Stereoselective synthesis of CF2-substituted phosphothreonine mimetics and their incorporation into peptides using newly developed deprotection procedures

Article abstract of DOI:10.1021/jo000169v

Stereoselective syntheses of all four stereoisomers of CF₂-substituted nonhydrolyzable phosphothreonine derivatives (33, 39, and their enantiomers) and their incorporation into peptides are described herein. Key to the synthesis of these amino acids was construction of secondary phosphate-mimicking difluoromethylphosphonate units along with generation of two stereocenters. The former was achieved using a Cu(I)-mediated cross-coupling reaction of BrZnCF₂P(O)(OEt)₂ (8) and β-iodo-α,β-unsaturated ester 12, with stereochemistry of both α- and β-stereocenters being established using bornane-10,2-sultam as a chiral auxiliary. Diastereoselective hydrogenation of a chiral α,β-unsaturated acylsultam (for the β-center) (e.g., 16a) and subsequent stereoselective bromination (for the α-center of the threo derivative) or amination (for the α-center of erythro (allo) derivative) were utilized. Transesterification of the bromide to the benzyl ester followed by azide displacement of the halogen, then reduction of the resulting azide, followed by Boc-protection and finally removal of the benzyl group, afforded protected both L- and D-phosphothreonine mimetics (39 and its enantiomer). On the other hand, protected both L- and D-allo-phosphothreonine mimetics (33 and its enantiomer) were synthesized via transesterification of the above-mentioned amination product, followed by hydrogenolytic removal of the benzyl group. Key to utilization of these amino acid analogues in peptide synthesis was removal of ethyl protection from the difluoromethylphosphonate moiety. A two-step deprotection methodology, consisting of a combination of a first-step reagent [0.3 M BSTFA-TBAI in CH₂Cl₂, BF₃·Et₂O] followed by a second-step reagent [1 M TMSOTf-thioanisole in TFA, m-cresol, EDT] was developed for use in solid-phase protocols. A 12-residue Cdc (cell division cycle) 2-peptide 41, possessing two nonhydrolyzable phosphoamino acid mimetics (F₂Pmab 6 and F₂Pmp 4), was subjected to this deprotection procedure and was obtained in 25% yield based on the protected resin. The present synthetic method affords nonhydrolyzable phosphoamino acid mimetics-containing peptides in high yield without accompanying side reactions.

Full text of DOI:10.1021/jo000169v

4888
J . Org. Chem. 2000, 65, 4888-4899
Ster eoselective Syn th esis of CF 2-Su bstitu ted P h osp h oth r eon in e
Mim etics a n d Th eir In cor p or a tion in to P ep tid es Usin g New ly
Develop ed Dep r otection P r oced u r es†,|
Akira Otaka,*,‡ Etsuko Mitsuyama,‡ Takayoshi Kinoshita,§ Hirokazu Tamamura,‡ and
Nobutaka Fujii‡
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, J apan and
Fujisawa Pharmaceutical Co., Ltd., Kashima, Yodogawa-ku, Osaka 532-8514, J apan
aotaka@pharm.kyoto-u.ac.jp
Received February 7, 2000
Stereoselective syntheses of all four stereoisomers of CF2-substituted nonhydrolyzable phospho-
threonine derivatives (33, 39, and their enantiomers) and their incorporation into peptides are
described herein. Key to the synthesis of these amino acids was construction of secondary phosphate-
mimicking difluoromethylphosphonate units along with generation of two stereocenters. The former
was achieved using a Cu(I)-mediated cross-coupling reaction of BrZnCF2P(O)(OEt)2 (8) and â-iodo-
R,â-unsaturated ester 12, with stereochemistry of both R- and â-stereocenters being established
using bornane-10,2-sultam as a chiral auxiliary. Diastereoselective hydrogenation of a chiral R,â-
unsaturated acylsultam (for the â-center) (e.g., 16a ) and subsequent stereoselective bromination
(for the R-center of the threo derivative) or amination (for the R-center of erythro (allo) derivative)
were utilized. Transesterification of the bromide to the benzyl ester followed by azide displacement
of the halogen, then reduction of the resulting azide, followed by Boc-protection and finally removal
of the benzyl group, afforded protected both L- and D-phosphothreonine mimetics (39 and its
enantiomer). On the other hand, protected both L- and D-allo-phosphothreonine mimetics (33 and
its enantiomer) were synthesized via transesterification of the above-mentioned amination product,
followed by hydrogenolytic removal of the benzyl group. Key to utilization of these amino acid
analogues in peptide synthesis was removal of ethyl protection from the difluoromethylphosphonate
moiety. A two-step deprotection methodology, consisting of a combination of a first-step reagent
[0.3 M BSTFA-TBAI in CH2Cl2, BF3‚Et2O] followed by a second-step reagent [1 M TMSOTf-
thioanisole in TFA, m-cresol, EDT] was developed for use in solid-phase protocols. A 12-residue
Cdc (cell division cycle) 2-peptide 41, possessing two nonhydrolyzable phosphoamino acid mimetics
(F2Pmab 6 and F2Pmp 4), was subjected to this deprotection procedure and was obtained in 25%
yield based on the protected resin. The present synthetic method affords nonhydrolyzable
phosphoamino acid mimetics-containing peptides in high yield without accompanying side reactions.
In tr od u ction
ing the difluoromethylene unit (CF2)4,5 as a replacement
for the phosphoryl ester oxygen have shown particular
utility. For example, 4-phosphonodifluoromethyl phenyl-
alanine (F2Pmp 4)6 as CF2-substituted pTyr (phospho-
tyrosine) 1 mimetic has served as a nonhydrolyzable pTyr
analogue where the acidity of F2Pmp is comparable to
that of pTyr, whereas the corresponding CH2-analogue
(phosphonomethyl phenylalanine, Pmp) is less acidic.
Similarly, CF2-substitution in pSer (phosphoserine) 2 or
pThr (phosphothreonine) 3 would seem to provide po-
tential nonhydrolyzable phosphoamino acid mimetic,
Phosphorylation and dephosphorylation of proteins
serve as posttranslational modifications that are critical
for intracellular signal transduction.1 Phosphopeptides2
have provided useful biochemical tools for evaluating the
roles of phosphorylation events in cellular signaling;
however, the phosphate moiety is easily hydrolyzed by
the action of protein phosphatases. For this reason,
nonhydrolyzable phosphoamino acid mimetic-containing
peptides3 have gained much attention as useful agents
for exploring signaling events. Among various nonhy-
drolyzable phosphoamino acid analogues, those employ-
(3) Burke, T. R., J r.; Russ, P.; Lim, B. Synthesis 1991, 1019-1020.
Shoelson S. E.; Chatterjee, S.; Chaudhuri, M.; Burke, T. R., J r.
Tetrahedron Lett. 1991, 32, 6061-6064. Cushman, M.; Lee, E.-S.
Tetrahedron Lett. 1992, 33, 1193-1196. Ojea, V.; Ruiz, M., Shapiro,
G.; Pombo-Villar, E. Tetrahedron Lett. 1994, 35, 3273-3276. Ruiz, M.;
Ojea, V.; Shapiro, G.; Weber, H.-P.; Pombo-Villar, E. Tetrahedron Lett.
1994, 35, 4551-4554. Nomizu, M.; Otaka, A.; Burke, T. R., J r.; Roller,
P. P. Tetrahedron 1994, 50, 2691-2702.
† This paper is dedicated to the memory of Prof. Toshiro Ibuka, who
unexpectedly passed away on J anuary 20, 2000.
‡ Kyoto University.
§ Fujisawa Pharmaceutical Co., Ltd.
| Abbreviations. TMSOTf ) trimethylsilyl trifluoromethanesulfonate,
TMSI ) trimethylsilyl iodide, TFMSA ) trifluoromethanesulfonic acid,
TFA ) trifluoroacetic acid, DMS ) dimethyl sulfide, EDT ) 1,2-
ethanedithiol, NaHMDS ) sodium bis(trimethylsilyl)amide, IS-MS )
Ion-spray mass spectrometry, ClZ ) 2-chlorobenzyloxycarbonyl, Cl2-
Bzl ) 2,6-dichlorobenzyl, PAM ) 4-(oxymethyl)phenylacetamidom-
ethyl, MBHA ) 4-methylbenzhydrylamine, DIPCDI ) N,N-diisopro-
pylcarbodiimide, HOBt ) 1-hydroxybenzotriazole, DIPEA ) N,N-
diisopropylethylamine, WSCDI ) water soluble carbodiimide (1-ethyl-
3-(3′-dimethylaminopropyl)carbodiimide), Cdc ) cell division cycle.
(1) Hunter, T. Cell 2000, 100, 113-127.
(4) Chambers, R. D.; O’Hagan, D.; Lamont, R. B.; J ain, S. C. J .
Chem. Soc., Chem. Commun. 1990, 1053-1054. O’Hagan, D.; Rzepa,
H. S. Chem. Commun. 1997, 645-652.
(5) For review of synthesis of gem-difluoromethylene compounds,
see: Trzer, M. J .; Herpin, T. F. Tetrahedron 1996, 52, 8619-8683.
(6) Burke, T. R., J r.; Smyth, M. S.; Nomizu, M.; Otaka, A.; Roller,
P. P. J . Org. Chem. 1993, 58, 1336-1340. Burke, T. R., J r.; Smyth, M.
S.; Otaka, A.; Roller, P. P. Tetrahedron Lett. 1993, 34, 4125-4128.
Smyth, M. S.; Burke, T. R., J r.; Tetrahedron Lett. 1994, 35, 551-554.
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D.; Hale, R. L.; Patel, D. V. J . Org. Chem. 1996, 61, 1537-1539.
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In Methods in Enzymology; Fields, G. B., Ed.; Academic Press: New
York, 1997; Vol. 289, pp 245-266.
10.1021/jo000169v CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/11/2000

