A novel high-yield synthesis of aminoacyl p-nitroanilines and aminoacyl 7-amino-4-methylcoumarins: Important synthons for the synthesis of chromogenic/fluorogenic protease substrates

Article abstract of DOI:10.3762/bjoc.7.117

Aminoacyl p-nitroaniline (aminoacyl-pNA) and aminoacyl 7-amino-4- methylcoumarin (aminoacyl-AMC) are important synthons for the synthesis of chromogenic/fluorogenic protease substrates. A new efficient method was developed to synthesize aminoacylpNA and aminoacyl-AMC derivatives in excellent yields starting from either amino acids or their corresponding commercially available N-hydroxysuccinimide esters. The method involved the in situ formation of selenocarboxylate intermediate of protected amino acids and the subsequent non-nucleophilic amidation with an azide. Common protecting groups used in amino acid/peptide chemistry were all well-tolerated. The method was also successfully applied to the synthesis of a dipeptide conjugate, indicating that the methodology is applicable to the synthesis of chromogenic substrates containing short peptides. The method has general applicability to the synthesis of chromogenic and fluorogenic peptide substrates and represents a convenient and high-yield synthesis of N^α-protected-aminoacyl-pNAs/AMCs, providing easy access to these important synthons for the construction of chromogenic/ fluorogenic protease substrates through fragment condensation or stepwise elongation.

Full text of DOI:10.3762/bjoc.7.117

A novel high-yield synthesis of aminoacyl p-nitro-
anilines and aminoacyl 7-amino-4-methylcoumarins:
Important synthons for the synthesis of chromo-
genic/fluorogenic protease substrates
Xinghua Wu1, Yu Chen1, Herve Aloysius1 and Longqin Hu*1,2,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2011, 7, 1030–1035.
1Department of Medicinal Chemistry, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey, Piscataway,
New Jersey 08854 and 2The Cancer Institute of New Jersey, New
Brunswick, New Jersey 08901
doi:10.3762/bjoc.7.117
Received: 02 March 2011
Accepted: 08 July 2011
Published: 27 July 2011
Email:
Longqin Hu* - LongHu@rutgers.edu
Associate Editor: D. Trauner
* Corresponding author
© 2011 Wu et al; licensee Beilstein-Institut.
License and terms: see end of document.
§ Phone: 732-445-5291. Fax: 732-445-6312
Keywords:
7-amino-4-methylcoumarin; p-nitroaniline; proteolytic substrate;
selenocarboxylate/azide amidation; synthon
Abstract
Aminoacyl p-nitroaniline (aminoacyl-pNA) and aminoacyl 7-amino-4-methylcoumarin (aminoacyl-AMC) are important synthons
for the synthesis of chromogenic/fluorogenic protease substrates. A new efficient method was developed to synthesize aminoacyl-
pNA and aminoacyl-AMC derivatives in excellent yields starting from either amino acids or their corresponding commercially
available N-hydroxysuccinimide esters. The method involved the in situ formation of selenocarboxylate intermediate of protected
amino acids and the subsequent non-nucleophilic amidation with an azide. Common protecting groups used in amino acid/peptide
chemistry were all well-tolerated. The method was also successfully applied to the synthesis of a dipeptide conjugate, indicating
that the methodology is applicable to the synthesis of chromogenic substrates containing short peptides. The method has general ap-
plicability to the synthesis of chromogenic and fluorogenic peptide substrates and represents a convenient and high-yield synthesis
of Nα-protected-aminoacyl-pNAs/AMCs, providing easy access to these important synthons for the construction of chromogenic/
fluorogenic protease substrates through fragment condensation or stepwise elongation.
Introduction
Chromogenic and fluorogenic amino acid/peptide conjugates acid/peptide conjugates liberates the free chromophore or fluo-
are widely used as substrates in enzyme assays for protease rophore, allowing the convenient determination of the rate of
activity and specificity [1]. Proteolytic cleavage of the amino enzyme catalysis with a UV or fluorescence spectrophotometer.
