Amino acid bromides: Their N-protection and use in the synthesis of peptides with extremely difficult sequences

Article abstract of DOI:10.1021/jo020280w

N^α-Protected α-amino acid bromides were easily generated in situ with 1-bromo-N,N-2-trimethyl-1-propenylamine from the corresponding amino acids under very mild conditions. o-Nbs and the azido moieties proved to be compatible with these overactivated halides and were successfully applied in difficult peptide bond formations. N-Deprotection methods and the total step-by-step solution synthesis of a peptide containing up to seven consecutive L-(αMe)Valine residues are also reported. The assembly of this homopeptide was achieved in a short time and in very high yields by the azido/bromide system in a single repetitive operation.

Full text of DOI:10.1021/jo020280w

Am in o Acid Br om id es: Th eir N-P r otection a n d Use in th e
Syn th esis of P ep tid es w ith Extr em ely Difficu lt Sequ en ces
Alma DalPozzo,*,† Minghong Ni,† Laura Muzi,† Andrea Caporale,† Roberto de Castiglione,†
Bernard Kaptein,‡ Quirinus B. Broxterman,‡ and Fernando Formaggio§
G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo 81, 20133 Milano, Italy,
Department of Fine Chemicals, Advanced Synthesis and Catalysis, DSM Research, P.O. Box 18,
6160 MD Geleen, The Netherlands, and Department of Organic Chemistry, University of Padova,
Institute of Biomolecular Chemistry, CNR, 35131 Padova, Italy
dalpozzo@ronzoni.it
Received April 19, 2002
NR-Protected R-amino acid bromides were easily generated in situ with 1-bromo-N,N-2-trimethyl-
1-propenylamine from the corresponding amino acids under very mild conditions. o-Nbs and the
azido moieties proved to be compatible with these overactivated halides and were successfully
applied in difficult peptide bond formations. N-Deprotection methods and the total step-by-step
solution synthesis of a peptide containing up to seven consecutive L-(RMe)Valine residues are also
reported. The assembly of this homopeptide was achieved in a short time and in very high yields
by the azido/bromide system in a single repetitive operation.
In tr od u ction
the in situ acylation, affording the amide bond formation
usually in a few minutes and in all cases without a loss
of chirality at the carboxylic site. This method allowed
us to introduce, for the first time, â-fluorine-substituted
R-amino acids at the C-terminus of peptides in excellent
yields.
The present work is aimed at (i) exploring alternative
NR-protecting groups, compatible with AA-Br, and (ii)
testing the performance of the acyl bromide method
under the repetitive coupling/deprotection steps normally
required for peptide synthesis.
As for the first target, it is worth reminding that, as
compared to chlorides and fluorides,5 AA-Brs undergo
very fast cyclization to Leuch’s anhydrides when NR-
protected as carbamates (e.g., with the Boc, Cbz, or Fmoc
group). NR-Protecting groups stable enough to survive to
an AA-Br generation are usually difficult to remove and
not generally suitable for peptide synthesis.
This is the case with phthaloyl, and also with tosyl-
and Pmc-sulfonamide groups.6 Therefore, to improve the
applicability of the acyl bromide method, there is a need
for NR-protecting groups that are unable to take part in
the cyclization and are removable under mild conditions.
In this paper, we report the successful application of
both o-Nbs7 and N38 as N-protecting groups in a difficult
peptide bond formation involving the (R-Tfm)Phe9 residue
Serious difficulties may be encountered during the
synthesis of peptides containing unusual R-amino acids,
the main problems arising from the incorporation of
sterically hindered or poorly electro- or nucleophilic
residues into the peptide chain. Even strongly activated
amino acid chlorides or fluorides fail to give condensation
with some extremely hindered CR-tetrasubstituted or NR-
substituted amino acids. This is also the case with
R-fluoroalkylamino acids, molecules in which researchers
have shown much biological interest,1,2 where, in addi-
tion, the nucleophilicity of the R-amino group is dramati-
cally decreased by the electron-withdrawing effect of the
R-trifluoromethyl substituent.
Recently, we described a new activating method for the
synthesis of particularly difficult peptide bonds,3 using
NR-phthaloyl-amino acid bromides as acylating agents.
Bromides were obtained from the corresponding NR-
phthaloyl amino acids by the readily available bromi-
nating reagent proposed by Ghosez.4 Amino acid bro-
mides (AA-Br), which had never been used before in
chemical practice, proved to be very efficient reagents for
* To whom correspondence should be addressed. Phone: +39-02-
70600223. Fax: +39-02-70641634.
† Ronzoni Institute.
