- Email: [email protected]
An alternative solid-phase approach to C1-oxytocin
Trtmhcdmn
ktten.
Vol. 36. No. 41. pp. 7387-7390, Ekvia
Pergamon
fwmd
in Glut
oo4c4039195
oo40-4039(95)0
1995
S&me
$9.504.00
1548-5
An Alternative Solid-Phase Approach To Cl-Oxytocin
John P. Mayer*, James R. Heil, Jingwen Zhang and Mark C. Munsxm Amgen Boulder Inc.. 3200 Walnut St.. Boulder, CO, 80301
Abstract. A general,solid-phasemethodbasedon in
siru
fomtationof a cystathionineresidueis described.The
procedure allows for the introduction of a thioether moiety in place of a native disulfide bond and is illustrated here by the synthesis of Cl-oxytocin 1.
CONHI
Cl-OXYTOCIN
1
Disulfide bonds function by covalently stabilizing the tertiary structures of polypeptide chains and as a result are critical to the biological activities of cystine containing peptides and proteins. The lack of stability of disulfides under reducing conditions has prompted the development of isosteric linkages where one or both sulfur atoms are replaced by a methylene group. ethylene
(-CH$H2-).
or “dicarba.”
modification
Both the thioether (-CH2-S-), have been employed
also called “monocarba.” and
as reductively
stable disulfide
surrogates. In the example of the neurohypophyseal hormones, while both modifications produced compounds
7387
L4d
Blimin
7388
with prolonged
biological
activities
with respect to the parent peptides, the dicarba analogs have been
characterized by markedly lower potencies. Rudinger* generally
to differentiate
1 Application of the thioether strategy, originally developed by
between the steric and functional
been more successful.
roles of the disultide
Reports of full or partial biological
activity
bond of oxytocin,
has
of deamino-Cl-oxytocin.2
vasopressin.3acalcitonin3h and insulin3c with respect to the parent peptides suggests that this modified linkage closely approximates the essential structural features of a native disulfide bond. this technique we have developed
a solid-phase, 4 Fmoc/t-Bu
In order to facilitate use of
protocol5 strategy which permits complete
assembly and cyclization of thioether containing peptides on solid support.6
Scheme 1
Legend. i. Fmoc-CVpyridine,
Commercially with Fmoc-CI.7
available L-homoserine
85%. ii. HBr, acetic acid, R.T.. Ihr.. 70%.
lactone 2 was protected on nitrogen as the Fmoc derivative 3
This material was converted to the bromoacid 4 by modification
of a published procedure8
(Scheme I). Assembly of the oxytocin sequence (Scheme 2) was accomplished by Fmoc/t-Bu basd chemistry on Rink amideo support (loading level = O.SSmmol/g) using DCC/HOBt well as the N-terminal
bromoacid 4 and 20% piperidine/DMF
by exposure to three successive IO minute treatments of 2% TFA.
exposure to 2% TFA
group was followed mercaptoethanol.
2% EtsSiH
DMF. 10 Determination
overnight cyclization in 5% N-methylmorpholine/ following
to activate the standard residues as
for Fmoc deprotection.
indicated no detectable resin cleavage.
by cleavage
and deprotection
Detritylation
in CH2Cl2
of cysteine
was followed
by
of resin loading levels prior to and Removal
under standard conditions
of the N-terminal
(80%
TFA.
Fmoc
5% H20,
5%
5% thioanisole. 5% phenol, 4 hrs) which produced the cyclized material in approximately
45% overall yield.11
The crude peptide was purified by high performance liquid chromatography12
and the
resulting product was characterized by electrospray mass spectrometry and amino acid analysis (electrospray M.S. analysis:
(exp) tr& = 989.2, (obs) = 989.5).
The stoichiometric level of cystathionine liberated during
amino acid analysis confirmed the high efficiency of cyclization. 13 A small amount of by-product (< 5%) in the crude material was identified
by M.S. and amino acid analysis as the octapeptide YIQNCPLG.
and its
presence was attributed to less than quantitative incorporation of the bromoacid 4. We believe this approach offers a convenient and straightforward containing
cyclic
peptides.
