A novel resin linker for solid-phase peptide synthesis which can be cleaved using two sequential mild reactions

Article abstract of DOI:10.1021/jo990770x

The interest in developing new linkers for solid-phase peptide and organic synthesis has increased tremendously as a result of the rapid development of combinatorial chemistry. Herein, we report the development of a new redox-sensitive linker for solid-phase peptide synthesis. This linker can be readily cleaved under mild conditions by using two sequential mild reactions, a reduction followed by a base (Bu₄N⁺F^-)-catalyzed cyclic ether formation. By using this new linker, two short peptides, a tetrapeptide [Boc- Trp-Ala-Gly-Gly-OH] and a pentapeptide [Boc-Asn-Ala-Ser(OBn)-Gly- Glu(OBn)OH)], were synthesized. Because the cleavage does not use acidic conditions, this resin linker provides an alternative when acidic conditions are not desirable. Furthermore, the cleavage conditions do not affect most of the side chain protecting group. Therefore, the peptides synthesized can be used for the segment synthesis of larger peptides without the need to reprotect the side chain functional groups.

Full text of DOI:10.1021/jo990770x

J . Org. Chem. 1999, 64, 7459-7466
7459
A Novel Resin Lin k er for Solid -P h a se P ep tid e Syn th esis Wh ich
Ca n Be Clea ved Usin g Tw o Sequ en tia l Mild Rea ction s
Ailian Zheng, Daxian Shan, Xuling Shi, and Binghe Wang*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
Received May 10, 1999
The interest in developing new linkers for solid-phase peptide and organic synthesis has increased
tremendously as a result of the rapid development of combinatorial chemistry. Herein, we report
the development of a new redox-sensitive linker for solid-phase peptide synthesis. This linker can
be readily cleaved under mild conditions by using two sequential mild reactions, a reduction followed
by a base (Bu4N+F-)-catalyzed cyclic ether formation. By using this new linker, two short peptides,
a tetrapeptide [Boc-Trp-Ala-Gly-Gly-OH] and a pentapeptide [Boc-Asn-Ala-Ser(OBn)-Gly-Glu(OBn)-
OH)], were synthesized. Because the cleavage does not use acidic conditions, this resin linker
provides an alternative when acidic conditions are not desirable. Furthermore, the cleavage
conditions do not affect most of the side chain protecting group. Therefore, the peptides synthesized
can be used for the segment synthesis of larger peptides without the need to reprotect the side
chain functional groups.
In tr od u ction
mild reaction conditions when needed. We have recently
reported the design and synthesis of a new redox-
sensitive resin linker for the synthesis of C-terminal
modified peptides.27 Herein, we report a new linker which
can be used for the synthesis of unmodified peptides and
can be cleaved under mild conditions.
Since its invention by Merrifield, solid-phase synthesis
has made an enormous impact not only in the peptide
chemistry field1-3 but also in the general field of organic
synthesis.4-8 With the rapid development of combinato-
rial chemistry, solid-phase synthesis is playing an even
more important role in the synthesis and search for
organic molecules of biological importance.5-7 The recent
rapid development of solid-phase synthesis also resulted
in an increased demand for new resin linkers that can
be cleaved under different conditions.7,9-26 These new
linkers should be stable and yet readily cleavable under
The design takes advantage of a “trimethyl lock”-
facilitated cyclic ether formation (Scheme 1),28-31 which
is different from the design of the linker reported earlier
from our group27 in that the earlier linker utilized a
facilitated lactone formation reaction. Consequently, the
linker reported in this study allows for the preparation
of peptides with free C-termini. In contrast, the linker
reported earlier27 only allows for the preparation of
C-terminally modified peptides. In the current design,
the first amino acid of the peptide is attached to the
linker through ester formation. Peptide synthesis can
then proceed from the C-terminus to the N-terminus. At
the end of the synthesis, the peptide can be cleaved from
the resin through a two-step process. First, the reduction
* To whom correspondence should be addressed. Phone: (919) 515-
2948. Fax: (919) 515-3757. E-mail: binghe•wang@ncsu.edu.
(1) Stewart, J . M.; Young, J . D. Solid-Phase Peptide Synthesis; Pierce
Chemical Co.: Rockford, IL, 1984.
(2) Atherton, E.; Sheppard, R. C. Solid-Phase Peptide Synthesis. A
Practical Approach; IRL Press: New York, 1989.
(3) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis;
Springer-Velag: New York, 1984.
(4) Bunin, B. A.; Ellman, J . A. J . Am. Chem. Soc. 1992, 114, 10997-
10998.
(5) Czarnik, A. W.; DeWitt, S. H. A Practical Guide to Combinatorial
Chemistry; American Chemical Society: Washington, DC, 1997.
(6) Terrett, N. K. Combinatorial Chemistry; Oxford University
Press: New York, 1998.
(7) Bunin, B. A. The Combinatorial Index; Academic press: San
Diego, 1998.
(19) J ung, K. W.; Zhao, X.; J anda, K. D. Tetrahedron Lett. 1996,
37, 6491.
(20) Sauerbrei, B.; J ungmann, V.; Waldmann, H. Angew. Chem., Int.
Ed. Engl. 1998, 37, 1143-1146.
(21) Plunkett, M. J .; Ellman, J . A. J . Org. Chem. 1995, 60, 6006-
(8) Zuckermann, R. N.; Kerr, J . M.; Kent, S. B. H.; Moos, W. H. J .
Am. Chem. Soc. 1992, 114, 10646-10647.
6007.
(22) Chenera, B.; Finkelstein, J . A.; Veber, D. F. J . Am. Chem. Soc.
1995, 117, 11999-12000.
(23) Deegan, T. L.; Gooding, O. W.; Baudart, S.; Porco, J . A., J r.
Tetrahedron Lett. 1997, 38, 4973-4976.
(24) Dressman, B. A.; Spangle, L. A.; Kaldor, S. W. Tetrahedron Lett.
1996, 37, 937-940.
(25) Gravert, D. J .; Datta, A.; Wentworth, J ., P.; J anda, K. D. J .
Am. Chem. Soc. 1998, 120, 9481-9495.
(26) Frechet, J . M.; Schuerch, C. J . Am. Chem. Soc. 1971, 93, 492-
496.
(9) Routledge, A.; Abell, C.; Balasubramanian, S. Tetrahedron Lett.
1997, 38, 1227-1230.
(10) Ngu, K.; Patel, D. V. Tetrahedron Lett. 1997, 38, 973-976.
(11) Zhao, X.; J ung, K. W.; J anda, K. D. Tetrahedron Lett. 1997,
38, 977-980.
(12) Backes, B. J .; Virgillio, A. A.; Ellman, J . A. J . Am. Chem. Soc.
