Oxybenzotriazole free peptide coupling reagents for N-methylated amino acids

Article abstract of DOI:10.1016/0040-4039(91)85015-W

The main product of N-methylated amino acid coupling, when oxybenzotriazole is present in the reagent (BOP, PyBOP, HBPyU) or as an additive (DCC/HOBt), is the weakly reactive benzotriazolyl ester. On the other hand, the corresponding new halogenated reage

Full text of DOI:10.1016/0040-4039(91)85015-W

Tet~ahedron Letters, Vo 32, No.IT pp 1967 1970, 1991
Prixltcd in Greal Britain
0040 4039/91 $3.00 ~Of)
Pergamon Press pie
Oxybenzotriazole Free Peptide Coupling Reagents
for N-Methylated Amino Acids
Jacques Cosle*, Eric Fr~rol, Patrick Jouln
Centre CNRS-INSERM de Pharmacologic Endocrinologie
Rue de la Cardonille, 34094 MonlpellierC~dex05, France
Bertrand Castro
SANOFLChimie, 32, Rue Marbeuf, 75008 Paris, France
Key Words" PeN de; Couplingreagent; N Me hy am no acid; Hydroxybenzotr azole.
Abstract: 7he main producl of N-methylated amino acid coupling, when oxybenzotriazoh> is presem in the
reagent (BOP, PyBOP, IIBPyU) or as an additive (DCC/IIOBt), is the weakly reactive benzotriazolyl ester. On the
other hand, the corresponding new halogenated reagents PyBroP, PyCIoP and PyClU give very good results and
can be recommended
The coupling of N methylated amino acids is a difficult reaction in which the usual reagents are often
inefficient1 5. These amino acids are present in many natural peptides exhibiting important biological
properties, such as cyclosporines3 and some pseudopeptides of marine origin including didemnins6 and
dolastatins7. The latter are currently being synthesized in our laboratory, which led us to test new coupling
reagents.
In a previous study1, we showed that the reagent BroP 28, unlike BOP 19, allows easy coupling of N-
methylated amino acids. This reagent compares favorably with BOP-CI 4,5,10,11 and Dpp-C112 and has the
advantage of being stable and easy to use. However, like BOP, it yields HMPT during the coupling
reaction, whose toxicity (see 13) could make its use impossible.
R = R ' =Ctt 3
Y = O B t
BOP
1
- O
L ~ ' / J3
PF 6
R ,R' = (CII2)4
R, R' = (CII2)4
R, R' = (CH2)4
Y = O B t
Y = Br
Y = C1
PyBOP
PyBroP
PyCloP
3
4
5
OBt
BOP can be favorably replaced by its pyrrolidino homologue, PyBOP® 313, in which case the
reaction produces tripyrrolidino phosphine oxide, an a priori noncarcinogenic by-product. This prompted us
to study the coupling of N methylated amino acids by pyrrolidino homologues of BroP: PyBroP 414 (bromo
tripyrrolidino phosphonium hexafluorophosphate) and PyCloP 514 (chloro tripyrrolidino phosphonium
hexafluorophosphate).
1967

