Convenient solid-phase synthesis of oligopeptides using pentacoordinated phosphoranes with amino acid residue as building blocks

Article abstract of DOI:10.1016/j.bioorg.2003.12.001

The reactive intermediates of pentacoordinated phosphoranes with amino acids (P(5)-AA) as building blocks, which were obtained by the reaction of O-phenylene phosphorochloridate with N,O-bis(trimethylsilyl)amino acids, were linked to a solid-phase support containing a hydroxymethyl polystyrene functional group. The first amino acid residue was coupled to the solid-phase support after washing the resin with organic solvent. Repeating the procedure led to oligopeptides linked on the resin. A series of free oligopeptides including tetra-Gly, di-Val, tri-Val, di-Leu, di-Phe, and Phe-Leu were obtained after cleavage from solid-phase support. The structure of these oligopeptides were determined by IR, ¹H NMR, FAB-MS, and HPLC.

Full text of DOI:10.1016/j.bioorg.2003.12.001

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 32 (2004) 170–177
www.elsevier.com/locate/bioorg
Convenient solid-phase synthesis
of oligopeptides using pentacoordinated
phosphoranes with amino acid residue
as building blocks
*
Zhaolong Li, Hua Fu, Hegui Gong, and Yufen Zhao
Key Laboratory of Bioorganic Phosphorus Chemistry, Ministry of Education, Department of Chemistry,
School of Life Sciences and Engineering, Tsinghua University, Beijing 100084, PR China
Received 9 August 2003
Abstract
The reactive intermediates of pentacoordinated phosphoranes with amino acids (P(5)-AA)
as building blocks, which were obtained by the reaction of O-phenylene phosphorochloridate
with N,O-bis(trimethylsilyl)amino acids, were linked to a solid-phase support containing a
hydroxymethyl polystyrene functional group. The first amino acid residue was coupled to the
solid-phase support after washing the resin with organic solvent. Repeating the procedure led
to oligopeptides linked on the resin. A series of free oligopeptides including tetra-Gly, di-Val,
tri-Val, di-Leu, di-Phe, and Phe–Leu were obtained after cleavage from solid-phase support.
1
The structure of these oligopeptides were determined by IR, H NMR, FAB-MS, and HPLC.
Ó 2003 Elsevier Inc. All rights reserved.
Keywords: Pentacoordinated phosphoranes; Amino acid; Oligopeptides; Solid-phase synthesis
1
. Introduction
Peptide chemistry is of great importance in pharmaceutical research having a ma-
jor impact on immunology such as in the preparation of synthetic vaccines and in the
study of biologically active molecules like enzymes and hormones [1]. The chemical
*
Corresponding author. Fax: +86-10-6277-1149.
E-mail address: zhlli@tsinghua.edu.cn (Z. Li).
0
045-2068/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2003.12.001

Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
171
Scheme 1. Self-activation of N-phosphorylamino acids into peptides.
synthesis of large peptides has been accomplished by two distinct approaches. Effi-
cient synthetic protocols to obtain pure peptides were first developed in solution
(
liquid-phase peptide synthesis LPPS) [2]. However, the stepwise addition of single
amino acids was revolutionized by the Merrifield solid-phase technique (solid-phase
peptide synthesis SPPS). Within the last thirty years this technique has assumed a
prominent place in peptide synthesis [3,4].
In our previous work, we have shown that a series of N-phosphoryl amino acids
formed oligopeptides in the absence of any kind of activator or coupling agent [5,6].
It has been reported that such organic phosphorous pentacoordinated phosphoranes
are efficient intermediates which react with nucleophiles [7]. Some models have been
proposed to explain the condensation process for peptide formation in aqueous
media. An intermolecular mixed carboxylic phosphoric anhydride intermediate
[
pentacoordinated phosphorane with amino acid P(5)-AA] can exist in the conver-
sion of N-dialkyloxy-phosphorylamino acid(DRPA) into oligopeptides (Scheme 1).
However, this intermediate could not be observed by any method in aqueous
media because of its rapid hydrolysis [8]. In order to shed light on the reaction mech-
anism of peptide formation by N-phosphorylamino acid, we found that the
N,O-bis(trimethylsilyl) amino acid (N,O-Bis-AA) could self-assemble into peptides
by treatment with O-phenylene phosphochloridate (O-PPC) in the absence of any
catalyst. The results showed that the pentacoordinated phosphoranes containing
amino acid (P(5)-AA) were key reactive intermediates in the process of peptide
formation [9–11]. In fact, under these conditions, it is not easy to control the length
of oligopeptide. Usually, a series of mass numbers was found, which corresponded to
oligopeptides ranging from monomers to different sized oligomers. For example,
when reacting O-PPC with N,O-Bis-Phe under aqueous conditions, different length
oligomers of Phe ranging from the monomer to the octamer were observed.
We report here that pentacoordinated phosphoranes can be used as building blocks
in the solid-phase synthesis of oligopeptides.
2
. Materials and methods
2
.1. Chemicals
4
Aldrich. Several amino acids, catechol, hexamethyldisilyldiamine, and other chemicals
-Hydroxymethyl polystyrene resin (HOCH
2
-R) was purchased from Sigma–