1996, 118, 3055-3056.
(13) Venkatesan, H.; Greenberg, M. M. J . Org. Chem. 1996, 61, 525-
529.
(14) Richter, L. S.; Desai, M. C. Tetrahedron Lett. 1997, 38, 321-
322.
(27) Zheng, A.; Shan, D.; Wang, B. J . Org. Chem. 1999, 64, 156-
161.
(15) Bannwarth, W.; Huebscher, J .; Barner, R. Bioorg. Med. Chem.
Lett. 1996, 6, 1525-1528.
(28) Borchardt, R. T.; Cohen, L. A. J . Am. Chem. Soc. 1972, 94,
9166-9174.
(16) Backes, B. J .; Ellman, J . A. J . Am. Chem. Soc. 1994, 116,
11171-11172.
(29) Borchardt, R. T.; Cohen, L. A. J . Am. Chem. Soc. 1973, 95,
8308-8313.
(17) Fukase, K.; Egusa, K.; Nakai, Y.; Kusumoto, S. Mol. Divers.
1997, 2, 182-188.
(30) Liu, S.; Wang, B.; Nicolaou, M. G.; Borchardt, R. T. J . Chem.
Crystal. 1996, 26, 209-214.
(18) Seitz, O.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 803-
805.
(31) Wang, B.; Nicolaou, M. G.; Liu, S.; Borchardt, R. T. Bioorg.
Chem. 1996, 24, 39-49.
10.1021/jo990770x CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/04/1999

7460 J . Org. Chem., Vol. 64, No. 20, 1999
Zheng et al.
Sch em e 2. Syn th esis of Ald eh yd es 7 a n d 9
Sch em e 1. Red ox-Sen sitive Resin Lin k er for
Solid -P h a se P ep tid e Syn th esis
of the quinone moiety to the dihydroquinone 2 can be
accomplished using a mild reducing agent. Then, treat-
ment with tetrabutylammonium fluoride (TBAF) ac-
complishes the cyclization with the concomitant release
of the peptide synthesized (Scheme 1).
Sch em e 3. Mod el Wittig Rea ction in Solu tion
P h a se
Resu lts a n d Discu ssion
The demonstration of the feasibility of the concept
requires (1) the synthesis of the quinone-linker moiety
with a pendent chain, (2) attachment of this linker moiety
to a resin material, and (3) peptide synthesis using the
resin linker prepared. We chose to use a polystyrene-
based resin as the solid-phase material because it is most
commonly used in the solid-phase synthesis of peptides
and organic compounds. In designing the approach used
for the attachment of the linker to the resin material,
we considered several options, including ester, amide,
and carbon-carbon bond formations. We chose a carbon-
carbon bond formation method because of the expected
stability of such an attachment, which will allow for the
necessary chemical transformations after the initial
attachment. We therefore chose to use a Wittig reaction
for the attachment because similar reactions have long
been used in solid-phase chemistry.32
P r ep a r a tion of Ald eh yd es 7 a n d 9. The design of
the synthesis requires the preparation of the linker
moiety with a pendent aldehyde functional group for the
subsequent Wittig reaction. Therefore, the synthesis
started with compound 5, which has been prepared in a
previous study.27 The phenol hydroxyl group of 5 was first
protected as a benzyl ether by using benzyl chloride in
the presence of potassium carbonate and a catalytic
amount sodium iodide in 90% yield (Scheme 2). There
are two ways that the protected lactone 6 could be
converted to an aldehyde. Ozonolysis of the side-chain
double bond of 6 gave aldehyde 7 in 61% yield. Alterna-
tively, hydroboration-oxidation of 6 followed by PCC
(pyridinium chlorochromate) oxidation in DCM (dichloro-
methane) gave aldehyde 9 in 75% overall yield for two
steps. Because the synthesis of the two aldehydes gave
comparable yields, we chose to use the aldehyde (9) with
a longer side chain for the attachment to a resin material
to minimize the potential interference of the solid-phase
material during the subsequent cleavage reaction. The
preparation of 9 involves a total of 6 steps from a
commercially available starting material, 2,5-dimethyl-
benzoquinone, with an overall yield of 38%.
Atta ch m en t of th e Lin k er Moiety to Resin Bea d s.
Before the attachment of aldehyde 9 to resin beads using
a Wittig reaction, we examined the corresponding solu-
tion-phase model reactions to understand the necessary
conditions for the solid-phase reactions. Therefore, Wittig
reagent 11 was prepared by following well-established
literature procedures starting from benzyl chloride
(Scheme 3).33 Subsequent Wittig reaction with 9 using
n-BuLi as the base gave the desired product 12 in 85%
yield. The double bond was reduced through hydrogena-
tion using Wilkinson’s catalyst [tris(triphenylphosphine)-
rhodium chloride] in quantitative yield. Wilkinson’s
catalyst was used because it is soluble in organic solvent.
An insoluble catalyst, such as Pd/C, will most likely not
work in the subsequent solid-phase synthesis because of
the heterogeneous nature of the reaction and the involve-
ment of two solid-phase materials, the resin beads and
Pd/C.
With the successful model reaction, we started the
procedure to attach the linker to polystyrene resin beads.
First, triphenylphosphine chloride in anhydrous benzene
was refluxed with a benzyl chloride resin 14 for 36 h to
give the resin-Wittig reagent 15 (Scheme 4). Subsequent
Wittig reactions with aldehyde 9 yielded 16 in about 60%
yield (Scheme 4). IR was used for monitoring the reac-
(32) Frechet, J . M.; Schuerch, C. J . Am. Chem. Soc. 1971, 93, 492-
496.
(33) Friedrich, K.; Henning, H.-G. Chem. Ber. 1959, 92, 2756-2760.

Novel Resin Linker for Solid-Phase Peptide Synthesis
J . Org. Chem., Vol. 64, No. 20, 1999 7461
Sch em e 4. Atta ch em en t of th e Resin Lin k er
Syn th esized to Resin Bea d s Usin g a Wittig
Rea ction
tions (Figure 1). The appearance of a peak at 1770 cm-1
(Figure 1b) after the Wittig reaction, corresponding to
the lactone absorption of 16, is indicative of a successful
coupling. The coupling yield was calculated on the basis
of the amount of triphenylphosphine oxide recovered from
the reaction mixture. The double bond was then hydro-
genated in the presence of Wilkinson’s catalyst by using
conditions similar to that of the solution-phase model
reaction. However, it needs to be noted that the IR
spectra before and after the hydrogenation did not show
major differences that would allow us to unequivocally
characterize the resin as the one with the double bond
saturated. The lactone was then reduced with lithium
aluminum hydride (LAH) to give the diol resin 18
(Scheme 4). The strong IR absorption at 1770 cm-1
disappeared (Figure 1c) after the reduction, indicating
the complete conversion of the lactone 17 to the diol 18.
