N-urethane-protected amino alkyl isothiocyanates: Synthesis, isolation, characterization, and application to the synthesis of thioureidopeptides

Article abstract of DOI:10.1021/jo900675s

(Chemical Equation Presented) Synthetically useful N-Fmoc amino-alkyl isothiocyanates have been described, starting from protected amino acids. These compounds have been synthesized in excellent yields by thiocarbonylation of the monoprotected 1,2-diamines with CS₂/TEA/p-TsCl, isolated as stable solids, and completely characterized. The procedure has been extended to the synthesis of amino alkyl isothiocyanates from Boc- and Z-protected amino acids as well. The utility of these isothiocyanates for peptidomimetics synthesis has been demonstrated by employing them in the preparation of a series of dithioureidopeptide esters. Boc-Gly-OH and Boc-Phe-OH derived isothiocyanates 9a and 9c have been obtained as single crystals and their structures solved through X-ray diffraction. They belong to the orthorhombic crystal system, and have a single molecule in the asymmetric unit (Z′ = 1). 9a crystallizes in the centrosymmetric space group Pbca, while 9c crystallizes in the noncentrosymmetric space group P2₁2₁2₁.

Full text of DOI:10.1021/jo900675s

pubs.acs.org/joc
N-Urethane-Protected Amino Alkyl Isothiocyanates: Synthesis, Isolation,
Characterization, and Application to the Synthesis of Thioureidopeptides
Vommina V. Sureshbabu,*,† Shankar A. Naik,† H. P. Hemantha,† N. Narendra,†
Ushati Das,‡ and Tayur N. Guru Row‡
†Peptide Research Laboratory, Department of Studies in Chemistry, Central College Campus, Bangalore
University, Dr. B. R. Ambedkar Veedhi, Bangalore 560 001, India, and ‡Solid State and Structural Chemistry
Unit, Indian Institute of Science, Bangalore 560 012, India
hariccb@rediffmail.com
Received March 31, 2009
Synthetically useful N-Fmoc amino-alkyl isothiocyanates have been described, starting from protected
amino acids. These compounds have been synthesized in excellent yields by thiocarbonylation of the
monoprotected 1,2-diamines with CS2/TEA/p-TsCl, isolated as stable solids, and completely characterized.
The procedure has been extended to the synthesis of amino alkyl isothiocyanates from Boc- and Z-protected
amino acids as well. The utility of these isothiocyanates for peptidomimetics synthesis has been demon-
strated by employing them in the preparation of a series of dithioureidopeptide esters. Boc-Gly-OH and
Boc-Phe-OH derived isothiocyanates 9a and 9c have been obtained as single crystals and their structures
solved through X-ray diffraction. They belong to the orthorhombic crystal system, and have a single
molecule in the asymmetric unit (Z0=1). 9a crystallizes in the centrosymmetric space group Pbca, while 9c
crystallizes in the noncentrosymmetric space group P212121.
Introduction
Thus, several classes of peptidomimetics that contain non-native
bonds such as ureas, carbamates, oligosulfonamides, peptoids,
hydrazino peptides, aminoxy peptides, and heterocycles have
been synthesized and employed for therapeutic applications.3,4
The synthesis of backbone modified peptides and their screen-
ing is an integral part of the present day drug development
processes owing to the dramatic improvement in the pharmaco-
kinetic properties of the bioactive peptides caused by the replace-
ment of one or more peptide bonds with unnatural linkages.1,2
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G.; Semetey, V.; Didierjean, C.; Aubry, A.; Briand, J. P.; Rodriguez, M. J.
Org. Chem. 1999, 64, 8702–8705. (b) Boeijen, A.; van Ameijde, J.; Liskamp,
R. M. J. J. Org. Chem. 2001, 66, 8454–8462. (ii) For carbamates see: (c) Cho,
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L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G. Science 1993, 261, 1303–1305.
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(e) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D.
A.; Banville, S.; Wang, S.; Rosenberg, S.; Marlowe, C. K.; Spellmeyer, D. C.;
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5260 J. Org. Chem. 2009, 74, 5260–5266
Published on Web 06/18/2009
DOI: 10.1021/jo900675s
r
2009 American Chemical Society

Sureshbabu et al.
JOCArticle
Among these compounds, oligoureas and ureidopeptides
have been extensively studied as inhibitors and antagonists
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tural motifs in de novo design.5,6 Isoelectronic replacement of
the oxygen atom of the uredio bond results in another
biologically, medicinally, and structurally relevant linkage,
the thioureido bond. The importance of the thioureido group
in conferring the required potency to bioactive molecules can
be seen in its insertion into many medicinally valuable com-
pounds. Many of the substituted thioureas are active as anti-
HIV,7 antiviral,8 antimicrobial,9 antituberculor,10 antitu-
mor,11 antihypertensive,12 and anticarcinogenic agents.13
The high acidity of the -NH-CS-NH- protons, in correla-
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and stabilization of secondary structures.14 Thiourea moieties
FIGURE 1. Amino/peptide acid ester derived isothiocyanates well
known in the literature.21,22
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(i) coupling of primary/secondary amines in the presence of
CS2,23 thiophosgene,24 or its equivalent,25 triphenylpho-
sphine thiocyanogen,26 thiourea, or H2S on substituted
guanidines27 [several thiocarbonylating agents such as
1-(methyldithiocarbonyl)imidazole,28 1,10-thiocarbonyldii-
midazole (TDI),29 di-2-pyridyl thionocarbonate (DPT),30
thiocarbonyl(bis-benzotriazole),31 1,3-diphenyl thiourea,32
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J. Org. Chem. Vol. 74, No. 15, 2009 5261

Sureshbabu et al.