The
original
solution
methods
as well
strategy for preparing as more
recent
thioether
solid-phase
7389
Scheme 2 H,N-Rink-
f\ 1) FmocAA/DCCXlOBt/DMF 2) 20% piperidine/DMF
0 P
0 P
H~N-Tyr(tBu)-lle-Gln()-Asn(Trt)-Cys(T~)-~o-~u-Gly-~-~~-
Fmoc-NH-CH(CHzCH2Br)C02H DCC/HOBt/DMF
CH2CH2Br
0
I
I
P
Fmoc-NH-CH-CO-Tyr(tBu)-Ile-Gln(Trt)-Asn(T~)-Cys(T~)-~o-Leu-Gly-~-R~-
I) 2 9 TFA and EtsSiH in CH2Clz 2) 5 %JNMM/DMF
24 hours, 25 C I
CH,CH,
SCH,
I
0
I
P
Fmoc-NH-CH-CO-Tyr(tBu)-lle-Gln(Tn)-Asn(Trt)-NH-CH-CO-Pro-Lcu-Gly-NH-Rink-
1) 20 8 piperidine/DMF 2) TFA cleavage I SCHz
CH,CH,
I
I
H~N-CH-CO-Tyr-lle-GIn-Asn-NH-CH-CO-Pro-~u-Gly-Nli~
approaches utilizing multiply protected cystathionine’4
or S-y-carboxypropyl-cysteinets
synthetically
of the thioether
challenging,
which
limits
application
strategy.
as building blocks are The present method is
advantageous because it allows for the complete assembly and cyclization of the peptide on resin support and requires only one final purification step. Once the thioether is formed, additional residues may be incorporated by solid-phase synthesis to extend the N-terminus. analysis with cyclization
efficiency
method is convenient for introduction polypeptides.
The ability to correlate cystathionine levels by amino acid
provides an additional advantage unique to this approach.
The present
of thioethers into not only small but also larger and more complex
It may also be useful in the construction of template constrained cyclic peptidesto.fdt9
for
examining the effect of conformation on biological activity.
Acknowledgement We thank Doug Lenz of Amgen Boulder Inc. for carrying out mass spectral and amino acid analyses, Dr. Theodore Jones for his help with the manuscript and The Peptide Technology group for their support.
7390
References and Notes 1969, z.
1239.
1.
Hase. S.; Morikawa.
2.
Rudinger, J.; Jest, K. Experientia.
3.
(a) Jost. K.; Prochazka, 2.; Cart, J.H.; Barth, T.; Skopkova, J.; Prusik. Z.; Sorm, F. Collection
T.; Sakakibara, S. Experientia.
Czechoslov. Cheer. Commun. Chem. CONSUL Naturforsch.
1964, m.
1974.3.2835.
197% $5, 1305.
1968, m.
570. (b) Rochazka, Z and Jest, K. Collection Czechoslov.
(c) Jest, K.; Rudinger, J.; Klostermeyer. H.; and Zahn, H.. Z.
1059.
4.
Merrifield,
5.
Fields, G.B.; Noble. R.L. fnt. J. Peptide Protein Res. 1990,2,
6.
For a discussion of synthetic approaches to cyclic peptides see: Kates. S. A.. Sole, N.A., Albericio,
R.B. J. Am. Chem. Sot. 1%3, a,
2149. 161. F..
and Barany, G. In Peptides: Design, Synthesis, and Biological Activity, Basava, C. and Anantharamaiah.
G.M., Eds.. Birkhauser, Boston,
7.
Son. J.-K.; Ramalingam.
8.
Jest, K.; Rudinger. J. Collection
9.
Rink, H. Tetrahedron Let?. 1987. ZH. 3787.
10.
1994,39.
K.; Woodard. R.W. Synthesis 1988,240. Czechoslov. Chem.Commun.
1%7,x,2485.
