Measuring wet bulb globe temperatures at point-of-exertion in worldwide UK military settings: a longitudinal observational study determining the accuracy of a portable WBGT monitor.

INTRODUCTION: Heat illness among the UK Armed Forces is usually exertional, and therefore preventable, yet the incidence has not reduced since 2011. JSP 539 explicitly states that wet bulb globe temperature (WBGT) should be measured 'at the location of greatest heat risk', not 'that of most convenience'. A handheld WBGT tracker used at point-of-exertion could reduce this incidence if proven to be as accurate as the current in-service device. METHODS: Longitudinal observational comparison and equipment feasibility study of the Kestrel 5400 and QUESTemp 34 (QT-34) in worldwide firm base and deployed UK Armed Forces locations. The locations chosen were Kenya, South Sudan, Belize, Tidworth, Aldershot and Brecon. Paired data points of WBGT readings were collected from November 2017 to August 2018 in all weather conditions. RESULTS: WBGT readings were comparable between the QT-34 and Kestrel 5400 across the UK and overseas. In addition, there was no change in accuracy between readings taken from the Kestrel 5400 when tripod-mounted and handheld. The Kestrel was easy to set up and far less susceptible to resupply or power supply limitations, as it requires no user input for wet bulb temperature, and runs on AA batteries. CONCLUSION: This equipment feasibility study has shown that the Kestrel 5400 gives an acceptable accuracy and is easier to use than the QT-34. The authors recommend that the Kestrel 5400 is introduced as an adjunct to the QT-34, and its use within the military setting monitored through ongoing comparative data collection in a large-scale proof-of-concept study.

Developing a multimodal biosensor for remote physiological monitoring.

INTRODUCTION: Several UK military expeditions have successfully used physiological sensors to monitor participant's physiological responses to challenging environmental conditions. This article describes the development and trial of a multimodal wearable biosensor that was used during the first all-female unassisted ski crossing of the Antarctic land mass. The project successfully transmitted remote real-time physiological data back to the UK. The ergonomic and technical lessons identified have informed recommendations for future wearable devices. METHOD: The biosensor devices were designed to be continuously worn against the skin and capture: HR, ECG, body surface temperature, bioimpedance, perspiration pH, sodium, lactate and glucose. The data were transmitted from the devices to an android smartphone using near-field technology. A custom-built App running on an android smartphone managed the secure transmission of the data to a UK research centre, using a commercially available satellite transceiver. RESULTS: Real-time physiological data, captured by the multimodal device, was successfully transmitted back to a UK research control centre on 6 occasions. Postexpedition feedback from the participants has contributed to the ergonomic and technical refinement of the next generation of devices. CONCLUSION: The future success of wearable technologies lies in establishing clinical confidence in the quality of the measured data and the accurate interpretation of those data in the context of the individual, the environment and activity being undertaken. In the near future, wearable physiological monitoring could improve point-of-care diagnostic accuracy and inform critical medical and command decisions.

The effect of added fat on the accuracy of Cobb angle measurements in CT SPR images: A phantom study.

INTRODUCTION: Adolescent idiopathic scoliosis (AIS) is a spinal deformity that mostly affects females aged between 10 and 17 years old. Cobb's method is the gold standard for assessing AIS. Being overweight is a common characteristic in AIS patients; therefore, the aim of this study is to investigate the effect fat mass has on the accuracy of Cobb angle measurements in 10-year-old female AIS patients. METHODS: A purpose-built phantom representing an AIS patient was scanned after adding several thicknesses of lard fat (0,2,4 and 8 cm). The phantom was scanned in an antero-posterior position using the scout mode of the CT scanner. 18 observers performed Cobb angle measurements on the images. RESULTS: The average Cobb angle at 0 cm of fat was 10.83° (SD = 3.06), at 2 cm it was 10.90° (SD = 3.16), at 4 cm it was 10.64° (SD = 3.06) and at 8 cm it was 10.88° (SD = 3.02). No significant difference was observed between the measurements at these thicknesses. CONCLUSION: Cobb angle measurements are not affected by the presence of fat. IMPLICATIONS FOR PRACTICE: When assessing overweight AIS patients, it not necessary to manipulate the acquisition parameters, which could lead to increased patient dose, in order to get more accurate Cobb angle measurement.

Effect of newly developed pigments and ultraviolet absorbers on the color change of pigmented silicone elastomer.

