Development of activity-based probes with tunable specificity for protein tyrosine phosphatase subfamilies

Article abstract of DOI:10.1016/j.tet.2010.04.065

Herein we describe the development of activity-based probes toward protein tyrosine phosphatase (PTP) subfamilies. A novel phosphotyrosine analog serving as the latent trapping unit has been designed and explored. It allows addition of various amino acid residues to its C- and N-termini to extend the recognition element. As a proof-of-concept, we have synthesized three tripeptide probes, which carry the phosphotyrosine analog in the middle position and a leucinamide residue at the C-terminus. The three tripeptide probes differed only in their N-terminal amino acid (Glu, Phe, and Lys). The labeling properties of these probes were determined and the results showed the newly synthesized probes could selectively label PTPs in an activity-dependent manner. In addition, the probes' target specificity was also shown to be influenced by the amino acid residues flanking the phosphotyrosine analog.

Full text of DOI:10.1016/j.tet.2010.04.065

Tetrahedron 66 (2010) 4521e4529
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Development of activity-based probes with tunable specificity for protein tyrosine
phosphatase subfamilies
,
,
,c,
*
Yu-Yen Huang b y, Chun-Chen Kuo c y, Chi-Yuan Chu b, Yung-Hsuan Huang b, Yi-Ling Hu b, Jing-Jer Lin a
,
,
Lee-Chiang Lo b
*
a Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
b Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
c Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the development of activity-based probes toward protein tyrosine phosphatase (PTP)
subfamilies. A novel phosphotyrosine analog serving as the latent trapping unit has been designed and
explored. It allows addition of various amino acid residues to its C- and N-termini to extend the rec-
ognition element. As a proof-of-concept, we have synthesized three tripeptide probes, which carry the
phosphotyrosine analog in the middle position and a leucinamide residue at the C-terminus. The three
tripeptide probes differed only in their N-terminal amino acid (Glu, Phe, and Lys). The labeling properties
of these probes were determined and the results showed the newly synthesized probes could selectively
label PTPs in an activity-dependent manner. In addition, the probes’ target specificity was also shown to
be influenced by the amino acid residues flanking the phosphotyrosine analog.
Received 17 December 2009
Received in revised form 11 April 2010
Accepted 16 April 2010
Available online 21 April 2010
Keywords:
Activity-based
Enzymes
Tyrosine phosphatase
Quinone methide
Peptides
Ó 2010 Elsevier Ltd. All rights reserved.
Biotin
1. Introduction
for PTPs (Scheme 1).12 Probe 1 consists of four structural units,
including a biotin reporter unit. The labeling event of PTPs with
Reversible protein tyrosine phosphorylation is one of the most
important post-translational modifications that is utilized by liv-
ing organisms to regulate protein functions.1,2 The phosphoryla-
tion state of a protein not only determines its function, subcellular
location, and distribution, but also relays the influence to other
proteins through proteineprotein interactions. The extent of ty-
rosine phosphorylation is tuned by the well-regulated actions of
protein tyrosine kinases (PTks) and protein tyrosine phosphatases
(PTPs). Dysfunctions of PTks and PTPs are usually associated with
human diseases.3 Although PTP-related research lags far behind
that of PTKs, there is an increasing interest, in recent years, in
PTPs as drug targets.4,5 For example, PTEN is implicated as
a product of a tumor suppressor gene6e8 and PTP1B is associated
with diabetes.9
probe 1 is a two-stage process, including activation and alkylation,
as shown in Scheme 1. When the PeO bond between the recogni-
tion unit and the latent trapping unit is selectively cleaved with the
Recently, development of activity-based probes for hydrolases
has proven to be a powerful tool in modern chemical biology
study.10,11 We have previously developed probe 1 as a general probe
* Corresponding authors. E-mail addresses: jjlin@ym.edu.tw (J.-J. Lin), lclo@ntu.
edu.tw (L.-C. Lo).
Scheme 1. The four structural units as well as the selective activation/labeling process
of the activity-based probe 1 for PTPs.
y
These two authors contribute equally to this work.
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.04.065

4522
Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
assistance of PTPs, it becomes activated with the release of
p-hydroxybenzylic fluoride intermediate 2. Intermediate 2 quickly
undergoes 1,6-elimination to produce highly reactive quinone
methide 3 (QM-3), which in turn alkylates nearby nucleophilic
residues to form the biotinylated adduct 4.13
85%). The benzylic alcohol 12 was then converted to the corre-
sponding fluoride bytreatment with DAST to afford the orthogonally
protected dipeptide intermediate 13 (yield 50%), which is a common
key intermediate for the preparation of probes 8aec.
The Boc group of the dipeptide intermediate 13 was removed by
treatment with TFA and the resultant ammonium salt was coupled
to the three respective amino acid residues via the N-terminus
(Scheme 4). The coupling reaction was accomplished with the
standard DCC/HOBt procedure to give the fully protected tripep-
tides 14aec (yields 75e80%). For 14a and 14c, Fmoc protecting
In this report, we describe the development of activity probes
that target subfamilies of PTPs. Since many PTPs showed different
substrate specificity,14 we envisioned that expansion of the recog-
nition region by adding suitable interacting elements to the latent
trapping unit would assist in tuning the target specificity.15 How-
ever, the latent trapping unit of probe 1 does not have free ends to
incorporate extra interacting elements. We thus designed phos-
photyrosine analog 5 to serve as a novel latent trapping unit
(Scheme 2), which would allow incorporation of various amino acid
residues to its C- and N-termini to extend the recognition unit. As a
proof-of-concept, we performed model synthesis on three tripep-
tide probes (8aec) and evaluated their labeling performances. As
shown in Figure 1, probes 8aec carry the phosphotyrosine analog in
the middle position and a leucinamide residue at the C-terminus of
the tripeptides. Probes 8aec differed only in the N-terminal residues
(Glu for 8a, Phe for 8b, and Lys for 8c). In this series of probes, a biotin
reporter unit was attached through a hydrophilic linker to the
N-terminus. The proposed activation/labeling mechanism is similar
to that of probe 1, except 1,4-elimination would occur for the for-
mation of QM-6 (Scheme 2).
group was used for the a-amino group, whereas for 14b, Boc was
used. These protecting groups could be removed under basic and
acidic conditions, respectively, allowing the attachment of a suc-
cinic acid spacer to offer compounds 15aec (yields 60e70%). The
biotin reporter group with an ethylene glycol-type linker (16)16 was
then coupled to compounds 15aec to give the fully protected form
of tripeptide probes 17aec (yields 60e65%). Final deprotection step
was successfully carried out with TMSBr and BSTFA to provide the
desired tripeptide probes 8aec (yields 80e90%).
To test if the newly designed and synthesized probes 8aec could
label PTPs, we used the catalytic domain of PTP1B, a 39 kDa protein
with 339 amino acid residues, as a model for the labeling experi-
ments. In a typical labeling reaction, 3 mg each of PTP1B proteins
were incubated with 0.25 mM of probe 8a at 25 ꢀC in the presence/
absence of 1 mM Na3VO4. The incubation mixtures were divided
Scheme 2. Novel phosphotryosine analog 5 serving as the latent trapping unit and the proposed activation/labeling process for PTPs.
Figure 1. The structures for probes 8aec.
2. Results and discussion
into two parts and then monitored using two parallel SDS-poly-
acrylamide gels (Fig. 2). The gel on the left was stained with Coo-
massie blue, which showed the relative amount of loaded proteins.
The gel on the right was visualized by immunoblotting analysis
after transferring the reaction products onto a nitrocellulose
membrane. As shown in Figure 2, intense biotinylated protein
bands were observed in PTP1B that was treated with probe 8a. In
contrast, no biotinylated adduct was observed when Na3VO4,
a phosphatase inhibitor, was present in the incubation mixture.
Similar results were obtained when TCPTP was used as the labeling
The syntheses of tripeptide probes 8aec began with commer-
cially available Boc-
methylation of 9 was accomplished by treating formaldehyde and
borax under alkaline condition to give diol 10 (yield 85%). -Leuci-
L
-Tyr (9). As shown in Scheme 3, ortho-hydroxy-
L
namide was then coupled to compound 10 under standard DCC/
HOBt condition to give the dipeptide unit 11 (yield 79%). Selective
phosphorylation of 11 at the phenolic OH was achieved with diallyl
phosphite/CCl4/DMAP to give the phosphorylated product 12 (yield

Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
4523
Figure 2. Activity-dependent labeling of PTP1B with probe 8a. Each of PTP1B (3 mg)
proteins were incubated with 0.25 mM of probe 8a at 25 ꢀC with or without 1 mM
Na3VO4. The reaction mixtures were separated by 10% SDS-PAGE and then subjected to
immunoblotting analysis using anti-biotin antibody. Labeled proteins were visualized
by chemiluminescence using an ECL kit (PerkineElmer). The Coomassie blue-stained
(left) and the ECL-developed gels (right) are presented.
