Fluorophore-labeled S-nitrosothiols

Article abstract of DOI:10.1021/jo015658p

A series of fluorophore-labeled S-nitrosothiols were synthesized, and their fluorescence enhancements upon removal of the nitroso (NO) group were evaluated either by transnitrosation or by photolysis. It was shown-that, with a suitable alkyl linker, the fluorescence intensity of dansyl-labeled S-nitrosothiols could be enhanced up to 30-fold. The observed fluorescence enhancement was attributed to the intramolecular energy transfer from fluorophore to the SNO moiety. Ab initio density functional theory (DFT) calculations indicated that the "overlap" between the SNO moiety and the dansyl ring is favored because of their stabilizing interaction, which was in turn affected by both the length of the alkyl linker and the rigidity of the sulfonamide unit. In addition, one of the dansyl-labeled S-nitrosothiols was used to explore the kinetics of S-nitrosothiol/thiol transnitrosation and was evaluated as a fluorescence probe of S-nitrosothiol-bound NO transfer in human umbilical vein endothelial cells.

Full text of DOI:10.1021/jo015658p

6064
J . Org. Chem. 2001, 66, 6064-6073
F lu or op h or e-La beled S-Nitr osoth iols
Xinchao Chen,† Zhong Wen,† Ming Xian,† Kun Wang,† Niroshan Ramachandran,‡
Xiaoping Tang,† H. Bernhard Schlegel,† Bulent Mutus,‡ and Peng George Wang*,†
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, and Department of
Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4
pwang@chem.wayne.edu
Received April 2, 2001
A series of fluorophore-labeled S-nitrosothiols were synthesized, and their fluorescence enhance-
ments upon removal of the nitroso (NO) group were evaluated either by transnitrosation or by
photolysis. It was shown that, with a suitable alkyl linker, the fluorescence intensity of dansyl-
labeled S-nitrosothiols could be enhanced up to 30-fold. The observed fluorescence enhancement
was attributed to the intramolecular energy transfer from fluorophore to the SNO moiety. Ab initio
density functional theory (DFT) calculations indicated that the “overlap” between the SNO moiety
and the dansyl ring is favored because of their stabilizing interaction, which was in turn affected
by both the length of the alkyl linker and the rigidity of the sulfonamide unit. In addition, one of
the dansyl-labeled S-nitrosothiols was used to explore the kinetics of S-nitrosothiol/thiol trans-
nitrosation and was evaluated as a fluorescence probe of S-nitrosothiol-bound NO transfer in human
umbilical vein endothelial cells.
In tr od u ction
responsible for the activity of RSNO in vivo8 and has been
suggested to be a signaling mechanism for the control of
cellular processes by NO.9
In recent years, S-nitrosothiols (RSNOs) have received
considerable attention because of their potential role in
nitric oxide (NO) storage and transfer in a range of
physiological processes.1 A number of S-nitroso adducts
have indeed been found in vivo2 and have been associated
with physiological functions.3 For example, RSNOs serve
not only as NO “pools”4 but also as intermediates in the
bioactivation of organic nitrates and nitrites5 and are
involved in altering the function and activity of proteins.6
Chemical understanding of RSNO formation, decomposi-
tion, and transport in biological processes is, therefore,
of fundamental importance. Analogous to alkyl nitrites
(RONOs), RSNOs may react with nitrogen, sulfur, or
oxygen nucleophiles to give free thiol and the correspond-
ing transnitrosation product.7 The transfer of NO be-
tween different thiols via transnitrosation (eq 1) is largely
RSNO + R'SH h R'SNO + RSH
(1)
The kinetics of the process in eq 1 has recently drawn
considerable interest.7,10 In addition, both photolytic
homolysis10d,11 and catalytic decomposition of RSNO in
aqueous solution,7a as represented in eq 2, directly relate
to the generation of NO free radical in vivo.
2RSNO f RS-SR + 2NO•
(2)
This general instability of RSNO should largely be
attributed to the low S-NO bond energies, as indicated
by our recent study of homolytic/heterolytic bond dis-
sociation energies (BDEs) of the RS-NO bonds.12
Recently, Mutus et al.13 reported that the fluorescence
emission of N-dansyl-S-nitrosohomocysteine was en-
hanced about 8-fold upon removal of the NO group
* Phone: (313) 933-6759. Fax: (313) 577-5831. E-mail: pwang@
chem.wayne.edu.
† Wayne State University.
‡ University of Windsor.
(1) (a) Stamler, J . S. In S-Nitrosothiols and the Bioregulatory
Actions of Nitric Oxide through Reactions with Thiol Groups: The Role
of Nitric Oxide in Physiology and Pathophysiology; Koprowski, H.,
Maeda, H., Eds.; Springer-Verlag: New York, 1993. (b) Butler, A. R.;
Rhodes, P. Anal. Biochem. 1997, 249, 1. (c) Hogg, N. Free Radical Biol.
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Demchenko, I. T.; Bonaventura, J .; Gernet, K.; Piantadosi, C. A. Science
1997, 276, 2034. (d) Stamler, J . S.; J araki, O.; Osborne, J . A.; Simon,
D. I.; Keaney, J .; Vita, J .; Singel, D. J .; Valeri, C. R.; Loscalzo, J . Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 7674.
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Chem. 1996, 271, 18596. (e) Pezacki, J . P.; Pelling, A.; Klugger, R. J .
Am. Chem. Soc. 2000, 122, 10734. (f) Wang, K.; Wen, Z.; Zhang, W.;
Xian, M.; Cheng, J .-P.; Wang, G. P. Bioorg. Med. Chem. Lett. 2001,
11, 433. (g) Rossi, R.; Lusini, L.; Giannerini, F.; Giustarini, D.;
Lungarella, G.; Di Simplicio, P. Anal. Biochem. 1997, 254, 215. (h)
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10.1021/jo015658p CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/10/2001

Fluorophore-Labeled S-Nitrosothiols
J . Org. Chem., Vol. 66, No. 18, 2001 6065
Ch a r t 1
Sch em e 1
Sch em e 2
(a) i. 1.0 equiv of NaH, THF, 0 °C, 30 min; ii. N-acetyl
homocysteine thiolactone, 5 h; (b) t-BuONO, MeOH, -20 °C, 2 h;
(c) homocysteine thiolactone, NaCN, MeOH/H2O, 4 h; (d) NaBH4,
MeOH, 0 °C, 3.5 h; (e) HNO2, -20 °C, 2 h.
(a) i. Benzyl mercaptan, piperidine, reflux, 6 h, 60%; ii, NaBH4,
MeOH, 0 °C, 72%; (b) i. BuLi, then TsCl, -78 °C, 1 h, 95%; ii.
NaCN, DMF, 90%; (c) i. LiAlH4, THF, reflux, 4 h, 55%; ii. Na, NH3,
-40 °C, 70%; (d) Dansyl chloride, DMAP, CH2Cl2, 0 °C, 2 h, 71%;
(e) NaNO2/HCl, MeOH/H2O, -20 °C, 92%.
(denitrosation). To explore the mechanism of fluorescence
enhancement as well as its potential for detecting thiols
(and free cysteines in proteins), we synthesized and
evaluated a series of S-nitroso compounds (FLSNOs
1-12, Chart 1) linked with different fluorophores. Struc-
tural factors relating to the fluorescence enhancements
in dansyl-labeled S-nitrosothiols (FLSNOs 7-12) were
examined using ab initio density functional theory (DFT)
calculations. The large fluorescence enhancement for
FLSNO 12 was used in the kinetic study of the S-
nitrosothiol/thiol transnitrosation (eq 1) as well as in the
fluorescence microscopic study of RSNO-bound NO trans-
port into the cellular membrane.
chloride and S-trityl penicillamine methyl ester, followed
by the routine procedure for deprotecting the sulfhydryl
group. The synthesis of tertiary S-nitrosothiol 6 is shown
in Scheme 2, starting from alcohol 15, obtained from a
two-step procedure reported by Pattenden et al.15 Scheme
3 shows a general approach for the synthesis of FLSNOs
7-11 as well as the synthesis of secondary S-nitrosothiol
12. In the final step of the synthesis of FLSNOs, either
tert-butyl nitrite (for 1) or nitrous acid (for 2-12) was
used for the S-nitrosation reaction. The FLSNO com-
pounds were moderately stable in the PBS buffer (1.0
mM EDTA, 20% MeOH), with half-lives ranging from 3
to 15 h.
Resu lts a n d Discu ssion
Syn t h esis of F lu or op h or e-La b eled S-Nit r oso-
th iols. Twelve S-nitrosothiols labeled with a variety of
fluorophores including 6-aminoquinoline, 2-aminopyri-
dine, 2-cyanoisoindole, and 5-(dimethylamino)-1-naph-
thalenesulfonamide (dansyl) were synthesized (Schemes
1-3). Homocysteine thiolactone and its N-acetyl deriva-
tive were used to introduce the sulfhydryl group in
FLSNOs 1-3 (Scheme 1).14 FLSNO 4 was prepared by
coupling dansyl chloride with homocysteine thiolactone,
followed by reducing the thiolactone with DIBALH.