JOCArticle
Glycosyl isothiocyanates are well known and highly useful
building blocks in carbohydrate chemistry.36 Their stereospe-
cific synthesis, stability and characterization aspects, and syn-
thetic applications are widely reported.37 They have been
employed in the preparation of thiourea-linked glyco-oligo-
mers that mimic the branching patterns of oligosaccharides and
multivalent glycosides, glycosyl heterocycles, and glyco-con-
jugates such as N-nucleosides.38
In peptidomimetic chemistry, while the bioisosteric replace-
ment of the -CdO/-C-O-C- bond with the -CdS/-C-
S-C-bond can be profoundly found in the case of the synthesis
of thiopeptides/thioamides and thiazoles with new proper-
ties compared to parent peptides/amides or oxazoles,3a,39
similar modification in the case of the other hetero bonds,
-NH-CO-NH- (ureido), -NCO (isocyano), and -NH-
CO-O-R (carbamate), is less commonly reported. Again,
when diversely substituted peptidyl ureas, e.g., N,N; N,N0,
linked oligoureas, R-peptidyl, β-peptidyl ureas etc.,40,41 have
been reported, the known types of peptidyl thioureas are
restricted to two examples, viz., R-amino acid esters linked
through thioureido linkages42 and Boc-NH-(CH2)2-
NHCSNH-R synthesized starting from ethylene diamine.
42,43
Similarly, there are few reports on the synthesis of
isothiocyanates derived from amino acids/peptides, and all
of these describe only R-isothiocyanato alkyl esters, SCN-
CHR-COOY, obtained by converting the R-amino group of
amino acid esters into the isothiocyano group under different
thiocarbonylation conditions. Halpern et al. synthesized
them by reacting R-amino acid esters with CS2 and subse-
quent decomposition of the dithiocarbamate intermediate
with chloroformate ester.44 Kunze et al. prepared R-isothio-
cyanato alkyl esters employing thiophosgene in water.21 The
products were purified by vacuum distillation and used for
the preparation of thiocarbamoylphosphines. Nowick et al.
prepared peptide ester isothiocyanates, SCN-CHR-
CONH-CHR-COOY by treating amino free peptidyl ester
with thiophosgene under biphasic conditions.22 Boas et al.
generated isothiocyanates by treating the resin bound Phe
and Tyr with CS2 in the presence of HBTU and PyBOP and
coupled with amino acid esters to obtain thioureas.42
ꢀ
ꢀ
(36) Jimenez Blanco, J. L.; Bootello, P.; Gutierrez Gallego, R.; Ortiz
ꢀ
Mellet, C.; Garcı
´
a Fernandez, J. M. Synthesis 2007, 2545–2558.
In this context, we envisaged the preparation of a hitherto
unreported class of N-urethane-protected amino alkyl iso-
thiocyanates. Owing to the vast diversity of synthetic appli-
cations of isothiocyanates, these novel compounds could
find utility as valuable intermediates in the synthesis of
several new classes of peptidomimetics including N-pro-
tected thioureidopeptide esters, amino acid analogues, and
peptide conjugates. Further, our interest in the design and
synthesis of Fmoc-amino acid derived novel monomeric
building blocks that possess a reactive functional group
inserted in place of the carboxyl moiety of amino acids and
useful in preparing backbone modified peptides with N as
well as C terminus41,45,46 led us to focus attention on the
synthesis of yet another new class of Nβ-Fmoc amino alkyl
isothiocyanates. Accordingly, we herein report the first
synthesis and isolation of N-Fmoc-β-amino alkyl isothio-
cyanates and demonstrate their utility in the preparation of
dithioureidopeptides which, to the best of our knowledge,
are hitherto unreported. The protocol has been extended to
obtain Boc and Z amino acid derived isothiocyanates as well.
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Defaye, J. Carbohydr. Res. 1995, 268, 57–71. (d) Camarasa, M. J.;
Fernandez-Resa, P.; Garcia-Lopez, M. T.; de las Heras, F. G.; Mendez-
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nucleosides and glycosylaminoheterocycles, see: (a) Reference 31. (b) Al-
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Bourgougnon, N.; Meslin, J. C.; Deniaud, D. J. Org. Chem. 2003, 68, 8583–
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(f ) Taylor, C. M. Tetrahedron 1998, 54, 11317–11362. (iii) Glycoconjugates:
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M. Chem. Commun. 2007, 831–833. (h) Walter, M.; Wiegand, M.; Lindhorst,
T. K. Eur. J. Org. Chem. 2006, 952–958. (i) Gomez-Garcia, M.; Benito, J. M.;
Rodriguez-Lucena, D.; Yu, J.-X.; Chmurski, K.; Ortiz Mellet, C.; Gutirrez-
Gallego, R.; Maestre, A.; Defaye, J.; Garcia Fernandez, J. M. J. Am. Chem.
Soc. 2005, 127, 7970–7971. ( j) Oshovsky, G. V.; Verboom, W.; Fokkens, R.