Scheme 3. Synthesis of the orthogonally protected dipeptide intermediate 13. Re-
agents and conditions: (a) CH2O, Na2B4O7, NaOH/H2O; (b) L-Leucinamide, DCC, HOBt,
DIEA, DMF; (c) (AllylO)2POH, CCl4, DMAP, DIEA, acetone; (d) DAST, CH2Cl2.
target (data not shown). Since the probes themselves are also the
substrates of the corresponding PTPs, the results clearly indicate
that the newly developed latent trapping unit well mimics the
phosphorotyrosine residue of the natural substrate. The results also
indicate that the long tail containing the linker and the biotin
reporter attached to the N-terminus of the tripeptide does not
prevent the probe from entering the active site. Significantly, the
labeling of PTPs with probe 8a was activity dependent. It should be
noted that the benzylic fluoride moiety in probes 8aec showed
reasonable stability in the labeling buffer. In the absence of PTPs,
they underwent hydrolysis slowly and the purity remained greater
than 90% even after 1 h (as determined by HPLC).
lysozyme. The results obtained showed that probes 8aec did not
label any of these proteins (data not shown). To test if probes 8aec
could differentiate PTPs from other phosphatases, we conducted
labeling experiments on nine phosphatases, including five PTPs,
alkaline phosphatase (ALP), PTEN (dephosphorylating the phos-
phatidylinositol 3,4,5-trisphosphate), and two serine/threonine
phosphtases (PPP1CA and PPM1A). The results showed that probes
8aec labeled all five PTPs and yet did not label any of the non-PTPs
(Fig. 3), confirming that probes 8aec are indeed highly specific for
PTPs.
In order to further study how well probes 8aec could dif-
ferentiate between the various PTPs, we compared the labeling
intensities on five PTPs (PTP1B, SHP2, TCPTP, VHR, and PTP-
PEST) with those obtained from probe 1. When the intensities of
To further confirm the group specificity of the activity probes,
we compared the effect of probes 8aec on other proteins, including
carbonic anhydrase,
g-globulin, phosphorylase b, RNase A, and
Scheme 4. Synthesis of probes 8aec for PTPs. Reagents and conditions: (a) for 14a: (i) TFA, CH2Cl2, (ii) Fmoc-Glu(OtBu), DCC, HOBt, DIEA, DMF; for 14b: (i) TFA, CH2Cl2, (ii) Boc-Phe,
DCC, HOBt, DIEA, DMF; for 14c: (i) TFA, CH2Cl2, (ii) Fmoc-Lys(Boc), DCC, HOBt, DIEA, DMF; (b) (i) 20% TFA (for 15b)/50% Et2NH (for 15a, 15c), (ii) succinic anhydride, DMAP, CH2Cl2; (c)
16, DCC, HOBt, DIEA, DMF; (d) BSTFA, TMSBr, CH3CN.

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Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
Figure 3. Probes 8aec specifically labeled PTPs. Each of (3
m
g) different phsophatases were incubated with 0.25 mM of the probes 8aec at 25 ꢀC for 20 min. For PPM1A, 60 mM of
MgCl2 was added to the reaction. The reaction mixtures were separated by 12% SDS-PAGE and then subjected to immunoblotting analysis using anti-biotin antibody.
Figure 4. Determination of PTP substrate preferences by probes 8aec. Each of (3 m
g) PTP1B, SHP2, TCPTP, VHR, or PTP-PEST were incubated with 0.1 mM probes at 25 ꢀC for 20 min.
The reaction mixtures were separated by 12% SDS-PAGE and subjected to immunoblotting analysis using anti-biotin antibody. Quantification of the western blots using Vilber
Lourmat quantification tool. The results show the average value of three to four independent experiments.
the probes 8aec-labeled bands in each set of experiments were
normalized relative to that of a probe 1-labeled band, we were
able to establish a quantitative comparison of their labeling
preference (Fig. 4). The result strongly suggests that the labeling
intensity also reflects the trend of substrate specificities for
these PTPs. Of the five PTPs tested, the substrate specificities of
PTP1B, TCPTP, and SHP2 were better studied than those of VHR
and PTP-PEST.17e21 The results for the former three PTPs showed
that both PTP1B and TCPTP preferred probes with sequence Phe-
pTyr-Leu over Glu-pTyr-Leu and Lys-pTyr-Leu, whereas SHP2
preferred Glu-pTyr-Leu and Phe-pTyr-Leu over Lys-pTyr-Leu,
clearly supporting the observation that these probes indeed
labeled different PTPs with varying efficiency. The trend of
substrate preference obtained from this study was similar to
those determined by other methods.17e21
PEST (a classical PTP). The results obtained suggest that PTP-PEST
showed a substrate preference similar to that of PTP1B and
TCPTP, whereas VHR did not show much substrate preference.
This provides evidence to support the concept that probes with
added amino acid residues flanking the latent trapping device
were able to influence their target specificities. Although probes
8aec have not yet achieved exclusive labeling on a designated
PTP, the underlying concept of expanding the recognition ele-
ment presented in this paper is consistent with the recent trend
in the development of inhibitors and chemical probes for
PTPs.22,23
3. Conclusion
We have developed
a novel phosphotyrosine analog as
We have also compared the labeling intensity data for the
other two PTPs, VHR (a dual-specificity phosphatase) and PTP-
a latent trapping unit. It was incorporated into three tripeptide
probes in the model synthesis. These probes could label PTPs in

Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
4525
an activity-dependent manner and the labeling was PTP-selec-
tive. The new trapping unit functioned as expected and that the
amino acids flanking the trapping device could affect the target
specificity. Therefore, our data support the concept that expan-
sion of the recognition unit would help differentiate PTPs. Our
ongoing studies aim to increase the variety of the recognition
elements at the flanking positions in order to obtain probes
capable of differentiating specific members in PTP subfamilies.
1H), 2.86 (dd, J¼7.9, 13.8 Hz, 1H), 1.78e1.50 (m, 3H), 1.35 (s, 9H,
t-Bu), 0.88 (d, J¼6.0 Hz, 3H), 0.86 (d, J¼5.7 Hz, 3H). 13C NMR
(100 MHz, acetone-d6):
d 175.5 (C), 172.6 (C), 156.4 (C), 154.8 (C),
129.7 (CH), 129.5 (CH), 128.9 (C), 128.0 (C), 115.9 (CH), 79.5 (C), 61.8
(CH2), 57.2 (CH2), 52.0 (CH), 41.8 (CH2), 37.8 (CH2), 28.5 (CH3), 25.1
(CH), 23.5 (CH3), 21.9 (CH3). IR (KBr): 3443, 3350, 3297, 3217, 2919,
2853, 1673, 1653, 1394, 1368, 1262, 990 cmꢂ1. HRMS calcd for
C21H33N3O6Na (MþNa)þ 446.2267, found 446.2289.
4. Experimental
4.1.3. tert-Butyl ((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)
amino)-3-(4-((bis(allyloxy)
phosphoryl)oxy)-3-(hydroxy-methyl)
4.1. Synthesis of the orthogonally protected dipeptide
intermediate 13
phenyl)-1-oxopropan-2-yl) carbamate (12). To an ice-cooled solu-
tion of compound 11 (2.00 g, 4.72 mmol), DIEA (3.1 mL, 19 mmol),
CCl4 (4.5 mL, 47 mmol), and DMAP (115 mg, 0.943 mmol) in 50 mL
of anhydrous acetone was added dropwise diallyl phosphite
(1.4 mL, 9.4 mmol). The mixture was allowed to warm to rt. After
stirring for 18 h the reaction mixture was concentrated under re-
duced pressure and the residual oil was dissolved in EtOAc and then
washed consecutively with 5% citric acid (ꢁ3), H2O (ꢁ3), and brine
(ꢁ2). The organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated. Compound 12 was obtained (2.34 g, 85%) as
a colorless oil after silica gel column chromatography eluted with
All reagents and starting materials were obtained from com-
mercial suppliers (Acros, Aldrich, and Merck) and were used
without further purification. IR spectra were recorded on a Nicolet
550 series II spectrometer. 1H, 19F, and 13C NMR were recorded
using a Bruker Avance 400 spectrometer. The proton and carbon
chemical shifts are given in parts per million using CDCl3 (d H 7.24
and 77.0) as internal standard. High resolution mass spectra were
recorded with a JEOL-102A mass spectrometer. Analytical TLC
(silica gel, 60F-54, Merck) were visualized under UV light and/or
phosphomolybdic acid/ethanol stain. Column chromatography was
performed with Kiesegel 60 (70e230 mesh) silica gel (Merck).
Melting points are reported without correction.