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Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
were commercially available from Beijing Chemical Company. All solvents were puri-
fied and dried before use.
2
.2. Synthesis of oligopeptide
Solid-phase synthesis was performed according to the following procedures.
Traces of oxygen were removed in a home-built reactor at room temperature. In
the first step, HOCH (R) (0.25 g) was swelled in dry benzene (5 ml) for 2 h. Elonga-
2
tion of the peptide was carried out by coupling pentacoordinated phosphorane (P(5)-
AA) (1.7 mmol) in benzene (5 ml) with solid support resin for 4 h. The resulting resin
2 2
was washed with CH Cl five times (5 ml each time). The elongation and washing
steps were repeated. Subsequently, the peptide was cleaved from resin in 10%
TFA containing the appropriate solvent. The crude peptide was obtained after re-
moval of solvent. Purification of oligopeptide is accomplished by aqueous workup
and/or reverse phase chromatography. The total yield of peptide varied between
5
0 and 70%.
A typical procedure for the synthesis of Phe–Leu was as follows. P(5)-Leu
(
0.1 mmol), dissolved in dry benzene (5 ml), was added to HOCH
resulting solution was stirred for 4 h at room temperature, and washed with 5 ml
CH Cl
2
(R) (0.25 g). The
2
2
(ꢀ5). After filtration, the resin was swelled in dry benzene (5 ml) and
P(5)-Phe (0.1 mmol) in benzene (5 ml) was added to the reactor. The resulting solu-
tion was stirred for 4 h at room temperature, washed and filtered as described above.
A solution of dioxane and acetonitrile containing 10% TFA was used to cleave the
oligopeptide from the solid-phase support. The solvent was removed in vacua and
the residue was dissolved in ethyl acetate. The solution was extracted with water
and the final product was obtained in 56% yield after lyophilization of aqueous
phase.
1
H NMR (200 MHz in CDCl ): d 7.1–7.3 (5H, m, aromatic H), 7.1 (1H NH of
3
a
a
b
Leu), 2.9–3.1 (2H m, C H of Leu and C H of Phe), 2.3–2.5 (5H, m C H of Phe,
b
c
ꢁ1
C H, C H of Leu), 0.5–0.6 (6H, d, 2 ꢀ CH of Leu). IR(cm , KBr) 3350/3225
3
(
NH), 3125/3300 (Ph–H and –CH –), 1670 (CONH), 1550/1500 (NH), 1530
2
þ
and 670 (benzene ring monosubstituted). FAB-MS [M + H] m/z 279. HPLC Rt
2
.690 min.
2
.3. Analytical methods
The IR spectra were measured as a KBr disc using a Nicolet 5DX FT-IR spec-
trometer. Positive ion FAB-MS data were obtained on a Finnigan MAT 90MS dou-
1
ble-focusing magnetic mass spectrometer. The H NMR spectra were recorded on a
Bruker AM 200 spectrometer. The analysis of oligopeptides was carried out on SHI-
MADZU model LC-9A (RPC ZORBOX ODS C18 column 4.6 ꢀ 150 mm). An aque-
2 4 3 4
ous solution containing 0.02 M of KH PO (adjusted to pH 2.5 using H PO ) buffer
was used to elute the oligopeptide formed in the solid-phase synthesis. Chromatog-
raphy was performed at 25 °C, and the elution profile was monitored at 210 nm using
a SHIMADZU SPD-6AV UV detector.

Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
173
3
. Results and discussion
.1. Procedures of solid-phase synthesis
In general, the typical solid-phase peptide synthesis involves multiple steps, in-
3
cluding the protection of the amino acid, activation, coupling, deprotection of the
amino acid, and the final cleavage of the peptide from the solid-phase support. In
our recent work, we found that (P(5)-AA) are reactive intermediates which react rap-
idly with (N,O-Bis-AA) to form a series of different length oligopeptides in aqueous
solution. On the other hand, P(5)-(AA) can also be attacked by nucleophiles, such as
ROH and RNH
selected a solid-phase support (4-hydroxymethyl polystyrene resin, resin 2 in Scheme
) as an efficient nucleophile. The solid-phase synthesis of oligopeptide by this pro-
2
[11]. In order to control the random elongation of oligopeptide, we
2
cedure is shown in Scheme 2.
As shown in Scheme 2, HOCH (R) (2) is a nucleophile that rapidly attacks
P(5)-AA (1) to form product 3. The experimental results show that the P–N bond
2
Scheme 2. Solid-phase synthetic process of oligopeptide.

