Oxathiaphospholane approach to N- and O-phosphorothioylation of amino acids

Article abstract of DOI:10.1021/jo026027d

A method of highly efficient synthesis of N- and O-phosphorothioylated amino acids was developed. N- and O-(2-Thiono-1,3,2-oxathiaphospholanyl)amino acid methyl esters (3) were prepared in high yields in reaction of amino acid methyl esters with 2-chloro-1,3,2-oxathiaphospholane in pyridine in the presence of elemental sulfur. Compounds 3 were converted in high yield into the corresponding methyl or benzyl phosphorothioamides 6 and 7 by DBU-assisted treatment with methanol or benzyl alcohol. When 3-hydroxypropionitrile was used instead of methanol or benzyl alcohol, the corresponding 2-cyanoethylphosphorothioamidates 4 were obtained in high yield, from which the 2-cyanoethyl group was removed with concentrated ammonium hydroxide. The oxathiaphospholane methodology was also applied for the phosphorylation of amino acids. Thus, 2-oxo-1,3,2-oxathiaphospholane derivatives 10 were prepared by oxidation of compounds 3 with SeO₂. Compounds 10 were transformed into the corresponding phosphate diesters or amidoesters upon treatment with 3-hydroxypropionitrile in the presence of DBU. The DBU-assisted oxathiaphospholane ring-opening process in 3 and 10 did not cause any measurable C-racemization of phosphorothioylated/phosphorylated amino acids.

Full text of DOI:10.1021/jo026027d

Oxa th ia p h osp h ola n e Ap p r oa ch to N- a n d O-P h osp h or oth ioyla tion
of Am in o Acid s
J anina Baraniak,* Renata Kaczmarek, Dariusz Korczy n´ ski, and Ewa Wasilewska
Department of Bioorganic Chemistry, Center of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Ł o´ d z´ , Poland
baraniak@bio.cbmm.lodz.pl
Received J une 7, 2002
A method of highly efficient synthesis of N- and O-phosphorothioylated amino acids was developed.
N- and O-(2-Thiono-1,3,2-oxathiaphospholanyl)amino acid methyl esters (3) were prepared in high
yields in reaction of amino acid methyl esters with 2-chloro-1,3,2-oxathiaphospholane in pyridine
in the presence of elemental sulfur. Compounds 3 were converted in high yield into the corresponding
methyl or benzyl phosphorothioamides 6 and 7 by DBU-assisted treatment with methanol or benzyl
alcohol. When 3-hydroxypropionitrile was used instead of methanol or benzyl alcohol, the
corresponding 2-cyanoethylphosphorothioamidates 4 were obtained in high yield, from which the
2
-cyanoethyl group was removed with concentrated ammonium hydroxide. The oxathiaphospholane
methodology was also applied for the phosphorylation of amino acids. Thus, 2-oxo-1,3,2-oxathia-
phospholane derivatives 10 were prepared by oxidation of compounds 3 with SeO2. Compounds 10
were transformed into the corresponding phosphate diesters or amidoesters upon treatment with
3
-hydroxypropionitrile in the presence of DBU. The DBU-assisted oxathiaphospholane ring-opening
process in 3 and 10 did not cause any measurable C-racemization of phosphorothioylated/
phosphorylated amino acids.
In tr od u ction
get a deeper insight into the influence of phosphorylation
on the structure and function of the peptides.
5
-9
Protein phosphorylation is widely recognized as an
important posttranslational modification that regulates
protein functions in living cells, by switching cellular
activities from one stage to another, and regulates gene
expression, cell proliferation, and cell differentiation. In
In general, introduction of phosphate moiety to esters
of amino acids or peptides has been accomplished em-
1
10
ploying dialkyl or diaryl phosphorochloridates. How-
ever, the most effective and versatile procedures for these
2
III
purposes were based on P chemistry, originally devel-
most cases tyrosine as well as serine/threonine kinases
and corresponding phosphatases are involved in this
oped for the solid-phase synthesis of oligodeoxynucle-
11
otides. A variety of different protecting groups of
3
process. N-Phosphorylated proteins and amino acids also
6a,7b
7c
5b
phosphate function (benzyl,
2
4-chlorobenzyl, allyl,
play important roles in the regulation of enzyme ac-
tivity and protein biosynthesis. The molecular basis of
9a
6a
-cyanoethyl, tert-butyl ) were introduced. Alterna-
4
tively, monoprotected amino acids or peptide phosphates
protein regulation induced by phosphorylation has at-
tracted interest recently. However, the isolation of phos-
phorylated peptides from biological sources for functional
studies is usually not feasible, and therefore efficient
chemical phosphorylation methods are still appreciated.
O-Phosphorylated derivatives of serine, threonine, and
tyrosine have been extensively studied, whereas limited
information is available for other amino acids modified
by a phosphate group due to paucity of such derivatives.
Therefore, many research groups are involved in the
development of synthetic methods for the preparation of
phosphoamino acids and phosphopeptides in order to
9b
were synthesized using H-phosphonate chemistry.
Phosphorothioate analogues of the phosphate were
originally developed for the modification of nucleotides
(
5) (a) Bannwarth, W.; Trzeciak, A. Helv. Chim. Acta 1987, 70, 175.
b) Kitas, E. A.; Knorr, R.; Trzeciak, A.; Bannwarth, W. Helv. Chim.
Acta 1991, 74, 1314.
6) (a) Perich, J . W.; J ohns, R. B. Tetrahedron Lett. 1988, 29, 2369.
b) Perich, J . W.; Ruzzene, M.; Pinna, L. A.; Reynolds, E. C. Int. J .
(
(
(
Pept. Protein Res. 1994, 43, 39.
(7) (a) Van Oijen, A. H.; Erkelenes, C.; van Boom, J . H.; Liskamp,
R. M. J J . Am. Chem. Soc. 1989, 111, 9103. (b) Dreef-Tromp, C. M.;
Lefeber, A. W. M.; van der Marel, G. A.; van Boom, J . H. Synthesis
1
992, 1269. (c) De Bont, D. B. A.; Moree, W. J .; van Boom, J . H.;
Liskamp, R. M. J . J . Org. Chem. 1993, 58, 1309.
8) (a) J i, G. I.; Xue, J . N.; Li, L. P.; Chai, W. G.; Zhao, Y. F. Synthesis
(
1
988, 444. (b) Ma, X. B.; Zhao, Y. F. J . Org. Chem. 1989, 54, 4005.
(9) (a) Kupih a´ r, Z.; V a´ radi, G.; Monostori, E.; T o´ th, G. K. Tetrahe-
*
To whom correspondence should be addressed. Fax +48-42-
815483.
1) Krebs, E. G. The Enzymes, 3rd ed.; Boyer, P. D., Krebs, E. G.,
Eds.; Academic: New York, 1986.
2) (a) Kamps, M. P.; Taylor, S. S.; Sefton, B. M. Nature 1984, 310,
89. (b) Yarden, Y.; Ullrich, A. Annu. Rev. Biochem. 1988, 57, 443.
6
dron Lett. 2000, 41, 4457. (b) Kupih a´ r, Z.; Kele, Z.; T o´ th, G. K. Org.
Lett. 2001, 3, 1033.
(10) (a) Alewood, P. F.; Perich, J . W.; J ohns, R. B. Synth. Commun.
1982, 12, 821. (b) Alewood, P. F.; Perich, J . W.; J ohns, R. B. Aust. J .
Chem. 1984, 37, 429. (c) J ohnson, T. B.; Coward, J . K. J . Org. Chem.
1987, 50, 1771.
(
(
5
(3) Hunter, T. Cell 2000, 7, 113.
(
4) Rosen, O. M.; Krebs, E. G. Protein Phosphorylation; Cold Spring
(11) McBride, L. J .; Caruthers, M. H. Tetrahedron Lett. 1983, 24,
245.
Harbor: Plainview, NY, 1981.
1
0.1021/jo026027d CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/17/2002
J . Org. Chem. 2002, 67, 7267-7274
7267

Baraniak et al.
and oligodeoxynucleotides.12 Only a few examples of
SCHEME 1a
phosphorothioylated amino acids or peptides obtained by
chemical synthesis have been reported.7
b,13,14.
Replace-
ment of the phosphate linked to an amino acid residue
with a phosphorothioate is expected to lead to derivatives
with increased stability, which may be useful in the
investigation of the process of protein phosphorylation/
dephosphorylation. Moreover, some of the well-known
inhibitors of metallopeptidases such as N-phospholeuci-
namide15 should increase their inhibitory activity by
substitution of one of the phosphate oxygen atoms with
sulfur. The basis for the enhanced inhibitory potency of
such compounds, especially against zinc metallopepti-
dases, is presumably due to favorable zinc-sulfur inter-
actions within the enzyme active sites.16
a
Reagents: (i) S8, pyridine; (ii) HOCH2CH2CN, DBU; (iii)
NH4OH.
