Development of a second generation coenzyme a analogue synthon

Article abstract of DOI:10.1021/jo981758s

We have previously reported a general synthetic approach to analogues of coenzyme A (CoA) which involves enzymatic synthesis of a general CoA analogue synthon having a thioester linkage in place of the amide bond nearest the thiol group (Martin et al. J. Am. Chem. Soc. 1994, 116, 4660). We report here the synthesis of a second CoA analogue synthon 1c which has the amide bond more distant from the thiol group replaced with a thioester. This analogue was prepared by nonenzymatic synthesis of a racemic phosphopantetheine analogue followed by enzymatic conversion to the corresponding CoA analogue. Stereochemical analysis showed that the natural enantiomer of the phosphopantetheine analogue was selectively converted to product by the enzyme phosphopantetheine adenylyltansferase, yielding a product that possessed the desired stereoconfiguration. Reaction of the new synthon 1c with a primary amine results in amide bond formation to form the CoA analogue of interest. This new methodology provides access to an even broader array of CoA analogues modified in the β-alanylcysteamine moiety. This has been demonstrated in the synthesis of an analogue having an extra methylene group in the β-alanine moiety and two analogues in which the amide bond nearest the thiol group is replaced with a pair of methylene groups.

Full text of DOI:10.1021/jo981758s

J . Org. Chem. 1999, 64, 2903-2909
2903
Develop m en t of a Secon d Gen er a tion Coen zym e A An a logu e
Syn th on
Richard T. Bibart,† Kurt W. Vogel,† and Dale G. Drueckhammer*
Department of Chemistry, Stanford University, Stanford, California 94305 and Department of Chemistry,
State University of New York at Stony Brook, Stony Brook, New York 11794-3400
Received J anuary 23, 1999
We have previously reported a general synthetic approach to analogues of coenzyme A (CoA) which
involves enzymatic synthesis of a general CoA analogue synthon having a thioester linkage in place
of the amide bond nearest the thiol group (Martin et al. J . Am. Chem. Soc. 1994, 116, 4660). We
report here the synthesis of a second CoA analogue synthon 1c which has the amide bond more
distant from the thiol group replaced with a thioester. This analogue was prepared by nonenzymatic
synthesis of a racemic phosphopantetheine analogue followed by enzymatic conversion to the
corresponding CoA analogue. Stereochemical analysis showed that the natural enantiomer of the
phosphopantetheine analogue was selectively converted to product by the enzyme phospho-
pantetheine adenylyltansferase, yielding a product that possessed the desired stereoconfiguration.
Reaction of the new synthon 1c with a primary amine results in amide bond formation to form the
CoA analogue of interest. This new methodology provides access to an even broader array of CoA
analogues modified in the â-alanylcysteamine moiety. This has been demonstrated in the synthesis
of an analogue having an extra methylene group in the â-alanine moiety and two analogues in
which the amide bond nearest the thiol group is replaced with a pair of methylene groups.
Sch em e 1
We have previously reported a general synthetic ap-
proach to analogues of coenzyme A (CoA) 1a and CoA
esters using a combination of enzymatic and nonenzy-
matic reactions.1,2 This method involves enzymatic syn-
thesis of a general CoA analogue synthon 1b having a
thioester linkage in place of the amide bond nearest the
thiol group of CoA (Scheme 1). For synthetic convenience,
the thiol group is replaced with a methyl group. An
aminolysis reaction is then performed to reform the
amide bond present in CoA and to introduce the func-
tionality of interest in place of the thiol group (Scheme
2). This methodology has been used to prepare mimics
of the enol or enolate intermediate in the reactions of
enzymes which catalyze deprotonation of acetyl-CoA2-4
and of the tetrahedral intermediate in the reactions of
acetyl-CoA-dependent acetyltransferases.5 More recently
this methodology has been employed in the synthesis of
an isomer of propionyl-CoA in which the orientation of
the thioester is reversed relative to that of a natural CoA
thioester.6 This compound was shown to be a time-
dependent inactivator of thiolase and served as a synthon
for certain other acyl-CoA analogues.
Despite its success as a facile route to a wide variety
of CoA analogues, the previously described synthetic
methodology permits modification only in the terminal
* To whom correspondence should be addressed. Phone: (516) 632-
7923, e-mail: dale.drueckhammer@sunysb.edu.
† Stanford University.
(1) Martin, D. P.; Drueckhammer, D. G. J . Am. Chem. Soc. 1992,
114, 7287-7288.
(2) Martin, D. P.; Bibart, R. T.; Drueckhammer, D. G. J . Am. Chem.
Soc. 1994, 116, 4660-4668.
acyl-thioethyl moiety. Reported here is the synthesis of
a new thioester analogue of CoA 1c, in which the more
central amide bond of CoA is replaced by a thioester, and
again the terminal thiol group is replaced by a methyl
group. Analogue 1c was prepared by enzymatic synthesis
from a newly prepared phosphopantetheine analogue 2c
(Scheme 1). While 2c was prepared in racemic form, the
(3) Usher, K. C.; Remington, S. J .; Martin, D. P.; Drueckhammer,
D. G. Biochemistry 1994, 33, 7753-7759.
(4) Schwartz, B. M.; Drueckhammer, D. G.; Usher, K. C.; Remington,
S. J . Biochemistry 1995, 34, 15459-15466.
(5) Schwartz, B. M.; Drueckhammer, D. G. J . Am. Chem. Soc. 1996,
118, 9826-9230.
(6) Vogel, K. W.; Drueckhammer, D. G. J . Am. Chem. Soc. 1998,
120, 3275-3283.
10.1021/jo981758s CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/31/1999

2904 J . Org. Chem., Vol. 64, No. 8, 1999
Bibart et al.
Sch em e 2
Sch em e 4
Sch em e 3
thioester 7.9 Reaction with N-propyl-â-mercaptopropion-
amide 8 and cesium fluoride resulted in thioester ex-
change and removal of one methyl group of the phosphate
triester to form 9. Further deprotection of the phosphate
with trimethylsilyl chloride and bromide ion gave 2c in
racemic form.2,10 Enzymatic conversion of 2c to 1c was
accomplished using the enzymes that catalyze the final
two steps of CoA biosynthesis (Scheme 1) immobilized
in polyacrylamide gel,11 as in the previously reported
synthesis of 1b.2,12 Phosphoenolpyruvate and pyruvate
kinase were added to regenerate the equivalent of ATP
used in the kinase step.13 The reaction was complete in
7 days, and the product was isolated by reverse-phase
HPLC.
