Characteristics of an RNA Diels-Alderase active site

Article abstract of DOI:10.1021/ja983989m

The active site components and substrate specificity of an RNA Diels- Alderase (DA22) were investigated. Diels-Alderase activity was found to be highly dependent on a unique 5-position pyridyl modified uridine. Even closely related pyridyl modifications failed to yield active catalysts. Substrate specificity of this Diels-Alderase was remarkable. Experiments with alternative diene and dienophile substrates showed the active site of the Diels-Alderase to be highly discriminating, even against molecules of similar reactivity and structure. Inhibition studies with a series of product analogues established that the RNA recognizes functional components in and around the reaction center of both the diene and the dienophile. Taken together, these results suggest that DA22 can fold into a structure that produces an intricate metal/ligand dependent active site capable of highly specific molecular recognition.

Full text of DOI:10.1021/ja983989m

3614
J. Am. Chem. Soc. 1999, 121, 3614-3617
Characteristics of an RNA Diels-Alderase Active Site
Theodore M. Tarasow, Sandra L. Tarasow, Chi Tu, Elizabeth Kellogg, and
Bruce E. Eaton*
Contribution from the Chemistry DiVision, NeXstar Pharmaceuticals, Inc., 2860 Wilderness Place,
Boulder, Colorado 80301
ReceiVed NoVember 19, 1998
Abstract: The active site components and substrate specificity of an RNA Diels-Alderase (DA22) were
investigated. Diels-Alderase activity was found to be highly dependent on a unique 5-position pyridyl modified
uridine. Even closely related pyridyl modifications failed to yield active catalysts. Substrate specificity of this
Diels-Alderase was remarkable. Experiments with alternative diene and dienophile substrates showed the
active site of the Diels-Alderase to be highly discriminating, even against molecules of similar reactivity and
structure. Inhibition studies with a series of product analogues established that the RNA recognizes functional
components in and around the reaction center of both the diene and the dienophile. Taken together, these
results suggest that DA22 can fold into a structure that produces an intricate metal/ligand dependent active
site capable of highly specific molecular recognition.
Introduction
Alderase represents a new mode of catalysis for this important
class of cycloaddition reaction.7 Understanding the specific
requirements of the catalyst and substrates will reveal important
new insights and could enhance our understanding of the
mechanistic possiblities for this [4 + 2] cycloaddition.8
Considerable effort has been devoted to the discovery of new
catalysts for Diels-Alder cycloadditions, with the primary
objective being to enhance reactivity or control selectivity.9
From a biological perspective, a key feature in any enzymatic
process is substrate selectivity. For a Diels-Alderase, selectivity
could be observed for either the diene or the dienophile and
ideally both. Even with the immense amount of Diels-Alder
catalyst research, on either macromolecules or small molecules,
discrimination between dienophile substrates of similar reactivity
has rarely been observed. Herein we describe the first examples
of substrate selectivity by an RNA Diels-Alderase and the
results from probing the active site through inhibition with a
series of product analogues. In addition, the importance of the
RNA modification10 was investigated through either molecular
replacement or rearrangement of the nitrogen contained in the
pyridyl modified uridines.
Among the most powerful of all synthetic organic transfor-
mations is the Diels-Alder [4 + 2] cycloaddition.1 Despite the
success of employing the Diels-Alder reaction to prepare
biologically active carbocycles,2 no naturally occurring Diels-
Alderases have been isolated and fully characterized.3 Recently,
our group reported on the in vitro selection and characterization
of the first RNA Diels-Alderase.4 Unlike any of the known
Diels-Alderase catalytic antibodies,5 or any other known RNA
catalyst,6 the RNA Diels-Alderase showed a specific require-
ment for cupric ion and pyridyl substituents.4 This Diels-
(1) Corey, E. J.; Cheng, X.-M. In The Logic of Chemical Synthesis;
Wiley: New York, 1989; pp 18-23.
(2) For some current examples, see: (a) Roush, W. R.; Sciotti, R. J. J.
Am. Chem. Soc. 1998, 120, 7411-7419. (b) Corey, E. J.; Letavic, M. A. J.
Am. Chem. Soc. 1995, 117, 9616-9617. (c) Corey, E. J.; Guzman-Perez,
A.; Loh, T- P. J. Am. Chem. Soc. 1994, 116, 3611-3612. (c) Kozmin, S.