CHCl3/MeOH (9/1). 1H NMR (400 MHz, CDCl3):
d
7.43 (d, J¼8.1 Hz,
1H, NH), 7.27 (s, 1H, aromatic), 7.09e7.00 (m, 2H, aromatic), 6.38
(s, 1H, NH), 6.26 (s, 1H, NH), 5.95e5.82 (m, 2H, ]CH), 5.67
(d, J¼8.1 Hz, 1H, NH), 5.34 (dt, J¼17.1, 1.3 Hz, 2H, ]CH2), 5.23 (dd,
J¼10.4, 1.1 Hz, 2H, ]CH2), 4.68e4.52 (m, 6H), 4.52e4.30 (m, 3H),
3.00e2.78 (m, 2H), 1.62e1.45 (m, 2H), 1.45e1.26 (m, 10H), 0.80
(d, J¼5.9 Hz, 3H), 0.79 (d, J¼8.7 Hz, 3H). 13C NMR (100 MHz, CDCl3):
4.1.1. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-hydroxy-3-(hydroxy-
methyl)phenyl) propanoic acid (10). To a solution of Boc- -Tyr (9)
L
(13.0 g, 46.2 mmol), 1 N NaOH (110 mL, 110 mmol), sodium borate
decahydrate (44.1 g, 116 mmol) in 180 mL of water was added 35%
formaldehyde (19.8 mL, 250 mmol). The reaction mixture was
stirred at 40 ꢀC for 3 days. When no more starting material was
detected by TLC, the pH was adjusted to three with 1 N HC1. The
solution was then extracted with EtOAc. The combined organic
layer was washed with brine (ꢁ2), dried over anhydrous Na2SO4,
filtered, and concentrated. Compound 10 was obtained (12.2 g,
d
174.7 (C), 171.3 (C), 155.4 (C), 146.7 (d, J¼7.1 Hz, C), 134.1 (C), 132.8
(d, J¼5.7 Hz, C), 131.8 (dd, J¼6.6, 3.1 Hz, C), 130.4 (CH), 129.7 (CH),
120.1 (CH), 118.8 (d, J¼3.0 Hz, CH2), 79.9 (C), 69.1 (d, J¼3.0 Hz, CH2),
59.4(CH2), 55.5 (CH), 51.3 (CH), 40.3 (CH2), 37.7 (CH2), 28.2 (CH3),
24.5 (CH), 22.8 (CH3), 21.7 (CH3). 31P NMR (162 MHz, CDCl3):
d
ꢂ4.99. IR (KBr): 3310, 2966, 2933, 2878, 1673, 1534, 1501, 1361,
1428, 1255, 1182, 1109, 1130 cmꢂ1. HRMS calcd for C27H42N3O9P Na
(MþNa)þ 606.2556, found 608.2368.
85%) as an oil. 1H NMR (400 MHz, acetone-d6):
d 7.13 (s, 1H, aro-
matic), 7.00 (dd, J¼1.5, 8.9 Hz, 1H, aromatic), 6.72 (d, J¼8.9 Hz, 1H,
aromatic), 4.69 (s, 2H, benzylic), 4.30 (m, 1H), 3.08 (dd, J¼5.0,
13.9 Hz, 1H), 2.91 (dd, J¼8.4, 13.9 Hz, 1H), 1.35 (s, 9H, t-Bu). 13C NMR
4.1.4. tert-Butyl ((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)
amino)-3-(4-((bis(allyloxy)
phosphoryl)oxy)-3-(fluoromethyl)phe-
nyl)-1-oxopropan-2-yl) carbamate (13). To an ice-cooled solution of
compound 12 (1.470 g, 2.52 mmol) in 25 mL of anhydrous CH2Cl2
(100 MHz, acetone-d6):
d 173.9 (C), 156.3 (C), 154.8 (C), 129.6 (CH),
129.3 (CH), 128.8 (C), 127.8 (C), 115.8 (CH), 79.3 (C), 61.7 (CH2), 55.9
(CH), 37.4 (CH2), 28.5 (CH3). IR (KBr): 3364, 2992, 2972, 1713, 1693,
1527, 1501, 1441, 1375, 1262, 1215, 1169, 1023 cmꢂ1. HRMS calcd for
C15H21NO6Na (MþNa)þ 334.1267, found 334.1275.
was slowly added DAST (463 mL, 3.78 mmol) through a syringe. The
reaction mixture was allowed to warm to rt. When no more starting
material was observed, it was cooled and quenched by adding
0.5 mL of MeOH and a small amount of silica gel. Silica gel was
filtered off and the filtrate was concentrated under reduced pres-
sure. The residual oil was dissolved in EtOAc and then washed
consecutively with 5% NaHCO3 (ꢁ3) and brine (ꢁ2). The organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated.
The desired product was purified by silica gel column chromatog-
raphy eluted with CHCl3/MeOH (95/5) to give compound 13
(738 mg, 50%) as a colorless oil. 1H NMR (400 MHz, CDCl3):
4.1.2. tert-Butyl ((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)
amino)-3-(4-hydroxy-3-(hydroxy-methyl)phenyl)-1-oxopropan-2-yl)
carbamate (11). To an ice-cooled solution of compound 10 (704 mg,
2.26 mmol), HOBt (61 mg, 0.45 mmol),
ride (374 mg, 2.26 mmol), and DIEA (1495
of anhydrous DMF was added solution of DCC (513 mg,
L-leucinamide hydrochlo-
mL, 9.05 mmol) in 20 mL
a
2.49 mmol) in 1 mL of DMF. The mixture was allowed to warm to rt
and stirred for another 16 h. The white DCU precipitate was filtered
off. The filtrate was concentrated to dryness and the residual oil was
dissolved in EtOAc and then washed consecutively with 5% citric
acid (ꢁ1), 5% NaHCO3 (ꢁ3), H2O (ꢁ3), and brine (ꢁ2). The organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated.
Compound 11 was obtained (760 mg, 79%) as a white solid after
silica gel column chromatography eluted with CHCl3/MeOH (94/6),
d
7.27e7.18 (m, 2H, aromatic), 7.14 (d, J¼8.4 Hz, 1H, aromatic), 8.30
(d, J¼8.3 Hz, 1H, NH), 6.22 (br s, 1H, NH), 6.06 (br s, 1H, NH),
5.97e5.79 (m, 2H, ]CH), 5.39 (d, J¼7.6 Hz, 1H, NH), 5.38
(d, J¼48.1 Hz, 2H, CH2F), 5.34 (d, J¼17.0 Hz, 2H, ]CH2), 5.23
(d, J¼10.4 Hz, 2H, ]CH2), 4.60 (t, J¼7.0 Hz, 4H, allylic), 4.50e4.25
(m, 2H), 3.03 (dd, J¼13.8, 7.0 Hz, 1H), 2.94 (dd, J¼13.0, 6.6 Hz, 1H),
1.66e1.49 (m, 2H), 1.45 (m, 1H), 1.35 (s, 9H, t-Bu), 0.84 (t, J¼6.3 Hz,
3H), 0.83 (t, J¼6.1 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 174.3 (C),
mp 168e170 ꢀC. 1H NMR (400 MHz, acetone-d6):
d
8.46 (br s, 1H,
171.2 (C), 155.5 (C), 147.2 (d, J¼11.0 Hz, C), 133.9 (C), 131.8 (dd, J¼6.8,
2.6 Hz, CH), 131.1 (CH), 130.5 (d, J¼5.8 Hz, CH), 127.6 (dd, J¼17.3,
6.6 Hz, C), 120.1 (CH), 118.9 (d, J¼1.4 Hz, CH2), 80.2 (C), 79.7
(d, J¼167 Hz, CH2F), 69.0 (dd, J¼5.6, 1.3 Hz, CH2), 55.7 (CH), 51.2
(CH), 40.5 (CH2), 37.2 (CH2), 28.2 (CH3), 24.6 (CH), 22.9 (CH3), 21.7
phenolic), 7.61 (d, J¼8.3 Hz, 1H, NH), 7.11 (s, 1H, aromatic), 6.99 (dd,
J¼1.7, 8.2 Hz, 1H, aromatic), 6.96 (s, 1H, NH), 6.71 (d, J¼8.2 Hz, 1H,
aromatic), 6.68 (s, 1H, NH), 6.20 (d, J¼7.6 Hz, 1H, NH), 4.67 (s, 2H,
benzylic), 4.63e4.43 (m, 2H), 4.30 (m, 1H), 3.03 (dd, J¼6.0, 13.8 Hz,

4526
Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
(CH3). 31F NMR (376 MHz, CDCl3):
d
ꢂ214.6 (d, J¼47.0 Hz). 31P NMR
(376 MHz, CD3OD):
CD3OD):
d
ꢂ214.7 (t, J¼48.0 Hz). 31P NMR (162 MHz,
(162 MHz, CDCl3):
d
ꢂ5.84. IR (KBr): 3297, 2959, 2912, 1653, 1501,
d
ꢂ5.38. IR (KBr): 3277, 2926, 1640, 1540, 1454, 1262, 1156,
1368, 1255, 1176, 1030, 963 cmꢂ1. HRMS calcd for C27H41FN3O8P Na
1043 cmꢂ1. HRMS calcd for C46H58FN4O11PNa (MþNa)þ 915.3721,
(MþNa)þ 608.2513, found 608.2518.
found 915.3680.