1968
Z-MeVal-VaI-OMe
Z-Val-MeVal OMe
Be,c-Pro MeVal OMe
Z MeVal MeVal OMe
6
7
8
9
Table I ." Coupling reactions a - % yield (% epimerization) b
Peptide
P y B O P
90 (0.2)
26 (_)c
P y B r o P
85 (0.3)
79 (0)
PyCIoP
87 (0.15)
85 (0.2)
H B P y U
87 (0.15)
21(-)c
PyCIU
6
8
9
90 (0.2)
82 (0.15)
< 5 c
6 1 ( < 0 . 3 )
5 9 ( < 0 . 2 )
<5 c
66(-<0.3)
58 (18) d
87 (-~ 0.3) e
64 (13)d
(a) Unless otherwise indicated, DIEA (3 eq) was added to a mixture (cooled to 0°C) of N protected amino acid (1 eq), C-
protected amino acid chlorhydeate (1.1 eq), and coupling reagent (1 eq) in CH2CI2 (1 ml/mmol), and stirred for 1 min cold and
lbr 1 h at room temperature. N-methylated amino acids were obtained &s described in21 . Elemental analysis and 1H-NMR spectra
were consistent with the structures proposed.
(b) Yields after column chromatography. Epimerization percentages were determined in crude products by comparison with the
dipeptide diastereoisomer22 using ttPLC (compounds 6 and 8), and IH NMR (360 MHz) (compound 9).
(c) The main product was the benzotriazolyl ester of the N-protected amino acidt8.
(d) Reaction time: 72 h.
(e) 1.5 eq of N-protected amino acid, 1 eq of C-protected amino acid chlorhydrate, 1.5 eq of PyBroP, and 4 eq of DIEA. Reaction
time 3 h.
Table II : Z-Val-MeVal-OMe 7 - % yield (% epimerization) a
Reaction
time (h)
Py BOP
l lbG)
P y B r o P
PyCloP
85 (-)
H B P y U
12b(-)
PyC1U
57 (0)
DCC/
HOBt
DCC
1
70 (0)
8b(-)
78(-)
100 (0.15)d
24
76 (0) c
96 (0.5)
96 (0.15)
77 (0) c
100 (0.8)
20 (0)c
87 (< 0.1)
(a) Same conditions as (a) in Table 1. In the case of DCC and DCC/HOBt : 1 eq of DIEA. Yield (internal standard) and
epimerization (comparison with dipeptide diastereoisomer22) were determined by HPLC in the reaction mixture.
(b) Main product: benzotriazolyl ester of Z-Val.
(c) A small % of benzotriazolyl ester remained. After total disappearance of this ester, the following yields were obtained:
PyBOP: 81% (48 h); HBPyU : 77% (90 h); DCC/HOBt : 22% (48 h)19.
(d) Excess reagent~ reaction time 3 h (see (e) in Table 1).

1969
The formation of the amide bond between the earboxyl of an N-methylated amino acid and the
primary amine of a C-protected amino acid does not present any particular difficulties: the reagents PyBOP,
PyBroP and PyCIoP showed comparable efficiency in the synthesis of dipeptide 6 (Table 1). In contrast,
when the C-protected amino acid was N-methylated or when both amino acids were N-alkylated
(compounds 7, 8, 9: Tables I and I1), PyBOP gave very low yields whereas PyBroP and PyCloP remained
efficient. With PyBOP, yields were only moderate after 24 h ( 7: 76%) or 72 h ( 9: 58%) and there was even
18% epirnerization in the last case. On the other hand, with the brominated homologue PyBroP 4, yields
were excellent after 24 h of reaction (compound 7: 96%) and even after only 3 h when excess (1.5 eq) N-
protected amino acid and reagent were used (compounds 7: llXI% and 9: 87%). In most cases epimerization
was absent or negligible (_< 0.3%).
" C - - Y
PF 6
Y = OBt
Y = C1
HBPyU
PyC1U
10
1 1
These results are similar to those we previously obtainedl when we compared the reagents BOP and
BroP15. They confirm the hypothesis of a deleterious effect of hydroxybenzotriazole in the coupling of N-
methylated amino acids, which has previously been observed for BOP CI5. To show the generality of this
effect, we compared DCC/HOBt with DCC and HBPyU 10 with PyCIU 11. Compounds 10 (2-(1H
benzotriazol-l-yl)-l,l,3,3-bis(tetramethylene)-uronium
hexafluorophosphate) and 11 (1,1,3,3
bis(tetramethylene)-chlorouronium hexafluorophosphate) are new reagents in the HBTU family16, which
we obtained by classical methods 17. PyBOP, HBPyU and DCC/HOBt showed low efficiency in the
synthesis of dipeptide 7 (Table II). The benzotriazolyl ester of Z-Va118 was formed rapidly and its reactivity
was weak. Even with long reaction times, the yields were unsatisfactory19. In the formation of dipeptides 8
and 9, HBPyU and PyBOP behaved in a similar manner (Table 1). On the other hand, with DCC or PyC1U,
as with PyBroP or PyCloP (i.e. in the absence of hydroxybenzotriazole), the yields of dipeptide 7 were high.
This result was confirmed with PyCIU in the synthesis of compounds 8 and 9 (Table I). Moreover,
comparable results obtained for compound 6 with HBPyU and PyCIU (]~ble 1) confirm that the coupling
reaction easily occurs if only the N-terminal amino acid is N methylated.
To summarize, the weak reactivity of the benzotriazolyl ester which is formed is responsible for tile
deficient coupling of N methylated amino acids carried out in the presence of HOBt or reagents containing
an oxybenzotriazole residue. In contrast, the halogenated reagents PyBroP, PyCIoP, and PyC1U proved to be
efficient in these couplings. Moreover, these compounds are solid, stable, and easy to use. PyBroP has the
advantage of being commercially available20. Lastly, these results raise the problem of the utility of HOBt
in peptide coupling reactions carried out on an amino acid protected in carbamate form.
REFERENCES AND NOTES
1.
2.
3.
Coste, J.; Dufour, M.-N.; Pantaloni, A.; Castro, B. Tetrahedron Lett. 1990, 31,669-672.
Cheung, S.T.; Benoiton, N.L. Can. J. Chem. 1977, 55, 911-915.
Wenger, R.M. Helv. Chim. Acta. 1983, 66, 2672-2702.