P ep tid e Syn th esis Usin g th e Resin Lin k er P r e-
p a r ed . To study the feasibility of using this linker for
peptide synthesis, we prepared two short peptides, a
tetrapeptide [Boc-Trp-Ala-Gly-Gly-OH] and a pentapep-
tide [Boc-Asn-Ala-Ser(OBn)-Gly-Glu(OBn)-OH)].
The solid-phase peptide synthesis followed well-
established procedures using Boc-protected amino acids
and either DCC (dicyclohexylcarbodiimide) or DIC (di-
isopropylcarbodiimide) as the activating reagent in the
presence of HOBt (1-hydroxybenzotriazole) and a cata-
lytic amount of DMAP (4-N,N-(dimethylamino)pyridine)
(Scheme 5).1 After the coupling of the first amino acid,
the dihydroquinone moiety of 19 was oxidized to a
quinone moiety by using NBS (N-bromosuccinimide) to
mask the phenol hydroxyl group.27,34 It should be noted
that methionine and cysteine are not compatible with the
NBS oxidation to give 20. In a separate solution-phase
study, we noticed that NBS oxidation with sulfur-
containing amino acids gave a complex mixture, presum-
ably through oxidation of the sulfur atom. These solid-
phase reactions were also followed by IR (Figure 1). The
IR after coupling of the first amino acid (Figure 1d)
F igu r e 1. The IR spectra of resins 15 (a), 16 (b), 18 (c), 19
(d), 20 (e), 21 (f), 22 (g), 23 (h), and 24 (i).
the progress of the reactions as designed. Unreacted free
hydroxyl groups were capped by reacting with acetic
anhydride in the presence of triethylamine (TEA) in
DCM. Cleavage of the Boc protecting group was achieved
by treatment with 25% TFA (trifluoroacetic acid) in DCM.
The ninhydrin test1 was used for the determination of
the degree of substitution of the resin, usually 0.3-0.5
mmol/g after coupling of the first amino acid. Therefore,
the overall yields from 14 and 16 were 37-63% and 73-
100%, respectively. The remaining amino acids were
showed new peaks at 1748 (ester) and 1719 (Boc) cm-1
,
indicating the attachment of the first amino acid. Sub-
sequent NBS oxidation resulted in a product 20 with new
a peak 1646 (quinone CdO) cm-1 (Figure 1e), indicating
(34) Wang, B.; Liu, S.; Borchardt, R. T. J . Org. Chem. 1995, 60, 539-
543.

7462 J . Org. Chem., Vol. 64, No. 20, 1999
Sch em e 5. Solid -P h a se P ep tid e Syn th esis Usin g th e Resin Lin k er P r ep a r ed
Zheng et al.
Sch em e 6. P r ep a r a tion of th e Sta n d a r d
assembled by using standard solid-phase synthesis pro-
cedure as described in the Experimental Section. Figure
1f shows the IR of 21, which lacks the peak corresponding
to the Boc group (1719 cm-1) indicating the successful
Boc deprotection. Figure 1g shows the IR of 22 with
multiple peaks around 1654 cm-1 indicating the presence
of amide bonds.
Tetr a p ep tid e
As designed (Schemes 1 and 5), the cleavage of the
peptide synthesized requires first the reduction of the
quinone moiety to dihydroquinone followed by base-
catalyzed cyclization. It is known that the reduction of
such a quinone moiety could be accomplished using
aqueous Na2S2O4.27,34 However, in this study it was
difficult to achieve complete reduction with Na2S2O4 to
give a satisfactory yield for the subsequent cleavage.
Therefore, the reduction was carried out using NaBH4.
For the based-catalyzed cyclization reaction, we studied
several methods including tetramethylguanidine (TMG)
in DMF,35 CsF, and tetrabutylammonium fluoride (TBAF).
The TBAF-catalyzed reactions gave the best results.
Therefore, after the reduction, the resin was treated with
1 M anhydrous TBAF in THF to achieve the desired
cleavage. The IR spectrum (Figure 1i) of the resin 24 after
the cleavage clearly indicated the disappearance of the
peaks corresponding to the peptide bonds. The crude
peptide was separated by using a mixed bed of Amberlyst
15 calcium sulfate resin and Amberlyst 15 sulfonic acid
resin36 followed by chromatography or recrystallization
to give the purified product in about 35% yield. It should
be noted that after the cleavage, most of the protecting
groups are left intact. Among the side chain protecting
groups encountered in this study, the formyl protecting
group of tryptophan was not stable under the cleavage
conditions. The side chain benzyl ester (glutamic acid)
and benzyl ether (serine) and the terminal N-Boc protect-
ing groups were unaffected during the cleavage when
anhydrous TBAF was used. This makes it possible for
these peptides to be used for the segment synthesis of
larger peptides.
Sch em e 7. P r ep a r a tion of th e Sta n d a r d
P en ta p ep tid e
For comparison purposes, both the standard tetrapep-
tide and the standard pentapeptide were synthesized by
using standard solution-phase peptide synthesis methods
with Boc-protected amino acids as described in Schemes
6 and 7.1 EDC [1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide] was used as the activating reagent in the
solution-phase approach because the urea side product
was water soluble and, therefore, the subsequent puri-
fication was easier. The synthesis of the protected tet-
rapeptide (Boc-Trp-Ala-Gly-Gly-OH) started with H-Gly-
OBn. The C-terminal benzyl protection was cleaved at
the end by catalytic hydrogenation (Scheme 6). For the
synthesis of the standard protected pentapeptide (Boc-
Asn-Ala-Ser(OBn)-Gly-Glu(OBn)-OH), the C-terminal pro-
tection cannot be a benzyl group because of the presence
of side chain benzyl protection of the desired peptide.
Therefore, a C-terminal allyl protection was used, which
was cleaved at the end by using tetrakis(triphenylphos-
phin)palladium(0) in the presence of morpholine (Scheme
7). The coupling of the last amino acid in the synthesis
of the pentapeptide gave a poor yield when EDC was used
as the activating reagent. Therefore, PyBOP (benzotria-
(35) Whitney, D. B.; Tam, J . P.; Merrifield, R. B. Tetrahedron 1984,
40, 4237-4244.
(36) Parlow, J . P.; Vazquez, M. L.; Flynn, D. L. Bioorg. Med. Chem.
Lett. 1998, 8, 2391-2394.

