A general strategy for the preparation of C-terminal peptide α-ketoacids by solid phase peptide synthesis

Article abstract of DOI:10.1039/b901198f

A new cyanosulfur-ylide based linker makes possible the synthesis of C-terminal peptide α-ketoacids by solid phase synthesis. The preparation of the requisite linker and its application to a variety of C-terminal peptide α-ketoacids with unprotected side

Full text of DOI:10.1039/b901198f

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
A general strategy for the preparation of C-terminal peptide a-ketoacids by
solid phase peptide synthesis†
Lei Ju and Jeffrey W. Bode*
Received 20th January 2009, Accepted 25th March 2009
First published as an Advance Article on the web 2nd April 2009
DOI: 10.1039/b901198f
A new cyanosulfur-ylide based linker makes possible the
synthesis of C-terminal peptide a-ketoacids by solid phase
synthesis. The preparation of the requisite linker and its
application to a variety of C-terminal peptide a-ketoacids
with unprotected side chains is reported.
Inspired by Rademann et al.’s work,8 which performed Wasser-
man et al.’s elegant phosphorus ylide9 chemistry on a solid
support, we hoped to enable the acylation of a cyanosulfur
ylide moiety and elongation of the peptide chain on a standard
solid support. We were encouraged by our previous results that
the cyanosulfur ylides could tolerate the standard procedures
employed for Fmoc-based N-terminal deprotection and extension
of the peptide chain in the C to N direction.6 This finding
anticipated an alkyl sulfide-containing linker as a precursor to
peptide cyanosulfur ylides for synthesis on a suitable solid support.
Tetrahydrothiophene-derived linker 5 was prepared in five
steps from commercially available tetrahydrothiophen-3-one
(Scheme 2). It contains a free acid for loading onto an amine
or alcohol derivatized solid support, and a four atom-spacer from
the acid to the tetrahydrothiophene core. Horner–Wadsworth–
Emmons reaction10 provided a,b-unsaturated ester 1, which was
subjected to 1,4-conjugate reduction to afford 2. All attempts at
metal catalyzed hydrogenation (Pd, Pt, Rh) failed due to catalyst
poisoning by the sulfur.11 We found that the conjugated ester
could be reduced by in situ generated nickel boride,12 accompanied
with a small amount of desulfurization as a side reaction.
Subsequent reduction of the ester with LiAlH4 provided alcohol
3.13 Elaboration of 3 presented the challenge of chemoselective O-
vs. S-alkylation with tert-butyl bromoacetate, which was achieved
under biphasic conditions to provide ester 4.14 A small amount
of recovered starting material 3 was resubjected to the alkylation.
Finally, removal of the tert-butyl protecting group under acidic
conditions provided the completed linker 5.
As part of the intense interest in the development of new methods
for native amide bond formation via chemoselective ligation
reactions,1,2 our group has identified a novel and unexpectedly
simple amide-forming ligation reaction that enables the coupling
of the peptide fragments by decarboxylative condensation between
a-ketoacids and N-alkyl hydroxylamines.3 This reaction offers
great promise as a general method for the chemoselective ligation
of two unprotected peptide fragments, but is currently limited
by the lack of practical methods for the preparation of ligation
partners bearing the requisite C-terminal a-ketoacids4 and N-
terminal hydroxylamines.5
In addressing this, we have recently reported a robust, chemos-
elective method to produce peptide a-ketoacids with minimal
epimerization via oxidation of cyanosulfur ylides.6 This method
is operationally friendly, high yielding and provides peptide a-
ketoacids in high enantiopurity. It is also compatible with all
unprotected amino acid side chains save cysteine and methionine.