The initial product 3c formed in the phosphopanteth-
eine adenylyltransferase catalyzed reaction of 2c with
ATP was isolated and subjected to stereochemical analy-
sis as shown in Scheme 4. Reaction with nucleotide
pyrophosphatase14 converted 3c back into the phospho-
pantetheine analogue 2c. Reaction of the resulting
sample of 2c with acid phosphatase liberated the pan-
tetheine analogue 10, which spontaneously lactonized to
form pantolactone 11. This lactone was converted to the
natural (R) isomer was selectively converted to product
as indicated by stereochemical analysis of 3c. Aminolysis
reactions of 1c have been used to prepare CoA analogues
with modifications more distant from the terminal thiol
than are available through the previously described
synthon 1b. This new CoA analogue synthon is expected
to be useful in the preparation of a variety of analogues
not available from synthon 1b.
1
Mosher ester derivative 12,15 and the H NMR spectrum
of 12 was compared with the spectra of the Mosher ester
derivatives of authentic racemic and natural (R)-panto-
lactone. The spectrum of 12 derived from 3c showed only
the natural (R)-isomer (90% ee), with no detectable
amount of the (S)-isomer.
Resu lts
Syn th esis of CoA An a logu es by Am in olysis Rea c-
tion s of 1c. The analogue 1c was used to prepare three
analogues not accessible via previously described meth-
odology. These include the CoA analogue 14, in which
an extra methylene group is inserted into the â-alanine
moiety (Scheme 5). For the preparation of 14, 4-amino-
butyrylcysteamine 13 was prepared as shown in Scheme
6. Reaction of cystamine dihydrochloride 16 with 4-bromo-
butyryl chloride 15 in a biphasic mixture of chloroform
Syn th esis of CoA An a logu e Syn th on 1c. The syn-
thesis of CoA analogue 1c was performed using the
enzymes catalyzing the final two steps of CoA biosyn-
thesis as reported previously for the synthesis of 1b
(Scheme 1).2 The phosphopantetheine analogue 2c was
prepared as shown in Scheme 3. Reaction of the aldehyde
4 with the lithium salt of tris(methylthio)methane fol-
lowed by workup with aqueous NaHCO3 formed the
racemic pantoic acid derivative 5, having the acid group
protected as a trithioortho ester.7 Conversion to the
dimethyl phosphate ester 6 was accomplished by reaction
with dimethyl chlorophosphate8 as described previously
for similar compounds.2 This was followed by hydrolysis
of the trithioortho ester with fluoboric acid to give the
(9) Barbero, M.; Cadamuro, S.; Degani, I.; Dughera, S.; Fochi, R. J .
Chem. Soc., Perkin Trans. 1 1993, 2075-2080.
(10) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M.
Tetrahedron Lett. 1977, 155-158.
(11) Pollak, A.; Blumenfeld, H.; Wax, M.; Baughn, R. L.; Whitesides,
G. M. J . Am. Chem. Soc. 1980, 102, 6324-6366.
(12) Martin, D. P.; Drueckhammer, D. G. Biochem. Biophys. Res.
Commun. 1993, 192, 1155-1161.
(13) Hirschbein, B. L.; Mazenod, G. M.; Whitesides, G. M. J . Org.
Chem. 1982, 47, 3765-3766.
(14) Kornberg, A.; Pricer, W. E. J . Biol. Chem. 1950, 182, 763-768.
(15) Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1973, 95, 512-
519.
(7) Denis, J . N.; Desauvage, S.; Hevesi, L.; Krief, A.Tetrahedron Lett.
1981, 22, 4009-4012.
(8) Shemin, D., Ed. Biochemical Preparations; J ohn Wiley and
Sons: New York, 1957; Vol. 5, pp 1-4.

Second Generation Coenzyme A Analogue Synthon
J . Org. Chem., Vol. 64, No. 8, 1999 2905
Sch em e 5
Sch em e 8
Sch em e 9
Sch em e 6
Ta ble 1. In h ibition of Citr a te Syn th a se by Ca r boxyla te
An a logu es of Acetyl-CoA
Km or Ki
24
25
26
260 µM (Ki)
16 nM (Ki)
16 µM (Km)
Sch em e 7
carboxymethylene group. Analogue 24 was prepared by
reaction of 1c with 8-aminooctanoic acid 23 as shown in
Scheme 9. The aminolysis reactions of Schemes 5 and 7
were performed at room temperature in acetonitrile
containing a minimum amount of water for solubilizing
1c. The reactions were monitored by analytical HPLC
and were complete in about 7 days. The reaction of
Scheme 9 was carried out in aqueous solution and was
complete in 4 days. The products were purified by
reverse-phase HPLC.
Analogue 24 was tested as an inhibitor of citrate
synthase, and the results are shown in Table 1. Also
shown in Table 1 are the Km for acetyl-CoA 26 and the
Ki for the related inhibitor 25 (Figure 1). These results
show that 24 is a very poor inhibitor of citrate synthase,
with a Ki more than 10-fold higher than the Km for acetyl-
CoA and more than 10 000-fold higher than the Ki for
25.
and aqueous sodium hydroxide formed the amide 17.
Displacement of the bromide with azide to form 18 was
followed by reduction of the azido and disulfide groups
using triphenylphosphine in aq THF to form 13.16,17 Also
prepared was CoA, analogue 20, in which the chain
length is unchanged relative to that of CoA but the amide
bond between the cysteamine and â-alanine moieties is
replaced with a pair of methylene groups (Scheme 7). For
the preparation of 20, 6-aminohexanethiol 19 was pre-
pared as shown in Scheme 8. 6-Bromohexanenitrile 21
was converted to the thiol 22 by displacement of the
bromide with thiophosphate followed by acid hydrolysis
of the resulting thiophosphate ester.18 Reduction of the
nitrile with the borane-dimethyl sulfide complex formed
the amine 19.19 The third analogue prepared was the
deamide carboxylate analogue 24. This is an analogue
of acetyl-CoA in which the amide bond nearest the
thioester is replaced with a pair of methylene groups (as
in 20) and the acetylthio moiety is replaced with a
Discu ssion
The extension of previous methodology for CoA ana-
logue synthesis as described here allows for the facile,
routine synthesis of CoA analogues not previously avail-
able. Synthesis of the analogue 1c required the develop-
ment of a method for synthesis of the corresponding
phosphopantetheine analogue 2c. In previous work,
generation of the unphosphorylated pantetheine ana-
logue 10 (shown in Scheme 4) from a hydroxyl-protected
derivative resulted in rapid cyclization to the lactone 11,
even when 10 was generated in the presence of reactive
phosphorylating agents.20 This cyclization is facilitated
by the geminal dimethyl groups, which are expected to
(16) Pilard, S.; Vaultier, M. Tetrahedron Lett. 1984, 25, 1555-1556.
(17) Humphrey, R. E.; McCrary, A. L.; Webb, R. M. Talanta 1965,
12, 727-732.
(18) Piper, J . R.; J ohnston, T. P. J . Org. Chem. 1967, 32, 1261-
1262.
(19) Brown, H. C.; Choi, Y.; Narasimhan, S. Synthesis 1981, 605-
606.