Synthesis of FLSNO 5 started from coupling dansyl
Exp er im en ta l Exa m in a tion of th e Str u ctu r a l Ef-
fect on F lu or escen ce En h a n cem en t th r ou gh Den i-
tr osa tion . Both transnitrosation (method A) and pho-
tolytic denitrosation (method B) were used to evaluate
the synthetic fluorescence probes. In method A, the
fluorescence intensities were measured before and after
(as A1 and A2, respectively) the transformation of FLSNO
to fluorophore-labeled thiol using excess glutathione
(GSH), and the fluorescence enhancement value was
obtained as the ratio of fluorescence intensities (A2/A1).
Similarly, the fluorescence enhancement value for method
B was obtained (as B2/B1) via the photolytic denitrosation
of FLSNO, which gives rise to disulfides (FL-S-S-FL)
(14) (a) Ramirez, J .; Yu, L. B.; Li, J .; Brauschweiger, P. G.; Wang,
P. G. Bioorg. Med. Chem. Lett. 1996, 6, 2575. (b) Hou, Y.-C.; Wang,
J .-Q.; Ramirez, J .; Wang, P. G. Methods Enzymology Nitric Oxide, Part
C: Biological and Antioxidant Activities; Packer, L., Ed.; Academic
Press: 1998, 301, 242.
(15) Pattenden, G.; Shuker, A. J . J . Chem. Soc., Perkin Trans 1 1992,
1215.

6066 J . Org. Chem., Vol. 66, No. 18, 2001
Chen et al.
Sch em e 3
the SNO moiety and the fluorophore emission for 2 (361
nm) is better than that for either 1 (∼380 nm) or 3 (∼433
nm), whereas this overlap appears most efficient between
the emission from dansyl (4) and SNO absorption at 540
nm. These observations are indeed consistent with the
intramolecular energy transfer mechanism described by
Forster’s formalism (eq 3).16
kT κ2J R-6
(3)
where kT is the rate of excited-state energy transfer from
a donor to an acceptor, J is the overlap integral of donor
emission and acceptor absorption, κ2 is the relative
spatial orientation of the transition dipoles of the donor
and the acceptor, and R is the donor-to-acceptor distance.
Here the kT value is equivalent to the “net” fluorescence
enhancement (A2/A1 - 1).17
The energy transfer rationale is further supported by
the low fluorescence enhancement value for dansyl-
labeled tertiary aliphatic S-nitrosothiols 5 and 6. As
recently suggested by Bartberger et al.,18 tertiary alkyl
substitution (R) prefers an anti conformation for the Rs
SsNdO moiety, thereby shifting the UV-vis absorption
of the SNO moiety to around 590 nm. This would lead to
poorer spectral overlap between dansyl fluorescence
emission and SNO absorption, and therefore, little fluo-
rescence enhancement is observed.
(a) Dansyl chloride, Et3N, CH2Cl2, rt, 83%; (b) i. TsCl, Et3N,
CH2Cl2, -20 °C to 0 °C, overnight, 92%; ii. AcSK, acetone, rt, 4 h,
100%; (c) DIBALH, CH2Cl2, -78 °C, 2 h, 70%; (d) HCl/NaNO2,
MeOH/H2O, -20 °C, 2 h, 85%; (e) i. PDC, CH2Cl2, rt, 3 h; ii. MeLi,
THF, -78 °C, 2.5 h, 60% (two steps).
Because the energy transfer mechanism (eq 3) predicts
large sensitivity of the fluorescence enhancement toward
the donor-to-acceptor distance (R), we further examined
dansyl-labeled compounds by varying the length of the
alkyl linker (FLSNOs 7-12). With a shorter linker
(number of methylene units n ) 2), FLSNO 7 exhibited
little fluorescence enhancement. FLSNO 8, similar to 4
in alkyl linker (n ) 3), had a correspondingly strong
enhancement. FLSNOs 9 and 12 (n ) 4) showed even
larger enhancements (up to 30-fold). As the length of
linker was increased further in 10 (n ) 5) and 11 (n )
6), however, the fluorescence enhancement decreased
somewhat. From a simple consideration that the dansyl-
to-SNO distance corresponds to the length of linker, the
above results are quite unexpected and thus merit
further investigation at the electronic structure level.
Th eor etica l Stu d y of th e Geom etr y a n d En er get-
ics of SNO In ter a ctin g w ith Da n syl Rin g. For an
intramolecular energy transfer process, the donor-to-
acceptor distance R strongly influences the kT value (eq
3), and the distance, in turn, depends on the flexibility
of the intramolecular donor-to-acceptor linkage. As a
simplified treatment, we assume that only conformers
with the closest donor-to-acceptor approach dominate the
energy transfer process, provided that these conformers
have reasonable populations (i.e., that they have energies
close to the global minimum). To probe the conforma-
tional energy and the fluorophore-to-SNO distance with
respect to the alkyl length, theoretical calculations were
performed for the dansyl-labeled FLSNOs 7-12 using ab
initio (HF/3-21G*) and density functional theory (B3LYP/
6-31G**) calculations.
F igu r e 1. Fluorescence emission spectrum of FLSNO 9 in
PBS buffer (curve 1), after transnitrosation with 50 molar
equiv of GSH for 40 min (curve 2), and after irradiation at
365 nm for 50 min (curve 3). The initial concentration of 9 is
40 µM, and the excitation wavelength (λex) is 323 nm.
and NO free radical (eq 2).11 Figure 1 illustrates the
fluorescence spectra of FLSNO 9 before and after the
removal of the NO group (with both methods). The
fluorescence enhancements of all FLSNOs as well as
relevant data are summarized in Table 1.
The fluorescence enhancement values obtained from
methods A and B overall demonstrate quite similar
trends; that is, larger A2/A1 values correspond to larger
B2/B1. With varied fluorophores (yet linked with roughly
similar flexible alkyl linkers), the enhancements for
FLSNOs 1-4 differ substantially. The fluorescence en-
hancements of both 6-aminoquionine-labeled 1 and 2-cy-
anoisoindole-labeled 3 were rather poor (close to unity),
whereas 2-aminopyridine-labeled 2 and dansyl-labeled
4 showed relatively large enhancements. Given that the
UV-vis absorption by primary or secondary aliphatic
S-nitrosothiol consists of a 340 nm band (n f π* transi-
tion) and a weaker 540 nm band (π f π* transition),
apparently the overlap between the absorption bands of
(16) Forster, Th. Ann. Phys. (Berlin) 1948, 2, 55.
(17) (a) Lakowicz, J . R. Principles of Fluorescence Spectroscopy;
Plenum Press: New York, 1983; Chapter 10. (b) Cheung, H. C. In
Topics in Fluorescence Spectroscopy: Principles; Lakowicz, J . R., Ed.;
Plenum Press: New York, 1991; Vol. 2, pp 127-176.
(18) Bartberger, M. D.; Houk, K. N.; Powell, S. C.; Mannion, J . D.;
Lo, K. Y.; Stamler, J . S.; Toone, E. J . J . Am. Chem. Soc. 2000, 122,
5889.

Fluorophore-Labeled S-Nitrosothiols
J . Org. Chem., Vol. 66, No. 18, 2001 6067
Ta ble 1. F lu or escen ce Excita tion a n d Em ission Wa velen gth s (λex a n d λem ), F lu or escen ce In ten sities,a a n d F lu or escen ce
En h a n cem en t Va lu esb d u r in g Den itr osa tion of F LSNOs 1-12
FLSNO
λ
ex (nm)
λ
em (nm)
10-5A1
10-5A2
A2/A1
10-5B1
10-5B2
B2/B1
1
2
340
300
350
368
370
330
323
327
323
325
326
330
381
361
433
532
540
530
530
532
525
530
530
525
50
5.5
12
37
22
11
25
17
38
56
66
63
54
31
40
0.7
4.3
0.9
6.1
1.0
1.8
1.2
5.1
20
49
5.3
12
48
28
1.0
5.2
1.6
20
3
19
4
4.0
16
4.3
17
86
5
16
0.9
1.4
6.3
15
6
21
19
27
7
47
37
234
232
92
8
13
15
9
3.2
3.2
2.1
2.0
3.1
2.8
1.6
1.9
30
10
11
12
17
81
29
15
20
45
28
26
14
a
Fluorescence intensities A1 and A2 are for transnitrosation between FLSNO and GSH (eq 1); B1 and B2 are for photolysis of FLSNO
(eq 2), where the subscripts 1 and 2 refer to the fluorescence intensity of FLSNO before and after denitrosation, respectively. Slit: 0.25
b
× 0.25. The estimated error bars for the ratio are (15% on the basis of repeated measurements.
F igu r e 2. Nonoverlapping (A) and overlapping (B) conforma-
tions of dansyl ethane.
F igu r e 3. Nonoverlapping (A) and overlapping (B) conforma-
tions for FLSNOs 7-11. For B, R is the closest dansyl-to-SNO
distance.
We first examined the geometries of the sulfonamide
unit in the dansyl moiety, using unsubstituted 5-(di-
methylamino)-1-naphthalenesulfonamide and its N-alkyl
derivative as models (Figure 2). Optimization at the HF/
3-21G* level revealed several features of the dansyl
sulfonamide group (SO2NH2): (i) both NsH bonds of the
sulfonamide nitrogen are eclipsed with the SdO bonds,
and (ii) the rotation of the SO2NH2 group about the CsS
bond is significantly constrained, with fairly high rota-
tional barriers due to the steric repulsion between NH2
and naphthalene hydrogens (∼11.6 kcal/mol for H8 and
4.0 kcal/mol for H2, respectively; see Supporting Informa-
tion). This suggests that the dansyl sulfonamide unit is
fairly rigid, with its SsN bond preferring an upright
orientation (dihedral angle C2-C1-S-N is ∼112°).
