Facile Synthesis of α,α-Diisobutylglycine and Anchoring Its Derivatives onto PAL-PEG-PS Resin

Article abstract of DOI:10.1021/jo034885j

α,α-Diisobutylglycine has been synthesized using a Pd-mediated dialkylation of ethyl nitroacetate as a key first step. The free ααAA is Nα-protected and has been applied to the assembly of conformationally constrained peptide analogues. Mixed anhydrides f

Full text of DOI:10.1021/jo034885j

F a cile Syn th esis of r,r-Diisobu tylglycin e
a n d An ch or in g Its Der iva tives on to
P AL-P EG-P S Resin
synthesis of Dibg via Pd-mediated diallylation of R-ni-
troacetate. Additionally, some of the peptides we are
currently making require a hindered RRAA at the
C-terminus. Such peptides are difficult to assemble as
coupling of the RRAA to the solid support generally
Yanwen Fu, Marcus A. Etienne, and
Robert P. Hammer*
9
affords low yields. Thus, we also report here an improved
method for anchoring RRAAs onto PAL-PEG-PS resin via
mixed anhydrides.
Department of Chemistry, Louisiana State University,
Baton Rouge, Louisiana 70803
We have found R-nitroacetate a very useful synthon
for the preparation of RRAAs with and without side chain
functionality, where the R,R-dialkylated nitroacetate was
rphammer@lsu.edu
3
produced as a key precursor to the target compound.
Received J une 23, 2003
Since direct dialkylation of nitroacetate with branched
alkyl halides proved to be nonproductive (Table 1, entry
Abstr a ct: R,R-Diisobutylglycine has been synthesized using
1
), we tried activated allyl halides for the alkylation
a Pd-mediated dialkylation of ethyl nitroacetate as a key
R
under different conditions. While allyl iodide gave the
diallylated acetate in a moderate yield (Table 1, entry
5), the branched allyl iodide, 3-iodo-2-methylpropene only
gave a low yield of the diallylated nitroacetate (Table 1,
entry 3), and the major product of this reaction was a
mixture of mono-C-allylated and O-allylated derivates.
In both cases, allyl bromides only gave trace amount of
desired compounds (Table 1, entries 2 and 4).
first step. The free RRAA is N -protected and has been ap-
plied to the assembly of conformationally constrained pep-
tide analogues. Mixed anhydrides from BOP-Cl and Fmoc-
RRAA-OH are used for anchoring RRAAs onto a trialkoxy-
benzyl linker on PEG-PS grafted support, upon which a
â-strand mimic with difficult sequence is assembled in a
superior quality.
In search for an efficient method for dialkylation of
R-nitroacetate with branched allyl halides or it equiva-
lents, we thought that use of “activated” allylic groups
as the electrophiles would be a feasible approach. Allylic
activation with palladium templates has long been used
for alkylation of preformed carbanions.10 The previously
reported Pd-catalyzed allylic alkylation of R-substituted
There is an increased interest in the synthesis and use
of symmetrically R,R-disubstituted amino acids (RRAAs)
in controlling peptide secondary structures.1,2 Recent
3
approaches to RRAAs via alkylation of R-nitroacetate and
4
Ni-Schiff base complex allow practical preparation of
a variety of RRAAs in good chemical yields. However,
highly hindered RRAAs that possess branched side
5
nitro carboxylic acids gave quarternary nitro esters in
chains, such as R,R-diisobutylglycine 3a (Dibg), have not
good yields.1
1,12
Muzart’s group and Lopez’s group re-
been readily synthesized.6,7 Though Dibg was tradition-
8
ported Pd-mediated alkylation of allyl acetate by nitro
ally prepared via the Bucherer-Bergs method, there
were major flaws associated with this approach due to
the high resistance to the formation and hydrolysis of
the hydantoin, which in turn gave a very low overall yield
of the desired product. In our effort to assemble a number
of â-strand mimics containing Dibg and other RRAAs, we
needed to develop a practical method for preparing a
large quantity of protected Dibg that is suitable for solid-
phase peptide synthesis. Herein we report an efficient
(
6) While alkylations of the Ni-Schiff base complex with un-
branched alkyl halides lead to the formation of RRAAs in high yields,
this method has not been extended for the synthesis of branched
RRAAs. The alkylation with branched alkyl halides gave predominately
mono-alkylated products under attempted conditions. Ellis, T. K.;
Martin, C. H.; Tsai, G. M.; Ueki, H.; Soloshonok, V. A. J . Org. Chem.
