Download CHAPTER 12 Solid-Phase Synthesis of Peptides Containing the

CHAPTER12
Solid-Phase
Synthesis
of Peptides Containing
the CHzNH Reduced Bond Surrogate
MichaeZ
W. Pennington
1. Introduction
There has been considerable interest in the past decade in the design
and development of competitive peptide agonists and antagonists for
numerous peptide-receptor systems. Systematic side-chain replacement
is often the first step in the design process of higher-affinity ligands.
Modification of the peptide backbone is another step in the design process, but requires more information about the stability and structure of
the peptide. The most common types of backbone alterations are the
incorporation of N-methylated amino acids and/or D amino acids into
the peptide. These changes can help stabilize the peptide against proteases, disrupt or induce secondary structural elements, and eliminate
hydrogen bonds. In addition to these approaches, replacement of peptide bonds by peptide bond surrogates, such as a CH,NH reduced peptide
bond, can also be performed. The initial approach to these type of compounds was exceedingly more difficult to accomplish and required
considerable solution synthesis (1). In this chapter, the protected dipeptide containing the reduced bond was prepared in solution by reductive
alkylation with the protected a-amino aldehyde and subsequently
coupled to the peptide chain. Low coupling yields were often described
when this method was attempted probably because of isosteric components present during the reaction (2).
Edited
From Methods
by: M. W. Pennington
in Molecular
Biology, Vol. 35: PeptIde Synthesis Protocols
and B. M. Dunn Copyright
01994
Humana Press Inc., Totowa,
241
NJ
242
Pennington
H
R
Fig. 1. Reductrve alkylation scheme for the synthesis of CHzNH isostere.
Since this early report, a procedure was developed by Sasaki and Coy
(2) that greatly simplified the entire process by exploiting the solid-phase
resin to eliminate the laborious postreaction work-up steps. This
approach relies on the same overall scheme of reductive alkylation using
N-protected a-amino aldehydes in the presence of NaBH,CN (Fig. 1).
However, the reduced bond is formed in situ on the resin with no need
for subsequent work-up.
This chapter is aimed at introducing this technology to peptide chemists who may have little or no experience working with or preparing
noncommercially available amino acid derivatives. This chapter employs
the same overall strategy as developed by Sasaki and Coy (2) to prepare
the reduced bond isostere CH,NH on a solid-phase support. In addition,
a description of the preparation of the starting material, N-protected a
amino aldehyde is presented. This chapter assumes some knowledge of
basic organic and peptide chemistry techniques.
2. Materials
1. Boc-amino acid derivatives may be obtained from Bachem Bioscience
Incorporated (King of Prussia, PA). Boc-a-amino alcohols can also be
purchased from the same vendors as custom synthesis products.
2. Dimethyl sulfoxide (DMSO), pyridine-sulfur trioxide complex (pyr.SO,),
isobutyl chloroformate, sodium cyanoborohydride, sodium borohydride, 1,2
dimethoxyethane (DME), N-methyl morpholine (NMM),O,N-Dimethylhydroxylamine HCl, methyl amine (TEA), dicyclohexylcarbodiimide (DCC),
lithium aluminum hydride (LAH), and potassium hydrogen sulfate benzotriazole-1-yloxytris-[dimethylaminol-phosphonium
hexafluorophosphate
(BOP) may be obtained from Aldrich (Milwaukee, WI).
3. All solvents, such as diethyl ether, tetrahydrofuran, drmethyl formamrde,
dichloromethane, and methanol, must be anhydrous. These can be purchased from Fisher (Fair Lawn, NJ), Burdick Jackson (McGaw Park, IL),
J. T. Baker (Phillipsburg, NJ), or Aldrich. These solvents should be dried
over Zeolite (molecular sieves) or redistilled prior to use.
Reduced Peptide Bond Surrogate
243
Fig. 2. Synthesis of N-protected a-amino alcohol from a-amino acids.
