Use of the dithiasuccinoyl (Dts) amino protecting group for solid-phase synthesis of protected peptide nucleic acid (PNA) oligomers

Article abstract of DOI:10.1021/jo9824394

'Peptide nucleic acid' (PNA) oligomers replace the oligonucleotide backbone of DNA with an achiral and neutral poly[N-(2-aminoethyl)glycine] backbone, and the four natural nucleobases are attached through methylene carbonyl linkages to the glycine nitrogens. The present work describes the efficient conversion of N(ω)-Boc/side-chain Z-protected PNA monomers to the corresponding derivatives protected by the thiolyzable N(ω)-dithiasuccinoyl (Dts) function. After acidolytic removal of Boc, treatment with bis(ethoxythiocarbonyl) sulfide gave the N(ω)-ethoxythiocarbonyl (Etc) derivatives, which were silylated at the α-carboxyl and converted to the heterocycle by reaction with (chlorocarbonyl)sulfenyl chloride. Net yields of homogeneous monomers were 71-78%. Conditions in the solid-phase mode for thiolytic removal of the Dts group, and for coupling of protected monomers, have been studied extensively and optimized. A protocol featuring (i) Dts removal with dithiothreitol (DTT) (0.5 M) in acetic acid (HOAc) (0.5 M)- CH₂Cl₂ (2 + 8 min); (ii) short neutralization with N,N- diisopropylethylamine (DIEA)-CH₂Cl₂ (1:19, 1 + 2 min); and (iii) coupling mediated by HBTU-DIEA (3:1) in N-methyl-2-pyrrolidinone (NMP) (3 h) was applied to the solid-phase synthesis of Dts-T₄-Gly-NH₂, Dts-G(Z)-G(Z)-T- A(Z)-Gly-NH₂, Dts-A(Z)-T-C(Z)-G(Z)-Gly-NH₂, and Dts-G(Z)-C(Z)-A(Z)-T-Gly- NH₂. The indicated protected PNA derivatives were released from the support, and their structures were verified by mass spectrometry.

Full text of DOI:10.1021/jo9824394

VOLUME 64, NUMBER 20
OCTOBER 1, 1999
© Copyright 1999 by the American Chemical Society
Articles
Use of th e Dith ia su ccin oyl (Dts) Am in o P r otectin g Gr ou p for
Solid -P h a se Syn th esis of P r otected P ep tid e Nu cleic Acid (P NA)
Oligom er s1-3
Marta Planas,4a,b Eduard Bardaj´ı,4a,b Knud J . J ensen,4c and George Barany*,4a
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
Received December 11, 1998 (Revised Manuscript Received J uly 9, 1999)
“Peptide nucleic acid” (PNA) oligomers replace the oligonucleotide backbone of DNA with an achiral
and neutral poly[N-(2-aminoethyl)glycine] backbone, and the four natural nucleobases are attached
through methylene carbonyl linkages to the glycine nitrogens. The present work describes the
efficient conversion of Nω-Boc/side-chain Z-protected PNA monomers to the corresponding derivatives
protected by the thiolyzable Nω-dithiasuccinoyl (Dts) function. After acidolytic removal of Boc,
treatment with bis(ethoxythiocarbonyl) sulfide gave the Nω-ethoxythiocarbonyl (Etc) derivatives,
which were silylated at the R-carboxyl and converted to the heterocycle by reaction with
(chlorocarbonyl)sulfenyl chloride. Net yields of homogeneous monomers were 71-78%. Conditions
in the solid-phase mode for thiolytic removal of the Dts group, and for coupling of protected
monomers, have been studied extensively and optimized. A protocol featuring (i) Dts removal with
dithiothreitol (DTT) (0.5 M) in acetic acid (HOAc) (0.5 M)-CH2Cl2 (2 + 8 min); (ii) short
neutralization with N,N-diisopropylethylamine (DIEA)-CH2Cl2 (1:19, 1 + 2 min); and (iii) coupling
mediated by HBTU-DIEA (3:1) in N-methyl-2-pyrrolidinone (NMP) (3 h) was applied to the solid-
phase synthesis of Dts-T4-Gly-NH2, Dts-G(Z)-G(Z)-T-A(Z)-Gly-NH2, Dts-A(Z)-T-C(Z)-G(Z)-Gly-NH2,
and Dts-G(Z)-C(Z)-A(Z)-T-Gly-NH2. The indicated protected PNA derivatives were released from
the support, and their structures were verified by mass spectrometry.
Sch em e 1
In tr od u ction
Peptide nucleic acids (PNAs) are DNA analogues with
an achiral, uncharged pseudopeptide backbone composed
of N-(2-aminoethyl)glycine monomer units; the nucleo-
bases are attached to the glycine nitrogens via methylene
(1) Preliminary accounts of this work have been reported. (a)
Working only with Dts-T-OH: J ensen, K. J .; Bardaj´ı, E.; Albericio, F.;
Coull, J . M.; Barany, G. In Peptides 1994: Proceedings of the Twenty-
Third European Peptide Symposium; Braga, Portugal, Sep 4-10, 1994;
Maia, H. L. S., Ed.; ESCOM Science Publishers: Leiden, The Neth-
erlands, 1995; pp 757-758. (b) Planas, M.; Bardaj´ı; Barany, G. In
Peptides 1998: Proceedings of the Twenty-Fifth European Peptide
Symposium; Budapest, Hungary, Aug 30-Sep 4, 1998; Bajusz, S.,
Hudecz, F., Eds.; Akade´mia Kiado´: Budapest, Hungary, 1999; pp 234-
235.
carbonyl linkers (Scheme 1).5 PNAs are chemically and
biologically stable, and bind with high affinity and
sequence specificity to both single-stranded DNA and
RNA, as well as to double-stranded DNA. These proper-
ties make PNAs attractive leads for the development of
gene therapeutics and biomolecular tools.6 The prepara-
10.1021/jo9824394 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/08/1999

7282 J . Org. Chem., Vol. 64, No. 20, 1999
Planas et al.
Sch em e 2
tion of PNA oligomers is based on standard solid-phase
peptide synthesis protocols. Two different protection-
schemes are currently used, Boc/Z7 and Fmoc/Bhoc,8 but
the need for new and mild strategies remains.
The dithiasuccinoyl (Dts) function was developed to
meet the requirements of an orthogonally removable NR-
amino protecting group,9a and has been studied thor-
oughly in our laboratories for the solid-phase synthesis
of peptides.9 The Dts group is stable to strong acids and
photolysis but is rapidly and specifically removed under
mild conditions by thiolysis.9b The present paper reports
the application of the Dts group to the solid-phase
synthesis of protected PNA oligomers. We describe
(2) The following abbreviations are used: Ac2O, acetic anhydride;
âME, â-mercaptoethanol; Bhoc, benzhydryloxycarbonyl; Boc, tert-
butyloxycarbonyl; BOP, benzotriazol-1-yl-N-oxytris(dimethylamino)-
phosphonium hexafluorophosphate; BSA, N,O-bis(trimethylsilyl-
acetamide); DECA, N,N-diethylcyclohexylamine; DIEA, N,N-diisoprop-
ylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMAP, N,N-
dimethyl-4-aminopyridine; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; Dts, dithiasuccinoyl; DTT, dithiothreitol; Etc,
ethoxythiocarbonyl; Et2O, diethyl ether; EtOAc, ethyl acetate; EtOH,
ethanol; FABMS, fast-atom bombardment mass spectrometry; Fmoc,
9-fluorenylmethoxycarbonyl; HAL, 5-(4-hydroxymethyl-3,5-dimeth-
oxyphenoxy) valeric acid handle; HATU, N-[(dimethylamino)-1H-1,2,3-
triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexa-
fluorophosphate N-oxide; HBTU, N-[1H-(benzotriazol-1-yl)(dimeth-
ylamino)methylene]-N-methylmethanaminium hexafluorophosphate
N-oxide; HOAc, acetic acid; HOAt, 7-aza-1-hydroxybenzotriazole (3-
hydroxy-3H-1,2,3-triazolo-[4,5-b]pyridine); HOBt, 1-hydroxybenzotria-
zole; HPLC, high-performance liquid chromatography; IRAA, “internal
reference” amino acid; MAc, N-methylmercaptoacetamide; MSNT,
1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole; NMM, N-methyl-
morpholine; NMP, N-methyl-2-pyrrolidinone; PAL, 5-[[(4-amino)-
methyl]-3,5-dimethoxyphenoxy]valeric acid handle (Peptide Amide
Linker); PEG-PS, poly(ethylene glycol)-polystyrene (graft resin sup-
port); TBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tet-
rafluoroborate; TFA, trifluoroacetic acid; TFFH, 1,1,3,3-tetramethyl-
2-fluoroformamidinium hexafluorophosphate; Z, benzyloxycarbonyl. All
solvent ratios are volume/volume unless stated otherwise.
