Solid-phase synthesis of amine-bridged cyclic enkephalin analogues via on-resin cyclization utilizing the Fukuyama-Mitsunobu reaction

Article abstract of DOI:10.1021/jo020447l

An efficient solid-phase synthetic route is described for the preparation of 13-membered amine-bridged cyclic enkephalin analogues (ABEs) 1a and 1c-1j (Figure 1) resulting from a sulfonamide-containing peptide whose backbone is bound to a resin. The Fukuyama-Mitsunobu reaction of the 2-nitrobenzenesulfonyl-protected amine bound to the solid support with protected aminoethanol in the presence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD) is utilized to prepare a resin-bound sulfonamide-protected secondary amine. After peptide cyclization, this protected amine functionality becomes the "amine bridge" of the target molecule. In addition, the reagent DIAD was found to be a superior reagent compared to diethyl azodicarboxylate (DEAD) in the solid-phase Fukuyama-Mitsunobu reaction.

Full text of DOI:10.1021/jo020447l

Solid -P h a se Syn th esis of Am in e-Br id ged Cyclic En k ep h a lin
An a logu es via On -Resin Cycliza tion Utilizin g th e
F u k u ya m a -Mitsu n obu Rea ction
Yosup Rew and Murray Goodman*
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive,
La J olla, California 92093-0343
mgoodman@ucsd.edu
Received J uly 3, 2002
An efficient solid-phase synthetic route is described for the preparation of 13-membered amine-
bridged cyclic enkephalin analogues (ABEs) 1a and 1c-1j (Figure 1) resulting from a sulfonamide-
containing peptide whose backbone is bound to a resin. The Fukuyama-Mitsunobu reaction of the
2-nitrobenzenesulfonyl-protected amine bound to the solid support with protected aminoethanol
in the presence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD) is utilized to prepare
a resin-bound sulfonamide-protected secondary amine. After peptide cyclization, this protected amine
functionality becomes the “amine bridge” of the target molecule. In addition, the reagent DIAD
was found to be a superior reagent compared to diethyl azodicarboxylate (DEAD) in the solid-
phase Fukuyama-Mitsunobu reaction.
In tr od u ction
activity relationships. In this paper we present a novel
combinatorial synthetic route for the derivatization of
amine-bridged cyclic enkephalins (ABEs) 1c-1j (Figure
1) on a solid support utilizing the Fukuyama-Mitsunobu
reaction.
The focus of our opioid project has been on the
synthesis of cyclic enkephalin analogues bridged by
heteroatoms such as sulfur or nitrogen.1-5 Amine-bridged
enkephalin analogues represent a new approach in the
design of cyclic peptide opioids.5 It is an advantage that
the trivalent amine offers a handle by which to function-
alize the bridge while maintaining the required array of
the pharmacophores. In a preliminary effort, a cyclic
analogue having a methylamine bridge, Tyr-c[(NâCH3)-
D-A2pr-Gly-Phe-NHCH2CH2-] [MABE(I), 1a ],5 and Tyr-
c[(NγCH3)-D-A2bu-Gly-Phe-NHCH2CH2-] [MABE(II), 1b]1,2
were synthesized. They were found to be potent but
nonselective opioid agonists (Figure 1).
The potencies of 1a and 1b make them promising new
lead compounds in our research to prepare enkephalin
analogues with selectivity for a specific receptor. How-
ever, the synthetic routes for the synthesis of 1a and 1b
described in the previous papers1,2,5 were not appropriate
to construct libraries of amine-bridged enkephalin ana-
logues, which are needed for the study of structure-
Resu lt a n d Discu ssion
Syn th etic Str a tegy. We have chosen a backbone
amide linker initially developed by J ensen and co-
workers6 which is versatile and does not require the
presence of side chain functionality. It is the key strategy
in the synthetic route of ABEs on the solid support to
utilize the Fukuyama-Mitsunobu reaction7-9 to prepare
a resin-bound sulfonamide-protected secondary amine
which becomes the tertiary amine bridge in the target
analogues. The Fukuyama-Mitsunobu reaction provides
an efficient route for the synthesis of various secondary
amines protected by the 2-nitrobenzenesulfonyl (nosyl)
group which can be deprotected under mild conditions.
Therefore, the derivatization is designed to be carried out
by removal of the nosyl protecting group and alkylation
on the cyclic peptide attached on the resin. The target
peptidomimetic structures are obtained after cleavage of
the resulting cyclic molecules from the solid support
(Scheme 1).
* To whom correspondence should be addressed. Phone: (858) 534-
4466. Fax: (858) 534-0202.
(1) Goodman, M.; Zapf, C.; Rew, Y. Biopolymers 2001, 60, 229-245.
(2) Baker, T. J .; Rew, Y.; Goodman, M. Pure Appl. Chem. 2000, 72,
347-354.
(3) Goodman, M.; Rew, Y.; Malkmus, S.; Svensson, C.; Yaksh, T.