A.; Rawal, V. H. J. Am. Chem. Soc. 1997, 119, 7165-7166.
(3) Laschat, S. Angew. Chem., Int. Ed. Engl. 1996, 35, 289-291.
(4) Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54-
57.
(5) (a) Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M. T. M.; J. Am.
Chem. Soc. 1989, 111, 9261-9262. (b) Braisted, A. C.; Schultz, P. G. J.
Am. Chem. Soc. 1990, 112, 7430-7431. (c) Suckling, C. J.; Tedford, M.
C.; Bence, L. M. Bioorg. Med. Chem. Lett. 1992, 2, 49-52. (d) Gouverneur,
V. E.; Houk, K. N.; de Pascual-Teresa, B.; Beno, B.; Janda, K. D.; Lerner,
R. A. Science 1993, 262, 204-208. (e) Yli-Kauhaluoma, J. T.; Ashley, J.
A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc.
1995, 117, 7042-7047.
Results and Discussion
The discovery of RNA Diels-Alderases came from in vitro
selection4 on large libraries (ca. 1014 sequences) of 5-(4-
methylpyridylcarboxamide)uridine10c modified RNA. Isolated
sequence families were identified that catalyze the Diels-Alder
cycloaddition shown in Scheme 1. One RNA from the largest
family of Diels-Alderases, DA22, was characterized in detail
(6) One example of a DNA ligase where Cu2+ could be substituted by
Zn2+ is given in: Cuenoud, B.; Szostak, J. W. Nature 1995, 375, 611-
614.
(7) Examples of pyridyl-cupric ion catalysis of a Diels-Alder reaction
have only been observed previously for dieneophiles with appended pyridyl
groups. Otto, S.; Bertoncin, F.; Engberts, J. B. F. N. J. Am. Chem. Soc.
1996, 118, 7702-7707.
(8) (a) Heine, A.; Stura, E. A.; Yli-Kauhaluoma, J. T.; Gao, C.; Deng,
Q.; Beno, B. R.; Houk, K. N.; Janda, K. D.; Wilson, I. A. Science 1998,
279, 1934-1939. (b) Houk, K. N.; Gonzalez, J.; Li, Y. Acc. Chem. Res.
1995, 28, 8, 81-90. (c) Breslow, R.; Zhu, Z. J. Am. Chem. Soc. 1995, 117,
9923-9924. (d) Otto, S.; Blokzijl, W.; Engberts, J. B. F. N. J. Org. Chem.
1994, 59, 5372-5376. (e) Blake, J. F.; Lim, D.; Jorgensen, W. L. J. Org.
Chem. 1994, 59, 803-805. (f) Breslow, R. Acc. Chem. Res. 1991, 24, 159-
164. (g) Grieco, P. A.; Garner, P.; He, Z.-M. Tetrahedron Lett. 1983, 24,
1897-1900.
(9) (a) Garner, P. P. In Organic Synthesis in Water, Diels-Alder
Reactions in Aqueous Media; Grieco, P. A., Ed.; Thomson Science: New
York, 1998; pp 1-42. (b) Pindur, U.; Lutz, G.; Otto, C. Chem. ReV. 1993,
93, 741-761. Oppolzer, W. In ComprehensiVe Organic Synthesis; Trost,
B. M., Ed.; Pergamon Press: Oxford, U.K., 1991; Vol. 5.
(10) (a) Tarasow, T. M.; Eaton, B. E. Biopolymers 1998, 48, 29-37.
(b) Eaton B. E. Curr. Opin. Chem. Biol. 1997, 1, 10-16. (c) Dewey, T.
M.; Zyzniewski, C.; Eaton, B. E. Nucleosides Nucleotides 1996, 15, 1611-
1617. (d) Dewey, T. M.; Mundt, A. A.; Crouch, G. J.; Zyzniewski, M. C.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 8474-8475. (e) Eaton, B. E.;
Pieken, W. A. Annu. ReV. Biochem. 1995, 64, 837-863.