Novel Resin Linker for Solid-Phase Peptide Synthesis
J . Org. Chem., Vol. 64, No. 20, 1999 7463
easier to reduce quinone resin 22 to the dihydroquinone
linker 23 with aqueous Na2S2O4, which is a much milder
reducing reagent and is not expected to cause any
problem for most of the functional groups commonly
encountered in peptides and organic compounds.27,34
Exp er im en ta l Section
Gen er a l. Mass spectra were obtained at the Mass Spec-
trometry Laboratory for Biotechnology at North Carolina State
University. HPLC studies were carried out by using a dual
pump system with a UV-vis detector (detection wavelength:
220, 245 nm; solvent: acetonitrile and water with 0.1% TFA).
For the tetrapeptide (Boc-Trp-Ala-Gly-Gly-OH) the mobile-
phase gradient was 70-50% H2O/CH3CN. For the pentapep-
tide (Boc-Asn-Ala-Ser(OBn)-Gly-Glu(OBn)-OH) the mobile-
phase gradient was 55-50% H2O/CH3CN. The column was a
C18 reversed-phase analytical column from YMC (4.6 mm ×
250 mm, ODS-A, S-5 micron 120A). Unless stated otherwise,
commercial reagents were from Aldrich and used without
further purification. Merrifield resin (1.0 mequiv Cl/g, 1% cross
linked) and protected amino acids were from Bachem Bio-
science. Acetonitrile and THF were of HPLC grade from Fisher
Scientific Inc. DCM was distilled from CaH2; THF, benzene,
and ethanol were distilled from sodium under N2. TBAF was
dried with P2O5 before the preparation of the solution. TFA
was of peptide synthesis grade.
7-Ally l-6-b e n zy lo x y -4,4,5,8-t e t r a m e t h y lh y d r o c o u -
m a r in 6. A mixture of compound 5 (635 mg, 2.44 mmol),
benzyl chloride (618 mg, 4.88 mmol), K2CO3 (674 mg, 4.88
mmol), and sodium iodide (36 mg, 0.244 mmol) in 8 mL of
acetone was refluxed for 20 h under N2 with stirring. To the
reaction mixture was added 20 mL of water, and then acetone
was removed under reduced pressure. The aqueous solution
was extracted with EtOAc and dried over MgSO4. Filtration,
evaporation, and purification by a silica gel column afforded
a white solid (772 mg, 90%). 1H NMR (CDCl3): δ 7.46-7.30
(5H, m), 5.96-5.93 (1H, m), 5.06-4.90 (2H, m), 4.74 (2H, s),
3.50 (2H, d, J ) 5.6 Hz), 2.59 (2H, s), 2.43 (3H, s), 2.23 (3H,
s), 1.47 (6H, s). 13C NMR (CDCl3): δ 168.4, 152.6, 146.3, 137.5,
136.2, 131.3, 129.3, 128.6, 128.1, 127.6, 126.8, 124.6, 115.6,
75.3, 46.0, 35.8, 31.5, 27.7, 15.2, 12.4. IR (film): 1768, 1245,
1120, 735, 696 cm-1. MS (FAB) m/z: 351 (M + H). Anal. Calcd
for C23H26O3: C, 78.80; H, 7.48. Found: C, 78.64; H, 7.39.
2-[7-(6-Ben zyloxy-4,4,5,8-tetr am eth ylh ydr o)cou m ar in yl]-
eth a n a l 7. Compound 6 (2.0 g, 5.71 mmol) was dissolved in a
mixture of 20 mL of DCM and 20 mL of methanol. Then, ozone
was passed through the reaction mixture at -78 °C for 5 h.
The reaction was stopped by flushing with N2 for 2 h followed
by the addition of dimethyl sulfide (1 mL). The reaction
mixture was then stirred for 10 h. Evaporation of the solvents
gave a pale yellow oil. Ether (70 mL) was added, and the
solution was washed with water and dried over MgSO4.
Filtration, evaporation, and purification by a silica gel column
(hexanes/EtOAc ) 2/1) gave product 7 (1.226 g, 61%) as white
F igu r e 2. HPLC profiles of the peptides synthesized: (a) the
standard tetrapeptide [Boc-Trp-Ala-Gly-Gly-OH] prepared
through solution-phase synthesis, (b) the tetrapeptide prepared
through solid-phase synthesis, (c) co-injection of the standard
and the sample (tetrapeptide), (d) the standard pentapeptide
[Boc-Asn-Ala-Ser(OBn)-Gly-Glu(OBn)-OH] prepared through
solution-phase synthesis, (e) the pentapeptide prepared through
solid-phase synthesis, and (f) co-injection of the standard and
the sample (pentapeptide).
zolyoxy-tris(pyrrolidino)-phosphonium hexafluorophos-
phate)37 was used for this step as the activating reagent,
and DIPEA (diisopropylethylamine) was used as the
base. The peptides synthesized by using the solid-phase
methods were identical to the standards from the solu-
1
tion-phase methods by H NMR and HPLC (Figure 2).
Con clu sion s
We have developed a new linker for the solid-phase
synthesis of peptides. This linker can be cleaved under
mild conditions by using two sequential reactions. Be-
cause the cleavage does not require acidic conditions,
which are commonly used for the cleavage of the con-
ventional Merrifield resin and Wang’s resin, this resin
provides an alternative when acidic conditions are not
desirable. Furthermore, the cleavage conditions do not
affect most of the side chain protecting groups. Therefore,
the peptides synthesized can be used for the segment
synthesis of larger peptides without the need to reprotect
the side chain functional groups. Although the feasibility
was only studied for polystyrene-based resin, it should
be feasible to attach this linker to other resin materials
for solid-phase synthesis. Furthermore, attachment of the
resin linker to a more hydrophilic resin could make it
1
solid. H NMR (CDCl3): δ 9.67 (1H, s), 7.38 (5H, s), 4.69 (2H,
s), 3.81 (2H, s), 2.60 (2H, s), 2.44 (3H, s), 2.16 (3H, s), 1.48
(6H, s). 13C NMR (CDCl3): δ 199.1, 168.1, 153.0, 146.4, 136.8,
130.8, 128.8, 128.4, 127.9, 127.1, 125.2, 124.8, 75.3, 45.8, 42.9,
35.9, 27.6, 15.3, 13.1. IR (film): 2719, 1768, 1719, 1244, 1121,
736, 696 cm-1. MS (FAB) m/z: 353 (M + H). Anal. Calcd for
C
22H24O4: C, 74.98; H, 6.86. Found: C, 74.69; H, 6.79.
