A convenient synthesis of N-Fmoc-N,N′-bis-Boc-7-guanyl-1,2,3,4-tetrahydro-isoquinoline-3- carboxylic acid (Fmoc-N,N′-bis-Boc-7-guanyl-Tic-OH, GTIC)

Article abstract of DOI:10.1016/S0040-4039(01)00491-9

Fmoc-N,N′-Bis-Boc-7-guanyl-Tic-OH (GTIC), a conformationally constrained amino acid with basic properties, has been synthesized in four steps. This amino acid can be incorporated into peptides using standard Fmoc solid phase synthesis, and to test its potential for biological activity applications, we prepared an analog of H-Dmt-Tic-NH₂.

Full text of DOI:10.1016/S0040-4039(01)00491-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 3507–3509
A convenient synthesis of
N-Fmoc-N,N%-bis-Boc-7-guanyl-1,2,3,4-tetrahydro-isoquinoline-3-c
arboxylic acid (Fmoc-N,N%-bis-Boc-7-guanyl-Tic-OH, GTIC)
Vincenzo Santagada,a,* Ferdinando Fiorino,a Beatrice Severino,a Severo Salvadori,b
Lawrence H. Lazarus,c Sharon D. Bryantc and Giuseppe Caliendoa
aDipartimento di Chimica Farmaceutica e Tossicologica, Universita` di Napoli, ‘Federico II’ Via D. Montesano, 49,
80131 Napoli, Italy
bDipartimento di Scienze Farmaceutiche, Universita` di Ferrara, via Fossato di Mortara, I-4100 Ferrara, Italy
cPeptide Neurochemistry, LCBRA, National Institutes of Environmental Health Science, Research Triangle Park,
NC 27709, USA
Received 15 January 2001; revised 14 March 2001; accepted 21 March 2001
Abstract—Fmoc-N,N%-Bis-Boc-7-guanyl-Tic-OH (GTIC), a conformationally constrained amino acid with basic properties, has
been synthesized in four steps. This amino acid can be incorporated into peptides using standard Fmoc solid phase synthesis, and
to test its potential for biological activity applications, we prepared an analog of H-Dmt-Tic-NH2. © 2001 Elsevier Science Ltd.
All rights reserved.
Providing conformational constraints is
a
major
the physical and pharmacological properties of bioac-
tive peptides and peptidomimetics including improve-
ment in their affinities and selectivities for biological
receptors/acceptors.1 Constrained amino acids have
been introduced into the sequences of bioactive pep-
approach to the modification of the chemical and bio-
logical properties of endogenous bioactive peptides,
hormones and neurotransmitters. Much evidence indi-
cates that such modifications can improve significantly
Scheme 1. Synthesis of GTIC.
Keywords: unconventional amino acid; synthesis; GTIC.
* Corresponding author. Tel.: 0039-81-678648; fax: 0039-81-678649; e-mail: santagad@unina.it
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00491-9