Barker. P.L.; Bullens. S.; Bunting, S.; Burdick, J.D.; Chart, K.S.; Deishe, T.; Eigenbrot, C.; Gadek. T.R.: Gantzos, R.; Lapari, M.T.; Muir. C.D.; Napier, M.A.; Pitti, R.M.: Padua. A.; Quart, C.; Stanley, M.; Struble, M.: Tom, J.Y.K.. and Burnier. J.P.. J. Med. Chem.
1 I.
1992, s,
2040.
The yield was calculated from initial loading of the Rink resin and integrated HPLC purity and weight of the crude material.
12.
Preparative HPLC conditions (+0.05%
13.
Amino acid analysis was carried out on an ABI 420 analyzer: Asx=l .O 1. Cystathionine=l. Glx=O.96, Gly=l.OO.
14.
: Vydac C4 column (25.4x 150mm). O-308 acetonitrile into Hz0
TFA) over 80 min.
Pro=O.99, T~0.77.
IO,
Ile=O.g2. Leu=O.92.
Safar, P.; Slaninova, J.; Lebl, M. In Peptides: Chemistry, Structure and Biology (Hodges, R.S. and Smith, J.A., Eds.) Escom. Leiden, The Netherlands, 1’994, 119.
15.
16.
Lebl. M.; Hruby. V.J. Tetrahedron
2067.
Cheng. S.; Craig, W.S.; Mullen, D.; Tschopp. J.F.; Dixon, D. and Pierschbacher, M.D. J. Med. Chem.
17.
Lert. 1984, a,
1’994,2,
1.
Jackson, S.; DeGrado, W.; Dwivedi, A.; Parthasarathy, A.; Higley. A.; Krywko. J.: Rockwell, A.;
Markwalder. J.; Wells, G.; Wexler. R.; Mousa, S. and Harlow, R. J. Am. Chem. Sot.
18.
Wood, S.W. and Wetzel, R. Int. J. Peptide Protein Res. 1992,3,533.
19.
Robey, F. A. and Fields, R.L.
Anal. Biochem. 1989. m.
(Received in USA 3 August 1995; accepted IO August 1995)
373.
1994, u,
3220.
ktten.
Vol. 36. No. 41. pp. 7387-7390, Ekvia
Pergamon
fwmd
in Glut
oo4c4039195
oo40-4039(95)0
1995
S&me
$9.504.00
1548-5
An Alternative Solid-Phase Approach To Cl-Oxytocin
John P. Mayer*, James R. Heil, Jingwen Zhang and Mark C. Munsxm Amgen Boulder Inc.. 3200 Walnut St.. Boulder, CO, 80301
Abstract. A general,solid-phasemethodbasedon in
siru
fomtationof a cystathionineresidueis described.The
procedure allows for the introduction of a thioether moiety in place of a native disulfide bond and is illustrated here by the synthesis of Cl-oxytocin 1.
CONHI
Cl-OXYTOCIN
1
Disulfide bonds function by covalently stabilizing the tertiary structures of polypeptide chains and as a result are critical to the biological activities of cystine containing peptides and proteins. The lack of stability of disulfides under reducing conditions has prompted the development of isosteric linkages where one or both sulfur atoms are replaced by a methylene group. ethylene
(-CH$H2-).
or “dicarba.”
modification
Both the thioether (-CH2-S-), have been employed
also called “monocarba.” and
as reductively
stable disulfide
surrogates. In the example of the neurohypophyseal hormones, while both modifications produced compounds
7387
L4d
Blimin
7388
with prolonged
biological
activities
with respect to the parent peptides, the dicarba analogs have been
characterized by markedly lower potencies. Rudinger* generally
to differentiate
1 Application of the thioether strategy, originally developed by
between the steric and functional
been more successful.
roles of the disultide
Reports of full or partial biological
activity
bond of oxytocin,
has
of deamino-Cl-oxytocin.2
vasopressin.3acalcitonin3h and insulin3c with respect to the parent peptides suggests that this modified linkage closely approximates the essential structural features of a native disulfide bond. this technique we have developed
a solid-phase, 4 Fmoc/t-Bu
In order to facilitate use of
protocol5 strategy which permits complete
assembly and cyclization of thioether containing peptides on solid support.6
Scheme 1
Legend. i. Fmoc-CVpyridine,
Commercially with Fmoc-CI.7
available L-homoserine
85%. ii. HBr, acetic acid, R.T.. Ihr.. 70%.