003, 68, 6208-6214.
(7) Synthetic approaches to Dibg were attempted via condensation
2
2 2
of symmetric ketones with PhCH NH or Hofmann degradation of
substituted cyanoacetic ester; however, both approaches gave a very
low overall yield of the desired product. See: (a) Freitas, A. M.; Maia,
H. L. S. Peptide, Proc. Eur. Pept. Symp. 1989, 13-15. (b) Lin, K.-H.;
Li, L.; Huang, Y.-T. Science & Technol. China 1948, 1, 5-10.
*
To whom correspondence should be addressed. Tel: (225) 578-
025. Fax: (225) 578-3458.
1) For recent reviews on use of RRAAs in peptide design, see: (a)
Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers 2001,
0, 396-419. (b) Wysong, C. L.; Yokum, T. S.; McLaughlin, M. L.;
Hammer, R. P. Chemtech 1997, 27, 26-33.
2) For a recent review on synthesis of RRAAs, see: Kotha, S. Acc.
Chem. Res. 2003, 36, 342-351.
3) Fu, Y.; Hammarstr o¨ m, L. G. J .; Miller, T. J .; Fronczek, F. R.;
McLaughlin, M. L.; Hammer, R. P. J . Org. Chem. 2001, 66, 7118-
124.
4) Ellis, T. K.; Martin, C. H.; Ueki, H.; Soloshonok, V. A. Tetrahe-
dron Lett. 2003, 44, 1063-1066.
5) Abbrevations: Aib, R-aminoisobutyric acid; Api, 4-aminopiperi-
4
R, R
(
(8) (a) Fu, Y. Artificial Peptides Containing C -disubstituted Amino
Acids: Synthesis, Conformational Studies, and Application as â-strand
Mimics. Dissertation, Louisiana State University, 2002, pp 22-66. (b)
Upham, S. D.; Dermer, T. C. J . Org. Chem. 1957, 22, 799-802.
(9) There are only a limited reports on assembling peptides with
an RRAA at the C-terminus using SPPS, where typically a less
sterically hindered RRAA such as Aib and Api, was coupled onto a resin
6
(
(
2 2
using Fmoc-RRAA-F in DMF or refluxing CH Cl . For examples, see:
7
(a) 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. (b) Yokum, T. S.; Gauthier, T. J .; Hammer, R. P.;
McLaughlin, M. L. J . Am. Chem. Soc. 1997, 115, 1167-1168. (c)
Wysong, C. L.; Yokum, T. S.; Morales, G. A.; Gundry, R. L.; McLaugh-
lin, M. L.; Hammer, R. P. J . Org. Chem. 1996, 61, 7650-7651.
(10) For reviews on Pd-catalyzed allylic alkylations, see: (a) Trost,
B. M. Acc. Chem. Res. 1996, 29, 355-364. (b) Trost, B. M. Acc. Chem.
Res. 1980, 13, 385-393. (c) Trost, B. M., Verhoeven, T. R. J . Am. Chem.
Soc. 1980, 102, 4730-4743. (c) Trost, B. M., Verhoeven, T. R. J . Am.
Chem. Soc. 1976, 98, 630-632.
