Resin-to-resin acyl- and aminoacyl-transfer reactions using oxime supports

Article abstract of DOI:10.1021/ja9818654

We describe a convergent approach to solid-phase synthesis in which two fragments of a molecule are synthesized on independent supports and then condensed in a key resin-to-resin transfer reaction. This approach has been utilized for the synthesis of amides and ureas by transferring acyl groups and aminoacyl groups from p-nitrophenyl(polystyrene)ketoxime resin to amino acid-functionalized Wang resins. Oxime resinderived esters of peptides undergo transacylation to a solution-phase nucleophilic activator which then transfers the peptide to another resin bearing a nucleophilic amine terminus, resulting in amide bond formation. Likewise, oxime resin-derived carbamates, prepared from phosgenated p-nitrophenyl(polystyrene)ketoxime resin, undergo thermolytic isocyanate liberation in solution, which reacts with a second resin bearing a nucleophilic amino terminus resulting in urea bond formation.

Full text of DOI:10.1021/ja9818654

1636
J. Am. Chem. Soc. 1999, 121, 1636-1644
Resin-to-Resin Acyl- and Aminoacyl-Transfer Reactions Using Oxime
Supports
Yoshitomo Hamuro,†,‡ Mark A. Scialdone,*,‡ and William F. DeGrado*,†
Contribution from the Department of Biochemistry and Biophysics, School of Medicine, UniVersity of
PennsylVania, Philadelphia, PennsylVania 19104-6059, and DuPont Central Research and DeVelopment,
P.O. Box 80328, Experimental Station, Wilmington, Delaware 19880-0328
ReceiVed May 28, 1998
Abstract: We describe a convergent approach to solid-phase synthesis in which two fragments of a molecule
are synthesized on independent supports and then condensed in a key resin-to-resin transfer reaction. This
approach has been utilized for the synthesis of amides and ureas by transferring acyl groups and aminoacyl
groups from p-nitrophenyl(polystyrene)ketoxime resin to amino acid-functionalized Wang resins. Oxime resin-
derived esters of peptides undergo transacylation to a solution-phase nucleophilic activator which then transfers
the peptide to another resin bearing a nucleophilic amine terminus, resulting in amide bond formation. Likewise,
oxime resin-derived carbamates, prepared from phosgenated p-nitrophenyl(polystyrene)ketoxime resin, undergo
thermolytic isocyanate liberation in solution, which reacts with a second resin bearing a nucleophilic amino
terminus resulting in urea bond formation.
Introduction
supports and then condensing the fragments together in a key
resin-to-resin transfer reaction (RRTR). Such an approach
would have a number of distinct advantages over existing linear
methods. This method should allow one to use conditions in
the synthesis of one fragment of a target molecule that might
not be tolerated during the elaboration of another fragment.
Furthermore, in some cases, this approach may allow the
purification of intermediates as in the solid-phase synthesis of
peptides via segment condensation.6,7 Finally, RRTRs might
have attractive possibilities for the synthesis of combinatorial
libraries; it should be possible to synthesize and store two
focused libraries on supports and then combine them in
appropriate combinations.
Merrifield’s method of solid-phase peptide synthesis has had
a revolutionary impact on organic chemistry,1 allowing the rapid
and convenient synthesis of a variety of biopolymers such as
peptides,2 oligonucleotides,3 and oligosaccharides4 as well as
organic compounds.5 In this method, a compound is synthesized
on a solid support, thereby facilitating the isolation of intermedi-
ates and allowing the use of excess reagents in solution. A
limitation of this method, however, is that it is linear in nature;
thus, the functionality and protecting groups introduced early
in the synthesis must be fully stable to all subsequent reaction
conditions. An alternate convergent approach might involve
synthesizing two fragments of a given molecule on separate
In the 1970s, triphasic reactions8 between two solid supports
were examined as a method to probe the mechanisms of acyl
transfer,9 phosphate transfer,10 and other reactions.11-13 For
† University of Pennsylvania.
‡ DuPont Central Research and Development.
(1) (a) Merrifield, R. R. J. Am. Chem. Soc. 1963, 85, 2149-2154. (b)
Merrifield, R. R. Angew. Chem., Int. Ed. Engl. 1985, 24, 799-810.
(2) For texts on solid-phase peptide synthesis, see: (a) Atherton, E.;
Sheppard, R. C. Solid-phase peptide synthesis; a practical approach; IRL
Press: Oxford, 1989. (b) Stewart, J. M.; Young, J. D. Solid-Phase Peptide
Synthesis, 2nd ed.; Pierce Chemical Co.: Rockford, IL, 1984. For reviews
on solid-phase peptide synthesis, see: (c) Jung, G.; Beck-Sickinger, A. G.
Angew. Chem., Int. Ed. Engl. 1992, 31, 367-383. (d) Pavia, M. R.; Sawyer,
T. K.; Moos, W. H. Bioorg. Med. Chem. Lett. 1993, 3, 387-396. (e) Gallop,
M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon, E. M. J.
Med. Chem. 1994, 37, 1233-1251.
(6) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982, 47, 3258-3261.
(7) Sasaki, T.; Findeis, M. A.; Kaiser, E. T. J. Org. Chem. 1991, 56,
3159-3168.
(8) Regen investigated triphase catalysis using solid supported catalysis
with aqueous and organic layers. See: (a) Regen, S. L. J. Am. Chem. Soc.
1975, 97, 5956-5957. (b) Regen, S. L. J. Am. Chem. Soc. 1976, 98, 6270-
6274. (c) Regen, S. L. J. Org. Chem. 1977, 42, 875-879.
(9) For acyl transfer, see: (a) Rebek, J.; Brown, D.; Zimmerman, S. J.
Am. Chem. Soc. 1975, 97, 454-455. For acyl transfer, see: (b) Rebek, J.;
Brown, D. In Peptides 1976, Proc. 14th Eur. Pep. Symp.; Loffet, A., Ed.;
Edition University of Brussels: Brussels, Belgium: Wepion, Belgium, 1976;
pp 61-64.
(3) For reviews on solid-phase oligonucleotide synthesis, see: (a)
Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223-2311. (b)
Montserrat, F. X.; Eritja, A. G.; Pedroso, E. Tetrahedron 1994, 50, 2617-
2622.
(10) For phosphate transfer, see: (a) Rebek, J.; Gavina, F. J. Am. Chem.
Soc. 1975, 97, 1591-1592. (b) Rebek, J.; Gavina, F. J. Am. Chem. Soc.
1975, 97, 3221-3222. (c) Rebek, J.; Gavina, F.; Navarro, C. Tetrahedron
Lett. 1977, 35, 3021-3022. (d) Rebek, J.; Gavina, F.; Navarro, C. J. Am.
Chem. Soc. 1978, 100, 8113-8117.
(11) For cyclobutadiene transfer, see: Rebek, J.; Gavina, F. J. Am. Chem.
Soc. 1974, 96, 7112-7114.
(12) For a mechanistic study on amide synthesis from acid and isocyanate,
see: (a) Rebek, J.; Brown, D.; Zimmerman, S. J. Am. Chem. Soc. 1975,
97, 4407-4408. (b) Rebek, J.; Brown, D.; Zimmerman, S. In Peptides:
Chem., Struct., Biol., Proc. 4th Am. Pept. Symp.; Walter, R., Meienhofer,
J., Eds.; Ann Arbor Sci.: Ann Arbor, MI, 1975; pp 371-374.
(13) For a mechanistic study on intramolecular nucleophilic catalysis,
see: Rebek, J.; Horton, J. A.; Brown, D. Isr. J. Chem. 1978, 17, 316-317.
(4) (a) Frechet, J. M.; Schuerch, C. Carbohydr. Res. 1972, 22, 399-
412. (b) Veeneman, G. H.; Notermans, S.; Liskamp, R. M. J.; Marel, G. A.
v. d.; Boom, J. H. v. Tetrahedron Lett. 1987, 28, 6695-6698. (c) Douglas,
S. P.; Whitfield, D. M.; Krepinski, J. J. J. Am. Chem. Soc. 1991, 113, 5095-
5097. (d) Danishefsky, S. J.; McClure, K. F.; Randolph, J. T.; Ruggeri, R.
B. Science 1993, 260, 1307-1309. (e) Schuster, M.; Wang, P.; Paulson, J.
C.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 1135-1136.