4.2.3. (9H-Fluoren-9-yl)methyl tert-butyl ((S)-6-(((S)-1-(((S)-1-amino-
4.2. Synthesis of probes 8aec
4-methyl-1-oxopentan-
2-yl)amino)-3-(4-((bis(allyloxy)phosphoryl)
oxy)-3-(fluoromethyl)phenyl)-1-oxopropan-2-yl)amino)-6-oxohexane-
1,5-diyl) dicarbamate (14c). The same procedure as that for com-
pound 14a was used, except Fmoc-Lys(Boc)-OH was used for the
coupling. Yield 78%, mp 148e150 ꢀC. 1H NMR (400 MHz, Ace-
4.2.1. tert-Butyl ((S)-1-(((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-
2-yl)amino)-3-(4-((bis (allyloxy)phosphoryl)oxy)-3-(fluoromethyl)
phenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenyl-
propan-2-yl)
carbamate (14b). To a solution of the fluorinated compound 13
(1.00 g, 1.71 mmol) in 17 mL CH2Cl2 was added 3.4 mL of TFA. After
stirring at rt for 30 min, the reaction mixture was concentrated
under reduced pressure and then kept under high vacuum to
remove the residual TFA. The resultant TFA salt was used for the
coupling reaction without further purification. To an ice-cooled
solution of the TFA salt, DIEA (1.2 mL, 6.8 mmol), HOBt (93 mg,
tone-d6):
d
7.87 (d, J¼7.6 Hz, 2H, aromatic), 7.72 (d, J¼7.2 Hz, 2H,
aromatic), 7.71 (s, 1H, NH), 7.56 (d, J¼5.9 Hz, 1H, NH), 7.45e7.36
(m, 3H, aromatic), 7.36e7.23 (m, 4H, aromatic), 6.89 (d, J¼5.9 Hz,
1H, NH), 6.68 (s, 1H, NH), 6.39 (s, 1H, NH), 6.04 (s, 1H, NH),
6.04e5.92 (m, 2H, ]CH), 5.47 (d, J¼49.1 Hz, 2H, CH2F), 5.39
(d, J¼19.0 Hz, 2H, ]CH2), 5.24 (d, J¼10.4 Hz, 2H, ]CH2),
4.72e4.60 (m, 5H), 4.45e4.33 (m, 2H), 4.30e4.17 (m, 2H), 4.11
(m, 1H), 3.21 (dd, J¼14.2, 5.1 Hz, 1H), 3.10e2.94 (m, 3H),
1.78e1.28 (m, 9H), 1.39 (s, 9H, t-Bu), 0.84 (d, J¼5.0 Hz, 3H), 0.83
0.68 mmol), and Boc-L-Phe (454 mg, 1.71 mmol) in 15 mL of anhy-
drous DMF was added a solution of DCC (423 mg, 2.05 mmol) in
3 mL of DMF. The mixture was allowed to warm to rt and stirred for
another 18 h. The white DCU precipitate was filtered off. The filtrate
was concentrated to dryness. The residual oil was dissolved in
CHCl3 and then washed consecutively with 5% citric acid (ꢁ3), H2O
(ꢁ3), and brine (ꢁ2). The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated. Compound 14b was obtained
(1.102 g, 80%) as a white solid after silica gel column chromatog-
raphy eluted with CHCl3/MeOH (92/8), mp 143e145 ꢀC. 1H NMR
(d, J¼5.0 Hz, 3H). 13C NMR (CD3OD, 100 MHz):
d 177.1 (C), 175.0
(C), 172.9 (C), 158.5 (C), 145.2 (C), 142.6 (C), 136.0 (C), 133.3 (CH),
132.5 (CH), 132.4 (CH), 128.8 (CH), 128.2 (CH), 126.3 (CH), 121.0
(CH), 119.2 (CH2), 80.8 (d, J¼164.2 Hz, CH2F), 79.9 (C), 70.4 (CH2),
68.1 (CH2), 56.7 (CH), 55.7 (CH) ,52.9 (CH), 48.4 (CH), 41.9 (CH2),
41.0 (CH2), 37.5 (CH2), 32.7 (CH2), 30.5 (CH2), 28.8 (CH3), 25.8
(CH), 24.1 (CH2), 23.5 (CH3), 21.9 (CH3). 19F NMR (376 MHz,
CDCl3):
d
ꢂ216.8 (t, J¼48.9 Hz). 31P NMR (162 MHz, Acetone-d6):
(400 MHz, CD3OD):
d
7.42 (s, 1H, aromatic), 7.38e7.15 (m, 7H, aro-
d
ꢂ5.77. IR (KBr): 3277, 3078, 2933, 2475, 2402, 1686, 1640, 1540,
matic), 6.08e5.92 (m, 2H, ]CH), 5.48 (d, J¼47.6 Hz, 2H, CH2F), 5.42
(d, J¼17.1 Hz, 2H, ]CH2), 5.30 (d, J¼10.5 Hz, 2H, ]CH2), 4.75e4.61
(m, 5H), 4.44 (t, J¼7.4 Hz, 1H), 4.36 (dd, J¼9.6, 4.7 Hz, 1H), 3.19 (dd,
J¼13.5, 5.4 Hz, 1H), 3.09e2.73 (m, 2H), 2.77 (dd, J¼13.7, 9.9 Hz, 1H),
1.77e1.57 (m, 3H), 1.38 (s, 9H, t-Bu), 0.97 (d, J¼6.3 Hz, 3H), 0.94
1428, 1255, 1176, 1030 cmꢂ1. HRMS calcd for C48H63FN5O11PNa
(MþNa)þ 958.4143, found 958.4147.
4.2.4. 4-(((S)-1-(((S)-1-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)
amino)-3-(4-((bis(allyloxy)
phosphoryl)oxy)-3-(fluoromethyl)phe-
(d, J¼6.2 Hz, 3H). 13C NMR (CD3OD, 100 MHz):
d
177.1 (C), 174.3 (C),
nyl)-1-oxopropan-2-yl)amino)-5-(tert-butoxy)-1,5-dioxopentan-2-yl)
amino)-4-oxobutanoic acid (15a). To a solution of compound 14a
(203 mg, 0.227 mmol) in 4 mL of CH2Cl2 was added 2 mL of
Et2NH. The mixture was stirred at rt for 30 min. When no more
starting material was observed by TLC analysis, the reaction
mixture was concentrated under reduced pressure and then kept
under high vacuum to remove the residual Et2NH. To a solution
of the resultant Et2NH salt in 2 mL of anhydrous CH2Cl2 was
added succinic anhydride (46 mg, 0.46 mmol) at rt. After stirring
for 18 h, CHCl3 (20 ml) was added to dilute the reaction mixture.
The diluted reaction mixture was then washed consecutively
with 5% citric acid (ꢁ2), H2O (ꢁ3), and brine (ꢁ2). The organic
layer was dried over anhydrous Na2SO4, filtered, and concen-
trated. Compound 15a was obtained (105 mg, 60%) as an oil after
silica gel column chromatography eluted with CHCl3/MeOH
172.7 (C), 157.6 (C), 148.8 (C), 138.5 (C), 135.8 (C), 133.3 (CH), 132.6
(CH), 132.5 (CH), 130.3 (CH), 129.4 (CH), 128.9 (C), 127.7 (CH), 120.9
(CH), 119.2 (CH2), 80.8 (d, J¼164.4 Hz, CH2F), 80.7 (C), 70.5 (CH2),
57.4 (CH), 55.5 (CH), 52.8 (CH), 42.0 (CH2), 39.2 (CH2), 38.1 (CH2),
28.7 (CH3), 25.8 (CH), 23.5 (CH3), 22.0 (CH3). 19F NMR (376 MHz,
CD3OD):
d
ꢂ214.4 (t, J¼48.0 Hz). 31P NMR (162 MHz, CD3OD):
d
ꢂ6.11. IR (KBr): 3284, 2919, 2853, 1640, 1554, 1123, 1036 cmꢂ1
.
HRMS calcd for C36H50FN4O9PNa (MþNa)þ 755.3197, found
755.3203.
