Angewandte
Chemie
property, however, that has not been studied is stereochem-
istry, whereby substitution of 2-nitrobenzylꢀs benzylic or a-
carbon results in a chiral center. For the case of nucleotide
synthesis, coupling of a racemic a-substituted 2-nitrobenzyl
alcohol would result in two diastereomers, which differ only
by the absolute configuration (R or S) at the benzylic carbon
(“*” in Figure 1). Here, we describe our efforts toward
improving the photochemical-cleavage properties by exam-
ining various ring-substituted, stereospecific a-isopropyl- and
a-tert-butyl-2-nitrobenzyl-modified reversible terminators.
Unlike our work with a-substituted HOMedU ana-
logues,[3] we identified chromatographic conditions to sepa-
rate C7-HOMedA analogues into single diastereomeric
nucleotides, with the first eluting isomer denoted as ds1 and
the second as ds2. To evaluate photochemical-cleavage
effects, three 2-nitrobenzyl-modified C7-HOMedA analogues
were synthesized and separated into single diastereomers,
dA.III.a (a-isopropyl), dA.III.b (a-isopropyl-4-OMe), and
dA.III.c (a-isopropyl-6-NO2), along with the parent dA.I (see
the Supporting Information). These C7-HOMedATP ana-
logues were applied in incorporation assays followed by
photochemical-cleavage experiments in sodium azide solu-
tion (Table 1). In all cases, the ds2 isomers of dA.III.a,
dA.III.b, and dA.III.c showed faster photochemical cleavage
4.5, 4.4, and 3.0, respectively, compared with their ds1
counterparts (Table 2). The photochemical-cleavage rates of
both 5-OMe ds1 and ds2 isomers were 1.4 times faster than
those of their respective 4-OMe isomers, demonstrating that
Table 2: Rates of photochemical cleavage for C7-HOMedG analogues.
C7-HOMedG Analogue
DT50 in 1 mm NaN3
No DTT
50 mm DTT
dG.I
9.2Æ0.3
11.0Æ0.4
3.6Æ0.3
4.9Æ0.3
1.1Æ0.1
3.5Æ0.3
0.8Æ0.1
2.4Æ0.1
0.8Æ0.1
8.1Æ0.2
10.7Æ0.2
3.5Æ0.3
4.6Æ0.3
1.3Æ0.2
3.0Æ0.1
0.8Æ0.1
2.3Æ0.2
0.8Æ0.1
dG.V.a ds1
dG.V.a ds2
dG.V.b ds1
dG.V.b ds2
dG.V.c ds1
dG.V.c ds2
dG.V.d ds1
dG.V.d ds2
ring position influences the cleavage rate. The bis-substituted
4,5-di-OMe ds1 isomer showed faster rates than the mono-
substituted 4-OMe (2.0-fold) and 5-OMe (1.5-fold) ds1
isomers. Conversely, both 5-OMe and 4,5-di-OMe ds2 isomers
exhibited the identical DT50 value of just 0.8 s. We note that in
the absence of an a substituent, Hasan et al. reported a rate
increase of only 1.2-fold for a 5-OMe-2-nitrobenzyl analogue
over its corresponding parent.[10b] Comparison of ds1 and ds2
isomers of dG.V.c with dG.V.a revealed higher rate increases
of 3.6-fold and 4.4-fold, respectively, suggesting that the
stereospecific tert-butyl group enhances the effect of the 5-
OMe group. With four-color CRT applications, this combina-
tion provides good flexibility for the utility of the ring system,
as a linker can be attached to the 4-position to create dye-
labeled analogues.[1a]
Table 1: Rates of photochemical cleavage for C7-HOMedA analogues.[a]
C7-HOMedA Analogue
DT50 in 1 mm NaN3
No DTT
50 mm DTT
dA.I
3.6Æ0.1
4.5Æ0.2
2.2Æ0.1
7.0Æ0.3
1.1Æ0.1
3.4Æ0.2
2.8Æ0.2
3.5Æ0.1
4.4Æ0.2
2.1Æ0.1
6.1Æ0.4
1.0Æ0.1
3.0Æ0.2
2.5Æ0.1
dA.III.a ds1
dA.III.a ds2
dA.III.b ds1
dA.III.b ds2
dA.III.c ds1
dA.III.c ds2
To determine the stereochemistry of these a-tert-butyl C7-
HOMedG analogues, the (1S)-camphanate of (R/S)-1-(5-
[a] A DT50 value is defined as the point in time at which 50% of the 2-
nitrobenzyl groups have been photochemically cleaved from the
extended primer/template complex. Lower DT50 values indicate faster
photochemical cleavage rates. DTT=dithiothreitol.
methoxy-2-nitrophenyl)-2,2-dimethyl-1-propanol
was
resolved into its enantiopure S alcohol by fractional crystal-
lization[11] (Figure S1 in the Supporting Information). This S
alcohol and (S)-a-tert-butyl-2-nitrobenzyl alcohol[3] were each
coupled to C7-HOMedG (Figure 1). RP-HPLC analysis of
their corresponding triphosphates revealed that both ds2
isomers of dG.V.a and dG.V.c have peak retention times
identical to those of dG.V and dG.VI, respectively, thus
indicating that both ds2 isomers have the same S configu-
ration at the a-carbon. By inference, the corresponding ds1
isomers of dG.V.a and dG.V.c have been assigned the R
configuration.
These S alcohols were then coupled to the remaining
nucleosides to examine the effect of the nucleotide leaving
group on the rate of photochemical cleavage. For example,
photochemical-cleavage experiments revealed that DT50
values for the parent 2-nitrobenzyl analogues ranged from
2.0 s for dC.I to 9.2 s for dG.I, suggesting that the leaving
group can influence the rate of photochemical cleavage
(Figure 2). Substitution of the benzylic carbon with a tert-
butyl group in the stereospecific S configuration, denoted
simply as (S)-a-tert-butyl, resulted in increased cleavage rates
by factors of 1.5–3.1, and the additional substitution with a 5-
rates (i.e., lower DT50 values) by factors of 2.0, 6.4, and 1.2,
respectively, compared to their ds1 counterparts. Interest-
ingly, the ds1 isomers exhibited similar (dA.III.c) or higher
(dA.III.a and dA.III.b) DT50 values than the parent dA.I.
These data provide evidence that the stereochemistry of the
a-substituted isopropyl group is an important determinant,
and coupled with a 4-OMe substitution, analogue dA.III.b ds2
produced the lowest DT50 value for the C7-HOMedA set.
Our previous work demonstrated that the a-tert-butyl
analogue dU.V exhibited excellent CRT properties, such as
single-base termination and high nucleotide selectivity.[3] This
allowed us to further examine the stereospecific effect using a
different a-substitution group coupled with various OMe ring
substitutions by synthesizing four a-tert-butyl C7-HOMedG
analogues, dG.V.a—dG.V.d, along with the parent dG.I
(Figure 1). Consistent with a-isopropyl-C7-HOMedATP ana-
logues, photochemical-cleavage experiments of the ds2 iso-
mers dG.V.a—dG.V.d showed faster rates by factors of 3.1,
Angew. Chem. Int. Ed. 2012, 51, 1724 –1727
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1725