7-(3-Hyd r oxy-1-p r op yl)-6-ben zyloxy-4,4,5,8-tetr a m eth -
ylh yd r ocou m a r in 8. To a cooled solution of compound 6 (8.07
g, 23.06 mmol) in freshly distilled THF (95 mL) was added
dropwise a borane solution (1.0 M in THF) (30.0 mL, 30.0
mmol) via a syringe under N2 at 0 °C with stirring. After
addition, stirring was continued for 2 h at 0 °C. The reaction
was stopped by addition of 10 mL of water in an ice bath. Then,
to the reaction mixture was added dropwise 3 N NaOH until
the pH of the solution reached 7-8. Hydrogen peroxide (3.5
mL, 30-35%) was added at 0 °C. Stirring was continued for
an additional 20 min. The reaction mixture was acidified with
1 N HCl to pH 6-7. Ether (200 mL) was added into the
(37) Coste, J .; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205-208.

7464 J . Org. Chem., Vol. 64, No. 20, 1999
Zheng et al.
mixture, the organic phase was separated, and the aqueous
phase was extracted with ether. The combined organic layer
was washed with brine and dried over MgSO4. Filtration and
evaporation gave an oil, which was purified by silica gel
column (EtOAc/hexanes ) 1/3-1/1 as eluent) to give a white
crude product was purified by using chromatotron (hexanes/
EtOAc ) 5/1) to give compound 13 as a colorless oil (19 mg,
quantitative). 1H NMR (CDCl3): δ 7.43-7.12 (10H, m), 4.73
(2H, s), 2.66 (4H, m), 2.57 (2H, s), 2.42 (3H, s), 2.22 (3H, s),
1.69 (2H, m), 1.54 (2H, m), 1.45 (6H, s). 13C NMR (CDCl3): δ
168.7, 152.7, 146.5, 142.7, 137.8, 134.6, 128.8, 128.6, 128.5,
128.4, 127.6, 126.8, 125.9, 124.0, 76.5, 46.2, 35.9, 32.0, 30.0,
27.9, 15.4, 12.5, 0.2. IR (film): 1768, 1244, 732, 697 cm-1. MS
(FAB) m/z: 442 (M+). Anal. Calcd for C30H34O3: C, 81.41; H,
7.74. Found: C, 81.33; H, 7.80.
1
solid (7.413 g, 87%). H NMR (CDCl3): δ 7.50-7.37 (5H, m),
4.75 (2H, s), 3.50 (2H, q, J ) 6.0 Hz), 2.81 (2H, t, J ) 7.2 Hz),
2.58 (2H, s), 2.45 (3H, s), 2.26 (3H, s), 1.77 (2H, m), 1.47 (6H,
s). 13C NMR (CDCl3): δ 168.4, 152.6, 146.6, 137.0, 133.2, 129.1,
128.8, 128.4, 127.9, 126.5, 124.0, 75.7, 61.59, 46.0, 35.8, 32.4,
27.7, 23.3, 15.3, 12.3. IR (film): 3442, 1766, 1246, 735, 697.
MS (FAB) m/z: 369 (M + H). Anal. Calcd for C23H28O4: C,
74.96; H, 7.66. Found; C, 74.94; H, 7.77.
Resin 15. A suspension of Merrifield resin (1 mequiv Cl/g)
(3.047 g, 3.047 mmol) and triphenylphosphine (2.358 g, 9.00
mmol) in 20 mL of anhydrous benzene was refluxed for 36 h
under N2. The mixture was allowed to cool to room tempera-
ture. The solid was filtered, washed with benzene and ether,
and dried in vacuo. The resin 15 (3.75 g, 99%) was obtained.
Resin 16. To a slurry of resin 15 (1.00 g, 1.00 mmol) in
anhydrous benzene (8 mL) was added dropwise BuLi in
hexanes (2.5 M) (400 µL, 1.00 mmol) under N2 via a syringe.
A red coloration developed immediately. After the mixture
stirred for 20 min at room temperature, a solution of aldehyde
9 (403 mg, 1.10 mmol) in anhydrous DCM (5 mL) was added
with stirring under N2. The red color of the resin vanished
immediately, and the reaction mixture was stirred for 2 h at
room temperature. Then, the reaction mixture was refluxed
for 5 h in an oil bath (bath temperature 80-85 °C). After
cooling to room temperature, the resin was filtered, washed
with ethanol, benzene, ether, and DCM, and dried in vacuo to
give a cream-colored resin (1.017 g). The filtrate was concen-
trated under reduced pressure to give a residue, which was
dissolved in 30 mL of benzene, washed with water, and dried
over MgSO4. After filtration, evaporation, and purification (by
silica gel column, DCM/MeOH ) 10/1) triphenylphosphine
oxide (172 mg, 0.62 mmol) was obtained. The yield of the
Wittig reaction was calculated (62%) on the basis of the
amount of triphenylphosphine oxide recovered. The substitu-
tion degree was determined to be 0.6 mequiv/g. IR (KBr): 1770,
3-[7-(6-Ben zyloxy-4,4,5,8-tetr am eth ylh ydr o)cou m ar in yl]-
p r op a n a l 9. A solution of compound 8 (3.04 g, 8.152 mmol)
in anhydrous DCM (15 mL) was added dropwise into a
suspension of PCC (3.315 g, 16.304 mmol) in anhydrous DCM
(45 mL) with stirring under N2 at room temperature. The flask
was washed with 10 mL of anhydrous DCM, and the washing
was added into the reaction flask. After addition, this mixture
was stirred for 2 h. The black material was filtered off through
a funnel with silica gel (70-230 mesh) and washed with DCM.
Evaporation of the solvent gave a brown solid, which was
purified on a silica gel column (hexanes/EtOAc ) 1/1) to afford
1
a white solid (2.57 g, 86%). H NMR (CDCl3): δ 9.75 (1Η, s),
7.49-7.34 (5H, m), 4.75 (2H, s), 2.98 (2H, t, J ) 7.5 Hz), 2.62
(4H, m), 2.43 (3H, s), 2.24 (3H, s), 1.47 (6H, s). 13C NMR
(CDCl3): δ 201.6, 168.3, 152.7, 146.4, 137.2, 132.3, 129.6, 128.8,
128.3, 127.7, 127.0, 123.8, 75.5, 46.0, 43.9, 35.9, 27.7, 20.2, 15.3,
12.4. ΙR (film): 2719, 1765, 1720, 1244, 737, 696 cm-1. MS
(FAB) m/z: 366 (M+). Anal. Calcd for C23H26O4: C, 75.58; H,
7.15. Found: C, 75.29; H, 7.16.
Ben zyltr ip h en ylp h osp h on iu m Ch lor id e 11.33 A mixture
of triphenylphosphine (4.039 g, 15.42 mmol) and benzyl
chloride (2.3 mL, 20.04 mmol) was heated without solvent
under N2 in an oil bath with stirring until it became a solution.
Heating was continued for an additional 15 min, and the
reaction was allowed to cool to room temperature. The result-
ing solid was recrystallized in EtOH to give a white crystalline
solid (5.98 g, 100%). 1H NMR (CD3OD): δ 7.91-6.98 (20Η, m),
3.31 (2H, s).