H.; Reinhoudt, D. N. Chem.;Eur. J. 2004, 10, 2739–2748. (iv) Thiourea-
bridged cluster glycosides from glycosyl isothiocyanates: (k) Lindhorst, T.
K.; Kieburg, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 1953–1956. (v) Linear
and dendritic thiourea-linked glycooligomers: (l) Jiminez, Blanco, J. L.;
Bootello, P.; Ortiz Mellet, C.; Garcia Fernandez, J. M. Eur. J. Org. Chem.
2006, 183–196. (vi) Antiviral, antibacterial and antitumour agents prepared
by using glucosyl isothiocyanates and biologically active amines: (m) Garcia
Fernandez, J. M.; Ortiz Mellet, C. Adv. Carbohydr. Chem. Biochem. 2000, 55,
35–135. (n) Todoulou, O. G.; Papadaki-Valiraki, A. E.; Filippatos, E. C.;
Ikeda, S.; De Clercq, E. Eur. J. Med. Chem. 1994, 29, 127–131. (vii) Glyco-
lipids for oral drug delivery: (o) Falconer, R. A.; Toth, I. Bioorg. Med. Chem.
2007, 15, 7012–7020.
Results and Discussion
Synthesis of Fmoc-Nβ-amino Alkyl Isothiocyanates Fmoc-
Xaa-ψ[CH2NCS] 2. The initial part of the study involved the
synthesis of title isothiocyanates 2 employing Fmoc chem-
istry. The synthesis was pursued with two different routes.
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cited therein.
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Engl. 1995, 34, 907–908. (b) Burgess, K.; Ibarzo, J.; Linthicum, D. S.; Russell,
D. H.; Shin, H.; Shitangkoon, A.; Totani, R.; Zhang, A. J. J. Am. Chem. Soc.
1997, 119, 1556–1564. (c) Tamilarasu, N.; Huq, I.; Rana, T. M. J. Am. Chem.
Soc. 1999, 121, 1597–1598. (d) Boeijen, A.; Liskamp, R. M. J. Eur. J. Org.
Chem. 1999, 2127–2135. (e) Guichard, G.; Semetey, V.; Didierjean, C.;
Aubry, A.; Briand, J. P.; Rodriguez,, M. J. Org. Chem. 1999, 64, 8702–
8705. (f ) Guichard, G.; Semetey, V.; Rodriguez, M.; Briand, J. P. Tetra-
hedron Lett. 2000, 41, 1553–1557. (g) Fischer, L.; Semetey, V.; Lozano, J.-
M.; Schaffner, A.-P.; Briand, J.-P.; Didierjean, C.; Guichard, G. Eur. J. Org.
Chem. 2007, 2511-2525 and references cited therein.
(41) For the synthesis, isolation, and characterization studies of isocya-
nates derived from Fmoc-R-amino acids/peptide acids, see: (a) Patil, B. S.;
Vasanthakumar, G. R.; Suresh Babu, V. V. J. Org. Chem. 2003, 68, 7274–
7280. (b) Sureshbabu, V. V.; Patil, B. S.; Venkataramanarao, R. J. Org.
Chem. 2006, 71, 7697–7705.
(43) A single example of the isothiocyanate, [[(1,1-dimethylethoxy)car-
bonyl]amino]ethylisothiocyanate, was prepared from ethylene diamine by
treating mono Boc-protected ethylene diamine with carbon disulfide and
DCC. The product was isolated after column purification in 92% yield: For
details see: Kneeland, D. M.; Ariga, K.; Lynch, V. M.; Huang, C.-Y.;
Anslyn, E. V. J. Am. Chem. Soc. 1993, 115, 10042–10055.
(44) (a) Halpern, B.; Close, V. A.; Wegmann, A.; Westley, J. W. Tetra-
hedron Lett. 1968, 27, 3119–3122. (b) Halpern, B.; Patton, W.; Crabbe, P. J.
Chem. Soc. B 1969, 1143–1145.
(45) For the synthesis of N-protected R-amino formamides from Fmoc/
Z-protected amino acids, see: (a) Sudarshan, N. S.; Narendra, N.;
Hemantha, H. P.; Sureshbabu, V. V. J. Org. Chem. 2007, 72, 9804–9807.
(b) Sureshbabu, V. V.; Narendra, N. Int. J. Pept. Res. Ther. 2008, 14, 201–207.
(46) For the synthesis of N-Fmoc-β-amino alkyl nitriles and their appli-
cation in the preparation of 1-substituted tetrazoles, see: Sureshbabu, V. V.;
Narendra, N.; Nagendra, G. J. Org. Chem. 2009, 74, 153–157.
5262 J. Org. Chem. Vol. 74, No. 15, 2009

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SCHEME 1
SCHEME 2
The direct thionation of known isocyanates (Fmoc-Aaa-
47
TABLE 1. Comparative Study of the Reagents for the Synthesis of 2f
ψ[CH2NCO] into isothiocyanates with P2S5 or Lawesson
reagent)48 and reaction of N-Fmoc-β-amino alkyl iodides
with isothiocyanate ions.49 But both these routes turned out
to be unsuccessful with the former resulting in very low yields
of the desired isothiocyanates and the later yielding a larger
product fraction as thiocyanate due to the ambident nature
of isothiocyanate ion.50 In view of these results, studies on
thiocarbonylation of amines 1 were undertaken (Scheme 1).