‡ DSM Research.
§ University of Padova.
(1) Ojima, I. In Organofluorine Compounds in Medicinal Chemistry
and Biochemical Applications; Filler, R., Ed.; Elsevier: Oxford, 1993;
pp 241-273.
(2) Koksch, B.; Sewald, N.; J akubke, H.-D.; Burger, K. In Biochemi-
cal Frontiers of Fluorine Chemistry; Ojima, I., McCarthy, J . R., Welch,
J . T., Eds.; ACS Symposium Series 639; American Chemical Society:
Washington, DC, 1996; pp 42-58.
(3) DalPozzo, A.; Bergonzi, R.; Ni, M.-H. Tetrahedron Lett. 2001,
42, 3925-3927.
(4) Devos, A.; Remion, J .; Frisque-Hesbain, A.; Colens, A.; Ghosez,
L. J . Chem. Soc., Chem. Commun. 1979, 1180-1181.
(5) Carpino, L. A.; Beyrmann, M.; Wenschuh, H.; Bienert, M. Acc.
Chem. Res. 1996, 29, 268-274.
(6) DalPozzo, A.; Bergonzi, R.; Ni, M.-H.; Cossettini, P. In Peptides:
The Wave of the Future; Lebl, M., Houghten, R. A., Eds.; Kluwer:
Dordrecht, The Netherlands, 2002; pp 93-94.
(7) Fukuyama, T.; J ow, C. K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6573-6574.
(8) Zaloom, J .; Roberts, D. C. J . Org. Chem. 1981, 46, 5173-5176.
(9) Sewald, N.; Burger, K. In Fluorine-Containing Amino Acids;
Kukhar, V. P., Soloshonok, V. A., Eds.; Wiley: New York, 1995; pp
130-220.
10.1021/jo020280w CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/14/2002
6372
J . Org. Chem. 2002, 67, 6372-6375

Amino Acid Bromides: N-Protection and Use in Peptide Synthesis
SCHEME 1
as a nucleophile. Indeed, the amino function of this amino
In Schemes 1 and 2, alternative methods for N-
acid is known to be extremely poorly reactive because of
the steric hindrance and the polarizing effect of the
trifluoromethyl group.
protection of AA-Br and deprotection of the products are
illustrated. The o-Nbs group is stable to both strongly
acidic and basic conditions but easily removed upon
treatment with potassium thiophenolate. The alkylazido
group is stable under basic conditions, but it can be
removed by two different methods: either by treatment
with Ph3P/H2O (Scheme 1) or by catalytic transfer
hydrogenation with HCOONH4 and Pd/C (Scheme 2b).
Under the latter conditions, the N3 function is converted
to NH2 in a few minutes with a 90% average yield,
independently of the length of the peptide.
As far as the second target of this work is concerned,
we chose to test the efficacy of the acyl bromide activation
method, in combination with the azido N-protecting
group, in the step-by-step solution synthesis of an
L-(RMe)Val (CR-methylvaline) homopeptide series. Pep-
tides based on CR-tetrasubstituted amino acids are
interesting because they show a great tendency to adopt
well-defined, rigid three-dimensional structures that can
be exploited as spacers or templates in bioorganic chem-
istry. In particular, L-(RMe)Val homopeptides form very
stiff â-turn or 310-helices depending on the number of
residues.10 However, the synthesis of these peptide mo-
lecular rulers is very difficult, as (RMe)Val is one of the
most sterically demanding â-branched tetrasubstituted
R-amino acids. Here, we compare the results obtained by
means of the azido/bromide method with those reported
for the same sequence, -[L-(RMe)Val]n- (n ) 2-8),11
prepared by using Cbz-(RMe)Val-F as the stepwise acy-
lating agent.
In Scheme 1, the synthesis of the model diastereomeric
dipeptide 2, obtained from two differently NR-protected
AA-Brs, is described. Due to the poorly reactive amino
function of (R-Tfm)Phe in peptide bond formation, a 5-fold
excess of the AA-Br was necessary for complete acylation.
To favor the coupling reaction over the competing de-
composition of the acyl bromide, we found it more
convenient to add the reagent solution in two portions.
In this way, we could obtain the protected dipeptides 1a
and 1b in about 90% yield after purification.
As expected, the coupling involving (RMe)Val residues
(Scheme 2) proved to be much easier as compared to that
observed for the (R-Tfm)-substituted amino acid: lower
amounts of acylating reagent, 2-azido-2,3-dimethylbu-
tyric acid (N3-DMB) bromide, and shorter reaction times
were required. A homopeptide containing seven residues
of L-(RMe)Val was prepared, each step giving good yields
in times ranging from a few minutes to 1 h (Table 1).
