Solid-Phase Synthesis of Arginine-Containing Peptides by Guanidine Attachment to a Sulfonyl Linker

Full text of DOI:10.1021/jo970736n

9
326
J . Org. Chem. 1997, 62, 9326-9330
Solid -P h a se Syn th esis of
of a suitable arenesulfonyl linker came to mind. In this
paper, we present our investigation of nitrogen attach-
ment to a resin-bound arenesulfonyl linker that is stable
to various reaction conditions and compatible with both
Boc and Fmoc peptide chemistry.5 Thus, four peptide
products (12, 15, 18, and 19) were constructed by addition
of protected amino acids to either or both termini of the
growing chain.
Ar gin in e-Con ta in in g P ep tid es by
Gu a n id in e Atta ch m en t to a Su lfon yl Lin k er
,6
†
H. Marlon Zhong, Michael N. Greco, and
Bruce E. Maryanoff*
Drug Discovery, The R. W. J ohnson Pharmaceutical
Research Institute, Spring House, Pennsylvania 19477
Resu lts a n d Discu ssion
Received April 24, 1997
Given the general utility of electron-rich N-sulfonyl
protecting groups for the guanidine moiety, we designed
4
Combinatorial chemistry methods have unleashed a
powerful strategy for the diversity-based discovery of new
research leads with desired chemical, biological, or physi-
cal properties.1 A centerpiece in this area is solid-phase
organic synthesis, which has been used for many years
in the effective preparation of oligopeptides and oligo-
nucleotides, but has only recently come to the forefront
in the preparation of small organic molecules. As a
critical part of the strategy of solid-phase organic syn-
thesis, one must be careful to select suitable polymer
supports and anchoring groups. Recent attention has
particularly focused on the development of new linker
technologies.1
a resin bearing a 4-(benzyloxy)benzenesulfonyl group, viz.
4, which could be readily obtained from the standard
Merrifield resin 1 (Scheme 1) and have useful loading
levels. Conditions for the solid-phase chemistry were
first established by performing a solution-phase model
reaction. Thus, benzyl chloride was successfully reacted
with sulfonate 2 in the presence of K CO and KI to give
2
3
the benzyl ether, and the corresponding sulfonyl chloride
was prepared by using PCl . By extension, Merrifield
5
resin 1 was reacted with 2 in a similar manner to
introduce the arenesulfonyl functionality onto the resin.
After any unreacted resin sites were capped with Et NH
b,e-g,2
2
In the course of a multistep resin-based synthesis, it
is necessary that the anchor and other protecting groups
be stable to the conditions employed, but be removable
through relatively mild or, at least, chemoselective
procedures. Typically, solid-phase synthesis of peptides
and related compounds is conducted by anchoring a
carboxy terminus to the resin and dressing up the amino
in DMF, sulfonic acid resin 3 was converted into desired
sulfonyl chloride resin 4 with PCl in DMF (Scheme 1).
5
Reaction of 4 with 1,3-propanediamine, followed by the
pentafluorophenyl ester of Fmoc-Gly, was intended to
provide 6 for assessing the resin-loading level. However,
the resin-loading of 0.14 mmol/g, determined spectro-
photometrically by liberation of the Fmoc chromophore
(dibenzofulvene) with piperidine in DMF (“Fmoc-echo”
procedure),7 was viewed as too low for further work,
considering the loading level for the commercial Merri-
field resin of 0.9 mmol/g.
terminus and/or various side chains with suitable pro-
tecting groups.1
b,e,g
Although linkage to the resin via an
amino terminus or side chain groups is also quite useful,
this approach is comparatively much less common.1b,3 For
the synthesis of arginine-containing derivatives, protec-
tion of the guanidine group with N-nitro, N-arenesulfo-
nyl, and N-carbalkoxy moieties is frequently required.4
However, it would seem expeditious to have a guanidine
blocking group that can simultaneously serve as an
anchor to the support. In seeking to develop an effective
means for mounting the guanidine group of an arginine
To improve the loading level of novel resin 4, we
8
changed the base in the alkylation of 1 to NaOMe. The
sodium phenoxide salt of 2 was prepared with 1.2 equiv
of NaOMe and reacted with 1 in N,N-dimethylacetamide
to afford 3, with a loading estimated at 0.70 mmol/g by
the increase in weight. Sulfonyl chloride resin 4 was
prepared from 3, and 4 now had a loading level of 0.63
mmol/g by weight increase and 0.51 mmol/g by elemental
analysis for Cl. Also, a loading level of 0.50 mmol/g was
realized after conversion of 4 to 6 and Fmoc-echo analy-
sis. This preferred NaOMe alkylation method (not
depicted in Scheme 1) is described in the Experimental
Section, and the improved batch of resin 4 was suitable
for solid-phase synthesis operations.