3508
V. Santagada et al. / Tetrahedron Letters 42 (2001) 3507–3509
tides in order to provide local constraints. Incorpora-
tion of these amino acids specifically restricts the rota-
tion of the NaꢀCa, CaꢀC(O), C(O)ꢀNH bonds, and side
chain conformations by covalent or noncovalent steric
interactions. Therefore, the design and synthesis of
conformationally constrained amino acids, bringing
selective chemical functions into their structure, may
provide a unique approach to obtain new insights into
the stereochemical, conformational and topographical
requirements of peptide ligand–receptor interactions
and for signal transduction that are not possible with
the 20 genetically coded (natural) residues. The cycliza-
tion between N and Cd of Phe and Tyr has led to highly
constrained amino acids such as 1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid (Tic) and 7-
hydroxy - 1,2,3,4 - tetrahydroisoquinoline - 3 - carboxylic
acid (HO–Tic). These structural analogues of the natu-
rally occurring amino acids phenylalanine and tyrosine
exhibit a diverse range of effects when introduced into
biological systems. In fact, Tic has been incorporated as
a phenylalanine replacement in many biologically active
peptides (e.g. opioid,2 substance P,3 FTase inhibitors,4
bradykinin,5 etc.).
endowed with potent and selective d opioid antagonist
activity.2c We were interested to test if a guanidino
function in the 7-position of 1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic acid (Tic) could modify the
opioid receptor occupation and activation. We there-
12
fore prepared the dipeptide, H-Dmt-GTIC-NH2 and
tested it for m and d receptor binding.2c
The result of the binding assay showed that GTIC
instead of Tic drastically modified the receptor affinity
and selectivity. The affinity to the d opioid receptor (Ki,
nM=26.0) is 213-fold lower than the reference com-
2c
pound H-Dmt-Tic-NH2, while binding to the m site
(Ki, nM=10.8) is 26-fold better than the same refer-
ence compound.
This result means that a guanidino function, as a
substituent in the 7 position of the aromatic ring of Tic,
induces a change of receptor affinity and selectivity.
In conclusion the Fmoc-N,N%-bis-Boc-7-guanyl-Tic-OH
(GTIC), a conformationally constrained amino acid,
will serve as a useful tool in peptide-based structure–
activity studies.
In this paper we describe a facile synthesis of Fmoc-
N,N%-bis-Boc-7-guanyl-Tic-OH, (GTIC), a new Tic
derivative substituted on the aromatic ring with a basic
guanidine group. This amino acid combines the basic
features of arginine with the aromatic features of
phenylalanine.
References
1. (a) Hruby, V. J. Biopolymers 1993, 31, 1073–1082; (b)
Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich,
M. Biopolymers 1997, 43, 219–266.
The synthetic procedure, summarized in Scheme 1,
2. (a) Schiller, P. W.; Weltrowska, T. M.; Nguyen, D.;
Lemieux, C.; Chung, N. N.; Marsden, B. J.; Wilkes, B. C.
J. Med. Chem. 1991, 34, 3125–3132; (b) Temussi, P. A.;
Salvadori, S.; Amodeo, P.; Bianchi, C.; Guerrini, R.;
Tomatis, R.; Lazarus, L. H.; Picone, D.; Tancredi, T.
Biochem. Biophys. Res. Commun. 1994, 198, 933–939; (c)
Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.;
Bryant, S. D.; Crescenzi, O.; Guerrini, R.; Picone, D.;
Tancredi, T.; Temussi, P. A.; Lazarus, L. H. Mol. Med.
1995, 1, 678–689; (d) Tancredi, T.; Salvadori, S.;
Amodeo, P.; Picone, D.; Lazarus, L. H.; Bryant, S. D.;
Guerrini, R.; Marzola, G.; Temussi, P. A. Eur. J.
Biochem. 1994, 224, 241–247.
3. Caliendo, G.; Calignano, A.; Grieco, P.; Mancuso, F.;
Perissutti, E.; Santini, A.; Santagada, V. Biopolymers
1995, 36, 409.
4. Caliendo, G.; Fiorino, F.; Greco, P.; Perissutti, E.; De
Luca, S.; Giuliano, A.; Santelli, G.; Severino, B.; Santa-
gada, V. Il Farmaco 1999, 54, 785.
started from commercially available
L-1,2,3,4-tetra-
hydro-3-isoquinolinecarboxylic acid (Tic, 1) which was
treated with fuming nitric acid and concentrated sulfu-
ric acid at −10°C to obtain the 7-nitro derivative (2) in
a mixture with the corresponding 6-nitro isomer in a
yield of over 90%.6 The direct nitration was very
regioselective, giving the 7-nitro-Tic isomer in over 90%
yield. The position of the nitro group was established
using NMR methods.
Reduction of the nitro compound to the amine (3) was
straightforward (yield 98%), although the final product
had to be protected from air to prevent oxidation.7
Next, the Fmoc- protection on the a-amino group of
the 7-amino Tic-OH (4),8 was introduced by treatment
with Fmoc–OSu in aqueous sodium carbonate. Under
these conditions, the amino group on the ring did not
react. The yield of this procedure was modest (63%),
presumably because of competition between the protec-
tion reaction and breakdown of the Fmoc- group by
the secondary amine followed by trapping of the amine
by dibenzofulvene.9 The amino group was converted
into the corresponding guanidine moiety (5) by reaction
with N,N%-bis-Boc-S-methyl-isothiourea, HgCl2 and
TEA in DMF.10 The product was purified using silica
gel chromatography and diethyl ether/hexane (yield
5. Guba, W.; Haessner, R.; Breiphol, G.; Henke, S.; Knolle,
J.; Santagada, V.; Kessler, H. J. Am. Chem. Soc. 1994,
116, 7532–7540.
6. Procedure for the synthesis of 2: (S)-(−)-1,2,3,4-tetra-
hydro-3-isoquinolinecarboxylic acid (2.5 g, 14.1 mmol)
was added slowly to 7.5 ml of concentrated sulfuric acid
maintained at a temperature of −10°C; 1.8 ml of concen-
trated nitric acid was then added dropwise. The reaction
mixture was stirred at this temperature for 3.5 h then
poured into 75 ml ice water. The product was precipi-
tated by neutralization with ammonium hydroxide,
filtered, washed with water, then dried to give a brown
solid (3 g). 1H NMR (D2O+DCl, 500 MHz): 3.10 (m,
1
58%).11 All new compounds were characterized by H
NMR and MS.
Recently, we reported that the di- and tripeptides of the
structure Tyr/Dmt-Tic-R (R=H, NH2, Ala) were