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Beilstein J. Org. Chem. 2011, 7, 1030–1035.
p-Nitroaniline (pNA) is one of the most commonly used aminoacyl-pNAs was also reported recently [17]. Although this
chromogenic reagents. The synthesis of peptide-pNAs usually method is free of racemization, it does not result in satisfactory
involves formation of aminoacyl-pNAs through the attachment yields. Because of these limitations of the existing methods,
of P1 amino acid to the amino group of p-nitroaniline, followed there is still a need to develop a generally applicable method for
by fragment condensation or stepwise elongation [P1 amino the synthesis of aminoacyl-pNAs. Conjugation of Nα-protected
acid refers to the residue immediately amino terminal to the amino acids with fluorogenic 7-amino-4-methylcoumarin
scissile bond in a proteinase substrate]. However, the synthesis (AMC) is similarly as problematic as p-nitroaniline due to the
of aminoacyl-pNAs is particularly problematic due to the poor low nucleophilicity of the 7-aromatic amino group. Thus, the
nucleophilicity of the aromatic amino group, which is further method developed for the synthesis of aminoacyl-pNAs could
deactivated by the electron-withdrawing nitro group at the para also be applied to the synthesis of aminoacyl-AMC derivatives.
position. Direct acylation of p-nitroaniline using common
coupling methods, such as DCC [2], HOBt activated ester [3], We previously described a novel selenocarboxylate/azide
acyl chloride [4], and mixed anhydride methods [5-8], did not amidation reaction [18,19] and its application to directly couple
afford satisfactory yields. A phosphorus trichloride method was amino acids with azides [20]. We demonstrated that our seleno-
developed to improve the acylation yield through a carboxylate/azide amidation does not involve reduction of the
phospho–azo mechanism [9]. However, this method is only azide to amine and subsequent standard coupling reaction
applicable to amino acids protected with acid resistant groups [18,19]. We also demonstrated that the selenocarboxylate/azide
such as Cbz and Pht. The use of phosphorus pentaoxide for amidation was highly chemoselective, mild, and free of ra-
peptide synthesis was also reported, but racemization could cemization in the coupling step [20]; was compatible with
occur under these reaction conditions [10,11]. Phosphorus common protecting groups used in peptide chemistry including
oxychloride was another condensing agent used to synthesize Fmoc, Boc, Cbz, and Trt; and more importantly, worked very
various N-protected aminoacyl-pNAs in yields between 70% efficiently for electron-deficient aromatic azides substituted
and 90% [12]. The proposed mechanism implies in situ acti- with an electron-withdrawing group [18-20]. We postulated that
vation of carboxylic acid by formation of the active mixed the selenocarboxylate/azide amidation could be a good solution
anhydride with phosphorodichloridic acid. Although the authors to the problem of synthesizing aminoacyl-pNAs and aminoacyl-
demonstrated that the synthesis was simple and free of ra- AMCs. Instead of p-nitroaniline and 7-amino-4-methyl-
cemization, this method required unfavorable anhydrous pyri- coumarin, p-nitrophenyl azide and 7-azido-4-methylcoumarin
dine as a solvent to suppress the potential removal of the Boc would be used to react with the selenocarboxylates derived
protecting group of amino acids. To increase nucleophilicity of from Nα-protected amino acids to form the desired amides. The
the aromatic amino group and hence improve the acylation azides can be readily prepared in excellent yields through diazo-
yield, p-(Boc-amino)aniline was used in place of p-nitroaniline tization of p-nitroaniline and 7-amino-4-methylcoumarin, fol-
as the starting material for amidation, followed by selective lowed by treatment with sodium azide, and, more importantly,
removal of the Boc protecting group and oxidation of the free the azides are stable and can be stored for a long period of time
amino group to the nitro group [13]. This method, however, is without significant degradation. More recently, we published a
not applicable to methionine, tyrosine, tryptophan and cysteine, facile procedure for the preparation of Nα-protected amino
all of which are sensitive to the oxidation conditions used. As selenocarboxylate that could be easily used in situ [21]. Herein,
an alternative, Nα-protected amino acids were used to condense we report a practical method for the preparation of aminoacyl-
with p-nitrophenyl isocyanate to form the Nα-protected pNA and aminoacyl-AMC derivatives through the selenocar-
aminoacyl-pNAs [14]. However, commercially available boxylate/azide amidation strategy, under aqueous or alcoholic
p-nitrophenyl isocyanate often contains impurities due to de- reaction conditions, with excellent yields.