To extend this strategy further in preparing larger peptide derived
ketoacids, we now report the synthesis and utility of a solid-
supported cyanosulfur ylide that makes possible the preparation
of C-terminal a-ketoacids using standard Fmoc-based solid phase
peptide synthesis (Scheme 1).7
Anchoring of the linker onto Rink amide MBHA resin (0.54–
0.72 mmol/g) was accomplished with a standard HBTU protocol
(Scheme 3).15 Following alkylation of the sulfide, the sulfonium salt
could be converted to its ylide in situ and coupled to N-terminal
Fmoc-protected amino acids in the presence of excess base. In
solution phase studies, the sulfonium bromide could be generated
Roy and Diana Vagelos Laboratories, Department of Chemistry, Univer-
sity of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA. E-mail:
bode@sas.upenn.edu; Tel: +1 215-573-1953
† Electronic supplementary information (ESI) available: Experimental
procedures and characterization data. See DOI: 10.1039/b901198f
Scheme 1 Preparation of a-ketoacid peptides via side-chain unprotected C-terminal sulfur ylides.
The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 2259–2264 | 2259
This journal is
©

View Article Online
Scheme 2 Preparation of linker 5 from tetrahydrothiophen-3-one.
Scheme 3 Generation of dipeptide derived sulfur ylide from sulfide-containing linker on a solid support.
by treating tetrahydrothiophene with neat bromoacetonitrile.
Although this reaction proceeded in almost quantitative yield,6
the bromide salt was extremely hygroscopic and we anticipated
that an alternative counterion would be necessary for a stable,
storable resin-bound sulfur-ylide precursor.
After evaluating a variety of sulfonium salts (BF4, TFA, OTf,
PF6, SbF6), we selected the hexafluorophosphate counterion for
further studies aimed at developing a reliable alkylation protocol
for the preparation of an easily handled solid-supported sulfur
ylide precursor. Cyanomethylation of the solid-supported linker
initially proved more challenging than anticipated, due in part to
the need to employ a co-solvent for the synthesis of the PF6, rather
than the bromide, salt. Optimization of the cyanomethylation
conditions was carried out by assaying the amount of sulfide 7
and Fmoc-Phe-sulfur ylide 11 generated after the first acylation,
as determined by 1H NMR after cleavage from Rink amide MBHA
resin (Table 1). Cyanomethylation of sulfide 7 proceeded cleanly in
1:2 CH3CN:PhCH3 upon heating with hexafluorophosphate salt 8,
2260 | Org. Biomol. Chem., 2009, 7, 2259–2264
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Table 1 Optimization of sulfide cyanomethylation
but could be used in subsequent steps. Following resin cleavage,
PTLC purification provided the desired dipeptide 13 with 65–74%
yield over five synthetic steps. Wang resin also gave similar results
for a carboxylic acid terminated peptide sulfur ylide.
Time/ Temp/ Sulfide 7:
Entry Reagentsa,b Solvent
h
◦C
Ylide 11c,d
We have previously documented that oxidative conversion of
peptide sulfur ylides could generate the corresponding a-ketoacids
in high enantiopurity.5 To confirm that the solid phase synthesis
protocol preserved the stereochemistry, our previously examined
model dipeptide derived sulfur ylide 13 was prepared and subjected
to standard aqueous Oxone18 oxidation (Scheme 4) to give a-keto-
acid 14 (92% yield by SFC analysis at 300 nm). Ligation of the
crude ketoacid to HONH-Gly-Phe-OtBu19 15 gave comparable
yield and diastereomeric ratio of the ligation product 16 (38%
yield, d.r. = 33:1) as obtained using a sulfur ylide prepared in solu-
tion phase. This confirms the practical extension of our peptide sul-
fur ylide methodology to a polymer support as a means of prepar-
ing C-terminal peptide a-ketoacids for chemoselective ligations.
To establish the utility of this strategy for the synthesis of
larger peptide a-ketoacids with unprotected amino acid residues,
a variety of different amino acids were loaded onto the polymer-
supported sulfur ylide. The peptide chain could be easily elongated
using standard Fmoc-based peptide coupling protocols. Scheme 5
shows detailed, standard conditions for the synthesis of C-terminal
peptide sulfur ylides, using the preparation of 21 as a representative
example. The loading of the first amino acid, which becomes the a-
ketoacid following ylide oxidation, was performed twice to ensure
complete loading due to the unreliability of Kaiser tests at this
stage. After the coupling of the second amino acid, the couplings
could be monitored by Kaiser tests and standard Fmoc-based
peptide synthesis protocols were applied. Cleavage from the resin
provided unprotected peptide sulfur ylide 21 in good yield (78%
mass yield, >70% HPLC yield), and could be readily purified by
preparative HPLC.