In this paper we present application of the oxathia-
1
7
phospholane methodology
to the synthesis of N-
TABLE 1. P h ysicoch em ica l Ch a r a cter istics of
Com p ou n d s 3a -l
phosphorothioylated amino acids as well as O-phospho-
rothioylated derivatives of hydroxyamino acids, i.e.,
serine, threonine and tyrosine. Although the approach
had been designed for the stereocontrolled synthesis of
oligo(nucleoside phosphorothioate)s, we demonstrated its
application to the synthesis of N3′ f P5′ dinucleotides.18
Recently, on the basis of this methodology, conjugates of
L-phenylalanine and L-tryptophan with AZT-5′-O-phos-
phorothioate and phosphorodithioate have been pre-
pared.19
FAB-MS
m/ z
(M - 1)
31
P NMR
(δ, ppm)a
yield
(%)b
entry
R
cmpd
1
2
3
4
5
6
7
8
9
CH3
3a 96.70; 97.34
3b 96.66; 97.91
3c 96.79; 95.61
3d 96.50; 95.59
3e 97.38; 96.52
97.66; 96.10
3g 96.71; 96.28
3h 96.54; 95.52
240
268
282
316
298
312
300
355
266
356
370
432
83
78
87
89
95
80
95
75
CH(CH3)2
CH2CH(CH3)2
CH2Ph
CH2COOMe
CH2CH2COOMe 3f
CH2CH2SCH3
CH2-indolyl-3
proline
CH2
CH(CH3)
CH2C6H4
3i
3j
96.50; 94.30
105.49; 105.55
87
34(83)
92
Resu lts a n d Discu ssion
c
1
1
1
0
1
2
3k 104.00; 103.89
3l 100.52; 100.50
Our synthetic strategy consists of three steps: (i) O-
or N-derivatization of the corresponding amino acid
methyl esters with oxathiaphosphitylating reagent, (ii)
opening of 2-thiono-1,3,2-oxathiaphospholane ring of
amino acid derivatives with 3-hydroxypropionitrile, and
75
a
In CDCl3. b Yield of isolated products. c Reaction was per-
formed in CH2Cl2 in a presence of N,N-diisopropylethylamine.
diastereomers in the ratio ca. 1:1 except for proline
derivative 3i, which was found to be a 41:59 mixture. In
the case of tryptophan derivative 3h , diastereomers were
separated by crystallization. The absolute configuration
at the phosphorus atom of individual species has been
assigned by X-ray analysis, and the diastereomer absorb-
ing at lower field in the 31P NMR spectrum has been
(
iii) removal of â-cyanoethyl protecting group. Attempts
at oxathiaphosphorylation of amino acids possessing free
carboxyl function were unsuccessful due to formation of
unknown byproducts.
N-Oxathiaphosphorothioylation of L-amino acid methyl
esters (1a -i) was achieved by reaction with 2-chloro-
1
presence of elemental sulfur (Scheme 1). The resulting
N-(2-thiono-1,3,2-oxathiaphospholanyl)amino acid methyl
esters (3a -i) were isolated from the reaction mixture by
silica gel column chromatography in satisfactory yields
,3,2-oxathiaphospholane (2) in pyridine solution in the
assigned as R
where.
P
. Relevant data will be published else-
To obtain N-[O-(2-cyanoethyl)phosphorothioyl]amino
acid methyl esters (4a -i), the oxathiaphospholane ring
in 3a -i was opened with 3-hydroxypropionitrile in the
3
1
(
75-95%) and characterized by P NMR and FAB-MS
2
0
presence of DBU
(Scheme 1). In this reaction the
analysis (Table 1). By virtue of asymmetry at phosphorus
formation of intermediates, N-[O-(2-cyanoethyl)-O-(2-
mercaptoethyl)-phosphorothioyl]amino acid methyl es-
ters, was expected. They should undergo fast elimination
of ethylene sulfide, providing the desired products
atom, compounds 3 appeared as the mixtures of two
(
(
(
12) Eckstein, F. Annu. Rev. Biochem. 1985, 54, 367.
13) Lindh, L.; Stawi n˜ ski, J . J . Org. Chem. 1989, 55, 1338.
14) (a) Lu, H.; Mlodnosky, K. L.; Dinh, T. T.; Dastgah, A.; Lam, V.
4
a -i. Indeed, examination of the reaction mixtures by
Q.; Berkman, C. E. J . Org. Chem. 1999, 64, 8698. (b) Lu, H.; Hu, Y.;
Choey, C. J .; Mallari, J . P.; Villanueva, A. F.; Arrozal, A. F.; Berkman,
C. E. Tetrahedron Lett. 2001, 42, 4313.
3
1
P NMR after 3 h revealed in each case full conversion
of 3a -i into a pair of diastereomeric organophosphorus
products with the chemical shifts characteristic for
O-alkyl-amidophosphorothioate.18 The crude compounds
4a -i were purified by silica gel column chromatography
and their structures were verified by FAB mass spec-
trometry.
(
15) Kam, C.-M.; Nishino, N.; Powers, J . C. Biochemistry 1979, 18,
032.
16) (a) Bell, C. F. In Principles and Application of Metal Chelation;
3
(
Atkins, P. W., Holker, J . S. E., Holliday, A. K., Eds.; Oxford Univer-
sity: Oxford, 1977. (b) Prasad, A. S. In Biochemistry of Zinc; Frieden,
E., Ed.; Plenum: New york, 1993.
(
17) Stec, W. J .; Grajkowski, A.; Karwowski, B.; Kobyla n´ ska, A.;
Koziołkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.; Guga, P.;
Boczkowska, M. J . Am. Chem. Soc. 1995, 117, 12019.
The derivatives 4a -i were converted into N-phospho-
rothioate amino acids (5a -i) by overnight treatment with
(18) Baraniak, J .; Korczy n˜ ski, D.; Stec, W. J . J . Org. Chem. 1999,
6
4
4, 4233.
(19) Baraniak, J .; Kaczmarek, R.; Stec, W. J . Tetrahedron Lett. 2000,
(20) Okruszek, A.; Olesiak, M.; Krajewska, D.; Stec, W. J . J . Org.
Chem. 1997, 62, 2269.
1, 9139.
7
268 J . Org. Chem., Vol. 67, No. 21, 2002

N- and O-Phosphorothioylation of Amino Acids
SCHEME 2a
SCHEME 3a
a
Reagents: (i) S8, pyridine; (ii) HOCH2CH2CN, DBU; (iii)
NH4OH.
concentrated aqueous ammonia at room temperature.
This process also converts carboxymethyl function into
carboxamide (Scheme 1). Compounds 5a -i were isolated
from the reaction mixture by means of Sephadex LH-20
gel filtration. Their structures were confirmed by FAB
mass spectrometry.
Analogous procedure has been applied for O-phospho-
rothioylation of N-(tert-butoxycarbonyl)-protected serine,
threonine, and tyrosine methyl esters (1j-l). The syn-
thesis of corresponding O-oxathiaphospholane deriva-
tives 3j-l together with their subsequent condensation
with 3-hydroxypropionitrile leading to compounds 4j-l,
and final deprotection providing 5j-l is depicted in
Scheme 2.
a Reagents: (i) DBU/CH CN; (ii) LiOH/MeOH.
3
then sulfur atom present in the resulting phosphorothio-
amino acids 5 originates from exocyclic sulfur in 3.
Replacement of this exocyclic sulfur atom by oxygen gives
rise to 10, which in the condensation reaction with 3-hy-
droxypropionitrile followed by elimination of episulfide
should provide compound possessing a monoalkyl-
phosphoramidate bond. Attempts at oxidation of inter-
III
mediately formed P 1,3,2-oxathiaphospholane deriva-
tives using anhydrous tert-butyl hydroperoxide in ben-
zene solution gave a mixture of products containing the
desired 10 and a considerable amount of unidentified side
products. Therefore, we turned our attention to the
oxidation of 2-thiono derivatives 3 into 2-oxo 10 by means
of selenium dioxide.22
It is noteworthy to mention that when serine derivative
1
j was oxathiaphosphitylated under standard conditions
with 2 in pyridine as a solvent and HCl scavenger,
desired compound 3j was isolated in 34% yield (Table 1).
However, when this reaction was performed in methylene
chloride in the presence of N,N-diisopropylethylamine,
the yield increased to 83%.
The methodology presented in this paper allowed us
to obtained not only amino acid derivatives with unpro-
tected phosphorothioic moiety but also their monoalkyl
phosphorothioate species. When the 2-thiono-1,3,2-oxa-
thiaphospholane ring in 3c and 3f has been opened by
means of methanol treatment for 3 h, the corresponding
N-(O-methylphosphorothioyl)amino acid methyl esters
When N-(2-thiono-1,3,2-oxathiaphospholanyl)amino acid
methyl esters (3) were treated in acetonitrile solution
with SeO2 for 15 min, quantitative transformation of
PdS compounds 3 into PdO derivatives 10 was ob-
served.23 Since the reaction was clean and no side prod-
ucts were formed, derivatives 10 were used for further
reactions after only filtration through a small volume of
silica gel in order to remove an excess of selenium dioxide.
