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SYNTHESIS OF N-PHOSPHOPEPTIDES COUPLED BYDICHLOROTRIPHENYLPHOSPHORANEShu-Zhi Dong a Hua Fu a amp Yu-Fen Zhao ba State Laboratory of Bioorganic Phosphorus Chemistry of Education Ministry Departmentof Chemistry School of Life Sciences and Engineering Tsinghua University Beijing100084 PR Chinab State Laboratory of Bioorganic Phosphorus Chemistry of Education Ministry Departmentof Chemistry School of Life Sciences and Engineering Tsinghua University Beijing100084 PR ChinaPublished online 09 Nov 2006

To cite this article Shu-Zhi Dong Hua Fu amp Yu-Fen Zhao (2001) SYNTHESIS OF N-PHOSPHOPEPTIDES COUPLED BYDICHLOROTRIPHENYLPHOSPHORANE Synthetic Communications An International Journal for Rapid Communication ofSynthetic Organic Chemistry 3113 2067-2075 DOI 101081SCC-100104428

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SYNTHESIS OF N-PHOSPHOPEPTIDES

COUPLED BY DICHLOROTRIPHENYL-

PHOSPHORANE

Shu-Zhi Dong Hua Fu and Yu-Fen Zhao

State Laboratory of Bioorganic Phosphorus Chemistry ofEducation Ministry Department of Chemistry School of

Life Sciences and Engineering Tsinghua UniversityBeijing 100084 PR China

ABSTRACT

Coupling of N-phosphoamino acids 2 and amino acid methylesters by dichlorotriphenylphosphorane 3 produced N-phos-phopeptide methyl esters 5 which were sequentially hydro-lyzed in triethylamine or dilute NaOH aqueous solutionThe target products N-phosphodipeptides 6 were obtainedin reasonable yields and high purity after isolation usingextraction method The convenient and efficient approachcould be generally used for synthesis of polypeptides

Derivatives of N-phosphopeptides are of pharmaceutical and biologi-cally active interest1ndash4 They are continuing to be increasingly important asmechanistic probes for proteases5 potent inhibitors of peptidases as tetra-hedral transition-state or high-energy intermediate analogs6 useful tools for

2067

Copyright amp 2001 by Marcel Dekker Inc wwwdekkercom

SYNTHETIC COMMUNICATIONS 31(13) 2067ndash2075 (2001)

Corresponding author Fax 86-10-62781695 E-mail tp-dchmailtsinghuaeducn

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the investigation of phosphorylation or as important posttranslationalmodification of peptides and proteins7

In order to decipher the role of many phosphopeptides and exploretheir biological activities a facile method to acqurie multi-gram quantitiesof phosphopeptides is in high demand In general N-(dialkoxyphosphor-yl)peptides were prepared by phosphorylating the peptides obtained by thedeprotection of N-protected peptides such as N-Boc-peptides4 HoweverN-(dialkoxyphosphoryl)amino acids have hardly been used for the synthesisof N-(dialkoxyphosphoryl)peptides probably because it is difficult tosynthesize and purify them Fortunately an efficient method to preparehighly pure and large quantities of N-phosphoamino acids has been devel-oped in our lab8

Furthermore it has been proved that the phosphinic carboxylic mixedanhydride method had the superiority in peptide synthesis9 than the mixedcarboxylic anhydride method for the faster coupling rate more regiospeci-ficity in nucleophilic attack and more thermal stability to disproportiona-tion Hence the methodology of N-phosphopeptide synthesis starting fromN-phosphoamino acid and using dichlorotriphenylphosphorane 3 as thecoupling reagent was investigated

RESULTS AND DISCUSSION

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters

5 (N-DIPP-dipeptide Methyl Esters)

Dichlorotriphenylphosphorane 3 which was assigned to the ionisedform according to 31P NMR spectrum (dPfrac14 611 ppm) is either commer-cially available or can readily be prepared by the reaction of tri-phenylphosphinewithhexachloroethanewithout anyundesirable phosphorusby-product (Scheme 1)10

15 equivalents of in-situ generated dichlorotriphenylphosphorane 3

was added to 1 equivalent of N-(diisopropyloxyphosphoryl)amino acid 2

(N-DIPP-amino acid 2) amino acid methyl ester and triethylamine(Et3N) in CH2Cl2 at room temperature The mixture was stirred for 1 h

2068 DONG FU AND ZHAO

Scheme 1 Synthetic Pathway of Dichlorotriphenylphosphorane 3

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and compound 2 and amino acid ester almost transferred into N-DIPP-dipeptide methyl ester 5 in quantitative amount (indicated by the singlesignal at about 31P NMR 68 ppm of compound 5) (Scheme 2) The activa-tion mechanism was proposed that dichlorotriphenylphosphorane 3 phos-phorylated N-DIPP-amino acid 2 to give the phosphinic carboxylic mixedanhydride 4 followed by the nucleophilic attack of amino acid methyl esterresulting into the formation of peptide bond11 Excessive dichlorotriphenyl-phosphorane 3 would consume the remnant water to guarantee the anhy-drous environment and make the coupling reaction complete Moreover itwould not bring in further by-products because 3 can react with H2O toform triphenylphosphine oxide which is the same as the product yielded incoupling reaction

Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

In order to avoid the side reactions on N-protecting group DIPP(diisopropyloxyphosphoryl) triethylamine as a weak base was the firstchoice to catalyze the hydrolysis of compound 5 (Scheme 3)12 Yet it wasonly efficient for the hydrolysis of compound 5a 5b and 5c Hydrolysis ofcompound 5d 5e 5f however was completed in a stronger base diluteNaOH aqueous solution with a longer time as shown in Table 1 Table 1

shows that the only difference between 5a 5b and 5d 5e 5f lies in the sidechain of the amino acid residue on C-terminal And the nuance betweencompound 5c and 5e is the addition of phenyl on the side-chain of the firstamino acid residue These show that the hydrolysis reaction is stronglydependent upon the dual side-chains

The hydrolysis reaction was carried out in aqueous-organic mixed sol-vents to facilitate the tracing of reaction process by 31P NMR techniquesN-DIPP-dipeptide methyl esters 5 are organic soluble species while the

N-PHOSPHOPEPTIDES 2069

Scheme 2 Synthetic Pathway of N-DIPP-dipeptide Methyl Esters 5

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basic hydrolyzed products 6 are the corresponding salts which are aqueoussoluble As the hydrolysis proceeding the signal at about 31PNMR 68 ppm ofcompound 5 in organic phase decreased At the end point there was no com-pound 5 left therefore no 31PNMR signal of compound 5 could be detected inthe organic phase Whilst triphenylphosphine oxide was imprisoned inorganic phase (the signal at about 31P NMR 280 ppm of Ph3Pfrac14O onlyexisted in the organic phase) So the major impurity was removed completelyDichlorotriphenylphosphorane 3 gives cleaner reactions due to the highsensitivity towards the atmosphere and the exclusive resultant triphenyl-phosphine oxide Hence after eliminating the main impurity Ph3Pfrac14O wegot highly pure N-phosphodipeptides 6andashf using simple extraction workup

The purity and structure of compounds 6andashf were determined by1H NMR 13C NMR 31P NMR and ESI-MS methods Each target productgave only one 31P NMR signal implying that no racemization occurredduring this peptide bond formation13 These highly optical pure N-DIPP-dipeptides 6andashf could be used for elongation of N-phosphopeptides orandtest for biological activities

