Application of azide-alkyne cycloaddition 'click chemistry' for the synthesis of Grb2 SH2 domain-binding macrocycles

Article abstract of DOI:10.1016/j.bmcl.2006.08.004

Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, 'Pmp-Ac₆c-Asn', where Pmp and Ac₆c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1 mM substrate concentrations, cyclization of monomeric units occurred. At 2 mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a K_d value of 0.23 μM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of 'click chemistry' to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.

Full text of DOI:10.1016/j.bmcl.2006.08.004

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5265–5269
Application of azide–alkyne cycloaddition ‘click chemistry’ for the
synthesis of Grb2 SH2 domain-binding macrocycles
Won Jun Choi,a Zhen-Dan Shi,a Karen M. Worthy,b Lakshman Bindu,b
Rajeshri G. Karki,a Marc C. Nicklaus,a Robert J. Fisherb and Terrence R. Burke, Jr.a,*
aLaboratory of Medicinal Chemistry, CCR, NCI, NIH, Frederick, MD 21702, USA
bProtein Chemistry Laboratory, SAIC-Frederick, Frederick, MD 21702, USA
Received 31 May 2006; revised 31 July 2006; accepted 1 August 2006
Abstract—Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing
macrocycles based on the Grb2 SH2 domain-binding motif, ‘Pmp-Ac6c-Asn’, where Pmp and Ac6c stand for 4-phosphonomethyl-
phenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1 mM sub-
strate concentrations, cyclization of monomeric units occurred. At 2 mM substrate concentrations the predominant products
were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a Kd value
of 0.23 lM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain
dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of ‘click chemistry’
to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.
Ó 2006 Elsevier Ltd. All rights reserved.
The growth factor receptor-bound protein 2 (Grb2) is an
SH2 domain-containing signal transducer that repre-
sents an attractive therapeutic target.1 For Grb2 SH2
domains, where open-chain ‘pTyr-Xxx-Asn’ sequences
are preferentially recognized in type-I b-turn conforma-
tions, induction of turn geometries through macrocycli-
zation using ring-closing olefin metathesis2 (RCM) has
resulted in a number of potent binding inhibitors.3 Re-
cent reports have highlighted the growing utility in drug
discovery of copper (I) promoted [3+2] Huisgen cyc-
loadditions of azides with terminal alkynes, which is
one example of ‘click chemistry’.4 This led us to examine
the potential usefulness of this approach to prepare
Grb2 SH2 domain-binding antagonists, including mac-
rocyclic inhibitors. To date few reports of ring closure
using azide–alkyne click chemistry have appeared for
peptides or peptide mimetics.5 Therefore, the current
study was undertaken to examine click chemistry in
the synthesis of Grb2 SH2 domain-binding inhibitors.
Macrocyclization using ring-closing [1,3] dipolar cyclo-
addition was undertaken using the protected monomeric
sequences 8 that contain the tripeptide motif, ‘Pmp-
Ac6c-Asn-amide’ (Scheme 1). (Here Pmp represents the
phosphatase-stable pTyr mimetic 4-phosphonomethyl-
phenylalanine6 and Ac6c stands for 1-aminocyclohexan-
ecarboxylic acid7). Preparation of 8 began with the
known protected amine 1, which was obtained by Mits-
unobu coupling of phthalimide with commercially avail-
able 4-pentyn-1-ol.8 Direct hydrazinolysis of 1 to yield
1-amino-4-pentyne had previously been reported to fail
or to require tedious ion exchange chromatographic
workup if an alternate reductive phthalimide cleavage
procedure was used.9 However, we were able to employ
1 as a source of 1-amino-4-pentyne by reacting this
material with hydrazine in aqueous EtOH (reflux,
2 h),10 then cooling the reaction mixture to room tem-
perature, filtering, and concentrating the volatile prod-
uct. Without further purification the crude amine was
coupled directly with N-Boc L-Asn using active ester
protocols [1-hydroxybenzotriazole (HOBT) and 1-(3-
dimethylaminopropyl)-3-ethyl carbodiimide hydrochlo-
ride (EDC)]. This provided the N-Boc-Asn[1-(5-penty-
nyl)] amide 2 in 48% yield from 1. Conversion of 2 to
the N-Fmoc protected dipeptide 4 required initial
TFA-mediated N-Boc deprotection followed by neutral-
ization and chromatographic purification. Active ester
Keywords: Macrocycle; Click chemistry; Grb2 SH2 domain; Cycload-
dition; Peptide mimetic.