Calculations with N-methyl substitution suggested that
an N-alkyl group would be tilted toward H2, because the
steric repulsion between N-alkyl and H2 is around 1 kcal/
mol smaller than that between N-alkyl and H8. Next, we
examined the conformations of N-ethyl substitution. As
shown in Figure 2, the terminal methyl group can be
oriented either away from the dansyl ring (conformer A)
or toward the dansyl ring (conformer B). The energy of
A is about 0.5 kcal/mol lower than that of B, suggesting
that the steric repulsion between the dansyl ring and the
alkyl chain would slightly favor a “nonoverlapping”
conformation.
On the basis of the geometries of conformers A and B
in Figure 2, a conformational search of dansyl-labeled
FLSNOs was carried out by replacing a CsH bond of the
N-alkyl substituent with the CsSNO moiety (Figure 3).
For all the conformers, the SNO moiety adopts a syn
orientation with its NdO bond eclipsing an RCsH bond,18
while the alkyl chains (for n g 4) adopt essentially anti
conformations to avoid gauche interactions. The non-
overlapping conformers (Figure 3A) were compared with
the overlapping forms (Figure 3B) in terms of electronic
energy ∆E and enthalpy ∆H, and the distance between
the nitrogen in the SNO moiety and the center of the
Ta ble 2. Com p u ted Closest Da n syl-to-SNO Dista n ce (R),
Nu m ber of Meth ylen e Un its (n ), a n d Cor r esp on d in g
Rela tive En er getic P r efer en ce (∆E a n d ∆H) in
Da n syl-La beled F LSNOs (7-12) a t 298 K
FLSNO
n
Ra
∆Eb
∆Hb
7
8
9
10
11
12
2
3
4
5
7.74
6.33
4.73c
4.79
5.57
4.71c
(0)
(0)
0.9
0.3
0.3
0.5
(0)
(0)
0.8
0.1
0.0
0.4
6
4d
a
Distance (Å) between the nitrogen in the SNO moiety and the
center of the naphthalene ring (Figure 3B), calculated from the
b
geometries optimized at the HF/3-21G* level. Energy difference
(∆E and ∆H, in kcal/mol) between non-overlapping and overlap-
ping conformations (Figure 3) calculated at the B3LYP/6-31G**//
HF3-21G* level, ∆E ) E - Eov. ∆H was obtained from ∆E and
the difference in thermal corrections to enthalpy. c Averaged value
d
of two conformers with nearly identical energies. (CH2)3C(CH3)
as the linker.
dansyl ring (R) was examined. The relevant results are
summarized in Table 2.
For FLSNOs 7 and 8, the SNO moiety is situated away
from the dansyl ring in the most stable conformation (∆E
) 0), and bringing the SNO group over the dansyl ring
leads to an energy rise of around 1 kcal/mol (Figure 4).
This is consistent with the results for N-ethyl substitu-
tion (Figure 2). As the length of the linker (n) increases,
however, the SNO moiety in FLSNOs 9 and 12 (n ) 4)
prefers to approach the dansyl ring. These overlapping
conformers are actually global minima (i.e., positive
values for ∆E or ∆H). Apparently, in the overlapping
conformations of 9 and 12, the inherent steric repulsion
between the alkyl chain and the dansyl ring is overcome
by the attractive interaction between the SNO moiety
and the dansyl ring.19 Even longer alkyl linkers (n ) 5,
6) lead to weaker preferences for the SNO-dansyl
overlap, as indicated by smaller ∆E values for 10 and
11. Consideration of solvent effects and entropy may alter

6068 J . Org. Chem., Vol. 66, No. 18, 2001
Chen et al.
F igu r e 5. Plot of the logarithm of “net” fluorescence enhance-
ment [ln(A2/A1 - 1)] versus the logarithm of the closet dansyl-
to-SNO distance (ln R) in FLSNOs 7-12.
F igu r e 4. Conformational preference for FLSNOs 8 (n ) 3)
and 9 (n ) 4) based on B3LYP/6-31G**//HF/3-21G* calcula-
tions.
sponds to an increase in fluorescence enhancement (Table
1). As shown in Figure 5, a roughly linear correlation can
be found between the logarithm of R and the logarithm
of “net” fluorescence enhancement (A2/A1 - 1).20 This
further supports the energy transfer rationale for the
described fluorescence enhancement effect.
the relative energies somewhat; nevertheless, the present
calculations indicate that the overlapping conformers
should play an important role in the intramolecular
energy transfer in FLSNOs 9-12.
The preceding survey of conformational preferences
allows a meaningful comparison of the dansyl-to-SNO
distances R in these structures (Table 2). It is interesting
that the preference for an overlapping conformation
(positive ∆E value) corresponds to a decrease in the R
value, which also reaches a minimum for n ) 4 (∼4.7 Å
for 9 and 12). This suggests that the dansyl-to-SNO
approach is indeed optimal at this linker length. The
relatively rigid Ar-S(O)2-NH unit is a contributing
factor in determining the optimal length. The upright
orientation of SO2NH allows the alkyl chain on the
sulfonamide nitrogen to be oriented either away from or
toward the dansyl ring, with only a small energy increase
for the latter (Figure 2). A short alkyl chain (n ) 2)
prevents the SNO moiety from reaching the dansyl ring
primarily because of steric repulsion, leading to a sig-
nificantly larger R value. In FLSNO 8, three methylene
units allows for closer dansyl-to-SNO approach (R ) 6.33
Å) than that in 7 (n ) 2). On the other hand, too long a
linker (n g 5) with an anti orientation slightly increases
the R values for 10 and 11, which weakens the attractive
interaction between the SNO moiety and the dansyl
ring.19 Clearly the length of the alkyl linker significantly
influences the dansyl-to-SNO approach by switching the
balance between steric repulsion and attractive interac-
tion.
Kin etic Stu d y of SNO/SH Tr a n sn itr osa tion w ith
F LSNO a n d Its P a r en t Th iol. The exchange of nitroso
group between S-nitrosothiol and thiol (eq 1) has been
regarded as an intracellular pathway of NO transfer and
also as a potential mechanism for the modification of
protein activity.21 It has been known that the SNO/SH
transnitrosation is accomplished through nucleophilic
attack of the nitroso nitrogen by the thiolate anion
generated from thiol, and therefore, factors including the
acidity of the sulfhydryl group, the pH of the reaction
medium, and the stereoelectronic effects (e.g., charge,
steric hindrance, inductive effect) of the substrates would
come into play.10,22 For example, it was reported that the
nucleophilic attack on the nitroso nitrogen atom is
favored by the electron-withdrawing substitution10c as
well as by a positive charge close to the SNO moiety.10i
Because the SNO/SH transnitrosation is rapid and
reversible, most of the previous kinetic studies of NO
transfer between different thiols have been based on
monitoring the UV-visible absorption changes of
RSNOs.10a-f Because of the low molar absorption coef-
ficients of the S-nitroso chromophore (ꢀ340 ) 800 M-1 cm-1
and ꢀ ) 20 M-1 cm-1 around 540-600 nm), however, high
concentrations of RSNOs are often required in these
direct spectroscopic methods.
The large fluorescence enhancement after NO transfer
from FLSNOs 9 and 12 to GSH demonstrates that
fluorescence emission of these FLSNOs is far lower than
that of the corresponding thiol (FL-SH), and this may
Moreover, for the most stable conformations of
FLSNOs 7-12, it is noted that a decrease in R corre-
(19) (a) The interaction between the SNO moiety and the dansyl
ring can be evaluated from the isodesmic reaction FL-SNO + CH3-
CH2-H* f FL-H* + CH3CH2-SNO, where the geometry of the
starred species is derived from the parent C-SNO compound (opti-
mized at the HF/3-21G* level of theory); the C-SNO moiety is replaced
by the C-H bond whose bond length is set at 1.08 Å, and other
geometry parameters (bond angles and dihedral angles) are the same
as those of the C-SNO moiety. The B3LYP/6-31G**//HF/3-21G*
calculation showed that, for FLSNOs 7-12, the stabilizing interactions
are 0.3 (7), 0 (8), 1.0 (9), 0.5 (10), 0.5 (11), and 1.3 (12) kcal/mol,
respectively. (b) Our recent experimental and theoretical study of the
interaction between the aromatic amino acid side chain and S-
nitrosothiols indicated that the binding energy between the aromatic
ring and the SNO moiety is fairly sizable (∼ 5 kcal/mol) and is probably
electrostatic by nature. Vignini, A.; Akhter, S.; Wen, Z.; Schlegel, H.
B.; Wang, P. G.; Mutus, B. Manuscript submitted for publication.
(20) It should be noted that Forster’s equation (eq 3) describes a
quantitative relationship between the energy transfer efficiency and
the average value of donor-to-acceptor distance, where the relative
spatial orientation of the transition dipoles of the donor and acceptor
also plays a role. Because of the flexibility of the alkyl linker in
FLSNOs 7-12 as well as the multiple polarizations for many chro-
mophores, a constant value of κ2 (2/3) is often adopted (Haas, E.;
Katchalski-Katzir, E.; Steinberg, I. Z. Biochemistry 1978, 17, 5064.),
and this allows direct correlation between ln(A2/A1 - 1) and ln(R).