AIM AND OBJECTIVE: The aim and objective of the study is to evaluate effect of ultraviolet (UV) stabilizer (UV absorber Chimassorb 81) on color change of pigmented silicone elastomer when commercially available (red and yellow), and newly developed pigments (sicotrans red and sicopal brown) were used. MATERIALS AND METHODS: Two commercially available pigments - red (P112 Brilliant Red) and yellow (P106 Yellow) and two newly developed pigments - sicotrans red and sicopal brown were studied. In total eight groups made up of nine samples each were fabricated using elastomer with the combinations of the above pigments and UV stabilizer (Chimassorb 81). Groups 1, 3, 5, and 7 contain elastomer in combination with sicotrans red, sicopal brown, yellow and red pigments, respectively. Similarly, groups 2, 4, 6, and 8 along with elastomer and pigments (sicotrans sed, sicopal brown, yellow and red, respectively) contain the UV stabilizer (Chimassorb 81). Samples were subjected to aging in an accelerated weathering chamber (Weather-Ometer). Color values CIE (Commission Internationale d'Eclairage) L*, a*, and b * were measured at baseline and after 1000 h of weathering. Change in color (Delta E) was calculated. RESULTS: All groups showed a significant color change at 1000 h. Groups 1, 2, 3, and 4 showed a statistically significant less change in both colors (sico trans red and sicopal brown) compared to groups 5,6,7, and 8 (commercial pigments-Red and Yellow). Overall, the change in the color in groups with the UV stabilizer (Chimassorb) was less when compared to the groups where the stabilizer was not used. CONCLUSION: The newly developed pigment led to increased color stability as compared to commercially available pigments. Addition of UV stabilizer, Chimassorb led to a further reduction in color change of the pigmented elastomer.

Myasthenia gravis: An emerging toxicity of immune checkpoint inhibitors.

The advent of immunotherapy has heralded a number of significant advances in the treatment of particular malignancies associated with poor prognosis (melanoma, non-small-cell lung, renal and head/neck cancers). The success witnessed with therapeutic agents targeting cytotoxic T-lymphocyte-associated protein 4, programmed cell death protein 1 and programmed cell death ligand 1 immune checkpoints has inevitably led to an explosion in their clinical application and the subsequent recognition of specific toxicity profiles distinct from those long recognised with chemotherapy. Consequently, as the utility of such therapies broaden, understanding the nature, timing and management of these immune-related adverse events (irAEs) becomes increasingly significant. Although neurological irAEs are considered relatively rare in comparison with hepatitis, colitis, pneumonitis and endocrinopathies, one emerging side-effect is myasthenia gravis (MG). Among the 23 reported cases of immune checkpoint inhibitor-associated MG, 72.7% were de novo presentations, 18.2% were exacerbations of pre-existing MG and 9.1% were exacerbations of subclinical MG. The average onset of symptoms was within 6 weeks (range 2-12 weeks) of treatment initiation. In addition, there was no consistent association with elevated acetylcholine antibody titres and the development of immune checkpoint inhibitor-related MG. Significantly, there was a 30.4% MG-specific-related mortality, which further emphasises the importance of early recognition and robust treatment of this toxicity. In addition to a review of the existing literature, we present a new case of pembrolizumab-induced MG and provide insights into the underlying mechanisms of action of this phenomenon.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance.

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface.

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

Multi-centre analysis of incidental findings on low-resolution CT attenuation correction images.

OBJECTIVE: To review new incidental findings detected on low-resolution CT attenuation correction (CTAC) images acquired during single-photon emission CT (SPECT-CT) myocardial perfusion imaging (MPI) and to determine whether the CTAC images had diagnostic value and warrant reporting. METHODS: A multicentre study was performed in four UK nuclear medicine departments. CTAC images acquired as part of MPI performed using SPECT were evaluated to identify incidental findings. New findings considered to be clinically significant were evaluated further. Positive predictive value (PPV) was determined at the time of definitive diagnosis. RESULTS: Of 1819 patients studied, 497 (27.3%) had a positive CTAC finding. 51 (2.8%) patients had findings that were clinically significant at the time of the CTAC report and had not been previously diagnosed. Only four (0.2%) of these were potentially detrimental to patient outcome. CONCLUSION: One centre had a PPV of 0%, and the study suggests that these CTAC images should not be reported. Two centres with more modern equipment had low PPVs of 0% and 6%, respectively, and further research is suggested prior to drawing a conclusion. The centre with best quality CT had a PPV of 67%, and the study suggests that CTAC images from this equipment should be reported. ADVANCES IN KNOWLEDGE: This study is unique compared with previous studies that have reported only the potential to identify incidental findings on low-resolution CT images. This study both identifies and evaluates new clinically significant incidental findings, and it demonstrates that the benefit of reporting the CTAC images depends on the type of equipment used.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer.