4.2.2. (S)-tert-Butyl 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-
5-(((S)-1-(((S)-1-amino- 4-methyl-1-oxopentan-2-yl)amino)-3-(4-((bis
(allyloxy)phosphoryl)oxy)-3-(fluoromethyl)phenyl)-1-oxopropan-2-yl)
amino)-5-oxopentanoate (14a). The same procedure as that for
compound 14b was used, except Fmoc-Glu(OtBu)-OH was used for
the coupling. Yield 75%, mp 149e150 ꢀC. 1H NMR (400 MHz,
(9/1). 1H NMR (400 MHz, CD3OD):
d 7.46 (s, 1H, aromatic), 7.38
(d, J¼8.4 Hz, 1H, aromatic), 7.29 (d, J¼8.4 Hz, 1H, aromatic),
6.06e5.86 (m, 2H, ]CH), 5.46 (d, J¼47.5 Hz, 2H, CH2F), 5.40
(d, J¼17.1 Hz, 2H, ]CH2), 5.28 (d, J¼10.5 Hz, 2H, ]CH2),
4.75e4.60 (m, 4H), 4.53 (dd, J¼10.2, 4.7 Hz, 1H), 4.37 (m, 1H),
4.18 (dd, J¼8.3, 5.4 Hz, 1H), 3.25 (dd, J¼14.1, 4.6 Hz, 1H), 3.09
(dd, J¼13.9, 10.5 Hz, 1H), 2.76 (m, 1H), 2.69e2.50 (m, 2H), 2.43
(m, 1H), 2.30e2.10 (m, 2H), 1.93 (m, 1H), 1.79 (m, 1H), 1.72e1.55
(m, 3H), 1.43 (s, 9H, t-Bu), 0.97 (d, J¼5.4 Hz, 3H), 0.90
CD3OD):
d
7.84 (d, J¼7.5 Hz, 2H, aromatic), 7.70 (d, J¼7.4 Hz, 2H,
aromatic), 7.46e7.38 (m, 3H, aromatic), 7.38e7.30 (m, 3H, aro-
matic), 7.27 (d, J¼8.4 Hz, 1H, aromatic), 6.04e5.90 (m, 2H, ]CH),
5.45 (d, J¼47.6 Hz, 2H, CH2F), 5.40 (d, J¼17.1 Hz, 2H, ]CH2), 5.29
(d, J¼10.5 Hz, 2H, ]CH2), 4.74e4.58 (m, 5H), 4.45e4.32 (m, 3H),
4.25 (t, J¼6.8 Hz, 1H), 4.10 (dd, J¼8.6, 5.5 Hz, 1H), 3.22 (dd, J¼13.9,
5.5 Hz, 1H), 3.03 (dd, J¼13.9, 8.2 Hz, 1H), 2.28 (t, J¼7.6 Hz, 2H), 2.03
(m, 1H), 1.85 (m, 1H), 1.73e1.55 (m, 3H), 1.48 (s, 9H, t-Bu), 0.93
(d, J¼6.2 Hz, 3H), 0.92 (d, J¼6.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
(d, J¼5.4 Hz, 3H). 13C NMR (100 MHz, CD3OD):
d 177.7 (C), 177.3
(C), 176.2 (C), 174.7 (C), 173.8 (C), 173.6 (C), 149.0 (C), 136.7 (C),
133.6 (CH), 132.6 (CH), 132.2 (CH), 129.3 (C), 121.3 (CH), 119.5
(CH2), 82.0 (C), 80.8 (d, J¼164.6 Hz, CH2F), 70.7 (CH2), 57.0 (CH),
55.4 (CH), 53.3 (CH), 41.8 (CH2), 36.9 (CH2), 32.7 (CH2), 31.4
(CH2), 30.3 (CH2), 28.6 (CH3), 27.8 (CH2), 26.0 (CH), 24.0 (CH3),
d
174.3 (C), 172.5 (C), 171.8 (C), 170.2 (C), 156.5 (C), 147.2 (C), 143.8
(C), 143.5 (C), 141.1 (C), 133.5 (C), 131.7 (CH), 131.1 (CH), 130.5 (CH),
127.6 (CH),127.0 (CH),125.1 (CH),120.1 (CH),119.8 (CH),118.8 (CH2),
80.8 (C), 79.7 (d, J¼165.5 Hz, CH2F), 69.0 (CH2), 67.1 (CH2), 54.7 (CH),
54.2 (CH), 51.3 (CH), 46.8 (CH), 40.7 (CH2), 37.7 (CH2), 31.6 (CH2),
27.9 (CH3), 27.9 (CH2), 24.7 (CH), 22.7 (CH3), 21.9 (CH3). 19F NMR
21.8 (CH3). 19F NMR (376 MHz, CD3OD):
31P NMR (162 MHz, CD3OD):
d
ꢂ214.5 (t, J¼48.9 Hz).
d
ꢂ6.11. IR (KBr): 3271, 2926, 1726,

Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
4527
1639, 1540, 1421, 1249, 1156, 1017, 937 cmꢂ1. HRMS calcd for
ice-cooled solution of 16 (386 mg, 0.819 mmol), 15b (600 mg,
0.819 mmol), DIEA (1.082 mL, 6.55 mmol) and HOBt (55 mg,
0.41 mmol) in 7 mL of DMF was added a solution of DCC (186 mg,
0.901 mmol) in 1 mL of DMF. The mixture was allowed to warm to
rt and stirred for another 18 h. The white DCU precipitate was fil-
tered off. The filtrate was concentrated to dryness. The residual oil
was dissolved in CHCl3 and then washed consecutively with 5%
citric acid (ꢁ1) and brine (ꢁ1). The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated. Compound 17b was
obtained (580 mg, 65%) as a oil after silica gel column chromatog-
raphy eluted with CHCl3/MeOH (9/1). 1H NMR (400 MHz, CD3OD):
C35H52FN4O12PNa (MþNa)þ 793.3201, found 793.3228.
4.2.5. 4-(((S)-1-(((S)-1-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)
amino)-3-(4-((bis(allyloxy)
phosphoryl)oxy)-3-(fluoromethyl)phe-
nyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-
4-oxobutanoic acid (15b). To a solution of compound 14b (607 mg,
0.828 mmol) in 5 mL of CH2Cl2 was added 1 mL of TFA. After stirring
at rt for 30 min, the solvent and acid were removed under reduced
pressure to give the TFA salt, which was used for the coupling
reaction without further purification. To a solution of the TFA salt in
8 mL of anhydrous CH2Cl2 was added TEA (465
mL, 3.35 mmol),
d
7.53 (s, 1H, aromatic), 7.46 (d, J¼8.3 Hz, 1H, aromatic), 7.35
DMAP (20 mg, 0.16 mmol), and succinic anhydride (166 mg,
1.66 mmol). After stirring at rt for 18 h, CHCl3 (50 ml) was added to
dilute the reaction mixture. The diluted reaction mixture was then
washed consecutively with 5% citric acid (ꢁ2), H2O (ꢁ3), and brine
(ꢁ2). The organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated. Compound 15b was obtained (426 mg, 70%) as
an oil after silica gel column chromatography eluted with CHCl3/
(d, J¼8.4 Hz, 1H, aromatic), 7.28e7.19 (m, 3H, aromatic), 7.19e7.12
(m, 2H, aromatic), 6.10e5.95 (m, 2H, ]CH), 5.52 (d, J¼47.7 Hz, 2H,
CH2F), 5.43 (d, J¼17.2 Hz, 2H, ]CH2), 5.32 (d, J¼10.5 Hz, 2H, ]CH2),
4.74e4.67 (m, 4H), 4.58e4.48 (m, 2H), 4.45e4.36 (m, 2H), 4.32
(m, 1H), 3.64 (s, 4H), 3.61e3.55 (m, 4H), 3.46 (m, 1H), 3.42e3.35
(m, 2H), 3.35e3.26 (m, 2H), 3.25e3.17 (m, 2H), 3.06 (m, 1H), 2.95
(m, 1H), 2.85e2.63 (m, 3H), 2.59e2.45 (m, 2H), 2.34 (m, 1H),
2.28e2.20 (m, 2H), 1.83e1.56 (m, 7H), 1.52e1.42 (m, 2H), 1.01
(d, J¼6.1 Hz, 3H), 0.94 (d, J¼5.9 Hz, 3H). 13C NMR (100 MHz,
MeOH (9/1). 1H NMR (400 MHz, CD3OD):
d 7.50 (s, 1H, aromatic),