(5) For reviews on solid-phase library synthesis, see: (a) Gordon, E.
M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop, M. A. J. Med.
Chem. 1994, 37, 1385-1401. (b) Fruchtel, J. S.; Jung, G. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 17-42. (c) Thompson, L. A.; Ellman, J. A. Chem.
ReV. 1996, 96, 555-607. (d) Nefzi, A.; Ostresh, J. M.; Houghten, R. A.
Chem. ReV. 1997, 97, 449-472.
10.1021/ja9818654 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/03/1999

Acyl- and Aminoacyl-Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1637
Scheme 1
instance, Rebek and Brown showed that the acyl group of a
resin-bound active ester may be transferred to a second amine-
containing polymer by addition of imidazole.9b Because of site
isolation between the two supports, it could be inferred that the
reaction occurred via nucleophilic catalysis, with imidazole
acting as a catalyst to assist the transfer of the acyl group from
the donor resin to the acceptor resin. In a similar manner it was
possible to show that phosphate-transfer reactions may occur
via a metaphosphate intermediate, which was sufficiently stable
to diffuse between two supports.10 Despite the elegance of these
mechanistic investigations, to the best of our knowledge, RRTRs
have not been fully utilized for synthetic purposes.14-16
In this paper, we explore the use of p-nitrophenyl(polysty-
rene)ketoxime polymer (1, Scheme 1)6,17 for resin-to-resin acyl-
and aminoacyl-transfer reactions. Oxime resin-derived esters 2
are designed to have a nucleophilic lability between that of
benzyl ester resins such as Merrifield’s1 or Wang’s18 supports
and other active ester polymers initially prepared by Patchornik
and others.19-25 These active esters contain acidic alcohol
leaving groups and have been used to transfer acyl groups to
amines in free solution. While one active ester resin has been
employed for RRTR, in practice it was too reactive to allow
many synthetic manipulations prior to the transfer reaction.14
Thus while they are suitable for the mechanistic studies,9,10 they
may have less utility for construction of libraries. However, the
oxime resin is stable enough to allow chemical transformations
including the synthesis of peptides by standard solid-phase
peptide methods6,17 or the construction of â-carbolines by the
Pictet-Spengler reaction.26 Once fully elaborated, the desired
compounds are removed under very mild condition by carbox-
ylic acid-catalyzed nucleophilic attack. Indeed, oxime resin-
derived peptide esters undergo transacylation by reaction with
excess N-hydroxypiperidine, suggesting it may serve as a
solution-phase nucleophilic activator27 or chaperone.14 However,
the resulting esters are of insufficient reactivity to allow facile
reaction with a variety of nucleophiles.28 Here we examine a
number of potential chaperones 3, such as HOBt and HOSu
for resin-to-resin acyl transfer reactions from oxime resin to
nucleophilic amine-containing resins 5. These derivatives are
nucleophilic enough to liberate the acyl group from an oxime
support to form active esters 4 in solution which then can react
with amine containing resins 5 resulting in amide bond
formation (Scheme 1).
In the second example of the use of the oxime resin in
RRTRs, we describe the transfer of aminoacyl groups to a
second amine-containing polymeric support via an isocyanate
intermediate. In previous work, we described the use of
phosgenated oxime resin, which reacts with amines to form
oxime-derived carbamates 9. Thermolysis of these carbamates
9 in the presence of amines in solution leads to the formation
of ureas 11 via freely diffusible isocyanate intermediates (10;
see Scheme 2).29,30 However, secondary amine-derived carbam-
ates of phosgenated oxime resin did not generate ureas,31
supporting in situ formation of the isocyanate by the mechanism
(14) Patchornik used resin-to-resin acyl transfer using o-nitrophenol resin
with imidazole as an acyl group carrier. However, transferred groups were
limited to N-protected monoamino acids. See: Patchornik, A. CHEMTECH
1987, 58-63.
(15) Patchornik published a multipolymer reaction for a preparative
purpose. However, one of the resins acts as a base not a substrate. See:
Cohen, B. J.; Kraus, M. A.; Patchornik, A. J. Am. Chem. Soc. 1977, 99,
4165-4167.
(16) Recently, Ouyang et al. published a two-resin system to generate
small-molecule libraries. However, the second resin acts as a acid scavenger.
See: Ouyang, X.; Armstrong, R. W.; Murphy, M. M. J. Org. Chem. 1998,
63, 1027-1032.
(17) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1980, 45, 1295-
1300.
(18) Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328-1333.
(19) Fridkin, M.; Patchornik, A.; Katchalski, E. J. Am. Chem. Soc. 1966,
83, 3164-3165.
(20) Fridkin, M.; Patchornik, A.; Katchalski, E. J. Am. Chem. Soc. 1968,
90, 2953-2957.
(21) Fridkin, M.; Patchornik, A.; Katchalski, E. Biochemistry 1972, 11,
466-471.
(26) Mohan, R.; Chou, Y.-L.; Morrissey, M. M. Tetrahedron Lett. 1996,
37, 3963-3966.
(22) Laufer, D. A.; Chapman, T. M.; Marlborough, D. I.; Vaidya, V.
M.; Blout, E. R. J. Am. Chem. Soc. 1968, 90, 2696-2698.
(23) Kalir, R.; Fridkin, M.; Patchornik, A. Eur. J. Biochem. 1974, 42,
151-156.
(27) Nakagawa, S. H.; Kaiser, E. T. J. Org. Chem. 1983, 48, 678-685.
(28) Jones, J. H.; Young, G. T. J. Chem. Soc. C 1968, 53-61.
(29) Scialdone, M. A. Tetrahedron Lett. 1996, 37, 8141-8144.
(30) Scialdone, M. A.; Shuey, S. W.; Soper, P. D.; Hamuro, Y.; Burn,
D. M. J. Org. Chem. 1998, 63, 4802-4807.
(24) Kalir, R.; Warshawsky, A.; Fridkin, M.; Patchornik, A. Eur. J.
Biochem. 1975, 59, 55-61.
(25) Recently, the polymer-bound active ester was revisited; see:
Dendrinos, K. G.; Kalivretenos, A. G. Tetrahedron Lett. 1998, 39, 1321.
(31) Hamuro, Y.; Marshall, W. J.; Scialdone, M. A. J. Combinatorial
Chem., in press.

1638 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Hamuro et al.
Scheme 2
Scheme 3
proposed by Levine and Frech.32,33 Furthermore, TGA-MS and
IR studies of carbamates derived from oxime resin also indicated
thermolytic isocyanate generation.34 These results led us to
attempt the thermolysis of oxime carbamate resins 9 in the
presence of polymer-supported amines (5; see Scheme 3).
assisting the transfer of an acyl group from oxime resin to an
amino group on Wang resin. A suspension of Z-Ala-oxime resin
2a, H-Phe-Wang resin 5a and a chaperone candidate 3 in CH2Cl2
was shaken overnight at room temperature. The resulting resin
mixture (1 and 6aa) was treated with 50% TFA in CH2Cl2 for
1 h, and then the resin mixture was filtered and washed with
CH2Cl2 (Scheme 4). The amount of Z-Ala-Phe-OH 7aa in the
combined filtrates was quantified using HPLC with nitrobenzene
as an internal standard. Several chaperone candidates, thiophenol
(PhSH), benzylthiol (BnSH), N,N-diethylhydroxyamine (Et2NOH),
p-nitrophenol (PnpOH), pentachlorophenol (PcpOH), pentafluo-
rophenol (PfpOH), 1-hydroxy-7-azabenzotriazole (HOAt), hy-
droxybenzotriazole (HOBt), ethyl cyanoglycoxylate-2-oxime
Results and Discussion
Resin-to-Resin Acyl Transfer Reactions. (a) Evaluation
of Chaperone Candidates for Resin-to-Resin Acyl Transfer.
We initially examined a number of chaperone candidates for
(32) Levine, A. W.; Frech, J. J. Org. Chem. 1972, 37, 1500-1503.
(33) Levine, A. W.; Frech, J. J. Org. Chem. 1972, 37, 2455-2460.
(34) Scialdone, M. A., unpublished results.