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

An unblinded, randomised phase II study of platinum-based chemotherapy with vitamin B12 and folic acid supplementation in the treatment of lung cancer with plasma homocysteine blood levels as a biomarker of severe neutropenic toxicity.

BACKGROUND: Vitamin B12 and folic acid (referred to as vitamin supplementation) improves the toxicity profile of pemetrexed containing regimens. Low baseline vitamin B12 and folate levels are reflected in a raised total homocysteine level (HC). Studies have suggested that pretreatment HC levels predict neutropenia toxicity. We have tested supplementation with vitamin B12 and folate in non-pemetrexed platinum-based regimens to decrease treatment-related toxicity and looked for a correlation between toxicity and change in homocysteine levels. PATIENT AND METHOD: Eighty-three patients with advanced lung cancer and malignant mesothelioma were randomly assigned to receive platinum-based chemotherapy with (arm A) or without (arm B) vitamin B12 and folic acid supplementation. The primary end point was grade 3/4 neutropenia and death within 30 days of treatment. Secondary end points included quality of life, overall survival (OS) and the relationship between baseline and post supplementation HC levels and toxicity. RESULTS: In the intention-to-treat population, no significant difference was seen between the two groups with respect to chemotherapy-induced grade 3/4 neutropenia and death within 30 days of chemotherapy (36% vs 37%; p=0.966, emesis (2% vs 6%; p=0.9) or OS (12.3 months vs 7 months; p=0.41). There was no significant difference in survival rates by baseline HC level (p=0.9). Decrease in HC with vitamin supplementation was less frequent than expected. High baseline HC levels decreased with vitamin supplementation in only 9/36 (25%) patients (successful supplementation). Post hoc analysis showed that patients in arm A who were successfully supplemented (9/36=25%) had less neutropenic toxicity (0% vs 69%; p=0.02) compared to unsupplemented patients. CONCLUSIONS: The addition of vitamin B12 and folic acid to platinum-containing regimens did not overall improve the toxicity, quality of life or OS. Rates of grade 3/4 neutropenia at 36/37% was as predicted. Further studies to increase the rate of successful supplementation and to further test the biomarker potential of post supplementation HC levels in predicting chemotherapy-induced neutropenia in platinum-based chemotherapy are warranted. TRIAL REGISTRATION NUMBER: EudracCT 2005-002736-10 ISRCTN8734355.

A study of the decision outcomes and financial costs of multidisciplinary team meetings (MDMs) in oncology.

BACKGROUND: The benefits of multidisciplinary working in oncology are now accepted as the norm and widely accepted as being pivotal to the delivery of optimal cancer care. Central to this are the multidisciplinary meetings (MDMs) and we have evaluated decision outcomes and financial costs of these. METHODS: We reviewed the electronic patient records of 551 newly referred patients, discussed at 14 tumour site-specific MDMs for adult solid tumours and lymphoma (paediatric oncology and acute leukaemia were excluded) over a 1-month period, a total of 52 MDMs were studied. In addition, the records of a further 81 patients from 10 different MDMs were reviewed where the treating consultant had clearly recorded their opinion of how the patient should be managed and this was compared with the final MDM's consensus view. We also costed the MDMs utilising two different methodologies. RESULTS: The mean age of the 551 patients in the study was 62 years. In all, 536 (97.3%) patients were treatment naive before MDM discussion and 15 (2.7%) had prior treatment. Median time to treatment after the MDM was 16 days. In 535 (97.1%) cases, the MDM discussions were clearly documented, 16 (2.9%) were not clearly documented. In total, 319 (57.9%) patients were discussed once, and 232 (42.1%) were re-discussed (one to six occasions). In 62 (12.7%) patients, there were delays in MDM discussion, 30 (48.4%) were related to radiology, 26 (41.9%) to histopathology and 6 (9.7%) a combination of both. Adherence to the MDM management plan decision occurred 503 times (91.3%) with 48 (8.7%) deviations. In the smaller cohort of 81 patients, the consultant management plan and MDM consensus was compatible 71 (87.6%) times. On four occasions, there were major alterations in management while six were minor. The cost per month of our MDMs ranged from £2192 to £10 050 (median £5136) with total cost of £80 850 per month and the cost per new patient discussed was £415. CONCLUSION: Adherence to MDM decisions by health-care professionals occurs in the majority of patients. MDMs are costly, which may have relevance in the currently challenged health-care financial environment. There is a need to improve MDM efficiency without losing the considerable benefits associated with regular MDMs.