V. Santagada et al. / Tetrahedron Letters 42 (2001) 3507–3509
3509
1H), 3.3 (m, 1H), 4.30 (m, 3H), 7.4 (m, 1H), 7.90 (m, 2H);
ESI: 223 (MH+); TLC (CHCl3/MeOH/H2O/HOAc: 6/4/2/
2) Rf=0.70; mp=277–278°C; yield: 90%.
amino-Tic-OH) (1.1 g 0.0027 mol) was dissolved in DMF
(40 ml). The mixture was cooled to 0°C, treated with
N,N%-bis-Boc-S-methyl-isothiourea (0.9 g, 0.0031 mol)
and HgCl2 (1.41 g 0.0052 mol), then after 10 minutes,
TEA (1.1 ml) was added. The reaction was warmed to
room temperature and after 2 hours the mixture was
filtered, the organic phase concentrated and loaded onto
a silica gel column. The column was eluted with diethyl
ether/hexane=6:4 yielding 1 g of N-Fmoc-N,N%-bis-Boc-
7 - guanyl - 1,2,3,4 - tetrahydro - 3 - isoquinolinecarboxylic
acid.
7. Procedure for the synthesis of 3: 7-Nitro-Tic-OH (1.12 g
0.0050 mol) was dissolved in 50 mL methanol. The
solution was degassed with nitrogen and 0.45 g of 10%
Pd/C was added. The reaction mixture was maintained
under a hydrogen balloon for 2 hours at room tempera-
ture. The mixture was then filtered to remove the Pd/C
and finally, the solvent was removed to give a white solid
(0.95 g). 1H NMR (D2O, 500 MHz): 3.21 (m, 1H), 3.4 (m,
1H), 4.40 (m, 3H), 7.20 (m, 1H), 7.5 (m, 2H); ESI: 193
(MH+); TLC (CHCl3/MeOH/H2O/HOAc: 6/4/2/2) Rf=
0.50; mp=220–221°C; yield: 98%.
8. Procedure for the synthesis of 4: 7-Amino-Tic-OH (0.9 g
0.0047 mol) was suspended in 10.5 mL of 9% Na2CO3
and cooled in ice water. A solution of 1.28 g (0.0038 mol)
of Fmoc-OSu in 11.2 mL of dioxane was then added
dropwise and the mixture was stirred at room tempera-
ture for 3 h. The solvent was evaporated, ethyl acetate
was then added and the water phase and organic phases
were separated. The organic phase was evaporated and
the residue loaded onto a silica gel column. The column
was eluted with diethyl ether. Fractions were checked by
TLC using the same solvent mixture for development.
Fractions containing the product were pooled and con-
centrated to obtain a white solid (1.18 g). 1H NMR
(CDCl3 500 MHz): 3.06–3.17 (m, 2H), 4.22–5.12 (m, 6H),
6.46–7.79 (m, 11H, aromatic); EI: 416 (MH+); TLC
(CHCl3/MeOH/H2O/HOAc: 6/4/2/2) Rf=0.85; mp=
159–160°C; yield: 63%.
1H NMR (CDCl3, 500 MHz), 1.50 (s, 9H), 1.54 (s, 9H),
3.10–3.23 (m, 2H), 4.34–4.80 (m, 6H), 7.10–7.79 (m, 11H
aromatic); ESI: 657.6 (MH+), TLC (hexane/diethyl ether,
9:1) Rf=0.85, mp=167–168°C, yield: 58%.
12. Procedure for the synthesis of H-Dmt-GTIC-NH2: The
dipeptide was obtained by standard solution methods. To
a solution of Boc-L-Dmt-OH (0.31 g, 1 mmol) and H-
GTIC-NH2 (0.43, 1 mmol) in DMF (10 mL) at 0°C were
added HOBt (0.18 g, 1.2 mmol), WSC (0.21 g, 1.2 mmol),
and TEA (0.17 mL, 1.2 mmol). The reaction mixture was
stirred for 3 h at 0°C and 24 h at room temperature.
After evaporation of DMF, the residue was solubilized in
EtOAc and washed with citric acid (10%), NaHCO3 (5%),
and brine. The organic phase was dried and evaporated
to dryness. The residue was crystallized from Et2O/
petroleum ether (1:1) to obtain the protected dipeptide
with a yield of 80%. The protected dipeptide (0.52 g, 0.72
mmol) was treated with TFA (2 mL) for 0.5 h at room
temperature. Et2O was added to the solution until the
product precipitated: yield 80%. The crude product was
purified by preparative RP-HPLC. The final compound
TFA·H-Dmt-GTIC-NH2 was characterized by FAB-MS.
The amino acid H-GTIC-NH2 was obtained from Fmoc-
GTIC-OH by reaction with DCC/BtOH·NH3 to obtain
Fmoc-GTIC-NH2 which was successively deprotected by
treatment with 33% diethylamine in THF.
9. The Chemical Synthesis of Peptides; Jones, J., Ed. Claren-
don Press: Oxford 1991.
10. Jones, R. M.; Hjorth, S. A.; Schwartz, T. W.; Portoghese,
P. S. J. Med. Chem. 1998, 41, 4911–4914.
11. Procedure for the synthesis of 5: N-Fmoc-7-amino-
1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (Fmoc-7-
.
.