In conclusion dichlorotriphenylphosphorane 3 is an effective couplingreagent for synthesis of N-phosphopeptides The present method enablesus to obtain N-phosphopeptides on a large-scale and highly pure productionbecause of its availability convenience and low racemization Furtherthe N-phosphopeptides could be deprotected under acid conditions14 toyield the corresponding peptides Thus the convenient and efficient approachcould be also applied for the synthesis of polypeptides

2070 DONG FU AND ZHAO

Table 1 Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

Final

Poduct 6 R1 R2 Base

Time

(h)

Overall

Yield ()

DIPP-Phe-Gly a -CH2-Ph -H Et3N 35 852

DIPP-Phe-Ala b -CH2-Ph -CH3 Et3N 35 607DIPP-Ala-Phe c -CH3 -CH2-Ph Et3N 35 600DIPP-Phe-Leu d -CH2-Ph -CH2-Pr

1 NaOH (017N) 70 803

DIPP-Phe-Phe e -CH2-Ph -CH2-Ph NaOH (017N) 70 758DIPP-Phe-Val f -CH2-Ph -Pr1 NaOH (065N) 90 882

Scheme 3 Synthetic Pathway of N-DIPP-dipeptides 6

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EXPERIMENTAL

General Procedure

All glassware was dried in an oven for at least 3 h at 120C prior to useAir sensitive materials were transferred under a nitrogen atmosphereDichloromethane and triethylamine were dried over P2O5 and CaH2

respectively 1H MNR and 13C NMR spectra were recorded on BrukerAM 500 spectrometer TMS (dfrac14 00) and CDCl3 (dfrac14 770 ppm) werereferences for 1H and 13C NMR spectra respectively 13C NMR spectrawere all taken under 1H decoupled and 31P coupled conditions 31P NMRspectra were taken on Bruker AC 200 spectrometer at 81MHz under1H decoupled conditions 31P NMR chemical shifts are reported in ppmdownfield (thorn) or upfield () from external 85 H3PO4 as reference Massspectra were conducted on Bruker Esquire-LC mass spectrometer in positiveand negative ion mode

Synthesis of Diisopropyl Phosphite (DIPPH) 1

The starting material DIPPH 1 was prepared according to the pub-lished procedure15 Yield 75 31PNMR 44 ppm

Synthesis of N-(Diisopropyloxyphosphoryl)amino Acids 2

N-DIPP-amino acid 2 was synthesized by adding diisopropylphosphite 1 to an aqueous-organic mixture containing the amino acidwater carbon tetrachloride triethylamine and ethanol well stirring themixture at 0ndash20C for a few hours and quenching the reaction by acidifica-tion8 Yields DIPP-Ala 89 DIPP-Phe 93 31P NMR 71 69 ppmrespectively

Synthesis of Dichlorotriphenylphosphorane 3

According to literature10 under a nitrogen atmosphere 2mmoltriphenylphosphine in 3mL dry CH2Cl2 was added dropwise to a stirred2mmol hexachloroethane in 3mL dry CH2Cl2 in an ice-water bathAfter five minutes the bath was removed The reaction was stirred continu-ously for 1 h at room temperature then dichlorotriphenylphosphorane 3 wasproduced Yield 90 (by 31P NMR spectral integral) 31P NMR 611 ppm

N-PHOSPHOPEPTIDES 2071

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Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters 5

Under a nitrogen atmosphere 12mmol dichlorotriphenylphosphor-ane 3 in CH2Cl2 was added dropwise to a stirred dry CH2Cl2 (5mL) solutionof 08mmol N-(diisopropyloxyphosphoryl)amino acid 2 08mmolamino acid methyl ester and 350 mL dry Et3N at room temperature Extraaddition of adequate Et3N was followed after half an hour The stirring wascontinued for 1 h After the removal of solvent the resultant residue wasdissolved in 15mL ethyl acetate then followed by the filtration of residuetriethylamine hydrochloride The filtrate was washed with 4mL 1molLcitric acid 4mL saturated NaCl solution 4mL 5 NaHCO3 solutionand 4mL H2O

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptides 6

Method A The above filtrate by ethyl acetate was evaporated to dry-ness A mixture of 35mL Et3N and 35mL H2O was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester 5

and stirred at 40C for 35 h The water layer was washed with Et3N(3 4mL) followed by addition of 15 equivalents NaOH The mixturewas stirred for ten minutes then washed with CH2Cl2 (3 5mL) and acid-ified to pH 3 with 1molL HCl in an ice-water bath The nepheloid solutionwas extracted with ethyl acetate (3 15mL) The organic layer was washedwith water brine and dried over anhydrous MgSO4 Removal of the solventafforded the pure products 6andashc as white solid

Spectral data for compounds 6andashc

N-DIPP-Phe-Gly 6a yield 852 31P NMR (810MHz AcOEt) d(ppm) 695 1H NMR (500MHz CDCl3) d (ppm) 109ndash125 (m 12H(P(OCH(CH3)2)2) 297ndash311 (m 2H CH2Ph) 378ndash383 (m 2HCH2COOH) 404ndash416 (br 1H CO-NH) 419 (q Jfrac14 57Hz3JP-Hfrac14 186Hz 1H CHCH2Ph) 427ndash438 (m 2H P(OCH(CH3)2)2)718ndash729 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 2352(br OCH(CH3)2) 4030 (d 3JP-Cfrac14 50Hz CH2Ph) 4149 ( CH2COOH) 5678(CHCH2Ph) 7107 (q 2JP-Cfrac14 61Hz P(OCH(CH3)2)2) 12680ndash13653 (Ph)17223 (COOH) 17240 (d 3JP-Cfrac14 44Hz CO-NH) Positive-ion ESI-MS(mz) 3871 (MthornH)thorn Negative-ion ESI-MS (mz) 3851 (M-H)

N-DIPP-Phe-Ala 6b yield 607 31P NMR (810MHz AcOEt) d(ppm) 721 1H NMR (500MHz CDCl3) d (ppm) 112 (d Jfrac14 60 Hz 3HCHCH3) 115ndash131 (m 12H (P(OCH(CH3)2)2) 301ndash308 (m 2H CH2Ph)374 (t 1H CHCH3) 406 (br 1H CO-NH) 428ndash441 (m 2H

2072 DONG FU AND ZHAO

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

N-PHOSPHOPEPTIDES 2073

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

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6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

N-PHOSPHOPEPTIDES 2075

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the investigation of phosphorylation or as important posttranslationalmodification of peptides and proteins7

In order to decipher the role of many phosphopeptides and exploretheir biological activities a facile method to acqurie multi-gram quantitiesof phosphopeptides is in high demand In general N-(dialkoxyphosphor-yl)peptides were prepared by phosphorylating the peptides obtained by thedeprotection of N-protected peptides such as N-Boc-peptides4 HoweverN-(dialkoxyphosphoryl)amino acids have hardly been used for the synthesisof N-(dialkoxyphosphoryl)peptides probably because it is difficult tosynthesize and purify them Fortunately an efficient method to preparehighly pure and large quantities of N-phosphoamino acids has been devel-oped in our lab8

Furthermore it has been proved that the phosphinic carboxylic mixedanhydride method had the superiority in peptide synthesis9 than the mixedcarboxylic anhydride method for the faster coupling rate more regiospeci-ficity in nucleophilic attack and more thermal stability to disproportiona-tion Hence the methodology of N-phosphopeptide synthesis starting fromN-phosphoamino acid and using dichlorotriphenylphosphorane 3 as thecoupling reagent was investigated