Chronic fatigue syndrome in the UK armed forces.

Chronic Fatigue Syndrome (CFS) is a debilitating condition that can have a significant impact on the lives of patients and those who care for them. In the UK Armed Forces this condition can also have a marked impact on a patient's career and their ability to function in the deployed environment. In this article the recognition and management of CFS will be discussed, as well as the occupational considerations within the UK Armed Forces.

A phase II study of ¹8F-fluorodeoxyglucose PET-CT in non-small cell lung cancer patients receiving erlotinib (Tarceva); objective and symptomatic responses at 6 and 12 weeks.

BACKGROUND: The aim of this study was to assess if (18)F-fluorodeoxyglucose (FDG) Positron Emission Tomography (PET)-CT scanning could minimise the time non-responding patients were exposed to erlotinib (Tarceva). METHODS: Patients were selected for clinical factors that would predict response to erlotinib. A FDG PET-CT and diagnostic contrast-enhanced (traditional) CT scan were carried out at baseline, and then a FDG PET-CT at 6 weeks and a traditional CT at 12 weeks were repeated. The primary end-point was rate of early progression in patients after 6 weeks, of which a minimum 12 out of 35 were required to make the study worthwhile. The responses at 6 (PET-CT) and 12 weeks (traditional CT) were compared and correlated with symptomatic response at both these time points. RESULTS: Forty seven patients were recruited with 38 and 33 patients assessable by FDG PET-CT at 6 weeks and traditional CT at 12weeks, respectively. There was good correlation between Partial response (PR) at both time points and all 10 patients who had a PR at 12 weeks had a PR at 6 weeks. Of the 13 patients with progressive disease (PD) at 12 weeks, seven had PD at 6 weeks and could have had their treatment stopped early. No evaluable patient with stable disease (SD) (8/38) or PD (9/38) on FDG PET-CT at 6 weeks went on to have a later response. Symptomatic response at 6 or 12 weeks did not correlate well with objective response on scanning at either time point. CONCLUSIONS: The primary end-point of this study was met as >12 (15/38) patients could have stopped treatment early on the basis of the FDG PET-CT scan result. A FDG PET-CT evaluable response of SD or PD at 6 weeks does predict future lack of response. No correlation was found between response and symptomatic response at either 6 or 12 weeks.

Time and chemotherapy treatment trends in the treatment of elderly patients (age>or=70 years) with non-small cell lung cancer.

AIMS: Palliative chemotherapy in non-small cell lung cancer (NSCLC) has been established since 1995 and little chemotherapy treatment was given to these patients before 1990. This retrospective study investigates the treatment outcome of elderly patients (age>or=70 years) with NSCLC over the past 13 years in a large UK cancer centre. MATERIALS AND METHODS: A comparison of all-cause survival between the time periods 1990-1994, 1995-1999 and 2000-2004 was adjusted for age, gender, stage and performance status. A comparison of survival was also made between three age groups: 70-74, 75-79 and 80+ years. RESULTS: Between 1990 and 2004, 302 patients>or=70 years had NSCLC. There were differences in age and performance status between the time periods. There was no improvement in median survival between the three time periods (P=0.6). There was little difference in outcome between the three age cohorts. CONCLUSIONS: The analysis shows that there has been no significant improvement in survival for elderly patients with advanced lung cancer treated with chemotherapy in the past 13 years.

Time and chemotherapy treatment trends in the treatment of elderly patients (age >/=70 years) with small cell lung cancer.