CF2-Substituted Phosphothreonine Mimetics
J . Org. Chem., Vol. 65, No. 16, 2000 4889
romethyl)zinc bromide (8) with alkenyl halides have been
reported,13 which prompted us to utilize similar meth-
odology in combination with Oppolzer’s sultam chemis-
try14 for the stereoselective synthesis of F2Pmab. We
chose to employ side chain ethyl protection of F2Pmab
similar to that used for F2Pmp and F2Pab,15 since the
requisite difluoromethylphosphonate reagent bearing
ethyl groups, BrCF2P(O)(OEt)2 (7),16 is easily obtainable
and cleanly converted to the corresponding Zn re-
agent.13,17 Furthermore, ethyl protection is stable toward
synthetic transformations employed in our study. How-
ever, incorporation of such ethyl-protected derivatives
into peptides should be done with extreme caution, since
this type of phosphonate protection may behave differ-
ently relative to other protecting groups normally em-
ployed in peptide chemistry.18 Increasing the acidity of
deprotection reagents typically leads to facile removal of
commonly used protecting groups; however, this is not
the case with phosphonate- or phosphate-alkyl protecting
groups, where low acidic SN2-type deprotecting method-
ologies have been shown to be critical for removal of side
chain protecting groups on phosphoamino acids. Thus,
we developed a two-step deprotecting methodology19
consisting of high acidic (1 M TMSOTf-thioanisole in
TFA20), followed by low acidic (1 M TMSOTf-thioanisole
in TFA + TMSOTf-DMS), treatment, which is suitable
for dimethyl-protected pTyr-, pSer-, and/or pThr-contain-
ing peptide resins. Application of this methodology to the
deprotection of ethyl-protected F2Pmp, or F2Pab-contain-
ing peptide resins affords fully deprotected peptides with
some success.12 However, incomplete removal of ethyl
groups, especially on F2Pmab, has been encountered.
Moreover, use of alternative low acidic treatment (TM-
SOTf-DMS-m-cresol-EDT), which provides effective
removal of ethyl groups, sometimes hampers the isolation
of fully deprotected peptide from reaction mixtures.10
We report herein the stereoselective synthesis of all
four isomers of protected F2Pmab as well as an examina-
tion of deprotecting reagent systems suitable for CF2-
substituted phosphoamino acid mimetics-containing pep-
tide resins.
F igu r e 1.
which led us to engage the synthesis of the pSer and pThr
mimetics (Figure 1).
Synthetic approaches toward CF2-substituted phos-
phoamino acid mimetics by us and others have provided
F2Pmp 46 and 2-amino-4,4-difluoro-4-phosphonobutanoic
acid (F2Pab 5)7 as nonhydrolyzable pTyr and pSer mi-
metics, respectively.8 Alternatively, synthesis of 2-amino-
4,4-difluoro-3-methyl-4-phosphonobutanoic acid (F2Pmab
6) as a CF2-substituted pThr mimetic initially was
lacking due to a paucity of efficient methodologies for
construction of the secondary CF2 phosphonate unit.
However, Berkowitz and co-workers9 did described a
stereoselective synthesis of this pThr mimetic through
the reaction of CH3MgBr onto a keto difluoromethylphos-
phonate. We have also accomplished the preparation of
racemic protected F2Pmab10 by the CeCl3-mediated con-
jugate addition of diethyl difluoromethylphosphonate
anion onto a nitroalkene.11 We then successfully incor-
porated these phosphoamino acid mimetics into peptides
using a combination of solid-phase peptide synthesis and
a two-step deprotection protocol, which consisted of high
acidic and low acidic treatments.12 Recently, copper(I)-
mediated coupling reactions of (diethylphosphonodifluo-
Resu lts a n d Discu ssion
Recently, Yokomatsu et al. have reported the CuBr-
mediated coupling of BrZnCF2P(O)(OEt)2 8 with alkenyl
halides to yield (R,R-difluoroallyl)phosphonates.13 We
undertook the stereoselective synthesis of all four F2-
(7) Otaka, A.; Miyoshi, K.; Burke, T. R., J r.; Roller, P. P.; Kubota,
H.; Tamamura, H.; Fujii, N. Tetrahedron Lett. 1995, 36, 927-930.
Berkowitz, D. B.; Shen, Q.; Maeng, J .-H. Tetrahedron Lett. 1994, 35,
6445-6448.
(13) Yokomatsu, T.; Suemune, K.; Murano, T.; Shibuya, S. J . Org.
Chem. 1996, 61, 7207-7211.
(14) Oppolzer, W. Pure Appl. Chem. 1990, 62, 1241-1250. Oppolzer,
W. Tetrahedron 1987, 43, 1969-2004.
(8) For biochemical aspects of nonhydrolyzable phosphopeptides,
see: Burke, T. R. J r.; Smyth, M. S.; Otaka, A.; Nomizu, M.; Roller, P.
P.; Wolf, G.; Case, R.; Shoelson, S. E. Biochemistry 1994, 33, 6490-
6494. Wange, R. L.; Isakov, N.; Burke, T. R., J r.; Otaka, A.; Roller, P.
P.; Watts, J . D.; Aebersold, R.; Samelson, L. E. J . Biol. Chem. 1995,
270, 944-948. Burke, T. R., J r.; Kole, H. K.; Roller, P. P. Biochem.
Biophys. Res. Commun. 1994, 204, 129-134. Chen, L.; Wu, L.; Otaka,
A.; Smyth, M. S.; Roller, P. P.; Burke, T. R., J r.; Hertog, J .; Zhang,
Z.-Y. Biochem. Biophys. Res. Commun. 1995, 216, 976-985.
(9) Berkowitz, D. B.; Eggen, M.; Shen, Q.; Shoemaker, R. K. J . Org.
Chem. 1996, 61, 4666-4675.
(10) Otaka, A,; Mitsuyama, E.; Oishi, S.; Miyoshi, K.; Tamamura,
H.; Fujii, N. In Peptides 1998, Proceedings of the Twenty-Fifth
European Peptide Symposium; Bajusz, S., Hudecz, F., Eds.; Akade´miai
Kiado´: Budapest, 1999; pp 264-265.
(11) Lequeux, T. P.; Percy, J . M. Synlett 1995, 361-362.
(12) Otaka, A.; Miyoshi, K.; Kaneko, M.; Burke, T. R. J r.; Roller, P.
P.; Tamamura, H.; Fujii, N. In Peptides 1996, Proceedings of the
Twenty Fourth European Peptide Symposium; Ramage, R., Epton, R.,
Eds.; Mayflower Scientific Ltd.: Kingswinford, 1998; pp 701-702.
(15) For the use of other protecting groups, see: Berkowitz, D. B.;
Sloss, D. G. J . Org. Chem. 1995, 60, 7047-7050.
(16) Burton, D. J .; Flynn, R. M. J . Fluorine Chem. 1977, 10, 329-
332.
(17) Burton, D. J .; Sprague, L. G. J . Org. Chem. 1989, 54, 613-
617. Burton, D. J .; Yang, Z.-Y. Tetrahedron 1992, 48, 189-275.
(18) Otaka, A.; Burke, T. R., J r.; Smyth, M. S.; Nomizu, M.; Roller,
P. P. Tetrahedron Lett. 1993, 34, 7039-7042.
(19) Otaka, A.; Miyoshi, K.; Roller, P. P.; Burke, T. R., J r.; Tama-
mura, H.; Fujii, N. J . Chem. Soc., Chem. Commun. 1995, 387-389.
Otaka, A.; Miyoshi, K.; Kaneko, M.; Tamamura, H.; Fujii, N.; Nomizu,
M.; Burke, T. R., J r.; Roller, P. P. J . Org. Chem. 1995, 60, 3967-3974.
(20) Fujii, N.; Otaka, A.; Ikemura, O.; Akaji, K.; Funakoshi, S.;
Hayashi, Y.; Kuroda, Y.; Yajima, H. J . Chem. Soc., Chem. Commun.
1987, 274-275. Fujii, N.; Otaka, A.; Ikemura, O.; Hatano. M.;
Okamachi, A.; Funakoshi, S.; Sakurai, M.; Shioiri, T.; Yajima, H. Chem.
Pharm. Bull. 1987, 35, 3447-3452. Yajima, H.; Fujii, N.; Funakoshi,
S.; Watanabe, T.; Murayama, E.; Otaka, A. Tetrahedron 1988, 44, 805-
819.

4890 J . Org. Chem., Vol. 65, No. 16, 2000
Otaka et al.
Sch em e 1. Syn th etic P la n for F 2P m a b
Der iva tives
Sch em e 2a
Pmab isomers by applying this coupling reaction to
â-halo-R,â-unsaturated carboxylic acid derivatives 9,
followed by diastereoselective hydrogenation and ami-
nation with the aid of a chiral auxiliary, as conceptually
outlined in Scheme 1.
a
Key: (i) NaI (1.0 equiv), AcOH; (ii) Ti(OiPr)4 (2.0 equiv), PMB-
OH (12 equiv); (iii) BrZnCF2P(O)(OEt)2 (2.0 equiv), CuBr (2.0
equiv), DMF; (iv) 95% TFA aq; (v) LiXR (or LiXS) (1.5 equiv), Piva-
Cl (1.2 equiv), Et3N (1.2 equiv), THF.
olefinic bond in the presence of Pd-C.24 Furthermore, 1,4-
hydride addition to olefinic sultam-imides using lithium
tri-s-butylborohydride (L-Selectride) has been reported
to proceed with high opposite π-face discrimination, as
compared with H2/Pd-C.25 These reports prompted us
to utilize similar methodologies for construction of the
stereogenic centers at the â-position. Hydrogenation of
the olefinic sultam-imide derivative 14 with H2/Pd-C in
EtOAc resulted in high stereoface discrimination, afford-
ing a mixture of major (16a ) and minor (16b) isomers
(96:4) in quantitative yield (Scheme 2). Following flash
chromatographic purification, 16a was crystallized from
EtOAc, with the stereochemistries of 16a and 16b being
determined as follows: X-ray analysis of 16a showed that
it possessed (3R-XR)-configuration. When 16a and 16b
were converted to their corresponding benzyl ester
derivatives (16a ′ and 16b′) using Ti(OiPr)4 in benzyl
alcohol,22,26 physical and chemical data for both com-
pounds were identical, except that the signs of optical
rotation were reversed. Based on this, we concluded that
16b possessed the (3S-XR)-configuration. Analogously,
Ethyl (Z)-3-iodo-2-butenoate (11) was employed as a
requisite â-halo alkenoic acid derivative, which could be
easily obtained in a highly regio- and stereoselective
manner by the reaction of ethyl 2-butynoate (10) with
NaI in AcOH (Scheme 2).21 Prior to introduction of the
difluoromethylphosphonate unit, 11 was converted to the
corresponding p-methoxybenzyl ester derivative 12
through transesterification using Ti(OiPr)4 in PMB-OH
(PMB ) p-methoxybenzyl).22 This was done to allow
subsequent selective removal of PMB groups without
affecting phosphonate ethyl esters. The CuBr-mediated
coupling of 8 with 12 in DMF at room temperature,
afforded adduct 13 in nearly quantitative yield, with
retention of the starting olefinic geometry.23 Alternative
attempts at coupling 8 with (Z)-3-iodo-2-butenoic acid did
not give a desired coupling product in satisfactory yield.
Treatment of 13 with 95% aqueous TFA, followed by
coupling with (2R)- or (2S)-bornane-10,2-sultam accord-
ing to published procedures,14 yielded sultam-imide
conjugated alkenes 14 or 15, respectively (Scheme 2).
Oppolzer et al. reported high diastereoface discrimina-
tion in the hydrogenation of a trisubstituted sultam-imide
(24) Oppolzer, W.; Mills, R. J .; Re´glier, M. Tetrahedron Lett. 1986,
27, 183-186.
(21) Marek, I.; Christophe, M.; Normant, J .-F. Org. Synth. 1997,
74, 194-204.
(22) Seebach, D.; Hungerbuhler, E.; Naef, R.; Schurrenberger, P.;
Weidmann, B.; Zuger, M. Synthesis 1982, 138-141.
(23) Double-bond geometries of 13 and 15 were confirmed by NOE
measurements in comparison with their isomers.
(25) Oppolzer, W.; Poli, G. Tetrahedron Lett. 1986, 27, 4717-4720.
Oppolzer, W.; Poli, G.; Starkermann, C.; Bernardinelli, G. Tetrahedron
Lett. 1988, 29, 3559-3562.
(26) Evans, D. A.; Britton, T. C.; Ellman, J . A.; Dorow, R. L. J . Am.
Chem. Soc. 1990, 112, 4011-4030.