Boc-arginine was mounted onto resin 4 by using 4.0 N
KOH in 1,4-dioxane (Scheme 2) to give resin 7 with a
loading of 0.40 mmol/g by weight increase and 0.34
mmol/g by elemental analysis for N. Cleavage of 7 was
studied under different acidic conditions, such as
(or a related molecule for that matter) onto a solid
support, for conducting subsequent chemical steps, and
for releasing the ultimate product from the resin, the idea
*
Address correspondence to this author. Fax: 215-628-4985.
J ohnson & J ohnson Excellence in Science Postdoctoral Scientist.
†
(1) (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) Thompson, L.
A.; Ellman, J . A. Chem. Rev. 1996, 96, 555-600. (c) Hermkens, P. H.
H.; Ottenheijm, H. C. J .; Rees, D. Tetrahedron 1996, 52, 4527-4554.
(d) Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J .; Steele,
J . Tetrahedron. 1995, 51, 8135-8173. (e) Fr u¨ chtel, J . S.; J ung, G.
Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (f) Balkenhohl, F.; von
dem Bussche-H u¨ nnefeld, C.; Lansky, A.; Zechel, C. Angew. Chem., Int.
Ed. Engl. 1996, 35, 2289-2337. (g) Fenniri, H. Curr. Med. Chem. 1996,
3
, 343-378.
2) (a) Chenera, B.; Finkelstein, J . A.; Veber, D. F. J . Am. Chem.
CF
hydrous HF in the presence of anisole as a cation
scavenger. Most of the conditions tried (except for CF
CO H, which gave minimal reaction) resulted in partial
3 2 3 2 3 2
CO H, HBr/CF CO H, triflic acid/CF CO H, and an-
(
Soc. 1995, 117, 11999-12000. (b) Plunkett, M. J .; Ellman, J . A. J . Org.
Chem. 1995, 60, 6006-6007. (c) Backes, B. J .; Virgilio, A. A.; Ellman,
J . A. J . Am. Chem. Soc. 1996, 118, 3055-3056. (d) Hughes, I.
Tetrahedron Lett. 1996, 37, 7595-7598. (e) Boehm, T. L.; Showalter,
H. D. H. J . Org. Chem. 1996, 61, 6498-6499.
3
-
2
(3) (a) Barlos, K.; Gatos, D.; Kallitsis, I.; Papaioannou, D.; Sotiriou,
(5) For a recent report on the use of a p-alkoxybenzenesulfonyl resin
for guanidine attachment, see: Bonnat, M.; Bradley, M.; Kilburn, J .
D. Tetrahedron Lett. 1996, 37, 5409-5412.
(6) For information on Boc- and Fmoc-based peptide synthesis, see:
Bodansky, M., Ed. Principles of Peptide Synthesis; Springer-Verlag:
Heidelberg, 1984.
(7) Chan, W. C.; Mellor, S. L. J . Chem. Soc., Chem. Commun. 1995,
1475. This method measures the amount of dibenzfulvene produced.
(8) Wang, S.-S. J . Am. Chem. Soc. 1973, 95, 1328-1333.
P. Liebigs Ann. Chem. 1988, 11, 1079-1081. (b) Hoekstra, W. J .; et
al. Bioorg. Med. Chem. Lett. 1996, 6, 2371-2376. (c) Hoekstra, W. J .;
Greco, M. N.; Yabut, S. C.; Hulshizer, B. L.; Maryanoff, B. E.
Tetrahedron Lett. 1997, 38, 2629-2632. (d) Dressman, B. A.; Spangle,
L. A.; Kaldor, S. W. Tetrahedron Lett. 1996, 37, 937-940.
(
4) For
Rzeszotarska, B.; Masinkiewicz, E. Org. Prep. Proced. Int. 1988, 20,
27-464.
a
review on different arginine protecting groups, see:
4
S0022-3263(97)00736-6 CCC: $14.00 © 1997 American Chemical Society

Notes
J . Org. Chem., Vol. 62, No. 26, 1997 9327
Sch em e 1
Sch em e 2
Sch em e 3
cleavage to form two products, 8 and 9, which were
identified by MS analysis. Only HF furnished a single
species, which was desired product 8. Apparently, the
benzyl ether used to attach the requisite arenesulfonyl
group is too labile to the stronger acids; however, the HF
cleavage procedure was certainly adequate. Examination
of 8 for stereochemical integrity by chiral HPLC revealed
virtually no racemization (see Experimental Section).
