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Synthetic Communications: AnInternational Journal for RapidCommunication of SyntheticOrganic ChemistryPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lsyc20

Direct Thiophosphorylation ofAmino Acids and PeptidesHai-Yan Lu a , Nan-Jing Zhang a , Xin Chen a , Hua Fua & Yu-Fen Zhao aa Bioorganic Phosphorus Chemistry Lab., Departmentof Chemistry , Tsinghua University , Beijing, 100084,ChinaPublished online: 20 Aug 2006.

To cite this article: Hai-Yan Lu , Nan-Jing Zhang , Xin Chen , Hua Fu & Yu-FenZhao (1998) Direct Thiophosphorylation of Amino Acids and Peptides, SyntheticCommunications: An International Journal for Rapid Communication of SyntheticOrganic Chemistry, 28:10, 1727-1736, DOI: 10.1080/00397919808007003

To link to this article: http://dx.doi.org/10.1080/00397919808007003

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SYNTHETIC COMMUNICATIONS, 28(10), 1727-1736 (1998)

DIRECT THIOPHOSPHORYLATION OF AMINO ACIDS AND PEPTIDES

Hai-Yan Lu, Nan-Jing Zhang, Xin Chen, Hua Fu, Yu-Fen Zhao*

Bioorganic Phosphorus Chemistry Lab., Department of Chemistry Tsinghua University, Beijing 100084, China

Abstract: N-thiophosphoryl amino acid and peptide are synthesized conveniently by the reaction of thiophosphoryl chloride with amino acid and peptide in aqueous solution.

Many phosphoryl amino acids and low molecular weight peptide are of

pharmaceutical interest.''2 The studies in our laboratory have shown that N-

phosphoryl amino acids play an important role in the origins of During the

past decades, studies on antisense nucleotides led to the introduction of

thiophosphate linkage instead of the naturally occurring phosphate linkage, which

resulted in the enhanced anti-virus activities and stabiity to n~clease."~ Moreover,

it is well-known that many thiophosphate and their analogues showed their

biological activities and were widely used as pesticide.6 All these facts make us

explore the studies on the thiophosphoryl amino acids and peptides.

* To whom correspondence should be addressed

1727

Copyright 8 1998 by Marcel Dekker, Inc.

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1728 LU ET AL.

Since amino acids and peptides are nearly insoluble in organic solvents, most of the

methods for the phosphorylation of amino acid and peptide are base on the

protection and deprotection strategies,”’ which was time-consuming. During the

past years, we have developed an efficient procedure for the phosphorylation of

amino acid in aqueous solution, using dialkylphosphite-CCb-Et3N systemg In this

communication, we report a convenient direct thiophosphorylation procedure for

amino acids and peptides in aqueous solution.

The thiophosphorylation procedure was carried out by adding thiophosphoryl

chloride into an aqueous solution containing amino acid or peptide, NaOH and

ethanol at room temperature. By stirring the mixture for 2 hrs, the

thiophosphorylated amino acid or peptide was produced, which was in turn

acidified with 1M HCl and extracted with ethyl acetate - tert-butyl alcohol (3:2).

The organic solvent was dried with MgS04 and then evaporated to afford the

product in 78-95% yield. (Scheme, Table) The product could be recrystalized in

ethyl ether - petroleum ether. It should be noted that the existence of ethanol is

very important. By using 31P NMR to trace the reaction procedure, we found that,

with the addition of ethanol, thiophosphoryl chloride could be converted into the

corresponding thiophosphoryl amino acid or peptide in >95% yield in 2 hrs.

However, without ethanol only 10% yield of N-thiophosphoryl products could be

obtained.

The purity and structure of compounds 2 were determined by ‘H N‘h-lR, I3C NMR,

P NMR, fast atom bombardment mass spectrometry (FAB-MS), microanalysis 31

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AMINO ACIDS AND PEPTIDES 1729

S II

(EtO)#'-NH-CH+XOH A I&N-CH-C:OOH I s R

NaOHEtOWyO or b or II

(EtOhPC1 +

Peptide S II

( E t O h P - N H - w

1 2

Scheme

Table Results of Thiophosphorylation of Amino Acids and Peptides

Entry Amino acid yield of2 "PNMR FAB-MS

or peptide 1 (Ppm) dzw-w-

1

Val

Ala

Ile

Phe

Glu

Tyr

CYS

LYS

Glycyl glycine

95

86

80

91

87

95

87

92

84

+72.0

+70.7

+70.9

+70.3

+71.2

+70.1

+71.0

+71.0

+72.5

+72.4

268

240

282

316

298

332

272

449

283

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1730 LU ET AL.

and high resolution mass spectrometry. The connection of thiophosphoryl group

and amino acid or peptide could be fbrther con6rmed by 13C NMR. In most cases,

the carboxyl carbon and the p carbon was splited to a doublet due to the coupling

of phosphorus. It is interesting to find out that in 'H NM& the two C B groups in

the ethoxy groups of compounds 2 were magnetically unequivalent. Every CH3

group was splited into a triplet but with different chemical shift. This may be

resulted fiom the unsymmetrical of the molecule. While for N-thiophosphoryl

glycyl glycine, the CHa group showed only one set of triplet because the molecular

is symmetrical.