3. Methods
To successfully introduce a CH2NH reduced peptide bond into a solidphase synthetic scheme, it is necessary to prepare the N-protected aamino aldehyde of the desired residue. The aldehyde derivative must be
freshly prepared and promptly used because of its limited stability. The
aldehyde derivative may be conveniently prepared from either the Boc-a
amino alcohol (3-5) or from the Boc amino acid directly (6). Each of these
procedures has been used successfully, and a detailed description of preparation from the protected a-amino alcohol as well as from the N-protected
a-amino acid is presented below because of their relative simplicity.
of N-Protected
&Amino
Alcohol
Using a mixed anhydride obtained from the Boc-a-amino acid by reaction
with isobutyl chloroformate in 1,2 dimethoxyethane, reactedwith 1.5 Eq of
aqueoussodium borohydride results in the Boc-a-amino alcohol (Fig. 2) (7).
3.1. Preparation
1. Cool 20 mL of DME to -15°C using a salt-ice bath.
2. Add 20 mmol of N-protected a-amino acid to the cold DME.
3. Successively add 2.2 mL (20 mmol) of NMM and 2.72 mL (20 mmol) of
isobutyl chloroformate.
4. After l-2 min, the precipitated NMM HCl salt is removed by filtration and
washed 5 times m 2 mL of cold DME. The filtrate and the washings are
combined in a 1-L flask and placed back in the salt-ice slurry.
5. Dissolve 1.140 g (30 mmol) of sodium borohydride in 10 mL of water, and
add this in one batch to the cold DME solution. This reaction will quickly
evolve gas.
6. Quickly add 500 mL of water. The resulting protected cl-amino alcohol
will usually precipitate, and can be collected over a Buchner funnel and
washed with water and hexane. Occasionally, the compound must be
extracted with ethyl acetate or n-butanol.
of the Protected
&Amino
AZdehyde
The protected a-amino alcohol must now be oxidized to the aldehyde
3.2. Preparation
before it can be coupled to the resin-bound peptide chain. Other proce-
Pennington
244
Pyrldme
P-N
(CH,),SO
l
0
SO3
P-N
(CZH&N
H
Fig. 3. Synthesis of N-protected amino aldehydes from a-ammo alcohol.
dures exist for accomplishing this oxidation (4,.5), but low yields resulting from a purification step over silica gel, which resulted in racemization of the amino aldehyde, hamper these methods. The method described
by Hamada and Shiori (3) utilizes a combination of sulfur trioxide-pyridine complex and DMSO in the presence of TEA (also known as the
Parikh-Doering oxidation) to accomplish this oxidation (Fig. 3).
1. Dissolve the protected a-amino alcohol (10 mmol) in 30 mL of anhydrous
DMSO containing TEA (3.035 g, 30 mmol). Stir this solution at room temperature, purge the vessel with nitrogen, and maintain an inert atmosphere.
2. Initiate the oxidation by adding a solution of sulfur trioxide-pyridme complex dissolved m 30 mL of anhydrous DMSO over 3-5 min.
3. The reaction progress can be conveniently monitored by TLC using chloroform-ethyl acetate (9:l) as the mobile phase.
4. Stir the reaction for 30-90 min under inert atmosphere, and follow reaction by TLC.
5. Pour entire reaction mixture into 300 mL of ice-water.
6. Extract this mixture with diethyl ether (4 x 200 mL) saving each of the
organic layers.
7. The combined organic layers are then washed successively with 10% aqueous citric acid (2 x 200 mL), water (2 x 200 mL), and saturated sodium
bicarbonate (2 x 200 mL), and subsequently dried over MgS04.
8. The solvent is removed by rotary evaporation under reduced pressure
resulting in the protected a-ammo aldehyde. Certain amino aldehydes can
be recrystallized, such as Boc-Phe-al, and Boc-Ala-al, Boc-Tyr(Bzl)-al,
whereas several are oils, such as Boc-Leu-al, Boc-Val-al, and Boc-Pro-al.