We carried out the syntheses of three different model
peptides starting with resin-bound arginine derivative
of the Boc group, Boc-Phe was introduced at the N-
terminus to provide resin 14. The yield of cleaved
tripeptide 15, Phe-Arg-Ala-OMe, was 0.20 mmol/g of
resin 7 (50% yield).9 The utility of resin 7 was further
demonstrated by the synthesis of tetrapeptide 18, Phe-
Gly-Arg-Ala-OMe (Scheme 5). Notably, one can perform
both standard Boc and Fmoc processes on the same resin,
an advantage which results from the high stability of the
sulfonyl linker. The three steps of coupling and three
steps of deprotection furnished cleaved tetrapeptide 18,
Phe-Gly-Arg-Ala-OMe, with a yield of 0.16 mmol/g of
resin 7 (40%).9
7
(Schemes 3-5). After HF cleavage, each target peptide
was inspected as the crude product for diastereomers due
to any stereomutation during coupling steps (HPLC/MS),
although this is expected to be insignificant under DIC/
HOBt conditions, and then purified. No diastereomeric
species were detected in any of the crude products. The
yield of dipeptide Arg-Phe, 12, was 0.29 mmol/g of resin
To test the versatility of our novel resin further, we
prepared Ser-Phe-Leu-Leu-Arg-Asn-NH (SFLLRN-NH ,
2 2
19), a biologically important hexapeptide representing
the activation sequence for the human thrombin receptor
(PAR-1).10 Our synthesis involved peptide chain exten-
sion in both directions, by amino acid addition to both
7
(72% yield). It is particularly attractive to be able to
build up the peptide chain by the coupling of amino acids
to either or both of the termini (C or N), as illustrated
by the preparation of tripeptide 15 (Scheme 4). Resin 7
was first coupled with Ala-OMe at the C-terminus in the
presence of DIC and HOBt in DMF. After deprotection
(9) ESI-MS examination of the minor constituents of the crude final
product (separated by reverse-phase HPLC) did not show the presence
of any diastereomeric peptides.

9
328 J . Org. Chem., Vol. 62, No. 26, 1997
Notes
Sch em e 4
Sch em e 5
the C- and N-terminals, as well as both Boc and Fmoc
protection, under standard, racemization-free coupling
conditions (DIC, HOBt; 24 h). The sequence was initi-
Fmoc peptide chemistry; however, it was labile to strong
acids such as HBr and triflic acid because of the benzyl
ether functionality. To have more convenient, mild
conditions for the final cleavage process, we have recently
2
ated by attachment of Asn-NH to resin 7. The two Leu
3
d,12
residues were installed via a Boc strategy, and then
Fmoc-Phe and Boc-(O-t-Bu)Ser were sequentially coupled.
Peptide-resin cleavage (HF, anisole) afforded the desired
hexapeptide (16% overall yield from 7), which was
identical with a sample prepared by using a Boc strategy
with a commercial methylbenzhydrylamine resin.11
employed the Wang p-nitrophenyl carbonate resin
for
anchoring amidines and guanidines, but this linker is not
amenable to Boc chemistry on account of its acid labil-
ity.13 For future improvements, one could also consider
modifying the arenesulfonyl linker, such as by adding
electron-releasing groups, to make it cleavable under
more convenient conditions.
Con clu sion
Our new resin potentially has other worthwhile uses.
Besides the role in anchoring guanidine groups, it would
be reasonable to apply the resin for nitrogen attachment
with amidines, heteroaromatics (e.g., indoles), and
We have developed a new Merrifield-type resin with
an electron-rich arenesulfonyl linker, having a reasonably
useful loading capacity of ca. 0.5 mmol/g, for the solid-
phase organic synthesis of compounds bearing guanidine
groups. In particular, we have applied this new resin to
the synthesis of compounds containing arginine, in which
the guanidine group of Arg was attached to the solid
support. A sequence of chemical manipulations culmi-
nated in release of the product with anhydrous HF. The
electron-rich arenesulfonyl linker was stable to various
reaction conditions and compatible with both Boc and
14
amines, although cleavage of a standard sulfonamide
group would most likely require harsher reaction condi-
15
tions. Also, one can envision exploiting this resin where
standard arenesulfonyl groups have found synthetic
utility, such as for the activation of alcohols, oximes, or
(12) This resin was purchased from NovaBiochem Corp.