The compounds 10 were then converted into the corre-
sponding phosphate derivatives 11 by treatment with
3-hydroxypropionitrile/DBU reagents (Scheme 4).
A major problem in amino acids chemistry is associated
with racemization of the amino acid or its derivative.
Since the oxathiaphospholane ring-opening process in 3
is catalyzed by a strong base such as DBU, it was
necessary to check if racemization of phosphorylated
derivatives occurred during this reaction. P NMR
technique was used for this purpose, and 2-thiono-1,3,2-
oxathiaphospholane derivatives of racemic alanine (3a ),
leucine (3c), and glutamic acid (3f) were prepared as
model compounds. 31P NMR spectra recorded for L-3 as
well as for the corresponding D,L-3 showed in both cases
two signals because L-3 exists as a pair of diastereomers
identical with two enantiomeric pairs present in D,L-3.
The ring-opening condensation of D,L-3 with 3-hydroxy-
propionitrile/DBU provided isomers of D,L-4, which also
(
(
6c, 6f) were obtained in almost quantitative yield
Scheme 3). Also N-(O-benzylphosphorothioyl)amino acid
methyl esters (7c, 7f) were obtained in high yield under
treatment of compounds 3c and 3f with benzyl alcohol,
but longer time (12h) was needed to complete this reac-
tion. Amino acid methyl esters in 6 and 7 were converted
into corresponding litium salts (8 and 9) by means of
LiOH/MeOH.14 Compound 6c, considered a potential
inhibitor of metallopeptidases, was synthesized earlier
in 75% yield by phosphorothioylation of leucinamide
followed by monodealkylation of the phosphorothioate
methyl ester.21 Hence, our strategy is highly efficient and
convenient for the synthesis of this class of compounds.
The introduction of a phosphate function into amino
acids based on the above-described methodology is also
possible. The oxathiaphospholane derivatives 3 possess
sulfur atom in the both exo- and endocyclic position, but
3
1
(22) Guga, P.; Doma n˜ ski, K.; Stec, W. Angew. Chem., Int. Ed. 2001,
4
0, 610.
23) Potencially this process can be used for stereospecific conversion
of pure diastereoisomer of 3 into analogue 10, as described for 3′-O-
(
2
2
(21) Thompson, C. M.; Lin, J . Tetrahedron Lett. 1996, 37, 8979.
(2-thiono-1,3,2-oxathiaphospholanyl)nucleosides.
J . Org. Chem, Vol. 67, No. 21, 2002 7269

Baraniak et al.
SCHEME 4a
a
Reagents: (i) SeO2 in CH3CN; (ii) HOCH2CH2CN, DBU.
SCHEME 5
precursors. These intermediates are obtained in a facile
and efficient way and are stable and versatile compounds
that can be stored for a long time. As can be seen from
the results summarized in Table 1, the oxathiaphospho-
rothioylation reaction works well not only for derivati-
zation of hydroxyl groups in the side chain of amino acids
but also for functionalization of the amino group. We
demonstrated that oxathiaphospholane derivatives 3 can
be considered as intermediates for condensation reaction
with various alcohols. This reaction, based on DBU-
assisted nucleophilic attack of alcohol on the phosphorus
atom of the oxathiaphospholane ring in 3, furnished
phosphorothioate derivatives 4, 6, and 7 in high yields.
The oxathiaphospholane strategy is of particular interest,
since it provides a facile route to compounds possessing
unprotected phosphorothioate moiety. If 3-hydroxypro-
pionitrile was used as the alcohol, the 2-cyanoethyl group
was easily removed from resulting 4 in the following
ammonolysis process, leading to phosphorothioamino
acids 5. We confirmed by 31P NMR analysis that the
presence of DBU in the oxathiaphospholane ring-opening
reactions did not induce the racemization of phospho-
rothioylated derivatives of amino acids.
formed two enantiomeric pairs. However, when deriva-
tization of D,L-3 was performed with AZT (Scheme 5), four
diastereomers of conjugates 12 were visible. The same
reaction performed for L-3 provided two diastereomers.
From these model experiments we concluded that the
oxathiaphospholane ring-opening process catalyzed by
DBU in 3 did not cause racemization in resulting phos-
phorothioylated amino acids 4. Another possibility of
racemization should be considered for the ammonolysis
process and the treatment with LiOH. To test whether
these steps may result in amino acid racemization,
conjugates 12 formed from L-3 were treated indepen-
We also demonstrated that the oxathiaphospholane
strategy employing 2-oxo-1,3,2-oxathiaphospholanes de-
rivatives 10 is suitable for phosphorylation of amino acids
and is a general route to the synthesis of phosphodiesters,
phosphoamidoesters, and phosphoamidates of amino acid
methyl esters, as well as their phosphorothioate conge-
ners. It is worthy to mention that it can also be expanded
to the preparation of phosphorylated amino acids labeled
dently with aqueous ammonia and LiOH/MeOH. 31
P
18
in phosphate moiety with oxygen isotope, since O-
NMR spectra recorded after 12 h did not reveal any extra
signals derived from D-analogues, indicating no racem-
ization during ammonolysis and lithium hydroxide treat-
ment. The result is in agreement with earlier literature
reports that the ammonia treatment does not cause
racemization of any amino acid.24
labeled 1,3,2-oxathiaphosphitylating reagent had been
synthesized in our laboratory.22
In summary, we developed a new strategy for facile
introduction of phosphate and phosphorothioate moiety
into amino acids. Application of the oxathiaphospholane
methodology to phosphorothioylation of peptides is cur-
rently in progress.
Con clu sion s
Exp er im en ta l Section
We developed a new strategy for the phosphorothioy-
lation of amino acids using 2-thiono-1,3,2-oxathiaphos-
pholane derivatives of amino acid methyl esters (3) as
Gen er a l Meth od s. 31P, 1H, and 13C NMR spectra (ppm)
were measured on a 200 MHz spectrometer unless stated
otherwise. 31P NMR shift values were assigned relative to
(24) Tung, C.-H.; Stein, S. Bioconjugate Chem. 2000, 11, 605.
3 4
H PO as an external standard. /Refers to the presence of
7
270 J . Org. Chem., Vol. 67, No. 21, 2002

N- and O-Phosphorothioylation of Amino Acids
diastereoisomeric peaks in the NMR spectrum. Analytical thin-
layer chromatography (TLC) was performed on precoated (0.25
mm thickness) glass plates (silica gel 60 F-254). Spots were
visualized by UV light (254 nm) if possible and by staining
13C NMR (CDCl
) δ: 172.80, 172.30, 68.62, 68.22, 54.67, 54.35,
3
52.35, 51.43, 36.81, 36.52, 29.34, 29.04, 28.41.
N-(2-Th ion o-1,3,2-oxa t h ia p h osp h ola n yl)m et h ion in e
1
Meth yl Ester (3g). H NMR (CDCl ) δ: 4.58-4.46 (m, 1H),
3
plates with either PdCl
2
or ninhydrin. Column chromatogra-
4.44-4.22 (m, 2H), 4.21-4.03 (m, 2H), 3.77* and 3.76* (s, 3H),
3.63-3.40 (m, 2H), 2.66-2.55 (m, 2H), 2.09* and 2.08* (s, 3H),
phy was performed using Kieselgel-60 230-400 mesh silica
gel. All nonvolatile compounds were routinely dried under high
vacuum. Anhydrous solvents and other liquid reagents were
2.04-1.89 (m, 2H). 13C NMR (CDCl ) δ: 172.74, 68.89, 68.51,
3
54.58, 54.08, 52.61, 37.15, 36.90, 33.13 (J ) 6.9 Hz), 32.86 (J
transferred by syringe. Acetonitrile was distilled from P
after being refluxed for several hours and stored over CaH2.
Pyridine was distilled from CaH after being refluxed for
several hours and stored over CaH . Other solvents were dried
according to known methods and distilled prior to use.2
2 5
O
) 6.9 Hz), 29.79, 15.31, 15.13.
N-(2-Th ion o-1,3,2-oxa t h ia p h osp h ola n yl)t r yp t op h a n
1
2
Meth yl Ester (3h ). H NMR (CDCl
(
4
1
1
) δ: 8.09 (bs, 1H), 7.57
3
2
d, 1H), 7.39-7.35 (m, 1H), 7.24-7.06 (m, 3H), 4.38-4.06 (m,
5
13
H), 3.67 (s, 3H), 3.44-3.26 (m, 4H). C NMR (CDCl ) δ:
3
2
-Chloro-1,3,2-oxathiaphospholane was prepared as previously
70.10, 123.40, 123.13, 122.22, 119.64, 118.55, 118.20, 111.27,
09.57, 68.70, 68.35, 56.37, 56.11, 52.50, 36.97, 30.02.