(3) In denoting PNA structures, both monomer building blocks and
oligomers, standard oligopeptide nomenclature is used. For example,
in the oligomer Dts-G(Z)-C(Z)-A(Z)-T-Gly-NH2, G, C, A, and T are the
G-acetyl, C-acetyl, A-acetyl,and T-acetyl N-(2-aminoethyl)glycyl units,
respectively; Gly-NH2 is the C-terminal glycinamide; Dts denotes the
N-terminal amino protecting group; and, Z is the N-amino protecting
group of the nucleobases. We also point out that while technically PNA
molecules are neutral rather than acidic, workers in the field have
retained the term “acid” in the name to emphasize the analogy to DNA
and RNA.
(4) (a) University of Minnesota. (b) Department of Chemistry,
University of Girona, 17751 Girona, Spain. (c) Department of Organic
Chemistry, Technical University of Denmark, 2800 Lyngby, Denmark.
(5) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1500.
procedures for the preparation of all four protected
monomer building blocks, followed by studies to optimize
conditions for deprotection/coupling cycles.
Resu lts a n d Discu ssion
P r ep a r a tion of Nω-Dts-P r otected Mon om er s. In-
troduction of the Dts moiety followed the general proce-
dures described for the synthesis of Dts-protected amino
acids and other Dts-protected amines (Scheme 2).9 Start-
ing from commercially available Boc-monomers 1-4, the
first step involved removal of the Boc group with TFA-
H2O (19:1), and this was followed immediately by treat-
ment with bis(ethoxythiocarbonyl) sulfide10 (5) in EtOH-
H2O (2:1), at pH 9-10, to provide the corresponding Etc
analogues 6-9 in high yields and purities after straight-
forward workup. Freshly prepared 6-911 were suspended
in CH3CN, treated with BSA (1.1-1.5 equiv) to effect
trimethylsilylation at the R-carboxyl group and concomi-
tant solubilization, and then treated in situ with (chlo-
rocarbonyl)sulfenyl chloride (10) (1.1-1.2 equiv)9c,11 to
achieve conversion to the Dts-protected monomers 11-
14. These reactions proceed rapidly and in good overall
yields (71-78%), with the caveat covered below.
(6) Reviews: (a) Nielsen, P. E. Methods Enzymol. 1996, 267, 426-
433. (b) Hyrup, B.; Nielsen, P. E. Bioorg. Biomed. Chem. 1996, 4, 5-23.
(c) M. Eriksson, M.; Nielsen, P. E. Quart. Rev. Biophys. 1996, 29, 369-
394. (d) Good, L.; Nielsen, P. E. Antisense and Nucleic Acid Drug Dev.
1997, 7, 431-437. (e) Knudsen, H.; Nielsen, P. E. Anti-Cancer Drugs
1997, 8, 113-118. (f) Dueholm, K. L.; Nielsen, P. E. New J . Chem.
1997, 21, 19-31. (g) Haaima, G.; Nielsen, P. E. Chem. Soc. Rev. 1997,
73-78. (h) Corey, D. R. TIBTECH 1997, 15, 224-229.
The conversion of trimethylsilyl thiocarbamate deriva-
tives of 6-9 to the corresponding Dts compounds 11-14
(9) (a) Barany, G.; Merrifield, R. B. J . Am. Chem. Soc. 1977, 99,
7363-7365. (b) Barany, G.; Merrifield, R. B. J . Am. Chem. Soc. 1980,
102, 2, 3084-3095 and references therein. (c) Slomczynska, U.; Barany,
G. J . Heterocycl. Chem. 1984, 21, 241-246. (d) Barany, G.; Albericio,
F. J . Am. Chem. Soc. 1985, 107, 4936-4942. (e) Albericio, F.; Barany,
G. Int. J . Peptide Protein Res. 1987, 30, 177-205. (f) Zalipsky, S.;
Albericio, F.; Slomczynska, U.; Barany, G. Int. J . Peptide Protein Res.
1987, 30, 740-783. (g) Hammer, R. P.; Albericio, F.; Gera, L.; Barany,
G. Int. J . Peptide Protein Res. 1990, 36, 31-45. (h) J ensen, K. J .;
Hansen, P. R.; Venugopal, D.; Barany, G. J . Am. Chem. Soc. 1996,
118, 3148-3155.
(7) (a) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J . Am.
Chem. Soc. 1992, 114, 1895-1897. (b) Egholm, M.; Nielsen, P. E.;
Buchardt, O.; Berg, R. H. J . Am. Chem. Soc. 1992, 114, 9677-9678.
(c) Egholm, M.; Behrens, C.; Christensen, L.; Berg, R. H.; Nielsen, P.
E.; Buchardt, O. J . Chem. Soc., Chem. Commun. 1993, 114, 800-801.
(d) Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen,
H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.; Buchardt,
O. J . Org. Chem. 1994, 59, 5767-5773. (e) Christensen, L.; Fitzpatrick,
R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.; Koch, T.; Egholm, M.;
Buchardt, O.; Nielsen, P. E.; Coull, J .; Berg, R. J . Peptide Sci. 1995, 3,
175-183. (f) Koch, T.; Hansen, H. F.; Andersen, P.; Larsen, T.; Batz,
H. G.; Otteson, K.; Ørum, H. J . Peptide Res. 1997, 49, 80-88. (g)
Angell, Y. M.; J ensen, K. J .; Gross, C. M.; Barany, G. In Innovation
and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries,
1998, Collected Papers, Fifth International Symposium, London, U.K.,
Sep 2-6, 1997; Epton, R., Ed.; Mayflower Scientific Ltd.: Kingswinford,
U.K., 1999; pp 59-62.
(10) Barany, G.; Fulpius, B. W.; King, T. P. J . Org. Chem. 1978, 43,
2930-2932.
(11) Compounds 6-9 should be prepared freshly because they are
prone to rearrange to the thermodynamically more stable S-alkyl
thiocarbamate isomers under a variety of conditions, including pro-
longed standing. Although 1 equiv of (chlorocarbonyl)sulfenyl chloride
(10) is sufficient for all of the Etc derivative to be converted to products,
the protocols reported here with (chlorocarbonyl)sulfenyl chloride (10)
always in excess were designed to minimize the isomerization problem.
See ref 9c.
(8) Thomson, S. A.; J osey, J . A.; Cadilla, R.; Gaul, M. D.; Hassman,
C. F.; Luzzio, M. J .; Pipe, A. J .; Reed, K. L.; Ricca, D. J .; Wiethe, R.
W.; Noble, S. A. Tetrahedron 1995, 51, 6179-6194.

Synthesis of Protected Peptide Nucleic Acid Oligomers
J . Org. Chem., Vol. 64, No. 20, 1999 7283
Sch em e 3a
Sch em e 4a
a
Rotamers of Dts-PNA monomers. The trans configuration is
preferred. When the protecting group on Nω is Boc or Etc
(structures not shown), the preference is the opposite (see text).
was usually carried out under ambient conditions, but
this mode gave problems when the target was Dts-C(Z)-
OH (12). Despite varying the ratio of 7:10, the product
mixture always included a substantial amount (as much
as 27% of the crude reaction mixture) of a byproduct in
addition to the expected 12. LC/MS analysis suggested
that the byproduct was the 1,2,4-thiadiazolidine-3,5-dione
heterocycle 15. Such heterocycles were identified previ-
ously in studies of Dts formation in simpler systems.9c
Here, formation of 15 was suppressed entirely by carry-
ing out the reaction of 10 with the trimethylsilyl ester of
7 at 0 °C.
a
Thiols and dithiols are listed in order of pKa (see Tables 1 and
2).
a and b observed for the major rotamer indicates that
methylene protons of the acyl side chain are close to the
glycine R-protons. The cis:trans ratio varies depending
on the nucleobase side-chain; values were 1:2 for Dts-
A(Z)-OH (11), 3:4 for Dts-C(Z)-OH (12), 1:2 for Dts-G(Z)-
OH (13), and 1:1.5 for Dts-T-OH (14).
Solid -P h a se Dep r otection Stu d ies. The Dts pro-
tecting group is removed by thiols9b,14 through the
intermediacy of an open-chain carbamoyl disulfide, which
reacts further with a second equivalent of the thiol to
give the free amino function (Scheme 4). The reaction is
driven to completion by loss of 2 equiv of gaseous carbonyl
sulfide, and is usually catalyzed by tertiary amines.
Optimized protocols for quantitative thiolytic removal of
the Dts group9d,e during solid-phase peptide synthesis
include (i) âME (0.5 M)-DIEA (0.5 M) in CH2Cl2, 2 × 2
min; (ii) MAc (0.5 M)-NMM (0.5 M) in CH2Cl2, 2 × 2
min; or (iii) MAc (0.5 M)-HOBt (0.1 M) in DMF, 2 × 2
min. When conditions (i) were applied to the solid-phase
synthesis of Dts-T5-Gly-NH2, the main product was the
expected one, but several byproducts were also noted.1a,15
For the present studies, we considered a variety of thiols
over a 4-order-of-magnitude pKa range,16 including ali-
phatic, aromatic, and bifunctional thiols, and focused on
the development of deprotection conditions that could be
applied in the absence of base and/or the presence of acid
(Scheme 4, Tables 1 and 2).