(13) (a) Such chemistry has been successfully executed in our
laboratory: Greco, M. N., unpublished results. Cleavage was easily
effected by using trifluoroacetic acid in methylene chloride. (b) For a
very recent paper in this area, see: Roussel, P.; Bradley, M.; Matthews,
I.; Kane, P. Tetrahedron Lett. 1997, 38, 4861-4864.
(
10) (a) Scarborough, R. M.; Naughton, M.; Teng, W.; Hung, D. T.;
Rose, J .; Vu, T.; Wheaton, V. I.; Turck, C. W.; Coughlin, S. R. J . Biol.
Chem. 1992, 267, 13146-13149. (b) Vu, T.-K. H.; Wheaton, V. I.; Hung,
D. T.; Coughlin, S. R. Cell 1991, 64, 1057-1068. (c) Coughlin, S. R.
Trends Cardiovasc. Med. 1994, 4, 77-83.
(14) For solid-phase synthesis with nitrogen attachment via an
acylsulfonamide “safety-catch” linker, see: (a) Backes, B. J .; Virgilio,
A. A.; Ellman, J . A. J . Am. Chem. Soc. 1996, 118, 3055-3056. (b)
Backes, B. J .; Ellman, J . A. J . Am. Chem. Soc. 1994, 116, 11171-
11172.
(15) (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Chemistry, 2nd ed.; Wiley-Interscience: New York, 1991. (b) Nyasse,
B.; Grehn, L.; Ragnarsson, U. Chem. Commun. 1997, 1017-1018 and
references therein.
(
11) The overall yield of SFLLRN-NH
on a commercial MBHA resin was 49%. We thank Kenway Hoey
RWJ PRI-La J olla) for this information, as well as a sample of the
SFLLRN-NH for comparison. Our hexapeptide sample was identical
with this “reference” material by H and C NMR.
2
from traditional synthesis
(
2
1
13

Notes
J . Org. Chem., Vol. 62, No. 26, 1997 9329
hydrazones.16 In this regard, colleagues in our labora-
tories have performed displacement reactions with di-
verse nucleophiles on alcohols activated by 4 as resin-
based sulfonates.17
50 °C for 24 h. A sample of this resin (0.20 g) was suspended in
DMF (15 mL) and treated with 1,3-propanediamine (0.074 g,
1
.0 mmol), HOBt (0.135 g, 1.0 mmol), and DIC (0.127 g, 1.0
mmol). After 16 h at 23 °C, the resin was washed with DMF (2
30 mL) and CH Cl
(2 × 30 mL), then suspended in DMF (15
mL), and treated with Fmoc-Gly-OC (0.278 g, 0.6 mmol) and
pyridine (0.079 g, 1 mmol) for 3 h. After sequential washings
with DMF and CH Cl , the resin was dried in vacuo, treated
×
2
2
6
F
5
Exp er im en ta l Section
2
2
with 20% piperidine-DMF (10 mL) for 30 min, collected, and
washed with DMF (2 × 5 mL). The combined filtrate was
measured by UV absorbance to quantitate Fmoc cleavage. The
Gen er a l. All chemicals and Merrifield resin were reagent
grade and used as purchased without further purification. R-N-
Boc-arginine was purchased from Aldrich Chemical Co.; the
other amino acid derivatives were purchased from Bachem
Bioscience Inc. Reactions involving resins were performed in
an hourglass reaction vessel (Peptides International, catalog
number SHG-21060-PI) with nitrogen gas being bubbled through
the sintered glass frit for agitation. Proton and carbon-13 NMR
spectra were obtained on a Bruker AC-300 or AC-400 spectrom-
7
loading was determined to be 0.40 mmol/g by Fmoc-echo, but
elemental analysis indicated 1.90% N for a loading of 0.34 mmol/
g. To assess the stereochemical integrity of the attached
arginine, we cleaved a sample of the N-Boc-Arg resin (HF/
anisole; see below) and triturated the crude product with ether.