3.3. Synthesis
of the Boc-Amino
Aldehyde
from the Boc-Amino
Acid
by Reduction
of the N’-Methoxy-N-MethyZ-ac
(Boc-AminoWarboxamide
As an alternative to preparation of the Boc-amino alcohol, one can
also prepare the aldehyde by reduction of the protected intermediate N’-
Reduced
245
Peptide Bond Surrogate
0
I)BOP
P-N
L
OH
P-N
2) H-r-CH3.HCI
OCH,
,Cf43
"'OCH
3
1) Ll AIH4
2) H20
0
P-N
+
H
*.
R
H
Fig. 4. Synthesis of Boc-amino aldehyde by reduction of the N’-methoxy-Nmethyl-a-(Boc-amino)-carboxamide.
methoxy-N-methyl-a-(Boc-amino)-carboxamide
with lithium aluminum
hydride (6). These products are obtained in a relatively good yield with a
high degree of optical purity (Fig. 4).
1. Dissolve 10 mmol of Boc-amino acid in 50-100 mL of DCM.
2. Add 10 mmol(l.012 g) of triethylamine to the stnred amino acid solution,
3. Dissolve 10 mmol(4.42 g) of the BOP reagent to this solution, and allow
to mix for 10 min.
4. Dissolve 11 mmol (1 .113 g) of O,N-dimethylhydroxylamine HCl to this
solution, and allow to mix for 2 h at room temperature. Monitor the pH of
the solution and maintain a pH of 7.0 by adding drops of TEA.
5. The reaction mixture is diluted to 250 mL with DCM and subsequently
extracted (3 x 75 mL) (3N HCl, followed by (3 x 75 mL) of saturated
NaHCOs solution, followed by (3 x 75 mL) of saturated NaCl solution.
6. The organic layer is retained and dried over MgSO+
7. The solvent is removed in VUCUO.
8. A portion of the resulting product the N-methyl-a-(Boc-amino)carboxamide (2.5 mmol) is dissolved in either 25 mL of diethyl ether or
tetrahydrofuran depending on the relative solubility.
9. Reduction of this carboxamide is initiated by adding 2.5 mmol(95 mg; 5
Eq) of lithium aluminum hydride to the stirred solution. The reduction IS
complete within 0.5 h.
10. The mixture is then hydrolyzed with 10 mL 0.35M KHS04 in water.
11. This solution is subsequently diluted with ether and extracted as described
previously in step 5.
12. The organic layer is retained and dried over MgSO,.
Pennington
246
13. The solvent is subsequently removed in vucuo resulting in the Bocamino aldehyde.
3.4. Synthesis of the Reduced Bond CHflH
on the Resin-Bound
Peptide
At this point, the reduced peptide bond may be introduced to the resinbound peptide. The N-terminal-protecting group of the resin-bound peptide
must be removed prior to performing the reductive alkylation step. The
CH2NH peptide bond is subsequentlyintroduced by the reductive alkylation
reaction between the protected a-amino aldehyde and the amine on the
resin-bound peptide using sodium cyanoborohydride in an acidified
DMF solution by the procedure developed by Sasaki and Coy (2).
1. Dissolve the protected a-amino aldehyde (4 Eq) in DMF containing 1%
acetic acid by volume.
2. Check the resin-bound peptrde by the Kaiser test (8) to ensure that the
N-terminal residue has been deblocked.
3. Add the above solution to the resin-bound peptrde and mrx for 5 min.
4. Add the sodium cyanoborohydride (4 Eq) portionwise, and continue to
mix the solution for 1.5 h.
5. Complete the synthetic cycle by washing peptrdyl-resin wrth DMF (2 x 20
mL), DCM (2 x 20 tnL), ETOH (2 x 20 mL), and DCM (2 x 20 mL).
Check for completeness of reaction by performmg a Kaiser test (8). A negative test indicates a successful coupling. A positive test requires a
recoupling step by repeating steps 1, 3,4, and 5.