(21) Stamler, J . S. Curr. Top. Microbiol. Immunol. 1995, 196, 19.
(22) (a) Butler, A. R.; Flitney, F. W.; Williams, D. L. H. Trends
Pharmacol. Sci. 1995, 16, 18. (b) Huok, J .; Singh, R.; Whitesides, G.
M. Methods Enzymol. 1987, 143, 129. (c) Whitesides, G. M.; Lilburn,
J . E.; Szajewski, R. P. J . Org. Chem. 1977, 42, 332.

Fluorophore-Labeled S-Nitrosothiols
J . Org. Chem., Vol. 66, No. 18, 2001 6069
Ta ble 3. Secon d -Or d er Ra te Con sta n ts (k2, M-1 s-1) for
th e RSNO/R′SH Tr a n sn itr osa tion (Eq 1) Deter m in ed by
Usin g F LSNO 12 a n d Its P a r en t Th iola
F igu r e 6. Quenching of intracellular fluorescence (λex ) 355
nm and λem ) 515 nm, A ) 0.501) in human umbilical vein
endothelial cells (HUVECs, preincubated with FLSNO 12)
through introduction of extracellular GSNO (100 µM). The
fluorescence intensity (intensity/µm2) was calculated by North-
ern Eclipse Software 5.0. The results were averaged from
quadruplicate studies.
a
All reactions were carried out in PBS buffer (containing 20%
MeOH, 1 mM EDTA, pH 7.4) at 25 °C.
be used for quantitative analysis of FL-SH. In the
present study, FLSNO 12 was selected for the investiga-
tion of the kinetics of NO transfer between different
thiols, including N-acetyl-L-cysteine, L-penicillamine, N-
acetyl-L-penicillamine (NAP), GSH, heptapeptide N-
acetyl-Glu-Ala-Leu-Phe-Gln-Cys-Gly (Ac-EALFQCG), and
bovine serum albumin (BSA). The transnitrosation kinet-
ics was also examined between the parent thiol of 12 and
S-nitrosothiols of biological significance, including S-
nitrosoglutathione (GSNO), S-nitroso-N-acetyl-L-penicil-
lamine (SNAP), and Glu-1-SNAP. By monitoring the
fluorescence change in the PBS buffer, the pseudo-first-
order rate constants (koff) were obtained in the presence
of a large excess of RSNO or RSH. The second-order rate
constants (k2) were obtained by plotting the koff values
as a function of [RSNO]0 or [RSH]0. In all of the kinetics
experiments, the concentration of FLSNO 12 or its parent
thiol was around 40 µM. The results of this study are
summarized in Table 3.
As shown in Table 3, the k2 values between FLSNO
12 and low molecular weight thiols were small, ranging
from 0.11 to 3.36 M-1 s-1. The magnitude of these data
is consistent with results obtained by other methods.10
The rate constant for the cysteine sulfhydryl in N-acetyl
heptapeptide was quite close to that for tripeptide GSH,
indicating their similar polar and steric environments.
The NO transfer from 12 to BSA showed the largest rate
constant in all of our experiments, comparable with the
reported value for S-nitrosocysteine to BSA (52 M-1
s-1).10i This relatively high reactivity of BSA in S-
nitrosation has been attributed to the fact that the free
sulfhydryl (Cys-34) is located in a hydrophobic pocket of
the amino terminal domain and is in close proximity to
anionic charge.
be large because of semirigid carbamide units on both
sides of the cysteine unit. The electrostatic repulsion
between the carboxylate in SNAP and anionic thiolate
(nucleophile) would, therefore, more strongly retard the
nucleophilic attack than in GSNO. A similar mechanism
may account for the much larger k2 value for Glu-1-
SNAP than for SNAP. Glu-1-SNAP is essentially neu-
tral, and therefore, the attack of its nitroso nitrogen by
thiolate would be easier than in SNAP despite their
similar steric hindrance (two methyl groups).
F lu or escen ce Micr oscop ic Stu d y of F LSNO a s a
P r obe for th e In flu x of RSNO-Bou n d NO th r ou gh
th e Cellu la r Mem br a n e. Because of its high reactivity,
NO cannot exist in vivo mainly as a free radical. Instead,
S-nitrosylated serum albumin and GSNO that are found
in high concentrations in vivo may serve as NO carriers
in the serum and in the cytosol.23 The transfer of RSNO-
bound NO conceivably should play a key role in physi-
ological functions of NO. In this work, the large fluores-
cence enhancement for FLSNO 12 was used for probing
the influx of NO in human umbilical vein endothelial cells
(HUVECs). After HUVECs were loaded with FLSNO 12
and the extracellular FLSNO was removed, the digitized
fluorescence images of the cells were obtained via a
fluorescence microscope, and the fluorescence intensity
of HUVECs was calculated and corrected for background.
Upon the introduction of extracellular GSNO (100 µM),
the changes in both the image and the intensity of
intracellular fluorescence were monitored with respect
to time (Figure 6).
As shown in Figure 6, HUVECs exhibit strong intra-
cellular fluorescence after being loaded with FLSNO 12,
indicating the formation of fluorescent thiol via denitro-
sation of FLSNO 12. As reported in our recent study, this
intracellular fluorescence was due to the transnitrosation
from FLSNO to intracellular thiols and, therefore, could
be an index of intracellular thiol level.13 On the other
hand, this also suggests that the HUVEC membrane is
Reaction kinetics of the transnitrosation between the
parent thiol of 12 and RSNO showed that the k2 values
of GSNO and Glu-1-SNAP are around 20-fold larger
than those of SNAP. This may be related to the difference
in the electrostatic interaction between the environment
of the SNO moiety and the attacking thiolate anion. At
pH 7.4, both SNAP and GSNO are anionic because of the
deprotonation of the carboxylic group. The anionic R-car-
boxylate group is close to the SNO moiety in SNAP,
whereas in GSNO this carboxylate-SNO distance would
(23) (a) Marks, D. S.; Vita, J . D. F.; Kneaney, J . F., J r.; Welch, G.
N.; Loscalzo, J . J . Clin. Invest. 1995, 96, 2630. (b) Liu, Z.; Rudd, M.
A.; Freedman, J . E.; Loscalzo, J . J . Pharmacol. Exp. Ther. 1998, 284,
526.

6070 J . Org. Chem., Vol. 66, No. 18, 2001
Chen et al.
stirred for 30 min, followed by the addition of N-acetyl
homocysteine thiolactone (481 mg, 3 mmol) in DMF (15 mL).
The solution was warmed to room temperature, stirred for
another 5 h, and quenched with water. The aqueous phase
was extracted with CHCl3 (50 mL × 3), and the combined
organic solution was dried over anhydrous Na2SO4, concen-
trated, and purified via flash chromatography (5% MeOH in
CHCl3) to give thiol 13 (545 mg, 60%). 1H NMR (300 MHz,
CDCl3): δ 9.89 (s, 1H), 8.48 (d, J ) 1.5 Hz, 1H), 8.12 (d, J )
1.5 Hz, 1H), 7.79 (d, J ) 9.0 Hz, 1H), 7.66 (d, J ) 9.0 Hz, 1H),
7.43-7.59 (m, 1H), 7.07 (m, 1H), 4.48-4.60 (m, 1H), 2.25-
2.38 (m, 2H), 1.80-1.95 (m, 2H), 1.75 (s, 3H). 13C NMR (75
MHz, CD3OD): δ 170.8, 170.7, 149.0, 145.2, 136.6, 135.8, 129.6,
128.7, 123.7, 121.5, 115.9, 52.7, 36.8, 23.0, 21.0. HRMS calcd
for C15H17O2N3S, 303.1041; found, 303.1042.
To a solution of 13 (150 mg, 0.5 mmol) in MeOH (5 mL)
was added tert-butyl nitrite (1.5 mL) dropwise over 5 min with
stirring at -20 °C in the dark. The solution was stirred at
-20 °C for 2 h and concentrated at room temperature in the
dark to give 1 as a red oil. 1H NMR (400 MHz, CD3OD): δ
8.77 (dd, J ) 4.5, 1.6 Hz, 1H), 8.38 (d, J ) 2.4 Hz, 1H), 8.36
(d, J ) 8.7 Hz, 1H), 7.96 (d, J ) 9.0 Hz, 1H), 7.86 (dd, J ) 9.0,
2.4 Hz, 1H), 5.40 (dd, J ) 8.1, 4.5 Hz, 1H), 4.69 (dd, J ) 9.0,
5.6 Hz, 1H), 2.54-2.68 (m, 2H), 2.07-2.20 (m, 2H), 2.05 (s,
3H). 13C NMR (100 MHz, CD3OD): δ 172.5, 171.6, 148.0, 143.2,
138.3, 137.4, 129.2, 127.3, 124.9, 121.8, 116.4, 53.4, 36.4, 21.3,
20.3. EIMS: 333 (M+ + 1).
permeable by FLSNO 12. Upon subsequent addition of
extracellular GSNO, the fluorescence of HUVECs was
rapidly quenched; the decrease in fluorescence intensity
in the first 2 s accounts for about half of the total loss.
This fluorescence quenching should be attributed to the
NO transfer from extracellular GSNO to fluorescent
dansyl-labeled thiol in the cytosol, where 12 is regener-
ated.