gradation during storage; these impurities must be removed
before use. In addition, a significant amount of hydantoin Results and Discussion
by-product was found in the reaction mixture. Although the An efficient method was developed to synthesize aminoacyl-
addition of DMAP improved the amidation yields, the forma- pNAs and aminoacyl-AMCs in excellent yields as shown in Ta-
tion of hydantoin by-product also increased [15]. A modified ble 1 and Table 2, through the reaction of N-hydroxysuccin-
procedure using Curtius rearrangement with diphenyl phosphor- imide (HOSu)-activated esters or mixed anhydrides of
azidate (DPPA) was reported to minimize the side reaction, Nα-protected amino acids with sodium hydrogen selenide to
affording moderate to excellent yields (54–95%) of the desired form their corresponding Nα-protected amino selenocarboxy-
Nα-protected aminoacyl-pNAs [16]. However, only Boc late, followed by the coupling of the selenocarboxylate with
protected amino acids were used in the reported study, with p-nitrophenyl azide and 7-azido-4-methylcoumarin. When
three examples provided. The enzymatic synthesis of Nα-protected amino acid-OSu activated ester is not commer-
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cially available, the mixed anhydrides of Nα-protected amino mation of Nα-protected amino selenocarboxylates through
acids were prepared from Nα-protected amino acids in situ, their deprotonated molecular ions and their characteristic Se
prior to use. Nα-Protected amino acid-OSu esters or mixed isotopic patterns with LC-MS in ES− mode as previously
anhydrides were mixed with a stoichiometric amount of freshly reported [21].
prepared NaHSe [22] to generate the corresponding selenocar-
boxylates in situ, which were then reacted with azides to form As shown in Table 1, Cbz, Boc, Fmoc and Trt protecting groups
the corresponding Nα-protected aminoacyl-pNAs or were all well tolerated under our conditions, with OSu-acti-
Nα-protected aminoacyl-AMCs. The amount of amino seleno- vated esters of Nα-protected amino acids as the starting ma-
carboxylates generated was slightly in excess (1.2 equiv) rela- terial, and excellent yields of aminoacyl-pNAs were obtained.
tive to the azide used. The reaction was typically complete We also found that the hydroxy group of tyrosine and threonine
within 0.5 h at 0 °C for OSu-activated esters or −10 °C for did not need to be protected. No significant intermolecular
mixed anhydrides. For sterically hindered amino acid OSu esterification was observed under the present reaction condi-
esters, such as isoleucine, valine and threonine, however, the tions and the desired p-nitroanilides were obtained in excellent
reaction was slower and required room temperature to reach yields (Table 1, entries 2 and 3). Furthermore, we prepared an
completion. After the completion of selenocarboxylation as aqueous solution of NaHSe and treated it with Cbz-Gly-OSu in
monitored by LC-MS, a solution of azide in THF was added by 50% aqueous THF to produce Cbz-Gly-SeNa. The subsequent
syringe or cannulation. The reaction started immediately with amidation with p-nitrophenyl azide afforded the desired Cbz-
the precipitation of gray selenium and the formation of Gly-pNA (1) in 95% yield (Table 1, entry 1), indicating that the
nitrogen gas, and was complete within 2 h at room temperature. reaction could be carried out under aqueous conditions. When
Although it was difficult to characterize the selenocarboxylate Fmoc-Ile-OSu reacted with NaHSe, a higher reaction tempera-
intermediates using NMR due to their short half-life and their ture (room temperature) was required to complete the selenocar-
sensitivity to ambient oxygen, we were able to monitor the for- boxylation, suggesting the presence of steric hindrance.
Table 1: Synthesis of Nα-protected aminoacyl-pNAs and aminoacyl-AMCs through selenocarboxylation of Nα-protected amino acid OSu esters with
NaHSe, followed by reaction with the azides (Procedure A).