7.42 (d, J¼8.5 Hz, 1H, aromatic), 7.33 (d, J¼8.4 Hz, 1H, aromatic),
7.28e7.17 (m, 5H, aromatic), 6.08e5.95 (m, 2H, ]CH), 5.50
(d, J¼47.5 Hz, 2H, CH2F), 5.42 (d, J¼15.6 Hz, 2H, ]CH2), 5.31
(d, J¼10.4 Hz, 2H, ]CH2), 4.75e4.65 (m, 4H), 4.56 (dd, J¼10.0,
4.7 Hz, 1H), 4.47 (dd, J¼9.5, 5.0 Hz, 1H), 4.40 (m, 1H), 3.27 (dd,
J¼14.1, 4.8 Hz, 1H), 3.13 (dd, J¼13.9, 10.2 Hz, 1H), 3.06 (dd, J¼14.1,
4.8 Hz, 1H), 2.83 (dd, J¼14.0, 9.7 Hz, 1H), 2.71 (m, 1H), 2.62e2.48
(m, 2H), 2.36 (m, 1H), 1.80e1.60 (m, 3H), 1.0 (d, J¼5.4 Hz, 3H), 0.95
CD3OD): d 177.5 (C), 176.5 (C), 176.2 (C), 174.8 (C), 174.7 (C), 173.5 (C),
166.1 (C), 148.7 (C), 138.3 (C), 136.8 (C), 133.4 (CH), 132.4 (CH), 132.0
(CH), 130.0 (CH), 129.5 (CH), 127.8 (CH), 121.0 (CH), 119.3 (CH2), 80.9
(d, J¼164.3 Hz, CH2F), 71.4 (CH2), 71.2 (CH2), 70.5 (CH2), 70.5 (CH2),
70.5 (CH2), 63.4 (CH), 61.6 (CH), 57.6 (CH), 57.1 (CH), 57.0 (CH), 53.1
(CH), 41.6 (CH2), 41.1 (CH2), 40.5 (CH2), 40.3 (CH2), 37.9 (CH2), 36.7
(CH2), 31.9 (CH2), 31.7 (CH2), 30.8 (CH2), 29.8 (CH2), 29.5 (CH2), 26.8
(CH2), 25.8 (CH), 23.7 (CH3), 21.5 (CH3). 19F NMR (376 MHz, CD3OD):
(d, J¼5.4 Hz, 3H). 13C NMR (100 MHz, CD3OD):
d 177.4 (C), 175.9 (C),
174.4 (C), 174.4 (C), 173.3 (C), 148.8 (C), 138.2 (C), 136.5 (C), 133.4
(CH), 132.4 (CH), 132.1 (CH), 130.0 (CH), 129.5 (CH), 129.1 (C), 127.8
(CH), 121.0 (CH), 119.3 (CH2), 80.3 (d, J¼164.4 Hz, CH2F), 70.5 (CH2),
57.2 (CH), 56.8 (CH), 53.1 (CH), 41.6 (CH2), 38.0 (CH2), 36.9 (CH2),
31.3 (CH2), 30.4 (CH2), 25.8 (CH), 23.7 (CH3), 21.6 (CH3). 19F NMR
d
ꢂ214.4 (t, J¼48.9 Hz). 31P NMR (162 MHz, CD3OD):
d
ꢂ6.12. IR
(KBr): 3430, 2913, 2853, 1646, 1554, 1467, 1255, 1096, 1036 cmꢂ1
.
HRMS calcd for C51H74FN8O13PSNa (MþNa)þ 1111.4715, found
1111.4746.
(376 MHz, CD3OD):
CD3OD):
1103, 1036 cmꢂ1
d
ꢂ214.4 (t, J¼48.9 Hz). 31P NMR (162 MHz,
4.2.8. (S)-tert-Butyl 4-(((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-
2-yl)amino)-3-(4-((bis(allyl- oxy)phosphoryl)oxy)-3-(fluoromethyl)
phenyl)-1-oxopropan-2-yl)carbamoyl)-6,9,20-trioxo-24-((3aS,4-
S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-13,16-dioxa-
5,10,19-triazatetracosan-1-oate (17a). The same procedure as that
for compound 17b was used. Yield 60%. 1H NMR (400 MHz, CD3OD):
d
ꢂ6.14. IR (KBr): 3271, 2919, 2853, 1640, 1554, 1428, 1255,
.
HRMS calcd for C35H46FN4O10PNa (MþNa)þ
755.2833, found 755.2809.
4.2.6. 4-(((10S,13S,16S)-13-(4-((Bis(allyloxy)phosphoryl)oxy)-3-
(fluoromethyl)benzyl)-16-
oxo-3-oxa-5,12,15-triazanonadecan-10-yl)amino)-4-oxobutanoic
carbamoyl-2,2,18-trimethyl-4,11,14-tri-
d
7.54 (s, 1H, aromatic), 7.46 (d, J¼8.4 Hz, 1H, aromatic), 7.33 (d,
J¼8.4 Hz, 1H, aromatic), 6.10e5.96 (m, 2H, ]CH), 5.51 (d, J¼47.4 Hz,
2H, CH2F), 5.43 (d, J¼15.7 Hz, 2H, ]CH2), 5.33 (d, J¼10.4 Hz, 2H, ]
CH2), 4.77e4.68 (m, 4H), 4.58e4.50 (m, 2H), 4.39 (m, 1H), 4.34 (dd,
J¼7.9, 4.4 Hz, 1H), 4.16 (dd, J¼8.4, 5.2 Hz, 1H), 3.70e3.63 (m, 4H),
3.63e3.54 (m, 4H), 3.54e3.35 (m, 4H), 3.28e3.20 (m, 2H), 2.96
(m, 1H), 2.83e2.70 (m, 2H), 2.65e2.52 (m, 2H), 2.43 (m, 1H),
2.34e2.13 (m, 4H), 1.98 (m, 1H), 1.90e1.55 (m, 9H), 1.55e1.40
(m, 11H), 1.01 (d, J¼6.2 Hz, 3H), 0.94 (d, J¼6.1 Hz, 3H). 13C NMR
acid (15c). The same procedure as that for compound 15a was
used. Yield 65%. 1H NMR (400 MHz, CD3OD):
d 7.52 (s,1H, aromatic),
7.45 (d, J¼8.5 Hz, 1H, aromatic), 7.32 (d, J¼8.4 Hz, 1H, aromatic),
6.10e5.95 (m, 2H, ]CH), 5.50 (d, J¼47.5 Hz, 2H, CH2F), 5.44
(d, J¼17.3 Hz, 2H, ]CH2), 5.32 (d, J¼10.5 z, 2H, ]CH2), 4.78e4.66
(m, 4H), 4.52 (dd, J¼10.7, 4.5 Hz, 1H), 4.40 (m, 1H), 4.11 (dd, J¼8.2,
4.5 Hz, 1H), 3.29 (m, 1H), 3.15 (m, 1H), 2.99 (t, J¼7.0 Hz, 2H), 2.81
(m, 1H), 2.70e2.55 (m, 2H), 2.44 (m, 1H), 1.78e1.53 (m, 5H),
1.53e1.44 (m, 2H), 1.47 (s, 9H, t-Bu), 1.44e1.33 (m, 2H), 1.02
(d, J¼5.6 Hz, 3H), 0.94 (d, J¼5.7 Hz, 3H). 13C NMR (100 MHz,
(100 MHz, CD3OD):
d 175.5 (C), 174.6 (C), 174.1 (C), 172.9 (C), 172.7
(C), 171.6 (C), 171.5 (C), 164.1 (C), 146.6 (C), 134.8 (C), 131.4 (CH),
130.2 (CH), 129.8 (CH), 127.1 (C), 119.0 (CH), 117.3 (CH2), 79.8 (C),
78.8 (d, J¼164.4 Hz, CH2F), 69.4 (CH2), 69.2 (CH2), 68.6 (CH2),
68.5 (CH2), 68.5 (CH2), 61.3 (CH), 59.6 (CH), 55.1 (CH), 55.0 (CH),
53.5 (CH), 51.2 (CH), 39.5 (CH2), 39.1 (CH2), 38.5 (CH2), 38.2
(CH2), 34.7 (CH2), 34.5 (CH2), 30.4 (CH2), 30.0 (CH2), 29.7 (CH2),
27.8 (CH2), 27.5 (CH2), 26.4 (CH3), 25.4 (CH2), 24.8 (CH2), 23.8
(CH), 21.8 (CH3), 19.5 (CH3). 19F NMR (376 MHz, CD3OD):
CD3OD): d 179.0 (C), 177.5 (C), 177.0 (C), 175.2 (C), 173.5 (C), 158.3(C),
148.6 (C), 136.7 (C), 133.3 (CH), 132.4 (CH), 132.1 (CH), 128.8 (C),
120.8 (CH), 119.2 (CH2), 80.8 (d, J¼164.4 Hz, CH2F), 79.7 (C), 70.4
(CH2), 57.0 (CH), 56.0 (CH) ,53.1 (CH), 41.6 (CH2), 40.8 (CH2), 36.5
(CH2), 32.4 (CH2), 32.4 (CH2), 31.6 (CH2), 30.5 (CH2), 28.8 (CH3), 25.7
(CH), 23.8 (CH2), 23.7 (CH3), 21.6 (CH3). 19F NMR (376 MHz, CD3OD):
d
ꢂ215.9 (t, J¼48.9 Hz). 31P NMR (162 MHz, CD3OD):
d
ꢂ6.15. IR
d
ꢂ214.6 (t, J¼48.9 Hz). 31P NMR (162 MHz, CD3OD):
d
ꢂ6.09. IR
(KBr): 3277, 2919, 2860, 1640, 1540, 1255, 1136, 1036 cmꢂ1. HRMS
(KBr): 3343, 2913, 2846, 1646, 1255, 1149, 1255, 1149, 1030 cmꢂ1
.
calcd for C37H57FN5O12PNa (MþNa)þ 836.3623, found 836.3593.