The mixture was filtered, treated with water, and filtered again
to remove the resin particles. Analysis of the aqueous solution
by HPLC (Beckman analytical system) on a chiral column (15
cm × 4.6 mm Crownpak CR+ from Chiral Technologies, Inc.),
eter, as indicated, in MeOH-d (s ) singlet, d ) doublet, t )
4
triplet, m ) multiplet). General MS analysis was performed on
a Micromass Platform-II spectrometer with electrospray ioniza-
tion (ESI); HR-MS data were obtained on a Finnigan MAT 900
double-focusing spectrometer with ESI. UV analyses for the
Fmoc-echo procedure were performed on a Hewlett-Packard
Model 8453 spectrometer. Optical rotations were determined
on Perkin-Elmer 241 polarimeter. HPLC separations were
performed on a Waters 600E instrument with three PrePak
cartridges in series charges with Bondapak C18 (12-20 µM, 125
Å, 40 mm × 100 mm; gradient elution with MeCN (w/0.1% TFA)
and water (w/2% TFA) starting at 5:95 and ending at 50:50, 40
mL/min over ca. 60 min; λ ) 220 nm). Elemental microanalysis
was performed by Robertson Microlit Laboratories, Inc. (% water
by Karl Fischer analysis). Abbreviations: DIC ) diisopropyl-
carbodiimide; HOBt ) 1-hydroxybenzotriazole, Fmoc ) 9-fluo-
renylmethoxycarbonyl, Boc ) tert-butoxycarbonyl, TFA ) tri-
fluoroacetic acid.
eluting isocratically with aqueous HClO at pH ) 1.5 (0.8 mL/
4
20
min, λ ) 200 nm), indicated a 97:3 ratio of L-Arg:D-Arg, which
was virtually identical with the enantiomeric purity (96%) of
1
8
the starting R-N-Boc-arginine.
Ar g-P h e Dip ep tid e 12.21 To a suspension of resin 7 (0.50
g, 0.2 mmol) in DMF (30 mL), agitated by nitrogen gas, was
added the HCl salt of Phe-O-t-Bu (0.294 g, 1.0 mmol), Et N
3
(0.101 g, 1.0 mmol), HOBt (0.135 g, 1.0 mmol), and DIC (0.127
g, 1.0 mmol). After 24 h at 23 °C, resin 11 was washed with
DMF (2 × 30 mL) and CH
2
Cl
2
(2 × 30 mL) and then dried in a
vacuum oven at 50 °C for 24 h. Resin 11 (0.30 g, 0.12 mmol)
was suspended in anhydrous anisole (2 mL) in a Teflon reaction
vessel, and HF (8 mL) was condensed into the vessel at -78 °C;
the vessel was sealed and warmed to 0 °C. The solution was
stirred at 0 °C for 4 h, and then the HF was removed under
2
reduced pressure. The residue was triturated with Et O (30 mL)
P r ep a r a tion of Novel Ar en esu lfon yl Resin 4 a n d Resin -
Bou n d Ar gin in e Der iva tive 7. To a suspension of Merrifield
resin (1) (5.0 g, 4.9 mmol) and anhydrous 4-hydroxybenzene-
sulfonic acid sodium salt (2.94 g, 15 mmol; commercial material
was dehydrated at 110 °C in vacuo for 8 h) in N,N-dimethylac-
etamide (50 mL) was added NaOMe (0.81 g, 15 mmol), and the
mixture was stirred at 90 °C for 2 days. The resin product (3)
was collected by filtration, washed (sequentially with DMF, 1.0
and filtered. The collected solid was treated with MeOH (25
mL) and filtered. The filtrate was concentrated to give the crude
product, which was purified by reverse-phase HPLC and lyoph-
2
3
ilized to give 12 (28.0 mg, 72%) as a white fluffy solid: [R]
D
1
+13.9° (c 1.00, H
2
O); H NMR (300.1 MHz) δ 7.27 (m, 5 H), 4.72
(
dd, J ) 9.3, 4.9 Hz, 1 H), 3.89 (t, J ) 6.0 Hz, 1 H), 3.20 (m, 3
H), 3.00 (dd, J ) 14.2, 9.3 Hz, 1 H), 1.90 (m, 2 H), 1.71 (m, 2 H);
1
3
C NMR (75.5 MHz) δ 174.5, 169.9, 158.8, 138.3, 130.2, 129.6,
N HCl, MeOH, and CH
C for 24 h. The resin was treated with 1:1 Et
mL) for 1 h and washed with DMF. To resin 3 in DMF (50 mL)
was added PCl (5.20 g, 25 mmol) in several portions, and the
resulting suspension was stirred at 23 °C for 4 h. After the
mixture was washed with DMF and CH Cl , the resin was dried
2
Cl
2
), and dried in a vacuum oven at 50
1
C
28.0, 55.4, 53.7, 41.9, 38.0, 29.7, 24.8; HR-MS calcd for
°
2
NH-DMF (50
+
15
H
24
N
5
O
3
(MH ) 322.1879, found 322.1876.