CF2-Substituted Phosphothreonine Mimetics
J . Org. Chem., Vol. 65, No. 16, 2000 4891
Sch em e 3a
Sch em e 4
Sch em e 5. Str a tegies for Dia ster eoselective
In tr od u ction of a n Am in o F u n ction a litya
a
Key: (i) nBuLi (2.2 equiv), (Boc)2O (1.55 equiv), THF; (ii)
nBu3Sn(nBu)Cu(CN)Li2 (1.3 equiv), EtOH, THF, (iii) I2(1.0 equiv),
CH2Cl2; (iv) BrZnCF2P(O)(OEt)2 (2.0 equiv), CuBr (2.0 equiv),
DMF, (v) 95% TFA aq.; (vi) LiXS (1.5 equiv), Piva-Cl (1.2 equiv),
Et3N (1.2 equiv), THF.
hexane as an NH2+ equivalent,29 while the other (Scheme
5, ii + iii) utilizes a diastereoselective enolate bromina-
tion followed by an SN2 substitution using azide anion.26,30
The former reaction is reported to proceed via a transition
state which features a chelated (Z)-enolate that is at-
tacked by the nitroso electrophile opposite to the lone pair
on the sultam nitrogen. The latter bromination reaction
proceeds from the same face. This implies that both
electrophilic amination and bromination of enolates
derived from XS-sultams should furnish (2S)-products.14
Thus, we speculated that the reaction of the enolate
derived from 17b (3R-XS) with 1-chloro-1-nitrosocyclo-
hexane, followed by hydrolysis of the resulting unisolated
nitrone, should afford 24 (2S,3R-XS), which is a precursor
to the L-F2Pmab derivative. However, attempted reac-
tions based on the above considerations resulted in
recovery of starting material. Alternatively, electrophilic
amination of Na-enolates derived from 17a (3S-XS) by
treatment with NaHMDS in THF at -78 °C, with
1-chloro-1-nitrosocyclohexane, followed by addition of
aqueous 1 N HCl, proceeded with high stereofacial
discrimination to afford hydroxylamine 27 (2S,3S-XS).
Without isolating, this was subjected to a sequence of
reactions to furnish Boc-protected L-allo-F2Pmab deriva-
tive en t-33. The difference in reactivity of Na-enolates
resulting from 17a and 17b potentially may be attributed
to differences in reactive conformations (TS (transition
state) 28 and TS 29) (Figure 2).
compounds 17a (major) and 17b (minor) obtained hy-
drogenolytically from 15, the antipode of 14, possessed
(3S-XS)- and (3R-XS)-configurations, respectively. To
obtain a diastereomer 16b (3S-XR) in a highly stereo-
selective manner, 1,4-hydride addition to 14 using L-
Selectride or K-Selectride was attempted, however high
diastereofacial discrimination was not observed (Scheme
2). This necessitated alternative synthetic approaches to
16b and 17b (Scheme 3).
Treatment of 1-bromo-1-propene (18) with nBuLi in
THF at -78 °C afforded propynyllithium, which was
subsequently carried forward in a one-pot reaction using
(Boc)2O to yield tert-butyl 2-butynoate (19).27 Kinetically
controlled stannyl cupration of 19 with nBu3Sn(nBu)Cu-
(CN)Li2 at -78 °C in the presence of EtOH, gave tert-
butyl (E)-3-tributylstannyl-2-butenoate (20),28 which upon
treatment with iodine in CH2Cl2 yielded tert-butyl (E)-
3-iodo-2-butenoate (21). Transformation of 21 to sultam-
imide conjugated alkene 23 was achieved in a fashion
similar to that employed in the conversion of 12 to 14.
The resulting conjugated alkene 23 was hydrogenated
over Pd-C in toluene with moderate π-face discrimina-
tion to afford a mixture of major 17b (3R-XS) and minor
17a (3S-XS) (89:11) isomers in quantitative yield. These
results indicated that hydrogenation of (Z)- or (E)-3-
diethylphosphonodifluoromethyl-2-butenimide deriva-
tives over Pd-C gave diastereoselectively (3R-XR)-16a
and its antipode 17a or (3S-XR)-16b and its antipode 17b,
respectively (Scheme 4).
In TS 29, both positioning of the difluoromethylphos-
phonate group on the Si-face due to 1,3-allylic strain31,32
and shielding of Re-face by the chiral auxiliary may
prevent attack of the nitroso electrophile from both sides,
thereby blocking product formation. On the other hand,
Having routes to the four stereochemically pure iso-
mers of 3-diethylphosphonodifluoromethylbutanimide in
hand, we next examined the diastereoselective introduc-
tion of amino functionality using two protocols (Scheme
5).
One approach toward diastereoselective amino intro-
duction (Scheme 5, i) employs a π-face selective electro-
philic enolate amination using 1-chloro-1-nitrosocyclo-
(29) Oppolzer, W.; Tamura, O.; Deerberg, J . Helv. Chim. Acta 1992,
75, 1965-1978.
(30) Nicola´s, E.; Russell, K. C.; Knollenberg, J .; Hruby, V. J . J . Org.
Chem. 1993, 58, 7565-7571.
(31) Hoffmann, R. W. Chem. Rev. 1989, 89, 1841-1860.
(32) Bromination (NaHMDS then NBS) of PMB ester, resulting from
treatment of 16a (3R-XR) with Ti(OiPr)4 in PMB-OH, proceeded with
moderate diastereoselectivity (de 79%) to mainly afford the (2R)-
product, which can be attributed to the Si-face shielding by dieth-
ylphosphonodifluoromethyl group.
(27) Suffert, J .; Toussaint, D. J . Org. Chem. 1995, 60, 3550-3553.
(28) Fleming, I. In Organocopper Reagents, A Practical Approach;
Taylor, R. J . K., Ed.; Oxford University Press: Oxford, 1994; pp 257-
292 and references therein.

4892 J . Org. Chem., Vol. 65, No. 16, 2000
Otaka et al.
F igu r e 2. Reagents are as follows: (i) NaHMDS, THF, 1-chloro-1-nitrosocyclohexane, -78 °C; (ii) 1 N HCl (aq).
Sch em e 6. Syn th esis of Non h yd r olyza ble
erythro) was achieved from 16a (3R-XR). Deprotonation
of 16a with NaHMDS in THF at -78 °C, followed by slow
addition of 1-chloro-1-nitrosocyclohexane (blue), instan-
taneously afforded a colorless solution of nitrone. Treat-
ment of this solution with aqueous 1 N HCl, followed by
an extractive workup, gave crude hydroxylamine 30,
which was taken to the next step without further
purification. Reduction of 30 with Zn-AcOH in THF,
followed by introduction of Boc protection onto the
resulting NH2 group using (Boc)2O, gave Boc-protected
31 (2R,3R-XR), with transformation of 31 to the corre-
sponding benzyl ester 32 being accomplished utilizing
Ti(OiPr)4-benzyl alcohol in toluene at 120 °C.26,33 Use
of toluene was critical due to low solubility of 31.
Hydrogenolytic debenzylation (H2/10% Pd-C in EtOAc)
gave the Boc-protected D-allo-amino acid 33. Application
of the same reaction sequence to 17a (3S-XS) afforded
the corresponding protected L-allo-amino acid en t-33
(2S,3S) (Scheme 6). Data obtained from NMR measure-
ments of 33 and en t-33 were identical to those published9
and of racemic allo-F2Pmab derivative synthesized by
us.10
a llo-p Th r Mim eticsa
In Scheme 7 is shown an alternative synthetic route
for the threo derivatives utilizing bromination and azide
displacement. Diastereoselective bromination with NBS
in THF of sodium enolates resulting from 16a , proceeded
with high diastereoselectivity (de 98.8%), to give bromide
34 (2R,3S-XR) in 96% yield. Conversion of 34 to the
corresponding benzyl ester 35 was achieved using Ti-
(OiPr)4-benzyl alcohol in toluene at 120 °C for 5 h.
Transformation at this stage is critical for accomplishing
the synthesis of threo derivatives.34,35 Treatment of 35
with tetramethylguanidinium azide (TMGA)36 in CH3CN
at room temperature gave azide derivative 36 (2S,3R)
in 50% yield accompanied by an E2-elimination product
37 (36:37 ) 3:1). It would be expected that repulsive
-CF2P(O)(OEt)2/-CO2Bzl interactions in 35 could make
C(R)-Br and C(â)-H groups antiperiplanar, which could
a
Key: (i) NaHMDS (1.1 equiv), 1-chloro-1-nitrosocyclohexane
(1.1 equiv), THF then 1 N HCl aq.; (ii) Zn (40 equiv), AcOH (50
equiv) then (Boc)2O (2.0 equiv), CH3CN; (iii) Ti(OiPr)4 (2.0 equiv),
Bzl-OH (44 equiv), toluene; (iv) H2/Pd-C, EtOAc.
shielding of the Re-face in TS 28 by both the difluoro-
methylphosphonate group and the sultam moiety should
permit facile attack by 1-chloro-1-nitrosocyclohexane
from the Si-face with high π-face discrimination. These
results show that while erythro (allo)-amino acids ((2R,3R)
and its antipode) could be obtained by direct amination
of 16a (3R-XR) and 17a (3S-XS) utilizing the nitroso
electrophile, similar procedures could not be applied to
the transformation of 17b (3R-XS) and its antipode 16b
(3S-XR) to yield the corresponding threo derivatives 24
(2S,3R-XS) and its antipode. Therefore, for the prepara-
tion of threo amino acid derivatives, we resorted to
successive transformations of 16a or 17a utilizing dia-
stereoselective bromination and azide substitution with
inversion of configuration.
(33) Oppolzer, W.; Schneider, P. Helv. Chim. Acta 1986, 69, 1817-
1820.
(34) Azide displacement of sultam-imide 34 gave much more amount
of an E2-elimination product than that resulting from benzyl ester 35.
(35) Transesterification of Boc-protected sultam-imide, resulting
from 34 via the following sequence (azide displacement, reduction, and
Boc protection), with Ti(OiPr)4-benzyl alcohol in toluene at 120 °C
recovered the starting material. Furthermore, the reaction at elevated
temperature gave decomposed products.
In Scheme 6 is summarized the synthesis of erythro
(allo) derivatives, in which the preparation of optically
pure protected D-allo-F2Pmab derivative 33 (2R,3R;
(36) Papa, A. J . J . Org. Chem. 1966, 31, 1426-1430.