(
(
dine-4-carboxylic acid; BOP-Cl, bis(2-oxo-3-oxazolidinyl)phosphinic
chloride; Dbzg, R,R-dibenzylglycine; DCE, 1,2-dichloroethane; Dibg,
R,R-diisobutylglycine; DIEA, N,N-diisopropylethylamine; Dpg, R,R-
dipropylglycine; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, O-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
HOAt, hydroxy-7-azabenzotriazole; MALDI-MS, matrix-assisted laser
desorption ionization mass spectrometry; PAL, 5-(4-aminomethyl-3,5-
dimethoxyphenoxy)valeric acid; PEG-PS, poly(ethylene glycol)polysty-
rene graft; PyAOP, 7-azabenzotriazol-1-yloxytris(pyrrolidino)phospho-
nium hexafluoro-phosphate; SPPS, solid-phase peptide synthesis; TPS,
triisopropylsilane; Z, benzyloxycarbonyl.
(11) Fornicola, R. S.; Oblinger, E.; Montgomery, J . J . Org. Chem.
1998, 63, 3528-3529.
(12) Lalonde, J . J .; Bergbreiter, D. E.; Wong, C.-H. J . Org. Chem.
1988, 53, 2323-2327.
1
0.1021/jo034885j CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/15/2003
9
854
J . Org. Chem. 2003, 68, 9854-9857

TABLE 1. Alk yla tion of Eth yl Nitr oa ceta te w ith Allyl
Ha lid es a n d Allyl Aceta tes
N
bipyramidal intermediate that must occur in an S 2
reaction. Trost demonstrated this difference in reactivity
by a competition reaction of malonate anions with a
substrate that had both primary bromide and an allylic
T
prod yieldb
methoda reagent (°C) no.
(%)
entry
electrophile
CHCH
50 1c tracec
60 1a tracec
60 1a <10
10
1
2
3
4
5
6
7
8
(CH
3
)
2
2
I
A
A
A
A
A
B
B
B
Bu
Bu
Bu
Bu
Bu
Pd(PPh
Pd(PPh
Pd(PPh
4
4
4
4
4
NI
NBr
NI
NBr
NI
acetate in the presence of Pd. Alkylation of the malonate
CH
CH
CH
CH
CH
CH
CH
2
dC(CH
dC(CH
3
)CH
)CH
2
Br
I
only occurred through the allylic acetate with the bromide
left unchanged. In contrast, alkylation of the nitroacetate
with alkyl halides (Table 1, entries 1-3) mainly goes
2
2
2
2
2
2
3
2
d
dCHCH
dCHCH
2
Br
I
60 1b trace
d
2
60 1b 45
dC(CH
dC(CH
3
)CH
)CH
2
2
OAc
OAc
3
3
3
)
)
)
4
4
4
50 1a 92 (90)e
25 1a 80
N
through a regular S 2 process.
3
To make the desired free RRAA, both the nitro group
and the side chain alkene in 1a needed to be reduced.
dCHCH
2
OAc
50 1b 93
a
Method A: solvent, DMF; 1.5 M nitroacetate, 0.15 M Bu4NI
or Bu4NBr when an alkyl bromide was the electrophile); 2.1 equiv
3
On the basis of our previous work, we found that the
(
tertiary NO
to the free amine under Raney nickel-catalyzed hydro-
genation. Thus, the reduction of the NO and CdC group
2
group in nitro esters could be easily reduced
of DIEA, 2.1 equiv of electrophile. Method B: solvent, THF;
0
2
.6 M nitroacetate, 5 mol % Pd catalyst, 2.1 equiv of electrophile,
b
.1 equiv of DIEA. All percentage yields refer to isolated prod-
2
c
ucts. Major products were mono-C-alkylated and O-alkylated
in 1a was carried out in one step under hydrogen (50 psi)
over T-1 Raney nickel to give the amino ester 2a in a
high yield (Scheme 1). This amino ester 2a was then
hydrolyzed in refluxing aq KOH-EtOH, followed by
isolation through a cation-exchange resin column to give
the free Dibg in a 91% yield. The free RRAA needed to
d
derivatives. Reaction results were previously reported in ref 3.
e
The number in parentheses represents the reaction yield when
2
mol % of Pd catalyst was added. Unless mentioned, 5 mol % Pd
catalyst was used for the activation.
esters and other nucleophiles, where both mono- and
diallylated products were usually obtained.13 Tsuji et al.