lactone 2 was protected on nitrogen as the Fmoc derivative 3
This material was converted to the bromoacid 4 by modification
of a published procedure8
(Scheme I). Assembly of the oxytocin sequence (Scheme 2) was accomplished by Fmoc/t-Bu basd chemistry on Rink amideo support (loading level = O.SSmmol/g) using DCC/HOBt well as the N-terminal
bromoacid 4 and 20% piperidine/DMF
by exposure to three successive IO minute treatments of 2% TFA.
exposure to 2% TFA
group was followed mercaptoethanol.
2% EtsSiH
DMF. 10 Determination
overnight cyclization in 5% N-methylmorpholine/ following
to activate the standard residues as
for Fmoc deprotection.
indicated no detectable resin cleavage.
by cleavage
and deprotection
Detritylation
in CH2Cl2
of cysteine
was followed
by
of resin loading levels prior to and Removal
under standard conditions
of the N-terminal
(80%
TFA.
Fmoc
5% H20,
5%
5% thioanisole. 5% phenol, 4 hrs) which produced the cyclized material in approximately
45% overall yield.11
The crude peptide was purified by high performance liquid chromatography12
and the
resulting product was characterized by electrospray mass spectrometry and amino acid analysis (electrospray M.S. analysis:
(exp) tr& = 989.2, (obs) = 989.5).
The stoichiometric level of cystathionine liberated during
amino acid analysis confirmed the high efficiency of cyclization. 13 A small amount of by-product (< 5%) in the crude material was identified
by M.S. and amino acid analysis as the octapeptide YIQNCPLG.
and its
presence was attributed to less than quantitative incorporation of the bromoacid 4. We believe this approach offers a convenient and straightforward containing
cyclic
peptides.
The
original
solution
methods
as well
strategy for preparing as more
recent
thioether
solid-phase
7389
Scheme 2 H,N-Rink-
f\ 1) FmocAA/DCCXlOBt/DMF 2) 20% piperidine/DMF
0 P
0 P
H~N-Tyr(tBu)-lle-Gln()-Asn(Trt)-Cys(T~)-~o-~u-Gly-~-~~-
Fmoc-NH-CH(CHzCH2Br)C02H DCC/HOBt/DMF
CH2CH2Br
0
I
I
P
Fmoc-NH-CH-CO-Tyr(tBu)-Ile-Gln(Trt)-Asn(T~)-Cys(T~)-~o-Leu-Gly-~-R~-
I) 2 9 TFA and EtsSiH in CH2Clz 2) 5 %JNMM/DMF
24 hours, 25 C I
CH,CH,
SCH,
I
0
I
P
Fmoc-NH-CH-CO-Tyr(tBu)-lle-Gln(Tn)-Asn(Trt)-NH-CH-CO-Pro-Lcu-Gly-NH-Rink-
1) 20 8 piperidine/DMF 2) TFA cleavage I SCHz
CH,CH,
I
I
H~N-CH-CO-Tyr-lle-GIn-Asn-NH-CH-CO-Pro-~u-Gly-Nli~
approaches utilizing multiply protected cystathionine’4
or S-y-carboxypropyl-cysteinets
synthetically
of the thioether
challenging,
which
limits
application
strategy.
as building blocks are The present method is
advantageous because it allows for the complete assembly and cyclization of the peptide on resin support and requires only one final purification step. Once the thioether is formed, additional residues may be incorporated by solid-phase synthesis to extend the N-terminus. analysis with cyclization
efficiency
method is convenient for introduction polypeptides.
The ability to correlate cystathionine levels by amino acid
provides an additional advantage unique to this approach.
The present
of thioethers into not only small but also larger and more complex
It may also be useful in the construction of template constrained cyclic peptidesto.fdt9
for
examining the effect of conformation on biological activity.
