Occurrence and Minimization of Cysteine Racemization during Stepwise Solid-Phase Peptide Synthesis

Article abstract of DOI:10.1021/jo9622744

Contrary to the conventional wisdom of the peptide synthesis field, N,S-protected derivatives of cysteine can undergo substantial levels of racemization with widely-used reagents and protocols for stepwise incorporation. A systematic study of this problem

Full text of DOI:10.1021/jo9622744

J . Org. Chem. 1997, 62, 4307-4312
4307
Occu r r en ce a n d Min im iza tion of Cystein e Ra cem iza tion d u r in g
Step w ise Solid -P h a se P ep tid e Syn th esis1
,2
Yongxin Han,3a,4 Fernando Albericio,3b and George Barany*,3a
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and
Department of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain
Received December 6, 1996X
Contrary to the conventional wisdom of the peptide synthesis field, N,S-protected derivatives of
cysteine can undergo substantial levels of racemization with widely-used reagents and protocols
for stepwise incorporation. A systematic study of this problem has been carried out as a function
of coupling conditions and â-thiol protecting groups, i.e., S-acetamidomethyl (Acm), S-triphenyl-
methyl (trityl or Trt), S-2,4,6-trimethoxybenzyl (Tmob), and S-9H-xanthen-9-yl (Xan), taking
advantage of a convenient and quantitative model system assay involving HPLC resolution of H-Gly-
2 2
L-Cys-Phe-NH from H-Gly-D-Cys-Phe-NH . For example, standard protocols for couplings mediated
by phosphonium and aminium salts, e.g., (benzotriazolyloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP), N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmetha-
naminium hexafluorophosphate N-oxide (HBTU), N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]-
pyridin-1-yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), and (7-
azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), typically involve
5
1
-min preactivation times and are conducted in the presence of suitable additives such as
-hydroxybenzotriazole (HOBt) or 7-aza-1-hydroxybenzotriazole (HOAt) plus a tertiary amine base
such as N,N-diisopropylethylamine (DIEA) or N-methylmorpholine (NMM). Under such conditions,
the levels of racemization in the model peptide, expressed as the ratio of D:L peptide formed, were
in the entirely unacceptable range of 5-33%. However, these levels were in general reduced by a
factor of 6- or 7-fold by avoiding the preactivation step. Additional strategies to reduce racemization
involved change to a weaker base, with 2,4,6-trimethylpyridine (TMP, collidine) being substantially
better than DIEA or NMM; 2-fold reduction in the amount of base; and change in solvent from
2 2
neat N,N-dimethylformamide (DMF) to the less polar CH Cl -DMF (1:1). Coupling methods for
the safe incorporation of cysteine with minimal racemization (<1% per step) in 9-fluorenylmethyl-
oxycarbonyl (Fmoc) solid-phase peptide synthesis include BOP (or HBTU or HATU)/HOBt (or HOAt)/
TMP (4:4:4) without preactivation in CH
preactivation, and preformed pentafluorophenyl (Pfp) esters in CH
2
Cl
2
-DMF (1:1), DIPCDI/HOBt (or HOAt) (4:4) with 5-min
Cl -DMF (1:1).
2
2
A series of careful experimental studies from the 1970’s
showed that during stepwise peptide synthesis both in
solution and solid-phase modes, activation/coupling of
protected amino acid building blocks proceeds with the
exquisite stereochemical fidelity that is an essential
requirement for the successful assembly of long chains.7
However, the special susceptibility of S-benzyl (Bzl)-
protected cysteine residues to racemization has been
noted in solution synthesis,8 and C-terminal cysteine
protected with any of a variety of groups, i.e., S-tert-butyl
5
6
(t-Bu), S-acetamidomethyl (Acm), S-triphenylmethyl (tri-
X
Abstract published in Advance ACS Abstracts, February 1, 1997.
(
1) Abbreviations used for amino acids and the designation of
tyl or Trt), S-tert-butylthio (S-t-Bu), S-(trimethylaceta-
mido)methyl (Tacm), and S-4-methylbenzyl (MeBzl), has
been shown to undergo racemization upon anchoring as
an ester in solid-phase synthesis; further epimerization
peptides follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J . Biol. Chem. 1972, 247, 977-983. The following
additional abbreviations are used: AA, amino acid; Acm, acetamido-
methyl; BOP, (benzotriazolyloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; DIEA, N,N-diisopropylethylamine; DCC, N,N′-
dicyclohexylcarbodiimide; DIPCDI, N,N’-diisopropylcarbodiimide; DMF,
R
is reported to occur during repetitive N -deprotection
N,N-dimethylformamide; Et
2
O, diethyl ether; Fmoc, 9-fluorenylmethyl-
steps in which treatment with the base piperidine
oxycarbonyl; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyri-
din-1-yl]methylene]-N-methylmethanaminium hexafluorophosphate N-
oxide; HBTU, N-[1H-(benzotriazol-1-yl)-(dimethylamino)methylene]-
N-methylmethanaminium hexafluorophosphate N-oxide; HOAt, 7-aza-
9
removes the 9-fluorenylmethyloxycarbonyl (Fmoc) group.
While applying our recently developed S-9H-xanthen-9-
1
2
-hydroxybenzotriazole; HOBt, 1-hydroxybenzotriazole; 2-Moxan,
-methoxy-9H-xanthen-9-yl; NMM, N-methylmorpholine; PAL, peptide
(6) Reviews: (a) Barany, G.; Merrifield, R. B. In The Peptides; Gross,
E., Meienhofer, J ., Eds.; Academic Press: New York, NY, 1979; Vol.
