3. Results and discussion
3.2. Synthesis of polymers by ring-opening polymerization of α-amino acid N-carboxy-
In any consideration of methods that may be employed for the union of two amino acids via a peptide linkage, the direct condensation of the amino group of the amino acid and the α-carboxyl group of the other, with the elimination of a single molecule of water, is most readily and simply visualized. Such an idealized synthesis may be represented by:
NH2CHRCO2H NH2CHR'CO2H NH2CHRCO NHCHR'CO2H H2O
Achievement of peptide bond synthesis through direct condensation, as formulated above, is difficult. However, this is difficult not only because of dipolar nature of amino acids, but also because of energy considerations as well.
In 1906, Hermann Leuchs, a student of Emil Fisher, discovered the class of N-carboxy-amino acid-anhydrides, also known as Leuchs’ anhydrides, or abbreviated NCAs. These compounds are, even 100 years after their discovery, valuable intermediates in organic synthesis due to their reactivity: NCAs polymerize with the elimination of carbon dioxide to yield polypeptides, model compounds for proteins. They can be used as starting material for a variety of pharmaceutical interesting products and for synthetic polymers (fibers and films).
Two basically different approaches exist for the synthesis of N-unsubstituted α-amino acid NCAs (Kricheldorf 1987):
1. Cyclization of N-alkoxycarbonyl-amino acid halogenides, the so-called “Leuchs Method”.
2. Phosgenation of free α-amino acids or suitable derivatives, the so-called “Fuchs-Farthing Method”.
The most widely used method for the preparation of NCAs is the phosgenation of free amino acids. This method was first applied by Fuchs and was later elaborated by Farthing for a broad variety of NCAs. The first step of the phosgenation seems to be the formation of an N-chloroformyl-amino acid (Scheme 23), because addition of aniline yields 5-phenyl-hydantoic acids.
OH O
NH2 R H
Cl O
Cl
O N O O
H R
H OH
O
NHCOCl R H
-HCl
-HCl
+Aniline
OH O
NHCONHC6H5 R H
Scheme 23. Synthesis of N-carboxy-anhydrides with phosgene.
α-Amino acid NCAs have four reactive sites – two electrophilic groups, CO-2 and CO-5, and two nucleophilic groups, NH and C-H after deprotonation.
The initiating of ring-opening polymerization can occur from action of different nucleophiles, which can be subdivided into two classes: protic nucleophiles and bases on the one hand and aprotic nucleophiles or bases on the other hand. Furthermore, it is useful to classify all initiators according to the following criteria (Kricheldorf 1990):
1. The reaction site of the NCA attacked by the initiator.
2. Reactivity of the initiator relative to that of the active chain end.
3. Incorporation of initiators into the peptide chain forming a dead chain end via a covalent bond.
Here, the two most important mechanisms will be presented. The detailed description of mechanisms with different initiators is found in (Kricheldorf 1990).
Initiation with primary amines
Primary amines are the most widely used initiators of all protic nucleophiles. They exclusively attack the carbonyl group C-5 of the NCAs. Because aliphatic primary amines are basic enough to stabilize the initially formed carbamic acid by deprotonation the chain growth can proceed in two ways (Figure 24). First, the active chain end is an amino group: a mechanistic pathway which is called “amine mechanism” or “normal propagation”. Second, the active chain end can be a carbamate ion: the “carbamate mechanism”. Because both protonation of carbamate ions and decarboxylation of carbamic acids are reversible reactions, it will be largely depend on the reaction conditions, in particular on temperature, solvent, CO2 pressure. Experimental observations suggest that the carbamate mechanism plays only a minor role (Kricheldorf 1990).
O O N
O
R R'-NH2
nucleophilic attack
HN HO
O
NH O
R'
R
N
O O O
R H
H H
N O
O
NH R' O
R
N
O O O
H R
H2N
R NH O
R'
NH O
O
O
O
HN
O
NH O
R' R
R
+H+
-CO2 N
HO H O
HN
O
NH O
R' R
R
-H+ -CO2
H2N
NH O
R'
R
+ n-2 NCA - n-1 CO2
n
"Carbamate-Mechanism" "Amine-Mechanism"
Scheme 24. Mechanism of polymerization of N-carboxyanhydrid by initiation with primary amines.
Primary alkylamines are more nucleophilic than active chain ends, so, initiation is more rapid than propagation. Therefore, polymers with high molecular weights (average degree of polymerization DP>150) are in general not possible.