reported decarboxylation-allylation of allyl methyl car-
bonate using Pd as a catalyst where the diallylated
R
be N -protected to allow for either solid-phase or solution-
R
phase peptide synthesis. The N -protection of the amine
group in 3a was carried out using an in situ silylation
16
procedure where Fmoc-Cl, (Boc)
2
O, or Z-Cl was added
1
4
nitroacetate was produced in a good chemical yield. Pd-
catalyzed cross-coupling reactions of nitro esters with
branched allyl acetate in an aqueous medium has also
been performed in which the diallylated acetate was
formed as a minor product in a low yield.15
to the silylated RRAA in CH Cl to provide Fmoc-Dibg-
2 2
OH, Boc-Dibg-OH, and Z-Dibg-OH in very good chemical
yields.
R
The main application for N -protected RRAAs in our
ongoing projects is for assembly of conformationally
constrained peptide mimics. In several designed peptides,
an RRAA was at the C-terminus and thus needed to be
directly attached to the solid support for C f N assembly
of the peptide. Attachment of the C-terminal RRAA with
a resin linker is typically difficult. For example, coupling
The synthetic approach to R,R-diisobutylglycine pro-
posed herein employes Pd(0)-mediated diallylation of
ethyl R-nitroacetate with 2-methylallyl acetate in the
presence of a tertiary amine (Scheme 1). In our experi-
ments, ethyl R-nitroacetate was treated with 2 equiv of
3
DIEA to form nitroacetate anion, which subsequently
2
of Fmoc-Dibg-OH onto PAL-PEG-PS resin (with NH as
the reactive functional group) using PyAOP, HATU, or
attacked the Pd(II)-allyl complex to give diallylated
nitroacetates in excellent yields (Table 1, entries 6-8).
amino acid fluorides, some of the conventional activation
Pd(Ph
3
P)
4
(5 mol %) was used for the formation of Pd-
17,18
methods for difficult couplings,
only afforded moder-
(II)-allyl complex in most attempts, and in one attempt
ate yields (Table 2, entries 1-3).
only 2 mol % of the Pd(0) catalyst was used for the
activation where a high yield of the desired product was
also obtained (Table 1, entry 6). It was observed that the
alkylation could be improved by heating, especially for
the reaction with branched allyl acetate, where a 92%
yield was obtained at 50 °C in comparison to a 80% yield
at room temperature (Table 1, entries 6 and 7). There
was no O-alkylated nitroacetate isolated under attempted
conditions. The Pd catalyst was easily removed by the
Similar results were obtained with Dpg couplings.
Anchoring of the first amino acid residue to the solid
support is one of the most critical steps in peptide
synthesis, and the inefficient attachment significantly
reduces the overall yield and also creates a deletion
sequence that could complicate subsequent peptide pu-
rification.
BOP-Cl-mediated coupling has been utilized for as-
sembly of peptide analogues with difficult sequences,
3
addition of polymer-supported PPh resin, and the dial-
lylated acetate 1 was obtained in an excellent yield.
The highly efficient dialkylation of the ethyl nitroac-
such as peptides containing N-substituted amino ac-
18,19
ids.
We have also observed that preformed mixed
anhydrides from BOP-Cl and Fmoc-L-amino acids could
etate occurred through a Pd-aided “net S
ment”. One of the reasons that the Pd-catalyzed allylation
is so efficient relative to a direct S 2 (or S 2′) reaction,
N
2 displace-
(16) (a) Bolin, D. R.; Sytwu, I.-I.; Humiec, F.; Meienhofer, J . J . Pept.
Protein Res. 1989, 33, 353-359. (b) Obrecht, D.; Lehmann, C.; Ruffieux,
N
N
R.; Sch o¨ nholzer, P.; M u¨ ller, K. Helv. Chim. Acta 1995, 78, 1567-1587.
even in sterically hindered cases, is that the transition
state for C-C bond formation involves exo attack on a
π-allyl-Pd complex rather than formation of a trigonal-
(
17) Fu, Y.; Hammer, R. P. Org. Lett. 2002, 4, 237-240 and
references therein.