The 1H and 13C NMR spectra of Etc and Dts monomers
were consistent with the presence of two rotamers.
Similar observations were reported previously12 for other
PNA monomers, as well as oligomers, and have been
ascribed to slow chemical exchange due to a cis-trans
equilibrium about the tertiary amide bond (torsion angle
ø1) (Scheme 3). To assign conformations of the amide
group, we first confirmed the literature conclusion from
1D and 2D 1H NMR experiments that in the Boc-
monomers 1-4 the side-chain carbonyl group of the
major rotamer points toward the C terminus12a,13 (cis
conformation, Scheme 3). Thus, in a 2D NOESY spec-
trum, cross-peaks between protons a and c-d were
observed for the major rotamer, while the minor con-
former revealed cross-peaks between protons a and b.
Similar results were observed with the Etc intermediates.
In contrast, the Dts-monomers showed peak intensities
opposite to Boc and Etc, suggesting that Dts protection
confers a preference for trans. This was confirmed by 2D
NOESY experiments, where cross-peaks between protons
Dts-T-Gly-PAL-PEG-PS served as a model system to
investigate deprotection rates and pathways; the glycine
residue was incorporated to facilitate solubility and
quantification. The Dts-resin was treated with the ap-
(14) (a) Barany, G.; Merrifield, R. B. Anal. Biochem. 1979, 95, 160-
170. (b) Barany, G. Anal. Biochem. 1980, 109, 114-122.
(15) In the preliminary work (ref 1a), formation of thiourethanes
due to âME were detected by HPLC and characterized by FABMS and
LC/MS. In the present work, with improved conditions, the thioure-
thanes were not observed.
(16) For references for the pKa of the thiols used see: (a) Bembrilla,
A.; Roizard, D.; Schoenleber, J .; Lochon, P. Can. J . Chem. 1984, 62,
2330-2336. (b) Barany, G. In Proceedings of the Sixth American
Peptide Symposium; Gross, E., Meienhofer, J ., Eds.; Pierce Chemical
Co.: Rockford, IL, 1979; pp 313-316. See also ref 9b, especially the
Supporting Information. (c) De Maria, P.; Fini, A.; Hall, F. M. J . Chem.
Soc., Perkin Trans. 2 1973, 1969-1971.
(12) (a) Chen, S.-M.; Mohan, V.; Kiely, J . S.; Griffith, M. C.; Griffey,
R. H. Tetrahedron Lett. 1994, 35, 5105-5108. (b) Leijon, M.; Gra¨slund,
A.; Nielsen, P. E.; Buchardt, O.; Norde´n, B.; Kristensen, S. M.;
Eriksson, M. Biochemistry 1994, 33, 9820-9825. (c) Meltzer, P. C.;
Liang, A. Y.; Matsudaira, P. J . Org. Chem. 1995, 60, 4305-4308. (d)
Howarth, N. M.; Wakelin, L. P. G. J . Org. Chem. 1997, 62, 5441-
5450.
(13) Reference 12c claims that in the major rotamer of the Boc
monomers the tertiary amide bond has a trans conformation, but these
results do not agree with ref 12a or with our own experiments.

7284 J . Org. Chem., Vol. 64, No. 20, 1999
Ta ble 1. Rep r esen ta tive Solid -P h a se Dep r otection s of Dts-T-Gly-P AL-P EG-P Sa
Planas et al.
deprotection conditionsb
additive/solvent
product distribution (% HPLC)c
entry
thiold
time (min)
H-T-Gly
26
Dts-T-Gly
thiourethane
25
1
2
3
4
5
6
7
8
9
10
11
12
13
16e
16
16
16
16
NMP
1
1
1
44
61
36
38
36
23
37
5
3
3
3
HOAc (0.1 M)-NMP
HOBt (0.1 M)-NMP
CH2Cl2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
2 + 8
20
79
HOAc (0.1 M)-CH2Cl2
HOAc (0.1 M)-NMP
HOAc (0.1 M)-NMP
HOAc (0.1 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (0.1 M)-CH2Cl2
HOAc (0.1 M)-CH2Cl2
HOAc (0.1 M)-CH2Cl2
17
55
52
69
10
89
38
42
38
27
31
18
6
18f
19g
19
7
9
67
21h
22i
23j
24k
2 + 8
1
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
27
25
23
4
3
7
5
3
4
a
Excerpted from extensive tables (1-10) in the Supporting Information. b See the Experimental Section for general procedures. c Relative
amounts of species reported are based on uncorrected total HPLC values, and do not necessarily add to 100%. [Thiol] ) 0.5 M. e pKa )
d
9.86 (ref 16a). f pKa ) 9.55 (ref 16a). pKa ) 9.45 (ref 16a). pKa ) 8.0 (ref 16b). pKa ) 6.8 (ref 16c). pKa ) 6.6 (ref 16c). pKa ) 6.1 (ref
g
h
i
j
k
16c).
Ta ble 2. Solid -P h a se Dep r otection s of Dts-T-Gly-P AL-P EG-P S w ith DTT (20)a
deprotection conditionsb
product distribution (% HPLC)c
entry
[DTT]d (M)
loading (mmol/g)
additive/solvent
time (min)
H-T-Gly
26
Dts-T-Gly
1
2
3
4
5
6
7
8
9
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.18
0.18
0.18
0.18
0.18
0.18
0.09
0.18
0.18
HOAc (0.1 M)-NMP
HOBt (0.1 M)-NMP
NMP
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
1 + 2 + 2
2 + 8
75
70
68
73
73
76
82
90
91
15
15
16
11
16
12
1
CH2Cl2
HOBt (0.1 M)-CH2Cl2
HOAc (0.1 M)-CH2Cl2
HOAc (0.1 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (1 M)-CH2Cl2
1
2 + 8
a
Excerpted from extensive tables (1-10) in the Supporting Information. b See the Experimental Section for general procedures. c Relative
d
amounts of species reported are based on uncorrected total HPLC values and do not necessarily add to 100%. pKa ) 8.6 (ref 16b).
Sch em e 5
(0.1 M)-NMP was used as solvent (e.g., Table 1, entries
1-3; no starting material was observed after 1 min
treatment). However, when CH2Cl2 or HOAc (0.1 M)-
CH2Cl2 was used as solvent, reactions were slower as
indicated by observation of unreacted Dts-T-Gly-NH2 in
some cases (e.g., Table 1, compare entries 1 and 4). These
observations agree with previous results that Dts thi-
olysis is promoted in polar aprotic media with high
dielectric constants.9b,16b A common byproduct during
these reactions was invariably noted, irrespective of the
thiol used, and accounted for as much as 42% of the
product mixture. The material was isolated and analyzed
by FABMS, from which the urea structure 26 was
proposed. Formation of 26 is plausibly explained by an
intersite nucleophilic attack of the amino terminus from
an already-deprotected H-T-Gly resin-bound unit onto the
carbonyl of the Dts group of another still-protected resin-
bound unit, with ultimate loss of carbonyl sulfide and
elemental sulfur (Scheme 6). The susceptibility of the Dts
heterocycle to aminolysis is precedented,9b,e although this
specific side reaction has not been described earlier.
propriate thiol for a particular time period, and then
cleaved with TFA-H2O (19:1, 2 h); the crude product
mixture was then dissolved in H2O and analyzed by
HPLC and mass spectrometry. The desired major product
from deprotection, H-T-Gly-NH2, was characterized by
mass spectrometry and co-injected with a sample pre-
pared by an Fmoc strategy. With certain thiols under
some solvent conditions, relatively low levels of thioure-
thane byproducts were observed (Scheme 5, Table 1,
entries 1-3, 7, and 11-13). As a control, the resin when
cleaved directly (without deprotection) provided Dts-T-
Gly-NH2, which was characterized further by mass
Based on the understanding of the source of byproduct
26, two strategies were devised to suppress its formation.
First, by working with resins of lower loading, the
effective concentration of reactive sites was expected to
be reduced. Indeed, the use of DTT (0.5 M) in HOAc (0.1
M)-CH2Cl2 to deprotect Dts-T-Gly-PAL-PEG-PS gave
12% of 26 at 0.18 mmol/g loading, but only 1% of 26 at
0.09 mmol/g loading (Table 2, compare entries 6 and 7).
Second, by increasing the acidity of the solvent milieu
for deprotection, it was expected that the liberated amino
groups after Dts removal would be protonated and less
1
spectrometry and H NMR. Finally, in some experiments
using âME (Table 1, entry 9) for deprotection, the
carbamoyl disulfide intermediate 25 was observed.
With all thiols tested, the Dts function was rapidly
transformed when NMP, HOAc (0.1 M)-NMP, or HOBt

Synthesis of Protected Peptide Nucleic Acid Oligomers
J . Org. Chem., Vol. 64, No. 20, 1999 7285
BOP reagent18/DIEA (3:3) was tested, giving similar
values (Table 3, entry 12). However, substantially better
results were achieved by reducing the amount of tertiary
amine during the coupling, i.e., HBTU/DIEA (3:1); this
led to Dts-T4-Gly-NH2 of 80% purity (Table 3, entry 5)
(Figure 1a). When NMM, DMAP, or DECA was used with
similar ratios, results were inferior (Table 3, entries 7-9).