So far, the chemistry of NO transport through the
cellular membrane has not yet been clarified. Previously,
it was suggested that the NO free radical, owing to its
charge neutrality and relatively low reactivity, diffuses
freely across the cell membrane.24 On the basis that the
cell membrane contains a significant thiol pool,25 it has
recently been shown that the cellular entry of NO
involves an SNO/SH transnitrosation that is catalyzed
by cell surface protein disulfide isomerase (PDI).26 The
present result demonstrated that the reversible transni-
trosation-induced change in fluorescence intensity be-
tween 12 and its parent thiol is a fairly sensitive probe
of the intracellular GSNO-bound NO transfer, and thus,
this type of FLSNO can be applied in further mechanistic
studies of RSNO-bound NO transport through cellular
membranes.
In summary, the present survey of twelve fluorophore-
labeled S-nitrosothiols revealed that the largest increase
in fluorescence intensity upon removal of the nitroso
moiety was found for dansyl-linked S-nitrosothiols
(FLSNOs 9 and 12) where the linker (alkyl chain)
contains four methylene units. The degree of fluorescence
enhancement depends not only on the spectral overlap
between the fluorophore emission and the SNO absorp-
tion but also on the structure of the linker. A theoretical
study revealed that the dansyl-to-SNO approach in 9 and
12 is indeed energetically most preferred, whereby the
stabilizing interaction between the SNO moiety and
dansyl ring compensates for the steric repulsion better
than with either longer or shorter linkers.
Using dansyl-linked FLSNO 12, the kinetic study of
SNO/SH transnitrosation suggested that a carboxylate
group that is in close proximity to a nitroso nitrogen
disfavors the nucleophilic attack in transnitrosation. The
fluorescence microscopic study showed that the reversible
change in fluorescence intensity for 12 could be a sensi-
tive probe for intracellular RSNO-bound NO transport.
F LSNO 2. This compound was prepared from 2-aminopy-
ridine and N-acetyl homocysteine thiolactone in a manner
similar to that described previously. 1H NMR (400 MHz, CD3-
OD): δ 8.21-8.31 (m, 1H), 7.81-8.05 (m, 2H), 7.15-7.30 (m,
1H), 4.67 (dd, J ) 8.8, 4.8 Hz, 1H), 2.54-2.68 (m, 2H), 2.03 (s,
3H), 1.99-2.19 (m, 2H). 13C NMR (100 MHz, CD3OD): δ 172.7,
172.3, 150.6, 145.0, 141.0, 120.3, 115.1, 53.4, 35.9, 21.2, 20.3.
F LSNO 3. 1,2-Phthalic dicarboxaldehyde (132 mg, 1 mmol),
homocysteine thiolactone hydrochloride (155 mg, 1 mmol), and
NaCN (54 mg, 1.1 mmol) were dissolved in MeOH (5 mL) and
water (5 mL). The mixture was stirred in the dark for 4 h,
followed by the addition of water. The aqueous phase was
extracted with CH2Cl2. The organic phase was dried over
anhydrous Na2SO4. After the removal of solvent, the residue
was purified by silica gel column chromatography (hexanes/
EtOAc, 8:2) to give 14 (118 mg, 50%). 1H NMR (400 MHz,
CDCl3): δ 7.65 (d, J ) 8.0 Hz, 1H), 7.60 (d, J ) 8.8 Hz, 1H),
7.33 (s, 1H), 7.24-7.28 (m, 1H), 7.11 (dd, J ) 8.8, 6.4 Hz, 1H),
5.33 (dd, J ) 13.2, 7.4 Hz, 1H), 3.59-3.52 (m, 1H), 3.48-3.42
(m, 1H), 3.06-2.99 (m, 1H), 2.77-2.66 (m, 1H). 13C NMR (100
MHz, CDCl3): δ 200.3, 131.7, 126.3, 124.8, 123.6, 121.0, 118.4,
118.3, 117.7, 114.1, 67.4, 33.1, 27.4.
To a solution of 14 (100 mg, 0.40 mmol) in MeOH (10 mL)
was added NaBH4 (40 mg, 1.2 mmol) in one portion at 0 °C.
The mixture was stirred for 3.5 h and was quenched with
water. The aqueous solution was extracted with EtOAc. The
extract was dried over anhydrous Na2SO4 and concentrated
to give the crude product. Purification was through a silica
gel column (hexanes/EtOAc, 5:5) to give the thiol (67 mg, 65%).
1H NMR (400 MHz, CDCl3): δ 7.64 (d, J ) 8.0 Hz, 1H), 7.62
(d, J ) 7.6 Hz, 1H), 7.48 (s, 1H), 7.26-7.23 (m, 1H), 7.11 (dd,
J ) 8.4, 6.8 Hz, 1H), 4.85-4.80 (m, 1H), 4.04 (dd, J ) 11.6,
6.4 Hz, 1H), 3.95 (d, J ) 11.6 Hz, 1H), 2.50-2.28 (m, 4H). 13C
NMR (100 MHz, CDCl3): δ 131.8, 125.9, 124.6, 123.2, 121.0,
118.2, 118.0, 114.9, 65.1, 61.4, 35.6, 20.9. To a solution of the
thiol (51 mg, 0.20 mmol) in MeOH (1.5 mL) and water (0.5
mL) was added HNO2 (1.20 mL, 1.0 M) dropwise over 5 min
at -20 °C in the dark. In the dark, the mixture was stirred
for another 2 h and concentrated at room temperature. The
residue was extracted with absolute anhydrous ethanol (1 mL).
The filtrate was evaporated in the dark to give pure FLSNO
3 in 78% yield. 1H NMR (400 MHz, CDCl3): δ 7.47 (m, 2H),
7.51 (s, 1H), 7.26 (dd, 1H, J ) 8.0, 7.2 Hz), 7.13 (dd, J ) 8.0,
7.2 Hz, 1H), 4.56-4.60 (m, 1H), 4.02 (dd, J ) 11.6, 6.0 Hz,
1H), 3.94 (dd, J ) 11.6, 4.4 Hz, 1H), 3.30-3.46 (m, 2H), 2.36-
2.47 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 131.8, 126.1,
124.7, 123.4, 121.1, 118.3, 117.9, 114.7, 65.0, 61.9, 31.3, 29.3.
Exp er im en ta l Section
Syn th esis of Flu or oph or e-Labeled S-Nitr osoth iols (FL-
SNO 1-12). All chemicals were obtained from commercial
suppliers and used without further purification. All NMR
spectra were recorded on a Varian Unity-500, Varian Unity-
400 MHz (1H), or Mercury 400 MHz spectrometer. NMR
chemical shifts were expressed in ppm relative to internal
solvent peaks, and coupling constants were measured in Hz.
MS spectra were obtained from a Kratos 80 MS spectrometer
(ionization mode: EI, ESI, or FAB). UV-visible spectroscopy
measurements were carried out on a HP 8453 UV-visible
spectrometer (Hewlett-Packard Co.).
F LSNO 1. To a solution of 6-aminoquinoline (436 mg, 3
mmol) in anhydrous DMF (20 mL) was added NaH (115 mg,
3.0 mmol, 60%) at 0 °C under argon. The brown mixture was
(24) Goretski, J .; Hollocher, T. J . Biol. Chem. 1988, 263, 2316.
(25) (a) Ammon, H.; Hagele, R.; Youssif, N.; Eujen, R.; El-Amri, N.
Endocrinology 1983, 112, 720-726. (b) Redelman, D.; Hudig, D. Adv.
Exp. Med. Biol. 1985, 184, 527.
(26) Zai, A.; Rudd, A.; Scribner, A. W.; Loscalzo, J . J . Clin. Invest.
1999, 103, 393.

Fluorophore-Labeled S-Nitrosothiols
J . Org. Chem., Vol. 66, No. 18, 2001 6071
F LSNO 4. This compound was prepared from dansyl
chloride and homocysteine thiolactone in a manner similar to
that described previously. 1H NMR (400 MHz, CD3OD): δ 8.94
(d, J ) 8.8 Hz, 1H), 8.70 (d, J ) 8.8 Hz, 1H), 8.43 (d, J ) 7.6
Hz, 1H), 8.11 (d, J ) 7.2 Hz, 1H), 7.85-7.92 (m, 2H), 3.48 (s,
6H), 3.27-3.40 (m, 3H), 3.05-3.31 (m, 2H), 1.41-1.75 (m, 2H).
13C NMR (100 MHz, CD3OD): δ 139.5, 138.0, 130.6, 129.5,
127.7, 127.5, 127.0, 126.1, 125.8, 119.5, 64.3, 54.8, 46.8, 30.3,
29.6.
F LSNO 5. This compound was prepared from dansyl
chloride and S-trityl penicillamine methyl ester. 1H NMR (400
MHz, CD3OD): δ 8.53-8.57 (m, 2H), 8.24 (d, J ) 6.4 Hz, 1H),
7.62-7.68 (m, 2H), 7.61 (d, J ) 7.6 Hz, 1H), 3.76 (s, 1H), 3.01
(s, 6H), 2.97 (s, 3H), 1.32 (s, 3H), 1.29 (s, 3H). 13C NMR (100
MHz, CD3OD): δ 169.7, 135.7, 130.1, 129.7, 129.3, 127.9, 123.9,
121.4, 116.1, 66.0, 50.7, 45.1, 44.7, 29.4, 27.6. EIMS: 426 (M+
+ 1).
necked round flask at -40 °C was added the above amine
(2.236 g, 10 mmol), followed by the addition of sodium metal
(1.25 g) in several portions to keep the reaction mixture blue.