RESULTS AND DISCUSSION

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters

5 (N-DIPP-dipeptide Methyl Esters)

Dichlorotriphenylphosphorane 3 which was assigned to the ionisedform according to 31P NMR spectrum (dPfrac14 611 ppm) is either commer-cially available or can readily be prepared by the reaction of tri-phenylphosphinewithhexachloroethanewithout anyundesirable phosphorusby-product (Scheme 1)10

15 equivalents of in-situ generated dichlorotriphenylphosphorane 3

was added to 1 equivalent of N-(diisopropyloxyphosphoryl)amino acid 2

(N-DIPP-amino acid 2) amino acid methyl ester and triethylamine(Et3N) in CH2Cl2 at room temperature The mixture was stirred for 1 h

2068 DONG FU AND ZHAO

Scheme 1 Synthetic Pathway of Dichlorotriphenylphosphorane 3

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and compound 2 and amino acid ester almost transferred into N-DIPP-dipeptide methyl ester 5 in quantitative amount (indicated by the singlesignal at about 31P NMR 68 ppm of compound 5) (Scheme 2) The activa-tion mechanism was proposed that dichlorotriphenylphosphorane 3 phos-phorylated N-DIPP-amino acid 2 to give the phosphinic carboxylic mixedanhydride 4 followed by the nucleophilic attack of amino acid methyl esterresulting into the formation of peptide bond11 Excessive dichlorotriphenyl-phosphorane 3 would consume the remnant water to guarantee the anhy-drous environment and make the coupling reaction complete Moreover itwould not bring in further by-products because 3 can react with H2O toform triphenylphosphine oxide which is the same as the product yielded incoupling reaction

Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

In order to avoid the side reactions on N-protecting group DIPP(diisopropyloxyphosphoryl) triethylamine as a weak base was the firstchoice to catalyze the hydrolysis of compound 5 (Scheme 3)12 Yet it wasonly efficient for the hydrolysis of compound 5a 5b and 5c Hydrolysis ofcompound 5d 5e 5f however was completed in a stronger base diluteNaOH aqueous solution with a longer time as shown in Table 1 Table 1

shows that the only difference between 5a 5b and 5d 5e 5f lies in the sidechain of the amino acid residue on C-terminal And the nuance betweencompound 5c and 5e is the addition of phenyl on the side-chain of the firstamino acid residue These show that the hydrolysis reaction is stronglydependent upon the dual side-chains

The hydrolysis reaction was carried out in aqueous-organic mixed sol-vents to facilitate the tracing of reaction process by 31P NMR techniquesN-DIPP-dipeptide methyl esters 5 are organic soluble species while the

N-PHOSPHOPEPTIDES 2069

Scheme 2 Synthetic Pathway of N-DIPP-dipeptide Methyl Esters 5

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basic hydrolyzed products 6 are the corresponding salts which are aqueoussoluble As the hydrolysis proceeding the signal at about 31PNMR 68 ppm ofcompound 5 in organic phase decreased At the end point there was no com-pound 5 left therefore no 31PNMR signal of compound 5 could be detected inthe organic phase Whilst triphenylphosphine oxide was imprisoned inorganic phase (the signal at about 31P NMR 280 ppm of Ph3Pfrac14O onlyexisted in the organic phase) So the major impurity was removed completelyDichlorotriphenylphosphorane 3 gives cleaner reactions due to the highsensitivity towards the atmosphere and the exclusive resultant triphenyl-phosphine oxide Hence after eliminating the main impurity Ph3Pfrac14O wegot highly pure N-phosphodipeptides 6andashf using simple extraction workup

The purity and structure of compounds 6andashf were determined by1H NMR 13C NMR 31P NMR and ESI-MS methods Each target productgave only one 31P NMR signal implying that no racemization occurredduring this peptide bond formation13 These highly optical pure N-DIPP-dipeptides 6andashf could be used for elongation of N-phosphopeptides orandtest for biological activities

In conclusion dichlorotriphenylphosphorane 3 is an effective couplingreagent for synthesis of N-phosphopeptides The present method enablesus to obtain N-phosphopeptides on a large-scale and highly pure productionbecause of its availability convenience and low racemization Furtherthe N-phosphopeptides could be deprotected under acid conditions14 toyield the corresponding peptides Thus the convenient and efficient approachcould be also applied for the synthesis of polypeptides

2070 DONG FU AND ZHAO

Table 1 Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

Final

Poduct 6 R1 R2 Base

Time

(h)

Overall

Yield ()

DIPP-Phe-Gly a -CH2-Ph -H Et3N 35 852

DIPP-Phe-Ala b -CH2-Ph -CH3 Et3N 35 607DIPP-Ala-Phe c -CH3 -CH2-Ph Et3N 35 600DIPP-Phe-Leu d -CH2-Ph -CH2-Pr

1 NaOH (017N) 70 803

DIPP-Phe-Phe e -CH2-Ph -CH2-Ph NaOH (017N) 70 758DIPP-Phe-Val f -CH2-Ph -Pr1 NaOH (065N) 90 882

Scheme 3 Synthetic Pathway of N-DIPP-dipeptides 6

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EXPERIMENTAL

General Procedure

All glassware was dried in an oven for at least 3 h at 120C prior to useAir sensitive materials were transferred under a nitrogen atmosphereDichloromethane and triethylamine were dried over P2O5 and CaH2

respectively 1H MNR and 13C NMR spectra were recorded on BrukerAM 500 spectrometer TMS (dfrac14 00) and CDCl3 (dfrac14 770 ppm) werereferences for 1H and 13C NMR spectra respectively 13C NMR spectrawere all taken under 1H decoupled and 31P coupled conditions 31P NMRspectra were taken on Bruker AC 200 spectrometer at 81MHz under1H decoupled conditions 31P NMR chemical shifts are reported in ppmdownfield (thorn) or upfield () from external 85 H3PO4 as reference Massspectra were conducted on Bruker Esquire-LC mass spectrometer in positiveand negative ion mode

Synthesis of Diisopropyl Phosphite (DIPPH) 1

The starting material DIPPH 1 was prepared according to the pub-lished procedure15 Yield 75 31PNMR 44 ppm

Synthesis of N-(Diisopropyloxyphosphoryl)amino Acids 2

N-DIPP-amino acid 2 was synthesized by adding diisopropylphosphite 1 to an aqueous-organic mixture containing the amino acidwater carbon tetrachloride triethylamine and ethanol well stirring themixture at 0ndash20C for a few hours and quenching the reaction by acidifica-tion8 Yields DIPP-Ala 89 DIPP-Phe 93 31P NMR 71 69 ppmrespectively

Synthesis of Dichlorotriphenylphosphorane 3

According to literature10 under a nitrogen atmosphere 2mmoltriphenylphosphine in 3mL dry CH2Cl2 was added dropwise to a stirred2mmol hexachloroethane in 3mL dry CH2Cl2 in an ice-water bathAfter five minutes the bath was removed The reaction was stirred continu-ously for 1 h at room temperature then dichlorotriphenylphosphorane 3 wasproduced Yield 90 (by 31P NMR spectral integral) 31P NMR 611 ppm

N-PHOSPHOPEPTIDES 2071

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Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters 5