2, pp 1-284. (b) Atherton, E.; Sheppard, R. C. Solid Phase Peptide
Synthesis: A Practical Approach; IRL Press: Oxford, 1989. (c) Fields,
G. B.; Tian, Z.; Barany, G. In Synthetic Peptides. A User’s Guide; Grant,
G. A., Ed.; W. H. Freeman and Co.: New York, 1992; pp 77-183. (d)
Merrifield, B. In Peptides: Synthesis, Structures, and Applications;
Gutte, B., Ed.; Academic Press, Inc.: San Diego, CA, 1995; pp 93-
169.
amide linker, 5-[4-[(9-fluorenylmethyloxycarbonyl)aminomethyl]-3,5-
dimethoxyphenoxy]valeric acid; PEG-PS, poly(ethylene glycol)-poly-
styrene graft resin support; Pfp, pentafluorophenyl; PyAOP, (7-
a z a b e n z ot r i a z ol -1 -y l ox y )t r i s (p y r r ol i d i n o)p h os p h on i u m
hexafluorophosphate; Tmob, 2,4,6-trimethoxybenzyl; TFA, trifluoro-
acetic acid; TFE, trifluoroethanol; Tmob, 2,4,6-trimethoxybenzyl; TMP,
2
9
,4,6-trimethylpyridine ≡ collidine; Trt, triphenylmethyl (trityl); Xan,
H-xanthen-9-yl. Amino acid symbols denote the L-configuration unless
indicated otherwise.
2) Taken in part from the Ph. D. thesis of Yongxin Han, University
of Minnesota, October 1996.
(7) Review: Kemp, D. S. In The Peptides; Gross, E., Meienhofer, J .,
Eds.; Academic Press: New York, NY, 1979; Vol. 1, pp 315-381.
(8) (a) Bodanszky, M.; Bodanszky, A. Chem. Commun. 1967, 591-
592. (b) Kovacs, J .; Mayers, G. L.; J ohnson, R. H.; Ghatak, U. R. Chem.
Commun. 1968, 1066-1067. (c) Mayers, G. L.; Kovacs, J . Chem.
Commun. 1970, 1145-1146. (d) Kovacs, J .; Mayers, G. L.; J ohnson,
R. H.; Cover, R. E.; Ghatak, U. R. J . Org. Chem. 1970, 35, 1810-1815.
(e) Barber, M.; J ones, J . H.; Witty, M. J . J . Chem. Soc., Perkin Trans.
1 1979, 2425-2428.
(
(
3) (a) University of Minnesota. (b) University of Barcelona.
(4) Present address: Hybridon, Inc., 155 Fortune Blvd., Milford, MA
0
1757.
5) Reviews: (a) Bodanszky, M. Principles of Peptide Synthesis, 2nd
ed.; Springer-Verlag: Berlin, 1993. (b) Sakakibara, S. Biopolymers
Pept. Sci.) 1995, 37, 17-28.
(
(
S0022-3263(96)02274-8 CCC: $14.00 © 1997 American Chemical Society

4
308 J . Org. Chem., Vol. 62, No. 13, 1997
Han et al.
yl (Xan) protecting group10 to the synthesis by Fmoc
chemistry of dihydrooxytocin with a (benzotriazolyloxy)-
tris(dimethylamino)phosphonium hexafluorophosphate
(
BOP)/1-hydroxybenzotriazole (HOBt)/N-methylmorpho-
1
1
10b
line (NMM) coupling protocol, we observed a satellite
to the major peak with the same mass as the desired
product. This surprising result led us to hypothesize
that, contrary to the conventional wisdom of the field,
racemization of internal cysteine residues incorporated
in amide linkages could be a serious concern in Fmoc
solid-phase synthesis.12 Given the increasing popularity
of a newer generation of in situ activating agents that
are phosphonium and aminium salts (details and litera-
ture vide infra), and considering advances in the protec-
1
3
tion and management of cysteine residues, we decided
to carry out a systematic study of this putative cysteine
racemization problem as a function of coupling conditions
and â-thiol protecting groups. Coupling strategies for the
safe incorporation of cysteine with minimal racemization
(
<1% per step) in Fmoc solid-phase peptide synthesis
have been identified in this work.
F igu r e 1. Detection of racemization during synthesis of
dihydrooxytocin. The crude peptide was analyzed by HPLC
on a Vydac C-18 column using a linear gradient of 0.1% TFA
in CH CN and 0.1% aqueous TFA from 1:19 to 2:3 over a period
3
of 30 min, flow rate 1.0 mL/min, detection at 220 nm.
Authentic dihydrooxytocin elutes at 16.7 min; and the pre-
Resu lts a n d Discu ssion
P ilot Obser va tion s of Ra cem iza tion d u r in g Solid -
P h a se Syn th esis of Dih yd r ooxytocin . The linear
oxytocin sequence was assembled on a PAL-PEG-PS
1
1b,15
1
support
(
by Fmoc chemistry with a BOP/HOBt/NMM
sumed racemized peptides all elute at 17.0 min. (A) Both Cys
6
and Cys were coupled by BOP/HOBt/NMM in DMF, D-peptide:
4:4:8 equiv with respect to peptide-resin; 5-min preac-
6
1
4
1
6
L-peptide ) 0.35. (B) Cys was coupled with DIPCDI/HOBt in
tivation) protocol. Both Cys and Cys were protected
by S-Xan.10 Upon completion of chain assembly, the
Fmoc group was removed, cleavage/deprotection was
carried out by reagent R1 (TFA-thioanisole-1,2-dithio-
ethane-anisole ) 90:5:3:2), and the crude peptide was
analyzed by HPLC (Figure 1A). The major peak was
identical to the expected dihydrooxytocin by several
criteria, including comparison to an authentic standard.
DMF (4:4 equiv with respect to peptide-resin) (5-min preac-
1
tivation), D-peptide:L-peptide ) 0.17. (C) Cys was coupled with
1
DIPCDI/HOBt, D-peptide:L-peptide ) 0.19. (D) Both Cys and
1b
Cys6 were coupled with DIPCDI/HOBt, racemization negli-
gible.
A second peak eluting immediately thereafter was ob-
served, at about one-third the intensity of the major peak.
Both peaks were isolated by preparative HPLC and
shown to have the same mass, matching the theoretical
value for dihydrooxytocin. Retaining the protection
scheme but substituting a coupling protocol using N,N′-
diisopropylcarbodiimide (DIPCDI)/HOBt (4:4 equiv with
respect to peptide-resin; 5-min preactivation) specifically
for the two Cys(Xan) residues, the satellite peak became
negligible (Figure 1D). Finally, when the Cys(Xan)
residue at position 1 was incorporated with BOP/HOBt/
NMM while that at position 6 was incorporated with
DIPCDI/HOBt, or vice versa, the amounts of the satellite
peak were about half of what they were in the all-BOP
assemblies (Figure 1B,C).