P h e-Ar g-Ala Tr ip ep tid e 15. To a suspension of resin 7 (0.50
g, 0.2 mmol) in DMF (30 mL), agitated by nitrogen gas, was
added alanine methyl ester hydrochloride (0.139 g, 1.0 mmol),
5
2
2
in a vacuum oven at 50 °C overnight to afford the novel
arenesulfonyl chloride resin, 4, which was suitable for further
applications. On the basis of weight increase, the loading level
was 0.63 mmol/g, but elemental analysis indicated 1.8% Cl for
a loading of 0.51 mmol/g. A sample of this resin (0.20 g) was
suspended in DMF and treated with 1,3-propanediamine and
pyridine (0.5 mL) at 23 °C for 18 h. The resin was washed with
Et
(0.127 g, 1.0 mmol). After 24 h at 23 °C, resin 13 was washed
with DMF and CH Cl . Resin 13 in CH Cl (15 mL) was treated
with TFA (15 mL) at 23 °C for 1 h, and the resulting resin was
washed sequentially with CH Cl , 20% Et N in DMF, and DMF.
3
N (0.101 g, 1.0 mmol), HOBt (0.135 g, 1.0 mmol), and DIC
2
2
2
2
2
2
3
The resin in DMF (30 mL) was treated with Boc-phenylalanine
(0.266 g, 1.0 mmol), HOBt (0.135 g, 1.0 mmol), and DIC (0.135
g, 1.0 mmol). After 24 h at 23 °C, resin 14 was washed with
DMF and CH Cl , and then dried in a vacuum oven at 50 °C for
2 2
24 h. Resin 14 (0.30 g, 0.12 mmol) in anhydrous anisole (2 mL)
DMF (2 × 30 mL) and CH
DMF (15 mL), and treated with Fmoc-Gly-OC
mmol) and pyridine (0.079 g, 1.0 mmol) for 3 h. After being
washed with DMF and CH Cl , 6 was dried in vacuo, treated
2
Cl
2
(2 × 30 mL), then suspended in
6
F
5
(0.278 g, 0.6
was treated with HF (8 mL) as described above. The residue
from evaporation of volatiles was triturated with Et O (30 mL).
2
2
2
with 20% piperidine in DMF (10 mL) for 30 min, filtered, and
washed with DMF (2 × 5 mL). The combined filtrate was
measured by UV absorbance to determine the amount of Fmoc
cleaved from resin. The loading of the resin was calculated as
After filtration, the collected solid was treated with MeOH (25
mL) and filtered. The filtrate was concentrated to give the crude
product, which was purified by reverse-phase HPLC and lyoph-
0
.50 mmol/g on the basis of this Fmoc-echo procedure.7
ilized to give 15 (24.2 mg, 50%) as a white fluffy solid: [R]23
D
1
-
9.7° (c 1.00, H
2
O); H NMR (300.1 MHz) δ 7.32 (m, 5 H), 4.39
The resin (4.00 g, 36.0 mmol) was treated with R-N-Boc-
1
8
arginine (2.14 g, 7.8 mmol) in the presence of 4.0 N KOH (5
1
9
mL) in dioxane (20 mL) at 75 °C for 2 days; the reaction was
(18) The commercial material had an enantiomeric purity of 96%
then cooled to 23 °C and filtered. The product resin (7) was
washed with DMF and CH Cl and dried in a vacuum oven at
2 2
(L/D ) 96:4) as determined by capillary electrophoresis (Beckman
P/ACE System 2100; 50 µM × 50 cm fused silica capillary; 0.1 N
ammonium acetate and 0.01 M hydroxypropyl-â-cyclodextrin; 18 kV).
The D isomer was analyzed for confirmation.
(
16) A polymeric “tosyl chloride” has been used as a polymer-bound
tosyl azide” reagent: Roush, W. R.; Feitler, D.; Rebek, J . Tetrahedron
Lett. 1974, 1391-1392.
17) (a) Reitz, A. B., and co-workers Tetrahedron Lett., in press. (b)
(19) Loading onto the resin required these reaction conditions. In a
separate experiment, the reaction was still incomplete (%Cl analysis)
after 2 d at 65 °C.
“
(
(20) The well-shaped, baseline-resolved peaks for D-Arg (t
2.6 min) and L-Arg (t ) 4.3-4.4 min) were corroborated by spiking
with authentic samples.