Fmoc-amino acids were initially reduced to the correspond-
ing β-amino alcohols via treatment of the mixed anhydride of
the acid with NaBH4.51 The alcohols were then converted
into the alkyl iodides 4 under Mitsunobu conditions, which
after isolation and purification through flash chromatogra-
phy were treated with NaN3 in DMF at room temperature
for 3-4 h to obtain the azides 5 as white solids.52 On
subjecting the azides to catalytic hydrogenation with Pd/C,
amines 1 were obtained quantitatively which were isolated as
HCl salts by the addition of CHCl3 to the reaction mixture
according to Liskamp et al.4b (Scheme 2). In the case of
Fmoc-Asp/Glu(Bzl)-OH and Fmoc-Ser(Bzl)-OH, the de-
sired amines were prepared by treating the corresponding
alkyl azides 5 with PPh3 in THF.53
reaction yield
time (h)
entry
reagent
(%)
1
2
3
4
5
6
7
thiophosgene
0.5
1.0
1.0
2.0
3.5
1.5
1.1
65
52
56
93
54
63
56
dipyridyl thionocarbonate (DPT)
1,10-thiocarbonyldiimidazole (TDI)
CS2/TEA/tosyl chloride
CS2/DCC
CS2/TEA/H2O2
CS2/PPh3 (for direct conversion of azide 5 into
isothiocyanate)
yielding, harsh and involved tedious workup with difficult
product isolation. Finally, the reaction of amine with CS2/
TsCl/TEA was found to be attractive in terms of furnishing
good yield, involving milder reaction conditions, less ex-
pense, and commercial availability of the reagents.
In a typical experiment, a solution of Fmoc-Ala-
ψ[CH2NH2 HCl] 1b in THF was treated with an equimolar
3
quantity of CS2 in the presence of TEA at 0 °C for 20 min,
and the in situ generated dithiocarbamic salt was decom-
posed with p-TsCl for about 30 min at the same temperature
(Scheme 1). Upon completion of the reaction (from TLC
analysis) isothiocyanate 2b was isolated by a simple workup
and purified through column chromatography as analyti-
cally pure solid in 92% yield (as evident by HPLC). The same
procedure was successfully extended to prepare a series of
novel isothiocyanates 2a-l from Fmoc amino acids includ-
ing those derived from side chain functionalized, viz., aspar-
tic acid, glutamic acid, and serine which were fully
characterized (Table 2). The 13C NMR of these compounds
contains a characteristic signal at around δ 132.0 ppm
corresponding to isothiocyanate carbon, and the IR spec-
trum exhibits a sharp and intense peak at around 2090 cm-1
characteristic of the isothiocyanato group. All the isothio-
cyanates showed exceptional stability; they were stable to-
ward column chromatography and long time storage (for
few months) at room temperature with no noticeable degra-
dation as analyzed by HPLC.
In the next step, conversion of the amine into isothiocya-
nate was undertaken. For this, the amine 1 was reacted with
thiocarbonylating agents thiophosgene, DPT, and TDI.
With thiophosgene, though the yield of the desired isothio-
cyanate was satisfactory, handling problems, difficulty in
removal of the excess of the reagent, and the toxicity
discouraged further usage. With both DPT and TDI the
reaction was slow and also the yields were unsatisfactory.
Consequently, generation of the dithiocarbamic salt by
reaction of the amine with CS2 followed by decomposition
into the isothiocyanate in the presence of TsCl,54 DCC,55 and
56
H2O2 was pursued. Alternatively, reduction of the alkyl
azide with PPh3 in the presence of CS2 was also carried out.57
The results of these studies are summarized in Table 1. The
reactions involving DCC and H2O2 were sluggish and less
(47) Scheeren, J. W.; Ooms, P. H. J.; Nivard, R. J. F. Synthesis 1973, 149–
150.
Synthesis of N-Boc/Z-Amino Alkyl Isothiocyanates Boc/
Z-Xaa-ψ[CH2NCS] 8 and 9. To obtain differentially N-
protected isothiocyanate derivatives of amino acids to be
useful in diverse conditions of peptide and peptidomimetic
synthesis, and also to demonstrate the generality of the
present protocol to yield the isothiocyanates containing the
other commonly employed urethane protections Boc and Z,
we extended the synthesis to prepare N-Boc- and Z-protected
isothiocyanates as well. The required alkyl diamines, Pg-
NH-Xaa-ψ[CH2NH2] 7 where Pg=Boc/Z, were synthesized
through a different route that involved the reduction of N-
Boc/Z-protected R-amino nitriles with LiAlH4, due to the
tolerance of Boc and Z groups toward LiAlH4, unlike the
Fmoc group. The resulting mono Boc/Z-protected alkyl
(48) Pedersen, U.; Thorsen, M.; El-Khrisy, E.-E. A. M.; Clausen, K.;
Lawesson, S.-O. Tetrahedron 1982, 38, 3267–3269.
(49) Johnson, T. B.; Bergman, W. J. Am. Chem. Soc. 1932, 54, 3360–
3362.
(50) Muller, A.; Wilhelms, A. Ber. Dtsch. Chem. Ges. 1941, 74, 698–703.
(51) (a) Kokotos, G.; Noula, C. J. Org. Chem. 1996, 61, 6994–6996. (b)
Rodriguez, M.; Linares, M.; Doulut, S.; Heitz, A.; Martinez, J. Tetrahedron
Lett. 1991, 32, 923–926.
