4382.
+
NO (M + H) 430.1502, found 430.1492. Anal. Calcd for
8

CR,R-Disubstituted R-Amino Acids
J . Org. Chem., Vol. 66, No. 21, 2001 7123
(
1
(
6
2
7
q, J ) 7.2 Hz, 2H), 3.18 (d, J ) 12.9 Hz, 2H), 2.77 (d, J )
trated in vacuo to provide a light yellow solid. The crude
2.9 Hz, 2H), 1.43 (s, 2H), 1.16 (t, J ) 7.2 Hz, 3H), 13C NMR
60 MHz, DMSO-d ) δ 174.8, 136.6, 130.0, 128.0, 126.6, 62.6,
product was purified by crystallization from Et
O-hexanes to
2
6
give the protected RRAA 7a as a white solid (1.5 g, 80% yield).
+
1
0.3, 45.9, 14.0; HRMS (FAB) calcd for C18
84.1650, found 284.1637. Anal. Calcd for C18
6.29; H, 7.47; N, 4.94. Found: C, 76.37; H, 7.31; N, 4.83.
Eth yl 2,2-Bis(ter t-bu tylca r boxylm eth yl)glycin e Ester
H
21NO
2
(M + H)
mp 184-185 °C; H NMR (250 MHz, CDCl ) δ 7.81-7.10 (m,
3
H
21NO
2
:
C,
18H), 5.50 (s, 1H), 4.49 (d, J ) 7.0 Hz, 2H), 4.29 (t, J ) 7.0
Hz, 1H), 3.90 (d, J ) 13.6 Hz, 2H), 3.26 (d, J ) 13.6 Hz, 2H);
1
3
C NMR (60 MHz, CDCl ) δ 177.2, 155.0, 144.2, 141.7, 136.1,
3
(
5e). To a solution of 4e (1.3 g, 3.92 mmol) in absolute ethanol
130.1, 128.8, 128.1, 127.5, 125.6, 120.4, 67.1, 66.9, 47.7, 41.5;
HRMS (FAB) calcd for C H NO (M + H) 478.2018, found
31 27 4
478.1996. Anal. Calcd for C H NO : C, 77.96; H, 5.70; N,
+
(10 mL) were added glacial acid (1 mL) and a 50% (w/w) slurry
3
1
27
4
of Raney nickel in water (1.0 g). The reaction was hydroge-
nated over H gas (50 psi) for 24 h. The resulting solution was
filtered carefully over Celite and the Celite cake washed with
EtOH (30 mL). The solvent was removed in vacuo, and the
crude was dissolved in diethyl ether (30 mL). After washed
with saturated sodium carbonate (30 mL) and brine, the
2
2.93. Found: C, 78.15; H, 5.85; N 2.99.
r
N -(9-F lu or en ylm eth yloxyca r bon yl)-2,2-bis(ter t-bu tyl-
ca r boxym eth yl)glycin e (7e). This compound was prepared
from 1e in 71% yield by using the same procedure as described
1
for 7a . H NMR (500 MHz, CDCl
(
3
) δ 7.76-7.24 (m, 8H), 6.33
organic fraction was dried (Na
2
SO
yield 5e in 92% yield. H NMR (250 MHz, CDCl
7.2 Hz, 2H), 2.68 (d, J ) 15.5 Hz, 2H), 2.48(d, J ) 15.5 Hz),
.26 (s, 2H), 1.37 (s, 18H), 1.20 (t, J ) 7.2 Hz, 3H); 13C NMR
60 MHz, CDCl ) δ 174.9, 169.7, 81.5, 61.5, 57.7, 45.0, 28.1,
4.2. Anal. Calcd for C16 : C, 57.99; H, 8.82; N, 4.23.
4
) and rotary evaporated to
s, 1H), 4.32 (d, J ) 7.0 Hz, 2H), 4.20 (t, J ) 7.0 Hz, 1H), 3.37
d, J ) 15.1 Hz, 2H), 2.85 (d, J ) 15.1 Hz, 2H), 1.38 (s, 18H);
C NMR (125 MHz, DMSO-d
40.7, 127.6, 127.0, 125.1, 120.1, 80.1, 65.6, 57.9, 46.5, 39.7,
7.6; Anal. Calcd for C29 : C, 66.27; H, 6.71; N, 2.66.
1
3
) δ 4.12 (q, J
(
)
2
(
1
13
6
) δ 171.9, 168.4, 153.9, 143.7,
1
2
3
H
35NO
8
H29NO
6
Found: C, 66.48; H, 6.87; N 2.56.
Found: C, 58.07; H, 8.86; N, 4.03.
Eth yl 2,2-Bis(ter t-bu tylcar boxyeth yl)glycin e Ester (5h ).