(20) Bibart, R. T.; Drueckhammer, D. G. Unpublished results.

2906 J . Org. Chem., Vol. 64, No. 8, 1999
Bibart et al.
phatase while CoA was a very poor substrate. Fortu-
nately, the dephospho-CoA analogue 3c resulting from
enzymatic coupling of 2c with ATP was found to have
the proper configuration as determined by the procedure
of Scheme 4, with no detectable amount of the (S)-isomer.
This demonstrates that the phosphopantetheine adenyl-
yltransferase is highly selective for the enantiomer of 2c
having the natural (R)-configuration. Some precedence
for this enantioselectivity is provided by the almost 20-
fold selectivity for the proper configuration at this posi-
tion of pantetheine pivalate thioesters by thiolase.23 It
is likely that the dephospho-CoA kinase may also selec-
tively utilize the (R)-isomer of 3c over any trace of (S)-
isomer that may be formed in the previous step, providing
further enantiomeric enrichment of 1c.
The utility of 1c as a versatile CoA analogue synthon
was demonstrated by the synthesis of the homo-â-alanyl
14 (Scheme 5) and “deamide” 20 (Scheme 7) analogues
of CoA, and the deamide carboxylate analogue 24. This
illustrates the utility of this methodology in manipulating
the structure of CoA at positions more distant from the
terminal thiol group. 14 is an isomer of a previously
prepared analogue having an extra methylene group in
the cysteamine portion of the molecule.24 These analogues
may be useful in elucidating details of the effects of the
â-alanine and amide moieties in orienting the thiol group
of CoA in the proper position in an enzyme active site
for enzyme catalysis to proceed. Initial insight into the
importance of these functionalities is provided by the
inhibition studies with 24. This analogue differs from the
previously studied carboxylate analogue of acetyl-CoA 25
only in the replacement of the amide bond functionality
with a pair of methylene groups (Figure 1). Such a
substitution is expected to have a minimal effect on the
position of the terminal carboxylate. This replacement
results in a 104-fold decrease in affinity of the inhibitor
for citrate synthase. As 25 is viewed as a mimic of the
enol or enolate intermediate, the greatly diminished
inhibition upon deletion of the amide functionality indi-
cates strong binding of the amide functionality by citrate
synthase in stabilization of the enol or enolate complex.
The aminolysis reactions of 1c in the synthesis of 14
and 20 were carried out in an aqueous acetonitrile mixed
solvent system due to the insolubility of the amines 13
and 19 in aqueous solution and the insolubility of 1c in
purely organic solvent. The aminolysis reactions required
7 days at room temperature for complete disappearance
of starting material, being much slower than similar
reactions of 1b which proceed to completion in 12-24 h.
This decreased reactivity was not predicted but may be
attributable to steric effects, which more than compen-
sate for any activating effect of the electronegative
R-hydroxyl group. As the amine 23 is water soluble, the
aminolysis reaction to form 24 was performed in aqueous
solution. The reaction appeared somewhat faster with
complete disappearance of starting material observed in
4 days, suggesting that the solvent conditions may be
partially responsible for the slower aminolysis rates with
13 and 19. However, the yield was lower than in the
slower reactions with 13 and 19 in mixed solvent and
the reaction was accompanied by the formation of several
F igu r e 1. Structures of acetyl-CoA 26 and a carboxylate
analogue 25.
induce a conformation favorable for lactonization.21,22 It
was thus concluded that the phosphate group in some
form must be introduced prior to formation of the
thioester. Use was made of a trithioortho ester as a
protected thioester that is resistant to reaction with
nucleophiles. The tris(methylthio)ortho ester of pantoic
acid 5 was prepared in racemic form by addition of the
tris(methylthio)methane anion to the aldehyde 4 follow-
ing general procedures for similar addition to other
aldehydes.7 This allowed for introduction of the phos-
phate group as the dimethyl ester prior to release of the
thioester by acid hydrolysis. The thioester exchange
reaction was driven by removal of volatile methanethiol
and was accompanied by nucleophilic reaction at a
phosphate methyl group. Two equivalents of the thiol
were thus required, with one equivalent being converted
to the methyl thioether. Phosphate deprotection was
accomplished by previously described methods, consisting
of conversion to the bis(trimethylsilyl) ester which was
converted to the phosphate monoester upon addition of
methanol.2,10
The phosphopantetheine analogue 2c was accepted as
a substrate by phosphopantetheine adenylyltransferase,
and the resulting dephospho-CoA analogue 3c was
subsequently accepted by dephospho-CoA kinase, as
indicated by monitoring of product formation by analyti-
cal HPLC and subsequent isolation and characterization
of the products 3c and 1c. Since the phosphopantetheine
analogue 2c prepared as in Scheme 3 was racemic, it was
necessary to address the stereochemistry of the product
of the enzyme-catalyzed conversion to the CoA analogue
1c. The degradation sequence of Scheme 4 was devised
to convert the product to the known compound panto-
lactone, for which stereochemical analysis could be
performed by standard procedures. This sequence in-
volved hydrolysis of 3c back into the phosphopantetheine
analogue 2c catalyzed by nucleotide pyrophosphatase
followed by further conversion to the known product
pantolactone 11. Nucleotide pyrophosphatase has been
previously shown to accept several dinucleotides as
substrates.14 The analysis was performed on the dephos-
pho-CoA analogue 3c rather than the CoA analogue 1c,
since preliminary studies showed that dephospho-CoA
was a good substate for the enzyme nucleotide pyrophos-
(23) Davis, J . T.; Moore, R. N.; Imperiali, B.; Pratt, A. J .; Kobayashi,
K.; Masamune, S.; Sinskey, A. J .; Walsh, C. T.; Fukui, T.; Tomita, K.
J . Biol. Chem. 1987, 262, 82-89.
(24) Gehring, A. M.; Lambalot, R. H.; Vogel, K. W.; Drueckhammer,
D. G.; Walsh, C. T. Chem. Biol. 1997, 4, 17-24.
(21) Wheeler, O. H.; Granell de Rodriguez, E. E. J . Org. Chem. 1961,
29, 1227-1229.
(22) Kirby, A. J . Adv. Phys. Chem. 1980, 18, 183-278.

Second Generation Coenzyme A Analogue Synthon
J . Org. Chem., Vol. 64, No. 8, 1999 2907
keep the temperature below -75 °C. The solution was stirred
for an additional 1 h at -78 °C, and a solution of 2,2-dimethyl-
3-hydroxypropionaldehyde 4 (5.00 g, 32.9 mmol, 70% pure) in
dry THF (20 mL) was added. The solution was allowed to
slowly warm to room temperature and was stirred at room-
temperature overnight. Aqueous NaHCO3 (100 mL, 1 M) was
added followed by ether (100 mL), and the layers were
separated. The ether layer was washed with aq NaHSO4 (50
mL, 5%), NaHCO3 (50 mL, 1 M), and saturated aqueous NaCl
(50 mL), dried over MgSO4, and evaporated. The residue was
purified by chromatography on silica gel (2:1 hexane/ethyl
acetate) to give 5 (Rf ) 0.40) as a colorless oil (6.09 g, 26.9
Sch em e 10
byproducts, one of which is likely the product resulting
from hydrolysis of the thioester.
