Synthesis of Protected L-4-[Sulfono(difluoromethyl)]phenylalanine and Its Incorporation into a Peptide

Article abstract of DOI:10.1021/ol0158664

matrix presented A protected form of L-4-[sulfono(difluoromethyl)]phenylalanine (F₂Smp), a novel non-hydrolyzable phospho- and sulfotyrosine mimetic, was synthesized via electrophilic fluorination of a benzylic sulfonate followed by a Pd-cataly

Full text of DOI:10.1021/ol0158664

ORGANIC
LETTERS
2001
Vol. 3, No. 10
1571-1574
Synthesis of Protected
L-4-[Sulfono(difluoromethyl)]phenylalanine
and Its Incorporation into a Peptide
Shifeng Liu, Chris Dockendorff, and Scott D. Taylor*
Department of Chemistry, UniVersity of Waterloo, 200 UniVersity AVenue West,
Waterloo, Ontario N2L 3G1, Canada
s5taylor@sciborg.uwaterloo.ca
Received March 19, 2001
ABSTRACT
A protected form of L-4-[sulfono(difluoromethyl)]phenylalanine (F2Smp), a novel non-hydrolyzable phospho- and sulfotyrosine mimetic, was
synthesized via electrophilic fluorination of a benzylic sulfonate followed by a Pd-catalyzed cross-coupling reaction between the fluorinated
sulfonate and the zincate of protected iodoalanine. F Smp was incorporated into a peptide using solid-phase peptide synthesis techniques.
2
The sulfation and phosphorylation-dephosphorylation of
tyrosine residues in proteins are important post-translational
events. Tyrosine sulfation is employed as a means of
mediating protein-protein interactions, while tyrosine phos-
phorylation and dephosphorylation act as on-off switches
in signal transduction pathways. Over the last several years
there has been tremendous interest in developing inhibitors
of proteins that are involved in these processes such as
protein tyrosine kinases (PTKs),1 protein tyrosine phos-
phatases (PTPases),2 SH2 domain binding proteins,3 and
proteins that bind sulfotyrosine (sTyr).4 Phosphono(difluo-
romethyl)phenylalanine (F2Pmp, 1) has been used extensively
as a non-hydrolyzable phosphotyrosine mimetic for the
development of inhibitors of enzymes that bind or hydrolyze
phosphotyrosine (pTyr).2 However, its sulfur analogue,
L-[sulfono(difluoromethyl)]phenylalanine (F2Smp, 2) has
never been reported. Recent studies with peptides bearing
sulfotyrosine5 and non-peptidyl compounds bearing the
difluoromethylenesulfonic acid moiety6 suggest that F2Smp
may be highly effective as a non-hydrolyzable, monoanionic,
pTyr mimetic for obtaining inhibitors of therapeutically
significant PTPases. It may also be useful as a pTyr mimetic
for obtaining inhibitors of PTKs and SH2 domain binding
proteins and as a sTyr mimetic for obtaining inhibitors of
proteins that bind sTyr. In this letter, we describe an
enantioselective synthesis of protected L-F2Smp and the
incorporation of L-F2Smp into a peptide.
(1) Al-Obeidi, F. A.; Lam, K. S. Oncogene 2000, 19, 5690-701.
(2) Ripka, W. C. Ann. Rep. Med. Chem. 2000, 35, 231-250.
(3) Cody, W. L.; Lin, Z.; Panek, R. L.; Rose, D. W.; Rubin, J. R. Curr.
Pharm. Des. 2000, 6, 59.
The key steps in our approach to the synthesis of protected
L-F2Smp are outlined in Scheme 1. One is a palladium-
(4) Kehoe, J. W.; Bertozzi, C. R.Chem. Biol. 2000, 7, R57.
10.1021/ol0158664 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/19/2001