Under a nitrogen atmosphere 12mmol dichlorotriphenylphosphor-ane 3 in CH2Cl2 was added dropwise to a stirred dry CH2Cl2 (5mL) solutionof 08mmol N-(diisopropyloxyphosphoryl)amino acid 2 08mmolamino acid methyl ester and 350 mL dry Et3N at room temperature Extraaddition of adequate Et3N was followed after half an hour The stirring wascontinued for 1 h After the removal of solvent the resultant residue wasdissolved in 15mL ethyl acetate then followed by the filtration of residuetriethylamine hydrochloride The filtrate was washed with 4mL 1molLcitric acid 4mL saturated NaCl solution 4mL 5 NaHCO3 solutionand 4mL H2O

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptides 6

Method A The above filtrate by ethyl acetate was evaporated to dry-ness A mixture of 35mL Et3N and 35mL H2O was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester 5

and stirred at 40C for 35 h The water layer was washed with Et3N(3 4mL) followed by addition of 15 equivalents NaOH The mixturewas stirred for ten minutes then washed with CH2Cl2 (3 5mL) and acid-ified to pH 3 with 1molL HCl in an ice-water bath The nepheloid solutionwas extracted with ethyl acetate (3 15mL) The organic layer was washedwith water brine and dried over anhydrous MgSO4 Removal of the solventafforded the pure products 6andashc as white solid

Spectral data for compounds 6andashc

N-DIPP-Phe-Gly 6a yield 852 31P NMR (810MHz AcOEt) d(ppm) 695 1H NMR (500MHz CDCl3) d (ppm) 109ndash125 (m 12H(P(OCH(CH3)2)2) 297ndash311 (m 2H CH2Ph) 378ndash383 (m 2HCH2COOH) 404ndash416 (br 1H CO-NH) 419 (q Jfrac14 57Hz3JP-Hfrac14 186Hz 1H CHCH2Ph) 427ndash438 (m 2H P(OCH(CH3)2)2)718ndash729 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 2352(br OCH(CH3)2) 4030 (d 3JP-Cfrac14 50Hz CH2Ph) 4149 ( CH2COOH) 5678(CHCH2Ph) 7107 (q 2JP-Cfrac14 61Hz P(OCH(CH3)2)2) 12680ndash13653 (Ph)17223 (COOH) 17240 (d 3JP-Cfrac14 44Hz CO-NH) Positive-ion ESI-MS(mz) 3871 (MthornH)thorn Negative-ion ESI-MS (mz) 3851 (M-H)

N-DIPP-Phe-Ala 6b yield 607 31P NMR (810MHz AcOEt) d(ppm) 721 1H NMR (500MHz CDCl3) d (ppm) 112 (d Jfrac14 60 Hz 3HCHCH3) 115ndash131 (m 12H (P(OCH(CH3)2)2) 301ndash308 (m 2H CH2Ph)374 (t 1H CHCH3) 406 (br 1H CO-NH) 428ndash441 (m 2H

2072 DONG FU AND ZHAO

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

N-PHOSPHOPEPTIDES 2073

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

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6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

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and compound 2 and amino acid ester almost transferred into N-DIPP-dipeptide methyl ester 5 in quantitative amount (indicated by the singlesignal at about 31P NMR 68 ppm of compound 5) (Scheme 2) The activa-tion mechanism was proposed that dichlorotriphenylphosphorane 3 phos-phorylated N-DIPP-amino acid 2 to give the phosphinic carboxylic mixedanhydride 4 followed by the nucleophilic attack of amino acid methyl esterresulting into the formation of peptide bond11 Excessive dichlorotriphenyl-phosphorane 3 would consume the remnant water to guarantee the anhy-drous environment and make the coupling reaction complete Moreover itwould not bring in further by-products because 3 can react with H2O toform triphenylphosphine oxide which is the same as the product yielded incoupling reaction

Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

In order to avoid the side reactions on N-protecting group DIPP(diisopropyloxyphosphoryl) triethylamine as a weak base was the firstchoice to catalyze the hydrolysis of compound 5 (Scheme 3)12 Yet it wasonly efficient for the hydrolysis of compound 5a 5b and 5c Hydrolysis ofcompound 5d 5e 5f however was completed in a stronger base diluteNaOH aqueous solution with a longer time as shown in Table 1 Table 1

shows that the only difference between 5a 5b and 5d 5e 5f lies in the sidechain of the amino acid residue on C-terminal And the nuance betweencompound 5c and 5e is the addition of phenyl on the side-chain of the firstamino acid residue These show that the hydrolysis reaction is stronglydependent upon the dual side-chains

The hydrolysis reaction was carried out in aqueous-organic mixed sol-vents to facilitate the tracing of reaction process by 31P NMR techniquesN-DIPP-dipeptide methyl esters 5 are organic soluble species while the

N-PHOSPHOPEPTIDES 2069

Scheme 2 Synthetic Pathway of N-DIPP-dipeptide Methyl Esters 5

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basic hydrolyzed products 6 are the corresponding salts which are aqueoussoluble As the hydrolysis proceeding the signal at about 31PNMR 68 ppm ofcompound 5 in organic phase decreased At the end point there was no com-pound 5 left therefore no 31PNMR signal of compound 5 could be detected inthe organic phase Whilst triphenylphosphine oxide was imprisoned inorganic phase (the signal at about 31P NMR 280 ppm of Ph3Pfrac14O onlyexisted in the organic phase) So the major impurity was removed completelyDichlorotriphenylphosphorane 3 gives cleaner reactions due to the highsensitivity towards the atmosphere and the exclusive resultant triphenyl-phosphine oxide Hence after eliminating the main impurity Ph3Pfrac14O wegot highly pure N-phosphodipeptides 6andashf using simple extraction workup

The purity and structure of compounds 6andashf were determined by1H NMR 13C NMR 31P NMR and ESI-MS methods Each target productgave only one 31P NMR signal implying that no racemization occurredduring this peptide bond formation13 These highly optical pure N-DIPP-dipeptides 6andashf could be used for elongation of N-phosphopeptides orandtest for biological activities

In conclusion dichlorotriphenylphosphorane 3 is an effective couplingreagent for synthesis of N-phosphopeptides The present method enablesus to obtain N-phosphopeptides on a large-scale and highly pure productionbecause of its availability convenience and low racemization Furtherthe N-phosphopeptides could be deprotected under acid conditions14 toyield the corresponding peptides Thus the convenient and efficient approachcould be also applied for the synthesis of polypeptides

2070 DONG FU AND ZHAO

Table 1 Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

Final

Poduct 6 R1 R2 Base

Time

(h)

Overall

Yield ()

DIPP-Phe-Gly a -CH2-Ph -H Et3N 35 852

DIPP-Phe-Ala b -CH2-Ph -CH3 Et3N 35 607DIPP-Ala-Phe c -CH3 -CH2-Ph Et3N 35 600DIPP-Phe-Leu d -CH2-Ph -CH2-Pr

1 NaOH (017N) 70 803

DIPP-Phe-Phe e -CH2-Ph -CH2-Ph NaOH (017N) 70 758DIPP-Phe-Val f -CH2-Ph -Pr1 NaOH (065N) 90 882