Another limitation in the synthesis of CoA analogues
using the first generation method of Scheme 2 is il-
lustrated by an oxoester analogue of a CoA ester.
Synthesis of such an analogue from 1b would require the
aminolysis reaction to be performed with an ethanol-
amine ester 27. However, this compound would undergo
intramolecular acyl transfer to form the amide 28 rather
than the desired reaction with the thioester of 1b, as
shown in Scheme 10. This problem has also been en-
countered in efforts to prepare other acyl CoA analogues
having electrophilic functionality in place of the thioester.
Availability of the synthon 1c should permit aminolysis
reactions to be performed with a â-alanylamide derivative
of 27, in which the oxoester and nucleophilic amine
groups are sufficiently separated such that intramolecu-
lar acyl transfer will not occur. Compound 1c is thus
expected to be valuable in the preparation of various CoA
ester analogues not accessible from 1b.
The work described here has solved most of the
limitations in the previously described method for CoA
analogue synthesis. Analogues are now accessible having
a wide range of functionality in place of the â-alanyl-
cysteamine moiety including previously inaccessible ana-
logues. This methodology should facilitate further mecha-
nistic and structural studies of CoA ester-utilizing en-
zymes using synthetic CoA analogues as probes.
1
mmol, 82% yield). H NMR (200 MHz, CDCl3) δ 3.89 (bs, 1H),
3.70 (s, 1H), 3.68 (d, J ) 11.2 Hz, 1H), 3.51 (d, J ) 11.2 Hz,
1H), 2.95 (bs, 1H), 2.27 (s, 9H), 1.28 (s, 3H), 1.21 (s, 3H). 13C
NMR (50 MHz, CDCl3) δ 80.8, 74.7, 72.9, 41.2, 24.0, 20.7, 14.2.
(R,S)-4,4,4-Tr is(m et h ylt h io)-2,2-d im et h yl-1,3-b u t a n e-
d iol, 1-Dim eth yl P h osp h a te (6). To a solution of dimethyl
phosphite (7.40 mL, 80.7 mmol) in dry toluene (150 mL) was
added N-chlorosuccinimide (10.8 g, 80.7 mmol) and the solu-
tion stirred at 0 °C for 1 h followed by 1 h at room temperature.
The resulting solution of dimethyl phosphorochloridate was
transferred (leaving behind precipitated succinimide) into a
solution of 5 (6.09 g, 26.9 mmol) in dry CH2Cl2 (200 mL) and
dry pyridine (100 mL) at -40 °C. The reaction was allowed to
warm to room temperature and stirred overnight, after which
water (100 mL) was added and stirring continued for an
additional 1 h. The solution was evaporated under reduced
pressure and the residue dissolved in ethyl acetate (100 mL).
The resulting solution was washed with water (50 mL),
aqueous H2SO4 (50 mL, 1 M), aqueous NaHCO3 (50 mL, 1 M),
and saturated aqueous NaCl (50 mL), dried over MgSO4, and
evaporated. The residue was purified by chromatography on
silica gel (ethyl acetate) to give 6 (Rf ) 0.42) as a colorless oil
1
(8.93 g, 24.5 mmol, 91% yield). H NMR (200 MHz, CDCl3) δ
4.22 (dd, J ) 9.3 Hz, 5.3 Hz, 1H), 3.79 (dd, J ) 9.4 Hz, 5.2 Hz,
1H), 3.78 (d, J ) 11.1 Hz, 6H), 3.77 (bs, 1H), 3.74 (s, 1H), 2.25
(s, 9H), 1.32 (s, 3H), 1.28 (s, 3H). 13C NMR (50 MHz, CDCl3)
δ 77.2, 77.6 (d, J ) 6.2 Hz), 74.7, 54.1 (d, J ) 5.9 Hz, 2C), 41.6
(d, J ) 6.9 Hz), 22.9, 19.8, 14.4.
(R,S)-P a n toth ioic Acid S-Meth yl Ester , 4-Dim eth yl
P h osp h a te (7). To a solution of 6 (8.93 g, 24.5 mmol) in dry
THF (50 mL) at 0 °C was added 48% HBF4 (3.52 mL, 27.0
mmol). The solution was allowed to warm to room temperature
with stirring and was stirred at room temperature for 2 h.
Ether (100 mL) was added, and the solution was washed with
aqueous NaHSO4 (50 mL, 5%), aqueous NaHCO3 (50 mL, 1
M), and saturated aqueous NaCl (50 mL), dried over MgSO4,
and evaporated. The residue was purified by chromatography
on silica gel (25:1 CH2Cl2/methanol) to give 7 (Rf ) 0.40) (6.64
g, 24.2 mmol, 95% yield) as a colorless oil. 1H NMR (200 MHz,
CDCl3) δ 4.42 (d, J ) 6.4 Hz, 1H), 4.20 (dd, J ) 10.2 Hz, 7.3
Hz, 1H), 4.16 (d, J ) 6.2 Hz, 1H), 3.80 (dd, J ) 10.1 Hz, 7.7
Hz, 1H), 2.26 (s, 3H), 1.15 (s, 3H), 0.90 (s, 3H). 13C NMR (50
MHz, CDCl3) δ 203.9, 79.1, 73.5 (d, J ) 6.0 Hz), 54.4 (d, J )