(
9) (a) Atherton, E.; Hardy, P. M.; Harris, D. E.; Matthews, B. H.
In Peptides 1990: Proceedings of the Twenty-First European Peptide
Symposium; Giralt, E., Andreu, D., Eds.; ESCOM Science Publishers:
Leiden, The Netherlands, 1991; pp 243-244. (b) Fujiwara, Y.; Akaji,
K.; Kiso, Y. In Peptide Chemistry 1993; Okada, Y., Ed.; Protein
Research Foundation: Osaka, 1994; pp 29-32.
(
10) (a) Sol e´ , N. A.; Han, Y.; V a´ gner, J .; Gross, C. M.; Tejbrant, J .;
Barany, G. In Peptides-Chemistry, Structure and Biology: Proceedings
of the Fourteenth American Peptide Symposium; Kaumaya, P. T. P.,
Hodges, R. S., Eds.; Mayflower Scientific Ltd.: Kingswinford, England,
1
3
996; pp 113-114. (b) Han, Y.; Barany, G. J . Org. Chem. 1997, 62,
841.
(
11) (a) Hudson, D. J . Org. Chem. 1988, 53, 617-624. (b) Albericio,
F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.;
Hudson, D.; Barany, G. J . Org. Chem. 1990, 55, 3730-3743.
(
12) Independent of our work, cysteine racemization in stepwise
Fmoc solid-phase peptide synthesis has been noted and discussed: (a)
Musiol, H.-J .; Siedler, F.; Quarzago, D.; Moroder, L. Biopolymer 1994,
The data in the previous paragraph are consistent with
the occurrence of racemization at Cys, tied to the use of
BOP/HOBt/NMM coupling. The all-L-dihydrooxytocin
elutes slightly before any of the three Cys-racemized
dihydrooxytocins, which do not appear to be resolved
further. When the dihydro species are oxidized to oxy-
tocin, the D-Cys-containing forms are not resolved from
the desired all-L species. Perhaps for this reason, race-
3
4, 1553-1562. (b) Kaiser, T.; Nicholson, G.; Kohlbau, H. J .; Voelter,
W. Tetrahedron Lett. 1996, 37, 1187-1190.
13) Review: Andreu, D.; Albericio, F.; Sol e´ , N. A.; Munson, M. C.;
Ferrer, M.; Barany, G. In Methods in Molecular Biology; Pennington,
M. W., Dunn, B. M., Eds.; Humana Press: Totowa, NJ , 1994; Vol. 35;
pp 91-169.
(
(
14) Assemblies of linear and cyclized oxytocin by Fmoc chemistry,
using a variety of coupling protocols, have been described in ref 10b
and several earlier publications from our laboratory: (a) Albericio, F.;
Hammer, R. P.; Garc ´ı a-Echeverr ´ı a, C.; Molins, M. A.; Chang, J . L.;
Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J . Peptide Protein
Res. 1991, 37, 402-413. (b) Munson, M. C.; Garc ´ı a-Echeverria, C.;
Albericio, F.; Barany, G. J . Org. Chem. 1992, 57, 3013-3018. (c) Han,
Y.; Bontems. S. L.; Hegyes, P.; Munson, M. C.; Minor, C. A.; Kates, S.
A.; Albericio, F.; Barany, G. J . Org. Chem. 1996, 61, 6326-6339.
1
4a,b
mization has been often overlooked in the past.
Develop m en t of Mod el Assa y for Cystein e Ra ce-
m iza tion . The literature lists several methods for the
quantification of racemization of amino acids in general,
and of cysteine in particular.16 In some variations, a test
or model peptide is hydrolyzed to its constituent amino
acids, followed by derivatization with a suitable chiral
reagent and chromatographic resolution of the resultant
mixture of diastereomers by HPLC or on an amino acid
(
15) (a) Barany, G.; Albericio, F.; Sol e´ , N. A.; Griffin,, G. W.; Kates,
S. A.; Hudson, D. In Peptides 1992: Proceedings of the Twenty-Second
European Peptide Symposium; Schneider C. H., Eberle, A. N., Eds.;
ESCOM Science Publishers: Leiden, The Netherlands, 1993; pp 267-
2
68. (b) Zalipsky, S.; Chang, J . L.; Albericio, F.; Barany, G. Reactive
Polymers 1994, 22, 243-258 and references cited therein.

Cysteine Racemization during Peptide Synthesis
J . Org. Chem., Vol. 62, No. 13, 1997 4309
peptide, expressed as the ratio of D:L peptide formed,
were in the entirely unacceptable range of 5-33%.
However, these levels were in general reduced by a factor
of 6- or 7-fold [e.g., Table 1, entries 6, 9, and 11; curiously
1
4b
no significant effect with S-trimethoxybenzyl (S-Tmob)
protection] by avoiding the preactivation step. This
expedient seems to be helpful because the presumed N,S-
protected C-activated intermediates are exposed to a base
in the absence of an amine nucleophile for a minimal
time; the activated species once formed becomes rapidly
acylated by the amine.
Additional strategies to reduce racemization involved
change to a weaker base, with 2,4,6-trimethylpyridine
(
TMP, collidine) being substantially better19 than DIEA
or NMM (compare Table 1, entries 8 and 9 vs 1-3 and
-7); 2-fold reduction in the amount of base (compare
Table 1, entry 2 vs 7), and change in solvent from neat
N,N-dimethylformamide (DMF) to the less polar CH Cl
DMF (1:1) (e.g., Table 1, entry 3 vs 9), or Sakakibara’s
5
2
2
-
mixture5
b,20
3
of 2,2,2-trifluoroethanol (TFE)-CHCl (3:7)
2 R
F igu r e 2. HPLC of a mixture of H-Gly-L-Cys-Phe-NH (t )
1
6.5 min) and H-Gly-D-Cys-Phe-NH
line separation was achieved on a Vydac C-18 column using a
linear gradient of 0.1% TFA in CH CN and 0.1% aqueous TFA
2
(t
R
) 18.8 min). Base-
(Table 1, entry 4; unfortunately, this solvent cannot be
recommended because couplings were found to be too
slow). These findings lead to the consensus optimal
conditions of BOP/HOBt/TMP (4:4:4) without preactiva-
3
from 0:10 to 3:2 over 30 min, flow rate 1.0 mL/min, detection
at 220 nm.