(18) Humphrey, J . M.; Chamberlin, A. R. Chem. Rev. 1997, 97,
2
243-2266 and references therein.
(19) For examples of synthesis of peptides containing N-substituted
(
13) (a) Ferroud, D.; Genet, J . P.; Muzart, J . Tetrahedron Lett. 1984,
5, 4379-4382. (b) Lopez, A.; Moreno-Manas, M.; Pleixats, R.; Roglans,
A. An. Quim. Int. Ed. 1997, 93, 355-362.
14) Tsuji, J .; Yamada, T.; Minami, I.; Yuhara, M.; Nisar, M.;
Shimizu, I. J . Org. Chem. 1987, 52, 2988-2995.
15) Genet, J . P.; Blart, E.; Savignac, M. Synlett 1992, 9, 715-717.
amino acids, see: (a) Tung, R. D.; Dhaon, M. K.; Rich. D. H. J . Org.
Chem. 1986, 51, 3350-3354. (b) Colucci, W. J .; Tung, R. D.; Petri, J .
A.; Rich, D. H. J . Org. Chem. 1990, 55, 2895-2903. (c) Van der Auwera,
C.; Anteunis, M. J . O. J . Pept. Protein Res. 1987, 29, 574-588. (d)
Anteunis, M. J . O.; Sharma, N. K. Bull. Soc. Chim. Belg. 1988, 97,
281-291.
2
(
(
J . Org. Chem, Vol. 68, No. 25, 2003 9855

SCHEME 1
TABLE 2. Atta ch m en t of (A) F m oc-Dibg-OH a n d (B)
F m oc-Dp g-OH w ith P AL-P EG-P S Resin
b
coupling yield (%)
entry
coupling methoda
base
A
B
c
c
46 (29)c
64
55
1
2
3
4
PyAOP
HATU
Fmoc-RRAA-F
BOP-Cl
DIEA
DIEA
DIEA
33 (25)
41 (26)
43
d
e
none
82
91
a
All couplings were performed in DCE-DMF (4:1) for 8 h.
Unless mentioned, all couplings were carried at 50 °C. b The
coupling yield was determined by UV analysis of the Fmoc-
c
deprotection. The number in parentheses represents the coupling
d
yield at 25 °C. Preformed mixed anhydride was used in the
coupling. The mixed anhydride was prepared by treatment of
1
equiv of Fmoc-RRAA-OH with 1 equiv of BOP-Cl with the
presence of 1 equiv of DIEA in CH2Cl2 at 0 °C for 2 h. The CH2Cl2
was removed in vacuo, and the concentrated anhydride was taken
up in DCE-DMF (4:1) for coupling. eDIEA was used in the
formation of the mixed anhydride; no additional base was used in
the coupling.
F IGURE 1. HPLC profile of crude peptide K7-INH: H-(Lys)
Dibg-Val-Dbzg-Phe-Dpg-NH . Column: Delta-Pak C18, 15 µm,
00 Å, 8 × 100 mm. Eluent: (A) H O and 0.1% TFA; (B) MeCN
and 0.1% TFA. Gradient: 10-70% B over 60 min. Flow rate:
mL/min.
7
-
SCHEME 2
2
3
2
1
result from the first amino acid attachment, we synthe-
sized a peptide K7-INH containing three different RRAAs,
7 2
H-(Lys) -Dibg-Val-Dbzg-Phe-Dpg-NH , using our previ-
ously reported symmetrical anhydride coupling proto-
1
7
col and PyAOP/HATU activation methods. The highly
efficient assembly of the difficult peptide was verified
by HPLC (Figure 1) and by MALDI-MS. Currently, this
peptide and other synthesized â-strand mimics are being
investigated for their inhibitory activities against â-amy-
loid assembly, and the results will be reported in due
course.
We have reported a practical synthesis of R,R-diisobu-
tylglycine via Pd-mediated diallylation of R-nitroacetate.