5.9 Hz, 2C), 39.6 (d, J ) 6.5 Hz), 20.8, 18.6, 11.1.
N-(3-Mer ca p top r op a n oyl)p r op yla m in e (8). To a solution
of acryloyl chloride (20.0 mL, 246 mmol) in dry CH2Cl2 (300
mL) at 0 °C was added dropwise a solution of propylamine
(21.8 mL, 270 mmol) in triethylamine (41.8 mL, 300 mmol),
and the resulting solution was stirred at room-temperature
overnight. The solution was washed with water (100 mL),
H2SO4 (100 mL, 1 M), NaHCO3 (100 mL, 1 M), and sat. aq
NaCl (100 mL), and dried with Na2SO4 and the solvent
evaporated to give N-acryloylpropylamine (24.3 g, 215 mmol,
87% yield). 1H NMR (200 MHz, CDCl3) δ 6.95 (bs, 1H), 6.26
(s, 1H), 6.23 (d, J ) 2.2 Hz, 1H), 5.60 (dd, J ) 7.1 Hz, 4.9 Hz,
1H), 3.27 (q, J ) 6.8 Hz, 2H), 1.45-1.65 (m, 2H), 0.93 (t, J )
7.4 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ 166.1, 131.2, 125.7,
41.0, 22.3, 11.0. The product (24.3 g, 215 mmol) was dissolved
in ether (500 mL), thiolacetic acid (17.2 mL, 240 mmol) was
added, and the reaction was stirred at room-temperature
overnight. The reaction was then washed with water (150 mL),
aqueous H2SO4 (150 mL, 1 M), aqueous NaHCO3 (150 mL, 1
M), and saturated aqueous NaCl (50 mL), dried with MgSO4,
Exp er im en ta l Section
Gen er a l Exp er im en ta l. Reagents were obtained from
Aldrich, Sigma, or Mallinckrodt and used as supplied while
commercial enzymes were from Sigma. Phosphopantethein
adenylytransferase and dephospho CoA kinase were isolated
and immobilized as described previously.2 Methylene chloride
(CH2Cl2), pyridine, toluene, and acetonitrile (CH3CN) were
distilled from calcium hydride, and tetrahydrofuran (THF) was
distilled from sodium. Analytical HPLC was performed using
a Microsorb C-18 column (4.6 mm × 25 cm) with monitoring
at 215 and 260 nm. Solvents used were aqueous potassium
phosphate (solvent A: 50 mM, pH 4.5) and methanol (solvent
B). Compounds were eluted at a flow rate of 1 mL/min with
5% solvent B for 2 min, followed by a linear gradient to 60%
solvent B over 12 min, and then maintenance at 60% solvent
B. Preparative HPLC was done on a Microsorb C-18 column
(21.4 cm × 25 cm) with monitoring at 215 and 280 nm and a
flow rate of 10 mL/min. Solvents were 0.2% trifluoroacetic acid
in water (solvent A) and 0.2% trifluoroacetic acid in methanol
(solvent B). Compounds were eluted with 5% B for 5 min
followed by a linear gradient to 80% B over 45 min. Product
CoA analogues eluted between 33 and 42 min. Mass spectral
analysis was performed at the University of Riverside Mass
Spectrometry Facility, Riverside, CA. Ki values were deter-
mined by computer fitting of data to the equation v ) (Vmax[S])/
(Km(1 + [I]/Ki) + [S]).25 The concentrations of CoA analogue
solutions were determined using ꢀ260 ) 15400 M-1 cm-1
.
4,4,4-Tr is(m eth ylth io)-2,2-d im eth yl-1,3-bu ta n ed iol (5).
To a solution of tris(methylthio)methane (23.1 g, 150 mmol)
in dry THF (500 mL) at -78 °C was added n-butyllithium (10
mL, 10M solution in hexanes, 100 mmol) slowly over 1 h to
(25) Cleland, W. W. Methods Enzymol. 1979, 63, 103-138.

2908 J . Org. Chem., Vol. 64, No. 8, 1999
Bibart et al.
and evaporated. The product was recrystallized from ether/
pentane to give 3-(acetylthio)propanoyl n-propylamine (39.4
g, 208 mmol) as white flakes in 97% yield. 1H NMR (200 MHz,
CDCl3) δ 5.83 (bs, 1H), 3.22 (q, J ) 6.9 Hz, 2H), 3.14 (t, J )
6.7 Hz, 2H), 2.48 (t, J ) 6.7 Hz, 2H), 2.34 (s, 3H), 1.41-1.62
(m, 2H), 0.93 (t, J ) 7.4 Hz, 3H). 13C NMR (50 MHz, CDCl3)
δ 196.7, 170.8, 41.1, 36.1, 30.4, 24.8, 22.5, 11.1. The product
(10.0 g, 52.8 mmol) was dissolved in sodium methoxide (116
mL, 0.5 M in methanol, 58.0 mmol) and stirred at room
temperature for 1 h. The solution was acidified with 1 M H2SO4
and extracted with ether (100 mL, 50 mL). The organic layers
were combined, dried with MgSO4, and evaporated to give 8
(7.34 g, 49.8 mmol) as a colorless oil in 94% yield with no
further purification required. 1H NMR (200 MHz, CDCl3) δ
5.88 (bs, 1H), 3.24 (q, J ) 6.9 Hz, 2H), 2.75-2.90 (m, 2H), 2.49
(t, J ) 6.7 Hz, 2H), 1.62 (t, J ) 8.3 Hz, 1H), 1.45-1.64 (m,
2H), 0.94 (t, J ) 7.4 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ
170.6, 41.2, 40.4, 22.8, 20.5, 11.3.
mL), and the solution was adjusted to pH 3.5 with 1 M
phosphoric acid. At 4 °C, the solution was loaded onto a DEAE
cellulose column (2.5 × 17 cm), which was previously equili-
brated with 3 mM HCl. The column was washed with 100 mL
of 3 mM HCl and then with a linear gradient (1 L) of lithium
choride (0-0.2 M) in 3 mM HCl. Individual fractions of 22 mL
were collected and analyzed by HPLC. Compound 1c eluted
between 0.10 and 0.12 M lithium chloride. Fractions containing
1c were adjusted to pH 4 with 1 M KH2PO4 and were
lyophilized and further purified by HPLC to give pure 1c as a
lyophilized powder (60 mg, 78 µmol, 25% yield based on (R)-
2c). Analytical HPLC: retention time ) 14.6 min, λmax ) 260
nm. 1H NMR (200 MHz, CDCl3) δ 8.56 (s, 1H), 8.33 (s, 1H),
6.10 (d, J ) 5.3 Hz, 2H), 4.75 (bs, 1H), 4.52 (bs, 1H), 4.15-
4.25 (m, 2H), 4.05 (s, 1H), 3.78 (dd, J ) 9.4 Hz, 4.6 Hz, 1H),
3.54 (dd, J ) 9.4 Hz, 4.3 Hz, 1H), 2.90-3.02 (m, 4H), 2.40 (t,
J ) 6.7 Hz, 2H), 1.30-1.40 (m, 2H), 0.84 (s, 3H), 0.75 (t, J )
7.6 Hz, 3H), 0.74 (s, 3H). 13C NMR (50 MHz, CDCl3) δ 208.6,
176.2, 152.7, 150.9, 148.1, 144.6, 120.9, 89.8, 85.9 (d, J ) 8.7
Hz), 81.7, 76.5, 76.5, 74.2 (d, J ) 5.7 Hz), 67.6 (d, J ) 5.1 Hz),
43.7, 41.3 (d, J ) 8.3 Hz), 37.5, 26.6, 24.2, 22.9, 20.9, 13.1.