Acyl- and Aminoacyl-Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1639
Scheme 4
The best yield obtained was 73% with 2.0 equiv of Z-Ala-
oxime resin and 30 equiv of HOBt (saturated) in CHCl3 at 50
°C for 16 h (entry 16). The major byproducts were a small
amount of unreacted phenylalanine as well as dipeptide that
was not cleaved from the Wang resin during the 1-h TFA
cleavage step. The yields at 50 or 70 °C with HOSu or HOBt
are over 60% and are practically useful (entries 9-12 and 14-
18). IR spectra of the recovered resins showed peaks at 1780
(oxime ester) and 1727 cm-1. This indicates that the excess
Z-Ala remained on the oxime resin and that the acyl oxime
group was not lost through side reactions.
(c) Epimerization during Resin-to-Resin Acyl Transfer.
For the resin-to-resin acyl transfer to be a useful method for
preparative purposes, the acyl transfer should proceed with very
little epimerization. Epimerization should be suppressed in the
case of urethane-protected amino acid transfer such as the
aforementioned Z-Ala transfer; however, other acyl derivatives
may present a problem. To clarify this issue, the transfer of
Boc-Leu-Ala from oxime resin to H-Phe-Wang resin 5a was
carried out (Table 3). The authentic samples, H-Leu-Ala-Phe-
OH (L-7ba) and H-Leu-D-Ala-Phe-OH (D-7ba) were prepared
by standard Fmoc chemistry on Wang resin. These two
diastereomers are fully resolved using a C18 column (acetonitrile
0-36% in water, linear gradient, 0.1% TFA buffered). Two
equivalents of Boc-Leu-Ala-oxime resin 2b and 6 equiv of a
chaperone 3 and 5a in 1,2-dichloroethane were stirred at 70 °C
for 21 h, and the crude product was quantified by HPLC
following removal from the Wang resin. When HOSu was used
as a chaperone, only a single major peak was observed by HPLC
and the L-7ba and D-7ba were obtained in 76 and 1.3% yield,
respectively. The purity of the crude product was calculated as
88% by dividing the HPLC-quantified yield by the actual
gravimetric yield. The reaction with HOBt also gave very little
epimerization (1.8%), but a lower yield (59%), and the reaction
with EACNOx gave a reasonable yield (67%), but epimerization
was more than doubled (3.9%). Therefore, HOSu was chosen
as the optimum chaperone for resin-to-resin acyl transfer
reactions.
(d) Use of HOSu as the Chaperone under Optimized
Conditions. The utility of acyl RRTR was exemplified through
the synthesis of a series of tri- and tetrapeptides. Three different
acylated oxime resins (Z-Ala-Oxime 2a, Boc-Leu-Ala-Oxime
2b, and Z-Leu-Ala-Oxime 2c) and three different Wang resins
(H-Phe-Wang 5a, H-Ala-Phe-Wang 5b, and H-Val-Phe-Wang
5c) were reacted in different combinations at 70 °C with HOSu
in 1,2-dichloroethane overnight. After TFA cleavage of the
peptides from the Wang resin, the crude peptides were dissolved
in MeOH/CH2Cl2 and then triturated with Et2O/hexanes. The
isolated products appeared pure by HPLC and NMR analysis
and the yields were good to excellent (see Table 4 and
Supporting Information).
Table 1. Transfer of Z-Ala from Oxime Resin to H-Phe-Wang
Resin with Various Chaperonesa
chaperone
solvent
CH2Cl2
CH2Cl2
CHCl3
CH2Cl2
CHCl3
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
ClCH2CH2Cl
temp
rt
rt
rt
rt
rt
rt
rt
rt
yield,b %
(1) HOSu
(2) HOBt
21
21
18
4.8
3.0
2.1
2.1
1.7
0
(3) EACNOx
(4) PfpOH
(5) HOAtc
(6) PnpOH
(7) PcpOH
(8) Et2NOH
(9) PhSH
rt
rt
70 °C
(10) BnSH
(11) none
0
0
a All resin-to-resin acyl-transfer reactions were carried out with 0.047
mmol of H-Phe-Wang resin, 1.6 equiv of Z-Ala-oxime resin and 6.4
equiv of chaperone in 5 mL of solvent. b The yield was obtained by
HPLC with nitrobenzene as internal standard. c This chaperone was
not completely soluble.
(EACNOx), and N-hydroxysuccinimide (HOSu), were tested
(Table 1). HOSu, HOBt, and EACNOx provided yields of 27,
23, and 18%, respectively, while the other candidates gave rise
to the dipeptide in less than 5% yield. Thus a further optimiza-
tion was carried out using HOSu, HOBt, and EACNOx as
chaperone candidates (Table 2).
(b) Optimization of Resin-to-Resin Acyl Transfer. Several
additives (DBU,35 DMAP, imidazole,9b AcOH) were tested with
HOSu in CH2Cl2 at room temperature, and none of them were
found to improve the yield (Table 2, entries 2-5). DMF was
also evaluated as a solvent without success in the hope that
increasing the polarity of the solvent might accelerate the
reactions (entry 6). Also, increasing the molar excess of the
chaperones beyond 6-fold failed to increase the yield, presum-
ably because of their limited solubilities in the solvents required
for these reactions (for example, compare entries 7 and 8 or
entries 9 and 10). A larger equivalent of acylated oxime resin
improved the yield slightly at 50 °C in chloroform (for example,
compare entries 7 and 9 or entries 8 and 10). Most significantly,
increasing the temperature greatly improved the yield (for
example, compare entries 1 and 7 or entries 9 and 11).
The optimized acyl-transfer method was also applied to more
practical targets, enkephalins.36,37 Enkephalins are natural ligands
for opiate receptors and known to consist of two subtypes, Leu-
enkephalin (7dd, H-Tyr-Gly-Gly-Phe-Leu-OH) and Met-
enkephalin (7de, H-Tyr-Gly-Gly-Phe-Met-OH). First, a tetra-
peptide, Boc-Tyr(But)-Gly-Gly-Phe was grown on oxime resin
using Boc-Phe-OH, Boc-Gly-Gly-OH and Boc-Tyr(But)-OH.
Then, the oxime resin 2d was split into two portions and the
peptide was transferred onto H-Leu-Wang 5d and H-Met-Wang
5e. After TFA cleavage, 7dd was obtained in reasonable purity
(36) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.;
Morgan, B. A.; Morris, H. R. Nature 1975, 258, 577-579.
(37) Akil, H.; Watson, S. J.; Young, E.; Lewis, M. E.; Khachaturian,
H.; Walker, J. M. Annu. ReV. Neurosci. 1984, 7, 223-255.
(35) DBU was used to facilitate the cleavage of peptides from oxime
resin by various oxygen nucleophiles. See: Pichette, A.; Voyer, N.;
Larouche, R.; Meillon, J.-C. Tetrahedron Lett. 1997, 38, 1279-1282.

1640 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Hamuro et al.
Table 2. Optimization of Transfer of Z-Ala from Oxime Resin 2d to H-Phe-Wang Resin 5aa
chaperoneb
additiveb
type
none
oximeb equiv
equiv
type
equiv
solvent
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CHCl3
DMF
CHCl3
CHCl3
CHCl3
CHCl3
ClCH2CH2Cl
ClCH2CH2Cl
CH2Cl2
CHCl3
CHCl3
CHCl3
ClCH2CH2Cl
ClCH2CH2Cl
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
ClCH2CH2Cl
ClCH2CH2Cl
temp, °C
time, h
yield,c %
(1) 1.6
(2) 1.6
(3) 1.6
(4) 1.6
(5) 1.6
(6) 1.6
(7) 1.6
(8) 1.5
(9) 2.0
(10) 2.0
(11) 2.0
(12) 2.5
(13) 1.6
(14) 1.5
(15) 2.0
(16) 2.0
(17) 2.0
(18) 2.5
(19) 1.6
(20) 1.6
(21) 1.5
(22) 2.0
(23) 2.0
(24) 2.0
(25) 2.5
6.4
6.4
6.4
6.4
6.4
6.4
6.4
30.0
6.0
30.0
6.0
6.0
6.4
30.0
6.0
30.0
6.0
6.0
6.4
6.4
30.0
6.0
HOSu
rt
rt
rt
rt
26
23
23
23
24
24
24
16
16
16
15
15
26
16
16
16
15
15
16
16
16
16
16
15
15
27
26
20
16
13
10
58
53
63
65
69
69
21
67
70
73
67
67
18
55
55
60
59
55
54
HOSu
HOSu
HOSu
HOSu
6.4
6.4
6.4
9.9
DBU
DMAP
Imidazole
AcOH
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
none
rt
HOSu
50
50
50
50
50
70
70
rt
50
50
50
70
70
rt
50
50
50
50
70
70
HOSu
HOSud
HOSu
HOSud
HOSu
HOSu
HOBt
HOBtd
HOBt
HOBtd
HOBt
HOBt
EACNOx
EACNOx
EACNOx
EACNOx
EACNOx
EACNOx
EACNOx
30.0
6.0
6.0
a All resin-to-resin acyl-transfer reactions were carried out with 0.047 or 0.050 mmol of H-Phe-Wang resin in 5 mL of solvent. b All equivalents
are relative to H-Phe-Wang resin. c The yield was obtained by HPLC with nitrobenzene as internal standard. d Chaperones were not completely
soluble.