*
Corresponding author. Tel.: +1 301 846 5906; fax: +1 301 846
6033; e-mail: tburke@helix.nih.gov
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.004

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W. J. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5265–5269
O
O
O
CONH2
O
N
H
N
CONH2
iii
N
H
HN
N
i
O
H
R
4
2 R = Boc
3 R = H
1
ii
Fmoc
N
H
O
O
O
O
P
OH
Fmoc
CONH2
O
N
H
ButO
CONH2
N
H
N
HN
5
ButO
HN
H
O
O
O
H
vi
viii
iv
N
N
H
N3
N
H
*
*
O
N
OBut
OBut
O
P
H
R
P
OR
OR
O
8a (*S), 8b (*R); R = But
9a (*S); R = H
6 (*R/S); R = Fmoc
7a (*S), 7b (*R); R = H
vii
v
O
OR
RO
O
N
P
CONH2
O
N
H
N
N
O
CONH2
O
HN
O
H
N
H
N
O
N
HN
*
H
N
O
N
N
N
H
+
H
N
N
*
N
O
H
N
N
O
O
NH
*
N
H
H
O
H2NOC
N
O
OR
P
OR
P
O
OR
O
OR
11a (*S), 11b (*R); R = But
13a (*S), 13b (*R); R = H
10a (*S), 10b (*R); R = But
12a (*S), 12b (*R); R = H
ix
ix
Scheme 1. Reagents and conditions: (i) a—N2H4ÆH2O, EtOH, H2O, reflux, 2 h; b—Boc-Asn-OH, EDC, HOBt, DMF, rt, 12 h (48% yield for two
steps); (ii) a—TFA, CH2Cl2, rt, 1 h (95% yield); (iii) a—Fmoc-1-amino-cyclohexanecarboxylic acid, EDC, HOBt, DMF, rt, 12 h (84% yield); (iv) a—
piperidine, CH3CN, rt, 2 h; b—EDCIÆHCl, HOBt, DMF, rt, 12 h (39% yield for two steps); (v) piperidine, CH3CN, rt, 2 h (37% yield for 7a; 38%
yield for 7b); (vi) a—(BrCH2CO)2O DMF, rt, 1 h; b—NaN3, DMF, rt, 24 h (two steps, 81% yield for 8a, 62% yield for 8b); (vii) TFA–HS(CH2)2SH–
H2O, rt, 1 h (69% yield); (viii) CuI, L-ascorbate, DIPEA, CH3CN/tBuOH/H2O, rt; (ix) TFA–HS(CH2)2SH–H2O, rt, 1 h.
coupling of the resulting free amine with N-Fmoc Ac6c
provided 4 in 80% yield from 2. Removal of N-Fmoc
protection followed by coupling with 4-[bis(tert-bu-
tyl)phosphonomethyl]-N-Fmoc-D,L-phenylalanine ([N-
Fmoc-D,L-(OBut)2Pmp], 5)11 gave the tripeptide 6 as
an inseparable mixture of (R/S) epimers at the Pmp a-
carbon. Following N-Fmoc removal, the epimeric free
amines could be separated by flash chromatography to
yield the (S)-Pmp- and (R)-Pmp-containing diastereo-
mers 7a and 7b, respectively. Assignment of relative con-
figurations was done by comparison with an authentic
sample of (S)-Pmp-containing tripeptide (corresponding
to the faster eluting material) prepared using enantio-
merically pure N-Fmoc-L-(OBut)2Pmp.12 Treatment of
7a and 7b with bromoacetic anhydride, followed after
1 h by the addition of sodium azide, yielded the cycload-
dition precursors 8a and 8b in 81% yield and 62% yield,
respectively.
tion reaction was conducted using a substrate concen-
tration of 2 mM, the expected monomeric cyclization
product 10 was obtained only as a minor component.