Scheme 3 Synthetic Pathway of N-DIPP-dipeptides 6

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EXPERIMENTAL

General Procedure

All glassware was dried in an oven for at least 3 h at 120C prior to useAir sensitive materials were transferred under a nitrogen atmosphereDichloromethane and triethylamine were dried over P2O5 and CaH2

respectively 1H MNR and 13C NMR spectra were recorded on BrukerAM 500 spectrometer TMS (dfrac14 00) and CDCl3 (dfrac14 770 ppm) werereferences for 1H and 13C NMR spectra respectively 13C NMR spectrawere all taken under 1H decoupled and 31P coupled conditions 31P NMRspectra were taken on Bruker AC 200 spectrometer at 81MHz under1H decoupled conditions 31P NMR chemical shifts are reported in ppmdownfield (thorn) or upfield () from external 85 H3PO4 as reference Massspectra were conducted on Bruker Esquire-LC mass spectrometer in positiveand negative ion mode

Synthesis of Diisopropyl Phosphite (DIPPH) 1

The starting material DIPPH 1 was prepared according to the pub-lished procedure15 Yield 75 31PNMR 44 ppm

Synthesis of N-(Diisopropyloxyphosphoryl)amino Acids 2

N-DIPP-amino acid 2 was synthesized by adding diisopropylphosphite 1 to an aqueous-organic mixture containing the amino acidwater carbon tetrachloride triethylamine and ethanol well stirring themixture at 0ndash20C for a few hours and quenching the reaction by acidifica-tion8 Yields DIPP-Ala 89 DIPP-Phe 93 31P NMR 71 69 ppmrespectively

Synthesis of Dichlorotriphenylphosphorane 3

According to literature10 under a nitrogen atmosphere 2mmoltriphenylphosphine in 3mL dry CH2Cl2 was added dropwise to a stirred2mmol hexachloroethane in 3mL dry CH2Cl2 in an ice-water bathAfter five minutes the bath was removed The reaction was stirred continu-ously for 1 h at room temperature then dichlorotriphenylphosphorane 3 wasproduced Yield 90 (by 31P NMR spectral integral) 31P NMR 611 ppm

N-PHOSPHOPEPTIDES 2071

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Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters 5

Under a nitrogen atmosphere 12mmol dichlorotriphenylphosphor-ane 3 in CH2Cl2 was added dropwise to a stirred dry CH2Cl2 (5mL) solutionof 08mmol N-(diisopropyloxyphosphoryl)amino acid 2 08mmolamino acid methyl ester and 350 mL dry Et3N at room temperature Extraaddition of adequate Et3N was followed after half an hour The stirring wascontinued for 1 h After the removal of solvent the resultant residue wasdissolved in 15mL ethyl acetate then followed by the filtration of residuetriethylamine hydrochloride The filtrate was washed with 4mL 1molLcitric acid 4mL saturated NaCl solution 4mL 5 NaHCO3 solutionand 4mL H2O

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptides 6

Method A The above filtrate by ethyl acetate was evaporated to dry-ness A mixture of 35mL Et3N and 35mL H2O was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester 5

and stirred at 40C for 35 h The water layer was washed with Et3N(3 4mL) followed by addition of 15 equivalents NaOH The mixturewas stirred for ten minutes then washed with CH2Cl2 (3 5mL) and acid-ified to pH 3 with 1molL HCl in an ice-water bath The nepheloid solutionwas extracted with ethyl acetate (3 15mL) The organic layer was washedwith water brine and dried over anhydrous MgSO4 Removal of the solventafforded the pure products 6andashc as white solid

Spectral data for compounds 6andashc

N-DIPP-Phe-Gly 6a yield 852 31P NMR (810MHz AcOEt) d(ppm) 695 1H NMR (500MHz CDCl3) d (ppm) 109ndash125 (m 12H(P(OCH(CH3)2)2) 297ndash311 (m 2H CH2Ph) 378ndash383 (m 2HCH2COOH) 404ndash416 (br 1H CO-NH) 419 (q Jfrac14 57Hz3JP-Hfrac14 186Hz 1H CHCH2Ph) 427ndash438 (m 2H P(OCH(CH3)2)2)718ndash729 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 2352(br OCH(CH3)2) 4030 (d 3JP-Cfrac14 50Hz CH2Ph) 4149 ( CH2COOH) 5678(CHCH2Ph) 7107 (q 2JP-Cfrac14 61Hz P(OCH(CH3)2)2) 12680ndash13653 (Ph)17223 (COOH) 17240 (d 3JP-Cfrac14 44Hz CO-NH) Positive-ion ESI-MS(mz) 3871 (MthornH)thorn Negative-ion ESI-MS (mz) 3851 (M-H)

N-DIPP-Phe-Ala 6b yield 607 31P NMR (810MHz AcOEt) d(ppm) 721 1H NMR (500MHz CDCl3) d (ppm) 112 (d Jfrac14 60 Hz 3HCHCH3) 115ndash131 (m 12H (P(OCH(CH3)2)2) 301ndash308 (m 2H CH2Ph)374 (t 1H CHCH3) 406 (br 1H CO-NH) 428ndash441 (m 2H

2072 DONG FU AND ZHAO

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

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6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

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Reprints of this article can also be ordered at

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Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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basic hydrolyzed products 6 are the corresponding salts which are aqueoussoluble As the hydrolysis proceeding the signal at about 31PNMR 68 ppm ofcompound 5 in organic phase decreased At the end point there was no com-pound 5 left therefore no 31PNMR signal of compound 5 could be detected inthe organic phase Whilst triphenylphosphine oxide was imprisoned inorganic phase (the signal at about 31P NMR 280 ppm of Ph3Pfrac14O onlyexisted in the organic phase) So the major impurity was removed completelyDichlorotriphenylphosphorane 3 gives cleaner reactions due to the highsensitivity towards the atmosphere and the exclusive resultant triphenyl-phosphine oxide Hence after eliminating the main impurity Ph3Pfrac14O wegot highly pure N-phosphodipeptides 6andashf using simple extraction workup

The purity and structure of compounds 6andashf were determined by1H NMR 13C NMR 31P NMR and ESI-MS methods Each target productgave only one 31P NMR signal implying that no racemization occurredduring this peptide bond formation13 These highly optical pure N-DIPP-dipeptides 6andashf could be used for elongation of N-phosphopeptides orandtest for biological activities

In conclusion dichlorotriphenylphosphorane 3 is an effective couplingreagent for synthesis of N-phosphopeptides The present method enablesus to obtain N-phosphopeptides on a large-scale and highly pure productionbecause of its availability convenience and low racemization Furtherthe N-phosphopeptides could be deprotected under acid conditions14 toyield the corresponding peptides Thus the convenient and efficient approachcould be also applied for the synthesis of polypeptides

2070 DONG FU AND ZHAO

Table 1 Hydrolysis of N-DIPP-dipeptide Methyl Esters 5

Final

Poduct 6 R1 R2 Base

Time

(h)

Overall

Yield ()

DIPP-Phe-Gly a -CH2-Ph -H Et3N 35 852

DIPP-Phe-Ala b -CH2-Ph -CH3 Et3N 35 607DIPP-Ala-Phe c -CH3 -CH2-Ph Et3N 35 600DIPP-Phe-Leu d -CH2-Ph -CH2-Pr

1 NaOH (017N) 70 803

DIPP-Phe-Phe e -CH2-Ph -CH2-Ph NaOH (017N) 70 758DIPP-Phe-Val f -CH2-Ph -Pr1 NaOH (065N) 90 882