1
7
reported.
Gen er a l P r oced u r e for th e Syn th esis of N- or O-(2-
Th ion o-1,3,2-oxa th ia p h osp h ola n yl)a m in o Acid Meth yl
Ester s (3). To a solution of amino acid methyl ester 1 (1 mmol)
in 5 mL of dry pyridine was added elemental sulfur (2 mmol)
followed by dropwise addition of 2-chloro-1,3,2-oxathiaphos-
pholane (2) (1 mmol). The reaction mixture was stirred at room
temperature for 12 h. Then the solvent was removed in vacuo
and the residue was triturated with acetonitrile (10 mL).
Undissolved sulfur was filtered and the filtrate was condensed
in vacuo. The residue was dissolved in 2-3 mL of chloroform
and applied to a silica gel column (2.5 × 18 cm). The column
was eluted with chloroform for compounds 3a ,b, 3d -l and with
methanol in chloroform (0 f 5%) for 3c.
N-(2-Th ion o-1,3,2-oxa th ia p h osp h ola n yl)p r olin e Meth -
1
yl Ester (3i). H NMR (CDCl ) δ: 4.58-4.29 (m, 3H), 3.74*
3
1
3
and 3.73* (s, 3H), 3.54-3.31 (m, 4H), 2.26-1.95 (m, 4H).
C
NMR (CDCl ) δ: 173.95, 68.47, 68.15, 62.31 (J ) 6.7 Hz), 61.74
3
(J ) 6.6 Hz), 52.25, 47.94, 47.67, 37.04, 36.67, 30.90 (J ) 9.2
Hz), 25.24 (J ) 8.2 Hz).
O-(2-Th ion o-1,3,2-oxa t h ia p h osp h ola n yl)-N-(ter t-b u -
1
toxyca r bon yl)ser in e Meth yl Ester (3j). H NMR (CDCl3)
δ: 4.57-4.37 (m, 5H), 3.74* and 3.73* (s, 3H), 3.55-3.41 (m,
2H), 1.45 (s, 10H). 13C NMR (CDCl ) δ: 169.76, 154.94, 80.32,
3
70.67, 68.12 (J ) 6.5 Hz), 53.65 (J ) 7.4 Hz), 52.71, 36.33,
28.20.
O-(2-Th ion o-1,3,2-oxa t h ia p h osp h ola n yl)-N-(ter t-b u -
1
Appropriate fractions were combined and evaporated in
t oxyca r b on yl)t h r eon in e Met h yl E st er (3k ). H NMR
vacuo to give desired compounds 3a -l. Their chemical shifts
(CDCl ) δ: 5.33-5.13 (m, 2H), 4.55-4.29 (m, 3H), 3.78* and
3
3
1
13
in P NMR, FAB-MS and yields are summarized in Table 1.
3.77* (s, 3H), 3.55-3.41 (m, 2H), 1.51-1.39 (m, 12H). C NMR
N-(2-Th ion o-1,3,2-oxa th ia p h osp h ola n yl)a la n in e Meth -
3
(CDCl )δ: 169.90, 155.38, 79.97, 70.31, 70.12, 57.54, 52.36,
36.33, 36.16, 27.88, 18.46, 17.95.
1
yl Ester (3a ). H NMR (CDCl
3
3
) δ: 4.46-4.28 (m, 2H), 4.17-
.99 (m, 2H), 3.75* and 3.73* (s, 3H), 3.51-3.38 (m, 2H), 1.46*
O-(2-Th ion o-1,3,2-oxa t h ia p h osp h ola n yl)-N-(ter t-b u -
1
3
1
and 1.41* (d, 3H, J ) 7.0 Hz). C NMR (CDCl
3
) δ: 173.41,
toxyca r bon yl)tyr osin e Meth yl Ester (3l). H NMR (CDCl
3
)-
1
70.64, 68.69, 68.38, 52.49, 51.42, 51.21, 36.98, 36.72, 20.60
δ: 7.27-7.11 (m, 4H), 4.98-4.41 (m, 3H), 3.70 (s, 3H), 3.48-
13
(J ) 6.8 Hz), 20.40 (J ) 6.4 Hz).
3.31 (m, 2H), 3.09-3.02 (m, 2H), 1.41 (s, 10H). C NMR
CDCl ) δ: 171.91, 154.80, 149.64 (J ) 10.0 Hz), 133.61,
130.26, 130.12, 121.42, 79.79, 70.95, 54.23, 52.08, 37.63, 36.49,
8.09.
Gen er a l P r oced u r e for Syn th esis of N- or O-[O-(2-
(
3
N-(2-Th ion o-1,3,2-oxa th ia p h osp h ola n yl)va lin e Meth yl
1
Ester (3b). H NMR (CDCl
(
2
3
) δ: 4.50-4.26 (m, 2H), 4.00-3.76
2
m, 2H), 3.73* and 3.72* (s, 3H), 3.55-3.37 (m, 2H), 2.13-
.00 (m, 1H), 0.96* and 0.92* (d, 6H, J ) 6.8 Hz). 1 C NMR
3
(CDCl
3
) δ: 172.61, 172.47, 68.79, 68.38, 61.11, 61.02, 52.11,
Cya n oeth yl)p h osp h or oth ioyl]a m in o Acid Meth yl Ester s
(4). To a solution of 3 (1 mmol) in 10 mL of dry acetonitrile
was added 3-hydroxypropionitrile (2 mmol) followed by DBU
3
1
7.02, 36.71, 31.88 (J ) 5.9 Hz), 31.64 (J ) 6.0 Hz), 18.82,
7.70.
(1.2 mmol). The reaction mixture was stirred for 3 h at room
N-(2-Th ion o-1,3,2-oxa th ia p h osp h ola n yl)leu cin e Meth -
1
temperature. Then the solvent was evaporated, and the
residue was dissolved in a mixture of chloroform/methanol (50:
yl Ester (3c). H NMR (CDCl
3
) δ: 4.49-4.36 (m, 1H), 4.28-
4
3
1
.21 (m, 1H), 4.13-4.08* and 4.04-3.96* (m, 1H), 3.72* and
1
, v/v) and chromatographed on a silica gel column (2.5 × 18
.71* (s, 3H), 3.54-3.49 (m, 1H), 3.45-3.40 (m, 1H), 1.83-
.74 (m, 1H), 1.63-1.49 (m, 2H), 1.00 (t, 1H, J ) 6.0 Hz), 0.94
cm; eluent 2% f 22% methanol in chloroform) to give
compounds 4a -l.
1
3
(
t, 6H, J ) 6.8 Hz). C NMR (CDCl ) δ: 173.58, 68.80, 68.42,
3
N-[O-(2-Cya n oeth yl)p h osp h or oth ioyl]a la n in e Meth yl
5
2
4.35, 54.08, 52.28, 42.96, 36.98 (J ) 5.9 Hz), 24.27, 22.64,
3
1
Ester (4a ). Yield 73%. P NMR (CDCl
5
3
/CD
OD) δ: 4.05-3.95 (m, 3H), 3.69*
and 3.67* (s, 3H), 3.36-3.30 (d, 2H), 2.72-2.62 (m, 2H), 1.33-
.30 (t, 3H, J ) 6.8 Hz). 13C NMR (CDCl
/CD OD) δ: 177.05,
3
OD) δ: 58.30,
1.84, 21.56.
1
3 3
8.20. H NMR (CDCl /CD
N -(2-Th ion o-1,3,2-oxa t h ia p h osp h ola n yl)p h e n yla la -
1
n in e Meth yl Ester (3d ). H NMR (CDCl
5
(
3
) δ: 7.39-7.12 (m,
1
3
3
H), 4.47-4.23 (m, 3H), 4.21-3.91 (m, 2H), 3.70* and 3.69*
13
176.24, 117.87, 59.98, 52.37, 52.24, 51.00, 50.20, 20.46 (J )
.0 Hz), 20.21, 19.36. FAB-MS m/z: (M - 1) 251.
s, 6H), 3.47-3.29 (m, 2H), 3.11-3.05 (m, 2H). C NMR
CDCl ) δ: 148.24, 136.99, 135.10, 129.10, 128.25, 126.87,
8.40, 68.13, 56.48, 52.06, 39.80 (J ) 4.5 Hz), 36.69, 36.43.