HRMS (FAB): [M - H]- calcd for C22H36N6O16P3S m/z 765.1124,
found 765.109. Compound 3c eluted between 0.08 and 0.10 M
lithium chloride from the DEAE column. Fractions containing
3c were adjusted to pH 4 with 1 M KH2PO4 and were
lyophilized and further purified by HPLC to give 3c as a
lyophilized powder (120 mg, 175 µmol, 56% yield based on (R)-
2c). Analytical HPLC: retention time ) 16.4 min, λmax ) 260
nm. 1H NMR (400 MHz, D2O) δ 8.51 (s, 1H), 8.32 (s, 1H), 6.03
(d, J ) 4.7 Hz, 1H) 4.60 (t, J ) 5.0 Hz, 1H) 4.44 (t, J ) 3.7 Hz,
1H), 4.31 (bs, 1H), 4.20 (bs, 2H), 4.03 (s, 1H), 3.78 (dd, J ) 9.5
Hz, 4.8 Hz, 1H) 3.54 (dd, J ) 9.2 Hz, 2.8 Hz, 1H) 2.88-3.01
(m, 4H), 2.39 (t, J ) 6.9 Hz, 2H) 1.32-1.39 (m, 2H), 0.84 (s,
3H), 0.74 (s, 3H), 0.74 (t, J ) 7.4 Hz, 3H). 13C NMR (100 MHz,
D2O) δ 208.5, 176.1, 152.5, 150.6, 147.9, 144.6, 120.7, 90.5, 86.4
(d, J ) 8.3 Hz), 81.7, 77.2, 74.2 (d, J ) 5.7 Hz), 72.5, 67.5,
43.7, 41.3 (d, J ) 7.7 Hz), 37.5, 26.6, 24.3, 22.9, 20.9, 13.2.
HRMS (FAB): [M - H]- calcd for C22H35N6O13P2 S m/z
685.1461, found 685.1488.
Ster eoch em ica l An a lysis of 3c. A 10 mg (15 µmol)
amount of 3c was dissolved in 800 µL of 0.1 M potassium
phosphate buffer, pH 7.4, and 4.5 units of nucleotide pyro-
phosphatase (EC 3.6.1.9, Crotalus atrox venom from Sigma)
was added. The reaction was monitored by analytical HPLC
and complete disappearance of 3c (retention time ) 16.4 min,
λmax ) 260 nm) and appearance of products (AMP, retention
time ) 8.71 min, λmax ) 260 nm, and 2c, retention time ) 16.48
min, λmax ) 240 nm) was observed in 4 h. 2c was purified,
and excess phosphate salts were removed by preparative
HPLC (5 min at 5% methanol in 0.2% aqueous TFA followed
by a linear gradient to 80% methanol in 0.2% aqueous TFA
over 45 min, 2c eluted between 30 and 38 min). Fractions
containing 2c were pooled and lyophilized and redissolved in
2.5 mL 0.1 M acetate, pH 5.0. Acid phosphatase (7.8 units)
(EC 3.1.3.2, potato from Sigma) was added, and the reaction
was monitored by analytical HPLC. After 6 h, an additional 7
units of phosphatase was added, and complete disappearance
of starting material was observed in 21 h. The reaction was
concentrated to approximately 500 µL under a stream of
nitrogen and extracted five times with 3 mL of ether. The
combined ether layers were concentrated by rotary evaporation
and purified via flash chromatography on a 20 mL silica gel
column (1:5 hexane/ethyl acetate, Rf ) 0.60). The residue
containing 11 was converted to the MTPA ester 12 using
standard procedures.15 Authentic MTPA esters of (R)- and (S)-
pantoyllactone were prepared separately. The NMR spectrum
of the MTPA ester of the sample of 12 derived from 3c showed
only the MTPA ester of (R)-pantoyllactone. (S)-MTPA-(R)-
pantoyllactone: 1H NMR (400 MHz, D2O): δ 0.956 (s, 3H),
1.179 (s, 3H), 3.634 (s, 3H), 4.066 (s, 2H), 5.596 (s, 1H), 7.42-
7.67 (m, 5H). (S)-MTPA-(S)-pantoyllactone: 1H NMR (400
MHz, D2O): δ 1.154 (s, 3H), 1.275 (s, 3H), 3.551 (s, 3H), 4.101
(s, 2H), 5.560 (s, 1H), 7.42-7.67 (m, 5H).
(R,S)-3-Hydr oxy-4-[[3-(pr opylam in o)-3-oxopr opyl]th io]-
2,2-d im eth yl-4-oxobu ta n -1-ol, 1-Meth yl P h osp h a te Ester
(9). To a solution of 8 (3.33 g, 22.6 mmol) and 7 (6.64 g, 24.2
mmol) in dry THF (20 mL) was added CsF (1.97 g, 12.9 mmol),
and the solution was stirred under a steady stream of nitrogen
vented to a 1 M aqueous NaOH bubbler to remove meth-
anethiol. After 1 day, an additional 1.5 g (9.87 mmol) of CsF
was added. After another day, the solvent was removed and
the residue dissolved in water (50 mL). The resulting solution
was extracted with ethyl acetate (25 mL). The aqueous solution
was lyophilized and purified by HPLC. This provided 9 (2.15
g, 5.79 mmol, 51% yield) and 2c (0.96 g, 2.69 mmol, 23% yield).
Analytical HPLC: retention time ) 16.8 min, λmax ) 240 nm.
1H NMR (400 MHz, D2O) δ 4.18 (s, 1H), 3.78 (dd, J ) 9.5 Hz,
4.0 Hz, 1H), 3.58 (d, J ) 10.7 Hz, 3H), 3.57 (dd, J ) 9.5 Hz,
4.0 Hz, 1H), 3.05-3.14 (m, 4H), 2.54 (t, J ) 6.7 Hz, 2H), 1.44-
1.53 (m, 2H), 1.01 (s, 3H), 0.93 (s, 3H), 0.88 (t, J ) 7.3 Hz,
3H). 13C NMR (100 MHz, D2O) δ 208.6, 175.6, 82.1, 73.7, 55.4,
43.8, 41.5 (d, J ) 7.1 Hz), 37.8, 26.8, 24.7, 23.4, 21.7, 13.6.
(R,S)-3-Hydr oxy-4-[[3-(pr opylam in o)-3-oxopr opyl]th io]-
2,2-d im eth yl-4-oxobu ta n -1-ol (2c). To a solution of 9 (2.15
g, 5.79 mmol) in dry CH3CN (30 mL) were added trimethylsilyl
chloride (6.35 mL, 50.0 mmol) and tetrabutylammonium
bromide (1.92 g, 5.95 mmol), and the resulting solution was
stirred at 40 °C for 15 h. The solvent was evaporated, the
residue dissolved in methanol, and the methanol evaporated.