10.1021/ja983989m CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/01/1999

Characteristics of an RNA Diels-Alderase ActiVe Site
J. Am. Chem. Soc., Vol. 121, No. 15, 1999 3615
Scheme 1
and demonstrated a rate acceleration of 800-fold over the
spontaneous reaction rate and an absolute dependence on the
presence of cupric ion. The absolute dependence of catalytic
activity on the presence of copper and the inability of isostruc-
tural metals to substitute for copper are highly suggestive of a
Lewis acid role for the bound copper in DA22.11 The DA22
sequence was also found to require the 5-(4-methylpyridylcar-
boxamide)uridine modification (4 of Scheme 1) for catalytic
activity.4
Table 1a
Pyridine Replacement and Rearrangement. To further
define the structural requirements for catalytic activity and to
establish the importance of the modified uridine, molecular
replacement experiments were conducted. The DA22 sequence
was transcribed12 with each of the alternative UTP’s shown in
Scheme 1 (3, 5-7) and the activity of each sequence evaluated
(see the Experimental Section for details). The DA22 sequence
transcribed with UTP 7 demonstrated no Diels-Alderase
activity. This is not surprising given the drastic loss of
functionality, both in hydrogen bonding and metal coordination,
relative to the selected Diels-Alderase. However, transcription
with the benzyl amide modified UTP 3 also gave RNA that
was catalytically inactive. The functionality of 3 is identical to
that of 4, except the pyridyl heterocyclic N is replaced by the
phenyl C-H. This result establishes an important role for the
pyridine in catalytic activity. Further, the precise orientation of
the pyridine nitrogen relative to the uridine ring was shown to
be critical for activity, as shown by the inactivity of the
sequences transcribed with either the 2- or the 3-methylpyridyl
modified UTP’s (5 and 6). When considered in the context of
the copper-dependent activity, these results suggest that DA22
has a unique cupric ion binding site comprised of one or more
pyridine ligands, which could serve as a Lewis acid to catalyze
the Diels-Alder cycloaddition. It appears that this RNA Diels-
Alderase can form a unique metal coordination geometry at the
active site; however, these results do not reveal if specific
substrate binding pockets are formed.
a Asterisk (*) indicates the values are background corrected and are
relative to the total amount of active catalyst. Dagger (‡) indicates values
below the assay detection limits.
Substrate Specificity. To explore the Diels-Alderase 22
(DA22) active site, the relative catalytic activities for alternative
(11) Cotton, F. A.; Wilkinson, G. In AdVanced Inorganic Chemistry, 5th
ed.; Wiley: New York, 1988.
(12) Wiegand, T. W.; Janssen, R. C.; Eaton, B. E. Chem. Biol. 1997, 4,
675-683.
substrates were determined as shown in Table 1 (1, 8-12).
Dienophile specificity was investigated using the acyclic diene
2, while activity in the prescence of the alternative diene 12

3616 J. Am. Chem. Soc., Vol. 121, No. 15, 1999
Tarasow et al.
Table 2
to measure the other inhibitors in the series. The minimal
product, 14, which only presented the core functionality of the
diene and dienophile, displayed the weakest inhibition (Kiapp
)
2.3 mM). As functional groups from both the diene and the
dienophile were restored, inhibition of DA22 catalysis increased,
as demonstrated by the Kiapp values for compounds 15 and 16
(Table 2). This trend clearly demonstrates that the RNA
recognizes functional group components of both the diene and
the dienophile. Furthermore, there is only a 2-fold difference
between the inhibition constants measured for 13 and 16,
suggesting that the functionality remote to the cyclohexane,
including the biotin substituent, does not contribute significantly
to the dienophile substrate binding. The product inhibition data,
in combination with the substrate specificity observed for DA22,
demonstrate that the active site recognizes substrate structure
surrounding the [4 + 2] reaction center, as well as attached
functionality.
Conclusions
It is now known that RNA Diels-Alderase 22 requires
specific pyridyl-uridine modifications to the extent that even
rearrangement of the pyridine nitrogen results in complete loss
of catalytic activity. These modifications are proposed to provide
unique cupric ion coordination environments. DA22 is an
intricate macromolecular catalyst that depends on the precise
presentation of its own functional groups for Diels-Alderase
activity. Furthermore, the active site created by this modified
RNA catalyst dictates precise interactions with both substrates,15
ultimately leading to high substrate selectivity.