Entry
Amino acid-OSu ester
Product
Yield (%)a
1
2
Cbz-Gly-OSu
Cbz-Gly-pNA (1)
98 (95b)
90 (88b)
91c
Cbz-Tyr-OSu
Cbz-Tyr-pNA (2)
3
Fmoc-Thr-OSu
Fmoc-Ile-OSu
Boc-Phe-OSu
Fmoc-Met-OSu
Fmoc-Trp-OSu
Fmoc-His(Trt)-OSu
Cbz-Gly-OSu
Fmoc-Thr-pNA (3)
Fmoc-Ile-pNA (4)
Boc-Phe-pNA (5)
4
90c
5
91
6
Fmoc-Met-pNA (6)
Fmoc-Trp-pNA (7)
Fmoc-His(Trt)-pNA (8)
Cbz-Gly-AMC (9)
Cbz-Tyr-AMC (10)
Boc-Phe-AMC (11)
Fmoc-Met-AMC (12)
Fmoc-Trp-AMC (13)
Fmoc-His(Trt)-AMC (14)
92
7
94
8
91
9
92 (89b)
86 (84b)
86
10
11
12
13
14
Cbz-Tyr-OSu
Boc-Phe-OSu
Fmoc-Met-OSu
Fmoc-Trp-OSu
Fmoc-His(Trt)-OSu
82
82
82
Conditions: aNaHSe (1.2 equiv) and Nα-protected amino acid-OSu (1.2 equiv) in THF/2-PrOH at 0 °C, then the azide (1.0 equiv); bNaHSe (1.2 equiv)
and amino acid OSu (1.2 equiv) in THF/H2O at 0 °C, then the azide (1.0 equiv); cNaHSe (1.2 equiv) and Nα-protected amino acid-OSu (1.2 equiv) in
THF/2-PrOH at r.t., then the azide (1.0 equiv).
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Beilstein J. Org. Chem. 2011, 7, 1030–1035.
However, once the Fmoc-Ile-SeNa was formed, it quickly with NaHSe; and 3) the in situ generated selenocarboxylate
reacted with p-nitrophenyl azide to provide Fmoc-Ile-pNA (4) reacted immediately with the azide. Isopropyl chloroformate
in an excellent yield. This indicated that the steric hindrance of was chosen to activate amino acids because the corresponding
selenocarboxylates did not play a significant role in the mixed anhydrides were relatively more resistant to hydrolysis or
amidation step (Table 1, entry 4), which is consistent with our alcoholysis. As shown in Table 2, Boc-Gln-pNA (15) and Cbz-
previous observation that the rate of selenocarboxylate/azide Ser-pNA (17) were successfully synthesized in excellent yields
amidation primarily depended on the electronic properties of the of 91% and 86%, respectively, without the need to protect the
azide [20]. Furthermore, the indole unit of tryptophan did not side chain functional groups (Table 2, entries 1 and 3).
need protection and was well-tolerated without affecting the Nα-protected arginine pNAs are of special interest since they are
yield of the amidation reaction (Table 1, entry 7). This newly often the starting materials for the synthesis of chromogenic
developed selenocarboxylate/azide amidation strategy also substrates for serine proteases. Due to the presence of the guani-
provided easy access to aminoacyl-AMC conjugates. 7-Azido- dino group, the synthesis of Nα-protected Arg-pNA was espe-
4-methylcoumarin was readily prepared from 7-AMC by the cially challenging. We used Boc-Arg-OH∙HCl directly without
standard diazotization/sodium azide protocol in a yield of 92%. protecting the guanidino group and found that Boc-Arg-
Under the same reaction conditions described above, the OH∙HCl could be activated with isopropyl chloroformate and
amidation of 7-azido-4-methylcoumarin with various amino the activated form was converted smoothly to the corres-
selenocarboxylates gave the desired Nα-protected aminoacyl- ponding selenocarboxylate followed by the reaction with
AMCs in excellent yields (82–92%) (Table 1, entries 9 to 14). p-nitrophenyl azide to give the p-nitroanilide 16 in 81% yield
The yields were slightly lower than the yields of the corres- (Table 2, entry 2). A dipeptide-pNA, Boc-Ser-Phe-pNA (18),
ponding p-nitroanilides prepared under the same conditions. was also prepared in an excellent yield of 89% in the same
This is consistent with the previously observed trend that azides manner, indicating a potential use of this methodology in the
that were more electron-deficient gave better yields.
synthesis of chromogenic substrates with short peptides. The
same procedure was applied to synthesize aminoacyl-AMC
For amino acids whose OSu-activated esters are not commer- conjugates 19, 20 and 11 starting from Boc-Gln-OH, Boc-Arg-
cially available or difficult to prepare, we developed a 3-step OH∙HCl and Boc-Phe-OH in excellent yields (Table 2, entries
one-pot procedure (Procedure B) starting from Nα-protected 5–7). It should also be pointed out that we previously deter-
amino acids: 1) The amino acid was first activated to form the mined the extent of racemization in the one-pot 3-step seleno-
corresponding mixed anhydride; 2) the resulting mixed anhy- carboxylate/azide amidation procedure using acid hydrolysis of
dride was converted in situ to the selenocarboxylate by reaction the product amide with 6 N HCl followed by derivatization with
Table 2: The 3-step one-pot selenocarboxylate/azide amidation to synthesize Nα-protected aminoacyl-pNAs and aminoacyl-AMCs (Procedure B).