Table 3. Epimerization Associated with Resin-to-Resin Transfer of
Boc-Leu-Ala 2b to H-Phe-Wang 5a by Three Chaperonesa
Resin-to-Resin Aminoacyl-Transfer Reactions. (a) Prepa-
ration of Oxime Carbamate Resins. Phosgenated oxime resin
8 can be reacted with either simple amines, amino acids, or
diamines. The reaction conditions for the amine attachment
depends on the solubilities of the amine. If the amine is soluble
in dichloromethane, this is the preferred solvent. In cases where
the amine is insoluble in dichloromethane, a solution of
DMF/dichloromethane, DMF, or pyridine can be used with
trimethylsilylating reagents (e.g., BSA,39 TMS-Cl40) if nec-
essary.
When the reaction is carried out with amino acids or diamines,
the resulting resin-bound intermediates may be further elaborated
by utilizing simple amide bond formation reactions for incor-
poration of additional functionalities31 (Scheme 5). In this
manner, many diverse functionalized oxime-derived carbamate
resins 9 can be generated.
(b) Thermolytic Resin-to-Resin Aminoacyl-Transfer Reac-
tions. The thermolytic resin-to-resin aminoacyl transfer was
typically carried out using N-terminally deprotected peptide-
Wang or amino acid-Wang resins 8 with 2 equiv of oxime
carbamate resin 9 in toluene at 80 °C for 24-48 h (Scheme 3
and Table 5). Following the resin-to-resin aminoacyl transfer,
the urea 13 was cleaved from the Wang resin by treatment with
50% TFA in CH2Cl2 for 1 h. When the aminoacyl group was
transferred to amino acid-Wang resins, the TFA cleavage gave
a mixture of hydantoic acid (13) and the corresponding
hydantoin (14; Scheme 6). Therefore, following acid cleavage,
the TFA solution was heated at 60 °C for 12 h to complete the
cyclization (entries 1, 6, and 9-12). In general, aryl carbamates
gave cleaner products in higher yield, presumably due to the
better stabilities of aryl isocyanates compared with alkyl
isocyanates.32,33,41 In the case of dipeptidyl Wang resin, good
chaperone
yield,b %
purity,c %
epimerization,d %
(1) HOSu
(2) HOBt
(3) EAcNOx
76
59
67
88
71
69
1.7
1.8
3.9
a Two equivalents of Boc-Leu-Ala-oxime resin 2b and 6 equiv of
chaperones were used for the reaction in ClCH2CH2Cl at 70 °C for 21
h. b The yield was obtained by HPLC with phenol as an internal
standard. c The purity was calculated by dividing the yields by
gravimetric yields. d The degree of epimerization was calculated from
the equation, epimerization ) mol (L-7ba)/(mol (L-7ba) + mol (D-
7ba)) after quantifying both isomers of L-7ba and D-7ba by HPLC
with phenol as internal standard.
and good yield (see Table 4, entry 10). However, Met-
enkephalin was obtained in very low purity with Met(SdO)-
enkephalin and H-Tyr-Gly-Gly-Phe-OH as major impurities after
TFA/thio-
anisole cleavage. The speculation was that methionine was
oxidized during RRTR due to the high temperature. Further,
thioanisole may catalyze the cleavage of excess peptide from
the oxime resin during the TFA cleavage reaction (2 equiv of
2d was used against 5e). Thus, for the synthesis of 7de,
thioanisole was added to RRTR reaction to minimize the
oxidation of methionine. Also, before TFA/thioanisole cleavage
of the peptide from the resin, diethyamine acetate was reacted
with the resin mixture to cleave excess peptide from the oxime
resin. In this modified manner, 7de was obtained in reasonable
purity and yield (see Table 4, entry 11). The degree of
epimerization in 7dd and 7de were found to be 4.0 and 3.9%,
respectively, by integration of the areas in their HPLC chro-
matograms (see Table 4).38
(38) Part of the racemization might have occurred during the attachement
of Boc-Phe onto oxime resin, as DMAP is known to cause racemization
for the coupling between Boc-Phe anhydride and Wang resin. See: Atherton,
E.; Benoiton, N. L.; Brown, E.; Sheppard, R. C.; Williams, B. J. Chem.
Commun. 1981, 336-337.
(39) Dressman, B. A.; Spangle, L. A.; Kaldor, S. W. Tetrahedron Lett.
1996, 37, 937-940.
(40) Bolin, D. R.; Sytwu, I.-I.; Humiec, F.; Meinhofer, J. Int. J. Pept.
Protein Res. 1989, 33, 353-359.

Acyl- and Aminoacyl-Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1641
Table 4. Resin-to-Resin Acyl Transfer with HOSu as the Chaperonea
acceptor group
on Wang resin
purity
(epimerization), %
acyl group transferred
(1) Z-Ala (2a)
(2) Z-Ala (2a)
product
yield, %
H-Phe (5a)
Z-Ala-Phe-OH (7aa)
69b
90d
75d
76b
70d
80d
83d
67d
68d
87b
53b
77c
H-Ala-Phe (5b)
H-Val-Phe (5c)
H-Phe (5a)
H-Ala-Phe (5b)
H-Val-Phe (5c)
H-Phe (5a)
H-Ala-Phe (5b)
H-Val-Phe (5c)
H-Leu (5d)
Z-Ala-Ala-Phe-OH (7ab)
Z-Ala-Val-Phe-OH (7ac)
H-Leu-Ala-Phe-OH (7ba)
H-Leu-Ala-Ala-Phe-OH (7bb)
H-Leu-Ala-Val-Phe-OH (7bc)
Z-Leu-Ala-Phe-OH (7ca)
Z-Leu-Ala-Ala-Phe-OH (7cb)
Z-Leu-Ala-Val-Phe-OH (7cc)
Leu-Enkephalin (7dd)
95e
(3) Z-Ala (2a)
87e
(4) Boc-Leu-Ala (2b)
(5) Boc-Leu-Ala (2b)
(6) Boc-Leu-Ala (2b)
(7) Z-Leu-Ala (2c)
71c (1.7)f
96e
93e
93e
(8) Z-Leu-Ala (2c)
97e
(9) Z-Leu-Ala (2c)
91e
(10) Boc-Tyr(But)-Gly-Gly-Phe (2d)
(11) Boc-Tyr(But)-Gly-Gly-Phe (2d)
71e (4.0)g
68e (3.9)g
H-Met (5e)
Met-Enkephalin (7de)
a
Two equivalents of acylated oxime resin and 6 equiv of HOSu were used for the resin-to-resin acyl-transfer reactions in ClCH2CH2Cl. b The
yields were obtained by HPLC with phenol as internal standard. c The degree of purity of the crude product was estimated by HPLC, comparing
the integrated intensity of the desired peak relative to the side products at 220 nm. d Isolated yields after triturating the crude product with MeOH/
CH2Cl2/Et2O/hexanes. e The purity was determined as in footnote c after triturating the crude product. The degree of epimerization was calculated
f
from the equation, epimerization ) mol (D-7ba)/(mol (L-7ba) + mol (D-7ba)) after quantifying both isomers of L-7ba and D-7ba by HPLC with
phenol as internal standard. g The degree of epimerization was calculated from the integrated intensities of the two epimers for enkephalins.