The potential of the a-ketoacid–hydroxylamine peptide ligation
lies in its application to fragment couplings at a variety of
C-terminal and N-terminal amino acid residues. To establish that
resin 9 and the optimized coupling procedures detailed in Scheme 5
were applicable to a variety of different peptide sequences, we
prepared four additional sulfur ylide sequences with distinct
C-terminal residues (Ala, Ile, Glu, Pro) and a full complement
of unprotected side chains. In all cases, the crude yields and
purities were within the expected range for tetrapeptides prepared
by Fmoc-based chemistry on other linkers (Fig. 1). These sulfur
ylides could be further purified by preparative HPLC without
difficulty.
1
2
3
4
5
6
7
BrCH2CN
BrCH2CN
PF6CH2CN MeCN:PhCH31:2 32
PF6CH2CN MeCN:PhCH31:2 18
PF6CH2CN MeCN:PhCH3 1:2
PF6CH2CN MeCN:PhCH3 1:4 14
PF6CH2CN MeCN:PhCH3 1:5 13
PhCH3
Neat
8
17
60
rt
rt
60
60
60
60
14:1
1.3:1
1:1.5
1:20
1:4
1:1.6
1:1.3
8
a The reactions were agitated in a glass vial filled under a N2 atmosphere
in an oven shaker. b The reactions were carried out with 5 equiv. alkylating
reagent (entries 1, 3–7). c The ratio of sulfide 7 : ylide 11 was determined
by 1H NMR. d The subsequent acylations were performed using a
standard procedure with Fmoc-Phe-OH (5 equiv.), HATU (5 equiv.),
HOBt (5 equiv.), DIPEA (20 equiv.) in DMF for 4 h.
which was generated by filtering the silver iodide precipitate from
the combination of AgPF6 and iodoacetonitrile. This procedure
was faster and cleaner than using bromoacetonitrile alone (Table 1,
entries 1–2) and left only a trace amount of sulfide unreacted.
We found it essential to carefully filter the silver iodide, as
contamination with silver caused aggregation of the resin and
darkened its colour. The alkylated sulfide gave a bright orange
colour to the beads. The polymer-bound sulfonium salt 9 was
bench stable, and its reactivity remained the same after storing
for months. In contrast, the corresponding resin-bound sulfonium
bromide salt was hygroscopic and prone to decomposition.
While related solid-supported phosphorus ylides are highly
water sensitive and require strong activation for the acylation step,
the resin-supported sulfonium ylides showed improved reactivity
and scope. For example, standard coupling conditions including
DIC/HOBt, TBTU, and PyBOP protocols failed for the phos-
phorous ylide,8 while these conditions were readily applied to the
synthesis of the C-terminal peptide cyanosulfur ylides. Acylation
of the resin-bound sulfur ylide could be easily accomplished
using HBTU/HOBT, HATU/HOBt,16 or carbodiimide reagents17
(DIC and EDC). The coupling efficiency of the first amino
acid to the resin was quantified by spectrophotometric Fmoc-
UV determination (0.37–0.42 mmol/g from 0.54 mmol/g Rink
amide MBHA resin) upon Fmoc deprotection with piperidine,
and by TFA cleavage and analysis of Fmoc-Phe-derived ylide 11.
The expected peptide sulfur ylide was obtained in 80% isolated
yield after purification. Following introduction of the first amino
acid, Fmoc-deprotection and subsequent elongation of the peptide
could be carried out under standard conditions. Kaiser tests after
the coupling of the first amino acid were not reliable due to trace
amounts of residual silver salts from the cyanomethylation step,
With purified, unprotected sulfur ylides 21–25, we confirmed
that oxidation to the a-ketoacid proceeds smoothly under our
previously reported, operationally simple conditions. Exposure of
Scheme 4 Oxone oxidation of solid phase generated dipeptide sulfur ylide and ligation with a hydroxylamine.