Water (2 mL) was added to quench the reaction after 1.5 h,
and the mixture was allowed to warm to room temperature,
while nitrogen was bubbled to evaporate ammonia gas. The
residue was dissolved in water. The aqueous solution was
extracted with CHCl3 (30 mL × 3), and the combined organic
phase was dried over Na2SO4. After the removal of solvent,
the crude product was purified with silica gel column chro-
matography (CH2Cl2/MeOH 10:1) to give 17 as a colorless oil
1
(0.904 g, 70%). H NMR (300 MHz, CDCl3): δ 2.64 (br s, 2H),
1.66 (br s, 3H), 11.44 (m, 4H), 1.29 (s, 6H). 13C NMR (75 MHz,
CDCl3): δ 44.7, 43.9, 42.5, 33.0, 29.6. EIMS: 103 (M+, 50), 89
(36), 74 (72), 67 (35), 59 (33).
To a solution of 17 (200 mg, 1.5 mmol) and 4-(dimethylami-
no)pyridine (365 mg, 3 mmol) in anhydrous CH2Cl2 (10 mL)
was added a solution of dansyl chloride (375 mg, 1.4 mmol) in
CH2Cl2 (10 mL) dropwise over 20 min at 0 °C. The mixture
was stirred for another 30 min from 0 °C to room temperature.
After the removal of the solvent, the residue was purified via
flash chromatography (hexanes/EtOAc 8:2) to give thiol 18 as
a green oil (370 mg, 71%). 1H NMR (300 MHz, CDCl3): δ 8.51
(d, J ) 8.4 Hz, 1H), 8.34 (d, J ) 8.4 Hz, 1H), 8.24 (dd, J ) 7.5,
1.2 Hz, 1H), 7.46-7.55 (m, 2H), 7.15 (d, J ) 7.2 Hz, 1H), 5.31
(t, J ) 6.3 Hz, 1H), 2.92 (td, J ) 6.6, 6.3 Hz, 2H), 2.85 (s, 6H),
1.37-1.45 (m, 2H), 1.23-1.32 (m, 2H), 1.1 (s, 6H). 13C NMR
(75 MHz, CDCl3): δ 152.2, 135.2, 130.6, 130.1, 129.9, 129.8,
128.6, 123.4, 119.2, 115.5, 45.7, 44.2, 43.7, 43.4, 32.6, 25.7.
ESIMS: 771 ((2M + K)+), 755 ((2M + Na)+), 405 ((M + K)+),
389 ((M + Na)+).
F LSNO 6. The mixture of 3,3-dimethyl acrylaldehyde (21.8
g), benzyl mercaptan (35 mL), and piperidine (1.5 mL) was
heated by steam bath for 6.5 h. The Michael addition product
was purified by distillation under reduced pressure. The
aldehyde (31.6 g, 60%) was collected at the bp 109-110 °C/15
mmHg. 1H NMR (CDCl3, 300 MHz): δ 9.82 (t, J ) 2.7 Hz,
1H), 7.23-7.35 (m, 5H), 3.79 (s, 2H), 2.545 (d, J ) 3.0 Hz,
2H), 1.46 (s, 6H). To a stirred slurry of NaBH4 (3.05 g) in ethyl
ether (200 mL) at 0 °C was added the above aldehyde (26.2 g)
in methanol (15 mL) dropwise over 2 h. The reaction mixture
was stirred at 0 °C for another 2 h, followed by the evaporation
of solvent, and the residue was dissolved in water. The aqueous
solution was extracted with Et2O (100 mL × 3), and the
combined ether solution was dried over anhydrous Na2SO4.
After the removal of solvent, the residue was purified by
distillation to give alcohol 15 (19.0 g, bp 135-137 °C/1.5
mmHg, 72%). 1H NMR (300 MHz, CDCl3): δ 7.22-7.35 (m,
5H), 3.82 (dt, J ) 6.6 Hz, 5.7 Hz, 2H), 3.77 (s, 2H), 2.40 (t, J
) 5.7 Hz), 1.85 (t, J ) 6.6 Hz), 1.36 (s, 6H). HRMS calcd for
Following procedures described previously, FLSNO 6 was
1
prepared from 18 (92%). H NMR (400 MHz, CD3OD): δ 8.89
(d, 1H, J ) 8.8 Hz), 8.75 (d, J ) 8.8 Hz, 1H), 8.32 (d, J ) 7.2
Hz, 1H), 8.19 (d, J ) 8.0 Hz, 1H), 7.82-7.88 (m, 2H), 3.54 (s,
6H), 2.94 (t, J ) 6.4 Hz, 2H), 1.90 (td, J ) 8.0, 4.0 Hz, 2H),
1.64 (s, 6H), 1.39-1.44 (m, 2H). 13C NMR (100 MHz, CD3OD):
δ 139.2, 137.9, 130.1, 129.4, 127.7, 127.6, 127.0, 126.0, 125.8,
119.8, 56.1, 46.9, 42.9, 40.1, 27.9, 25.0. ESIMS: 813 ((2M +
Na)+), 791 ((2M + H)+), 434 ((M + K)+), 418 ((M + Na)+), 396
((M + H)+).
C
12H18OS, 210.1078; found, 210.1082.
To a solution of 15 (6.05 g, 30 mmol) in anhydrous THF (20
mL) at -78 °C under nitrogen was added BuLi (19.0 mL, 1.6
M in hexane). The reaction mixture was stirred for another
0.5 h, followed by the addition of toluenesulfonyl chloride (5.75
g, 30 mmol) in THF (30 mL) in one portion, and warmed to
room temperature. After 0.5 h the solvent was removed at -78
°C. The residue was purified via flash chromatography (hex-
anes/EtOAc, 9:1) to give the tosylate as an oil (9.57 g, 95%).
1H NMR (300 MHz, CDCl3): δ 7.79 (d, J ) 7.8 Hz, 2H), 7.34
(d, J ) 7.8 Hz, 2H), 7.21-7.28 (m, 5H), 4.20 (t, J ) 7.2 Hz,
2H), 3.62 (s, 2H), 2.44 (s, 3H), 1.92 (t, J ) 7.2 Hz, 2H), 1.28 (s,
6H). A mixture of the above tosylate (11.6 g, 32.7 mmol) and
NaCN (2.450 g, 50 mmol) in DMF (60 mL) was stirred at 70
°C overnight. The solution was cooled to room temperature
and diluted with water (200 mL). The aqueous solution was
extracted with Et2O (100 mL × 3), and the combined organic
phase was dried over anhydrous Na2SO4. Removal of solvent
gave the crude product, which was purified with silica gel
column chromatography (hexane/EtOAc 20:1) to give 16 (6.25
g, 90%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3): δ
7.25-7.33 (m, 5H), 3.69 (s, 2H), 2.44 (t, J ) 7.8 Hz, 2H), 1.83
(t, J ) 7.8 Hz, 2H), 1.33 (s, 6H). 13C NMR (75 MHz): δ 138.1,
129.1, 128.9, 127.4, 120.3, 45.7, 37.5, 33.3, 28.8, 13.3. HRMS
calcd for C13H17NS, 219.1082; found, 219.1083.
To a solution of LiAlH4 (1.15 g, 30 mmol) in THF was added
the solution of 16 (6.25 g, 28.4 mmol) in THF (40 mL) at 0 °C
in 30 min. The mixture was refluxed for 6 h, cooled to 0 °C,
and quenched by water (1 mL), 15% NaOH (1.5 mL), and water
(5 mL). The mixture was filtered, and the solid residue was
washed three times with hot THF. The combined organic
solution was dried over anhydrous Na2SO4 and concentrated.
The crude product was purified via flash chromatography
(hexanes/EtOAc 4:1) to give the amine (3.32 g, 55%). 1H NMR
(300 MHz, CDCl3): δ 7.17-7.33 (m, 5H), 3.68 (s, 2H), 2.57-
2.62 (m, 2H), 1.49-1.52 (m, 4H), 1.28 (s, 6H), 1.20 (br s, 2H).
13C NMR (75 MHz, CDCl3): δ 138.8, 129.2, 128.6, 127.0, 46.3,
42.8, 39.8, 33.2, 29.3, 29.1. HRMS calcd for C13H21NS, 223.1395;
found, 223.1394. To anhydrous ammonia (60 mL) in a three-
Typ ica l P r oced u r e for t h e Syn t h esis of Da n syl-
La beled S-Nitr osoth iols (F LSNO 9). To a solution of
4-amino-1-butanol (100 mg, 1.1 mmol) and Et3N (0.35 mL) in
anhydrous CH2Cl2 (15 mL) was added dansyl chloride (270 mg,
1 mmol) in CH2Cl2 (10 mL) dropwise over 10 min. The solution
was stirred at room temperature for another 2 h. After the
removal of solvent, the residue was purified by silica gel
column chromatography (hexanes/EtOAc 8:2) to give pure 19
(264 mg, 83%). 1H NMR (300 MHz, CDCl3): δ 8.55 (d, J ) 8.7
Hz, 1H), 8.30 (d, J ) 8.7 Hz, 1H), 8.24 (dd, J ) 7.5, 1.2 Hz,
1H), 7.50-7.58 (m, 2H), 7.19 (d, J ) 7.2 Hz, 1H), 5.20 (t, J )
6.0 Hz, 1H), 3.51 (m, 2H), 2.91-2.93 (m, 2H), 2.90 (s, 3H),
1.46-1.54 (m, 4H).