HRMS calcd for C51H80FN8O15PSNa (MþNa)þ 1149.5083, found
1149.5045.
4.2.7. Diallyl (4-((2S,5S)-2-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)
carbamoyl)-5-benzyl-4,7,10,21-tetraoxo-25-((3aS,4S,6aR)-2-oxohex-
ahydro-1H-thieno[3,4-d]imidazol-4-yl)-14,17-dioxa-3,6,11,20-tetra-
azapentacosyl)-2-(fluoromethyl)phenyl) phosphate (17b). To an
4.2.9. tert-Butyl ((S)-21-(((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-
2-yl)amino)-3-(4-((bis
(allyloxy)phosphoryl)oxy)-3-(fluoromethyl)
phenyl)-1-oxopropan-2-yl)carbamoyl)-5,16,19-trioxo-1-((3aS,4S,6aR)-

4528
Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9,12-dioxa-6,15,20-
triazapentacosan-25-yl) carbamate (17c). The same procedure as
that for compound 17b was used. Yield 60%. 1H NMR (400 MHz,
Yield 80%. 1H NMR (400 MHz, CD3OD):
d 7.50e7.40 (m, 2H, aro-
matic), 7.31 (d, J¼7.4 Hz, 1H, aromatic), 5.57 (d, J¼47.6 Hz, 2H,
CH2F), 4.55e4.47 (m, 2H), 4.42e4.32 (m, 2H), 4.16 (m, 1H), 3.67
(s, 4H), 3.64e3.54 (m, 4H), 3.49 (m, 1H), 3.42e3.35 (m, 3H),
3.28e3.16 (m, 12H), 2.96 (dd, J¼12.7, 5.1 Hz, 1H), 2.82e2.70 (m, 2H),
2.65e2.52 (m, 2H), 2.43 (m, 1H), 2.32e2.13 (m, 4H), 2.00 (m, 1H),
1.88 (m, 1H), 1.80e1.55 (m, 8H), 1.55e1.40 (m, 2H), 1.34 (t, J¼7.3 Hz,
15H, TEA), 1.00 (d, J¼6.0 Hz, 3H), 0.93 (d, J¼6.0 Hz, 3H). 13C NMR
CD3OD):
d
7.55 (s, 1H, aromatic), 7.48 (d, J¼8.6 Hz, 1H, aromatic),
7.33 (d, J¼8.4 Hz, 1H, aromatic), 6.10e5.95 (m, 2H, ]CH), 5.51
(d, J¼47.5 Hz, 2H, CH2F), 5.44 (d, J¼17.1 Hz, 2H, ]CH2), 5.33
(d, J¼10.5 Hz, 2H, ]CH2), 4.77e4.67 (m, 4H), 4.57e4.48 (m, 2H),
4.41 (m, 1H), 4.34 (dd, J¼7.8, 4.4 Hz, 1H), 4.09 (m, 1H), 3.70e3.62
(m, 4H), 3.62e3.54 (m, 4H), 3.49 (m, 1H), 3.44e3.33 (m, 3H), 3.31
(m, 1H), 3.28e3.18 (m, 2H), 3.03e2.93 (m, 3H), 2.85e2.72 (m, 2H),
2.67e2.53 (m, 2H), 2.42 (m, 1H), 2.28e2.20 (m, 2H), 2.28e2.20
(m, 2H), 1.85e1.54 (m, 9H), 1.54e1.35 (m, 13H), 1.35e1.10 (m, 2H),
1.02 (d, J¼6.3 Hz, 3H), 0.94 (d, J¼6.2 Hz, 3H). 13C NMR (100 MHz,
(100 MHz, CD3OD):
d 178.2 (C), 177.6 (C), 176.6 (C), 176.2 (C), 175.1
(C), 174.6 (C), 173.9 (C), 166.1 (C), 151.0 (C), 134.0 (C), 130.8 (CH),
130.1 (CH), 129.7 (C), 121.7 (CH), 81.3 (d, J¼162.6 Hz, CH2F), 71.4
(CH2), 71.2 (CH2), 70.6 (CH2), 70.5 (CH2), 63.3 (CH), 61.6 (CH), 57.6
(CH), 57.0 (CH), 56.0 (CH), 53.1 (CH), 47.5 (CH2), 41.5 (CH2), 41.1
(CH2), 40.5 (CH2), 40.2 (CH2), 36.7 (CH2), 36.7 (CH2), 32.3 (CH2), 31.8
(CH2), 29.8 (CH2), 29.5 (CH2), 27.5 (CH2), 26.8 (CH2), 25.7 (CH), 23.8
CD3OD): d 177.3 (C), 176.6 (C),175.9 (C), 175.8 (C), 175.3 (C), 174.4 (C),
173.4 (C), 165.8 (C), 158.2 (C), 148.4 (C), 136.7 (C), 133.2 (CH), 132.1
(CH), 131.7 (CH), 128.8 (CH), 120.8 (CH),119.2 (CH2), 80.7
(d, J¼164.4 Hz, CH2F), 79.6 (C), 71.1 (CH2), 70.4 (CH2), 70.3 (CH2),
70.3 (CH2), 69.5 (CH2), 63.1 (CH), 61.4 (CH), 57.0 (CH), 56.9 (CH), 56.1
(CH), 53.0 (CH), 41.3 (CH2), 41.0 (CH2), 40.5 (CH2), 40.1 (CH2), 36.6
(CH2), 36.2 (CH2), 31.9 (CH2), 31.6 (CH2), 31.3 (CH2), 30.5(CH2), 29.6
(CH2), 29.3 (CH2), 28.8(CH3), 26.7 (CH2), 25.6 (CH), 23.8 (CH3), 23.6
(CH3), 21.4 (CH3), 9.15 (CH3). 19F NMR (376 MHz, CD3OD):
d
ꢂ218.4
(t, J¼48.9 Hz). 31P NMR (162 MHz, CD3OD):
ꢂ3.96. IR (KBr): 3278,
d
2919, 2846, 2661, 1733, 1633, 1547, 1262, 1030 cmꢂ1. HRMS calcd for
C41H64FN8O15PSNa (MþNa)þ 1013.3831, found 1013.3817.
4.2.12. 4-((2S,5S)-2-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)
carbamoyl)-5-(4-aminobutyl)- 4,7,10,21-tetraoxo-25-((3aS,4S,6aR)-
2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-14,17-dioxa-
(CH2), 21.4 (CH3). 19F NMR (376 MHz, CD3OD):
d
ꢂ214.6
(t, J¼48.9 Hz). 31P NMR (162 MHz, CD3OD):
d
ꢂ6.15. IR (KBr): 3284,
2913, 2860, 1646, 1547, 1467, 1249, 1156, 1036 cmꢂ1. HRMS calcd for
3,6,11,20-tetraazapentacosyl)-2-(fluoromethyl)phenyl
phosphate (8c). The same procedure as that for compound 8b was
used. Yield 80%. 1H NMR (400 MHz, D2O):
7.38 (s, 1H, aromatic),
dihydrogen
C53H85FN9O15PSNa (MþNa)þ 1192.5505, found 1192.5540.
d
4.2.10. 4-((2S,5S)-2-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)car-
7.37e7.22 (m, 2H, aromatic), 5.50 (d, J¼47.5 Hz, 2H, CH2F),
4.62e4.51 (m, 2H), 4.36 (m, 1H), 4.27 (m, 1H), 4.09 (m, 1H), 3.64 (s,
4H), 3.64e3.52 (m, 4H), 3.43 (m, 1H), 3.38e3.30 (m, 3H), 3.30e3.24
(m, 2H), 3.18 (dd, J¼14.7, 7.4 Hz, 1H, TEA), 3.19 (m, 1H), 2.93 (m, 1H),
2.79 (t, J¼7.7 Hz, 2H), 2.72 (m, 1H), 2.65e2.43 (m, 4H), 2.22
(t, J¼6.9 Hz, 2H), 1.75e1.30 (m, 15H), 1.25 (t J¼7.3 Hz, 3H, TEA), 0.92
(d, J¼5.7 Hz, 3H), 0.84 (d, J¼5.6 Hz, 3H). 13C NMR (100 MHz,
bamoyl)-5-benzyl-4,7,10,21-
tetraoxo-25-((3aS,4S,6aR)-2-oxohex-
ahydro-1H-thieno[3,4-d]imidazol-4-yl)-14,17-dioxa-3,6,11,20-tetraa-
zapentacosyl)-2-(fluoromethyl)phenyl dihydrogen phosphate (8b).