CF2-Substituted Phosphothreonine Mimetics
J . Org. Chem., Vol. 65, No. 16, 2000 4893
Sch em e 7. Syn th esis of Non h yd r olyza ble p Th r
Mim eticsa
with ether can be encountered. In developing the former
methodology, we have previously reported that operation
under SN2 conditions19,37 is crucial for removal of phos-
phate or phosphonate protecting groups. Additionally, we
have found that against dimethyl-protected phosphate-
containing peptides replacement of TMSOTf by TFMSA38
results in substantial formation of incompletely dem-
ethylated product. During the deprotecting reaction,
conversion of monoalkyl phosphate species to fully depro-
tected forms is rate determing. Therefore, differences
between TMSOTf- and TFMSA-based reagents may be
attributed to structural variations in intermediary
monoalkyl-protected forms. That is, silylated intermedi-
ates resulting from TMSOTf-based treatment may more
easily undergo nucleophilic attack by sulfides to form
fully deprotected products as compared to nonsilylated
intermediates.39 We speculated that a deprotecting sys-
tem capable of completely silylating monoalkyl phospho-
nates as well as inducing nucleophilic attack, could
potentially represent a highly efficient deprotective meth-
odology for ethyl protected CF2-phosphonate derivatives.
Gordeev et al. reported that N,O-bis(trimethylsilyl) tri-
fluoroacetamide (BSTFA)-TMSI in CH2Cl2 effectively
removes ethyl groups from protected Fmoc-F2Pmp amino
acid, and the ethyl-deprotected Fmoc amino acid was
successfully applied to practical F2Pmp peptide synthe-
sis.40 Removal of ethyl groups from protected Pmp
(phosphonomethyl phenylalanine) peptide was also
achieved TMSI in MeCN.41 However, direct application
of BSTFA-TMSI in CH2Cl2 or TMSI in MeCN to pro-
tected peptide resins has some disadvantage, which
might afford peptide mixtures involving incompletely
deprotected peptide and resin-bound peptide since TMSI
has ability to remove partly several protecting groups
including peptide-resin linkers. We therefore attempted
to develop a practical deprotecting system, followed by
easy workup, which could be directly used for protected
peptide resins. Since TMSI potentially serves as both an
activator of BSTFA and a nucleophile in Gordeev’s
method, we examined Lewis-acid-nucleophile combina-
tions as substituents for TMSI. For this purpose, an F2-
Pmab(OEt)2-containing model peptide resin 40, corre-
sponding to the partial sequence of the phosphrylated
domain of Cdc2,42 was prepared using standard Boc-
based solid-phase techniques (Figure 3).
a
Key: (i) NaHMDS (1.2 equiv), NBS (3.0 equiv), THF; (ii)
Ti(OiPr)4 (2.0 equiv), Bzl-OH (44 equiv), toluene; (iii) TMGA (3.0
equiv), CH3CN; (iv) Zn (40 equiv), AcOH (50 equiv) then Et3N,
(Boc)2O (2.0 equiv), CH3CN; (v) H2/Pd-C, EtOAc.
account for the formation of a significant amount of the
elimination product 37. Conversion of the azide to amine
(Zn-AcOH in THF), followed by introduction of Boc
protection using (Boc)2O/Et3N, gave Boc-protected threo
derivative 38 (2S,3R) in 82% yield. Catalytic hydrogena-
tion of 38 (H2/Pd-C) yielded the desired L-F2Pmab
derivative 39 in quantitative yield, suitable for solid-
phase peptide synthesis. The D-F2Pmab derivative en t-
39 (2R,3S) was also obtained from 17a using the same
reaction sequence. Data obtained from NMR measure-
ments of 39 and en t-39 were identical to those published9
and of racemic F2Pmab derivative synthesized by us.10
Next, we examined the synthesis of F2Pmab-containing
peptides. As mentioned above, ethyl phosphonate esters
are difficult to deprotect using reagent systems commonly
employed for peptide synthesis. We were therefore
prompted to examine reagent systems applicable to ethyl-
protected F2Pmp, F2Pab, and/or F2Pmab-containing pep-
tide resins, which lead to the development of two depro-
tecting protocols. The first method consisted of a one-
pot, two-step methodology which employed high acidic
(1 M TMSOTf-thioanisole in TFA) and low acidic (1 M
TMSOTf-thioanisole in TFA + TMSOTf-DMS) treat-
ments.19 The second method involved successive treat-
ment with 1 M TMSOTf-thioanisole in TFA followed by
ethyl deprotection of the ether-precipitated crude peptide
resulting from the first reaction mixture, using TMSOTf-
DMS-m-cresol-EDT.12 The use of the former procedure
sometimes resulted in incompletely deprotected peptide,
while the latter protocol has ethyl-removing ability
superior to that of the former system. It should be noted
however that partially deprotected peptide must be
precipitated and washed with ether prior to the second
step. Furthermore, difficulty in isolating fully deprotected
peptide from the second reaction mixture by precipitation
Starting from unsubstituted MBHA resin,43 the pro-
tected peptide resin was synthesized using manual Boc
methodology with a combination of TFA-mediated Boc
deprotection, followed by neutralization with DIPEA and
(37) Tam, J . P.; Heath, W. F.; Merrifield, R. B. J . Am. Chem. Soc.
1986, 108, 5242-5251. Tam, J . P.; Heath, W. F.; Merrifield, R. B. J .
Am. Chem. Soc. 1983, 105, 6442-6455. Tam, J . P.; Merrifield, R. B.
In The Peptide, Analysis, Synthesis, Biology; Meienhofer, J ., Uden-
friend, S., Eds.; Academic Press: New York, 1987; Vol. 9, pp 185-
248. Tam, J . P. In Macromolecular Sequencing and Synthesis, Selected
Methods and Applications; Schlesinger, D. H., Ed.; Alan R. Liss, Inc.:
New York, 1988; pp 153-184.
(38) Yajima, H.; Fujii, N. J . Am. Chem. Soc. 1981, 103, 5867-5871.
Yajima, H.; Fujii, N. In The peptides, Analysis, Synthesis, Biology;
Gross, E., Meienhofer, J ., Eds.; Academic Press: New York, 1983; Vol.
5, pp 65-109.
(39) Takeuchi, Y.; Demachi, Y.; Yoshii, E. Tetrahedron Lett. 1979,
1231-1232.
(40) Gordeev, M. F.; Patel, D. V.; Barker, P. L.; Gordon, E. M.
Tetrahedron Lett. 1994, 35, 7585-7588.
(41) Green, O. M. Tetrahedron Lett. 1994, 35, 8081-8084.
(42) Morgan, D. O. Nature 1995, 374, 131-134. Fisher, R. P. Curr.
Opin. Cell. Biol. Genet. 1997, 7, 32-38.
(43) Rivier, J .; Vale, W.; Burgus, R.; Ling, N.; Amoss, M.; Blackwell,
R.; Guillemin, R. J . Med. Chem. 1973, 16, 545-549.

4894 J . Org. Chem., Vol. 65, No. 16, 2000
Otaka et al.
Ta ble 1. Exa m in a tion of Dep r otectin g Con d ition s
ratio (%) of
components
run
reagent
40a 40b 40c
1
2
3
1 M TMSOTf in TFA, m-cresol, EDTa
1 M TMSOTf-thioanisole in TFAa
First step (run 2a) + Second step
(addition of DMS-TMSOTf (30:20)b)
First step (run 2a) + Second step
(TMSOTf-DMS-m-cresol-EDT
(4:6:0.5:0.5)b)
0
14 86
39 61
55 45
0
0
4
93
7
0
0
F igu r e 3. Protected peptide resin of a model peptide and
resulting deprotected peptides.
5
6
7
8
First step (0.3 M BSTFA-TBAI in CH2Cl2, 100
0
0
0
0
BF3‚Et2Oc) + Second step (run 2a)
First step (0.3 M BSTFA-TBAI in
CH2Cl2c) + Second step (run 2a)
First step (0.3 M BSTFA in CH2Cl2,
BF3‚ET2Oc) + Second step (run 2a)
First step (0.3 M TBAI in CH2Cl2,
BF3‚Et2Oc) + Second step (run 2a)
49 51
71 29
58 41
a
b
At 4 °C for 1.5 h then at room temperature for 0.5 h. At 4 °C
for 2 h. c At room temperature for 1.5 h.
ment of 40 with 1 M TMSOTf-thioanisole in TFA,
followed by TMSOTf-DMS-m-cresol-EDT (4:6:0.5:0.5,
v/v), predominantly gave 40a (Table 1, run 4). However,
difficulties in product isolation due to lack of ether-
induced peptide precipitate formation were encountered.
We next evaluated deprotection conditions based on the
silylation-deprotection concept. Here we initially planned
to use BSTFA as a silylating agent, BF3‚Et2O as an
activator of BSTFA, and (nBu)4NI (TBAI) as a soft
nucleophile. Dichloromethane (CH2Cl2) was used as a
reaction solvent to prevent hydrolysis of the TMS group
on monoethtyl intermediates. After preliminary experi-
ments, it was found that treatment of protected peptide
resins with 0.3 M BSTFA-TBAI (molar ratio 1:1) in CH2-
Cl2 (200 equiv), BF3‚Et2O (20 equiv), followed by filtration
of the resin and successive washing with solvent (CH2-
Cl2, DMF, and Et2O), provided peptide resin possessing
fully deprotected F2Pmab residues. Cleavage of resin with
1 M TMSOTf-thioanisole in TFA gave fully deprotected
F2Pmab peptides. Application of this sequence to 40 gave
fully deprotected peptide 40a without accompanying 40b
and 40c (Table 1, run 5). In this BSTFA-TBAI-BF3
methodology, the combination of the three reagents is
critical for efficient deprotection. As shown in Table 1,
omission of a single reagent resulted in the formation of
incompletely deprotected peptide (Table 1, runs 6-8). To
verify the general applicability of the newly developed
two-step deprotection protocol, we attempted the syn-
thesis of Ac-Lys-Ile-Gly-Glu-Gly-F2Pmab-F2Pmp-Gly-Val-
Val-Tyr-Lys-NH2 41, which is a partial sequence of Cdc2
and a potential candidate as an inhibitor of Cdc2545
phosphatase. Starting from unsubstituted MBHA resin,
the protected peptide resin was elongated using standard
Boc methodology. For side chain protection, the following
protecting groups were utilized: Bzl for Glu, Cl2Bzl for
Tyr, ClZ for Lys,46 and Et for F2Pmab and F2Pmp. This
peptide sequence contained two nonhydrolyzable CF2-
substituted phosphoamino acid mimetics (F2Pmab and
F2Pmp). Treatment of the completed peptidyl resin with
F igu r e 4. HPLC profile of crude 41. An asterisk denotes the
desired peptide: column, Cosmosil 5C18-AR (4.6 × 250 mm);
buffer A, 0.1% aqueous TFA; B, CH3CN (0.1% TFA); linear
gradient, 5-34% B over 50 min; flow rate, 1.0 mL/min,
detection at 220 nm.
DIPCDI/HOBt-mediated coupling of Boc-amino acids.
The following side chain protecting groups were uti-
lized: Bzl for Glu, Cl2Bzl for Tyr,44 and Et for the F2-
Pmab. To obtain incompletely deprotected standard
peptides (mono-Et form 40b and di-Et form 40c, Figure
3), the protected resin was treated with 1 M TMSOTf in
TFA, m-cresol, EDT (Table 1, run 1) or 1 M TMSOTf-
thioanisole in TFA, m-cresol, EDT (Table 1, run 2). The
former reagent system without sulfide as a nucleophile,
operates under SN1 conditions to yield a mixture of 40b
and 40c in a ratio of 14:86. Deprotection of 40 with the
latter thioanisole-mediated system may proceed by a
mixed mode consisting of SN1 and SN2 to afford crude
peptides in a ratio of fully deprotected peptide 40a :40b
(39:61).
Using standard peptides generated in this fashion for
HPLC analyses of crude deprotected mixtures, several
deprotection conditions were examined. Deprotection
with previously published one-pot two-step methodology
gave fully deprotected peptide as a main product; how-
ever, a significant amount of monoethyl peptide 40b was
obtained (40a :40b ) 55:45 Table 1, run 3). The use of
other low-acidic treatment, for example, successive treat-
(45) Strausfeld, U.; Labbe´, J . C.; Fesquet, D.; Cavadore, J . C.; Picard,
A.; Sadhu, K.; Russell, P.; Dore´e, M. Nature 1991, 351, 242-245.
Honda, R.; Ohba, Y.; Nagata, A.; Okayama, H.; Yasuda, H. FEBS Lett.
1993, 318, 331-334. Fauman, E. B.; Cogswell, J . P.; Lovejoy, B.;
Rocque, W. J .; Holmes, W.; Montana, V. G.; Piwnica-Worms, H.; Rink,
M. J .; Saper, M. A. Cell 1998, 93, 617-625.
(44) Erickson, B. W.; Merrifield, R. B. J . Am. Chem. Soc. 1973, 95,
3750-3756.
(46) Erickson, B. W.; Merrifield, R. B. J . Am. Chem. Soc. 1973, 95,
3757-3763.