Scheme 5
yields and purities were obtained for the p-anisidine isocyanate
transfer, although diketopiperazines were observed in the
washings after the thermolytic aminoacyl-transfer reactions
(entries 2 and 3). The aminoacyl-transfer reactions from oxime
carbamate resin to H-Phe-2-chlorotrityl resin was also attempted
(entries 13 and 14). Although small amounts of the desired
products were observed in mass spectroscopic analysis, the
reaction mixtures contained numerous impurities and were not
optimized further.
employed for the preparation of hydantoins via thermolytic
cyclitive cleavage of the carbamate amino acid amides with
triethylamine in methanol.39 To determine whether this reaction
occurs via an isocyanate intermediate or via direct nucleophilic
displacement of the Wang benzyloxy group, we attempted resin-
to-resin aminoacyl transfer of these substrates under the same
reaction conditions used for carbamates derived from phos-
genated oxime resin (entries 15-18). Surprisingly, neither the
carbamate of p-anisidine nor the carbamate of benzylamine
showed any transfer to the acceptor resin, H-Phe-Wang. Both
HPLC and mass spectroscopy showed phenylalanine as the
major product, and no hydantoins or hydantoic acids were
observed. This indicates that, unlike the carbamates derived from
phosgenated oxime resin, carbamates derived from Wang resin
do not undergo thermolytic isocyanate generation.
Also, carbamates of Wang resin, derived from the com-
mercially available p-nitrophenyl carbonate, have recently been
(41) Menger and co-worker studied the mechanism of aminolysis of
p-nitrophenyl N-phenylcarbamate in toluene and found this compound reacts
104 times faster than the N-methylated compound and 200 faster than
N-methylcarbamate; see: Menger, F. M.; Glass, L. E. J. Org. Chem. 1974,
39, 2469-2470.

1642 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Table 5. Resin-to-Resin Aminoacyl Transfera
Hamuro et al.
donor resin
(1) 8
(2) 8
(3) 8
(4) 8
(5) 8
(6) 8
(7) 8
(8) 8
(9) 8
(10) 8
(11) 8
(12) 8
(13) 8
(14) 8
(15) WangCOPnp
(16) WangCOPnp
(17) WangCOPnpc
(18) WangCOPnpc
first fragment
second fragment
acceptor resin
Phe-Wang (5a)
product
yield,b %
purity,b %
p-anisidine
p-anisidine
p-anisidine
p-anisidine
p-anisidine
benzylamine
benzylamine
benzylamine
p-nitroaniline
2-aminopyridine
p-Aba
14aa
13ab
13ac
13af
13ag
14ba
13bb
13bc
14ca
14da
14ea
14fa
80d
68
83
66
60
46d
37
33
79d
77d
23
22d
0e
93
89
87
78
46
76
61
80
89
87
50
49
Ala-Phe-Wang (5b)
Val-Phe-Wang (5c)
Val-Ala-Phe-Wang (5f)
Leu-Ala-Phe-Wang (5g)
Phe-Wang (5a)
Ala-Phe-Wang (5b)
Val-Phe-Wang (5c)
Phe-Wang (5a)
Phe-Wang (5a)
Phe-Wang (5a)
Phe-Wang (5a)
Phe-ClTrt
p-anisidine
p-Nbz-Cl
p-Pda
p-anisidine
benzylamine
p-anisidine
benzylamine
p-anisidine
benzylamine
Phe-ClTrt
0e
Phe-Wang (5a)
Phe-Wang (5a)
Phe-Wang (5a)
Phe-Wang (5a)
nrf
nrf
nrf
nrf
a p-Aba, p-aminobenzoic acid; p-Pda, p-phenylenediamine; p-Nbz-Cl, p-nitrobenzoyl chloride; ClTrt, 2-chlorotrityl resin; WangCOPnp, Wang
p-nitrophenylcarbonate resin. b Purities are HPLC area percent of crude products at 220 nm. Yields are isolated yields after purification. c Amide
transfer from p-nitrophenylcarbonate Wang resin with HOBt. d The isolated compounds were hydantoins, not hydantoic acids. e The desired products
were observed in the crude products by MS, but HPLC spectra were too complicated to identify the product peaks and the compounds could not
be isolated. f None of the desired products were observed.
Scheme 6
General Procedure for Peptidyl-Oxime Resin 2.42 (a) First Amino
Conclusion
Acid Attachment and Capping. A mixture of oxime resin 1 (5.26 g,
Although resin-to-resin transfer reactions were demonstrated
as early as 1975, they have had very limited applications for
preparative purposes. In this paper we demonstrate that, under
optimized conditions, acyl- and aminoacyl-transfer reactions
proceed in good yield and high chemical and stereochemical
purity. This method should be broadly generalizable to a number
of other transfer reactions and provides a convergent route for
the solid-phase synthesis of libraries of small and large
molecules.
0.76 mmol/g, 4.0 mmol), N-protected amino acid (8.0 mmol), DMAP
(0.98 g, 8.0 mmol), and DIC (1.25 mL, 8.0 mmol) in CH2Cl2 (80 mL)
was placed in a peptide synthesis apparatus, and the mixture was
bubbled with N2 at room temperature overnight. After the solution was
drained, the resin was washed with DMF × 3, MeOH × 3, CH2Cl2 ×
3, and DMF × 3. The unreacted oxime groups were capped by reaction
with pivaloyl anhydride or acetic anhydride (20 mmol) and DIPEA
(3.48 mL, 20 mmol) in DMF (30 mL) at room temperature for 2 h.
After the solution was drained, the resin was washed with DMF × 3,
MeOH × 3, and CH2Cl2 × 3.
(b) Second or after Amino Acid Attachment. The Boc group was
deprotected with 25% TFA in CH2Cl2 (60 mL) for 40 min. After the
solution was drained, the resin was washed with CH2Cl2 × 3, MeOH
× 3, and DMF × 3. The resin was coupled to N-protected amino acid
(8.0 mmol, only in the case of Boc-Tyr(But)-Gly-Gly-Phe-oxime resin
2d, Boc-Gly-Gly-OH was used instead of Boc-Gly-OH twice) with
HBTU (3.03 g, 8.0 mmol), HOBt‚H2O (1.22 g, 8.0 mmol), and DIPEA
(4.18 mL, 24.0 mmol) in DMF (30 mL) for 2 h. After the solution was
drained, the resin was washed with DMF × 3, MeOH × 3, and CH2Cl2
× 3 and then dried in vacuo.
Experimental Section
General Procedures. Oxime resin was prepared using Biobeads
SX-1 (1% cross-linked polystyrene) from Biorad.17 Leu-enkephalin and
met-enkephalin were purchased from Sigma. Other protected amino
acids and peptides were purchased from Bachem Bioscience Inc. and
used without purification. HBTU was purchased from Advanced
ChemTech, and amino acid-attached resins were purchased from
Novabiochem. All other reagents were purchased from Aldrich.
Thermolytic transfer reactions were carried out in 20-mL scintillation
vials from Kimble Glass Inc. of Vineland, NJ. IR spectra of the
functionalized resins were obtained with a Perkin-Elmer FT1600
infrared spectrometer using a KBr press. Low-resolution mass spectra
were obtained with a VG Trio-2000 quadruple mass spectrometer using
the atmospheric pressure chemical ionization (APCI) technique. High-
resolution mass spectra were obtained with a VG-70-VSE high-
resolution mass spectrometer. NMR experiments were carried out on
Bruker DRX-500 and DRX-300 spectrometers. HPLC analyses were
performed on a Hewlett-Packard 1090 liquid chromatography system
using a photodiode array detector and a Vydac C18 column, 2.1 × 150
mm. HPLC area ratios in the text were determined by integrating the
areas of the peaks at 220 nm.
General Procedure for Peptidyl Wang Resin 5.43 Fmoc-Xxx-Wang
resin (1.25 mmol) and 20% piperidine in DMF (20 mL) were placed
in a benchtop peptide synthesis apparatus, and N2 was bubbled at room
temperature for 20 min. In the case of monoamino acid-attached Wang
resin, after the solution was drained, the resin was washed with DMF
× 3, MeOH × 3, and CH2Cl2 × 3 and dried in vacuo to obtain 5. In
the case of dipeptidyl Wang resin, the second amino acid attachment
(42) Jackson, S.; DeGrado, W.; Dwivedi, A.; Parthasarathy, A.; Higley,
A.; Krywko, J.; Rockwell, A.; Markwalder, J.; Wells, G.; Wexler, R.; Mousa,
S.; Harlow, R. J. Am. Chem. Soc. 1994, 116, 3220-3230.
(43) NoVabiochem Catalog & Peptide Synthesis Handbook; 1997; pp
S58-S59.