1595, 1490, 1448, 1243 cm-1
.
Resin 17. To a slurry of resin 16 (5.844 mg, 3.506 mmol) in
anhydrous and degassed benzene (70 mL), ethanol (15 mL),
and glacial acetic acid (1 mL) was added tris(triphenylphos-
phine)rhenium chloride (540 mg, 0.584 mmol). This mixture
was flushed three times with hydrogen. Then, hydrogenation
was carried out under a hydrogen pressure of 55 psi for 24 h
at room temperature. The resin was filtered and washed with
ethanol, benzene, and DCM. Then, the resin was dried on an
oil pump overnight to give resin 17 (5.899 g).
Com p ou n d 12. To a solution of benzyltriphenylphospho-
nium chloride 11 (194 mg, 0.5 mmol) in anhydrous benzene
(4 mL) was added dropwise 2.5 M BuLi solution in hexanes
(200 µL, 0.5 mmol) via a syringe under N2 at room tempera-
ture. The resulting deep-red solution was stirred for 20 min
at room temperature. A solution of aldehyde 9 (201 mg, 0.55
mmol) in anhydrous DCM (2 mL) was added with stirring
under N2. The red color turned to an orange color. After
stirring for 2 h at room temperature, the reaction mixture was
refluxed for 3 h. After evaporation of the solvent, the residue
was dissolved in ether (80 mL), washed with water, and dried
over MgSO4. Filtration and evaporation gave a yellow oil,
which was separated with silica gel column (hexanes/EtOAc
) 4/1) to give compound 12 as a colorless oil (187 mg, 85%).
1H NMR (CDCl3): δ 7.50-7.17 (10H, m), 6.46-5.68 (2H, m),
4.78, 4.72 (2H, ss), 2.84 (2H, m), 2.59 (2H, s), 2.42 (5H, m),
2.29, 2.20 (3H, ss), 1.46 (6H, S); IR (film): 1768, 1598, 1244,
Resin 18. To a slurry of resin 17 (5.655 g, 3.393 mmol) in
anhydrous THF (40 mL) was added lithium aluminum hydride
(1.445 g, 10.179 mmol) in three portions with stirring under
N2 at 0 °C. Stirring was continued for 1 h at 0 °C and overnight
at room temperature. Hydrogen chloride (3 N HCl aqueous
solution, 30 mL) was slowly added into the mixture at 0 °C.
The resin was filtered, washed with 3 N HCl aqueous solution,
water, ethanol, and DCM, and dried on an oil pump overnight
to give the resin 18 (0.94 g). IR (KBr): 3400, 1601, 1492, 1451,
757, 690, 540 cm-1
.
Gen er a l P r oced u r e for th e Atta ch m en t of th e F ir st
Am in o Acid to Resin 18. The resin 18 (600 mg, 0.36 mmol)
was washed twice with DCM and swelled with DCM (7 mL).
Protected amino acid (1.08 mmol), DCC (222 mg, 1.08 mmol),
and DMAP (9 mg, 0.07 mmol) were added. The mixture was
shaken for 6 h at room temperature. The resin was filtered
and washed with DCM (7 × 7 mL), methanol and DCM (5 ×
7 mL), and DCM (3 × 7 mL). The resin was dried on an oil
1167, 736, 695 cm-1 13C NMR (CDCl3): δ 168.6, 152.8, 146.5,
.
137.9, 137.6, 133.7, 132.0, 130.5, 130.3, 129.7, 129.1, 128.8,
128.7, 128.3, 128.2, 127.6, 127.2, 126.9, 126.8, 126.2, 124.0,
75.4, 46.1, 35.9, 33.6, 29.2, 27.8, 15.4, 12.6. MS (FAB) m/z: 440
(M+). Anal. Calcd for C30H32O3: C, 81.78; H, 7.32. Found: C,
81.66; H, 7.37.
Com p ou n d 13. To a medium-pressure hydrogenation flask
was added compound 12 (17 mg, 0.039 mmol), 4 mL of
anhydrous and degassed benzene, 1 mL of ethanol, and 5 drops
of glacial acetic acid. Then, tris(triphenylphosphine)rhodium
chloride (6 mg, 0.0065 mmol) was added. The flask was flushed
with hydrogen gas five times. Then, hydrogenation was carried
out under 55 psi of hydrogen pressure for 24 h at room
temperature. After evaporation of solvent, ether (5 mL) was
added to the residue. The black solid was filtered off, and the
ether phase was washed with saturated NaHCO3 and water
and dried over MgSO4. After filtration and evaporation, the
pump. IR (KBr): 1748, 1719, 1161 cm-1
.
To a suspension of the resin in THF (8 mL) and H2O (4 mL)
was added NBS (158 mg, 0.89 mmol). This mixture was shaken
for 2.5 h. The resin was filtered, washed with H2O and THF
(3 × 7 mL), THF (3 × 7 mL), and DCM (3 × 7 mL), and dried
in vacuo. The free hydroxyl groups on the resin were capped
with Ac2O (132 mg, 1.29 mmol) and Et3N (131 mg, 1.294 mmol)
in 7 mL of anhydrous DCM for 2 h. The yellow resin was
filtered and washed with DCM and dried in vacuo. IR (KBr):
1748, 1719, 1645, 1163 cm-1
.

Novel Resin Linker for Solid-Phase Peptide Synthesis
J . Org. Chem., Vol. 64, No. 20, 1999 7465
The resin was washed with 25% TFA in DCM-indole (1 mg/
mL) (7 mL) and shaken for 30 min with 7 mL of a 25% TFA
solution. The resin was then washed with DCM, 10% TEA (2
× 7 mL), and DCM and dried on an oil pump for 2 h. Then a
quantitative ninhydrin test was performed to give a substitu-
tion level of approximately 0.31 mmol/g.
Gen er a l P ep tid e Cou p lin g P r oced u r e. To a suspension
of the resin (571 mg, 0.177 mmol) in 6.5 mL of anhydrous DCM
was added protected amino acid (0.531 mmol), DIC (67 mg,
0.531 mmol), HOBt (72 mg, 0.531 mmol), and DMAP (4.3 mg,
0.035 mmol). This mixture was shaken for 2 h at room
temperature. The resin was filtered and washed with DCM
(6 × 8 mL), and the ninhydrin test was conducted. When
positive ninhydrin test was obtained, the coupling reaction was
repeated.