To an ice-cooled solution of compound 17b (20.0 mg, 0.0184 mmol)
and BSTFA (99
mL, 0.37 mmol) in 1 mL of anhydrous CH3CN was
slowly added TMSBr (24
m
L, 0.183 mmol). The reaction mixture was
allowed to warm to rt and stirred further for 45 min. The reaction
was quenched with 50% TEA in MeOH (1 mL). The organic solvents
were removed under reduced pressure to give the crude product,
which was purified by chromatography over Sephadex LH-20
eluted with MeOH. The fractions containing the product were
pooled, concentrated and then lyophilized to afford compound 8b
(18 mg, 90%) as a colorless powder. 1H NMR (400 MHz, CD3OD):
CD3OD): d 177.7 (C), 177.2 (C), 176.1 (C), 175.2 (C), 174.6 (C), 174.0 (C),
166.1 (C), 151.3 (C), 133.8 (C), 131.3 (CH), 130.6 (CH), 129.0 (C),
121.3 (CH), 81.4 (d, J¼163.4 Hz, CH2F), 71.4 (CH2), 71.2 (CH2), 70.6
(CH2), 70.5 (CH2), 47.6(CH2), 41.5 (CH2), 41.1 (CH2), 40.6 (CH2),
40.2 (CH2), 36.7 (CH2), 36.3 (CH2), 32.1 (CH2), 31.8 (CH2), 30.7
(CH2), 29.8 (CH2), 29.5 (CH2), 28.0 (CH2), 26.9 (CH2), (CH2) 25.8
(CH), 23.8 (CH3), 22.5 (CH2), 23.4 (CH3), 9.16 (CH3). 19F NMR
d
7.44 (d, J¼8.7 Hz, 1H, aromatic), 7.43 (s, 1H, aromatic), 7.32
(376 MHz, CD3OD):
CD3OD):
d
ꢂ218.0 (t, J¼48.9 Hz). 31P NMR (162 MHz,
(d, J¼8.7 Hz, 1H, aromatic), 7.30e7.15 (m, 5H, aromatic), 5.58
(d, J¼47.6 Hz, 2H, CH2F), 4.57e4.48 (m, 2H), 4.45 (m, 1H), 4.42e4.18
(m, 2H), 3.64 (s, 4H), 3.62e3.52 (m, 4H), 3.47 (m, 1H), 3.42e3.36
(m, 3H), 3.28e3.13 (m, 9H), 3.10 (dd, J¼14.2, 4.4 Hz, 1H), 2.96 (dd,
J¼12.8, 4.9 Hz,1H), 2.83 (dd, J¼14.1,10.0 Hz,1H), 2.77e2.26 (m, 2H),
2.59e2.45 (m, 2H), 2.34 (m, 1H), 2.25 (t, J¼7.3 Hz, 2H), 1.83e1.53
(m, 8H), 1.53e1.37 (m, 3H), 1.33 (t, J¼7.3 Hz, 9H, TEA), 1.00 (d,
J¼5.4 Hz, 3H), 0.93 (d, J¼5.3 Hz, 3H). 13C NMR (100 MHz, CD3OD):
d
ꢂ3.28. IR (KBr): 3284, 2919, 2860, 1686, 1633, 1554,
1467, 1255, 1103 cmꢂ1. HRMS calcd for C42H69FN9O13PS (MþH)þ
990.4535, found 990.4563.
4.3. Typical procedures for the labeling of PTPs
4.3.1. Cloning of PTPs. To clone PTP1B and SHP-2, the cDNA
encoding the catalytic domain of human PTP-1B (1e321) and
SHP2 catalytic domains were obtained by RT-PCR using mRNA
prepared from human lung cancer cell line H1299. The PCR
products were then subcloned into pET6H to obtain plasmids
pET6H-PTP1B and pET6H-SHP2. To clone TCPTP (Swiss prot:
p17706) and VHR (BC002682), PPP1CA (BC00810), PPM1A
(BC026691), PTP-PEST (BC050008), and PTEN (BC005821), primer
pairs were used to PCR amplify cDNAs obtained from Meng TC
(TCPTP and VHR; Institute of Biochemistry, Academia Sinica, Tai-
pei, Taiwan) or Yang-Ming Genome Center (PPP1CA, PPM1A, PTP-
PEST, and PTEN; NYMU, Taipei, Taiwan). The PCR products were
then subcloned into pET6H. The DNA sequences of cloned genes
were confirmed by sequencing analysis using dideoxy chain-ter-
mination methods.
d
177.5 (C),176.4 (C),176.2 (C),174.7 (C),174.6 (C),173.7 (C),166.1 (C),
151.0 (C),138.4 (C),133.5 (C),131.1 (CH),130.1 (CH),130.0 (CH),129.7
(C), 129.5 (CH), 127.8 (CH), 121.8 (CH), 81.3 (d, J¼162.3 Hz, CH2F),
71.3 (CH2), 71.2 (CH2), 70.6 (CH2), 70.5 (CH2), 63.4 (CH), 61.6 (CH),
57.5 (CH), 57.0 (CH), 53.1 (CH), 53.0 (CH), 47.7 (CH2), 41.5 (CH2), 41.1
(CH2), 40.5 (CH2), 40.2 (CH2), 37.9 (CH2), 36.9 (CH2), 36.7 (CH2), 31.9
(CH2), 31.7 (CH2), 29.8 (CH2), 29.5 (CH2), 26.8 (CH2), 25.7 (CH), 23.7
(CH3), 21.5 (CH3), 9.16 (CH3). 19F NMR (376 MHz, CD3OD):
d
ꢂ217.5
ꢂ3.89. IR (KBr): 3377,
HRMS calcd for
(t, J¼48.9 Hz). 31P NMR (162 MHz, CD3OD):
d
3291, 2853, 1633, 1547, 1467, 1255 cmꢂ1
.
C45H66FN8O13PSNa (MþNa)þ 1031.4089, found 1031.4070.
4.2.11. (S)-4-(((S)-1-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)
amino)-3-(3-(fluoromethyl)-4- (phosphonooxy)phenyl)-1-oxopropan-
2-yl)carbamoyl)-6,9,20-trioxo-24-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)-13,16-dioxa-5,10,19-triazatetracosan-1-oic
acid (8a). The same procedure as that for compound 8b was used.
4.3.2. Expression and purification of PTPs. E. coli BL21(DE3) or BL21
(DE3)-pLysS (Novagen) was used as a host to express 6-His tagged
recombinant proteins. Similar procedures were used to purify these

Y.-Y. Huang et al. / Tetrahedron 66 (2010) 4521e4529
4529
phosphatases. Briefly, one-liter cultures of E. coli harboring
expression plasmids were grown at 37 ꢀC to an OD600 of 0.5 and
then induced with the addition of 1 mM IPTG. The cells were grown
at 25 ꢀC for another 4 h before being harvested by centrifugation.
Cells were resuspended in 10 mL of sonication buffer containing
50 mM NaH2PO4 pH 7.8, 300 mM NaCl, 1 mM DTT, 1ꢁ protease
inhibitors (Calbiochem), and sonicated to release the cell contents.
The sonicated cells were centrifuged at 13,000 g for 15 min at 4 ꢀC
to obtain total cell free extracts. The Ni SepharoseÔ (Amersham
Biosciences) was added to the total cell free extracts and incubated
at 4 ꢀC for 1 h. The resin was washed and eluted with buffer con-
taining 50 mM NaH2PO4 pH 8.0, 20% glycerol and varying amounts
of imidazole. Purified proteins were dialyzed against storage buffer
(50 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 50%
glycerol), aliquoted and frozen by dry ice/ethanol bath.
(Academia Sinica, Taipei). We also thank members of the In-
stitute of Biopharmaceutical Sciences for their help and support.
This research was supported by National Science Council
and National Health Research Institute grants (NSC 97-2311-B-
010-005, NSC 97-3112-B-010-013, NHRI-EX97-9625SI to J.-J.L.
and NSC 97-3112-B-002-005, NSC 98-2113-M-002-009-MY3
to L.-C.L.).
Supplementary data
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2010.04.065.
References and notes
4.3.3. Sources of other recombinant proteins. Lysozyme (Sigma
L6876), RNase from bovine pancreas (USB 21195), alkaline phso-
phatase (Sigma P7923), g-globulin from bovine (Sigma G5009), and
phsophorylase b (Sigma P6635) were purchased from Sigma.
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a nitrocelluose
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Acknowledgements
We thank Professor Tzu-Ching Meng for providing the SHP2
cDNA for protein purification and antibody against TCPTP