2 2
tion in CH Cl -DMF (1:1) (Table 1, entry 9). Results
with aminium salts (Table 1, entries 10-15) are very
comparable to those with BOP in all regards: conditions
using HBTU or HATU that are currently considered
standard give substantial racemization, whereas modi-
fied conditions along lines already described are ac-
companied by little or no racemization.
analyzer. The applicability of these methods to cysteine
is compromised by the instability and redox chemistry
of cysteine. Furthermore, procedures involving deriva-
tization of cysteine, as a residue incorporated in the
peptide (before hydrolysis) or as the free amino acid (after
hydrolysis), can be quite laborious and difficult. In
contrast, we have developed a convenient, quantitative,
and unambiguous model system assay involving solid-
phase assembly of a model tripeptide Gly-Cys-Phe on a
PAL support.11b After cleavage/deprotection, H-Gly-L-
Eva lu a tion of Cou p lin g Rea gen ts Th a t Do Not
Requ ir e Ad d ed Ba se. Classical coupling methods that
6
do not require added base were also evaluated, in view
of the known propensity of cysteine derivatives to race-
7,8
mize under basic conditions.
Use of N,N′-diisopropyl-
Cys-Phe-NH
2
was base-line separated from H-Gly-D-Cys-
carbodiimide (DIPCDI), either alone or in the presence
of HOBt or HOAt as additives, and in either neat DMF
or CH Cl -DMF (1:1), gave racemization levels of <4%,
2 2
and usually <2% (Table 2). Interestingly, a 5-min
preactivation usually provided a modest reduction in the
level of racemization. Results here did not change much
from varying the solvent. Several experiments showed
that couplings of symmetrical anhydrides formed in
Phe-NH by HPLC [Figure 2; the tripeptide model was
2
chosen after the corresponding des(Gly)-dipeptides were
shown to elute within 1 min of each other, which severely
limited the dynamic range of such a method]. This
system was used to study cysteine racemization as a
function of synthesis design and reaction conditions
(Tables 1-3).
Cystein e Ra cem iza tion d u r in g P h osp h on iu m or
Am in iu m Sa lt-Med ia ted Cou p lin g. The BOP reagent,
as well as N-[(1H-benzotriazol-1-yl)(dimethylamino)-
methylene]-N-methylmethanaminium hexafluorophos-
phate N-oxide (HBTU), N-[[(dimethylamino)-1H-1,2,3-
triazolo[4,5-b]pyridin-1-yl]methylene]-N-methylmetha-
naminium hexafluorophosphate N-oxide (HATU), and (7-
azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexa-
fluorophosphate (PyAOP)], all represent useful and de-
(17) The development and applications of the revelant coupling
reagents are reported by the following: (a) Castro, B.; Dormoy, J . R.;
Evin, G.; Selve, C. Tetrahedron Lett. 1975, 1219-1222. (b) Dourtoglou,
V.; Ziegler, J .-C.; Gross, B. Tetrahedron Lett. 1978, 1269-1272. (c)
Hudson, D. J . Org. Chem. 1988, 53, 617-624. (d) Fournier, A.; Wang,
C. T.; Felix, A. M. Int. J . Pept. Protein Res. 1988, 31, 86-97. (e) Knorr,
R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989,
30, 1219-1222. (f) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-
4
398. (g) Carpino, L. A.; El-Fahan, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201-203. (h) Kates, S. A.; Minor,
C. A.; Shroff, H.; Haaseth, R. C.; Triolo, S.; El-Faham, A.; Carpino, L.
A.; Albericio, F. In Peptides 1994: Proceedings of the Twenty-Third
European Peptide Symposium; Maia, H. L. S., Ed.; ESCOM Science
Publishers: Leiden, The Netherlands, 1995; pp 248-249.
servedly popular additions to the arsenal of coupling
reagents employed in solid-phase peptide synthesis.17,18
Standard coupling protocols typically involve 5-min pre-
activation times (e.g., Table 1, entries 1 and 2) and are
conducted in the presence of suitable additives such as
HOBt or 7-aza-1-hydroxybenzotriazole (HOAt) plus a
tertiary amine base as N,N-diisopropylethylamine (DIEA)
or NMM. The levels of racemization in the model
(18) Although initial reports (ref 17e-g) showed the structures of
HBTU and HATU as uronium salts, it has been shown more recently
that both compounds crystallize as aminium salts (guanidinium
N-oxides). See: (a) Abdelmoty, I.; Albericio, F.; Carpino, L. A.; Foxman,
B. M.; Kates, S. A. Lett. Pept. Sci. 1994, 1, 57-67. (b) Henklein, P.;
Costisella, B.; Wray, V.; Domke, T.; Carpino, L. A.; El-Faham, A.;
Kates, S. A.; Abdelmoty, I.; Foxman, B. M. In Peptides 1996: Proceed-
ings of the Twenty-Fourth European Peptide Symposium; Ramage, R.,
Epton, R., Eds.; Mayflower Scientific Ltd.: Kingswinford, England,
1996; in press.
(16) (a) Friedman, M.; Krull, L. H.; Cavins, J . F. J . Biol. Chem. 1970,
2
45, 3868-3871. (b) Nimura, N.; Ogura, H.; Kinoshita, T. J . Chro-
(19) Collidine (TMP) was recommended by Carpino as a useful base
to minimize racemization in HATU-mediated coupling of peptide seg-
ments: Carpino, L. A.; El-Faham, A. J . Org. Chem. 1994, 59, 695-
698.
(20) Kuroda, H.; Chen, Y.; Kimura, T.; Sakakibara, S. Int. J . Peptide
Protein Res. 1992, 40, 294-299.
matogr. 1980, 202, 375-379. (c) Kinoshita, T.; Kasahara, Y.; Nimura,
N. J . Chromatogr. 1981, 210, 77-81. (d) Br u¨ ckner, H.; Keller-Hoehl,
C. Chromatographia 1990, 30, 621-629. (e) Adamson, G. J .; Hoang,
T.; Crivici, A.; Lajoie, G. A. Anal. Biochem. 1992, 202, 210-214. (f)
Siedler, F.; Weyher, E.; Moroder, L. J . Pept. Sci. 1996, 2, 271-275.