N-(2-Th ion o-1,3,2-oxa th ia p h osp h ola n yl)a sp a r tic Acid
6
(
3
N-[O-(2-Cyan oeth yl)ph osph or oth ioyl]valin e Meth yl Es-
6
3
1
ter (4b). Yield 96%. P NMR (CDCl
3
/CD
OD) δ: 4.06-3.87 (m, 2H), 3. 65* and
.64* (s, 3H), 3.62-3.53 (m, 3H), 3.32-3.04 (m, 1H), 2.68-
/CD OD) δ:
3
OD) δ: 58.03, 57.71.
1
3 3
H NMR (CDCl /CD
1
Dim eth yl Ester (3e). H NMR (CDCl
3
): δ 4.51-4.28 (m, 4H),
.75* and 3.68* (s, 3H), 3.52-3.40 (m, 2H), 3.06-2.77 (m, 2H).
3
3
2
1
3
3
.60 (m, 2H), 0.89-0.79 (m, 6H). 13C NMR (CDCl
75.78, 175.46, 117.83, 60.37, 60.11, 59.84, 59.75, 51.84, 51.78,
1.84 (J ) 5.2 Hz), 19.41 (J ) 8.0 Hz), 18.80, 17.66. FAB-MS
1
3
3
C NMR (CDCl ) δ: 171.36, 171.12, 170.74, 170.58, 68.60,
3
6
8.38, 52.56, 52.50, 51.85, 51.70, 37.84 (J ) 3.9 Hz), 37.52 (J
)
4.9 Hz), 36.71, 36.41.
m/z: (M - 1) 279.
N-(2-Th ion o-1,3,2-oxa th ia p h osp h ola n yl)glu ta m ic Acid
N-[O-(2-Cya n oeth yl)p h osp h or oth ioyl]leu cin e Meth yl
1
Dim eth yl Ester (3f). H NMR (CDCl
4
2
3
) δ: 4.40-4.27 (m, 2H),
31
Ester (4c). Yield 96%. P NMR (CDCl
5
3
/CD
3
OD) δ: 59.14,
.17-4.09 (m, 2H), 3.74 (s, 3H), 3.66 (s, 3H), 3.51-3.39 (m,
H), 2.52-2.41 (m, 2H), 2.12-2.06 (m, 1H), 2.00-1.89 (m, 1H).
1
8.25. H NMR (CDCl /CD OD) δ: 4.06-3.96 (m, 3H), 3.77*
3 3
and 3.71* (s, 3H), 3.60-3.40 (m, 2H), 2.77-2.74 (m, 2H), 1.82-
1
6
.76 (m, 1H), 1.54-1.49 (m, 2H))0.94* and 0.93* (d, 9H, J )
.6 Hz). 13C NMR (CDCl
/CD OD) δ: 177.44, 176.41, 117.81,
(25) Perrin, D. D.; Armarego, W. L. F. In Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: New York 1988.
3
3
59.88, 53.96, 53.15, 52.08, 51.92, 43.54 (J ) 5.0 Hz),43.02 (J
J . Org. Chem, Vol. 67, No. 21, 2002 7271

Baraniak et al.
)
5.0 Hz), 24.32, 22.50, 22.39, 21.83, 21.46, 19.36 (J ) 8.4 Hz).
80.22, 72.67, 60.82, 60.46, 58.13, 52.44, 27.98, 19.32 (J ) 8.0
Hz), 18.31. FAB-MS m/z: (M - 1) 381.
FAB-MS m/z: (M - 1) 293.
N-[O-(2-Cya n oet h yl)p h osp h or ot h ioyl]p h en yla la n in e
O-[O-(2-Cya n oeth yl)p h osp h or oth ioyl]-N-(ter t-bu toxy-
ca r bon yl)tyr osin e Meth yl Ester (4l). Yield 74%. P NMR
3
1
31
Meth yl Ester (4d ). Yield 73%. P NMR (CDCl
3
/CD
3
OD) δ:
1
1
5
4
3
5
1
7.25, 56.73. H NMR (CDCl
3
/CD
3
OD) δ: 7.26-7.01 (m, 5H),
(CDCl
3
/CD
3
OD) δ: 53.17, 53.12. H NMR (CDCl
3
/CD
3
OD) δ:
.20-3.95 (m, 3H), 3.70-3.60 (m, 1H), 3.53* and 3.51* (s, 3H),
6.95 (dd, 4H, J ) 8.4 Hz), 4.42-4.36 (t, 1H, J ) 5.6 Hz), 4.23-
.45-3.29 (m, 1H), 2.94-2.69 (m, 2H), 2.37-2.28 (q, 2H, J )
4.04 (m, 2H), 3.61* and 3.60* (s, 3H), 3.59-3.52 (m, 2H), 2.94-
1
3
13
.8 Hz). C NMR (CDCl
3
/CD
3
OD) δ: 175.45, 174.82, 136.59,
2.89 (m, 2H), 2.67-2.59 (q, 2H, J ) 6.0 Hz), 1.31 (s, 9H).
C
29.12, 128.07, 126.48, 117.65, 59.33, 56.72, 55.94, 51.75, 39.96
3 3
NMR (CDCl /CD OD) δ: 172.36, 150.95, 131.60, 129.85,
(
J ) 7.0 Hz), 39.64 (J ) 7.0 Hz), 18.97 (J ) 8.2 Hz). FAB-MS
m/z: (M - 1) 327.
120.83, 117.51, 79.96, 61.16 (J ) 10.0 Hz), 54.34, 52.05, 37.24,
27.97, 19.33 (J ) 8.0 Hz). FAB-MS m/z: (M - 1) 443.
N-[O-(2-Cya n oet h yl)p h osp h or ot h ioyl]a sp a r t ic Acid
Gen er a l P r oced u r e for Syn th esis of N- or O-(P h os-
p h or oth ioyl)a m in o Acid Am id es (5). Compound 4 (1 mmol)
was suspended in 10 mL of 20% aqueous ammonia and left
for 24 h at room temperature in a tighthy closed vessel. Then
the ammonia was evaporated, and the residue was dissolved
3
1
Dim eth yl Ester (4e). Yield 96%. P NMR (CDCl
δ: 57.72, 56.82. H NMR (CDCl /CD
3 3
3
/CD
OD) δ: 4.30-4.19 (m,
H), 4.02-3.91 (m, 2H), 3.66* and 3.65* (s, 3H), 3.60* and
.58* (s, 3H), 3.55-3.50 (m, 2H), 2.81-2.74 (m, 2H), 2.65-
3
OD)
1
2
3
2
1
5
1
3
.63 (m, 2H). C NMR (CDCl
3
/CD
3
OD) δ: 174.05, 173.68,
in a mixture of MeOH/H
Sephadex LH-20 gel filtration column (eluent MeOH/H
2
O (6:1, v/v) and purified on a
O, 6/1,
71.87, 171.71, 117.84, 59.88, 59.82, 52.56, 52.50, 51.84, 51.72,
1.13, 38.32, 19.32 (J ) 8.4 Hz). FAB-MS m/z: (M - 1) 309.
N-[O-(2-Cya n oet h yl)p h osp h or ot h ioyl]glu t a m ic Acid
2
v/v). Compounds 5a -l were more than 92% pure according to
analytical HPLC [Econosphere C-18 column (4.6 × 250 mm),
1.0 mL/min flow rate; buffer A, 0.1 M TEAB pH 7.5; buffer B,
3
1
3 3
Dim eth yl Ester (4f). Yield 73%. P NMR (CDCl /CD OD) δ:
1
3
40% CH CN in 0.1 M TEAB; gradient from 0 f 50% B over
5
3
3
2
8.30, 58.20. H NMR (CDCl
3
/CD
3
OD) δ: 4.22 (bs, 1H), 4.00-
2
0 min, 50% f 100% over 3 min, then isocratic].
.92 (m, 2H), 3.66* and 3.64* (s, 3H), 3.61* and 3.58* (s, 3H),
N-(P h osp h or oth ioyl)a la n in a m id e (5a ). Yield 77%. 31P
.56 (bs, 1H), 3.28 (d, 1H), 2.69-2.58 (m, 2H), 2.43-2.33 (m,
H), 2.04-1.97 (m, 1H), 1.85-1.78 (m, 1H). 13C NMR (CDCl
3
/
2
1
3
NMR (D
O) δ: 47.49. H NMR (CD
OD) δ: 3.53-3.37 (m, 1H),
3
1
3
CD
OD) δ: 175.13, 175.08, 174.73, 174.19, 173.72, 117.73,
1.56 (d, 3H, J ) 7.2 Hz). C NMR (CD
21.32 (J ) 3.2 Hz). FAB-MS m/z: (M - 1) 183.
3
OD) δ: 188.12, 50.25,
5
2
9.74, 59.68, 54.06, 53.79, 52.08, 51.97, 51.61, 51.43, 29.93,
N-(P h osp h or oth ioyl)va lin a m id e (5b). Yield 84%. 31P
9.65, 29.06, 28.96 (J ) 6.0 Hz), 19.18 (J ) 8.0 Hz). FAB-MS
m/z: (M - 1) 323.