This is the first example of RNA exhibiting substrate
selectivity in a bimolecular transformation of small molecule
substrates. Taken together, these results indicate that DA22 can
fold in such a way as to create a sensitive and discriminating
active site analogous to protein enzymes. In fact, the substrate
molecular recognition of this RNA catalyst rivals its protein
counterparts.5 These results are surprising when considering the
structural and electronic differences in proteins versus nucleic
acids. Within DA22 and other isolated Diels-Alderase struc-
tures lies a wealth of information, which will improve our
limited understanding of oligonucleotide catalysts. Moreover,
structural studies on these new catalysts may be informative
with regard to the mechanism of Lewis acid catalysis of this
important cycloaddition reaction. Research is currently underway
to reveal the genetic, structural, and functional relationships that
exist within families of Diels-Alder catalysts.
was determined using maleimide 1. The extent of reaction was
determined after 3 min of incubation as described in the
Experimental Section. It should be noted that the spontaneous
cycloaddition rate for dienophiles 1, 8, 10, and 11 with dienes
2 or 12 gave relative rates within a factor of 2 of each other.
Therefore, the inherent reactivity of these diene and dienophile
substrates cannot be a determining factor in the substrate
specificity of DA22.13 If DA22 was simply acting as a general
Lewis acid, it might be expected that dieneophile 1 would react
with the cyclic diene 12 at a rate approaching that of the Diels-
Alderase substrates 1 and 2. Similarly, fumarate dienophile 8
reacting with diene 2 would also be expected to benefit from
general Lewis acid catalysis. Remarkably, DA22 showed
surprising selectivity, giving no rate enhancement over back-
ground for either 1 reacting with 12 or 8 reacting with 2. Even
more stunning, DA22 displayed a distinct selectivity against
the very similar dienophiles 10 and 11 reacting with 2. Both
10 and 11 are maleimide dienophiles and only begin to differ
at the second carbon center distal to the maleimide nitrogen.
The observed selectivity for DA22 was 100:6:4 for 1:10:11
reacting with 2. Not only are these alternative substrates identical
to 1 electronically, but the functionalities attached to the
maleimides are similar and, for 11, less sterically demanding.
Clearly, the active site of this catalytic RNA has an inherent
ability to discriminate between very similar substrates and the
Diels-Alderase activity observed can be ascribed to much more
than general Lewis acid catalysis.
Experimental Section
Molecular Replacement/Rearrangment Experiments. 5-Position
modified uridine nucleosides were synthesized using a previously
published procedure.16 Modified nucleotide triphosphates 3-6 were
prepared according to the protocol of Whitesides.17 Analytical data for
5′-monophosphate uridines, precursors to the 5′-triphosphates, 3-6 are
shown below. Detailed experimental procedures for the synthesis of
these compounds will be reported elsewhere. RNA Diels-Alder
reaction incubation were conducted as described previously at 500 µM
1.4 All modified nucleosides were characterized spectroscopically as
the nucleotide monophosphates.
Product Inhibition. To further survey the molecular recogni-
tion capabilities of the DA22 active site and access the
contribution of individual functional components within the
diene and dienophile, a product inhibition study was under-
taken.14 The apparent inhibition constant (Kiapp) was determined
for the series of cycloaddition products shown in Table 2.
Diels-Alderase 22 inhibition by 13 was previously reported,4
and since it contains all of the functionality present in the diene
and dienophile, its Kiapp value served as a benchmark by which
3: 1H NMR (D2O) δ 4.12 (m, 2 H), 4.30 (m, 2 H), 4.39 (t, J ) 4.7
Hz, 1 H), 4.53 (s, 2 H), 5.93 (d, J ) 5.0 Hz, 1 H), 7.30 (m, 5 H), 8.55
(s, 1 H); 13C NMR (D2O:MeOD ) 4:1) δ 44.0, 65.4, 71.0, 75.3, 85.0,
(15) Lightstone, F. C.; Bruice T. C. J. Am. Chem. Soc. 1996, 118, 2595-
2605.
(13) Only dieneophile 9 gave a significantly slower rate for the
spontaneous Diels-Alder reaction with 2.
(14) Because of the product-like transition state for Diels-Alder cy-
cloaddition, product analogues can serve as useful inhibitors.
(16) Dewey, T. M.; Zyzniewski, M. C.; Eaton, B. E. Nucleosides
Nucleotides 1996, 15, 1611-1617.
(17) Simon, E. S.; Grabowski, S.; Whitesides, G. M. J. Org. Chem. 1990,
55, 1834-1841.