Typ ica l Boc Dep r otection P r oced u r e Usin g Boc-Gly-
Gly-OBn a s a n Exa m p le. Boc-Gly-Gly-OBn (835 mg, 2.593
mmol) was treated with 4 mL of 25% TFA in DCM for 1 h at
room temperature. After evaporation of the solvent, the residue
was dried in vacuo. The resulting CF3CO2-‚+NH3-Gly-Gly-OBn
was used for the next coupling reaction step without purifica-
tion.
Boc-Ala -Gly-Gly-OBn . 1H NMR (CDCl3): δ 7.34 (s, 5H),
5.15 (s, 2H), 4.20-3.90 (m, 5H), 1.39 (s, 9H), 1.35 (d, J ) 7.0
Hz).
Boc-Tr p (F or )-Ala -Gly-Gly-OBn . 1H NMR (DMSO):
δ
9.60-7.00 (m, 6H), 5.12 (s, 2H), 4.31 (m, 2H), 3.93 (d, J ) 5.7
Hz, 2H), 3.76 (d, J ) 4.5 Hz, 2H), 3.13 (m, 1H), 2.85 (m, 1H),
1.24 (s, 9H), 1.17 (m, 3H). HRMS (FAB) for C31H37N5O8: calcd
608.2720, found 608.2753.
Boc-Tr p (F or )-Ala -Gly-Gly-OH. A suspension of Boc-Trp-
(For)-Ala-Gly-Gly-OBn (503 mg, 0.828 mmol) in 30 mL of
methanol was hydrogenated via a balloon in the presence of
10% Pd/C (75 mg) for 1 h at room temperature. The catalyst
was removed by filtration, and the reaction mixture was
evaporated to dryness. After purification using a silica gel
column (DCM/MeOH ) 10/1, 1% acetic acid), a white solid
product (385 mg, 90%) was obtained. 1H NMR (CD3OD): δ
9.50-7.09 (m, 6H), 4.46-4.25 (m, 2H), 3.98-3.78 (m, 4H), 3.28
(m, 1H), 3.02 (m, 1H), 1.36 (s, 9H), 1.23 (m, 3H). HRMS (FAB)
for C24H31N5O8: calcd 518.2251, found 518.2280.
Gen er a l P r oced u r e for th e Clea va ge of th e P ep tid es
fr om th e Resin . Red u ction w ith Na BH4. The quinone resin
22 (250 mg, 0.36 mmol/g, 0.09 mmol) was swelled in 5 mL of
THF. Sodium borohydride (35 mg, 0.9 mmol) and methanol
(0.5 mL) were added. The mixture was stirred for 0.5 h at room
temperature. The hydroquinone resin was filtered and washed
with methanol (3 × 10 mL), THF (3 × 10 mL), and anhydrous
THF (5 × 5 mL). The hydroquinone resin 23 was swelled in 3
mL of anhydrous THF. Anhydrous TBAF/THF (3 mL, 0.76 M)
was added, and the mixture was stirred for 20 h under N2. IR
showed that most of peptide had been cleaved. The resin was
filtered and washed with THF (3 × 5 mL), DMF (3 × 5 mL),
MeOH (3 × 5 mL), and THF (3 × 5 mL). The filtrate was
combined and solvent was removed at 30 °C and dried on oil
pump for 20 h.
Typ ica l P r oced u r e for th e P u r ifica tion of th e P ep tid es
Clea ved fr om th e Resin . The dried resin mixture from 250
mg of resin 22 was swelled in 20 mL of anhydrous THF,
Amberlyst-15 calcium sulfonate resin (8.5 g, 29.75 mmol,
approximately 1.76 mequiv/g of Ca2+) and Amberlyst-15 sul-
fonic acid resin (300 mg, 14.1 mmol, 4.7 mequiv/g of H+) were
added, and the mixture was stirred for 4 h at room tempera-
ture.36 The Amberlyst-15 resin was filtered and washed with
DMF (10 × 10 mL), and the solvent was removed at 30 °C.
The residue was recrystallized in a suitable solvent to afford
a pure peptide. The filtrate was further purified on a silica
gel column to afford another part of the pure peptide.
Boc-Tr p -Ala -Gly-Gly-OH. To the residue obtained follow-
ing the general procedures described above from 0.25 g of resin
was added 1 mL of ethyl acetate. The solution was stored at
about -12 °C for 6 h. The white precipitates were filtered and
washed with ethyl acetate to afford 4 mg of the product. TLC
showed a single spot. The filtrate was further purified with a
silica gel column (DCM/CH3OH ) 10/1, 0.1% HOAc) to afford
12 mg of a white solid. The total yield was 16 mg (37%). 1H
NMR and HPLC studies indicated that this peptide was
identical to the standard prepared by the solution-phase
method.
Boc-Asn -Ala -Ser (OBn )-Gly-Glu (OBn )-OH. The residue
from 50 mg of resin was recrystallized using methanol to afford
a white solid (3 mg, 34%). 1H NMR and HPLC studies
indicated that this peptide was identical to the standard
prepared using solution phase method.
Boc-Tr p -Ala -Gly-Gly-OH . Boc-Trp(For)-Ala-Gly-Gly-OH
(52 mg, 0.10 mmol) was treated with 20% piperidine in DMF
(2 mL) for 3 h at 0 °C and 19 h at room temperature under
N2. After evaporation of the solvent, the residue was dissolved
in 10 mL of EtOAc. This solution was washed with 10% citric
acid (2 × 5 mL) and brine (2 × 5 mL) and dried over MgSO4.
Filtration and evaporation gave an oil, which was purified by
radial preparative TLC (DCM/MeOH ) 10/1, 1% CH3CO2H).
1
A white solid was obtained (18 mg, 36%). H NMR (CD3OD):
δ 8.20-6.99 (5H, m), 4.36-4.24 (2H, m), 3.89-3.60 (4H, m),
3.23-3.05 (2H, m), 138 (9H, s), 1.23 (3H, d, J ) 6.42 Hz).
HRMS (FAB) for C23H31N5O7: calcd 489.2223, found 489.2235.
Boc-Glu (OBn )-OCH2CHdCH2. A solution of Boc-Glu-
(OBn)-OH (709 mg, 2.1 mmol) in 4 mL of methanol was added
to a solution of cesium carbonate (330 mg, 1.01 mmol) in 2
mL of water with stirring at room temperature. This solution
was stirred for 30 min, and then the solvent was evaporated.
Benzene (3 × 5 mL) was added to the mixture and then
removed by evaporation to give a white solid. To the cesium
salt was added DMF (6 mL) and allyl bromide (1 mL). After
reaction for 1 h, the white solid was filtered off and washed
with ethyl acetate several times. Evaporation of the filtrate
under reduced pressure and purification of the residue using
a silica gel column (hexanes/EtOAc ) 2/1-1/1) gave a white
solid (740 mg, 94%).1H NMR (CD3OD): δ 7.35 (s, 5H), 5.91-
5.23 (m, 3H), 5.12 (s, 2H), 4.62 (d, J ) 5.8 Hz, 2H), 4.38 (m,
1H), 2.50-2.46 (m, 2H), 2.24 (m, 1H), 1.96 (m, 1H), 1.43 (s,
9H).