Purification by HPLC provided 2c (1.81 g, 5.06 mmol, 87%
yield). Analytical HPLC: retention time ) 15.5 min, λmax
)
240 nm. 1H NMR (400 MHz, D2O) δ 3.85 (s, 1H), 3.45 (dd, J )
9.6 Hz, 4.5 Hz, 1H), 3.23 (dd, J ) 8.8 Hz, 4.6 Hz, 1H), 2.70-
2.81 (m, 4H), 2.20 (t, J ) 6.5 Hz, 2H), 1.10-1.17 (m, 2H), 0.65
(s, 3H), 0.56 (s, 3H), 0.52 (t, J ) 7.4 Hz, 3H). 13C NMR (100
MHz, D2O) δ 208.3, 175.0, 81.9, 73.2, 43.5, 41.2 (d, J ) 7.5
Hz), 37.4, 26.6, 24.2, 22.9, 21.2, 13.2.
Ad en osin e 5′-(Tr ih yd r ogen d ip h osp h a te) 3′-(Dih yd r o-
gen p h osp h a te) 5′-[(R)-3-Hyd r oxy-4-[[3-(p r op yla m in o)-3-
oxop r op yl]th io]-2,2-d im eth yl-4-oxobu tyl] Ester (1c). To
a solution of racemic 2c (0.90 g, 2.52 mmol) in HEPES buffer
(0.1 M, 16 mL, pH 7.5) were added phosphopantetheine
adenylyltransferase and dephosphocoenzyme A kinase co-
immobilized in polyacrylamide as described previously.2 ATP
(3.00 mmol, dissolved in 10 mL of water, pH adjusted to 7 with
KOH), MgCl2 (0.54 mmol), and inorganic pyrophosphatase (10
units) were added, and the reaction mixture was gently swirled
in a rotary shaker at room temperature. Pyruvate kinase (22
units) and phospho(enol) pyruvate (0.5 mmol, as the potassium
salt, pH adjusted to 7 with KOH) were added daily. The
progress of the reaction was monitored by HPLC. The reaction
was stopped after 7 days, at which time HPLC showed
unreacted 2c (retention time ) 15.5 min) along with new peaks
at 14.6 min (1c) and 16.4 min (3c). The reaction mixture was
centrifuged, the supernatant was decanted, and the im-
mobilized enzymes were washed twice with HEPES buffer (0.1
M, 5 mL). The combined washes and supernatant were filtered
through a 0.45 µm nylon filter and were lyophilized. One-
fourth of the crude material was dissolved in 3 mM HCl (80
N,N-Bis(4-ch lor obu tyr yl)cysta m in e (17). To a solution
of cystamine dihydrochloride 16 (6.76 g, 30.0 mmol) in aqueous

Second Generation Coenzyme A Analogue Synthon
J . Org. Chem., Vol. 64, No. 8, 1999 2909
NaOH (200 mL, 2 M) was added chloroform (200 mL).
4-Chlorobutyryl chloride 15 (10.0 mL, 89.2 mmol) was slowly
added to the gently stirred biphasic system, and stirring was
continued overnight at 40 °C. The layers were then separated,
and the organic layer was washed with aqueous H2SO4 (100
mL, 1 M), aqueous NaHCO3 (100 mL, 1 M), and saturated
aqueous NaCl (50 mL), dried with Na2SO4, and evaporated.
The product was recrystallized from ether/pentane to give N,N-
bis(4-chlorobutyryl)cystamine 17 (8.53 g, 23.6 mmol, 79% yield)
as a colorless solid. 1H NMR (200 MHz, CDCl3) δ 6.48 (bs, 2H),
3.62 (t, J ) 6.2 Hz, 4H), 3.59 (q, J ) 6.3 Hz, 4H), 2.84 (t, J )
6.4 Hz, 4H), 2.42 (t, J ) 7.4 Hz, 4H), 2.05-2.20 (m, 4H). 13C
NMR (50 MHz, CDCl3) δ 172.3, 44.4, 38.4, 37.6, 33.0, 28.1.
N,N-Bis(4-a zid obu tyr yl)cysta m in e (18). To a solution of
17 (4.25 g, 11.8 mmol) in DMSO (50 mL) was added sodium
azide (1.95 g, 30.0 mmol), and the solution was stirred at room-
temperature overnight. Saturated aq NaCl (50 mL) was added,
and the solution was extracted with ether (3 × 25 mL). The
combined ether layers were washed with water (2 × 25 mL),
dried with MgSO4, and evaporated to give N,N-bis(4-azido-
butyryl)cystamine 18 (4.27 g, 11.4 mmol, 97% yield) as a
a gentle reflux under N2. BH3‚Me2S (2.8 mL, 28 mmol) was
slowly added, and after several minutes a gel formed. The
reaction was cooled to 0 °C, and 2 M HCl in CH3OH (15 mL)
was added slowly. After the gel had dissolved, the reaction
was heated and the majority of the solvent removed by
distillation. The reaction was filtered, the solid rinsed with
CH3OH, and the filtrate concentrated under rotary evapora-
tion. The resulting oil was dissolved in H2O (10 mL), and
impurities were removed by extracting with CH2Cl2 (3 × 20
mL). The aqueous solution was adjusted to pH 10 with 1 N
NaOH and extracted with CH2Cl2 (3 × 20 mL), the combined
organic extracts were dried over MgSO4, and the solvent was
removed by rotary evaporation to yield 0.67 g (38%) of
1
6-aminohexanethiol 19 as a white solid. H NMR (200 MHz,
CDCl3): δ 1.35 (m, 10H), 1.60 (m, 2H), 2.55 (t, 2H, J ) 6.8
Hz), 2.70 (t, 2H, J ) 6.8 Hz). 13C NMR (50 MHz, CDCl3): δ
24.52, 26.32, 28.17, 33.60, 33.92, 42.11.
Ad en osin e 5′-(Tr ih yd r ogen d ip h osp h a te) 3′-(Dih yd r o-
gen p h osp h a te) 5′-[(R)-3-Hyd r oxy-4-[(6-m er ca p toh exyl)-
a m in o]-2,2-d im eth yl-4-oxobu tyl] Ester (20). A solution of
1c (10 mg, 13 µmol) and 19 (0.27 g, 2 mmol) in CH3CN (1 mL)
was stirred at room temperature. Water (100 µL) was added
and the pH adjusted to 10.5 daily. The reaction was complete
in 7 days as determined by HPLC. Water (20 mL) was added
and the solution extracted with EtOAc (3 × 10 mL). The
aqueous phase was adjusted to pH 4.5 and lyophilized and
purified by HPLC to give 20 (8 mg, 10 µmol) as a lyophilized
powder in 80% yield. Analytical HPLC: retention time ) 15.5
1
colorless oil. H NMR (200 MHz, CDCl3) δ 6.56 (bs, 1H), 3.58
(q, J ) 6.3 Hz, 2H), 3.37 (t, J ) 6.6 Hz, 2H), 2.83 (t, J ) 6.5
Hz, 2H), 2.34 (t, J ) 7.2 Hz, 2H), 1.85-2.01 (m, 2H). 13C NMR
(50 MHz, CDCl3) δ 172.3, 50.7, 38.4, 37.6, 32.9, 24.7.