Characteristics of an RNA Diels-Alderase ActiVe Site
J. Am. Chem. Soc., Vol. 121, No. 15, 1999 3617
90.7, 106.8, 128.2, 128.5, 129.8, 147.4, 151.5, 164.5, 164.6; HRMS
(FAB+) m/z 719.1488 (M (triphosphate) + Et3NH+, calcd ) 719.1496
for C23H38N4O16P3).
by flash silica gel chromatography (62%), and 1.4 mmol was dissolved
in 28 mL of anhydrous THF, cooled to 0 °C, and treated with
ethylenediamine (20 mmol). The reaction mixture was allowed to warm
to ambient temperature overnight. A white precipitate was removed
and the filtrate concentrated by rotary evaporation. Continued evapora-
tion under high vacuum gave the desired monoamide/monamine in 90%
yield from the NHS ester. The methacroylyl amine (161 mg, 1.26 mmol)
was then combined with NHS-biotin (357 mg, 1.05 mmol) in 5 mL
of ethanol. The heterogeneous mixture was stirred at ambient temper-
ature for 18 h followed by removal of the ethanol under reduced
pressure. The remaining white residue was triturated with ethyl acetate
(5 × 5 mL) to give compound 9 as a white solid in 42% yield from
the biotin-NHS ester: 1H NMR (DMSO-d6) δ 1.30-1.60 (m, 6 H),
1.84 (s, 3 H), 2.05 (t, J ) 7.4 Hz, 2 H), 2.56 (d, J ) 12.4 Hz, 1 H),
2.81 (dd, J ) 12.4, 5.07 Hz, 1 H), 3.13 (m, 5 H), 4.13 (m, 1 H), 4.29
(m, 1 H), 5.32 (s, 1 H), 5.65 (s, 1 H), 6.37 (s, 1 H), 6.44 (s, 1 H), 7.88
(s, 1 H), 7.93 (s, 1 H); 13C NMR (DMSO-d6) δ 18.6, 25.3, 28.1, 28.2,
35.2, 38.0, 38.1, 45.7, 55.4, 59.1, 60.9, 119.2, 139.8, 162.7, 167.5, 172.4;
HRMS (FAB+) m/z 355.1810 (M + H+, calcd ) 355.1804 for
C16H28N4O3S).
1
4: H NMR (D2O) δ 4.0 (m, 2 H), 4.23 (m, 1 H), 4.28 (t, J ) 4.5
Hz, 1 H), 4.43 (t, J ) 5.2 Hz, 1 H), 4.60 (s, 2 H), 5.93 (d, J ) 5.1 Hz,
1 H), 7.34 (d, J ) 4.8 Hz, 2 H), 8.42 (d, J ) 4.8 Hz, 2 H), 8.55 (s, 1
H); 13C NMR (D2O:MeOD ) 100:1) δ 43.2, 64.9, 71.1, 74.9, 85.1,
90.7, 106.9, 123.4, 147.7, 149.4, 150.2, 152.1, 165.0, 165.5; 31P NMR
(MeOD) δ 8.2; HRMS (FAB+) m/z 459.0909 (M + H+, calcd )
459.0917 for C16H20N4O10P).
1
5: H NMR (D2O) δ 4.0 (m, 2 H), 4.25 (m, 2 H), 4.39 (t, J ) 4.8
Hz, 1 H), 4.52 (s, 2 H), 5.89 (d, J ) 4.9 Hz, 1 H), 7.38 (dd, J ) 7.7,
5.1 Hz, 1 H), 7.75 (d, J ) 7.9 Hz, 1 H), 8.36 (d, J ) 4.7 Hz, 1 H),
8.42 (s, 1 H), 8.50 (s, 1 H); 13C NMR (D2O:MeOD ) 100:1) δ 43.3,
65.0, 71.1, 74.9, 85.1, 90.8, 106.9, 125.4, 135.5, 137.7, 147.6, 148.4,
151.9, 165.0; 31P NMR (MeOD) δ 2.2; HRMS (FAB+) m/z 459.0913
(M + H+, calcd ) 459.0917 for C16H20N4O10P).
6: 1H NMR (D2O) δ 3.98 (m, 2 H), 4.23 (m, 2 H), 4.41 (t, J ) 5.1
Hz, 1 H), 4.62 (s, 2 H), 5.91 (d, J ) 5.1 Hz, 1 H), 7.31 (dd, J ) 7.4,
5.6 Hz, 1 H), 7.80 (td, J ) 7.8, 1.7 Hz, 1 H), 8.42 (d, J ) 4.7 Hz, 2
H), 8.51 (s, 1 H); 13C NMR (D2O:MeOD ) 100:1) δ 43.5, 65.7, 72.0,
76.0, 85.6, 91.4, 106.8, 123.1, 124.0, 139.0, 147.6, 150.0, 152.5, 159.0,
165.0,165.9; 31P NMR (MeOD) δ 3.7; HRMS (FAB+) m/z 459.0909
(M + H+, calcd ) 459.0917 for C16H20N4O10P).