(21) Appel, R.; Hiester, E. Chem. Ber. 1981, 114, 2649.
R
) 2.5-
This type of chemistry has recently been executed with a different
Merrifield resin-based sulfonyl chloride; see: Hunt, J . A.; Roush, W.
R. J . Am. Chem. Soc. 1996, 118, 9998-9999.
R

9
330 J . Org. Chem., Vol. 62, No. 26, 1997
Notes
(
2
m, 2 H), 4.15 (dd, J ) 8.1, 6.0 Hz, 1 H), 3.72 (s, 3 H), 3.21 (m,
washed (CH
2
Cl
2
; 20% Et
3
N in DMF; DMF). The resin in DMF
H), 3.02 (dd, J ) 14.2, 8.3 Hz, 1 H), 1.82 (m, 1 H), 1.71 (m, 3
(30 mL) was retreated in the same manner with Boc-Leu‚H
The washed resin was suspended in CH Cl (15 mL), treated
with TFA (15 mL) at 23 °C for 30 min, washed (CH Cl ; 20%
Et N in DMF; DMF), and treated in DMF (30 mL) with Fmoc-
2
O.
1
3
H), 1.41 (d, J ) 7.3 Hz, 3 H), 1.27 (m, 1 H); C NMR (100.6
MHz) δ 174.6, 172.8, 169.5, 163.0, 158.7, 135.5, 130.5, 128.9,
5
C
2
2
2
2
5.5, 54.1, 52.8, 42.1, 38.6, 30.6, 26.0, 17.2; HR-MS calcd for
3
+
19
H
31
N
6
O
4
(MH ) 407.2406, found 407.2409.
Phe (0.387 g, 1.0 mmol), HOBt (0.162 g, 1.2 mmol), and DIC
(0.151 g, 1.2 mmol). After 24 h at 23 °C, the resin was washed
with DMF, treated with 20% piperidine in DMF (20 mL) for 30
min, washed with DMF, and treated in DMF (30 mL) with Boc-
(O-t-Bu)Ser (0.261 g, 1.0 mmol), HOBt (0.162 g, 1.2 mmol), and
DIC (0.151 g, 1.2 mmol). After 24 h at 23 °C, the resin was
P h e-Gly-Ar g-Ala Tetr a p ep tid e 18. To a suspension of resin
(0.50 g, 0.2 mmol) in DMF (30 mL), agitated by nitrogen gas,
7
3
was added Ala-OMe‚HCl (0.139 g, 1.0 mmol), Et N (0.101 g, 1.0
mmol), HOBt (0.135 g, 1.0 mmol), and DIC (0.127 g, 1.0 mmol).
After 24 h at 23 °C, the resin product was washed with DMF
and CH
2
Cl
2
. The resin in CH
2
Cl
2
(15 mL) was treated with TFA
Cl ; 20% Et N in DMF;
2 2
washed (DMF; CH Cl ) and dried in a vacuum oven at 23 °C for
24 h. The resin product (0.50 g, 0.17 mmol) in anhydrous anisole
(2 mL) was treated with HF (8 mL) as described above. After
(15 mL) at 23 °C for 1 h, washed (CH
2
2
3
DMF), suspended in DMF (30 mL), and treated with Fmoc-
glycine (0.297 g, 1.0 mmol), HOBt (0.135 g, 1.0 mmol), and DIC
2
evaporation of volatiles, the residue was triturated with Et O
(
0.135 g, 1.0 mmol). After 24 h at 23 °C, resin 16 was washed
with DMF and treated with 20% piperidine in DMF (20 mL) for
h. After the mixture was washed with DMF, the resin in DMF
30 mL) was treated with Boc-phenylalanine (0.266 g, 1.0 mmol),
HOBt (0.135 g, 1.0 mmol), and DIC (0.135 g, 1.0 mmol). After
4 h at 23 °C, resin 17 was washed (DMF; CH Cl ) and dried in
(30 mL) and filtered. The solid was treated with MeOH (25 mL)
and filtered. The filtrate was concentrated to yield the crude
product, which was purified by reverse-phase HPLC and lyoph-
1
(
2
3
ilized to give 19 (28.8 mg, 16%) as a white fluffy solid: [R]
D
1
2
-6.3° (c 0.50, H O); H NMR (400.1 MHz) δ 0.94 (m, 12 H), 1.65
2
2
2