Acyl- and Aminoacyl-Transfer Reactions
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1643
is as follows. The solution was drained and the resin was washed with
DMF × 3, i-PrOH × 3, and DMF × 3. A solution of Fmoc-Xxx-OH
(2.5 mmol, 2.0 equiv), HBTU (948 mg, 2.5 mmol, 2.0 equiv), and
HOBt‚H2O (383 mg, 2.5 mmol, 2.0 equiv) in DMF (10 mL) was added
to the resin. DIPEA (0.87 mL, 5.0 mmol, 4.0 equiv) was added to the
mixture, which was then bubbled with N2 at room temperature for 1 h.
The solution was drained and washed with DMF × 3, 20% piperidine
in DMF (20 mL) was added to the resin, and N2 was bubbled at room
temperature for 20 min. After the solution was drained, the resin was
washed with DMF × 3, MeOH × 3, and CH2Cl2 × 3 and dried in
vacuo to obtain 5.
General Procedure for the Attachment of Amines to Phosgenated
Oxime Resin 9. To a solution of amine (15 mmol) in CH2Cl2 (75 mL)
was added phosgenated oxime resin (8, 0.711 mmol/g, 7.03 mg, 5.0
mmol), and the reaction mixture was stirred at room temperature for 2
h. The resin was collected on a glass filter, washed with CH2Cl2 × 2,
DMF × 3, MeOH × 4, and CH2Cl2 × 4, and dried in vacuo. The
reaction condition for the amine attachment depends on the solubility
of the amine. If the amine is soluble in CH2Cl2, this is the preferred
solvent. In case the amine is not soluble in CH2Cl2, DMF/CH2Cl2, DMF,
or pyridine can be used with a trimethylsilylating reagent (e.g., BSA,39
TMS-Cl40) if necessary.
General Procedure for Thermolytic Aminoacyl-Transfer Reac-
tion 12. A mixture of the peptidyl Wang resin (5, 0.20 mmol) and
oxime carbamate resin (9, 0.40 mmol) in toluene (16 mL) was shaken
at 80 °C for 44 h. The resin mixture was collected on a glass filter,
washed with DMF × 4, MeOH × 4, and CH2Cl2 × 3, and dried in
vacuo.
General Procedure for the Cleavage of Ureas from Wang Resin
13. To the above resin mixture 12 was added CH2Cl2 (5 mL) and TFA
(5 mL), and the mixture was shaken at room temperature for 1 h. The
resin was removed by filtration and washed with 25% TFA in CH2Cl2
× 2 and CH2Cl2 × 2. The filtrates were combined and evaporated in
vacuo to give the crude cleavage product.
General Procedure for the Optimization of Resin-to-Resin Acyl
Transfer. A suspension of Z-Ala-oxime resin 2a, H-Phe-Wang resin
5a (0.05 mmol), a chaperone candidate, and an additive in 5 mL of
solvent was prepared in a vial, which was then shaken overnight at the
designated temperature. The resin mixture was collected and washed
with the reaction solvent × 3, MeOH × 3, and CH2Cl2 × 3. The
collected resin mixture was treated with 50% TFA in CH2Cl2 for 1 h,
and then the resin mixture was filtered and washed with CH2Cl2 × 3.
The filtrates were combined and evaporated. The amount of Z-Ala-
Phe-OH generated was quantified by HPLC with nitrobenzene as an
internal standard.
General Procedure for the Epimerization Test of Resin-to-Resin
Acyl Transfer. A suspension of Boc-Leu-Ala-oxime resin 2b (302 mg,
0.66 mmol/g, 0.20 mmol), H-Phe-Wang resin 5a (172 mg, 0.58 mmol/
g, 0.10 mmol), and a chaperone candidate (0.60 mmol) in ClCH2CH2-
Cl (10 mL) was prepared in a vial, which was then shaken at 70 °C for
21 h. The resin mixture was collected and washed with CH2Cl2 × 3,
DMF × 3, MeOH × 3, and CH2Cl2 × 3. The collected resin mixture
was treated with 50% TFA in CH2Cl2 (6 mL) for 70 min, and then the
resin mixture was filtered off and washed with CH2Cl2 × 3. The filtrates
were combined and evaporated. The amounts of L-7ba and D-7ba
generated were quantified by HPLC with phenol as an internal standard.
General Procedure for Thermolytic Resin-to-Resin Acyl-Transfer
Reaction from Oxime Resin to Wang Resin and Cleavage of Peptide
7. A suspension of acylated oxime resin (2, 0.50 mmol), peptidyl Wang
resin (5, 0.25 mmol), and HOSu (173 mg, 1.50 mmol) in ClCH2CH2-
Cl (15 mL) was prepared in a 20-mL vial, which was then shaken
overnight at 70 °C. The resins were collected on a glass filter and
washed with CH2Cl2 × 3, DMF × 3, MeOH × 3, and CH2Cl2 × 3. To
the resin mixture was added 50% TFA in CH2Cl2 (15 mL), and the
mixture was shaken at room temperature for 75 min. The resin was
filtered and washed with CH2Cl2 × 3. The filtrate and washings were
combined and evaporated in vacuo to give the crude cleavage product.
The crude product was dissolved in MeOH/CH2Cl2 and filtered through
a pipet with cotton. The solution was triturated with Et2O and hexanes
to obtain the product.
Procedure for Thermolytic Resin-to-Resin Acyl-Transfer Reac-
tion from Oxime Resin to Wang Resin and Cleavage of Methionine-
Containing Peptide. A suspension of acylated oxime resin (2, 0.40
mmol), peptidyl Wang resin (5, 0.20 mmol), HOSu (138 mg, 1.2 mmol),
and thioanisole (0.47 mL, 4.0 mmol) in ClCH2CH2Cl (15 mL) was
prepared in a 20-mL vial, which was then shaken overnight at 70 °C.
The resins were collected on a glass filter and washed with CH2Cl2 ×
3, DMF × 3, MeOH × 3, and CH2Cl2 × 3. To the resin mixture was
added diethylamine (103 µL, 1.0 mmol) and AcOH (57 µL, 1.0 mmol)
in CH2Cl2 (15 mL), and the resultant mixture was stirred at room
temperature overnight to cleave excess peptide from the oxime resin.
To the resin mixture above was added 5% thioanisole in 50% TFA/
50% CH2Cl2 (15 mL), and the mixture was shaken at room temperature
for 90 min. The resin was filtered and washed with CH2Cl2 × 3 and
MeOH × 3. The filtrate and washings were combined and evaporated
in vacuo to give the crude cleavage product. The crude product was
dissolved in MeOH/CH2Cl2 and filtered through a pipet with cotton.
The solution was triturated with Et2O and hexanes to obtain the product.
Preparation of Phosgenated p-Nitrophenyl(polystyrene)ketoxime
(8, Phoxime Resin). The phosgenated oxime resin was prepared from
oxime resin 1 by the procedure in ref 30. The loading of the resin was
calculated as 0.711 mmol/g by the method described in the same
reference: IR 3025, 2922, 1944, 1871, 1800, 1601, 1525, 1492, 1451,
1347 cm-1. Anal. found Cl, 2.52%.
General Procedure for Hydantoin (14) Formation. The above-
mentioned cleavage procedure gives a mixture of hydantoin (14) and
hydantoic acid (13) when an amino acid Wang resin was used. To
complete the cyclization reaction, 2 mL of TFA was added to the crude
product and the mixture was shaken at 60 °C for 12 h. The TFA was
evaporated in vacuo to give the crude hydantoin product.
3-[(4-Methoxy)phenyl]-5-[(S)-phenylmethyl]hydantoin (14aa). The
HPLC purity of the crude product was 93%. The crude product was
purified by column chromatography (SiO2, 1/20 ) MeOH/CH2Cl2) to
obtain the titled compound as a pale yellow solid (47 mg, 80%): mp
121-122 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.43 (s, 1H, Phe-
NH), 7.4-7.2 (m, 5H, aromatics), 6.94 (d, J ) 8.9 Hz, 2H, anisidine),
6.86 (d, J ) 8.9 Hz, 2H, anisidine), 4.53 (t, J ) 4.5 Hz, 1H, NHCH),
3.76 (s, 3H, CH3O), 3.07 (m, 2H, PhCH2); 13C NMR (125 MHz,
DMSO-d6) δ 173.7, 156.7, 136.1, 130.9, 129.1, 128.9, 127.9, 125.6,
115.0, 58.0, 56.4, 37.7; HRMS m/e calcd for C17H16N2O3 (M+)
296.1161, found 296.1139.