4
310 J . Org. Chem., Vol. 62, No. 13, 1997
Han et al.
Ta ble 1. Ra cem iza tion d u r in g P h osp h on iu m - or Am in iu m Sa lt-Med ia ted Syn th esis of Mod el P ep tid e
H-Gly-Cys-P h e-NH2 a s a F u n ction of Cou p lin g Con d ition s a n d Cystein e P r otectin g Gr ou p a
coupling conditionsa
base
D-Peptide:L-Peptide × 100
no.
reagent
additive
solvent
DMF
preact
Xanb
Tmob
Trt
Acm
1
2
3
4
5
6
7
8
9
BOP (4)
BOP (4)
BOP (4)
BOP (4)
BOP (4)
BOP (4)
BOP (4)
BOP (4)
BOP (4)
HBTU (4)
HBTU (4)
HATU (4)
HATU (4)
HATU (4)
HATU (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOBt (4)
HOAt (4)
HOAt (4)
DIEA (8)
NMM (8)
NMM (8)
NMM (8)
NMM (8)
NMM (8)
NMM (4)
TMP (8)
TMP (4)
NMM (8)
TMP (4)
NMM (8)
TMP (4)
NMM (8)
TMP (4)
5
5
5
5
2
0
5
5
0
5
0
5
0
5
0
18.9
17.2
11.4
<0.2
9.2
2.1
6.6
0.8
0.8
14.3
0.6
19.6
0.9
32.8
0.8
6.0
7.3
5.9
0.2
5.6
4.8
4.6
3.5
0.9
3.7
0.6
5.2
0.8
7.9
0.6
17.1
22.2
5.9
<0.2
15.2
3.1
10.4
2.2
0.8
24.7
0.8
36.6
0.7
51.3
0.6
11.0
11.0
3.3
<0.2
4.0
1.0
3.3
1.0
0.4
8.8
0.4
16.1
0.6
29.0
0.3
DMF
c
CH2Cl2-DMF
TFE-CHCl3
DMF
DMF
DMF
DMF
CH2Cl2-DMF
DMF
c,d
c
c
c
1
1
1
1
1
1
0
1
2
3
4
5
e
e
e
CH2Cl2-DMF
DMF
CH2Cl2-DMF
DMF
CH2Cl2-DMF c
a
See Experimental Section for general procedures; special details are presented in subsequent footnotes. Conditions that are considered
“
safe” for introduction of cysteine, i.e., acceptably low levels of racemization, are highlighted in bold . b A few experiments used S-2-
methoxy-9H-xanthen-9-yl (2-Moxan) in place of Xan. With BOP/HOBt/NMM (4:4:8)-mediated-coupling in DMF (compared to entries 2,
5
, and 6), racemization levels (ratio of D:L) were 2.1% for no preactivation, 11.2% for 2-min preactivation, and 21.2% for 5-min preactivation.
c
d
CH2Cl2-DMF mixtures were 1:1 (v/v); TFE-CHCl3 mixture was 3:7 (v/v) (see refs 5b and 20). Extent of coupling during the indicated
e
time was ∼50%. Final coupling concentration was 0.05 M due to the poor solubility of HBTU and HATU in CH2Cl2-DMF (1:1).
Ta ble 2. Ra cem iza tion Du r in g N,N′-Diisop r op ylca r bod iim id e (DIP CDI)-Med ia ted Syn th esis of Mod el P ep tid e
H-Gly-Cys-P h e-NH2 a s a F u n ction of Cou p lin g Con d ition s a n d Cystein e P r otectin g Gr ou p a
coupling conditionsa
additive solvent
D-Peptide:L-Peptide × 100
no.
reagent
preact
Xan
Tmob
Trt
Acm
1
2
3
4
5
6
7
8
9
DIPCDI (4)
DIPCDI (4)
DIPCDI (4)
DIP CDI (4)
DIP CDI (4)
DIP CDI (4)
DIPCDI (4)
DIPCDI (4)
DIPCDI (4)
DIP CDI (4)
DIP CDI (4)
DIP CDI (4)
DMF
DMF
DMF
DMF
DMF
DMF
CH2Cl2-DMF
CH2Cl2-DMF
CH2Cl2-DMF
CH2Cl2-DMF
CH2Cl2-DMF
CH2Cl2-DMF
0
0
0
5
5
5
0
0
0
5
5
5
1.3
1.3
1.6
0.6
0.4
0.3
1.2
1.0
1.4
0.3
0.3
0.3
3.5
2.9
3.2
3.0
3.4
2.4
1.8
1.2
1.4
0.5
0.2
0.4
0.8
0.9
0.8
0.6
0.6
0.6
0.9
0.9
0.8
0.5
0.5
0.5
0.4
0.4
0.4
0.6
0.5
0.5
0.7
0.7
0.6
0.4
0.2
0.3
HOBt (4)
HOAt (4)
HOBt (4)
HOAt (4)
HOBt (4)
HOAt (4)
1
1
1
0
1
2
HOBt (4)
HOAt (4)
a
See Experimental Section for general procedures. CH2Cl2-DMF mixtures were 1:1 (v/v). Conditions that are considered “safe” for
introduction of cysteine, i.e., acceptably low levels of racemization, are highlighted in bold . Special considerations with S-Tmob discussed
in text.