Scheme 1
Scheme 2
catalyzed cross-coupling reaction between suitably protected
sulfonate 3 and the organozincate 4 to produce the fully
protected L-F2Smp. A similar approach has been used
successfully for the enantioselective synthesis of protected
L-F2Pmp.7 The other key step involves the synthesis of 3.
We anticipated that this could be accomplished using a
procedure recently developed in our laboratory that involves
reacting R-carbanions of sulfonate esters with N-fluoroben-
zenesulfonimide (NFSi), an electrophilic fluorinating agent.8
Indeed, to our knowledge, this is the only method available
for the preparation of benzylic R-fluorosulfonates.
During our earlier studies on electrophilic fluorination of
benzylsulfonates it was found that the use of the neopentyl
(nPt) protecting group was essential for obtaining good yields
and that other protecting groups examined, ethyl and
isopropyl, yielded only decomposition products.8 The results
with the ethyl and isopropyl esters were most likely due to
deprotonation at the â-position of the protecting group
followed by elimination of the sulfonate salt. However, at
the time the present study was initiated, we were concerned
that the nPt protecting group might be problematic since the
conditions that had previously been used for its removal,
tetramethylammonium chloride, DMF, 160 °C, 16 h9 or LiBr
in refluxing butanone for 48 h,8 are quite harsh. Therefore,
in addition to the nPt group, it was decided to also examine
the trichloroethyl (TCE) group as a protecting group for the
sulfonate moiety. The TCE group was chosen because it lacks
â-protons that could interfere with the fluorination reaction,
can be readily removed under mild conditions (e.g., Zn/
HOAc, Cd/DMF) and has been used successfully as a
phosphate and carboxylate protecting group in peptide
synthesis.10
of the nPt ester was achieved in a single step by adding 3.0
equiv of NaHMDS in THF dropwise to a solution of 1.0
equiv of 7 and 2.5 equiv of NFSi in THF at -78 °C. After
stirring at -78 °C for 2 h and room temperature for 2 h, the
fluorinated product 9 was obtained in an excellent 90% yield.
The fluorination of the TCE ester using similar conditions
was less successful. In addition to the desired ester 10 (20-
30% yield), an equivalent quantity of the chloride elimination
product, 11, was also obtained. None of the non-fluorinated
or monofluorinated elimination products were formed. This
suggested to us that the elimination reaction occurred only
with the difluorinated product 10. Therefore, we reasoned
that the amount of byproduct could be reduced by performing
the reaction in a stepwise manner by reacting 8 with 1.0
equiv of NaHMDS followed by the addition of a slight excess
of NFSi and then repeating this process. When the reaction
was performed in this manner, the yield of the desired ester
10 was increased to a respectable 71%, while the elimination
product was obtained in a 15% yield.
Compounds 9 and 10 were coupled to Fmoc-3-iodo-L-
alanine methylester using conditions similar to those devel-
oped by Burke and co-workers for the synthesis of protected
L-F2Pmp (Scheme 3).7 Thus, a solution of 1 equiv of 9 or
10 in THF/DMAC (1:1) containing 5 mol % Cl2Pd(PPh3)2
and 10 mol % DIBAL11 in THF/DMAC was added to a
solution of 2 equiv of the zincate 127,12 in THF/DMAC
(1:1), and the mixture was stirred at 65-70 °C for 6 h. This
gave the nPt-protected and TCE-protected amino acids 13
The synthesis of protected F2Smp is outlined in Scheme
2. p-Iodobenzylbromide was transformed into the sulfonate
salt 5, which was then converted into the sulfonyl chloride
6 in overall high yield. Treatment of 6 with neopentyl alcohol
or trichloroethanol in the presence of Et3N in THF gave the
nPt ester 7 and TCE ester 8 in excellent yields. Fluorination
(5) Desmarais, S.; Jia, Z.; Ramachandran, C. Arch. Biochem. Biophys.
1998, 345, 225.
(6) Kotoris, C. C.; Chen, M.-J.; Taylor, S. D. Bioorg. Med. Chem. Lett.
1998, 8, 3275-3280.
(7) Smyth, M. S.; Burke, T. R. Tetrahedron Lett. 1994, 35, 551.
(8) Kotoris, C. C.; Chen, M.-J.; Taylor, S. D. J. Org. Chem. 1998, 63,
8052.
(9) Roberts, J. C.; Gao, H.; Gopalsamy, A.; Kongsjahju, A.; Patch, R. J.
Tetrahedron Lett. 1997, 38, 335.
(10) For examples see: Paquet, A. Int. J. Pept. Protein Res. 1992, 39,
82. Mora, N.; Lacombe, J. M.; Pavia, A. A. Int. J. Pept. Protein Res. 1995,
45, 53.
(11) We found that the yields of the cross-coupling reactions could be
slightly improved using Cl2Pd(PPh3)2/DIBAL (Negishi method) instead of
Cl2Pd(PPh3)2 alone; see: Negishi, E. Acc. Chem. Res. 1982, 15, 340. For
a recent example of this see: Amat, M.; Hadida, S.; Pshenichnyi, G.; Bosch,
J. J. Org. Chem. 1997, 62, 3158-3171.
(12) Jackson, R. F. W.; James, K.; Wythes, M. J.; Wood, A. J. J. Chem.
Soc., Chem. Commun. 1989, 644.
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Org. Lett., Vol. 3, No. 10, 2001