N,N-Bis(4-a m in obu tyr yl)cystea m in e (13). To a solution
of 18 (2.20 g, 5.87 mmol) in THF (50 mL) were added water
(0.72 mL, 40 mmol) and triphenylphosphine (5.11 g, 19.5
mmol), and the solution was stirred overnight at 40 °C. The
solvent was evaporated, and the residue was dissolved in water
(40 mL) and extracted with ethyl acetate (20 mL). The aqueous
layer was lyophilized to give 13 (1.75 g, 10.8 mmol, 92% yield)
as a colorless oil. 1H NMR (200 MHz, CDCl3) δ 3.49 (t, J ) 6.2
Hz, 2H), 2.98 (t, J ) 7.7 Hz, 2H), 2.82 (t, J ) 6.2 Hz, 2H), 2.35
(t, J ) 7.4 Hz, 2H), 1.82-2.00 (m, 2H). 13C NMR (50 MHz,
CDCl3) δ 177.5, 41.6, 40.8, 39.4, 35.3, 25.8.
1
min, λmax ) 260 nm. H NMR (400 MHz, D2O) δ 8.67 (s, 1H),
8.41 (s, 1H), 6.20 (d, J ) 5.8 Hz, 1H) 4.58 (bs, 1H), 4.24 (bs,
2H), 4.13 (s, 1H), 3.84 (dd, J ) 9.5 Hz, 4.4 Hz, 1H), 3.60 (dd,
J ) 9.1 Hz, 3.8 Hz, 1H), 2.94 (t, J ) 7.4 Hz, 2H), 1.61 (t, J )
6.5 Hz, 2H), 1.50 (t, J ) 6.1 Hz, 2H), 1.33 (bs, 4H), 0.91 (s,
3H), 0.84 (s, 3H). MS (FAB): [M - H]- calcd for C22H38N6O15P3S
m/z 751, found 751.
Ad en osin e 5′-(Tr ih yd r ogen d ip h osp h a te) 3′-(Dih yd r o-
gen p h osp h a te) 5′-[(R)-3-Hyd r oxy-4-[(6-ca r boxyh exyl)-
a m in o]-2,2-d im eth yl-4-oxobu tyl] Ester (24). A 2 mL vol-
ume of a saturated solution of ω-aminocaprylic acid 19, pH
adjusted to 10.0 with sodium hydroxide (the solution was about
2 M in the amino acid), was added to 1c (10 mg, 13 µmol).
The reaction was monitored by HPLC, and disappearance of
starting material 1c and appearance of product 24 (retention
time ) 16.6 min) were complete in 4 days. Several other
unidentified products were observed in the analytical HPLC
traces. The solution was adjusted to pH 4.5 with aqueous HCl
and purified by preparative reverse-phase HPLC to give 2.03
Ad en osin e 5′-(Tr ih yd r ogen d ip h osp h a te) 3′-(Dih yd r o-
gen p h osp h a t e) 5′-[(R)-3-H yd r oxy-4-[[3-[2-(m er ca p t o-
e t h yl)a m in o]-3-oxob u t yl]a m in o]-2,2-d im e t h yl-4-oxo-
bu tyl] Ester (14). A solution of 1c (10 mg, 13 µmol) and 13
(0.32 g, 2 mmol) in acetonitrile (1 mL) was stirred at room
temperature. The reaction was checked daily by HPLC for
disappearance of starting material. Water (100 µmol) was
added daily and the pH adjusted to 10.5. The reaction was
complete in 7 days. Water (20 mL) was added and the solution
extracted with EtOAc (3 × 10 mL). The aqueous layer was
adjusted to pH 4.5, lyophilized, and purified by HPLC to give
14 (8 mg, 10 µmol) as a lyophilized powder in 80% yield.
Analytical HPLC: retention time ) 13.1 min, λmax ) 260 nm.
1H NMR (400 MHz, D2O) δ 8.66 (s, 1H), 8.41 (s, 1H), 6.20 (d,
J ) 5.8 Hz, 1H), 4.58 (bs, 1H), 4.23 (bs, 2H), 4.02 (s, 1H), 3.84
(bs, 1H), 3.57 (bs, 1H), 3.31 (t, J ) 6.5 Hz, 2H), 3.19 (t, J )
6.7 Hz, 2H), 2.60 (t, J ) 6.4 Hz, 2H), 2.24 (t, J ) 7.4 Hz, 2H),
1.70-1.80 (m, 2H), 0.92 (s, 3H), 0.81 (s, 3H). MS (FAB): [M -
H] calcd for C22H37N7O13P3S m/z 780, found 780.
1
mg of 24 (20%). λmax ) 260 nm. H NMR (400 MHz, D2O): δ
0.72 (s, 3H), 0.84 (s, 3H), 0.87 (t, 2H, J ) 7.4 Hz), 1.15 (m,
6H), 2.20 (m, 4H), 3.10 (m, 2H), 3.50 (m, 1H), 3.80 (m, 1H),
3.95 (s, 1H), 4.18 (br s, 2H), 4.53 (s, 1H), 6.13 (d, 1H, J ) 5.6
Hz), 8.29 (s, 1H), 8.55 (s, 1H). HRMS (FAB): [M - H]- calcd
for C24H40N6O17P3 m/z 777.166, found 777.169.
6-Mer ca p toh exa n en itr ile (22). To a solution of sodium
thiophosphate dodecahydrate (12 g, 30.4 mmol) dissolved in
H2O (80 mL) was added 6-bromohexanenitrile 21 (2 mL, 15.2
mmol) in DMF (10 mL) and the resulting suspension stirred
overnight. The next morning H2O was added (15 mL), the pH
lowered to 4 with 10% HCl, and the solution stirred an
additional 4 h. The product was extracted with pentane (2 ×
80 mL), and the combined extracts were washed with brine
(20 mL) and dried over MgSO4. Solvent was removed via rotary
evaporation to yield 1.7 g (84%) of 6-mercaptohexanenitrile
22. 1H NMR (200 MHz, CDCl3): δ 1.37 (t, 1H, J ) 7.9 Hz),
1.57 (m, 6H), 2.37 (t, 3H, J ) 6.9 Hz), 2.48 (m, 2H).
Ack n ow led gm en t. This work was supported by
National Institutes of Health Grant GM45831 and
National Science Foundation Grant MCB 9722936.
D.G.D. is a fellow of the Alfred P. Sloan Foundation
(1996-1998).
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 1c, 2c, 3c, 5-9, 12-14, 17-20, 22, and 24. This
material is available free of charge via the Internet at
http://pubs.acs.org.
6-Am in oh exa n eth iol (19). 6-mercaptohexanenitrile 22 (1.7
g, 13 mmol) was dissolved in dry THF (20 mL) and heated to
J O981758S