1
NMR (D
2
O) δ: 47.05. H NMR (CD
3
OD) δ: 3.30-3.07 (m, 1H),
1
3
N-[O-(2-Cyan oeth yl)ph osph or oth ioyl]m eth ion in e Meth -
2.15-2.00 (m, 1H), 0.95-0.83 (m, 6H). C NMR (CD OD) δ:
187.15, 58.45, 33.21 (J ) 6.5 Hz), 21.08, 18.13. FAB-MS m/z:
(M - 1) 211.
3
3
1
yl Ester (4g). Yield 89%. P NMR (CDCl
3
/CD
3
OD) δ: 58.57,
1
5
7.85. H NMR (CDCl /CD OD) δ: 4.18-3.97 (m, 3H), 3.72*
3 3
N-(P h osp h or oth ioyl)leu cin a m id e (5c). Yield 81%. 31P
and 3.69* (s, 3H), 3.38 (bs, 1H), 3.24 (bs, 1H), 2.75-2.62 (m,
1
2
1
H), 2.57-2.52 (q, 2H, J ) 7.4 Hz), 2.04* and 2.03* (s, 3H),
.99-1.94 (m, 1H), 1.91-1.82 (m, 1H). 13C NMR (CDCl
OD) δ: 176.73, 175.48, 117.99, 60.24, 60.11, 54.84, 53.65,
NMR (D
2
O) δ: 49.34. H NMR (CD
3
OD) δ: 3.54-3.45(m, 1H),
13
/
2.95-2.87 (m, 1H), 1.72-1.63 (m, 2H), 0.99-0.91 (m, 6H).
NMR (CD OD) δ: 186.02, 55.13, 47.21 (J ) 6.1 Hz), 26.10,
24.31, 22.05. FAB-MS m/z: (M - 1) 225.
N-(P h osph or oth ioyl)ph en ylalan in am ide (5d). Yield 72%.
C
3
CD
3
3
5
1
2.61, 52.38, 33.42, 33.87, 29.95, 29.79, 19.47 (J ) 8.0 Hz),
5.12. FAB-MS m/z: (M - 1) 311.
N-[O-(2-Cyan oeth yl)ph osph or oth ioyl]tr yptoph an Meth -
3
1
1
P NMR (D
2
O) δ: 46.01. H NMR (CD
3
OD) δ: 7.25-7.15 (m,
3
1
13
yl Ester (4h ). Yield 93%. P NMR (CDCl
3
/CD
3
OD) δ: 57.15,
5H), 3.95 (m, 1H), 2.95 (m, 2H). C NMR (CD
3
OD) δ: 183.12,
1
5
7
4
2
1
1
6.11. H NMR (CDCl
3
/CD
3
OD) δ: 8.15 (bs, 1H), 7.49 (d, 1H),
136.21, 129.42, 127.92, 126.31, 55.73, 40.21. FAB-MS m/z: (M
- 1) 259.
.46-7.31 (m, 1H), 7.28-7.01 (m, 3H), 4.34-4.23 (m, 2H),
.15-4.05 (m, 1H), 3.64* and 3.59* (s, 3H), 3.28-3.20 (m, 2H),
N-(P h osp h or oth ioyl)a sp a r ticd ia m id e (5e). Yield 83%.
1
3
31
1
.38-2.13 (m, 2H). C NMR (CDCl
3
/CD
3
OD) δ: 176.16,
P NMR (D
1H), 2.12-1.97 (m, 2H). C NMR (CD
2
O) δ: 47.35. H NMR (CD
3
OD) δ: 3.50-3.38 (m,
1
3
75.60, 123.41, 122.43, 121.54, 118.91, 118.14, 117.89, 117.76,
11.24, 109.58, 59.53, 55.96, 54.95, 52.02, 30.07, 29.62, 18.92
3
OD) δ: 191.05, 187.12,
52.31, 38.34. FAB-MS m/z: (M - 1) 226.
(
J ) 8.0 Hz). FAB-MS m/z: (M - 1) 366.
N-(P h osp h or oth ioyl)glu ta m icd ia m id e (5f). Yield 77%.
3
1
O) δ: 47.83. 1H NMR (CD
N-[O-(2-Cya n oeth yl)p h osp h or oth ioyl]p r olin e Meth yl
P NMR (D
(m, 1H), 2.18-2.04 (m, 2H), 1.84-1.60 (m, 2H). C NMR
(CD OD) δ: 184.21, 182.12, 53.18, 36.45, 34.02. FAB-MS m/z:
2
3
OD) δ: 3.48-3.37
3
1
13
Ester (4i). Yield 88%. P NMR (CDCl
3
/CD
3
OD) δ: 58.77,
1
5
3
3
1
1
5
2
6.99. H NMR (CDCl
3
/CD
3
OD) δ: 4.23-4.18 (m, 1H), 4.07-
3
.97 (m, 2H), 3.65* and 3.63* (s, 3H), 3.59-3.54 (m, 2H), 3.31-
(M - 1) 240.
.17 (m, 1H), 2.70-2.59 (m, 2H), 2.10-2.03 (m, 1H), 1.85-
N-(P h osp h or oth ioyl)m eth ion in a m id e (5g). Yield 80%.
1
3
31
1
.75 (m, 3H). C NMR (CDCl
3
/CD
3
OD) δ: 177.39, 176.46,
P NMR (D
1H), 2.35-2.21 (m, 2H), 2.13 (s, 3H), 1.97-1.91 (m, 2H).
NMR (CD
2
O) δ: 41.97. H NMR (CD OD) δ: 3.49-3.35 (m,
3
13
17.92, 117.79, 61.65 (J ) 7.0 Hz), 59.71 (J ) 4.0 Hz), 52.12,
1.92, 47.53, 46.65, 31.33 (J ) 6.8 Hz), 30.97 (J ) 7.5 Hz),
5.50 (J ) 7.4 Hz), 25.29 (J ) 7.2 Hz), 19.33 (J ) 8.0 Hz).
C
3
OD) δ: 185.12, 53.00, 34.61, 29.27, 15.61. FAB-MS
m/z: (M - 1) 243.
FAB-MS m/z: (M - 1) 277.
N-(P h osph or oth ioyl)tr yptoph an ylam ide (5h ). Yield 80%.
3
1
1
O-[O-(2-Cya n oeth yl)p h osp h or oth ioyl]-N-(ter t-bu toxy-
P NMR (D
3H), 7.05-6.95 (m, 2H), 3.92 (m,1H), 3.18 (m, 2H). C NMR
(CD OD) δ: 187.15, 135.90, 127.61, 124.12, 122.30, 119.00,
2
O) δ: 49.78. H NMR (CD OD) δ: 7.51-7.10 (m,
3
3
1
13
ca r bon yl)ser in e Meth yl Ester (4j). Yield 97%. P NMR
1
(
CDCl
3
/CD
.40 (bs, 1H), 4.25-4.09 (m, 2H), 4.07-3.94 (m, 2H), 3.68* and
.67* (s, 3H), 3.57 (bs, 1H), 3.29-3.25 (qw, 1H, J ) 1.6 Hz),
/CD OD) δ:
3
OD) δ: 59.17, 58.22. H NMR (CDCl
3
/CD
3
OD) δ:
3
4
3
2
1
1
118.42, 111.22, 108.89, 55.18, 30.72. FAB-MS m/z: (M - 1)
298.
.68-2.61 (m, 2H), 1.35 (s, 9H). 13C NMR (CDCl
71.03, 155.74, 117.53, 80.21, 65.73, 60.74, 53.86, 52.43, 28.00,
9.23 (J ) 7.0 Hz). FAB-MS m/z: (M - 1) 367.
N-(P h osp h or oth ioyl)p r olin a m id e (5i). Yield 81%. 31P
3
3
1
NMR (D
2
O) δ: 47.17. H NMR (CD
3
OD) δ: 3.95-3.87 (m, 1H),
1
3
3.64-3.52 (m, 2H), 3.29-3.15 (m, 2H), 2.08-1.97 (m, 2H).
C
O-[O-(2-Cya n oeth yl)p h osp h or oth ioyl]-N-(ter t-bu toxy-
NMR (CD OD) δ: 182.13, 63.21, 49.21, 30.59 (J ) 9.2 Hz),
3
3
1
ca r bon yl)th r eon in e Meth yl Ester (4k ). Yield 70%.
P
25.14. FAB-MS m/z: (M - 1) 209.
1
NMR (CDCl
3
/CD
3
OD) δ: 58.22, 57.36. H NMR (CDCl
3
/CD
3
-
O-(P h osp h or ot h ioyl)-N-(ter t-b u t oxyca r b on yl)ser in a -
m id e (5j). Yield 83%. 31P NMR (D
O) δ: 46.83. H NMR (CD
1
-
OD) δ: 4.82 (bs, 1H), 4.18-4.00 (m, 2H), 3.74 (bs, 2H), 3.68*
2
3
and 3.67* (s, 3H), 3.27-3.24 (qw, 1H, J ) 1.6 Hz), 2.67-2.60
OD) δ: 3.31-3.17 (m, 1H), 2.95-2.80 (m, 2H), 1.44 (s, 9H).