The reaction rates and yields tend to decrease when the
length of the oligomer increases. The improvement of this
synthetic procedure over the acylation via NR-Cbz-amino
acid fluoride, previously used for the same peptide,10,11
Resu lts a n d Discu ssion
The synthesis of NR-protected AA-Br was achieved by
simple addition of the corresponding N-protected AA to
a solution of the bromoenamine, as described in the
Experimental Section.
(10) Polese, A.; Formaggio, F.; Crisma, M.; Valle, G.; Toniolo, C.;
Bonora, G. M.; Broxterman, Q. B.; Kamphuis, J . Chem. Eur. J . 1996,
2, 1104-1111.
(11) Rainaldi, M. Chemistry Doctor Degree Thesis, , University of
Padova, Padova, Italy, 1999.
J . Org. Chem, Vol. 67, No. 18, 2002 6373

DalPozzo et al.
SCHEME 2
TABLE 1. Rea ction Con d ition s for th e Syn th esis of
N3-Dm b-[L-(rMe)Va l]n -OtBu (n ) 1-7 for 4a -g,
r esp ectively) P ep tid es
equiv of
N3-DMB
reaction time
(min)
yield
(%)
entry
4a
4b
4c
4d
4e
4f
1
1
2
2
2
3
3
10
10
20
20
60
98.6
90.2
99.0
86.9
76.8
68.5
67.0
120
120
4g
is dramatic. As a matter of fact, the reaction via Cbz/
acyl fluoride was very sluggish, each coupling taking 1
week on the average, with modest yields (total yield for
the final peptide) equaling about 5%.
Another great advantage of the acyl bromide method
comes from the in situ generation of the acylating species,
thus favoring a rapid step-by-step solution-phase peptide
assembly. Indeed, in most cases, the isobutyramide
formed from the bromoenamine reagent is easily removed
by simple filtration through silicagel at the end of the
coupling reaction. On the contrary, R-amino acid fluorides
need to be isolated before use to achieve satisfactory
results.
F IGURE 1. FT-IR absorption spectra in the conformationally
informative region 3500-3200 cm-1 for the N3-DMB-[L-(RMe)-
Val]n-OtBu (n ) 1-7 for 4a -g, respectively) homopeptide
series in CDCl3 solution. Peptide concentration: 0.1 mM.
decreases. This behavior clearly indicates that the â-turn
type of folding, already observed at the level of the
tetrapeptide (n ) 3 in Figure 1), evolves into a 310-helical
structure in the longest oligomers.12
Con clu sion s
The N3 group at the N-terminus, besides preventing
the aminoacyl bromide intramolecular cyclization, en-
dows the intermediate and final oligomers with higher
solubility as compared to the Cbz-protected analogues.
Moreover, this group does not interfere with the tendency
of L-(RMe)Val homopeptides to adopt turn/helical confor-
mations, as clearly shown in Figure 1. As the peptide
main-chain length increases, the relative intensities of
the IR band associated with the hydrogen-bonded amide
N-H stretching modes (below 3400 cm-1) grow and,
concomitantly, the frequency of the absorption maximum
R-Amino acid bromides, when suitably NR-protected,
have been shown to be superior reagents for the solution
synthesis of difficult peptide sequences. Because of the
mild conditions involved, the bromides can be generated
in situ and directly used for coupling, thus avoiding the
disadvantages associated with their isolation and puri-
fication.
(12) Toniolo, C.; Bonora, G. M.; Barone, V.; Bavoso, A.; Benedetti,
E.; Di Blasio, B.; Grimaldi, P.; Lelj, F.; Pavone, V.; Pedone, C.
Macromolecules 1985, 18, 895-902.
6374 J . Org. Chem., Vol. 67, No. 18, 2002

Amino Acid Bromides: N-Protection and Use in Peptide Synthesis
mg, 0.672 mmol), under Ar. After 10 min, the substrate
The different protecting groups used in combination
with amino acid bromides provide enough orthogonality
for application to the synthesis of a variety of peptide
sequences. In particular, R-azido acids appear to be ideal
substrates for conversion to the corresponding acyl
bromides and their subsequent utilization in the coupling
reactions.