Boc-Gly-Glu (OBn )-OCH 2CHdCH2. 1H NMR (CD3OD): δ
7.34 (s, 5H), 5.94-5.12 (m, 3H), 5.11 (s, 2H), 4.61 (d, J ) 5.6
Hz, 2H), 4.53 (m, 1H), 3.72 (brs, 2H), 2.48 (t, J ) 7.5 Hz, 2H),
2.24 (m, 1H), 2.00 (m, 1H), 1.43 (s, 9H).
Boc-Ser (OBn )-Gly-Glu (OBn )-OCH2CHdCH2. 1H NMR
(CD3OD): δ 7.34 (m, 10H), 5.93-5.15 (m, 3H), 5.10 (s, 2H),
4.60 (d, J ) 5.4 Hz, 2H), 4.51-4.47 (m, 3H), 4.28-4.22 (m,
1H), 3.88 (m, 2H), 3.70 (m, 2H), 2.49-2.42 (m, 2H), 2.22-2.13
(m, 1H), 1.99-1.90 (m, 1H), 1.43 (s, 9H).
Syn th esis of th e Sta n d a r d Tetr a p ep tid e [Boc-Tr p -Ala -
Gly-Gly-OH] a n d P en ta p ep tid e [Boc-Asn -Ala -Ser (OBn )-
Gly-Glu (OBn )-OH] by th e Solu tion -P h a se Meth od . Typ i-
ca l Cou p lin g P r oced u r e Usin g Boc-Gly-Gly-OBn a s a n
Exa m p le. To a cooled solution of Boc-Gly-OH (525 mg, 3.00
mmol) in 25 mL of anhydrous DCM was added EDC (634 mg,
3.30 mmol), HOBt (446 mg, 3.30 mmol), H-Gly-OBn‚HCl (495
mg, 3.00 mmol), DMAP (73 mg, 0.60 mmol), and TEA (636
mg, 6.20 mmol). This mixture was stirred under N2 for 2 h at
0 °C and 3 h at room temperature. Evaporation of the solvent
under reduced pressure gave a residue, which was dissolved
in EtOAc (70 mL), washed with 10% citric acid, saturated
NaHCO3, and brine, and dried over MgSO4. Filtration, evapo-
ration, and drying in vacuo gave a white solid (835 mg, 86%).1H
NMR (CDCl3): δ 7.35 (s, 5H), (5.17 (s, 2H), 4.09 (d, J ) 5.3
Hz, 2H), 3.84 (d, J ) 5.3 Hz, 2H), 1.45 (s, 9H).
Boc-Ala -Ser (OBn )-Gly-Glu (OBn )-OCH 2CH dCH 2. 1H
NMR (CD3OD): δ 7.34 (m, 10H), 5.93-5.19 (m, 3H), 5.10 (s,
2H), 4.60 (d, J ) 5.4 Hz, 2H), 4.52 (s, 2H), 4.48 (m, 2H), 4.03-
3.72 (m, 5H), 2.47 (t, J ) 7.5 Hz, 2H), 2.23-2.17 (m, 1H), 2.02-
1.95 (m, 1H), 1.39 (s, 9H), 1.27 (d, J ) 7.1 Hz, 3H).
Boc-Asn -Ala -Ser (OBn )-Gly-Glu (OBn )-OCH 2CH dCH 2.
CF3CO-‚+NH3-Ala-Ser(Bn)-Gly-Glu(OBn)-OCH2CHdCH2 was
coupled with Boc-Asn-OH (121 mg, 0.523 mmol) in 5 mL of
anhydrous DCM and 2 mL of DMF in the presence of the
PyBOP (272 mg, 0.523 mmol) and DIPEA (202.8 mg, 1.569
mmol). This mixture was stirred for 30 min at 0 °C and
overnight at room temperature under N2. After workup and

7466 J . Org. Chem., Vol. 64, No. 20, 1999
Zheng et al.
purification by a silica gel column (DCM/MeOH ) 15/1-10/
3.76 (d, J ) 5.3 Hz, 2H), 3.62 (d, J ) 5.6 Hz, 2H), 2.41 (m,
4H), 2.00 (m, 1H), 1.82 (m, 1H), 1.37 (s, 9H), 1.19 (d, J ) 6.8
Hz, 3H). HRMS (FAB) for C36H48N6O12: calcd 757.3408, found
757.3402.
1), the protected peptide was obtained as a white solid (278
1
mg, 67%). H NMR (CD3OD): δ 7.31 (m, 10H), 5.90-5.15 (m,
3H), 5.07 (s, 2H), 4.58 (d, J ) 5.4 Hz, 2H), 4.48 (s, 2H), 4.46-
4.23 (m, 4H), 3.87-3.71 (m, 4H), 2.68-2.57 (m, 2H), 2.46-
2.40 (m, 2H), 2.20-2.09 (m, 1H), 2.00-1.89 (m, 1H), 1.38 (s,
Ack n ow led gm en t . Financial support from the
National Institutes of Health (GM-52515) is greatly
appreciated. We would also like to thank Professor
David Shultz for allowing us to use his FTIR instru-
ment. High-resolution mass spectra were obtained at
the Mass Spectrometry Laboratory for Biotechnology,
North Carolina State University. Partial funding for the
facility was obtained from the North Carolina Biotech-
nology Center and the National Science Foundation
(9111391).
9H), 1.31 (d, J ) 6.9 Hz, 3H). HRMS (FAB) for C39H52N6O12
calcd 797.3721, found 797.3740.
:
Boc-Asn -Ala -Ser (OBn )-Gly-Glu (OBn )-OH. Under argon,
tetrakis(triphenylphosphine) palladium(0) (44 mg, 0.0384
mmol) was added to a solution of Boc-Asn-Ala-Ser(OBn)-Gly-
Glu(OBn)-OCH2CHdCH2 (306 mg, 0.384 mmol) in anhydrous
THF (26 mL) and DMSO (2.6 mL), followed by the addition of
morpholine (335 mg, 3.84 mmol). This pale yellow solution was
stirred for 4.5 h under argon at room temperature. After
evaporation of the solvent, the crude product was washed with
2 N HCl, chloroform, and ethyl acetate and dried in vacuo. A
white solid was obtained (233 mg, 80%). 1H NMR (DMSO-d6):
δ 7.34 (m, 10H), 5.08 (s, 2H), 4.48 (s, 2H), 4.40-4.22 (m, 4H),
J O990770X