1
3
(
6
t, 2H, J ) 6.4 Hz), 1.35 (s, 9H), 1.27* and 1.23* (s, 3H, J )
C NMR (CD
3
OD) δ: 183.99, 155.17, 84.07, 65.12, 56.42,
1
3
.4 Hz). C NMR (CDCl
3 3
/CD OD) δ: 172.16, 150.75, 117.65,
28.01. FAB-MS m/z: (M - 1) 299.
7
272 J . Org. Chem., Vol. 67, No. 21, 2002

N- and O-Phosphorothioylation of Amino Acids
N-(P h osp h or ot h ioyl)-N-(ter t-b u t oxyca r b on yl)t h r eo-
N-(O-Ben zylp h osp h or oth ioyl)glu ta m ic Acid Dim eth yl
Ester (7f). Reaction of compound 3f (1 mmol) with benzyl
alcohol (1.5 mmol) in a presence of DBU (1.1 mmol) was
performed as described for 4 except that the reaction mix-
ture was stirred for 12 h at room temperature. Yield 96%.
3
1
1
n in a m id e (5k ). Yield 80%. P NMR (D
CD OD) δ: 3.43-3.33 (m, 1H), 3.01-2.86 (m, 1H), 1.44 (s,
OD) δ: 183.12,
2
O) δ: 48.52. H NMR
(
3
9
1
1
H), 1.32 (d, 3H, J ) 6.2 Hz). 13C NMR (CD
3
56.12, 84.00, 68.15, 59.47, 28.15, 19.31. FAB-MS m/z: (M -
) 313.
3
1
1
P NMR (CDCl
3
) δ: 59.37, 58.34. H NMR (CDCl ) δ: 7.28-
3
N-(P h osp h or ot h ioyl)-N-(ter t-b u t oxyca r b on yl)t yr osi-
7.16 (m,5H), 4.96-4.80 (t, 1H), 4.81-4.76 (t, 1H), 4.99 (d,
1H), 3.58-3.55 (m, 6H), 3.37-3.12 (m, 1H), 2.36 (t, 2H), 1.81
3
1
1
n a m id e (5l). Yield 77%. P NMR (D
2
O) δ: 43.92. H NMR
(bs, 1H), 1.79 (bs, 1H). 13C NMR (CDCl
) δ:175.12, 174.51,
(
(
1
CD
m, 2H), 1.37 (s, 9H). C NMR (CD
55.47, 130.12, 126.74, 115.32, 86.22, 56.14, 40.12, 28.00. FAB-
MS m/z: (M - 1) 375.
3
OD) δ: 7.21-6.88 (m,4H), 3.80-3.62 (m, 1H), 2.90-2.85
3
1
3
OD) δ: 183.77, 156.21,
137.50, 137.12, 128.15, 127.32, 127.10, 126.92, 68.43 (J ) 5.9
Hz), 52.47, 51.43, 51.03, 29.92, 29.14. FAB-MS m/z: (M - 1)
360.
3
N-(O-Meth ylph osph or oth ioyl)leu cin e Meth yl Ester (6c).
Reaction of compound 3c (1 mmol) with methanol (1.5 mmol)
in the presence of DBU (1.1 mmol) was performed as described
N-(O-Ben zylp h osp h or oth ioyl)glu ta m ic Acid Tr ilith i-
u m Sa lt (9f). The reaction was carried out according to the
3
1
procedure described in the case of 8c. Compound 9f: P NMR
3
1
1
1
for 4. Yield 96%. P NMR (CDCl
CDCl ) δ: 3.97 (bs, 1H), 3.80 (bs, 1H), 3.62* and 3.55* (s, 3H),
.44* and 3.38* (d, 3H, J ) 12.7 Hz), 1.88-1.45 (m, 1H), 1.39-
3
) δ: 63.64, 60.10. H NMR
(CD
3
OD) δ: 59.14, 58.37. H NMR (CD
3
OD) δ: 7.47-7.33 (m,
(
3
5H), 4.91-4.84 (m, 3H), 3.75-3.58 (m, 1H), 2.26-2.16 (m, 2H),
1.90-1.80 (m, 2H). 13C NMR (CD
OD) δ: 131.47, 130.80,
3
1
1
2
3
1
3
.33 (m, 2H), 0.79-0.75 (qw, 6H). C NMR (CDCl
3
) δ: 178.29,
130.55, 69.82, 59.71, 59.54, 36.58, 34.62. FAB-MS m/z: (M -
76.26, 52.57, 52.28 (J ) 4.8 Hz), 51.61, 43.40 (J ) 6.0 Hz),
4.07, 22.59, 21.78. FAB-MS m/z: (M - 1) 254.
Li) 344.
Gen er a l P r oced u r e for th e Syn th esis of N- or O-[O-
(2-Cya n oeth yl)p h osp h or yl]a m in o Acid Meth yl Ester s
(11). (A) Syn th esis of N- or O-(2-Oxo-1,3,2-oxa th ia p h os-
p h ola n yl)a m in o Acid Meth yl Ester s (10). Compound 3 (1
N-(O-Meth ylp h osp h or oth ioyl)leu cin e Dilith iu m Sa lt
(
8c). Compound 6c (O.5 mmol) was dissolved in methanol (3.0
mL), into which was added a 1.0 M aqueous solution of
LiOH (2.5 mL). The solution was stirred at room temperature
for 12 h and then filtered. The solvent was evaporated in
vacuo, and the residue was resuspended in anhydrous metha-
nol and filtered through 0.2 µm Teflon membrane to pro-
mmol) was dissolved in dry acetonitrile (10 mL), and then SeO
2
(2 mmol) was added. The reaction mixture was stirred for 15
min at room temperature. At this point, the 31P NMR control
showed full disappearance of the substrate and quantitative
formation of 2-oxo derivative 10 (two signals absorbing at 44-
48 ppm). Then the reaction mixture was passed through a dry
3
1
1
vide 8c. P NMR (CD
3
3
OD) δ: 59.43, 59.04. H NMR (CD OD)
δ: 3.68-3.57 (m, 1H), 3.51* and 3.49* (d, 3H, J ) 12.9 Hz),
1
.69-1.59 (m, 2H), 1.46-1.37 (m, 2H), 0.91-0.86 (qw, 6H).
2
silica gel column (ca. 1 g) to absorb traces of dissolved SeO .
1
3
C NMR (CD
3
OD) δ: 58.96 (J ) 4.6 Hz), 55.43, 54,0.81, 47.17,
(B) Con d en sa tion of 10 w ith 3-Hyd r oxyp r op ion itr ile.
Crude compound 10 in acetonitrile solution was reacted with
3-hydroxypropionitrile and DBU, providing 11. The reaction
was carried out according to general procedure described for
compounds 4.
2
2
7.11, 26.06, 24.96, 24.70. FAB-MS m/z: (M+1) 254, (M-Li)
46.
N-(O-Meth ylp h osp h or oth ioyl)glu ta m ic Acid Dim eth yl
E st er (6f). Reaction of compound 3f (1 mmol) with meth-
anol (1.5 mmol) and DBU (1.1 mmol) was performed as
N-(2-Oxo-1,3,2-oxa t h ia p h osp h ola n yl)a la n in e Met h yl
3
1
31
described for 4. Yield 96%. P NMR (CDCl
3
) δ: 60.59, 59.18.
Ester (10a ). P NMR (CD CN) δ: 46.26, 45.84. FAB-MS
3
1
H NMR (CDCl
3
) δ: 3.96 (bs, 1H), 3.67* and 3.65* (s, 3H), 3.61*
m/z: (M - 1) 224.
and 3.59* (s, 3H), 3.58-3.56 (m, 1H), 3.47* and 3.44* (d, 3H,
N-[O-(2-Cya n oeth yl)p h osp h or yl]a la n in e Meth yl Ester
J ) 13.0 Hz), 2.45-2.37 (m, 2H), 2.04-1.97 (m, 1H), 1.87-
31
1
(
(
(
11a ). Yield 70%. P NMR (CDCl
CDCl /CD OD) δ: 4.05-3.95 (m, 3H), 3.69 (s, 3H), 3.36-3.30
d, 2H), 2.72-2.62 (m, 2H), 1.33-1.30 (t, 3H, J ) 6.8 Hz).
/CD OD) δ: 177.05, 176.24, 117.87, 59.98, 52.37,
3 3
/CD OD) δ: 4.92. H NMR
1
3
1
3
.79 (m, 1H). C NMR (CDCl ) δ: 174.13, 173.79, 53.86, 52.32
3
3
(
J ) 4.9 Hz), 51.72, 51.58, 30.14, 29.78. FAB-MS m/z: (M -
13
C
1
) 284.