(52) Mondal, S.; Fan, E. Synlett 2006, 306–308.
(53) Pal, B.; Jaisankar, P.; Giri, V. S. Synth. Commun. 2004, 34, 1317–
1323.
(54) Wong, R.; Dolman, S. J. J. Org. Chem. 2007, 72, 3969–3971.
(55) Jochims, J. C.; Seelinger, A. Angew. Chem., Int. Ed. Engl. 1967, 6,
174–175.
(56) Hartmann, A. Methoden Org. Chem. (Houben-Weyl) 1983, E4, 834.
(57) Garcia-Moreno, M. I.; Diaz-Perez, P.; Benito, J. M.; Ortiz Mellet,
C.; Defaye, J.; Garcia Fernandez, J. M. Carbohydr. Res. 2002, 337, 2329–
2334.
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Sureshbabu et al.
JOCArticle
TABLE 2. List of N-Fmoc-Aaa-ψ[CH2NCS]s Prepared
TABLE 3. List of Z/Boc-Aaa-ψ[CH2NCS]s Synthesized
HRMS
(M þ Naþ)
HRMS
(M þ Naþ)
[R]25
yield mp (c 1,
D
yield mp [R]25D (c 1,
(%) (°C) CHCl3)
entry
isothiocyanates
(%) (°C) CHCl3) calcd
obsd
272.9a
entry
R1
calcd
obsd
8a Z-Ala-ψ[CH2NCS]
8b Z-Val-ψ[CH2NCS]
8c Z-Leu-ψ[CH2NCS]
8d Z-Phe-ψ[CH2NCS]
8e Z-Phg -ψ[CH2NCS]
8f Z-Pro-ψ[CH2NCS]
9a Boc-Gly-
88
85
86
88
85
80
89
112 -95.0 273.0
119 -103.0 301.0987 301.0980
2a
2b CH3
H
90 171
347.0830 347.0830
92 183
90 164
88 168
90 154
93 187
85 148
-67.0 361.0987 361.0974
-71.0 389.1300 389.1314
-87.0 403.1456 403.1470
-71.0 403.1456 403.1453
-17.0 437.1300 437.1274
-16.0 423.1143 423.1140
þ15.0 423.1143 423.1142
-52.0 387.1143 387.1140
-44.0 475.1667 475.1689
-36.0 495.1354 495.1352
-23.0 509.1511 509.1502
-17.0 467.1405 467.1402
108 -110.0 315.1
128 -23.0 349.0
315.0a
349.0a
2c CH(CH3)2
2d CH2CH(CH3)2
2e CH(CH3)C2H5
2f CH2C6H5
2g C6H5
132 þ11.8 335.0830 335.0837
gum -88.0 299.0830 299.0822
65
225.0674 225.0680
ψ[CH2NCS]
2h C6H5 (D-amino acid) 82 145
9b Boc-Val-
82
87
85
80
82
78 -45.0 267.1143 267.1140
2i -(CH2)3- (Proline)
2j (CH2)2COOtBu
2k CH2COOBzl
2l (CH2)2COOBzl
2m CH2OBzl
87 Oil
87 123
91 175
88 186
84 142
ψ[CH2NCS]
9c Boc-Phe-
ψ[CH2NCS]
9d Boc-D-Phg-
107 -40.0 315.1
75 -30.0 301.0
315.0a
300.9a
ψ[CH2NCS]
9e Boc-Met-
ψ[CH2NCS]
9f Boc-Cys(Bzl)-
68 -46.7 299.0864 299.0867
gum -39.0 361.1020 361.1029
SCHEME 3
ψ[CH2NCS]
aESI-MS of the isothiocyanates.
SCHEME 4. Synthesis of Thioureidodipeptides
diamines were then converted to isothiocyanates by follow-
ing the same procedure used for the preparation of Fmoc-
protected isothiocyanates (Scheme 3). In these studies also,
the desired isothiocyanates Boc/Z-Xaa-ψ[CH2NCS] 8 and 9
were obtained in 80-89% yields. All of these compounds
(Table 3) were isolated as stable solids after column purifica-
tion, completely characterized, and found to be shelf-stable
like their Fmoc counterparts.
Synthesis of Thioureidodipeptides. Dithioureidopeptides
were synthesized by coupling the isothiocyanates 2 with amino
acid esters. Compounds 2a-d and 2f were reacted with amino
acid methyl/ethyl esters (obtained by deprotonation of the HCl
salt of the amino ester with activated Zn)58 in the presence of
DIEA to obtain the thioureidodipeptides 10a-g in 65-74%
yields after column purification, which were adequately char-
acterized (Scheme 4, Table 4). The 13C NMR of thioureas 10
showed a signal at around δ 181.00 corresponding to thiocar-
bonyl carbon. No significant difference was observed with
respect to reaction time or yields upon changing the base from
DIEA to TEA or pyridine or N-methylmorpholine (NMM).
These isothiocyanates take a longer time (4 h) to couple with
amino acid esters compared to the related class of isocyanates
which readily couple with the amines within 30 min.6a
Isothiocyanates derived from Boc and Z amino acids were
also employed in the synthesis of dithioureidopeptides.