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Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
is unstable in compound 3. Phosphate 5 could be removed to give 4 by washing with
CH Cl . Product 4 was analyzed by IR spectra showing amide bond formation
Fig. 1). Compound 5 was a phosphate triester corresponding to the P NMR signal
2
2
31
(
at 3.5 ppm, which was determined by comparison with a control sample and identi-
fied by high resolution electron ionization mass spectrometry (EI-MS) [11]. Similar
nucleophilic attack by the amino group in 4 to the carbonyl group in 1 led to dipep-
tide linking with resin as the leaving compound 5. Repetition of the process above
yielded oligopeptide linked with resin 6. The final oligopeptide was obtained after
cleavage from the resin by trifluoracetic acid (TFA).
3
.2. Infrared spectra of oligopeptides
We have made a series of oligopeptides using our novel solid-phase synthesis
method, including Phe–Leu and Leu–Leu. Since IR spectroscopy is a very powerful
technique for determination of functional groups linked to a solid support, we deter-
mined the IR spectra of HOCH (R) (I), oligopeptide (Phe–Leu) (II) and (Leu–Leu)
2
(
sin (I) showed a series of typical absorption peaks at 3125–3000 cm (Ph–H and
III) linked to the resin, respectively. It can be seen in Fig. 1 that the polystyrene re-
1
ꢁ
ꢁ
1
–
CH
The new peaks in their IR spectra appeared at 1640 and 1670 cm for (II) and
III) respectively, which are different from those observed in (I). This result shows
2
–stretch region); 1500, 1050, 740, and 690 cm (1,4-disubstituted benzene).
ꢁ
1
(
amide bond absorption consistent with peptide bond formation. It is interesting that
the IR spectra of (II) showed an obviously monosubstituted benzene ring with char-
ꢁ
1
acteristic absorption peaks at 1530 and 670 cm , which is due to Phe and is not seen
in the resin or in the Leu–Leu oligopeptide spectra.
2
Fig. 1. The IR spectra of HOCH (R)(I), oligopeptide (Phe–Leu)(II) and (Leu–Leu)(III) linked the resin,
respectively.

Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
175
Table 1
FAB mass spectral data of oligopeptides
Oligopeptides
tetra-Gly
247
di-Val
217
tri-Val
316
di-Leu
245
di-Phe
313
Phe–Leu
279
þ
[
MH] m/z
3
.3. FAB mass spectrometric measurements
The oligopeptides were identified by FAB mass spectrometry, and comparing re-
sults with the corresponding authentic samples. The positive ion FAB mass spectral
data of oligopeptides formed in the solid-phase synthesis are listed in Table 1.
It is seen from Table 1 that the FAB mass spectral data of corresponding oligo-
peptides obtained in SPPS are all agreement with those of the authentic oligopep-
þ
tides. It is found that the peak [M + H] at m/z 247 (65%) in tetra-Gly was
þ
protonated tetra-Gly. There are still di-Gly [M + H] at m/z 133 and a few hexa-
þ
Gly [M + H] at m/z 361 in the synthesis of oligo-Gly in addition to the tetra-Gly.
This is due to the rapid interaction of the produced higher peptide and neighboring
group effects in the presence of polymer solid-phase support. However, except for
Gly, our results show that the formation of the target molecular is the major product
in these reactions.
3
.4. HPLC analysis of oligopeptides
The reverse-phase HPLC analysis confirmed that the correct oligopeptides were
formed in the solid-phase synthesis (Fig. 2). The samples synthesized by SPPS had
retention times of 2.65 min (di-Phe) and 2.69 min (Phe–Leu) (Fig. 2C and D). The
retention time of the synthesized di-Phe is compared to that of an authentic sample
(
2.67 min) (Fig. 2B). Phe has a retention time at 1.6 min (Fig. 2A).
Fig. 3 shows the HPLC profiles of products obtained using our solid-phase pep-
tide synthesis (SPPS) and products obtained using liquid-phase peptide synthesis
LPPS). Fig. 3A corresponds to di-Phe obtained by SPPS (- - -) compared to an au-
thentic sample (—).
(
There is very good agreement between the retention times of di-Phe synthesized
by our SPSS with that of LPPS (Fig. 3B). These results indicated that the self-cata-
lytic formation of peptide was better controlled in SPPS than the LPPS method. It
appears to be a more efficient method for oligopeptide formation.
4
. Conclusion
The solid-phase synthesis of oligopeptides using reactive pentacoordinated phos-
phoranes P(5)-AA as building blocks has been described. This strategy represents a
modified solid-phase peptide synthesis method, which is different from the conven-
tional approach, involving the protection of amino acid, activation, coupling and
deprotection.

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Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
Fig. 2. HPLC profiles of oligopeptides and authentic samples: (A) phenylalanine; (B) di-Phe; (C) di-
Phe(SPPS) and (D) Phe–Leu(SPPS).
Fig. 3. HPLC profiles of oligopeptide synthesized by SPPS and LPPS method.

Z. Li et al. / Bioorganic Chemistry 32 (2004) 170–177
177
Acknowledgments
The authors thank the financial supports from the National Natural Science
Foundation of China (No. 20132020 and 20175026).
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