The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 2259–2264 | 2261
This journal is
©

View Article Online
Scheme 5 Representative conditions for Fmoc-based synthesis of C-terminal peptide sulfur ylides on solid support.
Fig. 1 Peptide sulfur ylides prepared from resin 9. The crude purities of sulfur ylides (22–25) were assayed by HPLC; yields refer to mass yields of crude
peptides after washing with cold ether; isolated yields obtained after purification by HPLC are given in parentheses.20
a dilute (0.02–0.05 M in 2:1 DMF:H2O) solution of the peptide
sulfur ylide to Oxone (2 equiv.) effected rapid formation of the
desired a-ketoacid, as determined by LCMS. The oxidations were
generally completed within 10 to 30 minutes, and were monitored
by HPLC; extended reaction times led to decomposition of the a-
ketoacid to the corresponding carboxylic acid. For the purposes
of this study, the reactions were quenched by the addition of
dimethylsulfide and taken directly to the ligation reaction with
simple peptide hydroxylamines (Scheme 6).21 The conversion of
these diverse, unprotected peptide ylides to a-ketoacids and their
subsequent ligation confirms the remarkable tolerance of these two
reactions to unprotected side chains including amines, carboxylic
acids, alcohols, and electron-rich aromatics. As noted in our
prior studies, they are not, however, compatible with unprotected
cysteine or methionine residues.
of ligation pairs that will inform the application of this process
to larger fragment coupling reactions. When amino acids with
bulkier side chains were placed adjacent to the sulfur ylide (Ile,
Glu), the ligation proceeded more slowly with sterically hindered
hydroxylamines. A notable amount of ketoacid still persisted after
stirring for 48 hours in DMF at 60 ◦C when the peptide a-
ketoacids were subjected to ligation with phenylalanine derived
hydroxylamines. Ligation with glycine derived hydroxylamines, in
contrast, proceeded more smoothly under similar conditions.
In conclusion, we have documented the preparation and utility
of sulfonium resin 9 for the Fmoc-based, solid phase synthesis
of enantiopure C-terminal peptide sulfur ylides. These products
serve as immediate precursors to C-terminal peptide a-ketoacids
via chemoselective oxidation under simple conditions. The ready
availability of C-terminal peptide a-ketoacids using this method
will enable further research and application of the a-ketoacid–
hydroxylamine ligation to complex peptide synthesis.
While the ligation reactions performed for this study were not
optimized, the results provide qualitative data on the viability
2262 | Org. Biomol. Chem., 2009, 7, 2259–2264
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Scheme 6 Chemoselective oxidation of sulfur ylides to a-ketoacids and subsequent ligations with peptide hydroxylamines.
4 A. J. L. Cooper, J. Z. Ginos and A. Meister, Chem. Rev., 1983, 83,
321–358.
Acknowledgements
5 H. C. J. Ottenheijm and J. D. M. Herscheid, Chem. Rev., 1986, 86,
This work was supported by the NIH NIGMS [R01-GM076320].
Unrestricted support from Bristol Myers Squibb, Eli Lilly, and
Roche is greatly appreciated. J.W.B. is a fellow of the Beckman
Foundation, the Packard Foundation, and the Sloan Foundation.
697–707.
6 L. Ju, A. R. Lippert and J. W. Bode, J. Am. Chem. Soc., 2008, 130,
4253–4255.
7 (a) R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149–2154; (b) R. B.
Merrifield, Angew. Chem., Int. Ed., 1985, 24, 799–810.
8 (a) S. Weik and J. Rademann, Angew. Chem., Int. Ed., 2003, 42, 2491–
2494; (b) A. El-Dahshan, S. Weik and J. Rademann, Org. Lett., 2007,
9, 949–952.
Notes and references
9 (a) H. H. Wasserman and W.-B. Ho, J. Org. Chem., 1994, 59, 4364–
4366; (b) H. H. Wasserman, C. M. Baldino and S. J. Coats, J. Org.