Scheme 3 Synthetic Pathway of N-DIPP-dipeptides 6

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EXPERIMENTAL

General Procedure

All glassware was dried in an oven for at least 3 h at 120C prior to useAir sensitive materials were transferred under a nitrogen atmosphereDichloromethane and triethylamine were dried over P2O5 and CaH2

respectively 1H MNR and 13C NMR spectra were recorded on BrukerAM 500 spectrometer TMS (dfrac14 00) and CDCl3 (dfrac14 770 ppm) werereferences for 1H and 13C NMR spectra respectively 13C NMR spectrawere all taken under 1H decoupled and 31P coupled conditions 31P NMRspectra were taken on Bruker AC 200 spectrometer at 81MHz under1H decoupled conditions 31P NMR chemical shifts are reported in ppmdownfield (thorn) or upfield () from external 85 H3PO4 as reference Massspectra were conducted on Bruker Esquire-LC mass spectrometer in positiveand negative ion mode

Synthesis of Diisopropyl Phosphite (DIPPH) 1

The starting material DIPPH 1 was prepared according to the pub-lished procedure15 Yield 75 31PNMR 44 ppm

Synthesis of N-(Diisopropyloxyphosphoryl)amino Acids 2

N-DIPP-amino acid 2 was synthesized by adding diisopropylphosphite 1 to an aqueous-organic mixture containing the amino acidwater carbon tetrachloride triethylamine and ethanol well stirring themixture at 0ndash20C for a few hours and quenching the reaction by acidifica-tion8 Yields DIPP-Ala 89 DIPP-Phe 93 31P NMR 71 69 ppmrespectively

Synthesis of Dichlorotriphenylphosphorane 3

According to literature10 under a nitrogen atmosphere 2mmoltriphenylphosphine in 3mL dry CH2Cl2 was added dropwise to a stirred2mmol hexachloroethane in 3mL dry CH2Cl2 in an ice-water bathAfter five minutes the bath was removed The reaction was stirred continu-ously for 1 h at room temperature then dichlorotriphenylphosphorane 3 wasproduced Yield 90 (by 31P NMR spectral integral) 31P NMR 611 ppm

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Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters 5

Under a nitrogen atmosphere 12mmol dichlorotriphenylphosphor-ane 3 in CH2Cl2 was added dropwise to a stirred dry CH2Cl2 (5mL) solutionof 08mmol N-(diisopropyloxyphosphoryl)amino acid 2 08mmolamino acid methyl ester and 350 mL dry Et3N at room temperature Extraaddition of adequate Et3N was followed after half an hour The stirring wascontinued for 1 h After the removal of solvent the resultant residue wasdissolved in 15mL ethyl acetate then followed by the filtration of residuetriethylamine hydrochloride The filtrate was washed with 4mL 1molLcitric acid 4mL saturated NaCl solution 4mL 5 NaHCO3 solutionand 4mL H2O

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptides 6

Method A The above filtrate by ethyl acetate was evaporated to dry-ness A mixture of 35mL Et3N and 35mL H2O was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester 5

and stirred at 40C for 35 h The water layer was washed with Et3N(3 4mL) followed by addition of 15 equivalents NaOH The mixturewas stirred for ten minutes then washed with CH2Cl2 (3 5mL) and acid-ified to pH 3 with 1molL HCl in an ice-water bath The nepheloid solutionwas extracted with ethyl acetate (3 15mL) The organic layer was washedwith water brine and dried over anhydrous MgSO4 Removal of the solventafforded the pure products 6andashc as white solid

Spectral data for compounds 6andashc

N-DIPP-Phe-Gly 6a yield 852 31P NMR (810MHz AcOEt) d(ppm) 695 1H NMR (500MHz CDCl3) d (ppm) 109ndash125 (m 12H(P(OCH(CH3)2)2) 297ndash311 (m 2H CH2Ph) 378ndash383 (m 2HCH2COOH) 404ndash416 (br 1H CO-NH) 419 (q Jfrac14 57Hz3JP-Hfrac14 186Hz 1H CHCH2Ph) 427ndash438 (m 2H P(OCH(CH3)2)2)718ndash729 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 2352(br OCH(CH3)2) 4030 (d 3JP-Cfrac14 50Hz CH2Ph) 4149 ( CH2COOH) 5678(CHCH2Ph) 7107 (q 2JP-Cfrac14 61Hz P(OCH(CH3)2)2) 12680ndash13653 (Ph)17223 (COOH) 17240 (d 3JP-Cfrac14 44Hz CO-NH) Positive-ion ESI-MS(mz) 3871 (MthornH)thorn Negative-ion ESI-MS (mz) 3851 (M-H)

N-DIPP-Phe-Ala 6b yield 607 31P NMR (810MHz AcOEt) d(ppm) 721 1H NMR (500MHz CDCl3) d (ppm) 112 (d Jfrac14 60 Hz 3HCHCH3) 115ndash131 (m 12H (P(OCH(CH3)2)2) 301ndash308 (m 2H CH2Ph)374 (t 1H CHCH3) 406 (br 1H CO-NH) 428ndash441 (m 2H

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

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6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

N-PHOSPHOPEPTIDES 2075

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Order now

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081SCC100104428

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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014

ORDER REPRINTS

EXPERIMENTAL

General Procedure

All glassware was dried in an oven for at least 3 h at 120C prior to useAir sensitive materials were transferred under a nitrogen atmosphereDichloromethane and triethylamine were dried over P2O5 and CaH2

respectively 1H MNR and 13C NMR spectra were recorded on BrukerAM 500 spectrometer TMS (dfrac14 00) and CDCl3 (dfrac14 770 ppm) werereferences for 1H and 13C NMR spectra respectively 13C NMR spectrawere all taken under 1H decoupled and 31P coupled conditions 31P NMRspectra were taken on Bruker AC 200 spectrometer at 81MHz under1H decoupled conditions 31P NMR chemical shifts are reported in ppmdownfield (thorn) or upfield () from external 85 H3PO4 as reference Massspectra were conducted on Bruker Esquire-LC mass spectrometer in positiveand negative ion mode

Synthesis of Diisopropyl Phosphite (DIPPH) 1

The starting material DIPPH 1 was prepared according to the pub-lished procedure15 Yield 75 31PNMR 44 ppm

Synthesis of N-(Diisopropyloxyphosphoryl)amino Acids 2

N-DIPP-amino acid 2 was synthesized by adding diisopropylphosphite 1 to an aqueous-organic mixture containing the amino acidwater carbon tetrachloride triethylamine and ethanol well stirring themixture at 0ndash20C for a few hours and quenching the reaction by acidifica-tion8 Yields DIPP-Ala 89 DIPP-Phe 93 31P NMR 71 69 ppmrespectively

Synthesis of Dichlorotriphenylphosphorane 3

According to literature10 under a nitrogen atmosphere 2mmoltriphenylphosphine in 3mL dry CH2Cl2 was added dropwise to a stirred2mmol hexachloroethane in 3mL dry CH2Cl2 in an ice-water bathAfter five minutes the bath was removed The reaction was stirred continu-ously for 1 h at room temperature then dichlorotriphenylphosphorane 3 wasproduced Yield 90 (by 31P NMR spectral integral) 31P NMR 611 ppm

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Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters 5

Under a nitrogen atmosphere 12mmol dichlorotriphenylphosphor-ane 3 in CH2Cl2 was added dropwise to a stirred dry CH2Cl2 (5mL) solutionof 08mmol N-(diisopropyloxyphosphoryl)amino acid 2 08mmolamino acid methyl ester and 350 mL dry Et3N at room temperature Extraaddition of adequate Et3N was followed after half an hour The stirring wascontinued for 1 h After the removal of solvent the resultant residue wasdissolved in 15mL ethyl acetate then followed by the filtration of residuetriethylamine hydrochloride The filtrate was washed with 4mL 1molLcitric acid 4mL saturated NaCl solution 4mL 5 NaHCO3 solutionand 4mL H2O