Initiation with tertiary amines
Tertiary amines develop a new mechanistic concept – the activated monomer mechanism AMM:
instead of nucleophilic attack the deprotonation of unsubstituted NH group takes place. This means that the initiator reacts exclusively as base and not as nucleophile (Figure 25). The formed
anion then attacks the next N-carboxyanhydride at CO-5 and forms a dimer. This dimer can be attacked by the next deprotonated N-carboxyanhydride, and so on. As a result of these condensations, a polymer with high molecular weight can be form. The initiator is not incorporated to the polymer chain, but acts only as a catalyst in protonation/deprotonation equilibrium. Therefore, the control of molecular weight or molecular weight distribution is not possible.
R'3N O N O
O R
H
O N
O
O
R R'3NH+
O N
O O
R H
O N O
O R
O NH
O O R
R'3NH+
+NCA H+ Transfer
-CO2 O N
O
O
R R'3NH+
O N O
O R
O NH2 R N-Aminoacyl-NCA
+ n-1 NCA - n-1 CO2
O N O
O R O
HN
O
NH2 R
R
n
Scheme 25. Mechanism of polymerization of N-carboxyanhydrid by initiation with tertiary amines.
Both described mechanisms are always in competition to each other and give in each case side reactions. Exchanging between amine- and AM- mechanisms during polymerization reaction is also possible.
For synthesis of boron containing polymers S-(5-amino-5-carboxylpentyl)-thio-undecahydro-closo-dodecaborate(2-) bis-tetraphenylphosphonium salt 9 was chosen. To receive Leuchs’
anhydride we used “Fuchs-Farthing Method” with phosgene. We showed that gaseous phosgene can be used as well as phosgene dissolved in toluene as 20% solution (Scheme 26). Both ways gave the product 29 with high yield (90-95%).
S
(CH2)4 COOH NH2
2Ph4P+
2-O
Cl Cl
20% in toluene S
(CH2)4
2Ph4P+
2-HN O O
O O Cl Cl
gas
tetrahydrofuran acetonitrile
9 29
Scheme 26. Synthesis of Leuchs’ anhydride 29.
As initiators for polymerization n-butylamine (the more frequently used for polymerization of natural amino acids) and the fluorescent dansyl amine (see paragraph 2.2) were chosen. The polymerization reaction was carried out with n-butylamine in dry acetonitrile. The molar ratio 10:1 (NCAs:n-butylamine) was used. The reaction mixture was stirred at room temperature for 4 days. After elimination of the solvent in vacuum, the residue was analyzed.
In the positive mode of elecrospray ionization mass spectrum we observed only one peak, which corresponds to the tetraphenylphosphonium cation. In the negative mode – a series of peaks, but no one belongs to the structure of the desired polymer (Scheme 27). The negative mode of MALDI-TOF showed the peak of highest mass at m/z 1860 amu, but it was difficult to determine whether this peak has a boron isotopic pattern. Since each monomer of the synthesized polymer contains a anion with charge “2-”, in the case of successful polymerisation we should receive a very polar compound. In the literature we did not find any examples of analysis of such kind of systems with ESI or MALDI mass spectrometry.
HN
NH O
S B12H11
2-2Ph4P+
29 H2N
H
n
n=10
Scheme 27. Polymerization with n-butylamine.
At the same time polymerization with the fluorescent dansyl amine as initiator was made. This time polymerization was carried out with the simplest combination of molar ratio 2:1 (NCA:initiator) (Figure 28).
H2N
HN S
O O
N
H2N
NH O
HN
O S
S
NH S
O O
B12H11
2-B12H11
2-N 2Ph4P+
2Ph4P+ 30
29
Scheme 28. Polymerization with dansyl amine.
From the mass spectrum of synthesized material we did not determine any peaks for the desired dimer, but comparing the negative spectra of ESI with the previous one we found similarities of most peaks present. This fact gave to us the idea that polymerization, probably, goes without interaction of primary amines and the initiator “plays role” only as catalyst. Then polymerization goes via the AM mechanism. The reason of this could be the sterical hindrance: the dodecaborate cluster with two tetraphenylphosphonium cations around prevents nucleophilic attack of the primary amine and, therefore, the last one deprotonate unsubstituted NH group of the anhydride 29.
Hydrogen
Boron Sulfur
Carbon Oxygen
Nitrogen
Figure 12. 3D structure of the anhydride 29 (structure shown without tetraphenylphosphonium cation).
In the Table 1 we present common peaks in the negative MS of synthesized materials. ESI-MS spectrum and the calculated isotopic pattern of probable anions are shown in Appendix 2.
Common peaks of negative ESI-MS (m/z)
Probable structure of the anion
R=-(CH2)4-S-B12H11
2-100 m/2 ?
141 m/1 [B12H11]
-(see paragraph 3.3) 157 m/1 [B12H11]--H++OH-
180 m/2 ?
420 m/2 ?