This method may be extended for the synthesis of a
variety of sterically hindered symmetrical and asym-
metrical RRAAs. Also, we have reported an improved
method for coupling RRAAs to PAL-linker on PEG-PS
resin using preformed mixed anhydrides. This coupling
efficiently acylate the N-terminus of sterically hindered
RRAAs.17 Here we report our preliminary study on
coupling RRAAs onto a solid support using mixed anhy-
drides. It was found that preformed mixed anhydrides
from BOP-Cl and Fmoc-RRAA-OH residues could readily
acylate the NH
2
group on PAL-PEG-PS resin (Scheme
2
; Table 2, entry 4). There was no additional base added
to the coupling, and the PS resin was suspended in the
relatively nonpolar DCE-DMF (4:1) solvent at 50 °C to
help the resin swell. On the basis of the excellent coupling
9
856 J . Org. Chem., Vol. 68, No. 25, 2003

method may be useful for anchoring other sterically
demanding amino acids to solid supports. Based on our
previous results and the findings described herein, we
believe that BOP-Cl-mediated coupling may be generally
used for the synthesis of conformationally constrained
peptides having RRAAs at different sequence positions.
as a solid support. Except for the last six Lys residues that were
incorporated into the sequence via the PyAOP protocol (Fmoc-
Lys(Boc)-OH, 4 equiv; PyAOP, 4 equiv; DIEA, 8 equiv; solvent
DMF; 2 h for each coupling at 50 °C) on a Perseptive Biosystem
Peptide Synthesizer, all amino acids were incorporated into the
sequence manually. Dpg was attached to the solid support using
the preformed mixed anhydride (8 equiv, 0.3 M) in DCE-DMF
(4:1) at 50 °C. Capping of the resin was carried out by treating
the resin with excess 0.2 M of Ac O in 0.28 M of DIEA (16 equiv)
2
Exp er im en ta l Section
in DMF for 2 h. Phe and Dbzg were incorporated into the
sequence using PyAOP with DIEA in DCE-DMF (1:1); Val
residue was coupled to N-terminus of Dbzg via amino acid
symmetrical anhydride method.17 Dibg was coupled to Val using
HATU (4 equiv), HOAt (4 equiv), DIEA (8 equiv), and Fmoc-
Dibg-OH (4 equiv, 0.3 M) in DCE-DMF (4:1) at 50 °C. Lys
residue was coupled to Dibg via symmetrical anhydride at
50 °C. Removal of Fmoc protection group before each coupling
step was performed using DBU-piperidine-DMF (2:5:93) at
50 °C for 15 min. After each coupling step, coupling efficiency
was examined by UV analysis of the Fmoc-deprotection (∼8 mg
of resin in 1 mL of deblocking solution for 10 min). After the
assembly, the resin was treated with a cleavage solution of
Eth yl 2,2-Bis(2-m eth yla llyl)-2-n itr oa ceta te (1a ). To a
solution of ethyl nitroacetate (10.0 g, 75.1 mmol) in dry THF
(
110 mL) were added 2-methylallyl acetate (18.0 g, 157.1 mmol)
and Pd(PPh (9.0 g, 7.8 mmol). After 15 min DIEA (20.1 g,
57.1 mmol) was added, and the reaction was stirred under
3 4
)
1
argon for 8 h at 50 °C. The resulting solution was filtered over
Celite while washing with THF (2 × 120 mL). The filtrate was
concentrated, dissolved in 75 mL of EtOAc and washed with 10%
K
(
2
2
CO
1.3 mmol/g, Argonaut) resin and the mixture was shaken for
5 min. The resin was filtered off and the filtrate was purified
via column chromatography (EtOAc-Et O, 2:1) to provide a light
yellow oil: yield 16.5 g (92%); H NMR (250 MHz, CDCl ) δ
.96-4.94 (m, 2H), 4.81-4.79 (m, 2H), 4.24 (q, J ) 7.2 Hz, 2H),
.08-2.95 (m, 4H), 1.72-1.71 (m, 6H), 1.28 (t, J ) 7.2 Hz, 3H);
3 3
(50 mL). To the organic layer was added 12 g of PS-PPh
2
1
5
1
3
2
TFA-TPS-phenol-H O (88:2:5:5, v/v/v) at room temperature
4
3
for 2 h. After filtration, the filtrate was concentrated and added
to cold ether and the precipitate was isolated by centrifugation.