NMR (CDCl
3
3
N-(O-Meth ylp h osp h or oth ioyl)glu ta m ic Acid Tr ilith i-
5
2.24, 51.00, 50.20, 20.46 (J ) 6.0 Hz), 20.21, 19.36. FAB-MS
m/z: (M - 1) 235.
u m Sa lt (8f). The reaction was carried out according to the
3
1
procedure described in the case of 8c. Compound 8f: P NMR
N-(2-Oxo-1,3,2-oxath iaph osph olan yl)leu cin e Meth yl Es-
1
(
CD
3
OD) δ: 60.52, 60.32. H NMR (CD
H), 3.45* and 3.42* (d, 3H, J ) 13.1 Hz), 2.23-2.15 (m, 2H),
.87-1.76 (m, 4H). 13C NMR (CD
OD) δ: 59.74 (J ) 4.7 Hz),
3
OD) δ: 3.68-3.56 (m,
31
ter (10c). P NMR (CD
3
CN) δ: 47.73, 46.80. FAB-MS m/z:
1
1
5
(
M - 1) 266.
3
N-[O-(2-Cya n oeth yl)p h osp h or yl]leu cin e Meth yl Ester
7.70, 54.84, 42.05, 36.61, 34.36. FAB-MS m/z: (M - 1) 274,
1
(
(
(
2
1
5
11c). Yield 71%. 31P NMR (CDCl
CDCl /CD OD) δ: 4.18-4.01 (m, 3H), 3.71 (s, 3H), 3.75-3.52
m, 2H), 2.78-2.71 (m, 2H), 1.90-1.82 (m, 1H), 1.60-1.53 (m,
3 3
/CD OD) δ: 5.12. H NMR
(M - Li) 268.
3
3
N-(O-Ben zylph osph or oth ioyl)leu cin e Meth yl Ester (7c).
Reaction of compound 3c (1 mmol) with benzyl alcohol (1.5
mmol) in the presence of DBU (1.1 mmol) was performed as
described for 4 except that the reaction mixture was stirred
H), 0.93 (d, 6H, J ) 6.6 Hz). 13C NMR (CDCl
/CD OD) δ:
3 3
77.40, 176.61, 118.11, 59.88, 53.15, 52.08, 51.92, 44.14 (J )
.2 Hz), 43.02 (J ) 5.0 Hz), 24.32, 22.90, 22.39, 21.83, 19.36
3
1
for 12 h at room temperature. Yield 90%. P NMR (CDCl
3
) δ:
) δ: 7.29-7.17 (m, 5H), 4.88 (bs,
H), 4.78 (bs, 1H), 4.01* and 3,83* (bs, 1H), 3.58* and 3.54*
(
J ) 8.2 Hz). FAB-MS m/z: (M - 1) 277.
1
5
1
9.15, 57.30, H NMR (CDCl
3
N-(2-Oxo-1,3,2-oxa th ia p h osp h ola n yl)a sp a r tic Acid Di-
m eth yl Ester (10e). 31P NMR (CD
MS m/z: (M - 1) 282.
CN) δ: 47.36, 47.16. FAB-
3
(
s, 3H), 3.33 (s, 1H), 1.69-1.60 (m, 1H), 1.43-1.35 (m, 2H),
.80 (t, 6H, J ) 6.0 Hz). 1 C NMR (CDCl
) δ: 176.63, 137.82,
3
0
1
5
3
37.76, 128.07, 127.56, 127.37, 127.29, 68.71 (J ) 5.9 Hz),
2.00, 51.80, 43.56 (J ) 6.0 Hz), 22.51, 22.35, 21.98, 21.57.
N-[O-(2-Cya n oet h yl)p h osp h or yl]a sp a r t ic Acid Di-
3
1
m eth yl Ester (11e). Yield 72%. P NMR (CDCl
3
/CD
OD) δ: 4.36-4.25 (m, 2H), 4.11-
.99 (m, 2H), 3.66 (s, 3H), 3.61 (s, 3H), 3.58-3.54 (m, 2H),
3
/CD -
3
OD) δ:
1
FAB-MS m/z: (M - 1) 330.
4.45. H NMR (CDCl
3
/CD
3
3
2
N-(O-Ben zylp h osp h or ot h ioyl)leu cin e Dilit h iu m Sa lt
.88-2.76 (m, 2H), 2.66-2.61 (m, 2H). 13C NMR (CDCl
(
9c). The reaction was carried out according to the procedure
3
3
1
OD) δ: 174.32, 173.99, 172.17, 171.71, 117.64, 59.88, 59.77,
described in the case of 8c. Compound 9c: P NMR (CD
3
OD)
OD) δ: 7.36-7.27 (m, 5H), 4.82
s, 2H), 3.61-3.56 (m, 2H), 1.58-1.51 (m, 1H), 1.32-1.29 (m,
1
52.76, 52.61, 51.84, 51.90, 51.75, 38.32, 19.32. FAB-MS m/z:
δ: 58.49, 57.54. H NMR (CD
3
(
M - 1) 293.
(
2
1
2
H), 0.79-0.76 (q, 6H, J ) 3.0 Hz). 13C NMR (CD
3
OD) δ:
N-(2-Oxo-1,3,2-oxath iaph osph olan yl)tr yptoph an Meth -
31.37, 130.70, 69.63, 56.77, 51.60, 47.24, 27.04, 24.96, 24.81,
yl Ester (10h ). 31P NMR (CD
m/z: (M - 1) 339.
3
CN) δ: 47.42, 46.66. FAB-MS
4.68. FAB-MS m/z: (M - 1) 322, (M - Li) 316.
J . Org. Chem, Vol. 67, No. 21, 2002 7273

Baraniak et al.
N-[O-(2-Cyan oeth yl)ph osph or yl]tr yptoph an Meth yl Es-
117.82, 80.56, 72.67, 60.88, 60.36, 58.43, 52.44, 28.05, 19.48,
18.30. FAB-MS m/z: (M - 1) 365.
ter (11h ). Yield 68%. 31P NMR (CDCl
NMR (CDCl /CD
m, 1H), 7.27-7.00 (m, 3H), 4.32-4.25 (m, 2H), 4.18-4.08 (m,
/CD
OD) δ: 8.10 (bs, 1H), 7.50 (d, 1H), 7.48-7.32
OD) δ: 5.10. H
1
3
3
3
3
(
1
Ack n ow led gm en t. Authors are grateful to Prof.
Wojciech J . Stec and Dr. Piotr Guga for stimulating
discussion and helpful suggestions and wish to thank
Dr. Kyoichi A. Watanabe for careful reading of the
manuscript. This project was financially assisted by the
State Committee for Scientific Research (KBN), grants
1
3
H), 3.60 (s, 3H), 3.28-3.21 (m, 2H), 2.36-2.15 (m, 2H).
/CD OD) δ: 176.10, 175.83, 123.01, 122.13,
21.56, 119.01, 118.34, 118.07, 117.78, 111.34, 109.78, 59.53,
C
NMR (CDCl
3
3
1
5
1
5.72, 54.85, 52.34, 30.56, 29.62, 18.73. FAB-MS m/z: (M -
) 350.
O-(2-Oxo-1,3,2-oxa t h ia p h osp h ola n yl)-N-(ter t-b u t oxy-
4
P05F 006 17 and PBZ KBN 008/T09/98 (to W.J .S.).
3
1
ca r b on yl)t h r eon in e Met h yl E st er (10k ).
P
NMR
(
CD
3
CN) δ: 45.36, 45.30. FAB-MS m/z: (M - 1) 354.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
O-[O-(2-Cya n oeth yl)p h osp h or yl]-N-(ter t-bu toxyca r bo-
31
cedure and P NMR spectra for compounds 12a , 12c, and 12f
obtained in DBU catalyzed reactions of L-3a , D,L-3a , L-3c,
D,L-3c, L-3f, and D,L-3f with AZT. This material is available
free of charge via the Internet at http://pubs.acs.org.
3
1
n yl)th r eon in e Meth yl Ester (11k ). Yield 62%. P NMR
1
(
1
(
3
CDCl
H), 4.17-4.02 (m, 2H), 3.74 (bs, 2H), 3.65 (s, 3H), 3.25-3.20
m, 1H), 2.64-2.55 (t, 2H, J ) 6.2 Hz), 1.32 (s, 9H), 1.24 (s,
3
/CD
3
OD) δ: 0.39. H NMR (CDCl
3 3
/CD OD) δ: 4.79 (bs,
H, J ) 6.2 Hz). 13C NMR (CDCl
/CD
OD) δ: 172.56, 151.00,
J O026027D
3
3
7
274 J . Org. Chem., Vol. 67, No. 21, 2002