L.; Chung, N. N.; Schiller, P. W.; Daubert, J . D.; Cassel, J . A.; DeHaven,
R. Abstract of Papers, 221st ACS National Meeting, San Diego, April
1-5, 2001; American Chemical Society: Washington, DC, 2001; ORGN-
109.
(4) Rew, Y.; Malkmus, S.; Svensson, C.; Yaksh, T. L.; Chung, N. N.;
Schiller, P. W.; Cassel, J . A.; DeHaven, R. N.; Goodman, M. J . Med.
Chem. 2002, 45, 3746-3754.
(5) Shreder, K.; Zhang, L.; Dang, T.; Yaksh, T. L.; Umeno, H.;
DeHaven, R.; Daubert, J .; Goodman, M. J . Med. Chem. 1998, 41, 2631-
2635.
Syn th esis. The development of the synthetic route was
initiated by the preparation of the orthogonally protected
(6) J ensen, K. J .; Alsina, J .; Songster, M. F.; Vagner, J .; Albericio,
F.; Barany, G. J . Am. Chem. Soc. 1998, 120, 5441-5452.
(7) Fukuyama, T.; J ow, C.-K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6373-6374.
(8) Fukuyama, T.; Cheung, M.; J ow, C.-K.; Hidai, Y.; Kan, T.
Tetrahedron Lett. 1997, 38, 5831-5834.
(9) Han, Y.; Albericio, F.; Barany, G. J . Org. Chem. 1997, 62, 4307-
4312.
10.1021/jo020447l CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/20/2002
8820
J . Org. Chem. 2002, 67, 8820-8826

Synthesis of Amine-Bridged Enkephalin Analogues
F IGURE 1. Structures of 1a , 1b, and ABEs 1c-1j.
SCHEME 1. Retr osyn th etic An a lysis of On -Resin
Syn th esis of Am in e-Br id ged Cyclic En k ep h a lin
An a logu es
backbone with an allyl ester and a Boc group as the acid
and amine protecting groups. The first amino acid was
anchored to the aldehyde resin by on-resin reductive
amination of phenylalanine allyl ester. The next residue
was incorporated with the Trt protecting group protocol,
which circumvents diketopiperazine formation upon ad-
dition of the third residue, 4.6 Coupling of Trt-Gly-OH
to the secondary amine was mediated by 2-chloro-1-
methylpyridinium iodide (CMPI), 1-hydroxy-7-azaben-
zotriazole (HOAt), and diisopropylethylamine (DIEA) in
CH2Cl2/DMF (v/v ) 9/1). Selective removal of the Trt
group was accomplished by treatment with CH2Cl2/TFA/
triisopropylsilane (TIS) (v/v/v ) 96/2/2).
The third residue, 2, was found to be very susceptible
to racemization. The building block 2 was introduced
successfully by an in situ neutralization/coupling protocol
mediated by HBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate), HOBt (1-hydroxy-
benzotriazole monohydrate), and a mild base, 2,6-lutidine
in CH2Cl2/DMF (v/v ) 1/1). Under the same coupling
conditions with DIEA, the epimerization of 1a occurred
at an unacceptable level (>10%).11 Finally, after removal
of the NR-Fmoc protecting group, addition of Boc-Tyr-
(tBu)-OH was achieved using the same conditions men-
tioned above to yield a linear tetrapeptide backbone, 5.
Fmoc-D-A2pr(nosyl)-OH (2), where A2pr represents 2,3-
diaminopropionic acid (Scheme 2).
The subsequent alkylation to the sulfonamide 5 with
N-(allyloxycarbonyl)ethanolamine (6) was achieved using
the Fukuyama-Mitsunobu reaction (diisopropyl azodi-
carboxylate (DIAD), Ph3P, THF, 0 °C, 10 min, then room
temperature, 12 h). The reaction was monitored by
periodic resin cleavage, with product analysis by HPLC,
and was complete after 12 h. Interestingly, the solid-
phase Fukuyama-Mitsunobu reaction under the same
conditions using diethyl azodicarboxylate (DEAD) always
gave the ethylated product 10 in more than 40% yield in
addition to the desired product 9 (Scheme 4).
Attempts to protect the amine directly in Fmoc-D-A2pr-
OH with 2-nitrobenzenesulfonyl chloride (nosyl chloride)
under various conditions were unsuccessful because of
the presence of the free carboxylic acid. Thus, we tried
to mask the carboxylic acid with a temporary protecting
group. The intermediate Fmoc-D-A2pr-OH was first con-
verted into the silyl ester, which is soluble in CH2Cl2
using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MST-
FA) and triethylamine (TEA) in refluxing CH2Cl2 under
argon.10 The sulfonylation was then achieved at room
temperature without isolation of the silylated amino acid
by adding a slight excess of nosyl chloride and 1 equiv of
TEA. After the hydrolysis of the silyl ester of the
orthogonally protected amino acid with methanol, the
target amino acid building block 2 was readily obtained.