Diels-Alderase Inhibition by Product Analogues. The Diels-
Alder products were synthesized from the appropriate diene and
maleimide as described in the following general procedure. The diene
(4.62 mmol) and maleimide (4.62 mmol) were dissolved in 10 mL of
benzene and heated to 45 °C for 2-7 days. The reaction mixture was
then evaporated to dryness and the cycloaddition product purified by
flash silica gel chromatography using hexane/ethyl acetate at the eluent.
Alternative Substrate Experiments. Biotin Maleimides 1 and 10
were purchased from Pierce while compound 11 was purchased from
Sigma. All were used without further purification. Diene 12 was
prepared and characterized as previously described.18 Fumarate 8 and
methacrylamide 9 were synthesized as described below. Incubations
were conducted as described previously4 at 500 µM dienophile, 500
nM RNA, and 25 °C.
1
Products were analyzed by H NMR, 13C NMR, and MS.
14: 1H NMR (CDCl3) δ 1.07 (t, J ) 7.2 Hz, 3 H), 1.48 (d, J ) 7.2
Hz, 3 H), 2.40 (m, 1 H), 2.56 (m, 1 H), 3.03 (dd, J ) 8.8, 6.5 Hz, 1
H), 3.32 (m, 2 H), 3.45 (q, J ) 7.2 Hz, 2 H), 3.95 (m, 2 H), 5.67 (dt,
J ) 9.1, 3.1 Hz, 1 H), 5.73 (dt, J ) 9.1, 2.8 Hz, 1H); 13C NMR (CDCl3)
13.5, 17.3, 32.2, 34.5, 40.0, 43.9, 46.4, 63.3, 131.1, 135.9, 180.4, 180.5;
HRMS (FAB+) m/z 224.1284 (M + H+, calcd ) 224.1286 for C12H18-
NO3).
8. Maleic anhydride (10.2 mmol) and 2-methoxyethanol (6.3 mmol)
were combined and heated to 100 °C for 18 h. The reaction mixture
was then added to 100 mL of hexanes and agitated, and the hexane
layer was decanted. The remaining oily residue was washed once more
with hexanes (100 mL) and then refluxed for 2 h with thionyl chloride
(12.7 mmol). The excess thionyl chloride was then removed under
reduced pressure, and the desired acid chloride was added dropwise
over 30 min to a solution of N-hydroxysuccinimde (12.7 mmol) and
triethylamine (12.7 mmol) in 30 mL of anhydrous THF at 0 °C. The
reaction mixture was allowed to warm to ambient temperature for 2 h
followed by removal of the THF by rotary evaporation and addition of
diethyl ether (100 mL). The oraganic mixture was washed with brine
(3 × 75 mL) and dried over MgSO4. The product was purified by flash
silica gel chromatography to give a 65% yield of the NHS fumarate
ester. The fumarate ester (1.05 mmol), N-(2-aminoethyl)biotinamide
(1.2 mmol, Molecular Probes), and triethylamine (1.2 mmol) were
combined in 5 mL of ethanol at ambient temperature. The mixture was
stirred for 18 h followed by removal of the solvent under reduced
pressure. The remaining white residue was triturated with ethyl acetate
(10 × 5 mL) to give a 67% yied of the desired biotinylate fumaramide
8: 1H NMR (MeOD) δ 1.41-1.60 (m, 6 H), 2.19 (t, J ) 7.5 Hz, 2 H),
2.69 (d, J ) 12.9 Hz, 1 H), 2.91 (dd, J ) 12.9, 5.0 Hz, 1 H), 3.37 (m,
7 H), 3.63 (t, J ) 4.7 Hz, 2 H), 4.31 (m, 3 H), 4.46 (m, 1 H), 6.71 (d,
J ) 15.5 Hz, 1 H), 7.00 (d, J ) 15.5 Hz, 1 H); 13C NMR (MeOD) δ
26.8, 29.5, 29.8, 36.8, 39.8, 40.4, 41.1, 57.0, 59.1, 61.7, 63.4, 65.2,
71.4, 130.6, 138.0, 166.2, 166.4, 166.8, 176.5; HRMS (FAB+) m/z
443.1968 (M + H+, calcd ) 443.1964 for C19H32N4O6S).