This one-step procedure can be applied to a broad range of amino acids such as

acidic, basic, aliphatic, aromatic amino acids and to a number of peptides. For basic

amino acid, such as lysine, the two amino group can be thiophosphorylated at the

m e time by two molar-equivalent thiophosphoryl chloride. For cysteine, the

thiophosphorylation occurred selectively on amino group with less than 5% on

thiol group. For tyrosine, the thiophosphorylation occurred only on amino group.

But, when this procedure was applied to serine and threonine, the products became

very complex. In addition to the N-thiophosphorylated product, there were side

products which might be 0-thiophosphorylated products.

Due to the ready availability of the starting material thiophosphoryl chloride and

amino acid or peptide as well as its general applicability, our method is a general

convenient method for the preparation of N-thiophosphoryl amino acid and N-

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AMINO ACIDS AND PEPTIDES 1731

thiophosphoryl peptide. Further studies on their chemical activities and biological

activities are in progress in our laboratory.

EXPERIMENTAL

FAB-MS were recorded on a KYKYZP-5 MS spectrometer. FAB-HRMS were

recorded on a F e g a n MAT 90 double-focusing mass spectrometer. 'H and 13C

NMR spectra were recorded on a Bruker AC 200P spectrometer at 200.13 MHz

and 50.32 MHz respectively and reported in ppm relative to TMS. 31P NMR

spectra were recorded at 81.01 M H z and the chemical shifts values are referenced

to 85% H3P04 with negative shifts upfield.

General procedure for synthesis of 2a-2i:

To a stirring solution of amino acid or peptide (20 mmol), 1N NaOH (40 ml) and

ethanol (10 ml) was added 20 mmol thiophosphoryl chloride at room temperature.

After the addition, the mixture was stirred for 2 hrs. The ethanol was evaporated

with the rotatory evaporator. The aqueous solution was extracted with ether ( 25

rnl x 3) and then acidfied to pH=3 with 1M HCI. The resulted solution was

extracted with ethyl acetate - text-butyl alcohol (3:2). The organic layer was dried

with MgSO4. The solvent was then evaporated under reduced pressure to afford

the product, which could be recrystdied from ether - petroleum ether.

2a: "P NMR (81 MHz, CDCb): 6= +72.0 ppm; 'H NMR(200 MHz, CDC13): 6=

1.245(t, 3M.8Hz, 3H, CH~CHZO), 1.211(t, '&6.8Hz, 3H, CH~CHZO), 4.046(111,

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1732 LU ET AL.

4H, ~ X C H ~ C H ~ O ) , 0.951(d, 3J=6.8HZ, 3H, CH(CH3)2), 0.882(d, 3s-6.8Hz, 3H,

CH(CH&), 2.040(m, lH, CH(CH&), 3.531(t, 3J=ll.lHz, lH, NH), 3.790(dxt,

3J=10.8Hz, 3J=4.9 Hz, NHCH), 11.50(br s, lH, COOH). 13C NMR (50 MHq

CDCl3): 6=15.7(d, 3J=6.8HZ, m&H20), 15.5(d, 3J=6.8Hz, m3CH20),

63.0(2xCH3CH20), 59.8(NHCH), 3 1.65(d, 3J=6.3Hz, CH(CH3)2), 18.8

(CH(a3)2), 17.3(CH(m3)2), 178.8(br s, COOH); FAB-MS d z : 268 (M-H)'.

Anal. Calcd. for CSH~ONOPS: C: 40.12, H: 7.43, N: 5.20; Found: C: 39.96, H:

7.43, N: 5.17.