Omission of base entirely led to no product (Table 3,
entries 10 and 15). Generation of in situ acyl fluorides
with TFFH19/DIEA (3:1) was not successful (Table 3,
entry 17), nor was the classical DIPCDI/HOBt method
(Table 3, entry 11). Finally, results with HBTU were
slightly better than those with TBTU, HATU, or BOP
(Table 3, compare entries 13, 14, and 16).
Sch em e 6
In summary, a repetitive deprotection/coupling cycle
for incorporation of a Dts-PNA monomer at 25 °C
involves: (i) thiolytic deprotection with DTT (0.5 M) in
HOAc (0.5 M)-CH2Cl2, 2 + 8 min; (ii) neutralization with
DIEA-CH2Cl2 (1:19, 1 + 2 min), and (iii) coupling of Dts-
PNA-monomer (3 equiv) using HBTU/DIEA (3:1) in NMP,
3 h.
Solid -P h a se Syn th esis of Dts-P r otected P NA Tet-
r a m er s. The methodology summarized above for Dts-
T4-Gly-NH2, was extended to the synthesis of Dts-
protected tetramers containing the four nucleobases.
Thus, Dts-G(Z)-G(Z)-T-A(Z)-Gly-NH2 (54% purity, Figure
1b), Dts-A(Z)-T-C(Z)-G(Z)-Gly-NH2 (54% purity, Figure
1c), and Dts-G(Z)-C(Z)-A(Z)-T-Gly-NH2 (62% purity, Fig-
ure 1d) were prepared and characterized by LC/MS.
likely to serve as nucleophiles. Again, this prediction was
borne out experimentally, particularly with MAc or DTT
as thiols (Table 1, entry 10; Table 2, compare entry 6 with
8 and 9). The greater acidity made it necessary to apply
longer treatments to achieve complete Dts removal.
Suitable conditions for quantitative removal of the Dts-
amino protecting group at 25 °C, estimated on the basis
of the considerations outlined in the previous paragraphs,
include (i) DTT or MAc (0.5 M) in HOAc (0.5 M)-CH2Cl2
for 2 + 8 min; (ii) DTT (0.5 M) in HOAc (1 M)-CH2Cl2
for 2 + 8 min. These conditions were studied further, as
described in the next section.
Con clu sion s
We have described an efficient procedure for prepara-
tion of Dts-protected PNA monomers and defined proto-
cols for their application to the synthesis of Dts-protected
oligomers under mild, orthogonal conditions. Since Dts
removal can be achieved under acidic conditions followed
by a short neutralization step, previously described PNA
synthesis side reactions6b,f,8 such as N-acyl migration or
N-terminal deletion products, might be minimized in this
system. Ongoing work is directed at the use of protected
tetramers as building blocks20 for segment condensation
synthesis of longer PNA.
Eva lu a tion of Con d ition s for Solid -P h a se Syn -
th esis of Dts-T4-Gly-NH2. Initial studies focused on the
model Dts-T4-Gly-NH2 (Table 3). Optimized conditions for
Dts removal were applied, and a short neutralization step
with DIEA-CH2Cl2 (1:19, 1 + 2 min) was interpolated
to follow the deprotection. Couplings of the Dts-T-OH
monomer were mediated by a variety of reagent/additive
protocols, always with NMP as solvent. After chain
assembly was completed, the Dts-protected tetramers
were cleaved from the resin with TFA-H2O (19:1), and
the crude product was analyzed by HPLC and mass
spectrometry.
Exp er im en ta l Section
For syntheses of PNA by Boc chemistry, HATU and
HBTU with in situ neutralization were reported to give
the best results.7e,f However, standard coupling protocols
with aminium salts require a tertiary amine for optimal
efficiency,17 leading to a concern that the applicability of
such methods to Dts chemistry might be compromised
by the known lability of the Dts group to amines.
Activation at each step of Dts-T-OH (3 equiv) with HBTU/
DIEA (3:3) provided crude Dts-T4-Gly-NH2 with substan-
tial heterogeneity (Table 3, entries 1 and 3). Results did
not improve upon changing to a weaker base, such as
collidine (Table 3, entry 2). Also, a combination of the
Materials, solvents, instrumentation, and general methods
were essentially as described in previous publications from our
laboratories.9,10,14 HATU, PEG-PS resins23 (0.15-0.18 mmol/g
and containing Nle as the IRAA), protected PNA monomers,
and TFFH were obtained from the BioSearch division of
PerSeptive Biosystems (Framingham, MA). DIEA, HOBt,
NMP, piperidine, and TFA were from Fisher (Pittsburgh, PA).
(19) (a) Carpino, L. A.; El-Faham, A. J . Am. Chem. Soc. 1995, 117,
5401-5402. (b) Carpino, L. A.; Beyermann, M.; Wenschuh, H.; Bienert,
M. Acc. Chem. Res. 1996, 29, 268-274.
(20) Ideally, these building blocks should have a free carboxyl group.
Toward this end, we synthesized Boc-T4-OH at a stage in the research
program where other aspects had not been optimized. Dts-T-OH (2
equiv) was anchored onto HAL-PEG-PS (0.20 g, 0.23 mmol/g) (ref 21)
by a protocol including MSNT (2 equiv) (ref 22) activation in the
presence of N-methylimidazole (5 equiv) in CH2Cl2-THF (1:2, 3 mL).
Deprotection/couplings introduced two further Dts-T-OH, and the
terminal T was introduced with Nω-Boc protection. The protected PNA
tetramer was released from the HAL anchor with TFA-CH2Cl2
(1:999, 2 mL) for 2 h at 25 °C and characterized by HPLC (>60% purity)
and ESMS (calcd for C49H66N16O19 1182.5, found m/z 1205.4 [M +
Na]+).
(17) (a) Dourtoglou, V.; Ziegler, J . C.; Gross, B. Tetrahedron Lett.
1978, 1269-1272. (b) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gilless-
en, D. Tetrahedron Lett. 1989, 30, 1927-1930. (c) Carpino, L. A. J .
Am. Chem. Soc. 1993, 115, 4397-4398. (d) Carpino, L. A.; El-Faham,
A.; Minor, C. A.; Albericio, F. J . Chem. Soc., Chem. Commun. 1994,
201-203. (e) Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron
Lett. 1994, 35, 2279-2282.
(18) Castro, B.; Dormoy, J . R.; Evin, G.; Selve, C. Tetrahedron Lett.
1975, 1219-1222.
(21) Albericio, F.; Barany, G. Tetrahedron Lett. 1991, 1015-1018.
(22) Nielsen, J .; Lyngsø, L. O. Tetrahedron Lett. 1996, 8439-8442.

7286 J . Org. Chem., Vol. 64, No. 20, 1999
Planas et al.
a
Ta ble 3. Eva lu a tion of Con d ition s for Solid -P h a se Syn th esis of Dts-T4-Gly-NH2
coupling conditionsb
deprotection conditions
c
entry
reagent
base or additive
thiol
solvent
time (min)
Dts-T4-Gly-NH2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
HBTU (3 equiv)
DIPCDI (3 equiv)
BOP (3 equiv)
DIEA (3 equiv)
collidine (3 equiv)
DIEA (3 equiv)
DIEA (1 equiv)
DIEA (1 equiv)
DIEA (1 equiv)
NMM (1 equiv)
DMAP (1 equiv)
DECA (1 equiv)
DTT (0.5 M)
DTT (0.5 M)
DTT (0.5 M)
DTT (0.5 M)
DTT (0.5 M)
MAc (0.5 M)
DTT (0.5 M)
HOAc (1 M)-CH2Cl2
HOAc (1 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (1 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
2 + 15
2 + 15
2 + 8
2 + 8
2 + 8
2 + 8
2 + 8
46
36
57
68
80
70
36
NPd
55
DTT (0.5 M)
HOAc (0.5 M)-CH2Cl2
2 + 8
NPd
NPd
45
HOBt (3 equiv)
DIEA (3 equiv)
DIEA (1 equiv)
DIEA (1 equiv)
DTT (0.5 M)
DTT (0.5 M)
DTT (0.5 M)
HOAc (1 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
2 + 15
2 + 8
2 + 8
BOP (3 equiv)
67
TBTU (3 equiv)
TBTU (3 equiv)
HATU (3 equiv)
TFFH (3 equiv)
69
NPd
60
DIEA (1 equiv)
DIEA (1 equiv)
DTT (0.5 M)
DTT (0.5 M)
HOAc (0.5 M)-CH2Cl2
HOAc (0.5 M)-CH2Cl2
2 + 8
2 + 8
15
a
b
See the Experimental Section for general procedures. Dts-T-OH (3 equiv); solvent, NMP; time, 3 h. Upon completion of coupling,
ninhydrin test was negative. c Percent determined by HPLC. No product was formed; syntheses were aborted after the first coupling
d
step, since positive ninhydrin tests indicated that coupling was incomplete.
were visualized by UV. Elemental analyses were conducted
by M-H-W Laboratories, Phoenix, AZ.