Peptide purification was performed by reversed-phase HPLC on
a C18 column to yield 188 mg of crude product (81% crude purity)
1
3
C NMR (75 MHz, CDCl
1.8, 23.2, 13.7. Anal. Calcd for C12
3
) δ 166.6, 138.4, 117.2.3, 95.1, 62.7,
4
H
19NO
4
: C, 59.73; H, 7.94;
+
N, 5.81. Found: C, 59.42; H, 7.69; N, 5.56.
which was identified by MALDI-MS, m/z calcd [M + H] 1710.4,
r
r
An Exa m p le of Syn th esis of N -P r otected Dibg. N -(9-
F lu or en ylm et h oxyca r b on yl)-2,2-d iisob u t ylglycin e (4a ).
TMSCl (8.8 g, 81.0 mmol) was added to a suspension of amino
obsd 1710.7. Semipreparative HPLC gave an overall 43% yield
of pure peptide (> 99% purity).
acid 3a (7.4 g, 39.6 mmol) in dry CH
under N for 8 h. The mixture was cooled to 0 °C, and DIEA
10.5 g, 81.0 mmol) and Fmoc-Cl (10.0 g, 38.6 mmol) were added.
The reaction was allowed to warm to 25 °C and stirred for 20 h.
The resulting mixture was concentrated in vacuo to provide a
yellow solid which was then dissolved in EtOAc (80 mL). To this
mixture, water was added (40 mL), and the solution was acidified
to pH 2.0 with 2 N HCl. The separated organic layer was dried
2 2
Cl (80 mL) and refluxed
Ack n ow led gm en t. We thank Dr. Dale Treleaven,
Dr. Tracy McCarley, and Ms. Martha M. J uban for
expert technical support and Dr. Sidney Val for helpful
discussions. We also thank the Nebraska Center for
Mass Spectrometry for technical assistance. We thank
the Louisiana Board of Regents for a graduate fellow-
ship to ME. This work was supported by a grant from
the National Institute on Aging (AG 17983) of the
National Institutes of Health.
2
(
(
4
MgSO ) and concentrated in vacuo to provide a light yellow
solid. The crude product was triturated in hexanes to afford a
1
white solid (13.7 g, 88% yield): mp 132-133 °C; H NMR (250
MHz, DMSO-d ) δ 7.90-7.28 (m, 8H), 6.48 (s, 1H), 4.33 (d, J )
6
6
1
d
6
7
.7 Hz, 2H), 4.20 (t, J ) 6.7 Hz, 1H), 2.03-1.97 (m, 2H), 1.63-
.48 (m, 4H), 0.80-0.76 (m, 12 H); 13C NMR (75 MHz, DMSO-
) δ 175.7, 153.4, 143.7, 140.7, 127.6, 127.0, 125.0, 120.1, 64.9,
1.5, 46.7, 43.6, 23.8, 23.7, 23.1. Anal. Calcd for C25 : C,
3.32; H, 7.63; N, 3.42. Found: C, 73.50; H, 7.76; N 3.52.
Solid -P h a se P ep tid e Syn th esis. The peptide H-(Lys) -Dibg-
Val-Dbzg-Phe-Dpg-NH was prepared using Fmoc methodology
with Fmoc-PAL-PEG-PS resin (0.17 mmol, 0.16 mmol/g loading)
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for 1b, 2a , 3a , 5a , 6a ; H and C NMR spectra for 1a ,
a , 3a , 4a , 5a , and 6a ; methods for anchoring Dpg and Dibg
onto the resin; MALDI mass spectrum of peptide K7-INH. This
material is available free of charge via the Internet at
http://pubs.acs.org.
1
13
6
2
H
31NO
4
7
2
J O034885J
J . Org. Chem, Vol. 68, No. 25, 2003 9857