2b, "P NMR (81 MHz, CDCb): 6= +70.7 ppm; 'H NMEt(200 MHz, CDCl3): 6=

1.300(t, 3&7.0HZ, 3H, CH3CH20), 1.272(t, 'J=7.OHz, 3H, CH3CH20), 4.04(1~

4H, 2xCH3CH20), 3.67(br s, lH, NH), 4.04(m, lH, NHCH), 1.424(d, '&.8Hz,

3H, CHj), 9.72(br s, lH, COOH); "C NMR (50 MHz, CDCl3): 6=15.7(d,

3J=6.6Hz, m&H20), 15.6(d, 3 J = 6 . 6 ~ CH~CHZO), 63.0(d, *&4.4&,

~ x C H ~ C H ~ O ) , SO.l(NHCH), 20.6(d, 3J=4.9Hz, CHCH3), 178.8(d, 3&.7Hz,

COOH); FAB-MS m / ~ : 240 (M-H)-; HRMS(M-w: 240.0453 (Calcd. 240.0459)

ZC, 31P NMR (81 MHz, CDCC): 6= +70.9 ppm; 'H NMR(200 MHz, CDCb): 6=

1.270(t, 3J=6.8Hz, 3H, CHjCH20), 1.238(t, '-6.8Hz, 3H, CHjCHzO), 4.048(1~

4H, ~ x C H ~ C H ~ O ) , 3.54(t, 'J=lO.SHz, lH, NH), 3.90(dt, 'J=10.6Hz, 3k5.6Hz,

lH, NHCH), 1 . 7 7 2 ( ~ lH, CH3CH), 0.933(d, 3J=6.7Hz, 3H, CHKH), 1.444(m,

2H, CHXH,), 0.890(t, 3&7.3Hz, 3H, CHzCHj), 10.69(br s, lH, COOH); 13C

NMR (50 MHz, CDCb): 6=15.7(d, 3J=6.8Hz, m3cH20), 15.6(d, 3 & 6 . 8 ~

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AMINO ACIDS AND PEPTIDES 1733

a&H20) , 63.0(2xCH3CH20), 58.9(NHCH), 38.7(d, 'H.OHz, CH'CH),

15.2(a3CH), 24.6(CH3CH2), 11.3(CHXH2), 178.6(COOH). FAB-MS 4 2 : 282

(M-q ; HRMS(M-m: 282.0926 (Calcd. 282.0928).

2d, 31P NMR (81 MHz, CDCls): 6= +70.3 ppm; 'H NMR(200 MHz, CDCI'):

6=1.236(t, 3 5 = 7 . 0 ~ 3H, CH&H20), 1.187(t, 3 .+7.0~, 3H, CH3CH20), 3.87(%

2H, CHdYhO), 3.73(m, 2H, CHd.Yf20), 3.52(t, 'ill.OHZ, 1% NH), 4.272(m,

lH, NHCH), 9.02(br s, lH, COOH), 3.062(dd, 3J=13.6Hz, 2J=28.1Hz, lH,

PhCHz), 3.031(dd, 'fi13.6, 2.&28.1, lH, PhCHZ), 7.19-7.34(~ 5H, Ph-H) ; "C

NMR (50 MHz, CDCI'): 6=15.8(d, ' M . 0 - m&H20), 15.7(d, '&S.OW

m&&O), 63.03(d, 2 & S . 0 ~ CH3CH20), 62.93(d, 2 J . 5 . 0 ~ C H ~ ~ Z O ) ,

5 5 . 8 5 0 , 40.0(d, '&6.2Hz, P h m ) , 127.0, 128.4, 129.5, 135.8(Ph-C),

177.4(d, %.OHZ, COOH); FAB-MS d z : 316 (M-w, HRMS(M-H)-: 316.0770

(Calcd. 316.0772).

2e, "P NMR (81 MHz, CDCh): 6= +71.2 ppm; 'H NMR(200 MHz, CDC13):

6=1.260(t, 'J=7.0HZ, 3H, CHjCH20), 1.270(t, 3 & 7 . 0 ~ 3H, CH3CH20), 4.08(m,

4H, ~ x C H ~ C H ~ ) , 2.05(m, 2H, CHCH2), 3.78(t, 3 i 1 0 . 5 ~ 1% NH), 3.93(n

IH, NHCH), 2.53(t, 'J=7.OHz, 2H, CH&OOH), 10.14(br s, 2% 2xCOOH); 13C

NMR (50 MHz, CDC13): 6=15.7(d, ' 7 . 6 - a3CH20), 15.65(d, 3 J = 7 . 6 ~

a&&O) , 63.3(d, '&4.5HZ, ~xCH~CH~O), 53.5(NHCH), 28.6(d, '&6.9HZ,

CHCH2), 29.5(C&COOH), 177.8(d, 'J=S.OHz, CHCOOH), 178.7(CH2COOH);

FAB-MS m / ~ : 298 (M-H)i HRMS(M-w: 298.0519 (Calcd. 298.0513).

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1734 LU ET AL.