The synthesis of 1a on a solid support is summarized
in Scheme 3. We employed 2-(4-formyl-3-methoxy)phe-
noxyethyl polystyrene resin (3), which is a commercially
available aldehyde resin, to prepare a linear peptide
The ethylated product was formed presumably because
of the ease in the hydrolysis of DEAD. We did not observe
this side reaction when DIAD was used. Thus, the use
of DIAD is preferred to DEAD in the solid-phase Mit-
sunobu reaction, while it is reported that both DIAD and
DEAD work equally well as oxidants and can be used
interchangeably for reactions in solution.12 This result
indicates that even a simple reaction such as the Mit-
(11) Epimerization was confirmed by analytical HPLC after cleavage
of the target cyclic peptide 1a from the solid support.
(12) Wisniewski, K.; Koldziejczyk, A. S.; Falkiewicz, B. J . Pept. Sci.
1998, 4, 1-14.
(10) Raillard, S. P.; Mann, A. D.; Baer, T. A. Org. Prep. Proced. Int.
1998, 30, 183-186.
J . Org. Chem, Vol. 67, No. 25, 2002 8821

Rew and Goodman
SCHEME 2. P r ep a r a tion of 2a
a
Reagents and conditions: (a) (i) IBTFA (1.1 equiv), pyridine (3.0 equiv), CH2Cl2, rt, 24 h; (ii) 1 N aqueous HCl; (b) (i) TEA (1.0 equiv),
MSTFA (2.2 equiv), CH2Cl2, reflux for 1 h; (ii) nosyl chloride (1.1 equiv), TEA (1.0 equiv), rt, 4 h; (c) MeOH, rt, 1 h.
SCHEME 3. On -Resin Syn th esis of 1a a
a
Reagents and conditions: (a) (i) H-Phe-OAll‚p-tosylate (8.0 equiv), NaBH(OAc)3 (8.0 equiv), 1,2-dichloroethane, 18 h; (ii) Trt-Gly-OH
(5.0 equiv), CMPI (5.0 equiv), HOAt (5.0 equiv), DIEA (8.0 equiv), CH2Cl2/DMF (8/1), 12 h; (b) (i) CH2Cl2/TFA/TIS (96/2/2), 1 min, three
times; (ii) 2 (3.0 equiv), HBTU (3.0 equiv), HOBt (3.0 equiv), 2,6-lutidine (4.0 equiv), CH2Cl2/DMF (1/1), 8 h; (iii) 20% piperidine in NMP,
10 min, two times; (vi) Boc-Tyr(tBu)-OH (3.0 equiv), HBTU (3.0 equiv), HOBt (3.0 equiv), 2,6-lutidine (4.0 equiv), CH2Cl2/DMF (1/1), 8 h;
(c) (i) 6 (5.0 equiv), THF 12 h; (ii) PhSiH3(24 equiv), Pd(Ph3)4 (0.1 equiv), CH2Cl2, 10 min, two times; (iii) HBTU (3.0 equiv), HOBt (3.0
equiv), 2,6-lutidine (4.0 equiv), CH2Cl2/DMF (1/1), 8 h; (d) DBU (5.0 equiv), mercaptoethanol (10 equiv), DMF, 30 min; (e) (i) 38%
formaldehyde (40 equiv), THF/TMOF (1/1), 12 h; (ii) NaBH(OAc)3 (20 equiv), 1,2-dichloroethane, 12 h; (iii) TFA/TIS/H2O (92/5/3), 2 h.
sunobu reaction may result in different products and
should be carefully considered when applied to solid-
phase synthesis. This variability in solid-phase reactions
arises from the requirement of great excess for the
amounts of reagents necessary to “push” the reactions
forward to satisfactory yields.
On-resin derivatization commenced with selective re-
moval of the nosyl group with 2-mercaptoethanol and
DBU, a nonionic strong base, to produce 8.15 Attempts
to cleave the nosyl group with other well-known condi-
tions such as Fukuyama’s thiophenol/K2CO3 and 2-mer-
captoethanol/LiOH conditions failed.7 Methylation of the
on-resin cyclic peptide with a secondary amine bridge was
successfully achieved by a two-step reductive amina-
tion: (1) immonium ion formation using 38% aqueous
formaldehyde (40 equiv) in THF/trimethylorthoformate
(TMOF) (v/v ) 1/1) and (2) NaBH(OAc)3 (20 equiv) in 1,2-
dichloroethane. The target compound 1a was obtained
by the acidolytic cleavage of the resulting cyclic molecule
from the resin using TFA/TIS/H2O (v/v/v ) 92/5/3) with
94% purity as evidenced by analytical HPLC, and in 78%
yield based on the aldehyde resin (Scheme 3). The NMR
spectrum of the compound is identical with that reported
Deprotection of the allyl ester and Alloc groups was
easily carried out under mild conditions, using excess
PhSiH3 as an allyl acceptor and a catalytic amount of
Pd(PPh3)4 in CH2Cl2.13,14 The nosyl-protected cyclic pep-
tide attached to the solid support, 7, was prepared by
cyclization with HBTU/HOBt in the presence of 2,6-
lutidine in CH2Cl2/DMF (v/v ) 1/1). No detectable
epimerization product was found in the analytical HPLC
(Scheme 3).