6. Following successful coupling, solid-phase assembly of the remainder of
the peptide can be resumed by conventional Merrifield strategy (9) (see
Notes l-5).
4. Notes
1. Capping protocols should not be employed from this point to prevent capping the reactive secondary amine that has been created at the CHzNH
bond. The secondary amine generated has poor reactivity because of steric
factors. However, a small reactive acylating species, such as acetic anhydride, will react much more readily than a Boc-amino acid derivative.
2. The unmasked secondary amine has been shown not to be a serious problem in the synthesis of several peptides using a DCC-mediated acylation
(10-12). However, a less hindered amino acid, such as Boc-Gly, was reactive at this site. Sasaki and Coy (2) found that Boc-Gly coupling at the
secondary amine was reduced by using the HOBT ester instead of the
DCC-generated symmetric anhydride.
Reduced
Peptide Bond Surrogate
247
3. Amino acid analysis results will reflect a loss of the dipeptide generated by
incorporating the CHzNH bond. This a nonhydrolyzable bond that is stable
to acid as well as to proteases.
4. The CHzNH bond is stable to HF cleavage conditions (see Chapter 4) and
requires no special scavengers.
5. Protection of the reduced bond secondary amme can be accomplished if
desired by reacting the peptidyl resin with benzyl chloroformate
(carbobenzoxy-chloride) prior to deprotection of the Boc group.
References
1. Szelke, M., Leckie, B., Hallet, A., Jones, D. M., Sueiras, J., Atrash, B., and Lever,
A. F. (1982) Potent new inhibitors of human renin. Nature 299,555-557.
2 Sasaki, Y. and Coy, D. H. (1987) Solid phase synthesis of peptides containing the
CH2NH peptide bond isostere. Peptides ?3,119-121.
3 Hamada, Y. and Shiori, T (1982) New methods in organic syntheses 29: a practical method for the preparation of optically active N-protected a-amino aldehydes
and peptide aldehydes. Chem. Pharm. Bull. 30,1921-1924.
4. Pfitzner, K and Moffatt, J (1970) Sulfoxide-carbodiimide
reactions I A facile
oxidation of alcohols. J. Am. Chem. Sot. 87,5661-5670
5 Stanfield, C. F., Parker, J. E., and Kanellis, P. (1981) Preparatron of protected
amino aldehydes J. Org. Chem 46,4797-4798
6. Fehrentz, J. and Castro, B. (1983) An efficient synthesis of optically active a-(tbutyloxycarbonylamino)-aldehydes
from a-amino acids. Synthesis 676-678.
7 Rodriguez, M., Llinares, M., Doulut, S., Heitz, A , and Martinez, J. (1991) A facile
synthesis of chiral N-protected a-ammo alcohols. Tetrahedron Lett 32,923-926.
8. Kaiser, E., Colescott, R , Bossinger, C., and Cook, P. (1970) Color test detection of
free terminal amine groups in the solid-phase synthesis of peptides Anal. B&hem.
34,595-598.
9. Merrifield, R. B. (1963) Sohd phase peptide synthesis: the synthesis of a tetrapeptide. J Am. Chem Sot. 85,2149-2153.
10 Martinez, J., Bali, J , Rodriguez, M., Castro, B., Magous, R., Laur, J., and Ligon,
M. (1985) Synthesis and biological activities of some pseudo-peptide analogues of tetragastrin. the importance of the peptide backbone. J. Med. Chem.
28, 1874-1879.
11. Coy, D. H., Heinz-Erian, P., Jiang, N., Sasaki, Y., Taylor, J , Moreau, J., Wolfrey,
W. T., Gardner, J., and Jensen, R. (1988) Probing peptlde backbone function in
bombesin.J Biol. Chem. 263,5056-5060.
12. Haffar, B., Hocart, S., Coy, D., Mantey, S., Chiang, V., and Jansen, R. (1991)
Reduced peptide bond pseudopeptide analogues of secretin. J. Biol. Chem. 266,
316-322.