This compound was prepared from 4h in 92% yield by using
Solid -P h a se P ep tid e Syn th esis. (1) P r ep a r a tion of
R esin -Bou n d H-Dbg-Ala -Dp g-Glu (OtBu )-P AL-P E G-P S.
1
6
1
The peptide was prepared using Fmoc methodology with
Fmoc-PAL-PEG-PS resin (0.20 mmol/g loading) as a solid
support. To ensure a high degree of coupling efficiency, all
amino acids were coupled using PyAOP in the manual protocol
as a coupling reagent. The peptide chain was assembled at
the same procedure as described for 5e. H NMR (250 MHz,
DMSO-d
s, 2H), 1.38 (s, 18H), 1.18 (t, J ) 7.2 Hz, 3H); C NMR (60
MHz, DMSO-d ) δ 175.2, 172.0, 79.5, 60.4, 59.2, 34.2, 29.8,
7.6, 14.0; MS (FAB) calcd for C18 359.46, found 359.35.
Eth yl 2,2-Bis(ter t-bu tylca r boxyeth yl)-N-h yd r oxygly-
6
) δ 4.09 (q, J ) 7.2 Hz, 2H), 2.34-1.64 (m, 8H), 1.81
13
(
6
2
H33NO
6
0
.4 mmol scale, and each coupling consisted of the following
steps: (i) removal of Fmoc protection group with DBU-
piperidine-DMF (2:5:93; 20 mL) at room temperature for 15
min. (ii) Coupling of the Fmoc-amino acid (1.6 mmol, 4 equiv)
onto resin using PyAOP (1.6 mmol, 4 equiv) and DIEA (3.2
mmol, 8 equiv) in the solvent of DCE-DMF (1:1; 8 mL) at 50
cin e E st er (6h ). To a solution of 4h (1.0 g, 2.57 mmol) in
absolute ethanol (15 mL) was added carefully 10% (w/w)
palladium on carbon (0.1 g). The reaction was hydrogenated
over H
carefully over Celite and the Celite cake washed with EtOAc
30 mL). The organic filtrate was dried (Na SO ) and rotary
evaporated to yield 5k in 97% yield. H NMR (250 MHz,
DMSO-d ) δ 7.23 (s, 1H), 5.69 (s, 1H), 4.06 (q, J ) 7.2 Hz,
H), 2.17-2.13 (m, 4H), 1.74-1.70 (m, 4H), 1.37 (s, 18H), 1.17
2
gas (50 psi) for 24 h. The resulting solution was filtered
°
C for 2 h. After each coupling step, coupling efficiency was
(
2
4
1
examined by UV analysis of the Fmoc-deprotection (∼8 mg of
resin in 1 mL of deblocking solution for 10 min).
6
2
(
1
Cou p lin g of F m oc-Lys(Boc)-OH on to H-Dbg-Ala -Dp g-
Glu (OtBu )-P AL-P EG-P S. Coupling of Fmoc-Lys(Boc)-OH
onto resin-bounded peptide was performed on 0.1 mmol scale
resin under different conditions. (i) Coupling using PyAOP and
DIEA was carried out under the same conditions as described
above. Acylation yield, 20%. (ii) Coupling of Fmoc-Lys(Boc)-
OH (0.4 mmol, 4 equiv) using PyAOP (0.4 mmol, 4 equiv),
HOAt (0.4 mmol, 4 equiv), and DIEA (0.8 mmol, 8 equiv) in
DCE-DMF (1:1; 2 mL) at room temperature for 8 h gave a
25% coupling yield. (iii) The acylation of N-terminal Dbg with
Fmoc-Lys(Boc)-OH (0.2 mmol, 4 equiv) using HATU (0.2 mmol,
4 equiv) and DIEA (0.4 mmol, 8 equiv) in DMF (2 mL) at room
temperature gave an 18% coupling yield. (iv) The symmetrical
anhydride method was performed by adding preformed (Fmoc-
1
3
t, J ) 7.2 Hz, 3H); C NMR (60 MHz, DMSO-d
6
) δ 173.6,
72.0, 79.5, 66.6, 60.5, 29.1, 27.7, 26.3, 14.0; MS (FAB) calcd
+
for C18
7
H33NO (M + H) 376.47, found 376.77.
2
,2-Diben zylglycin e (1a ). A suspension of 5a (2.0 g, 7.1
mmol) in 2 M KOH (50 mL) and ethanol (25 mL) was refluxed
under N for 24 h. The resulting mixture was concentrated to
0 mL in vacuo, and the mixture was acidified to pH 6.5 with
2
2
concentrated HCl. The white precipitate was filtered, washed
with water (5 mL), and evaporated to provide the amino acid
6
(
1
a (1.58 g, 6.2 mmol) in 88% yield (mp > 300 °C). H NMR
250 MHz, CD
3
OD) δ 7.27 (bs, 10H), 3.36 (d, J ) 13.3 Hz, 2H),
2
.85 (d, J ) 13.3 Hz, 2H), 1.27 (s, 2H), HRMS (FAB) calcd for
+
C
16
H
17NO
Calcd for C16
5.34; H, 6.64; N, 5.30.