(13) Thieriet, N.; Alsina, J .; Giralt, E.; Guibe, F.; Albericio, F.
Tetrahedron Lett. 1997, 38, 7275-7278.
(14) Dessolin, M.; Guillerez, M.-G.; Thieriet, N.; Guibe, F.; Loffet,
A. Tetrahedron Lett. 1995, 36, 5741-4.
(15) Miller, S. C.; Scanlan, T. S. J . Am. Chem. Soc. 1998, 120, 2690-
2691.
8822 J . Org. Chem., Vol. 67, No. 25, 2002

Synthesis of Amine-Bridged Enkephalin Analogues
SCHEME 4. F u k u ya m a -Mitsu n obu Rea ction of th e P ep tid e Atta ch ed on th e Resin , 5, w ith 6 in th e
P r esen ce of DEAD
SCHEME 5. On -Resin Der iva tiza tion of Am in e-Br id ged Cyclic En k ep h a lin s
earlier.5 On the basis of this chemistry, we have created
a general route for the solid-phase synthesis of diverse
amine-bridged opioid structures and accomplished the
derivatization of ABEs by reductive amination, alkyation,
or acylation (Scheme 5).
lations of the analogues are in progress and will be
reported elsewhere.
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Notes. The Kaiser test16 was
used for the qualitative test for the presence or absence of the
free amino group. The final products were purified and
analyzed by RP-HPLC using protein peptide C18 columns.
Column dimensions were 4.5 × 250 mm (90 Å silica, 5 µm) for
analytical and 22 × 250 mm (90 Å silica, 10 µm) for prepara-
tive HPLC, and UV absorbance was monitored at 220 nm. A
binary system of water and acetonitrile, both containing 0.1%
TFA, was used throughout. Purity analysis of the crude final
The quality of the synthesis was verified at several
stages, by cleaving portions of resins 4, 5, 7, and 8 with
TFA and analyzing the products by HPLC and MS
techniques. HPLC profiles of crude peptides 1a , 1c, and
1d are given in Figure 2.
Con clu sion s
products was carried out on a PDA system using a linear
We have devised efficient routes for the synthesis of
ABEs using an aldehyde resin and the Fukuyama-
Mitsunobu reaction and demonstrated that this synthetic
route is appropriate for the parallel synthesis of an array
of target structures for structure-activity relationship
studies. A variety of amine-bridged molecules were
prepared in high purity by these techniques. In addition,
we found that the use of DIAD is preferred to DEAD for
the solid-phase Fukuyama-Mitsunobu reactions.
a
gradient of 10-90% acetonitrile over 30 min (condition A, tR
)
at a 1 mL/min flow rate. Two analytical HPLC profiles of
purified products were obtained using a linear gradient of 10-
50% acetonitrile over 30 min (condition B, tRb) and one of the
following isocratic conditions: an isocratic elution of 16%
acetonitrile (condition C, tRc), an isocratic elution of 20%
acetonitrile (condition D, tRd), an isocratic elution of 23%
acetonitrile (condition E, tRe), an isocratic elution of 27%
f
acetonitrile (condition F, tR ) at a 1 mL/min flow rate. Prepara-
The in vitro and in vivo biological test results and the
conformational analysis using NMR and computer simu-
(16) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-8.
J . Org. Chem, Vol. 67, No. 25, 2002 8823

Rew and Goodman
F IGURE 2. HPLC profiles of crude peptides cleaved from resin-bound peptides: nosyl-ABE(I) (1c) cleaved from the peptide-
bound resin 7 (top), H-ABE(I) (1d ) cleaved from the peptide-bound resin 8 (middle), and 1a cleaved from the peptide-bound resin
8 after reductive amination (bottom) using formaldehyde with a gradient of 10-90% (0.1% TFA/CH3CN in 0.1% TFA/H2O) over
30 min at 1 mL/min.
tive HPLC was carried out at a 10 mL/min flow rate using
condition B, and the materials so obtained were further
purified by one of the isocratic conditions mentioned above at
a 10 mL/min flow rate.