Reaction of 8 or 9 with amino acid esters in the presence of
DIEA readily yielded the corresponding Boc- and Z-pro-
tected dithioureidopeptides 11 and 12 in 67-74% yield
(Figure 2, Table 4). Again, all the thioureas thus obtained
were well characterized.
tides 13a,b (Fmoc protected), 14a,b (Boc protected), and 15a,b
(Z protected) were synthesized by coupling Fmoc/Boc-Phe-
ψ[CH2NCS] and Z-Phg-ψ[CH2NCS] with (R)-1-phenylethy-
lamine and (S)-1-phenylethylamine separately (Figure 3).
1
In each set, H NMR of the particular epimer contained a
single distinct methyl group doublet. Observed δ values for the
-CH3 group of the compounds are as follows: 13a 1.40, 1.38
and 13b 1.49, 1.47; 14a 1.42, 1.41 and 14b 1.44, 1.43; and 15a
1.28, 1.27 and 15b 1.31, 1.32 ppm. Further, the samples 13c,
14c, and 15c prepared by coupling the isothiocyanates with
racemic 1-phenylethaneamine showed methyl group reso-
nances at δ values of 1.37, 1.40, 1.47, 1.48; 1.45, 1.44, 1.41;
and 1.13, 1.15, 1.17, respectively, indicating the presence of two
isomers with well-separated methyl group doublets. Also, the
HPLC profile of the two epimers, 13a and 13b, had peaks at
Rt values of 17.8 and 19.2 min, respectively. Similarly, HPLC
of the crude samples of compound 10b and its epimer Fmoc-
Phe-ψ[CH2NHCSNH]-D-Ala-OMe had major peaks at Rt
values of 20.13 and 21.17 min, respectively, and the equimolar
mixture of these epimers that was prepared by coupling
racemic alanine methyl ester with Fmoc-Phe-ψ[CH2NCS]
showed two well-separated peaks corresponding to the thiour-
eas at Rt 20.14 and 21.21 min. Thus, from the above studies it
was evident that the samples analyzed were optically pure and
the synthesis of isothiocyanates as well as their coupling with
amino acid esters takes place with retention of configurations
at the chiral center of the isothiocyanate as well as that of the
newly coupled amino acid ester residue.
Racemization Studies. The optical purity of the isothiocya-
nates as well as that of the thioureidopeptides was evaluated by
1H NMR studies of the model dithioureidopeptides prepared
via the present protocol starting from Fmoc, Boc, as well as Z
amino acids. For this, three sets of epimeric dithioureidopep-
(58) Sureshbabu, V. V.; Ananda, K. J. Pept. Res. 2001, 57, 223–226.
5264 J. Org. Chem. Vol. 74, No. 15, 2009

Sureshbabu et al.
JOCArticle
TABLE 4. List of Dithioureido Peptides
Hence, the present crystal structure studies would also be
helpful in evaluating the structural parameters of N-blocked
amines functionalized at the β position. The compounds 9a
and 9c belong to the orthorhombic crystal system, and have a
single molecule in the asymmetric unit (Z0 =1) (Figure 4).
However, while 9a crystallizes in the centrosymmetric space
group Pbca, 9c crystallizes in the noncentrosymmetric space
group P212121. Selected bond lengths and torsion angles,
hydrogen bonding geometries, and parts of the crystal
structures are given in the Supporting Information.
S1 no.
dithioureido peptides
yielda (%)
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
12a
12b
12c
12d
12e
Fmoc-Gly-ψ[CH2-NH-CS-NH]-Phe-OMe
Fmoc-Phe-ψ[CH2-NH-CS-NH]-Ala-OMe
Fmoc-Leu-ψ[CH2-NH-CS-NH]-β-Ala-OMe
Fmoc-Phe-ψ[CH2-NH-CS-NH]-β-Ala-OMe
Fmoc-Ala-ψ[CH2-NH-CS-NH]-β-Ala-OMe
Fmoc-Ala-ψ[CH2-NH-CS-NH]-β-Ala-OEt
Fmoc-D-Phg-ψ[CH2-NH-CS-NH]-β-Ala-OEt
Z-Phe-ψ[CH2-NH-CS-NH]-Leu-OMe
Z-Cys(Bzl)-ψ[CH2-NH-CS-NH]-Ala-OMe
Z-Phe-ψ[CH2-NH-CS-NH]-β-Ala-OEt
Z-Phg-ψ[CH2-NH-CS-NH]-β-Ala-OMe
Boc-Gly-ψ[CH2-NH-CS-NH]-Phe-OMe
Boc-Ala-ψ[CH2-NH-CS-NH]-Val-OEt
Boc-Ala-ψ[CH2-NH-CS-NH]-β-Ala-OMe
Boc-Leu-ψ[CH2-NH-CS-NH]-β-Ala-OMe
Boc-Phe-ψ[CH2-NH-CS-NH]-Phe-OMe
73
71
74
65
67
69
73
72
70
67
71
73
74
70
70
73
Conclusion
In summary, we describe the synthesis of Nβ-Fmoc/Boc/Z-
amino isothiocyanates by the reaction of N-Fmoc/Boc/Z-
amino acid derived alkyl amines with CS2 in the presence of
TEA and p-TsCl. The isothiocyanates were isolated as stable
solids and characterized through NMR, IR, and mass spec-
trometry. These isothiocyanates 2, 8, and 9 were conveni-
ently used as building blocks for the synthesis of thioureido-
linked dipeptidomimetics 10, 11, and 12. Crystal structures
of isothiocyanates 9a and 9c that are synthesized from Boc-
Gly-OH and Boc-Phe-OH, respectively, have been solved,
which constitutes the first report on the crystal structure of
amino acid derivatives containing an isothiocyanate moiety.