668 m/1
HN O O
O
R Ph4P+
-684 m/1
O N
R O
O
O R NH2
3*Na+
-HN N R
O
O
COOH R 3*Na+
-699 m/1
O N
R O
O
O R NH2
2*Na+
-K+
HN N R
O
O
COOH R 2*Na+
-K+
1181 m/1
O N
R O
O
O
R N
H O
R NH2 -H+ 4*K+ 2*Cu+
-Table 1. Common peaks in ESI-MS of synthesized polymers.
The presumable structures given in Table 1 are based on the known capacity of amino acids to form metal complexes (e.g. biuret reaction).
IR spectra of the synthesized materials did not show signals in the area of 1650-1690 cm-1, which correspond to the peptide bond. Comparison of IR spectra of synthesized materials and Leuchs’
anhydride (Figure 13) made it clear that signals at 1846 and 1780 cm-1 (2-CO and 5-CO of 2,5-oxazolidinedione cycle) did not disappear. These facts confirm the presence of an anhydride group in the synthesized products.
wavenumber (cm-1)
500 1000
1500 2000
2500 3000
3500 4000
transmission (%)
Amino acid 9
Leuchs' anhydride 29
Polymerization with n-butylamine Polymerization with dansyl amine
Figure 13. IR spectra of synthesized compounds.
Gel filtration chromatography is a separation based on size. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated. Molecules that are small enough can fit inside all the pores in the beads and are said to be included. These small molecules have access to the mobile phase inside the beads as well as the mobile phase between beads and elute last in a gel filtration separation. Molecules that are too large to fit inside any of the pores are said to be excluded.
They have access only to the mobile phase between the beads and, therefore, elute first.
Molecules of intermediate size are partially included - meaning they can fit inside some but not all of the pores in the beads. These molecules will then elute between the large ("excluded") and small ("totally included") molecules.
For the gel filtration experiment the sample of polymerization with dansyl amine was used. The sample was dissolved in acetonitrile and injected to the Phenomenex Phenogel 5µ 10*3A column. Since dansyl amine was used as the initiator of polymerization, detection of UV chromatogram signals at 337 nm was done. In UV chromatogram (Figure 14, curve B) several peaks are present. The first peak –peak of the biggest size molecule in mixture comes at 10.0-12.0 minutes and the last one – peak of the smallest size molecule (probably the starters) comes at 14.5 minutes.
2712DG02.D: TIC ±All
2712DG02.D: UV Chromatogram, 337 nm
2712DG02.D: EIC 1180 ±All
2712DG02.D: EIC 1090 ±All 0.5
1.0 x105 Intens.
0 20 40 60
0 2000 4000 6000 8000
0 500 1000 1500
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min]
A
B
C
D
Figure 14. UV chromatogram (B) of gel filtration, total ion current chromatogram (A) and mass traces of peaks m/z 1180 (C) and m/z 1090 (D).
Mass spectrum on Figure 15 shows only a fragment peak at m/z 141, which belongs to the [B12H11]- ion and a peak at m/z 1181. The peak at m/z 1181 could belong to the structure
determined above (see Table 1), or to a fragment peak of polymer. But the absence of peptide bond signal in IR spectra does not confirm the suggestion of presence big molecules (polypeptides) in the synthesized mixture.
Gel chromatography was done also with the same sample with detection at 220 nm. This experiment did not clarify the way of polymerization and information about synthesized products. The conditions of gel chromatography and MS spectrum of measurements at 220 nm are present in Appendix 2.
In conclusion of this chapter we can say that dodecaborate-containing amino acids are able to form N-carboxy anhydrides, but polymerization with primary amines goes via the AM mechanism. Since the control of molecular weight or molecular weight distribution is not possible, we tried to determine this with available technique (ESI-MS, MALDI-TOF, gel chromatography). It is difficult to make final conclusion since presumable anions from ESI-MS spectrum could be also a fragment ions of big size molecule.
141.1
317.0 699.4
839.5 1084.8
1180.8
1366.5 1509.3 1799.0
All, 10.3-11.6min (#388-#439)
141.1 299.7
479.9 575.5 940.6 1181.01280.9
All, 8.2-8.9min (#308-#336)
141.1
252.1
435.6 629.1 778.4 898.6
1090.6
1328.9
1459.5 1740.6 1848.1
All, 12.0-12.5min (#452-#471)
140.1 195.1
435.1514.2594.4 689.5 954.4 1089.6 1361.5 1508.6 1931.1
All, 12.9-13.4min (#485-#507) 0
200 400 600 800 Intens.
0 500 1000 1500
0 100 200 300
0 100 200
250 500 750 1000 1250 1500 1750 2000 m/z
Figure 15. Mass spectra (ESI-MS, negative mode) of separated by gel filtration products (time of separation is indicated in the right corner of the spectra).