Platinum-based treatment for small cell lung cancer (SCLC) has been established since 1995. This study investigates treatment outcome of elderly patients (age >/=70 years) with SCLC over the past 20 years in a large UK cancer centre. Comparison of all-cause survival was assessed in patients presenting between two predefined time periods: 1982-1994 and 1995-2003. All the survival analysis were adjusted for stage and performance status and age if appropriate. Survival between different chemotherapy treatment regimens was compared. A total of 322 elderly patients (31% of all) registered between 1982-2003 received chemotherapy for SCLC. Patients presenting in 1995-2003 had an overall better median survival (43 vs 25 weeks) and a 1-year survival (37 vs 14%) than patients presenting in 1982-1994 (P<0.001). This applied to patients with both limited and extensive stage disease and all age groups. There was a trend towards the use of more platinum-based treatments in the later cohort but the use of radiotherapy remained constant. Patients who received platinum combinations (Carboplatin or Cisplatin) had significantly improved survival over those who received single agents or other combinations (P<0.001) and there was no significant difference between carboplatin and cisplatin (P=0.7). The analysis demonstrates that there has been a significant improvement in survival for elderly patients with lung cancer treated by chemotherapy in the past 20 years despite more very elderly patients being treated with a poorer performance status. This change is probably multifactorial and may be due to the increased use of platinum-based treatment and improved supportive care.

Electrophilic fluorination of pyroglutamic acid derivatives: application of substrate-dependent reactivity and diastereoselectivity to the synthesis of optically active 4-fluoroglutamic acids.

Electrophilic fluorination of enantiomerically pure 2-pyrrolidinones (4) derived from (L)-glutamic acid has been investigated as a method for the synthesis of single stereoisomers of 4-fluorinated glutamic acids. Reaction of the lactam enolate derived from 9 with NFSi results in a completely diastereoselective monofluorination reaction to yield the monocyclic trans-substituted alpha-fluoro lactam product 21. Unfortunately, a decreased kinetic acidity in 21 and other structurally related monofluorinated products renders them resistant to a second fluorination. In contrast, the bicyclic lactam 12 is readily difluorinated under the standard conditions described to yield the alpha,alpha-difluoro lactam 24. The difference in reactivity between the two types of related lactams is attributed mainly to the presence or lack of a steric interaction between the base used for deprotonation and the protecting group present in the pyrrolidinone substrates. This conclusion was reached based on analysis of the X-ray crystal structure of 21, molecular modeling, and experimental evidence. The key intermediates 21 and 24 are converted to (2S,4R)-4-fluoroglutamic acid and (2S)-4,4-difluoroglutamic acid, respectively.

Phosphinic acid pseudopeptides analogous to glutamyl-gamma-glutamate: synthesis and coupling to pteroyl azides leads to potent inhibitors of folylpoly-gamma-glutamate synthetase.

Several routes to a complex phosphinate phosphapeptide analogous to the gamma-glutamyl peptide Glu-gamma-Glu have been investigated. Formation of gamma-phosphono glutamate derivatives via addition of a phosphorus-based radical to protected vinylglycine was found to be of limited value because of the elevated temperatures required. Alkylation and conjugate addition reactions of trivalent phosphorus (P(III)) species were investigated. In situ generation of bis-trimethylsilyl esters of phosphinous acids proved to be an effective route to phosphinates of modest structural complexity. However, this chemistry could not be extended to the incorporation of an amino acid moiety at the N-terminal side of the desired phosphinate. A successful synthesis of the target phosphinate phosphapeptide was effected using P(III) chemistry and dehydrohalogenation to yield an alpha,beta-unsaturated phosphinic acid ester, following which conjugate addition of diethylacetamido malonate and acid-mediated hydrolysis afforded the desired phosphinate phosphapeptide. Coupling of the unprotected phosphinate phosphapeptide with two acyl azides derived from folic acid and methotrexate led to the corresponding pteroylphosphapeptides of interest as possible mimics of tetrahedral intermediates in the reaction catalyzed by folylpolyglutamate synthetase.

N-Me-pAB-Glu-gamma-Glu-gamma-Tyr(3-NO(2)): an internally quenched fluorogenic gamma-glutamyl hydrolase substrate.

A gamma-glutamyl tripeptide containing an internally quenched fluorophore has been synthesized and shown to be a substrate for recombinant rat gamma-glutamyl hydrolase. HPLC, LC-MS, and fluorescence spectra support the conclusion that selective hydrolysis occurs at the penultimate peptide bond. Preliminary data indicate that hydrolysis of this substrate can be monitored continuously to yield steady-state kinetic data.