Analytical HPLC was performed at 1.2 mL/min using a
Vydac C18 reversed-phase column (0.46 × 25 cm; 5 µm particle
size) with UV detection at 220 and 265 nm. Linear gradients
of 0.1% aqueous TFA and 0.1% TFA in CH3CN were run from
1:0 to 1:1 over 15 min and then 1:1 to 1:3 over 5 min. Low-
resolution fast-atom bombardment mass spectroscopy (FABMS)
was carried out in glycerol-H2O or 3-nitrobenzyl alcohol
(MNBA) matrices on a high-resolution double-focusing mass
spectrometer operated at a resolution of 2000. Liquid chro-
matography/mass spectrometry (LC/MS) was performed using
a Zorbax SB-C18 narrowbore reversed-phase column (3.0 mm
× 25 cm; 5 µm particle size) and detection at 220 nm. This
system was connected to a PE-Sciex API III triple-quadrupole
mass spectrometer equipped with an ionspray interface.
1H NMR spectra were recorded at 300 MHz or at 500 MHz.
Assignments of spectra were based on 2D homonuclear chemi-
cal-shift correlation spectroscopy. 2D NOESY spectra were
acquired at 298 K with 300 ms mixing time. 13C NMR spectra
were recorded at 75 or 125 MHz, and assignments were based
on heteronuclear multiple quantum coherence (HMQC) spec-
troscopy.
N -[2-(N -E t h oxyt h ioca r b on yl)a m in oe t h yl]-N -[[6-N -
(ben zyloxyca r bon yl)a d en in -9-yl]a cetyl]glycin e (6). A so-
lution of Boc-A(Z)-OH (1) (409 mg, 0.78 mmol) in TFA-H2O
F igu r e 1. Analytical HPLC of crude product from synthesis
of (A) Dts-T4-Gly-NH2, (B) Dts-G(Z)-G(Z)-T-A(Z)-Gly-NH2, (C)
Dts-A(Z)-T-C(Z)-G(Z)-Gly-NH2, and (D) Dts-G(Z)-C(Z)-A(Z)-T-
(19:1, 16 mL) was stirred for 60 min. Concentration in vacuo
Gly-NH2. Chromatography followed conditions listed in the
(1 mm) gave an oil that was redissolved in EtOH-H2O (2:1)
Experimental Section. Ordinate is absorbance at 265 nm.
(15 mL). Next, bis(ethoxythiocarbonyl) sulfide (5) (246 mg, 1.17
o-Benzenedimethanethiol, benzylmercaptan, âME, BSA, DECA,
DIPCDI, DMAP, DTT, p-chlorothiophenol, collidine, p-meth-
oxybenzylmercaptan, p-methylthiophenol, pyridine, and thiophe-
nol were from Aldrich (Milwaukee, WI), and MAc was obtained
from Fluka (Ronkonkoma, NY). BOP, HBTU, and TBTU were
from Advanced ChemTech (Louisville, KY). Bis(ethoxythio-
carbonyl) sulfide (5) was prepared as described previously,10
or the product purchased from Fairfield Chemical (Blythewood,
SC) was recrystallized from ethanol-water (3:1). (Chlorocar-
bonyl)sulfenyl chloride (10) was prepared as described previ-
ously.24
All transformations (solution and solid-phase) and washes
were done at 25 °C unless noted otherwise. PNA-resin samples
were hydrolyzed in 12 N aqueous HCl-propionic acid (1:1) at
160 °C for 2 h, and analyses for Gly and NLeu were carried
out on an amino acid analyzer. Melting points are uncorrected.
Silica gel chromatography was performed with silica gel 60
(230-400 mesh) from EM Science (Gibbstown, NJ ). Thin-layer
chromatography was performed on either Polygram SIL
G/UV254 plates (250 mm, 40 × 80 mm, Macherey-Nagel) or
Kieselgel 60 F254 (0.2 mm, 40 × 80 mm, EM Science); spots
mmol, 1.5 equiv) was added and the resulting suspension
brought to a pH of 9-10 by addition of 2.5 M aqueous NaOH
(2.0 mL). Additional 2.5 M aqueous NaOH (0.6 mL) was added
dropwise over a period of 4 h in order to maintain the pH; at
this point, all of 5 was dissolved and the pH remained constant
overnight. The solution was diluted with H2O (18 mL),
acidified to pH 2 with 12 N HCl, and extracted with EtOAc (5
× 20 mL). The organic layers were combined, dried (MgSO4),
and evaporated in vacuo to provide a colorless oil. Trituration
(23) (a) Barany, G.; Albericio, F.; Sole´, N. A.; Griffin, S. W.; Kates,
S. A.; Hudson, D. In Peptides 1992: Proceedings of the Twenty-Second
European Peptide Symposium; Schneider, C. H., Eberle, A. N., Eds.;
ESCOM Science Publishers: Leiden, The Netherlands, 1993; pp 267-
268. (b) Zalipsky, S.; Chang. J . L.; Albericio, F.; Barany, G. React.
Polym. 1994, 22, 243-258. (c) Barany, G.; Albericio, F.; Kates, S. A.;
Kempe, M. In Chemistry and Biological Application of Polyethylene
Glycol; ACS Symposium Series 680; Harris, J . M., Zalipsky, S., Eds.;
American Chemical Society Books: Washington, DC, 1997; pp 239-
264.
(24) Barany, G.; Schroll, A. L.; Mott, A. W.; Halsrud, D. A. J . Org.
Chem. 1983, 48, 4750-4761.

Synthesis of Protected Peptide Nucleic Acid Oligomers
J . Org. Chem., Vol. 64, No. 20, 1999 7287
of the oil with Et2O yielded 6 as a white solid that was dried
over P2O5 (385 mg, 95%): mp 118-120 °C; H NMR (DMSO-
slow. The combined organic phases were dried (MgSO4) and
concentrated to dryness in vacuo to provide 9 as a colorless
foam, which was crystallized from EtOAc-hexane to provide,
after drying in vacuo, white crystals (1.07 g, 96%): mp 98-
1
d6) δ (two rotamers) 9.26 (m, 0.6H), 9.02 (m, 0.4H), 8.58 (s,
1H), 8.33 (s, 0.4H), 8.32 (s, 0.6H), 7.45 (d, J ) 7.0 Hz, 2H),
7.38 (d, J ) 7.0 Hz, 2H), 7.33 (t, J ) 7.0 Hz, 1H), 5.36 (s, 1.2H),
5.21 (s, 2H), 5.16 (s, 0.8H), 4.41 (q, J ) 7.0 Hz, 1.2H), 4.36 (s,
0.8H), 4.35 (q, J ) 7.0 Hz, 0.8H), 4.01 (s, 1.2H), 3.70-3.34 (m,
4H), 1.26 (t, J ) 7.0 Hz, 1.8H), 1.20 (t, J ) 7.0 Hz, 1.2H); 13C
NMR (DMSO-d6) δ25 (190.3, 189.9), (170.8, 170.3), (167.2,
166.6), 152.4, 152.2, 151.5, 149.3, 145.2, 136.4, 128.4, 128.0,
127.9, 122.8, 66.3 (65.6, 65.3), (49.2, 47.6), (45.7, 45.4), (44.1,
43.9), (42.7, 41.9), (14.2, 14.1); FABMS m/z 516.2 [M + H]+,
514.1 [M - H]-. Anal. Calcd for C22H25N7O6S‚3/2H2O: C, 48.70;
H, 5.16; N, 18.07; S, 5.91. Found: C, 48.57; H, 4.88; N, 17.79;
S, 5.20.
1
100 °C; H NMR (DMSO-d6) δ 11.29 (s, 1H), 9.13 (m, 0.6H),
9.01 (m, 0.4H), 7.29 (s, 0.6H), 7.25 (s, 0.4H), 4.65 (s, 0.6H),
4.45 (s, 0.4H), 4.41-4.33 (m, 2H), 4.21 (s, 0.4H), 3.99 (s, 0.6H),
3.62-3.26 (m, 4H), 1.73 (s, 3H), 1.23 (m, 3H); 13C NMR
(DMSO-d6) δ25 (190.2, 189.9), (170.7, 170.4), (167.7, 167.2),
164.4, 150.9, 142.0, (108.2, 108.1), (65.6, 65.3), (49.0, 46.4),
(47.7, 47.6), (45.6, 45.2), (42.2, 41.9), (14.3, 14.2), (12.0, 11.9);
FABMS m/z 373.3 [M + H]+, 371.3 [M - H]-. Anal. Calcd for
C
14H20N4O6S‚H2O: C, 43.07; H, 5.67; N, 14.35; S, 8.21. Found:
C, 43.24; H, 5.10; N, 13.53; S, 8.09.
N-[2-(N-Dit h ia su ccin oyl)a m in oet h yl]-N-[[6-N-(b en z-
yloxyca r bon yl) a d en in -9-yl]a cetyl]glycin e (11). BSA (59
µL, 0.24 mmol, 1.5 equiv) was added to a suspension of freshly
prepared 6 (81 mg, 0.16 mmol) in CH3CN (9 mL) and the
mixture stirred for 30 min. Next, Cl(CdO)SCl (10) (14 µL, 0.18
mmol, 1.1 equiv) was added, resulting in a yellow solution;
the reaction was complete after 20 min as indicated by HPLC.