The major product under these conditions proved to
be the dimeric macrocycle 11 (26% yield for 11a, 56%
yield for 11b). The dimerization reaction was highly con-
centration dependent, since repeating the procedure at
1 mM substrate concentration reversed the product ra-
tios and provided monomeric 10 (29% yield for 10a
and 19% yield for 10b) as the predominant product with
dimeric 11 appearing as a minor component. Similar
peptide cyclodimerization by copper-catalyzed azide–al-
kyne cycloaddition has recently been reported.5a Cleav-
age of the phosphonate tert-butyl esters (TFA–
ethanedithiol–H2O) and purification by HPLC gave
the final monomeric (12) and dimeric (13) products,
respectively.
For comparison purposes the (S)-Pmp-containing open-
chain monomer (9a) (Scheme 1) and dimer (18) (Scheme
2) were prepared. In the latter case, starting from the
(S)-Pmp-containing tripeptide 7a, cycloaddition precur-
sors 15 and 16 were synthesized bearing C-terminal
alkyne and N-terminal azide functionality, respectively.
Performing [3+2] cycloaddition of 15 and 16 using CuI
Initial attempts at [1,3] Huisgen cycloaddition using CuI
and L-ascorbate13 proceeded poorly and yielded mainly
unreacted starting material. However, addition of N,N-
diisopropylethylamine (DIEA) significantly facilitated
the reaction, apparently by promoting formation of
the copper-(I)-alkyne complex.14 When the cycloaddi-

W. J. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5265–5269
5267
O
O
CONH2
O
N
CONH2
O
N
H
H
i
HN
HN
O
O
O
P
O
P
N
H
N
ButO
ButO
H
NH2
NH2
14
ButO
ButO
7a
ii
iii
O
O
CONH2
N
CONH2
N
H
H
HN
O
HN
O
O
O
H
N
H
N
N
H
N3
N
H
O
O
OBut
OBut
OBut
OBut
P
P
O
O
16
15
iv
RO
RO
O
P
O
O
H
N
CONH2
O
N
N
H
H
O
HN
O
NH
O
H
H
N
N
H2NOC
N
N
N
H
N
O
O
17; R = But
18; R = H
v
OR
P
O
OR
Scheme 2. Reagents and conditions: (i) Pd/C, H2, EtOAc; (ii) a—(BrCH2CO)2O DMF, rt, 1 h; b—NaN3, DMF, rt, 24 h (93% yield from 7a); (iii)
1-acetylimidazole, DMF, 24 h (quantitative); (iv) CuI, L-ascorbate, DIPEA, CH3CN/tBuOH/H2O, rt (73% yield); (v) TFA–HS(CH2)2SH–H2O, rt,
1 h (76% yield).
and L-ascorbate in the presence of DIEA gave the pro-
tected product 17, which was converted to 18.
O
NH2
N
H
O
O
Evaluation of Grb2 SH2 domain-binding affinities was
conducted using surface plasmon resonance (SPR) as
previously described.15 A key feature of this technique
was its use of Biacore S51 instrumentation, which
allowed direct measurement of synthetic peptide binding
to surface-bound Grb2 SH2 domain protein. In similar
studies conducted on the reference macrocycle 19
(Fig. 1) using a Biacore 3000 instrument, close agree-
ment was observed between the SPR binding affinity
(Kd = 0.9 nM) and affinity determined by ELISA meth-
ods (IC50 = 1.4 nM).3d
HN
O
O
N
H
HO
P
CO2H
19
OH
Figure 1. Structure of reference macrocycle examined by both SPR
and ELISA binding experiments.
Table 1. Grb2 SH2 domain-binding affinities of synthetic peptides
a
Compound
Binding affinity Kd (lM)
As shown in Table 1, the (R)-Pmp-containing monomer-
ic and dimeric macrocycles (12b and 13b, respectively)
exhibited binding constants above 1 lM. However, the
monomeric (S)-Pmp-containing macrocycle (12a)16
bound with sub-micromolar affinity. Of particular note
were the affinities of the dimeric (S)-Pmp-containing
peptides, which provided low nanomolar Kd values for
9a
>1
0.23
12a
12b
13a
13b
18
>1
Kd1 = 0.0018; Kd2 = 0.0040
>1
Kd1 = 0.0011; Kd2 = 0.087
a Values were determined as described in Ref. 15.