15: 1H NMR (CDCl3) δ 0.90 (m, 2 H), 1.15 (m, 3 H), 1.49 (d, J )
7.2 Hz, 3 H), 1.50-1.78 (m, 7 H), 2.45 (m, 1 H), 2.60 (m, 1 H), 3.05
(dd, J ) 8.5, 6.5 Hz, 1 H), 3.30 (d, J ) 6.9 Hz, 2 H), 3.34 (dd, J )
8.5, 7.2 Hz, 1 H), 3.90 (dd, J ) 12.0, 9.4 Hz, 1 H), 4.02 (dd, J ) 12.0,
5.7 Hz, 1 H), 5.70 (dt, J ) 9.1, 3.1 Hz, 1 H), 5.76 (dt, J ) 9.1, 3.1 Hz,
1 H); 13C NMR (CDCl3) δ 16.8, 25.5, 26.1, 30.6, 30.6, 31.2, 36.1,
38.1,42.7, 44.7, 62.9, 128.4, 135.3, 177.7, 179.3; HRMS (FAB +) m/z
292.1920 (M + H+, calcd ) 292.1912 for C17H26NO3).
16: 1H NMR (CDCl3) δ 0.88 (m, 2 H), 1.13 (m, 3 H), 1.44 (d, J )
7.4 Hz, 3 H), 1.48-1.73 (m, 7 H), 2.43 (m, 1 H), 2.60 (m, 1 H), 3.01
(dd, J ) 8.5, 7.1 Hz, 1 H), 3.20 (dd, J ) 8.7, 6.0 Hz, 1 H), 3.27 (d, J
) 6.9 Hz, 2 H), 3.40 (broad q, J ) 5.2 Hz, 2 H), 3.57 (t, J ) 5.0 Hz,
4 H), 3.73 (t, J ) 4.9 Hz, 2 H), 4.51 (dd, J ) 11.0, 8.1 Hz, 1 H), 4.63
(dd, J ) 11.0, 7.4 Hz, 1 H), 5.25 (broad t, 1 H), 5.74 (m, 2 H); 13C
NMR (CDCl3) δ 16.6, 25.5, 26.2, 30.6, 31.3, 36.1, 36.2, 40.8, 42.2,
44.5, 44.6, 61.7, 64.9, 70.0, 72.2, 129.2, 135.2, 156.5, 177.4, 177.5;
HRMS (FAB +) m/z 423.2505 (M + H+, calcd ) 423.2495 for
C22H35N2O6).
Product Inhibition. Apparent Ki values for the cycloaddition
products 13-16 were determined at 500 µM 1 by fitting the observed
first-order rate constants to the following equation for inhibition: kobs
) (kobs0/2)(RE - I - Ki + ((Ki + RE - I)2 + 4Ki)1/2), where kobs is the
measured rate constant in the presence of inhibitor, kobs0 is the observed
rate constant in the absence of inhibitor, RE represents the fractional
(R) concentration of functional active sites (E), I is the concentration
of inhibitor, and Ki is the apparent inhibition constant.19
9. To a solution of N-hydroxysuccinimide (5.2 mmol) and triethyl-
amine (5.2 mmol) in 13 mL of anyhydrous THF at 0 °C was added
dropwise over 30 min methacrylyl chloride (5.2) in 13 mL of THF.
The reaction was allowed to warm to room temperature over 2 h
followed by removal of the THF under reduced presuure. The remaining
residue was dissolved in diethyl ether (100 mL), washed with brine (3
× 75 mL), and dried over MgSO4. The resulting NHS ester was purified
Acknowledgment. The authors are grateful to Sarah Way-
land and Jeff Beckvermit for the synthesis of modified tri-
phosphates 3 and 4. Special thanks is given to the Medicinal
Chemistry group at NeXstar Pharmaceuticals for insightful and
stimulating discussions.
(18) Tarasow, T. M.; Tinnermeier, D.; Zyzniewski, C. Bioconjugate
Chem. 1997, 8, 89-93.
(19) Williams, J. W.; Morrison, J. F. Methods Enzymol. 1979, 63, 437-
467.
JA983989M