N-((4-Methoxy)phenylaminocarbonyl)alanylphenylalanine (13ab).
The HPLC purity of the crude product was 89%. The crude product
was purified by column chromatography (SiO2, 1/6/60 ) AcOH/MeOH/
CH2Cl2) to obtain the titled compound as a pale yellow solid (52 mg,
1
68%): mp 194-197 °C; H NMR (500 MHz, DMSO-d6) δ 8.44 (s,
1H, Ph-NH), 8.05 (d, J ) 7.7 Hz, 1H, Phe-NH), 7.2-7.0 (m, 7H,
aromatics), 6.73 (d, J ) 8.9 Hz, 2H, anisidine), 6.22 (d, J ) 7.7 Hz,
1H, Ala-NH), 4.31 (m, 1H, NHCH), 4.15 (m, 1H, NHCH), 3.61 (s,
3H, CH3O), 3.02 (dd, J ) 13.8, 4.9 Hz, 1H, Ph CHH), 2.84 (dd, J )
13.8, 8.8 Hz, 1H, Ph CHH), 1.11 (d, J ) 6.9 Hz, 3H, Ala-CH3); 13C
NMR (125 MHz, DMSO-d6) δ 173.0, 172.5, 154.5, 153.9, 137.8, 133.4,
129.1, 128.0, 126.2, 119.2, 113.8, 55.1, 53.7, 40.0, 36.7, 19.5; HRMS
m/e calcd for C20H23N3O5 (M+) 385.1638, found 385.1649.
N-((4-Methoxy)phenylaminocarbonyl)valylphenylalanine (13ac).
The HPLC purity of the crude product was 87%. The crude product
was purified by column chromatography (SiO2, 1/6/60 ) AcOH/MeOH/
CH2Cl2) to obtain the titled compound as a yellow solid (68 mg,
1
83%): mp 189-191 °C; H NMR (500 MHz, DMSO-d6) δ 8.50 (s,
1H, Ph-NH), 8.22 (d, J ) 7.7 Hz, 1H, Phe-NH), 7.3-7.1 (m, 7H,
aromatics), 6.80 (d, J ) 9.0 Hz, 2H, anisidine), 6.17 (d, J ) 9.0 Hz,
1H, Val-NH), 4.42 (m, 1H, NHCH), 4.14 (m, 1H, NHCH), 3.69 (s,
3H, CH3O), 3.07 (dd, J ) 13.9, 5.1 Hz, 1H, Ph CHH), 2.91 (dd, J )
13.9, 9.0 Hz, 1H, Ph CHH), 1.97 (m, 1H, CH(CH3)2), 0.86 (d, J ) 6.8
Hz, 3H, Val-CH3), 0.78 (d, J ) 6.8 Hz, 3H, Val-CH3); 13C NMR (125
MHz, DMSO-d6) δ 172.9, 171.5, 155.0, 153.8, 137.7, 133.5, 129.0,
128.0, 126.2, 119.0, 113.8, 57.0, 55.0, 53.5, 36.6, 31.1, 19.1, 17.3;
HRMS m/e calcd for C22H27N3O5 (M+) 413.1951, found 413.1962.
N-((4-Methoxy)phenylaminocarbonyl)valylalanylphenylalanine
(13af). The HPLC purity of the crude product was 78%. The crude
product was purified by repeated trituration with Et2O to obtain the
titled compound as an ivory solid (63 mg, 66%): mp 222-225 °C; 1H

1644 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Hamuro et al.
NMR (500 MHz, DMSO-d6) δ 8.47 (s, 1H, Ph-NH), 8.08 (d, J ) 7.6
Hz, 1H, Phe-NH), 7.97 (d, J ) 7.8 Hz, 1H, Ala-NH), 7.3-7.1 (m, 7H,
aromatics), 6.79 (d, J ) 12.5 Hz, 2H, anisidine), 6.18 (d, J ) 8.9 Hz,
1H, Val-NH), 4.41 (m, 1H, Phe-NHCH), 4.31 (quintet, J ) 7.2 Hz,
1H, Ala-NHCH), 4.12 (dd, J ) 5.3, 8.8 Hz, 1H, Val-NHCH), 3.67 (s,
3H, CH3O), 3.03 (dd, J ) 14.0, 5.4 Hz, 1H, Ph CHH), 2.90 (dd, J )
13.9, 8.3 Hz, 1H, Ph CHH), 1.93 (m, 1H, CH(CH3)2), 1.18 (d, J ) 7.1
Hz, 3H, Ala-CH3), 0.84 (d, J ) 6.9 Hz, 3H, Val-CH3), 0.76 (d, J )
6.8 Hz, 3H, Val-CH3); 13C NMR (125 MHz, DMSO-d6) δ 172.6, 172.0,
171.3, 155.2, 153.9, 137.4, 133.6, 129.1, 128.1, 126.4, 119.1, 114.0,
57.2, 55.2, 53.3, 47.9, 36.7, 31.2, 19.3, 18.2, 17.5; HRMS m/e calcd
for C25H33N4O6 (M + H+) 485.2400, found 485.2394.
N-((4-Methoxy)phenylaminocarbonyl)leucylalanylphenyl-
alanine (13ag). The HPLC purity of the crude product was 46%. The
crude product was purified by repeated trituration with Et2O to obtain
the titled compound as an ivory solid (60 mg, 60%): mp 217-220
°C; 1H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H, Ph-NH), 8.11 (d, J
) 7.6 Hz, 1H, Phe-NH), 7.93 (d, J ) 7.7 Hz, 1H, Ala-NH), 7.3-7.1
(m, 7H, aromatics), 6.79 (d, J ) 9.0 Hz, 2H, anisidine), 6.20 (d, J )
8.4 Hz, 1H, Leu-NH), 4.39 (m, 1H, Phe-NHCH), 4.3-4.2 (m, 2H, Ala-
NHCH + Val-NHCH), 3.68 (s, 3H, CH3O), 3.04 (dd, J ) 13.9, 5.2
Hz, 1H, Ph CHH), 2.89 (m, 1H, Ph CHH), 1.62 (m, 1H, CH(CH3)2),
1.5-1.3 (m, 2H, CH2CH(CH3)2), 1.19 (d, J ) 7.1 Hz, 3H, Ala-CH3),
0.87 (d, J ) 6.0 Hz, 6H, Leu-CH3); 13C NMR (125 MHz, DMSO-d6)
δ 173.0, 172.8, 172.3, 155.3, 154.3, 137.7, 133.8, 129.5, 128.4, 126.7,
119.5, 114.2, 55.5, 53.8, 51.4, 48.2, 37.0, 24.5, 23.5, 22.1, 18.4; HRMS
m/e calcd for C26H35N4O6 (M + H+) 499.2557, found 499.2585.
3-(Phenylmethyl)-5-[(S)-phenylmethyl]hydantoin (14ba). The HPLC
purity of the crude product was 76%. The crude product was purified
by column chromatography (SiO2, 1/20 ) MeOH/CH2Cl2) to obtain
the titled compound as an ivory solid (26 mg, 46%): mp 131-133
°C; 1H NMR (500 MHz, DMSO-d6) δ 8.22 (s, 1H, Phe-NH), 7.2-7.1
(m, 10H, aromatics), 6.69 (m, 2H, aromatics),4.38 (t, J ) 4.3 Hz, 1H,
Phe-NHCH), 4.31 (d, J ) 16.2 Hz, 1H, PhCHHN), 4.21 (d, J ) 15.6
Hz, 1H, PhCHHN), 2.90 (d, J ) 4.7 Hz, 1H, Ph CH2CH); 13C NMR
(125 MHz, DMSO-d6) δ 173.9, 156.9, 137.0, 135.7, 130.6, 129.0, 128.8,
127.6, 127.5, 127.3, 57.9, 41.4, 36.8; HRMS m/e calcd for C17H16N2O2
(M+) 280.1212, found 280.1211.