,2-Bis(ter t-bu tylca r boxym eth yl)glycin e (1e). To a solu-
2
Na (M + Na) 278.1157, found 278.1159. Anal.
H
17NO
2
: C, 75.27; H, 6.71; N, 5.48. Found: C,
2
Lys(Boc)) O (0.3 mmol, 3 equiv) to the resin-bound peptide in
7
DCE-DMF (9;1; 4 mL) in the absence of base. The reaction
was carried out at 50 °C for 2 h to give a 95% coupling yield
determined by UV analysis. After acylation, the Fmoc group
on Lys was removed, and the resin was suitable for cleavage.
2
tion of 5e (2.0 g, 6.03 mmol) in ethanol (20 mL) was added
LiOH (0.4 g, 18.1 mmol) in water (10 mL). The reaction was
stirred at 50 °C for 3 h followed by the same workup procedure
as described for 1a to provide the amino acid as a white solid.
Yield, 1.53 g (82%). H NMR (250 MHz, CD
P ep tid e Clea va ge a n d P u r ifica tion . The resin was
treated with a cleavage solution of TFA/TPS/phenol/water (88:
1
3
OD) δ 2.78 (d, J
2
:5:5, v/v/v, 10 mL) at room temperature for 2 h. After
)
17.2 Hz, 2H), 2.69 (d, J ) 17.2 Hz), 1.37 (s, 18H); 13C NMR
filtration, the filtrate was diluted with cold 30% acetic acid
(
(
125 MHz, CD
FAB) calcd for C14
3
OD) δ 175.8, 173.7, 85.7, 62.7, 43.3, 30.5; HRMS
(
50 mL) and extracted with cold ether (4 × 30 mL). The
+
H
25NO
6
(M + H) 304.1760, obsd 304.1760.
aqueous fraction was then lyophilized overnight to provide the
crude peptide. Peptide purification was performed by reversed-
phase HPLC on C18 column. The peptide homogeneity (>99%)
was determined by analytical HPLC using the same solvents
with a gradient of 10-40% (v/v) acetonitrile over 30 min.
r
N -(9-F lu or e n ylm e t h oxyca r b on yl)-2,2-d ib e n zylgly-
cin e (7a ). TMSCl (0.85 g, 7.84 mmol) was added to a
suspension of amino acid 1a (1.0 g, 3.92 mmol) in dry CH Cl
20 mL) and refluxed under N for 8 h. The mixture was cooled
to 0 °C, and DIEA (1.01 g, 7.84 mmol) and Fmoc-Cl (1.01 g,
2
2
(
2
+
Peptide was identified by MALDI-MS, m/z calcd [M + Na]
7
3
2
.92 mmol) were added. The reaction was allowed to warm to
5 °C and stirred for 30 h. The resulting mixture was
46.9, obsd 747.1.
concentrated in vacuo to provide a yellow solid which was
dissolved in aqueous 10% NaHCO . This solution was washed
with Et
O (3 × 15 mL), and the aqueous layer was acidified
to pH 2.0 with HCl and extracted with EtOAc (3 × 35 mL).
The combined EtOAc layers were dried (MgSO ) and concen-
3
Ack n ow led gm en t . We gratefully recognize the
expert technical assistance of Dr. Dale Treleaven, Dr.
Zhe Zhou, Ms. Martha M. J uban, Dr. Tracy McCarley
2
4

7
124 J . Org. Chem., Vol. 66, No. 21, 2001
Fu et al.
and Ms. Renee Sims. We also thank the Nebraska
Center for Mass Spectrometry for technical assistance.
This work was supported by grants from the National
Institute on Aging (AG17983) and Institute of General
Medical Sciences (GM56835) of the National Institutes
of Health. We thank the NSF for funding to purchase
NMR (CHE-9809187) and mass spectrometry (CHE-
Su p p or tin g In for m a tion Ava ila ble: NMR spectra for 4a -
4i, 5a , 5e, 5h , 6h , 1a , 1e, 7a , and 7e; ORTEPs of 4e and 5e,
tables of crystallographic data, bond lengths and angles,
atomic coordinates and anisotropic thermal parameters for
structures for 4e and 5e; analytical HPLC profile for isolated
2
pure peptide H-Lys-Dbg-Ala-Dpg-Glu-NH . This material is
available free of charge via the Internet at http://pubs.acs.org.
9
809178) instrumentation used in this work.
J O015809O