F m oc-D-A2p r (n osyl)-OH (2). To a solution of Fmoc-D-Asn-
OH (11.3 g, 31.9 mmol) in DMF/water (2/1, v/v, 250 mL) was
added bis(trifluoroacetoxy)iodobenzene (IBTFA) (15.1 g, 35.1
mmol) at 0 °C. The reaction was then stirred for 10 min at 0
°C, and pyridine (7.75 mL, 95.8 mmol) was added. After being
stirred for 24 h at room temperature, the reaction mixture was
concentrated under reduced pressure. The concentrated reac-
tion mixture was dissolved in 1 N aqueous hydrochloric acid
solution (100 mL) and then extracted with ether to remove
the organic impurity (100 mL). The aqueous layer was lyoph-
ilized, and the resulting crude product was recrystallized from
ether/EtOH (3/1, v/v). The crystals were filtered and washed
with cold ether to give Fmoc-D-A2pr-OH‚HCl (10.3 g, 28.4
mmol, 89%) as a yellow solid: mp 145-149 °C (lit.17 mp 146
1
°C); [R]25 ) 19.5° (c ) 0.96, MeOH); H NMR (DMSO-d6) δ
D
8.13 (br s, 3H, NH3+), 7.89 (d, J ) 7.6 Hz, 2H), 7.81 (d, J ) 8.4
8824 J . Org. Chem., Vol. 67, No. 25, 2002

Synthesis of Amine-Bridged Enkephalin Analogues
Hz, 1H, NH), 7.73 (d, J ) 6.8 Hz, 2H), 7.42 (t, J ) 7.2 Hz,
2H), 7.33 (t, J ) 7.6 Hz, 2H), 4.40-4.20 (m, 4H), 3.20 (br, 1H),
3.00 (br, 1H); 13C NMR (DMSO-d6) δ 170.7, 156.1, 143.6, 140.6,
127.6, 127.1, 125.2, 120.1, 66.1, 51.9, 46.7, 34.3; MS (ESI) m/z
327 [M + H]+, 325 [M - H]-; HRMS (MALDI) m/z [M + H]+
calcd for C18H19N2O4 327.13448, found 327.13480; IR (KBr
pellet, cm-1) 3326, 3038, 2964, 1701, 1540, 1450, 1306, 1261,
1107, 761, 739.
P r ep a r a tion of Nosyl-P r otected Cyclic P ep tid e At-
ta ch ed to th e Resin , 7. To an argon-agitated suspension of
the peptide-bound resin 5 (2.06 mmol) and N-(allyloxycarbon-
yl)ethanolamine (6) (5 equiv) in THF (80 mL) were added
carefully DIAD (5.0 equiv) and Ph3P (5.0 equiv) at 0 °C, and
the reaction was agitated at room temperature for 12 h. After
successive washings with DMF, MeOH, and CH2Cl2 (three
times), a solution of PhSiH3 (24 equiv) and a solution of Pd-
(PPh3)4 (0.1 equiv) in CH2Cl2 (20 mL) were added to the resin
under Ar. The resin was shaken for 10 min, the peptide-resin
was washed with DMF, MeOH, and CH2Cl2 (three times), and
then the deprotection process was repeated once. The depro-
tected peptide-resin was suspended again in DMF/CH2Cl2
(1/1, v/v, 15 mL) and treated with HBTU (3.0 equiv), HOBt
(3.0 equiv), and 2,6-lutidine (4.0 equiv) for 12 h. After being
washed successively with DMF, MeOH, and CH2Cl2 (three
times), the resin was dried in vacuo to provide the peptide-
resin 7 (5.67 g, 1.95 mmol, theoretical yield 5.71 g, 95% from
peptide-bound resin 4). The loading level of the resin 7 was
0.34 mmol/g.
To a suspension of Fmoc-D-A2pr-OH‚HCl (8.00 g, 22.0 mmol)
in CH2Cl2 (100 mL) were added TEA (3.07 mL, 22.0 mmol)
and MSTFA (8.56 mL, 46.2 mmol) successively at 0 °C, and
the reaction was refluxed until a clear solution was obtained.
The clear reaction mixture was then cooled to room temper-
ature, and nosyl chloride (5.36 g, 24.2 mmol) was added
followed by TEA (3.07 mL, 22.0 mmol) and stirred for 4 h. After
the addition of MeOH (70 mL), the reaction mixture was
stirred for 1 h and then concentrated under reduced pressure.
The reaction was diluted with EtOAc (100 mL) and washed
with 10% aqueous citric acid solution (100 mL) and brine (100
mL). The organic layer was dried over MgSO4 and concen-
trated under reduced pressure. Flash chromatography (CH2-
Cl2/MeOH/AcOH ) 70/1/0.1 to 30/1/0.1, v/v/v) afforded 7.01 g
(13.8 mmol, 63%) of the target compound 2 as a light yellow
solid. A further analytical sample was recrytallized from ether/
EtOH: mp 137-141 °C; Rf ) 0.15 (CH2Cl2/MeOH/AcOH ) 20/
Syn th esis of Nosyl-ABE(I) (1c) fr om th e P ep tid e-
Resin 7. Target peptide was cleaved from the peptide-bound
resin 7 (0.50 g, 0.17 mmol) by treatment with TFA/TIS/H2O
(v/v/v ) 92/5/3, 12 mL) at room temperature for 2 h. The
filtrate from the cleavage reaction was collected and combined
with TFA washes (2 × 10 mL) of the cleaved peptide-resin.