CF2-Substituted Phosphothreonine Mimetics
J . Org. Chem., Vol. 65, No. 16, 2000 4895
benzyl alcohol (50 mL, 362 mmol) was stirred under vacuum
for 1 h. To the solution at room temperature under argon was
added 7.5 g (31.2 mmol) of 11, and the mixture was allowed
to warm 70 °C and to stir at this temperature for 2 h. The
reaction was quenched at room temperature by addition of
aqueous 1 N HCl. The mixture was extracted with Et2O, and
the extract was washed with brine, dried over MgSO4, and
concentrated in vacuo to yield an oil. Flash chromatographic
purification of the oil over silica gel with n-hexanes-EtOAc
(3:1) gave 8.7 g (83.6% yield) of the title compound 12 as a
colorless oil: 1H NMR (270 MHz, CDCl3) δ 2.72 (d, J ) 1.4
Hz, 3H), 3.81 (s, 3H), 5.13 (s, 2H), 6.31 (q, J ) 1.4 Hz, 1H),
6.86-6.92 (m, 2H), 7.28-7.36 (m, 2H); HRMS (FAB) m/z calcd
for C12H13O3I (MH+) 331.9911, found 331.9917.
0.3 M BSTFA-TBAI in CH2Cl2, BF3‚Et2O at room
temperature for 1.5 h, followed by washing with solvent,
and exposure to 1 M TMSOTf-thioanisole in TFA,
m-cresol, EDT at 4 °C for 1.5 h with additional stirring
for 0.5 h at room temperature, resulted in the release of
completely deprotected peptide. Analyses of resulting
crude peptide by HPLC and IS-MS showed that no
significant side reactions had occurred.47 After HPLC
purification, the purified peptide was obtained in 25%
yield based on the protected peptide resin. These results
indicate that the newly developed two-step protocol,
utilizing a BSTFA-TBAI-BF3‚Et2O in CH2Cl2 system,
has wide applicability for the synthesis of CF2-substituted
nonhydrolyzable phosphoamino acid mimetics-containing
peptides.
That no loss of peptide from the resin occurred during
the first deprotection treatment was supported by model
experiments using Boc-Leu-PAM-Gly resin (Gly, internal
standard for amino acids analyses), where cleavage under
the deprotection condition was not observed. PAM linker48
is known to be sensitive to acidic treatment as compared
to MBHA linker used in this work, indicating that resin
cleavage would be highly unlikely using MBHA resin.
In conclusion, we have achieved the stereoselective
synthesis of all four isomers of protected F2Pmab. Fur-
thermore, deprotection methodology operating under SN2
conditions based on a silylation-deprotection concepts,
which is suitable for removal of Et groups on F2Pmab
residues, was developed. A combination of this new
system and 1 M TMSOTf-thioanisole in TFA provides a
general and efficient procedure for the practical synthesis
of F2Pmab-, F2Pmp-, and/or F2Pab-containing peptides.
Biological evaluation of synthetic Cdc2 peptide is now in
progress.
4-Met h oxyb en zyl (Z)-3-(Diet h ylp h osp h on od iflu or o-
m eth yl)bu t-2-en oa te (13) fr om 12. To a stirred suspension
of 7.5 g (115 mmol) of Zn dust, activated according to published
procedure,50 in DMF (150 mL) at room temperature under
argon was slowly added 18.3 mL (104 mmol) of diethyl
(bromodifluoromethyl)phosphonate (7). An exothermic reaction
occurring with the addition of 7 was maintained around 55
°C by controlling the addition rate. After addition was com-
pleted, additional stirring for 3 h at room temperature gave a
solution of (diethylphosphonodifluoromethyl)zinc bromide (8).
To the solution at room temperature was added a solid of 14.9
g (104 mmol) of CuBr in one portion. After an additional 30
min of stirring, a solution of 17.3 g (52.1 mmol) of 12 in DMF
(50 mL) was added to the above solution. The reaction was
stirred for 3 h at room temperature and then quenched at 4
°C by addition of aqueous 1 N HCl. The mixture was passed
through Celite and extracted with EtOAc. The extract was
successively washed with 1 N HCl, saturated NaHCO3, and
brine, dried over MgSO4, and concentrated in vacuo. Flash
chromatographic purification of the crude materials over silica
gel with n-hexanes-EtOAc (2:1) gave 19.6 g (95.9% yield) of
the title compound 13 as a pale yellow oil: 1H NMR (270 MHz,
CDCl3) δ 1.34 (t, J ) 7.0 Hz, 3H), 2.02 (t, J ) 1.8 Hz, 3H),
3.80 (s, 3H), 4.18-4.32 (m, 4H), 5.10 (s, 2H), 6.05 (t, J ) 1.8
Hz, 1H), 6.84-6.91 (m, 2H), 7.28-7.34 (m, 2H); HRMS (FAB)
m/z calcd for C17H24O6F2P (MH+) 393.1267, found 393.1278.
(2R)-N-[(Z)-3′-(Dieth ylp h osp h on od iflu or om eth yl)bu t-
2-en oyl]bor n a n e-10,2-su lta m (14) fr om 13. Treatment of
10 g (25.5 mmol) of 13 with TFA-H2O (95:5, 50 mL) at 4 °C
for 1 h, followed by concentration in vacuo, afforded a crude
carboxylic acid derivative as a gelatinous residue. The residue
was dissolved with EtOAc. The organic phase was washed with
brine (5 times), dried over MgSO4, and concentrated to leave
a gelatinous residue, which was redissolved with THF (100
mL). To the solution were successively added 4.3 mL (30.6
mmol) of Et3N and 3.8 mL (30.6 mmol) of pivaloyl chloride at
-78 °C under argon. After 15 min of stirring at -78 °C, the
solution was allowed to warm to 4 °C and stirred for 1 h to
yield a mixed anhydride. In separate flask lithiated sultam
(LiXR) was prepared by treatment of 8.2 g (38.3 mmol) of
sultam (HXR) in THF (100 mL) with 29.8 mL (45.9 mmol) of
1.54 M n-BuLi in hexane at -78 °C under argon followed by
an additional 15 min of stirring. To recooled solution of the
mixed anhydride to -78 °C was added the solution of lithiated
sultam, and stirring was continued for 1 h at -78 °C. The
reaction was quenched with saturated NH4Cl solution and
extracted with EtOAc. The extract was successively washed
with saturated NaHCO3 and brine, dried over MgSO4, and
concentrated in vacuo to yield an yellow oil. The oily material
was purified by flash chromatography over silica gel with
n-hexanes-EtOAc (1:1) to give 9.65 g (81.1% yield) of the title
Exp er im en ta l Section
Gen er a l Meth od s. All reactions were carried out under a
positive pressure of argon except for deprotective reactions for
protected peptide resins. All glassware and syringes were dried
in an electric oven at 100 °C prior to use. All melting points
are uncorrected. All NMR spectra were recorded in CDCl3.
Chemical shifts are reported in parts per million downfield
from internal Me4Si (s ) singlet, d ) doublet, dd ) double
doublet, t ) triplet, q ) quadruplet, m ) multiplet). For flash
chromatographies, silica gel 60H (silica gel for thin-layer
chromatography, Merck) or silica gel 60 (finer than 230 mesh,
Merck) was employed. All HPLC separations were carried out
using either Cosmosil 5C18-AR analytical (4.6 × 250 mm) or
Cosmosil 5C18-AR preparative column (20 × 250 mm). Eluting
products were detected by UV at 220 nm. Solvent A was 0.1%
TFA (v/v) in water, and solvent B was 0.1% TFA in CH3CN
unless otherwise specified. A flow rate of 1 mL/min was used
for all analytical separations and a 7 mL/min flow rate for
preparative runs. Both isomers of bornane-10,2-sultam were
prepared according to the literature.49 Protected amino acids,
MBHA resin and other peptide synthesis chemicals were
purchased from Nova Biochmicals or Watanabe Chemical Inc.
All other chemicals were purchased from either Nacalai
Tesque, Wako Pure Chemical Industries, Kanto Chemicals,
Aldrich, or Merck.
compound 14 as an yellow oil: [R]23 -59.3° (c 1.87, CHCl3);
D
1H NMR (270M Hz, CDCl3) δ 0.97 (s, 3H), 1.16 (s, 3H), 1.37 (t,
J ) 7.1 Hz, 6H), 1.34-1.45 (m, 2H), 1.85-1.93 (m, 3H), 2.09
(t, J ) 1.7 Hz, 3H), 2.14-2.26 (m, 2H), 3.40 (d, J ) 7.0 Hz,
1H), 3.48 (d, J ) 7.0 Hz, 1H), 3.87 (dd, J ) 8.0, 5.0 Hz, 1H),
4.25-4.38 (m, 4H), 6.33 (t, J ) 1.2 Hz, 1H); HRMS (FAB) m/z
calcd for C19H31O6NF2PS (MH+) 470.1578, found 470.1570. The
4-Meth oxyben zyl (Z)-3-Iod obu t-2-en oa te (12) fr om 11.
A solution of 18.4 mL (62.5 mmol) of Ti(OiPr)4 in 4-methoxy-
(47) The crude peptide was ascertained not to accompany Et-
remaining peptides by co-injection HPLC analysis.
(48) Tam, J . P.; Kent, S. B. H.; Wong, T. W.; Merrifield, R. B.
Synthesis 1979, 955-957.
(49) Vandewalle, M.; Eycken, V.; Oppolzer, W.; Vullioud, C. Tetra-
hedron 1986, 42, 4035-4043.
(50) Yeh, M. C. P.; Knochel, P. Tetrahedron Lett. 1988, 29, 2395-
2396.