The suspension was distributed between EtOAc (10 mL) and
H2O (15 mL), and the aqueous phase was extracted with
EtOAc (5 × 15 mL). The organic layers were combined, dried
(MgSO4), and concentrated to a small volume that upon cooling
to -20 °C gave 11 as an off white solid (70 mg, 80%): mp 138-
140 °C; 1H NMR (DMSO-d6) δ (two rotamers) 8.59 (s, 1H), 8.39
(s, 0.3H), 8.27 (s, 0.7H), 7.45 (d, J ) 7.0 Hz, 2H), 7.39 (d, J )
7.0 Hz, 2H), 7.33 (t, J ) 7.0 Hz, 1H), 5.29 (s, 0.6H), 5.21 (s,
2H), 5.10 (s, 1.4H), 4.32 (s, 1.4H), 4.01 (t, J ) 6.8 Hz, 0.6H),
3.99 (s, 0.6H), 3.81 (t, J ) 5.3 Hz, 1.4H), 3.73 (t, J ) 6.8 Hz,
0.6H), 3.53 (t, J ) 5.3 Hz, 1.4H); 13C NMR (DMSO-d6) δ25
(170.9, 170.6), (169.6, 169.2), 167.9, 166.9, 152.7, 152.6, 151.9,
149.6, (145.6, 145.5), 136.8, 128.9, 128.4, 128.3, 122.9, 66.7,
(48.6, 48.2), (44.8, 44.4), (44.3, 44.2), (43.3, 42.7); FABMS m/z
546.2 [M + H]+, 544.0 [M - H]-. Anal. Calcd for C21H19N7O7S2‚
3/2H2O: C, 44.05; H, 3.87; N, 17.12; S, 11.19. Found: C, 43.76;
H, 3.46; N, 16.80; S, 10.73.
N -[2-(N -E t h oxyt h ioca r b on yl)a m in oe t h yl]-N -[[4-N -
(ben zyloxyca r bon yl)cytosin -1-yl]a cetyl]glycin e (7). Start-
ing with Boc-C(Z)-OH (2) (314 mg, 0.62 mmol), compound 7
was prepared as described for compound 6, except for the
workup. Following overnight reaction, the cloudy solution was
diluted with H2O (16 mL) and acidified to pH 2 with 12 N HCl.
The precipitate that formed immediately was collected by
filtration, washed with Et2O, and dried in vacuo over P2O5 to
yield a white solid, 7 (257 mg, 84%): mp 201-202 °C; 1H NMR
(DMSO-d6) δ (two rotamers) 9.24 (m, 0.6H), 9.15 (m, 0.4H),
7.90 (d, J ) 7.5 Hz, 0.6H), 7.87 (d, J ) 7.5 Hz, 0.4H), 7.41-
7.31 (m, 5H), 6.99 (d, J ) 7.5 Hz, 0.6H), 6.98 (d, J ) 7.5 Hz,
0.4H), 5.17 (s, 2H), 4.81 (s, 1.2H), 4.60 (s, 0.8H), 4.38 (q, J )
7.0 Hz, 1.2H), 4.35 (q, J ) 7.0 Hz, 0.8H), 4.01 (s, 0.8H), 3.94
(s, 1.2H), 3.63-3.24 (m, 4H), 1.22 (t, J ) 7.0 Hz, 1.8H), 1.20
(t, J ) 7.0 Hz, 1.2H); 13C NMR (DMSO-d6) δ25 (190.5, 190.1),
(168.1, 168.0), (167.3, 167.2), (163.5, 163.4), (155.5, 155.4),
153.6, 151.4, 136.4, 128.9, 128.6, 128.4, 94.3, 66.9, (65.9, 65.7),
(51.5, 48.7), 50.0, (46.4, 45.8), 42.5, 14.7; FABMS m/z 492.3
[M + H]+, 514.2 [M + Na]+, 490.2 [M - H]-. Anal. Calcd for
C
21H25N5O7S‚H2O: C, 49.50; H, 5.34; N, 13.74; S, 6.29. Found:
C, 49.60; H, 5.56; N, 13.94; S, 6.20.
N -[2-(N -E t h oxyt h ioca r b on yl)a m in oe t h yl]-N -[[2-N -
(ben zyloxyca r bon yl)gu a n in -9-yl]a cetyl]glycin e (8). Start-
ing with Boc-G(Z)-OH (3) (402 mg, 0.74 mmol), compound 8
was prepared as described for compound 6, except for workup.
Following overnight reaction, the cloudy solution obtained was
diluted with H2O (18 mL), and a white precipitate formed. The
resulting suspension was acidified to pH 2 with 12 N HCl,
whereupon the solid redissolved. EtOAc (20 mL) was added
to the solution, and a white precipitate formed that was
collected by filtration. The aqueous phase was extracted
further with EtOAc (3 × 20 mL), and the organic layers were
combined, dried (MgSO4), and concentrated under reduced
pressure. The white solids were combined, washed with Et2O,
and dried in vacuo over P2O5 to yield 361 mg of 8 (92%): mp
184-186 °C; 1H NMR (DMSO-d6) δ 11.67 (s, 0.45H), 11.55 (s,
0.55H), 11.36 (s, 0.45H), 11.35 (s, 0.55H), 9.22 (m, 0.55H), 9.00
(m, 0.45H), 7.81 (s, 0.55H), 7.79 (s, 0.45H), 7.43-7.34 (m, 5H),
5.24 (s, 2H), 5.11 (s, 0.9H), 4.92 (s, 1.1H), 4.43-4.31 (m, 2H),
4.33 (s, 0.9H), 4.01 (s, 1.1H), 3.68-3.37 (m, 4H), 1.25-1.18
(m, 3H); 13C NMR (DMSO-d6) δ25 (190.2, 189.9), (170.9, 170.4),
(167.2, 167.1), 150.2, (154.6, 154.5), (149.6, 149.5), 147.2, 140.5,
(135.5, 135.4), 128.5, 128.3, (128.2, 128.1), 119.2, (67.3, 67.2),
(65.6, 65.4), (49.2, 47.8), (45.7, 45.4), (44.0, 43.9), (42.4, 41.9),
(14.3, 14.1); FABMS m/z 532.2 [M + H]+, 530.2 [M - H]-. Anal.
Calcd for C22H25N7O7S‚H2O: C, 48.08; H, 4.91; N, 17.84; S, 5.83.
Found: C, 47.96; H, 4.87; N, 17.65; S, 6.12.
N-[2-(N-Dit h ia su ccin oyl)a m in oet h yl]-N-[[4-N-(b en z-
yloxyca r bon yl)cytosin -1-yl]a cetyl]glycin e (12). Starting
with 7 (80 mg, 0.16 mmol), compound 12 was prepared as
described for compound 11, except the workup and that
heterocyclization with Cl(CdO)SCl (10) (16 µL, 0.19 mmol, 1.2
equiv) was performed at 0 °C. The reaction was complete after
20 min as indicated by HPLC. Next, the solution was distrib-
uted between EtOAc (10 mL) and H2O (15 mL), and the
aqueous phase was extracted with EtOAc (5 × 15 mL). The
combined organic phases were concentrated to a small volume
that upon cooling to -20 °C gave 12 as a white solid (70 mg,
1
84%): mp 198-200 °C; H NMR (DMF-d7) δ (two rotamers)
8.10 (d, J ) 12.0 Hz, 0.4H), 7.91 (d, J ) 12.5 Hz, 0.6H), 7.50-
7.36 (m, 5H), 7.15 (d, J ) 12.0 Hz, 0.4H), 7.14 (d, J ) 12.5 Hz,
0.6H), 5.25 (s, 2H), 4.98 (s, 0.8H), 4.81 (s, 1.2H), 4.41 (s, 1.2H),
4.18 (s, 0.8H), 4.15 (t, J ) 12.0 Hz, 0.8H), 3.98 (t, J ) 9.5 Hz,
1.2H), 3.81 (t, J ) 12.0 Hz, 0.8H), 3.71 (t, J ) 9.5 Hz, 1.2H);
13C NMR (DMF-d7) δ25 (170.9, 170.7), (169.4, 169.2), 168.7,
167.8, (163.8, 163.6), (155.5, 155.3), 153.9, (151.2, 150.9), 136.7,
128.8, 128.5, 128.3, 94.2, 67.1, (49.9, 49.7), (48.7, 48.1), (44.8,
44.5), (43.2, 42.9); FABMS m/z 522.2 [M + H]+, 520.1 [M -
H]-. Anal. Calcd for C20H19N5O8S2: C, 46.06; H, 3.65; N, 13.43;
S, 12.28. Found: C, 45.82; H, 3.90; N, 13.23; S,12.04.