Ta ble 3. Ra cem iza tion d u r in g Syn th esis of Mod el P ep tid e H-Gly-Cys-P h e-NH2 Usin g P r ea ctiva ted Sp ecies, a s a
F u n ction of Cou p lin g Con d ition s a n d Cystein e P r otectin g Gr ou p a
coupling conditionsa
additive
D-Peptide:L-Peptide × 100
no.
reagent
solvent
Xan
Tmob
Trt
Acm
1
2
3
4
5
sym a n h y (4)
P fp ester (4)
P fp ester (4)
P fp ester (4)
P fp ester (4)
CH2Cl2-DMF
DMF
DMF
DMF
CH2Cl2-DMF
0.4
0.2
0.3
0.2
0.3
2.0
0.2
0.8
0.2
0.8
0.3
0.2
0.6
0.4
0.6
0.4
0.4
0.2
0.2
0.2
HOBt (4)
HOAt (4)
HOBt (4)
a
See Experimental Section for general procedures. CH2Cl2-DMF mixtures were 1:1 (v/v). Conditions that are considered “safe” for
introduction of cysteine, i.e., acceptably low levels of racemization, are highlighted in bold .
situ,21 and of pentafluorophenyl (Pfp) active esters,22,23
resulted in negligible racemization (Table 3). These
results are consistent with expectations from the litera-
ture.6
Effect of Cystein e P r otectin g Gr ou p . As opti-
mized, none of the S-protecting groups evaluated provides
much risk of racemization (always <1% in model studies),
with the exception of S-Tmob, which for DIPCDI/HOAt,
DIPCDI/HOBt, and symmetrical anhydride procedures
gives slight racemization (∼2-3%; e.g., Table 2, entries
5 and 6; Table 3, entry 1). However, S-Tmob is preferred
to S-Xan, S-Trt, and S-Acm when phosphonium and
aminium salts are used (e.g., Table 1, entries 1, 10, 12,
and 14).
,7
(21) (a) Rich, D. H.; Singh, J . In The Peptides; Gross, E., Meienhofer,
J ., Eds.; Academic Press: New York, 1979; Vol. 1, pp 241-261. (b)
Merrifield, R. B.; Vizioli, L.D.; Boman, H. G. Biochemistry 1982, 21,
Mech a n ism s. It is well understood that the racem-
ization process is a competition between activation,
rearrangement of the activated species to a 5(4H)-
oxazolone, base-catalyzed abstraction of the R-proton
(enolization) from either the activated species directly or
5
020-5031.
(
22) Kisfaludy, L.; Sch o¨ n, I. Synthesis 1983, 325-427.
(23) Fmoc-Cys(Trt)-OPfp and Fmoc-Cys(Acm)-OPfp were commer-
cially available, and Fmoc-Cys(Xan)-OPfp and Fmoc-Cys(Tmob)-OPfp
were prepared as described in the Experimental Section.

Cysteine Racemization during Peptide Synthesis
J . Org. Chem., Vol. 62, No. 13, 1997 4311
from the oxazolone, and acylation of the activated species
and/or the oxazolone with an incoming amine nucleo-
phile, which is also a base. The more activated species
couple more rapidly, but are also more prone to racemize.
The presence of base gives dual and opposite effects,
because while base catalyzes more rapid acylation, it also
leads to more rapid epimerization.
All of the phenomena described in the above paragraph
are exacerbated with protected activated cysteine; no
further epimerization occurs once cysteine is incorporated
within the peptide chain. Those activation protocols that
involve the presence of base are likely to result in
measurable cysteine racemization. As might be expected,
the level of racemization decreases by using less base,
weaker base, and eliminating preactivation. J ones and
Kovacs, with their respective co-workers, have suggested
that the â-sulfur of cysteine stabilizes, possibly via a five-
OSu (1.15 g, 3.4 mmol) in CH
min to a yellowish solution of H-D-Cys(Xan)-OH (0.96 g, 3.2
mmol) in H O-CH CN (1:1, 40 mL) plus Et N (1.06 mL, 7.6
3
CN (20 mL) was added over 30
2
3
3
mmol). The reaction mixture was stirred at 25 °C for 3 h,
adjusted to pH 3.5 by addition of 10% aqueous citric acid
solution, and concentrated under reduced pressure. The
resultant precipitate was extracted with EtOAc (3 × 100 mL),
2
washed with H O (2 × 100 mL) and brine (1 × 100 mL), and
2 4
dried (Na SO ). Evaporation of organic solvent gave a white
solid that was purified further by silica gel chromatography
CHCl -MeOH ) 30:1, R ) 0.11): yield 1.21 g (72%); mp 117-
20 °C; R [CHCl -MeOH (10:1)] 0.48; H NMR (CD
δ 12.87 (broad s, COOH), 7.87 (d, J ) 7.5 Hz, 2H), 7.75 (d, J
8.4 Hz, 1H), 7.70 (d, J ) 7.5 Hz, 2H), 7.10-7.47 (m, 12H),
.48 (s, 1H), 4.18-4.30 (m, 3H), 4.03-4.10 (m, 1H), 2.81 (dd, J
) 4.5 and 13.5 Hz, 1H), 2.64 (dd, J ) 9.9 and 13.5 Hz, 1H).
Anal. Calcd. for C31 S (523.61): C, 71.11; H, 4.81; N,
(
1
3
f
1
f
3
3 3
SOCD )
)
5
H
25NO
5
2.68; S, 6.12. Found: C, 70.96; H, 4.76; N, 2.65; S, 6.00.
r
N -(9-F lu or en ylm eth yloxyca r bon yl)-S-(9H-xa n th en -9-
yl)cystein e P en ta flu or op h en yl Ester [F m oc-Cys(Xa n )-
OP fp ]. N,N′-Dicyclohexylcarbodiimide (DCC) (0.77 g, 3.7
membered ring, an anion at the R-carbon of an activated
cysteine derivative.8
b-e
Such insights are of peripheral
mmol) was added in small portions to a suspension of Fmoc-
value in providing a semiquantitative rationalization of
the experimental results reported in the present work.
10b
Cys(Xan)-OH
(1.86 g, 3.6 mmol) and 2,3,4,5,6 pentafluoro-
Cl (70 mL)
phenol (0.79 g, 4.3 mmol) in freshly distilled CH
2
2
over 10 min. A clear solution formed immediately, followed
by precipitation about 2 min later. The reaction mixture was
stirred under N at 25 °C overnight, filtered to remove the
2
Con clu sion s
Our work provides some cautionary information that
is of both mechanistic and practical significance. We are
able to define effective compromise conditions that may
be adopted to manual and automated solid-phase syn-
thesis. Racemization of cysteine by whatever coupling
protocol can be minimized by the following: (i) using as
insoluble urea, concentrated to ∼10 mL under reduced pres-
sure, and then stored at 4 °C for 2 h. A new filtration removed
further urea, and the filtrate was diluted with hexane (20 mL)
and stored at 4 °C for 4 h. The resultant TLC-pure white
2 2
precipitate was collected by filtration, washed with CH Cl -
hexane (1:2, 3 × 5 mL), and dried in vacuo: yield 2.32 g (94%);
1
mp 151-153 °C; R
f
[hexane-EtOAc (3:1)] 0.61; H NMR
solvent CH
2
Cl
2
-DMF, with the minimal amount of DMF
(CDCl ) δ 7.77 (d, J ) 7.5 Hz, 2H), 7.58 (d, J ) 7.5, 2H), 7.22-
3
consistent with solubility of derivatives; (ii) avoiding
preactivation in BOP, HATU, or HBTU-mediated (and
related) protocols; (iii) replacing the traditional DIEA or
NMM base with TMP. Once a cysteine residue has been
incorporated safely, there appears to be little risk for
further racemization at steps to couple additional amino
acids.