1970
4.
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6.
7.
Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107, 4342 4343.
Van der Auwera, C.; Anteunis, M. J. O. Int. J. Peptide Protein Res. 1987, 29, 574-588.
Jouin, P.; Poncet, J.; Dufour, M. N.; Pantaloni, A.; Casia'o, B. J. Org. Chem. 1989, 54, 617-627.
Pettit, G. R.; Kamano, Y.; Herald, C. L.; Dufresne, C.; Bates, R. B.; Schmidt, J. M.; Cerny, R. L.;
Kizu, H. J. Org. Chem. 1990, 55, 2989-2990.
8.
9.
Castro, B.; Dormoy, J.-R. Tetrahedron Lett. 1973, 3243 3246.
Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron Left. 1975, 1219-1222.
10. Diago-Meseguer, J.; Palomo-Coll, A. L.; Fernandez-Lizarbe, J. R.; Zugaza-Bilbao, A. Synthesis
1980, 547-551.
I 1. Colucci, W. J.; Tung, R.D.; Petri, J. A.; Rich, D. A. J. Org. Chem. 1990, 55, 2895-2903.
12. Galpin, I. J.; Mohammed, A. K.; Patel, A. Tetrahedron 1988, 44, 1685-1690.
13. Coste, J.; Le~Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31,205-208.
14. Castro, B.; Coste, J. Fr. Patent 89 02 361.
15. In the formation of compound 7, the difference in yields between BOP (67%)1 and PyBOP (11%)
Ipresent communicationl can be attributed to different experimental conditions. Under the
conditions used here (Table 1), a yield of 10% was obtained with BOP after 1 h of reaction, and the
main product was the benzotriazolyl ester. Compound 7 was then obtained with a 73% yield after
24 h of reaction.
16. a) Dourtoglou, V.; Ziegler, J. C.; Gross, B. Tetrahedron Lett. 1978, 1269-1272.
b) Knott, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927-1930.
17. Compound 10 (F = 2(X/-2°C(dec)) was obtained by the action of HOBt/NEt3 on compound 11. We
designate it as HBPyU to comply with16. Its homologue tetrafluoroborate (TgPyU) was recently
described by Henklein, P.; Beyermann, M.; Biene17, M.; Knott, R. (Poster at the 21st European
Peptide Symposium, Platja d'Aro, Spain, September 2-8, 1990). Compound 11 (F - 151-3°C) was
obtained by the action of POCI3 and then KPF6/H20 on the corresponding urea. We designate it as
PyCIU (Pyrrolidino ChloroUronium) to comply with names used for homologues of the BOP
fanfily. This reagent is not hygroscopic, in contrast with the corresponding chloride (Henklein, P.;
Beyermann, M.; Bienert, M.; Knorr, R. Poster at the 21st European Peptide Symposium, Platja
d'Aro, Spain, September 2-8, 1990). Elemental analysis, Fab MS, and 1H-NMR of compounds 10
and 11 are consistent with the proposed structures.
18. The benzotriazolyl esters were obtained by the action of BOP (1 eq) and DIEA (2 eq) on the N
protected anfino acid, followed by the usual treatment. Although these esters are sensitive to
hydrolysis they were detected by TLC, HPLC and 1H NMR.
19. The very low yield obtained with DCC/HOBt could be due to hydrolysis of the benzotriazolyl ester
by water contained in commercial HOBt.
20. Novabiochem (Switzerland).
21. McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973,51, 1915 1919.
22. The diastereoisomers of compounds 6, 7 and 9 have previously been obtained1; that of compound 8
was synthesized with PyBroP.
(Received in France 22 January 1991)