4896 J . Org. Chem., Vol. 65, No. 16, 2000
Otaka et al.
enantiomer 15 ([R]23 +58.0° (c 1.89, CHCl3)) of the title
with that described above, the title compound 17a (major) and
17b (minor) were prepared. For 17a (3S-XS): colorless crystals;
D
compound 14 described above was prepared in an identical
fashion.
[R]26 +49.0° (c 1.14, CHCl3). For 17b (3R-XS): colorless
D
(2R,3′R)-N-[3′-(Dieth ylp h osp h on od iflu or om eth yl)bu t-
a n oyl]bor n a n e-10,2-su lta m (16a , 3R-XR) a n d (2R,3′S)-N-
[3′-(dieth ylph osph on odiflu or om eth yl)bu tan oyl]bor n an e-
10,2-su lta m (16b, 3S-XR) fr om 14. A suspension of 10%
Pd-C (200 mg) and 14 (1.0 g, 2.13 mmol) in EtOAc was
subjected to catalytic hydrogenation at room temperature for
10 h. The catalyst was removed by filtration, and the filtrate
was concentrated under reduced pressure to give a mixture
of major (16a ) and minor (16b) isomers. Diastereomer analysis
[HPLC; H2O-CH3CN (52:48)] of the unpurified product gave
a 16a (tR ) 31.8 min):16b (tR ) 29.2 min) ratio of 96:4. Flash
chromatographic purification of the mixture over silica gel with
n-hexanes-EtOAc gave 16a (891 mg, 88.8% yield) and 16b
(36.8 mg, 3.7% yield), respectively. Recrystallization of 16a
from Et2O gave colorless crystals. An X-ray structural analysis
of the crystals (obtained from EtOAc) indicated that 16a
crystals; [R]21 +74.0° (c 2.15, CHCl3). Other physical and
D
chemical data for 17a and 17b, except for optical rotation, were
identical to those of 16a and 16b, respectively.
ter t-Bu tyl Bu t-2-yn oa te (19) fr om 18. To a solution of
38.0 g (314 mmol) of 18 in THF (250 mL) at -78 °C under
argon was added 285 mL (440 mL) of 1.54 M n-BuLi in hexane.
After the mixture was stirred at -78 °C for 2 h, a solution of
44 g (220 mmol) of (Boc)2O in THF (100 mL) was added. The
reaction was allowed to warm to 4 °C, stirred for 10 h, and
quenched with saturated NH4Cl solution. The mixture was
extracted with Et2O, and the extract was successively washed
with 5% NaHCO3 and brine, dried over MgSO4, and concen-
trated under reduced pressure to leave residues. Distillation
(20 mmHg, 77 °C) of the crude materials gave 22.2 g (72.1%
yield) of the title compound 19 as a colorless oil: 1H NMR (270
MHz, CDCl3) δ 1.49 (s, 9H), 1.96 (s, 3H); HRMS (CI) m/z calcd
for C8H13O2 (MH+) 141.0915, found 141.0914.
possesses (3′R)-configuration. For 16a : mp 130-131 °C; [R]27
D
1
-49.0° (c 1.14, CHCl3); H NMR (270 MHz, CDCl3) δ 0.98 (s,
ter t-Bu tyl (E)-3-Iod obu t-2-en oa te (21) fr om 19. To a
stirred suspension of CuCN (8.6 g, 96.3 mmol) in THF (300
mL) at -78 °C was added by syringe 131 mL (202 mmol) of
1.54 M n-BuLi in hexane, and the mixture was allowed to
warm to 4 °C so that a homogeneous solution was obtained.
The mixture was recooled to -78 °C, where neat n-Bu3SnH
(49.9 mL, 185 mmol) was added, and the mixture was stirred
for 15 min. To the solution of resulting stannylcuprate was
added 20 mL (354 mmol) of EtOH. After 5 min, a solution of
19 (10.0 g, 71.3 mmol) in THF (50 mL) was added by syringe
at -78 °C, and the mixture was stirred at the same temper-
ature for 2 h. The reaction was quenched with saturated NH4-
Cl-5% NH4OH (1:1) solution. The mixture was extracted with
EtOAc, and the extract was washed with brine, dried over
MgSO4, and concentrated in vacuo to give an oily product.
Without further purification, solid iodine (27 g, 106 mmol) was
added piecemeal at room temperature over 5 h to a solution
of the above crude material (20) in CH2Cl2 (100 mL). After 24
h, the reaction mixture was concentrated to yield a reddish
brown oil. Distillation (8 mmHg, 86 °C) of the crude materials
gave 11.5 g (59.9% yield) of the title compound 21 as a colorless
oil: 1H NMR (270 MHz, CDCl3) δ 1.47 (s, 9H), 2.95 (d, J ) 1.4
Hz, 3H), 6.55 (q, J ) 1.4 Hz, 1H); HRMS (CI) m/z calcd for
C8H14O2I (MH+) 269.0040, found 269.0049.
3H), 1.16 (s, 3H), 1.17 (d, J ) 7.5 Hz, 3H), 1.38 (t, J ) 7.0 Hz,
6H), 1.34-1.45 (m, 2H), 1.85-1.93 (m, 3H), 2.08-2.13 (m, 2H),
2.79 (dd, J ) 16.0, 9.5 Hz, 1H), 2.83-3.07 (m, 1H), 3.16 (dd, J
) 16.0, 2.5 Hz, 1H), 3.43 (d, J ) 13.5 Hz, 1H), 3.51 (d, J )
13.5 Hz, 1H), 3.88 (dd, J ) 7.0, 5.9 Hz, 1H), 4.26 (q, J ) 7.0
Hz, 2H), 4.29 (q, J ) 7.0 Hz, 2H). Anal. Calcd for C19H32NO6-
SPF2: C, 48.40; H, 6.84; N, 2.97. Found: C, 48.29; H, 6.77; N,
2.90. Crystal data for 16a : orthorhombic, a ) 13.0428(8) Å, b
) 22.948(1) Å, c ) 7.6546(8) Å, V ) 2291.3(2) Å3, Z ) 4, space
group P212121, Dcalc ) 1.367 mg m-3. Data collection: A total
of 2243 reflections were measured by Rigaku AFC7R diffrac-
tometer with graphite monochromated Cu KR radiation (λ )
1.54178 Å) at room temperature. Structure analysis and
refinement: The crystal structure was solved by the direct
methods with the program SnB51 and refined by full-matrix
least squares. Non-hydrogen atoms were refined with aniso-
tropic temperature factors, and hydrogen atoms were included
at calculated positions and refined atoms with isotropic
temperature factors. The final R value for 2119 reflections with
I > 2σ(I) was 0.056 and 0.069. The maximum and minimum
peaks in final difference Fourier map were 0.46 and -0.48
eÅ-3, respectively. Keeping of the purified 16b in refrigerator
for several days afforded colorless crystals: mp 58-60 °C;
[R]21 -68.2° (c 2.05, CHCl3); 1H NMR (270 MHz, CDCl3) δ
ter t-Bu t yl (E)-3-(Diet h ylp h osp h on od iflu or om et h yl)-
bu t-2-en oa te (22) fr om 21. By a procedure identical with that
described for the synthesis of 13 from 12, 11.5 g (42.9 mmol)
of 21 was converted to 14.0 g (99.4% yield) of the title
compound 22 as a colorless oil: 1H NMR (270 MHz, CDCl3) δ
1.39 (t, J ) 7.3 Hz, 6H), 1.50 (s, 9H), 2.24-2.27 (m, 3H), 4.27
(q, J ) 7.3 Hz, 2H), 4.30 (q, J ) 7.3 Hz, 2H), 6.13-6.18 (m,
1H); HRMS (FAB) m/z calcd for C26H47O10F4P2 (2M + H+)
657.2580, found 657.2565.
D
0.97 (s, 3H), 1.15 (s, 3H), 1.20 (d, J ) 7.0 Hz, 3H), 1.38 (t, J )
7.0 Hz, 6H), 1.34-1.47 (m, 2H), 1.84-1.94 (m, 3H), 2.01-2.18
(m, 2H), 2.73 (dd, J ) 17.3, 8.4 Hz, 1H), 2.83-3.10 (m, 1H),
3.20 (dd, J ) 17.3, 4.1 Hz, 1H), 3.43 (d, J ) 13.8 Hz, 1H), 3.51
(d, J ) 13.8 Hz, 1H), 3.88 (dd, J ) 7.3, 5.1 Hz, 1H), 4.26 (q, J
) 7.0 Hz, 2H), 4.29 (q, J ) 7.0 Hz, 2H). Anal. Calcd for C19H32
-
NO6SPF2: C, 48.40; H, 6.84; N, 2.97. Found: C, 48.59; H, 7.04;
N, 2.96; HRMS (FAB) m/z calcd for C19H33O6NF2PS (MH+)
472.1734; found 472.1726. To reconfirm the 3′-configuration
of 16b, 16a and 16b were respectively converted to the
corresponding benzyl ester derivatives using a procedure
similar to that described for the conversion of 11 to 12. Benzyl
(2S)-N-[(E)-3′-(Dieth ylp h osp h on od iflu or om eth yl)bu t-
2-en oyl]bor n a n e-10,2-su lta m (23) fr om 22. By a procedure
identical with that described for the synthesis of 14 from 13,
10.0 g (30.5 mmol) of 22 was converted to 10.9 g (75.5% yield)
of the title compound 23. For 23: colorless crystals from
ester 16a ′ from 16a : a colorless oil; [R]21 +7.23° (c 1.23,
D
CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.18 (d, J ) 6.8 Hz, 3H),
1.37 (t, J ) 7.0 Hz, 6H), 2.33 (dd, J ) 15.9, 9.7 Hz, 1H), 2.70-
2.90 (m, 1H), 2.95 (dd, J ) 15.9, 3.8 Hz, 1H), 4.25 (q, J ) 7.0
Hz, 2H), 4.28 (q, J ) 7.0 Hz, 2H), 5.14 (s, 2H), 7.33-7.38 (m,
5H); HRMS (FAB) m/z calcd for C16H24O5F2P (MH+) 365.1329;
found 365.1338. Benzyl ester 16b′ from 16b: a colorless oil;
[R]21D -6.31° (c 1.39, CHCl3). Both NMR and FABMS analyses
of 16b gave the same results as those of 16a . From these
results, we concluded that the minor isomer (16b) possesses
(3′S)-configuration.
EtOAc-n-hexane; mp 113-115 °C; [R]23 +58.0 (c 1.89,
D
CHCl3); 1H NMR (270 MHz, CDCl3) δ 0.98 (s, 3H), 1.17 (s, 3H),
1.34-1.41 (m, 6H), 1.35-1.45 (m, 2H), 1.87-1.97 (m, 3H),
2.10-2.16 (m, 2H), 2.27 (t, J ) 1.6 Hz, 3H), 3.43 (d, J ) 13.8
Hz, 1H), 3.51 (d, J ) 13.8 Hz, 1H), 3.93 (dd, J ) 6.8, 5.7 Hz,
1H), 4.22-4.37 (m, 4H), 6.80-6.86 (m, 1H). Anal. Calcd for
C
19H30NO6SPF2: C, 48.61; H, 6.44; N, 2.98. Found: C, 48.52;
H, 6.30; N, 2.92.
Ca ta lytic Hyd r ogen a tion of 23. A suspension of 10%
Pd-C (500 mg) and 23 (8.0 g, 17.0 mmol) in toluene (80 mL)
was subjected to catalytic hydrogenation at room temperature
for 5 h. The catalyst was removed by filtration, and the filtrate
was concentrated under reduced pressure to give a mixture
of minor (17a ) and major (17b) isomers. Diastereomer analysis
[HPLC; H2O-CH3CN (52:48)] of the unpurified product gave
a 17a (tR ) 33.0 min):17b (tR ) 29.8 min) ratio of 11:89. Flash
chromatographic purification of the mixture over silica gel with
(2S,3′S)-N-[3′-(Dieth ylp h osp h on od iflu or om eth yl)bu t-
a n oyl]bor n a n e-10,2-su lta m (17a , 3S-XS) a n d (2S,3′R)-N-
[3′-(Dieth ylph osph on odiflu or om eth yl)bu tan oyl]bor n an e-
10,2-su lta m (17b, 3S-XS) fr om 15. By a procedure identical
(51) Miller, R.; Gallo, S. M.; Khalak, H. G.; Weeks, C. M. J . Appl.
Cryst. 1994, 27, 613-621.