2f, 31P NMR (81 MHz, CDCl'): 6= +70.1 ppm; 'H NMR(200 MHz, CDCb):

6=1.152(t, '&~.OHZ, 3H, CH3CH20), 1.187(t, 3&7.0HZ, 3H, CH3CH20),

3 . 8 9 % ~ 4H, ~XCH~CH~O), 3.634(t, 3.bll.0HZ, lH, NH), 4.16(m, lH, NHCH),

2.89(m, 2H, PhCH2), 6.69-7.01(q, 4H, Ph-N), 6.65(br s, lH, Ph-OH), 8.44(br s,

lH, COOH); I3C NMR (50 MHZ, cDcl3): 6=15.7(d, '&S.OHZ, CH~CHZO),

15.6(d, 3&5.0HZ, m&H20), 63.0(d, '&4.5HZ, C H ~ ~ Z O ) , 62.9(d, 2 & 4 . 5 ~

C H ~ ~ Z O ) , 55.9(NHcH), 39.8(d, ' H . O H z , PhCHz), 155.1, 130.5, 127.3,

115.4(Ph-C), 175.8(d, '.H.OHZ, COOH); FAB-MS m/z: 332 (M-W, HRMS(M-

€I)-; 332.0726 (Calcd. 332.0721).

2g, 31P NMR (81 MHZ, CDCL): 6= +71.0 ppm; 'H NMR(200 MHZ, CDCl3):

S=1.253(t, '&7.OHZ, 3H, CH~CHZO), 1.236(t, '&7.O& 3% CH3CH20),

3.922-4.136(111, SH, 2xCH3CH.0, WCH), 3.786(t, 'J=10.6&, lH, NH),

2.836(dd, 3J=8.9Hz, 2&18.6Hq lH, CHzSH), 2.859(dd, 'J=8.9HZ, '&18.6&

lH, 0, 9.5390r s, lH, COON); "C NMR (50 MHz, CDCC): 6=

15.8 ~ ( ~ x C H ~ C H ~ O ) , 63.25(d, 'J=4.0Hz, ~xCH~CH~O) , 55.88(NHCH), 28.80(d,

H . s & , ~ Z S I I ) , 174.54(d, 'H.5&, COOH); FAB-MS m/z: 268 (M-H)-; 3

HRMS(M-H)-: 272.0189 (Calcd. 272.0179).

2b, "P NMR (81 M H q (CD')C=O): 6= +71.0, +72.5 ppm; 'H NMR(200 MHz,

(CD3)C=O): 6=1.236(t, '&7.2HZ, 3H, CH3CH20), 1.217(t, '&7.1H~, 3H,

CHSHzO), 1.180(t, 3 J = 7 . 1 ~ , 6H, ~xCH~CH~O) , 3.830-4.05 l ( ~ 8% 4~

CH~CHZO), 1.720(5, 'fi7.2 HZ, 2H, CHCHZCH~CH~), l.SOO(m, 4H,

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AMINO ACIDS AND PEPTIDES 1735

CHCHSHZCH~), 2.919(dt, '&S.OHZ, 3.J=6.4Hz, 2H, CH,NH), 4.471(m, 3 6

2xNH+NHCH); 13C NMR(50 MHz, (CB)C=O): 6=16.l(d, '&8.OHz,

~xCH~CHZO), 63. ~(~xCH~CHZO), 6 2 . 7 ( 2 ~ C & ~ z o ) , 55.1S(NHCH), 34.1S(d,

'.&.OH4 CHCHz), 31.2(CHCHzCHz), 31.54(d, '&5.6Hz, CHzCHzNH), 41.88

(CHzCHzNH), 175.0(d, COOH); FAB-MS d z : 449 (M-w;

HRMS(M-H)-: 449.1092 ( C d d 449.1098).

2i: 31P NMR(81 MHq CDCl3): 6= +72.4 pprn. 'H NMR(200 MHq CDCl3): 6=

1.309(t, 3&7.0& 6H, ~xCHSHZO), 4.077(m, 4H, ~xCH~CHZO), 4.44(br s, lH,

NH), 3.76(br d, 'J-13.1Hz. 4H, 2xNHCHz), 7.51@r s, lH, NH), ll.lO(br s, lH,

COOH). 13C NMR(50 MHq CDCl3): 6=15.6(d, 'h7.8- ~xCH~CHZO), 63.3(d,

'&4.6H~, ~xCH~CHZO), 44.4(NHCH2), 41.1(CH2COOH), 172.0(0=cNy

COOH); FAB-MS d!: 283 (M-H)-; Anal. Calcd. for CIH~~NZO~PS: C133.77,

H5.98, N:9,85, Found: C:33.87, H:6.22, N:9.85.

Acknowledgment: This work was supported by the National Natural Science

Foundation of China, the Chinese National Committee of Science and Technology

and Science Grant fiom Tsinghua University.

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1736 LU ET AL.

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