Concentration of the combined filtrates under reduced pres-
sure, precipitation in IPE (10 mL), and centrifugation yielded
a crude peptide‚TFA salt as a yellow solid (94 mg, 0.12 mmol,
71% from the peptide-bound resin 7) with 88% purity from
1/0.1); [R]25 ) 17.1°(c ) 0.95, MeOH); 1H NMR (DMSO-d6) δ
D
8.16 (br, 1H, NH), 8.00 (m, 2H), 7.88 (d, J ) 7.6 Hz, 2H), 7.84
(m, 2H), 7.70 (d, J ) 7.6 Hz, 2H), 7.57 (d, J ) 8.8 Hz, 1H,
NH), 7.41 (t, J ) 7.6 Hz, 2H), 7.32 (t, J ) 7.6 Hz, 2H), 4.25
(m, 3H), 4.08 (dd, J ) 12.8 and 7.6 Hz, 1H), 3.26 (m, 2H); 13
C
a
NMR(DMSO-d6) δ 171.1, 155.7, 147.5, 140.6, 134.0, 132.7,
132.5, 129.4, 127.6, 127.0, 125.2, 124.5, 120.1, 65.9, 54.0, 46.7,
43.8; MS (ESI) m/z 512 [M + H]+, 534 [M + Na]+, 510 [M -
H]-; HRMS (MALDI) m/z [M + Na]+ calcd for C24H21N3O8NaS
534.0949, found 534.0942; IR (KBr pellet, cm-1) 3325, 3068,
2955, 2893, 1704, 1541, 1450, 1344, 1166, 1085, 760, 740, 587.
the analytical HPLC (tR ) 15.24 min) and in 67% overall yield
based on the aldehyde resin 3. Compound 1c was further
purified by RP-HPLC as described in the General Procedures
and Notes: MS (ESI) m/z 682 [M + H]+, 704 [M + Na]+, 680
[M - H]-, 716 [M + Cl]-; HRMS (MALDI) m/z [M + Na]+ calcd
for C31H35N7O9NaS 704.21092, found 704.20867; RP-HPLC
b
d
P r ep a r a tion of Tr t-Gly-(r esin )-P h e-OAll (4). To a stirred
suspension of 2-(4-formyl-3-methoxy)phenoxyethyl polystyrene
resin 3 (10.0 g, 0.51 mmol/g, 5.10 mmol) in 1,2-dichloroethane
(200 mL) with a mechanical stirrer were added H-Phe-OAll‚
TsOH (8.0 equiv) and NaBH(OAc)3 (8.0 equiv) successively at
room temperature. After being stirred for 9 h at room tem-
perature, the reaction was quenched with MeOH (40 mL)
carefully. The resin was then filtered, washed with MeOH,
CH2Cl2, 20% piperidine in DMF, MeOH, and CH2Cl2 (three
times), and dried in vacuo. The dried intermediate resin was
suspended in CH2Cl2/DMF (v/v, 8/1, 200 mL) and allowed to
react with Trt-Gly-OH (5.0 equiv), CMPI (5.0 equiv), HOAt
(5.0 equiv), and DIEA (8.0 equiv) for 12 h. After being washed
with DMF, MeOH, and CH2Cl2 (3 times), the resin product
was dried in vacuo to provide the dipeptide attached on the
solid support, 4 (12.4 g, 5.06 mmol, theoretical yield 12.5 g,
99%). The loading level of resin 5 was 0.41 mmol/g.
P r ep a r a tion of Boc-Tyr (tBu )-D-A2p r (n osyl)-Gly-(r esin )-
P h e-OAll (5). The peptide-bound resin 4 (5.02 g, 2.06 mmol)
was treated with CH2Cl2/TFA/TIS (v/v/v ) 96/2/2, 80 mL, 3 ×
1 min) and then washed again with DMF, MeOH, and CH2-
Cl2 (three times). The resin was suspended in DMF/CH2Cl2
(1/1, v/v, 80 mL), treated with Fmoc-D-A2pr(nosyl)-OH (2.5
equiv), HBTU (2.5 equiv), HOBt (2.5 equiv), and 2,6-lutidine
(3.5 equiv) for 8 h. After being washed with DMF, MeOH, and
CH2Cl2 (three times), the resin was treated with 20% piperi-
dine in NMP (10 mL, 2 × 10 min) and washed with CH2Cl2,
MeOH, CH2Cl2, and DMF (three times). The washed resin was
suspended again in DMF/CH2Cl2 (1/1, v/v, 80 mL) and treated
with Boc-Tyr(tBu)-OH (2.5 equiv), HBTU (2.5 equiv), HOBt
(1.0 equiv), and 2,6-lutidine (3.5 equiv) for 8 h. After being
washed successively with DMF, MeOH, and CH2Cl2 (three
times), the resin was dried in vacuo to provide 5.
tR ) 20.97 min, tR ) 22.21 min.
Syn th esis of H-ABE(I) (1d ) fr om th e P ep tid e-Resin
7. The peptide-bound resin 7 (0.50 g, 0.17 mmol) was sus-
pended in DMF (6 mL) and treated with DBU (5 equiv) and
mercaptoethanol (10 equiv) for 30 min at room temperature.
A bright yellow solution was indicative of nosyl cleavage. After
the deprotected peptide-bound resin 8 was extensively washed
with DMF, MeOH, and CH2Cl2 (three times), the target
peptide was cleaved from the resin and isolated using the
described procedure for the synthesis of compound 1c. A crude
peptide‚2TFA salt was obtained as a yellow solid (82 mg, 0.11
mmol, 65% from the peptide-bound resin 7) with 89% purity
a
from the analytical HPLC (tR ) 11.31 min). Compound 1d
was further purified by RP-HPLC as described in the General
Procedures and Notes: MS (ESI) m/z 497 [M + H]+, 519 [M +
Na]+, 495 [M - H]-, 531 [M + Cl]-; HRMS (MALDI) m/z
[M + H]+ calcd for C25H33N6O5 497.25070, found 497.24916;
b
c
RP-HPLC tR ) 13.29 min, tR ) 7.62 min.