CF2-Substituted Phosphothreonine Mimetics
J . Org. Chem., Vol. 65, No. 16, 2000 4897
n-hexanes-EtOAc gave 17a (0.23 g, 2.9% yield) and 17b (7.0
g, 87.1% yield), respectively. Each compound obtained was
identical to that derived from 15 by catalytic hydrogenation
over 10% Pd-C in EtOAc.
5H); HRMS (FAB) m/z calcd for C16H33O9N3F2P (MH+) 480.1922,
found 480.1953.
(2R,3R)-2-(ter t-Bu toxyca r bon yl)a m in o-3-(d ieth ylp h os-
p h on od iflu or om eth yl)bu ta n oic Acid (33, D-a llo-F or m )
fr om 32. A solution of 150 mg (0.31 mmol) of 32 in EtOAc (3
mL) in the presence of 10% Pd-C was subjected to debenzy-
lation under H2 at room temperature for 2 h. The catalyst was
removed by filtration, and the filtrate was concentrated under
reduced pressure to give 120.5 mg (98.9% yield) of the title
(2R,2′R,3′R)-N-[2′-(ter t-Bu t oxyca r b on yl)a m in o-3′-(d i-
eth ylp h osp h on od iflu or om eth yl)bu ta n oyl]bor n a n e-10,2-
su lta m (31, 2R,3R-XR) fr om 16a . To a solution of 500 mg
(1.2 mmol) of 16a in THF (8 mL) at -78 °C under argon was
added by syringe 1.4 mL (1.4 mmol) of 1 M NaHMDS in THF.
After the mixture was stirred at this temperature for 1 h,
where a blue solution of 205 mg (1.4 mmol) of 1-chloro-1-
nitrosocyclohexane in THF (1.4 mL) was added with additional
stirring at -78 °C for 30 min. To the mixture at -78 °C was
added aqueous 1 N HCl (6.6 mL). The mixture was allowed to
warm to room temperature, and the stirring was continued
for 30 min. The mixture then was concentrated in vacuo to
leave residues, to which n-hexane (40 mL) and aqueous 1 N
HCl (40 mL) were added. The organic phase was washed with
aqueous 1 N HCl (40 mL). The combined aqueous phase was
alkalinized with solid NaHCO3 and extracted with EtOAc. The
extract was dried over MgSO4 and concentrated to give an oil
of a hydroxylamine derivative 30. Without further purification,
the residue was dissolved with THF (5 mL). To the solution
at room temperature were successively added AcOH (3.3 mL),
aqueous 1 N HCl (6.6 mL), and Zn dust (3.03 g, 46.4 mmol).
After the suspension was stirred for 2 h, the reaction was
alkalinized with solid NaHCO3. The reaction mixture was
passed through Celite, and then the filtrate was extracted with
EtOAc. The extract was washed with 5% NaHCO3 and brine,
dried over MgSO4, and concentrated to yield an oil of a crude
amine derivative. To a solution of the amine in CH3CN (5 mL)
was added at room temperature a solution of 506 mg (2.3
mmol) of (Boc)2O in CH3CN (5 mL). After the reaction was
stirred at room temperature for 12 h, the mixture was directly
purified by flash chromatography over silica gel with n-hex-
anes-EtOAc (2:3) to yield 372 mg (56% yield) of the title
compound 31 as a colorless oil. Diastereomer analysis [HPLC;
H2O-CH3CN (50:50)] of the unpurified product gave a 31 (tR
) 40.6 min):(2′S) isomer (tR ) 48.7 min) ratio of 97.7:2.3. For
compound 33 as a colorless oil: [R]19 -13.7° (c 1.41, CHCl3);
D
1H NMR (270 MHz, CDCl3) δ 1.19 (d, J ) 7.3 Hz, 3H), 1.38 (t,
J ) 7.0 Hz, 3H), 1.39 (t, J ) 7.0 Hz, 3H), 1.44 (s, 9H), 2.79-
3.05 (broad, 1H), 4.26-4.36 (m, 4H), 4.86 (dd, J ) 9.0, 1.5 Hz,
1H), 5.17 (d, J ) 9.0 Hz, 1H); HRMS (FAB) m/z calcd for
C
14H27O7NF2P (MH+) 390.1493, found 390.1481.
The antipode (L-allo-form, en t-33) of 33 was also prepared
from 17a according to the sequence of reactions employed for
the conversion of 16a to 33. L-allo-form: [R]19 +14.4° (c 1.32,
D
CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.20 (d, J ) 7.3 Hz, 3H),
1.38 (t, J ) 7.0 Hz, 3H), 1.39 (t, J ) 7.0 Hz, 3H), 1.45 (s, 9H),
2.80-3.05 (broad, 1H), 4.26-4.37 (m, 4H), 4.87 (dd, J ) 8.9,
1.5 Hz, 1H), 5.18 (d, J ) 8.9 Hz, 1H), 7.90-8.60 (broad, 1H);
HRMS (FAB) m/z calcd for C14H27O7NF2P (MH+) 390.1493,
found 390.1485. Data obtained from NMR measurements of
33 and en t-33 were identical to those published in the
literature9 and of racemic allo-F2Pmab derivative synthesized
by us.10 Enantio- and diastereomeric purities of 33 were
ascertained by comparison of NMR data between D-allo-form
(33) and other isomers derivatized as Mosher amides, obtained
via the following sequence: (i) TMSCH2N2, MeOH, benzene;52
(ii) TFA then Et3N, THF; (iii) (S)-R-methoxy-R-(trifluorometh-
yl)phenylacetic acid ((S)-MTPA),53 WSCDI, HOBt, THF. From
this experiment, no other isomers were detected in synthetic
D-allo-form 33.
(2R,2′R,3′S)-N-[2′-Br om o-3′-(d ieth ylp h osp h on od iflu o-
r om eth yl) bu ta n oyl]bor n a n e-10,2-su lta m (34, 2R,3S-XR)
fr om 16a . To a solution of 3.0 g (6.40 mmol) of 16a in THF
(30 mL) at -78 °C under argon was added by syringe 7.7 mL
(7.70 mmol) of 1 M NaHMDS in THF. After the mixture was
stirred at this temperature for 1 h, a precooled (-78 °C)
suspension of 3.42 g (19.2 mmol) of NBS in THF (30 mL) was
added with additional stirring at -78 °C for 90 min. The
reaction was quenched at -78 °C by addition of aqueous 0.5
M NaHSO3. After warming to room temperature, the mixture
was extracted with EtOAc. The extract was washed with 0.5
M NaHSO3 and brine, dried over MgSO4, and concentrated
under reduced pressure to give an oil. Flash chromatography
of the crude material over silica gel with n-hexanes-EtOAc
(1:1) yielded 3.37 g (95.7% yield) of the title compound 34 as
31: [R]25 -27.2° (c 0.74, CHCl3); 1H NMR (270 MHz, CDCl3)
D
δ 0.96 (s, 3H), 1.18 (s, 3H), 1.25 (d, J ) 7.3 Hz, 3H), 1.34-
1.41 (m, 6H), 1.41 (s, 9H), 1.38-1.45 (m, 3H), 1.85-1.95 (m,
3H), 2.02-2.30 (m, 2H), 3.08-3.32 (broad, 1H), 3.41 (d, J )
13.5 Hz, 1H), 3.49 (d, J ) 13.5 Hz, 1H), 3.92 (dd, J ) 7.0, 5.8
Hz, 1H), 4.26 (q, J ) 7.3 Hz, 2H), 4.29 (q, J ) 7.3 Hz, 2H),
4.91-5.00 (m, 1H), 5.26 (d, J ) 7.3 Hz, 1H); HRMS (FAB) m/z
calcd for C24H40O8N2F2PS (M-H-) 585.2211, found 585.2215.
The (2′S) isomer for diastereomer analysis was prepared from
bromide 34 via the following sequence: (i) azide displacement;
(ii) reduction; (iii) Boc protection.
a pale yellow oil: [R]23 -42.7° (c 1.28, CHCl3); 1H NMR (270
D
Ben zyl (2R,3R)-2-(ter t-Bu t oxyca r b on yl)a m in o-3-(d i-
eth ylp h osp h on od iflu or om eth yl)bu ta n oa te (32) fr om 31.
A solution of 0.34 mL (1.14 mmol) of Ti(OiPr)4 in benzyl alcohol
(2.4 mL, 22.8 mmol) was stirred under vacuum for 1 h. To the
solution at room temperature under argon was added a
solution of 334 mg (0.57 mmol) of 31 in toluene (3 mL), and
the mixture was allowed to warm to 120 °C and to stir at this
temperature for 5 h. The reaction was quenched at room
temperature by addition of aqueous 1 N HCl. The mixture was
extracted with EtOAc, and the extract was washed with 1 N
HCl and brine, dried over MgSO4, and concentrated in vacuo
to leave an oil containing 32, benzyl alcohol, and the sultam.
The oil was treated with 4.3 mL (45.6 mmol) of Ac2O and 0.92
mL (11.4 mmol) of pyridine at room temperature for 20 min.
The reaction was quenched at 4 °C by slow addition of
saturated NaHCO3, followed by extraction with EtOAc. The
extract was successively washed with saturated NaHCO3, 1
N HCl, and brine, dried over MgSO4, and concentrated to give
crude materials. Flash chromatographic purification of the
crude materials over silica gel with n-hexanes-EtOAc (1:1)
gave 204 mg (74.5% yield) of the title compound 32 as a
colorless oil: 1H NMR (270 MHz, CDCl3) δ 1.14 (d, J ) 7.3
Hz, 3H), 1.36 (t, J ) 7.3 Hz, 6H), 1.43 (s, 9H), 2.75-3.00 (m,
1H), 4.24 (q, J ) 7.3 Hz, 2H), 4.27 (q, J ) 7.3 Hz, 2H), 4.85
(dd, J ) 8.9, 2.7 Hz, 1H), 5.10-5.25 (m, 3H), 7.33-7.37 (m,
MHz, CDCl3) δ 0.98 (s, 3H), 1.20 (s, 3H), 1.38 (t, J ) 7.0 Hz,
6H), 1.37-1.45 (m, 2H), 1.49 (d, J ) 7.0 Hz, 3H), 1.87-1.97
(m, 3H), 2.07-2.20 (m, 2H), 3.19 (m, 1H), 3.45 (d, J ) 14.0
Hz, 1H), 3.53 (d, J ) 14.0 Hz, 1H), 3.94 (dd, J ) 7.5, 5.0 Hz,
1H), 4.27 (q, J ) 7.0 Hz, 2H), 4.30 (q, J ) 7.0 Hz, 2H), 5.17 (d,
J ) 7.0 Hz, 1H); HRMS (FAB) m/z calcd for C19H32O6NBrF2-
PS (MH+) 550.0840, found 550.0831. Diastereomer analysis
was carried out after conversion of the crude products to the
corresponding p-methoxybenzyl esters using Ti(OPMB)4 in
toluene. An HPLC analysis of the PMB (H2O-CH3CN (55:45))
afforded 99.4:0.6 ratio of (2R) and (2S) diastereomers (tR 61.8
and 53.5 min, respectively). Authentic samples were prepared
as follows: (i) conversion of 16a to the corresponding PMB
ester, (ii) bromination, and (iii) separation of diastereomers
on HPLC.
Ben zyl (2S,3R)-[2-Azid e-3-(d iet h ylp h osp h on od iflu o-
r om eth yl)]bu ta n oa te (36, 2S,3R) fr om 34. Conversion of
34 to the corresponding benzyl ester derivative 35 was
achieved according to a procedure identical with that described
for the transesterification of 31 to 32. Treatment of 8.0 g (14.5
(52) Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull.
1981, 29, 1475-1478.
(53) Dale, J . A.; Dull, D. L.; Mosher, H. S. J . Org. Chem. 1969, 34,
2543-2549.