Entry
Amino acid
Product
Yield (%)a
1
2
3
4
5
6
7
Boc-Gln-OH
Boc-Gln-pNA (15)
Boc-Arg-pNA (16)
Cbz-Ser-pNA (17)
Boc-Ser-Phe-pNA (18)
Boc-Gln-AMC (19)
Boc-Arg-AMC (20)
Boc-Phe-AMC (11)
91
81
86
89
84
82
87
Boc-Arg-OH·HCl
Cbz-Ser-OH
Boc-Ser-Phe-OH
Boc-Gln-OH
Boc-Arg-OH·HCl
Boc-Phe-OH
Conditions: aNα-protected amino acid (1.2 equiv), isopropyl chloroformate (1.2 equiv) and N-methylpiperidine (1.2 equiv) in THF at −15 °C for 30 min;
isopropanolic NaHSe (1.2 equiv) solution at < −10 °C for additional 30 min; then the azide (1.0 equiv), r.t., 2 h.
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Beilstein J. Org. Chem. 2011, 7, 1030–1035.
o-phthalaldehyde/Nα-tert-butyloxycarbonyl-L-cysteine and ture. The mixture was stirred under a nitrogen atmosphere to
HPLC analysis, and we concluded that the obtained amide provide a colorless solution of NaHSe in isopropanol, which
product contained a small amount (1.7%) of the D-isomer was ready to be used.
resulting from the first step of carboxylate activation rather than
the last two steps of selenocarboxylate generation and Preparation of an aqueous solution of sodium hydrogense-
amidation [20].
lenide [22]. To a suspension of selenium (40 mg, 0.5 mmol) in
deaerated distilled water (5 mL) was added sodium borohy-
In summary, amino acyl conjugates of pNA and AMC were dride (40 mg, 1.0 mmol) in one portion at 5 °C. The mixture
readily synthesized through the selenocarboxylate/azide was vigorously stirred under a nitrogen atmosphere to provide a
amidation reaction. This method has many advantages, colorless aqueous solution of NaHSe, which was ready to be
including high yields, reproducibility, short reaction time, used.
mild reaction conditions, and compatibility with a range
of organic and aqueous solvents. Most importantly, the Synthesis of aminoacyl-pNAs and aminoacyl-AMCs starting
protecting groups commonly used in amino acid and peptide from amino acid-OSu esters (procedure A). In an ice-water
chemistry, such as Fmoc, Boc, Cbz, and Trt, are all well-toler- bath, to a 0.05 M isopropanolic or aqueous solution of NaHSe
ated under the present conditions. This selenocarboxylate/azide (0.5 mmol in 10 mL), freshly prepared as described above, was
amidation strategy represents a convenient and high-yield syn- added a solution of Nα-protected amino acid-OSu (0.5 mmol) in
thesis of Nα-protected-aminoacyl-pNAs/AMCs, providing easy THF (10 mL) by syringe. The resulting mixture was stirred at
access to these important synthons for the construction of 0–5 °C for 1 h under nitrogen atmosphere to afford the corres-
chromogenic/fluorogenic protease substrates through fragment ponding Nα-protected amino selenocarboxylate. Then, a solu-
condensation or stepwise elongation. The 3-step one pot proce- tion of the azide (0.42 mmol) in THF (2 mL) was added to the
dure was also successfully applied to the synthesis of a dipep- above solution of amino selenocarboxylate by a syringe. The
tide conjugate indicating that this methodology is applicable to reaction was carried out at room temperature under a nitrogen
the synthesis of chromogenic substrates containing short atmosphere for 2 h. The organic solvents were removed under
peptides.
reduced pressure, and the residue was suspended in a saturated
NaHCO3 aqueous solution followed by extraction with CH2Cl2.