Syn th esis of MABE(I) (1a ) fr om th e P ep tid e-Resin 7.
The nosyl protecting group of the peptide-bound resin 7 (0.50
g, 0.17 mmol) was removed using the described procedure for
the synthesis of compound 1d . The deprotected peptide-bound
resin 8 was treated with 37% formaldehyde (40 equiv) in THF/
TMOF (v/v, 1/1, 10 mL) at room temperature for 12 h and
washed again with 1,2-dichloroethane. The resin was then
suspended in 1,2-dichloroethane and treated with NaBH(OAc)3
(20 equiv) at room temperature for 12 h. After the peptide-
bound resin 8 was washed with DMF, MeOH, and CH2Cl2
(three times), the target peptide was cleaved from the resin
and isolated using the described procedure for the synthesis
of compound 1c. A crude peptide‚2TFA salt was obtained as a
yellow solid (72 mg, 0.098 mmol, 57% from the peptide-bound
a
resin 7) with 94% purity from the analytical HPLC (tR ) 11.35
min). Compound 1a was further purified by RP-HPLC as
described in the General Procedures and Notes: MS (ESI) m/z
511 [M + H]+, 533 [M + Na]+, 509 [M - H]-, 545 [M + Cl]-;
(17) Zhang, L.-h.; Kauffman, G. S.; Pesti, J . A.; Yin, J . J . Org. Chem.
1997, 62, 6918-6920.
J . Org. Chem, Vol. 67, No. 25, 2002 8825

Rew and Goodman
HRMS (MALDI) m/z [M + H]+ calcd for C26H35N6O5 511.26635,
washed again with DMF, MeOH, and CH2Cl2 (three times).
The target peptide was cleaved from the resin and isolated
using the described procedure for the synthesis of compound
1c. A crude peptide‚TFA salt was obtained as a yellow solid
(65 mg, 0.10 mmol, 59% from the peptide-bound resin 7) with
b
c
found 511.26507; RP-HPLC tR ) 13.47 min, tR ) 7.85 min.
Syn th esis of Allyl-ABE(I) (1e) fr om th e P ep tid e-Resin
7. The nosyl protecting group of the peptide-bound resin 7 (0.50
g, 0.17 mmol) was removed using the described procedure for
the synthesis of compound 1d . The deprotected peptide-bound
resin 8 was treated with allyl bromide (20 equiv) and NaHCO3
(40 equiv) in DMF (10 mL) at room temperature for 24 h and
washed again with DMF, H2O, and benzene (three times). The
target peptide was cleaved from the resin and isolated using
the described procedure for the synthesis of compound 1e. A
crude peptide‚2TFA salt was obtained as a yellow solid (80
mg, 0.10 mmol, 59% from the peptide-bound resin 7) in 96%
a
72% purity from the analytical HPLC (tR ) 11.61 min).
Compound 1h was further purified by RP-HPLC as described
in the General Procedures and Notes: MS (ESI) m/z 539
[M + H]+, 561 [M + Na]+, 537 [M - H]-, 573 [M + Cl]-; HRMS
(MALDI) m/z [M + H]+ calcd for C27H35N6O6 539.26126, found
b
c
539.26228; RP-HPLC tR ) 15.01 min, tR ) 10.07 min.
Syn th esis of Ben zoyl-ABE(I) (1i) fr om th e P ep tid e-
Resin 7. The nosyl protecting group of the peptide-bound resin
7 (0.50 g, 0.17 mmol) was removed using the described
procedure for the synthesis of compound 1d . The deprotected
peptide-bound resin 8 was treated with benzoyl chloride (5
equiv) and DIEA (5 equiv) in CH2Cl2 (10 mL) for 24 h and
washed again with DMF, MeOH, and CH2Cl2 (three times).
The target peptide was cleaved from the resin and isolated
using the described procedure for the synthesis of compound
1c. A crude peptide‚TFA salt was obtained as a yellow solid
(75 mg, 0.11 mmol, 65% from the peptide-bound resin 7) with
a
purity as determined by analytical HPLC (tR ) 12.42 min).
Compound 1e was further purified by RP-HPLC as described
in the General Procedures and Notes: MS (ESI) m/z 537
[M + H]+, 559 [M + Na]+, 535 [M - H]-, 571 [M + Cl]-; HRMS
(MALDI) m/z [M + H]+ calcd for C28H37N6O5 537.28200, found
b
c
537.28494; RP-HPLC tR ) 15.72 min, tR ) 12.18 min.