Scheme 3
Scheme 4
and 14 in yields of 45% and 44%, respectively.13 Selective
hydrolysis of the carbon esters to give 15 and 16 was
achieved in high yield using aqueous LiOH/THF.14,15
For peptide synthesis, the hexapeptide DADE-F2Smp-
LNH2, 17, was chosen as a model system. The DADE-X-
LNH2 sequence has been used extensively for examining
various moieties as phosphotyrosine mimetics16 for obtaining
inhibitors of PTP1B, a therapeutically significant PTPase.17
Manual solid-phase peptide synthesis was performed using
Fmoc-protected amino acids, the Rink amide AM resin, and
HATU/HOAT as coupling agents. The peptide was cleaved
from the support by subjecting the resin-bound material to
Reagent K (82.5% TFA, 2.5% EDT, 5% H2O, 5% thioani-
sole, and 5% phenol) for 2 h. When the TCE-protected amino
acid 16 was used, we were surprised to find that the HPLC
chromatogram of the crude peptide consisted of numerous
peaks and, therefore, no attempt was made to obtain the pure
peptide from the mixture.18 However, when the nPt-protected
amino acid 15 was used, the HPLC chromatogram of the
crude peptide consisted mainly of two peaks. On the basis
of electrospray mass spectral analysis, these peaks were
attributed to the nPt-protected peptide 18 (major product)
and the completely deprotected peptide 17 (minor product)
(Scheme 4). Further studies with the resin-bound peptide
revealed that the loss of the nPt group was occurring during
the treatment with Reagent K. Although complete removal
of the nPt group could be achieved by prolonged treatment
with Reagent K, this resulted in an increase in the formation
of impurities. Nevertheless, it was found that the nPt group
could be removed cleanly under very mild conditions by
stirring a solution of the crude peptide in 1:1 acetonitrile/
water containing 0.1% TFA for 4-5 days. Pure peptide was
obtained by preparative HPLC of the crude reaction mixture
(32% yield). It was also found that the nPt group could be
removed by refluxing the crude peptide in a solution of
butanone containing 2 equiv of LiBr for 48 h. However, this
reaction did not proceed as cleanly as the above procedure.
In summary, we have described the first synthesis of
protected L-F2Smp and developed a straightforward proce-
dure for incorporating F2Smp into peptides, which should
be readily adaptable to automated solid-phase peptide
synthesis. Inhibition studies with PTP1B and peptide 17
indicate that it is almost 2 orders of magnitude more potent
than the analogous peptides bearing other16 monoanionic
pTyr mimetics.19 F2Smp may also be useful as a monoanionic
pTyr mimetic for the development of inhibitors and probes
of other enzymes that bind phosphotyrosine such as PTKs
and SH2 domain binding proteins. It has recently been shown
that tyrosine sulfation on CCR5 is important for efficient
adhesion of HIV and sulfated peptides that mimic the sulfated
tyrosine sequences inhibit HIV infection of macrophages.20,21
F2Smp may also prove to be useful in the development of
hydrolytically stable forms of such peptides.
(13) The coupling reaction was also attempted using the Boc-protected
zincate. However, separation of Boc-alanine methylester, a byproduct of
the reaction, from the coupled product proved to be quite difficult and, as
a result, provided considerably lower yields than when the Fmoc derivative
was used.
(14) (a) Burke, T. R.; Smyth, M. S.; Otaka, A.; Roller, P. P. Tetrahedron
Lett. 1993, 34, 4125. (b) Ye, B.; Akamatsu, M.; Shoelson, S. E.; Wolf, G.;
Giorgeti-Peraldi, S.; Yan, X.; Roller, P. P.; Burke, T. R. J. Med. Chem.
1995, 38, 4270.
(15) Only the ee of compound 15 was determined. This was achieved
by preparing two dipeptides, F2Smp-L-Leucine and F2Smp-DL-leucine,
followed by analytical HPLC analysis of the dipeptides. The ee of 15 was
found to be >97%. Since the procedure used to prepare 16 from 10 was
identical to that used for preparing 15 from 9, it is reasonable to assume
that 16 was obtained with an ee comparable to that of 15.
(16) See: Gao, Y.; Wu, L.; Luo, J. H.; Guo, R.; Yang, D.; Zhang, Z.
Y.; Burke, T. R. Bioorg. Med. Chem. Lett. 2000, 10, 923 and references
therein.
(17) (a) Kennedy, B. P.; Ramachandran, C. Biochem. Pharmacol. 2000,
60, 877-883. (b) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.;
Collins, S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan,
C. C.; Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy, B P.
Science 1999, 283, 1544-48.
(18) Studies with 16 in 20% piperidine in DMF suggested that some
loss of the TCE group may have occurred during peptide synthesis, which
may have resulted in an increase in the formation of impurities.
Acknowledgment. This work was supported by the
Natural Sciences and Engineering Research Council (NSERC)
(19) Details of the kinetic studies will be reported elsewhere.
(20) Farzan, M.; Mirzabekov, T.; Kolchinsky, P.; Wyatt, R.; Cayabyab,
M.; Gerard, N. P.; Gerard, C.; Sodroski, J.; Choe, H. Cell 1999, 96, 667.
(21) Farzan, M.; Vasilieva, N.; Schnitzler, C. E.; Chung, S.; Robinson,
J.; Gerard, N. P.; Gerard, C.; Choe, H.; Sodroski, J. J. Biol. Chem. 2000,
275, 33516.
Org. Lett., Vol. 3, No. 10, 2001
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of Canada in the form of an operating grant to S.D.T., who
also gratefully acknowledges Merck-Frosst Canada & Co.,
The Canadian Institutes of Health Research (CIHR), the
NSERC University-Industry Projects program, and The
University of Waterloo for additional support.
Supporting Information Available: Preparation proce-
dures and characterization data for 5-17. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0158664
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Org. Lett., Vol. 3, No. 10, 2001