Acknowledgement We thank Doug Lenz of Amgen Boulder Inc. for carrying out mass spectral and amino acid analyses, Dr. Theodore Jones for his help with the manuscript and The Peptide Technology group for their support.
7390
References and Notes 1969, z.
1239.
1.
Hase. S.; Morikawa.
2.
Rudinger, J.; Jest, K. Experientia.
3.
(a) Jost. K.; Prochazka, 2.; Cart, J.H.; Barth, T.; Skopkova, J.; Prusik. Z.; Sorm, F. Collection
T.; Sakakibara, S. Experientia.
Czechoslov. Cheer. Commun. Chem. CONSUL Naturforsch.
1964, m.
1974.3.2835.
197% $5, 1305.
1968, m.
570. (b) Rochazka, Z and Jest, K. Collection Czechoslov.
(c) Jest, K.; Rudinger, J.; Klostermeyer. H.; and Zahn, H.. Z.
1059.
4.
Merrifield,
5.
Fields, G.B.; Noble. R.L. fnt. J. Peptide Protein Res. 1990,2,
6.
For a discussion of synthetic approaches to cyclic peptides see: Kates. S. A.. Sole, N.A., Albericio,
R.B. J. Am. Chem. Sot. 1%3, a,
2149. 161. F..
and Barany, G. In Peptides: Design, Synthesis, and Biological Activity, Basava, C. and Anantharamaiah.
G.M., Eds.. Birkhauser, Boston,
7.
Son. J.-K.; Ramalingam.
8.
Jest, K.; Rudinger. J. Collection
9.
Rink, H. Tetrahedron Let?. 1987. ZH. 3787.
10.
1994,39.
K.; Woodard. R.W. Synthesis 1988,240. Czechoslov. Chem.Commun.
1%7,x,2485.
Barker. P.L.; Bullens. S.; Bunting, S.; Burdick, J.D.; Chart, K.S.; Deishe, T.; Eigenbrot, C.; Gadek. T.R.: Gantzos, R.; Lapari, M.T.; Muir. C.D.; Napier, M.A.; Pitti, R.M.: Padua. A.; Quart, C.; Stanley, M.; Struble, M.: Tom, J.Y.K.. and Burnier. J.P.. J. Med. Chem.
1 I.
1992, s,
2040.
The yield was calculated from initial loading of the Rink resin and integrated HPLC purity and weight of the crude material.
12.
Preparative HPLC conditions (+0.05%
13.
Amino acid analysis was carried out on an ABI 420 analyzer: Asx=l .O 1. Cystathionine=l. Glx=O.96, Gly=l.OO.
14.
: Vydac C4 column (25.4x 150mm). O-308 acetonitrile into Hz0
TFA) over 80 min.
Pro=O.99, T~0.77.
IO,
Ile=O.g2. Leu=O.92.
Safar, P.; Slaninova, J.; Lebl, M. In Peptides: Chemistry, Structure and Biology (Hodges, R.S. and Smith, J.A., Eds.) Escom. Leiden, The Netherlands, 1’994, 119.
15.
16.
Lebl. M.; Hruby. V.J. Tetrahedron
2067.
Cheng. S.; Craig, W.S.; Mullen, D.; Tschopp. J.F.; Dixon, D. and Pierschbacher, M.D. J. Med. Chem.
17.
Lert. 1984, a,
1’994,2,
1.
Jackson, S.; DeGrado, W.; Dwivedi, A.; Parthasarathy, A.; Higley. A.; Krywko. J.: Rockwell, A.;
Markwalder. J.; Wells, G.; Wexler. R.; Mousa, S. and Harlow, R. J. Am. Chem. Sot.
18.
Wood, S.W. and Wetzel, R. Int. J. Peptide Protein Res. 1992,3,533.
19.
Robey, F. A. and Fields, R.L.
Anal. Biochem. 1989. m.
(Received in USA 3 August 1995; accepted IO August 1995)
373.
1994, u,
3220.