The successful results reported here suggest that this
new coupling procedure is not restricted to special
situations, but rather should find extensive application
in current peptide synthesis. Moreover, it might be
applied to other interesting classes of peptidomimetics
hitherto practically unavailable.
disappeared (TLC, 1:1 hexanes-EtOAc). The solvent was
evaporated and the residue redissolved in 30 mL of EtOAc and
washed with water (2 × 20 mL) and brine (20 mL). After
evaporation of the solvent, the residue was purified by flash
chomatography (99:1 CHCl3-MeOH), affording 54.5 mg of the
pure NR-deprotected peptide 2 (yield 80%). Dep r otection of
1b: After flash chromatography (80:20 hexanes-EtOAc),
2-azido-3-phenylpropionyl-(R,S)-(R-Tfm)Phe-OEt was obtained
as a yellowish syrup (yield 90%). Compound 1b (100 mg, 0.23
mmol) was dissolved in 1.5 mL of THF; Ph3P (181 mg, 0.69
mmol) in 1.5 mL of THF was slowly added and the mixture
left under stirring at room temperature overnight. Then, 150
µL of water was added and the solution refluxed for 8 h. After
evaporation of the solvent, the residue was purified by flash
chromatography (60:40 hexanes-EtOAc) giving 2 (yield 85%).
2 (Dia ster eom er ic Mixtu r e): RP-HPLC (50% CH3CN in
water + 0.1% TFA); diastereomer I, Rt 12.89 min; diastereomer
II, Rt 13.62 min.; 1H NMR (CDCl3) δ 7.30-6.90 (m), 4.44-
4.25 (m), 4.15 and 3.48 (2 dd, J ) 12.87 and 11.38), 3.61-3.55
(m), 3.30, 2.68 and 2.40 (dd, J ) 9.54, 13.79), 1.34 (2t); 19F
NMR (CDCl3, TFA) δ 1.59 and 1.57. Anal. Calcd. for C21H23F3O3‚
H2O: C, 59.15; H, 5.86; N, 6.57; F; 13.38. Found: C, 60.37; H,
5.89; N, 6.52; F, 13.18.
Exp er im en ta l Section
Gen er a l. DCM was distilled over P2O5 and stored under
argon. Optically pure L-(RMe)Val was prepared according to
a published chemoenzymatic method13 by DSM Research.
Thin-layer chromatography (TLC) was routinely carried out
to monitor reactions, and the products were located with UV
light (254 nm), ninhydrin, or KMnO4 according to their
chemical structure. Analytical liquid chromatography (RP-
HPLC) was carried out with a Ultrasphere ODS (5µ, 10 × 250)
Beckmann column. H-(R,S)-(R-Tfm)Phe-OEt was synthesized
following published methods.14
N3-DMB-L-(RMe)Va l-OtBu (4a ). To a solution of HCl‚H-
L-(RMe)Val-OtBu (3a )10 (295 mg, 1.32 mmol) and collidine (525
µL, 3.96 mmol) in 6 mL of DCM, at 0 °C, was added 2.5 mL of
the N3-DMB bromide solution (1.32 mmol). After 10 min at
room temperature, the substrate disappeared (TLC, 1:1 hex-
anes-EtOAc). After evaporation of the solvent, 25 mL of 5%
NaHCO3 and 12 mL of THF were added to the residue and
the mixture was stirred for 30 min. After evaporation of THF,
the aqueous solution was extracted with DCM and the
combined organic phases were washed with water, 1 N HCl,
and water to neutrality. The solvent was dried and evaporated,
and the crude residue was dissolved in 70:30 hexanes-EtOAc
and filtered through silica gel. The title compound 4a was
obtained as a colorless oil (yield 98.6%): [R]2D0 -17.0° (c 0.3,
MeOH); IR (KBr) 3360, 2113, 1713, 1669, 1514 cm-1; 1H NMR
(CDCl3) δ 7.13 (s), 2.22 and 2.16 (2 m), 1.54 and 1.51 (2s), 1.47
(s), 1.00 and 0.92 (2 m).
o-Nbs-Phe-OH was prepared from H-Phe-OtBu and o-
nitrobenzene-sulfonyl chloride by the method described by
Fukuyama et al.,7 followed by treatment with TFA. R-Azido
acids were synthesized by diazotransfer reaction from the
corresponding R-amino acids as described by Alper et al.15
1-Bromo-N,N-2-trimethyl-1-propenylamine was obtained by
the method of Ghosez et al.16 The reagent can be stored in
vials containing an approximately 0.5 M solution in DCM,
under Ar, at -18 °C, for several months. The title of the
solution was determined before use by converting Pht-Phe-
OH (Pht, phthaloyl) into its bromide and quenching with dry
methanol: disappearance of the acid and appearance of the
methyl ester was monitored by TLC, under UV light. NR-
protected amino acid bromides: in a typical procedure 1.15
mmol of amino acid was dissolved into 2.4 mL of a 0.5 M
solution of bromoenamine in DCM and stirred under Ar for
15 min. The solution of the bromide was used immediately
for acylation of the desired R-amino acid ester or peptide.