The combined organic phase was washed with water and brine,
Experimental
General methods. Solvents were either ACS reagent grade or and dried over anhydrous Na2SO4. After removal of Na2SO4 by
HPLC grade and used directly without further purification. All filtration, the filtrate was concentrated to dryness. The crude
reaction mixtures were magnetically stirred and monitored by product was purified by flash column chromatography (FCC)
thin-layer chromatography (TLC) with polymer-backed F254 on silica gel. The physical and spectroscopic data of all amides
silica gel plates and/or LC-MS system. Flash column chroma- were consistent with their structures.
tography (FCC) was performed on a Teledyne ISCO Combi-
Flash Companion Automated Flash Chromatographic System Synthesis of aminoacyl-pNAs and aminoacyl-AMCs starting
with pre-packed silica gel columns. Yields were based on the from amino acids by activation with isopropyl chlorofor-
chromatographically pure compounds whose structures were mate (procedure B). To a solution of Nα-protected amino acid
assigned by NMR and mass spectral data. All 1H and 13C NMR (0.5 mmol) and N-methylpiperidine (61 µL, 0.5 mmol) in THF
spectra were recorded on a 200 MHz Varian or a 400 MHz (5 mL) was added a 1.0 M solution of isopropyl chloroformate
Bruker spectrometer at ambient temperature and calibrated in toluene (0.5 mL, 0.5 mmol) at –15 °C under a nitrogen
using residual undeuterated solvents as the internal reference. atmosphere. The resulting mixture was stirred for 20 min at
The LC-MS monitoring was performed on a Shimadzu LCMS –15 °C. Then, the resulting mixed anhydride solution was added
2010 system with Chromolith SpeedROD RP-18e column (50 × into the freshly prepared NaHSe (0.5 mmol) solution by cannu-
4.6 mm) at 1 mL/min with a 10-min gradient of 10–90% aceto- lation over a period of 5 min. The reaction mixture was stirred
nitrile containing 0.1% formic acid. High resolution mass spec- for an additional 30 min below –10 °C under a nitrogen atmos-
trometry (HRMS) was performed on a Finnagen LTQ OrbiTrap phere. Then, a solution of azide (0.42 mmol) in THF was added
mass spectrometer.
into the above selenocarboxylate solution by cannulation. The
reaction was carried out at room temperature under a nitrogen
Preparation of an isopropanol solution of sodium hydro- atmosphere for 2 h. The organic solvents were removed at
genselenide [22]. To a suspension of selenium (40 mg, reduced pressure, and the residue was suspended in a saturated
0.5 mmol) in deaerated isopropanol (5 mL) was added sodium NaHCO3 aqueous solution followed by extraction with CH2Cl2.
borohydride (24 mg, 0.6 mmol) in one portion at room tempera- The combined organic phase was washed with water and brine,
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Beilstein J. Org. Chem. 2011, 7, 1030–1035.
14.Nishi, N.; Tokura, S.; Noguchi, J. Bull. Chem. Soc. Jpn. 1970, 43,
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and dried over anhydrous Na2SO4. After removal of Na2SO4
through filtration, the filtrate was concentrated to dryness. The
crude product was purified by FCC on silica gel. Yields and
physical and spectroscopic data of all amides were consistent
with their structures.
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Supporting Information
18.Wu, X.; Hu, L. Tetrahedron Lett. 2005, 46, 8401–8405.
doi:10.1016/j.tetlet.2005.09.145
Analytical data and yields for compounds 1–20, among
which compounds 10 and 12–14 are new compounds.
19.Surabhi, P.; Wu, X.; Hu, L. Tetrahedron Lett. 2006, 47, 4609–4613.
doi:10.1016/j.tetlet.2006.04.144
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Supporting Information File 1
Analytical data and yields for compounds 1–20.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-7-117-S1.pdf]
21.Wu, X.; Chen, Y.; Hu, L. Tetrahedron Lett. 2009, 50, 5585–5588.
doi:10.1016/j.tetlet.2009.07.080
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Acknowledgments
We gratefully acknowledge the financial support of a research
grant from the State of New Jersey Commission on Cancer
Research and the Elsa U. Pardee Foundation, and a research
scholar grant from the American Cancer Society. We thank
Prof. Lawrence Williams, Prof. Spencer Knapp and Dr. Yiyu
Ge for helpful discussions.
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