7.45 (m, 8H), 7.11 (dd, J ) 3.3 and 7.8 Hz, 4H), 5.38 (s, 1H),
5.14 (d, J ) 8.1 Hz, 1H), 4.62 (dd, J ) 6.0 and 13.5 Hz, 1H),
4
6
.41 (d, J ) 6.9 Hz, 2H), 4.21 (t, J ) 6.9 Hz, 1H), 2.88 (d, J )
.3 Hz, 2H). Anal. Calcd for C37
24 5 5
H F NO S (689.66): C, 64.44;
H, 3.51; F, 13.77; N, 2.03; S, 4.65. Found: C, 64.57; H, 3.71;
F, 13.01; N, 2.26; S, 4.30.
r
N -(9-Flu or en ylm eth yloxycar bon yl)-S-(2,4,6-tr im eth ox-
yben zyl)cystein e P en ta flu or op h en yl Ester [F m oc-Cys-
(
Tm ob)-OP fp ]. DCC (0.88 g, 4.3 mmol) was added in five
1
4b
portions over 10 min to a solution of Fmoc-Cys(Tmob)-OH
Exp er im en ta l Section
(
2.13 g, 4.1 mmol) and 2,3,4,5,6-pentafluorophenol (0.90 g, 4.8
mmol) in freshly distilled CH Cl (35 mL). The reaction
mixture was stirred under N at 25 °C overnight, and further
Most of the materials and general synthetic and analytical
procedures have been described in our earlier publica-
2
2
2
1
0b,11b,14,15
14b
tions.
OH
Fmoc-Cys(Tmob)-OH
and Fmoc-Cys(Xan)-
steps followed very closely the above procedure for the corre-
sponding Cys(Xan) derivative: yield 2.53 g (90%); mp 108-
110 °C; R
(d, J ) 7.5 Hz, 2H), 7.61 (d, J ) 7.5 Hz, 2H), 7.26-7.41 (m,
6H), 6.13 (s, 2H), 5.94 (d, J ) 7.8 Hz, 1H), 4.97 (m, 1H), 4.49
(d, J ) 6.9 Hz, 2H), 4.26 (t, J ) 6.9 Hz, 1H), 3.79 (s, 9H), 3.10
(dd, J ) 4.5 and 14.4 Hz, 1H), 2.95 (dd, J ) 7.8 and 14.4 Hz,
1H). Anal. Calcd for C34
4.09; F, 13.77; N, 2.03; S, 4.65. Found: C, 59.00; H, 4.16; F,
13.50; N, 2.20; S, 4.81.
1
0b
were made in our laboratory as described previously,
and other protected amino acid derivatives were commercially
available. H NMR spectra were recorded at 300 MHz using
3 3
either CDCl or CD OD as solvents. Fast atom bombardment
mass spectrometry (FABMS) to characterize synthetic peptides
was carried out with glycerol or thioglycerol matrices being
used to obtain both positive and negative ion spectra. El-
emental analyses were performed by M-H-W Laboratories,
Phoenix, AZ.
1
f
[hexane-EtOAc (3:1)] 0.40; H NMR (CDCl
3
) δ 7.74
1
28 5 7
H F NO S (689.66): C, 59.21; H,
Thin-layer chromatography was performed on Analtech or
Merck silica gel GF plates (250 µm, 10 × 20 cm), and
compounds were observed by fluorescence quenching and by
spraying with a dilute ethanolic ninhydrin solution. Analytical
HPLC were carried out on C-18 columns.
Dih yd r ooxytocin (H-Cys-Tyr -Ile-Gln -Asn -Cys-P r o-Leu -
Gly-NH ). The detailed experimental design, HPLC docu-
2
mentation (Figure 1), and essential conclusions are described
in the text. In brief, Fmoc-Cys(Xan)-Tyr(tBu)-Ile-Gln-Asn-Cys-
(Xan)-Pro-Leu-Gly-PAL-PEG-PS (300 mg, 0.15 mmol/g initial
loading) was assembled by Fmoc chemistry, with 1-h couplings
in DMF of all residues [except that Asn and Gln were
incorporated as their corresponding Pfp esters (with HOBt,
1:1 ≡ 6 equiv), and Cys as specified] mediated by BOP/HOBt/
NMM (4:4:8 equiv with respect to peptide-resin; 5-min preac-
tivation). Cleavage/deprotection with reagent R was carried
out for 1 h at 25 °C. The filtrate from the cleavage reaction
S-(9H -Xa n t h en -9-yl)-D-cyst ein e [H -D-Cys(Xa n )-OH ].
.
.
Starting with D-cysteine H
previously described
2
O HCl (2.0 g, 11.4 mmol), the
procedure for the corresponding
compound was followed exactly: yield 2.87 g (84%); mp 175
1
0b
L
1
°
C dec; R
f
2 3
[MeOH-H O (4:1)] 0.78; H NMR (CD OD) δ 7.51-
7
7
1
1
.58 (dd, J ) 7.5 and 12.9 Hz, 2H), 7.31 (t, J ) 7.5 Hz, 2H),
.11-7.17 (m, 4H), 5.48 (s, 1H), 3.52 (dd, J ) 4.2 and 7.8 Hz,
H), 2.97 (dd, J ) 4.2 and 14.4 Hz, 1H), 2.85 (dd, J ) 7.8 and
was evaporated partially under a stream of N
CH Cl (1 mL for 20 mg peptide-resin), and then precipitated
with anhydrous Et O (10 mL). The procedure of adding CH
Cl followed by Et O precipitation was repeated a total of three
2
, diluted with
4.4 Hz, 1H). Anal. Calcd. for C16
H
15NO
3
S (301.36): C, 63.77;
2
2
H, 5.02; N, 4.65; S, 10.64. Found: C, 63.18; H, 5.93; N, 4.45;
S, 9.96.