Application of the present isothiocyanates in the synthesis of
eoligothioureidopeptides, peptide heterocycles, and thiocar-
bamate-linked peptides is being investigated.
aIsolated yield after column purification.
FIGURE 2. Thiourea-linked dipeptidomimetics.
Experimental Section
General Procedure for the Synthesis of N-Fmoc Amino Alkyl
Isothiocyanates 2. To a chilled solution of N-Fmoc-amino alkyl
amine 3 (1.3 mmol) in dry THF was added CS2 (1.3 mmol)
followed by TEA (3.5 mmol). The ice bath was removed and
stirring was continued for another 10 min. The reaction mixture
was again chilled; p-toluene sulfonyl chloride (1.5 mmol) was
added and the solution was stirred for another 25 min or until the
completionofreactionasjudgedbyTLC. Anexcess ofCH2Cl2 (20
mL) was added and the organic layer was washed twice with citric
acid solution (10%, 15 mL), followed by water (15 mL) and brine
(10 mL). It was then dried over anhydrous sodium sulfate and
concentrated under vacuum. The resulting crude product was
subjected to column chromatography (silica gel 100-200 mesh,
20% ethyl acetate in hexane) to afford the title compound as a
white solid. Batch reactions up to 50 mmol were safely carried out
and the products were isolated in excellent yield.
N-Fmoc-Thioureido Dipeptidyl Esters 10. A solution of amino
acid ester (1.2 mmol, obtained by neutralizing the hydrochlor-
ide salt by treatment with zinc dust) in dry CH2Cl2 was added
to a stirred solution of N-Fmoc-amino alkyl isothiocyanate 2
(1 mmol) followed by DIEA (1.5 mmol) at 0 °C. The solution
was then allowed to warm to room temperature and stirred for
3 h. After completion of the reaction, the solution was diluted
with 10% citric acid (8 mL) and the layers were allowed
to separate. The organic phase was washed with 10% Na2CO3
(10 mL), water (2 ꢀ 10 mL), and brine (10 mL) and dried over
anhydrous sodium sulfate. After the removal of the solvent
under vacuum, the crude was purified through column chro-
matography (silica gel 100-200 mesh, 30-40% ethyl acetate in
hexane) to afford the thioureido dipeptidyl thiourea esters as
off-white to white semisolids.
FIGURE 3. Epimeric dithioureidopeptides synthesized for racemi-
zation studies.
Crystal Structures of Boc-Gly-ψ[CH2-NCS] 9a and Boc-
Phe-ψ[CH2-NCS] 9c. X-ray diffraction of single crystals of
amino acid derivatives generates valuable information on the
structural properties which would influence the reactivity and
chemical behavior of these compounds during peptide coupling
and further in peptidomimetic synthesis. Nevertheless, only a
small fraction of the vast number of reported amino acid
derivatives have been crystallized, and generally these com-
pounds are the ones with conformationally restricted CR,R
disubstituted glycine residue that show higher crystallinity.59
In the present study, we have obtained single crystals of two
isothiocyanates 9a and 9c containing proteinogenic amino
acids, Gly and Phe, respectively. To the best of our knowledge,
this is the first report on the crystal structure analysis of amino
acid derivatives containing an isothiocyanato group.
The crystal structures indicate the stability of this class of
isothiocyanates compared to their isocyanate counterparts
which are not stable and hence have not been isolated.4a,40f
Spectral Characterization Data of Representative Compounds
2b and 10c. (S)-(9H-Fluoren-9-yl)methyl 1-isothiocyanatopro-
pan-2-ylcarbamate (N-Fmoc-Ala-ψ[CH2NCS]) (2b): yield 92%
of white solid; mp 183 °C; Rf (10% EtOAc:hexane) 0.32;
(59) For a review on X-ray diffraction analysis of N-protected amino acid
halides, esters, anhydrides, UNCAs, azides, and amides see : Toniolo, C.;
Crisma, M.; Formaggio, F. Biopolymers Peptide Science 1996, 40, 627-651
and references cited therein.
J. Org. Chem. Vol. 74, No. 15, 2009 5265

Sureshbabu et al.
JOCArticle
FIGURE 4. The molecular structure of Boc-Gly-ψ[CH2-NCS] 9a (left) and Boc-Phe-ψ[CH2-NCS] 9c (right) showing the atom-labeling
scheme. Displacement ellipsoids are drawn at the 50% probability level. H atoms are shown as small spheres of arbitrary radii.
[R]25D -67.0 (c 1.0, CHCl3); 1H NMR (CDCl3) δ 1.26 (d, J=6.4
Hz, 3H), 3.94 (br, 1H), 4.21 (d, J = 6.4 Hz, 2H), 4.35-4.49
(m, 3H), 4.83 (d, J=6.0 Hz, 1H), 7.30-7.42 (m, 4H), 7.58 (d, J=
7.2 Hz, 2H), 7.76 (d, J=7.6 Hz, 2H); 13C NMR (CDCl3) δ 18.4,
47.3, 47.7, 50.5, 67.3, 120.5, 125.6, 127.6, 128.3, 133.0, 141.8,
144.2, 155.9; HRMS calcd for C19H18N2O2S m/z 361.0987 (Mþ þ
Na), found 361.0974.