3-(4-Nitrophenyl)-5-[(S)-phenylmethyl]hydantoin (14ca). The HPLC
purity of the crude product was 89%. The crude product was purified
by column chromatography (SiO2, 1/20 ) MeOH/CH2Cl2) to obtain
the titled compound as an yellow solid (48 mg, 79%): mp 188-189
°C; 1H NMR (500 MHz, DMSO-d6) δ 8.75 (br, 1H, NH), 8.30 (d, J )
8.8 Hz, 2H, NO2Ph-3H and 5H), 7.46 (d, J ) 8.8 Hz, 2H, NO2Ph-2H
and 6H), 7.4-7.2 (m, 5H, Ph), 4.62 (m, 1H, NHCH), 3.11 (m, 2H,
PhCH2); 13C NMR (125 MHz, DMSO-d6) δ 171.9, 154.2, 145.7, 137.5,
134.8, 129.5, 128.0, 126.8, 126.2, 123.9, 57.0, 36.5; HRMS m/e calcd
for C16H13N3O4 (M+) 311.0906, found 311.0905.
N-(2-Pyridylaminocarbonyl)phenylalanine (14da). The HPLC
purity of the crude product was 87%. The crude product was purified
by column chromatography (SiO2, 1/5/100 ) AcOH/MeOH/CH2Cl2)
to obtain the titled compound as a pale yellow oil (42 mg, 77%): 1H
NMR (500 MHz, DMSO-d6) δ 12.87 (br, 1H, COOH), 9.33 (br, 1H,
NH-Py), 8.59 (br, 1H, NH-Ala), 8.11 (dd, J ) 4.9, 1.2 Hz, 1H, Py-
5H), 7.66 (td, J ) 7.8, 1.9 Hz, Py-4H), 7.4-7.2 (m, 6H, Py-3H and
Ph), 6.91 (td, J ) 5.0, 0.7 Hz, 1H, Py-5H), 4.46 (m, 1H, NHCH), 3.13
(dd, 1H, J ) 13.7, 5.0 Hz, PhCHH), 2.99 (dd, 1H, J ) 13.6, 7.4 Hz,
PhCHH); 13C NMR (125 MHz, DMSO-d6) δ 173.1, 154.2, 153.1, 146.3,
138.0, 137.0, 129.2, 128.0, 126.4, 116.7, 111.4. 53.8, 37.2; HRMS m/e
calcd for C15H13N3O2 (M+) 267.1008, found 267.1013.
3-(4-(4-Methoxy)phenylcarbamoyl)phenyl-5-[(S)-phenylmethyl]-
hydantoin (14ea). The HPLC purity of the crude product was 50%.
The crude product was purified by column chromatography (SiO2, 1/50
) MeOH/CH2Cl2) to obtain the titled compound as a pale yellow solid
1
(18 mg, 23%): mp 221-223 °C; H NMR (500 MHz, DMSO-d6) δ
10.13 (s, 1H, anisidine-NH), 8.60 (s, 1H, Phe-NH), 7.94 (d, 9.7 Hz,
2H, p-Aba), 7.66 (d, 9.2 Hz, 2H, p-Aba), 7.4-7.2 (m, 5H, Phe), 7.17
(d, J ) 8.6 Hz, 2H, anisidine), 6.92 (d, J ) 9.1 Hz, 2H, anisidine),
4.58 (td, J ) 4.9, 1.0 Hz, 1H, NHCH), 3.75 (s, 3H, CH3O), 3.09 (m,
2H, Ph CH2); 13C NMR (125 MHz, DMSO-d6) δ 172.3, 164.4, 155.6,
154.9, 135.0, 134.4, 134.1, 132.1, 129.8, 128.1, 127.9, 126.9, 125.8,
121.9, 113.7, 57.1, 55.1, 36.6; HRMS m/e calcd for C24H22N3O4 (M +
H+) 416.1611, found 416.1598.
3-[4-(4-Nitrophenyl)carbonylamino)phenyl]-5-[(S)-phenylmethyl]-
hydantoin (14fa). The HPLC purity of the crude product was 49%.
The crude product was purified by column chromatography (SiO2, 1/50
) MeOH/CH2Cl2) to obtain the titled compound as a pale yellow solid
N-(Phenylmethylaminocarbonyl)alanylphenylalanine (13bb). The
HPLC purity of the crude product was 61%. The crude product was
purified by column chromatography (SiO2, 1/6/60 ) AcOH/MeOH/
CH2Cl2) to obtain the titled compound as an ivory solid (28 mg, 37%):
1
(18 mg, 22%): mp 253-256 °C; H NMR (500 MHz, DMSO-d6) δ
10.65 (s, 1H, p-Pda-NH), 8.50 (s, 1H, Phe-NH), 8.37 (d, 8.8 Hz, 2H,
p-Nba), 8.19 (d, 8.8 Hz, 2H, p-Nba), 7.77 (d, 8.8 Hz, 2H, p-Pda), 7.4-
7.2 (m, 5H, Phe), 6.99 (d, J ) 8.8 Hz, 2H, p-Pda), 4.55 (t, J ) 4.6 Hz,
1H, NHCH), 3.07 (m, 2H, Ph CH2); 13C NMR (125 MHz, DMSO-d6)
δ 172.7, 164.1, 155.5, 149.4, 140.5, 138.2, 135.2, 129.9, 129.4, 128.2,
127.8, 127.6, 126.9, 123.7, 120.7, 57.2, 36.8; HRMS m/e calcd for
C23H19N4O5 (M + H+) 431.1356, found 431.1377.
1
mp 195-197 °C; H NMR (500 MHz, DMSO-d6) δ 7.90 (d, J ) 7.5
Hz, 1H, Phe-NH), 7.3-7.0 (m, 10H, aromatics), 6.46 (t, J ) 6.0 Hz,
1H, BnNH2), 6.10 (d, J ) 7.8 Hz, 1H, Ala-NH), 4.27 (m, 1H, NHCH),
4.10 (m, 3H, PhCH2NH + NHCH), 3.01 (dd, J ) 13.7, 5.1 Hz, 1H, Ph
CHH), 2.84 (dd, J ) 13.7, 8.1 Hz, 1H, Ph CHH), 1.07 (d, J ) 7.0 Hz,
3H, Ala-CH3); 13C NMR (125 MHz, DMSO-d6) δ 173.0, 172.7, 157.2,
140.6, 137.9, 129.2, 128.2, 128.0, 126.9, 126.5, 126.2, 53.8, 48.6, 42.8,
36.8, 19.5; HRMS m/e calcd for C20H23N3O4 (M+) 369.1689, found
369.1704.
Acknowledgment. We thank Linda Cox (DuPont CR&D)
for technical assistance and Mr. Richard McKay, Mr. Jeff
Krieger, and Mr. Jim Valentine (DuPont CCAS) for mass
spectrometry. This work was supported by National Science
Foundation GOALI Grant CHE9634646. Contribution No. 7782
from DuPont Central Research and Development.
N-(Phenylmethylaminocarbonyl)valylphenylalanine (13bc). The
HPLC purity of the crude product was 80%. The crude product was
purified by column chromatography (SiO2, 1/5/100 ) AcOH/MeOH/
CH2Cl2) to obtain the titled compound as an ivory solid (26 mg, 33%):
1
mp 190-192 °C; H NMR (500 MHz, DMSO-d6) δ 8.02 (d, J ) 7.1
Hz, 1H, Phe-NH), 7.4-7.1 (m, 10H, aromatics), 6.55 (t, J ) 6.0 Hz,
1H, BnNH2), 6.06 (d, J ) 9.2 Hz, 1H, Ala-NH), 4.35 (m, 1H, NHCH),
4.20 (m, 2H, PhCH2NH), 4.08 (m, 1H, NHCH), 3.06 (dd, J ) 13.8,
5.3 Hz, 1H, Ph CHH), 2.84 (dd, J ) 13.8, 8.3 Hz, 1H, Ph CHH), 1.94
(m, 1H, CH(CH3)2), 0.83 (d, J ) 6.8 Hz, 3H, Val-CH3), 0.75 (d, J )
6.8 Hz, 3H, Val-CH3); 13C NMR (125 MHz, DMSO-d6) δ 173.0, 171.7,
157.8, 140.7, 138.0, 129.2, 128.2, 128.0, 126.9, 126.5, 126.2, 57.6,
53.8, 48.6, 42.8, 36.8, 31.1, 19.3, 17.4; HRMS m/e calcd for C22H27N3O4
(M+) 397.2002, found 397.1995.
Supporting Information Available: Preparation and char-
acterization of resins (2a-2d and 5a-5e) and peptides 7aa-
7de as well as HPLC chromatograms of peptides 7aa-7de
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
JA9818654