(m, 8 H), 1.77 (m, 1 H), 1.88 (m, 1 H), 2.73 (d, J ) 6.4 Hz, 2 H),
2.94 (dd, J ) 14.2, 9.5 Hz, 1 H), 3.19, (m, 3 H), 3.90 (m, 3 H),
4.29 (m, 1.5 H), 4.37 (m, 1.5 H), 4.67 (m, 2 H), 7.21 (m, 1 H),
7.26 (m, 4 H); 13C NMR (100.6 MHz) δ 21.99, 23.49, 23.53, 25.85,
25.93, 26.04, 29.64, 37.64, 38.30, 41.39, 41.68, 42.00, 51.41, 53.46,
53.67, 54.60, 55.87, 56.71, 61.86, 127.99, 129.64, 130.25, 138.20,
158.65, 168.82, 173.44, 173.51, 174.81, 174.98, 175.14, 175.57;
a vacuum oven at 50 °C for 24 h. Resin 17 (0.30 g, 0.12 mmol)
in anhydrous anisole (2 mL) was treated with HF (8 mL) as
described above. The residue from evaporation of volatiles was
2
triturated with Et O (30 mL). After filtration, the collected solid
was treated with MeOH (25 mL) and filtered. The filtrate was
concentrated to give the crude product, which was purified by
reverse-phase HPLC and lyophilized to give 18 (22 mg, 40%) as
+
11
HR-MS calcd for C34
Anal. Calcd for C34
N, 14.81; F, 12.05; H
N, 14.47; F, 11.64; H
H
58
N
11
O
8
(MH ) 748.4470, found 748.4453.
‚2.2TFA‚2.3H O: C, 44.34; H, 6.18;
O (KF), 3.98. Found: C, 44.09; H, 5.84;
O (KF), 3.82.
2
3
1
a white fluffy solid: [R]
MHz) δ 7.31 (m, 5 H), 4.40 (m, 2 H), 4.10 (dd, J ) 8.1, 5.2 Hz,
H), 3.90 (d, J ) 16.6 Hz, 1 H), 3.83 (d, J ) 16.6 Hz, 1 H), 3.70
s, 3 H), 3.30 (m, 2 H), 3.20 (m, 3 H), 3.07 (m, 1 H), 1.86 (m, 1
D
-8.8° (c 0.27, H
2
O); H NMR (300.1
H
57
N
11
O
8
2
2
1
(
2
1
3
H), 1.69 (m, 3 H), 1.39 (d, J ) 7.3 Hz, 3 H); C NMR (75.5 MHz)
Ack n ow led gm en t. We thank Ralph Rivero for
assistance with the “Fmoc-echo” procedure, Richard
Dunphy for high-resolution mass spectrometry data,
and Gregory Leo and Diane Gauthier for NMR spectral
data. We are particularly grateful to Rekha Shah for
chiral HPLC and capillary electrophoresis analyses.
δ 174.6, 173.4, 170.8, 170.3, 158.7, 135.6, 130.5, 130.2, 128.9,
5
5.8, 54.0, 52.8, 49.6, 43.2, 42.2, 38.5, 30.5, 26.0, 17.2; HR-MS
+
calcd for C21
Ser -P h e-Leu -Leu -Ar g-Asn -NH
(0.80 g, 0.28 mmol) in DMF (30 mL) in a bubbler, agitated by
nitrogen gas, was added Asn-NH ‚HCl (0.168 g, 1 mmol), Et
0.102 g, 1 mmol), HOBt (0.162 g, 1.2 mmol), and DIC (0.151 g,
34 7 5
H N O (MH ) 464.2621, found 464.2633.
1
0
2
(19). To a suspension of
7
2
3
N
(
1
CH
.2 mmol). After 24 h at 23 °C, the resin was washed (DMF;
Cl ), suspended in CH Cl (15 mL), treated with TFA (15
mL) at 23 °C for 30 min, washed sequentially with CH Cl , 20%
Et N in DMF, and DMF, suspended in DMF (30 mL) in a
O (0.25 g, 1.0 mmol), HOBt
0.162 g, 1.2 mmol), and DIC (0.151 g, 1.2 mmol). After 24 h at
Su p p or tin g In for m a tion Ava ila ble: Proton and carbon-
3 NMR spectra for 12, 15, 18, and 19 (9 pages). This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
2
2
2
2
1
2
2
3
bubbler, and treated with Boc-Leu‚H
(
2
2
3 °C, the resin was washed (DMF; CH
2 2 2
Cl ), suspended in CH -
Cl
2
(15 mL), treated with TFA (15 mL) at 23 °C for 30 min, and
J O970736N