5268
W. J. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5265–5269
both the macrocyclic (13a)17 and open-chain (18)18
forms. These high affinities were somewhat unexpected
based on the low affinity of the open-chain precursor
9a (Kd > 1 lM).
The binding kinetics of 13a and 18 were complex and
consistent with multiple binding components. The qual-
ity of the Biacore data was very high and this was not
felt to be an artifact of the data. Multiple binding com-
ponents have been reported previously for Grb2 SH2
domain-binding macrocycles, however the physical
interpretation is not clear.19 Fitting data to a two-com-
ponent system (Fig. 2) provided the Kd1 and Kd2 values
shown in Table 1.
High Grb2 SH2 domain-binding affinity has been
reported previously for a peptide containing two pTyr
mimetic residues.20 However, in this latter case both res-
idues were situated in the proximal pTyr and pTyr+1
positions with the pTyr+1 residue participating in a
non-canonical interaction with the Grb2 Arg142.21
Molecular modeling indicates that this type of interac-
tion is not possible for dimeric peptides 13a and 18
(Fig. 3).
Peptides based on immunoreceptor tyrosine activation
motifs (ITAMs) also present tandem pTyr-containing
Figure 3. Potential Grb2 SH2 domain-binding interactions of syn-
thetic ligands as determined by molecular modeling. (A) Dimeric
macrocycle 13a; (B) open-chain dimer 18.
motifs that bind with high affinity to targets such as the
ZAP-70 tyrosine kinase that contain multiple SH2 do-
mains. X-ray analysis has confirmed that this binding in-
volves simultaneous interaction of each pTyr-containing
motif with a separate SH2 domain.22 To our knowledge,
peptides 13a and18are among the first examples of dimer-
ic pTyr-mimetic-containing peptides that show high affin-
ity against a single SH2 domain construct. The structural
basis for the more than two orders of magnitude affinity
enhancement in going from the monomer 9a to the dimers
13a and 18 is not apparent from docking studies with the
isolated Grb2 SH2 domain.
Reported herein is the first application of [3+2] Huisgen
cycloaddition click chemistry for the synthesis of SH2
domain-binding peptides. By appending azide/alkyne
functionality to the N- and C-termini, respectively, mac-
rocyclization could be effected in monomeric or dimeric
fashion depending on substrate concentration. Open-
chain dimeric analogues could also be prepared by react-
ing monomers that contained a single azide/alkyne
group each. Although the open-chain (S)-Pmp-contain-
ing monomer exhibited very low binding affinity, dimer-
ic analogues based on the same motif bound with very
Figure 2. Representative SPR data for 13a and 18 showing response in
RU units versus time. Traces in black are for 2-fold dilutions of
inhibitor from 62 to 0.98 nM. Curve fitting for two-component models
is shown in red.

W. J. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5265–5269
5269
J. Org. Chem. 2005, 70, 9595; (d) Bock, V. D.; Perciacc-
ante, R.; Jansen, T. P.; Hiemstra, H.; van Maarseveen, J.
H. Org. Lett. 2006, 5, 919.
high affinity and displayed complex kinetics. These re-
sults support the potential utility of click chemistries
for the preparation of novel SH2 domain-binding
antagonists.
6. Marseigne, I.; Roques, B. P. J. Org. Chem. 1988, 53, 3621.
7. Garcia-Echeverria, C.; Gay, B.; Rahuel, J.; Furet, P.
Bioorg. Med. Chem. Lett. 1999, 9, 2915.
8. Tiecco, M.; Testaferri, L.; Temperini, A.; Bagnoli, L.;
Marini, F.; Santi, C.; Terlizzi, R. Eur. J. Org. Chem. 2004,
3447.