Syn th esis of Ben zyl-ABE(I) (1f) fr om th e P ep tid e-
Resin 7. The nosyl protecting group of the peptide-bound resin
7 (0.50 g, 0.17 mmol) was removed using the described
procedure for the synthesis of compound 1d . The deprotected
peptide-bound resin 8 was treated with benzyl bromide (20
equiv) and NaHCO3 (40 equiv) in DMF (10 mL) at room
temperature for 24 h and washed again with DMF, H2O, and
benzene (three times). The target peptide was cleaved from
the resin and isolated using the described procedure for the
synthesis of compound 1c. A crude peptide‚2TFA salt was
obtained as a yellow solid (95 mg, 0.12 mmol, 59% from the
peptide-bound resin 7) with 91% purity from the analytical
a
81% purity from the analytical HPLC (tR ) 14.31 min).
Compound 1i was further purified by RP-HPLC as described
in the General Procedures and Notes: MS (ESI) m/z 601
[M + H]+, 623 [M + Na]+, 599 [M - H]-; HRMS (MALDI) m/z
[M + H]+ calcd for C32H37N6O6 601.27691, found 601.27624;
b
d
RP-HPLC tR ) 20.01 min, tR ) 17.60 min.
Syn th esis of 1-Na p h th ylm eth yl-ABE(I) (1j) fr om th e
P ep tid e-Resin 7. The nosyl protecting group of the peptide-
bound resin 7 (0.50 g, 0.17 mmol) was removed using the
described procedure for the synthesis of compound 1d . The
deprotected peptide-bound resin 8 was treated with 1-naph-
thylmethyl bromide (20 equiv) and NaHCO3 (40 equiv) in DMF
(10 mL) at 80 °C for 24 h and washed again with DMF, H2O,
and benzene (three times). The target peptide was cleaved from
the resin and isolated using the described procedure for the
synthesis of compound 1c. A crude peptide‚2TFA salt was
obtained as a yellow solid (100 mg, 0.12 mmol, 71% from the
peptide-bound resin 7) with 92% purity from the analytical
a
HPLC (tR ) 15.91 min). Compound 1f was further purified
by RP-HPLC as described in the General Procedures and
Notes: MS (ESI) m/z 587 [M + H]+, 609 [M + Na]+, 585 [M -
H]-, 621 [M + Cl]-; HRMS (MALDI) m/z [M + H]+ calcd for
C
32H39N6O5 587.29764, found 587.29928; RP-HPLC tRb ) 22.00
e
min, tR ) 14.33 min.
Syn th esis of Cyclop r op ylm eth yl-ABE(I) (1g) fr om th e
P ep tid e-Resin 7. The nosyl protecting group of the peptide-
bound resin 7 (0.50 g, 0.17 mmol) was removed using the
described procedure for the synthesis of compound 1d . The
deprotected peptide-bound resin 8 was treated with cyclopro-
pylmethyl bromide (20 equiv) and NaHCO3 (40 equiv) in DMF
(10 mL) at 80 °C for 24 h and washed again with DMF, H2O,
and benzene (three times). The target peptide was cleaved from
the resin and isolated using the described procedure for the
synthesis of compound 1c. A crude peptide‚2TFA salt was
obtained as a yellow solid (90 mg, 0.12 mmol, 59% from the
peptide-bound resin 7) with 85% purity from the analytical
a
HPLC (tR ) 18.10 min). Compound 1j was further purified
by RP-HPLC as described in the General Procedures and
Notes: MS (ESI) m/z 637 [M + H]+, 659 [M + Na]+, 635 [M -
H]-, 671 [M + Cl]-; HRMS (MALDI) m/z [M + H]+ calcd for
C
36H41N6O5 637.31330, found 637.31250; RP-HPLC tRb ) 25.81
f
min, tR ) 20.28 min.
Ack n ow led gm en t. This work was supported by
NIH Grant R01-DA 05539 (M.G.). We also thank Dr.
Chiara Perazzolo and Dr. Antonia DeCapua for the
interpretation of NMR spectra and Sandra Blaj Moore
for her helpful assistance in preparation of this manu-
script.
a
HPLC (tR ) 14.00 min). Compound 1g was further purified
by RP-HPLC as described in the General Procedures and
Notes: MS (ESI) m/z 551 [M + H]+, 573 [M + Na]+, 549 [M -
H]-; HRMS (MALDI) m/z [M + H]+ calcd for C29H39N6O5
b
d
551.29764, found 551.29825; RP-HPLC tR ) 17.49 min, tR
)
9.12 min.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra
and their peak assignments, HR mass spectra, and analytical
HPLC profiles of both crude and purified final products. This
material is available free of charge via Internet at
http://pubs.acs.org.
Syn th esis of Acetyl-ABE(I) (1h ) fr om th e P ep tid e-
Resin 7. The nosyl protecting group of the peptide-bound resin
7 (0.50 g, 0.17 mmol) was removed using the described
procedure for the synthesis of compound 1d . The deprotected
peptide-bound resin 8 was treated with acetyl chloride (5
equiv) and DIEA (5 equiv) in CH2Cl2 (10 mL) for 24 h and
J O020447L
8826 J . Org. Chem., Vol. 67, No. 25, 2002