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptides 6

Method A The above filtrate by ethyl acetate was evaporated to dry-ness A mixture of 35mL Et3N and 35mL H2O was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester 5

and stirred at 40C for 35 h The water layer was washed with Et3N(3 4mL) followed by addition of 15 equivalents NaOH The mixturewas stirred for ten minutes then washed with CH2Cl2 (3 5mL) and acid-ified to pH 3 with 1molL HCl in an ice-water bath The nepheloid solutionwas extracted with ethyl acetate (3 15mL) The organic layer was washedwith water brine and dried over anhydrous MgSO4 Removal of the solventafforded the pure products 6andashc as white solid

Spectral data for compounds 6andashc

N-DIPP-Phe-Gly 6a yield 852 31P NMR (810MHz AcOEt) d(ppm) 695 1H NMR (500MHz CDCl3) d (ppm) 109ndash125 (m 12H(P(OCH(CH3)2)2) 297ndash311 (m 2H CH2Ph) 378ndash383 (m 2HCH2COOH) 404ndash416 (br 1H CO-NH) 419 (q Jfrac14 57Hz3JP-Hfrac14 186Hz 1H CHCH2Ph) 427ndash438 (m 2H P(OCH(CH3)2)2)718ndash729 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 2352(br OCH(CH3)2) 4030 (d 3JP-Cfrac14 50Hz CH2Ph) 4149 ( CH2COOH) 5678(CHCH2Ph) 7107 (q 2JP-Cfrac14 61Hz P(OCH(CH3)2)2) 12680ndash13653 (Ph)17223 (COOH) 17240 (d 3JP-Cfrac14 44Hz CO-NH) Positive-ion ESI-MS(mz) 3871 (MthornH)thorn Negative-ion ESI-MS (mz) 3851 (M-H)

N-DIPP-Phe-Ala 6b yield 607 31P NMR (810MHz AcOEt) d(ppm) 721 1H NMR (500MHz CDCl3) d (ppm) 112 (d Jfrac14 60 Hz 3HCHCH3) 115ndash131 (m 12H (P(OCH(CH3)2)2) 301ndash308 (m 2H CH2Ph)374 (t 1H CHCH3) 406 (br 1H CO-NH) 428ndash441 (m 2H

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

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6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

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Order now

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081SCC100104428

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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Synthesis of N-(Diisopropyloxyphosphoryl)dipeptide Methyl Esters 5

Under a nitrogen atmosphere 12mmol dichlorotriphenylphosphor-ane 3 in CH2Cl2 was added dropwise to a stirred dry CH2Cl2 (5mL) solutionof 08mmol N-(diisopropyloxyphosphoryl)amino acid 2 08mmolamino acid methyl ester and 350 mL dry Et3N at room temperature Extraaddition of adequate Et3N was followed after half an hour The stirring wascontinued for 1 h After the removal of solvent the resultant residue wasdissolved in 15mL ethyl acetate then followed by the filtration of residuetriethylamine hydrochloride The filtrate was washed with 4mL 1molLcitric acid 4mL saturated NaCl solution 4mL 5 NaHCO3 solutionand 4mL H2O

Synthesis of N-(Diisopropyloxyphosphoryl)dipeptides 6

Method A The above filtrate by ethyl acetate was evaporated to dry-ness A mixture of 35mL Et3N and 35mL H2O was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester 5

and stirred at 40C for 35 h The water layer was washed with Et3N(3 4mL) followed by addition of 15 equivalents NaOH The mixturewas stirred for ten minutes then washed with CH2Cl2 (3 5mL) and acid-ified to pH 3 with 1molL HCl in an ice-water bath The nepheloid solutionwas extracted with ethyl acetate (3 15mL) The organic layer was washedwith water brine and dried over anhydrous MgSO4 Removal of the solventafforded the pure products 6andashc as white solid

Spectral data for compounds 6andashc

N-DIPP-Phe-Gly 6a yield 852 31P NMR (810MHz AcOEt) d(ppm) 695 1H NMR (500MHz CDCl3) d (ppm) 109ndash125 (m 12H(P(OCH(CH3)2)2) 297ndash311 (m 2H CH2Ph) 378ndash383 (m 2HCH2COOH) 404ndash416 (br 1H CO-NH) 419 (q Jfrac14 57Hz3JP-Hfrac14 186Hz 1H CHCH2Ph) 427ndash438 (m 2H P(OCH(CH3)2)2)718ndash729 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 2352(br OCH(CH3)2) 4030 (d 3JP-Cfrac14 50Hz CH2Ph) 4149 ( CH2COOH) 5678(CHCH2Ph) 7107 (q 2JP-Cfrac14 61Hz P(OCH(CH3)2)2) 12680ndash13653 (Ph)17223 (COOH) 17240 (d 3JP-Cfrac14 44Hz CO-NH) Positive-ion ESI-MS(mz) 3871 (MthornH)thorn Negative-ion ESI-MS (mz) 3851 (M-H)

N-DIPP-Phe-Ala 6b yield 607 31P NMR (810MHz AcOEt) d(ppm) 721 1H NMR (500MHz CDCl3) d (ppm) 112 (d Jfrac14 60 Hz 3HCHCH3) 115ndash131 (m 12H (P(OCH(CH3)2)2) 301ndash308 (m 2H CH2Ph)374 (t 1H CHCH3) 406 (br 1H CO-NH) 428ndash441 (m 2H

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

Dow

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ORDER REPRINTS

6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

N-PHOSPHOPEPTIDES 2075

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Order now

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081SCC100104428

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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(P(OCH(CH3)2)2) 441ndash454 (m 1H CHCH2Ph) 719ndash748 (m 6H P-NHPh) 13C NMR (125MHz CDCl3) d (ppm) 1813 (CHCH3) 2351ndash2360(m (P(OCH( CH3)2)2) 3959 (d 3JP-Cfrac14 55Hz CH2Ph) 4814 (CHCOOH)5654 (CHCO-NH) 7200 (q 2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12683ndash13625(Ph) 17199 (d 3JP-Cfrac14 44Hz CO-NH) 17516 (COOH) Positive-ion ESI-MS (mz) 401 (MthornH)thorn Negative-ion ESI-MS (mz) 399 (M-H)

N-DIPP-Ala-Phe 6c yield 600 31P NMR (810MHz AcOEt) d(ppm) 685 1H NMR (500MHz CDCl3) d (ppm) 121 (d Jfrac14 45Hz 3HCHCH3 126ndash130 (m 12H (P(OCH(CH3)2)2) 293 (d Jfrac14 75Hz 2HCH2Ph) 454 (m 1H CHCOOH) 454 (br 1H CO-NH) 458ndash461 (m2H (P(OCH(CH3)2)2) 480 (m 1H CHCH3) 719ndash726 (m 6H P-NH Ph)13C NMR (125MHz CDCl3) d (ppm) 2067 (CHCH3) 2357ndash2373((P(OCH(CH3)2)2) 3745 (CH2Ph) 5142 (CHCOOH) 5327 (CHCH3)7206 (q 2JP-Cfrac14 53Hz (P(OCH(CH3)2)2) 12677ndash13655 (Ph) 17312 (d3JP-Cfrac14 55Hz CO-NH) 17420 (COOH) Positive-ion ESI-MS (mz) 401(M+H)+ Negative-ion ESI-MS (mz) 399 (M-H)