Acknowledgments
The authors thank Drs. James A. Kelley and Christo-
pher Lai of the Laboratory of Medicinal Chemistry,
NCI, for mass spectral data. This research was support-
ed in part by the Intramural Research Program of the
NIH, Center for Cancer Research, NCI-Frederick. This
project has been funded in whole or in part with federal
funds from the National Cancer Institute, National
Institutes of Health, under contract N01-CO-12400.
The content of this publication does not necessarily re-
flect the views or policies of the Department of Health
and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorse-
ment by the US Government.
9. Bellier, B.; Dugave, C.; Etivant, F.; Genet, R.; Gigoux, V.;
Garbay, C. Bioorg. Med. Chem. Lett. 2004, 14, 369.
10. L’abbe, G.; Leurs, S.; Sannen, I.; Dehaen, W. Tetrahedron
1993, 49, 4439.
11. Burke, T. R., Jr.; Russ, P.; Lim, B. Synthesis 1991, 11, 1019.
12. Li, P.; Zhang, M.; Peach, M. L.; Liu, H.; Yang, D.; Roller,
P. P. Org. Lett. 2003, 5, 3095.
13. Rostovtsev, V. V.; Green, L. G.; fokin, V. V.; Sharpless,
K. B. Angew. Chem. Int. Ed. 2002, 41, 2596.
14. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrhedron
Lett. 1975, 16, 4467.
15. Oishi, S.; Karki, R. G.; Shi, Z.-D.; Worthy, K. M.; Bindu,
L.; Chertov, O.; Esposito, D.; Frank, P.; Gillette, W. K.;
Maderia, M. A.; Hartley, J.; Nicklaus, M. C.; Barchi, J. J.,
Jr.; Fisher, R. J.; Burke, T. R., Jr. Bioorg. Med. Chem.
2005, 13, 2431.
16. Compound 12a 1H NMR (CD3OD) d 8.21 (s, 1H), 8.00 (d,
J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.68 (d, J = 4.8 Hz, 1H), 7.21
(dd, J = 8.0 and 2.4 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 5.18
(d, J = 16.0 Hz, 1H), 4.99 (d, J = 16.0 Hz, 1H), 4.66 (m,
1H), 4.31(t, J = 6.4 Hz, 1H), 3.33 (m, 1H), 3.05 (d,
J = 21.6 Hz, 2H), 3.06–2.94 (m, 2H), 2.73 (t, J = 6.0 Hz,
2H), 2.51 (dd, J = 15.6 and 7.2 Hz, 1H), 2.39 (dd, J = 15.6
and 5.6 Hz, 1H), 2.03–1.90 (m, 3H), 1.70 (m, 2H), 1.58 (m,
1H), 1.48–1.32 (m, 4H), 1.15 (m, 1H), 0.79 (m, 1H).
MALDI-MS (+ve) m/z: 647 [MH+].
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.08.004.
References and notes
17. Compound 13a 1H NMR (400 MHz, d6-DMSO) d 8.70 (d,
1H, J = 7.8 Hz), 8.54 (s, 1H), 7.97 (d, 1H, J = 8.0 Hz), 7.45
(s, 1H), 7.42–7.39 (m, 2H), 7.22–7.16 (m, 4H), 6.96 (s, 1H),
4.95 (d, 1H, J = 16.0 Hz), 4.80 (d, 1H, J = 16.2 Hz), 4.75
(m, 1H), 4.36 (dd, 1H, J = 5.4 Hz and 13.2 Hz), 3.23–3.16
(m, 2H), 3.05 (m, 1H), 2.95 (dd, 1H, J = 6.5 Hz and
21.2 Hz), 2.76–2.66 (m, 2 H), 2.58 (t, 2H, J = 7.5 Hz), 2.43
(dd, 1H, J = 4.7 Hz and 15.6 Hz), 2.02–1.14 (m, 12H).
FABMS (-ve) m/z 1291.8 [(MÀH)À].
1. (a) Feller, S. M.; Lewitzky, M. Curr. Pharm. Des. 2006, 12,
529; (b) Dharmawardana, P. G.; Peruzzi, B.; Giubellino,
A., ; Burke, T. R., Jr.; Bottaro, D. P. Anti-Cancer Drugs
2006, 17, 13.
2. Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. J. Am.
Chem. Soc. 1996, 118, 9606.
3. (a) Gao, Y.; Wei, C.-Q.; Burke, T. R., Jr. Org. Lett. 2001,
3, 1617; (b) Wei, C.-Q.; Gao, Y.; Lee, K.; Guo, R.; Li, B.;
Zhang, M.; Yang, D.; Burke, T. R., Jr. J. Med. Chem.
2003, 46, 244; (c) Dekker, F. J.; de Mol, N. J.; Fischer, M.
J. E.; Kemmink, J.; Liskamp, R. M. J. Org. Biomol. Chem.
2003, 1, 3297; (d) Shi, Z.-D.; Wei, C.-Q.; Lee, K.; Liu, H.;
Zhang, M.; Araki, T.; Roberts, L. R.; Worthy, K. M.;
Fisher, R. J.; Neel, B. G.; Kelley, J. A.; Yang, D.; Burke,
T. R., Jr. J. Med. Chem. 2004, 47, 2166; (e) Shi, Z.-D.; Lee,
K.; Wei, C.-Q.; Roberts, L. R.; Worthy, K. M.; Fisher, R.
J., ; Burke, T. R., Jr. J. Med. Chem. 2004, 47, 788; (f)
Oishi, S.; Karki, R. G.; Kang, S.-U.; Wang, X.; Worthy,
K. M.; Bindu, L. K.; Nicklaus, M. C.; Fisher, R. J.; Burke,
T. R., Jr. J. Med. Chem. 2005, 48, 764; (g) Shi, Z.-D.;
Karki, R. G.; Worthy, K. M.; Bindu, L. K.; Fisher, R. J.;
Burke, T. R., Jr. Chem. Biodiversity 2005, 2, 447; (h)
Burke, T. R., Jr. Int. J. Pept. Res. Ther. 2006, 12, 33.
4. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003,
8, 1128.
18. Compound 8 1H NMR (400 MHz, CD3OD) d 8.06 (d, 1H
J = 15.2 Hz), 7.69 (s, 1H), 7.24–7.14 (m, 9H), 5.00 (dd, 2H,
J = 45.6 and 16.0 Hz), 4.63 (m, 2H), 4.49 (dd, 1H, J = 6.4
and 5.2 Hz), 4.41 (t, 1H, J = 6.0 Hz), 3.21–3.02 (m, 5H),
3.05 (d, 4H, J = 21.2 Hz), 2.93 (dd, 1H, J = 14.0 and
9.2 Hz), 2.85–2.63 (m, 6H), 1.84 (s, 3H), 2.00–1.66 (m,
8H), 1.52–1.06 (m, 18H), 0.85 (t, 3H, J = 7.2 Hz). FABMS
(-ve) m/z 1254.5 [(MÀH)À].
19. Oishi, S.; Shi, Z.-D.; Worthy, K. M.; Bindu, L. K.; Fisher,
R. J.; Burke, T. R., Jr. ChemBioChem 2005, 6, 668.
20. Liu, W. Q.; Vidal, M.; Gresh, N.; Rogues, B. P.; Garbay,
C. J. Med. Chem. 1999, 42, 3737.
21. Nioche, P.; Liu, W.-Q.; Broutin, I.; Charbonnier, F.;
Latreille, M.-T.; Vidal, M.; Roques, B.; Garbay, C.;
Ducruix, A. J. Mol. Biol. 2002, 315, 1167.
22. Hatada, M. H.; Lu, X.; Laird, E. R.; Green, J.; Morgen-
stern, J. P.; Lou, M.; Marr, C. S.; Phillips, T. B.; Ram, M.
K.; Theriault, K.; Zoller, M. J.; Karas, J. L. Nature 1995,
377, 32.
5. (a) Punna, S.; Kuzelka, J.; Wang, Q.; Finn, M. G. Angew.
Chem. Int. Ed. 2005, 44, 2215; (b) Billing, J. F.; Nilsson, U.
J. J. Org. Chem. 2005, 70, 4847; (c) Angell, Y.; Burgess, K.