The synthesis of Dts-C(Z)-OH (12) was also carried out at
25 °C with a range of excesses of Cl(CdO)SCl (10) (1-2.2
equiv). HPLC analysis of the crude reaction mixtures showed
12 as the major product, but a significant amount (17-27%)
of 1,2,4-thiadiazolidine-3,5-dione 15 was also detected. Com-
pound 15 was characterized by LC/MS analysis of the crude
mixtures: ESMS (m/z) calcd for C38H38N10O14S 890.3, found
N-[2-(N-Eth oxyth ioca r bon yl)a m in oeth yl]-N-[(th ym in -
1-yl)a cetyl]glycin e (9). Starting with Boc-T-OH (4) (1.15 g,
3 mmol), compound 9 was prepared as described for compound
6, except that less of bis(ethoxythiocarbonyl) sulfide (5) was
used (0.76 g, 3.6 mmol, 1.2 equiv) and the workup was
different. Following overnight stirring, the solution was diluted
with water (50 mL) and washed with CH2Cl2 (2 × 50 mL).
The aqueous phase was acidified to pH 2 with 12 N HCl and
extracted with EtOAc (3 × 80 mL); the phase separations were
890.5 [M + H]+, 446.0 [M + 2H]2+
.
N-[2-(N-Dit h ia su ccin oyl)a m in oet h yl]-N-[[2-N-(b en z-
yloxyca r bon yl) gu a n in -9-yl]a cetyl]glycin e (13). Starting
with 8 (80 mg, 0.15 mmol), compound 13 was prepared as
described above for compound 11, except for workup. The
reaction was complete after 30 min as indicated by HPLC. The
solution was distributed between EtOAc (10 mL) and H2O (15
(25) Each carbon has two resonances, as confirmed by HMQC.

7288 J . Org. Chem., Vol. 64, No. 20, 1999
Planas et al.
mL), and the aqueous phase was extracted with EtOAc (5 ×
15 mL). The combined organic phases were concentrated to a
small volume, which upon cooling to -20 °C gave 13 as an
off-white solid (71 mg, 85%): mp 158-160 °C; 1H NMR (DMF-
d7) δ (two rotamers) 11.75 (s, 0.7H), 11.56 (s, 0.3H), 11.43 (s,
1H), 7.96 (s, 0.3H), 7.81 (s, 0.7H), 7.54-7.38 (m, 5H), 5.35 (s,
1.4H), 5.33 (s, 0.6H), 5.31 (s, 0.6H), 5.06 (s, 1.4H), 4.51 (s,
1.4H), 4.23 (t, J ) 7.5 Hz, 0.6H), 4.19 (s, 0.6H), 3.99 (t, J )
5.5 Hz, 1.4H), 3.89 (t, J ) 7.5 Hz, 0.6H), 3.73 (t, J ) 5.5 Hz,
1.4H); 13C NMR (DMF-d7) δ25 (171.0, 170.6), 169.3, 168.3,
167.2, 155.5, (155.3, 155.2), 150.0, 148.4, (140.8, 140.5), (136.2,
136.1), 128.9, (128.7, 128.6), (128.5, 128.3), 118.7, (67.9, 67.8),
(49.0, 48.7), (45.2, 44.7), 44.3, (43.4, 43.0); FABMS m/z 562.1
[M + H]+, 560.1 [M - H]-. Anal. Calcd for C21H19N7O8S2‚2H2O:
C, 42.17; H, 3.85; N, 16.41; S,10.73. Found: C, 42.04; H, 3.99;
N, 15.73; S, 9.95.
together with any additive, for a given amount of time (Tables
1 and 2), and washed with CH2Cl2 (8 × 1 min). The resin was
cleaved with TFA-H2O (19:1) for 2 h, and the filtrates were
expressed from the vessels with positive nitrogen pressure.
The cleaved resin was washed with TFA-H2O (19:1) (2 × 0.5
mL), and the filtrates were combined and evaporated to
dryness. The resulting residue was dissolved in water and
analyzed by analytical HPLC (conditions at the beginning of
this Experimental Section) and studied further by mass
spectrometry.
Dts-T-Gly-NH2 was characterized by mass spectrometry and
1H NMR prior to the deprotection studies: FABMS calcd for
C15H18N6O7S2 458.4, found m/z 459.1 [M + H]+, 458.1 [M‚]-
;
1H NMR (DMF-d7) δ (two rotamers) 11.28 (s, 0.3H), 11.22 (s,
0.7H), 8.45 (m, 0.7 H), 8.22 (m, 0.3H), 7.50 (bs, 1H), 7.42 (s,
0.3 H), 7.24 (s, 0.7H), 7.08 (bs, 1H), 4.77 (s, 0.6H), 4.63 (s,
1.4H), 4.29 (s, 1.4H), 4.11 (s, 0.6H), 4.08 (m, 0.6H), 3.93 (t, J
) 6.0 Hz, 1.4H), 3.86 (d, J ) 6.0 Hz, 1.4H), 3.76 (d, J ) 6.0
Hz, 0.6H), 3.77 (m, 0.6H), 3.67 (t, J ) 6.0 Hz, 1.4H), 1.78 (s,
3H).
N-[2-(N-Dith ia su ccin oyl)a m in oeth yl]-N-[(th ym in -1-yl)-
a cetyl]glycin e (14). Meth od A. BSA (136 µL, 0.55 mmol, 1.1
equiv) was added to a solution of 9 (186 mg, 0.5 mmol) in
CH3CN (5 mL), and the mixture was stirred for 30 min. Next,
Cl(CdO)SCl (10) (45 µL, 0.55 mmol, 1.1 equiv) was added,
resulting in a yellow solution. After 2 h, the suspension was
distributed between EtOAc (20 mL) and water (5 mL), and
the aqueous phase was extracted with EtOAc (4 × 20 mL).
The organic layers were combined, dried (MgSO4), and con-
centrated to a small volume that upon cooling to -20 °C gave
14 as an off-white solid (161 mg, 80%). Meth od B (P r efer r ed
on La bor a tor y Sca le). The resultant foam from the synthesis
of Etc-T-OH (9) (1.21 g, 3.0 mmol) was taken up directly in
CH3CN (15 mL), and BSA (0.81 mL, 3.3 mmol, 1.1 equiv) was
added. After 30 min, Cl(CdO)SCl (10) (0.27 mL, 3.3 mmol,
1.1 equiv) was added, and the solution turned yellow. After a
further 2 h, a white precipitate was observed, and HPLC
analysis indicated completion of the reaction. The suspension
was distributed between EtOAc (80 mL) and water (30 mL),
and the aqueous phase was extracted with EtOAc (4 × 80 mL).
The organic layers were combined, dried (MgSO4), and con-
centrated to a small volume that upon cooling to -20 °C gave
14 as an off-white solid (0.91 g, 76% from 4) (sometimes, a
yellow solid was obtained, but it could be decolorized by
suspending in diethyl ether, followed by overnight stirring and
H-T-Gly-NH2 was characterized by mass spectrometry and
co-injected with a sample prepared by an Fmoc-strategy:
FABMS calcd for C13H20N6O5 340.4, found m/z 341.2 [M + H]+,
363.2 [M + Na]+.
When deprotection was carried out with p-methoxybenzyl-
mercaptan (16), benzylmercaptan (18), p-methylthiophenol
(22), thiophenol (23), and p-chlorothiophenol (24), relatively
low but nonzero levels of thiourethane formation were ob-
served (Scheme 5, Table 1, entries 1-3, 7, and 11-13).
Thiourethane corresponding to thiol 16: FABMS calcd for
C
22H28N6O7S 520.5, found m/z 521.2 [M + H]+, 520.3 [M‚]-,
633.2 [M - H + TFA]-. Thiourethane corresponding to thiol
18: FABMS calcd for C21H26N6O6S 490.5, found m/z 491.2 [M
+ H]+, 513.2 [M + Na]+, 489.0 [M - H]-, 603.2 [M - H +
TFA]-. Thiourethane corresponding to thiol 22: ESMS calcd
for C21H26N6O6S 490.5, found m/z 491.0 [M + H]+. Thioure-
thane corresponding to thiol 23: ESMS calcd for C20H24N6O6S
476.5, found m/z 476.8 [M + H]+. Thiourethane corresponding
to thiol 24: ESMS calcd for C20H23ClN6O6S 510.9, found m/z
511.0 [M + H]+.
The carbamoyl disulfide intermediate 25 was observed in
some experiments (e.g., Table 1, entry 9) using âME for
deprotection; it was characterized by mass spectrometry:
ESMS calcd for C16H24N6O7S2 476.5, found m/z 477.0 [M + H]+,
499.0 [M + Na]+.
The urea derivative 26 was also noted during the deprotec-
tion reactions (Tables 1 and 2) and characterized by mass
spectrometry: FABMS calcd for C27H38N12O11 706.6, found m/z
707.1 [M + H]+, 705.1 [M - H]-.