A solution of 19 (325 mg, 1.0 mmol) and Et3N (1.5 mL) in
anhydrous CH2Cl2 (25 mL) was cooled to -20 °C under
nitrogen atmosphere and stirred for 30 min, followed by the
addition of toluenesulfonyl chloride (195 mg, 1 mmol) in one
portion. The solution was kept in the refrigerator overnight.
After the removal of solvent, the crude product was purified
via flash chromatography (hexanes/EtOAc 8:2) to give the
tosylate as a viscous oil (431 mg, 92%). 1H NMR (300 MHz,
CDCl3): δ 8.51 (d, J ) 8.4 Hz, 1H), 8.27 (d, J ) 8.4 Hz, 1H),
8.18 (d, J ) 7.5 Hz, 1H), 7.68 (d, J ) 7.8 Hz, 2H), 7.45-7.52
(m, 2H), 7.27 (d, J ) 7.8 Hz, 2H), 7.14 (d, J ) 7.8 Hz, 1H),
5.31 (t, J ) 6.3 Hz, 1H), 3.82 (t, J ) 5.7 Hz, 2H), 2.85 (s, 6H),
2.80 (m, 2H), 2.38 (s, 3H), 1.47-1.56 (m, 2H), 1.34-1.43 (m,
2H). 13C NMR (75 MHz, CDCl3): δ 152.2, 145.1, 134.9, 132.9,
130.7, 130.2, 130.0, 129.7, 129.6, 128.7, 128.0, 123.4, 118.9,
115.5, 70.1, 45.6, 42.6, 25.9, 25.8, 21.9. A solution of the
tosylate (256 mg, 0.55 mmol) and potassium thioacetate (250
mg, 2.2 mmol) in acetone (10 mL) was stirred at room
temperature for 4 h, followed by evaporation of solvent. The
residue was purified with silica gel column chromatography
to give 20 with quantitative yield. 1H NMR (300 MHz,

6072 J . Org. Chem., Vol. 66, No. 18, 2001
Chen et al.
CDCl3): δ 8.47 (d, J ) 8.7 Hz, 1H), 8.31 (d, J ) 8.7 Hz, 1H),
8.19 (d, J ) 7.5 Hz, 1H), 7.42-7.49 (m, 2H), 7.10 (d, J ) 7.5
Hz, 1H), 5.61 (t, J ) 6.3 Hz, 2H), 2.84-2.87 (m, 2H), 2.80 (s,
6H), 2.58 (t, J ) 6.3 Hz, 2H), 2.20 (s, 3H), 1.33-1.39 (m, 4H).
13C NMR (75 MHz, CDCl3): δ 196.2, 152.1, 135.2, 130.5, 130.0,
129.8, 129.6, 128.6, 123.4, 119.2, 115.4, 45.6, 42.8, 30.8, 28.7,
28.49, 26.61.
crude product was dissolved in anhydrous THF (10 mL). To
this solution was added MeLi solution (1.0 M in hexane, 4 mL,
4.0 mmol) dropwise at -78 °C under nitrogen. The solution
was stirred for another 2.5 h at -78 °C and quenched by
MeOH. After workup, the crude product was purified by silica
gel chromatography to give 22 as a green oil (207 mg, 60%,
two steps). 1H NMR (300 MHz, CDCl3): δ 8.49 (d, J ) 8.7 Hz,
1H), 8.32 (d, J ) 8.4 Hz, 1H), 8.20 (dd, J ) 7.2, 1.2 Hz, 1H),
7.50-7.44 (m, 2H), 7.12 (d, J ) 8.1 Hz, 1H), 5.87 (t, J ) 7.6
Hz, 1H), 3.58-3.52 (m, 1H), 2.89-2.81 (m, 2H), 2.83 (s, 6H),
2.49 (br s, 1H), 1.46-1.39 (m, 2H), 1.28-1.20 (m, 2H), 0.95 (d,
J ) 6.0 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 152.0, 135.1,
130.5, 130.0, 129.8, 129.6, 128.6, 123.4, 119.2, 115.4, 67.6, 45.6,
43.4, 40.0, 36.0, 23.6.
FLSNO 12 can be obtained from alcohol 22 by following the
procedure for the synthesis of 9. 1H NMR (300 MHz, CD3OD):
δ 8.90 (d, J ) 9.3 Hz, 1H), 8.78 (d, J ) 8.7 Hz, 1H), 8.34 (d, J
) 6.6 Hz, 1H), 8.22 (d, J ) 7.6 Hz, 1H), 7.84-7.92 (m, 2H),
4.05-4.15 (m, 1H), 3.56 (s, 1H), 2.87 (t, J ) 6.3 Hz, 2H), 1.20-
1.28 (m, 2H), 1.33-1.51 (m, 2H), 1.156 (d, J ) 7.2 Hz, 3H).
13C NMR (75 MHz, CD3OD): δ 139.1, 137.8, 130.2, 129.4,
127.7, 127.6, 127.0, 126.0, 125.8, 119.9, 46.8, 42.2, 32.4, 26.9,
19.2. ESIMS: 785 ((2M + Na)+), 763 ((2M + H)+), 421 ((M +
K)+), 404 ((M + Na)+), 382 ((M + 1)+ ).
To a solution of 20 (195 mg, 0.51 mmol) in anhydrous CH2-
Cl2 (10 mL) at -78 °C under argon atmosphere was added
DIBALH (2.2 mL, 1 M in hexanes) dropwise over 10 min. The
solution was stirred at -78 °C for another 2 h and quenched
with cold, diluted hydrochloric acid. The aqueous phase was
extracted with Et2O, and the combined organic solution was
dried over anhydrous Na2SO4 and concentrated. The crude
product was purified via flash chromatography (hexane/EtOAc
1
8:2) to give thiol 21 in 70% yield. H NMR (300 MHz, CDCl3):
δ 8.53 (d, J ) 8.7 Hz, 1H), 8.30 (d, J ) 9.0 Hz, 1H), 8.23 (dd,
J ) 7.5 Hz, 1.2 Hz, 1H), 7.48-7.56 (m, 2H), 7.16 (d, J ) 6.9
Hz, 1H), 5.28 (t, J ) 6.3 Hz, 1H), 2.87-2.92 (m, 2H), 2.87 (s,
6H), 2.23-2.30 (m, 2H), 1.40-1.45 (m, 4H). 13C NMR (75 MHz,
CDCl3): δ 152.2, 135.0, 130.7, 130.1, 129.8, 129.7, 128.7, 123.4,
119.0, 115.5, 45.7, 42.9, 30.9, 28.4, 24.2. HRMS calcd for
C
16H22O2N2S2, 338.1123; found, 338.1126.
To a solution of thiol 21 (121 mg, 0.35 mmol) in MeOH (1.5
mL) and water (0.5 mL) at -20 °C in the dark was added
HNO2 (1.30 mL, 1.0 M) dropwise over 5 min. The mixture was
stirred for another 2 h, followed by evaporation at room
temperature in the dark. The residue was extracted with
absolute anhydrous ethanol (1 mL). The filtrate was evapo-
rated in the dark to give pure 9 in 85% yield. 1H NMR (400
MHz, CD3OD): δ 8.88 (d, J ) 8.8 Hz, 1H), 8.67 (d, J ) 8.8 Hz,
1H), 8.35 (d, J ) 6.4 Hz, 1H), 8.16 (d, J ) 8.4 Hz, 1H), 7.83-
7.91 (m, 2H), 3.53 (s, 6H), 3.35-3.54 (m, 2H), 2.89 (t, J ) 6.6
Hz, 2H), 1.28-1.42 (m, 4H). 13C NMR (100 MHz, CD3OD): δ
139.3, 137.9, 130.1, 129.4, 127.5, 127.4, 126.9, 126.0, 125.6,
119.6, 46.7, 42.1, 37.6, 28.4, 25.9. ESIMS: 368 (M+ + 1).
F LSNO 7. This compound was prepared in a manner
Gen er a l P r oced u r e for Eva lu a tin g th e F lu or escen ce
En h a n cem en t of Syn th etic F lu or op h or e-La beled S-Ni-
tr osoth iols. The fluorescence spectra were recorded with a
SPEX FluoroMax spectrometer. All reactions were carried out
in PBS buffer (1.0 mM EDTA, 20% MeOH). In the experiment
of transnitrosation with free thiols (method A), the fluorescence
emission spectrum of the substrate (40 µM) in the buffer was
measured to record the emission intensity (A1), then excess
GSH (50 equiv) was added, and the fluorescence emission was
monitored over time, from which the maximum intensity (after
around 40 min) was recorded (A2) to derive fluorescence
enhancement ratio (A2/A1). Photolytic denitrosation (method
B) was performed as follows: the solution of FLSNO was
exposed to UV (365 nm) irradiation, during which the time-
dependent fluorescence intensity was recorded. The maximum
emission intensity (B2) was compared with the intensity of
fluorescence emission recorded before irradiation (B1) to derive
the fluorescence enhancement ratio (B2/B1).