H-P h e-(R,S)-(R-Tfm )P h e-OEt (2). A solution of H-(R,S)-
(R-Tfm)Phe-OEt14 (100 mg, 0.383 mmol) and collidine (50 µL,
0.383 mmol) in 1 mL of DCM at 0 °C was treated with the
desired acyl bromide solution (1.15 mmol of the in situ, freshly
prepared o-Nbs-Phe-Br or 2-azido-3-phenylpropionyl bromide)
and, after 10 min, with an additional amount (0.766 mmol) of
the bromide solution. After 3 h at room temperature, the
substrate disappeared (monitoring by HPLC). The solvent was
evaporated and the residue redissolved in 60 mL of a 1:2
mixture of THF-5% NaHCO3 and stirred for 20 min. After
removal of THF, the aqueous solution was extracted with DCM
(1 × 40 mL, 2 × 20 mL). The combined organic phases were
washed with water (10 mL), 1 N HCl (10 mL), and water and
then evaporated to dryness. Dep r otection of 1a : After flash
chromatography (75:25 hexanes-EtOAc), o-Nbs-Phe-(R,S)-(R-
Tfm)Phe-OEt was obtained as a thick oil (yield 92%). Com-
pound 1a (100 mg, 0.168 mmol) was dissolved in 1 mL of DMF
containing thiophenol (35 µL, 0.336 mmol) and K2CO3 (92.8
N3-DMB-[L-(RMe)Va l]n -OtBu (n ) 2-7; 4b-g). To a
solution of 4a (1.3 mmol) in 7 mL of MeOH were added 5.2
mmol of ammonium formate and 250 mg of 10% Pd/C, and
the mixture was stirred for 30 min at rt. The reaction mixture
was filtered through Celite (2.5 g) and the filtrate evaporated
to dryness; the residue was redissolved in 30 mL of DCM and
the solution washed with brine. The organic phase was dried
and evaporated affording 354.7 mg of the dipeptide H-[L-(RMe)-
Val]2-OtBu (3b) (97%), which was immediately coupled to N3-
DMB bromide, as described above for 4a , to give 4b. Further
iterative synthetic steps to build the azido-peptide 4g were
performed using the same procedure (Scheme 2b). When
required, the intermediates were purified by flash chroma-
tography. 4g: mp 239-240 °C; [R]2D0 +6.3° (c 0.3, MeOH); IR
1
(KBr) 3334, 2106, 1727, 1660, 1520 cm-1; H NMR (CDCl3) δ
7.70, 7.27, 7.22, 7.15, 6.95 and 6.21 (s), 2.40-1.70 (m), 1.62-
1.20 (m), 1.07-0.83 (m); 13C NMR (CDCl3) δ 173.58, 173.17,
173.16, 172.72, 172.39, 172.23, 171.23, 170.23, 79.92, 70.47,
63.60, 63.46, 63.43, 63.38, 62.51, 62.44, 37.10, 36.32, 36.11,
35.91, 35.90, 35.63, 33.84, 27.91, 20.29, 19.27, 19.24, 19.18,
18.17, 17.81, 17.75, 17.77, 17.59, 17.56, 17.54, 17.50, 17.42,
17.37, 17.27, 17.14, 17.12, 17.08, 17.06, 17.04, 16.86; MS calcd
for (M + Na+) 1028, found 1028; MS calcd for (M + K+) 1044,
found 1044.
(13) Sonke, T.; Kaptein, B.; Boesten, W. H. J .; Broxterman, Q. B.;
Kamphuis, J .; Formaggio, F.; Toniolo, C.; Rutjes, F. P. J . T.; Schoe-
maker, H. E. In Stereoselective Biocatalysis; Patel, R. N., Ed.; Dekker:
New York, 2000; pp 23-58.
(14) Burger, K.; Gaa, K. Chem. Zeit. 1990, 114, 101-104.
(15) Alper, P. B.; Hung, S. C.; Wong, C. H. Tetrahedron Lett. 1996,
37, 6029-6032
(16) Haveaux, B.; Dekoker, A.; Rens, M.; Sidani, A. R.; Toye, J .;
Ghosez, L. Org. Synth. 1980, 59, 26-34.
Ack n ow led gm en t. The authors are indebted to
Prof. Claudio Toniolo (University of Padova) for helpful
suggestions and discussions.
J O020280W
J . Org. Chem, Vol. 67, No. 18, 2002 6375