(S)-(9H-fluoren-9-yl)methyl (S)-3-phenyl-1-(3-((S)-1-phenyl-
ethyl)thioureido)propan-2-ylcarbamate (Fmoc-Phe-ψ[CH2NHCSNH]-
(S )-1-phenethylamine) (13b) was obtained in 74% yield:
1
Rt (HPLC) 19.2 min; H NMR (CDCl3) δ 1.48 (d, J=6.6 Hz,
3H), 2.73 (m, 2H), 3.53 (m, 1H), 3.85 (m, 1H), 4.14 (t, J=6.9 Hz,
1H), 4.35 (t, J=7.2 Hz, 2H), 5.29 (m, 1H), 6.04 (br, 2H), 7.07 (d,
J=6.9 Hz, 2H), 7.24-7.44 (m, 14H), 7.51 (t, J=7.5 Hz, 2H), 7.77
(d, J=7.2 Hz, 2H). Finally, 2f (0.3 g, 0.72 mmol) was treated
with racemic-(1)-phenylethylamine (95 mg, 0.79 mmol) and
DIEA (0.12 mL, 1.5 mmol). After the usual workup, the product
13c isolated was found to be a 1:1 mixture of both diastereo-
mers (Fmoc-Phe-ψ[CH2NHCSNH]-(R and S )-(þ)-phenylethy-
lamine): Rt (HPLC) 17.8 and 19.2 min.
(S)-Methyl-3-(3-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-
methylpentyl)thio ureido)propanoate (N-Fmoc-Leu-ψ[CH2-
NHCSNH]-β-Ala-OMe) (10c): yield 74%; Rf (30% EtOAc:hexane)
D
1
0.32; [R]23 -12.4 (c 1.0, CHCl3); H NMR (CDCl3) δ 0.92
(d, J=6.8 Hz, 6H), 1.37 (m, 2H), 1.66 (m, 1H), 2.58 (br, 2H), 3.58
(d, 3H), 3.65-3.80 (m, 4H), 4.02 (br, 1H), 4.19 (t, J=6.8 Hz,
1H), 4.35 (m, 2H), 5.21 (br, 1H), 6.96 (br, 2H), 7.22-7.76
(m, 8H); 13C NMR (CDCl3) δ 22.4, 23.5, 25.3, 34.0, 42.3, 44.4,
47.6, 50.2, 52.3, 53.9, 67.4, 120.5, 125.6, 127.6, 128.2, 141.7,
144.2, 157.8, 173.5, 183.2; HRMS calcd for C26H33N3O4S m/z
506.2089 (Mþ þ Na), found 506.2112.
Acknowledgment. We thank the Department of Science
and Technology (grant No. SR/S1/OC-26/2008) and Council
of Scientific and Industrial Research, Government of India,
for financial support. We also thank the Departments of
Sophisticated Instrument Facility, Organic Chemistry, Inor-
ganic & Physical Chemistry, I.I.Sc., Bangalore, for recording
NMR and mass spectra and thank Professor H. Suryaprakash
Rao of Pondichery University, Pondichery, Dr. S. Chandra-
sekhar of IICT, Hyderabad and Dr. T. Narasimhaswamy of
CLRI, Chennai, India for useful assistance.
Test for Racemization. To a solution of isothiocyanate 2f
(300 mg, 0.72 mmol) in dry CH2Cl2 (5.0 mL) at 0 °C was added
(R)-1-phenylethylamine (95 mg, 0.79 mmol) followed by DIEA
(0.12 mL, 1.5 mmol) and the reaction mixture was stirred
for 2.5 h, then it was diluted with CH2Cl2 (10 mL), washed with
5% citric acid (10 mL), water (2 ꢀ 10 mL), and brine (10 mL),
dried over anhydrous sodium sulfate, and concentrated under
reduced pressure to afford the product as a single diastereomer
of the thiourea. (S)-(9H-fluoren-9-yl)methyl (S)-3-phenyl-1-(3-
((R)-1-phenylethyl)thioureido)propan-2-ylcarbamate (Fmoc-Phe-ψ-
[CH2NHCSNH]-(R)-1-phenethylamine) (13a): yield 72%;
Supporting Information Available: General experimental
procedure for N-protected amino alkyl amines and the product
characterization data of all the compounds discussed along with
copies of 1H NMR, 13C NMR, and mass spectra for 4d-g, 4i,
4k-m, 5d-e, 5g, 5k-m, 1a-c, 1e, 1g, 1k, 1m, 2a-m, 10a-g,
13a-c, 8a-f, 11a-d, 15a,b, 9a-f, 12a-e, 14a,b, and CIFs of
compounds 9a and 9c. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
Rt (HPLC) 17.8 min; H NMR (CDCl3) δ 1.39 (d, J=6.0 Hz,
3H), 2.75 (m, 2H), 3.51 (m, 1H), 3.80 (m, 1H), 4.17 (t, J=6.9 Hz,
1H), 4.31 (t, J=7.2 Hz, 2H), 5.23 (m, 1H), 6.06 (br, 2H), 7.08
(d, J=6.9 Hz, 2H), 7.26-7.43 (m, 14H), 7.53 (t, J=7.2 Hz, 2H),
7.76 (d, J = 7.2 Hz, 2H). When the experiment was repeated
with 2f and (S)-1-phenylethylamine, the other diastereomer
5266 J. Org. Chem. Vol. 74, No. 15, 2009