1
then filtration): mp 158-160 °C; H NMR (DMSO-d6) δ (two
rotamers) 11.29 (s, 0.4H), 11.27 (s, 0.6H), 7.34 (s, 0.4H), 7.12
(s, 0.6H), 4.59 (s, 0.8H), 4.42 (s, 1.2H), 4.19 (s, 1.2H), 3.97 (s,
0.8H), 3.91 (t, J ) 6.8 Hz, 0.8H), 3.79 (t, J ) 5.5 Hz, 1.2H),
3.57 (t, J ) 6.8 Hz, 0.8H), 3.50 (t, J ) 5.5 Hz, 1.2H), 1.74 (s,
3H); 13C NMR (DMSO-d6) δ25 (170.9, 170.7), (169.4, 169.2),
168.7, 167.7, (164.9, 164.8), (151.4, 151.3), (142.5, 142.1),
(108.7, 108.6), (48.6, 48.1), (47.9, 47.8), (44.5, 44.3), (43.1, 42.8),
14.5; FABMS m/z 403.1 [M + H]+, 402.2 [M‚]-. Anal. Calcd
Solid -P h a se Dep r otection Stu d ies on Resin w ith Re-
d u ced Loa d in g. Fmoc-PAL-PEG-PS resin (208 mg, 0.18
mmol/g) was treated with piperidine-NMP (3:7, 2 + 8 min),
and after washings with NMP (5 × 2 min), Fmoc-Gly-OH (8
mg, 0.7 equiv) was coupled to the resin using HBTU (10 mg,
0.7 equiv) and DIEA (4.5 µL, 0.7 equiv) in NMP for 1 h. The
resin was washed with NMP (5 × 1 min) and CH2Cl2 (5 × 1
min), acetylated with Ac2O-pyridine-CH2Cl2 (1:9:9, 2 + 30
min), washed with CH2Cl2 (5 × 2 min), and dried in vacuo. A
sample of the dried resin (6 mg) was treated with piperidine-
for
C13H14N4O7S2: C, 38.80; H, 3.48; N, 13.93; S, 15.92.
Found: C, 38.90; H, 3.70; N, 13.61; S, 16.16.
Solid -P h a se Dep r otection Stu d ies. Fmoc-PAL-PEG-PS
resin (0.44 g, 0.18 mmol/g) was washed with NMP (3 × 2 min)
and CH2Cl2 (3 × 2 min), treated with piperidine-NMP (3:7, 2
+ 8 min), and finally washed with NMP (5 × 2 min). Fmoc-
Gly-OH (119 mg, 5 equiv) was coupled to the resin with HBTU
(152 mg, 5 equiv) and DIEA (70 µL, 5 equiv) in NMP for 3 h.
After washes with NMP (5 × 2 min) and CH2Cl2 (5 × 2 min),
the resin was acetylated with Ac2O-pyridine-CH2Cl2 (4 mL,
1:9:9, 2 + 30 min) and washed with CH2Cl2 (5 × 2 min) and
NMP (3 × 2 min). Following Fmoc removal and washes
described above, Dts-T-OH (95 mg, 3 equiv) was incorporated
onto the resin with HBTU (91 mg, 3 equiv) and DIEA (14 µL,
1 equiv) in NMP at 25 °C for 3 h (Kaiser ninhydrin test26
negative after this time). The resin was washed with NMP (3
× 2 min) and CH2Cl2 (3 × 2 min), and dried in vacuo.
NMP (3:7, 2 + 8 min) and analyzed on the basis of UV (301
27
nm, ꢀ ) 7800 cm-1 M-1
)
absorbance of the resultant piperi-
dine-dibenzofulvene adduct. A second sample (8 mg) of resin
was similarly deprotected, hydrolyzed, and evaluated by amino
acid analysis. These complementary methods showed a final
loading of 0.09 mmol/g. Dts-T-OH (23 mg, 3 equiv) was coupled
onto the resin with HBTU (21 mg, 3 equiv) and DIEA (3 µL,
1 equiv) in NMP at 25 °C for 3 h (Kaiser ninhydrin test26
negative after this time). The resin was washed with NMP (5
× 1 min) and CH2Cl2 (5 × 1 min), and dried in vacuo. Aliquots
of this resin were taken to test Dts deprotection, which were
carried out as outlined in the preceding paragraph.
Portions of this resin (10-15 mg per experiment) were
subjected to various Dts deprotection conditions, as follows:
Dts-T-Gly-PAL-PEG-PS resin was swollen in CH2Cl2 (2 × 5
min), treated with a freshly prepared solution of the thiol,
(26) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Anal.
Biochem. 1970, 34, 595-598.
(27) Grant, G. A. In Synthetic Peptides: A User’s Guide; Grant, G.
A., Ed.; W. H. Freeman and Co.: New York, 1992; p 119.

Synthesis of Protected Peptide Nucleic Acid Oligomers
J . Org. Chem., Vol. 64, No. 20, 1999 7289
Eva lu a tion of Con d ition s for Solid -P h a se Syn th esis
of Dts-T4-Gly-NH2. Fmoc-Gly-PAL-PEG-PS resin (40 mg, 0.18
mmol/g) was treated with piperidine-NMP (3:7, 2 + 8 min)
to remove Fmoc, washed with NMP (5 × 2 min), and used for
evaluation of coupling procedures for introduction of Dts-T-
OH (Table 3). Upon completion of coupling (3 h), the resin was
washed with NMP (3 × 2 min) and CH2Cl2 (3 × 2 min).
Removal of the Dts protecting group was accomplished using
the conditions shown (Table 3). After washes with CH2Cl2
(3 × 1 min), neutralization was achieved with DIEA-CH2Cl2
(1:19, 1 + 2 min), followed by washing with CH2Cl2 (3 × 1
min) and NMP (3 × 1 min). The coupling/deprotection cycle
was repeated for the remaining Dts-T-OH monomers, followed
by final washes with NMP (3 × 1 min) and CH2Cl2 (3 × 1 min)
and drying the resin in vacuo. The resin was treated with
TFA-H2O (19:1) for 2 h, filtered, and washed (2 × 1 mL) with
TFA-H2O (19:1). The combined filtrates were evaporated to
dryness under a stream of N2, and the resulting residue was
dissolved in H2O, lyophilized, and analyzed by analytical
HPLC.
(matrix: glycerol-TFA): calcd for C48H60N18O19S2 1257.2,
found m/z 1257.5 [M]+.
Dts-G(Z)-G(Z)-T-A(Z)-Gly-NH 2. Manual chain assembly
was carried out exactly as described for Dts-T4-Gly-NH2. The
crude product was analyzed by analytical HPLC (tR 16.12 min,
54% purity, 88% cleavage yield) and final product confirmed
by FABMS (matrix: glycerol-TFA): calcd for C72H75N27O21S2
1718.6, found m/z 1718.2 [M]+.
Dts-A(Z)-T-C(Z)-G(Z)-Gly-NH2. Using the same proce-
dures, the crude product was analyzed by analytical HPLC
(tR 16.53 min, 54% purity, 83% cleavage yield) and final
product confirmed by FABMS (matrix: glycerol-TFA) calcd
for C71H75N27O21S2 1678.6, found m/z 1678.02 [M]+.
Dts-G(Z)-C(Z)-A(Z)-T-Gly-NH2. Using the same proce-
dures, the crude product was analyzed by analytical HPLC
(tR 16.67 min, 62% purity, 90% cleavage yield) and final
product confirmed by FABMS (matrix: glycerol-TFA): calcd
for C71H75N27O21S2 1678.6, found m/z 1678.24 [M]+.
Dts-T4-Gly-NH2. Manual chain assembly was carried out
starting with Fmoc-Gly-PAL-PEG-PS (40 mg, 0.18 mmol/g).
The resin was treated with piperidine-NMP (3:7, 2 + 8 min)
and washed with NMP (5 × 2 min). Dts-T-OH monomers (9
mg, 3 equiv) were coupled to the support using HBTU (3 mg,
3 equiv) and DIEA (1.2 µL, 1 equiv) in NMP at 25 °C for 3 h.
After washings with NMP (3 × 1 min) and CH2Cl2 (3 × 1 min),
the Dts protecting group was cleaved with DTT (0.5 M) in
HOAc (0.5 M)-CH2Cl2 (2 + 8 min). The resin was washed with
CH2Cl2 (5 × 1 min), neutralized with DIEA-CH2Cl2 (1:19, 1
+ 2 min), and washed with CH2Cl2 (3 × 1 min) and NMP (3 ×
1 min). Further aspects to assemble, cleave, and evaluate the
Dts-protected PNA tetramers were as described in the previous
“Evaluation of Conditions”. The crude product was analyzed
by analytical HPLC (tR 9.06 min, 80% purity, 88% cleavage
yield), and the final product was confirmed by FABMS
Ack n ow led gm en t. M.P. thanks NATO for a post-
doctoral fellowship, E.B. acknowledges a fellowship from
CIRIT-DGU and QFN94-4620 for financial support, and
K.J . thanks the Alfred Benzon Foundation for a post-
doctoral fellowship. We are grateful to NIH and NIST
for financial support.
Su p p or tin g In for m a tion Ava ila ble: Tables 1-10, which
are solid-phase deprotection studies of Dts-Gly-PAL-PEG-PS
with the thiols mentioned in the text. 1H NMR and NOESY
spectra of compounds 4 and 14 and NOESY spectra of
compounds 1 and 11. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O9824394