1
similar to that described above. H NMR (300 MHz, CD3OD):
δ 8.88 (d, J ) 8.4 Hz, 1H), 8.67 (d, J ) 8.7 Hz, 1H), 8.33 (d, J
) 7.2 Hz, 1H), 7.84-7.93 (m, 2H), 3.52 (s, 6H), 3.45-3.25 (m,
2H), 3.05 (t, J ) 6.6 Hz, 2H). 13C NMR (75 MHz, CD3OD): δ
139.4, 137.4, 130.2, 129.5, 127.6, 127.4, 126.9, 125.8, 119.6,
46.7, 41.5, 37.1. ESIMS: 378 (M+ + K), 362 (M+ + Na), 340
(M+ + 1).
Gen er a l P r oced u r e for th e Kin etic Stu d y of Tr a n sn i-
tr osa tion Usin g F LSNO 12. All materials were purified
before use. Following the literature procedure, S-nitroso-N-
acetylpenicillamine (SNAP),27 Glu-1-SNAP,14a and N-Ac-
EALFQCG28 were synthesized. Preparation of GSNO is de-
scribed in the next section. All reactions were carried out in
PBS buffer (containing 20% methanol and 1 mM EDTA, pH
F LSNO 8. This compound was prepared in a manner
1
similar to that described above. H NMR (300 MHz, CD3OD):
δ 8.92 (d, J ) 8.7 Hz, 1H), 8.64 (d, J ) 8.7 Hz, 1H), 8.36 (d, J
) 7.5 Hz, 1H), 8.15 (d, J ) 7.5 Hz, 1H), 7.86-7.93 (m, 2H),
3.51 (s, 6H), 3.25-3.55 (m, 2H), 2.87 (t, J ) 6.9 Hz, 2H), 1.54-
1.64 (m, 2H). 13C NMR (75 MHz, CD3OD): δ 139.4, 137.5,
130.2, 129.4, 127.6, 127.4, 126.9, 126.1, 125.7, 119.6, 46.7, 41.5,
7.4). The fluorescence emission spectra (λex ) 330 nm, λem
)
540 nm) were obtained using a SPEX FluoroMax spectrometer.
First, it was confirmed that the fluorescence intensity of the
parent thiol of 12 responds linearly to the concentration of thiol
in the presence of excess GSH (Supporting Information). The
pseudo-first-order rate constant k0 was determined by reacting
FLSNO 12 with a ∼15-35-fold amount of thiol or by reacting
the thiol of 12 with a ∼15-35-fold amount of S-nitrosothiol.
All reactions were carried out at 25 °C, and the initial
concentration of 12 or its thiol was 40 µM. During the reaction,
the fluorescence intensity of the mixture was measured at 525
nm every 2 min. Very good first-order behavior was generally
found in this study. The second-order rate constant k2 for each
reaction was obtained by plotting k0 as a function of [RSNO]0
or [RSH]0.
34.7, 28.9. ESIMS: 392 (M+ + K), 376 (M+ + Na), 354 (M+
1).
+
F LSNO 10. This compound was prepared in a manner
1
similar to that described above. H NMR (300 MHz, CD3OD):
δ 8.92 (d, J ) 8.4 Hz, 1H), 8.64 (d, J ) 9.0 Hz, 1H), 8.55 (dd,
J ) 7.6, 1.2 Hz, 1H), 8.16 (d, J ) 7.6 Hz, 1H), 7.85-7.91 (m,
2H), 3.51 (s, 6H), 3.30-3.45 (m, 2H), 2.86 (t, J ) 6.6 Hz, 2H),
1.29-1.36 (m, 4H), 1.10-1.28 (m, 2H). 13C NMR (75 MHz, CD3-
OD): δ 139.5, 137.8, 130.0, 129.5, 127.5, 127.4, 126.9, 126.1,
125.5, 119.5, 46.7, 42.4, 38.0, 28.3, 28.2, 25.4. EIMS: 382 (M+
+ 1).
F LSNO 11. This compound was prepared in a manner
1
similar to that described above. H NMR (300 MHz, CD3OD):
δ 8.93 (d, J ) 9.0 Hz, 1H), 8.76 (d, J ) 8.7 Hz, 1H), 8.35 (d, J
) 7.2 Hz, 1H), 8.21 (d, J ) 7.8 Hz, 1H), 7.85-7.92 (m, 2H),
3.54 (s, 6H), 3.35-3.45 (m, 2H), 2.84 (t, J ) 7.0 Hz, 2H), 1.27-
1.38 (m, 2H), 1.07-1.17 (m, 2H). 13C NMR (75 MHz, CD3OD):
δ 139.0, 137.8, 130.1, 129.5, 127.7, 127.6, 127.0, 125.9, 125.7,
119.8, 46.8, 42.6, 32.7, 29.2, 28.6, 27.9, 25.7.
Flu or escen ce Micr oscopic Stu dy of In tr acellu lar Flu o-
r escen ce Qu en ch in g w ith th e Use of F LSNO 12. (A)
P r ep a r a tion of GSNO. To a solution of GSH in ice cold 0.5
M HCl was added equimolar sodium nitrite in the dark at 4
°C over 40 min. The pH of the reaction was adjusted to 7.0,
F LSNO 12. To a solution of 19 (350 mg, 1.1 mmol) in
anhydrous CH2Cl2 (20 mL) was added pyridinium dichromate
(1.6 equiv) in several portions over 3 h at 0 °C until the
substrate disappeared. The mixture was filtered through a
short Celite column, and the filtrate was concentrated. The
(27) Field, L.; Dilts, R.; Ravichandran, R.; Lenhert, P. G.; Carnahan,
G. E. J . Chem. Soc., Chem. Commun. 1978, 249.
(28) Xian, M.; Wang, Q. M.; Chen, X.; Wang, K.; Wang, P. G. Bioorg.
Med. Chem. Lett. 2000, 10, 2097.

Fluorophore-Labeled S-Nitrosothiols
J . Org. Chem., Vol. 66, No. 18, 2001 6073
and the product GSNO was crystallized by the slow addition
of cold acetone.
analysis was performed to verify the nature of each structure
and to obtain zero-point energies (ZPEs) and thermal correc-
tions to enthalpy. For conformations within 2 kcal/mol of the
most stable structure, electron correlation effects were incor-
porated by single-point calculations at the B3LYP/6-31G**
level to identify the minimum energy conformation in the gas
phase. The structures, electronic energies, and thermal cor-
rections to enthalpies of all compounds (FLSNO 7-12) are
provided in the Supporting Information.
(B) Cell Cu ltu r e. Human umbilical vein endothelial cells
(HUVEC-CRL 1730) were grown in Gibco’s Ham F12K with
L-glutamine (2 mM), sodium carbonate (1.5 g/L), heparin (100
µg/mL), endothelial cell growth supplement (50 µg), and fetal
bovine serum (10%). The HUVECs were grown to confluence
on slides (Erie Scientific) at 37 °C with atmospheric CO2 (10%).
(C) F lu or escen ce Micr oscop y. HUVECs were loaded with
FLSNO 12 (A ) 0.501) for 2 min. Following a 4 × 30 s wash,
GSNO (100 µM) was introduced. Fluorescence quenching
images were obtained every 300 ms via a Zeiss Axiovert 35
microscope. The fluorescence intensity (intensity/µm2) was
calculated and corrected for background by a data acquisition
system (Northern Eclipse 5.0).
Ack n ow led gm en t. This work is generously sup-
ported by a grant from the NIH (GM 54074). Z.W.
thanks Mr. J ason Cross and Dr. J oanne M. Wittbrodt
for their generous technical support. H.B.S. acknowl-
edges support from the NSF (Grant CHE 9874005).
Th eor etica l Ca lcu la tion s. All calculations were performed
using the Gaussian 98 program package.29 A conformational
search was carried out for each molecule by full optimization
at the HF/3-21G* level as mentioned in the text. A frequency
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C NMR spec-
tra for FLSNOs 1-12; mass spectra for FLSNOs 1, 5-10, and
12; plot of fluorescence intensity versus the concentration of
parent thiol of FLSNO 12 in the presence of excess GSH; plots
of the time course of fluorescence intensity in the transnitro-
sation between 12 and GSH as well as between GSNO and
the parent thiol of 12; plot of the pseudo-first-order rates
versus the initial concentrations of GSH in the transnitrosa-
tion between 12 and GSH; relaxed potential energy surface
for the rotation of the SO2NH2 group around the C-S bond in
5-(dimethylamino)-1-naphthalenesulfonamide (HF/3-21G*); HF/
3-21G* optimized structures, total electronic energies com-
puted at the B3LYP/6-31G** level and thermal corrections to
enthalpy for FLSNOs 7-12. This material is available free of
charge via the Internet at http://pubs.acs.org.
(29) Frisch, M. J .; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J . R.; Zakrzewski, V. G.; Montgomery, J . A.,
J r.; Stratmann, R. E.; Burant, J . C.; Dapprich, S.; Millam, J . M.;
Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J .;
Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo,
C.; Clifford, S.; Ochterski, J .; Petersson, G. A.; Ayala, P. Y.; Cui, Q.;
Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J . B.; Cioslowski, J .; Ortiz, J . V.; Baboul, A. G.; Stefanov,
B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.;
Martin, R. L.; Fox, D. J .; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; J ohnson, B.; Chen,
W.; Wong, M. W.; Andres, J . L.; Gonzalez, C.; Head-Gordon, M.;
Replogle, E. S.; Pople, J . A. Gaussian 98, rev. A.9; Gaussian, Inc.:
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