2
2
-
2
2
r
N -(9-F lu or en ylm eth yloxyca r bon yl)-S-(9H-xa n th en -9-
times, in order to remove all low molecular weight organic
compounds. The residue was dissolved in the mixture of
yl)-D-cystein e [F m oc-D-Cys(Xa n )-OH]. A solution of Fmoc-

4
312 J . Org. Chem., Vol. 62, No. 13, 1997
Han et al.
HOAc-H
Figure 1). Diastereomeric dihydrooxytocins were separated
by semipreparative HPLC (Waters Delta Prep 3000, using a
linear gradient of 0.1% TFA in CH CN and 0.1% aqueous TFA
2
O (1:4) (1 mg/mL) and applied to HPLC analysis
side-chain protection were incorporated by 1-h couplings
mediated by the coupling reagents, additives, and bases
indicated in Tables 1-3 [no. of equiv in parentheses; concen-
tration of Cys derivative always 0.18 M unless indicated
otherwise]; solvent and preactivation time (expressed in min)
as specified. After completion of the chain assembly, the
(
3
from 1:19 to 2:3 over 50 min, flow rate 5.0 mL/min, detection
at 220 nm) and analyzed by mass spectrometry: FABMS calcd
monoisotopic mass of dihydrooxytocin 1008.44, found positive
tripeptide H-Gly-Cys-Phe-NH
by treatment with Reagent R at 25 °C for 1 h, precipitated by
cold diethyl ether, dissolved in deionized H O, lyophilized, and
2
was released from the support
+
and negative of isolated dihydrooxytocin [M + H] 1009.6,
-
[
M-H] 1007.7, Found positive and negative of racemized
2
+
-
dihydrooxytocin [M + H] 1009.6, [M-H] 1007.6.
Syn th esis a n d An a lysis of Sta n d a r d H-Gly-L-Cys-P h e-
analyzed by HPLC (see legend of Figure 2 for details).
However, for those experiments using Cys(Acm), the following
steps were carried out after completion of chain assembly, but
2 2
NH a n d H-Gly-D-Cys-P h e-NH . Starting with Fmoc-PAL-
PEG-PS (1.0 g, 0.15 mmol/g), peptide chain assembly was
carried out using 1-h DIPCDI/HOBt (4:4 equiv with respect
to peptide-resin, 0.18 M, 5-min preactivation)-mediated cou-
plings in DMF (3.3 mL). In separate syntheses, Fmoc-Cys-
before cleavage: peptide-resin was treated with Hg(OAc)
2
solution (0.06 M) in DMF in the dark at 25 °C for 3 h and
then washed with â-mercaptoethanol-DMF (1:19, v/v, 3 × 2
min).
(Xan)-OH and Fmoc-D-Cys(Xan)-OH were incorporated; oth-
Exa m in a tion of Cystein e Ra cem iza tion d u r in g Step -
w ise Solid -P h a se P ep tid e Syn th esis of H-Gly-Cys-P h e-
erwise, Fmoc-Phe-OH and Boc-Gly-OH were used. Fmoc
removal was carried out with piperidine-DMF (1:4, 2 + 8
min), followed by washes with DMF (5 × 2 min). Final release
of peptides was achieved with reagent R at 25 °C for 1 h.
Crude peptides were purified on semipreparative HPLC
NH
2
w ith P r efor m ed Sym m etr ica l An h yd r id es (Ta ble 3,
en tr y 1). The general outline described above was followed.
Symmetrical anhydrides6
ing AA/DIPCDI (8:4 ) 0.16 M) in CH
Fmoc-, S-Tmob-, S-Trt-, and S-Acm-protected cysteine deriva-
tives and by combining AA/DIPCDI (8:4 ) 0.12 M) in CH Cl
DMF (2:1) for Fmoc-Cys(Xan)-OH. After 5 min, these reaction
mixtures were diluted with CH Cl to provide a final solvent
milieu of CH Cl -DMF (1:1) and a concentration of 0.09 M
b,21
were prepared in situ by combin-
R
2 2
Cl -DMF (10:1) for N -
(
Waters Delta Prep 3000, using a linear gradient of 0.1% TFA
in CH CN and 0.1% aqueous TFA from 0:10 to 3:2 over 50
min, flow rate 5.0 mL/min, detection at 220 nm). Purified
H-Gly-L-Cys-Phe-NH and H-Gly-D-Cys-Phe-NH were char-
acterized by HPLC (Figure 2) and FABMS analyses (matrix:
glycerol-TFA): calcd monoisotopic mass of C14 S 324.4,
found positive and negative of H-Gly-L-Cys-Phe-NH
3
2
2
-
2
2
2
2
H
20
N
4
O
3
2
2
+
2
[M + H]
activated species for coupling.
-
m/ z 325.2, [M - H] m/ z 323.2, found positive and negative
Exa m in a tion of Cystein e Ra cem iza tion d u r in g Step -
w ise Solid -P h a se P ep tid e Syn th esis of H-Gly-Cys-P h e-
+
-
of H-Gly-D-Cys-Phe-NH
23.1.
Exa m in a tion of Cystein e Ra cem iza tion d u r in g Step -
w ise Solid -P h a se P ep tid e Syn th esis of H-Gly-Cys-P h e-
NH (Ta bles 1 a n d 2). Each experiment started with Fmoc-
2
[M + H] m/ z 325.2, [M - H] m/ z
3
NH
2
w it h P en t a flu or op h en yl E st er s (Ta b le 3, en t r ies
2
-5). The general outline described above was followed;
concentrations of the active species were 0.18 M. Additives,
when used in Pfp ester couplings, were introduced to the
peptide-resin ahead of other reagents.
2
PAL-PEG-PS (50 mg, 0.15 mmol/g ) 1 equiv resin) and
followed the overall experimental outline of the above descrip-
tion of standards. Fmoc-Cys derivatives with the indicated
J O9622744