Method B The above filtrate by ethyl ecetate was evaporated todryness A mixture of 6mL NaOH aqueous solution (017molL for6d and 6e 065molL for 6f ) and 3mL CH2Cl2 was added to the resultingresidue containing N-(diisopropyloxyphosphoryl)dipeptide methyl ester5 and stirred at room temperature for 7 h The water layer was washedwith CH2Cl2 (3 4mL) The following isolation procedure was the sameas that of method A

Spectral data for compounds 6dndashf

N-DIPP-Phe-Leu 6d yield 803 31P NMR (810MHz AcOEt) d(ppm) 728 1H NMR (500MHz CDCl3) d (ppm) 089 (q Jfrac14 65 165Hz6H CH2CH(CH3)2) 113ndash125 (m 12H P(OCH(CH3)2)2) 144ndash155 (m2H CH2CH(CH3)2) 159ndash164 (m 1H CH2CH(CH3)2) 300ndash315 (m2H CH2Ph) 358 (t 1H CHCOOH) 407 (br 1H CO-NH) 432ndash443(m 2H P(OCH(CH3)2)2) 459 (q Jfrac14 78Hz 3JP-Hfrac14 138Hz 1HCHCH2pH) 719ndash738 (m 6H P-NH Ph) 13C NMR (125MHz CDCl3)d (ppm) 2229 2265 (CH2CH(CH3)2) 2350ndash2365 (P(OCH(CH3)2)2) 2452(CH2CH(CH3)2) 3930 (d 3JP-Cfrac14 45Hz CH2Ph) 4151 (CH2CH(CH3)2)5083 (CHCOOH) 5662 (CHCH2Ph) 7192ndash7213 (q 2JP-Cfrac14 59HzP(OCH(CH3)2)2) 12684ndash13626 (Ph) 17185 (d 3JP-Cfrac14 54Hz CO-NH)17526 (COOH) Positive-ion ESI-MS (mz) 4433 (M+H)+ Negative-ionESI-MS (mz) 4413 (M-H)

N-DIPP-Phe-Phe 6e yield 758 31P NMR (810MHz AcOEt) d(ppm) 693 1H NMR (500MHz CDCl3) d (ppm) 103ndash123 (m 12H(P(OCH(CH3)2)2) 289ndash307 (m 4H 2(CH2Ph)) 383 (t Jfrac14 108Hz 1H

N-PHOSPHOPEPTIDES 2073

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

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6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

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Order now

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All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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CHCOOH) 399 (br 1H CO-NH) 426ndash438 (m 2H (P(OCH(CH3)2)2)479 (q Jfrac14 65Hz 3JP-Cfrac14 125Hz 1H CHCO-NH) 707ndash739 (m 11HP-NH 2Ph) 13C NMR (125MHz CDCl3) d (ppm) 2345ndash2354((P(OCH(CH3)2)2) 3752 (PhCH2CHCOOH) 3951 (d 3JP-Cfrac14 49HzPhCH2CHCONH) 5346 (CHCOOH) 5683 (CHCONH) 7100 (q2JP-Cfrac14 61Hz (P(OCH(CH3)2)2) 12669ndash12648 (2 Ph) 17206 (d3JP-Cfrac14 51Hz CO-NH) 17370 (COOH) Positive-ion ESI-MS (mz) 477(M+H)+ Negative-ion ESI-MS (mz) 475 (M-H)

N-DIPP-Phe-Valf 6f yield 882 31 P NMR (810MHz AcOEt) d(ppm) 689 1H NMR (500MHz CDCl3) d (ppm) 086 (dd Jfrac14 70140Hz 6H CHCH(CH3)2) 108ndash119 (m 12H (P(OCH(CH3)2)2)213ndash218 (m 1H CHCH(CH3)2) 296ndash304 (m 2H CH2Ph) 410 (m 1HCHCOOH) 412 (br 1H CO-NH) 418ndash437 (m 2H (P(OCH(CH3)2)2)445 (q Jfrac14 50Hz 3JP-Hfrac14 85Hz 1H CHCH2Ph) 712ndash740 (m 6HP-NH Ph) 13C NMR (125MHz CDCl3) d (ppm) 1772 1853(CHCH(CH3)2) 2335ndash2346 ((P(OCH(CH3)2)2) 3092 (CHCH(CH3)2) 3967(d 3JP-Cfrac14 51Hz CH2Ph) 5658 (CHCOOH) 5734 (CHCH2Ph) 7164 (q2JP-Cfrac14 58Hz (P(OCH(CH3)2)2) 12659ndash 13641 (Ph) 17285 (d3JP-Cfrac14 43Hz CO-NH) 17496 (COOH) Positive-ion ESI-MS (mz) 429(M+H)+ Negative-ion ESI-MS (mz) 427 (M-H)

ACKNOWLEDGMENTS

The authors would like to thank the financial supports from theChinese National Natural Science Foundation (No 29902003) the Ministryof Science and Technology the Chinese Education Ministry andTsinghua University

REFERENCES

1 Nordmann J Mattioda G Loiseau G French Patent 7690 (1970)Ugine Kuhlmann CA 1972 76 144808

2 Nordmann J Mattioda G French Patent 3936 19683 French Patent 6430 (1968) Laboratories Sobio CA 1971 74 677124 Sakakibara S Yugari Y Hashimoto S European Patent 0085448

(1983) Ajinomoto Co CA 1984 100 520185 Camp NP Hawkins PCD Hitchcock PB Gani P Bioorg Med

Chem Lett 1992 2 1047

2074 DONG FU AND ZHAO

Dow

nloa

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vers

ity o

f So

uthe

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ORDER REPRINTS

6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

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Order now

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081SCC100104428

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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ctob

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014

ORDER REPRINTS

6 (a) Bartlett PA Marlowe CK Science 1987 235 569 (b) McleodDA Brinkworth RI Ashley JA Janda KD Wirshing PBioorg Med Chem Lett 1991 1 653

7 Tholey A Pipkorn R Zeppezauer M Reed J Lett in PeptideScience 1998 5 263

8 Ji GJ Xue CB Zeng JN Li LP Chai WG Zhao YFSynthesis 1988 6 444

9 Ramage R Hopton D Parrott MJ Richardson RS KennerGW Moore GA J Chem Soc Perkin Trans I 1985 461

10 (a) Appel R Scholer H Chem Ber 1977 110 2382 (b) Appel RWillms L Chem Ber 1979 112 1057

11 Wieland T Seeliger A Chem Ber 1971 104 399212 Valette G Pompon A Girardet JL Cappellacci L Franchetti

P Grifantini M LaColla P Loi AG Perigaud C Gosselin GImbach JL J Med Chem 1996 39 1981

13 Zeng JN Xue CB Chen QW Zhao YF Bioorg Chem 198917 434

14 Zhao YF Xi SK Song AT Ji GJ J Org Chem 1984 49 454915 Arbusov BA et al Iivestiya Acad Nauk SSSR okhn 1947 617

Received in the Netherlands September 14 2000

N-PHOSPHOPEPTIDES 2075

Dow

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014

Order now

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081SCC100104428

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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d] a

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014

Order now

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081SCC100104428

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request PermissionReprints Here link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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