Chem., 1995, 60, 8231–8235; (c) H. H. Wasserman and J. Parr, Acc.
Chem. Res., 2004, 37, 687–701; (d) H. H. Wasserman, D. S. Ennis, P.
L. Power and M. J. Ross, J. Org. Chem., 1993, 58, 4785–4787; (e) H. H.
Wasserman and C. B. Vu, Tetrahedron Lett., 1990, 31, 5205–5208.
10 M. J. Fisher, W. J. Hehre, S. D. Kahn and L. E. Overman, J. Am. Chem.
Soc., 1988, 110, 4625–4633.
11 R. A. W. Johnstone, A. H. Wilby and I. D. Entwistle, Chem. Rev., 1985,
85, 129–170.
12 (a) J. M. Khurana and P. Sharma, Bull. Chem. Soc. Jpn., 2004, 77,
549–552; (b) B. Ganem and J. O. Osby, Chem. Rev., 1986, 86, 763–780.
13 A. Srikrishana and G. Satyanarayana, Tetrahedron, 2006, 62, 2892–
2900.
1 For recent reviews of peptide ligations, see: (a) P. E. Dawson and S. B.
H. Kent, Annu. Rev. Biochem., 2000, 69, 923–960; (b) D. Macmillan,
Angew. Chem., Int. Ed., 2006, 45, 7668–7672; (c) C. P. R. Hackenberger
and D. Schwarzer, Angew, Chem., Int. Ed., 2008, 47, 10030–10074.
2 For selected peptide-forming ligation reactions, see: (a) P. E. Dawson,
T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science, 1994, 266, 776–
779; (b) B. L. Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett., 2001,
3, 9–12; (c) A. Tam, M. B. Soellner and R. T. Raines, J. Am. Chem.
Soc., 2007, 129, 11421–11430; (d) E. Saxon, J. I. Armstrong and C. R.
Bertozzi, Org. Lett., 2000, 2, 2141–2143; (e) D. Crich, K. Sana and K.
Guo, Org. Lett., 2007, 9, 4423–4426; (f) Q. Wan, J. Chen, Y. Yuan and
S. J. Danishefsky, J. Am. Chem. Soc., 2008, 130, 15814–15816.
3 (a) J. W. Bode, R. M. Fox and K. D. Baucom, Angew. Chem., Int. Ed.,
2006, 45, 1248–1252; (b) N. Carrillo, E. A. Davalos, J. A. Russak and
J. W. Bode, J. Am. Chem. Soc., 2006, 128, 1452–1453.
14 K. B. Sloan and S. A. M. Koch, J. Org. Chem., 1983, 48, 3777–3783.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 2259–2264 | 2263
©

View Article Online
15 Loading of the linker and solid phase peptide synthesis follows the
general procedures described in Novabiochem catalogue, 2008.
16 F. Albericio, J. M. Bofill, A. El-Faham and S. A. Kates, J. Org. Chem.,
1998, 63, 9678–9683.
17 L. A. Carpino and A. El-Faham, Tetrahedron, 1999, 55, 6813–6830.
18 R. J. Kennedy and A. M. Stock, J. Org. Chem., 1960, 25, 1901–1906.
19 Hydroxylamines were prepared according to Fukuyama’s method: H.
Tokuyama, T. Kuboyama and T. Fukuyama, Org. Synth., 2003, 80,
207–212.
20 Fmoc-His-Ser-Phe-Pro sulfur ylide 25 was prepared by loading dipep-
tide Fmoc-Phe-Pro-OH onto sulfonium resin 9 followed by Fmoc-
deprotection and elongation. Stepwise coupling with Fmoc-Pro-OH
also provided the desired tetrapeptide sulfur ylide with somewhat lower
yield.
21 Peptide targets 26–30 are fragments chosen from either a Fuzeon
or human prostate-specific antigen protein with the sole purpose of
demonstrating disconnection at ligation site with different amino acid
residues.
2264 | Org. Biomol. Chem., 2009, 7, 2259–2264
This journal is
The Royal Society of Chemistry 2009
©