4898 J . Org. Chem., Vol. 65, No. 16, 2000
Otaka et al.
mmol) of 34 in toluene (60 mL) with Ti(OiPr)4 (4 equiv) in
benzyl alcohol (32 equiv) at 120 °C for 5 h, followed by the
usual workup gave a mixture of 35, benzyl alcohol, and the
sultam (HXR) as an oil. The oil was treated with 110 mL (1.16
mol) of Ac2O and 23.3 mL (0.29 mol) of pyridine at room
temperature for 20 min. The reaction was quenched by slow
addition of saturated NaHCO3, followed by extraction with
EtOAc. The extract was successively washed with saturated
NaHCO3, 1 N HCl, and brine, dried over MgSO4, and concen-
trated to give an oil. Flash chromatographic purification of the
oil over silica gel with n-hexanes-EtOAc (3:2) gave a ca. 1:1
mixture of 35 and the sultam (HXR). For 35: 1H NMR (270
MHz, CDCl3) δ 0.87 (d, J ) 7.0 Hz, 3H), 1.26 (s, 9H), 1.39 (t,
J ) 7.3 Hz, 6H), 2.98-3.15 (m, 1H), 4.24 (q, J ) 7.3 Hz, 2H),
4.27 (q, J ) 7.3 Hz, 2H), 4.78 (d, J ) 6.2 Hz, 1H), 5.20 (s, 2H),
7.35-7.40 (m, 5H); HRMS (FAB) m/z calcd for C16H23O5BrF2P
(MH+) 443.0435, found 443.0428. Without further purification,
the mixture was subjected to next step. To a solution of the
mixture (containing ca. 9.8 mmol of 35) in CH3CN (40 mL)
was added at room temperature a solution of 4.6 g (29.3 mmol)
of tetramethylguanidinium azide in CH3CN (30 mL). After the
reaction was stirred for 12 h, quenched by addition of aqueous
saturated NaHCO3. The mixture was extracted with EtOAc.
The extract was washed with saturated NaHCO3 and brine,
dried over MgSO4, and concentrated to give a mixture of azide
displaced product 36, an elimination product 37, and HXR.
Flash chromatography of the mixture over silica gel with
n-hexanes-EtOAc (3:2) yielded, in order of elution, 1.65 g of
synthesized by us.10 Optical purity of 39 was judged to be
>99% ee from an HPLC analysis of its Mosher amide, obtained
via the following sequence: (i) TMSCH2N2, MeOH, benzene;
(ii) TFA then Et3N, THF; (iii) (S)-MTPA, WSCDI, HOBt, THF.
Syn th esis of P r otected P ep tid e Resin 40. Protected
peptide resin was manually constructed using the Boc-based
solid-phase method on MBHA resin (0.10 mmol, NH2 0.46
mmol/g). Boc deprotection was achieved using 50% TFA-2%
anisole in toluene (1 × 1 min, 1 × 15 min); 5% DIPEA in
toluene (2 × 1 min) was used for neutralization of the TFA
salts. Boc-protected amino acids including Boc-F2Pmab-
(OEt)2-OH 39 (2.5-fold molar excess, 0.25 mmol) were sequen-
tially condensed using DIPCDI (0.25 mmol)-HOBt (0.25
mmol) in DMF. Standard Boc protocols were applicable to the
incorporation of F2Pmab derivative into protected peptide
resins.
Tr ea tm en t of P r otected P ep tid e Resin 40 w ith Acid ic
Dep r otectin g Rea gen ts. Ru n 1: 1 M TMSOTf in TF A,
m -Cr esol, EDT System . To 10 mg (3.12 µmol) of the protected
resin 40 were added m-cresol (32 µL) and EDT (32 µL). After
stirring for 5 min at room temperature, the reaction mixture
was cooled to 4 °C with an ice-chilled bath, and then precooled
TFA (0.5 mL) and TMSOTf (121 µL, 0.624 mmol) were
successively added, and the stirring was continued at 4 °C (1.5
h) and then at room temperature (0.5 h). After removal of the
resin by filtration, ether was added to the filtrate to precipitate
the crude product. The precipitate was collected by centrifuga-
tion and washed with ether (3 times) to remove scavengers.
The crude peptides were dissolved in 0.1% TFA in H2O-CH3-
CN (3:1, 2.0 mL). The peptide solution was subjected to an
HPLC analysis using analytical HPLC (B; 15-30% over 40
min). Components, corresponding to 40b (tR ) 23.5 min) and
40c (tR ) 41.0 min) obtained by analytical HPLC purification,
were identified using IS-MS analyses. The ratio of components
was determined by comparison of peak areas, where no
significant side reactions were observed which could prevent
the comparison of the peak areas. IS-MS (reconstructed): m/z
calcd for C48H69O17N10PF2 (40b) 1127.11, found 1127.99; m/z
calcd for C50H73O17N10PF2 (40c) 1155.17, found 1154.49.
Ru n 2: 1 M TMSOTf-Th ioa n isole in TF A, m -Cr esol,
EDT System . To 10 mg (3.12 µmol) of 40 were added m-cresol
(32 µL), thioanisole (73 µL, 0.624 mmol), and EDT (32 µL).
After 5 min of stirring at room temperature, the reaction
mixture was cooled to 4 °C with an ice-chilled bath, and then
precooled TFA (0.43 mL) and TMSOTf (121 µL, 0.624 mmol)
were successively added. The reaction was stirred at 4 °C for
1.5 h with additional stirring at room temperature for 0.5 h.
A procedure identical to that described above was utilized to
obtain the crude peptides and to determine the ratio of
an oil consisting of 37 and HXR, and 1.98 g (35% yield from
1
34) of 36 as an oil. For 36: [R]23 -2.91 (c 2.54, CHCl3); H
D
NMR (270 MHz, CDCl3) δ 1.24 (d, J ) 7.3 Hz, 3H), 1.35 (t, J
) 7.3 Hz, 3H), 1.37 (t, J ) 7.3 Hz, 3H), 2.80-3.05 (m, 1H),
4.14 (d, J ) 6.5 Hz, 1H), 4.22 (q, J ) 7.3 Hz, 2H), 4.28 (q, J )
7.3 Hz, 2H), 5.23 (s, 2H), 7.33-7.42 (m, 5H); HRMS (FAB) m/z
calcd for C16H23O5N3F2P (MH+) 406.1343, found 406.1349.
Ben zyl (2S,3R)-[2-(ter t-Bu t oxyca r b on yl)a m in o-3-(d i-
eth ylp h osp h on od iflu or om eth yl)]bu ta n oa te (38, 2S,3R)
fr om 36. To a solution of 1.97 g (4.9 mmol) of 36 in THF (20
mL) were successively added Zn dust (6.35 g, 97.2 mmol) and
AcOH (2.8 mL, 49 mmol) at room temperature. After 2 h of
stirring, the suspension was neutralized by addition of Et3N.
To the mixture was added a solution of 2.1 g (9.7 mmol) of
(Boc)2O in THF (5 mL). After the stirring was continued for
12 h, the solid precipitate was removed by filtration. The
filtrate was extracted with EtOAc, followed by the usual
workup. The crude material was subjected to flash chromato-
graphic purification over silica gel with n-hexanes-EtOAc (1:
1) to yield 1.9 g (81.9% yield) of the title compound 38 as a
colorless oil: [R]23D +15.9° (c 3.53, CHCl3); 1H NMR (270 MHz,
CDCl3) δ 1.29 (d, J ) 7.6 Hz, 3H), 1.33 (t, J ) 7.0 Hz, 3H),
1.35 (t, J ) 7.0 Hz, 3H), 1.44 (s, 9H), 3.03-3.25 (m, 1H), 4.22
(m, 4H), 4.56 (dd, J ) 10.0, 2.2 Hz, 1H), 5.16 (s, 2H), 5.47 (d,
J ) 10.0 Hz, 1H), 7.32-7.38 (m, 5H); HRMS (FAB) m/z calcd
for C21H33O7NF2P (MH+) 480.1963, found 480.1953.
products. IS-MS (reconstructed): m/z calcd for C46H65O17N10
PF2 (40a ) 1099.06, found 1099.99.
-
Ru n 3: 1 M TMSOTf-Th ioa n isole in TF A, m -Cr esol,
EDT System (fir st step ), F ollow ed by Ad d ition of TM-
SOTf-DMS (secon d step ). Protected peptide resin 40 (10
mg, 3.12 µmol) was treated with 1 M TMSOTf-thioanisole in
TFA (0.624 mL) in the presence of m-cresol (32 µL) and EDT
(32 µL) at 4 °C for 1.5 h with additional stirring at room
temperature for 0.5 h. The reaction mixture was recooled to 4
°C, where DMS (187 µL) and TMSOTf (125 µL) were succes-
sively added. The reaction was stirred for at 4 °C for 2 h. A
procedure identical to that described above was utilized to
obtain the crude peptides and to determine the ratio of
products.
Ru n 4: 1 M TMSOTf-Th ioa n isole in TF A, m -Cr esol,
EDT System (fir st step ), F ollow ed by TMSOTf-DMS-
m -Cr esol-EDT Tr ea tm en t (secon d step ). Protected pep-
tide resin 40 (10 mg, 3.12 µmol) was treated with 1 M TMSOTf-
thioanisole in TFA (0.624 mL) in the presence of m-cresol (32
µL) and EDT (32 µL) at 4 °C for 1.5 h with additional stirring
at room temperature for 0.5 h. Crude deprotected peptide were
obtained by a procedure identical to that mentioned above. To
the crude deprotected peptide was successively added at 4 °C
m-cresol (15 µL), EDT (15 µL), DMS (182 µL), and TMSOTf
(121 µL, 0.624 mmol). After 2 h of stirring at 4 °C, the reaction
was quenched by successive addition of Et2O (5 mL) and H2O
(2S,3R)-2-(ter t-Bu toxyca r bon yl)a m in o-3-(d ieth ylp h os-
p h on od iflu or om eth yl)bu ta n oic Acid (39, L-F 2P m a b De-
r iva tive) fr om 38. A suspension of 10% Pd-C (50 mg) and
38 (200 mg, 0.42 mmol) in EtOAc (2 mL) was subjected to
catalytic hydrogenation at room temperature for 2 h. The
catalyst was removed by filtration, and the filtrate was
concentrated under reduced pressure to give 161 mg (98.9%
yield) of the title compound 39 as a colorless oil: [R]22D +22.7°
(c 1.00, MeOH) [lit.9, [R]D +21.0° (c 1.10, MeOH)]; 1H NMR
(270 MHz, CDCl3) δ 1.27 (d, J ) 7.3 Hz, 3H), 1.35-1.45 (m,
6H), 1.45 (s, 9H), 2.97-3.23 (broad, 1H), 4.24-4.40 (m, 4H),
4.61 (dd, J ) 8.9, 3.0 Hz, 1H), 5.47 (d, J ) 8.9 Hz, 1H); HRMS
(FAB) m/z calcd for C14H26O7NF2PNa (MNa+) 412.1313, found
412.1308.
The antipode (D-F2Pmab derivative en t-39) of 39 was also
prepared from 17a according to the sequence of reactions
employed for the conversion of 16a to 39. For the D-form: [R]22
D
-23.7° (c 0.99, MeOH); HRMS (FAB) m/z calcd for C14H27O7-
NF2P (MH+) 390.1493, found 390.1497. Data obtained from
NMR measurements of 39 and en t-39 were identical to those
published on literature9 and of racemic F2Pmab derivative

CF2-Substituted Phosphothreonine Mimetics
J . Org. Chem., Vol. 65, No. 16, 2000 4899
(2 mL). The aqueous phase was washed with Et2O (3 times)
and filtered. The filtrate was subjected to an HPLC analysis
to determine the ratio of products.
E xa m in a t ion of St a b ilit y of P ep t id e-R esin Bon d
u n d er Tr ea tm en t w ith 0.3 M BSTF A-TBAI in CH2Cl2,
BF 3‚Et2O. To each suspension of Boc-Leu-PAM-Gly resin (10
mg each, 7.8 µmol) in CH2Cl2 (2.4 mL) was successively added
TBAI (288 mg, 0.78 mmol), BSTFA (207 µL, 0.78 mmol), and
BF3‚Et2O (9.6 µL, 78 µmol) at room temperature. Each reaction
mixture was shaken at room temperature for 30 min, 1 or 2
h, and passed through a filter. Each remaining resin on the
filter was washed as described above, and a part of the resin
was hydrolyzed with 6 N HCl at 110 °C for 18 h. The resulting
hydrolysates were subjected to an amino acid analysis. Re-
mained Leu on the resin was quantified compared with amino
acid analysis of hydrolysate of the intact resin. Leu remained
on the PAM resin; 95.3% (30 min), 91.1% (1 h), and 94.1% (2
h).
Ru n 5: 0.3 M BSTF A-TBAI in CH2Cl2, BF 3‚Et2O Sys-
tem (fir st step ), F ollow ed b y 1 M TMSOTf-Th ioa n isole
in TF A, m -Cr esol, EDT System (secon d step ). To a
suspension of 40 (10 mg, 3.12 µmol) in CH2Cl2 (1.9 mL) were
successively added TBAI (230 mg, 0.624 mmol), BSTFA (166
µL, 0.624 mmol), and BF3‚Et2O (7.7 µL, 62.4 µmol) at room
temperature. The mixture was shaken at room temperature
for 1.5 h, and then passed through filter. The remaining resin
on the filter was successively washed with CH2Cl2, DMF,
MeOH, and Et2O, and dried in vacuo. The peptide attached
resin was treated with 1 M TMSOTf-thioanisole in TFA (0.624
mL) in the presence of m-cresol (32 µL) and EDT (32 µL) at 4
°C for 1.5 h and then at room temperature for 0.5 h, followed
by a usual workup and HPLC analysis. As other runs (runs
6-8), protected resin 40 was treated under conditions similar
to the title condition (run 5), where only one of the above three
agents (TBAI, BSTFA, or BF3‚Et2O) was omitted.
Syn th esis of Ac-Lys-Ile-Gly-Glu -Gly-F 2P m a b-F 2P m p -
Gly-Va l-Va l-Tyr -Lys-NH2 (41). A protected peptide resin
corresponding to the Cdc2 peptide 41 was prepared by
standard Boc-based solid-phase techniques on MBHA resin
(0.23 mmol, NH2 0.46 mmol/g) as previously described, where
2.5-fold excess of Boc-F2Pmab(OEt)2-OH 39 and Boc-F2Pmp-
(OEt)2-OH were used for incorporation of F2Pmab and F2Pmp
residues, respectively. The completed resin (56 mg, 14.7 µmol)
was treated with 0.3 M BSTFA-TBAI in CH2Cl2 (8.6 mL, 200
equiv) in the presence of BF3‚Et2O (72.8 µL, 40 equiv) at room
temperature. After 1.5 h of shaking, the resin was washed with
solvents described above and subjected to the second step with
1 M TMSOTf-thioanisole in TFA (2.95 mL), m-cresol (141 µL),
and EDT (141 µL) with stirring for 1.5 h at 4 °C and then for
0.5 h at room temperature. A workup similar to that already
mentioned above was utilized to obtain the crude peptide.
HPLC purifications (linear gradient of B, 15-40% over 30 min)
of the crude product gave the pure title peptide 41 as a white
powder: 3.7 mg (25.2% yield, based on the protected peptide
resin); IS-MS (reconstructed) m/z calcd for C65H101O22N15P2F4
41 1582.56, found 1582.49.
Ackn owledgm en t. We thank Dr. Terrence R. Burke,
J r., NCI, NIH, Bethesda, MD 20892, for reading the
manuscript and providing useful comments. This work
was supported in part by The J apan Health Sciences
Foundation and Grands-in-Aid for Scientific Research
from the Ministry of Education, Science and Culture of
J apan, to which the authors’ thanks are due.
Su p p or tin g In for m a tion Ava ila ble: Tables of atomic
coordinates, anisotropic displacement parameters, bond length
and angles, and ORTEP diagram for 16a . HPLC charts of the
diastereomer analysis of products obtained from following
reactions; (i) hydrogenation of 14 and 23, (ii) amination of 16a
(including supporting NMR charts of resulting isomers), and
(iii) bromination of 16a (including supporting NMR charts of
resulting isomers). NMR charts of the enantiomer and dias-
tereomer analysis of 33 based on MTPA method. HPLC charts
of the enantiomer and diastereomer analysis of 39 based on
1
MTPA method. Copies of H NMR spectra of compounds 13,
its (E)-isomer, 16a , 16b, 15, 23, 33, and 39. HPLC charts of
deprotection reactions shown in Table 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O000169V